Chitosan Manufacture Properties and Usage

BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE
CHITOSAN: MANUFACTURE,
PROPERTIES, AND USAGE
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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE
PROPERTIES, AND USAGE
SAMUEL P. DAVIS
EDITOR
Nova Science Publishers, Inc.

New York
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Chitosan : manufacture, properties, and usage / editor, Samuel P. Davis.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-61942-780-8 (eBook)
1. Chitosan--Biotechnology. I. Davis, Samuel P.
[DNLM: 1. Chitosan--chemistry. 2. Chitosan--therapeutic use. 3.
Biocompatible Materials. 4. Drug Delivery Systems. QU 83 C5436 2010]
TP248.65.C55C55 2010
660.6--dc22
2010022688
Published by Nova Science Publishers, Inc. † New York

CONTENTS
Preface vii
Chapter 1 Chitosan from Fungi 1
Mirko Trutnau, Thomas Bley and Jelka Ondruschka
Chapter 2 Chitosan: Modifications and Applications in Dosage Form Design 71
Ashok Kumar Tiwary, Bharti Sapra, Gurpreet Kaur and Vikas Rana
Chapter 3 Chitosan: Manufacture, Properties and Uses 133
C. K. S. Pillai, Willi Paul and Chandra P. Sharma
Chapter 4 Interpolyelectrolyte Complexes of Chitosan 217
N. A. Samoilova, M. A. Krayukhina and I. A. Yamskov
Chapter 5 N-Carboxyethylchitosan-Based Polymer Materials 261
Dilyana Paneva, Rosica Mincheva, Elena Yancheva,
Nevena Manolova, Olya Stoilova, Philippe Dubois
and Iliya Rashkov
Chapter 6 Chitosan Nanoparticles for Biomedical Applications 321
Paula Pereira, Vera Carvalho, Reinaldo Ramos and Miguel Gama
Chapter 7 Chitosan-Based Nanocarriers: Effective Vehicles for Mucosal
Protein Delivery 365
Luis Braz, Marita Dionísio and Ana Grenha
Chapter 8 Chitosan: A Potential Bio-Polymer for Drug Delivery 413
Sanjay K Jain, Piush Khare, Arvind Gulbake and Satish Shilpi
Chapter 9 Current Status of Chitosan on Dermal/Transdermal Drug Delivery
Systems 449
Ipek Ozcan, Taner Senyigit, Evren Homan Gokce and Ozgen Ozer
Index 485
PREFACE
Chitosan is a partially deacetylated derivative of chitin, a natural polysaccharide extracted

from crustaceans, insects and certain fungi. Owing to its unique properties such as
biodegradability, biocompatability, biological activity, and capacity of forming polyel-
ectrolyte complex with anionic polyelectrolytes, chitosan has been widely applied in the food
and cosmetics industry as well as the biomedical field in relation to tissue engineering, and
the pharmaceutical industry relating to drug delivery. This book gathers current research from
around the globe in the study of chitosan, including its manufacture, properties and usage
across a broad spectrum.
Chapter 1 - Chitosan was initially discovered in the mid-18th century, but remained little-
known until preliminary clarification of its crystalline structure in 1934 and further pioneering
studies by Muzzarelli and Hirano aroused further interest. Discovered in molds, and
commercially produced from crustacean shells, chitosan is now used in diverse applications.
Due to the seasonal lulls in fishery industries and the still-growing demand for high quality
chitosan, sources like mushrooms and other fungi are being re-evaluated. However, the crab
shells currently used to make chitosan are waste materials of the fishery industry. Hence,
chitosan production from fungi can only be economically competitive if waste mycelia from
the industrial use of fungi as bio-catalysts in “white biotechnology” , or waste carbon sources,
e.g. from food processing industries, are used as substrates for cultivating high chitosan-
yielding fungi. Many fungi are known to produce sufficiently high amounts of chitosan for
commercial production, and many treatments can reportedly enhance chitosan yields without
applying metabolic or genetic engineering techniques. However, although there many
potential sources, applications, and manufacturers, of chitosan - and many chemical and
physical techniques have been established for its characterization and quality control - it is
still very difficult to obtain chitosan that is fully standardized with respect to molecular
weight and degree of deacetylation, especially for pharmaceutical research.
Despite this problem, the chitosan research “community” is still growing, accompanied
by exponential growth in the annual number of publications on chitosan. Further, chitosan
was initially almost entirely used in macro-scale applications, but in recent decades many
micro- and nano-scale applications of chitosan in the form of nano-particles and composite
materials have emerged, and current foci are largely on such small-scale uses.
This chapter considers: the economic values of chitosan itself and the raw materials that
are potentially available for chitosan production (apart from the fishery industry); sources of
substrates and potential chitosan-producing fungi; cultivation techniques that can be used to
viii Samuel P. Davis
increase chitosan production; extraction methods; and laboratory protocols that can be used to
determine the quality of the extracted chitosan. In addition, current and future applications are
summarised and some results from the authors’ studies are presented on chitosan adsorption
of copper and 17ß estradiol, and the application of chitosan-containing substrates for
biomimetic coatings in tissue engineering.
Chapter 2 - Chitosan is the deacetylated form of chitin. Generally, the substance becomes
soluble in dilute acids when the degree of deacetylation is more than 50%. The solubility of
chitosan in dilute acids is often needed to be modified when specific drug release properties
have to be tailored into the dosage form. Chitosan carries free amine functionalities on the
deacetylated units and hydroxyl groups on the acetylated as well as deacetylated units.
Derivatization by introducing small functional groups such as, alkyl or carboxymethyl groups
can increase the solubility of chitosan at neutral and alkaline pH without affecting its cationic
character. In addition, chitosan can be grafted with other molecules through covalent binding.
The amino groups can be used for acetylation, quaternization, reactions with aldehydes and
ketones, chelation of metals etc. The hydroxyl groups can lend to o-acetylation, H-bonding
with polar atoms etc. Primary derivatization followed by grafting improves the solubility,
antibacterial, antioxidant, chelating, complexing, bacteriostatic and adsorbing properties
while maintaining its mucoadhesivity, biodegradability and biocompatibility. Functionalities
can also be used for interaction of chitosan with ions.
Chapter 3 - Chitin and Chitosan are natural polymers belonging to aminopolysaccharides
having interesting structural features for chemical modifications to generate novel properties,
functions and applications. Despite its huge availability, the utilization of chitin has been
restricted by its intractability and insolubility. Chitosan because of its improved solubility and
enhanced functions and properties find innumerable applications because of its
biocompatibility, biodegradability and non-toxicity together with its antimicrobial activity
and low immunogenicity. This review covers the production, properties and applications of
chitosan and their derivatives.
Chapter 4 - The results of studies of interpolyelectrolyte complexes of chitosan and
different polyanions are summarised and described systematically. Specific properties of
chitosan as polyelectrolyte are described. The general concept of the formation of
polyelectrolyte complexes is developed. Separate parts of the review are dedicated to
investigation of polyelectrolyte complexes of chitosan with:
-biopolyelectrolytes, including polysaccarides (plant, animal, bacterial polysaccarides and
lipopolysaccarides), proteins, nucleic acids and also modified natural polyanions
(carboxymethylchitin, carboxymetylcellulose, etc.),
-synthetic polyanions.
The data on application of polyelectrolyte complexes of chitosan in medicine and
biotechnology, particularly for creation of hemocompatible, thromboresistant materials,
bioconstruction materials for replacement of coverlets, blood vessels, bone tissue,
immobilization of biological active compounds, non-viral vectors of genetic information
transfer and so on are generalized.
Chapter 5 - N-carboxyethylchitosan (CECh) is a chitosan derivative. It preserves the
valuable properties of its precursor such as biocompatibility and biodegradability. Moreover,
CECh possesses some advantages such as larger variety of functionalities and solubility in
neutral and alkaline medium. The gained up to date knowledge on the synthesis of CECh, its
solution properties and biological behavior in respect to cells and pathogenic microorganisms
Preface ix
are emphasized in the chapter. Due to its amino- and carboxyl groups CECh behaves as a
polyampholyte/polyzwitterion in aqueous solutions. This chitosan derivative self-assembles
into nanoparticles in a pH range close to its isoelectric point. The conditions (pH of the
medium, ionic strength and polymer concentration) and CECh molecular weight for
nanoparticles preparation are described in details. Electrospinning represents a unique tool to
produce nanofibrous materials that resemble the extracellular matrix and thus are promising
for wound healing and tissue engineering applications. The fabrication of CECh-containing
nanofibrous materials by electrospinning is described in the chapter. Similarly to the case of
chitosan, the electrospinning of CECh from its aqueous solutions is rendered feasible by the
presence of a non-ionogenic polymer. In addition, CECh has been used for preparation of
organic/inorganic hybrid nanofibers containing superparamagnetic iron oxide or silver
nanoparticles by combination of the sol-gel technique and electrospinning. Some properties
and possible applications of these hybrids are detailed. As a polyampholyte CECh forms
polyelectrolyte complexes (PECs) with polyacids and polybases. The pH-dependent
formation of PECs; the preparation of hydrogel materials; as well as nanoparticles from
CECh-based complexes are discussed in the chapter. Finally, the design of non-woven textiles
by combining PEC formation and electrospinning is reported.
Chapter 6 - Chitosan is a rather abundant material with exquisite properties, which may
be processed into a variety of materials including hydrogels, fibres, membranes, etc. The
production of chitosan-based nanogels, also known as macromolecular miceles, has been
successfully achieved using different techniques, which will be reviewed. This chapter covers
the properties and applications of chitosan nanogels in the biomedical field, namely as a drug
delivery vehicle for biopharmaceuticals. The main achievements and recent developments
will be addressed.
Chapter 7 - Many newly designed therapeutic biomacromolecules, including protein-
based drugs, are characterised by reduced capacity to permeate biological membranes and/or
low stability in physiological environments. Over recent years, a major challenge of the
pharmaceutical industry has concerned the necessary development of suitable non-injectable
drug carriers that permit overcoming these limitations, opening the possibilities for the
administration of the referred molecules through routes which are alternative to the
parenteral. In this regard, nanocarriers have emerged as one of the most exciting tools to
circumvent these drawbacks, mainly owing to their increased surface-to-volume ratio, which
results in improved interaction with epithelial surfaces and, in some cases, in the ability to
cross epithelial barriers. Moreover, nanoparticles further enable the protection of the
encapsulated molecules, which retain their biological activity, permitting their administration
through routes that were previously unviable. The compelling need to design biocompatible,
biodegradable and non-toxic vehicles has turned polysaccharides into a very attractive class
of materials. In this group, chitosan has reached a position of evidence, due to its interesting
physicochemical and biopharmaceutical properties. Chitosan nanoparticles have in fact
proven to be very effective vehicles for systemic mucosal protein delivery, demonstrating
high capacity for protein association, enabling their protection from harsh environments and,
owing to chitosan mucoadhesive character, improving the proteins’ residence time in contact
with the absorptive epithelia. Overall, chitosan nanocarriers have been demonstrating the
ability to increase the bioavailability of encapsulated drugs. In this chapter, we present
chitosan-based nanoparticles that have been proposed for the administration of proteins
through mucosal routes such as the oral, buccal, nasal and pulmonary. More specifically, the
x Samuel P. Davis
various techniques and materials applied on the elaboration of the carriers, along with
chitosan, are analysed and the efficacy of these carriers is discussed, analysing the various
mechanisms proposed for their effectiveness.
Chapter 8 - Chitosan is a natural biopolymer derived from chitin found in the cell walls of
fungi, molds, yeasts and exoskeletons of crustaceans, mollusks, crabs and shrimps etc.
Chitosan is generally partially deacetylated polymer of N-acetylglucosamine which is second
most abundant polymer on earth after cellulose. Chitosan is biocompatible, biodegradable,
non-toxic and mucoadhesive polymer which has attracted the attention of the scientists and
researchers in the pharmaceutical and biomedical fields. Chitosan has many properties which
make it a suitable candidate to be utilized in various applications. Besides being biosafe it
possesses antimicrobial activity where it is effective against the gram negative bacteria and
also possesses antifungal activities. It has wound healing property. It also finds application in
the cosmetics and biomedical arena. Major impetus is being laid in the field of
pharmaceuticals where the polymer is being utilized for formulation development and novel
drug delivery research. The polymer has been deployed in the formulation of the extended
release tablets and various sustained release matrix tablets which have been utilized for the
treatment of gastrointestinal related ailments such as amoebiasis. The same has been used to
formulate the biofilms which have been developed for wound dressings and in manifestations
such as mucositis. Chitosan has been engineered to transform into novel drug delivery
systems such as nanoparticles, microspheres, gastrointestinal patches, buccal patches etc.
through a number of reported methods. There are a variety of published reports where the
chitosan microspheres have been utilized for colonic delivery and colon associated
malfunctions. The same have also been used for other gastrointestinal related manifestations
such as in H. pylori infection, duodenal ulcers etc. Moreover, these have been utilized for
targeted drug delivery for the cure of a particular diseased organ. In continuation to this the
nanoparticles have been utilized for the delivery of the antigen and protein i.e. in vaccine
delivery wherein the immunoadjuvant property of the polymer has been harnessed to increase
the potential of the vaccine since chitosan also improves the transfection efficiency and hence
the absorption of drug or protein is enhanced. The gastrointestinal patches and other delivery
systems have also been developed against a variety of malfunctions. This chapter is directed
towards the elucidation of the use and development of the chitosan in the drug delivery field
and includes a detailed discussion of the same alongwith a focus on the future prospects of the
polymer in drug delivery.
Chapter 9 - In case of targeting the drug to the desired part of the skin, vehicles play an
important role, beside the characteristics of the drug. Many natural and synthetic vehicles
have been used for various topical dermal/transdermal preparations. However, chitosan has
been standing out with its many advantages based mainly on its biological and
physicochemical properties. Chitosan is a unique hydrophilic biopolymer obtained by partial
deacetylation of chitin, which is one of the most abundant polysaccharide. It is a natural
product widely found in crustacean shells, fungal cell walls, insect exosceletons, and
mollusks. Chitosan is a linear glycosaminoglycan made up of N-acetyl-D-glucosamine units.
Characteristics of chitosan, such as the molecular weight, viscosity and the degree of
deacetylation, greatly influence the properties of formulations. The by-products formed after
the biodegradation of the polymer does not cause immune responses making it biocompatible.
Due to the specific cationic glucosamine groups of chitosan, it can be interacted with anionic
proteins in the skin providing the bioadhesive characteristics. These properties result in
Preface xi
improved efficacy, enhanced bioavailability and reduced toxicity -generally recognized as

safe (GRAS). Furthermore, the antimicrobial/ antibacterial and skin hydrating effects of
chitosan have been received considerable attention for dermal/transdermal applications. It
plays an important role in the cell regulation, tissue regeneration and collagen production.
Chitosan and some of its complexes were approved by FDA for use in wound dressing
products.
Chitosan also provides the controlled release of numerous active agents used for the
treatment of skin diseases such as corticosteroids, antifungal agents, nonsteroidal anti-
inflammatory drugs, hormones, local anesthetics, antiviral and antiseptic agents, etc.
Regarding to the good bioadhesive property of chitosan and its ability to sustain the release of
the active compounds, it has found many practices in the formulation of gels,
dermal/transdermal patches, sponges, micro- and nanoparticulate systems as drug carriers.
Particularly, chitosan has been used in the preparation of mucoadhesive formulations, for
improving the dissolution rate of the poorly soluble drugs, drug targeting and enhancement of
peptide absorption.
This paper is focused on the use of chitosan for dermal/transdermal drug delivery systems
following a general overview of chitosan. This natural polymer is a promising carrier or
excipient as a delivery system and remarkable advances have been made about its potential
applications in skin delivery.
In: Chitosan: Manufacture, Properties, and Usage ISBN 978-1-61728-831-9
Editor: Samuel P. Davis © 2011 Nova Science Publishers, Inc.
Chapter 1
CHITOSAN FROM FUNGI
Mirko Trutnau1,∗, Thomas Bley1 and Jelka Ondruschka2,•

1
TU Dresden, Institute of Food Technology and
Bioprocess Engineering, Dresden, Germany
2
SIAB, Saxon Institute of Applied Biotechnology,
Leipzig, Germany
ABSTRACT
Chitosan was initially discovered in the mid-18th century, but remained little-known
until preliminary clarification of its crystalline structure in 1934 and further pioneering
studies by Muzzarelli and Hirano aroused further interest. Discovered in molds, and
commercially produced from crustacean shells, chitosan is now used in diverse
applications. Due to the seasonal lulls in fishery industries and the still-growing demand
for high quality chitosan, sources like mushrooms and other fungi are being re-evaluated.
However, the crab shells currently used to make chitosan are waste materials of the
fishery industry. Hence, chitosan production from fungi can only be economically
competitive if waste mycelia from the industrial use of fungi as bio-catalysts in “white
biotechnology” , or waste carbon sources, e.g. from food processing industries, are used
as substrates for cultivating high chitosan-yielding fungi. Many fungi are known to
produce sufficiently high amounts of chitosan for commercial production, and many
treatments can reportedly enhance chitosan yields without applying metabolic or genetic
engineering techniques. However, although there many potential sources, applications,
and manufacturers, of chitosan - and many chemical and physical techniques have been
established for its characterization and quality control - it is still very difficult to obtain
chitosan that is fully standardized with respect to molecular weight and degree of
deacetylation, especially for pharmaceutical research.
Despite this problem, the chitosan research “community” is still growing,
accompanied by exponential growth in the annual number of publications on chitosan.
Further, chitosan was initially almost entirely used in macro-scale applications, but in
recent decades many micro- and nano-scale applications of chitosan in the form of nano-
∗ Bergstraße 120, D-01062 Dresden, Germany.

• Permoserstraße 15, D-04318 Leipzig, Germany.
2 Mirko Trutnau, Thomas Bley and Jelka Ondruschka
particles and composite materials have emerged, and current foci are largely on such
small-scale uses.
This chapter considers: the economic values of chitosan itself and the raw materials
that are potentially available for chitosan production (apart from the fishery industry);
sources of substrates and potential chitosan-producing fungi; cultivation techniques that
can be used to increase chitosan production; extraction methods; and laboratory protocols
that can be used to determine the quality of the extracted chitosan. In addition, current
and future applications are summarised and some results from the authors’ studies are
presented on chitosan adsorption of copper and 17ß estradiol, and the application of
chitosan-containing substrates for biomimetic coatings in tissue engineering.
1. INTRODUCTION
Chitin was first isolated from mushrooms and named fungine in 1811 [1]. About 12 years
later chitin was also discovered in insects [2]. Not until 1859 However, chitosan was not
isolated (by boiling chitin in a potassium hydroxide solution) until 1859 [3]. Since then it has
been clear that chitosan can be derived from chitin by deacetylation.
Chitin (β-1,4-N-acetyl-D-glucosamine) is a component of the cell walls of fungi, insects
and crustaceans, and after cellulose it is the most abundant natural biopolymer, with at least
1010 tons constantly present in the biosphere [4]. Chitin represents an essentially inexhaustible
(gigaton), permanently available source of chitosan; arthropods (which account for more than
106 species of the 1.2x106 total of recognized species in the animal kingdom) alone constitute
a massive source [5]. In contrast, chitosan is only found in the cell walls of certain groups of
fungi, especially Zygomycetes.
Chitosan is easily extractable with solutions of bulk chemicals like HCl and NaOH from
crab fishery wastes. The global amount of chitin annually extracted from waste seafood
material is ca. 40,000 tonnes, and currently a few thousand tonnes of chitosan is produced per
year (mostly in Japan).
The main problem associated with obtaining chitosan from seafood waste is the seasonal
availability, which does not affect fungal chitosan. Thus, to meet the global demand for
chitosan continuous production is necessary, which necessitates use of other sources, like
mushrooms or other fungi. Hence, chitosan from fungi is unlikely to replace, and is more
likely to supplement, current marine sources.
The great advantage of fungal chitosan is that it can be extracted directly from fungal
biomass, independently of seasonal fluctuations, while for fungi that only produce chitin in
their cell walls a more complex extraction procedure is required, very similar to the one used
to obtain chitosan from crustaceans. Hence it might be more economical to use only fungi that
already provide chitosan..
A wide variety of fungi are able to produce chitosan, at contents and yields varying from
0.3-12.5% of cell dry weight. Furthermore, they can be cultivated on virtually any substrate,
although for sustainability the best substrates are clearly by-products or waste products of
other industrial processes, such as rinse washes from distilleries, molasses, whey retentate, or
soybean and mungbean residues. Alternatively, chitin and/or chitosan can be produced
directly from waste mycelia of common industrial fungi, e.g. Trichoderma reesei mycelia
from cellulase and hemicellulase production or Penicillum verruculosum, Aspergillus niger or
Rhizopus oryzae mycelia from antibiotic, citric acid or lactic acid production, respectively. In
Chitosan from Fungi 3
this context it is worth noting that more than 80,000 tons of waste Aspergillus niger
mycelium per year is generated from citric acid production alone [6].
A further source is the mushroom industry. The total global production of mushrooms in
2006 reached 20 million tonnes, of which 14 million tonnes was produced in China [7]. 5-
20 % is wasted in the form of stalks or irregular dimensions and shapes, depending on the size
of mushroom farm [6]. Hence, 1-4 million tons of waste mycelia is available for
chitin/chitosan production from mushrooms alone, which are generally not used, especially in
the United States [8].
2. OVERVIEW OF CURRENT INDUSTRIAL PRODUCTION AND

ECONOMICAL ASPECTS
Most of the commercially available chitin and chitosan is produced in Asian countries.
There are more than 200 suppliers and manufacturers of chitosan globally, and about 50 % of
total capacity is in China. Conventionally, chitosan is derived from chitin in seafood waste. In
2008 Japan was the largest market for chitin derivatives, consuming 20,000 tonnes, and by
2012 this is expected to rise to more than 51,400 metric tons. In addition, there have been ca.
21.5 % and 24.5 % compound annual growth rates in the US market and Asian-Pacific
markets for chitin derivatives in the first decade of this century [9]. The largest chitosan
production facility, with an annual output of about 1000 tonnes came on stream in Tangshan
in 2002. However, only a few companies, e.g. KitoZyme, are currently producing chitin and
chitosan from fungi.
The development that has had the most positive effect on chitosan production has been its
use as a dieting aid, since it promises a convenient way to lose weight via its fat-absorbing
effect. Lifestyle and wellness products enjoy great popularity; in 2002 the European market
for dieting supplements was worth about 15.2 bn dollar, and this had increased to 17.5 bn by
2006 [10]. €7bn of the chitosan produced globally, mostly sourced from shellfish (the world’s
first fungal chitosan was ready for release on the weight loss market in 2009), is produced for
weight management [11]. Currently, there are more than 40 different dietary supplements on
the market that include chitosan as a fat-absorbing agent, with end-user prices ranging from
180-370 EUR/kg. In 2009 the market price of chitosan was about 16$/kg [12], but it is
decreasing with increases in the number of factories producing it..
Companies like Advanced Biopolymers, Biothera, CarboMer, Dalian Xindie Chitin,
HaloSource Inc, Heppe GmbH, Kunpoong Bio, Meron Biopolymers, Navamedic, Primex Ehf,
Qbas, Taizhou Candorly Sea Biochemical and Health Products, United Chitotechnologies
and V-Labs, Inc. are the key global players [9].
3. USABLE FUNGI AND SUBSTRATES

In order to further consider chitosan production we need to differentiate between sources
and discuss possible extraction and processing options in more detail. Chitosan can be
extracted directly from various, specific fungi or chitin can be extracted and then
deacetylated. Chitin is a more widely synthesized polymer in the fungi kingdom than
chitosan, being produced by Zygomycetes, Ascomycetes, Basidomycetes, Deuteromycetes

and Phycomycetes. In contrast, chitosan is found only in the cell walls of certain groups of
fungi, especially Zygomycetes. The great advantage of producing chitosan from fungi is that
it can be directly extracted from fungal biomass at any time, avoiding seasonal fluctuations.
For fungi that only provide chitin in their cell walls an almost identical extraction procedure
to that used to extract chitosan from crustaceans must be applied. Hence, it is more
economical to use only fungi that already provide the target product. For instance, ready-to-
use chitosan can be reportedly produced from Absidia coerulea very simply by boiling in
25 % NaOH without extracting with hot acetic acid because the chitosan is not intermingled
with glucan [13].
Table 1. Reported chitosan production potential from selected yeasts, mushrooms and
other fungi. The listed chitosan contents are total amounts of extract, including
deacetylated chitin
1
Fungal strains Chitosan content [% Productivity Comment Reference
CDW] [mg/L/d]
Absidia atrospora 3 61.20 [14]
barley-buckwheat-shochu
2.1 26.46 distillery wastewater [14]
sweet potato-shochu
5.8 59.16 wastewater [14]
A. glauca 7.4 325.23 [15]
A. coerulea 1.3 46.41 [15]
6.3 n.a. [16]
0.16 (of dry weight
of substrate) 4571.4 [mg/kg/d] Cotton seed hulls [17]
0.20 (of dry weight
of substrate) 5571.4 [mg/kg/d] Corn residue [17]
0.22 (of dry weight
of substrate) 6257.1 [mg/kg/d] Soybean residue [17]
0.23 (of dry weight
of substrate) 6285.7[mg/kg/d] Potato pieces [17]
Agaricus bisporus 0.9 29.02 [15]
Ashbya gossypii 0.3 4.61 [15]
Aspergillus clavatus 0.9 12.39 [15]
A. flavus 2 56.70 [15]
A. nidulans 3.9 100.43 [15]
A. niger 0.8 37.16 [15]
11 165.00 [18]
1.7 (of dry weight of
substrate) 1417.7 [mg/kg/d] soybean sst [19]
A. oryzae 1.1 23.42 [15]
A. terreus 0.5 12.37 [15]
A. terricola 3.4 70.38 [15]
A. usamii 1.3 42.64 [15]
Blakeslea trispora 1.4 30.38 [15]
Botrytis cinerea 1.9 13.13 [15]
Ceratocystis ips 2.4 70.80 [15]
1
Fungal strains Chitosan content [% Productivity Comment Reference
CDW] [mg/L/d]
Cladosporium
cucumerinum 1.99 50.75 [15]
C. cladosporioides 4.1 19.56 [15]
Candida albicans 4.4 39.60 [20]
Epicoccum nigrum 0.7 7.59 [15]
Gibberella fujikuroi 1.3 30.33 [15]
Gliocladium
catenulatum 1.6 51.52 [15]
Gongronella butleri 1.4 38.22 [15]
5714.3 [mg/d/kg
4-6 wet potatoes] potatoes [21]
6.3-7.9 96.6-105.8 [15]
barley-buckwheat-shochu
0.9-1.3 8.2-15.4 distillery wastewater [14]
sweet potato-shochu
7.3 70.40 wastewater [14]
Humicola grisea 1 29.75
Lentinus edodes 3.3 5.13 [20]
Mucor hiemalis 2.4 47.18 [15]
M. rouxii 3.8 105.45 [15]
12.5 10.10 [22]
5-7 100.1-139.9 [23]
4-8 108-627 [24]
1.2 - [25]
7.3 145.90 [26]
7.7 457.50 [27]
8 450.00 [27]
1-4.4 111.6-484.9 [28]
0.8-0.9 37.5-86.0 [15]
Myrothecium
verrucaria 1.3 10.45 [15]
Penicillium
chrysogenum 0.4 10.96 [15]
P. digitatum 2.9 27.84 [15]
P. blakesleeanus 2.7 29.90 [15]
Pleurotus sajo-caju 1.2 5.71 [20]
Rhizopus oryzae 0.9 45.23 [15]
14 59.15 [20]
8-10 277.25 molasses [29]
7.5-8.8 7.1-8.25 [30]
R. pusillus 8 [31]
R. stolonifer 1.9 33.92 [15]
Sclerotinia
sclerotiorum 1 5.27 [15]
Trichoderma viride 0.9 24.64 [15]
T. roseum 0.9 12.99 [15]
Zygosaccharomyces
rouxii 3.6 52.80 [20]
1
Substrate used for cultivation; if none mentioned a synthetic medium was used.
Table 1 shows reported chitosan and chitin synthesis capacities of a range of fungi. It
should be noted that it provides biased comparisons in some cases, since almost all of the
cited investigations were laboratory scale, i.e. the estimated yields were obtained from
shaking flask experiments with synthetic media.
Table 2. Industrial waste products that can be used as substrates for cultivating
fungal biomass
Fungal strains Source Waste product Reference

Absidia atrospora Distillery barley-buckwheat-shochu distillery wastewater [14]
Distillery sweet potato-shochu wastewater [14]
A. coerulea Agriculture Cotton seed hulls [17]
A. coerulea Agriculture Corn residue [17]
A. coerulea Agriculture Soybean residue [17]
A. coerulea Agriculture Potato pieces [17]
A. niger soybean
Agriculture [19]
Gongronella butleri Agriculture potatoes [21]
G. butleri Distillery barley-buckwheat-shochu distillery wastewater [14]
G. butleri Distillery sweet potato-shochu wastewater [14]
Rhizopus oryzae Distillery molasses [29]
Furthermore, widely differing extraction protocols were employed, making it difficult to

compare all the results in a valid manner. Nevertheless, it clearly illustrates the broad
spectrum of chitosan producers and yields. So, the question is not whether we can produce
chitosan from fungi, but how can we do it in the most economical and effective way. There
are two possible answers to this question, since the main factors affecting production costs are
the substrate used to cultivate the organism and the cultivation energy required (basically for
aeration and mixing). We can choose between the following two options:
I. to use by-products or waste products of some other industrial process, such as rinse
washes from distilleries, molasses, whey retentate, or soybean and mungbean
residues, or
II. waste mycelia of common industrial fungi e.g. Trichoderma reesei mycelia from
cellulase and hemicellulase production or Penicillum verruculosum, Aspergillus
niger or Rhizopus oryzae mycelia from antibiotic, citric acid or lactic acid
production, respectively. As noted above, citric acid production alone generates
about 80,000 tonnes of waste A. niger mycelium per year [6].
In both cases the first step following initial biomass production is to generate the main
target product (e.g. sugar or cellulase). Then, using either a waste product or waste biomass
from the first production process as a substrate, a second value-adding product (chitosan) can
be created, thereby raising the profitability of the entire process.
When using shellfish waste from shrimp, crab or lobster there is a similar, second value
addition. However, a deacetylation step is required because only chitin can be extracted from
the exoskeleton of crustaceans. Since most industrially used fungi and yeast strains also
contain chitin, production of chitosan from their biomass will not have any clear advantage
relative to crustacean chitosan, it merely provides another way to satisfy market demand. In
contrast, using chitosan-producing fungi directly does have advantages in terms of the savings
of energy and chemicals required for the deacetylation and washing steps. A further
possibility is to convert chitin to chitosan under mild conditions using the enzyme chitin
deacetylase (CDA) [32,33], which can be produced in several ways. Moulds and other fungi
are obvious sources of this enzyme, since it participates in their metabolism. However, plants
[34] and insects [33] are also able to synthesize CDA, and in addition to the many natural
sources CDA could be produced in the future by well understood recombinant organisms,
such as Escherichia coli [35].
As shown in Table 1, a wide variety of fungi can produce chitosan, at contents and yields
varying from 0.3-12.5% of cell dry weight. Furthermore, they can be cultivated on many
substrates, but (as mentioned above) for sustainability the ideal substrates are clearly by-
products or waste products of other industrial processes, such as rinse washes from
distilleries, molasses, whey retentate, or soybean and mungbean residues (Table 2). In many
studies refined carbon sources, like glucose and sucrose, have been used. The final price of
products of cultivations with such carbon sources will depend on the market price of the raw
material (e.g. sugar or some other agricultural product). However, these sources are also used
for generating “green” biofuels, like ethanol, so market prices are driven by industrial energy
demands. Thus, chitosan production in this manner is only viable if waste products are used.
Therefore investigations of the growth of candidate fungi on such substrates seem to be of
most relevance in attempts to develop commercial processes from scientific studies.
3.1. Factors Influencing Chitosan Yields
In order to optimise productivity all production stages have to be considered, both

upstream and downstream of chitin/chitosan extraction. The upstream stages are mainly the
cultivation processes. There are many factors that influence the growth of fungi, but the most
critical are pH, oxygen supply, energy input for stirring (which also affects the oxygen
supply) and of course the cultivation medium. To optimise the upstream processes (and
others) various methods can be used, as outlined below.
3.1.1. Optimisation of Experimental Conditions by Statistical Experimental Design

Optimal experimental design (OED) involves model-based evaluation of process
parameters to maximise or minimise outputs and target parameters, as appropriate, robustly
and using as few experiments as possible (accounting for interactions between parameters
where feasible). This procedure was applied by Göksungur [29] to optimise chitosan yields of
Rhizopus oryzae, using a face central statistical design to select experiments. The effects of
the parameters agitation rate, aeration rate and sugar concentration on the chitosan yield were
investigated by varying each of them at three levels, and a polynomial response surface for
the chitosan concentration (in mg dm−3) was generated from the modelled data. The optimal
process conditions were found to be a sugar concentration of 45.4 g/L, with an aeration rate of
2.1 vvm and an agitation speed of 340 rpm.
3.1.2. Enhancement of Chitosan Content by Using Plant Growth Hormones

Plant growth hormones (phytohormones) are not true hormones, as defined for animal
hormones, since they do not act solely as intercellular signal molecules that are active at sites
other than their production site, but rather they transport information to other cells [36].
However, phytohormones are like animal hormones in several other ways, e.g. they are active
at very low concentrations and do not act as nutrients. They can be classified in five groups
[37]:
1. auxins
2. cytokinins
3. gibberellins
4. ethylene
5. abscisic acid
Auxins, gibberellins and cytokinins are the most widely used phytohormones to influence
fungi [38,39].
In plant cells auxins (e.g. indoleacetic acid, indolebutyric acid, phenylacetic acid and
indole-3-acetamide), in combination with cytokinins, promote the growth of calli and regulate
morphogenetic development [37].
Gibberellins (e.g. gibberellic acid) play a major role in the promotion of elongation
growth in stems and leaves [36]. They comprise a large family of structurally related
diterpenoid acids that occur in plants, fungi and bacteria, of which giberellic acid (especially)
can be produced in large amounts by some fungi, notably Gibberella fujikuroi [40].
Cytokinins (e.g. kinetin) stimulate protein synthesis and participate in cell cycle control in
intact plants [41].
Recent studies have shown that hormones (gibberellic acid, indole-3-acetic acid, indole-
3-butyric acid and kinetin) can increase mycelial growth of Rhizopus oryzae by 19–32% [38].
However, the accompanying increases in chitosan content of the mycelia were generally
relatively small. The strongest effect was observed when using 0.1 mg/L gibberellic acid,
which resulted in a 50% increase in chitosan production, but other tested phytohormone
treatments resulted in 1.7–14.3% increases. Higher hormone concentrations inhibited growth
and reduced chitosan contents. The average molecular weight was also increased, which
represents an increase in quality. Further, all tested hormones enhanced the CDA activity of
Rhizopus oryzae by 6.7 to 26.7%, which was held responsible for the increased chitosan
production.
The effects of the same hormones on Mucor rouxii have also been examined [39].
The mycelial yield was increased by 12% to 17.4%, while the chitosan yield could be
increased by 34% to 69%. Again, gibberellic acid was the most effective, enhancing chitosan
production by 69% compared to controls when supplied at 3mg/L. In both studies the degree
of acetylation was not affected by the addition of hormone to the medium, but the average
molecular weight of chitosan increased by more than 50%.
3.1.3. Use of Fungal Properties to Enhance the Chitosan Yield

From the literature it is known that some microorganisms seem to have an inherent ability
to adapt to a certain level of mechanical stress with time [42].
10
9
batch (F4.1, 22.25 h)
batch (F4.2, 24 h)
batch (F7.2, 23.25 h)

batch (F7.1, 23.75 h)
8
Yield of chitosan [%]

7
batch (F11.3, 25 h)
batch (F11.1, 40 h)
batch (F11.2, 25 h)
repeated
6
repeated
batch (F3, 49.25 h)

batch (F5, 24 h)
repeated
5
batch (F2, 24 h)
repeated
batch (F10, 38 h)
4
3
2
1
0
Experiment
From Trutnau et al. [28] with permission.
Figure 1. Comparison of chitosan yield per gram cell dry weight from various batch and semi-
continuous experiments. Data from semi-continuous experiments are shown by the dashed outlines.
Batch F11.1 was inoculated with a spore suspension whereas 600 mL portions of a 24 h subculture
were used in the other batches. The repeated batches where inoculates with 500 mL of the previous
batch. All experiments were carried out under the same conditions in terms of pH and temperature set
point, aeration, agitation rate and medium composition.
10
9
batch (F4.1, 22.25 h)
batch (F4.2, 24 h)
batch (F7.2, 23.25 h)

batch (F7.1, 23.75 h)
8
Yield of chitosan [%]
7
batch (F11.3, 25 h)
batch (F11.1, 40 h)
batch (F11.2, 25 h)
repeated
6
repeated
batch (F3, 49.25 h)

batch (F5, 24 h)
repeated
5
batch (F2, 24 h)
repeated
batch (F10, 38 h)
4
3
2
1
0
Experiment
Figure 2. Comparison of chitosan production rates [mg Chitosan/L/d] in various batch and semi-
continuous experiments. Data from semi-continuous experiments are shown by the dashed outlines.
Batch F11.1 was inoculated with a spore suspension whereas 600 mL portions of a 24 h subculture
were used in the other batches. The repeated batches were inoculated with 500 mL of the previous
batch (See Figure 1). All experiments were carried out under the same conditions in terms of pH and
temperature set points, aeration, agitation rate and medium composition.
For this, the cell wall has to become more stable to prevent rupture. The structural
components of fungal cell walls are chitin and chitosan. Hence, such adaption should increase
the contents of these components. Repeated batch cultivations of Mucor rouxii have indicated
that this seems to be true (Figure 1, from Trutnau et al. [28] with permission).
The data clearly show that fresh batch cultures gave lower chitosan contents of the cell
dry weight and production rates than semi-continuous cultures (in which previous cultures
were reduced in volume and re-started with fresh media). Hence, the productivity of semi-
continuous cultures was also higher, as depicted in Figure 2 (from Trutnau et al. [28] with
permission) and calculated from:
C X ⋅ Ychitosan / X
rP = (1)
tCult
Since the chitosan yield in single batch experiments is generally lower than the yield
from repeated batches a longer cultivation period combined with a sufficiently high substrate
concentration appears to be required for significant adaptation. Neither of these conditions are
usually met, especially towards the end of single batch cultures. Hence, fed-batch or semi-
continuous processes are necessary to provide these two requirements for high chitosan yields
(Figure 1), as indicated by the hyphal length and chitosan contents of two batch cultures (F10
and F11.1) compared in Figure 3.
A batch culture with short hyphae and low numbers of tips (small mycelia) gives a lower
chitosan yield (compare F10/F11.1 in Figure 2 and Figure 3) than a culture with long hyphae
and a high number of branches, i.e. tips. Hence the stability of the mycelium, the chitosan
content and the length of hyphae are directly connected.
Figure 3. Comparison of data from two batch experiments (F10 and F11.1) with Mucor rouxii. On the
left the estimated and simulated average numbers of tips per hyphal element, represented as integers
with branching rate constants of 25 tips/µm/h (F10) and 41 tips/µm/h (F11.1). On the right,
measurements and simulation of the average total length per hyphal element with tip extension rate
constants of 46 µm/tip/h (F10) and 30 µm/tip/h (F11.1). The cultures were inoculated with a spore
suspension overnight and the first samples were taken after 12 h.
Thus, by recycling stress-adapted cultures in a semi-continuous batch mode, it has been

found that chitosan yields as well as productivity can be almost doubled. Other researchers
suggest that the optimal time for harvesting batch cultures is at the end of the exponential
phase [43], which agrees with these observations.
4. EXTRACTION OF CHITOSAN
The cell wall provides cells mechanical and chemical stability and possibilities to interact
with their environment via exchanges of nutrition and metabolites. It is a complex structure of
proteins, lipids and polysaccharides beside many other minor components. Polysaccharides,
proteins and lipids account for up to 80%, 3-20% and up to 5% of the cell walls’ dry weight,
respectively [44]. Depending on the fungal species, chitin, chitosan and glucans are the most
abundant carbohydrates. A detailed description can be found in the literature, e.g. [44].
Different stepwise procedures are required to extract chitosan and chitin from the cell
wall since different compounds are associated with them, and they have different chemical
properties, inter alia differences in solubility and other characteristics in acidic and basic
milieu.
The most resistant compounds are the polymers chitin, cellulose, chitosan and glucan.
Hence for a successful extraction one has to remove all other compounds like proteins and
lipids. Various protocols have been used for this in published studies, which can be divided
into chemical and enzymatic procedures, as illustrated in Figure 4 (where the enzymatic
method is on the right). The chemical procedures can be further divided into alkali- and acid-
based methods. The extraction of chitin from fungi is similar to its extraction from crustacean
shells. The main difference is the higher fraction of CaCO3 in the latter, which is a major
component of crustacean waste. For chitin 5% NaOH is usually used in the first step to
remove proteins and lipids, while 30% HCl is used to remove the CaCO3, which dissolves in
acid. The insoluble fraction remaining after these treatments is chitin. To obtain chitosan from
shells 5% of HCl or EDTA is initially used to dissolve or bind the calcium carbonate, then
40% NaOH at 110 °C to remove the proteins and lipids, with parallel deacetylation of chitin
to chitosan. Chitin normally accounts for 14-27 % of the dry weight of shells, of which 60-
80 % can be converted to chitosan. A great advantage of fungal chitosan is its very low
contents of inorganic material. Hence, demineralization is not required..
Many protocols have been proposed and applied for fungal chitosan extraction in
published studies. The most common general procedure (for which there are many variations
in process parameters, especially in concentrations of NaOH and acetic acid, temperature and
treatment times, depending on the biomass source) is shown on the left in Figure 4. For
deproteination, NaOH is the caustic agent of choice although other hydroxides could also be
used. Chitosan is soluble in many acids, but not sulphuric acid [31].
Extraction experiments have shown that use of hydrochloric acid instead of acetic acid
has advantages in terms of the final yield [24], but in most protocols acetic acid is used as
extraction agent, due to the higher degree of deacetylation of the extracts and the lower rate of
depolymerization. Of the three main extraction options the filtration protocol is simplest,
since the others require numerous steps, including washing, neutralisation and centrifugation.
The filtration protocol requires less steps by exploiting the solubility of chitosan in sulphuric
122 Mirko Trutnau,
T Thom
mas Bley and Jelka
J Ondruschka
accid at temperratures above 90°C (like proteins p and lipids) and itsi insolubilityy at lower
teemperatures [3 31]. Chitin is not soluble att either above or below 90°°C, so it can be b removed
byy hot filtration
n. Subsequent cooling of thhe filtrate makes the chitosann precipitate, but not the
lippids and proteins. Hence chitosan cann be extractedd by just onee “chemical” step. The
reesulting filter cake, which contains
c fractiions of chitin, can be furtheer deacetylatedd (Figure 4
daashed box) an nd filtered, finally yielding chitosan.
c Com mmonly, myceelial waste of Aspergillus
A
niiger from citrric acid prodduction is treaated by the trraditional acidd-alkali method but the
chhitosan obtainned is of poor quality
q [45].
The enzym matic method is i applied under physiologiccal conditions with no harshh treatment
exxcept the aciddic extraction. Stepwise treaatments with lyysozyme, snaiilase, proteasee and chitin
deeacetylase aree used to cut outo the chitosaan from the associated
a celll wall materiaal [32]. The
grreat advantagees of this metthod are that the reactions are performed under mild conditions
annd no highly caustic
c wastewwater is producced. In principple it might alsso be possiblee to recover
thhe used enzym mes for further use, but problems might arrise from the relatively
r longg enzymatic
trreatments.
Fiigure 4. Compaarison of differeent protocols forr extracting chittosan from funggi. Dashed boxees indicate a
sttep (deacetylatio
on) that is not necessary
n if chittosan is directlyy extracted from
m biomass withoout using the
chhitin which can be further convverted to chitosaan by deacetylaation.
5. QUALITY ASPECTS
Commercially available chitosan is mainly obtained from crustacean chitin by chemical
deacetylation under strongly alkali conditions. The physico-chemical properties of the
chitosan obtained by this method can be inconsistent due to the variability of the raw
materials and the harshness of the isolation and conversion processes [24].
In order to produce chitosan of a more consistent quality Zygomycetes have been
considered as alternative sources. The most important advantage of these organisms is that
their cell walls already contain large quantities of chitosan. Further, the physico-chemical
properties of this chitosan can be manipulated and standardized by controlling the
fermentation parameters. For instance, chitosan of different molecular weights is produced
when these fungi are grown on media differing in pH and composition [46] as well as when
different growth hormones are applied (section 3.1.2.). Finally, the chitosan can be extracted
from fungi by simpler and milder treatments than the chemical process required to extract it
from crustacean shells (section 4).
The preparation method affects the molecular weight and degree of deacetylation of the
chitosan obtained, which also depend on the process conditions.
Two of the main limitations in the use of unmodified chitosan in several applications are
its high viscosity when dissolved in acetic acid and low solubility at neutral pH.
5.1. General Properties of Chitosan
Chitosan is a mucopolysaccharide and, ideally, a linear polymer of 1,4-ß glycosidically

linked glucosamine (2-amino-2-deoxy-β-d-glucopyranose), but usually there are also residues
of acetylglucosamine (2-acetamido-2-deoxy-ß-d-glycopyranose). If the content of these
acetylglucosamines is lower than 50 % the substance is classified as chitosan, otherwise
chitin. Chitin is known to be the most abundant polymer on earth after cellulose.
The great potential for using both chitin and chitosan is not solely based on their
abundance. Their two-fold, helical, cellulose-like structure [47]. combined with the functional
amino groups and acetyl amino groups of chitosan and chitin, also affords an extraordinary
range of possible applications. From a chemical perspective, chitosan from fungi is the same
as that from of other sources, i.e. crustaceans and insects. However, the average molecular
weight of fungal chitosan is lower (<200 kDa) than derivatives from crustaceans (up to
1000 kDa), as shown in Table 5. Low molecular weight chitosan in the range of 5–10 kDa is
attracting increasing attention since it offers higher specific activity compared to highly
polymerized glucosamine chains [17].
The degree of polymerisation of the final product depends on the extraction parameters,
i.e. the reaction time, the concentration of the acids used and the temperature. Use of milder
conditions increases the average molecular weight [48]. While chitin is insoluble in acidic
solutions unmodified chitosan is only soluble in acidic liquids. The solubility is influenced by
the degree of acetylation and the distribution of acetylated glucosamines as well as the
stiffness of the polymer, which increases with increasing homogeneity of their distribution
[49,50]. Chitosan has an orthorhombic crystalline structure with cell dimensions of the fibre
axis of 8.9, 17 and 10.25 Â [51].
Chitosan is a weak alkaline polyelectrolyte [52]. A cross-linked unmodified chitosan

membrane is almost non-conductive in dry state, but is ionic-conductive when fully hydrated
[53].
5.2. Characterization Methods
Despite the large numbers of commercial manufacturers who offer chitosan of various
quantities and qualities, and increasing interest in potential applications of chitosan in drug
delivery and other medical contexts, it is still very difficult to obtain chitosan that is fully
standardized with respect to molecular weight and degree of deacetylation for pharmaceutical
research. Hence, for any successful application key characteristics - e.g. degree of
deacetylation (DD), molecular weight (MW), polydispersity and crystallinity - of the material
must be thoroughly classified and validated to ensure it is of sufficient quality. The degree of
deacetylation also provides important information about the solubility of chitosan, which is
essential for optimising the manufacturing process and the scope for modifying the reactive
amino group. The molecular weight determines the viscosity of solutions and increases with
increasing MW. Hence, this is a parameter that influences procedures such as spraying
chitosan coatings. The crystallinity influences the stiffness of the polymer and knowledge of
the crystal structure provides further information about incorporated metal ions and salts,
which correlate with the ash content. Basically, high ash contents interfere with further
processing and modification. The protein content is especially important in medical uses since
proteins may cause inflammatory reactions.
As summarized in Table 3, various methods have been used to determine of chitosan
characteristics.
Table 3. Methods for determining physico-chemical characteristics of chitosan

(modified from Aranaz [54])
Physico-chemical Determinations methods Reference

characteristics
Degree of • Infrared spectroscopy [50,54-56]
deacetylation • First derivative UV-spectroscopy [57,58]
• Nuclear magnetic resonance spectroscopy [48,54,59-61]
• Titration (alkalimetric, conductometric, [28,59,62]
potentiometric)
• Differential scanning calorimetry [63]
Molecular weight / • Viscosimetry [48,64]
Mw distribution • Gel permeation chromatography [49,65]
• Light scanning [27]
• Electrophoresis [13]
Crystallinity • X-ray diffraction [27,65,66]
Moisture content • Gravimetric analysis [67]
Ash content • Gravimetric analysis [67]
Protein content • Bradford method [68]
In the next section selected protocols that have already been applied are briefly described,
and references are given for further details. At the end of the section properties of chitosans
from various fungi are summarised and compared to crustacean chitosan (Table 5).
5.2.1. Commonly Used Protocols in Laboratory Practice
5.2.1.1. Degree of Deacetylation
5.2.1.1.1. First derivative UV-spectroscopy

Molecules are stimulated by electromagnetic waves in the UV range (1-380 nm), which
raise their energy level by activating valence electrons. This can be used to determine the
degree of deacetylation in chitin or chitosan by measuring the absorbance of the acetyl group
[57]. A detailed description of the usual protocol is provided by Wu and Zivanovic [58].
Standard solutions of N-acetylglucosamine (GlnNAC) and glucosamine (GlcN), each at
concentrations between 0 and 50 µg/mL are prepared by dissolving them in 0.85%
phosphoric acid. The first derivative UV values are determined at 203 nm and correlated to
the concentrations using equation 1:
1
CGlcNAC = ⋅ H 203nm (2)
slope
To analyse samples a 100 mg portion of each sample is dissolved in 20 mL of 85 %

H3PO4 and heated at 60°C for 40 min. A 1 mL portion of the sample solution is then diluted
with 100 mL dist. water and incubated at 60°C for 2 hours and finally measured by UV at
203 nm. The degree of deacetylation (DD) is calculated according to equation 2:
mGlcNAC
⋅100
DD[%] = 100 − 203.21 (3)
msample
− mGlcNAC
mGlcNAC
+ 20
203.21 161.17
5.2.1.1.2. Nuclear Magnetic Resonance Spectroscopy

In a magnetic field nuclei that have odd numbers of protons and/or neutrons (and hence
“spin”) have an intrinsic magnetic moment, angular moment and resonance frequency. Thus,
by scanning a sample for these frequencies the elements present can be quantified, as briefly
described below.
Liquid-State NMR
A 100 mg sample is dissolved in 10 mL deutered water with 0.07 M HCl (pH 4) and
shaken overnight at room temperature. After adding 2-4 mg NaNO2 the sample is stored at
room temperature for 4 hours and then freeze dried. The residues are dissolved in deuterated
water. This procedure is repeated three times [60,61], then NMR spectra of the dissolved
samples are recorded at 99.6 MHz at 90°C.
Solid-State NMR
To analyze solid-state samples Cross Polarized Magic Angle Spinning (CP-MAS) 13C
NMR spectra are often acquired, by a spectrometer operating at 50.3 MHz, from a 200 mg
sample placed in a zirconia rotor spinning at 4 kHz. After 2,000-10,000 scans the following
signals are analysed -- C1 (δ104.5-104.6 ppm), C2 (δ55.6-55.7 ppm), C3 (δ74.0 ppm), C4
(δ83.6-83.8 ppm), C5 (δ76.1 ppm), C6 (δ60.9-61.5 ppm), CH4 (δ23.2-23.2 ppm) -- and the
degree of deacetylation is calculated from [48,54,59-61]:
⎛ I CH 3 ⎞
DDA = 100 ⋅ ⎜1 − ⋅6⎟ (4)
⎝ I C1 + I C 2 + I C 3 + I C 4 + I C 5 + I C 6 ⎠
5.2.1.1.3. Titration
Titration exploits the removal of protons from the liquid phase that occurs when chitosan
dissolves in acid solutions due to protonation of the amino groups, which can be quantified by
adding base and monitoring the changes in pH using a pH indicator, pH meter or conductivity
meter. For this, a sample of 200-500 mg is dissolved in 20 mL 0.1 M HCl, then the solution is
titrated to the endpoint using NaOH at 0.1 M or some other appropriate concentration. The
degree of deacetylation can be calculated by using different equations. The latter one is
derived from dimensional analysis whereas the molecular weights M [g/mol], the sample
mass m [g], and the molarity c [mol/L] and volume V [L] of the NaOH and HCl solutions are
used [28].
X
DDA = ⋅100 (5)
(cNaOH ⋅ VNaOH + cHCl ⋅ VHCl ) mSample − X
+
M glucosamine M Acethylglucosamine
(cNaOH ⋅ VNaOH + cHCl ⋅ VHCl )

with X =
M glucosamine
or
mSample ⋅ M acetyl - glucosamine ⋅ f ⋅ c ⋅ (VNaOH + VHCl )

DDA = ⋅100
f ⋅ c ⋅ (VNaOH + VHCl ) ⋅ ( M acetyl - glucosamine + M glucosamine )
(6)
2
VNaOH V
with f = 331.310 2
− 26.243 NaOH + 0.96
[ L] [ L]
5.2.1.1.4. Infrared Spectroscopy

Many procedures involving use of infrared (IR) or Fourier Transform infrared (FTIR)
spectroscopy have been described. Infrared spectroscopy exploits the absorption of energy by
molecules and molecular bonds at the rotation and oscillation levels, respectively, which
induces stimulation of bondings that can be detected by a spectrometer.
Therefore, permanent or IR-inducible dipoles in the investigated substance are needed,
and each molecule has a characteristic absorption maximum. High wave numbers are typical
for small atoms and strong bondings, and low wave numbers typical for large atoms and weak
bondings. Various characteristic wave numbers have been published that can facilitate
analysis (Table 4). Figure 5 shows illustrative spectra of different chitosans derived from
fungi and crustacean shells.
Summarizing overviews have been published by Van de Velde and Kasaai [54-56]. In
practice, approximately 2 mg of a thoroughly dried (1 h at 105°C) sample is mixed with
200 mg ground potassium bromide (kept permanently at 50°C) and re-dried at 105°C for at
least an hour. After grinding again, 13 mm diameter pellets (40 mg) are formed by applying a
100-120 kN force. A KBr reference pellet is also made by the same procedure.
Table 4. Overview of useful absorbance peaks for the determination and calculation of
the degree of deacetylation (modified from Van de Velde and Kasaai [54-56])
Wave Type of peak Explanation Baseline Calculation of Degree Range

number of band of Deacetylation (DD) DD [%]
[cm-1] [%]
3450 Reference OH stretching 1950-3840
peak
2878 Reference C-H 2670-3010
peak stretching
1420 Reference C-H 1402-1478
peak deformations
1550 Measurement C=O 1242-1850 A1550 5-90
100 − 35.461
peak stretching in A2878
secondary
amide
(amide II)
1655 Measurement C=O 1242-1850 A1655
100 − 78.188
peak stretching in or A3450 40-60
secondary 1602-1750
amide or
(amide I) A1655
100 − 115
A3450 30-60
1320 Measurement C-N 1276-1348 A1320 0-100

100 − 442.48 + 13.92
peak stretching in A3450
secondary
amide or
(amide III) A1320
100 − 31.918 + 12.20
A1420 0-100
188 Mirko Trutnau,
T Thom
mas Bley and Jelka
J Ondruschka
Fiigure 5. FT-IR spectra of chitoosan from Mucoor rouxii, Trichooderma reesei and
a commerciall
(ccrustacean) shelll, with permissiion of Groeger et al. [69].
There pelleets are transferred to a desicccator, which is placed in a 95°C oven for
f 3 hours,
affter which IR spectra of the pellets betw ween 4000-4000 cm-1 are acqquired and thee degree of
deeacetylation iss interpreted according
a to Table 4.
5..2.1.2. Moleccular Weightt/Mw Distribution
5..2.1.2.1. Viscoosity
The solubiility of chitossan in acidic solution depends on the fraction of deeacetylated
ammino groups; the higher thee fraction the higher
h the soluubility and heence the viscosity. Chitin
w
with a zero deegree of deaccetylation is insoluble. It is also know w that there isi a strong
coorrelation bettween the average polymeer length andd viscosity [664]. Hence thhe average
m
molecular weig
ght can be calcculated from thhe intrinsic visscosity.
Hence, thee average molecular weightt can be calcuulated from thhe intrinsic visscosity. To
m
measure this prroperty, chitosan solutions (0.1-2 %) aree prepared in 0.3 M acetic acid/0.2 M
soodium acetatee. The dynam mic viscosity is determinedd for each soolution in a Ubbelohde
caapillary viscossimeter (Ø= 0.5 mm) at 25°°C and the intrrinsic viscosityy is determineed from the
inntercept of a linear regressioon with the y-axis of the reesulting η-c diagram (c=conncentration
off chitosan). The average molecular
m weigght can be dettermined from m the followinng equation
[448]:
1
⎛ [η ] ⎞ 0.76
MW = ⎜ ⎟ (7)
⎝ 0.078
0 ⎠
5..2.1.2.2. Ligh
ht Scattering
The size diistribution of small
s particles or polymers in suspensionns or solutionss is usually
deetermined, in various physiical and chem mical contexts,, by dynamic light scatterinng analysis
(aalso known as quasi-elastic light scatterinng analysis or photon correlation spectrosscopy). The
baasic principle is that particcles scatter ligght and the inntensity of thee scattered ligght changes
with time due to the movement of particles in the solution across the incident light path. The
velocity (depending on the molecule size) and the time-dependent changes in scattered light
intensity provide information about the molecular weight of the particles. In practice, a 1 %
chitosan solution is prepared in 0.3 M acetic acid/0.2 M sodium acetate and filtered using a
0.22 µm filter, then analysed at a suitable wavelength (often 632.8 nm) at 28 °C [27].
5.2.1.2.3. Gel Permeation Chromatography

Gel permeation chromatography (GPC) is based on the differential exclusion from paths,
within a suitable gel, of molecules with different molecular weights (MW). The passage is
hindered more, and hence the retention time within the gel is longer, for molecules with a
high degree of polymerisation and high molecular weight.
For analysis a chitosan solution (0.5 g/L) is prepared in 0.3 M acetic acid/0.2 M sodium
acetate, and the system is calibrated by determining the retention times of dextran standards
with a range of molecular weights, typically 40, 181, 270, 482, 679 and 2000 kDa [65].
Columns are packed with Sephacryl S-300 HR, S-400 HR/ or S-500 HR, and the samples are
applied in, and eluted, using 0.3 M acetic acid/0.2 M sodium acetate solvent as the mobile
phase, with a flow rate of ca. 0.25-1 mL/min. Fractions (1-10 mL) of the eluent are collected
and the concentration of polymers in them is determined either by light scattering analysis or
by the phenol-sulphuric acid method [70].
5.2.1.2.4. Electrophoresis
A similar principle of gel permeation is also applied in electrophoresis. This method is
less sensitive than GPC, but allows the MW of chitosan samples to be estimated if
chromatographic equipment is not available. A protocol has been published by Muzzarelli et
al. [71]. The system applied can be calibrated using dextran standards with different
molecular weights, as mentioned in section 5.2.1.2.3.
In practice, a slab gel of 25% polyacrylamide, 7 M urea and 5.5% acetic acid is prepared,
together with a 1 % chitosan solution in 0.3 M acetic acid/0.2 M sodium acetate, filtered
using a 0.22 µm filter. The gel is placed in 5.5 % acetic acid and loaded with 10 µL of the
prepared chitosan solution then a constant current of 30 mA is applied for 7 h, after which the
gel is stained by a 0.125% solution of Coomassie Brilliant Blue in methanol:acetic acid:water
(50:10:40), and destained with a 10:10:80 methanol:acetic acid:water mixture.
5.2.1.3. Protein Determination by Bradford Assays

Depending on the source of chitosan and the thoroughness of the deproteination step,
varying amounts of protein residues may be left in the chitosan polymer, which lowers its
quality. Hence, protein determination is needed to validate the purity of the extracts. A
commonly used method is that of Bradford [68]. For this, standard solutions ranging from 5-
100 µg protein (e.g. albumin, gamma globulin) per 100 µL are prepared to generate a
calibration curve, then they and 50 mg chitosan samples are incubated in 1 mL of 5 M urea
solution at 95°C for 30 min [27]. After cooling, 100 µL portions of the supernatants from the
centrifuged samples (and standards) are incubated for 5 min with 5 mL of Bradford reagent
and the absorbance of the solutions is measured at 595 nm. Bradford reagent is prepared by
dissolving 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol, adding 100
ml of 85% (w/v) phosphoric acid, diluting to 1 litre when the dye has completely dissolved,
and filtering through Whatman #1 paper just before use.
5.2.1.4. Crystallinity by X-Ray Diffraction

In principle X-ray diffraction is similar to light scattering, but much shorter wavelengths
are used. Hence, the beam can be scattered by much smaller scale features, i.e. atomic planes
of a crystal lattice. Further, since each crystalline material has a characteristic atomic
structure X-rays will be diffracted in a unique, characteristic pattern. Hence, it provides
accurate measurements of crystalline contents, which affect the physical and biological
properties of the polymer.
For chitosan analysis wide-angle X-ray diffraction (WAXD) with Cu/Ni radiation at
40 kV and 30 mA with a scattering range (2θ) of 2°-100° is generally used. Either of two
equations can be used to calculate the crystallinity index (CrI). The first (which does not
require use of an amorphous standard) was proposed by Struszczyk [72]:
I crystal (2θ = 20°) − I amorph (2θ = 12°30 ')

CR[%] = ⋅100 (8)
I crystal (2θ = 20°)
Here, the index is calculated from the peak height intensities, I, in contrast to the equation
of Goikhman [73], which uses the integral of intensities corrected by data from an amorphous
standard:
40
∫ ⎡⎣ I amorph (2θ ) ⎤⎦ d (2θ )

CR[%] = 40
5
⋅100 (9)
∫ ⎡⎣ I (2θ ) − I
5
amorph (2θ ) ⎤⎦ d (2θ )
The advantage of this equation is its higher degree of accuracy, but problems may arise
from the low availability of appropriate standards.
5.2.1.5. Moisture Content

The moisture content of chitosan samples can be determined according to the standard
methods [67]. A wet sample of 200-500 mg is dried at 103-105°C for 1 hour in a drying oven
on a preweighed glass-fibre filter. The filter disk is then transferred to a desiccator, cooled
and the moisture content of each sample is estimated from the difference between its wet and
dry weights.
5.2.1.6. Ash Content

The ash content is determined according to the standard method [67]. A dried sample of
200-500 mg is ignited to constant weight on a preweighed glass-fibre filter at 550°C in a
muffle furnace. A blank glass fibre filter is also ignited. Usually, 15 to 20 min ignition is
required for 200 mg residue. The filter disks are transferred to a desiccator after being
partially cooled in air until most of the heat has dissipated.
Table 5. Overview of physico-chemical properties of chitosan isolated from the indicated

fungi (by the authors and other researchers) and crustacean chitosan
Degree of
Chitosan deacetylation Viscosity Mw Ash Protein Reference
sample from (%) (mPa*s) (kDa) (%) (%)
A. coerulea 84.5 6.6
M. rouxii 80.4 56 [74]
R. oryzae 81.6 190
M. rouxii 83-89 42 0.9 0.1
[27]
crustacean 89.7 1000 0.6 0.05
crab shell (Sigma) 96.8 316
A. niger 84.2 5.9
P. citrium 78.5 4.6 [75]
F. oxysporum 73.4 2.7
R. oryzae 90.2 6.8
crab shell (Sigma) 97.9 373 940
A. niger 90 6.2 140
R. oryzae 87.9 3.5 69
[20]
L. edodes 86.5 5.8 190
P. sajo-cuju 83.8 5.6 110
M. rouxii 85.1 3.3 27
C. albicans 83.8 3.1 110
crab / shrimps 82-99 8.3-1260 [48]
crab shell (Fluka) 75-95 35 0.5-1.2 0.2-1.2
A. coerulea 83.0-84.9 1.9-9.2 2.2-5.6 0.02 Own
M. rouxii 83.4-86.8 2.9-6.2 1.8-4.2 0.01 studies
R. oryzae 79.4-85.3 3.3-3.6 1.1-2.9
T. reesei 84.4-87.2 2.1-2.6 0.4-3.5 0.01
Ignition, cooling, desiccation and weighing are repeated until a constant weight is
obtained or the weight change is less than 4% or 0.5 mg, whichever is less. The ash content of
each sample is estimated from the difference in the weights of the glass fibre filters before
and after the heat treatment.
6. APPLICATIONS
Chitosan has several characteristics that are exploited in different applications. As a
polycation it is similar to metal ions in that it can bind and form complexes with negatively
charged molecules such as proteins, lipids or hormones. It also has antibacterial and
antifungal activities, but less toxicity to higher organisms, e.g. the LD50 for mouse is 16 g/kg.
In the body it can be slowly hydrolysed by non-specific enzymes, such as lysozyme. Hence it
can be used for purposes, e.g. as a composite material in tissue engineering [76].
3500
Annual number of publications

3000
2500
2000
1500
1000
500
0
'91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02 '03 '04 '05 '06 '07 '08 '09
Year of publication
Figure 6. Exponential growth in the annual number of publications regarding chitosan production,
applications and characterisation since 1991 (sciencedirect.com).
Its water retention capability is also used in healthcare and cosmetic applications [77].
Furthermore, chitosan can be easily processed. For instance, foils can be simply formed from
powders by dissolution in an acidic solvent and subsequent spraying and drying, e.g. on plant
seeds. A thin layer of chitosan is formed, which wraps the sprayed article and protects it,
especially against microbial infection [78-80].
6.1. Summary of Current Commercial Chitosan Applications

and Future Perspectives
When discussing chitosan no distinction is generally drawn regarding the source, i.e.
whether it is crustacean or fungal, since the chemical structure of chitosan from both sources
is the same. The main difference is that fungal chitosan usually has a lower degree of
polymerisation and hence a lower molecular weight.
Based on the number of annual publications since the 1990’s there has been increasing
interest in chitosan (Figure 6). Due to the similarities of its structure and properties to those of
cellulose (see section 5.1) it has similar fields of application to cellulose, but additional
applications due to its further polar functional groups. Many applications have been proposed
and assessed in published studies, and many articles have summarized the “potential”
applications of chitosan based on the state of the art at the time [9,81-85]. Figure 7 provides a
short review of the applications (actual and potential) that have been described
Chitosan from Funggi 23
Fiigure 7. Overvieew of current annd prospective applications off chitosan due too its inherent prroperties and
veersatile modificcation possibilitiies.
However, only
o a few of these
t applicattions are well established
e annd in commerccial use. As
m
mentioned in section 2, theere are more than 200 suuppliers of raaw chitosan or o products
including chitosan. In Table 6 current fields of commercial chitosan application, together with
the purposes of specific products and some manufacturers, are listed. Most of the products are
applied in fields with less restricted approval criteria than medical applications, e.g. as bone
replacement materials, artificial skins and tumour therapy, which are regulated by authorities
such as the FDA or EC (e.g. Directive 2001/83/EG). In the authorisation procedure the
pharmaceutical quality, potency, harmlessness and benefit vs. risk ratio have to be rigorously
demonstrated in laboratory and clinical studies, although fast-track procedures for approval
are possible within six months for pandemic vaccines, since they are not regarded by
regulatory authorities as entirely “new” vaccines, because “...they build on the technology
used to produce vaccines for seasonal influenza, established procedures for testing and
regulatory control, and an extensive body of safety data.” [86].
Table 6. Overview of current products and suppliers of chitosan products categorized

by application fields (comprehensiveness not guaranteed)
Field of
application Product purpose Manufacturers
Healthcare immune booster/cholesterol JA KWANG Co. LTD.
supplements lowener
immune anti-fatigue Shanghai Yuanpai Trading Developing Co., Ltd.
Capwork Nutrition Ltd.
cholesterol/ fat blocking USA laboratories Inc.
weight loss/ dietary Fujian Newgift Enterprisies Co., Ltd.

THE d.o.o.
Softgel capsules (packaging) Qingdao Sunrise Trading Co., Ltd.
iummune booster Samsung Chitopia Co., Ltd.
cholesterol/fat blocking Shop for you Health Products Ltd.

Zendu Alimentos y Complementos Nutritionales SL
diet pills GMF Corp.
Greatwallbuy International Tade CO.LTD
Best Way Well Health Products Limited
Anny Int'l Health Beauty (HK)Dev.Ltd
HUAYI INT’L Ltd
Consava International Co., Ltd
Shanghai Leeab Industrial Co., Ltd.
Omana Nutraceuticals (USA)
nourisher for stomach and liver 21st Century Products Sdn. Bhd.
medical face mask Pannarajintertrade
immune booster Capistone International, Inc.
immune anti-fatigue (advanced Private Label Nutraceuticals Llc.

Fat, Sugar and Carb Blocker)
immune anti-fatigue Laboratorio Farmaceutico Vitamed Ltda.
health care supplement China Apollo Group International.Co., Ltd
slimming coffee KangBo International Biological Development

Field of
Cosmetic cleansing cream YesKorea Corporation
soap Eastar Holding Group Co., Ltd
Claypia Co., Ltd
shampoo Yes Pet Service Center
moisture-keep of cosmetics Xiamen Top Helth Biochem Tech. Co., Ltd.
vaginal douche Alta Care Laboratoires
toothpaste TripleLife Co., Ltd
cosmetic cream Kim Jeong Moon Aloe Co., Ltd.
analogous hyaluronic acid Sino-Strong (Xiamen) Co., Ltd.

chitosan
Nutrition packaging in nutrients capsules Sunrise Nutrachem Group Co., Ltd
nutrition supplement and food Xian Medicines and Health Products Imp. and
additive Exp.Corp
A and Z Food Additives Co., Ltd
Water flocculant FRANCE - CHITINE SARL
treatment
Agriculture fungicides BEIJING MULTIGRASS FORMULATION CO.,
LTD.
Qingdao Jingling Marine Scientific andtechnological
Co., Ltd
Qingdao Olympic Chemestry Co., Ltd.
Natagri Co., Ltd.
insecticides Qingdao Whale Spirituality Ocean Technology Co.,
Ltd
bactericides China Ocean University Organism Project
Development Company
pesticides Qingdao Whale Spirituality Ocean Technology Co.,
Ltd.
germicides Beijing Kingbo Biotech Co., Ltd
fertilizer/ insecticides Qingdao Whale Spirituality Ocean Technology Co.,

Ltd.
plant fertilizer Ideal Marketing and Consultant Co. Ltd
Huzhou Nanxun Hongquan Organic Fertilizer Factory
China Ocean University Organism Project
Development Company
Qingdao Jingling Marine Scientific andTechnological
Co., Ltd
animal fertilizer Pannarajintertrade
biostimulation FRANCE - CHITINE SARL
Fruit and vegetable preservation Xiamen Top Helth Biochem Tech. Co., Ltd.
Table 7. Overview of current products and suppliers of chitosan products categorized

by application fields (comprehensiveness not guaranteed) (Continued)
Field of
Textile functional socks SUNSHINE TRADING CO, LTD
industry
chitosan fibres/yarn KOREA YOUNGDEOK CHITOSAN (WEIFANG)
CO., LTD
Nantec Textile Co., Ltd
Xiamen Zanglong Import Export Co., Ltd.
Quigdao United Tongsung Trading Co., Ltd.
Dezhou Huayuan Eco-Technology Co., Ltd.
chitosan blended yarn Quigdao United Tongsung Trading Co., Ltd.
knitting yarn Quigdao United Tongsung Trading Co., Ltd.
weaving yarn Quigdao United Tongsung Trading Co., Ltd.
Paper paper stability enhancer Xiamen Top Health Biochem Tech. Co., Ltd.
industry
mulching paper SOC Co., Ltd
Medicine sterile dressing Anhui Xiaoshan Medical Material Co., Ltd.
wound dressing Zhejiang Kanglidi Medical Articles Co,. Ltd.
hospital mask/ Uniko Enterprise Co., Ltd.

anti-virus mask
clinical dressing Qingdao Dongwen Xing Trading Co., Ltd
anti hypertension BIOTECH CO., LTD.
Hygenics odour neutralizer KEITI
detox foot patch/ Era Medical Technology Co., Ltd

foot plaster/ New Era Adhesives Technology Co., Ltd
cleansing Shanghai Saint-koms Inc
ONFARMS CO., LTD
IonFarms Korea - Follow Nature
IANSH DAIRY COMPANY LTD
Wenzhou Yijia Light Industrial Co. Ltd
air freshener Danhwang Gonyangcho Co., Ltd
sanitary napkin Seol Design Co., Ltd.

panties for urinal incontinence TripleLife Co., Ltd
pet spray for odour removal/ Yes Pet Service Center
antibiotic action
Depending on the application, authorisation for commercial use of a new medical product
or therapy may take up to 10 years [87], but new applications are continually emerging, and
there is still intense activity in the fields summarized in Table 6 and Figure 7. Further, in the
past the focus was on macro-scale applications but current research interest is focused more
on nano-particle and chitosan composite materials for use as thin layers in sensor systems and
drug/DNA delivery systems or in regenerative energy systems. Most of the potential

applications have been generally summarized in previous reviews [81-83,88-96,84]. In the
following sections concrete novel applications described in literature published in 2009/2010
will be surveyed to summarise future perspectives for commercial uses based on recent
research
6.1.1. Application in Microbial Fuel Cells

Chitosan has been used to enhance the performance of Enterobacter cloacae microbial
fuel cells [97], with anodes containing multi-walled carbon nanotubes (MWCNTs) dispersed
in 0.1% chitosan or 1% Nafion. The maximal power output was observed after four hours; the
fuel cell with the modified anode delivering 252.6% more power than counterparts with
unmodified anodes (13.8 μW). MWCNTs dispersed in chitosan yielded nearly 50% greater
power outputs than those dispersed in Nafion, due to increased aggregation of Nafion.
Another fuel cell application tested involved use of anion-exchange membranes composed of
quaternized-chitosan derivatives for alkaline fuel cells [53]. The main properties of these
membranes (such as crystallinity, swelling index, ion exchange capacity, ionic conductivity
and thermal stability) could be effectively regulated by adjusting the degree of quaternization
and crosslinking density. Another strategy has been applied in membranes of direct methanol
fuel cells, in which titanate nanotubes have been incorporated in a chitosan matrix [98]. The
homogeneously distributed nanotubes (15 % w/w), about 10 nm in diameter and 200-600 nm
long, increased the pressure tolerance (to 85.0 MPa) and thermal stability of the membranes,
which were less permeable to methanol (0.497 10-6 cm2 s-1) due to the free fractional volume
afforded by the nanotube incorporation. This afforded conductivity of 0.0151 S cm-1.
Substantially lower conductivity was obtained by using blended polymer membranes
consisting of chitosan and polyvinyl alcohol (PVA), which are used in proton batteries [99].
The highest conductivity (1.60 × 10-3 S cm-1) was achieved by using 70 % (w/w) ethylene
carbonate and 30 % (w/w) PVA-chitosan-NH4NO3 (60 % of PVA–chitosan in a 60:40
mixture to 40% NH4NO3, by weight). The open circuit potential of Zn/MnO2 batteries
fabricated with these membranes was between 1.6 and 1.7 V. In addition, electrical
conductivity and thermal shielding properties of polypyrrole can be reportedly improved by
using a conductive polymer composite of polypyrrole-chitosan [100]. Further, use of thiourea
chitosan as an eco-friendly inhibitor of corrosion of mild steel in sulphuric acid medium has
been reported by Fekry and Mohamed [101], who found that 0.76 mM acetyl thiourea
chitosan reduced corrosion in 0.5 M H2SO4 by 94.5%.
6.1.2. Micro- and Nano-Scale Fibres and Particles

Protonized chitosan has been used in chitosan nano-particles/plasticized-starch
composites [102]. The tensile strength, storage modulus, glass transition temperature, water
vapour barrier and thermal stability that could be attributed to the filler/matrix interactions
were positively influenced when low proportions of chitosan nanoparticles were dispersed
uniformly in the glycerol plasticized-starch matrix. The polycationic properties of chitosan
have also been exploited to construct nanofibrous scaffolds based on polyelectrolyte
complexes for delivering DNA [103]. For this purpose a chitosan solution was dropped into
polyacrylic acid, a polyanion, which led to the formation of nanofibrous scaffolds with
average diameters of 140 nm that were obtained after lyophilization. Experiments with
transgenic expression in human dermal fibroblasts seeded on these nanofibrous scaffolds have
shown that these nanofibrous scaffolds have favourable characteristics for nonviral gene
delivery to mammalian cells. Although chitosan is regarded as a potential gene carrier its poor
solubility is a major factor limiting its utilization for this purpose. Hence, chitosan has to be
made soluble and DNA sequences have to be connected. This has been done by preparing
chitosan phosphate, a substance that is readily soluble in aqueous solutions, which was then
mixed with antisense oligonucleotides [104].
It should be noted that chitosan nanoparticles can be functionalised with inorganic as well
as organic substances. For instance, magnetic Fe3O4-chitosan nano-particles have been
recently synthesized [105] and they can be used for various applications involving a
subsequent biomagnetic separation step, e.g. sorption processes of dyes and heavy metals in
suspensions, from which the loaded chitosan can be separated by a magnetic field. Further
possible applications of such modified chitosan carriers are in drug targeting, since they can
be moved to the desired area by a controlled magnetic field and subsequently release a
specific drug. In addition, chitosan-magnetide nano-powders with particle sizes of 80 nm
have been synthesized by Bhatt [106],. and shown by impedance measurements to ease the
electron transfer between solutions and electrodes. In immunization studies chitosan-based
nano-particles with a recombinant hepatitis B surface antigen (rHBsAg) have induced 9-fold
higher anti-HBsAg IgG levels than the standard alum-adsorbed vaccine [107].
6.1.3. Intelligent Drug Delivery Systems and Hydrogels

Intelligent delivery systems can make a therapy or drug delivery more efficient and save
time, costs and even lives.
For instance, if an auto-regulating system is to be used to supply insulin for diabetics it
must permanently supply sufficient amounts. For this purpose glucose-responsive composite
microparticles based on chitosan, concanavalin A and dextran can be used [108], in which
concanavalin A is coupled to chitosan microparticles and covered with a dextran layer via
specific affinity. Via electrostatic and intermolecular interactions insulin can be loaded into
the 2.5 μm microparticles with a loading capacity of 9.1% and an entrapment efficiency of
92.2%. Insulin is reportedly released in a glucose concentration-dependent manner, and the
glucose sensitivity is reversible, with no loss of insulin activity. In addition, pH-sensitive
carrier systems can be used to allow peptide and protein drugs to pass through the harsh, acid
environment of the stomach and be preferentially released in the intestine. In vitro tests with
carrier systems based on ionotropically cross-linked mixtures of sodium alginate and
chemically modified carboxymethyl chitosan coated with poly(ethylene glycol) and entrapped
model protein drugs have been developed that show consistent swelling patterns and high
entrapment efficiency [109]. A temperature and pH responsive microhydrogel has also been
prepared, by Jocic et al. [110], from a temperature responsive poly-N-isopropylacrylamide
and pH-responsive chitosan as an innovative strategy for functional finishing of cotton. The
responses to pH, temperature and humidity stimuli promise good applicability. The
thermosensitivity of chitosan/poly(vinyl alcohol) hydrogels containing hydroxyapatite has
also been exploited for protein delivery [111]. In addition, a self-hardening calcium phosphate
composite scaffold for bone tissue engineering has been developed by Liu and Zhou [112].
Although calcium phosphate cement forms solid hydroxyapatite in situ, offers
osteoconductive behaviour, forms complex cavity shapes without machining and is resorbed
and replaced by new bone, it has weak mechanical strength and is not macroporous. These
limitations can be addressed by using chitosan dissolved in a weak citric acid solution. The
acid environment increases the degradation rate and the incorporated chitosan introduces a
macroporous structure after dissolution, which enhances the strength, and osteoblast
migration muscle pouches in rabbit thighs. The cationic behaviour of chitosan has also been
used to make rechargeable long-term antimicrobial and biofilm-controlling systems using the
antibiotic rifampin to control the growth of Staphylococcus epidermidis and S. aureus [113].
Refampin release was observed over periods exceeding 30 days and the system could be
recharged.
Finally, for this section poly(l-lactide) and biodegradable chitosan/poly(l-lactide)
composites have been shown by Meng [114] to have shape memory properties arising from
the viscoelastic properties of poly(l-lactide). Due to the incompatibility between chitosan and
poly(l-lactide) the shape recovery ratio of the composites decreased significantly with
increasing chitosan contents, and the cited authors proposed a chitosan content below 15%
(w/w) to obtain good shape memory effects.
6.1.4. Hydrogel Cartilage and Scaffolds for Tissue Engineering

Due to the biocompatibility and high fluid uptake of chitosan it is highly suitable for use
in cartilage substitution. Temperature-responsive chitosan hydrogels can be prepared by
combining chitosan, β-sodium glycerophosphate (GP) and hydroxyethyl cellulose (HEC). A
study of the use of chitosan hydrogel in a sheep model as a scaffold for chondrocytes has
shown that chondrocytes in reconstructed cartilage survived and retained their ability to
secrete matrix when cultured in vitro [115]. Cartilage defects can be repaired within 24 weeks
and the use of hydrogels without chondrocytes also has marked effects on regeneration.
Hence, in both case it is possible to repair articular cartilages within 24 weeks. Other
composite scaffolds, consisting of alginate dialdehyde cross-linked with chitosan/calcium
polyphosphate, can be used for meniscus tissue engineering [116]. Calcium phosphate can be
used to enhance the low mechanical stability of pure chitosan. A disadvantage is that the
degradation rate is reduced by the calcium cross-linking, but the composite reportedly
remains biocompatible without cytotoxic effects. Experiments with chitosan/polyester-based
scaffolds for cartilage tissue engineering have shown similar results [117]. No cytotoxic
effects have been observed in the response of osteoblasts of rats seeded onto
polycaprolactone/chitosan blend porous scaffolds either [118]. Analyses of adhesion,
proliferation, cell morphology and alkaline phosphatase activity during in vitro cultivation
showed that incorporating chitosan improved both the adhesion and growth of osteoblasts.
In peripheral nerve regeneration chitosan tubes coated with maggot homogenates have
been used as artificial nerve guides [119]. Here the chitosan was applied as a biocompatible
and biodegradable bilateral guide while the maggot product promoted wound nerve
regeneration and the release of neuropeptides.
6.1.5. Biosensors
The applicability of chitosan for biosensors has been known for several decades, and it
has been used (inter alia) in immuno, electrochemical and amperometric sensors.
6.1.5.1. Sensors Based on Specific Molecule Binding

Disposable electrochemical immunosensors have been developed based on carbon paste
electrodes constructed from chitosan nanoparticles modified with colloidal gold for assaying
human immunoglobulin [120]. Antibodies immobilized on the electrode provide a

measurement range of 0.3 to 120 µg/L and a detection limit of 0.1 µg/L at a signal to noise
(S/N) ratio of 3. The same analytical range has been achieved for the detection of
Carcinoembryonic antigen (CEA), a marker for colorectal cancer [121]. In this amperometric
device Prussian blue nanoparticles were used as redox probes, immobilized on a three
dimensionally structured, homogeneous, composite membrane of gold colloidal nanoparticles
doped with chitosan-multiwall carbon nanotubes. CEA antibodies were immobilized on the
reduced gold surface and the device provided good accuracy combined with high sensitivity
and stability. The CAE system has also been combined with a disposable glassy carbon
electrode coated with a nano-Au/multi-walled carbon nanotubes-chitosan nanocomposite film
[120]. The mixture of multi-walled carbon nanotubes and chitosans was dissolved in acetic
acid, then gold nano-particles were synthesized in situ and dried as a thin film on a glassy
electrode. The CEA antibody was subsequently immobilized in the film. This configuration
was sensitive to CEA at concentrations ranging from 0.3-2.5 and 2.5-20 µg/L with a detection
limit of 0.01 µg/L (S/N = 3). Immuno sensors have also been used to detect whole
microorganisms e.g. Campylobacter jejuni [122], by immobilizing anti-FlaA 2D12
monoclonal antibodies on O-carboxymethylchitosan surface-modified Fe3O4 nanoparticles
then measuring changes in impedance before and after attachment of C. jejuni to the surface.
The measurement range has been reported to be between 1.0×103 and 1.0×107 colony forming
units (CFU)/mL. The biosensor can be simply regenerated by cleaning with glycine-HCl
buffer solution (pH 2.8), which makes its use and maintenance very convenient. Similarly,
Shigella flexneri can be detected with multi-wall carbon nanotubes/chitosan modified
electrodes, on which HRP-anti-S. flexneri is printed without any crosslinking.[123]. Under
optimal conditions the reported detection range of this system is from 107 to 1013cfu/L with a
detection limit of 2.3 × 107 cfu/L. The great advantages of this system are that it is
inexpensive and easy to make.
Double strand DNA dsDNA has been immobilized on a thin layer of dendrimer-
encapsulated bimetallic nanoparticles (Au-Pd) in a chitosan composite which was then spread
on a glassy carbon electrode to determine the antioxidant capacity of sericin [124]. In this
application it is possible to investigate diseases that are caused by oxidative stress and the
effects of antioxidants. Of course, it is not only limited to medical applications since many
biotechnological processes also depend on oxidative processes, e.g. in wastewater treatment
plants [125]. DNA sensors have also been used to detect single-strand DNA (ssDNA), for
instance of Yersini enterocolitica by using a carbon ionic liquid electrode modified with V2O5
nanobelts, multi-walled carbon nanotubes and chitosan nanocomposite materials. Specific
ssDNA sequences can be detected at concentrations from 1.0×10-11 to 1.0×10-6 mol/L with a
detection limit of 1.76×10-12 mol/L [126]. A further sensor has been developed for the
detection of α-fetoprotein (AFP), a marker for the diagnosis of hepatocellular carcinoma
[127]. For this purpose a dinuclear copper complex was absorbed on a glassy carbon
electrode then a nano gold/chitosan composite was immobilized by adsorption of horseradish
peroxidase and α-fetoprotein antibody. AFP detection was reported to be highly selective and
robust, with a range of 0.2 to 120.0 µg/L and a detection limit of 0.05 µg/L.
6.1.5.2. Enzyme Based Sensors

An amperometric biosensor has been used for hydrogen peroxide detection [128], in
which horseradish peroxidase is trapped in an organic-inorganic hybrid material composed of
zirconia-chitosan sol-gel and Au nano-particles. The sensitivity was enhanced by a polymer-

copper nanostructure composite (pPA-FCu). Under optimal conditions the measurement
range and detection limit are reportedly 7.8 x 10-4 - 3.7 mM/L and 3.2 × 10-4 mM,
respectively. Another system, based on a MgO nanoparticles-chitosan composite matrix has
an even lower measurement range of 1.0x10-4-1.3 mM, with a detection limit of 0.05× 10-3
mM at a signal to noise ratio of 3 [129]. There are many other horse radish peroxidise systems
with different immobilization methods that provide similar performance [130].
A tyrosinase-based amperometric carbon fibre microbiosensor has been used to detect
dopamine in vivo [131]. In this system the enzyme is trapped in chitosan and ceria-based
metal oxides deposited on the surface of a carbon fibre microelectrode. The dopamine
detection limit is reportedly 1 nM, with a linear range of 0.010 µM- 220 μM, and it provides
good selectivity versus ascorbic acid, uric acid, serotonin, norepinephrine, epinephrine and
3,4-dihydroxy-L-phenylalanine. Preliminary in vivo experiments have detected dopamine at
levels up to 1.69 μM in brains of rats. Hence, the system provides an alternative to fast scan
cyclic voltammetry for in vivo dopamine monitoring.
Although several kinds of glucose sensors are being routinely used, further developments
are still being made. A novel type is based on a graphene/Au nano-particles/chitosan
nanocomposite film deposited on a gold electrode in combination with glucose oxidase [132].
Oxalate biosensors have also been prepared, from a mucin/chitosan matrix in which oxalate
oxidase is immobilized, using glutaraldehyde as crosslinking agent and then applied in an
amperometric system [133,134]. The sensitivity of this system is mainly dependent,
reportedly, on the swelling index of the matrix, which is largely influenced by mucin. High
sensitivities were obtained with highly swollen matrices. Hence, a hydrophilic environment is
necessary for a strong enzymatic response. The system has proved to be quite stable,
displaying 75% of the initial response after two months. Ascorbic acid can be detected with
ferricyanide, trapped in a chitosan sol-gel matrix, as an electron mediator, with a
measurement range from 1.0 × 10-6 to 2.143 × 10-3 mol/L, a response time of <6s and a
detection limit of 7.0 × 10-7 mol/L [135]. An electrochemiluminescent biocompatible
biosensor for detecting choline in biological samples has also been prepared, based on a
choline oxidase/titanate nanotubes/chitosan composite-coated glassy carbon electrode [136].
In analyses of milk samples the sensitivity could be improved by adding luminal.
Biocompatibility also proved to be essential for measurements of extracellular nitric oxid
production by living cells [137], utilising a system in which an NO fluorescent probe (4,5-
diaminofluorescein) was encapsulated by a combination of a layer-by-layer technique and
acid-free chitosan dissolving techniques, offering high sensitivity with a range of 5-
500 nmol/L. An amperometric biosensor for nitrophenol has also been created, using co-
immobilization of horseradish-peroxidase and methylene blue with chitosan on Au-modified
TiO2 nanotube arrays [138]. The titania nanotube arrays were directly grown on a Ti substrate
and a thin gold film was then coated onto them. In tests with several phenols, the detection
range for 3-nitrophenol was found to be 3×10-4 to 1.2×10-1 mmol/L, with a detection limit of
9 × 10-8 mol/L.
An interesting application for obesity control is the development of cholesterol sensors
[139,140], in which a metal oxide (nano CeO2 or SnO2) nanocomposite film is deposited on
an indium-tin-oxide coated glass plate to immobilize cholesterol oxidase. A detection range
of 0.1-4.0 g/L cholesterol has been achieved by this approach, with a detection limit of
50 mg/L. The presence of NanoCeO2 in CH-CeO2 nanocomposites increases the electroactive
surface area for cholesterol oxidase loading, and hence enhances electron transfer to the
electrode [140]. The total cholesterol content in human serum samples has also been
determined using a haemoglobin-cholesterol oxidase-cholesterol esterase-encapsulated
chitosan-modified glassy carbon electrode [141], providing an analytical range from 0.1 to
1.1 g/L.
Sensitive amperometric sensing of reduced nicotinamide adenine dinucleotide (NADH)
has also been demonstrated using a novel nanocomposite of glutaraldehyde-crosslinked
chitosan-dopamine with multiwalled carbon nanotubes [142], providing a linear response
range between 0.1 and 600 μmol/L with a detection limit of 0.012 µmol/L. The cited authors
reported that acidified nanotubes provided better performance than non-acidified
counterparts. Further, they noted that this system could be potentially used for in vivo NAD+-
dependent dehydrogenase enzyme-based biosensing. NADH is quite an expensive bulk
material, hence when it is used biotechnologically the scope for regenerating it should be
considered, and such NADH sensors could be useful for production control in such contexts.
In addition, a creatine sensor has been developed by Tiwari and Shukla [143], from a novel
chitosan-graft-polyaniline matrix with covalently immobilized creatine amidinohydrolase
using glutaraldehyde as a linker. It was reported that the matrix has high ability to immobilize
creatine amidinohydrolase.
1.2. Experimental Use Fungal Chitosan
In the following sections we present findings we have obtained in studies of copper and
17ß-estradiol adsorption, and use of chitosan as a bone replacement material in medicine.
6.1.6. Adsorption of Copper

Copper is an essential trace element, since it is a component of many enzymes. In the
human body its concentration is normally homeostatically adjusted, but oral or subcutaneous
exposure to large quantities of copper can lead to metabolic cycle disturbances, liver and
kidney damage or, even, mortality. Its toxicity is due (inter alia) to its ability to bind to the
thiol-groups of proteins and thus inhibit the activity of sensitive enzymes. It may also cause
peroxidation of lipids in cell membranes, via several intermediate steps in which free radicals
are produced that can further damage membranes, and DNA.
In addition, even at low concentrations copper may disturb the ecological equilibrium of
aquatic systems and increase the mortality of fishes and invertebrates. A further undesirable
side effect of such ecological disturbance is the enrichment of copper and other heavy metals
in plants, especially agricultural products, and hence in the food chain. Higher concentrations
have toxic effects on the flora.
Consequently, it is important to remove copper and other heavy metals from drinking
water and industrial wastewater. Several methods can be used to do this, e.g. filtration,
precipitation, electrodialysis, membrane separation or ion exchange procedures. However,
most of the applied processes are expensive, inefficient or limited by low throughputs. A
better choice is to use adsorption processes, in which the metal ions are bound to the surface
of a solid phase consisting of activated carbon, silica gel or ion exchange resin, but
production of these phases is often very complex. Therefore, more economical alternatives
are required that are available in high quantities, can be used after just a few processing steps
Chitosan from Funggi 33
annd (for sustainnability) ideallly can be maade from wastte products or natural sourcces that are
inndependent off the market prrice of other bulk
b chemicalss. Such materiials could incllude lignin,
m
moss, biomass or chitosan byy-products from other indusstries.
Chitosan iss known to bee the best heavvy metal adsoorbent of all poolymers charaacterized to
daate as chelatiing polymers [144], especcially fungal chitosan whicch has higherr chelating
abbility than cru
ustacean chitossan, mainly duue to its high content
c of amiino groups and abundant
hyydroxyl group ps. Therefore, chitosan is highly suitablee for the remooval of heavy metals and
raadioactive isootopes from contaminated waters, andd for recoverring noble metals. m The
coomplexation between
b crustaacean chitosann and metal ioons is well documented. Thhe utility of
fuungal biomasss as an adssorbent has also a been invvestigated. However,
H therre is little
innformation on fungal chitosan as an adsorrbent or chelaator, and thesee materials havve not been
innvestigated commparatively [15].
6..1.6.1. Reactiion Mechaniism

Various th
heories regardding chitosan reactions havve been preseented by severral authors
[666,145,146]. The
T most reacctive group off chitosan is the amino grooup, which exxplains the
immportance of the
t degree off deacetylationn and pH. Theere is also eviidence that thhe hydroxyl
grroups of the molecule
m play a role in sorpttion processess [15]. If chitoosan is added to
t an acidic
soolution the am
mino group willl become prottonated and thhe pH will incrrease slightly.
Due to pro
otonation, paraallel chelationn of copper (III) ions occurs.. Three basic models for
thhis have been proposed:
p
a) The brridge model: involving

i coordination of copper
c with foour amino grooups of one
or seveeral chitosan molecules
m [1455]
b) The peendant model:: involving cooordination of copper withh one amino group,
g two
OH-ion ns and water and/or the hyydroxyl groupp at the C3-atom of the gllucosamine
monom mer [66,147]
c) Chelattion between two
t amino andd two C3-hydrroxyl groups of
o two monom mers of one
or seveeral moleculess of chitosan [146].
Alternativeely, accordingg to Schmuhl et al. [148] the complexattion is dependdent on the

pH H; At values between
b a 5.8 a compplex with the structure ([cu (- NH2)]2+, 2 OH, H2O)
5.3 and
iss formed, whille at pH valuees above 5.8 copper
c (II) ionns coordinate with
w two aminno and two
O groups.
OH
Fiigure 8. Protonaation of the freee amino groups of Chitosan in acidic solutionss.

Several models describing the equilibrium of sorption to solids from solutions have also
been proposed. The most common models for sorption isotherms are the LANGMUIR, BET
(Brunauer, Emmett and Teller) and FREUNDLICH models, which are described in detail in
section 6.1.7.
The LANGMUIR model is probably the most widely used and provides good agreement
with numerous sets of experimental data [149-154]. Due to its common application results
obtained by different research groups can be compared. Nevertheless, its applicability is
limited. For instance, in the sorption of heavy metal ions to chitosan a mixture of forces and
energies are involved, arising from ion exchange and chelation reactions, that are not
considered in the LANGMUIR equation. Hence, new approaches have been developed that
take into account more complex reaction mechanisms, e.g. the REDLICH, PETERSON,
TEMKIN, DUBIN and RADUSHKEVICH isotherms [154,155].
6.1.6.2. Influential Factors

Various values for chitosan capacities to sorb copper ions have been published, ranging
between 5 and 1100 mg Cu/g chitosan [155]. There are several reasons for the variations,
including differences in the:
a) Source of raw material

b) Production and characteristics of the extracted chitosan
c) Experimental setup
Sorption processes are strongly influenced by the polymer structure, i.e. the degree of
polymerization. Hence, there are differences between fungal and crustacean chitosan since the
extraction from fungi usually releases low molecular weight chitosan. This is also why the
manufacturing process is the most significant step influencing the characteristics of the
chitosan produced. For example, air-dried chitosan has a different supra-molecular structure,
including greater swelling tendencies (i.e. higher liquid uptake per unit mass) and lower
sorption capacities than lyophilised (i.e. sublimation-dried) chitosan [156]. In addition, the
sorption capacity depends on the degree of deacetylation since the amino groups are the most
reactive groups and act as binding sites for copper ions. The higher the proportion of
glucosamine in the polymer the higher the amount of copper that can be taken up. Since
adsorption is a process that occurs on the surface of certain materials the form of chitosan
(e.g. flakes, beads or powders), its particle size and crystallinity all influence its adsorption
kinetics, as well as the maximum loading.
As mentioned above, the pH plays an important role since it determines the charge of the
amino group of the glucosamine moieties. Under acidic conditions the hydrogen ions compete
with cu (II) ions for the amino group, but the copper has higher affinity and hence greater
tendencies to form complexes with the amino group. At pH > 6 copper hydroxide is formed,
which is insoluble. Hence, in published studies of copper sorption by chitosan, recommended
pH values for efficient sorption are between 4 and 6 [150,153,154,157]. The capacity of
chitosan to sorb Cu (II) ions also depends on the anions present, e.g. chitosan can adsorb
more copper from CuSO4 solutions than from either Cu(ClO4)2 or Cu(NO3)2 solutions [157].
The presence of other (heavy metal) ions or complexion agents also influences the sorption
process with respect to a specific ion since they compete for the binding sites [154,158,159].
Partial chemical modifications to chitosan have been made to increase the selectivity, stability
and effectiveness of the sorption in basic and acidic milieu by linking additional reactive
groups [150,158,160]. However, the price of greater stability is a reduction in the maximum
sorption capacity since the crosslinking and coupling of additional reactive groups inevitably
results in binding of a fraction of the reactive amino groups.
1.1
1.0
Wofatit
0.9 Chitosan
0.8
C(t) / C0[-] of Copper
0.7
0.6
0.5
0.4
0.3
0.2
0 20 40 60 80 100 120 140 160 180 200

Time [min]
Figure 9. Time courses of changes in copper concentrations after addition of 2 g/L chitosan and wofatit
to 5 mmol CuSO4.
1
C(t) / C0[-] of Copper
0.5 g/L
0.1 2.0 g/L
5.0 g/L
10.0 g/L
20.0 g/L
0 10 20 30 40 50 60
Time [min]
Figure 10. Time courses of changes in copper concentrations (initial concentration 5 mmol CuSO4)
after addition of chitosan with final concentration of 0,5 -20 g/L.
366 Mirko Trutnau,
T Thom
mas Bley and Jelka
J Ondruschka
In a compaarison of the adsorption

a parrameters of coopper to chitossan and wofattit (a cation
exxchange resin n) we found thhat the biopollymer has a higher
h initial sorption rate and higher
soorption capacity. In additiion it takes longer
l to reach the adsorpption equilibrrium using
chhitosan (2 houurs in the experimental systeem, compared to 60 minutess for wofatit). (Figure 9).
The depend dence of the soorption processs on the amouunt of fungal chitosan
c emplloyed (0.5 -
200 g/L) with a copper
c sulphaate (5 mmol) was
w also investtigated. The fiinal concentrations of Cu
raanged between n 0.1 and 4.6 mmol, depennding on the amount
a of chittosan used, annd the time
neeeded to reacch equilibrium m ranged bettween 5 min and more thhan 2 hours. Under the
exxperimental conditions aboout 98% of coopper could bee removed froom the solutioon if 5 g or
m
more of chitosaan was used.
6..1.6.3. Immobilized Chitoosan

For effectiive industrial application of o any sorbennt it is obviouusly importannt to use it
opptimally. Imm mobilization iss an appropriaate way to asssure a maxim mum contact areaa from a
giiven material input. Thereffore, chitosan has been linkked to numerrous carrier materials
m by
vaarious method ds. In tests off the effects off chitosan immmobilization on
o copper sorrption three
m
methods were used
u to immoobilize it to vaarious substrattes (glass beadds, several sannd particles
annd silica gel).
In the firstt method the substrate surfface was activvated with NaaOH then silaanized with
A
APTES (3-Amiinopropyltiretthoxysilan), byy the method of [161] exceppt that a glyciidol anchor
w attached to
was o the amino group of the AP PTES, to bind the chitosan Figure
F 11.
Fiigure 11. Immo

obilization of chhitosan accordinng to the modifiied method of Liu
L [161].
Table 8. Portion of the adsorbed quantity of copper (II) - ions and sorption capacities of
the immobilized chitosan (4% chitosan on quartz sand)
Reference Liu et al., Rashidova et Wan et Chitosan pur

2002 al., 2004 al., 2004
[163] [162] [152]
adsorbed copper [% intial 80,3 72,5 69 78
value]
Degree of sorption 9,8 15,7 9,6
[mg Cu / g carrier]
Degree of sorption 141 129 114 138
[mg Cu / g chitosan]
Rashidova [162] was used, i.e. chitosan films were formed by dissolving it in acetic acid,
then adding carriers (glass beads, sand or silica gel) and drying the products. Thirdly, using
the method of Wan et al. [152] the carriers were mixed with chitosan and HCl. The
permutations of immobilization methods and substrates were then evaluated by calculating
the sorption capacities of the resulting immobilisates and the proportions of added copper that
they bound. The acquired data also provide information about the number of free reactive
amino groups.
The degree of sorption was determined by measuring the concentrations of the metal ions
in the aqueous phase before and after contact with the chitosan material and expressed as
metal uptake:
q=
( C0 − C∞ ) ⋅Vreaction (10)
madsorbent
where C0 are C∞ the initial and equilibrium (final) concentration of cu-ions (mg/l);
Vreaction is the volume of the sample (l), and madsorbent is the dry weight of the adsorbent added
(g).
The amounts of copper (II) ions that bound to the immobilisates were similar to the
quantity that bound to non-immobilised chitosan; they removed between 50 and 98% of the
added heavy metal ions.
The sorption capacities corresponded with the capacities of non immobilized chitosan, for
which an immobilized mass of 4 % (m/m) was determined. The amount of immobilisate
produced using the method of Wan Ngah et al. [150] was lower. The maximum copper uptake
of fungal chitosan from a copper solution (pH 5-5.5; 25°C) was 130-140 mg/g, and the
capacity for copper removal was found to decline in the order: fungal chitosan > Wofatit >
fungal biomass.
6.1.7. Sorption of 17ß-Estradiol

17ß-estradiol is a steroid hormone that is the primary product of follicular estrogen
synthesis in mammals.
388 Mirko Trutnau,
T Thom
mas Bley and Jelka
J Ondruschka
For the pro operties of steeroid hormonees a second messenger

m mecchanism is nott necessary
siince the stero oid receptor complex
c is abble to connecct to the DNAA directly annd act as a
trranscription faactor which is inducing a mR RNA [164].
Fiigure 12. Structture of 17ß-estraadiol, selected derivatives

d and 17α-ethinylestrradiol. 17α-ethiinylestradiol
is not natural butt is one of a num
mber of synthetiic estrogens useed as contracepttives.
It’s a so caalled “nucleus receptor“, whhich steroid reeceptors represent intracelluular soluble
prroteins which h are responssible for horrmone recognnition and forrwarding the accordant
innformation [16 65]. Particularrly this is thee reason for thhe comprehennsive behaviouur to other
sppecies. Also a non receptor dependingg impact e.gg. ATP-syntheesis in mitocchondria is
poossible. The ability
a of steroid hormones to t induce totallly different efffects in differrent organs
arre explained byb the growth factor
f coding of the steroid complex [1655].
The degrad dation of 17ß-estradiol, to avoid its releease into the environment,
e is of great
immportance, du ue to the posssible formation of free radiicals and mettabolites with estrogenic
pootency; 17ß-estradiol (E2) has the highest potency folllowed by 17α α-ethinylestraddiol (EE2),
esstron (E1) and d estriol (E3) [166].
The potenccy of 17ß-estrradiol dependds on both thee species invoolved and thee timing of
exxposure [167]. However, every minor persistent natural n and annthropogenic input can
pootentially disturb the hormoonal equilibriuum, and thus feminization or o virilizationn of diverse
orrganisms, such h as fish, snaills and crocodiiles [168].
Due to thee natural excrretion of wom men of 2-12 µg/day 17ß-eestradiol (E2)) and 3-20
µg/day estron (Belfroid et al., a 1999) com mbined with thheir stability in wastewaterr treatment
pllants and poteency in the ng//L range it is necessary to remove
r estroggens and substtances with
siimilar potency y (e.g. bisphennol A, nonylphhenol etc.).
There are several approaches for removing estrogenic substances from both drinking and
waste water. They can be divided into biological treatment, oxidative treatment and filtration,
adsorption and hybrid techniques.
In biological treatment biological filters or an activated sludge process are used. Of these
two options activated sludge reportedly offers the highest removal efficiencies; up to 99 % for
E2 and 78 % for EE2 compared to 64 % and 83 %, respectively, by a biofilter [169]. Clearly,
the activated sludge process is more efficient than a biofilter, but such removal efficiencies
are only achieved in laboratory-scale experiments. In a municipal wastewater treatment plant
it would take ca. five months to get the same results [170], but the average hydraulic retention
time (HRT) is only 4-14 hours, depending on the capacity and flowrate (10³ to 106 m3d-1). In
rural regions the HRT is even shorter, as low as 0.5 h when using biological filters [171].
Hence, the removal efficiency of biofilters seems to be much higher for practical applications.
A great advantage of biofilters is that one can choose microorganisms which that are
appropriate for specific tasks.
In 2003 an estradiol-degrading bacterium, Novosphingobium tardaugens, was isolated
from a Japanese treatment plant [172]. Another, named Denitratisoma oestradiolicum, has
also been isolated that is able to completely metabolize 17ß-Estradiol to carbon dioxide and
water [173]. Another option is to engineer a suitable organism instead of searching for natural
candidates [174]. For instance, from published studies it is known that the “old yellow
enzyme” of Kluyveromyces lactis is able to bind estrogens, the yeast Pichia pastoris has been
genetically transformed to express it, and the transformants are capable of removing
bisphenol A from aqueous solutions [175].
Oxidative, ozone-based treatments have been used in drinking water processing for
decades for disinfection and to remove persistent pollutants via reactions with unsaturated
compounds and by cracking aromatic connections. During these reactions ozone is reduced to
oxygen, which supports algal growth and does not cause wastewater contamination, unlike
the residues from chlorine-based treatments. The dosage is in the range of 10 mg/L and
hydrogen peroxide may enhance the degradation efficiency[176]. Ozonation has been used in
large-scale practice (2x105 m³) in the wastewater treatment plant Tubli Tse in Bahrain since
2002. Many researchers have investigated its efficiency both alone and in combination with
UV radiation and pH [177-180]
Modern filtration processes include membrane processes such as micro (MF), nano (NF)
and ultra filtration (UF) as well as reverse osmosis (RO). NF and UO are usually used for pre-
treatment of water and drinking water. Using NF in the last step of wastewater treatment
allows 70%-100% of the xeno-estrogenic substances bisphenol and nonylphenol (the smallest
endocrine disrupter) to be removed [181].
However, filtration treatment is relatively expensive since it is limited to low
throughputs, and it consumes high amounts of energy due to the pressure drop across
currently available membranes.. For these reasons absorption by activated carbon is more
widely applied than membrane processes in drinking water treatments [182].
6.1.7.1. Physico-Chemical Properties of 17ß-Estradiol

To calculate boundary conditions required for efficient removal using sorption processes
one has to know the physical and chemical properties of the target compound. Table 9 shows
characteristics of 17ß-estradiol (E2).
Table 9. Physico-chemical properties of 17ß-estradiol
Molecular weight Solubility (H2O) Polarity

3.6 mg L-1
272.4 g mol-1 4.1 (J cm-3)0.5
Charge at pH 7 pKa Log Kow

4.01
neutral 10.71
Dipole moment Henry constant Length

0.798 debye 3.64x10-11 atm m³ mol-1 1.258 nm
As can be seen in Table 9, E2 has a pKa of 10.7, much higher than that of chitosan,
which is 6.3 [183]. In order to assess the possibility of using chitosan to eliminate E2 we
should first consider the implications of this difference in pKa values. Therefore, let’s
examine the charge distribution of chitosan and E2 across a broad pH range. The fractions of
protonized amino groups (NH3+) and negatively charged E2- can be described by their
dissociation constants using the relationship of Katchalsky [184].
⎛ 1− a ⎞
pKa=pH + log ⎜ ⎟ (11)
⎝ a ⎠
The protonation is given by:
⎯⎯
→ R-NH 2 +H 3+ O
R-NH 3+ +H 2 O ←⎯
⎯
Glucosamine
(1-a) a a
The dissociation behaviour of primary and secondary hydroxyl groups is similar to that of
E2, and correlates with changes in the NH2-fraction. Hence, the relationship between the
hydroxyl groups of chitosan and E2 involves both attraction and repulsion. The dissociation
of E2 is therefore given by:
⎯⎯
→ 2R-O - +2H 3+ O
2R-OH+2H 2 O ←⎯
⎯
17ß-estradiol (E2)
(1-a) a a
Theoretically (Figure 13, bottom left and right), both the amino groups of chitosan and
hydroxyl groups of E2 should be uncharged in the pH range from 8 to 9. Hence, the degree of
electrostatic interaction is very low in this range, resulting in a very low loading capacity.
The model predictions correlate very well with the experimental results shown in Figure
14, indicating that the sorption is substantially lower at pH 8 than at the other tested pH
values. With increasing pH the net charge of E2 is becoming more negative. At decreasing
pH values the mentioned charge decreases until the isoelectric point at pH 10.71. With
increasing H+ concentration E2 becomes almost neutral. However, the amino groups are
further protonated and the interaction potential increases. At pH 6.3 chitosan is at its
isoelectric point and the net charge is becoming positive, which also explains the solubility of
chitosan in weak acids. Hence, the maximum loading capacity increases independently of the
charge of E2.
1.0 1.0
0.8 NH2 0.8 E2-OH

-
Fraction a [-]
Fraction a [-]
NH3
+ E2-O
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
pH pH
1.0 + 1.0
NH3
- NH2
0.8 E2-O 0.8
Fraction a [-] E2-OH
Fraction a [-]
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
pH pH
Figure 13. Model of the degree of protonation of chitosan (top left) und 17ß-estradiol (top right) across
the pH range from 0 to 14. Comparison of charged (bottom right) and uncharged (bottom right) amino
and hydroxyl groups of chitosan and 17ß-estradiol.
0.8
X [µg/mg]
0.6
8 0.4
0.2
0
0 2 4 6 8 10 12
pH
Figure 14. Dependence of E2 binding to chitosan on pH after 120 hours equilibration (T=20°C,
n=150 min-1 agitation, CE2,0 =200 µg L-1).
10 10
KD
X00
8 8
-1.73
- - - - KD = 0.0484 CSorb
2
6 R = 0.937 6
X00 [µg mg ]
-1
KD [L g ]
-1.41
-1
X00 = 0.0169 CSorb

4 2 4
R = 0.907
2 2
0 0
0.1 0.2 0.3 0.4 0.5

-1
Chitosan concentration CSorb [g L ]
Figure 15. Effects of chitosan concentration on the sorption constant KD and loading (pH=6 after 120 h,
T=20°C, 150 min-1 agitated, CE2,0 =200 µg L-1).
The variation in equilibrium loading ( X ∞ ) as a function of pH shows that the sorption

process becomes more efficient with decreasing pH. In theory the sorption should increase
with decreasing pH because of the increasing electrostatic interaction between the protonized
primary amino groups (R-NH3+) of the glucosamine and the two depolarized terminal OH-
groups of E2. However, the solubility of chitosan also increases with increasing degree of
protonation of the amino groups. At pH values lower than 4 the loading capacity declines, due
to the mentioned solubility of chitosan at low pH. This is described as the adsorbance-
concentration effect.
6.1.7.2. Effect of the Chitosan Concentration on the Sorption Process

To design a cost-effective removal process it is necessary to know the optimal sorbent-
adsorbent ratio. Therefore the concentration of chitosan was varied between 0.07
and 0.4 g L-1 with a constant E2 concentration of 200 µg/L. The sorption constant with
respect to the sorbens concentration is calculated from:
X∞
KD = ⋅1000 (12)
C∞
with the loading at equilibrium X ∞ from equation 12.

K D describes the sorption with respect to the sorbens concentration, and in isotherm
models the sorption constant is affected by changes in the sorptive concentration. The
sorption constant and the loading decrease with increasing chitosan concentration (Figure 15).
This is due to the adsorbent-concentration effect [185]. It has been observed that hydrophobic
organic compounds adsorb to the carbonaceous matrix while some of the adsorbate
(adsorptive-adsorbens-complex) is present as colloidal, non-fractionable micro-particles. The
higher the chitosan concentration the higher the fraction with adsorbed E2 that will dissolve.
Hence, if the E2 is detected by UV-spectroscopy an increasing concentration of E2 will be
observed. The fraction that will be dissolved is fixed with respect to the total concentration of
the solid phase [185]. This effect is only observed if the loading is determined by the E2
concentration in the liquid phase.
Furthermore, the E2 removal efficiency is reduced by the presence of ions like NaCl.
Increasing the electrolyte concentration significantly decreases the capacity to sorb E2, due to
the higher affinity and mobility of small ions competing with E2.
6.1.7.3. Sorption Isotherms

Sorption isotherms are used to obtain information about the contact time (time required to
reach equilibrium) and the maximum loading in order to define boundary conditions for
large-scale applications. The most common models for this purpose are Langmuir, BET
(Brunauer, Emmett and Teller) and Freundlich models.
According to Langmuir, the loading at equilibrium X ∞ depends on the maximal
monolayer capacity Xmax, the sorptive concentration in the liquid phase at equilibrium C∞
and the sorption constant, which provides information about the affinity.
k L ⋅ C∞
X ∞ = X max ⋅ (13)
1 + k L ⋅ C∞
Langmuir [186] assumed a uniform surface with energetically equal binding sites, a
monolayer of adsorptive, at most, and no interaction between adjacent adsorbed molecules.
Hence, Langmuir models neglect the possibility that the first layer may provide a substrate for
a further layer. This possibility is considered in the model of [187], in which an infinite
number of layers is possible.
C∞
X∞ = (14)
⎛ 1 (kB − 1) ⎞
⎜ + ⎟ ⋅ ( Cmax − C∞ )
⎝ kB ⋅ X max kB ⋅ X max ⋅ Cmax ⎠
For a monolayer the equation is reduced to that of Langmuir. Xmax is the maximum
monolayer capacity, kB is the sorption constant and Cmax is the maximum solubility of the
sorptive. The maximum solubility of E2 in water is 3.6 mg/L at 20°C [188].
As mentioned above, a Langmuir model does not consider either the heterogeneity of the
binding sites or the interaction (attraction and repulsion) of adsorbed molecules. However, the
sorption enthalpy often decreases with increasing loading. Hence, Langmuir principles are not
valid for systems with high changes in enthalpy.
The empirical Freundlich model describes the logarithmic reduction in sorption enthalpy
with increasing loading, which is given by.
Table 10. Model linearization of sorption isotherms
Model Linearization
C∞ 1 1
Langmuir = + ⋅ C∞
X ∞ k L ⋅ X max X max
C∞
=
1 ( k − 1) ⋅ C∞
+ B
BET
X ∞ ( Cmax − C∞ ) k B ⋅ X max k B ⋅ X max Cmax
1
Freundlich log X ∞ = log k F + ⋅ log C∞
n
0.10
280
C /(X (Cmax-C )) [mg µg ]

-1
240
0.08
C /X [mg L ]
-1
200 0.06
8
160 0.04
8
120 y = 0.675x+135.519 y = 0.747x+0.038
8 2 8 0.02 2
80 R = 0.583 R = 0.615
0.00
20 40 60 80 100 120 140 160 8
0.00 0.01 0.02 0.03 0.04 0.05
C [µg/L] C /Cmax
8
8
-0.1 y = 0.617x-1.536
2
-0.2 R = 0.861
log |X |
8 -0.3
-0.4
-0.5
-0.6
1.2 1.4 1.6 1.8 2.0 2.2
log |C |
8
Figure 16. Sorption isotherms at 20 °C and pH 6 generated by Langmuir (left), BET (middle) and
Freundlich (right) models (duplicate** samples, agitation rate, 150 min-1, CCHI=67 mg L-1).
The empirical Freundlich model describes the logarithmic decreasing sorption enthalpy
with increasing loading which is given by:
nF
X ∞ = k F ⋅ C∞ (15)
where kF is the Freundlich sorption constant and nF the empirical Freundlich exponent,
which accounts for the concentration dependency of the reaction. However, the missing limit
for increasing equilibrium concentrations is problematic, since it implies that a surface may
have an infinite number of binding sites at high equilibrium concentrations. This results in an
overestimation of the sorption because the binding sites are actually limited. By model
linearization (Table 10) the parameters can be estimated.
Table 11. Summary of estimated model parameters from Langmuir, BET and
Freundlich isotherms at pH=6
⎡L⎤ ⎡ µg ⋅ LnF ⎤ ⎡ µg ⎤
Isotherm kL ⎢ ⎥ kB [ −] kF ⎢ nF ⎥
nF [-] X max ⎢ ⎥ R2
⎣ µg ⎦ ⎣ mg ⋅ µg ⎦ ⎣ mg ⎦
Langmuir 0.005 - - - 1.481 0.583
BET - 46.775 - - 0.573 0.615
Freundlich - - 0.029 1.620 - 0.861
Figure 17. Sorption hypothesis derived from the results of sorption experiments based on the
Freundlich model (E2 on chitosan at pH 6 and 20°C). The hydrophobic areas are coloured yellow, H+
red, oxygen dark blue and nitrogen light blue. The arrows represent the direction of interaction while
the thickness of the arrows qualitatively indicates the intensity of interaction.
Since the aim of the investigation was to evaluate the use of chitosan in wastewater and
drinking water treatment to remove estrogenic compounds, which may cause ecological
problems, as noted above, and the pH of wastewater and drinking water usually ranges
between 6 and 8, pH 6 was chosen for this parameterization experiment.
From the model linearization we can determine all parameters, as summarized in Table
11. The Freundlich isotherm has a significantly higher correlation coefficient (R²=0.861) than
both the Langmuir and BET isotherms (R²=0.583 and 0.615, respectively).
The modelling results indicate that E2 forms a monolayer of molecules on chitosan, in
which the E2 molecules interact with each other. In Figure 17 a model-based hypothesis is
depicted in which the E2 molecules adsorb vertically to chitosan, because in this
configuration the interaction potential is assumed to be maximal. At pH 6 the ratio of
protonized and unprotonized amino groups is about 0.5. Due to the strong affinity of E2 for
NH+ the binding will occur first at these sites and possibly subsequently at the nonprotonized
amino groups.
0.10
280
C /(X (Cmax-C )) [mg µg ]

-1
0.08
240
[mg L ]
-1 200 0.06
8
160 0.04
8
120
C /X
y = 1.292x+0.118 0.02 y = 1.364x+0.010

8 8
2
80 2
R = 0.965 R = 0.967
8 0.00
20 40 60 80 100 120 140 160 0.00 0.01 0.02 0.03 0.04 0.05
C [µg/L] C /Cmax
8
8
y = 0.281x-0.792
-0.1 2
R = 0.883
-0.2
log |X |
8 -0.3
-0.4
-0.5
-0.6
1.2 1.4 1.6 1.8 2.0 2.2
log |C |
8
Figure 18. Sorption isotherms obtained at pH 1.5 and 20°C from Langmuir (left), BET (middle) and
Freundlich (right) models (duplicate samples, agitation rate, 150 min-1, CCHI=67 mg L-1).
Figure 19. Illustration of sorption hypothesis derived from the results of sorption experiments based on
the Freundlich model (E2 on chitosan at pH 6 and 20°C). The hydrophobic areas are coloured yellow,
H+ red, oxygen dark blue and nitrogen light blue. The arrows indicate the direction of interaction while
the thickness of the arrows qualitatively indicates the intensity of interaction.
Interactions between hydrophobic areas lead to forces between neighbouring molecules

and those that are close to them but still in solution. Furthermore, it is possible that the
terminal OH groups of E2 may interact with each other. Depending on the orientation, both
attraction and repulsion forces may occur.
If the pH is set to 1.5 (Figure 18) the results differ according to the considered sorption
theories.
As shown in Table 12 the correlation coefficients of the Langmuir and BET isotherms are
almost identical and significantly higher than those of the Freundlich model.
Table 12. Summary of estimated model parameters from Langmuir, BET and
Freundlich isotherms at pH=1.5
⎡L⎤ ⎡ µg ⋅ LnF ⎤ ⎡ µg ⎤
Isotherm kL ⎢ ⎥ kB [ −] kF ⎢ nF ⎥
nF [-] X max ⎢ ⎥ R2
⎣ µg ⎦ ⎣ mg ⋅ µg ⎦ ⎣ mg ⎦
Langmuir 10.987 - - - 0.774 0.965
BET - 235.943 - - 0.423 0.967
Freundlich - - 0.162 3.563 - 0.883
Hence it can be concluded that a change in pH leads to a change in the sorption behaviour
to a Langmuir type. This indicates there are negligible differences in surface energy at the
binding sites, implying that there are no interactions between adsorbed molecules that are
orientated flatly on chitosan, and the sorption of molecules is independent of the occupancy
of neighbouring binding sites(Figure 19).
As mentioned above, the BET model is identical to the Langmuir model if there is only
one layer of adsorbed molecules. However, the Xmax value for the Langmuir monolayer
capacity is almost as twice as high as the BET capacity. Hence, development of more than
one sorption layer can be assumed since the Langmuir model allows only one layer, in
contrast to the BET model, and the first layer may act as the substrate for the next layer.
According to the BET model, interactions between adsorbed E2 molecules are not
allowed Figure 19, but in reality there will be interactions between the hydrophobic
components and between the OH groups of E2. The estimated monolayer capacities indicate a
partially developed double layer. Figure 19 depicts optimum adsorption, i.e. one E2 molecule
binds to one glucosamine monomer. However, the results show that only about 1/1000 of the
theoretically possible loading is reached. There may be several reasons for this, including the
abovementioned solubility of chitosan as well as the size of the E2 molecule, which may
affect the stability of adsorption and physisorption, respectively. The reasons for this are still
unclear and require further investigation.
6.1.7.4. Sorption Kinetics

In this section the kinetics of sorption E2 to chitosan are considered, which were
addressed in both short and long term studies. Two models were used to describe the
dynamics of the mass transfer in the first seven hours from the liquid to the solid phase: the
pseudo 1st order of Lagergren,
dX La (t )
= k La ( X ∞ , La − X La (t ) ) (16)
dt
and the pseudo 2nd order model of Ho [189],
dX Ho (t )
= k Ho ( X ∞ , Ho − X Ho (t ) )
2
(17)
dt
where X ∞ ,La and X ∞ ,Ho describe the concentration reached at equilibrium, while k La and
k Ho are the maximum sorption rates. Both parameters were estimated by fitting the models to
the data (Figure 20). The results are summarized in Table 13.
Both models describe the kinetic data obtained in the first seven hours very well, with
correlation coefficients close to one. In section 6.1.7.1. it was shown that the maximal loading
at equilibrium is slightly lower at pH 1.5 than at pH 6, but in Figure 20 it can be seen that
loading is significantly higher at pH 1.5 than at pH 6. This may be explained by the
increasing number of protonized amino groups, which promotes rapid adsorption of E2.
These findings indicate that the dissolution of chitosan is slower than the sorption rate
since in section 6.1.7.1. it was pointed out that the maximum loading declines with decreasing
pH due to the increasing chitosan solubility, and the E2 is indeed adsorbed but the adsorbate
complex has partially colloidal character and cannot be separated from the liquid phase by
0.45 µm filtration.
During long-term observations, up to 120 hours (Figure 21), we found that the adsorption
was not stable, but instead there was a dynamic equilibrium involving both adsorption and
desorption. This was not due to the solubility of chitosan since it was also observed when
using immobilisates.
This effect of reductions in pH may be due to the repulsive forces between unfavourably
oriented dipoles. It is assumed that the attraction is based on the interactions between the
amino groups of the glucosamine and the OH groups of E2. At first an attraction occurs
between the dipoles at a far distance. However, at less than a critical distance repulsive forces
dominate the interaction. This short distance interaction of dipoles is called Keesom
interaction. It is possible that an E2 molecule may be attracted and repulsed by two opposite
amino groups, causing permanent migration of the sorptive, thus preventing stable attachment
and adsorption. On the other hand, the structure of the glucosamine monomer of the chitosan
polymer and the E2 molecule may prevent any possible alignment. In addition, weak
interactions with other molecules (e.g. heavy metals or other organics) may impart destructive
momentum to the sorbed E2. The likelihood of such an effect increases with increases in the
fraction of protonized amino groups, which could explain the stronger desorption at pH 1.5
than at pH 6 and 10. These effects are unlikely to occur following the sorption of metal ions
since the ions will adsorb in an optimal orientation due to their small molecular size.
Table 13. Summary of estimated parameters of the kinetic pseudo 1st and 2nd order
models at pH 1.6, 6 and 10 (20°C, 150 min-1 agitation, CE2,0 =200 µg L-1, mCHI=67 mg L-1)
k La kHo X ∞, La X ∞, Ho 2 2
Parameter -1 -1 -1 -1
RLa RHo
[h ] [mg µg h ] [ µg mg ] [ µg mg -1 ]
pH 1.5 0.843 0.905 1.024 1.195 0.996 0.993
pH 6 0.918 1.358 0.792 0.913 0.987 0.996
pH 10 0.733 1.745 0.388 0.470 0.990 0.981
pH 1.5
1.5 pH 6
pH 10
------- pseudo 1. order
pseudo 2. order
1.0
X(t) [µg mg ]
-1
0.5
0.0
0 1 2 3 4 5 6 7 8
Time [h]
Figure 20. Effect of pH on the loading capacity of chitosan for duplicate E2 samples (agitation
rate,150 min-1, CCHI=67 mg L-1).
Figure 21. Kinetics of E2 sorption to fungal chitosan powder at pH 1.5, 6 and 10 during 120 h of
sampling.
6.1.7.5. Influence of Immobilisation onto Glass Beads

Chitosan was immobilised according to [163] with glass beads ranging from 150 to
212 µm in diameter, and the method of Svennerholm was used, with modification, to quantify
the amounts of immobilised glucosamine and chitosan [190]. The average mass fraction was
determined to be 0.02 % (mChitosan/mGlassBeads), regardless of the chitosan concentration used
during the immobilisation procedure.
The immobilisation provided a 30-fold enhancement of the loading at equilibrium with a
sorption time of 7 hours (Table 13 and Table 14).
It was possible to decrease the E2 concentration in the supernatant to 33 % at pH 7 and
27 % at pH 6. The enhanced performance of the E2 removal is due to several reasons. Firstly,
due to immobilisation no colloidal fraction of chitosan occurs that is inseparable from the
supernatant and lowers the observed adsorption. Secondly, the immobilisation orientates the
chitosan polymers in a uniform direction, thus reducing the probability that attachment of E2
molecules will be hindered by two opposite amino groups.
The behaviour of the sorption process at pH=10 could not be explained (Figure 21).
Furthermore, after 24 h of treatment unidentified peaks were observed using UV fluorescence
spectroscopy that interfered with the signal analysis, causing the apparent E2 concentration to
increase to more than the initial concentration.
60 1.0
pH 1,5 pH 1,5
pH 6
50 pH 10
pH 6
pH 10
------- pseudo 1. order 0.9 ------- pseudo 1. order
40 pseudo 2. order pseudo 2. order
X (t) [µg m g -1 ]
30
C (t)/C 0
0.8
20
0.7
10
0 0.6
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time [h] Time [h]
Figure 22. E2 loading capacity of immobilised chitosan (duplicate samples, 20°C, 150 min-1 agitation
rate, mGlassBeads=11 g L-1 equals mCchitosan=22 mg L-1).
Table 14. Parameters of the pseudo 1st and pseudo 2nd order kinetic models at pH 1.5, 6
and 10, obtained using chitosan immobilised on glass beads (20°C, 150 min-1 agitation
rate, CE2,0 =200 µg L-1, mGlassBeads=11 g L-1 equals mCchitosan=22 mg L-1)
k La kHo X ∞ , La X ∞, Ho 2 2
Parameter -1 -1 -1 -1
RLa RHo
[h ] [mg µg h ] [ µg mg ] [ µg mg -1 ]
pH 1.5 0.613 0.019 30.984 37.336 0.967 0.986
pH 6 0.661 0.029 21.382 25.776 0.975 0.988
pH 10 - - - - - -
1.0 1.0
0.8 ideal BT curve

0.8 -1
F = 2,7 ml min , CE2,0=100µg L
-1
-1 -1 real curve
F = 41,6 ml min , CE2,0=200µg L
-1 -1
0.6
C /C 0
F = 2,3 ml min , CE2,0=200µg L
C/C 0
0.6
0.4
0.4 0.2
0.0
0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160
Bed volumes Bed volumes
Figure 23. Breakthrough curves for E2 at different concentrations and flow rates (left). Comparison of
an ideal and a real breakthrough curve (right).
Figure 24. Schematic diagram of the sorption column as an ideal, anisotropic, stationary column
reactor. F is the flow rate, C the concentration of E2, VBed the bed volume, X the loading capacity, z the
filter bed length and m the mass flow.
6.1.7.6. Filter Bed Columns with Immobilised Chitosan for 17ß-Estradiol Removal
To evaluate E2 sorption in columns, 5.5 g portions of chitosan-coated glass beads were
placed in glass columns. The bed volume was estimated to be 3.6 cm³, with a bulk density, ρB,
of 1.53 g cm-3 and a bed porosity, υ, of 38.8 % . The filter bed was 0.9 cm wide and 5.6 cm
high with a calculated surface of about 734 cm², assuming an average bead diameter of
180 µm. The prepared column was equilibrated by flushing first with 100 mL of 0.05 M
HNO3 and then 400 mL distilled water.
For laboratory-scale experiments breakthrough was evaluated at pH 6 with E2
concentrations varied in the 100 µg range (in accordance with the sensitivity of the
fluorescence spectrometer used for E2 analysis) and different flow rates (Figure 23).
No typical breakthrough curves were obtained, as shown in Figure 23, due to the
unfavourable relationships between bed volume, flow rate and E2 concentration. The bed
volume to flow rate ratio determines the contact time between sorptive and sorbent. The
minimum flow rate was limited by the pump used to 2.3 mL min-1 and the bed volume by
limited available resources for the production of glass bead immobilisates. The E2
concentration was chosen according to the detection limit of the analytical procedure. Under
these circumstances a circulating use of the sorption column was preferred. Therefore,
500 mL of a solution with 200 µg L-1 E2 was pumped several times through the filter bed to
achieve an adequate contact time.
To draw general conclusions regarding the performance of the sorption process inside the
column several assumptions were made for modelling.
Firstly, ideal plug flow was assumed, i.e. it was assumed that no diffusion and dispersion
occurred. Hence, radial concentration gradients were neglected (Figure 24). These
assumptions are usually valid [191]. If so, a concentration gradient only occurs along the
length of the column. The change in temperature was also neglected.
∂C ∂C ∂C ∂T ∂T ∂F ∂F
= = 0, = f ( z ), = = 0, = =0
∂t ∂r ∂z ∂t ∂z ∂t ∂z
In the first hours of the process under constant boundary conditions it can be regarded as
irreversible, as known from previous experiments.
Sorptiv + Sorbens ⎯⎯→

k BA
Sorbat
(18)
CE 2 + NChitosan ⎯⎯→
k BA
E 2 − Chitosan
Therefore, it was assumed that an amount of sorbens with a residual capacity N [µg L-1]
decreases over time by the rate kBA. From the rate of decreasing residual capacity and E2 we
can obtain Bohart-Adams’ 1st order equations, which were initially developed for the
characterisation of charcoal columns [192].
1 ⎛ ∂m0 ⎞
⎜ ⎟ =r (19)
V ⎝ ∂t ⎠ Adsorption
with r = −kBA ⋅ CE 2 ⋅ N (20)
and ∂V = ABed ⋅ ∂z (21)
∂m
= − k BA ⋅ CE 2 ⋅ N ⋅ ABed (22)
∂z
∂CE 2 k ⋅ C ⋅ N ⋅ ABett
= − BA E 2 (23)
∂z F
∂N
= −k BA ⋅ CE 2 ⋅ N (24)
∂t
In circulated operation the filter bed is regarded as a single finite unit and the sorption
zone as the product of its length multiplied by the number of passages of the model solution.
VE 2− Solution
n= = 139 (25)
VBed
For example, three passages of the original volume (500 mL) results in 3x139 mL bed
volumes. Since the column reactor is used in circulation mode and (hence) the return ratio is
RV → ∞ the reactor operates as an ideal stirred batch reactor. The process time can then be
expressed by the cumulative contact time and the number of bed volumes.
VE 2 − solution VBed
tcontact = = ⋅n (26)
VBed F
By using Bohart-Adams’ equations we can obtain the change in the residual capacity and
the E2 concentration depending on the number of passed bed volumes (Figure 25).
∂CE 2 ∂N k ⋅ C ⋅ N ⋅ VBed
= = − BA E 2 (27)
∂n ∂n F
Contact time [h]
0 5 10 15 20 25 30 35 40 45
0 1 2 3 4 5 6 7
0.0 0.5 1.0 1.5 2.0 2.5
-1
F=2,3 mL min
1.0 -1
F=15 mL min
-1
F=41,6 mL min
0.9
C/C0
0.8
0.7
0 250 500 750 1000 1250 1500 1750

Bed volumes
Figure 25. Time course of changes in the E2 concentration (initial conc. 200 µg L-1) during the filter
bed contact volumes and time. Each data point is the mean of five determinations.
By balancing around the filter bed the loading after each run is calculated from:
∂m1 ∂X
= mBed = F ⋅ CE 2,0 − F ⋅ CE 2,i (28)
∂t ∂t
X ( n) =
(C E 2,0 − CE 2,i +1 ) ⋅VE 2− solution
mBed ⋅ 0, 0002
⎧⎪CE 2,i ; a ⋅ i ≤ n ≤ a ⋅ (i + 1) for a = 139

CE 2,i = ⎨ (29)
⎪⎩CE 2,i +1 ; a ⋅ (i + 1) ≤ n ≤ a ⋅ (i + 2) and i = 0...11
The factor 0.0002 represents the mass fraction (0.02 %) of chitosan relative to the mass
of modified glass beads used (mChitosan/mGlassBeads).
Using the dynamic sorption models of Lagergren and Ho (see previous section) maximal
loadings of 41.5 µgE2/mgChitosan (Ho, pseudo 2nd order) or 30.0 µgE2/mgChitosan (pseudo 1st
order) at a flow rate of 15 mL min-1 can be estimated. With an increased flow rate of
41.6 mL min-1the max. loading decreases to 29.7 µgE2/mgChitosan (pseudo 2nd order) and
20.6 µgE2/mgChitosan (pseudo 1st order).
Contact time [h]
0 5 10 15 20 25 30 35 40 45
0 1 2 3 4 5 6 7
0.0 0.5 1.0 1.5 2.0 2.5
30 F=2,3 mL min
-1
-1
F=15 mL min
-1
25 F=41,6 mL min
20
X(n) [µg mg ]
-1
15
10
0
0 250 500 750 1000 1250 1500 1750
Bed volumes
Figure 26. Time course of the column loading mE2/mChitosan at different flow rates, with an E2
concentration of 200 µg L-1 and 500 mL test solution. Each data point is the mean of five
determinations.
Table 15. Key parameters for characterising the column performance
Residual Sorption rate

Flow rate velocity v X∞,La X∞,Ho
capacity kBA
F [mL min-1] [mL cm-2 min-1] [µg mg-1] [µg mg-1]
N [µg L-1] [mL µg-1 min-1]
2.3 44.5 0.026 3.6 20.1 22.4
15 70.4 0.035 23.6 30.0 41.5
41.6 48.1 0.057 65.4 29.7 20.6
The residual capacities are quite different from those of Bohart-Adams, because the
solution volume is considered in the models of Lagergren and Ho, here 500 mL. If this is
accounted for the three models give similar results, as shown in Table 15.
The amount of adsorbed E2 which is due to the glass beads was determined to be 7 % at
equilibrium, due to the porosity of the glass particles which was estimated to be 9 %.
At a flow rate of 2.3 mL min-1 release of E2 occurs (marked by arrows in Figure 25 and
Figure 26) which may be due to desorption, which was also observed in previous
experiments. The exact time and conditions are unclear. However, after desorption the
original loading is reached again. This phenomenon was observed in all long-term
experiments and was also reported by Zhang et al. [193]. Compared to chitosan the adsorption
by activated carbon is more stable. However, if we understood the conditions that result in
desorption it could be used in a self-cleaning sorption process with cyclic adsorption and
desorption intervals.
6.1.8. Chitosan as Bone Replacement Material in Medicine - Biomimetic Coating of

Chitosan Films
In renewable medicine chitosan has gained increasing acceptance due to its outstanding
biocompatibility and biodegradability, notably as a component of drug delivery systems
[84,194,195]. Chitosan stimulates the formation of connective tissue [84], promotes natural
wound healing [196], acts as an antibacterial and antiviral agent [197,198] and provides an
osteoconductive stimulus [199].
Given these characteristics and knowledge about the composition of bones
(approximately 70% hydroxyapatite and 30% collagen) it appears feasible to develop a spare
bone material from chitosan and hydroxyapatite. This could be highly valuable since bone can
be spontaneously regenerated, but this capacity decreases, in humans, from the age of 40
years by ca. 2-3% per year[200],
and the current annual demand for bone replacement operations in Europe is about ½
million [201].
In Germany there are 71,000 bone replacements annually, usually relying on autologous
bone tissue [202]. A disadvantage of bone replacement by withdrawing autologous bone
material is the severe limitations on the amount that can be removed. Furthermore, the
demand for bone replacement material is increasing due to the increasing life expectancy of
the population [202].
Therefore, research is focusing on the development of suitable implant materials which
are sufficiently available and induce no toxic or carcinogenic reactions, infections or
repulsion reactions.
566 Mirko Trutnau,
T Thom
mas Bley and Jelka
J Ondruschka
Bone replaacement mateerials promotte the bone healing proccess either allone or as
coomposites witth other mateerials. In the context of tissue t engineeering, three-ddimensional
caarriers (scaffolds) are usually colonised with
w autologouus cells and finally
fi implantted into the
paatient [203]. Chitosan
C seem
ms to be suitablle for use in sttructured mateerials to close large areas
[1195,204,205]. Its outstandinng biocompattibility and sccope for modiification are particularly
p
vaaluable in thiss context.
The osteo oconductivity of chitosann can also be increasedd by coatingg it with
hyydroxyapatite, which enhannces its interaaction with boone material. This T causes a significant
inncrease in celll differentiation along thee osteogenic line, resultingg in neoplasm m of bone
m
material [206,2
207].
During bio omimetic coaating the im mplant materiaal (polymer, ceramics or metal) is
inncubated in a simulated body fluid (SBF F) with similaar ion concenttrations to huuman blood
[2208]. Importan nt factors are a short periodd of incubatioon (Barrere et al., 2002) [2009] and the
immprovement of o hydroxyapaatite formed onn the implant surface durinng the incubatiion. As yet
thhe interactionss with biomoleecules, aspectss of crystal deevelopment rattes and the conntrol of the
crrystal morphology are not well
w understood.
Renewablee chitosan seeems to be an a adequate material for developing biomimetic b
cooatings, using g a solution of 2% chitossan in 0.1 M acetic acid.. Calcium phhosphate is
prrecipitated as hydroxyapatitte onto chitosaan films. The precipitated crystals
c can bee identified
annd characterizzed by energy dispersive X-ray spectrosccopy (EDX), slot s electron microscopy
m
(RREM), Fourieer transformaation infrared spectroscopyy (FTIR) andd transmissioon electron
m
microscopy (TE EM). Hydroxyyapatite has been
b detected after just sevven days incubbation both
onn the surface and
a within thee film of chitossan at physioloogical pH Figuure 27.
In experim
ments with mouuse osteoblastts cells were completely
c sprread on the chhitosan film
w
within 24 hourss of incubatioon. The cultivaated cells exhhibited a stretcched actin-skeeleton, thus
prroviding a largge contact areaa between the chitosan filmms and cells (Fiigure 28).
In additionn, the cells foormed a focal adhesion which
w enabled them to bindd the actin
skkeleton mech hanically to thet surface within
w 24 hoours. Further cell contactts between
neeighbouring cells
c occurred which boundd the cells to each other and a allowed inntercellular
coommunication n. No preferreed orientation of mouse 7F2 osteoblasts was observedd, which is
inndicative of a planar,
p unstruuctured surfacee. The bundledd actin filamennts were visibble as stress
fibres, which alllow the cells to migrate by contraction.
Fiigure 27. TEM image of the HA

AP-surface of a fracture and chhitosan-HAP innterface [210].
Figure 28. FM (Fluorescence microscope) images of mouse 7F2 osteoblasts on a chitosan film after 2 h
(left) and 24 h (right) incubation. Cell cores (blue); actin skeleton (green) [210].
CONCLUSION
Since chitosan was initially isolated from mushrooms ca. 150 years ago, following the
pioneering efforts of Muzzarelli and Hirano, many potential applications have been
investigated and some have been developed into well-established, environmentally friendly
and sustainable uses. Chitosan has also had a great impact on polymer chemistry, but its
potential is still far from fully exploited. Although there are more than 200 chitosan
manufacturers and suppliers, and thousands of proved applications, it is still very difficult to
obtain chitosan that is fully standardized with respect to molecular weight and degree of
deacetylation, e.g. for pharmaceutical use. Furthermore, not all possible sources of good
quality chitosan are economical, e.g. cultivation of chitosan-producing fungi using waste
substrates or waste mycelia from other processes such as citric acid production. However, the
number of manufacturers of fungal chitosan is increasing as well as the demand for raw
polymer, and many techniques have been developed to ensure a high quality product. Process
parameters influencing both the quality and productivity have been identified, including
technical process parameters (aeration rate, agitation rate etc.), cultivation medium
supplements like plant hormones and aspects of the extraction procedure. The demand for
chitosan has been greatly boosted due to its fat binding capacity (although there is some
scepticism in this respect), and the demand is rapidly growing for both this and other
purposes. Many more applications will surely emerge in coming decades, possibly with
similar (or greater) groundbreaking character.
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Chapter 2
CHITOSAN: MODIFICATIONS AND APPLICATIONS

IN DOSAGE FORM DESIGN
Ashok Kumar Tiwary∗, Bharti Sapra, Gurpreet Kaur

and Vikas Rana
Product Development Group,
Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala, India
Chitosan is the deacetylated form of chitin. Generally, the substance becomes soluble in
dilute acids when the degree of deacetylation is more than 50%. The solubility of chitosan in
dilute acids is often needed to be modified when specific drug release properties have to be
tailored into the dosage form. Chitosan carries free amine functionalities on the deacetylated
units and hydroxyl groups on the acetylated as well as deacetylated units. Derivatization by
introducing small functional groups such as, alkyl or carboxymethyl groups [Jayakumar et al.,
2006; Lu et al., 2007] can increase the solubility of chitosan at neutral and alkaline pH
without affecting its cationic character. In addition, chitosan can be grafted with other
molecules through covalent binding. The amino groups can be used for acetylation,
quaternization, reactions with aldehydes and ketones, chelation of metals etc. The hydroxyl
groups can lend to o-acetylation, H-bonding with polar atoms etc. Primary derivatization
followed by grafting improves the solubility, antibacterial, antioxidant [Xie et al., 2001; Xie
et al., 2002a], chelating [Yang and Yuan, 2001], complexing [Chen and Wang, 2001],
bacteriostatic [Jung et al., 1999] and adsorbing [Thanou et al., 2001a] properties while
maintaining its mucoadhesivity [Hoffman et al., 1997], biodegradability [Singh and Ray,
1998] and biocompatibility [Tasker et al., 1998; Ono et al., 2000]. These functionalities can
also be used for interaction of chitosan with ions [Sugunan et al., 2005; Piyakulawat et al.,
2007], polymers [Prabaharan and Gong, 2008] and drugs [Puttipipatkhachorn et al., 2001] for
obtaining materials useful for various applications. In its putative form, chitosan’s positive
∗ E-mail: aktiwary2@rediffmail.com.
72 Ashok Kumar Tiwary, Bharti Sapra, Gurpreet Kaur et al.
charge allows it to interact with macromolecules like exogenous nucleic acids, negatively
charged mucosal surfaces and plasma membranes [Danielsen et al., 2005; Bowman and
Leong, 2006]. It is used as a support for gene delivery [Mao et al., 2010] and cell culture
[Riske et al., 2007]. In addition, chitosan is reported to possess antibacterial, anti-fungal, anti-
viral, anti-acid and anti-ulcer properties [Tokura et al., 1997; Kweon et al., 2003; Khnor et al.,
2003]. The ability of chitosan to swirl across the membrane lipids is reported to be associated
with its property to perturb the paracellular pathway for enhancing the permeation of
hydrophilic drugs and to act as tight junction opener [Ward et al., 2000]. This contributes to
its role as a percutaneous and intestinal absorption enhancer. Chitosan derivative (N,O-
carboxymethyl chitosan) and alginate blended with genipin was developed for site-specific
protein drug delivery in the intestine that requires protection of drug release in the gastric pH.
Tablets [Nunthanid et al., 2004], microspheres [Anal et al., 2006], nanoparticles [Trapani et
al., 2009], intradermal vaccines [Bal et al., 2009] etc. are being formulated using chitosan for
modifying the drug release characteristics.
The available toxicological data on chitosan and its modified forms appears to indicate its
safety for oral use because high doses have been found to be tolerated well in rodents and
rabbits. However, its local action as a haemostatic together with its ability to activate
macrophages and cause cytokine release may require a careful assessment of its safety for
parenteral use.
WHY CHITOSAN DERIVATIVES

The accumulated information about the physicochemical and biological properties of
chitosan led to the recognition of this biopolymer as a promising biomaterial. Chitosan is a
partially deacetylated polymer of N-acetyl glucosamine obtained after alkaline deacetylation
of chitin, which is found in the shells of crustaceans, the exoskeletons of insects, and the cell
walls of fungi. Chitosan, an unbranched cationic biopolymer, has three types of reactive
functional groups that allow further chemical modification, i.e., amino or amido groups at C-2
positions as well as both primary and secondary hydroxyl groups at C-6 and C-3 positions,
respectively (Figure 1).
It displays interesting properties such as biocompatibility and biodegradability [Felt et al.,
1998; Kumar et al., 2003], and its degradation products are nontoxic, nonimmunogenic, and
noncarcinogenic [Bersch et al., 1995; Muzzarelli, 1997]. Chitosan is insoluble in water,
aqueous alkaline solutions, and common organic solvents, but it readily dissolves in aqueous
inorganic and organic acid media. Therefore, special attention has been paid to the chemical
modification of chitosan. Cross-linking and graft copolymerization are well-known methods
for the modification of chitosan and represent convenient and effective ways for improving
the physical and mechanical properties for practical uses. Due to its polymeric character,
chitosan has been used in the development of drug delivery systems [Prabaharan and Mano,
2005].
Chitosan: Modifications and Applications in Dosage Form Design 73
Figure 1. Structure of chitosan.
Table 1. Physicochemical and biological proprieties of chitosan
Physicochemical properties of chitosan Biological properties of chitosan
Molecular weight (MW) of 104 Da. Biocompatible, Biodegradable

Possess three types of reactive functional groups; an Haemostatic, bacteriostatic,
amino group as well as both primary and secondary and fungistatic
hydroxyl groups.
After heating decompose prior to melting, thus has Antitumor
no melt points.
Nearly all aqueous acids dissolve chitosan; most Anticholesteremic
commonly used are formic acid and acetic acid.
Multitude of cationic sites formed due to Safe and nontoxic
protonation of amino groups by acids along the
chitosan chain increases its solubility by increasing
both the polarity and the degree of electrostatic
repulsion.
Forms salts with organic and inorganic acids. Spermicidal
When protonated, adheres to negatively charged Mucoadhesive
surfaces (muco-adhesive and forms gels with
polyanions).
High charge density at pH < 6.5. Binds to mammalian cells aggressively
High molecular weight linear polyelectrolyte. Regenerative effect on connective gum
tissue
Aminable to chemical modification. Accelerates bone formation
Viscosity, high to low. Immunoadjuvant
Chelates certain transitional metals.
Gupta and Ravi Kumar, 2000; Dutta et al., 2004.
In all the studies enhanced absorption of chitosan has been found only in acidic
environments in which the pH was less or of the order of the pKa value of chitosan (5.5–6.5).
Chitosan, a weak base, requires a certain amount of acid to transform the glucosamine units
into the positively charged water-soluble form. Due to the loss of charge in neutral and basic
environments, chitosan precipitates from solution rendering it unsuitable as an absorption
enhancer. At this pH, the molecule is most likely to exist in a coiled configuration [Artursson
et al., 1997].
Further, the apical incubation of caco-2 cell monolayers with chitosan hydrochloride or
chitosan glutamate at a pH of 6.2 resulted in reduction of Transepithelial Electrical resistance.
At this pH, both chitosan salts (chitosan hydrochloride and chitosan glutamate) did not form
clear solutions. In agreement with the results of the TEER experiments, no increase in the
transport of the hydrophilic model compound [14C]-mannitol was observed at pH of 6.2 and
7.4 after incubation with these chitosan salts [Kotze et al., 1999]. The peroral route is
considered to be the most convenient way of drug application for the patient. Most
macromolecular pharmaceuticals such as peptide and protein drugs are indicated for chronic
administration and therefore the peroral route will be the most suitable way of administering
these drugs. The potential use of chitosans, as absorption enhancer in the more basic
environments of the large intestine and colon, are limited.
In this regard it would be worthy to consider chitosan derivatives with different
physicochemical properties, especially water solubility at neutral and basic pH values as they
might prove to be useful as absorption enhancers in these environments. It was hypothesized
by Kotze´ et al. (1999a) that polymers derivatives with different substituents, different
basicities, or different charged densities could have the same or even increased efficacy in
opening tight junctions than unmodified chitosan, which has a primary amino group. Chitosan
is a versatile polymer with many functional groups available for chemical modification.
Physicochemical and biological properties of chitosan are summarized in Table 1.
CHITOSAN FUNCTIONALIZATION
Thiolated Chitosan
Thiol-containing chitosan, also called thiolated chitosan, is obtained through the reaction
between chitosan and thiolactic acid. In this reaction, carbodiimide, N-(3-dimeth-
ylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) can be used to graft these two
materials. EDC is a water-soluble carbodiimide that is typically employed in the 4.0–6.0 pH
range. It is a zero-length crosslinking agent that has been widely used to couple carboxylic
acid groups to primary amines. Thiolactic acid gets covalently attached to the primary amino
group of chitosan. The carboxylic acid moieties of thiolactic acid are activated by EDC
forming a O-acylurea derivative as an intermediate product that reacts with the primary amino
groups of chitosan [Jayakumar et al., 2007].
The primary amino group at the 2-position of the glucosamine subunits of chitosan is the
main target for the immobilization of thiol groups. Sulfhydryl bearing agents can be
covalently attached to this primary amino group via the formation of amide or amidine bonds.
In the case of the formation of amide bonds the carboxylic acid group of the ligands, cysteine
and thioglycolic acid react with the primary amino group of chitosan mediated by a water
soluble carbodiimide. The formation of disulfide bonds by air oxidation during the synthesis
is avoided by performing the process at a pH below 5. At this pH-range the concentration of
thiolate anions, representing the reactive form for oxidation of thiol groups, is low, and the
formation of disulfide bonds can be almost excluded. Alternatively, the coupling reaction can
be performed under inert conditions. For formation of amidine bonds, 2-iminothiolane is used
as a coupling reagent. It offers the advantage of a simple one step coupling reaction.
Table 2. Preparation
P of thiolated chitosan derivatives
d and th
heir uses
Derivative/Mo odification Reeaction with Exaample P

Property Application References
Thiolated chittosan Deerivatization of Polyycarbophil or nsufficient stability of
In Mucoadhesive propeerties can further be Hassan and Galllo, 1990;
priimary amino thio
omers e.g. th
hiomer improved by immobiilization of thiol groups on Bernkop-Schnu urch et al.,
grooups of chitosan chittosan-cysteine, the polymer. Improveed permeation enhancinng 1999; Schipper et al., 1999;
witth coupling chittosan-thioglycolic properties. Enhanced d and sustained gene Bernkop-Schnu urch and
ageents bearing acid
d, chitosan-4-thio delivery. Due to exceellent cohesive propertyy, Hopf, 2001; Kaast and
thiiol functions ethyylamidine prolonged controlled d release of embedded Bernkop-Schnu urch, 2001;
conj
njugate ingredients. Leitner et al., 20
003; Kast et
In situ gelling or crosss linking can be observved al., 2002; Guggi et al.,
within a range of 5-66 pH therefore absorptioon 2003; Krauland d et al., 2004;
of thiolated chitosan on vaginal, nasal, buccal Maculotti et al.,, 2005; Lee
(chitosan–thioethylamidine) and ocular mucus beccomes possible. Enzym me et al., 2006; 20007
inhibitory properties can be beneficial for orral
administration of pep ptide and protein drugs.
Im
midoester Chiitosan- By the immobilization
B New polymeric excip pient for various drug Kafedjiiski et all., 2005;
reaaction with thio
oethylamidine of thiol groups delivery systems; imp provement of both the Bravo-Osuna ett al., 2008
iso
opropyl-S- deriivative; chitosan– m
mucoadhesion was zinc-binding capacity y and the antiprotease
aceetylthioacetimid 4-th
hio-butyl-amidine sttrongly improved due effect of chitosan waas observed when the
atee to
o the formation of biopolymers (chitosaan
disulfide bonds with and thiolated chitosan n) were used as coatingg
m
mucus glycoproteins. component of the corre–shell nanoparticles, iin
comparison with theiir behaviour in solution.
Chiitosan 2- Immproved Promising tool for th

he non-invasive Maculotti et all., 2005
iminnothiolane m
mucoadhesive and administration of hyd
drophilic macromolecullar
conj
njugate permeation enhancing drugs.
prroperties, more stable
in
n aqueous media with
reespect to unmodified
chhitosan
Table 3. Quaternized chitosan derivatives and their applications
Derivative/Modification Reaction with Example Property Application References

Quaternized chitosan Methyl iodide in N,N,N-trimethyl chitosan Much higher aqueous Absorption enhancer Kim et al., 2002;
alkaline solution chloride solubility in much Improvement of Merwe et al., 2004;
of N-methyl broader pH and mucoadhesive Atyabi et al., 2007;
pyrrolidone concentration range property, antibacterial Sajomsang et al.,
activity 2009
Cross linked Cross linked N- Gene carrier in Sieval et al., 1998;
with Trimethylated Chitosan epithelial call line Thanou et al.,1999;
glutaradehyde Microsphere 2000a; 2002; Kean
fabrication et al., 2005
N-octyl- N-trimethyl Increased solubility Polymeric micelle Peng and Zhang,
choitosan derivatives formation, 2005
solubilization and
controlled release of
10-
hydroxycamptothecin
Schiff’s reaction N-alkyl derivative Antibacterial activity Jia et al., 2001;
with aldehydes/ (quaternisation) Avadi et al., 2004
ketones N,N,N-Trimethyl
Chitosan
N-propyl- N,N-dimethyl
chitosan
N-furfuryl- N,N-dimethyl
chitosan
N- diethyl-methyl amino
chitosan
N,N,N-Trimethylated chitosan Methylation Quaternised alkyl Improved polycationic Antifungal
chitosan properties
Salicylaldehyde N-arylidene chitosan Antioxidant similar to
derivatization chitosan
Chitosan-schiff’s base
reaction with methoxy Impart insolubility to
phenyl aldehyde e.g. chitosan, biodegradable
vanillin, o- vanillin, and mechanically
syringaldehyde, resistant
veratraldehyde
Reaction with Example Property Application Reference
Reductive N,N dialkyl chitosan Vesicles (for drug Li and Xin, 2006; Li
alkylation with release experiment) et al., 2007
octyl, decyl and
dodecyl
aldehydes
With alkyl Alkyl Chitosan e.g. Improved solubility in DNA delivery Liu et al., 2003
halides under isobutyl chitosan neutral aqueous solution (dodecyl chitosan)
The methylated N-aryl chitosan derivative ;methylated N-(4-N,N- basic conditions due to reduction in
dimethylaminocinnamyl) chitosan chloride) crystallinity of chitosan
N-Carboxybenzyl, Chromatographic Gupta and Kumar,

glycine-glucan, N- media and metal ion 2000
carboxy- collection
(aryl) chitosans methyl
chitosan,
Folate N-trimethyl chitosan The enhancement of Zheng et al., 2009

conjugated (folate-TMC) cellular uptake.
Nanoparticles were
promising carrier for
protein
By reacting N- N-(1-Carboxymethyl-2- Water-soluble quaternary Holappa, et al., 2006
chloroacyl-6-O- pyridinium)chitosan chitosan derivative, Full
triphenylmethylc chloride, N-[1- degree of substitution of
hitosans with Carboxymethyl-2-(1- the quaternization step
tertiary amines. methylimidazolium)]chito was
san chloride, N-(1- obtained
Carboxymethyl-2-
tributylammonium)chitos
an chloride
Investigations with all these thiolated chitosans showed that a degree of modification of 25–
250 mmol thiol groups per gram of chitosan leads to the highest improvement in the
mucoadhesive and permeation enhancing properties [Leitner et al., 2003]. Table 2 describes
the preparation of thiolated chitosan derivatives along with their uses.
The improved mucoadhesive properties of thiolated chitosans are explained by the
formation of covalent bonds between thiol groups of the polymer and cysteine – rich sub
domains of glycoproteins in the mucus layer [Andreas et al., 2003; Leitner et al., 2003]. These
covalent bonds are supposedly stronger than noncovalent bonds, such as ionic interactions of
chitosan with anionic substructures of the mucus layer. This theory was supported by the
results of tensile strength studies using tablets of thiolated chitosans, which demonstrated a
positive correlation between the degree of modification with thiol bearing moieties and the
adhesive properties of the polymer [Kast and Bernkop-Schnürch, 2001]. These findings were
confirmed by another in vitro mucoadhesion test system, where the time of adhesion of
tablets on intestinal mucosa was determined. The contact time of the thiolated chitosan
derivatives increased with increasing amount of immobilized thiol groups [Kast and Bernkop-
Schnürch, 2001; Kast and Andreas, 2001]. With chitosan-thioglycolic acid conjugates, a 5–
10-fold increase in mucoadhesion in comparison to unmodified chitosan was achieved. The
mucoadhesive properties of chitosan-4-thio-butyl-amidine (chitosan-TBA) conjugates were
even better. This phenomenon could be attributed to additionally increased mucoadhesive
properties due to improved ionic interactions between the additional cationic amidine
substructure of the chitosan-TBA conjugate and anionic substructures of the mucus layer.
The permeation enhancing capabilities of chitosan for the first time were revealed by
Illum et al., 1994. Chitosan was able to enhance the paracellular route of absorption, which is
important for the transport of hydrophilic compounds such as therapeutic peptides and
antisense oligonucleotides across the membrane. Various studies carried out on Caco-2 cell
monolayers demonstrated a significant decrease in the TEER after the addition of chitosan
[Artursson et al., 1997; Dodane et al., 1999; Martien et al., 2008]. The mechanism underlying
this permeation enhancing effect seems to be due to the positive charges of the polymer,
which interact with the cell membrane resulting in a structural reorganization of tight
junction-associated proteins [Schipper et al., 1997]. In the presence of the mucus layer,
however, this permeation enhancing effect is comparatively lower, as chitosan cannot reach
the epithelium because of size limited diffusion and/or competitive charge interactions with
mucins [Schipper et al., 1999]. Nevertheless, the results obtained on Caco-2 cell monolayers
were confirmed by in vivo studies that revealed an enhanced intestinal absorption of the
peptide drug buserelin in rats after co-administration with chitosan hydrochloride [Thanou et
al., 2000]. The permeation enhancing effect of chitosan can be strongly improved by the
immobilization of thiol groups. This effect of thiolated chitosans was shown in various
permeation studies in Ussing type chambers using freshly excised intestinal mucosa. The
uptake of fluorescence labeled bacitracin was improved 1.6-fold utilizing 0.5% of chitosan-
cysteine conjugate instead of unmodified chitosan [Bernkop-Schnu¨rch et al., 1999].
The uptake of the cationic marker compound rhodamine 123 was 3-fold higher in the
presence of thiolated chitosan as compared to that in the presence of unmodified chitosans
[Langoth et al., 2004]. This improved permeation enhancement has been ascribed to the
inhibition of protein tyrosine phosphatase. This enzyme is believed to be involved in the
opening and closing process of the tight junctions due to its involvement in the
dephosphorylation of tyrosine subunits of occludin that represents an essential transmembrane

protein of the tight junctions. When these tyrosine subunits of occludin are dephosphorylated,
the tight junctions are closed. In contrast, when these tyrosine subunits are phosphorylated,
the tight junctions are opened. The inhibition of protein tyrosine phosphatase by compounds
such as phenylarsine oxide, pervanadate or reduced glutathione leads consequently to
phosphorylation and opening of the tight junctions [Clausen et al., 2002; Barrett et al., 1999;
Staddon et al., 1995]. In contrast to the stable but toxic protein tyrosine phosphatase inhibitors
phenylarsine oxide and pervanadate, the inhibitory effect of glutathione is lower as it is
rapidly oxidized on the cell surface loosing its inhibitory activity [Grafstrom et al., 1980].
However, in combination with thiolated chitosans, the oxidation of glutathione on the
membrane is restricted because thiomers are capable of reducing oxidized glutathione
[Clausen et al., 2002].
The chemical modification of chitosan via derivatization with various reagents bearing
sulfhydryl groups causes a dramatic change in the polymer’s properties. Mucoadhesiveness
and cohesiveness are strongly improved. A comparatively stronger permeation enhancing
effect can be achieved, which can be further improved by combining thiolated chitosans with
the permeation mediator glutathione. Furthermore, thiolated chitosans display in situ-gelling
features and facilitate controlled drug release. Due to these advantageous features thiolated
chitosans have been successfully used for peroral administration of peptide drugs. They seem
to represent a promising new generation of polymeric excipients in particular for the delivery
of hydrophilic macromolecular drugs. Thiolated chitosans might also prove successful as
scaffold material in tissue engineering and as coating material for stents.
Tri Methylated Chitosan
Tri methylated chitosan (TMC), a partially quaternised derivative of chitosan, has

intensely been studied and described by Kotze´ et al. [1999a] for its absorption enhancing
effects. It was concluded that the potential use of TMC, in neutral and basic environments
where normal chitosan salts are ineffective as absorption enhancers, could contribute
significantly to the effective delivery of hydrophilic compounds such as protein and peptide
drugs. Recently, another derivative of chitosan, mono-N-carboxymethyl chitosan (MCC) was
also evaluated for its absorption enhancing ability [Thanou et al., 2001b].
TMC is a partially quaternised derivative of chitosan which is prepared by reductive
methylation of chitosan with methyl iodide in a strong basic environment at an elevated
temperature (Table 3). The degree of quaternisation can be altered by increasing the number
of reaction steps or by increasing the reaction time [Sieval et al., 1998; Snyman et al., 2002].
The chitosan used for synthesizing TMC was only soluble in acidic solutions, but after
quaternisation it became perfectly soluble in water (Figure 1). This enhanced solubility, either
in basic or acidic medium, was observed for low degree of quaternisation of 10% [Kotze´ et
al., 1998]. TMC exhibited superior solubility and basicity, even at low degrees of
quaternisation as compared to chitosan salts. The increase in solubility was attributed to the
replacement of the primary amino group on the C-2 position of chitosan with quaternary
amino groups. It should be noted that the molecular weight of the polymer chain increases
during the reductive methylation process due to the addition of methyl groups to the amino
group of the repeating monomers. However, a net decrease in the absolute molecular weight
was observed due to degradation of the polymer chain caused by exposure to the specific
reaction conditions during the synthesis [Snyman et al., 2002]. The mucoadhesive properties
of TMC with different degrees of quaternisation, ranging between 22 to 49%, were
investigated by Snyman et al [2003]. TMC was found to have a lower intrinsic
mucoadhesivity as compared to the chitosan salts, chitosan hydrochloride and chitosan
glutamate. However, as compared to the reference polymer pectin, TMC possessed superior
mucoadhesive properties. The decrease in the mucoadhesivity of TMC could be explained by
a change in the conformation of the TMC polymers due to interactions between the fixed
positive charges on the quaternary amino groups, which possibly also decreased the flexibility
of the polymer molecules. The interpenetration into the mucus layer by the polymer was
influenced by a decrease in flexibility consequently resulting in decreased mucoadhesivity
[Snyman et al., 2003].
Incubation of intestinal epithelial cells (Caco-2) with TMC (1.5, 2.0 or 2.5% w/v with a
degree of quaternisation of 12% resulted in an immediate reduction in TEER values. The
reduction in TEER was 9 ± 4, 52 ± 3 and 79 ± 0.3%, respectively, after 20 min. Prolonged
incubation only resulted in a gradual decrease in resistance compared with the initial
reduction in TEER after 20 min. The highest reduction in TEER was measured at a
concentration of 2.5% w/v thus indicating that the reduction in TEER was concentration
dependent [Schipper et al., 1997]. With removal of the polymer solutions, repeated washing
and substitution of the apical medium with fresh Dulbecco’s Modified Eagles Medium,
reversibility of the effects was noticed, especially at 1.5 and 2.0% w/v concentrations of
TMC. The decrease in TEER 20 min after incubation, at 0.25% w/v concentration, followed
the order chitosan hydrochloride (71 ± 4% reduction), chitosan glutamate (56 ± 1%
reduction), TMC (28 ± 1% reduction), suggesting that the chitosan salts were more effective
than TMC at similar weight concentrations [Kotze´ et al., 1998].
Exposure of the apical side of the caco-2 cells monolayers to 0.25% w/v of the polymers
resulted in a 34-fold (chitosan hydrochloride), 25-fold (chitosan glutamate) and 11-fold
(TMC) increase in the absorption rate of [14C]-mannitol, compared to the control group.
Similar results were obtained for [14C] PEG 4000. At higher concentrations, TMC was able to
increase the partition coefficient and transport values further for both [14C]-mannitol and [14C]
PEG 4000. An increase of 17- and 21-fold in transport values were observed for [14C]-
mannitol at 2.0 and 2.5% w/v concentrations of TMC, respectively. The same tendency was
observed with [14C] PEG 4000 [Kotze´ et al., 1998]. From these results, it was evident that
TMC was not as effective at similar weight concentrations as chitosan hydrochloride and
chitosan glutamate. Further, TMC with a degree of quaternisation of 12% was found to
increase the transport of fluorescein isothiocyanate-labelled dextran (FD-4) across Caco-2 cell
monolayers. The transport of this large hydrophilic model compound (4400 Da) was
increased ~ 167, ~ 274- and ~ 373-fold with 1.5, 2.0 and 2.5% w/v concentrations of TMC,
respectively [Kotze´ et al., 1997a].
Figure 1. Approaches for preparing chitosan derivatives.
Table 4. In vitro studies of TMC derivatives as absorption enhancers
TMC Degree of Drug Enhancement Ref.

Derivative Quaternization
(%)
TMC 12.28 Buserelin 28-73 fold Kotzé et al., 1997a
FITC- 167-373 fold Kotzé et al., 1997a
Dextran
Insulin Increased transport Kotzé et al., 1997b
Mannitol Increased transport Kotzé et al., 1998
PEG 4000 Increased transport Kotzé et al., 1998
60 Octreotide Increased PC (34-121 fold) Thanou et al., 2000a
acetate
12-59 Mannitol PC Increased with increase Hamman et al., 2003
in degree of quaternization
(max. with TMC 49)
TMC-H 61.2 Mannitol Increased PC (31-48 fold) Kotzé et al., 1999c
19.9 Mannitol Increased PC (97 fold) Kotzé et al., 1999c
TMC-L 12.3 Mannitol Increased PC Kotzé et al., 1999c
12.6 Mannitol Increased PC (51 fold) Kotzé et al., 1999c
TMC 40 40 Mannitol Increased PC Thanou et al., 2000b
Buserelin Increased PC (21 fold) Thanou et al., 2000c
TMC 60 60 Mannitol Increased PC Thanou et al., 2000b
Buserelin Increased PC (60 fold) Thanou et al., 2000c
TMC 22 22 Mannitol PC Increased with increase Hamman et al., 2003
TMC 38 38 in degree of quaternization
TMC 43 43 (max. with TMC 49)
TMC 49 49
*PC: Partition coefficient.
TMC was also able to increase the transport of insulin at a pH of 4.4 compared to the
control where no transport was observed. The transport of insulin was increased to 0.3% and
0.8% of the total dose applied at 1.5% and 2.5% w/v concentrations of TMC, respectively. An
increase in the transport of buserelin (1300 Da) was also observed at a pH of 6.2. The
transport was increased to 1.4% and 2.7% of the total dose applied with 1.5 and 2.5% w/v
solutions of TMC, respectively, as compared to the control group where only 0.04% of total
dose applied was transported [Kotze´ et al., 1997a]. The different in vitro studies performed
with TMC are summarized in (Table 4).
The different variables that can influence the enhancing properties of these polymers
have been included. TMC derivatives are especially effective in enhancing the transport of
small hydrophilic compounds (e.g., mannitol), though they also improve the transport of large
molecules such as buserelin, insulin, and octreotide acetate.
Highly Cationic Derivatives
Chitosan can be modified in different ways to introduce a cationic charge. Modifications,

such as alkylation or acylation, can be executed at the amino function of chitosan to obtain a
quaternized amino group carrying the cationic charge (Table 5).
Table 5. Derivatization of chitosan for enhancing its cationic character

Highly cationic Amino alkylation (i.e. Partially substituted Enhanced penetration of Lee et al., 2002; Je
derivatives with dialkyl amino N,O-[(N,N- diethyl hydrophobic/hydrophilic and Kim, 2005;
alkyl chloride in amino methyl diethyl materials across porcine 2006;
alkaline condition) di- methylene epithelium
ammonium)] methyl
chitosan
Glcydyl-tri-methyl N-(2- Nanoparticles for protein Xu et al., 2003
ammonium chloride hydroxyl)propyl-3- delivery (ionic gelation
trimethyl ammonium with sodium
chitosan chloride tripolyphosphate)
Cross-linking with Chitopearls for
bifunctional reagents chromatographic purposes
e.g. diisocyanate or and as enzyme supports
diepoxy derivatives
DMA-LiCl or Chitin Flexible, opaque material Microencapsulation of Grobouillot et al.,
anhydrous pyridine insoluble in aqueous and lactic acid bacteria 1993
with excess 1,6- organic solvents,
diisocyanotohexane remarkable crystallinity,
typical IR but no
thermoplasticity
Chitosan Methoxy Polycationic Effective gene delivery Xu et al., 2009
conjugated with poly(ethylene polyethylenimine was and package molecule.
polyethylenimine and glycol)– introduced onto chitosan
methoxy poly(ethylene polyethylenimine– for the purpose of
glycol). chitosan enhancing the positive
charge of the copolymer
Alternatively, a moiety itself carrying cationic charge can be introduced. This moiety can
be covalently linked to one of the chitosan functionalities such as the hydroxyl or the amino
group. Modifications which result in enhanced permanent cationic charge on the chitosan
molecule are highly preferred, as they exhibit a pH-independent positive charge. Depending
on the magnitude of cationic charge introduced on the chitosan molecule, water solubility at
neutral to basic pH values can be achieved, in contrast to unmodified chitosan which is
soluble only under acidic conditions. A simple way to introduce a permanent cationic charge
to the chitosan molecule is the methylation of chitosan's amino groups, resulting in N-
trimethyl chitosan salts, notably the chloride form [Figure 1] [Sieval et al., 1998]. The
cationic polymers are prepared by reaction of chitosan and dialkylaminoalkyl chloride in
alkaline condition [Je et al., 2006]. Chitosan derivatives of dialkylaminoalkyl type with N-
aminoethyl, N-diethylaminoethyl, N-dimethylaminoethyl, N-dimethylaminoisopropyl etc.
have exhibited the ability to enhance the penetration of both hydrophobic and hydrophilic
molecules across the excised porcine cheek epithelium (Table 5). This effect was observed to
be stronger than that of the known absorption enhancer TMC [Zambito et al., 2006].
A water-soluble derivative of chitosan, N-(2-hydroxyl) propyl-3-trimethylammonium
chitosan chloride (HPTCC) was synthesized by its reaction between glycidyl-trimethyl-
ammonium chloride [Xu et al., 2003]. HPTCC was employed for making nanoparticles for
protein delivery utilizing its ionic gelation with sodium tripolyphosphate [Xu et al., 2003].
Highly cationic chitosans find applications in cosmetics for hair and skin care too. Chitopearl
products (Fuji Spinning Co., Japan) belong to the class of highly cationic derivatives of
chitosan. They comprise of chitosan porous beads cross-linked by bifunctional reagents such
a diisocyanate or diepoxy derivatives [Seo et al., 1989a]. Chitopearl spherical chitosan
particles produced from diisocyanates are suitable for chromatographic purposes and as
enzyme supports [Yoshida et al., 1994]. Treatment of chitin solution in solvents such as
DMA–LiCl or anhydrous pyridine with excess 1,6-diisocyanatohexane and exposure to water
vapor for two days produced flexible, opaque materials whose main characteristics included
insolubility in aqueous and organic solvents, remarkable crystallinity, typical infrared
spectrum and degree of substitution but no thermoplasticity. Microencapsulation of lactic acid
bacteria based on the cross-linking of chitosan by 1,6-diisocyanatohexane has been performed
[Grobouillot et al., 1993]. The cationic character of the chitosan is pivotal to many of its
applications such as bioadhesion, absorption enhancement, transfection efficiency as well as
biological activities such as antitumor, antimicrobial, antiinflammatory, and antihype-
rcholesterolemic effect.
Hydroxyalkyl Chitosan
Hydroxyalkyl chitosans are obtained on reacting chitosan with epoxides (ethylene oxide,
propylene oxide, butylenes oxide) and glycidol (Table 6). Depending on the epoxide and
conditions (e.g. solvent and temperature), the reaction may take place predominantly at the
amino or alcohol group, yielding N-hydroxyalkyl chitosan or O-hydroxyalkyl chitosan or a
mixture of both [Lang et al., 1988; 1989; 1990; Donges et al., 2000]. The choice of catalyst
(NaOH or HCl) and reaction temperature determines the ratio of O/N-substitution
(hydroxypropylation of chitosan by propylene oxide) [Maresh et al., 1989]. Hydroxypr-
opylation of chitosan with propylene oxide is controlled by the pH of the solution.
Table 6. Derivatization of hydroxyl alkyl chitosans
Derivative/ Reaction with Example Property Application Ref.

Modification
Hydroxy Epoxides N-hydoxy Products having Roberts et
alkyl (ethylene alkyl chitosan marked surface al., 1992
chitosans oxide, activity and foam
propylene enhancing
oxide, butylene properties
oxide) and
glycidol
2- O-hydoxy Oligomers at a Maresh et
chloroethanol alkyl chitosan temperature higher al., 1989
in alkaline than 40ºC
medium Hydroxy Antimicrobial Peng et al.,
propyl temperature 2005; Dang
chitosan sensitive injectable et al., 2006
carrier for cells
Glycol Nanoparticles as As stabilizer for Kim et al.,
chitosan carrier for protein encapsulated 2006; Park et
paclitaxel, into poly (lactide-co- al., 2006;
doxorubicin glycolide) Lee et al.,
microparticles 2007
The substitution reaction occurred preferentially at the amino groups or hydroxyl groups
under neutral or alkaline conditions, respectively [Maresch et al., 1989; Lang et al., 1997].
Hydroxyalkylations with 2-chloroethanol and 3-chloropropane-1,2-diol were also reported
[Tokura et al., 1983]. Synthesis of hydroxypropylchitosan and its evaluation for use as an
antimicrobial agent [Peng et al., 2005] or temperature sensitive injectable carrier for cells
[Dang et al., 2006] has been carried out by different researchers. O-hydroxyethylchitosan
(glycol chitosan) was synthesized by reaction with 2-chloroethanol in alkaline medium
[Ronghua et al., 2003]. The epoxides used for hydroxyalkylation of chitosan can be
substituted for example with carboxylic groups [Gruber et al., 1995; 1997].
Self-assembled nanoparticles based on glycol chitosan were prepared as a carrier for
paclitaxel and doxorubicin [Kim et al., 2006; Park et al., 2006]. Glycol chitosan was also used
as stabilizer for protein encapsulated into poly(lactide-co-glycolide) microparticle [Lee et al.,
2007].
Carboxyalkyl Chitosan
The process of carboxyalkylation introduces acidic groups on the polymer backbone. On

introduction of carboxyl groups on the amino groups of chitosan, amphoteric polyelectrolytes
containing both cationic and anionic fixed charges are obtained (Table 7). By varying the
degree of substitution of the carboxyl bearing group, various charge densities on the
molecular chain can be obtained, which provide a convenient way to control pH-dependent
behaviour. The other synthetic route that is selective in the formation of N-carboxyalkylation
uses carboxyaldehydes in a reductive amination sequence [Muzzarelli et al., 1982].
Table 7. Derivatization of chitosans into carboxyalkyl chitosans along with the properties

Carboxyalkyl chitosans Monohalocarboxylic N-carboxy Introduction of Kim et al., 1997;
acids alkylated carbonyl groups Liu et al., 2001
chitosan onto amino groups
of chitosan
N-carboxyalkylation O-carboxyalkyl Control pH Muzzarelli et al.,
with chitosan dependent behavior 1982
carboxyaldehydes in
a reductive amination
sequence
Glycoxylic acid N-carboxy Water soluble, high Used in food products and Muzzarelli et al.,
methyl chitosan viscosity, large cosmetics. Development of 1994; Pavlov et
hydrodynamic different protein drug al., 1998; Chen et
volume, film and delivery systems e.g. al., 2004a; Chen
gel forming superporous hydrogels, pH et al., 2004b; Lin
capabilities sensitive hydrogels, cross- et al., 2005; Yin
linked hydrogels et al., 2007
N,N –dicarboxy Good chelating Favored osteogenesis while Muzzarelli et al.,
methyl chitosan properties promoting bone 1998; Zhu and
mineralization Fan, 2005
O-carboxy Antibacterial activity, Zhu et al., 2006;
methyl chitosan modifies adhesive 2007; Liu et al.,
properties, enhanced 2006; 2007a
chondrocyte adhesion;
surface modification of
vascular grafts, enhance
blood compatibility; as
carrier for delivering drugs
such as gatifloxacin,
campoththecin, ibuprofen,
adriamycin
o- Molecular sieves, viscosity Gupta and Ravi
Carboxymethyl, builders, and metal ion Kumar, 2000
cross-linked o- collection
carboxymethyl
3-halopropionic acids Higher homolog Antioxidant and Skorik et al.,
under mild alkaline of carboxymethyl antimutagenic activity 2003; Kogan et
conditions and chitosan i.e. N- al., 2004
ambient temperature (2-carboxyethyl)
chitosan
Arylonitrile Cyanoethyl
chitosan
Ethyl acrylate in N-carboxyethyl Can be easily
aqueous acidic ester hydrolysed to free
medium (intermediate) acid or used as
intermediate to
substitute with
various hydrophilic
amines, without
requiring protecting
groups
Reductive amination N-carboxy- pH-sensitive Colon specific drug delivery Lin et al., 2007
with 2-carboxy benzyl chitosan hydrogel of 5-FU
benzaldehyde and
cross linked with
glutaraldehyde
Carboxyalkylation 4-hydroxyphenyl Stable self
(with pyruvic acid sustaining gels
carboxyaldehydes) Modified Hydrolysed by Kumar et al.,
chitosan lysozyme, lipase, 2004
papain
Chitosan-α- Theophylline loaded iron Ding et al.,
ketoglutaric acid (III)-cross-linked polymeric 2007a; 2007b
and beads for prolonged drug
hydroxamated release, as well as in
chitosan- α- augmenting adsorption
ketoglutaric acid properties
By using glyoxylic acid, water-soluble N-carboxymethyl chitosan is obtained, which is

soluble in water, has high viscosity, large hydrodynamic volume, film and gel-forming
capabilities [Muzzarelli et al., 1994] (Table 7). Due to these properties it has emerged as an
attractive option for its use in food products and cosmetics [Muzzarelli et al., 1988; Pavlov et
al., 1998]. Carboxymethyl chitosan is used for developing different protein drug delivery
systems as super porous hydrogels, pH-sensitive hydrogels and cross-linked hydrogels [Chen
et al., 2004a; 2004b; Lin et al., 2005; Yin et al., 2007]. N,N-dicarboxymethyl chitosan
possesses good chelating abilities and its chelate with calcium phosphate favour osteogenesis
while promoting bone mineralization [Muzzarelli et al., 1998]. O-carboxymethyl chitosan
exhibits antibacterial activity and modified adhesive properties. In addition, it exhibits
enhanced blood compatibility [Zhu and Fan, 2005]. Carboxymethyl chitosan and modified
carboxymethyl chitosan have been employed as a carrier for delivering drugs like
gatifloxacin, camptothecin, ibuprofen, and adriamycin [Zhu et al., 2006; Liu et al., 2006; Liu
et al., 2007a; Zhu et al., 2007]. N-carboxyalkyl derivative of chitosan was synthesized and
tested for antioxidant and antimutagenic activity [Skorik et al., 2003; Kogan et al., 2004].
Quaternized carboxymethyl chitosan (QCMC) was used for obtaining carboxymethyl
chitosan (CMC). N-quaternary ammonium group was introduced by the reaction of CMC
with 2, 3-epoxypropyl trimethylammonium. Degree of substitution (DS) of CMC does not
significantly affect the antimicrobial activity. However, the antimicrobial activity of QCMC
was found to be enhanced with increase of their DS of quaternization or the decrease of their
molecular weight. QCMC was complexed with calcium hydroxide. Animal experiment results
indicated that QCMC could induce reparative dentine formation and showed better ability to
induce dentin as compared to calcium hydroxide [Sun et al., 2006].
Microwave technique was successfully used for crosslinking CMC films for possible use
in wound care applications [Wongpanit et al., 2005]. FT-IR spectroscopy studies indicated
that crosslinking of microwave-treated CM-chitosan films involved the carboxylate and the
amino groups. Pure CM-chitosan films appeared to be amorphous. Based on both direct and
indirect cytotoxicity assays, microwave treated CM-chitosan films were not found to be
cytocompatible. Despite their observed cytotoxicity, biological response of human fibroblasts
for microwave treated CM-chitosan films appeared to be normal, as evidenced from the
amount of protein synthesized. Lastly, it was observed that human fibroblasts adhered on the
surface of microwave-treated CM-chitosan films, which indicated the potential of these films
in wound healing.
CMCS can be obtained from the reaction of chloroacetic acid and chitosan in alkaline
condition with easy building blocks for application in drug delivery, tissue engineering, and
viscosupplementation [Chen et al., 2004a; Liang et al., 2004]. Because of cationic amine
groups and anionic carboxyl groups, the swelling, drug permeation, and release properties of
CMC could be controlled by pH change [Chen et al., 2004b]. The major problem encountered
with this degradable polysaccharide was its high solubility in aqueous media [Chen and Park,
2003]. As a consequence, film coatings consisting of this polymer alone would be unable to
effectively control the drug release. However, the incorporation of CMC in water-insoluble
film-forming polymers, such as poly(estersulfone) (PES) or poly(vinyl alcohol) (PVA), could
provide a promising alternative [Zhao et al., 2003; Wang et al., 2007].
The CMC-coated capillary has been successfully applied to separate basic proteins and
recombinant human erythropoietin (rhEPO). Furthermore, several experimental parameters,
such as the concentration and pH of the running buffer, temperature, and applied voltage,
were optimized for the separation of rhEPO glycoforms. Comparison of an uncoated capillary
with chitosan- or CMC - coated capillaries for the separation of rhEPO glycoforms demonstr-
ated that rhEPO glycoforms could be well separated by a CMC-coated capillary within 8 min
with good reproducibility and resolution [Fu et al., 2007].
CMC injection spots did not show erythema or oedema after 24, 48 or 72 h suggesting
complete hydrolysis within 3 days of implantation possibly due to the carboxymethyl groups
in their structure. When carboxymethyl groups were synthesized with chitosan, the
polysaccharide became an ampholyte, which was easy to combine with lysozyme [Chen et al.,
2005]. CMC films showed mild inflammatory reactions, fast in vivo degradation and
complete disappearance of inflammatory reactions in the end. These results demonstrated
good biocompatibility of CMC. The carboxyl groups introduced in CM-chitosan destroy their
sub-structure, reduce the crystal degree and increase the solubility. Moreover, CMC exhibited
polymeric property of zwitterions. Hence, they have different dimensional structures and
configurations, which endow them with several biologic activities [Chen et al., 2000]. CMC
at low concentrations revealed stimulative effect on fibroblasts’ growth, and exhibited good
biodegradability and biocompatibility [Chang et al., 2008].
CMC with DS ranging from 0.25 to 1.19 was synthesized by alkalization of chitosan,
followed by carboxymethylation with monochloroacetic acid. CMC alone could be
electrospun into fibers and required the addition of a water-soluble polymer. Cross-linking by
heat-induced esterification (at 140º C for 30 min) rendered CMC/PVA fibrous membranes
insoluble in water. The mass retention and fiber morphology confirmed their highly
substituted nature, which favored inter-molecular crosslinking leading to a more stable and
water-insoluble fibrous membrane. However, the membrane from the less substituted CMC
(DS = 0.36) was more hydrophilic and retained the desirable amine functionality, which is
recognized to be responsible for antibacterial properties and biocompatibility [Du and Hsieh,
2008].
Guangyuan et al. [2009] used a combination of carboxymethylation and a bimodal
molecular weight distribution for synthesizing chitosan derivatives. Specifically, chitosan was
carboxymethylated to a theoretical extent of ∼30% at physiological pH. CMC was used to
form films and constructed by varying the ratio of high to low molecular weight (MW)
fractions while maintaining the mechanical properties of the polymer. The rate of degradation
of these films was found to be dependent upon both the carboxymethylation and the ratio of
high to low MW polymer. Subsequently, biocompatibility was examined to determine the
effects of the modifications upon Neuro-2a cells cultured on the films. Neuro-2a cells adhered
and proliferated on the modified films at a rate comparable to those cultured on unmodified
films. This data indicated that CMC exhibited tunable degradation rates and hence, could be
used for neural tissue engineering.
With the development of biomaterial and biomedicine industry, CMC is expected to play
an important role in both research and application fields.
Cyclodextrin Linked Chitosan
Chitosans bearing cyclodextrin (CD) have been developed to form non-covalent inclusion
complexes with a number of guest molecules altering their physicochemical properties for
improved drug delivery, cosmetics, and analytical chemistry [Tanida et al., 1998; Prabaharan
and Mano, 2006]. There are different means to link CD to chitosan e.g using 2-O-
formylmethylated CD; inclusion complexes with 4-tert-butyl benzoic acid; chloroacyl CD in
organic basic solvents or using 1,6- hexamethylene diisocyanate, 2-hydroxypropyl moiety,
reducing sugar derivatives as spacer (Table 8). Chitosan derivative bearing β-CD moiety was
also synthesized by the reaction between succinated chitosan and mono-amino- β-CD (Figure
2). The reaction was carried out in water at room temperature with water soluble
carbodiimide. The obtained material was insoluble in water and contained 50% of
cyclodextrin moiety [Aoki et al., 2001].
Chitosan microspheres obtained by cross-linking with glutaraldehyde were further
reacted with chloroacyl CDs in organic basic solvents. Higher quantity of acyl CD could be
linked to the microspheres through spacer and C–N bonds with a smaller cross-linking
degree. The inclusion efficiency was checked with nalidixic acid, piroxicam, and p-
nitrophenol [Mocanu et al., 2004]. The CD-linked chitosan can also be prepared by reaction
with monochlorotriazinyl derivative of CD. Triazinyl moiety acts as a spacer [Martel et al.,
2001]. This compound was used for decontamination of water containing textile dyes. El-
Tahlawy et al. [2006] used a novel technique for preparation of β-CD grafted chitosan by
reacting β-CD citrate with chitosan dissolved in formic acid solution and evaluated these
polymers as antimicrobial agents. They also reported analogous synthesis with β-CD-
itaconate and chitosan along with its utility as ion exchange resin [Gaffar et al., 2004]. The β-
CD linked chitosan using 1,6-hexamethylene diisocyanate as spacer was also prepared
[Sreenivasan, 1998; Chen et al., 2007]. This material interacted with cholesterol and was
found to be useful as an adsorbent. The spacer can also be 2-hydroxypropyl moiety
introduced by grafting β-CD onto chitosan using epoxyactivated chitosan [Zhang et al., 2004]
or a reducing sugar derivative [Auzely-Velty and Rinaudo, 2002]. Aime et al. [2006]
functionalized CD by means of a maleic spacer, whose free carboxyl group was subsequently
activated with a carbodiimide to form amide linkages with amino groups of chitosan. The
regioselectivity of the coupling could be accurately controlled if the 6-monotosyl-CD
derivative was used as substrate for nucleophilic substitution with sodium maleate. An
insoluble cross-linked chitosan bearing β-CD was prepared using N-succinyl chitosan and
aminated-β-CD (mono-6-aminomono-6-deoxy-β-cyclodextrin) via amide bond formation in
the presence of the water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
under homogeneous conditions [Aoki et al., 2003].
The chitosan derivative bearing β-CD and Schiff-base was prepared by reacting chitosan
with glutaric diketone, functionalizing with epichlorohydrin followed by reaction with β-CD.
The synthesized polymer could adsorb metallic ions and phenolic compounds simultaneously
[Zha et al., 2007].
Chitosan and β-CD/grafted chitosan, having different molecular weights, were evaluated
as antimicrobial agents for different microorganisms such as, Bacillus megaterium,
Pseudomonas fragi, Bacillus cereus, Staphylococcus aureus, Escherichia ecoli and
Aeromonas hydra [El-Tahlawy et al., 2006]. In order to develop a treatment method for
industrial wastewater, the adsorption of 4-nonylphenol ethoxylates (NPEs), non-ionic
surfactants used in the industry onto chitosan beads having cyclodextrin (CDC beads) was
investigated. The CDC-beads with CD content above 30% w/w were prepared by the
condensation of carboxymethylated α, β, or γ -CD and chitosan beads. Among the different
sizes of the CD cavities, β–CD was the best for the adsorption of NPE.
ation, properties and applicationss of cyclodextrin linked
Table 8. Prepara l chitosan
Derivative/Moodification R
Reaction with Example Property Application Referencess
Cyclodextrin linked chitosan 2--O-formyl-methyl-α-CD D α-CD linked chittosan Tanida et al.,
a 1998;
byy reductive N-alkylationn Tojimaa et al., 1999
R
Reductive amination witth hitosan bearing pendant
p Auzely-Veelty and
4--tert-butylbenzoic acid chitosan Rinaudo, 2001
2
Supermolecular Auzely-Veelty and
assemblies of chiitosan Rinaudo, 2002
2
Mono chloro triazinyl
M CD linked chitossan For deccontamination of water Martel et al.,
a 2001
deerivative of CD contain
ning textile dyes
T
Tosylated β-CD CD-linked chitossan Enhanced soolubility Improvved in vivo release Chen and Wang,
W 2001
C
Chloroacyl CD in organiic Higher amoounts of acyl Improvved Inclusion efficiencyy of Mocanu et al., 2004
baasic solvents CD are linkeed to the Nalidix
xic acid, piroxicam, p-nnitro
microspherees through phenoll
spacer
β--CD itaconate Ion excchange resin Gaffar et all., 2004
1,,6- hexamethylene β-CD linked chittosan Interacts witth Adsorbbent Sreenivasaan, 1998;
diiisocyanate as spacer cholesterol Chen et al., 2007
2--hydroxypropyl moiety as Zhang et all., 2007
sp
pacer
R
Reducing sugar derivativ
ve Regioselectiivity of Auzely-Veelty and
ass spacer coupling couuld be Rinaudo, 2002
2
controlled by
b using 6-
monotosyl-C CD
derivative as spacer
N-succinyl chitosan and

N Cross linked chittosan Aoki et al.,, 2003
am
midated β-CD via amidde bearing β-CD
bo
ond formation
- and -CDs are linkeed Bitter taste masked
m Binello et al.,
a 2004
to
o chitosan through
su
uccinyl bridges
Table 8. Preparation, properties and applications of cyclodextrin linked chitosan (Continued)

Chitosan microspheres, Chitosan derivatives Conjugates used as supports for Georgeta et al., 2004
obtained through containing cyclodextrin chromatographic separations or
crosslinking with moieties as pendant controlled release drug systems
glutaraldehyde of an acetic groups
acid solution of chitosan,
in an organic suspension
medium, were reacted with
chloroacyl cyclodextrins in
organic basic solvents
Reaction of chitosan Chitosan microspheres Grafting and inclusion Wang et al., 2005
microspheres and mono- grafted with β-CD was found to be stable
(6-p-tosyl)- β-CD
Coupling β-CD citrate with β-CD-grafted chitosan Antimicrobial activity for El-Tahlawy et al.,
chitosan dissolved in microorganisms such as, Bacillus 2006
different formic acid megaterium, Pseudomonas fragi,
solutions having different Bacillus cereus, Staphylococcus
concentrations aureus, Escherichia ecoli and
Aeromonas hydra.
SOCl2 and CD were heated Carboxymethyl- β-CD Introduction of chitosan Application in separation, Xiao et al., 2006
with chitosan (in DMF) grafted chitosan enhanced the adsorption concentration and
and product was dried with ability and adsorption analysis of nucleotides in biological
5% acetic acid. selectivity of β- CD for sample
guanosine 5'-
monophosphate.
CD-monoaldehyde CD-chitosan derivative Mucoadhesive Venter et al., 2006

β-CD citrate with chitosan β-CD grafted chitosan Antimicrobial agent El-Tahlawy et al.,
dissolved in formic acid 2006
solution
Figure 2. Approaches for modifying chitosan.
The adsorption of NPE depended on the ethoxy chain in the NPE molecules. The used β -
CDC beads could be recovered by ethanol treatment. The NPE could be continuously
removed from a water solution using the β -CDC beads packed in a glass column [Aoki et al.,
2007].
Zhang et al. [2009] investigated the possibility of using chitosan bearing β-CD
nanocomplexes for controlled protein release. Synthesis was done by reacting N-succinylated
chitosan with mono (6-(2-aminoethyl) amino-6-deoxy)-β-cyclodextrin in the presence of the
water-soluble carbodiimide. The amount of β-CD grafted was up to 62.1% w/w. In vitro
cytotoxicity study against NIH 3T3 cells showed that the complex was not cytotoxic. Self-
assembled nanocomplexes containing insulin ranging from 190 nm to 328 nm, electrical
charge from +3.7 to +25.5 mV and high loading efficiency of 37.7% could be prepared.
Insulin release in vitro was affected by the medium pH and the composition of copolymer.
The results demonstrated that complexed copolymer was a new promising vehicle for
controlled protein release.
Nanoparticles made of chitosan and carboxymethyl-β-CD (CM-β–CD) were evaluated
for their potential for delivery of macromolecular drugs. Chitosan and CM-β-CD or mixtures
of CM-β-CD/tripolyphosphate (TPP) were processed to nanoparticles via the ionotropic
gelation technique. The resulting nanoparticles ranged from 231 nm to 383 nm and showed a
positive zeta potential ranging from +20.6 to +39.7mV. These nanoparticles were stable in
simulated intestinal fluid pH 6.8 at 37º C for at least 4 h. Insulin and heparin (macromolecular
model drugs) were incorporated into the different nanocarriers with association efficiencies of
85.5–93.3 and 69.3–70.6%, respectively. The association of these compounds led to an
increase of the size of the nanoparticles (366–613 nm), with no significant modification of
their zeta potentials (+23.3 to +37.1 mV). The release profiles of the associated
macromolecules were highly dependent on the type of molecule and its interaction with the
nanomatrix. Insulin release was observed to be high (84-97% released within 15 min)
whereas, heparin remained highly associated to the nanoparticles for several hours (8.3–9.1%
released within 8 h). Hence, these nanoparticles were suggested to be suitable for the fast or
slow delivery of macromolecules [Krauland and Alonso, 2007].
Moses et al. [2000] attempted to complex insulin with β-CD followed by encapsulation in
the chitosan matrix. Insulin complexed with β-CD for 20 min was used for encapsulation in
chitosan. The results showed that the matrix yielded different drug release profiles in
simulated intestinal medium (pH 7.4). The change in the loading character of the matrix was
found to be inversely related to the concentration of β-CD when it was above the
stoichiometric equivalent of the drug. In an attempt to increase the payload of the drug in the
matrix, the pH of the processing medium consisting of calcium chloride and chitosan was
varied. It was found that the encapsulation efficiency increased with decrease in pH from pH
6 to pH 4. Loading efficiency was also increased by reducing the concentration gradient
between the crosslinking medium and the alginate solution containing the drug.
Another study revealed that the supramolecular assembly of the polymers and α-CD
molecules led to hydrogel formation in aqueous media. The poly(ethylene glycol) side chains
on the chitosan backbones were found to form inclusion complexes with α-CD, creating
hydrophobic micro-domains with channel-type crystalline structure, which play an important
role as physical junctions in the hydrogels [Huh et al., 2004].
Table 9. Preparation, properties and applications of acyl derivatives of chitosan

N-acyl chitosan Acyl chlorides and These polymers form a Targeted drug delivery to Zong et al., 2000;
anhydrides (acylation) layered structure in solid cancerous cells (acylation Lee et al., 2006
state and layer spacing with folic acid)
increases with increasing
length of side chains. Better
solubility
Thermolysis of acyl Improved mechanical Toffey and
ammonium salts in solid properties of tablets. The Glasser, 2001;
state release was found to be Tien et al., 2003
controlled by diffusion or by
swelling followed by
diffusion, depending on acyl
chain length and degree of
acylation
Carboxy methylation Hexanoyl chitosan Excellent water absorption Liu et al., 2006
and water retention abilities
under neutral conditions and
used as a carrier for
delivering amphiphatic
agents.
Chitosan with hexanoyl Hexanoyl chitosan Winie et al., 2004.

chloride in a mixture of
pyridine
and THF
Acrylic acid and NaOH N-carboxyethyl Weng et al., 2008
chitosan
Table 9. Preparation, properties and applications of acyl derivatives of chitosan (Continued)

Formyl, acetyl, propionyl, Textiles, membranes, and Gupta and
butyryl, hexanoyl, medical aids Kumar,
acetanoyl, decanoyl, dodecanoyl, 2000
tetradecanoyl, lauroyl, myristoyl,
palmitoyl, stearoyl, benzoyl,
monochloroacetoyl,
dichloroacetyl,
trifluoroacetyl, carbamoyl,
succinyl,
acetoxybenzoyl
Pyridine/chloroform with N-acyl chitosan Hydrophobic derivatives Expected to be good models Hirano and
carboxylic acid chlorides; formation. Swelling index of of drug delivery systems. Ohe, 1975;
and acylchitosan has been chitosan derivatives decreasd Fujii et
prepared by chemical with the increase of the degree al.,1980;
N-acylation of the of substitution hence, tighter Hirano and
chitosan with the and more compact structure for Midorikawa
corresponding fatty acid limiting water uptake , 1998;
anhydride. Acylation Hirano et al.,
reactions are carried out 2002;
frequently in aqueous Rodrigues,
acetic acid/methanol 2005
Pyridine and chloroform N,N-Diacyl Chitosan Ming-chun
followed by hydrolysis et al., 2005
N,N-Diacyl Chitosan
Carboxy methylation Hexanoyl chitosan Excellent water absorption and Liu et al.,
water retention abilities under 2006
neutral conditions and used as
a carrier for delivering
amphiphatic agents.
O-acyl chitosan Introduction of O,O-didecanoyl- Hydrophobic groups contribute organo Sashiwa et
hydrophobic moiety with chitosan, O-succinyl solubility al., 2002a;
an ester linkage chitosan The ester linkage is hydrolysed by enzyme 2002b; Kim
like lipase. Therefore are biodegradable et al., 2003
coating material
Alkanoic acid derivatives O-(butyroyl) chitosan Fungicidal activity against the Badawy et
with chitosan in the grey mould Botrytis cinerea al., 2005
presence of H2SO4 as a (Leotiales: clerotiniaceae) and
O-(acyl) chitosan catalyst the rice leaf blast pathogen
Pyricularia grisea
CHITOSAN DERIVATIZATION
EPOXY ACTIVATED SUCCINYLATION OF GLUTARALDEHYDE ACYLATION OF ACYLATION OF ACYLATION OF
ACYLATION OF
CHITOSAN CHITOSAN CHITOSAN CHITOSAN CHITOSAN CHITOSAN
CHITOSAN
cyclodextrin Succinyl chitosan Glutaraldehyde Cyclic Phthalic

DMF/H2O
chitosan anhydride RCOOH, anhydride
Lactome 120° C
Carbodimnide
Animated Chloroacyl
Chitosan-2-Hydroxy Carboxy acyl N-phthaloyl chitosan
cyclodextrin cyclodextrin Hydroxy acyl
propyl cyclodextrin Chitosan Acyl chitosan
chitosan
Chitosan Succinic Chitosan Pyridine, Trityl
90° C chloride
acid cyclodextrin glutaraldehyde
cyclodextrin
N-phthaloyl-O-
triphenyl methyl
chitosan
NH2NH2H2O
O-Triphenyl methyl
chitosan
RCOCl,
N-Acylation
Pyridine, RT
N-acyl O-triphenylmethyl
chitosan
Aq. HCl,
RT
N-Acyl chitosan
Figure 3. Approaches for modifying chitosan.

It was reported that these supramolecular hydrogels could be useful for biomedical
applications because of their biocompatible constituents and supramolecular functionality,
such as a thermo-reversible gel–sol transition property.
Chitosan-CD hybrid nanoparticles were obtained by the ionic gelation process in the
presence of glutathione, chosen as a model drug. The nanoparticles were proven to efficiently
encapsulate glutathione in their inner cores, thus showing promising perspectives as drug
carriers [Ieva et al., 2009].
N-Acyl Chitosan
N-Acyl derivatives of chitosan can be easily obtained from acyl chlorides and anhydrides
(Figure 3). In a general way, acylation reactions occur easily in aqueous acetic acid/methanol,
pyridine, pyridine/chloroform, trichloroacetic acid/dichloroethane, ethanol/methanol mixture,
methanol/formamide or Dimethylacetamide (DMA) (containing 5-9% LiCl i.e. DMAc/LiCl)
solvent systems (Table 9) [Shigemasa et al., 1999]. Due to fairly different reactivities of the
two hydroxyl and the amino group on the repeating unit of chitosan, acylation can be
controlled at the expected sites, i.e. on either amino [Seo et al., 1989; Hirano et al., 2002; Tien
et al., 2003], hydroxyls [Sashiwa et al., 2002], or on both groups [Grant et al., 1990; Zong et
al., 2000; Seo et al., 2001; Wu et al., 2005]. The introduction of hydrophobic branches
generally endows new physicochemical properties such as the formation of polymeric
assemblies, including gels [Martin et al., 2002], polymeric vesicles [Wang et al., 2001],
Langmuir–Blodgett films [Nishimura et al., 1993; Xu et al., 1996], liquid crystals [Rout et al.,
1993; Wu et al., 2003], membranes [Seo et al., 1995], and fibers [Hirano et al., 2000; Hirano
and Moriyasu, 2004]. Hydrophobic associating water-soluble polymers have emerged as a
new class of industrially important macromolecules. Some of these are intended to mimic the
endotoxins [Desbrieres et al., 1996]. The introduction of hydrophobic branches also endows
the polymers with a better soluble range than chitosan itself. Zong et al. [2000] synthesized
acylchitosan with longer chains by reacting chitosan in pyridine/chloroform with hexanoyl,
decanoyl, and lauryl chlorides. These acylated chitosans with 4 degree of substitution per
monosaccharide ring (disubstitution at amino and monosubtitution each at hydroxyl groups)
exhibited an excellent solubility in organic solvents such as chloroform, benzene, pyridine,
and THF. The analysis indicated that these polymers form a layered structure in solid state
and the layer spacing increases linearly with increasing length of side chains.
The presence of such layered structure was elucidated with N-aliphatic acyl chitosans and
N-aliphatic-O-dicinnamoyl-chitosans with acyl as acetyl, butyryl, octanoyl, lauroyl and
stearoyl moieties. Interestingly, none of these polymers could be dissolved in CHCl3, CH2Cl2,
THF, (Me)2CO, DMAc, DMF, DMSO, DMSO/ CHCl3. The reason for the striking stability of
N-aliphatic acyl against solvents was attributed to the compact arrangement of both the main
chains and the side chains of N-aliphatic acyl to form a crystal with strong hydrogen bond
interactions together with strong interactions between closely packed hydrophobic side
chains. On the other hand, the polymers belonging to the series of N-aliphatic- O-
dicinnamoyl-chitosans displayed solubilities strongly related to the length of the flexible side
chains. In general, increasing length of the flexible side chains reduced the solubility [Wu et
al., 2006]. N-acylated chitosans with saturated (e.g. C2–C18) and unsaturated acyl groups of
different chain length (e.g. oleic, linoleic, elaidoic, erucoyl) as well as aromatic acyl groups
(e.g. phthaloyl, p-nitrobenzoyl, cinnamoyl) had been successfully synthesized to obtain

randomly distributed substituents along the chitosan chain [Hirano and Ohe, 1975; Hirano et
al., 1976; Hirano and Moriyasu, 1981].
Cyclic acid anhydrides too are used for acylation purpose via ring-opening reactions
giving N-carboxyacyl chitosans (e.g. succinic, maleic, glutaric, itaconic, phthalic, cis-1,2,3,6-
tetrahydrophthalic, 5-norbornyl-endo-2,3-dicarboxylic, cis-1,2-cyclohexyl dicarboxylic,
trimellitic anhydride, (2-octen-1-yl)succinic, citraconic, trimellitic, pyromellitic) [Sashiwa
and Shigemasa, 1999; Hirano and Moriyasu, 2004]. Thermolysis has been used for
synthesizing acylated chitosan derivatives [Toffey and Glasser, 2001]. This method was used
to prepare chitosan amides derived from acids, such as acetic, acrylic, methacrylic,
trifluoroacetic, and myristic [Vasnev et al., 2006]. N-acylation of chitosan with longer chain
acid chlorides increased its hydrophobic character and made important changes in its
structural features, which were reflected in improved mechanical properties of tablets
prepared using these derivatives. The release characteristics of the drug suggested that release
was controlled by diffusion or by swelling followed by diffusion, depending on both the acyl
chain length and the degree of acylation [Tien et al., 2003].
Hexanoyl chitosan with carboxymethylation was developed into amphiphatic hydrogel
with excellent water-absorption and water retention abilities under neutral conditions and then
employed as a carrier for delivering amphiphatic agents [Liu et al., 2006]. The hexanoyl
substitution significantly improved the water-absorption ability of hydrogel by altering the
number of water-binding sites under low humidity. In addition, the state of water in fully
swollen state retarded the water mobility during deswelling, and enhanced amphiphatic drug
encapsulation efficiency compared to pristine chitosan.
The acylated chitosan are being applied for stabilization of nanoparticles of iron oxide,
and gold [Remantbahadur et al., 2006; Bhattarai et al., 2007]. N-succinyl-chitosan obtained
by introduction of succinyl groups into N-terminal of the glucosamine units of chitosan could
be modified easily with respect to succinylation degree by changing reaction conditions and
the molecular weight using hydrochloric acid [Yamaguchi et al., 1981; Kato et al., 2002].
Although, N-succinyl-chitosan was initially developed as wound dressing material
[Kuroyanagi et al., 1994], it is currently also used as a cosmetic material [Izume, 1998]. New
wound dressings composed of N-succinyl-chitosan and gelatin have also been developed
[Tajima et al., 2000]. N-succinyl-chitosan has unique in vitro and in vivo characteristics due
to many carboxyl groups. For example, ordinary chitosan can be dissolved in acidic but not in
alkaline water, whereas, N-succinyl-chitosan exhibits the opposite behaviour [Kato et al.,
2002]. Further, it is valuable as a carrier as it can readily be used for conjugating with various
drugs due to –NH2 and –COOH groups in its structure e.g. carbodiimide and mitomycin C
[Song et al., 1992; Kato et al., 2004]. N-succinyl chitosan can form well-dispersed and stable
nanospheres that exhibit great potential in the controlled drug delivery e.g oxymatrine [Zhu et
al., 2006; Yan et al., 2006]. The succinyl chitosan can be adapted for solubility by addition of
a long alkyl moiety as hydrophobic function to the amino group given that succinyl moiety
provides hydrophilic and alkyl moiety provides the hydrophobic properties. This adapted
derivative was used as a carrier for doxorubicin [Xu et al., 2007]. For liver-targeting lactose
group was introduced [Kato et al., 2001a; 2001b]. N-acetyl, N-propionyl and N-hexanoyl
with different degrees of substitution were synthesized and evaluated for in vitro antibacterial
activity [Hu et al., 2007]. The nanoparticles prepared from N-acyl chitosan were found to be
compatible with blood [Lee et al., 2004]. The betaine derivatives of chitosan have two major
advantages over chitosan. They are water-soluble at physiological pH, and they have a
permanent positive charge on the polysaccharide backbone. Condensation with carbodiimide
of N-acylated chitosan was performed with amino acids (lysine, arginine, aspartic acid,
phenylalanine) and these moieties were subsequently entrapped in PLA surfaces that
demonstrated good cyto-compatibility to chondrocytes [Zhu et al., 2002]. Acylated chitosan
when complexed with DNA revealed enhanced transfection efficiency due to enhanced cell
membrane–carrier interaction and/or destabilization of cell membrane [Kim et al., 2001; Yoo
et al., 2005] making it feasible to target the DNA delivery to cancerous cells [Mansouri et al.,
2006; Lee et al., 2006].
Graft Copolymerization of Chitosan
Graft copolymerization is well-known method for the modification of chitosan and

represents convenient and effective way of improving the physical and mechanical properties
for practical uses. Graft copolymerization is one of the important techniques used to modify
chitin and chitosan chemically as well as for widening their therapeutic and other uses.
Different reagents/initiators have been developed for graft copolymerization e.g. ammonium
persulphate [Lv et al., 2009], potassium persulphate [Akgün et al., 2007], cerric ammonium
nitrate [Joshi and Sinha, 2007], thiocarbonation potassium bromate [El-Tahlawy and Hudson,
2001], potassium diperiodatecuprate [Liu et al., 2002], bisisobutyronitrile [Deng et al., 2009]
and ferrous ammonium sulphate [Lagos and Reyes, 2003]. In addition graft copolymerization
can also be prepared via radical generation [Lv et al., 2009], polycondensation [Yang et al.,
2005], oxidative coupling [Romaškevič et al., 2007], ring opening [Liu et al., 2005], grafting
onto method [Huang et al., 2005] or photochemical reaction [David and Samuel, 2002]. The
parameters like grafting percentage or the efficiency depends upon type and concentration of
initiator, monomer, concentration, reaction temperature and time. Many studies have been
conducted by different researchers to study the effects of the above mentioned variables on
grafting parameters and on the grafted chitosan [Kim et al., 2000; Sun et al., 2003; Xie et al.,
2002a].
Graft Copolymerization by Free Radicals

Various redox reagents have been reported for carrying out graft polymerization onto
chitosan e.g. cerric ammonium nitrate, potassium persulphate, ammonium persulphate or
Fenton’s reagent. Several studies have been conducted on copolymerization after primary
derivatization of chitosan e.g. carboxymethyl chitosan [Joshi and Sinha, 2007], N-
carboxymethyl chitosan [Singh and Ray, 1994; Kang et al., 2006], maleolyl chitosan [Don
and Chen, 2005], hydroxypropyl chitosan [Xie et al., 2002b], or trimethyl chitosan [Mao et
al., 2005] etc. Chitosan was modified with polyacrylic acid, using a grafting reaction in a
homogeneous phase [Yazdani-Pedram et al., 2000]. The grafting was carried out in the
presence of potassium peroxydisulfate (PPS) and ferrous ammonium sulfate (FAS) as the
combined redox initiator system. The efficiency of grafting was found to depend on
monomer, initiator, and ferrous ion concentrations, as well as on the reaction time and
temperature. It was observed that the level of grafting could be controlled to some extent by
varying the amount of ferrous ion as a co-catalyst in the reaction. The maximum efficiency of
grafting attained was 52%. The results revealed that in homogeneous systems the grafting
reactions take place not only on the surface but also in the molecules of the whole substrate.
A graft material based on chitin and poly acrylic acid has been prepared by using potassium
peroxodisulfate as a redox initiator. The water sorption ability of the films revealed that the
films possessed a network structure and exhibited enhanced water sorption ability
[Tanodekaew et al., 2004].
Grafting Via Radiation

Grafting with the aid of radiation has been of great interest. Pengfei et al. [2001] used
60
Co-γ-radiation for grafting polystyrene onto chitosan at ambient temperature. The study
revealed the effect of various variables such as adsorbed dose, type of solvent and effect of
oxygen. Grafting yield was found to increase with increase in the adsorbed dose. Other
researchers reported the use of N, N’-dimethyl amino ethyl methacrylate for radiation grafting
[Kang et al., 2006], butyl acrylate using γ-radiation [Li et al., 2005; Huang et al., 2005],
polyhydroxy ethyl methacrylate by UV light [Ng et al., 2001] and polyacrylonitrile by
microwave radiation [Singh et al., 2004]. The grafting percentage was increased with the dose
but decreased with the increase in chitosan concentration or reaction temperature [Yu et al.,
2003]. The grafted chitosan films were found to exhibit more hydrophobicity and impact
strength. Microwave irradiation using poly acrylonitrile revealed good grafting yield (70%) in
1.5 min [Singh et al., 2005].
Grafting by Polycondensation
Lactic acid [Bhattarai et al., 2006] or 4-dimethyl aminopyridine has been used for
condensation polymerization. Lactic acid has been used to prepare a pH sensitive hydrogel
whereas 4-dimethyl amino pyridine was used as grafting polymer so as to improve the
adhesion, compatibility or to support the profileration of human endothelial cells [Feng and
Dong, 2007].
Nanoparticles of ~10 nm in diameter made with chitosan or lactic acid-grafted chitosan
were developed for high drug loading and prolonged drug release. Encapsulation efficiency of
92% and release rate of 28% from chitosan nanoparticles over a 4-week period were
demonstrated with bovine serum protein. To further increase drug encapsulation, prolong the
drug release and increase its solubility at neutral pH, chitosan was modified with lactic acid
by grafting D,L-lactic acid onto amino groups in chitosan without using a catalyst. The lactic
acid-grafted chitosan nanoparticles demonstrated drug encapsulation efficiency of 96% and
protein release rate of 15% over 4 weeks. With increased protein concentration, the drug
encapsulation efficiency decreased and drug release rate increased. Unlike chitosan, which is
generally soluble only in acid solution, the chitosan modified with lactic acid could be
dissolved in solutions of neutral pH, offering an additional advantage of allowing proteins or
drugs to be uniformly incorporated in the matrix structure with minimal degradation
[Bhattarai et al., 2006].
Grafting by Epoxy-Terminated Polydimethylsiloxane

Epoxy-terminated polydimethylsiloxane (PDMS) was grafted onto chitosan using UV
irradiation at room temperature without using a catalyst. The product was a pH-sensitive
hydrogel without any chemical cross-linking. In fact, the PDMS substituents provided the
basis for hydrophobic interactions that contributed to the formation of the hydrogel network.
The hydrogels exhibited high equilibrium water content in the range of 82–92% [Kim et al.,
2002].
Chitosan mini-emulsions were used for the synthesis of epoxy particles by polyaddition.
For the preparation of mini-emulsions, chitosan composed of two different molecular weights
(low and high) was dissolved in 1% v/v aqueous acetic acid, which was used as aqueous
phase in the mini-emulsion polymerization. The monomer phase consisting of a mixture of
styrene, hexadecane as hydrophobe, and 2,2-azobis-(2,4-dimethylvaleronitrile) as oil-soluble
initiator was added to the chitosan solution. The mixture obtained was stirred at room
temperature and mini-emulsification was obtained by ultrasonication. As chitosan bears
amine and alcohol functions, it can react with the epoxide and can be grafted onto the
particles, which are obtained by polyaddition reaction. This is a convenient technique to
modify water-soluble chitosan with water insoluble reaction partners, thus resulting in new
chitosan derivatives [Marie et al., 2002].
Miscellaneous Graft Copolymers of Chitosan

The term PEGylation usually refers to a process involving the conjugation of PEG with a
substrate. The conjugation of PEG to drugs, especially protein drugs, is well known to
enhance the solubility and stability of the protein in solution, to alter bioavailability,
pharmacokinetics, immunogenic properties, and biological activities, and also to protect the
drugs from recognition by the immune system, prolonging circulation time and efficacy in
vivo [Saito et al., 1997]. Several methods have been reported on the PEGylation of
chitin/chitosan using PEGs with various terminal reactive groups [Harris et al., 1984]. Several
other techniques are also reported for grafting of polymers e.g. the conductive polymers were
prepared by grafting polyaniline onto chitosan by oxidative coupling. Copolymerization by
the mechanism of ring opening of polymer was carried out by Jenkins and Hudson [2001]
using N-carboxyanhydride. The polymerization of aniline, an air stable organic conductive
material, in the presence of chitosan resulted in a graft copolymer that was soluble in a
slightly acidic aqueous solution and formed self-supporting materials, including thick films
and fibers, that were conductive when protonically doped. The reaction of chitosan in
aqueous acetic acid solution with aniline in the presence of ammonium persulfate (APS)
enables the introduction of polyaniline side chains at the amino groups. Chitosan-graft-
polyaniline was fabricated into films and fibers, but the properties varied according to the
ratio of the amino group to aniline in the grafting reaction. With the ratio of 1:1 to 1:5, the
products were sturdy and flexible, while those with a ratio of 1:6 to 1:10 were brittle. Optical
microscopic observations indicated that the products prepared at a ratio below 1:5 were
homogeneous, but those above 1:6 had crystalline regions [Yang et al., 1989].
Biomedical Applications of Graft Copolymers
Controlled Release Drug Delivery Systems

Chitosan has interesting biopharmaceutical characteristics such as pH sensitivity,
biocompatibility and low toxicity. Moreover, chitosan is metabolised by certain human
enzymes, especially lysozyme, and is biodegradable. Due to these favorable properties, the
interest in chitosan and its derivatives in drug delivery applications has increased in recent
years. Kumbar and Amabhavi [2003] used the microspheres of polyacrylamide grafted
chitosan cross-linked with glutaraldehyde to encapsulate indomethacin, a nonsteroidal anti-
inflammatory drug, used in the treatment of arthritis. The microspheres with a mean particle
size of 525 nm were produced by the water/oil emulsion technique, and encapsulation of
indomethacin was carried out before cross-linking the matrix. The release of indomethacin
depended on the cross-linking of the network and also on the amount of drug loaded.
Microspheres of grafted chitosan cross linked with glutaraldehyde were prepared to
encapsulate nifidifine. N-laurylcarboxymethyl chitosan with both hydrophobic and
hydrophilic groups was studied in connection with the delivery of taxol to cancerous tissues
[Yoshioka et al., 1995]. Other examples are related to the production of polymeric vesicles
for encapsulation of hydrophobic compounds like bleomycin [Miwa et al., 1998]. Chitosan
has also been modified with polyacrylonitrile through ceric-initiated graft copolymerization
[Pourjavadi et al., 2003]. The chitosan-graft-poly(acrylonitrile) product was then saponified
using sodium hydroxide aqueous solution to prepare a novel super-adsorbent hydrogel, H-
chitosan-polyacrylonitrile. It was observed that both modified chitosans exhibited enhanced
thermal stability over chitosan. Swelling capacity of the hydrogel was observed to be affected
by NaOH concentration. The lower concentration of alkali resulted in higher water
absorbency. The super-absorbent swelling gel exhibited a high sensitivity to pH. Sharp and
large volume changes were observed with small pH variation. This super-adsorbent
polyampholytic network intellingently responding to pH may be considered as an excellent
candidate to design novel drug delivery systems. The chitosan-grafted poly (vinyl alcohol)
(PVA) copolymer matrix containing prednisolone exhibited sustained release charactoristics
[Kweon and Kang, 1999]. In particular, systems that employed combination of chitosan and
poly(N-isopropylacrylamide) showed drug release profiles that could be controlled by both
pH and temperature [Lorenzo et al., 2005; Bhattarai et al., 2005]. Chitosan-based systems
bearing β-CD cavities have been proposed as a matrix for controlled release [Prabaharan and
Mano, 2005; Krauland and Alonso, 2007]. Due to the presence of the hydrophobic β-CD
rings, the systems exhibited slower release of the entrapped hydrophobic drug. Stimuli-
responsive hydrogels with improved drug loading capacity were observed to exhibit sustained
release behaviour [Prabaharan and Mano, 2006].
Tissue Engineering
The recent tissue engineering research is based on the seeding of cells onto porous
biodegradable polymer matrixes. A primary factor is the availability of good biomaterials to
serve as the temporary matrix. Recently, chitosan and its derivatives have been reported as
attractive candidates for scaffolding materials because they degrade as the new tissues are
formed, eventually without inflammatory reactions or toxic degradation. Possible applications
of chitosan in spine tissue engineering encompass three different fields, namely spine fusion,
gene therapy, and nucleus pulpous regeneration. When a bone graft alternative is applied
during spinal fusion procedures, several local biomechanical factors are considered,
depending on the type and position of the chosen graft. Anterior inter-body grafts are exposed
to high compressive forces and need to possess load-bearing ability. In contrast, a posterior-
applied bone graft is placed along the tension side of the spinal column in the absence of local
compressive stimuli, and thus bone graft incorporation is less likely to be affected by local
biomechanical factors [Mwale et al., 2005]. Materials such as PLA (poly-d,l-lactic acid) or
PLA-PEG (polyethylene glycol) have been tested for spinal fusion and are considered good
candidates due to their plasticity, stiffness, biodegradability, and ability to support cells and
growth factor [Saito et al., 1999; Hoshino et al., 2007]. A possible application of chitosan
could be a composite graft material with a predictable degradation rate and macroporous
structure that would allow linking of growth factors and support osteogenic cells for spinal
fusion [Prabaharan et al., 2006]. Intervertebral disks possess different functional and anatomic
regions: the inner nucleus pulpous, a hydrated gelatinous tissue rich in proteoglycans, and the
outer annulus fibrosis made of concentric collagen lamellae. Loss of proteoglycans and water
content in the inner nucleus pulpous initiates degenerative spinal disease. Biologic disk
regeneration is considered a promising approach to restore biological integrity and function of
a pathologically altered disk [Boden, 2002]. Another approach regarding the chemical
modification of chitosan for tissue engineering applications has been to introduce the specific
recognition of cells by sugars. The synthesis of sugar-bound chitosan can be related to
preparation of mannosylated chitosan having specific recognition to antigen presenting cells
such as β-cells, dendritic cells and macrophages [Kim et al., 2006].
Wound Healing and Antimicrobial Properties

Grafted chitosan presents interesting properties for wound-healing applications, because
chitosan derivatives can exhibit enhanced bacteriostatic activity with respect to pure chitosan.
Ethylene diamine tetra acetic acid (EDTA) grafted on chitosan increases the antibacterial
activity of chitosan by chelating magnesium that under normal circumstances stabilizes the
outer membrane of gram-negative bacteria [Valenta et al., 1998]. The increase in chitosan
antimicrobial activity is also observed with carboxymethyl-chitosan, which makes essential
transition metal ions unavailable for bacteria [Muzzarelli, 1989] or binds to the negatively
charged bacterial surface to disturb the cell membrane [Liu et al., 2001]. Carboxymethyl-
chitosan is used for reducing periodontal pockets in dentistry [Bernkop-Schnu¨rch et al.,
1997] and chitosan grafted with EDTA as a constituent of hydro-alcoholic gels for topical use
[Muzzarelli, 1989]. Poly(ethylene terephthalate) (PET) fiber was treated with 60Co-c-ray and
grafted with acrylic acid. The resulting fibers were further grafted with chitosan via
esterification. Collagen was then immobilized on chitosan grafted fibers. The antibacterial
activity of chitosan against Staphylococus aureus, Escherichia coli, and Pseudomonas
aeruginosa was preserved after collagen immobilization. The results indicated that by
grafting with chitosan and immobilizing with collagen, PET fibers exhibited both
antibacterial activity against four pathological bacteria and improved the proliferation of
fibroblast [Jou et al., 2007]. Similar work done by Huh et al. [2001] reported that PET
treatment with oxygen plasma glow discharge, followed by a graft copolymerization of
acrylic acid could be used to prepare carboxylic acid group–containing PET. Chitosan and
quaternized chitosan were then ionically or covalently immobilized on PET–acrylic acid. The
experiments of antibacterial activity of chitosan graft-PET against Staphylococus aureus
showed high growth inhibition in the range of 75–86% and still maintaind 48–58% bacterial
growth inhibition after laundering. Hu et al. [2003] reported the grafting of acrylic acid on
ozone-treated poly(3-hydroxybutyric acid) and poly(3-hydroxybutyric acid-co-3-
hydroxyvaleric acid) membranes. The resulting membranes were further grafted with chitosan
via esterification. These chitosan-grafted membranes showed antibacterial activity against
Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus
(MRSA), and Staphylococcus aureus. Acrylic acid grafting increased the biodegradability
with Alcaligens faecalis, whereas chitosan and chitooligosaccharide grafting reduced the
biodegradability. In addition, chitosan-grafted-poly (3- hydroxybutyric-acid-co-3-

hydroxyvaleric acid) membrane showed higher antibacterial activity and lower
biodegradability than chitooligosaccharide-grafted membrane. Two anionic soluble
monomers, mono(2-methacryloyloxyethyl) acid phosphate and vinyl sulfonic acid sodium
salt can be grafted on chitosan to obtain copolymers with zwitterionic property [Jung et al.,
1999]. The grafting reaction improved the antimicrobial activities of chitosan against Candida
albicans, Trichophyton rubrum, and Trichophyton violaceum, which depended on the amount
and type of grafted chains, as well as on the changes in pH [Yang et al., 1989]. The
antibacterial activity of the polypropylene was enhanced by the modification of radiation–
induced grafting of acrylic acid and the immobilization of chitosan onto the polypropylene-
graft-acrylic acid-modified polymer [Yang and Lin, 2004].
Chitosan possess the characteristics favorable for promoting rapid dermal regeneration
and accelerated wound healing. It was observed that chitosan oligosaccharides possessed a
stimulatory effect on macrophages, and both chitosan and chitin were chemo attractants for
neutrophils in vitro and in vivo. Chitin and chitosan may further facilitate wound healing by
stimulating granulation tissue formation or re-epithelization [Campos et al., 2006]. The argon
plasma–treated chitosan membranes exhibited excellent attachment, migration, and
proliferation of the human skin–derived fibroblasts compared to the untreated ones. To
improve the healing process, chitosan has been combined with a variety of modified materials
such as growth factors, extracellular matrix components, and antibacterial agents. The
incorporation of basic fibroblast growth factor with chitosan accelerated the rate of healing
[Mizuno et al., 2003; Liu et al., 2007b]. Hence, it has been revealed that chitosan derivatives
have great potential to be used in other biomedical applications.
Miscellaneous Applications
Chitosan-grafted systems have also been investigated for use in the cardiovascular
science. Mao et al. [2004b] studied the blood compatible properties of O-butyrylchitosan-
grafted poly(vinyl chloride) and polyethylene. The grafted polymers was observed to be
compatible with blood [Mao et al., 2004a; 2004b]. The amphiphillic polymers poly(ethylene
glycol) (PEG) or poly(ethylene oxide) (PEO) have been used with natural and synthetic
macromolecules to obtain hydrophilic biocompatible materials. The surface modification of
natural and synthetic macromolecules with water-soluble polymers such as PEG or PEO can
prevent plasma protein adsorption, platelet adhesion, and thrombus formation by stearic
repulsion mechanism [Ikada, 1984]. The chitosan membrane grafted with hydroxyethyl
methacrylate by plasma polymerization in water did not exhibit significantly different bulk
properties as compared to modified membrane, although the surface hydrophilicity was
improved. It was found that the permeability of chitosan membrane could be controlled
through plasma treatment, having the potential to be used in the dialysis process [Li et al.,
2003]. Colon-targeted delivery of bioactives has recently gained importance in addressing
specific needs in the therapy of colon-based diseases. Many approaches have been
investigated for developing colon-specific delivery systems without much success. Recent
research into the utilization of the metabolic activity and the colonic flora the lower
gastrointestinal tract has attained great value in the design of novel colon-targeted delivery
systems based on natural biodegradable polymers. Chitosan is a promising polymer for colon
drug delivery since it can be biodegraded by the colonic bacterial flora, and it has muco-
adhesive character [Kaur et al., 2009a; 2009b]. Table 10 summarizes the different
formulations prepared using various chitosan derivatives.
Table 10. A summary of different formulations prepared by employing chitosan

derivatives
Derivative System/formulation Drug Reference

Chitosan cross-linked with Transdermal delivery Propanolol HCl Thacharodi and
Dialdehyde, Rao, 1995
glutardehyde Membranes for controlled Riboflavin Aly, 1998
delivery
Transdermal delivery Oxprenolol HCl Bolgul et al., 1998
Fibers 5-fluorouracil Denkbas et al.,
2000
Freeze-dried microspheres Goserelin Illum et al., 2000
Chemical crosslinking Nanoparticles 5-fluorouracil Ohya et al., 1994
of the chitosan in the
presence of glutaraldehydes
N-trimethylene chloride Octreotide Thanou et al.,
Chitosan 2000a
Chitosan succinate and Matrices for the colon- Sodium Sinha and Kumria,
Chitosan phthalate specific oral delivery diclofenac 2001
Chitosan cross-linked with Injectable microspheres Mi et al., 2002b
Genipin Gel beads for the Indomethacin Mi et al., 2002a
controlled release
chitosan glutamate Nanoparticles Insulin Dyer et al., 2002
Chitosan cross-linked with Mucoadhesive vaginal Metronidazole Kamel et al., 2002
glutaraldehyde, together with tablets
sodium alginate with or
without microcrystalline
cellulose
Polyacrylamide grafted Microspheres Indomethacin Kumbar and
chitosan crosslinked with Amabhavi, 2003
glutaraldehyde
N-octyl-O-sulfate chitosan Micelle Taxol Zhang et al., 2003
Chitosan cross-linked with Microspheres with Glipizide Patel et al., 2005
glutaraldehyde mucoadhesive property
Ionically cross-linking N-[(2- Microgels Methotrexate Zhang et al., 2006
hydroxy-3-
trimethylammonium) propyl]
chitosan chloride in the
presence of
tripolyphosphate
N-palmitolyl Chitosan Micelle Ibuprofen Jiang et al., 2006
Hydroxylpropylcyclodextrins Nanoparticles for Triclosan and Maestrelli et al.,
(ionic cross linking of transmucosal delivery furosemide 2006
chitosan with sodium
tripolyphosphate)
Chitosan and randomly Nasal administration Estradiol Leng et al., 2006
methylated b-cyclodextrin
Derivative System/formulation Drug Reference

50% N-acetylated low Renal targeted intravenous Prednisolone Yuan et al., 2007
molecular weight chitosan injection
(LMWC)
N-trimethyl chitosan Nanoparticles Cisplatin Cafaggi et al.,
2007
Chitosan–poly Hollow nanosphere Doxorubicin Hu et al., 2007a
(acrylic acid)
Succinyl chitosan Eudragit coated Prednisolone Oosegi et al., 2008
microspheres
Chitosan-g-poly(N- Magnetite nanoparticles Doxorubicin Yuan et al., 2008

isopropylacrylamideco-
N,N-dimethylacrylamide)
Quaternized derivatives of Nanoparticles Insulin loaded Sadeghi et al.,
chitosan [trimethyl chitosan 2008
(TMC), dimethylethyl
chitosan (DMEC),
diethylmethyl chitosan
(DEMC) and triethyl
chitosan (TEC)]
Methoxy poly (ethylene Polyion complex micelles Diammonium Yang et al., 2009a
glycol) (PEG)-graftchitosan for liver-targeted glycyrrhizinate
and lactose-conjugated PEG- delivery
graft-chitosan
Chitosan-modified Nanoparticles Paclitaxel Yang et al., 2009b
poly(lactic-coglycolic
acid)
Carboxymethyl chitosan Nanoparticles Adriamycin Tan and Liu, 2009
modified with linoleic acid
PEGylated chitosan Nanoparticles Paclitaxel, Qu et al., 2009b;
Camptothecin, Opanasopit et al.,
Methotrexate, and 2006; Yang et al.,
trans- retinoic acid 2008; Jeong et al.,
2006
N-octyl-chitosan and N- Tablets Calcein, FITC- Green et al., 2009
octyl-O-sulfate chitosan dextran
Safety and Toxicological Studies
The regulatory and toxicological status of chitosan has already been established. Chitosan
is widely regarded as a non-toxic, biologically compatible polymer [Thanou et al., 2001a]. It
is approved for dietary applications in Japan, Italy and Finland [Illum, 1998] and it has been
approved by the FDA for use in wound dressings [Wedmore et al., 2006]. The modifications
made in chitosan could make it more or less toxic and any residual reactants should be
carefully removed. It is important to consider that the formulation of chitosan with a drug
may alter the pharmacokinetic and biodistribution profiles. For instance, in the case of
chitosan/plasmid DNA nanoparticles, the in vivo kinetics and distribution are mainly
controlled by the nanoparticle properties (size and charge). Further, cellular uptake kinetics
may be altered due to the charge interaction (e.g. in the case of DNA complexes). This
balancing, or reduction, of the positive charges on the chitosan molecule influences its
interaction with cells and the microenvironment, often leading to decreased uptake and a
decrease in toxicity. Schipper et al. described the effect of chitosans with differing molecular
weights and degree of deacetylation [DA] on CaCo-2 cells, HT29-H and in situ rat jejunum.
Toxicity was found to be dependent on DA and molecular weight. At high DA the toxicity
was related to the molecular weight and the concentration, while at lower DA, toxicity was
less pronounced and less related to the molecular weight. However, most of the chitosans
tested did not increase dehydrogenase activity significantly in the concentration range tested
(1–500 μg/ml) on Caco-2 cells. The in situ rat jejunum study showed no increase in LDH
activity with any of the chitosans tested (50 μg/ml) [Schipper et al., 1996; 1999]. Trimethyl
chitosan has relatively low cytotoxicity. It is an oligomer (Mw 3–6 kDa) with IC 50 >10
mg/ml for degrees of quaternisation below 55%. However, it was shown that trimethyl
chitosan of increasing degree of trimethylation possessed increased cytotoxicity as did higher
molecular weight derivatives (100 kDa) [Kean et al., 2005]. Chitosan and its derivatives seem
to be toxic to several bacteria [Jumaa et al., 2002], fungi [Guo et al., 2006] and parasites
[Pujals et al., 2008]. This pathogen related toxicity is an effect which could aid in infectious
disease control. When emulsions containing chitosans were tested, bacterial inhibition took
place in acidic solutions (pH 5–5.3), and the 87 kDa having 92% degree of deacetylation
(DD) chitosan was more effective than a 532 kDa having 73% DD chitosan against both
Pseudomonas aeruginosa and Staphylococcus aureus. In vivo toxicity particularly after long
term administration is of high importance for the design of drug delivery systems based on
chitosan. Succinimidyl chitosan was not significantly toxic at 2 g/kg in mice [Song et al.,
1993]. A photo cross linkable chitosan was developed as tissue glue. No toxic effect was
noted after subcutaneous implantation of 200 μl of 30 mg/ml photo-cross linked azide–
chitosan–lactose gel. This photo cross linkable gel exhibited the same or greater sealing
capabilities in comparison to fibrin glue [Ono et al., 2000]. Wound contraction and faster
healing were observed using both chitosan [Azad et al., 2004] and photo cross linked chitosan
[Ono et al., 2000]. Slight toxicity at high dose was observed after oral administration of
trimethyl chitosan/pDNA nanoparticles, causing light diarrhoea, which was relieved by
stopping its administration [Zheng et al., 2007]. In a relatively long study (65 days), no
detrimental effect on body weight was found when chitosan oligosaccharides were injected
(7.1–8.6 mg/kg over 5 days). An increase in lysozyme activity was apparent on the first day
post injections [Hirano et al., 1991]. However, 50 mg/kg intravenously administered chitosan
caused death, probably due to blood aggregation [Hirano et al., 1988].
Self-assembled nanoparticles, formed by polymeric amphiphiles, have been demonstrated
to accumulate in solid tumors due to enhanced permeability and retention effect following
intravenous administration. Irrespective of the dose, a negligible quantity of self-aggregates
was found in heart and lung, whereas a small amount (3.6–3.8% of dose) was detected in liver
for 3 days after intravenous injection of self-aggregates. The distributed amount of self-
aggregates gradually increased in tumor as blood circulation time increased. When self-
aggregates loaded with doxorubicin were administered into the tumor-bearing mice via the
tail vein, they exhibited lower toxicity but comparable anti-tumor activity in comparison to
free doxorubicin [Parka et al., 2006].
A chitosan derivative micelle system was developed for delivering a novel anti-tumor
drug, gambogic acid (GA).
Table 11. Safety indicators of chitosan derivatives
Particular (in Dose range Formulation Result Reference

vitro / in
vivo)
Chitosan oligomers Mouse 1000-10000 No deaths/ clinical Qin et al.,
(produced by mg/kg toxicity signs 2006
depolymerization of
chitosan)
N-acetylglucosamine Rats 0%, 0.625%, Dietary No obvious toxicity Lee et al.,
(chitosan’s copolymer) (males and 1.25%, 2.5%, administration observed 2004a
females) 5%
Glucosamine Rats 300-2700 Dietary No adverse effects Anderson
derivative of chitosan mg/kg/day administration et al.,
Dogs 159-2149 Dietary No adverse effects 2005
mg/Kg administration
Chitosan oligomers Male and 0, 750, 1500, Dietary No signs of toxicity Qin et al.,
(produced by female rats 3000 administration 2006
depolymerization of mg/Kg/Day
chitosan)
Oligo-N-glucosamine Male and 641 or 3640 Dietary No obvious toxixity, Tago et
(consists of the female rats mg/kg/Day administration findings were limited to al., 2007
monomer and occasional increase in
oligomers of N-acetyl- food consumption
D- glucosamine)
Biocompatibility study In vitro, 1 μm/ml-3 Microspheres Cytotoxicity (IC50 of 0.21 Carreno-
of Chitosan polymers murine mg/ml mg/ml range), damage to Gomez
melanoma erythrocyte membrane and
B16F10 cells Ducan,
1997
N-[(2-hydroxy-3- Concentration Complete bacterial Lim and
trimethylammonium)pr of 10 ppm reduction (with Hudson,
opyl]chitosan chloride, Staphylococcus aureus 2004
further modified by and Escherichia coli)
introducing functional
(acrylamidomethyl)
groups
Aminated chitosan In vitro 1 ml of 1 % Bacteriostatic and Eldin et
(for chitosan bactericidal al., 2008
bactericidal derivatives
activity)
Using caco 2 0.6 x 105 cells Viability of the live cell Eldin et
Aminated chitosan cell line with each was decreased by al., 2008
the direct were increase the amine group
connect established on substitution, however, the
method (for Petri-dishes toxicity of aminated
cytotoxicity) chitosan is negligible.
N-trimethyl chitosan Intestinal Polymers of No substantial cell Thanou et

Caco-2 cell different membrane damage al., 1999
monolayers degrees of
substitution
(20, 40 and
60%) at 1.0%
(w/v
concentration)
Table 11. Safety indicators of chitosan derivatives (Continued)
Particular (in Dose range Formulation Result Referenc

vitro / in e
vivo)
N,N,N- Minimal local toxicity Hagenaa
trimethylchitosan rs et al.,
2010
Glycol chitosans Mice Nanoparticles Lower toxicity than but Parka et
comparable anti-tumor al., 2006
activity to free doxorubicin
Trimethyl chitosan IC50 >10 Low cytotoxicity Kean et
mg/ml al., 2005
The physicochemical and pharmaceutical properties of GA-loaded micelles (GA-M) were

evaluated and compared with the formulation GA-L-arginine (GA-L) injection. This
preparation has entered phase I clinical trials. Biodistribution study indicated that 67% of
GA in the GA-M was distributed in the liver, while the value of the GA-L group was 55%.
Additionally, GA amount in the kidney was greatly reduced in the GA-M group. Also, GA-M
was shown to reduce the acute toxicity after i.v. administration in mice compared with GA-L.
The study indicated that GA was rapidly eliminated from the blood and transferred to the
tissues, especially the liver. Moreover, GA acute toxicity and vein irritation were decreased
[Qu et al., 2009a]. The study conducted by Hagenaars et al. [2010] showed that both whole
inactivated influenza virus (WIV) and WIV adjuvanted with N,N,N-trimethylchitosan (TMC)
formulations induced minimal local toxicity. These results provided more insight into the
safety of TMC and justifed further research to develop TMC-adjuvanted nasal vaccines.
Table 11 summarizes the in vitro and in vivo data of safety and toxicological studies of
different chitosan derivatives.
CONCLUSION
The availability of chitosan in abundance and its physiochemical properties have made it
a versatile polymer for use in pharmaceutical sciences. The amino groups present in chitosan
make it amenable to cross-linking with anionic polymers. These cross-linked molecules are
insoluble in water. Further, trimethyl, acyl, alkyl, carboxymethyl, cyclodextrin or grafted
derivatives of chitosan can be made, which possess unique property of mucoadhesivity,
biocompatibility or perturbing the paracellular pathway. Chitosan being biodegradable has
been found to be suitable for use in tissue engineering. To improve the healing process,
chitosan has been combined with a variety of modified materials such as growth factors,
extracellular matrix components and antibacterial agents. The novel properties of chitosan
make it one of the most promising biopolymers for cell therapy, tissue engineering and gene
therapy. It is hoped that these diverse approaches for regenerative medicine will translate
from research laboratories to the clinical usage in the future. The relatively safe toxicity
profile of chitosan and majority of its derivatives suggest a great promise for using it to
develop a wide variety of pharmaceutical dosage forms with tailor made properties.
However, it is important to note that the issue of residual solvents should be critically
addressed while using chitosan products obtained through catalytic reactions. In addition,
long term toxicity, especially of products using modified chitosan molecules for parenteral
use needs to be ruled out. Nevertheless, chitosan possesses a great potential for further
development as a dosage form ingredient on the basis of the promising reports of dosage form
performance as reviewed in this article.
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based noveel gatifloxacin delivery systeem. Carbohyddr. Polym., 68,, 693-700.
Zhu, A., Liu, J.., and Ye, W. (2006). Effecctive loading and a controlledd release of cam mptothecin
by O-carbo oxymethylchittosan aggregattes. Carbohyddr. Polym., 63, 89-96.
Zhu, H.U., Ji, J., Lin, R. G., Gao, C. G., Feng, L. X. and Shen, J.C. (2002) Surface
engineering of poly(D,L-lactic acid) by entrapment of chitosan-based derivatives for the
promotion of chondrogenesis. J. Biomed. Mater. Res., 62, 532-539.
Zong, Z., Kimura, Y., Takahashi, M. and Yamane, H. (2000). Characterization of chemical
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In: Chitosan: Manufacture, Properties, and Usage ISBN: 978-1-61728-831-9
Editor: Samuel P. Davis ©2011 Nova Science Publishers, Inc.
Chapter 3
PROPERTIES AND USES
C. K. S. Pillai, Willi Paul and Chandra P. Sharma*

Division of Biosurface Technology, Biomedical Technology Wing,
Sree Chitra Tirunal Institute for Medical Sciences Technology,
Biomedical Technology Wing, Poojappura Trivandrum
ABSTRACT
Chitin and Chitosan are natural polymers belonging to aminopolysaccharides having
interesting structural features for chemical modifications to generate novel properties,
functions and applications. Despite its huge availability, the utilization of chitin has been
restricted by its intractability and insolubility. Chitosan because of its improved solubility
and enhanced functions and properties find innumerable applications because of its
biocompatibility, biodegradability and non-toxicity together with its antimicrobial
activity and low immunogenicity. This review covers the production, properties and
applications of chitosan and their derivatives.
Keywords: Chitin, Chitosan, Production, Fibre Formation, Properties, Applications
INTRODUCTION
Chitin and its derivative, chitosan, are biopolymers of crustacean origin having structural
similarity to cellulose. Fourty years have lapsed since this biopolymer had aroused the interest
of the scientists, as a unique biopolymer based on the N-acetyl-glucosamine monomer. They
are amino polysaccharides having the rare amide/amino functionality and hydroxyl groups
that can undergo chemical modifications to give a variety of materials in a number of
* to whom correspondence should be addressed, E-mail: sharmacp@sctimst.ac.in

134 C. K. S. Pillai, Willi Paul and Chandra P. Sharma
applications in healthcare/biomedicine, biotechnology, water treatment, cosmetics and

toiletries, food and beverages, agrochemicals, pulp and paper, textile finishes, photography
products, product seperation and recovery, membranes, and other miscellaneous [1-10]. Many
of their applications depend on their excellent adhesion characteristics, biocompatibility and
admirable biodegradability with ecological safety and low toxicity with versatile biological
activities such as antimicrobial activity and low immunogenicity [1-5]. Chitin, in fact, is one
of the most abundant polysaccharides (next only to cellulose) found in nature, making
chitosan a plentiful and relatively inexpensive product. Coupled with the possibility of
preparing a variety of chemically or enzymatically modified products and processes, these
biopolymers having the rare amino functionality and two hydroxyl groups for chemical
modifications are potential materials in a variety of applications in biomedical,
biotechnological and pharmaceutical areas [6-12]. Despite its huge annual production and
easy availability, chitin still remains an under utilized resource primarily because of its
intractable molecular structure. However, chitosan derived from chitin because of its
improved solubility and enhanced functionality is better placed for a variety of applications.
The promise for this biomaterial is vast and will continue to increase as the chemistry to
extend its capabilities and new applications are investigated [13].
Recent findings that chitosan is a good candidate as a support material for gene delivery
[14], cell culture [15], and tissue engineering [16, 17] indicates its potential as novel
functional materials. This review focuses its attention to discuss the available data on the
manufacture, properties and uses.
LITERATURE
The emergence of chitin as a material to be reckoned with started during the 1950s. A
staggering volume of publications, reports and patents on chitin and chitosan reveals the great
interest generatred by these materials in various areas of research and development. One of
the earliest bibliography on chitin is the book written by Richards in 1951 [18]. Although
written “primarily for entomologists and other invertebrate zoologists”, the book also contains
much information on chemistry with emphasis on the possibilities of chemical modification
on chitosan. The first book fully dedicated to Chitin and chitosan was written by Muzzarelli
[19]. This book provides substantial information on chemistry, enzymatic synthesis of chitin,
structure and properties and applications pertaining to that period. References on chitin
appeared as chapters in a number of books published prior to 1977 [20-23]. Several books
and chapters have appeared since then [24-26]. Muzzarelli’s contributions in this area are
immense. The first international conference on chitin and chitosan was held at Boston, MA,
the proceedings of which appeared in 1978. He was chairman and organizer of a series of
symposia at Senigallia, Italy, on chitin chemistry and chitin enzymology (1985, 1993, 1996,
and 2001). In 1980, Paiser and Lombardi brought out a source book on chitin [27]. The
chemistry, biochemistry, properties and applications were discussed by Yalpani et al. in 1992
[28]. A similar book was published by Skryabin, et al. in 2002. Ito and Hidaka have produced
a handbook in 1997. Since then, several other books have come up.
Chitosan: Manufacture, Properties and Uses 135
HISTORICAL
The use of chitin has been known from very ancient times in India, China and Korea.
Chitin was used in a Chinese herb formula, Chantui, for treating chiefly to dispel wind-heat
and relieve spasms and convulsions [29]. Many ancient practices in folk medicine passed
down through the generations illustrate that Indian and Korean ancestors recognized some of
the healing properties of chitosan, although they certainly couldn't isolate the ingredient or
mass produce it. Koreans sprinkled squid bone powder on open wounds. Crabs were boiled
and used as medicine as well [30]. The protein components of the cuticle break down quickly
but chitin lasts much longer. Prof. Briggs and his colleagues at the Department of Earth
Sciences, University of Bristol [31] analysed fossils of fossils of 20,000 year old beetles from
tar pits at La Brea in California and found components of chitin in fossils up to 25 million
years old!
The credit for the discovery of chitin in the modern era goes to Henri Braconnot, who
was director of the botanical garden in Nancy, France. He noticed in 1821 that there was a
material in the cell walls of mushrooms that did not dissolve in sulfuric acid. He called it
fungine [32]. However, it was left to Odier in 1823 to rename Henri Braconnot fungine as
chitin (meaning ‘tunic-envelop’ in Greek) almost 3 decades before the isolation of cellulose
[33]. It was, however, 36 years later (1859) that another French scientist, C. Roget, isolated
chitosan from chitin. He noted that that chitin which was insoluble became soluble in acids
when boiled in a very concentrated potassium hydroxide solution. In 1878, Ledderhose
clearly indicated that chitin is composed of glucosamine and acetic acid and Gilson confirmed
it in 1894. The same year Hoppe-Seyler studied modified chitin and named it as chitosan.
Sutures are probably the largest groups of material implants used in human body and the
suture market so huge with tally exceeding $ 1.3 billions annually [34-37]. Physicians have
used sutures for at least 4,000 years. Archaeological records from ancient Egypt show that
Egyptians used linen and animal sinew to close wounds. In ancient India, physicians used the
heads of beetles or ants to effectively staple wounds shut. Other natural materials doctors
used in ancient times were flax, hair, grass, cotton, silk, pig bristles, and animal gut. [38]. In
ancient India, Susruta [38] is reported to have used suture materials of bark, tendon, hair and
silk in surgery [39-42]. Although chitin fibres could be made into textile materials [16] chitin
sutures have remarkable properties over other fibres for biomedical applications. Chitin
sutures resist attack in bile urine and pancreatic juice, which are problem areas with other
absorbable sutures [43].
OCCURRENCE
Chitin is a structural biopolymer which has a role in nature analogous to that of collagen
in the higher animals and cellulose in terrestrial plants. Plants produce cellulose in their cell
walls and insects and crustaceans produce chitin in their shells. Cellulose and chitin are, thus,
two important and structurally related polysaccharides that provide structural integrity and
protection to plants and animals respectively. Chitin occurs in nature as ordered crystalline
microfibrils forming structural components in the exoskeleton of arthropods or in the cell
walls of fungi and yeast. Fungal chitin occurs as randomly oriented microfibrils intertwined
and embeded in an amorphous matrix to provide the frame work in cell wall morphology.
Chitin is the second most abundant organic compound in nature after cellulose. Chitin, thus,
occurs in the shells of crustaceans such as crabs, shrimps, lobsters, mollusks etc., in the
exoskeleton of marine zoo-plankton, including coral, jellyfish and squid pens and in the
cuticle of insects. It forms about 20-40% of the exoskeleton of these animals. In fungal cell
walls, it is present as polysaccharide linked to glucans covalently linked with proteins and
lipids forming the rest. In crustaceans, chitin occurs as a component of a composite structure
consisting of a complex network of proteins onto which calcium carbonate is embedded to
generate a rigid shell. Chitin is strongly bound to proteins giving a polysaccharide-protein
complex. Except in some fungal strains (Mucoraceae), chitosan does not occur natively and is
derived from chitin by a process called deacetylation using sodium hydroxide.
Chitin is known to be present in smaller quantities in insects such as butter flies,
ladybugs, and the cell walls of yeast, mushrooms, in algae, fungi, protozoa (flagellates,
ameba, ciliates), coelenterates (skelett, stolones), bryozoa, entoprocta, mollusca (radula,
schulp), annelida (gut), arthropoda (cuticle, peritrophic membrane), echiurida, brachiopoda,
phoronida, pogonophora, bacteria (rhizobia, nod-factors), vertebrates (homologues to nod-
factors) etc. Chitin from diatoms such as Cyclotella cryptica and Thalassiosira fluviatilis are
the only form reported to be 100 % pure (polyN-acetyl glucosamine) with out impurities such
as proteins and hence is termed as chitan. A small number of fungal strains are known to
produce chitosans directly instead of chitin.
The principal source for the production of chitin commercially is shellfish waste such as
shrimps, crabs, and crawfish [44] including Pandalus borealis [45]. It also is extracted from a
few species of fungi. But since the crustacean shells (crabs etc) are waste products (now
byproducts) of food industry, these are commercially employed for production of chitin and
chitosan. It is believed that at least ten gigatons (1013 Kgs.) of chitin are synthesized and
degraded and it is also estimated that over 1,50,000 tons of chitin is available for commercial
use annually. One of the main concerns in the utilization of chitin is the the variation in
composition and properties arising out of the variations in the source raw material differing
with sex, age and habitat of the animal. It has been estimated that worldwide production of
chitin could potentially yield 1,18,000 tonne annually from shell fish, krill, clams, oysters etc.
against present production of approximately 1,600 tonne. The potential markets are Japan,
USA, UK, and Germany. The market is expected to exceed US $ 2 billion soon. In India
starting with a modest two tonnes export of chitin earning Rs.1.47 lakh in 1991-92, the export
market has grown over the past six to seven years. India has potential of 2000 tonnes
production of chitin. During 1996-97, it is estimated that around 350 tonnes of chitin worth
nearly 85 lakh (approx.) have been exported. In the year 1997-98 around 350 tonnes of chitin
and 50 tonnes of chitosan have been exported fetching a foreign exchange of nearly Rs.90
lakh. Table 1 provides an estimate of the available waste and chitin content in them.
Chitosan is produced commercially by deacetylation of chitin, which is the structural
element in the exoskeleton of crustaceans (crabs, shrimp, etc.) and cell walls of fungi. The
degree of deacetylation (%DA) can be determined by NMR spectroscopy, and the % DA in
commercial chitosans is in the range 60-100 %.
Table 1. Estimate of the available waste and chitin content in various species
Resource Landings Potential waste Estimated waste Dry waste Chitin content
(MT) (MT) (MT) (MT) (MT)
Shrimp 2,647,345 1,058,938 710,000 177,500 44,375
Squid 1,991,094 389,219 99,531 24,882 1,244
Crabs 1,348,323 943,826 482,744 144,823 28,964
Oyster Clam 2,547,287 1,783,100 304,948 274,453 12,350
Krill 232,700 93,080 93,080 23,270 1,629
Total 8,766,749 88,652
PRODUCTION OF CHITIN AND CHITOSAN

Methods of production of chitin and chitosan vary considerably in different parts of the
world. However, all these methods follow the general principle of deproteinization and
demineralization of the crustacean shells. Generally, the crustacean shells obtained as food
processing waste mainly consists of 30-40% protein, 30-50% calcium carbonate and calcium
phosphate, and 20-30% chitin on a dry basis of [46, 47]. Table 2 provides the composition of
the crustacean shells based on dry weight. These proportions vary with species and seasons
[49] as provided in table 3. Some lipids and traces of pigment will also be there. The proteins
are removed by treatment with dil.sodium hydroxide (about 10%) at about 85o C to 100o C or
digested enzymatically by proteases or micro-organism. The shells are then demineralized to
remove calcium carbonate by the treatment with dil. hydrochloric acid (about 10%) at room
temperature. Lipids are extracted by soaking in organic solvents such as acetone or ethanol.
An oxidative bleaching treatment with hydrogen peroxide or sodium hypochlorite is also
given to obtain a white powder [48].
Table 2. Composition of the crustacean shells based on dry weight.
Crawfish/Crabs Shrimp % Fungi %

Chitin 25-30 30-40 15-40
Protein 15 35 5-10
CaCO3 55 30 Glycans
Lipids 2-5 5-10 5-10
Table 3. Chitin contents variation in different species.
Source Crab Shrimp Snow crab Aspergillus niger Mucor rouxiiPencillium notatum
Chitin 13-15 14.27-17.0 18.7-32.2 42 44.5 18.5
content %
Chitin represents one third of the shell composition, and is highly hydrophobic and
insoluble in water and most organic solvents. Chitosan, the deacetylated product of chitin, is
soluble in very dilute acids such as acetic acid or formic acid. Traditional isolation of chitin
from crustacean shell waste consists of three basic steps: demineralization (DM-calcium
carbonate and calcium phosphate separation), deproteinization (DP-protein separation), and
decolorization (DC-removal of pigments). These three steps are the standard procedure for
chitin production [50]. Protein extraction is usually accomplished with a mild alkaline
solution, such as 1 or 2% sodium hydroxide, at 60-70° C, for a few hours, and the extracted
proteins can be recovered for other uses, such as animal feed. Removal of calcium carbonate
and calcium phosphate is accomplished by dissolution with dilute acids. Hydrochloric acid is
used most commonly, but other acids can also be used. Chitosan is produced from chitin by
deacetylation with highly concentrated (40-50%) solutions of sodium hydroxide at high
temperatures (100-150°C), exclusive of air, for about an hour. The subsequent conversion of
chitin to chitosan (DA, deacetylation) is generally achieved by treatment with concentrated
sodium hydroxide solution (40-50%) at 100ºC or higher to remove some or all of acetyl group
from the chitin.The process of deacetylation involves the removal of acetyl groups from the
molecular chain of chitin, leaving behind a compound (chitosan) with a high degree chemical
reactive amino group (-NH2). This makes the degree of deacetylation (DD) an important
property in chitosan production as it affects the physicochemical properties, hence determines
its appropriate applications [51]. Deacetylation also affects the biodegradability and
immunological activity [52].
The deacetylation process is a harsh treatment usually performed with concentrated
sodium hydroxide solution. Chitin flakes are treated in suspension with aqueous 30 - 60%
caustic solution at 80 - 1200C with constant stirring for 4 - 6 hours and this treatment is
repeated for once or more times for obtaining high amino content product. To avoid
depolymerization due to oxidation, sodium borohydrate is added. Deacetylation of chitin can
also be done enzymatically. Here powdered chitin is treated with N-deacetylase (EC 3.5.1.41)
or with microbes which secrete N-deacetylase. The enzymatic method yields chitosan with
low degree of N-acetylation and low degree of polymerization. The transition from the
arthopods to chitin is given in figure 1.
Arthropods Shell Fungi Chitin

Figure 1. The transition from the arthopods to chitin.
Detailed flow sheet diagram for chitosan production is given in figure 2 . Essentially, the
manufacturing process involves the following steps: (1) Cleaning and washing the shells,
drying, grinding and sieving, (2) Demineralization: removal of mineral matter from th shell
by treatment with 6% commercial hydrochloric acid followed by washing and removal of
sand and other exreneous matter present in the shell by floatation; (3) Deproteinization:
Removal of protein by treatment with 0.5 % sodium hydroxide solution at 80°C for 30 min.
and recovery of protein by neutralization and concentration by evaporation of the excess
water. A second treatment with 3% caustic soda solution at 80°C for 30 min. is required to
remove fully the remaining proteins. Chitin is obtained after washing and drying. (4)
Deacetaylation of chitin: chitin is converted to chitosan by deacetylation by treating with 50%

sodium hydroxide at 90-95 °C for 1-2 hrs. The chitosan is recovered by centrifuging, drying
and later pulverized to get the desired particle size. The unused alkali can be recoverd. Chitin
recovered in stage can be extracted with acetone to get astaxanthen pigment. Another stage
can balso be introduced to bleach the chitin using 0.315 NaOCl for five min. at room
temperature so that the chitin obtained is perfectly white in color.
Wet shrimp fish Washing and drying Grinding and sieving Demineralization
shells
6% HCl for 1
hr. at room
temp.
Filtering Demineralised shelll Washing, floatation Deproteinization

0.5 % NaOH at 80 °C
Filtrate Protein recovery for 30 min, and 3 %
Astaxanthin pigment NaOH at °C for 30 Min.
1h t t
Filtering Washing Extraction with Acetone Drying
Bleaching Washing and drying CHITIN Deacetylation
0.315 % NaOCL %50 NaOH for 30

for 5 min. at min. at 90-95 °C .
room temp.,
solid
Centrifuging, drying CHITOSAN

and pulverising
Figure 2. Chitosan production flow sheet
The mineralization stage can be omitted in the cases of fungi and squid bone etc. In the
case fungi, after deproteinization satge, the product is extraxted with solvents such as
LiCl/DMAc and precipitated in water and dried to obtain chitin. The resulting chitin is
deacetylated in 40% sodium hydroxide at 120°C for 1–3 h. This treatment produces 70%
deacetylated chitosan. In this way, α-chitin is produced while squid pens are used to produce
β-chitin.
CHITIN DEACETYLATION
Chitosan is prepared by hydrolysis of acetamide groups of chitin. This is normally
conducted by severe alkaline hydrolysis treatment due to the resistance of such groups
imposed by the trans arrangement of the C2-C3 substituents in the sugar ring. Thermal
treatments of chitin under strong aqueous alkali are usually needed to give partially
deacetylated chitin (DA lower than 30%), regarded as chitosan. Usually, sodium or potassium
hydroxides are used at a concentration of 30-50% w/v at high temperature (100ºC). In
general, two major different methods of preparing chitosan from chitin with varying degree of
acetylation are known. These are the heterogeneous deacetylation of solid chitin and the
homogeneous deacetylation of pre-swollen chitin under vacuum (by reducing pressure) in an

aqueous medium. Heterogeneous deacetylation, which is the preferred industrial treatment,
involves preferential reaction in the amorphous regions of the polymer, leaving almost intact
the intractable crystalline native regions in the parent chitin.
Alternatively, homogeneous modification is conducted by use of moderately concentrated
alkali (13% w/w) acting on pre-swollen chitin to improve the interaction with the alkali and
left to react at 25-40ºC for 12-24 hours. In both, heterogeneous or homogeneous conditions,
the deacetylation reaction involves the use of concentrated alkali solutions and long
processing times which can vary depending on the heterogeneous or homogeneous conditions
from 1 to nearly 80 hours. Factors that affect the extent of deacetylation include concentration
of the alkali, previous treatment, particle size and density of chitin. The last two factors affect
penetration rate of the alkali into the amorphous region and to some extent also into the
crystalline regions of the polymer, needed for the hydrolysis to take place. In practice, the
maximal DD that can be achieved in a single alkaline treatment is about 75-85%. In general,
during deacetylation, conditions must be the proper ones to deacetylate, in a reasonable time,
the chitin to yield a chitosan soluble in diluted acetic acid. Thiophenol and NaBH4 have been
used as oxygen scavenger and reducing agents, respectively, thus effectively resulting in a
product of greater viscosity. Also, treatments with concentrated NaOH in the presence of
water-miscible diluents such as 2-propanol, 2-methyl-2-propanol, polyethylene glycol
dimethyl ether, and acetone or paraffin oil have enabled the volume of concentrated NaOH
required to be reduced by at least 85%. Several alternative processing methods have also been
developed to reduce the long processing times and large amounts of alkali typically needed to
deacetylate chitin to an acid-soluble derivative. Examples of these include the use of
successive alkali treatments using thiophenol in DMSO; thermo-mechanical processes using a
cascade reactor operated under low alkali concentration; flash treatment under saturated
steam; use of microwave dielectric heating; and intermittent water washing. Improved
production techniques have been recently reported [53-56]. When a 4-5 % solution of
chitosan flakes in acetic acid is filtered and is reprecipiatated in sodium bicarbonate solution,
the chitosan obtained is called “activated chitosan”. If the filtered acetic solution of chitosan
is degraded at 70 °C and “sheared in NaOH”, the chitosan obtained is called “microcrystalline
chitosan” [57]. Granulated chitosan is prepared in a wet-dry spinning process [58].
ENZYMATIC DEACETYLATION OF CHITIN

Deacetylation of chitin can also be done enzymatically [59,60]. Powdered chitin is treated
with N-deacetylase (or with microbes which secrete N-deacetylase. The enzymatic method
yields chitosan with low degree of N-acetylation and low degree of polymerization. In fact
there are two advantages of chitosan over chitin. In order to dissolve chitin, highly toxic
solvents such as lithium chloride and dimethylacetamide are used whereas chitosan is readily
dissolved in diluted acetic acid. The second advantage is that chitosan possesses free amine
groups which are an active site in many chemical reactions [61].
CHITOSAN DEPOLYMERIZATION
Several methods for the rapid determination of the degree of acetylation of chitin and
related polymers have been evaluated, including the use of the infrared and the mass spectra.
Chitin and chitosan have characteristic degradation temperatures and it is possible to
determine the acetylation degree by the use of empirical correlations based on the weight
losses associated with the main decomposition peaks. The main limitations in the use of
chitosan in several applications are its high viscosity and low solubility at neutral pH. Low
molecular weight (Mw) chitosans and oligomers can be prepared by hydrolysis of the
polymer chains. For some specific applications, these smaller molecules have been found to
be much more useful. Chitosan depolymerization can be carried out chemically,
enzymatically or physically. Chemical depolymerization (Fig. 2) is mainly carried out by acid
hydrolysis using HCl or by oxidative reaction using HNO2 and H2O2. It has been found to be
specific in the sense that HNO2 attacks the amino group of D-units, with subsequent cleavage
of the adjacent glycosidic linkage. In the case of enzymatic depolymerization, low molecular
weight chitosan with high water solubility were produced by several enzymes such as
chitinase, chitosanase, gluconase and some proteases. Non-specific enzymes including
lysozyme, cellulase, lipase, amylase and pectinase that are capable of depolymerizing
chitosan are known. In this way, regioselective depolymerization under mild conditions is
allowed. Physical depolymerization yielding dimers, trimers and tetramers has been carried
out by radiation (Co-60 gamma rays) but low yields have been achieved.
FUNCTIONAL GROUPS
Chitin and chitosan are of commercial interest due to their high percentage of nitrogen
(6.89%) compared to synthetically substituted cellulose (1.25%). The unique structural
feature of chitosan is the presence of the primary amine at the C-2 position of the
glucosamine. Few biological polymers have such a high content of primary amines, and these
amines confer important functional properties to chitosan that can be exploited for chemical
modification. As some amount of deacetylation might take place during extraction, chitin may
also contain a small portion of amino groups [62,63]. In addition to its unique polysaccharide
architecture, the presence of a few amino groups (5-15%) in chitin is highly advantageous for
providing distinctive biological functions and for conducting modification reactions [64-66].
As N-deacetylation is almost never complete, chitosan will have some acetamodo groups
also. Apart from the amino group, Chitosan possesses a primary and a secondary hydroxyl
group for chemical modification so that its properties can be altered to incorporate improved
physical, mechanical, solution, biological properties. So, the presence of the acetamodo and
amine groups in chitin and chitosan influences their properties and make them different in
many properties from those of cellulose. Chemical modifications of chitin are generally
difficult owing to the lack of solubility, and the reactions under heterogeneous conditions are
accompanied by various problems such as the poor extent of reaction, difficulty in selective
substitution, structural ambiguity of the products, and partial degradation due to severe
reaction conditions. Therefore, with regard to developing advanced functions, much attention
had been paid to modification of chitosan rather than chitin [67]. The physical properties of
chitosan depend on the degree of N-acetylation and the distribution of N-acetyl groups [68].
Chitosan is a high molecular weight polysaccharide. It is a natural biodegradable polymer
derived from chitin. It is very similar in structure to cellulose, with the hydroxyl group in 2-
position is replaced by amino group. It is a copolymer of glucosamine and N-acetyl
glucosamine, the polymer is considered as chitosan if the ratio of glucosamine to N-acetyl
glucosamine is greater than 1. So, in chitin the degree of N-acetylation i.e. the ratio of 2-
acetamido-2-deoxy-D-glucopyranose to 2-amino-2-deoxy-D-glucopyranose structural units
has a striking effect on chitin solubility and solution properties. Chitosan is the fully or
partially N-deacetylated derivative of chitin with a typical degree of acetylation of less than
0.35. It is is positively charged due to the acidic environment and, therefore, it can interact
with mucin by electrostatic forces. The mechanism underlying the behaviour permeation
enhancer is also based on the interaction of positively charged chitosan and the cell
membrane resulting in a reorganization of the tight junction-associated proteins.
Antimicrobial effect: the interaction with anionic groups on the cell surface, due to its
polycationic nature, causes the formation of an impermeable layer around the cell, which
prevents the transport of essential solutes. The second mechanism involves the inhibition of
the RNA and protein synthesis by permeation into the cell nucleus. It is suggested that their
wound-healing properties are due to their ability to stimulate fibroblast production by
affecting the fibroblast growth factor. As a result of its cationic character, chitosan is able to
react with polyanions giving rise to polyelectrolyte complexes.
PHYSICO-CHEMICAL CHARACTERIZATION OF CHITOSAN
Degree of N-Acetylation
An important parameter to examine closely is the degree of N-acetylation in chitin, i.e.

the ratio of 2-acetamido-2-deoxy-D-glucopyranose to 2-amino-2-deoxy-D-glucopyranose
structural units. This ratio has a striking effect on chitin solubility and solution properties.
Chitosan is the universally accepted non-toxic N-deacetylated derivative of chitin, where
chitin is N-deacetylated to such an extent that it becomes soluble in dilute aqueous acetic and
formic acids. In chitin, the acetylated units prevail (degree of acetylation typically 0.90).
Chitosan is the fully or partially N-deacetylated derivativeof chitin with a typical degree of
acetylation of less than 0.35.
Solubility and Molecular Weight
The selection of the solvent is also important when molecular weight has to be calculated
from intrinsic viscosity using the Mark–Houwink relation (η =KMα where η is the intrinsic
viscosity, M is the molecular weight, K and α are constants.). The values of the parameters K
depend on the nature of the solvent and polymer. For example, one solvent system first
proposed (0.1M AcOH/0.2M NaCl) for molecular weight characterization was shown to
promote aggregation and the values of molecular weights calculated were overestimated
Chittosan: Manufaacture, Properrties and Uses 143
[669,70]. Rinauddo et al. propoosed that 0.3M M acetic acid//0.2M sodium m acetate (pH = 4.5) as a
soolvent can be sued to overrcome the prooblem of aggregation as theere was no evvidence for
agggregation in this mixture [71]. Using acid-soluble
a chhitosans of DA
D varying froom 0.02 to
0..61, they conccluded that thhe stiffness off the chain waas nearly indeependent of thhe DA and
deemonstrated th hat the variouus parameters depended onlly slightly on the DA [71]. In contrast
too this proposittion, Kasai et al indicate thhat a and K area inversely related
r and thhat they are
innfluenced by DA,
D and pH and a ionic strenngth of the soolvent [72]. They studied thhe intrinsic
viiscosity–molecular weight relationship for f chitosan in i 0.25 M acetic acid/0.255M sodium
accetate. Chitossan samples with
w a degreee of acetylatioon (DA) betw ween 20 and 26% were
prrepared from shrimp-shell chitosan by acid a hydrolysiis (HCl) and oxidative fraggmentation
(NNaNO2). Abso olute molecullar weights were
w measuredd by light sccattering and membrane
ossmometry. Sizze exclusion chromatograph
c hy (SEC) wass used to deterrmine averagee molecular
w
weights (Mn, Mv,
M and Mw) and a polydispeersity. The datta of K determ mined by varioous authors
(rrefer Table 3 of
o reference 722) can be plotted against DA A which indiccates that theree cannot be
anny relationshipp between DA A and K value.. Kasai has sinnce modified his h work [73].
As the valu
ues of K and α differ, it is poointed that it would
w always be better to foollow those
vaalues where thhe authors havve used a standdard referencee for comparinng the molecuular weights
annd a standard method such as light scatteering or gel peermeation chroomatographgyy [74,75] to
deetermine the absolute
a molecular weights. The relativelly high valuess for the param meter α are
inn agreement with
w the semiriigid character of chitosan. The T charged nature
n of chitoosan in acid
soolvents and chhitosan’s propeensity to form
m aggregation complexes
c reqquire care wheen applying
thhese constants [76]. The weiight-average molecular
m weiight of chitin isi 1.03 x 106 too 2.5 x 106,
5 5
buut the N-deaceetylation reacttion reduces thhis to 1x10 too 5x10 [77].
E
Effect of Deg
gree of Deaccetylation and Molecullar Weight
The relatioonship betweeen solubility, molecular weeight and deggree on N-acyylation has
beeen establisheed by severall groups [78--89]. X-ray diffraction
d and deamination analyses
suuggested that the distributioon of N-acetyyl groups in thhe chitin molecule was moore random
thhan those in th he regeneratedd chitin [80]. At
A 50 % N-accetylation, chiitosan solubility in water
diid not show anya change wiith an increasee in the moleccular weight [81]. Howeverr, a notable
crrystal structurre transition from
f crystal "Form
" II" witth constrainedd chain confoormation to
"FForm I" havin ng a more exteended chain strructure to a crrystalline formm similar to thhat of chitin
w observed on
was o increasing acetyl group [85]. The acettyl group depeendent transfoormation in
crrystal structurre indicates thhat control off the degree ofo acetylation can be used to control
soolubility. Thiss has led to the preparation of WSC by controlling
c thee degree of deeacetylation
(DDD) and molecular weight of o chitin throuugh alkaline annd ultrasonic treatment [90]. The WSC
w found to be
was b more efficient than chitinn or chitosan as a wound-hhealing acceleerator when
teested in rats. Homogeneous
H ly deacetylateed samples weere obtained by b this alkalinee treatment
off chitin under dissolved connditions [90,91]. The homogeneity of thee deacetylatedd chitin was
laater assured by the recetylaation of highlyy deacetylatedd chitosan [922]. The solubility of the
paartially deacettylated chitinss had a close reelationship wiith their crystaal structure, crrystallinity,
annd crystal im mperfection as well as the glucosamine content [91].. The wide-anngle X-ray
diiffractometry (WAXD) revvealed that thhe chitin witth ca. 28% DD D retained the crystal
sttructure of -cchitin with siggnificantly redduced crystalliinity and perfeection of the crystallites.
c
1444 C. K. S.
S Pillai, Willii Paul and Chaandra P. Sharm
ma
The water-solu uble chitin of ca.

c 49% DD hadh a new crysstal structure similar
s to that of β-chitin
raather than eitther -chitin or chitosan, suggesting that t the hom
mogeneous deeacetylation
trransformed thee crystal struccture of chitinn from the too the β-form [91].
[ Physicall properties
suuch as crystalllinity and pollymermorphicc forms are reeported to be affected by the t process
coonditions of preparation
p [93-97]. The cryystalline state of the samplees was said to be the key
paarameter on which
w depended the rate connstants of bothh alkaline hyddrolysis and deeacetylation
prrocess [98]. So olubility couldd be enhanced by appropriatte chemical moodification [999-113]. The
vaarious analyticcal techniques employed for the determinattion molecularr weight, viscoosity etc are
prrovided in refeerences [114-1222].
C
Crystallinity
y
Chitin occuurs in nature as

a ordered cryystalline microofibrils forminng structural components
c
inn the exoskeleton of arthroppods or in the cell walls of fungi
f and yeasst. It is also prroduced by
a number of oth her living organisms in the lower plant and a animal kinngdoms, servinng in many
fuunctions wherre reinforcemeent and strenngth are requiired. Dependinng on its souurce, chitin
occcurs as two allomorphs,
a naamely the α annd β forms. A third allomorrph γ-chitin haas also been
deescribed but from
fr a detailedd analysis, it seems
s that it iss just a variannt of the α fammily. Chitin
iss by far the most
m abundant;; it occurs in fungal and yeeast cell wallss, in krill, in lobster
l and
crrab tendons annd shells, and in shrimp sheells, as well as in insect cuticcle. α-chitin iss by far the
m
most abundant;; it occurs in fungal and yeeast cell wallss, in krill, in lobster
l and crrab tendons
annd shells, and in shrimp sheells, as well as in insect cuticcle.
PROPE
ERTIES OF CHITIN AN
ND CHITOS
SAN
B
Biocompatib
bility
Chitin and chitosan havee been found to t have diversse uses as a biiopolymer in many
m areas
off medical sciences and tecchnology. Thiis calls for ceertain essentiaal requiremennts such as
biiocompatibilitty and biodegrradability. Being a part of the structural element of innnumerable
annimal speciess, biocompatibbility of chitiin and chitosaan must be structurally
s inncorporated
duuring biologiccal synthesis and
a in fact waas found to be so. So, chitinn and chitosann have been
heeavily exploreed as a suitable functional material
m in biomedical applications due mainly
m to its
biiocompatibilitty, biodegradaability, and noon-toxicity. Thhey are not only biocompaatible [123-
1229] and non toxic
t [130], but antigenicallly inactive, inn both animall and plant tisssues [131-
1333]. The biocoompatibility and
a safety of chitosan
c have been revealedd through testss involving
m
mutagenicity, acute
a and sub acute toxicityy, pyrogens, heemolysis, andd sensitization [130]. The
U Food and Drug
US D Administration considder chitosan ass a food addittive in animal feed when
ussed as a preciipitating agentt for proteinecceous materials [134]. Seo has shown thhat chitosan
w
when orally ad
dministrated too rabbits, brooilers and hens at a doasagge of 0.7 -0.8 g/kg body
w
weight /day fro
o upto 239 dayys, no abnorm mal symptoms were observeed. Rabbits diggested upto
288-38% chitin and 38-79% % chitosan whhile broilers and a hens diggested them completely.
c
R
Rabbits also did
d not exhibbit any abnorrmal symptom m when chitoosan was inttravenously
injected. It was also observed that the presence of chitosan enhanced the absorption of drugs
when administrated orally [135]. There are a few studies that implicated that chitosan was
toxic and the toxicity was dependent upon their molecular weight, degree of deacetylation and
salt form [136,137]. However, further investigations revealed that the toxicity of chitosan was
negligible [138-140].
The Immunological, Antibacterial and Wound-Healing Activity
The immunological [141,142], antibacterial [143,144], and wound-healing activity [145-

153], of chitosan have been established by many authors. Sharma et al. showed that chitosan
exhibits excellent biocompatibility when used as a hemoprofusion membrane for blood [154].
Muzzarelli et al demonstrated the high biocompatibility of dibutyryl chitin in the form of
films and non-wovens for human, chick and mouse fibroblasts by the viability/cytotoxicity
assay, in situ cell proliferation assay, neutral red retention assay, lactate dehydrogenase
release assay, MTS cytotoxicity assay, and scanning electron microscopy.
Effect of Chemical Modification on Biocompatibility
Chitin and chitosan have been modified via a variety of chemical modifications to
improve solubility and hence biocompatibility and impart functional properties [155-162].
Graft copolymerisation has been suggested as an efficient tool to introduce functional
properties [163]. Chitosan can be functionalized rather easily through its hydroxyl and/or
amine groups. Chemical modifications such as acylation, alkylation and phthaloylation etc.
has been investigated to alter solubility and biocompatibility. Among the various acylated
chitosans, N-hexanoyl chitosan (H-chitosan) was found to exhibit the best blood
compatibility [164,165].
Effect of Complexation, Blending and Composite

Formation on Biocompatibility
It has been showed that addition of 20% collagen and 0.5% sodium hyaluronate improved
the biocompatibility of the chitosan complex when tested as substrates for cultivating rabbit
corneal cells and and a feasible material as a scaffold of tissue-engineered cornea. Zhang et al
reported excellent adhesion between osteoblast and PLLA/CHS fabrics indicating good
biocompatibility of the fabrics with osteoblast; poly(l-lactic acid) (PLLA) fibers were
prepared by the dry-wet-spinning method, while chitosan (CHS) fibers were prepared via the
wet-spinning method. The two fibers were blend spun and then fabricated into PLLA/CHS
fabrics [166]. Li et al. studied a series of PLA/chitosan composite materials for Chondrocyte
cultivation, subcutaneous and intramuscular implantation. The results showed that the cells
could well adhere, grow and multiplicate on the surface of the materials. The plantation test
revealed that the tissue response toward the implanted PLA/chitosan composite materials was
mild, regardless of the ratio of PLA and chitosan. PLA materials were degraded and absorbed
2 months later in the body, while the composite materials could still keep certain strength and
had far less inflammation [167]. Studies with polycaprolactone scaffolds indicate that the
chitosan-modified PCL scaffolds are more favorable for cell proliferation by improving the
scaffold biocompatibility [168,169].
Effect of Molecular Weight on Biocompatibility
Yao, et al. studied the effect of molecular weight of chitosan on biocompatibility and
showed that low molecular weight chitosans are more effective than high molecular weight
polymers in biocompatibility. Chitosan membranes were prepared with different molecular
weight (MW) of 130KDa, 220KDa, 300KDa and 500KDa respectively. The degree of
deacetylation (DD) for the all kinds of chitosan is 95%. The keratocytes were cultured on the
surface of different chitosan membranes. The morphological characteristic, the cell -
adhesion, the cells proliferation and the activity of LDH in the medium were investigated.
The results showed that the chitosan membrane with molecular weight of 500KDa and
130KDa caused damages to corneal cells, so the cell could not adhere or grow well on the
surface of such chitosan membrane. The chitosan membrane with molecular weight of
200KDa and 300KDa could support the keratocytes growing into monolayer that indicated
that such membranes were polemical scaffolds for corneal cell culture [170]. Molinaro et al
showed that a higher degree of deacetylation of the chitin chain is desirable for superior
biocompatibility. They used chitosan solutions containing glycerol-2-phosphate (β-GP) which
undergoes sol-gel transition at a temperature close to 37°C that makes them suitable for the
parenteral administration of drugs. Four chitosan/β-GP solutions tested triggered a non-
specific response, with solutions prepared with chitosans of higher deacetylation degrees
yielding a lesser inflammatory reaction and (ii) systemic pretreatment of animals with
icatibant, apafant and diphenhydramine did not significantly diminish this response;
dexamethasone practically abolished it for all solutions and ketanserine only slightly
decreased it in one preparation at two different times [171].
Effect of Irradiation on Biocompatibility
Irradiation of the surface of chitosan has been shown to alter biocompatibility. The results
of recent FT-IR and Raman studies indicated the formation of new carbon structures and new
functional groups by ion beam irradiation. Chitosan film irradiated with each ion at fluence of
1×1015 ions/cm 2 exhibited excellent cell adhesion [172].
Biodegradability
Today the medical applications of polymers are at an all time high and the need to have
biocompatibility and biodegradability in the same molecule has enhanced the search for
biodegradable polymers from renewable resources. It is well known that polymers containing
hydrolysable linkages along the chain or easily oxidisable functional groups on the chain are
susceptible to biodegradation by microorganisms and hydrolytic enzymes. The hydrolytically
degradable glucose moieties and the amide/amino functionalities ensure biodegradability in

chitin and chitosan. Thus, when attacked by natural fungi, chitosan films have a built-in
source of nitrogen to enhance biodegradation. Surprisingly, information on the in vivo
biodegradability of chitosans with differing chemistries and structures, and which are utilized
in multiple applications, is lacking. It is generally believed that lysozyme is mainly
responsible for chitosan degradation in the human body [173]. Lysozyme is present in many
tissues and secretions such as tears, saliva, blood and milk, and is released and utilized by
phagocytic cells during the inflammatory response to a foreign implant [174-176].
Onishi et al studied the biodegradability, body distribution and urinary excretion of 50%
deacetylated chitin after the intraperitoneal (ip) administration to mice using fluorescein
isothiocyanate labeling [177]. The labeled chitosan (FTC-Chi) moved fast to the kidney and
urine, and was scarcely distributed to the liver, spleen, abdominal dropsy and plasma. Most of
FTC-Chi was excreted into urine after 14 h, and the molecular weight of the excreted FTC-
Chi was as small as that of the product obtained by the long in vitro incubation. Therefore,
chitosan is considered to be highly biodegradable and easily excreted in urine, and further it is
suggested to have no problem on accumulation in the body; however, at the same time,
chitosan is found not to operate as a polymer support showing long retention in the body.
Effect of Chemical Modification
Biodegradability has been shown to be altered by appropriate chemical modifications on

chitin and chitosan. Acylation has been shown to enhance biodegradability [178-181].
Winursito et al. correlated the glucosamine content in the polymer chain with
biodegradability. Using a series of partially N-acylated and dicarboxylated chitosans, they
concluded that chitosans having more than 7 mol% glucosamine content exhibited good
biodegradation [182]. In an interesting study of several N-acyl and N-acetyl derivatives, Lee
et al. established that N-Acyl chitosans had as high a susceptibility to lysozyme as N-acetyl
chitosans. It was considered that the amount of derivatized groups and the physical form of
N-acyl chitosans contributed to biodegradability [183]. In another study designed to improve
the solubility of chitosan, Park et al. showed that chitosan modified with gluconic acid
exhibited enhancement in the susceptibility to lysozyme. They pointed that it was possible to
control the biodegradability of chitosan by adjusting the amounts of gluconyl and N-acetyl
groups in the chitosan backbone [184,185].
Xu et al showed that biodegradation tests on nonmodified or hexanoylated chitosan films
exhibited no apparent weight loss for 35 day exposures in laboratory-scale aerobic
thermophilic test reactors [186]. In contrast, by regenerating chitin at film surfaces via N-
acetylation, the rate of film biodegradation was dramatically enhanced. Chitosan films were
acylated under heterogeneous conditions in methanol with acetic and hexanoic anhydrides
and characterized by proton nuclear magnetic resonance, elemental analysis, and multiple
internal reflective Fourier transform infrared spectroscopy. Biodegradation studies of the
acylated chitosan films carried out in laboratory-scale aerobic thermophilic compost reactors
revealed that the formation of chitin at the film surface enhanced the biodegradability of the
films. Specifically, the 3 h acetylated chitosan film (0.045 mm thickness) showed 100%
weight loss during a 28 day exposure, whereas unmodified chitosan showed no significant
weight loss after 35 days. Similarly, introduction of dendrimer groups in chtosan was found
to delay biodegradation in comparison to that of chitosan itself [187]. Sashiwa et al have

shown that the Michael reaction of chitosan with acrylic acid gave N-carboxyethylchitosan
which exhibited good solubility and hence excellent biodegradability with standard activated
sludge [188]. Chitosan was modified by Kumar et al. using the enzyme tyrosinase to convert
a low molecular weight phenolic substrate (p-cresol) into a reactive o-quinone that undergoes
subsequent reaction with chitosan. Incubation of these gels with the hydrolytic enzyme
chitosanase showed that the cresol-modified chitosan remained biodegradable [189].
The Effects of Temperature and Molecular Weight on the

Biodegradation of Chitosan
The effects of temperature and molecular weight on the biodegradation of chitosan were
examined by Maria Ratajska et al. [190,191]. They studied the biodegradation process of
three chitosan samples characterised by similar values of deacetylation degree but different
molecular weights in an aqueous medium using FTIR spectrophotometry, GPC
chromatography and x-ray (WAXS) methods.. The biodegradation process was caused by
microorganisms present in activated sludge from the waste-water treatment station of a
cellulose plant. The range of the most appropriate temperatures for chitosan biodegradation
was estimated as 30-36°C. The shortest time of biodegradation and the induction time was
observed in the chitosan sample with the lowest molecular weight. In this article, the changes
to biodegraded chitosan structure as estimated by FTIR spectrophotometry, GPC
chromatography and x-ray (WAXS) methods are presented. Apart from the structure, the
origin and properties of the samples, as well as the degree of deacetylation and conditions of
the biodegradation process affect biodegradation of chitosan. Research into the
biodegradation of mixtures of synthetic and natural polymers with the use of the method of
light transmittance led to the general conclusion that the biodegradation process was largely
dependent on the type the synthetic polymer as well as on conditions of the process [192].
Effect of Copolymerisation
Copolymerisation especially grafting is a very useful method to control the

biodegradation of chitosan polymers [193-195]. Studies by Gisha and Pillai showed that the
physico-chemical properties and the rate of degradation of chitosan–polylactide graft
copolymers can be controlled by adjusting the amount of LLA in the CL graft copolymers,
with biodegradation decreasing with increase in LLA content [196]. This may find wide
applications in wound dressing and in controlled drug delivery systems. Hydrolytic
degradation takes place preferentially on the amorphous portion of graft copolymer and the
resulted short chains are dissolved out into water by creating pores on the surface.
Effect of Complexation, Blending and Composite

Formation on Biodegradability
Chitosan-gelatin film is found biodegrade faster than chitosan and it has good
biocompatibility. Lysozyme may promote biodegradation of the mixed film [197]. Similarly,
incorporation of pectin makes chitosan more biodegradable [198]. Han et al developed an
injectable thermosensitive hydrogel for local drug delivery to treat cancers clinically based on
chitosan as a polymer matrix because of its biocompatibility and biodegradability [199].
Glycerol 2-phosphate disodium salt hydrate (β-GP) was used to neutralize the chitosan
solution to physiological pH. The chitosan solution displayed a sol-gel phase transition in a
pH- and temperature-dependent manner and formed an endothermic hydrogel after
subcutaneous injection into mouse in the presence of β-GP. Additionally, we evaluated the
biodegradation of chitosan hydrogel in mice by measuring the volume of injected chitosan
hydrogel after subcutaneous injection. The injected chitosan hydrogel in mice was sected and
stained with hematoxylin-eosin reagent for histological observation to confirm biodegradation
of the hydrogel by the infiltrated cells. Addition of chitosan to other polymers as blend might
improve mechanical property, permeability and biodegradability. This has been shown to be
the case PHB/chitosan films [200]. Ohkawa et al. showed that seven species of soil
filamentous fungi such as Aspergillus oryzae, Penicillium caseicolum, P. citrinum, Mucor sp.,
Rhizopus sp., Curvularia sp., and Cladosporium sp. grew on two kinds of polyion complex
(PIC) fibers, chitosan-gellan (CGF), and poly(L-lysine)-gellan (LGF) fibers [201].
Microscopic observation of the biodegradation processes revealed that P. caseicolum on the
CGF and LGF grew, along with the accompanying collapse of the fiber matrices. In the
biochemical oxygen-demand (BOD) test, the biodegradation of the LGF by P. caseicolum and
Curvularia sp. exceeded 97% carbon dioxide generation and the biodegradation of the CGF
by A. oryzae was 59%.
Nanotubes fabricated through the layer-by-layer assembly technique of alternate
adsorption of alginate and chitosan exhibited good biodegradability and low cytotoxicity
[202]. Kuo et al showed that the extent of biodegradability of chitosan was strongly affected
by the addition percentage of chitosan in blend consisting of nylon 11 and chitosan [203].
Zhuang et al. showed that gelatin/montmorillonite- chitosan intercalated nanoco-mposite in
vitro degradation tests had a lower degradation rate than gelatin-chitosan composite [204].
Effect of Size and Shape, Cross Linking and Processing

Conditions on Biodegradability
When chitosan microspheres were used for drug administration, Yoshino et al. report a
gradual degradation of chitosan indicating the effect of size and shape on biodegradability
[205]. In another study, Wang et al. showed that degradation occurred during the 8th to the
12th week and degraded completely within 24 weeks when a novel surgery mesh made of
polydioxanone (PDO) with collagen and chitosan coating was used. They concluded that
biodegradable polydioxanone mesh has an excellent biocompatibility, a friendly tissue-
material interface and a proper degradation rate matching to the tissue regeneration rate [206].
Kofuji et al prepared chitosan (CS) gel beads in 10% amino acid solution (pH 9) and
implanted into air pouches (AP) prepared subcutaneously on the dorsal surface of mice [207].
No inflammatory response was observed, and degradation of the beads in the AP increased as
their degree of deacetylation decreased. Degradation could be altered by changing the nature
of the CS or by increasing the CS concentration. They concluded that degradation of CS gel
beads can be controlled by changing the structure of the gel matrix, which appears to make
these beads a promising biodegradable vehicle for sustained delivery. Zhang et al. found that
the degradation rate of dry-wet-spun poly(l-lactic acid) fibers was higher than those of the
melt-spun or dry-spun ones in a study on degradation and biocompatibility of poly(l-lactic
acid)/chitosan fiber composites fabrics [208]. In an histomorphometric analysis with chitosan-
tripolyphosphate beads, Durkut et al. showed that beads made from of chitosan alone
degraded faster than those modified either by coating with sodium alginate or by cross-
linking with glutaraldehyde, the cross linked beads showing the least biodegradability [209].
In a study on a novel injectable, chitosan based implant; Mi et al. demonstrated that genipin-
crosslinked chitosan microspheres have a superior biocompatibility and a slower degradation
rate than the glutaraldehyde-crosslinked chitosan microspheres [210]. Accordingly, the
genipin-crosslinked chitosan microspheres may be a suitable polymeric carrier for long-acting
injectable drug delivery. In another study of glutaraldehyde cross-linked chitosan
microspheres as a long acting biodegradable drug delivery vehicle, Jameela and Jayakrishnan
did not notice any significant biodegradation of the material over a period of 3 months in the
skeletal muscle of rats [211]. Makarios-Laham et al. studied the biodegradation of
polyethylene-chitin (PE-chitin) and polyethylene-chitosan (PE-chitosan) films, containing
10% by weight chitin or chitosan, by pure microbial cultures and in a soil environment [212].
Three soil-inhabited organsims, Serratia marcescens, Pseudomonas aeruginosa, and
Beauveria bassiana were able to utilize chitin and chitosan after eight weeks of incubation at
25°C in a basal medium containing no source of carbon or nitrogen. In soil placed in the lab,
73.4% of the chitosan and 84.7% of the chitin in the films were degraded, while 46.5% of the
starch in a commercial film was degraded after six months of incubation. In an open field,
100% of the chitin and 100% of the chitosan in the films were degraded, but only 85% of the
starch in the commercial film was degraded after six months of incubation. The weight of
controls, (polyethylene films), remained mainly stable during the incubation period. A low
toxicity of chitin was demonstrated to be mostly due to biodegradability and the fast
metabolization of hydrolysate in animal body [213]. Table 4 provides the key specifications
of chitin and chitosan.
Table 4. Key specifications for chitins and chitosans
Property Chitin Chitosan

Mol. Wt daltons 105-106 105-106
Deacetylation % ~10 60-90
Viscosity of 1% solution in acetic acid , cps 200-2000
Moisture content % <10 <10
Dissociation constant, Ka 6.0-7.0
A number of characteristics make chitosan important. They are: very strong antibacterial
effect, total biodegradability, biocompatibility (anallergicity), high humidity absorption,
solubility in acids etc. The molecular weights of chitin are usually larger than 1 million
Daltons, while chitosan products fall between 100.000 and 1.2 million Daltons. The viscosity
of chitosan tends to increase with decreasing pH in acetic acid but decrease with decreasing
pH in HCl. The bulk density of chitin differs from 0.06 g/cm³ to 0.39 g/cm³ according to the
source. Water binding capacity of chitosan is ranged from 5.81% to 11.50%.
There have been a number of earlier attempts at reviewing the area on chitin and CS
fibres covering certain aspects of their importance, properties and applications [214-222].
Rathke and Hudson [214] pointed out that chitin's microfibrillar structure indicated its
potential as fiber- and film-former, but as chitin was found to be insoluble in common organic
solvents, the N-deacetylated derivative of chitin, CS, was developed. After Rinaudo et al.
[222] who described the production of chitin and CS fibers by wet spinning method in 2001
and Rajendran and Anand [221] who discussed briefly the properties of chitin and chitin
fibres in 2002, there have been no serious attempts at reviewing the production, properties
and applications of chitin and CS fibres. Considering the potential applications of chitin and
CS fibres, it appears that a consolidation of the data relating the chemistry, solubility and
fibre formation of chitin and CS polymers is required. Chitin fibres stand apart from all the
other biodegradable natural fibres in many inherent properties such as biocompatibility, non-
toxicity, biodegradability, low immunogenicity, non-toxicity etc. [214, 223-225]. These
properties in combination with good mechanical properties make them good candidate
materials for sutures that form the largest groups of material implants used in human body
[214, 226-228]. It was reported that the chitin suture was absorbed in about four months in rat
muscles [229]. Application in 132 patients proved satisfactory in terms of tissue reaction and
good healing indicating satisfactory biocompatibility. Toxicity tests, including acute toxicity,
pyrogenicity, and mutagenicity were negative in all respects. The persistence of the tensile
strength of the chitin was better than DexonTM or catgut in bile, urine and pancreatic juice but
weakening occurred early in the presence of gastric juice [229]. Apart from sutures, chitin and
CS fibers have been found to be useful in other medical textiles [230-235], wound dressing
[236-244] and haemostatic materials [245-252] and several other prosthetic devices such as
haemostatic clips, vascular and joint prostheses, mesh and knit abdominal thoracic wall
replacements and as antimicrobial agents [253-256].
Chitosan carry a strong positive charge that allows them to chemically bond with certain
compounds. This binding action had drawn the interest of numerous industries, ranging from
food and cosmetics (chitinosans can stabilize colors and retain moisture) to wastewater
treatment (chitinosans can be spread on water to absorb grease and other potentially toxic
substances). These claimed fat-fighting properties have made chitinosan products wildly
popular as weight-loss agents in Japan, where annual sales are estimated to approach $1
billion. Sales have also boomed in recent years in the U.S., spurred in part by aggressive
advertising on the part of a few companies. For example, television infomercials featuring
former Los Angeles Dodger baseball player Steve Garvey extol the wonders of the chitinosan
product Fat Trapper. Testimonials abound about how one chitinosan product or another
helped the user lose weight while not having to make any changes to his or her diet or
lifestyle.
Chitosan has a history of about three decades of use in processes like detoxifying water.
When is spread over the surface of water, it literally absorbs greases, oils, heavy metals and
other potentially toxic substances. Like a "fat magnet," it attracts these bio-hazardous
substances from drinking water to such an extent that a scum forms in the water, which can be
easily removed. Water purification plants throughout the nation use Chitosan for this purpose.
What this indicates to scientists was that Chitosan can selectively absorb fats even in a water
medium.
Chitin is a structural biopolymer which has a role in nature analogous to that of collagen
in the higher animals and cellulose in terrestrial plants. Plants produce cellulose in their cell
walls and insects and crustaceans produce chitin in their shells. Cellulose and chitin are, thus,
two important and structurally related polysaccharides that provide structural integrity and
protection to plants and animals respectively [257]. As they are seen in many organisms
widely spread throughout the biosphere, they are two of the most abundant biopolymers on
earth. Crustacean shells contain 30% Chitin, 50% of inorganic salts like calcium and 20% of
protein. The isolation includes two steps: demineralization with HCl and deproteination with
aqueous NaOH. Lipids and pigments may also be extracted. These operations may vary with
the differently mineralized shells, seasons, and presence of different crustaceans in the catch.
Chitin isolates differ from each other in many respects, including degree of acetylation,
typically close to 0.90; elemental analysis, with nitrogen content typically close to 7%; N/C
ratio, 0.146 for fully acetylated chitin; molecular size; and polydispersity. The resulting chitin
is deacetylated in 40% sodium hydroxide at 120 °C for 1–3 h. This treatment produces 70%
deacetylated chitosan.
It is now well established that the difficulty in solubilisation of chitin results mainly from
the highly extended hydrogen bonded semi crystalline structure of chitin [258-261]. Chitin is
a structural biopolymer, which has a role analogous to that of collagen in the higher animals
and cellulose in terrestrial plants [259-261]. Plants produce cellulose in their cell walls and
insects and crustaceans produce chitin in their shells. Cellulose and chitin are, thus, two
important and structurally related polysaccharides that provide structural integrity and
protection to plants and animals respectively [262,263]. Chitin occurs in nature as ordered
crystalline micro fibrils forming structural components in the exoskeleton of arthropods or in
the cell walls of fungi and yeast [264,265]. In crustaceans, Chitin is found to occur as fibrous
material embedded in a six stranded protein helix. Chitin may be regarded as cellulose with
hydroxyl at position C-2 replaced by an acetamido group [266]. Both are polymers of
monosaccharide made up of β-(1-4)-2-acetamido-2-deoxy- β –D-glucose and β-(1-4)-2-
deoxy- β –D-glucopyranose units respectively (Figure 3). Thus, chitin is poly (β-(1-4)-N-
acetyl-D-glucosamine) [267] (Figure 4).
Figure 3. Structure of glucosamine (Monomer of chitosan) and glucose (Monomer of cellulose)

Figure 4. Structure of chitin and chitosan (Reproduced from reference [51] by permission of Elsevier
Science, Amsterdam).
In fact, as in the case of cellulose, chitin exists in three different polymorphic forms (α, β
and γ) [268-271]. Recent studies have reported that the γ form is a variant of α family [272].
The polymorphic forms of chitin differ in the packing and polarities of adjacent chains in
successive sheets; in the β -form, all chains are aligned in a parallel manner, which is not the
case in α-chitin. The molecular order of chitin depends on the physiological role and tissue
characteristics. The grasping spines of Sagitta are made of pure α -chitin, because they should
be suitably hard to hold a prey, while the centric diatom Thalassiosira contains pure β-chitin.
A simple treatment with 20% NaOH followed by washing with water is reported to convert α-
chitin β chitin [273,274]. In both structures, the chitin chains are organized in sheets where
they are tightly held by a number of intra-sheet hydrogen bonds with the α and β chains
packed antiparallel arrangements [275-281]. This tight network, dominated by the rather
strong C–O--NH hydrogen bonds (Figure 5), maintains the chains at a distance of about
0.47nm [276]. Such a feature is not found in the structure of β-chitin, which is therefore more
susceptible than α-chitin to intra-crystalline swelling [277, 280]. The current model for the
crystalline structure of α -chitin indicates that the inter-sheet hydrogen bonds are distributed
in two sets with half occupancy in each set [276]. These aspects make evident the insolubility
and intractability of chitin.
Figure 5. Molecular structure and hydrogen bonding in (a) α–chitin and (b) β-chitin (Reproduced from
[51] by permission of Elsevier Science, Amsterdam).
In chitin, the degree of acetylation is typically 0.90 indicating the presence of some
amino groups (As some amount of deacetylation might take place during extraction, chitin
may also contain a small portion of amino groups (5-15%) [282,283]). So, the degree of N-
acetylation i.e. the ratio of 2-acetamido-2-deoxy-D-glucopyranose to 2-amino-2-deoxy-D-
glucopyranose structural units has a striking effect on chitin solubility and solution properties
[283]. Chitosan is the N-deacetylated derivative of chitin with a typical degree of acetylation
of less than 0.35. It is, thus, a copolymer composed of glucosamine and N-acetylglucosamine.
The physical properties of chitosan depend on a number of parameters such as the molecular
weight (from approximately 10,000 to 1 million Dalton), degree of deacetylation (in the range
of 50-95%), sequence of the amino and the acetamido groups and the purity of the product
[284-287]. The crustacean shells (crabs etc.) which are waste products (now byproducts) of
food industry are commercially employed for production of chitin and chitosan. It is believed
that at least 1011 tons (1013 Kgs) of chitin are synthesized and degraded, but only over
1,50,000 tons of chitin is made available for commercial use [288].
Chemical Modifications
Chitin and chitosan are interesting polysaccharides because of the presence of the amino
functionality, which could be suitably modified to impart desired properties and for providing
distinctive biological functions including solubility [259,260,281,282,289-292]. Apart from
the amino groups, they have two hydroxyl functionalities for effecting appropriate chemical
modifications to enhance solubility. The possible reaction sites for chitin and chitosan are
illustrated in Figure 6.
Figure 6. Illustration of the possible reaction sites in chitin and chitosan.
As with cellulose [293], chitin and chitosan can undergo many of the reactions such as
etherification [293,294], esterification [292-295], cross linking [287,293], graft
copolymerization [297, 298] etc. [288, 292-299]. Muzzarelli [261], Hon [294] and Zhang and
Ren [295] have summarized the possible chemical modification reactions. A number of
authors have reviewed the area emphasizing various aspects of chemical modification of
chitosan [7-9,11-13,292,297-308]. The amino functionality gives rise to chemical reactions
such as acetylation, quaternization, reactions with aldehydes and ketones (to give Schiff's
base) alkylation, phthaloylation, grafting, chelation of metals etc to provide a variety of
products with properties such as such as anti bacterial, anti fungal, anti viral, anti acid, anti
ulcer, non toxic, non allergenic, total biocompatibility and biodegradability etc. The hydroxyl
functional groups also give various reactions such as o-acetylation, H-bonding with polar
atoms, grafting etc. [6,11,14,15,260,297,298,301,303,304]. As chemical modification of
chitin is generally difficult owing to the lack of solubility and intractability [6,260,261],
attention has been given to chitosan with regard to developing advanced properties and
functions [11-13,18,292,309]. Grafting on to chitosan has been adopted a very useful
methodology to impart specific properties and fumctions. Recent trends are to design the
macromolecule to meet certain functions such as receptor-mediated gene delivery [308],
adhesion promoter, cell penetration enhancer, site specific tracking [310,311], enhancing
biological activity [312], etc. to cite a few examples. Specific examples of modifications
effected on chitin and chitosdan to enhance solubilty will be discussed under section on
enhanced solubility by chemical modification.
CRITERIA FOR POLYMER SOLUBILITY

It is well known that polymer solubility depends on polarity, molecular weight,
branching, degree of cross-linking and crystallinity [310,311].
Chitin having a semi crystalline structure with extensive hydrogen bonding, the cohesive
energy density and hence the solubility parameter will be very high and so will be insoluble in
all the usual solvents [8,260,266,286,316,317]. The solubility parameter of chitin and
chitosan was determined by group contribution methods (GCM) and the values were
compared with the values determined from maximum intrinsic viscosity, surface tension, the
Flory-Huggins interaction parameter and dielectric constant values [318]. The values, thus,
1556 C. K. S.
ma
obbtained were confirmed by b values obttained from GCM. The solubility s paraameters of
chhitosan determined by thhese methodss are more or less equaal and the average a is
appproximately 41 J1/2/cm3/2 [318].
[ Using these
t and sim
milar approaches, the overalll solubility
paarameter of chitosan
c havinng any degreee of deacetylaation can be estimated andd the shear
prroperties evalluated [286,3118,319]. The solubility of chitin can bee enhanced byy treatment
w strong aqu
with ueous HCl whereby a solid-state transform mation of β-chhitin into α-chitin occurs.
β--chitin is repoorted to be moore reactive thhan the α-form m, an importaant property inn regard to
ennzymatic and chemical traansformations of chitin [8,3320-322]. Aibba demonstareed that the
diistribution off acetyl grouups influenced the solutioon properties and showedd that the
diistribution of acetyl groupss must be ranndom to achievve the higher water solubillity around
500% acetylation n [323].
The structu ural similarityy of chitin too cellulose has induced many m authors to try the
soolvents used for
f cellulose [3324-326]. As in the case off cellulose, the existence off both intra
annd intermoleccular hydrogenn bonds for chitinc in the solid
s state strrongly resist dissolution.
d
[3327-329]. But, many of theese solvents arre toxic, corroosive or degraadative or muttagenic and
heence cannot be b used in medicinal appliication and allso have diffiiculties in takking up for
inndustrial prodduction. For each e solvent system polyymer concentrration, pH, counter c ion
cooncentration, temperature effects,
e degreee of acetylatioon and moleccular weights are known
innfluence the dissolution
d prrocess and soolution viscossity. The disssolution in manym cased
innvolves severaal days of penetration, swelling prior to going
g into soluution. In manyy cases, the
soolvents are strong
s acids, fluoroalcohools, chloroalccohols and certain c hydrotropic salt
soolutions, which degrade the chitin or are inconvenient
i t use [8,18,3330-332].
to
The first systematic
s stuudy on the solubility of chhitin and chitoosan was carrried out by
A
Austin who intrroduced the soolubility param meters for chiitin in various solvents [3333,334]. The
chhoice of solveent in a particuular situation involves manny more factorrs, including evaporation
e
raate, solution viiscosity, or ennvironmental and
a health conncerns, and oftten the effectivveness of a
soolvent depend ds on its abiility to adequuately dissolvve one material while leaaving other
m
materials unafffected [335,336].
C
Chitin and Chitosan
C Sollubility
While chitin is insoluble in most orgganic solventss, chitosan is readily solublle in dilute
accidic solutionss below pH 6.0. This is because chitosan can be considdered a strongg base as its
prrimary amino o groups (pKaa is 6.3). Thee presence off the amino groupsg indicattes that pH
suubstantially alters the charged state and properties
p of chitosan [12]. At
A low pH, thhese amines
geet protonated and becomee positively chargedc and that
t makes chitosan
c a waater-soluble
caationic polyelectrolyte. On the other hannd, as the pH increases aboove 6, chitosaan’s amines
beecome deproto onated and thhe polymer losses its charge and becomess insoluble. Thhe soluble-
innsoluble transiition occurs att its pKa valuee around pH between
b 6 and 6.5. As the pK Ka value is
hiighly dependeent on the deggree of N-acetylation, the solubility
s of chitosan
c is deppendent on
thhe degree of deeacetylation and the methodd of deacetylattion used [3377].
It can easilly form quaternary nitrogenn salts at low pH values. So,S organic aciids such as
accetic, formic, and lactic acidds can dissolvve chitosan [3338-340]. The best solvent for
f chitosan
w found to be formic acid,, where solutioons are obtainned in aqueouss systems-conntaining 0.2
was
too 100% of forrmic acid [3411]. The most commonly
c useed is 1% acettic acid solutioon at about
Chittosan: Manufaacture, Properrties and Uses 157
pH H 4.0 as a refference. Chitosan is also solluble in 1% hydrochloric

h accid and dilutee nitric acid
buut insoluble in n sulfuric and phosphoric acids.
a But concentrated acettic acid solutioons at high
teemperature can n cause depollymerization of o chitosan [342]. Solubilizzation of chitoosan with a
loow DA occurss for an averagge degree of ionization α off chitosan arouund 0.5; in HC Cl, when α
=0:5, it corresp ponds to a pH of 4.5–5. It is reported that at higher pH, precipitation or gelation
teends to occur and the chitosan solution tends t to form gels with aniionic hydrocollloids [14].
The concentration of the acid plays a greaat importance to impart dessired functionaality [343].
Soolubility also depends on the t ionic conccentration andd a salting-outt effect was observed
o in
exxcess of HCl (1M HCl), making m it posssible to preparre the chlorhyydrate form of o chitosan.
W
When the chlorrhydrate and acetate
a forms of chitosan arre isolated, theey are directlyy soluble in
w
water giving ann acidic soluttion with pKoo = 6 ± 0.1 [3339] in agreem ment with preevious data
[3344] and correesponding to the t extrapolation of pK forr a degree of α = 0. Thus, chitosan,c as
sttated above, iss soluble at pH H below 6. It is known that the t amount off acid needed depends
d on
thhe quantity of chitosan to bee dissolved [338]. The conccentration of protonsp neededd is at least
eqqual to the co oncentration of o -NH2 unitss involved. The T solubility is thus a verry difficult
paarameter to co ontrol as it invvolves a compplex array of controlling
c facctors [8]. Chittosan is not
sooluble in any y organic solvvents such ass dimethylforrmamide and dimethyl sullfoxide. Its
soolubility in accidified polyool is substanttially good. ThereT are seveeral critical factors
f that
coontribute to ch hitosan solubiility. They maay include facctors such as temperature and a time of
deeacetylation, alkali
a concenttration, prior treatments
t appplied to chitin isolation, ratiio of chitin
too alkali solutioon, particle sizze etc. The solution propertties of chitosaan depend not only on its
avverage DA bu ut also on the distribution
d off the acetyl grroups along thhe main chain in addition
off the moleculaar weight [3222,345-349]. Appart from the degree d of deaccetylation, thee molecular
w
weight is also an
a important parameter
p thatt controls signnificantly the solubility
s [3500-358]. The
accid-soluble ch hitosans with >95%
> solubiliity in 1% acettic acid at a 0.5% concentraation could
bee obtained by treatment of the original chhitin with 45--50% NaOH for f 10-30 min.[359-361].
Itt is reported th hat [362] a reeaction time of o 5 min withh 45% NaOH may not be enough e for
chhitin particless to be sufficciently swolleen. This is crritical as furthher reductionn in NaOH
cooncentration in ncreased time for dissolutioon >30 min. [3363]. Further, the microstruccture of the
poolymer is said d to have a rolle in the dissolution [322]. It I is also reporrted that the crystallinity
c
inndex decreases on treating chtin with HC Cl, NaOH etcc. [364]. Glyccerol 2-phosphhate is also
reeported to aid in the preparaation of waterr soluble chitosan at neutral pH for use ass injectable
biiomaterial [36 65-367].
Chitosan becomes
b solubble with the entire
e pH rangge with increaasing substituution of the
ammino groups by b carboxylicc groups, which became neegatively charrged above pH H 6.0. The
soolubility of th he partially deacetylated
d c
chitins has a close relationnship with thheir crystal
sttructure, crysttallinity, and crystal imperrfection as well w as the gllucosamine coontent. For
exxample, chitin n with ca. 28% % DD is repoorted to retain the crystal sttructure of -chitin with
siignificantly reduced crystalllinity [330,3677,368]. As thee DD increasess to ca. 49%, chitin c has a
neew crystal sttructure similaar to that off β-chitin rathher than eitheer -chitin orr chitosan,
suuggesting thatt the homogeeneous deacetyylation transfformed the crrystal structure of chitin
frrom the to th he β-form [3588] and it is waater soluble.
Chitin Solubility: Dissolution by Inorganic Chemicals
There were several attempts at dissolution of chitin using inorganic bases such as sodium
hydroxide and inorganic salts. Kunike kept chitin in 5% caustic soda at 60 °C for 14 days or
in autoclave for 3 hrs at 180°C and 10 atm pressure and there was of course deacetylation
[369] and the product was soluble in acetic acid. von Weimarn prepared for the first time in
1926 solutions of chitin in inorganic salts capable of strong hydration such as LiCNS,
Ca(CNS), CaI, CaBr, CaCl, etc. [370]. Clark and Smith dissolved chitin in presaturated
solution of lithium thiocyante at 95 °C, but it was difficult to remove the solvent even at
200°C [371], but the films produced were readily dispersed in water. Threads extruded from
lithium thiocyanate with tension applied during their formation were said to develop
orientation, but an x-ray pattern of a chitin sheet supported on a glass plate reprecipitated
from lithium thiocyanate solution, showed only the broad diffuse nodes of a strained,
noncrystalline material.
Varum and coworkers who studied the solution properties of α-chitin dissolved in 2.77 M
NaOH [359] obtained second virial coefficients in the range 1 to 2 × 10-3 mL-mol-g-2
indicating that 2.77 M NaOH is a good solvent to chitin. The Mark - Houwink - Sakurada
equation and the relationship between the z-average radius of gyration (Rg) and the weight-
average molecular weight (Mw) were determined to be [η] = 0.10Mw0.68 (mL-g-1) and Rg =
0.17Mw0.46 (nm), respectively, suggesting a random-coil structure for the chitin molecules in
alkali conditions. These random-coil structures have Kuhn lengths in the range 23-26 nm
[359]. Danilov and Plisko tried repeated freezing and thawing in alkali solution for several
attempts and thought that chitin structure becomes friable [372]. Kennedy and coworkers
showed that addition of urea enhances solubility of chitin with 8 wt% NaOH and 4 wt% urea
concentrations at -20 °C [373]. In addition, the rheological properties suggested that chitin
aqueous solution in high concentration is a pseudoplastic fluid and that chitin aqueous
solution in low concentrations is a Newtonian fluid [373]. Vincendon noted a decrease in the
viscosity and molecular weight with time with no change in the degree of acetylation on
dissolving chitn in concentrated phosphoric acid at room temperature and reported the NMR
spectra of chitin dissolved in a fresh saturated solution of lithium thiocyanate [374,375].
Chitin Dissolution by Strong Acids and Polar Solvents
Strong polar protic solvents such as trichloro acetic acid, dichloroacetic acid etc. have
been found to dissolve chitin. In 1975, Brine and Austin dissolved chitin in trichloro acetic
acid as a solvent [376,377] after pulverization with two parts by weight of chitin added to 87
parts by weight of a solvent mixture containing 40% TCA, 40 % chloral hydrate and 20%
methylene chloride. Kifune and co-workers tried dissolving chitin in TCA containing
chlorinated hydrocarbons such as chloromethane, dichloromethane, and 1,1,2-trichloroethane
[378,379]. A number of similar patents have also been reported wherein a mixture of water
and dichloroacetic acid [380] and mixtures of TCA/dichloromethane or TCA/ chloral hydrate
/ dichloroethane solvent system [381-383] have been used. Tokura and co-workers used a
combination of formic acid (FA), DCA and diisopropyl ether as a solvent system [384]. But,
TCA and DCA are corrosive and degrade the polymer lowering the molecular weight to such
levels where the strength of the fibres will get affected. Although dry tenacities of above 3 g/d
were obtained, the low wet tenacities were still undesirable. In addition, chlorohydrocarbons
are solvents that are increasingly becoming environmentally unacceptable. Austin and Brine
[385] describe high tensile strength chitin fibers are obtained when chitin dope prepared by
dissolving chitin in a trichloroacetic acid-containing solution followed by wet spinning and
cold stretching. The chitin fibers obtained, however, are very thick. Filaments having a tensile
strength of 63 kg/mm2 were obtained. This value is correspondent to 5 g/d when calculated
assuming that the density is 1.4. Although it is apparent that high tensile strength chitin fibers
can be obtained, the diameter thereof is 0.25 mm. When calculated with the density as 1.4, it
corresponds to 618 denier. When chitin was dissolved in dichloroacetic acid to prepare a
chitin dope solution, the fibres obatined after wet-spinned and stretching gave only low
tensile strength [168]. It is described that 3.0 to 3.5 denier of chitin fibers were obtained, but
that the tensile strength was 1.2 to 1.5 g/d (a knot tensile strength of 0.6 to 0.7 g/d).
Solubilisation of chitin has also been reported using higly polar solvents such as as
methane sulphonic acid, hexafluoroisopropyl alcohol and hexafluoracetone sesquihydrate
[387-389]. Capozza used hexafluoroisopropyl alcohol or hexafluoroacetone sesquihydride as
solvents for chitin and the resulting solution could be wet spun or dry spun into fiber,
filaments, or cast into films or solid articles, which may be used as absorbable surgical
sutures, or other absorbable surgical elements. As chitin is enzymatically degradable in living
tissue, and is resistant to hydrolytic degradation, surgical elements prepared from this
polymer have good storage characteristics under a wide variety of conditions. It should,
however, be noted that these solvents are toxic.
SOLUBILITY AND MOLECULAR WEIGHT

The selection of the solvent is also important when molecular weight has to be calculated
from intrinsic viscosity using the Mark–Houwink relation (η =KMα where η is the intrinsic
viscosity, M is the molecular weight, K and α are constants.). The values of the parameters K
depend on the nature of the solvent and polymer. For example, one solvent system first
proposed (0.1M AcOH/0.2M NaCl) for molecular weight characterization was shown to
promote aggregation and the values of molecular weights calculated were overestimated
[390,391]. Rinaudo et al. proposed that 0.3M acetic acid/0.2M sodium acetate (pH = 4.5) as a
solvent can be sued to overcome the problem of aggregation as there was no evidence for
aggregation in this mixture [392]. Using acid-soluble chitosans of DA varying from 0.02 to
0.61, they concluded that the stiffness of the chain was nearly independent of the DA and
demonstrated that the various parameters depended only slightly on the DA [392]. In contrast
to this proposition, Kasai et al indicate that a and K are inversely related and that they are
influenced by DA, and pH and ionic strength of the solvent [393]. They studied the intrinsic
viscosity–molecular weight relationship for chitosan in 0.25 M acetic acid/0.25M sodium
acetate. Chitosan samples with a degree of acetylation (DA) between 20 and 26% were
prepared from shrimp-shell chitosan by acid hydrolysis (HCl) and oxidative fragmentation
(NaNO2). Absolute molecular weights were measured by light scattering and membrane
osmometry. Size exclusion chromatography (SEC) was used to determine average molecular
weights (Mn, Mv, and Mw) and polydispersity. The data of K determined by various authors
can be plotted against DA which indicates that there cannot be any relationship between DA
and K value. Kasai has since modified his work [394].
As the values of K and α differ, it is pointed that it would always be better to follow those
values where the authors have used a standard reference for comparing the molecular weights
and a standard method such as light scattering or gel permeation chromatographgy [395,396]
to determine the absolute molecular weights. The relatively high values for the parameter α
are in agreement with the semirigid character of chitosan. The charged nature of chitosan in
acid solvents and chitosan’s propensity to form aggregation complexes require care when
applying these constants [334]. The weight-average molecular weight of chitin is 1.03 x 106 to
2.5 x 106, but the N-deacetylation reaction reduces this to 1x105 to 5x105 [286].
THE DIBUTYRYL CHITIN

Another major development for chitin dissolution was the synthesis of alkyl derivatives
of chitin whereby butyryl chitin was found to be soluble in normal solvents as reported by
Szosland [397-400]. Chitin has been known to form microfibrillar arrangements in living
organisms [401]. These fibrils are usually embedded in a protein matrix and have diameters
from 2.5 to 2.8 nm. Crustacean cuticles possess chitin microfibrils with diameters as large as
25 nm. The presence of microfibrils suggests that chitin has characteristics, which make it a
good candidate for fibre spinning. To spin chitin or chitosan fibres, the raw polymer must be
suitably redissolved. This was resolved through alkyl chitin route [397, 401-407].
Figure 7. Synthesis of dibutyryl chitin (Reproduced from Ref 222 with permission of Wiley
Interscience).
Dibutyrylchitin (DBCH) was obtained from native krill chitin by its esterification with
butyric anhydride in the presence of perchloric acid [402, 406-409] as shown in figure 7.
DBCH fibres were manufactured from a polymer solution in ethyl alcohol by extrusion
[410,411]. Because a dry-wet formation method was applied, the fibres obtained had a porous
core [412]. Alkaline treatment was adopted to improve upon the properties. The microporous
DBCH fibres were then treated with aqueous KOH solutions [413-417] whose SEM
micrograph is as shown in Figure 8. Structure analysis and degree of substitution of chitin,
chitosan and dibutyrylchitin were studied by FT-IR spectroscopy and solid state13C NMR
[418].
Figure 8. (a) SEM micrograph of the surface of DBCH fibres (x 500), (b) SEM micrograph of the
surface of regenerated fibres (x 500), (c) SEM micrograph of the cross section of DBCH fibres (x 1000)
(Reproduced from Ref. 228 with permission from Fibres and Textiles in Eastern Europe Poland).
The wet spinning of a 14.5% solution in dimethylformamide created dibutyrylchitin

filaments, which were treated with an alkali solution for chitin regeneration. Fiber samples
with different degrees of chitin restoration were obtained. The restoration of the chitin
structure resulted in a gradual increase in the degree of crystallinity, the density of the
structured area, the tensile strength, and the average elongation at rupture and in a decrease in
the diameter of the fibers. The physico-chemical properties were investigated [419-422]. The
crystallinity degree of fully regenerated chitin, the final product of alkaline hydrolysis,
reached a value close to that of native chitin [423-425].
A new method of manufacturing nonwoven products made from dibutyrylchitin, based on
fleece manufacturing directly from the polymer solution using electrospinning (e-spinning)
technology which allows fibres of transverse dimensions of below 0.4 μm to be obtained was
developed [426]. DBCH or regenerated chitin (RC) was used for coating a trade
polypropylene non-woven material. Biological evaluation indicated that DBCH or
regenerated chitin (RC have positive influence on the wound healing process [426-430].
Wawro et al reported the manufacture of fibres from dibutyrylchitin (DBC) using the wet
spinning method using solvents sucha as dimethyl formamide, dimethyl sulphoxide, n-methyl-2-
pyrrolidone and ethyl alcohol [431]. They also studied the effect of polymer content in the
solution and the spinning conditions (temperature of coagulation bath, fibre take-up speed and
stretching ratio) on the mechanical properties of DBC fibres. The conditions of DBC fibre
spinning on a semi-technical scale were defined for the DMSO and RMF spinning dopes. DBC
fibres from DMSO and DW solutions were characterised by a tenacity of 10.5 to 14.6 cN/tex and
elongation at break from 10% to 22%. Batches of DBC fibres (from DMF and DMSO solutions)
were prepared for further processing into nonwovens for medical use.
The WAXS measurements of the krill chitin showed that its supermolecular structure is
ordered and has a high degree of crystallinity [417,419,424]. The butyrylation process leading
to dibutyrylchitin disrupts the supramolecular structure of chitin. The diffraction reflexes in
the ordered area disappear followed by a broadening of the remaining reflexes (Figure 9).
Dibutyrylchitin is, thus, characterized by significantly lower crystallinity degree as well as by
the smaller size of the crystalline regions, which results from a small structural ordering of the
polymer.
Figure 9. WAXS diffraction pattern of DBCH and krill chitin fibres (Reproduced from Ref. 228 with
permission from Fibres and Textiles in Eastern Europe Poland).
It was interesting to note that the alkaline treatment of dibutyrylchitin (5% KOH and at
20 °C - series A, at 50 °C - series B, at 70 °C - series C and at 90 °C - series D) to obtain the
regenerated chitin brings about a reverse chemical process in which the supermolecular
structure of chitin is gradually being regained and thus the configuration of the polymer
macromolecules becomes similar to the crystalline network of the krill chitin [417,419]. The
process as a whole looks to be a case of disruption and reformatuion of the hydrogen bonded
supramolecular structure during butyrylation and debutyrylation respectively. Spectroscopic
examinations carried out using different techniques gave support to these observations. The
characteristic changes of Amide I band of krill chitin, dibutyrylchitin and regenerated chitin
indicated extensive hydrogen bonds between the C=O and the NH for every second C=O
group in chitin [424].
Studies by fluorescent microscopy have revealed a specific skin-core structure of DBCH
fibres, preserved in the whole course of the alkaline treatment [416]. See figure 10 for the
fluorescent microphotographs of the DBCH fibres and before and after alkali treatment. The
fluorescence was intensified by the specific sorption of Rhodamine B used as a dye. As
Rhodamine B reveals no affinity to the examined fibres, it is accumulated in microcapillaries
of the fibres by adhesion. DBCH fibres in the absence of Rhodamine B revealed a specific
greenish fluorescence in UV light when the blue filters are used (Figure 10a) indicating
homogeneity of the fibre surface topography. The fibres are smooth and homogeneous with
no impairments or defects. In the photograph of the cross-section of DBCH fibres (Figure

10b), a clear fluorescence effect of a thin surface layer of a fibre can be seen. The authors
explain this phenomenon as due to the specific supermolecular structure of the fibres formed
using a wet-dry spinning method. The fibres were then subjected to the alkaline treatment
which resulted in obtaining fibres from the regenerated chitin and finally chitosan fibres
(Figure 10c). As a result of the partial N-deacetylation, a distinct skin-core structure can be
observed. The cytotoxicity of the DBCH was evaluated and no agglutination, vacuolization, and
cell membrane lysis was observed [400]. The number of cells separated from the matrix was found
to be the same as in the control cultures.
Figure 10. (a) the surface of DBCH fibres (x 180), (b) the cross-section of DBCH fibres (x 620), (c) the
cross-section of chitosan fibres (DD=84) (x 320), (Reproduced from Ref. 227 with permission from
Institute of Biopolymers and Chemical Fibres, Łódź, Poland)
Dutkiewicz et al. prepared a series of chitinwhich were used to coat glass surfaces and
studied with respect to their wetting properties and thrombogenic characteristics [432]. All
materials had much better wetting properties than siliconized glass surface. The surface
tension of these chitin derivatives was calculated and found to correlate with substrate
thrombogenicity. The clotting time of whole blood depended on the chemical modification of
the polyaminosaccharide structure and was significantly higher than the silicone surface.
Gamma radiation treatment of chitosan led to a decrease in intrinsic viscosity but a small
influence on the clotting time of blood in contact with the polymer samples. Solid coatings of
acetate, butyrate and citrate salts of chitosan exhibited the greatest thromboresistance of all
the investigated derivatives. The kaolin clotting time of plasma shaken in tubes coated with
the salts did not show any leachable substances that affected blood coagulation factors except
for chitosan-poly(acrylic acid) complex.
Włochowicz et al. obtained dibutyrylchitin from krill chitin by esterification with butyric
anhydride in the presence of perchloric acid [433]. The wet spinning of a 14.5% solution in
dimethylformamide created dibutyrylchitin filaments, which were treated with an alkali
solution for chitin regeneration. Fiber samples with different degrees of chitin restoration
were obtained. The restoration of the chitin structure resulted in a gradual increase in the
degree of crystallinity, the density of the structured area, the tensile strength, and the average
elongation at rupture and in a decrease in the diameter of the fibers. The crystallinity degree
of fully regenerated chitin, the final product of alkaline hydrolysis, reached a value close to
that of native chitin [433].
The effect of varying the molecular weight of butyrylchitin membranes cast from
methylene chloride on their properties has been investigated by Urbanczyk et al. [239d]. The
crystal structure of the butyrylchitin was shown by X-ray and scanning calorimetry to consist
of fringed micelles, irrespective of the molecular weights examined, and of lower crystal
volume fraction than usually found for chitin membranes, 0.3 versus 0.5, respectively. The
membranes exhibit greater ductility and less swelling in physiological salt solution with
increasing molecular weight [434].
Chilarski et al. obtained DBCH [435] from shrimp chitin by reaction with with butyric
anhydride, carried out under heterogeneous condition, in which perchloric acid was used as a
catalyst of reaction. DBCH with molar mass of 132 × 103 daltons was used for the
manufacturing of DBCH fibres and DBCH non-woven materials. DBCH non-woven fabrics
after γ-sterilisation were applied to a group of nine patients with different indications.
Satisfactory results of wound healing were achieved in most cases, especially in cases of burn
wounds and postoperative/posttraumatic wounds and various other conditions causing
skin/epidermis loss [435].
Marszalek et al. reported the morphological and physical properties of polymer blends
containing DBCH [436]. Two different synthetic polymers, polystyrene (PS) and poly(vinyl
acetate) (PVAc) were blended with dibutyrylchitin in common solvent (dimethylformamide
or methanol) in various weight fractions of DBCh. The films of the blends were prepared by
casting method. Microscopic studies indicate heterogeneous structures of both blends. DSC
studies show that a characteristic endothermic peak observed for DBCH influenced by water
moisturizing effect is shifting toward lower temperatures with an increasing content of the
synthetic polymers. The values of glass transition temperatures Tg of both synthetic polymers
were found weakly affected by the presence of DBCH. The structure and morphology of
DBCH with poly(ethylene oxide) was also reported [437].Figure 10d shows a scaffold model
from DBCH (maincomponent) and polyhydroxybutyrate.
Muzzarelli et al. demonstrated that DBCH is highly biocompatible for contacting intact
and wounded human tissues, chick and mouse fibroblasts by the viability/cytotoxicity assay,
in situ cell proliferation assay, neutral red retention assay, lactate dehydrogenase release
assay, MTS cytotoxicity assay, and scanning electron microscopy [437]. DBCH was hardly
degradable by lysozyme, amylase, collagenase, pectinase and cellulase over the observation
period of 48 days at room temperature, during which no more than 1.33% by weight of the
DBCH filaments (0.3 mm diameter) was released to the aqueous medium. DBCH non-
wovens were incorporated into 5-methylpyrrolidinone chitosan solution and submitted to
freeze-drying to produce a reinforced wound dressing material. The latter was tested in vivo
in full thickness wounds in rats. The insertion of 4×4 mm pieces did not promote any adverse
effect on the healing process. Muzzarelli et al. further showed that DBCH fibres and their on-
woven exhibited depressed crystallinity, the peak at 0.46-0.47 nm, typical of chitin, being
hardly detectable, while the one usually at ca. 1.00 nm was present at ca. 1.20 nm [438,439].
Both DBC fibres and non-woven were highly oriented. When exposed to porcine pancreatic
lipase or wheat germ lipase, the DBC fibres gained improved crystallinity with peaks at 1.14-
1.18 and 0.41 nm, due to partial regain of chitin structure as a consequence of partial
enzymatic removal of butyryl groups, as confirmed by ATR-FTIR. The RC fibres exhibited
broad XRD peaks at 0.96 and 0.36 nm; sharper peaks at 0.34, 0.46-0.49 and 0.96 were
observed after exposure to lipases, due to removal of a disordered polymer fraction
susceptible to the unspecific enzymatic depolymerization. In fact the RC fibres were found to
have 8% lower degree of acetylation compared to parent chitin, as a consequence of the
alkaline regeneration treatment. They concluded that these modified chitins were scarcely
susceptible to degradation by lipases (besides to lysozyme, as already reported in the
literature); therefore their biochemical significance in wound management seemed limited.

They, however, appear to be the ideal textile materials for providing mechanical support to
freeze-dried chitosan sponges having amply documented activity in wound healing, and for
the preparation of specialty textiles.
WATER SOLUBLE ALKALI CHITIN

Treatment with alkali has been used by many authors to prepare water soluble chitin
(WSC) [13,329,440,441]. Alkali is known to deacetylate and degrade chitin. Both these
processes are expected to improve solubility. Deacetylation reduces crystallinity and
degradation reduces the molecular weight [329]. One gets alkali chitin when reacted with
concentrated NaOH. Alkali chitin is highly reactive and can give rise many water/organo
soluble derivatives [13]. For example, it reacts with 2-chloroethanol to yield O-(2-
hydroxyethyl) chitin, known as glycol chitin. Alkali chitin with sodium monochloroacetate
yields the widely used water-soluble O-carboxymethylchitin sodium salt [441]. It was found
by Guo et al. that regenerated chitin obtained by a concentrated alkali treatment at a low
temperature is water-soluble [442].
In one process, chitin is first dispersed in concentrated NaOH and allowed to stand at
25°C for 3 h or more; the alkali chitin obtained is dissolved in crushed ice around 0°C [363].
The resulting chitin is amorphous and under some conditions, it can be dissolved in water,
while chitosan with a lower degree of acetylation (DA) and ordinary chitin are insoluble.
Sannan et al showed that the regenerated chitin with around 50% of deacetylation isolated at
low temperature from an alkali chitin solution left at 25°C for 48 to 77h has very good
solubility in water at 0°C. The X-ray diffraction diagrams showed that these were amorphous,
although both chitin with lower degree of deacetylation and chitosan had crystallinity. The
improved solubility of chitin with about 50% of deacetylation would be attributed to the
partial deacetylation which probably brought about the destruction of secondary structure and
also the increase of the hydrophilic property on account of the increased number of amino
groups [442,443]. This phenomenon could also be related to the decrease of molecular weight
under alkaline conditions; they confirmed that to get water solubility, the acetyl groups must
be regularly dispersed along the chain to prevent packing of chains resulting from the
disruption of the secondary structure in the strong alkaline medium. The alkali solubility was
used to spun cellulose-chitin-silk fibroin filaments which had 3.9-5.0 deniers for the titer
value (for fibre containing less than 43% silk fibroin), 0.70-0.93 g/denier for the tenacity
value and 20.6-28.6% for the elongation value [443]. A recent study by Liu et al has shown
that hydrogen bonds in chitin were weakened by the alkali treatment and the crystallinity of
chitin decreased significantly when soaked in higher concentration alkali solutions at room
temperature. The molecular weight and DA of chitin decreased significantly at treatment
temperatures higher than 20 °C or treatment times longer than 4 h. Wang et al. prepared WSC
by N-acetylation in mild conditions. The results showed that the surface tension decreased
with increasing WSC concentration in the high dilute solution, the critical aggregation
concentration was about 0.010 g/ml. Introduction of acetyl group favored aggregation
formation. The possible aggregation mechanism of WSC in water was attributed to the
molecules' conformation transition, resulting in change of intra- and inter-molecular
interactions such as hydrogen bonding and hydrophobic interaction. The time of
deacetylation when treated with NaOH was dramatically reduced when the alkali chitin was
heated in an auto clave. When chitin was cross linked with diisocyanatohexane, trimellitic
anhydride or dibromodecane, prior to deacetyaltion with alkali, the product was insoluble.
CHITIN FIBRE FORMATION

Sutures are probably the largest groups of material implants used in human body and the
suture market is very huge with a total tally exceeding $ 1.3 billions annually [444].
Physicians have used sutures for the past at least 4,000 years [445]. Archaeological records
from ancient Egypt and India show use of linen, animal sinew, flax, hair, grass, cotton, silk,
pig bristles, and animal gut to close wounds [445,446]. The famed Susruta is reported to have
used suture materials of bark, tendon, hair and silk as sutures in surgery [447]. Although
chitin fibres could made into textile materials [26,331,448,449], chitin sutures have
remarkable properties over other fibres for biomedical applications [5,11,13,15,26,274,450-
452]. Chitosan-poly(L-glutamic acid) and chitosan-polyacrylic acid fiber are finding potential
industrial applications due to their enhanced tensile strength and environmental
biodegradability [450].One study reports that chitin fibres have comparable properties to
those of collagen and lactide fibres [452]. Chitin sutures resist attack in bile urine and
pancreatic juice, which are problem areas with other absorbable sutures [274]. The polymeric
linear chain structure of chitin is expected to give rise to fibre formation and film forming
ability similar to those of cellulose [18]. Thus, the presence of the micro fibrils of chitin with
diameters from 2.5 to 2.8 nm which are usually embedded in a protein matrix indicates that
chitin can be spun into fibres [453,454]. The polyamide-type structure should be broken up to
enable solubilisation of chitin into a solvent [384]. This requires either melting or dissolution
in appropriate solvents. Melt spinning is ruled out as chitin decomposes prior to melting.
There have been many attempts at dissolution of chitin and spinng of chitin and chitosan into
fibre form.
Hirano et al reported novel chitin-silk fibroin fibres and chitin fibres prepared by an
environmental friendly wet-spinning method [466]. Each aqueous solution of sodium chitin
(N-acetylchitosan) salt and its blends of silk fibroin in aqueous 14% sodium hydroxide was
spun through a viscose-type spinneret into an aqueous 10% sulfuric acid solution saturated
with ammonium sulfate (about 43%), and the corresponding white filament was obtained. The
tenacity and elongation values of the chitin -silk fibroin filament decreased with an increase
of fibroin content up to 33% by weight. A scanning electron microscopy analysis revealed
that both the chitin filament and the chitin silk fibroin (67:33, w/w) filament had vertical
strips with faint scale structures on their surfaces. Some applications of these staple fibres
were also reported. The preparation of chitin threads for use in the fabrication of absorbable
suture materials, dressings, and biodegradable substrates for the growth of human skin cells
fibres has been reported [386,468].
BLENDING WITH OTHER FIBRES/POLYMERS

The incorporation of chitin fibres in synthetic composites and blends is proposed to give
interesting properties [469]. The concept of fibres as composites, where hard and stiff phases
are combined with softer polymeric materials especially the deformation mechanism of chitin
fibres in comparison to other natural and synthetic polymers has been recently discussed by
Young and Eichhorn [470]. In the preparation of blends containing alginate and water-soluble
chitin prepared by spinning their mixture solution through a viscose-type spinneret into a
coagulating bath containing aqueous CaCl2 and ethanol, the strong interaction from the
intermolecular hydrogen bonds and electrostatic forces were used to ensure good miscibility
[467]. Best values for the dry tensile strength and breaking elongation were obtained when
the water-soluble chitin content was 30 wt%. The wet tensile strength and breaking
elongation decreased with the increase of water-soluble chitin content. Additionally, the
introduction of water-soluble chitin in the blend fiber can improve the water-retention
properties of the blend fiber compared to pure alginate fiber. Chitin fibers when treated with
aqueous solution of silver nitrate were found to have good antibacterial activity to
Staphylococcus aureus [467]. Significant improvement in properties have been reported for
blends of chitin/chitosan fibres with various natural fibres/synthetics to get chitin-cellulose,
chitin-silk fibroin, chitin-glycosaminoglycans, chitin-cellulose-silk fibroin, chitosan-
tropocollagen, and chitin-cellulose-silk fibroin, chitin-natural rubber blends [471-473]. Cross-
linking with crosslinking agents such as epichlorohydrin improves properites. [474-476].
Chitin fibers incorporated as reinforcement in poly(lactic acid) polymer showed suitable
mechanical properties and retention for fixing cancerous bone fractures, but likely had
insufficient stiffness for applications such as bone plates for fixing cortical bone fractures
[477].
Fan et al. reported alginated /water-soluble chitin (half-deacetylated chitin, prepared from
chitosan by N-acetylation with acetic anhydride) blend fibers prepared by spinning their
mixture solution through a viscose-type spinneret into a coagulating bath containing aqueous
CoCl2 and ethanol [478]. Structure analysis by IR, XRD, SEM indicated good miscibility
existed between alginate and water-soluble chitin, due to the strong interaction from the
intermolecular hydrogen bonds and electrostatic interactions. Best values for the dry tensile
strength and breaking elongation were obtained when the water-soluble chitin content was 30
wt%. The wet tensile strength and breaking elongation decreased with the increase of water-
soluble chitin content. The introduction of water-soluble chitin in the blend fiber can improve
the water-retention properties of the blend fiber compared to pure alginate fiber. The fibers
treated with aqueous solution of silver nitrate have good antibacterial activity to
Staphylococcus aureus.
Special properties could be built by appropriate chemical chemical modification to
generate a series of chemically modified fibers such as N-acylchitosans, N-arylidene- and N-
alkylidene-chitosans, N-acetylchitosan, chitin-tropocollagen and chitosan-transition metal
complexes [470,479-481]. Hirona et al. prepared novel fibers of N-acylchitosan and N-
acylchitosan-cellulose composite using an aq. 14% NaOH solution of sodium N-acylchitosan
xanthate [O-(sodium thio) thiocarbonyl N-acylchitosan] and of its mixture with sodium
cellulose xanthate [O-(sodium thio) thiocarbonyl cellulose) [478]. The fibers were spun at 45-
50°C through a viscose-type spinneret into a coagulating bath containing aq. 10% H2SO4,
1668 C. K. S.
ma
322% Na2SO4 anda 1.3% ZnS SO4. All the fibers obtaineed were whitte. N-Propionylchitosan-
ceellulose compposite filamennt had better mechanical properties
p thaan did N-acetyylchitosan-
ceellulose composite filamentt. These fiberss were digestedd by chitinasee and lysozyme, in which
thhe hydrolysis rate was conntrolled by thee N-acyl struccture of chitoosan. Figure 111 shows a
scchematic preseentation of a tyypical wet-spiinning producttion line.
Fiigure 11. Schem

matic depiction of a typical wett-spinning prodduction line (Repproduced from Ref. 20
w permission of Wiley IneterrScience).
with
The crystalllinity and surrface charge deensity of the deacetylated

d chhitin can be inncreased on
trreatment with hydrochloric acid treatmennt to improve the t fibre propeerties [257]. Itt should be
nooted that East and Qin empployed heat treeatment for prreparing regennerated chitin by b reaction
(NN-acetylation)) between chhitosan and accetic acid [463]. The besst properties for tensile
sttrength (4g deen-1) and moduulus (100g deen-1) for chitinn were reporteed by the mixxed ester of
chhitin or chitoosan acetate/fformate polym mer. Use of chitin wiskerr route may be of use
prreparing highh strength fibbres [482]. Fuurther improvvement in fibbre propertiess could be
acchieved with the applicatioon of spinninng fibre from lyotropic liquuid crystallinee solutions
[4483].
The properrty of spontanneous orientattion from lyootrpoic liquid crystals was utilized to
drraw fibres froom 2 wt% chiitin/LiCl/DMA Ac that gave best spinnabiility and best quality of
fibre after spin
nning [484]. Both
B thermotroopic and lyotrropic liquid crrystalline behaaviors have
beeen reported on
o chitin/chitoosan based pollymers [485-4487], but theree are no attem mpts at fibre
sppinning. Fibree spinning frrom liquid cry rystalline soluutions has siggnificant advaantages for
inncreased stren
ngth and otherr properties [488]. Irradiation of chitin--fiber-reinforcced poly( -
caaprolactone) composite
c shoowed 45 % im mprovement inn tensile strenngth and tensille modulus
w
with respect to those of the unttreated specimens [489]]. Polymers such as
poolyvinylpyrrollidone, methyyl cellulose, annd sulfite celluulose are reporrted to be usedd to modify
thhe properties of
o chitin fibrees added to thhe spinning solution [490]. Further improovement in
fibre propertiess could be effeected through appropriate cchemical moddifications [4991-493].
APPLICATION IN WOUND HEALING: BIOMEDICAL TEXTILES FROM

DBCH FIBRES (CHITOMED)
There is a lack of innovative biomaterials that aid in regeneration of wound tissue.
Chitomed as novel biomaterials and medical items that accelerate wound healing with no scar
and undesirable effects, that are easy to handle and could be prepared as self-adhering
dressings was developed by Profs. Gustaaf Schoukens and Paul Kiekens at The Department
of Textiles are coordinator of this project which is financed by the European Commission.
Chitin from Marseille (France) is appreciated for the delivery of shrimp’s chitin, the Institute
of Dyes and Organic Products from Zgierz (Poland) for the preparation of DBC from
shrimp’s chitin and the Institute of Chemical Fibres from Łódź (Poland) for the spinning of
DBC fibres.
BIODEGRADABILITY OF CHITIN FIBRES

Chitin is considered to be highly biodegradable and easily excreted in urine, and further it
is suggested to have no problem on accumulation in the body as shown by Onishi et al in a
study on the biodegradability, body distribution and urinary excretion of 50% deacetylated
chitin after the intraperitoneal (ip) administration to mice using fluorescein isothiocyanate
labeling [494]. When attacked by natural fungi, chitosan films have a built-in source of
nitrogen to enhance biodegradation. Surprisingly, information on the in vivo biodegradability
of chitosans with differing chemistries and structures, and which are utilized in multiple
applications, is lacking. It is generally believed that lysozyme is mainly responsible for
chitosan degradation in the human body. Lysozyme is present in many tissues and secretions
such as tears, saliva, blood and milk, and is released and utilized by phagocytic cells during
the inflammatory response to a foreign implant [495-498].
Water-soluble succinyl chitin and chitosan find application for treatment of arthritis
[499]. The biocompatibility and safety of chitosan have been revealed through tests involving
mutagenicity, acute and sub acute toxicity, pyrogens, hemolysis, and sensitization [500]. The
US Food and Drug Administration consider chitosan as a food additive in animal feed when
used as a precipitating agent for proteineceous materials [501]. Seo has shown that chitosan
when orally administrated to rabbits, broilers and hens at a dosage of 0.7 -0.8 g/kg body
weight /day for upto 239 days, no abnormal symptoms were observed [500]. Rabbits digested
upto 28-38% chitin and 38-79% chitosan while broilers and hens digested them completely.
Rabbits also did not exhibit any abnormal symptom when chitosan was intravenously
injected. It was also observed that the presence of chitosan enhanced the absorption of drugs
when administrated orally [502-508].
Blasinska and Drobnik studied the effects of nonwoven mats of DBCH on the process of
wound healing [509]. The results showed that DBCH implanted subcutaneously to the rats
increased weight of the granulation tissue. Increased cell number isolated from the wound and
cultured on the DBCH films was also revealed. The DBCH was proved to reduce also the necrotic
cells number in the culture. DBCH elevates the glycosaminoglycans (GAG) level in the
granulation tissue. The total collagen content in the wound was not influenced by all applied
dressing materials. However, a low level of the poorly polymerized soluble collagen in the
wounds treated with DBCH and butyryl chitin indicated better polymerization of the remaining
part of that protein. Both DBCH and chitosan increased the weight of granulation tissue. However,
chitosan contrary to DBCH lowered GAG content and increased water capacity in the wound. The
study documents the beneficial influence of DBC on the repair, which could be explained by the
modification of the extracellular matrix and cells number [509].
FIBRE FORMATION FROM CHITOSAN

Development of fibres from chitosan was comparatively easy as it was soluble in dilute
acids such as acetic acid. Formation of the fibre was reported as early as 1926 [36]. But
chitosan fibres were found to be expensive due to high production cost [445]. This induced
researchers to look into blends or composites with other existing yarns. Production of fibres
with chemical modification such as grafting has also been reported.
Chitosan fibres having similar strength to viscose fibres can be obtained by treating chitin
with alkali [455]. Shear precipitation is employed by some researchers to the orientation and
crystallinity of the fibres [455]. Structural studies on chitin, chitosan and butyryl chitin have
shown that the three types of filaments differed in their crystalline structure, degree of
crystallinity and average lateral crystallite sizes [510]. Reaceylation have been employed to
generate chitin which could be spun into fibres [24,302,463,511,512]. Chitosan is dissolved in
acetic acid solution and then extruded through the spinneret into a caustic coagulation bath to
obtain a regenerated fibre. However, such fibres have poor wet strength (tenacity 2.0 g/d).
The acetylation process was affected by the reaction temperature, the treatment time, and the
molar ratio of anhydride to amine groups. The fibre properties are affected by spinning conditions,
such as spin-stretch ratio, coagulation bath concentration and drying conditions. Fibre can be
produced with tenacities up to 0.24mN/tex. The acetylated chitosan fibres, or regenerated chitin
fibres, showed good thermal stability and improved dry and wet strengths. It was found that, after
acetylation, the fibers had an improved thermal stability and tensile strength. Investigations have
shown the improved tenacity of up to 4.4 g/d by incorporation of surfactants into the
coagulation bath [513]. Such fibres find use in the production of textiles having antimicrobial,
antithrombogenic, haemostatic, deodorizing, moisture controlling, and non allergenic
properties which are intern used as bandages for wound- dressing, as sutures, as perfume
releasing fabrics [513]. N-Acylation with longer hydrocarbon side chain resulted in a higher
spatial organization of the chain to the long axis and showed lower moisture retention [514]. The
chemical structure of chitosan fibers was gradually altered from hydrated form (anti-parallel
structure) to dehydrated form (parallel structure) with the treatment of carboxylic anhydrides. The
dry and wet tenacity of chitosan were improved by addition of a cross linking agent and
sodium acetate to the coagulation bath [474].
Liu et al reported the effect of hydroxyethylation of chitosan on the fiber properties
[514] chitosan was modified with epoxyethane in the presence of sodium hydroxide to
enhance the capability of water absorbency. This property offers potential application as
wound dressings with enhanced capability to absorb wound exudates. The FTIR and 13C
NMR spectra indicated that hydroxyethyl groups were mainly attached to C-6 positions. The
inter- and intra-hydrogen bonding were broken while the crystallinity remained unchanged,
indicated by FTIR and XRD results. TGA analysis showed that thermal stability decreased as
DS increased. The modified chitosan fibers with increased hydrophilic hydroxyethyl groups
exhibited lower tensile strength and elongation than initial chitosan fibers. Hydroxyethylation
with lower DS enhanced the water absorbency of the fibers.
Improved tenacity of up to 4.4 g/d was obtained by incorporation of surfactants into the
coagulation bath. Such fibres find use in the production of textiles having antimicrobial,
antithrombogenic, hemostatic, deodorizing, moisture controlling, and non-allergenic
properties. A composite material of chitin/chitosan and cellulose produced by mixing powder
chitin/chitosan with viscose pulp and then wet spun showed higher moisture keeping property
than cellulosic fibres and has dyeability towards direct and reactive dyes [461,513].
Knaul et al prepared a highly deacetylated chitosan from shrimp with a degree of
deacetylation of 95 ± 3% was prepared and spun into a monofilament chitosan using a
solution of 5% by weight chitosan in 5% by volume aqueous acetic acid [515]. Samples of the
spun fibers were immersed in separate solutions containing phosphate ions and phthalate ions,
and subsequently washed and dried. The various solutions ranged in pH from 4.12 to 7.75.
The highest dry mechanical properties resulted from solutions containing phthalate ions
between 4.5-5.5 pH, and from solutions containing phosphate ions at pH 5.4. Immersion time
was varied between 1 and 60 min at 25.8°C, and temperature was varied between 25.8 and
70.0°C, in the phosphate ion solutions at a pH of 5.8. Dry mechanical properties were highest
at 25.8°C and after 1 h of treatment. Chitosan films were subjected to similar treatments in
phosphate and phthalate ion solutions. Fourier transform infrared data (FTIR) on the films
suggested that some interaction was occurring between the phosphate ions and the amine
group on the chitosan backbone. An additional experiment was performed whereby the same
chitosan was used to prepare a dope of 4% by weight chitosan in 4% by volume aqueous
acetic acid with 30% by volume methanol. This solution was spun into fibers, but was
subjected to a 'final draw' by increasing the speed of the winder. With increasing the final
draw, denier and elongation-at-break decreased, while the other mechanical properties
showed a marked increase.
These fibres have the property of keeping skin from drying with out giving no irritation
to skin. These clothes are recommended, therefore, babies and old aged people who have
weak and sensitive skin [461,513]. The properties of chitin and chitin fibres have been
recently been reviewd by Rajendran [517]. Apart from their use as sutures, there are several
applications such as antimicrobial wound dressings [518-520] and as reinforcement
in hydroxyapatite bone cement [521]. The use of chitosan use as antimicrobial agents and
textile chemicals has been brought by Lim and Hudson. Synergistic effects were observed by
combining random suture filaments and chitosan in calcium phosphate cement [521]. Li et al.
has used chitosan fibres for reinforcing porous bone scaffolds and the porosity and pore size
of the reinforced scaffolds were both satisfactory [521]. The prospects of chitosan and
chitosan blended with other fibers have been discussed recently by Struszczyk [522-524].
Application studies of chitosan fibres in 3-D fiber mesh scaffolds for tissue engineering
showed that both types of structures (fibers and scaffolds) were found to be non-cytotoxic to
fibroblasts [525]. Figure 12 shows the appearance of osteoblast-like cells proliferating over
chitosan based fibers after seven days of culture. Qin et al describes that the antimicrobial
properties of chitosan can significantly improved by introducing silver into chitosan
[526,527]. Shin et al. have shown that chitosan oligomer imparts antimicrobial finishing to
polypropyleneA long ripening time is shown to decrease mechanical properties [528].
Properties of chitosan fibres and properties of chitosan non-woven fibres are given Tables 5
and 6 [523].
Figure 12. Osteoblast-like cells proliferating over chitosan based fibers after 7 days of culture
(Reproduced from Ref. 419 with permission of Wliey-VCH- Verlag).
Table 5. Some properties of chitosan fibres
Property Specification
A) Mechanical
Titre, dtex 1.5-3.0
Tenacity in standard conditions, cN/tex 10-15
Tenacity in wet conditions, cN/tex 3-7
Loop tenacity, cN/tex 3-7
Elongation in standard conditions, % >10
B) Structural
Av. molecular weight (Mv), kD 150-300
Polydispersity (Pd) 3.6-6
WRV, % ~150
Crystallinity index (CrI), % 35 - 50
Table 6. Some properties of multi-layer non-woven
Parameter Specification
Composition Fibres PP, CS fibres (not more than 15
wt%) in active layer.
Specific weight, g/m2 80
- total 40
- active layer
Tenacity of supporting layer, N/cm 10
Air permeability l/m2s 1200
WRV, % 400
Bacteriostatic activity >0
Urbanczyk studied the fine structural properties such as degree of crystallinity,

dimentions of the lattice unit cell and average lateral crystallite sizes as well as morphological
features viz. appearance of the skin core structure, occurrence of spherulite crystalline
aggregations and shape, area and length of cross-section of chitin, chitosan and butyryl chitin
filaments and showed that that the three types of filaments differed in their crystalline
structure, degree of crystallinity and average lateral crystallite sizes [510]. A polypropylene-
chitosan non-woven prepared according to a wet paper method by Niekraszewicz [530]
showed stimulation of fibroblast division and accelerates wound healing in animal testing.
They can be easily processed into nonwoven structures and also the fibre surface can be
modified by graft copolymerisation of vinyl monomers. The crystallinity and surface charge
density of the deacetylated chitin have been increased after hydrochloric acid treatment. It has
been proved that the degree of acetylation is significantly lowered when acids other than
acetic acid such as formic, propionic and butyric acids are used for derivatisation. The
properties of chitosan-fibroin composite fibres [531] are given in Table 7.
Table 7. Properties of CS-fibroin Composites [531]
Fibre Content of fibroin Tenacity cN/dtex Elongation at break %

CS 0 17.2 9.2
CS-fibroin -1 4 15.9 8.5
CS-fibroin -11 6 15.2 8.1
Stęplewski et al. produced alginate–chitosan fibres by two methods [532]. The first
method consists in fibre spinning by feeding chitosan into a coagulation bath produced
alginate-chitosan fibres with a maximum chitosan content of about 3.1%. The second method
used chitosan in the finishing process producing alginate-chitosan fibres with a chitosan
content of up to 9.2% wt. A maximum tenacity of 22.2 cN/tex and an elongation at break of
19% were obtained for the fibre composite when chitosan content was as high as 11.6%
obtained in the presence of polyvinylpyrrolidione. Qian et al. reports that chitosan and
alginate can be coated uniformly onto the chemical fibers by using their special dissolving
properties to get the hydrophobic surface of chemical fibers to be modified to hydrophilic
[426,427]. The properties of blends of chitosan with various other fibres such as cellulose,
silk fibroin, tropocollagen etc. have been evaluated [472,473]. Figure 13 shows bundles of
commercial chitosan fibre. Figure 14 shows ChittoSan® Fibers and chitin socks.
Figure 13. Bundles of chitosan fibre.

Figure 14. ChitoSan® fibers, chitin socks and chitosan wound dressings
BIODEGRADABILITY OF CHITOSAN FIBRES

When chitosan is proposed for large-scale use as textile and suture materials, it is
important to know its degradation behavior. Several studies have been reported [535]. In a
study on the use of chitin as a new absorbable suture material, Szosland and others concluded
that the chitin fibres fulfill the basic biological requirements set up for the bio-medical
devices [427]. Tachibana et al. carried out a comparative study of four absorbable suture
materials, namely; chitin, polyglycolic acid (PGA), plain catgut and chromic catgut [536].
The straight pull strength of USP 3-0 size chitin was over 2.6 kg, compared with 3.4 kg of
PGA, and 2.0 kg of the catguts [536]. Chitin showed the lowest elongation among the four.
The tensile strength retention (TSR) of Chitin in muscle was 45 per cent at 14 days and 7 per
cent at 25 days, which was similar to that of PGA. The TSR of Chitin was maintained by 35
per cent in gastric juice, 97 per cent in bile and 100 per cent in pancreatic juice after
immersion for 30 days. The corresponding values for PGA were 54 per cent, 0 per cent and 0
per cent, respectively, whereas both catguts had dissolved within 30 days. The tissue reaction
of chitin was similar to that of PGA, whereas the catguts caused more intense tissue reaction
[536]. Chitin is considered an appropriate absorbable suture material because it also possesses
suitable mechanical properties. Nakajima et al. observed good healing which provided
evidence for a satisfactory biocompatibility and could not notice any specific tissue reaction.
Onishi and Machida examined the biodegradability, body distribution and urinary excretion
of randomly 50% deacetylated chitin after the intraperitoneal administration to mice [494].
The in vitro biodegradability studies by incubation with lysozyme and murine plasma and
urine using fluorescein isothiocyanate (FITC) labeled chitosan showed accelerated
degradation of chitosan. Most of labeled chitosan was excreted into urine after 14 h giving
low molecular weight products. Therefore, chitosan is considered to be highly biodegradable
and easily excreted in urine with no problem of accumulation in the body. A study on the
influence of physical parameters such as porosity and fibre diameter on the degradation of
chitosan fibre-mesh scaffolds, as a possible way of tailoring the degradation of such scaffolds
has shown that the scaffolds with higher porosity degrade faster and that, within the same
range of porosity, the fibres with smaller diameter degrades slightly faster. Furthermore, the
morphological differences between the scaffolds did not affect the degree of cell adhesion,
and the cells were observed throughout the thickness of all four types of scaffolds [537]. It is
reported that once its role is over, general lysosomes in the body degrade CS into a common
aminosugar, N-acetyl glucosamine, which is incorporated into the synthetic pathway of
glucoproteins and is subsequently excreted as CO2 [538]. In anotherv study, Four chitosan
samples with different molecular weight Mw and DD were used to investigate the absorption
and distribution in mice after oral administration afterv labelling fluorescein isothiocyanate.
The results indicated that the absorption and distribution of chitosan was significantly
influenced by its Mw and water-solubility [539].
The biological properties, toxicity, skin physiology etc. of chitosan have been reported by
several authors [540-546]. Qin et al. showd that intoroducing silver ions can significantly
enhance the antimicrobial properties of the chitosan fibers [533]. Modification with gelatin
showed that the modified chitosan fibers have an improved mechanical property and
biocompatibility [542]. The lysozyme biodegradation test on collagen/chitosan scaffolds
demonstrated that the presence of chitosan, especially the high-molecular-weight species,
could significantly prolong the biodegradation. In vitro culture of L929 mouse connective
tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote
and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The
results elucidated that the blends of collagen with low-molecular-weight chitosan have a high
potential to be applied as new materials for skin-tissue engineering [543]. Nanofibrous
composite of poly(lactide-co-glycolide) (PLGA) and chitosan/ poly(vinyl alcohol) (PVA)
membranes prepared by simultaneously electrospinning PLGA and chitosan/PVA from two
different syringes showed that the introduction of chitosan/PVA component changed the
hydrophilic/hydrophobic balance and, thus, influenced degradation behavior and mechanical
properties of the composite membranes during degradation [545]. The cells could not only
favorably attach and grow well on the composite membranes, but were also able to migrate
and infiltrate the membranes. Therefore, the results suggest that the composite membranes
can positively mimic the structure of natural extracellular matrices and have the potential for
application as three-dimensional tissue-engineering scaffolds for human embryo skin
fibroblasts (hESFs) culture [545].
1776 C. K. S.
ma
Studies by Gisha and Piillai showed that

t the rate of
o degradationn of chitosan–p –polylactide
grraft copolymeers can be conttrolled by adjuusting the amoount of lactidee content in thhe CL graft
coopolymers, with biodegradation decreasiing with increease in LLA content whichh may find
w applicatio
wide ons in wound dressing
d and in controlled drug
d delivery systems
s [547]. Figure 15
shhows the porous character of
o chitosan scaaffold.
Fiigure 15. Porou

us character of chitosan
c scaffoldd
STRUCTURA
AL MODIFIICATION
Researcherrs are focusingg on the modiffication of struucture of chitin polysaccharrides with a

viiew to enhance the mechaanical and cheemical properrties. Agnihottri et al has shown s that
chhemical modification of chhitosan has im mproved the stability
s of thee polymer [5448]. Chitin
w enhanced tensile strenggth (4g/d) and modulus (1100g/d) was produced
with p from
m chitin or
chhitosan acetatte/formate poolymer. Fibress spun from lyotropic liqquid crystallinne solution
poossess highly oriented chainns both in amoorphous as weell as crystallinne regions andd thus offer
hiigher breaking g strength and modulus [4822].
Knual et all introduced improvements in the dryingg process for wet-spun
w chitosan fibers
ussing a 6% by weight chitossan in 3% by volume aceticc acid solutionn [549]. The fibers f were
coollected as a 20 filament yarn
y intendedd for use as a chaff substraate. The yarnn had to be
suufficiently dryy following sppinning to alllow for windiing and subseequent separation of the
filaments. Dryiing of the yarrn was attemppted using varrious techniquues including direct and
raadiant heat, fo
orced air, and chemical dryiing agents. Prooduct yarns were
w analyzed for ease of
seeparation of thhe filaments, as
a well as com mparison of mechanical
m propperties. Indiviidual fibers
w
were evaluatedd on the basiss of moisturee content, surfface morphology and fiberr diameter.
R
Results indicatte that the parrticular dryingg method or agent
a used haas a consideraable impact
uppon all of th he characteristtics listed aboove. A methaanol dry bathh was found to provide
opptimum dryin ng of the chitosan yarn, prroducing filam ments with low w moisture content that
seeparated easilly from one another.
a Methhanol drying yielded chitoosan fibers wiith smaller
diiameter, superior surface smoothness
s annd superior mechanical
m prroperties to fibers
fi dried
ussing forced aiir, heat, or otther tested dryying agents suuch as acetonne and isopropanol. The
prroperties of chhitin producedd by microwavve-medicated reaction are at a par with thoose derived
frrom conventional chemicaally modifiedd ones [550]]. A blend of o chitosan withw konjac
glucomannan (KGM) fibers showed good antibacterial activity to Staphylococcus aureus. The
structure analysis by FTIR, SEM and XRD indicated that there were strong interaction and good
miscibility between the chitosan and KGM molecule which resulted from strong intermolecular
hydrogen bonds [551]. Coating cellulose with chitosan, it was shown that novel bioactive
cellulosic-chtosan fibres could be developed [552]. Liu et al used chitosan for coating onto
cotton fiber by the oxidation of a cotton thread with potassium periodate at 60 °C in water and
subsequent treatment with a solution of chitosan in aqueous acetic acid A new cotton [553].
Infrared spectra of the CCCF suggested the formation of Schiff's base between the chitosan
and the oxidized cellulose. Kjeldahl nitrogen analysis of the CCCF showed that the maximum
percentage of chitosan introduced into the cotton fiber was 1.58% (w/w). Treatment of the
fiber with 2′,7′-difluoro fluorescein (an amino group-specific probe) followed by fluorescent
microscopic analysis revealed that the modification with chitosan occurred on the surface of
the cotton fiber. Scanning electron microscopy (SEM) photographs showed that the surface of
the treated fiber was slightly changed after the series reaction. However, the mechanical
strength of the cotton thread, which was oxidized by the potassium per iodide solution at a
concentration of less than 2.0 mg/ml, was found to be almost the same as the original cotton
thread. Furthermore, a model experiment for the controlled release of the drug was preformed
using shikonin, a component of a Chinese medicine, suggested potential usefulness of the
chitosan treated fiber as a supporter for the controlled release of drugs. The post chemical
modification of chitosan fiber gives rise to a series of chemically modified fibers: N-
acylchitosans, N-arylidene- and N-alkylidene-chitosans, N-acetylchitosan (chitin)-
tropocollagen, and chitosan-transition metal complexes with significant property changes
[554].
The antibacterial activity of the modified fibers showed great enhancement as shown by
Liu et al. [555]. Chitosan /N,O-carboxymethylated chitosan /viscose rayon antibacterial fibers
were prepared by blending chitosan emulsion, N,O-carboxymethylated chitrosan (N,O-CMC),
and viscose rayon together for spinning. TEM micrographs showed that chitosan
microparticles dispersed uniformly along the oriented direction with the mean size ranging
from 0.1 to 0.5 μm. Although the addition of chitosan slightly reduced the mechanical
properties, the antibacterial fibers properties were found to meet commercial requirements.
The chitosan blends exhibited excellent antibacterial activity against E. coli, S. aureus, and C.
albicans. The antibacterial activity increased along with the chitosan concentration and was
not greatly affected by 15 washings in water. SEM micrographs demonstrated that greater
amounts of bacteria could be adsorbed by the antibacterial fiber than by the reference fiber;
these bacteria were overwhelmingly destroyed and killed. Another work reported a novel
fiber -reactive chitosan derivative synthesized in two steps from a chitosan of low molecular
weight and low degree of acetylation. First, a water-soluble chitosan derivative, N-[(2-
hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC), was prepared by
introducing quaternary ammonium salt groups on the amino groups of chitosan. This
derivative was further modified by introducing functional (acrylamidomethyl) groups, which
can form covalent bonds with cellulose under alkaline conditions, on the primary alcohol
groups (C-6) of the chitosan backbone. The fiber -reactive chitosan derivative, O-
acrylamidomethyl-HTCC (NMA-HTCC), showed complete bacterial reduction within 20min
at the concentration of 10ppm, when contacted with Staphylococcus aureus and Escherichia
coli (1.5-2.5×105 colony forming units per milliliter [CFU/mL]).
GENE DELIVERY SYSTEM

Gene therapy provides a promising strategy for cell-based therapy, and the prospect for
gene therapy has progressed rapidly. There are two main types of vectors that are used in
gene therapy based on viral or nonviral gene delivery systems. The viral gene delivery system
shows a high transfection yield but it has many disadvantages, such as oncogenic and
immunogenic effects. Nonviral delivery systems for gene therapy have been proposed as safer
alternatives to viral vectors and have attracted increasing attention. Nonviral delivery systems
have advantages such as ease of preparation, cell=tissue targeting, low immune response,
unrestricted plasmid size, and large-scale reproducible production. Chitosan-based gene
delivery systems are promising candidates for nonviral gene therapy. Deoxycholic acid,
which is the main component of bile acids, was used to modify chitosan hydrophobically and
to obtain self-assembling macromolecules for a nonviral gene delivery system. The self-
aggregate-DNA complex from deoxycholic acid–modified chitosan was shown to enhance the
transfection efficiency over monkey kidney cells. Chitosan containing 5.1 deoxycholic acid
groups per 100 anhydroglucose units was synthesized by a 1-ethyl-3-(3-dimethyamino
propyl) carbodiimide (EDC)-mediated coupling reaction. The feasibility of chitosan
selfaggregates for the transfection of genetic material in mammalian cells was also
investigated. Self-aggregates can form charge complexes when mixed with plasmid DNA.
These self-aggregate DNA complexes are considered to be useful for transfer of genes into
mammalian cells in vitro and served as a good delivery system composed of biodegradable
polymeric material.
NOVEL APPLICATIONS
Porous chitosan fibres have been shown to be useful as reinforcement in chitosan based
nerve conduits fabricated from chitosan yarns and a chitosan solution by combining an
industrial braiding method with a mold casting/lyophilization technique. The compressive
load of the reinforced conduits was significantly higher than that of a non-reinforced control
conduit at equal levels of strain. The tensile strength of the reinforced conduits was also
increased from 0.41 ± 0.17 to 3.69 ± 0.64 MPa. An in vitro cytotoxicity test showed the
conduits were not cytotoxic to Neuro-2a cells. Preliminary in vivo implantation testing
indicated that the conduits were compatible with the surrounding tissue [556,557]. Another
significant development is in the area of cartilage engineering. A novel approach involving a
replica molding technique for the production of fibres with controlled dimensions in the
micron regime from chitosan as fibrous chitosan scaffolds was demonstrated recently [558].
A three-dimensional scaffold fabricated from the chitosan-based hyaluronic acid hybrid
polymer fibers whose porous structure could be controlled was also recently developed [559].
These scaffolds showed high mechanical properties compared with liquid and gel materials.
The data derived from this study suggest great promise for the future of a novel fabricated
material with relatively large pore size as a scaffold for cartilage regeneration. In another
interesting development, chitosan and cellulose acetate (CA) blend hollow fibers with high
chitosan contents were prepared through the use of a non-acidic organic dope solvent. The
chitosan/CA blend dope solution for spinning the blend hollow fibers was prepared by the
addition of CA into nano particles of chitosan (about 50-150 nm) prepared using a surfactant,
sodium dodecyl sulfate (SDS) and dispersed in N-methyl-2-pyrrolidone (NMP). FTIR
analysis indicated that SDS interacted with chitosan. The blend hollow fibers were highly
porous and gave a tensile stress at break greater than 1-2 MPa [560]. Chitosan hollow fibers
were prepared by wet spinning, taking advantage of the unique rheological properties of
highly viscous chitosan in acetic acid [561]. Yet another interesting work reports that the
surface of poly (ethylene terephthalate) (PET) textiles was modified by electrospinning a
blend of PET/chitosan nanofibrous mats. The method introduced antibacterial activity and
biocompatibility to the surface of poly (ethylene terephthalate) textiles [562]. In combination
with alginate fibres, chitosan could be fabricated into a fibrous scaffold for annulus fibrous
(AF) cell culture using a wet-spinning and lyophilization technique. The work also
demonstrated the feasibility of using this scaffold for application for intervertebral disc tissue
engineering [562]. Chitin fibres are also finding applications in wool knitted fabrics [563].
Novel methods have been recently devised for the preparation of chitin threads for the
fabrication of absorbable suture materials, dressings, and biodegradable substrates for the
growth of human skin cells (keratinocytes and fibroblasts) [564]. Chitin fibers have been
extracted recently using ultrasonic techniques to obtain fibres with uniform diameters in the
range of 25-120 nm and possessing the optimized hierarchical supramolecular structures
[565]. This methodology might be valuable to provide a convenient, versatile, and
environmentally benign fabrication method for producing bionanofibers at an industrial scale.
A recent article reports the finding of the occurrence of silica-chitin fibre composite in
skeletons of marine sponges. This is the first report of a silica-chitin's composite biomaterial
found in nature. From this perspective, the view that silica-chitin scaffolds may be key
templates for skeleton formation [566]. This structural information could be useful in
developing scaffolds for tissue engineering and other applications. In an vitro study on the
degradation and biocompatibility of poly(l-lactic acid)/chitosan (PLLA/CHS) fiber
composites, excellent adhesion between osteoblast and PLLA/CHS fabrics was observed,
indicating good biocompatibility of the fabrics with osteoblast and its possible use as
supporting materials for chest walls and bones [567]. Chitin fiber is also employed in a novel
degradable biomaterial-short chitin fiber reinforced polycaprolactone (PCL) as chest wall
prosthesis. The results of the study showed that degradable Chitin fiber reinforced PCL has
fine biocompatibility and can provide effective support for chest walls, making it a promising
biomaterial for chest wall reconstruction. [568]. Chitin fiber is also employed to fabricate
novel biomimetic nanostructured bicomponent scaffolds consisting of chitin and silk fibroin
(SF) nanofibres by an electrospinning process. Cytocompatibility and cell behavior studies on
this system indicated that the hybrid matrix with 25% chitin and 75% SF could be a potential
candidate for tissue engineering scaffolds [569].
FUTURE OF CHITIN NANO FIBERS IN NANO MEDICINE

There is a growing interest in integrating nanotechnology with medicine, creating so-
called nanomedicine aiming for disease diagnosis and treatment with unprecedented precision
and efficacy [570,571]. According to the US National Science Foundation, the global nano-
products market will register a turnover of over 1,000 billion dollars a year in the next 10-15
years! In addition to novel materials, nano-enabled products are also likely to include highly
efficient catalysts, supercapacitors, fast charge/discharge high-energy batteries, membranes
with novel permeation characteristics, and various biometric structures that could have
applications in medicine as well as security and identification. New products are being
designed, or have been designed based on chitin, which present such innovative features, also
in terms of their applications, as to have already influenced our current lifestyle. Figure 16
shows a comparison of chitin nano fibres wit other nano structures.
Figure 16. The nanometric scale.
With the use of the newly developing technological platforms, it is possible to obtain thin
nanostructured films organized as nets, capable of providing a huge surface that is available
for interaction with the skin tissue and the external environment (Figure 17) [572]. This was
achieved by MAVI with the production of 240 nm chitin nanofibrils capable of accelerating
in a physiological manner the reparation of damaged skin (Figure 18). Chitin nanofibrils
(Figure 19), of an average size of 240 nm, can also be used as carriers, since they can release
in a controlled manner active principles for pharmacological or cosmetic use, such as lutein,
for instance. Chitin is a known natural polyglucoside that is easily recognized and hydrolized
by the skin’s cutaneous enzymes, while lutein, a natural oxicarotenoid, is an antioxidant
capable of enriching the skin’s antioxidant system. If these two molecules are properly
treated, the complex resulting from their bonds can surely perform an interesting protective
role on the skin and mucosae. In fact, it is capable of penetrating very easily through the
skin’s layers, if well dosed and vehicled, without causing toxic side effects and serving, on
the contrary, as an energy deposit (Figure 20). It is interesting to underscore the ease with
which these chitin nanofibrils can be included both in the natural and in the artificial fibers to
generate entirely innovative tissues (Figure 21).
Figure 17. Nanostructure of chitosan films.

Figure 18. Skin repair through the use of a particular gel containing chitin nanofibrils.
Figure 19. Chitin nanofibril, a natural product obtained from the chelae of shellfish.
Figure 20. Transcutaneous penetration of chitin nanofibrils.
Figure 21. Chitin nanofibrils for innovative tissues.

In comparison to product “created” in the lab such as the carbon nanotub, natural
nanostructures like chitin nanofibril [573,574] (Figure 22), like polyglucoside, are rapidly
catabolized and reduced to glucose and glucosamine by the enzymes of the skin following the
normal catabolic process [575] (Figure 23). In fact, these nanofibrils, on which
chemicalphysical as well as biological studies have already been conducted, appear to be
useful as active carriers to be employed in cosmetics as well as in the area of smart bio
tissues [576]. Bhatarai and his co-workers used electrospun chitosan nanofibers in studying
the behavior of chondrocytes and osteoblasts [577]. It was observed that adhesion of cells was
promoted and cell morphology was good, showing the potential of these nanofibers as good
carriers in bone tissue engineering.
Figure 22. Chitin nanofribrils.
Figure 23. The cell activity carried out by chitin nanofibtrils corresponds perfectly with the activity
carried out by a normal vehicle
CONCLUSION
Chitin and chitosan are aminopolysaccharides that have the structural requirements to be
processed into fibres having mechanical and biological properties suitable for medicals
textiles such as sutures. Despite its huge availability, the utilization of chitin has been
restricted by its intractability and insolubility. This report summarises the several attempts
that have been reported on solving these problems. The problems associated with the use of
the corrosive and degradative solvents used for dissolution are discussed. With all problems,
fibres with excellent properties equal to or better than cellulose have emerged. The best
properties for tensile strength (4g/d) and modulus (100g/d) for chitin were reported by the
mixed ester of chitin or chitosan acetate/formate polymer. The emerging electrospinning
techniques might be employed to control parameters for better properties. Further
improvement in fibre properties could be achieved with the application of spinning fibre from
lyotropic liquid crystalline solutions.
ACKNOWLEDGMENT
We are grateful to the Prof. K. Radhakrishnan, Director, and Dr. G.S. Bhuvaneshwar,
Head, BMT Wing of Sree Chitra Tirunal Institute for Medical Sciences & Technology for
providing facilities and support for preparing this reort. We are thankful to the laboratory staff
and library staff for their assistance. This work was partially supported by the Department of
Science & Technology, Govt. of India through the project 'Facility for nano/microparticle
based biomaterials - advanced drug delivery systems' #8013, under the Drugs &
Pharmaceuticals Research Programme.
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Editor: Samuel P. Davis, pp. © 2011 Nova Science Publishers, Inc.
Chapter 4
INTERPOLYELECTROLYTE COMPLEXES
OF CHITOSAN
N. A. Samoilova∗, M. A. Krayukhina
and I. A. Yamskov
A.N.Nesmeyanov Institute of Organoelement Compounds
Russian Academy of Sciences,
Moscow, Russia
ABSTRACT
The results of studies of interpolyelectrolyte complexes of chitosan and different
polyanions are summarised and described systematically. Specific properties of chitosan
as polyelectrolyte are described. The general concept of the formation of polyelectrolyte
complexes is developed. Separate parts of the review are dedicated to investigation of
polyelectrolyte complexes of chitosan with:
− biopolyelectrolytes, including polysaccarides (plant, animal, bacterial poly-

saccarides and lipopolysaccarides), proteins, nucleic acids and also modified natural
polyanions (carboxymethylchitin, carboxymetylcellulose, etc.),
− synthetic polyanions.
The data on application of polyelectrolyte complexes of chitosan in medicine and

biotechnology, particularly for creation of hemocompatible, thromboresistant materials,
bioconstruction materials for replacement of coverlets, blood vessels, bone tissue,
immobilization of biological active compounds, non-viral vectors of genetic information
transfer and so on are generalized.
∗
To whom any correspondence should be addressed. A.N.Nesmeyanov Institute of Organoelement Compounds
Russian Academy of Sciences, 119991, Moscow, Vavilova str., 28, Russia. Tel./fax.: 495-135-50-37; E-mail
address: samoilova.nadezhda@gmail.com.
218 N. A. Samoilova, M. A. Krayukhina and I. A. Yamskov
I. INTRODUCTION
In recent years, several publications reviewed data on complexes of the polysaccharide
chitosan (CS) with anionic polyelectrolytes (mainly of natural origin) and considered the
potential applications of chitosan compositions in bio-medicine, pharmacy, food industry, etc.
[1-4]. In the present chapter is smmarized literature data on the synthesis, study and
application of polyelectrolyte complexes of chitosan with natural, natural modified and
synthetic polyanions.
Chitosan has unique physicochemical and biological properties such as biocompatibility,
antimicrobial activit, the ability to activate macrophages and enhance fibroblast proliferation,
etc. Due to the diverse, recyclable and unlimited sources of the raw material, this biopolymer
offers some important benefits [5, 6].
One of the attractive characteristics of chitosan is its ability to form non-covalent
complexes with other polyelectrolytes. These complexes possess potentially useful specific
properties [2]. Complexation is greatly affected by molecular mass of chitosan, degree of
polydispersity, degree of deacetylation (DD), dissociation constant (pKa) of the protonated
amino groups of chitosan and the distribution of functional (amino and acetamido) groups
along the polymer chain. The degree of deacetylation of chitosan determines the value of pKa,
which can vary from 6.46 to 7.32 [7]. The conformation state and size of chitosan
macromolecules in solution also depend on the DD and solution ionic strength.
For DD > 80%, the Coulomb repulsion of the protonated amino groups of chitosan
dominates within a certain range of pH and ionic strength. For 50% < DD < 80%, hydrogen
bonds formed with participation of acetamide groups of chitosan make an additional
contribution to intermolecular interactions. The bulky acetamide residues and the hydrogen
bond network create steric hindrance and decelerate the rotation of the pyranose units around
the β-glycosidic bond, increasing the rigidity of the polysaccharide macromolecule. As DD
decreases, these effects become more important, and hydrophobic interaction increase; all this
leads to increased self-aggregation of chitosan molecules in solutions, which also depend on
the polymer concentration [4]. Thus, the ability of chitosan to form polyelectrolyte complexes
depends on the nature of interactive polyelectrolytes and reaction conditions.
II. GENERAL CONCEPT OF THE FORMATION OF POLYELECTROLYTE

COMPLEXES
The complex systems formed by macromolecules (interpolymer complexes) can be
classified according to the dominant type of intermolecular interaction:
− complexes formed by the van der Waals interactions;

− complexes formed by hydrogen bonding between macromolecules;
− coordination complexes;
− covalent complexes;
− polyelectrolyte complexes stabilised by intermolecular ionic bonds.
Interpolyelectrolyte Complexes of Chitosan 219
− sterically matching (‘key – lock’ type) complexes.
In addition, there are combined types of interpolymer complexes, which owe their
stability to cooperative interactions involving the above-mentioned types of bonding. The
overwhelming majority of publications are devoted to polyelectrolyte interactions at the level
of linear, dendritic, protein, etc. macromolecules. Moreover, complexation of polyelectrolytes
with oppositely charged surfaces (e.g., with polymer ‘brushes’ [8], latex particles and
monolamellar liposomes [9, 10]) and macroscopic, weakly crosslinked hydrogels [11, 12] was
described.
Several recent publications dealt with the properties of systems formed by the interaction
of oppositely charged polyelectrolyte networks (microgel particles) [13] and nano-sized
particles [14].
The ionogenic groups of the macromolecular chain of polyelectrolytes dissociate in polar
media into charged units on the polymer chain and low-molecular-mass counterions. The
electrostatic attraction of oppositely charged polymer units leads to the formation of polyion
pairs, namely, interpolyelectrolyte complexes. In this review for simplicity we will use the
therm polyelectrolyte complex and abbreviation PEC. The foundations of the theory of PEC
formation and organization were laid in works [15 - 19].
Polyelectrolyte complexes have attracted the attention of researchers because of their
unique properties and ease of preparation. PEC solutions can be regarded as smart polymer
systems due to their ability to change their phase state after insignificant changes in the
external factors (pH, ionic strength, temperature, etc.) [1, 20]. Some of the possible
approaches to the classification of PEC are as follows:
− according to the nature of polyions involved in the formation of PEC, one can
distinguish complexes based on polymers of only natural or synthetic origin and PEC
of mixed type, involving synthetic polymers and biomacromolecules;
− complexes can be formed by strong or weak polyelectrolytes;
− according to their composition, PEC may be stoichiometric and non-stoichiometric;
− soluble and insoluble PEC can be formed depending on the reaction conditions.
A distinction of the interactions of polyelectrolytes (compared with processes involving

their low molecular mass analogues) is the cooperative character of bonds formed between
the electrostatically complementary anionogenic and cationogenic macromolecules involved
in complexation. This type of bond imparts high stability to PEC within wide ranges of pH
and ionic strength of the medium.
The formation of PEC and variation of its composition (i.e., the polyelectrolyte reaction)
is a reversible process controlled by many factors such as the nature of polyelectrolytes, their
molecular mass and molecular mass distribution, ionic strength, pH of polyelectrolyte
solutions, etc. The same factors and the sequence in which the components are mixed regulate
the formation of soluble and insoluble as well as stoichiometric and non-stoichiometric PEC.
There are several procedures for the preparation of PEC. The simplest method is mixing of
the aqueous solutions of polycations and polyanions. Another way is polymerisation of ionic
monomers on oppositely charged polyionic matrices [15].
Upon mixing of solutions containing oppositely charged polyions, PECs are formed by
an ionexchange reaction, occurring at a high rate even in highly dilute starting solution.
If complexation involves weak polyelectrolytes (for example, polyacrylic acid and
poly(dimethylaminoethyl methacrylate)), the extent of reaction (conversion of ionogenic
groups to the so-called interchain ‘salt’ bonds between two polyions) can be varied by
changing the pH of the reaction system. In this case, the measure of the free energy of
complexation (ΔGcs) is ΔpH, which is the function of ionicity (α) and conversion (θ) of
polyelectrolytes that defines the multicentre cooperative interaction between the oppositely
charged polyion units for θ = α (i.e., if each ionogenic group of co-polyelectrolytes of PEC
is assumed to form, after being charged, an ionic bond with the corresponding oppositely
charged group) [15]:
ΔрН(α,θ) = [ΔG(α) – ΔG(θ)]/2.3RT = ΔGcs/2.3RT,
where ΔG(α) and ΔG(θ) are the free energies of ionisation of a weak polyelectrolyte in the
absence and the presence of an oppositely charged polyion for the given α and θ.
If a PEC is formed upon matrix polymerisation of charged monomers on the oppositely
charged polyion (matrix), the change in the free energy (motive force of complexation) is
described by the equation:
ΔGtp= ΔGp + ΔGcs,
where ΔGp – is the free energy of polymerisation in the absence of a matrix. When monomer
polymerisation is thermodynamically forbidden (ΔGp > 0), but ΔGcs is negative and и |ΔGcs| >
|ΔGp|, the matrix polyion shifts the equilibrium toward the oppositely charged polyion. Note
that the same factor is responsible for clusterisation of protein globules with oppositely
charged polyions to form protein polyelectrolyte complexes. When the ratio of the numbers of
ionised groups (Z = m: n) of the starting polyelectrolytes is 1 and θ → 1, stoichiometric PEC
are formed in the system, which are insoluble in any solvents, but can swell in water to a
certain extent. These insoluble PECs resemble cross-linked hydrogels in their properties.
They have found wide use as binding agents to prevent wind and water erosion of soil in
agriculture; as effective coagulants of colloidal dispersions in in agriculture; as effective
coagulants of colloidal dispersions in industry; as sorbents and matrices for enzyme
immobilisation and bio/hemocompatible coatings for medical materials contacting with blood
and other biological media in medicine and biotechnology [1, 2, 5, 15].
Despite the long history of PEC studies, the structure and properties of insoluble PEC are
much less defined than the structure and properties of soluble PEC. In insoluble PEC, more or
less extended regions with interchain ion pairs co-exist with separate loops and ‘tails’, which
can be regarded as structural defects. The higher the conversion θ the higher is the number of
‘paired’ units in PEC. The free hydrophilic units localised in loops and ‘tails’determine the
degree of PEC swelling in solvents [19].
If Z = 1 but θ < 1 (incomplete reaction), water-soluble PECs are formed as equilibrium
products of complexation. The units of the starting polyelectrolytes not involved in the
formation of interchain ionic bonds play the role of lyophilic (hydrophilic) fragments of
macromolecules due to which PEC particles are held in solution. When θ → 1, water-soluble
PECs are formed if the molar ratio between the units (φ) of the polyelectrolyte taken in
deficiency (blocking polyelectrolyte) and polyelectrolyte taken in excess (lyophilising
polyelectrolyte, LPE) in PEC is smaller than 1, i.e., if the LPE is present in excess in the
system. In the work [20], these pair-forming polyelectrolytes are called guest polyelectrolyte
(GPE) and host polyelectrolyte (HPE), respectively. Below we use these abbreviations.
As a rule, reactions of polyelectrolytes can form equilibrium systems in which soluble
and insoluble PEC co-exist. The equilibrium can be shifted toward the soluble PEC by
introducing an excess of HPE as the lyophilising high-molecular-mass component in the
system.
If the degree of polymerisation of HPE is not lower than that of GPE, soluble products
are formed. The critical value (φc) is the limiting degree of occupation of GPE chains in HPE
above which the hydrophilicity of the excess single-stranded segments is not sufficient for
PEC particles to be held in solution; as a consequence, phase separation occurs in the system
[19]. For the majority of defined systems, φc is 0.2 - 0.6 and depends on the chemical nature
of polyelectrolytes. Soluble equilibrium PEC with 0 < φ < φc behave in dilute solutions as if
they were individual polyelectrolytes with different charge densities.
The behaviour of non-stoichiometric PEC formed by the linear polyions of HPE with a
high degree of polymerisation and small charged GPE particles (cationic dendrimers, globular
proteins, etc.) in solution is similar to the behaviour of non-stoichiometric PEC consisting of
linear polyelectrolytes. The non-stoichiometric PEC containing hydrophilic single-chain
segments of HPE and the relatively hydrophobic GPE - HPE double-chain domains are
regarded as some kind of block copolymers [21].
Yet another family of soluble PECs are the so-called block ionomer complexes (BIC),
including a block copolymer of ionic and non-ionic blocks as one of the components [22-26].
These complexes remain soluble even when Z = φ = 1. For example, a BIC based on the
diblock co-polymer of polymethacrylate - block polyethylene oxide and polyvinylpyrrolidone
(PVP) is known.[24] For Z = φ = 1, the BIC unimer contains a hydrophobic domain of the
stoichiometric polymethacrylate - PVP complex covalently bound with the hydrophilic block
of polyethylene oxide and is thus an amphiphilic macromolecule. The behaviour of these
compounds is similar to that of surfactants. As in the latter case, polymer micelles are formed
at concentrations higher than the critical micelle concentration. A micelle has a water-
insoluble nucleus (PEC block) and a hydrophilic ‘crown’ (polyethylene oxide block), due to
which it is soluble in water and has aggregation stability. Unlike classical polymer micelles,
they disaggregate when low-molecular-mass salts are added because of nucleus
decomposition caused by the electrostatic screening of the charge.
The above tendencies in the formation and behaviour of PEC are characteristic of
systems in which the Coulomb interactions are dominant. More complex behaviour is shown
by PEC where electrostatic interactions co-exist with hydrophobic, donor - acceptor and
dipole - dipole interactions and hydrogen bonding. The regularities of complexation of these
multifactor polyelectrolyte systems are not discussed here.
Major attention in this chaptr is paid to structure organization, physicochemical

properties and applications of chitosan PECs with different polyanions.
III. POLYELECTROLYTE COMPLEXES OF CHITOSAN WITH

BIOPOLYELECTROLYTES AND MODIFIED NATURAL POLYANIONS
1. Interpolyelectrolyte Complexes of Chitosan with Polysaccarides
The majority of publications are devoted to studies of chitosan polycomplexes with

polysaccharides of another nature. All used polysaccharides are conventionally divided into
four groups: plant, animal, bacterial polysaccharides, and synthetic polysaccharides. The
typical representatives of plant polysaccharides are cellulose, hemicelluloses, xylans,
glucomannans, galactans, pectins, starch, and algal polysaccharides (agar, carrageenan, fucan,
galactans, etc.); animal polysaccharides include chitin, glycogen, and glycosaminoglycans of
connective tissue (heparin, hyaluronic acid, chondroitin sulfates and keratan sulfates); the
well-known polysaccharides of microorganisms include dextrans and mannans, hyaluronic
acid from streptococci A and C, etc.
Polysaccharides exhibit high biological activities; at the same time, they are low-toxic,
biocompatible and biodegradable. Due to this combination of properties, they have many
useful scientific and practical applications; in particular, they are used for constructing
various biomaterials by complexation with oppositely charged biomolecules.
1.1. Interpolyelectrolyte Complexes of Chitosan with Animal Polysaccarides and

Modified Animal Polysaccarides
The best studied chitosan PEC include complexes with animal polysaccharides, including
those with glycosaminoglycans (GAG). As it is known GAGs play an important role in living
organisms. Thus the mucopolysaccharide keratan sulfate is part of the proteoglycan complex,
which can form complexes with hyaluronic acid and is involved in the formation of blood
vessel walls [27, 28]. Heparin can accelerate multiplication of some cells and inhibit growth
of others, it stabilize the growth factor; moreover, it possesses anticoagulant, antisclerotic and
antiinflammatory properties and is one of the most widely used medicines for treatment and
prevention of cardiovascular diseases. Chitosan, in turn, is a biocompatible and bioresorptive
polymer; it is cytocompatible with keratinocytes and fibroblasts the adhesion and
proliferation of which on chitosan depends on DD (the higher the DD the higher the cell
adhesion). Fibroblasts adhere better than keratinocytes, but proliferation of the latter is faster
when the DD of chitosan is higher [29]. In vivo, chitosan can be bound to GAG (in particular,
to chondroitin sulfate and hyaluronic acid) [27] to form stable PEC.
The PECs of chitosan with hyaluronic acid, chondroitin sulfate, chondroitin 6-sulfate,
heparin and sulfated cellulose and methods of their analysis were described [30-40]. It was
found that the stoichiometric PEC of glycol chitosan with hyaluronic acid [31] could be
obtained at pH 6.0. In the structure of chitosan, there is one weakly basic amino group per
pyranose ring; in the structure of hyaluronic acid, there is one weakly acidic carboxyl group
per two pyranose rings (i.e., the number of amino groups is not equivalent to the number of
carboxyl groups). In addition, there is steric hindrance for the rotation of pyranose rings,
which rationalises the deviation from the stoichiometric composition at low pH values. In
contrast to hyaluronic acid, the heparin molecule has two strongly acidic (sulfate) and one
weakly acidic (carboxyl) groups per two pyranose rings; therefore, in the case of the chitosan
PEC formed at low pH values, the calculated and experimental data were in agreement [31].
The physicochemical aspects of the interactions of highly deacetylated samples of
chitosan (DD 98%) with chondroitin 4-sulfates (55% and 70% sulfo groups, molecular mass
45.5 kDa), chondroitin 6-sulfate (90% sulfo groups, 59 kDa) and hyaluronic acid were
considered [32,33]. As could be expected, the degree of electrostatic binding depended on the
degree of ionisation of polyelectrolytes, the nature of ionogenic groups and charge density.
For chitosan PEC with chondroitin 4- or 6-sulfate, the result was independent of the
position of the sulfo group in the polysaccharide units. When the salt was formedt (sodium)
of chondroitin sulfate was mixed with chitosan, maximum precipitation was observed at a
molar ratio (ρ) of 1 between the glucosamine residues and the residues of the sulfo and
carboxylic groups. The results agreed with previously published data [30, 31]. For the acidic
form of chondroitin sulfate, the equivalence point corresponded to ρ = 0.75; in this case, the
NH3 groups of chitosan formed ionic bonds with all sulfo groups of chondroitin sulfate
(which completely dissociated) and only half of all carboxylic groups. Stable polyelectrolyte
complexes of chondroitin sulfate – chitosan (potencial colon-targeted drug carriers) [34] were
formed at different pH values with various ratios or at a fixed molar ratio ρ = 0.5 under
various pH conditions and quantitated with the use of solid-state 13-C CP MAS NMR and
element analysis. FTIR and 13-C NMR clearly showed H-bond formation at low pH,
indicating that in addition to the electrostatic interaction, H-bonding may be involved in
complex formation. Chondroitin sulfate – chitosan complex yield [34], FTIR spectra [34],
thermogravimetric [34 -36] and dielectric [36] analysis were used to study the degree of
interactive strength between polyions.
Complexation of chitosan with both salt and acid forms of hyaluronic acid led to the
formation of a 1: 1 PEC [32]. The effect of the DD of chitosan on the stability of the resulting
complexes with hyaluronate showed that as the DD (charge density) increased, the stability of
PEC, as well as the cooperativity of the electrostaic interactions, increased [33]. In the course
of the formation of chitosan PEC with acid forms of GAG, the carboxylic residues of
polyelectrolytes were deprotonated. Depending on the pKa value, deprotonation was either
partial (for chondroitin sulfate) or complete (for hyaluronic acid, whose pKa is lower) [32].
The order of the addition of polyelectrolytes did not affect the complexes formed. PEC
characterised by high stability over wide ranges of pH and ionic strength were obtained by the
interaction of chitosan with GAG in both acid and salt forms.
Recently nanosized particles (NPs) of GAG-based PEC were described [37-39]. The one-
shot addition of polycation and polyanion solutions (ρ = 0.09-19.2) used for NPs permitted
formation of both cationic and anionic particles from both polysaccharide pairs [38]. For most
conditions studied, colloidally stable, nonstoichiometric NPs were formed in solution.
However, NPs formation was inhibited by flocculation at charge mixing ratios near 1. When
adsorbed to surfaces and dried, some formulations resulted in discrete NPs, while others
partially or completely aggregated or coalesced, leading to different surface morphologies.
Nanosized (average diameter 162.8 ± 18.9 nm) and stable chitisan – g-PEG/heparin PEC
micelles were produced by a self-assembly process [38]. PEC micelles facilitated the
intracellular delivery of heparin, triggered the caspase activation, and consequently promoted
apoptotic death of cancer (mouse melanoma 13 16F10). In the work [39] self-assembled
enoxaparin (low molecular weight heparin) PECs were formed with chitisan and chitosan
derivatives (CS-cystein, trimethyl CS (TMC), PEGylated TMC copolymers). Soluble NPs in
the size range of 200-500 nm with spherical morphology could be obtained in the pH range of
3.0 - 6.5, with positive charge and drug encapsulation efficiency of approximately 90%. The
PECs of chitosan derivatives were rather stable against ionic strength even when ionic
strength was as high as 300 mM, as a consequence of the steric effect of methyl groups,
polyethylene glycol segments and the formation of disulfide bonds for TMC, PEGylated
TMC and chitosan-cystein conjugates, respectively.
Complexation of heparin, chondroitin sulfate A, chondroitin sulfate C with N-acylated
chitosan derivatives (N-benzoylchitosan, N-myristoylchitosan, N-propanoylchitosan, etc.)
with different degrees of substitution is reported [41]. It was noted that maximum turbidity of
the system increased with the degree of acylation of chitosan and decreased to minimum
because of PEC precipitation after the system had been stored for 24 h. It was shown that at
pH 2.8 and 4.5 stoichiometric PEC of chondroitin sulfates A and C with chitosan and N-
benzoylchitosan could be formed.
Materials based on biodegradable polymers such as chitosan and GAG possesses useful
biological properties. They are cytocompatible and can stimulate wound healing processes
and induce cell proliferation for the recovery of cartilage, skin tissue, etc. Novel
mucoadhesive nasal inserts based on chitosan/hyaluronate polyelectrolyte complexes for
vancomycin or insulin delivery were obtained at different pH and molar ratios [35]. Chitosan
beads with encapsulated gentamicin sulfate were used as templates for layer-by-layer
assemble with hyaloronic acid [40]. These beads showed slow sustained drag release in vitro
and could be used as a drug delivery system.
Hydrolysis of complexes with specific enzymes (chondroitinase, hyaluronidase) and the
ability of biomaterials based on chitosan and GAG to be cytocompatible with keratinocytes
and chondrocytes were studied [27, 42]. It was shown that chitosan in PEC acted as a
protector against hydrolytic cleavage of GAG. However, chitosan showed better results in
experiments on cell adhesion and proliferation and in the series of in vivo experiments, which
demonstrated its higher efficiency as a wound healing agent compared with the efficiency of a
PEC of chitosan with GAG.
Films of chitosan – hyaluronic acid PEC in NaCl solutions can reversibly change their
form under the action of an electric field [43]; the bending angle can be adjusted by varying
the ionic strength of the salt solution and the electric field strength. These ‘electrosensitive’
PECs are promising for use as the components of artificial organs (e.g., as contractive
structures mimicking the muscle work), sensors, on-off switches, etc.
Porous constructions [44] (membranes, blocks, tubes, etc.) with controllable (by varying
the freezing - drying conditions) pore sizes (1 - 250μm) were obtained on the basis of
chitosan. These structures were later used for the formation of chitosan - GAG (heparin)
PECs. Complexation did not violate the inner microstructure of pores.
Glycosaminoglycans such as heparin and heparane sulfate are capable of binding the
growth factors and cytokines and also initiate or enhance their synthesis. Thus, constructions
containing chitosan – GAG complexes can be used as systems capable of retaining and
accumulating the necessary factors secreted by the colonising cells and even growth factors
from liquid around tissues. These constructions are suggested as potential implants in tissue
engineering [44-46].
Highly selective membranes were obtained from films of the PEC of chitosan (550 kDa,
DD 87%) with carboxymethylchitin (CMCS, 60 kDa, 1.17 carboxymethyl groups per unit)
possessing a specific structure stabilised by a system of ionic and hydrogen bonds; the
membranes could be used for separating water - organic mixtures and for hemodialysis [47].
The composition of CS – CMCS PEC used for casting of films varied from 0.5 to 4.0 (CS:
CMCS); this determined the conditions of additional hydrothermal treatment and the basic
physicochemical characteristics of films such as swelling, sorption capacity, mechanical and
transport properties, etc. At higher chitosan contents in films, the degree of swelling in water
increased drastically. Films with a stoichiometric composition had the lowest degree of
swelling. Thermal treatment of films led to dehydration of interchain salt bonds and resulted
in covalent cross-links (amide bonds). The resulting partially cross-linked PEC films were
characterised by poorer swelling in all solvents and lost solubility in acidic solvents (in
contrast to thermally nonprocessed PEC).
The system of ionic and hydrogen bonds in CS – CMCS polyelectrolyte complexes
ensured the formation of a more rigid (compared with the chitosan structure), strained and
fragile structure. When the composition of the PEC film approached stoichiometric, their
sorption capacity decreased, while the stability and rigidity slightly increased. Thermally
treated stoichiometric PECs having greater numbers of ionic and covalent cross-links than
non-stoichiometric PECs are demonstrated the highest selectivity in the pervaporative
separation of water - organic mixtures, and high permeability. It was noted, however, that CS
– CMCS PEC films are inferior to chitosan – poly(acrylic acid) (CS – PAA) PEC films in
ability to separate water-enriched water - propan-2-ol mixtures [48, 49]. This effect was
explained [47] by the presence of two bulky substituents in the CMCS macromolecule
because of which the structure of CS – CMCS PEC probably had lower packing density than
the structure of CS – PAA PEC, despite the higher steric complementarity of co-
polyelectrolytes in the case of chitosan and CMCS.
In the work [50] investigated the elaboration of nanoparticles from the pH-induced self-
complexation of the amphoteric polysaccharide N-sulfated chitosan. The acidification of
aqueous solutions of chitosan having a degree of acetylation of 24% and a degree of sulfation
of 34% or 56% was followed stepwise by turbidimetry, dynamic light scattering, and
electrophoresis. With the highest sulfated chitosan, no turbidity was recorded between pH =
7.8 and 2.0, traducing a high apparent solubity of the polymer chains in this domain of pH.
With the lowest sulfated chitosan, a steady increase in turbidity was monitored from pH =
6.90 to 6.15 followed by the flocculation of the polymer at pH approximately 6.0. In this
range of pH, the polymer phase separated to yield particles having hydrodynamic diameters
decreasing from 350 to 260 nm and an almost constant negative charge. These particles were
assembled by electrostatic interactions between the protonated amino residues and the sulfate
functions and stabilized by an excess of surface sulfate groups. The particles could be
separated from the reaction medium and concentrated by centrifugation-redispersion cycles

without alteration of their structure.
The two derivatives of chitosan were used to prepare stable multilayer polyelectrolyte
films through a layer-by-layer (LbL) deposition technicue [51]. Low molecular weight
chitosan was modified by glycidyltrimethylammonium chloride to obtain the cationic form of
the polymer. The anionic form of chitosan was obtained by sulfonation of
carboxymethylchitosan with trimethylamine - sulfur trioxide. The films revealed smooth
surfaces and linear growth of the thickness during LbL adsorption as measured by atomic
force microscope. Contact angle measurements revealed a very hydrophilic nature of the
formed films. These films thanks to biocompatibility and bacteriostatic properties of chitosan
and its derivatives may be potentially useful for many biomedical and environmental
protection applications.
Composite of carbonate-containing low-cristallinity nanoparticle hydroxyapatite (HA)
and chitisan – phosphorilated chitosan PEC was evaluated in vitro and in vivo. The HA-PEC
nanocomposite with complicated poros structure was prepared by a biomimetic method. The
biodegradable composite promoted osteoblast adhesion, proliferation [52].
1.2. Interpolyelectrolyte Complexes of Chitosan with Plant Polysaccarides and

Modified Plant Polysaccarides
Chitosan PEC with carrageenans (anionic linear sulfated polysaccharides containing D-
galactopyranose residues linked by regularly alternating α (1 → 3) and β (1 → 4 ) bonds)
have been considered [53-57]. The ι- and κ-carrageenan molecules include 3,6-anhydro-D-
galactopyranose residues and have a double helix conformation in aqueous solutions, which
determines their gel-forming ability. λ- Carrageenan has a high content of sulfo groups and
no anhydrogalactose residues; in addition, it cannot form gels. Carrageenans are non-toxic
and biocompatible and can therefore be useful in food and pharmacological industries.
When chitosan forms complexes with κ-, ι-, and λ-carrageenans, the type of the
resulting PEC is dictated by the charge density on polysaccharide molecules, their
concentration, and conformation (globular or helical) of carrageenan macromolecules [57].
Chitosan PEC with ι-, κ-, and λ-carrageenans [54] were prepared for medical and biological
applications. It was noted that at concentrations higher than 0.1% for aqueous chitosan and
higher than 0.3% for λ-carrageenan, PEC were formed as gels in the bulk; insoluble PEC
were formed at lower polymer concentrations. Complexes can be obtained from concentrated
(4%) and highly concentrated (20%) polysaccharide systems, their mixing generally led to the
precipitation of complexes [55-57].
Structural organisation of gels and consequently their mechanical and rheological
properties depended on the chitosan content in PEC and on the nature of carrageenans [54].
λ-Carrageenan did not substantially affect the properties of PEC; it played an auxiliary role
of a co- polyelectrolyte required for the preparation of PEC with chitosan. ι- And κ-
carrageenans were more important for the complexation; their gels with chitosan were
thermally sensitive, which was explained by the thermally reversible helix - knot
conformation transition of carrageenan molecules, occurring as a sol - gel transition at low

temperatures.
λ-carrageenan. Static viscosity increased
The least viscous gels were prepared by using
substantially on passing to ι- and κ-carrageenans. As a result, chitosan gels with κ-
carrageenan were more rigid. The reason for these distinctions is possibly the formation of
additional cross-links, which formed in systems with ι- and κ-carrageenans, but were absent
in the case of λ-carrageenan.
pH-Sensitive polyelectrolyte complex hydrogel systems of chitosan with
carboxymethylcellulose (CMC) and κ-carrageenan were described [53]. The homogeneity
and swelling ability of these hydrogels depended on the chitosan: carrageenan: CMC ratio
and the content of low-molecular-mass salts in polymer solutions. The threshold
polyelectrolyteto salt ratios were determined at which homogeneous or heterogeneous gels
formed or not. The degree of swelling of chitosan – carrageenan – CMC tripolymer gels
decreased proportionally to the decreasing content of carrageenan in PEC because of the
decrease in the osmotic pressure and reached maximum at pH 11 - 12; a similar dependence
was also observed for chitosan – carrageenan bipolymer PEC.
Direct mixing of chitosan and alginate solutions would readily coagulate or form gels,
leading to unfeasibility in the production of defined form of PEC particles or fibers. Alginic
acid – linear copolymer of homopolymeric blocks of mannouronat and its epimer guluronate
residues, covalently linked together. Alginates are extracted mainly from seeweeds
(Laminaria) or bacteria’s.
Alginate – chitosan beads [58, 59] or microparticles [60] were synthesized by chitosan
treating of calcium alginate particles. Chitosan – alginate beads were prepared by dropping
chitosan solution into a sodium alginate solution without any salt [61, 62]. Alginate –
chitosan – alginate microcapsules were used as implantat in the subcutaneous space of mice
[63]. Proper selection of the reaction pH, polymer concentration and hence charge density,
and hardening time was important and determines the characteristics of PEC particles. Based
on alginate – chitosan PEC fibers [64], fibrous scaffolds [65] were described. Gelation
problem associated with the direct production of alginate – chitosan hybridized fibers has
been overcome by the use of chitosan in the form of the chitosan – citrate emulsion, which
had been prepared from olive oil and sodium dodecyl sulfate aqueous solution [64]. The
chitosan – citrate emulsion was mixed with the alginate aqueous solution to obtain the
spinning dope suspension for the preparation into alginate – chitosan hybridized fibers by wet
spinning. The results showed that the tensile property values were greatest at the lowest
content of incorporated chitosan (0.5% w/w). But it was shown that absorbtion of the Amido
Black 10B dye (model of anionic drug) in the fibers increased with an increase in the chitosan
content. Alginate – chitosan PEC- based films [66-68], membranes [69-70], hydrogel coatings
on silicon wafers or paper [71] and titanium surface [72] were obtained. For preparation of
good film, it is desirable that the polyelectrolyte complex coacervate suspension, which is
cast to form the film, must be fine and uniform. The reaction rate of chitosan to alginate is
very high, it can be controlled by addition water miscible solvent like acetone and methanol
[66]; propylene glycol (0.5%) was used to obtain flexible film and for good peeling of film.
Besides precipitate method, layer-by-layer technique was used for film formation [65].
PEC of chitosan with its sulfated derivative [73] (its biological properties are similar to
those of heparin) and alginic acid [74, 75] were obtained. The effects of the DD and
molecular mass of chitosan on the stability of the complexes formed were studied. The higher
the DD and the lower the molecuar mass, the more stable the complexes. Yan et al. [75]
studied the PEC of sodium alginate (100 kDa) with chitosan samples (DD 95%) with low
(low-molecular-mass chitosan, LM CS, 130 kDa), medium (530 kDa), and high (HM CS,
1000 kDa) molecular mass. The HM CS complex with alginate formed large gel-like
coacervates, while the LM CS – alginate polyelectrolyte complexes were more finely
dispersed. The most homogeneous films with the highest water permeability in neutral media
were obtained from PEC of this type.
Interactions in chitosan – pectin (plant polyanionic saccaride) systems and the properties
of the PEC obtained were studied in many works [76-82]. The binding parameters of LMW
chitosan to pectin in solutions and multilayers were evaluated by Hill’s equation and
Karisson’s model respectively [81]. In both systems, the binding of chitosan to pectin was
anti-cooperative, and the binding constant was 196 ± 8. Membranes and fungistatic laminated
films based on chitosan – pectin PEC [79, 80] possessed pervaporative properties. The
swelling ability of the films of these PEC [78] can be controlled by varying the composition
and pH of the medium; the highest values were obtained at pH < 2 and >7. Chitosan - pectin
complex prepared in molar ratio 1:9 showed the highest mucoadhesive properties and pH-
dependent swelling sensitivity suitable for colon delivery of vancomycin [82].
Complexation of chitosan (230 kDa, DD 80%) with polygalacturonic acid (main
component of pectin) (PGA, 102 kDa, 87.5% galacturonic acid) was studied by
conductometry [83]. The conductivity of the system was measured during titration of sodium
polygalacturonate with solutions of chitosan hydrochloride; the change in the slope of the
conductometric curve suggested complete PEC formation. A stoichiometric PEC formed at
the equivalence point (CS: PGA = 0.98 ± 0.04), which was confirmed by several independent
methods (viscosimetry, potentiometry, turbidimetry and gravimetry); the position of the
equivalence point was independent of the order of mixing of polyelectrolytes. The conversion
of polyelectrolytes (degree of complexation) was determined, which depended on the molar
ratio of the reactants. The dependences found suggested that under the chosen conditions
complexation in the CS – PGA system was cooperative.
Stoichiometric complexes of chitosan with carboxymethylcellulose, alginic and
polygalacturonic acids were obtained at equimolar ratios of components [84-87]; the highest
degree of swelling of PEC was observed at pH 4.0 - 5.8 [87]. Polyelectrolyte complexes of
chitosan with carboxymethylcellulose revealed clot-inhibition in vitro [88], were used for
pulp tissue regeneration [89], based on such PEC hydrogels were pH-sensitive and undergo
enzymatic degradation [90].
Chitosan (370 kDa, DD 82%) PECs with carboxymethylated cashew gum (Anacardium
occidentale) [91] and carboxymethylglucomannan extracted from tubers of Amorphophallus
konjac were described [92]. The highest product yield was achieved at a ratio of chitosan:
cashew gum of 25: 75 [91]. Thermogravimetric and IR spectrometric analyses of the PEC
were performed and the activation energies of thermal destruction and the degradation
temperatures of polysaccharides and their complexes were determined. It was found that the
thermal stability of PEC decreased relative to that of the starting polysaccharides. Based on
chitosan – carboxymethylglucomannan PEC [92], nano- and microparticles were obtained,

their size varied from 50 nm to 1.2 μm depending on the initial concentrations of the
polysaccharides, pH and ionic strength of solutions. It was shown that nanoparticles based on
chitosan – carboxymethyl glucomannan PEC could effectively be used as systems for
encapsulating water-soluble drugs.
Polyelectrolyte complexes of chitosan and gum kondagogu (Cochlospermum gossypium),
free exudate gum that belong to the family Bixacoae, were prepared by mixing polymeric
solutions of concentrations 0.02-0.18% w/v. The complexes formed were loaded with
diclofenac sodium. PECs showed increase in relative bioavailability compared to the free
drug when administered orally to the rats [93].
Polyelectrolyte complexes of chitosan with plant polysaccharides (alginic acid, pectin,
carrageenan and so on) are extensively used for the creation of systems with controllable
release of drugs [58-60, 66, 82, 92-106], for stability improvement of enzymes [61], as
materials for wound healing [66, 71, 107], for cells incapsulating [72], as biocompatible
materials and biological construct for tissue engineering applications [63, 65] were used.
In the work [65] the blood compatibility of the chitosan – alginate PEC fibrous scaffold
could be significantly improved by incorporation a small amount of heparin in the
polyelectrolyte solution during fiber formation. The platelet production and platelet adhesion
on the chitosan – alginate – heparin fibrous scaffold were comparable to those on the resting
control. In vitro cytotoxicity test showed that the scaffold was not toxic to human
mesenchymal stem cells, and in vivo (rats) scaffold was biocompatible
Immobilised indometacin was obtained from genipin – cross-linked chitosan – sodium
alginate PEC [105]. The swelling ability of these systems decreased with pH and alginate
concentration in the gel-forming solution. Moreover, this parameter also depended on the
degree of cross-linking. As could be expected, the degree of cross-linking decreased with the
increasing degree of protonation of the amino groups of chitosan because the nucleophilic
attack of the dihydropyran ring of genipin was inhibited. As a result, the rate of indometacin
release from PEC particles with low degrees of cross-linking (formed at lower pH) was
higher.
Films [101], microcapsules [102], and pellets [96, 103, 104] of chitosan – carrageenan
complexes were prepared for the starting concentrations of carrageenan of 0.1% - 4.0% in
solutions. One of the most important characteristics of these systems is the swelling ability,
depending on structure of PEC, pH of the media, and determines the degree of release of
drugs. The swelling ability of drug matrices based on chitosan – alginate and chitosan –
carrageenan PEC was analysed using the Hopfenberg model [96] for evaluating the
contributions of various physicochemical processes (diffusion, relaxation, sorption -
penetration, etc.). Because of the better water-binding ability of carrageenans (e.g., compared
with the same ability of hydroxypropylmethylcellulose [104] or alginate [96] for the same
polyanion content in PEC), the pelletised forms were liable to undergo fast (in less than 30 s)
disintegration after the swelling stage. Thus, chitosan – carrageenan systems proved less
favourable matrices for the preparation of drugs with a prolonged activity than chitosan –
alginate PEC [96].
1.3. Interpolyelectrolyte Complexes of Chitosan with Microbial Polysaccarides and

Modified Microbial Polysaccarides
A study of chitosan PEC with sodium dextran sulfate (SDS) was reported [108-112]. The
PECs obtained were sensitive to changes in pH and the solution ionic strength [108]. At pH
10.5, the swelling was maximum and decreased when the solution ionic strength increased.
Variation of pH, molecular mass and the rate and order of mixing of polyelectrolytes led to
PEC with a molar ratio of CS: SDS from 0.8 to 2.5 [109, 110]. Two types of polyelectrolyte
complexes [110] with different molecular structures and properties could be formed; In the
first series (A), PECs were obtained by mixing solutions of chitosan and SDS of the same
concentration (1 g/ litre) in 2% acetic acid at volume ratios of 4 : 1 and 1 : 4; in the second
series (B), equal volumes of polymer solutions of the same concentration were mixed in 1%
hydrochloric acid. As a result, PECs of the series A were characterised by a greater number of
-
salt bonds between –NH3+ and –OSO3 groups than PECs of the series B. Moreover,
hematologic tests showed that PEC of the series A had anticoagulant (antithrombogenic)
properties, while PEC of the series B enhanced blood coagulation. Complexes with low-
molecular-mass SDS inhibited hemocoagulation to a greater extent than PEC with high-
molecular-mass SDS [109]. The order of mixing also produced a certain effect on the
anticoagulant properties of PEC; polyelectrolyte complexes formed by the addition of
chitosan to SDS had a more pronounced thrombogenic activity than complexes obtained by
mixing the components in the reverse order.
The ability of the obtained PEC to bind lipoproteins and their anticoagulant activity were
studied; a non-linear dependence of these parameters on the PEC composition was found. The
CS–SDS polyelectrolyte complexes possess selective complexing ability with respect to low-
density lipoproteins (LDL); the sorption selectivity decreased as the PEC: LDL ratio
increased. The effects of the PEC concentration and PEC: heparin ratio on the interaction of
PEC with heparin were follows: at PEC concentrations smaller than 10 mg/ml [111] and the
same or lower content of heparin in blood plasma, the activity of heparin was enhanced; when
the PEC concentration increased to 100 mg/ml, the antiheparin effect was observed. With the
optimum ratio of the components, the CS–SDS complex showed higher anticoagulant and
anticholesterol activities compared with those of the individual SDS. The effect of higher
activity of SDS in its complex with chitosan was explained by the optimisation of its
physicochemical parameters (when SDS macromolecules were chemically modified with
chitosan), in particular, by the decreased density of the negative charge and the flexibility of
SDS macromolecules.
Kikuchi and Takewayashi [112] studied insoluble PECs of chitosan and
carboxymethyldextran, their antithrombogenic properties depended on the pH of the polymer
solutions; at pH 5.5 less thrombogenic products were obtained than at pH 3.5. These effects
were explained by differences in the molecular structures of the PEC and the presence of
residual carboxylic groups in them.
Thus, the efficiency of modification of anionic polysaccharides with chitosan through
non-stoichiometric complexation and the possibility of controlling certain biological
properties of PEC were shown.
Xanthan gum, a microbial exopolysaccharide consisting of a cellulossic backbone with
two mannose and one glucuronic acid side chains on every second glucose residue, formed
PEC gels with chitosan [113-118]. Molecular weight of xanthan can reach up to 6 millions
Daltons, which makes it possible to create extremely viscous solutions at very low
concentrations; xanthan is enzymatic resistant, stable over a wide range of temperatures and
pH. Xantan concentration was found to be the most critical parameter in PEC network
formation. The hydrogel capsules were completely cross-linked at pH 6.2 [118] when initial
xanthan solution concentration was at 1.5% (w/v). The swelling data also show that there is a
significant decrease in the degree of swelling (SD) when pH is increased from 4.5 (SD =
2164) to 6.2 (SD = 1187), which can be the increase in the crosslinking density. pH-Sensitive
swelling characteristics enable the controlled release of entrapped materials such as
therapeutic agents, enzymes and bacteria [113-116], the targeted delivery and controlled
release of encapsulated products for oral administration [117].
PECs were suggested for immobilisation of enzymes. For example, chitosan – alginate
PEC with immobilised urease [119] and ternary chitosan – polyanion – glucoamylase PECs
[120] were described. SDS, polyvinyl sulfate and heparin [120] were used as polyanions.
Insoluble PECs were prepared under certain conditions by co-precipitation of enzymes by
mixing chitosan solutions with polyanions. The presence of an enzyme did not affect the
composition of the resulting PEC. The enzyme activity depended on the type of PEC. Thus,
in insoluble chitosan–SDS PEC, glucoamylase exhibited 100% activity, while in chitosan–
polyvinyl sulfate and chitosan–heparin PEC, the activity decreased by 16% and 54%,
respectively [120]. Immobilisation of urease in the chitosan–alginate PEC led to increased
thermal stability of the enzyme and stability on storage; after 70 days, the catalytic activity
was 48% of the initial level, while native urease was deactivated by 50% in 7 days and by
100% in 20 days. The observed effects were explained by the appearance of a multipoint
ionic interaction between the immobilised enzyme and PEC, which is the scaffold that
imparts high conformation stability to the urease macromolecule [119].
1.4. Interpolyelectrolyte Complexes of Chitosan with Lipopolysaccarides

Yet another interesting type of chitosan complex is PEC with lipopolysaccharides (LPS),
which are the components of the outer membrane of gram-negative bacteria.
Lipopolysaccharides consist of regions differing in structure: lipid A having low structural
variability, a highly variable O-specific polysaccharide chain and a less variable
oligosaccharide segment (core). In rough mutants (R forms), there are no polysaccharide
chains. Lipid A is the most conservative part of polysaccharides, which is invariable in
structure in the majority of microorganisms studied. Lipopolysaccharides play an important
role in the physiological functioning of bacteria; they are involved in transmembrane
transport of various compounds and are endotoxins, antigens (the so-called O-antigens) and
bacteriophage receptors, they are the primary target of the attack by antibacterial drugs and
immunoglubulins. The polysaccharide chain of LPS is oriented toward the environment, and
its fine structure is responsible for the recognition of bacteria and the specificity of immune
reponse in higher animals and humans. Lipid A, which is closely associated with proteins,
ensures the entity and stability of the cell membrane by decreasing its permeability and
increasing its stability against hydrophobic agents; it is also responsible for the toxic
properties of lipopolysaccharides [4]. The death of gram-negative bacteria as a result of
inflammation, antibiotic activity, etc. liberates large amount of endotoxin. As is known,
endotoxins (LPS, in particular, lipid A) have a pyrogenic action on the infected living
organism; in particular, they result in severe intoxication (the so-called endotoxemy) and
endotoxic shock [121]. Therefore, of particular interest is seeking the substances capable of
reducing the toxicity of LPS.
LPS molecules bearing the negative charge on the surface (due to the presence of
phosphate, pyrophosphate and carboxyl groups) interact with polycations (e.g., chitosan
macromolecules) to form low-toxic chitosan–LPS complexes with different stoichiometry,
which are promising as immunostimulants [122-125]. The mechanism of this complexation
was studied by various techniques. It was found that the binding of chitosan to an endotoxin
is a complicated process, largely depending on the macromolecular organisation of
endotoxins, the molecular mass and the DD of chitosan, reactant concentration and reaction
temperature and time [122-125]. The complex is formed by means of not only ionic binding,
but also other types of interaction. Stable chitosan–LPS complexes were obtained only after
preliminary incubation of the components at 37oC. Presumably, the complexation was
accompanied by additional dissociation of LPS aggregates and formation of a chitosan
complex, which was quite stable over prolonged periods of time in solutions with high ionic
strength [122-124]. An electron microscopic study of the complexes revealed a considerable
effect of chitosan on the morphology of LPS. This effect manifested itself as the loss of the
ultrastructural organisation of the endotoxin and dissociation of aggregates formed by the
granular PEC particles. It was noted that morphological inhomogeneity depended on the
concentrations and ratio of the starting components and was probably related to the
concentration dependence of the supramolecular structure of the LPS, which in turn affected
their binding to chitosan.
It was found that chitosan specimens with lower molecular mass and degree of
acetylation had higher affinity for LPS, which could be explained by the higher flexibility of
chitosan macromolecules with a lower molecular mass. As a result, steric hindrance to the
binding of high-molecular ligands diminished and hence the sites for binding became more
accessible. The affinity for chitosan increased with the increase in the length of the O-specific
polysaccharide chains of LPS; this enhanced the interaction between the components
(increased the binding constants), but decreased the number of binding sites; this resulted in a
decreased amount of bound LPS. As the LPS concentration in a solution decreased, the
amount of the endotoxin capable of binding to chitosan increased. The stoichiometry of the
complexes depended strongly on the LPS concentration in a solution [123-125].
In a series of biological experiments, it was shown that complexation of LPS with
chitosan effectively decreased the acute toxicity of endotoxins [123, 124].
2. Polyelectrolyte Complexes of Chitosan with Nucleic Acids
One of the promising directions of research in biotechnology is the creation of non-viral

vectors for transferring the specific genetic information to cells. These artifial viruses can
successfully be used in gene therapy for the treatment of diseases caused by gene
malfunction. The curing action is achieved by the expression of the gene vector, which
replaces the defective gene or, vice versa, inhibits the excess activity, e.g., of the transforming
gene or introduces a new gene for vaccination purposes.
To incorporate DNA into a cell, one can use [126] viral vectors, varieties of liposomal
transport, specially designed protein or peptide vectors, including those addressed by the
ligand - receptor or antigen - antibody interactions to certain cell lines, and vectors with
positively charged HIV−TAT type peptides.
To obtain effective non-toxic and biocompatible DNA carriers, new systems are being
developed that use polymers of natural origin, in particular, chitosan and its derivatives. In the
resulting PEC, the positive charge of the amino groups of chitosan is neutralised by the
negative charge on DNA; this leads to DNA compaction, protects the molecule from cleavage
by endonucleases, and ensures gene penetration through cell membrane. Recent studies [127]
on models of the membrane bilayer showed that chitosan could penetrate into the cell
breaking the double lipid layer of the cell membrane and could also induce the formation of
transport channels in the latter, thus prolonging the supply of DNA and drugs to the cell.
Chitosan–DNA complexes has been used to prepare poros scaffold for tissue engineering
[128], micro- [129] nanoparticles [130-134] as non-viral delivery vehicles or membrane
[135].
DNA–chitosan complexes were stable, possesses high porosity (more than 80%) [128].
PECs were not toxic to MG-63 osteoblast-like cells and coused only a mild tissue response
when implanted subcutaneously in the backs of rats.
Chitosan/DNA-derived material surface properties such as charge, wettability, roughness
have be studed to affect cell functions such as attachment, spreading, migration, proliferation,
differentiation and aggregation.
Transfection of the chitosan−luciferase plasmid (pGL3-Luc) complex was studied by
laser microscopy using FITC-labelled plasmid (FITC is fluorescein isothiocyanate) and Texas
Red-labelled chitosan (Texas Red is sulforhodamine 101) [129]. The transfection activity and
efficiency of incorporation in the cell were found to be affected by the molecular mass of
chitosan, the stoichiometry of complexes and pH of the medium. The highest level of
transfection of the chitosan - plasmid complex was observed when using chitosan with a
molecular mass of 40 and 84 kDa, the ratio of chitosan nitrogen to DNA phosphorus (N : P)
equal to 5, the serum content in the tranfection medium 10% and pH 7. The study of the
transfection mechanism showed that the chitosan–plasmid complex formed aggregates of the
size 5 - 8μm, which were adsorbed on the surface of the cell, penetrated in it as a result of
endocytosis and were further liberated from endosomes under the action of the lysosomal
hydrolytic enzymes. Moreover, CS–DNA complexes in the form of nanosized particles (38 ±
4 nm [130]), 200 - 400 nm [131]) were proposed for the use in gene therapy. It was noted
[131] that using chitosan with a molecular mass of 10 kDa formed large particles of the size
600 - 1000 nm. When the molecular mass of chitosan increased to 40 - 150 kDa, PEC
particles of the size 200 - 400 nm could be obtained; in this range of molecular masses, the
DD of chitosan and the N : P ratio did not appreciably affect the size of the aggregates.
For improving the release of DNA from CS–DNA complexes their internal structure was
modified by incorporating a negatively charged poly(γ−glutamic acid)
(γ−PGA) [132]. The analysis of small angle X-ray scattering results revealed that DNA and
γ−PGA formed complexes with CS separately to yield two tipes of domains, leading to the
formation of ‘compaunded NPs’. Such ‘compaunded NPs’ mightly disintegrate into a number
of small sub-particles after cellular internalization, thus improving the dissociation capacity of
CS and DNA, increasing their transfection efficiency and cellular uptake.
Plasmid DNA (pDNA) and siRNA were encapsulated in CS nanoparticles (NPs) using a
complex coacervation process [133]. It was shown that CS in CS–DNA NPs could delay the
DNA release (pH 7.4) and protect the encapsulated DNA from nuclease degradation.
Morphology of HeLa cells transfected in vitro with CS–siRNA complexes was studied using
AFM. The results suggest that CS may be more capable than liposome (at used molecular
weight, charge ratio and NA concentration in CS–NA NPs) in delivery siRNA to target cells.
Direct force measurements between small interfering RNA (siRNA) and chitosan
molecules were utilize using force spectroscopy (AFM) [134]. To prepare a feasible model
system for the chitosan–siRNA interaction, siRNA was immobilized to AFM tip, and
chitosan was grafted onto gold coated surface via a linker of glutarealdehyde. The force
measurements revealed that the adhesive interactions decreased in force strength and force
frequency as the pH was increased from 4.1 to 6.1, 7.4 and 9.5, exhibiting distinct multimodal
distribution of the interaction forces between siRNA and chitosan moleculos at acidic pH and
only negligible adhesive forces were observed at neutral or high pH. The strong pH
dependence of siRNA – chitosan interaction can provide a convincing rationale for siRNA–
chitosan complex formation and nanoparticle stability under low acidic conditions.
Three kinds of membranes for controlling cell spreading and aggregation were prepared
by mixing chitosan and DNA at different ratios [135]. The membrane with the high ratio of
chitosan had a less hydrophilic surface (contact angle measurement data). The surface of the
polyelectrolyte complex membranes became rough as the DNA content increased (SEM
observations). Human mesenchymal stem cells adhered and spread on membranes prepared at
chitosan–DNA ratios of 1:1 and 3:1, while they did not on membranes prepared at chitosan–
DNA ratio of 1:3. Cells aggregated on the membrane prepared at a chitosan–DNA ratio of
1:3. Cell viability was also higher on membranes prepared at chitosan – DNA ratios of 1:1
and 3:1 than that on the membrane prepared at a chitosan–DNA ratio of 1:3. The membrane
with a high content of chitosan facilitated cell adhesion and spreading, while a high content of
DNA suppressed cell adhesion and spreading.
Chitosan and polygalactosamine were used as non-viral vectors for the transfection of
luciferase plasmid pGL3 in tumour cells [136]. Chitosan was several times more effective
than polygalactosamine in supplying the plasmid complex to the target cells. The efficiency
of the transfection depended on the stoichiometry of the chitosan - plasmid complex, pH of
the culture medium and the molecular mass of chitosan. The optimum parameters were the
chitosan: plasmid ratio 5: 1 and pH 6.9. The luciferase activity and gene expression efficiency
for systems containing chitosan with molecular masses of 15 and 52 kDa were higher than for
systems with chitosan with a molecular mass of 5100 kDa. The chitosan heptamer (1.3 kDa)
did not show any gene expression.
Studies on DNA complexation with N-alkylated [137, 138] and non-modified chitosan
(50 kDa, DD 99%) showed [137] that CS–DNA PEC formed at a charge ratio of 1: 1, while
the hydrophobically modified CS–DNA complex formed at a ratio of 1: 4. The introduction
of an alkyl substituent (C4 − C16 ) favoured easier penetration of DNA through the cell
membrane due to hydrophobic interactions and easier ‘unpacking’ of DNA from the carrier as
a result of a weakening of electrostatic interactions between hydrophobically modified

chitosan and DNA. The efficiency of transfection increased with the length of the
hydrocarbon fragment in the range C4 − C8, whereafter its level changed insignificantly.
While facilitating the penetration of plasmid into the cell, the hydrophic substituent of the
resulting plasmid complex also performed the protecting function, namely, it protected the
DNA molecule from the action of deoxyribonucleases. A methodological approach is now
being developed that uses modification of chitosan with low-molecular-mass specific ligands
to achieve the specific supply of DNA complexes to the target cells. Thus, artificial viruses
become ‘recognisable’ by definite cells. For example, low-molecular-mass chitosan was
modified [139] with galactose residues to make the vector ‘recognised’ by the liver cells
having specific receptors for galactose. Galactosylated chitosan was used in vitro for the
transfer of the reporter β-galactosidase in gene pSV to human hepatocarcinoma cells (HepG2,
SMMC-7721) and hepatocytes of the L-02 line. The complex obtained possessed low
cytotoxicity and high selectivity for hepatocytes, while the efficiency of gene transfection
increased with the degree of chitosan galacto-sylation.
It is worthy to note the studies on the properties of liquid-crystalline complexes formed
by chitosan with linear double-stranded molecules of nucleic acids (NA) of the B and A
families and with synthetic double-stranded polyribonucleotides (PRN) [140-142]. In the
reaction with NA molecules, chitosan converts the system into the liquid-crystalline state. Its
form depends on the molecular mass of chitosan and the values of chitosan DD, pH and ionic
strength of solutions.
The CS–NA complex was obtained by adding aqueous solutions of low-molecular-mass
fractions of chitosan (2 − 31 kDa) with different contents of amino groups (46% − 85%) to
the aqueous salt solutions of NA with stirring. Spectroscopy detected the formation of PEC
dispersions. Circular dichroism (CD) studies of the dispersions showed that the interaction of
chitosan with DNA resulted in the formation of liquid-crystalline dispersions with a
characteristic helically twisted (cholesteric) spatial structure. The classical cholesterics
formed by the phase exclusion of the double-stranded DNA molecules of the form B
exhibited an abnormal negative band in the CD spectrum, which pointed to the levorotary
form of the spatial cholesteric structure. The CD spectra of dispersions of the CS – DNA PEC
showed an abnormal positive band, which corresponded to the cholesteric dextrorotary
structure. PEC of chitosan and double-stranded DNA molecules of the form A, as well as
‘pure’ DNA molecules formed cholesteric dispersion with a levorotary spatial structure.
Studies of the formation conditions of CS–NA PEC liquid-crystalline dispersions showed that
a definite value of the molecular mass of chitosan is exist, starting from which the PEC
formed in a cooperative way. The abnormal optical activity of CS–DNA and CS–PRN PEC
dispersions depended differently on the molecular mass of chitosan. At pH near 6.8
(corresponding to the pKa of chitosan in aqueous salt solutions), the optimum cholesteric
packing of the CS–DNA PEC formed. Under the given conditions, the degree of ionisation of
chitosan was 50%. At pH < 7 and pH > 7, two types of CS–DNA liquid-crystalline
dispersions formed, which differed in the molecular packing; dispersions of the first type
were characterised by an abnormal positive band in their CD spectra, while dispersions of the
second type did not show any changes in the CD spectra. For each chitosan sample with a
definite DD, there exists a definite range of ionic strength values, within which the chitosan–
DNA PEC with an abnormal band in the CD spectra can form. The liquid-crystalline
dispersion was not formed outside these limits. Moreover, abnormal optical properties of the
liquid crystals in question depended on the distance between the amino groups in chitosan
molecules and the solution ionic strangth; varying these two parameters affords liquid-
crystalline dispersions that showed a negative or positive band in the CD spectra or did not
show any abnormal optical activity at all.
To evaluate the steric and energy characteristics of chitosan complexation with DNA in
solutions with variable ionic strength, a theoretical approach was developed, which allowed
the construction of a mathematical model of binding of these polyelectrolytes that adequately
described the experimental data [142]. The binding constant was calculated within this
approach and was shown to decrease when the solution ionic strength increased. Note that
treatment of the chitosan–DNA PEC dispersion with daunomycin (DAU), the molecules of
which intercalate between the DNA base pairs, led to the formation of a cholesteric
dispersion, in which the initial dextrorotary spatial structure changed to the levorotary
structure. However, despite the reversal of the sign of the abnormal band in the CD spectrum,
the X-ray parameters of the CS–DNA and CS–DAU–DNA PEC phases coincided; i.e., the
introduction of DAU in PEC did not affect the distances between DNA molecules in the
resulting dispersion.
The dependence of the optical properties of CS–DNA PEC liquid-crystalline dispersions
on a number of parameters makes it possible to use the optical properties of these PECs as
quality testers for chitosan samples employed in medicine and biotechnology. Studies of the
mechanism of the transiton of plasmid DNA to the liquid-crystalline state upon complexation
with chitosan and the properties of the resulting PEC with different twist of the cholesteric
structure for their future use for gene transfection are in progress.
3. Polyelectrolyte Complexes of Chitosan with Proteins
Association of protein and polysaccharide molecules through Coulomb and non-Coulomb

interactions leads to the formation of PEC [143]. Under certain conditions, proteins (natural
polyampholites) can show the properties of both polyacids and polybases; for complexation
with chitosan, the protein should act as an acid.
The PECs of completely deacetylated chitosan with bovine atelocollagen were obtained
[144, 145]. The structure of PEC in which co-polyelectrolytes were bound through
electrostatic interactions was proved by various methods. It was noted that under the
conditions chosen, gel formation of collagen competed with collagen complexation with
chitosan. It was impossible to choose the conditions that provided exclusively the ionic
complexation mechanism of the interaction of collagen with chitosan. At elevated
temperatures at which protein denaturing was induced, it was possible to approach the
synthesis of a stoichiometric polycation - polyanion complex. When chitosan was present in a
large excess, the complexation was dominant, but there was hydrogen bonding between the
protein and polysaccharide in addition to the electrostatic interaction. Biocompatible
membranes were prepared from nanofibres of the chitosan PEC with collagen [146] cross-
linked with glutaraldehyde and exceeded the widely used collagen sponges in their wound-
healing properties.
Chitosan PEC with legumin of fodder beans and of its partial trypsinolysis product,
legumin-T, were described [147, 148]. It was shown that the functional properties of plant
proteins could be regulated by complexation with charged polysaccharides; complexation
imparted solubility to the precipitated protein molecules near their isoelectric point, which
allowed control over the solubility of precipitated globulins in weakly acidic and weakly
basic media. Moreover, the formation of chitosan PEC with legumins considerably increased
the stability of protein emulsions.
Chitisan–gelatin PECs are the perspective biomaterials. Gelatin is obtained through a
controlled denature of the fibrous insoluble protein – collagen, which is the major component
of skin, bone and connective tissue. It is characterized of no antigenicity in comparision to
collagen.
Chitosan–gelatin polyelectrolyte complex formation was described in work [149] with
use of conductometric and pH titration. The chitosan interact with ampholitic gelatin (Gel)
electrostatically and the interaction mainly depends on net negative charge on Gel
macromolecular chains.
A combination of flexible protein and rigid polysaccharide results in biomimetic CS–Gel
based biomaterials, drug delivary systems, surface modifers, sponges and scaffolds for tissue
engineering and non-viral vectors for gene therapy [150-160].
Porous scaffolds, mono- and bilayer biomimetic structures were developed on the basis
of chitosan PEC with gelatin; due to their properties, they are recommended for use in tissue
engineering [150-152,155,158, 159].
An asymmetric CS–Gel scaffold was prepared via controlling both the freezing
temperature and heat transfer rate [150]. Bilayer scaffolds were fabricated with variance in
the mean pore size from 30μm to 250μm. Scaffold was used as a temple for fibroblast and
keratinocytes co-cultivation and provided suitable microenvironment for keratinocytes
growthing and differentiating. The presence of hyaluronic acid in scaffold enhanced its water
retention ability and elongation. Such scaffold implanted subcutaneously degradated at 4th
week in vivo.
In vitro it was shown [155] that gelatin-containing scaffolds had faster degradation rate
and significant loss of material than CS in the presence of lysozyme. Mechanical properties of
CS are affected by the addition of Gel althougt there was no clear trend. Three-dimentional
CS and CS–Gel scaffolds supported fibroblast viability equally. However, CS membranes
decreased cell-spreading areas, disrupted F-actin and localized FAK in the nucleus of
HUVECs. Importantly, the lowest shear stress tested (4.5dyn/cm2) for 3 h washed away cells
on CS suggesting weak cell adhesion. No significant differences were observed in PECAM-1
expression.
Tubular sandwich structures based on chitosan–gelatin (4: 1 by mass) PEC with a wall
thickness of 1mm and porosity of 81.2% were designed for tissue reconstruction of blood
vessels [160]. It was shown that vascular smooth muscle cells of blood vessels spread and
grew effectively on these polyelectrolyte structures.
Collagen-contained hybrid membranes [156] and scaffolds [161] were used with aim of
investigatin of cells adhesion, growth, proliferation and so on. The chitosan–collagen–gelatin
(CCG) membranes with surface micropatterns were fabricated by soft lithography [156]. The
C3A cells on the CCG membranes with micropatterns have higher metabolism and growth
rates than those on the flat CCG membranes and on T-flask disces.
Tanabe et al. [162] prepared chitosan–keratin PEC films having enhanced mechanical
properties (flexibility, elasticity and stability) compared with the properties of films from the
starting polyelectrolytes and swelling in acid, neutral, and alkaline media (this was shown for
pH 4.0, 6.3 and 8.9) due to the properties of the biopolymers of the PEC, namely, the acidic
protein keratin having pI 4.9 − 6.1 and precipitating in acidic media and the basic
polysaccharide chitosan having pK 6.3 and precipitating in alkaline media. Chitosan–keratin
films have an antibacterial activity inherent in chitosan films, stimulate the attachment and
proliferation of fibroblasts and serve as substrates for mammalian cells.
Self-assembled polyelectrolyte nanocomplexes (size range 200-500 nm) between
methylated or PEGylated chitosan and insulin were prepared, and parameters influencing on
complex formation were characterized [163]. Turbidimetric titration in combination with
dynamic light scattering and laser Doppler anemometry were used to study the complexation
process; the morphology of the PECs was determined using atomic force microscopy. Soluble
PECs with spherical or subspherical morphology and smooth surface structure were obtained
at pH 6.5−8.0 and MW of the polymers less or equal 25 kDa. PECs were stable and could
protect insulin from degradation even at 50o C for at least 6h. All complexes could be
lyophilized without influencing the particle size and stability of insulin.
A method for selective removel of undenatured β-lactoglobulin from cheese whey was
described [164]. At pH 6.2, addition of 1.9 to 3.0 mg/ml of chitosan led to complete removal
of β-lactoglobulin, ehereas at least 80% of the rest of whey proteins remained in solution. The
production of cheese whey without β-lactoglobulin could help to expand the applications of
dairy by-products in food processing and to isolate hypoallergenic whey protein concentrates.
The complexation of chitosan and musin was described in works [165, 166].
Mucins representatives of a special subclass of high-molecular-mass glycoproteins with
high carbohydrate content (up to 50% − 80% by mass). They possess branched
oligosaccharide chains linked by the O-glycosidic bonds with the protein moiety of the
macromolecule. Mucins are characterised by high content of such amino acids as serine,
threonine and proline and such monosaccharides as D-galactose, L-fucose, N-
acetylglucosamine and N-acetylgalactosamine as the main carbohydrates and sialic acids. The
monosaccharide residues form oligosaccharide chains linked to the protein by the O-
glycosidic bond of N-acetyl-galactosamine and the hydroxyl group of the side chain of serine
or threonine [165].
The effects of the molecular mass of chitosan (70, 750, 2000 kDa), pH (2.9 − 5.0) and
ionic strength (0.01 – 1.0M NaCl) of a medium on the complexation with bovine
submaxillary mucin (400 kDa) containing 12% sialic acid residues were studied by
fluorescence polarization [166]. Dextran sulfate or albumin was used as reference co-
polyelectrolytes instead of mucin. For fluorescent polarisation studies of the molecular
mechanism of the interactions of chitosan with mucins, FITC-labelled chitosan (FITC−CS)
was synthesized. For FITC−CS complexes with mucin, the degree of polarisation increased
with the mucin: chitosan molar ratio and the solution ionic strength. For example, at a ratio of
mucin: FITC−CS = 19: 1 (750 kDa) and pH 2.9 (0.1M acetic acid), the degree of polarisation
increased by 61%. In addition, it was found that the increased molecular mass of chitosan, as
well as the increased mucin content in the complex, also led to the increased degree of
polarisation. Thus, the number of sites capable of adding mucin increased with the molecular
mass of chitosan. It was assumed that the chitosan molecule might have more than one mucin
molecule bound to it, while binding of chitosan to dextran sulfate occurred according to the
‘one-to-one’ principle, and complexation of chitosan with albumin did not occur at all under
these conditions. The maximum change in the degree of polarisation in the interaction of
chitosan with mucin was observed at lower pH (to pH 2.9 where the degree of ionisation of
chitosan was almost 100% and the degree of ionisation of sialic acid residues was the least,
20%), which points to the dominant effect of chitosan on the interaction with mucin.
These studies allowed the researchers to conclude that the binding of two polymeric
molecules involved not only electrostatic interaction between the D-glucosamine residues of
chitosan and the sialic acid residues of mucin, but also hydrogen bonds and hydrophobic
interactions.
Qaqish and Amiji [166] intended to use chitosan - mucin complexes for the creation of
pH-sensitive drugs for localised suppy of drugs to the alimentary canal.
IV. POLYELECTROLYTE COMPLEXES OF CHITOSAN

WITH SYNTHETIC POLYANIONS
In the field of chitosan PEC with synthetic polyacid anions, the largest number of
publications was devoted to chitosan PEC with polyacrylic acid (PAA) [48, 49, 167-185].
Insoluble PECs of chitosan (220 kDa) with PAA (202 kDa) of different compositions
obtained at pH 3 − 6 were studied [168]. It was found that the solution ionic strength in the
range 0.025 − 0.3 did not affect the formation of PEC. At pH < 3, insoluble chitosan–PAA
PECs were not formed. The pH value affected the ionisation of the reactant (charge density
distribution along the chain) and hence the composition of PEC. The content of
polysaccharide in the insoluble fractions of PEC increased with pH. Thus, PEC with
controllable composition and properties could be prepared with consideration of the
tendencies found for complexation.
The formation and structure of chitosan PEC (190 kDa, DD 80%) with PAA (500 kDa)
was reported [169]. The dependences of the degree of complexation and the composition of
PEC on pH were studied by potentiometric titration. It was shown that insoluble PEC
enriched in PAA formed at low conversion. As the reaction proceeded further, PEC particles
became enriched in chitosan; in neutral media, stoichiometric PECs with quantitative amounts
of both polyelectrolytes were formed. In alkaline media, the complex decomposed with
liberation of polyacrylate anions from PEC particles and isolation of chitosan-enriched
insoluble PEC fraction.
The behaviour of the PEC of water-soluble chitosan (DD 50%) with PAA was described
[170]. Hydrodynamic studies showed that an increase in the chitosan fraction in PEC led to
compaction of complex particles and hence to a decrease in the intrinsic viscosity of
solutions; the minimum viscosity corresponded to the composition [CS]: ([CS] + [PAA]) = 1:
2. In acidic media (at pH < 2), PEC completely dissociated into the starting polyelectrolytes;
in basic solutions (at pH > 8), optical transmission decreased from 100% to 70% because of
partial precipitation of chitosan, which is characteristic of the behaviour of water-soluble
chitosan PEC. It was emphasised that hydrogen bonding also made a considerable
contribution to complex stabilisation along with electrostatic interactions, which played the
major role in the formation of PEC.
One of the major applications of CS–PAA PEC is the reparation of semi-permeable
membranes, which are widely used in food industry, medicine (in blood, plasma and blood
substitute purification), pharmaceutical industry, waste treatment, oil products purification,
etc. Semi-permeable membranes possess high separating ability (selectivity), high
permeability, stability against the action of the medium being separated and sufficient
mechanical stability. Note that semi-permeable membranes can serve as models of biological
membranes characterised by high biological and chemical selectivity; therefore, their studies
are also important for scientific purposes.
The structure of CS–PAA PEC’s films, their mechanical and physicochemical properties,
as well as the PECs potential applications as membranes for pervaporation, sensors,
membranes for fuel cells, ets. were described [48, 49, 171-173, 181-183]. Studies of the
mechanical properties of CS−PAA films showed [172] that tensile strength increased, while
the relative lengthening during sample destruction decreased with respect to those of the
starting polymer films. This result is explained by ionic cross-linking between the
macromolecular chains in PEC. The lability of the PEC obtained at different polyelectrolyte
ratios (depending on the pH of the medium) was evaluated from the change in the degree of
swelling of films and IR spectroscopic data. Thus for CS: PAA = 1: 1 PEC, swelling
increased at pH 3 and 9 and was minimum at pH 7. In the IR spectra of films after swelling at
pH 3, the characteristic absorption band of the carboxyl group of PAA (1610 cm- 1) vanished;
at pH 9, the band corresponding to the vibrations of the carboxylate ion of PAA was observed
at 1590 cm- 1; and at pH 7, the absorption peak characteristic of the C=O stretching vibrations
shifted because of the formation of the CS–PAA complex. The pervaporation properties of
the obtained membranes were studied using various aqueous-organic mixtures; it was shown
that the efficiency of pervaporation depended on the PEC composition [49, 172]. The sample
films of chitosan (190 kDa, DD 80%) PEC with PAA (200 kDa) of different compositions
(CS: PAA ratio 0.25 − 0.67) were obtained by casting from formic-acid solitions of polymers
(pH 2). The properties of pervaporation membranes prepared, which were designed for
separating water - organic mixtures were studied [49]. The mechanical stability of films was
found to increase as PEC was enriched in rigid chains of chitosan. In contrast to PEC based
on flexible-chain synthetic polymers, the films retained their stability in neutral and weakly
alkaline media and demonstrated high transport and sorption characteristics. Materials with
these properties are extensively used for the design of systems with controllable release of
drugs in pharmacology, for the creation of constructions with definite chemomechanical
properties [143, 174, 180-184], etc.
Wang et al. [173] obtained PEC by mixing equimolar amounts of an acetic acid solution
of chitosan (300 kDa, DD 86%) with aqueous solution of PAA (230 kDa). After covalent
cross-linking with glutaraldehyde, pH-sensitive membranes were obtained from PEC. A
series of tests showed that the starting CS–PAA complex dissociated at pH < 2 and
manifested high swelling at pH > 7. At the same time, the degree of swelling of CS–PAA
membranes was high at pH < 2, decreased drastically as pH increased to 8 and then increased
to a maximum at pH 11. Swelling of membranes in salt solutions increased with the ionic
strength and depended on the valence of cations at constant values of the ionic strength; i.e., it
was maximum in solutions of trivalent metal salts (Me = Al) and gradually decreased on
passing to divalent (Me = Ca, Mg, etc.) and univalent (Me = K, Na) metals. Note that the
mechanical properties of membranes became reversible when they were treated alternately
with KCl and CaCl2 solutions at equal ionic strengths; when membranes were treated with a
CaCl2 solution, their size increased, but diminished after treatment with KCl solution. This
behaviour is determined by the ion-exchange properties of PAA.
Of particular note are works where PECs were obtained by the free-radical
polymerisation of ionised monomers on a polymer matrix [176-179]. For example, ‘self-
polymerising’ CS–PAA membranes were prepared using free-radical polymerisation of
acrylic acid on a chitosan matrix at moderate temperatures (37o C) [170]. The structure of
polyelectrolyte membranes was confirmed by IR spectroscopy. The degree of swelling of
membranes was found to depend on pH and composition; it increased with pH (from 5 to 10)
and the PAA content in PEC. Thermogravimetric studies showed that interchain amide bonds
formed in membranes at high temperatures (starting from 100o C). Subsequent thermal
treatment at elevated temperatures led to further dehydration of ionic bonds between the
chitosan and PAA macromolecules in PEC, mass loss and eventually PEC degradation.
CS–PAA composite nanofibrous membrane with 3D network nano-structure was
prepared using an electrostatic process by adding succinic acid as branch promoter [181]. The
smallest fiber diameter size distribution of the scaffold can be obtained when the PAA/CS
ratio was in the range of 2:1 − 1:2 in a pH 3 environment. Negative charge nanofibers
prepared using PAA/CS ratio 2:1 and pH 3 had an average diameter of 215 nm. The
formation of the interconnecting 3D self-organized network structure can be build up with
limited parasitic branching by crystallized siccinic acid. The results of sensing experiments
indicated that the sensitivity of nanofibrous membrane-coated sensors was eight times higher
that of continuous film-coated sensors. Sensitivity towards ammonia was 50 ppm at a relative
humidity of 45%.
PEC CS–PAA was used as proton exchange ionically cross-linked membranes for fuel
cells [182]. Membranes were characterized for morphology, their intermolecular interactions,
thermal stability, and physicomechanical properties using SEM, FTIR, DSC, sorption studies,
and tensile testing, respectively. Among the blends synthesized, the membrane blend with 50
wt. % of CS and 50 wt. % of PAA, was identified as ideal for direct methanol fuel cell
applications as it exhibited methanol permeability 3.9×10-8 cm2/s, excellent
physicomechanical properties and comparatively high proton conductivity (0.038 S × cm-1).
PEC CS–PAA membranes [183] and hydrogels [184] with high content of chitosan were
used as drug release systems. Hydrogels with CS/PAA ratio 1:0.25 showed greater
mucoadhesive property and maximum swelling in 0.1 N HCl [184] and complete release of
amoxicillin and metronidazole in 10h. Permeability studies in relation of CS–PAA PEC
membranes to a drug model (sodium sulphamerazine) were discussed in work [183]. Two
membranes with CS/PAA ratio 95:5 were obtained: one prepared using PAA solution in 3.5%
formic acid (CS–PAA 3.5) and another one using a PAA solution in 10% formic acid (CS–
PAA 10). Water uptake results showed that PEC membranes were more hydrophilic than pure
chitosan. Permeability values decreased with complex formation and were lower for CS –
PAA 10 than CS–PAA 3.5. Diffusion coefficient values indicated that CS–PAA 3.5 had a
higher drug retention capacity than CS-PAA 10.
For complexation with chitosan besides of proteins may be also involved synthetic
polyamino acids [132, 186-189]. Poly(aspartic acid), recognized as green material, widely
applied in agriculture, medicine, etc [188, 189]. Nonstoichiometric polyelectrolyte complex
nanoparticles were prepared based on chitosan and polyaspartic acid sodium salt (PAsp). The
physicochemical properties of the complexes were investigated by means of turbidity,
dynamic light scattering, transmission electron microscopy and zeta potential. The results
indicated that the slow dropwise addition of chitosan solution into PAsp one allowed
elaborate either anionic or cationic particles in the size range of 85-300 nm with proper CS
and PAsp unit molar ratios; microstructure of the nanoparticles depended strongly on the unit
molar ratio. Nanoparticles containing 5-fluorouracil were prepared by mixing and absorption
method. In vitro and in vivo experiments indicated that drug-loaded nanoparticles presented a
sustained release of drug compared to the 5-fluorouracil solution and the areas under curve
were increased by about fout times. Polyelectrolyte complex hydrogel composed of chitosan
and poly(γ-glutamic acid) was prepared for biological application, physical properties and
cytocompatibility were investigated [186]; poly(γ-glutamic acid) – chitosan/DNA complex
nanoparticles [132] were described in Part 2.
Other synthetic polyanions such as polyvinyl sulfate [120,190], polystyrene sulfonate
[191, 192] and poly(2-acrylamido-2-methylpropansulfonic acid [193] were also introduced in
complexes; the conditions influencing on complexes formation were discussed [190, 192].
The water-soluble nonstoichiometric polyelectrolyte complexes have been first prepared via
the interaction of chitosan with excess of polystyrene sulfonate anions in acid media [192].
The region of existence of soluble complexes can be narrowed down through a decrease in
the degree of polymerization of polyanion until fully degenerates as in the case of oligomeric
anions.
In the work [193] it was found that enzymatic chitosan hydrolysis was not suppressed by
combining it with poly(2-acrylamido-2-methylpropansulfonic acid. The results on the soil
fungus Trichoderma viride immobilized on beads imply that chitosan, as well as polymer
complexes are appropriate carriers for development of ecologically safe phytosanitary
preparations.
Complexation of chitosan with tripolyphosphate (TPP) was described [194-196], and
PECs were used for immobilisation of enzymes such as lipases [194, 195]. Lipases are widely
employed in biotechnology for separation of enantiomers, hydrolysis of fats to fatty acids,
catalysis of cleavage of glycerol esters at the lipid - water interface, etc. The applications of
these enzymes are limited by their instability and narrow range of the conditions (pH and
temperature) of their biocatalytic activity. Lipases can destroy the outer layer of the microbial
cell membranes. However, their shell contains waxlike lipids, and hence phagocytosis of
bacteria cannot be affected by macrophages. One of the solutions to this problem is
immobilisation of lipases on polymer carriers capable of penetrating through the cell barrier.
For this purpose, ionic gelation of chitosan solutions with tripolyphosphate was performed to
obtain microcapsules based on the chitosan PEC used for immobilisation of lipase from
Candida rugosa. It appeared that the enzymatic activity of the lipase enclosed in chitosane
microcapsules was fivetimes higher than the activity of the enzyme in alginate microcapsules.
The molecular mass of the chitosan samples used for microencapsulation did not substantially
affect the enzymatic activity of the enzyme. For maximum efficiency of lipase liberation from
capsules, the DD of chitosan should be >90%.
It was demonstrated [196] that polyanion initiated gelation process in fabrication CS–
TPP nanoparticles in the size range of 100−250 nm intended to be used as carriers for the
delivery of gene or protein macromolecules. The critical chitosan concentrations for
spontaneous formation of large particle aggregates were found to be 0.65%, 0.25%, 0.15%
(w/v) at pH 4.0, and 1.00%, 0.85%, 0.75% (w/v) at pH 5.0 for LMW, MMW and HMW
chitosan, respectively. The isoelectric point of the CS–TPP nanoparticles was found at around
pH 9.0. Morphological study of the nanoparticles formed under different conditions revealed
the formation of regularly shaped polyhydrone particles (pentagon and hexagon), indicating a
similar crystallization mechanism during the particle formation and growth process. The
bovine serum albumine incorporated particles had a size range of 300-350 nm, doubling the
size of CS–TPP particles. The protein incorporated particles possessed a tipical spherical
shape and smooth surface.
Interpolymer complexes of chitosan with poly(metacryloylamidobenzoic acid) were
described [197]. They were obtained by mixing equimolar amounts of polyelectrolytes with
subsequent evaporation of solvents. Further thermal treatment of these complexes at 120oC
led to the formation of covalent interchain amide bonds. The complexes had physicochemical
properties typical of cross-linked gels, in particular, their degree of sweeling correlated with
the degree of dehydration of the system in the course of thermal treatment.
The PECs of chitosan and alkylene oxide (ethylene oxide or copolymer of ethylene oxide
with propylene oxide) co-polymers with maleic acid were obtained [198-200] and used to
obtain pH-and thermally labile films (on-off switches) for selective transport of drugs [198,
199]. These films swell at low pH and temperatures and are capable of reversibly and rapidly
changing the degree of swelling in response to the change in the ambient pH and temperature.
The introduction of model drugs (salicylic acid, phenol and glucose) into films and their
isolation from the latter, depending on the pH (3.8, 6.2, 7.2) and temperature (25 and 50o C),
as well as the molecular mass of a drug, were examined. Upon increase in temperatures,
decrease in pH from 7.2 to 3.8, and decrease in molecular mass of model substances, the rate
of their penetration in films increased, while the rate of release of the drugs increased with pH
and temperature. This effect was explained by an increase (with pH) in the electrostatic
repulsion between the carboxylic groups of the copolymers of alkylene oxide with maleic
acid and the anionic groups of the model compounds (phenol or salicylic acid), and also by an
increase in the diffusion coefficient. Moreover, the rate of liberation of active substances
depended on the microscopic structure of films and the degree of hydrophobicity of alkylene
oxide copolymer samples.
Stoichiometric and nonstoichiometric PECs were prepared with polyethylene glycol-
monosuccinate (1:1 mol. ratio) and chitosan [201]. The physicochemical properties of the
synthesized PEC’s were characterized by using elemental analysis, FTIR, H-1 and C-13
NMR, dissolution behavior, and phase transition phenomenon. Furthermore, some properties
of the PECs obtained were analyzed by UV-visible spectrometry, wide-angle X-ray
diffraction, DSC, SEM and estimated solubility, and cell viability assay, respectively. The
study of MTT assay suggested that the viability of HepG2 human hepatoblastoma cells on
PEC were increased significantly in accordance with mole ratio of CS.
Table. Use of chitosan PEC in medicine and biotechnology
Application Polyanion in PEC Ref.

Specific (bio)sorbents Copolymers of maleic acid with N- 202, 204,205
vinylpyrrolidone, styrene, or ethylene
Alginate 61
Immobilisation of BAC* Antigenes 206, 207
according to the β -Lactoglobulin 164
polyelectrolyte interaction Insulin 163
principle Tripolyphosphate 196
Films and pervaporation Poly(4-styrene sulfonate) 73,191
membranes Sodium alginate 75, 209
Pectin 78-80
Carboxymethylchitin 47
Polyacrylic acid 48, 49, 168-173
Carrageenan 101
Hemocompatible and Copolymer of N-vinylpyrrolidone with Nα-
thromboresistant materials lysine monomaleamide 204, 205
Carboxymethyldextran 112, 210
Carboxymethylcellulose 88
Dextran sulfate 110
Substrates for BAC or drugs Dextran sulfate 120
immobilisation with selective Tripolyphosphat 194, 195
and prolonged transport of Alginic acid 58, 59, 66, 75, 94, 96-98,
BAC or drugs 105, 106, 119
Pectin 95, 99, 82, 99
Carrageenan 96, 102-104
Carboxymethylglucomannan 92
Mucin 165
Alkylene oxide - maleic acid copolymer 198, 199
Polyvinyl sulfate 120
Heparin 120
Poly(ethylene glycol)/heparin 38
Hualuronic acid 35, 40
Xanthan 113,116
Gum komdagogu 93
Gelatin 155,157
Collagen/hyaluronan 153
Poly(acrylic) acid 183,184
Poly(γ-glutamic) acid 186
Polyaspartic acid 187
Application Polyanion in PEC Reference
Tissue scaffolds, wound Glycosaminoglycans 27,30-33,42-46,211-213

dressing materials, Gelatin 151, 152,158-160
bioconstraction materials for Keratin 162
replacement of coverlets, Carboxymethylcellulose 89
blood vessels, bone tissue, Collagen 161
pulp cells regeneration DNA 128
Alginate 69, 107
Alginate/heparin 65
Substrates for cell Alginic acid 63, 72, 215

immobilisation or cell Poly (γ−glutamic acid) 215
adhesion Carrageenan 215
Carboxymethylcellulose 215
Phosphorylated chitosan 52
Xanthan 115
DNA 135
Gelatin 154
Collagen/gelatin 156
Polyethyleneglycol-monosuccinate 201
Non-viral vectors of genetic DNA 129-131,137-139, 214

information transfer
Complexation to reduce Endotoxin 122-125
toxicity
* − biologically active compounds.
The conditions of synthesis, as well as the properties and structure of chitosan PECs with
polyanionic co-polymers based on maleic acid were studied by Samoilova et al. [202-205]. N-
Vinylpyrrolidone, styrene and ethylene were used as comonomers of maleic acid. The
complex was also modified by introducing a modified polymer derivative, namely, the
copolymer of N-vinylpyrrolidone with monomaleamide containing an amino acid residue of
lysine. New approaches were suggested to the preparation of biospecific polymeric materials
(affinity sorbents for the isolation of wheat germ lectin [202], thromboresistive coatings for
devices contacting blood [205]).
The choice of maleic acid copolymers as co-polyelectrolytes of chitosan for the
preparation of PEC was dictated by the regular structure of these polymers (strictly
alternating monomer units) and the possibility of transforming maleic acid residues into
reactive anhydride groups, which react (under mild conditions) with amino- and hydroxyl-
containing ligands, thus providing easy modification of the polyacid before or after
complexation. The above properties are not inherent in the chitosan co-polyelectrolytes
described in the literature. Moreover, it is certainly of interest to study the formation of these
PEC at the molecular (polymer unit) level, taking into account interatomic distances in
reactant groups and the conformations of the polymers used [203].
CONCLUSIONS
This review convincingly demonstrates that chitosan-based PECs are of great practical
value and widely used in scientific research, medicine, biotechnology, food and textile
industries, etc. The major medical and biotechnological applications of these PECs are
summarised in Table.
Chitosan PECs with a wide range of polyanions of natural and synthetic origin have been
studied. It was found that complexation between oppositely charged polyelectrolytes is a
complicated process, which depends on many parameters. It should be emphasised that
chitosan PECs generally have different types of binding, e.g., hydrogen bonding and
intermolecular hydrophobic interactions in addition to electrostatic interactions.
The structure and properties of chitosan PECs depend on the nature and stucture of
polyanions (molecular masses, conformations of macromolecules, charge density and
distribuiton of ionised groups along the polymeric chain), and also on the conditions of PEC
formation (pH and solution ionic strength, temperature, concentration, and polymer ratio).
Soluble or insoluble and stoichiometric or non-stoichiometric PECs are formed at certain
combinations of the above factors.
By varying the type of co-polyelectrolyte and the conditions of the polyelectrolyte
reaction, one can obtain chitosan PEC in the form of gels, nano- and microparticles, films,
membranes, porous structures and liquid-crystalline dispersions. PECs with natural anionic
polyelectrolytes are best studied, but PECs with synthetic polyanions, including block
copolymers, are also of great interest because they expand the possibilities for selectively
adjusting the properties of the obtained PECs.
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Chapter 5
N-CARBOXYETHYLCHITOSAN-BASED
POLYMER MATERIALS
Dilyana Paneva,1 Rosica Mincheva,2 Elena Yancheva,1

Nevena Manolova,1 Olya Stoilova,1 Philippe Dubois,2
and Iliya Rashkov1∗
1
Laboratory of Bioactive Polymers, Institute of Polymers,
Bulgarian Academy of Sciences, Sofia, Bulgaria
2
Laboratory of Polymeric and Composite Materials,
University of Mons-UMONS, Mons, Belgium
ABSTRACT
N-carboxyethylchitosan (CECh) is a chitosan derivative. It preserves the valuable
properties of its precursor such as biocompatibility and biodegradability. Moreover,
CECh possesses some advantages such as larger variety of functionalities and solubility
in neutral and alkaline medium. The gained up to date knowledge on the synthesis of
CECh, its solution properties and biological behavior in respect to cells and pathogenic
microorganisms are emphasized in the chapter. Due to its amino- and carboxyl groups
CECh behaves as a polyampholyte/polyzwitterion in aqueous solutions. This chitosan
derivative self-assembles into nanoparticles in a pH range close to its isoelectric point.
The conditions (pH of the medium, ionic strength and polymer concentration) and CECh
molecular weight for nanoparticles preparation are described in details. Electrospinning
represents a unique tool to produce nanofibrous materials that resemble the extracellular
matrix and thus are promising for wound healing and tissue engineering applications. The
fabrication of CECh-containing nanofibrous materials by electrospinning is described in
the chapter. Similarly to the case of chitosan, the electrospinning of CECh from its
aqueous solutions is rendered feasible by the presence of a non-ionogenic polymer. In
addition, CECh has been used for preparation of organic/inorganic hybrid nanofibers
containing superparamagnetic iron oxide or silver nanoparticles by combination of the
sol-gel technique and electrospinning. Some properties and possible applications of these
∗ Corresponding author’s e-mail: rashkov@polymer.

262 Dilyana Paneva, Rosica Mincheva, Elena Yancheva et al.
hybrids are detailed. As a polyampholyte CECh forms polyelectrolyte complexes (PECs)

with polyacids and polybases. The pH-dependent formation of PECs; the preparation of
hydrogel materials; as well as nanoparticles from CECh-based complexes are discussed
in the Chapter. Finally, the design of non-woven textiles by combining PEC formation
and electrospinning is reported.
LIST OF ABBREVIATIONS
αeq equilibrium water content
2-Cl-PA 2-chloropropionic acid
3-Х-PA 3-halopropionic acid (X = Cl, Br, I)
AA acrylic acid
AFM atomic force microscopy
AFS applied field strength
AgNPs silver nanoparticles
AMPS 2-acrylamido-2-methylpropanesulfonic acid
ATRP atom transfer radical polymerization
CECh N-carboxyethylchitosan
D diffusion coefficient
DDA degree of deacetylation
Dh hydrodynamic diameter
Dh,app apparent hydrodynamic diameter
DLS dynamic light scattering
DS degree of substitution
FCECh molar fraction of CECh-units
FDMAEMA molar fraction of DMAEMA-units
Fe3O4 magnetite
FF(Fe3O4) ferrofluid of magnetite (Fe3O4)
FF(Fe3O4×CECh) ferrofluid of Fe3O4 prepared in the presence of CECh
GA glutaraldehyde
HMMCECh high molecular weight CECh
I ionic strength
IEP isoelectric point
LMMCECh low molecular weight CECh
PAA poly(acrylic acid)
PAAm polyacrylamide
PAMPS poly(2-acrylamido-2-methylpropanesulfonic acid)
PDMAEMA poly[2-(dimethylamino)ethyl methacrylate]
PDMAEMAQ100 fully quaternized PDMAEMA
PDMAEMAQ50 50% quaternized PDMAEMA
PEC polyelectrolyte complex
PEG poly(ethylene glycol)
PEO poly(ethylene oxide)
PLLA poly(L-lactide)
N-Carboxyethylchitosan-Based Polymer Materials 263
PVA poly(vinyl alcohol)

q2 absolute value of the scattering vector
SEM scanning electron microscopy
TEM transmission electron microscopy
WAXRD wide angle X-ray diffraction
INTRODUCTION
Chitosan is a deacetylated derivative of chitin which is the second in abundance
renewable biopolymer after cellulose. Chitin is a basic structural component in the
crustaceans’ exoskeleton, in insects’ shells, as well as in the cell walls of some fungi and
bacteria. Since chitosan is an enzymatically biodegradable, biocompatible and non-toxic
polymer much interest has been paid to its biomedical, agricultural, environmental, food
processing and other industrial applications during the past two decades. Regarding particular
applications chitosan is not a suitable candidate due to its insolubility in aqueous solutions at
pH values higher than 6.0. One of the most applied approaches for imparting chitosan water-
solubility in neutral and alkaline media is its modification into carboxyethylated derivatives.
In contrast to carboxymethyl- and carboxybuthylchitosan derivatives, the data on N-carboxy-
ethylchitosan (CECh) has been limited up to the recent few years. Despite its main advantage
to be soluble in neutral and alkaline media CECh has additional beneficial properties. The
presence of carboxyl groups in its structure together with amino groups is a prerequisite this
chitosan derivative to self-assemble in aqueous solutions at pH values near its isoelectric
point giving credit to biopolymer nanoparticles preparation. Similarly to chitosan, CECh
preserves its ability to form polyelectrolyte complexes with polyanions in acidic medium. A
peculiarity of CECh is that it can form polyelectrolyte complexes also with polycations in
neutral and alkaline medium. In very recent studies the polyelectrolyte complex approach has
been successfully applied for the design of pH-sensitive hydrogels, core-shell nanoparticles as
well as for modification of the surface biological behavior of non-woven textiles prepared by
electrospinning. The latter technique being considered as the most promising one for
fabrication of 3D-scaffolds for wound healing dressings as well as for cell and tissue
engineering scaffolds has also been applied for preparation of CECh-containing nanofibrous
membranes by electrospinning of common solutions of CECh and a non-ionogenic polymer.
Besides, CECh is a suitable polymer for stabilizing dispersions of inorganic nanoparticles,
such as superparamagnetic iron oxide or silver nanoparticles. This benefit of the natural
polymer has been utilized for preparation of hybrid nanofibrous materials by means of
electrospinning.
In the present chapter, the synthetic routes for modification of chitosan into CECh are
outlined. Special attention is focused on the physicochemical characteristics and the
biological behavior of this modified natural polymer. The gained up to date knowledge on the
preparation of CECh-based nanofibrous materials by electrospinning is summarized. The
application of the polyelectrolyte complex formation as a tool for tailored design of diverse
polymer materials from CECh is discussed in detail.
1. SYNTHESIS AND PROPERTIES OF

N-CARBOXYETHYLCHITOSAN
This Section deals in brief with the methods for CECh synthesis and the available data on
the physicochemical characteristics and the biological behavior of this modified natural
polymer.
1.1. Synthesis of N-Carboxyethylchitosan (CECh)
Until 2003, the studies on CECh synthesis were limited. In 2003 Skorik et al. [1] and
Sashiwa et al. [2] reported two procedures for CECh synthesis: (i) alkylation of chitosan by 3-
halopropionic acid (3-Х-PA; X = Cl, Br, I); (ii) alkylation of chitosan by acrylic acid (AA)
via Michael reaction.
1.1.1. Synthesis of CECh through Alkylation of Chitosan by 3-Halopropionic Acid

Hayes [3] has carried out alkylation of chitosan by chloroacetic acid in strong alkaline
medium and has obtained N,O-carboxymethylchitosan. Similarly, Shigemasa et al. [4] have
alkylated chitosan by 2-chloropropionic acid (2-Cl-PA) in strong alkaline medium and
introduced 2-carboxyethyl groups. They have synthesized N,O-(3-O, 6-O)-carboxyethyl-
chitosan. Orienti et al. [5] have reported the synthesis of CECh with a degree of substitution
(DS) of 0.43 through alkylation by 3-bromopropionic acid in a mixed water/pyridine solvent.
The first regioselective alkylation of the chitosan amino groups by 3-Х-PA has been
performed by Skorik et al. [1]. The reaction has been conducted in mild alkaline aqueous
medium (pH = 8 - 9, NaHCO3) at 60°C. Low molecular weight chitosan with an acetylation
degree of 0.08% has been subjected to alkylation by halogenated acids Х-CH2CH2COOH
(where X = Cl, Br and I) (Scheme 1). The 1H NMR analyses have indicated mono- and
disubstituted amino groups. No O-alkylation has been detected. The impact of the reaction
time on the degree of carboxyethylation of chitosan is presented in Figure 1. Initially (until
the 40th h), DS substantially increases with reaction time. The alkylation rate corresponds to
the reactivity order of the alkylating agents: Cl < Br < I.
Scheme 1. Carboxyethylation of chitosan with 3-halopropionic acids. Different residues are randomly
distributed. Reproduced from Skorik et al. [1] by permission of Elsevier.
Figure 1. DS values of 2-carboxyethyl groups as a function of the reaction time of chitosan with 3-Cl-
PA (1), 3-Br-PA (2) and 3-I-PA (3). The DSs were determined by 1H NMR spectra. Reproduced from
Skorik et al. [1] by permission of Elsevier.
After that the carboxyethylation degree of chitosan stabilizes and inversion of Cl and Br
reactivity is detected. The authors have made the assumption that a competitive reaction
proceeds concurrently with substitution: elimination of НХ from the alkylating agent leading
to AA formation. The rate of the elimination reaction slightly depends on the nature of the
halogen atom and follows the hydratation ability. The obtained AA is capable of reacting with
chitosan amino groups, thus increasing DS. The following advantages of the method have
been emphasized: (i) the alkylation regioselectivity with respect to the amino groups of
chitosan is 100%; (ii) DS can be tuned depending on the reaction time. As most efficient
alkylating agent, Cl-CH2CH2COOH has been pointed out. Another asset of this acid is its
lower price. However, this approach for CECh synthesis is applicable only to chitosans which
are soluble at pH ≥ 7 (low molecular weight chitosans or chitosan oligomers).
H2C CH
OH OH NH2
NH2
O O COOH
O HO O HO
HO O O HO O O
NH2 U/H2O
NHCOCH3 OH OH
CH2COOH HOOCH2C
OH H2C NH OH H2C CH2CH2COOH
N
O HO O O HO
O
HO O O HO O O
NHCOCH3 NH2 OH
OH
Scheme 2. Synthesis of N-carboxyethylchitosan via Michael reaction.

2666 Dilyana Paaneva, Rosica Mincheva, Ellena Yanchevaa et al.
1..1.2. Synthesiis of CECh th hrough Alkyllation by Acryylic Acid via Michael Reaaction
Another method
m for regiioselective synnthesis of CEECh applicablee to chitosans of various
m
molecular weigghts has been proposed
p by Sashiwa
S et al. [2,6,7].
[ Originnally, they havve used AA
essters (methyl or ethyl acryylate) for alkyylation of chiitosan [6,7]. However,
H thiss approach
reequires hydroolysis of the ester groupps to carboxyyl ones. Furrther, the autthors have
deemonstrated thhat AA is an appropriate reeagent for seleective carboxyyethylation off the amino
grroups of chito
osan (via Michhael reaction) [2]. Moreoveer, AA as a prroton donor iss a suitable
aggent for disso
olving chitosann. The CEChh synthesis byy direct carboxxyethylation of o chitosan
w AA is show
with wn in Schemee 2.
Fiigure 2. 1H NM
MR (A) and 13C NMR
N (B) spectra of CECh. Reeproduced from
m Mincheva et all. [8] by
peermission of Elssevier.
Figure 3. IR spectra of CECh thin films cast from aqueous solutions of CECh at different pH values.
Reproduced from Mincheva et al. [8] by permission of Elsevier.
Commercial chitosans with degree of deacetylation (DDA) varying from 60 to 95% and M w
values in the range from 68 000 to 530 000 g/mol have been used [2]. DS increases upon
increasing AA content in the reaction mixture, as well as upon increasing reaction
temperature and time. The obtained results confirm the applicability of CECh synthesis via
selective carboxyethylation of the amino groups of chitosan with AA to chitosans of various
molecular weights and DDA value, and therefore this method is the preferred one [8-13]. The
use of water as a solvent is an additional advantage. Similarly to the studies performed by
Sashiwa et al. [2], the highest degree of substitution (73 - 76%) has been achieved at
[aminoglucoside units]/[AA-units] molar ratio of 1/2, 90ºС and reaction time - 24 h [8-10].
Mincheva et al. [8] have demonstrated that the alkylation by AA leads to the formation of
mono- and disubstituted aminoglucoside units. The 1Н and 13С NMR spectra of CECh with a
total DS of 73% are presented in Figure 2. In the 1H NMR spectrum (D2O) the following
chemical shifts are observed (Figure 2A) (δ, ppm): 4.52-5.09 (H-1), 3.19 [H-2 of N-alkylated
aminoglucoside units (Glc-NH-CH2CH2COOH)], 3.45-4.15 [H-3, H-4, H-5 and H-6 of N-
aminoglucoside (Glc–NH2) and N-acetylated aminoglucoside units (Glc–NH–COCH3), H-2
of Glc–NH–COCH3 and Glc–NH–CH2CH2COOH), 2.89 (H-2 of Glc–NH2), 2.58 (Glc-NH-
CH2CH2COOH), 2.68 [Glc-N(CH2-CH2COOH)2] and 2.06 ppm (Glc-NH-COCH3). The
intensity ratio of the signals at δ = 2.58 ppm and δ = 2.68 ppm shows that the content of
mono- and disubstituted units is 71.6% and 1.4%, respectively. The 13C NMR spectrum
(D2O) shows chemical shifts (Figure 2B) (δ, ppm) at: 97.5 (C-1), 77.3 (C-4), 75.5 (C-5), 70.5
(C-3), 62.4 (C-6), 61.3 (C-2), 175 (C-7) and 22.8 (C-8), which is in good agreement with data
concerning the 13C NMR spectrum of pure chitosan (DCl/D2O) [14]. The appearance of new
signals at δ = 178.8 ppm (Glc-NH-CH2-CH2-COOH), 45.3 ppm (C-9, C-11) and 32.3 ppm
(C-10, C-12) is observed.
The IR spectra of thin CECh films differ from the spectrum of chitosan. A strong
characteristic band corresponding to νC=O of non-ionized carboxyl group appears at 1719 cm-1
at pH = 1.2. The intensity of this band depends to a great extent on medium pH value because
of the carboxyl groups ionization. It decreases on increasing medium pH value up to pH 7.0
where the band disappears (Figure 3). At the same time, a change in the band intensity at
1575 cm-1 (Amide II and νC=O of the ionized carboxyl groups) is observed: the intensity of
this band increases on increasing pH. These results show that on increasing medium pH value
the ionization degree of CECh carboxyl groups increases.
Pan et al. [12] have evaluated the impact of the carboxyethylation on CECh crystallinity
by means of wide angle X-ray diffraction (WAXRD). Chitosan is a semi-crystalline polymer
and in its XRD spectrum two diffraction peaks are observed at 2θ = 11º and 2θ = 20º, which
correspond to crystal form І and crystal form ІІ, respectively. In the XRD spectrum of CECh
one broad peak is recorded at about 2θ = 22º. This is an indication that the crystalline phase of
chitosan has been destroyed to a great extent after its modification. The peak broadening has
been attributed by Pan et al. [12] to inter- and intramolecular hydrogen bonding in CECh.
Further, CECh is characterized by higher degradation temperature as compared to the starting
chitosan [12]. This result is in conformity with the data reported by Mincheva et al. [15]. Pan
et al. [12] have assumed that this is due to ionic interaction between the N-containing groups
and the carboxyl groups in the structure of CECh.
1.2. CECh Behavior in Aqueous Solutions; CECh-Based Nanoparticles
The macromolecules of CECh contain carboxyl, primary and secondary amino groups,
and in the samples with higher alkylation degrees tertiary amino groups are present, as well
(Scheme 2). This determines the extremely interesting behavior of CECh in aqueous solutions
depending on medium pH value. It is outlined schematically in Figure 4. In strongly acidic
medium CECh amino groups are protonated and it behaves as a polycation similarly to
chitosan. In alkaline medium CECh carboxyl groups are ionized and it behaves as a
polyanion. The behavior of CECh as a polyelectrolyte in strongly acidic and alkaline media
determines its ability to form water-insoluble polyelectrolyte complexes (PECs) with
polyanions or polycations, respectively. The formation of PECs with the participation of
CECh is discussed in Section 3 of the present Chapter. Because of the presence of both
protonated amino and ionized carboxyl groups CECh self-assembles at certain pH values
(near its isoelectric point, IEP) as a result of intra- and intermolecular interactions between
the oppositely charged groups. Similar behavior has been reported for other chitosan
derivatives containing anionic functional groups, such as O-carboxymethylchitosan [16] and
mono-(2-acryloyloxyethyl)phosphonate-modified chitosan [17].
Figure 4. Schematic presentation of the structure of CECh at different medium pH values. The pH
range in which CECh interacts electrostatically with polyanions or polycations is outlined as well.
Reproduced from Yancheva et al. [9] by permission of ACS.
10.0
viscosity number, dl/g
7.5
HMMCECh
5.0
2.5
1.5
1.0
LMMCECh
0.5
0.0
2 3 4 5 6 7 8 9 10
pH
Figure 5. Dependence of the viscosity number of 0.1 wt % LMMCECh and HMMCECh solutions on
pH of the medium (I = 0.1; 25 ± 0.1 ºC). Mincheva et al. [10], Self-assembly of N-carboxyethylchitosan
near the isoelectric point, J. Polym. Sci. Part A: Polym. Chem., 2008, 46, 6712–6721, Copyright Wiley-
VCH Verlag GmbH and Co. KGaA. Reproduced with permission.
At the IEP the macromolecules of these polymers exist in zwitterionic form and in aqueous
solutions their conformation represents a compact coil [18]. At рН values different from the
IEEP the cation nic or anionicc groups preddominate and the macromoolecules havee expanded
coonformation. At pH valuess close or eqqual to the IE EP depending on the polyaampholyte/
poolyzwitterion concentrationn self-assembblies, such as a nano- or microparticlees or their
agggregates, cann be formed.
The IEP of o CECh has been determ mined viscometrically [8-10], by turbiddity and ζ-
pootential measurements [13]. It is know wn that the deependence off the viscosityy of dilute
soolutions of polyampholyte
p es and polyzzwitterions onn pH at a given g ionic strength
s is
chharacterized by
b a minimum m at pH value equal to the IEP. The dependence of thhe viscosity
nuumber of CECh with a tootal DS of 766 % obtained from low molecular weigght or high
m
molecular weigght chitosan onn medium pH H value is showwn in Figure 5.It
5 has been determined
d
thhat the IEP off CECh is witthin the pH raange from 5.00 to 5.6 (~5.2). The viscosiity number
vaalues obtained d for CECh in i acidic meddium are low wer than thosee determined in alkaline
m
medium. This observation is i in accordaance with literature data [118] and is atttributed to
foormation of hy ydrogen bondds between thee carboxylate ions and H+ of o the type CO OO-···H+···-
O
OOC which leaads to a decreease of the hyydrodynamic volume
v of polybetaines as well as by
sppecific bindinng of the low molecular weeight counteriions to the prrotonated amiino groups.
Lower viscositties of aqueoous CECh soolutions at acidic рН valuues (4.0-4.5) have been
obbserved by Paan et al. [12], as
a well.
Recently Peng
P et al. [13] have reporrted on the deetermination ofo the IEP off CECh by
tuurbidity measurements (Figgure 6). At either low or high рН values v the sollutions are
prractically transparent (optical transmittannce: 95%). Thee light transmiittance sharplyy decreases
inn the рН rangee from 6 to 6.8. The IEP off CECh with a DS of 0.21 has h been founnd to be ca.
6..65. By meassuring the ζ-potential of CECh aqueouus solutions, Peng et al. [13] have
deemonstrated th hat the IEP of
o the studied CECh is situaated within thhe рН range fromf 5.2 to
6..8. The ζ- poteential providees information about the chaarge density of
o the particless as well as
abbout the electrrostatic interacctions betweenn charged partticles.
Fiigure 6. The opttical transmittannce (T%) of CE

ECh aqueous soolution at λ = 5000 nm under varrious pHs.
R
Reproduced from
m Peng et al. [133] by permissioon of Elsevier.
The increase in the рН values provokes a decrease of ζ-potential from + 34 mV at рН 3.0 to -

40 mV at рН 10.45; i.e. the net surface charge changes from a positive to a negative one. At
рН values near the IEP (рН 5.2-6.8), where phase separation occurs, the determined value of
the ζ-potential tends to zero. This indicates that at the IEP of CECh charge compensation
occurs as a result of ionic interactions between the oppositely charged groups in the structure
of the polyampholyte/polyzwitterion.
It is known that polyampholytes exhibit an antipolyelectrolyte effect (the aqueous
solution viscosity increases with increasing ionic strength) at pH values near the IEP [18-20].
Yancheva et al. have studied the impact of the ionic strength on the viscosity of CECh
aqueous solutions at рН value close to the IEP [9].The dependence of the reduced viscosity of
CECh aqueous solution on NaCl concentration is shown in Figure 7. As seen, the increase in
the ionic strength from 0.1 to 1.5 leads to an increase in the reduced viscosity of the solution
of CECh. At low concentrations of the low molecular weight salt, the intramolecular
interactions between the positive and negative charges in CECh macromolecule predominate
as a result of which the polymer chains collapse into a coiled structure [18]. Due to the
polyzwitterionic nature of CECh the coiling may also be due to dipole-dipole interactions
between the oppositely oriented dipoles in its structure [20]. The increase in the solution
viscosity is related to conformational changes of CECh macromolecules: from coiled
conformation driven by dipole-dipole interactions to individual macromolecules with
expanded conformation.
During the last two years, the investigations on the possibilities of CECh to form self-
assemblies have evoked increasing interest [10,12,13]. Methods such as dynamic light
scattering (DLS), atomic force microscopy (AFM) and fluorescence spectroscopy using a
pyrene probe have been employed.
7
Reduced viscosity, dl/g
0
0.0 0.5 1.0 1.5 2.0
Ionic strength
Figure 7. Dependence of the reduced viscosity of CECh solution on the ionic strength (25°C, 0.1 wt %).
Figure 8. AFM micrograph (in height) of HMMCECh nanoparticles obtained at pH 5.0 from aqueous
solutions with concentration 0.05 mg/ml (I = 0.1; 25 ± 0.1ºC). Mincheva et al. [10], Self-assembly of N-
carboxyethylchitosan near the isoelectric point, J. Polym. Sci. Part A: Polym. Chem., 2008, 46, 6712–
6721, Copyright Wiley-VCH Verlag GmbH and Co. KGaA. Reproduced with permission.
By means of DLS analyses Mincheva et al. [10] have demonstrated for the first time the
formation of nanosized self-assemblies of CECh (DS = 76%) in the рН range around its IEP
(рН 5.0 - 5.6). It has been found that out of this pH range the aqueous solutions of CECh
behave as real solutions. The studies have been carried out using HMMCECh ( M w = 6.7 ×
105) and LMMCECh ( M w = 4.5 × 103) at polymer concentrations ranging from 0.01 to 0.1
mg/ml. In the above-mentioned concentration range both LMMCECh and HMMCECh
aqueous solutions have remained transparent regardless of the polymer molecular weight and
concentration as well as medium pH value. These observations indicate that the size of the
formed self-assemblies is below the critical value at which macroscopic phase separation
occurs and are in accordance with data obtained for aqueous solutions of synthetic
polyzwitterions [21]. It has been found that at polymer concentration of 0.1 mg/ml the size of
the obtained structures depends to a great extent on the medium рН near the IEP, and the
largest structures are formed at рН 5.2 and 5.4. At these рН values CECh molecular weight
does not exert a significant effect on the size of the self-assemblies. This is explained by the
formation of large secondary aggregates. The latter are formed as a result of complete charge
compensation in CECh macromolecules which leads to the occurrence of hydrophobic
interactions and hydrogen bonding between CECh macromolecules. At рН 5.0 and 5.6 the
effect of CECh molecular weight is more pronounced and the self-assemblies formed by
LMMCECh are smaller as compared to the structures obtained from HMMCECh. This result
is in conformity with the reported data for formation of smaller particles from
polyelectrolytes with shorter chains as compared to particles formed from polyelectrolytes
having longer chains [22]. At these conditions CECh has been found to form spherical self-
assemblies as attested by DLS [10]. Their size has been found to decrease on decreasing
CECh concentration and at polymer concentration of 0.01% HMMCECh nanoparticles with
average diameters of 45 and 30 nm have been obtained at pH 5.0 and 5.6, respectively. In
Figure 8 the formation of spherical HMMCECh nanoparticles at pH 5.0 and polymer
concentration of 0.05 mg/ml is shown. LMMCECh forms aggregates of spherical particles at
рН 5.0 and 5.6 in the whole concentration range (from 0.01 to 0.1 mg/ml). The hydrodynamic
diameter (Dh) of HMMCECh and LMMCECh self-assemblies has also been found to depend
on the polymer concentration at pH 5.2 (Figure 9А). As known, for structures formed by
aggregation, Dh decreases on decreasing polymer concentration because of reduction of the
interparticle interactions [23]. It has been established that this dependence is valid for CECh,
as well. As seen in Figure 9А, on decreasing polymer concentration (from 0.1 to 0.01 mg/ml)
Dh decreases from 980 to 120 nm and from 880 to 120 nm for HMMCECh and LMMCECh,
respectively. By using DLS in combination with AFM it has been found that at рН 5.2 and
polymer concentration of 0.1 mg/ml HMMCECh forms rod/worm-like self-assemblies
whereas the structures obtained from LMMCECh are spherical ones. On decreasing
HMMCECh concentration, the angular dependence of the diffusion coefficient (D) on the
absolute value of the scattering vector (q2) becomes less pronounced, and at CECh
concentration of 0.01 mg/ml D is independent of q2 (Figure 9B). This is an indication that at
this concentration of HMMCECh spherical self-assemblies are formed. The obtained results
are in good agreement with the data reported by Zhu et al. [16], who have shown that
carboxymethylated chitosan derivatives tend to form aggregated structures in aqueous
solutions at polymer concentrations higher than 0.05 mg/ml.
Based on the systematic study performed by Mincheva et al. [10], it has been concluded
that the size and shape of the self-assembled structures obtained from CECh at pH values
close to IEP depend on CECh molecular weight, medium рН value and polymer
concentration. The aggregation behavior of CECh in vicinity of the IEP has been verified by
data obtained by means of fluorescence spectroscopy using a pyrene probe [12,13].
980 4
1000 HMMCECh
880
LMMCECh 0.1
3
750
Dx10-7, cm2/sec
680
2
Dh, nm
0.05
500 440
1
0.1
250
120120 0.01
0 0.0
0.010 0.050 0.100 0.0 2.5 5.0 7.5 10.0
Concentration, mg/ml 2 10
q x10 ,cm -2
A B
Figure 9. Hydrodynamic diameter (Dh) of nanosized structures obtained by self-assembly of

HMMCECh and LMMCECh in aqueous solution as a function of polymer concentration (A) and
dependence of the diffusion coefficient (D) on the absolute value of the scattering vector (q2) for self-
assembled structures obtained from HMMCECh at different concentrations (B) (pH 5.2; I = 0.1; 25 ±
0.1ºC). The corresponding concentration values (in mg/ml) are shown above each curve (B). Mincheva
et al. [10], Self-assembly of N-carboxyethylchitosan near the isoelectric point, J. Polym. Sci. Part A:
Polym. Chem., 2008, 46, 6712–6721, Copyright Wiley-VCH Verlag GmbH and Co. KGaA.
Reproduced with permission.
1.3. Biological Behavior of CECh in Respect to Cells or Pathogenic

Microorganisms
The data concerning the biological behavior of CECh are still scarce. Sashiwa et al. have
studied the biodegradability of CECh using standard activated sludge according to a
procedure reported in [24]. They have found that CECh degrades faster as compared to the
pristine chitosan. This has been attributed to the fact that in contrast to chitosan CECh is
soluble in the medium used for the biodegradability tests (pH 7, adjusted by carbon-free
inorganic buffer). The authors have also established that the biodegradability of CECh
decreases with increasing DS, which is most probably due to structural resistance to the attack
of enzymes. Recently it has been demonstrated that CECh is compatible with fibroblast cells
[12]. The performed studies on the behavior of hydrogels based on CECh and oxidized
dextran in respect to dermal fibroblasts have also indicated good compatibility with the cells
[11].
Chitosan possesses inherent antibacterial and hemostatic activity [25-29]. It is attributed
to ionic interactions between the protonated amino groups of chitosan and negatively charged
moieties of the cell membranes of pathogenic microorganisms and blood cells, respectively. It
is expected that the introduction of carboxyl groups by N-carboxyethylation whereby CECh is
obtained will lead to the preparation of a modified product whose biological behavior differs
from that of its precursor. Yancheva et al. [9] have studied ex-vivo the behavior of CECh in
contact with the pathogenic microorganism Escherichia coli (E. coli). They have found that
CECh does not possess any antibacterial activity even at a concentration as high as 20 mg/ml
(for comparison, chitosan induces complete inhibition of the bacterial growth at
concentrations as low as 0.25 mg/ml).
White blood cells Red blood cells Platelets
100
blood cells (% of control)
80
60
40
20
0
cech cech cech net-cech net-cech net-cech
CECh net-CECh
Figure 10. Dependence of the blood cell counts on the crosslinking of CECh (polymer concentration 5
mg/ml). Crosslinked CECh is designated as net-CECh. Yancheva et al. [30], Polyelectrolyte complexes
based on (quaternized) poly[(2-dimethylamino)ethyl methacrylate]: behavior in contact with blood,
Macromol. Biosci., 2007, 7, 940-954, Copyright Wiley-VCH Verlag GmbH and Co. KGaA.
Solution Gel
CECh Net-CECh
Figure 11. Schematic representation of the interaction of the blood cells with a CECh solution and a
film of crosslinked CECh (designated as net-CECh). Yancheva et al. [30], Polyelectrolyte complexes
based on (quaternized) poly[(2-dimethylamino) ethyl methacrylate]: behavior in contact with blood,
Macromol. Biosci., 2007, 7, 940-954, Copyright Wiley-VCH Verlag GmbH and Co. KGaA.
The lack of antibacterial activity of CECh has been attributed to the substitution of the amino
groups which are responsible for the antimicrobial activity of chitosan. Investigations have
been carried out to assess the effect of CECh on human plasma hemostatic parameters and
blood cells, as well [30]. In the studies performed using human pool plasma the impact of
CECh on the following hemostatic parameters: prothrombin time (PT), activated partial
thromboplastin time (APTT), thrombin time (TT), fibrinogen and antithrombin III (АТІІІ)
levels, has been monitored. It has been found that CECh exerts a weak effect on them. At
higher concentrations of CECh (10 and 16 mg/ml, respectively), slight prolongation of PT,
APTT, and TT has been observed. The behavior of CECh upon contact with blood plasma
does not essentially differ from that of its precursor (chitosan), which does not exert any
effect on the hemostatic parameter values [27]The behavior of CECh in contact with blood
has been assessed using blood cell counting tests [30]. These tests allow quantitative
evaluation of the changes that occur in the blood cell counts after contact of the polymers
with whole blood [31]. In addition, tests of this type have been applied to films of CECh
crosslinked with glutaraldehyde (GA). Figure 10 shows the changes in the white blood cells,
red blood cell and platelet counts after a 30-min incubation of CECh and crosslinked CECh in
whole blood. As seen, the blood cell counts decrease significantly in the presence of CECh.
This result reveals that, similarly to chitosan [27,28], CECh induces alterations in the blood
cells due to ionic interactions of the positively charged amino groups in its structure with the
negatively charged moieties on the surface of the blood cells. The engagement of CECh
amino groups in covalent bonds by crosslinking with GA results in a polymer material which
is characterized by considerable compatibility with the blood cells. As seen from Figure 10,
the cell counts are almost unaffected by the presence of crosslinked CECh. It should be noted
that in this case the polymer is in a swollen state whereas the behavior of non-crosslinked
CECh upon contact with blood has been evaluated in solution. This also accounts for the
difference in the behavior of (crosslinked) CECh upon contact with whole blood. It can be
assumed that the contact between crosslinked CECh and the blood cells occurs mostly on the
film surface. A schematic representation of the interactions between (crosslinked) CECh and
the blood cells is shown in Figure 11. In the case when a CECh solution is used, all of the
amino groups (which are responsible for the low compatibility of CECh with the blood cells)
are accessible for ionic interactions with the blood cells. For crosslinked CECh film, the
blood cells need to penetrate into the film in order to come in contact with the free (i.e., non-
engaged in covalent bonds) amino groups located in the bulk of the film. In this case, the
porosity of the material that determines the ability of the blood cells to penetrate into the bulk
of the film is of crucial importance.
Kogan et al. [32] have demonstrated that CECh possesses antioxidant and antimutagenic
activity. The antioxidant activity of CECh has been assessed by quantitative evaluation of
quenching of color produced by ABTS·+ chromophore using a known procedure [33]. The
comparison of the antioxidant activity of CECh with that of a reference antioxidant, trolox [a
derivative of α-tocopherol (vitamin E)], has indicated that depending on the DS (varying from
0.27 to 1.61) CECh is 100-300 times more potent. The antimutagenic activity of CECh has
been assessed using the unicellular flagellate Euglena gracilis subjected to the action of
genotoxic agents - acridine orange and ofloxacin. Euglena gracilis has been selected for
performance of the tests because its chloroplasts represent a certain type of “model
intracellular bacteria” used as indicators of genotoxicity in complex bacterial mutagenicity
assays. The chloroplast genome is extremely sensitive to various chemical and physical
factors which can lead to damage or complete loss of chloroplast DNA. Using this property of
the chloroplast DNA of Euglena gracilis it has been found that CECh acts as an antimutagen
and the mechanisms of its antigenotoxicity against acridine orange and ofloxacin are
different. The antimutagenic activity of CECh toward ofloxacin has been assigned to the
antioxidant activity of the polymer, while its antigenotoxicity against acridine orange has
been attributed to interaction of CECh with the cell membrane of Euglena gracilis, which
results in hindered access of acridine orange to chloroplasts.
2. FABRICATION OF CECH-CONTAINING NANOFIBERS BY

ELECTROSPINNING
Recently, the preparation of nanofibrous materials with high surface area-to-volume and
aspect ratios has evoked great interest. Such materials are considered applicable in numerous
areas, such as medicine, pharmacy, cosmetics, design of military protective clothing, filters,
electronics, nanosensors, etc. [34-37]. The processing techniques used up to date are template
synthesis [38], self-assembly [39], phase separation [40], drawing [41], melt blowing [42],
and electrospinning [34-37,43,44]. Among them electrospinning is regarded as the most
promising one for fabrication of fibers having diameters within the micro- and nanoscale,
while reaching tens of meters and more in length. This method involves the application of
high electric field to a polymer in a solution or melt. The high electric field induces electrical
charges creating a force acting oppositely to the surface tension. When this force overcomes
the surface tension a charged polymer jet of small diameter is ejected, which is stretched
because of the electrostatic repulsion between the charges on its surface. The small diameter
of the charged jet allows fast mass exchange and the solvent usually evaporates between the
electrodes, thus dry polymer fibers are deposited on the collector. The fiber diameter and
morphology depend mainly on: (i) solution concentration, viscosity and conductivity; (ii)
solvent surface tension; and (iii) the applied electric field strength [35,36,45].
Regarding biomedical applications, special attention is paid to the electrospinning of
natural polymers, such as collagen, fibrinogen, cellulose, chitosan and its derivatives [35-
37,44,46-50]. In this Section, the existing knowledge on the preparation of CECh-containing
nanofibrous materials by electrospinning is discussed. The approaches applied for
stabilization of such nanofibers against dissolution in water and their potential biomedical
applications are summarized. In addition, the preparation of hybrid organic/inorganic
nanofibers based on CECh is highlighted.
2.1. Preparation of Nanofibers in the Presence of a Non-Ionogenic Polymer

Partner
Similarly to its precursor, chitosan [51], all attempts for electrospinning of CECh alone
from its aqueous solutions have failed [15]. In such cases only electrospraying with formation
of micro- and nanoparticles occurs. Electrospinning of CECh aqueous solutions is only
feasible in the presence of a non-ionogenic water-soluble polymer: polyacrylamide (PAAm)
or poly(vinyl alcohol) (PVA) [15,52,53]. For CECh/PAAm pair the applied field strength
(AFS, ranging from 0.7 to 1.6 kV/cm) has no significant effect on the morphology and mean
fiber diameter [15]. Fiber morphology and diameter are strongly affected by the composition
of the spinning solutions (Figure 12). Upon increasing CECh content (up to 50 wt.%), fiber
morphology changes from continuous defect-free fibers to fibers having spindle-like defects.
The size of the defects has been found to increase on increasing CECh content, while the
average distance between two neighboring defects decreases, and at CECh/PAAm = 9/1
(w/w) few beads which are not connected to the fibers are observed. Moreover, the higher the
polyelectrolyte content, the smaller the fiber diameter and the narrower the diameter
distribution. Thus, defect-free nanofibers with a diameter of 215 nm have been obtained for
CECh/PAAm = 1/9 (w/w), while fibers with spindle-like defects with a diameter of 50 nm in
the defect-free part have been electrospun from mixed CECh/PAAm = 9/1 (w/w) solutions.
These observations have been explained by the combined effects of decreasing the solution
viscosity, and increasing the concentration of the ionizable groups with increasing CECh
content [15].
CECh-containing nanofibers with ribbon-like shape have been obtained by
electrospinning of mixed CECh/PVA aqueous solutions at CECh/PVA = 1/75 (w/w) and low
AFS (1.2 kV/cm) [52]. The formation of such flat nanofibers has been found to be related to
the formation of a thin skin on the jet surface due to fast solvent evaporation [54]. At
CECh/PVA = 1/75 (w/w), the morphology of the nanofibers changes to cylindrical on
increasing the AFS to 2.2 kV/cm. Meanwhile, their average width decreases from 820 to 420
nm for AFS 1.2 and 2.2 kV/cm, respectively. The tendency of the average fiber diameters to
decrease on increasing AFS has also been observed for CECh/PVA = 1/8 - 1/1 (w/w), thus
supposing that in this case AFS has a significant effect on fiber morphology and diameter.
Another parameter, found to affect the morphology and diameter of CECh-containing
naanofibers obtained by elecctrospinning of CECh/PVA mixed aquueous solutionns is their

coomposition (F Figure 13). Att a constant AFS,
A the increease of CEChh content in CECh/PVA
C
m
mixed solution
ns results in fibers
fi of smaller diameters and narrowerr diameter distributions,
siimilarly to thee CECh/PAAm m system. Again, spindle-liike defects aloong the fibers have been
obbserved at CE ECh/PVA ≥ 1/3. However, the electrosppinning of CE ECh/PVA at weight
w ratio
hiigher than 1 iss not feasible,, and leads to the formationn of “tailed” beads.
b These results have
beeen attributedd to the decrreased solutioon viscosity and a explainedd by the semmi-empirical
m
methodology reported
r by Shenoy
S et al. [55]. Additional informattion on the size
s of the
C
CECh/PVA naanofibers has been obtainned by transm mission electrron microscoopy (TEM)
(F
Figure 14). Th he obtained TEM
T micrograaphs have alloowed provingg the formatioon of fibers
w
with average diameters
d rannging from 5 to 20 nm, not detectablle by scanninng electron
m
microscopy (SEEM).
In view of o some potenntial applicatiions of CECh-containing fibrous mateerials, their
sttabilization ag
gainst dissoluttion in aqueouus solutions iss of significannt importance.. Mincheva
ett al. have perrformed crosslinking of eleectrospun CEC Ch/PAAm [155] and CEChh/PVA [52]
naanofibrous materials
m by heat-treatment of the mats in solid statte. Melting annd thermal
deegradation of all polymers have
h been stricctly avoided.
Fiigure 12. Effectt of the composition of the spinnning solution on

o the morpholoogy of CECh/PAAAm
naanofibers. Weigght ratio CECh//PAAm=1/4 (A)); 1/1 (B); 4/1 (C) and 9/1 (D).. Total polymerr
cooncentration: 3%
%, AFS 1.1 kV//cm. Reproduceed from Mincheeva et al. [15] byy permission off SAGE.
Figure 13. SEM micrographs of CECh/PVA nanofibers. Weight ratio CECh/PVA = 1/75 (a), 1/8 (b),
1/2 (c) and 1/1 (d); AFS 1.2 kV/cm. Reproduced from Mincheva et al. [52] by permission of Elsevier.
Figure 14. TEM micrographs of CECh/PVA = 1/1: (a) nanofibers and (b) defects. AFS 1.2 kV/cm.
Reproduced from Mincheva et al. [52] by permission of Elsevier.
It has been demonstrated that the water-resistance of the thermally crosslinked nanofibers
depends on the crosslinking conditions and fiber composition. It has been found that in the
case of the CECh/PAAm system at high PAAm content [CECh/PAAm < 4/1 (w/w)] no
crosslinking of the fibrous materials takes place [15]. Nanofibers from CECh/PAAm = 1/1
(w/w), heated at 100ºC for 10 h swell and partly merge after contact with water vapor, and
dissolve after 1h contact with water (Figure 15). Fibers, having the same composition, but
heated at higher temperature (120°C) for 10 h are partly resistant to water and water vapor.
The increase in CECh content [CECh/PAAm = 4/1 (w/w)] in the fiber composition allows the
preparation of crosslinked fibrous materials by heat treatment at 120 °C for 5 h. As seen from
Figure 15D, after being in contact with saturated water vapor for 1 h, the nanofibers do not
merge. In addition, they preserve their morphology and size and do not dissolve in water. This
is an indication that the crosslinking of CECh/PAAm system proceeds mainly with the
participation of CECh and is driven by interaction between its amino and carboxyl groups.
For nanofibers with the highest CECh content [CECh/PAAm = 9/1 (w/w)] the contact with
water leads do dissolution of the defect-free part of the nanofibers, while the defects remain
unchanged. This has been attributed to phase-separation of the polymers during the
electrospinning at large CECh excess. Since the crosslinking induced by heat treatment is due
to interactions between the amino and carboxyl groups of CECh (PAAm does not participate
in the crosslinking process), it has been suggested that PAAm content is higher in the defect-
free part of the nanofibers and the content of CECh is higher in the defects.
Figure 15. CECh/PAAm nanofibers. CECh/PAAm=1/1 (w/w), heat-treated at 100°C for 5h (A) and
after subsequent contact with water vapor for 1 hour (B). CECh/PAAm = 4/1 (w/w), heat treated at 120
°C for 5 h (C) and after subsequent contact with water vapor for 1 h (D). AFS 1.2 kV/cm. Reproduced
from Mincheva et al. [15] by permission of SAGE.
Figure 16. A simplified sketch of the crosslinking reactions, occurring during heat-treatment of
nanofibers of CECh and PVA. Reproduced from Mincheva et al. [52] by permission of Elsevier.
Similarly to CECh/PAAm nanofibrous materials, CECh/PVA mats have been crosslinked

successfully by heat-treatment [52]. Again, the water-resistance of the nanofibers is
dependent on the composition of the spinning solution and increases on increasing CECh
content. In contrast to CECh/PAAm system, in which crosslinked mats have only been
obtained at CECh/PAAm ≥ 4/1 (w/w) [15], in the case of CECh/PVA nanofibers efficient
crosslinking has been achieved at CECh/PVA ≥ 1/3 (w/w). This implies that for CECh/PVA
fibers the non-ionogenic polymer also participates in the crosslinking reaction. It has been
suggested that the possible crosslinking reactions proceed similarly to the reported reactions
between –COOH, –OH, and –NH2-containing polymers [56-58]. A simplified sketch of these
interactions is presented in Figure 16. As seen, they include crosslinking between CECh
chains (by amidation or dehydration), and between CECh and PVA chains (esterification). It
is noteworthy that the heat-induced crosslinking of CECh/PAAm and CECh/PVA nanofibers
is attained in the temperature range that is suitable for dry sterilization. This fact is important
and it paves the way to shortening the production stages of sterile materials with potential
biomedical applications. The use of purposely designed collectors consisting of conductive
strips has enabled the preparation of aligned nanofibers from CECh/PVA nanofibers [52]. It
has been shown that the degree of fiber alignment depends on the number and the
configuration of the conductive strips-collector type, as well as on the type of the used
insulating material.
Zhou et al. [53] have prepared water-insoluble CECh/PVA fibrous mats [CECh/PVA =
50/50 (w/w)] by crosslinking under GA vapors. The SEM analyses have revealed that the
fibrous materials retain their morphology after a 48-h stay in aqueous solution.
As aforementioned, the interest in obtaining nanofibrous materials from CECh is based
on their potential applications in the biomedical field as wound dressing materials, controlled
drug delivery systems, and tissue engineering scaffolds. Mincheva et al. have shown the
preparation of CECh/PAAm nanofibrous materials containing the broad spectrum
antimicrobial and antimycotic agent 7-iodo-8-hydroxyquinoline-5-sulfonic acid (SQ) [15]. In
the presence of SQ the conductivity of the spinning solution increases, resulting in a more
than two-fold decrease in fiber diameter. Concurrently, the diameter distribution narrows and
spindle-like defects are observed along the fiber length (Figure 17). The antibacterial and
antimycotic activity of these mats against Gram(–) bacteria (E. coli), Gram(+) bacteria
(Staphylococcus aureus; S. aureus) and the fungus Candida albicans (C. albicans) has been
tested. Well-defined wide sterile zones with diameters of 3, 2 and 3 cm (for S. aureus, E. coli
and C. albicans, respectively) have been observed around the SQ-containing mats. Sterile
zones have not appeared in the case of CECh/PAAm nanofibers without SQ.
Figure 17. SEM-micrographs of nanofibers electrospun from CECh/PAAm mixed solutions and
average diameter distribution of the nanofibers without (A) and in the presence of 1% SQ (B).
CECh/PAAm=1/4; AFS 1.25kV/cm. Reproduced from Mincheva et al. [15] by permission of SAGE.
Figure 18. SEM images of L929 cell seeded on nanofibrous membrane of CECh/PVA (50/50) after 48
h culture. Reproduced from Zhou et al. [53] by permission of ACS.
It has been demonstrated that CECh/PVA nanofibrous materials represent suitable

scaffolds for attachment and proliferation of mouse fibroblasts (L929) (Figure 18). Thus,
Zhou et al. [53] have concluded that these electrospun membranes have the potential to be
used as wound dressings for skin regeneration.
2.2. Preparation of Hybrid Nanofibers Containing Inorganic Nanoparticles
After the possibilities for fabrication of CECh-based nanofibers have been discussed in
Section 2.1., in this Section the existing up to now knowledge on the preparation of hybrid
organic/inorganic nanofibrous materials based on CECh will be surveyed. By combining the
properties of CECh with those of inorganic additives (metal oxides, metal nanoparticles, etc.)
hybrid polymer-inorganic materials with tailored mechanical, optical, magnetic and biological
properties, intended for targeted applications, can be fabricated. Moreover, the improved
functionality of CECh as compared to chitosan allows the preparation of stable dispersions of
metal nanoparticles in aqueous solutions and therefore offers possibilities of obtaining novel
hybrid materials. In this Section the preparation of polyelectrolyte-stabilized magnetic
nanoparticles and the preparation of nanofibers responding to changes in an external magnetic
field is discussed. The incorporation of silver nanoparticles (AgNPs) in CECh-based

nanofibrous materials by electrospinning is outlined as well.
2.2.1. Fabrication of CECh/Magnetite Hybrid Nanofibers by Electrospinning

The increasing interest in the preparation of polyelectrolyte-stabilized magnetic
nanoparticles (the term “nanoparticle” usually refers to a particle with a diameter smaller than
100 nm) is attributed to the wide range of their potential applications: in environmental
protection (wastewater purification), biotechnology and medicine (separation of
microorganisms, blood purification, targeted drug delivery, magnetic resonance imaging,
hyperthermia, etc.) [59-69]. In this respect, magnetite (Fe3O4) is one of the most thoroughly
studied magnetic materials since it is resistant to oxidation and its preparation on a laboratory
scale is an easily feasible one. In addition, Fe3O4 preserves high magnetization and possesses
proven biocompatibility.
Typically, magnetite nanoparticles are obtained by the sol-gel method through co-
precipitation of aqueous solutions of ferrous (Fe3+) and ferric (Fe2+) salts in alkaline medium
(usually, NaOH or NH4OH) at room temperature [70-72] according to the equation:
2+ 3+ _
Fe + 2 Fe + 8OH Fe 3O4 + 4 H2O
(1)
The precipitated Fe3O4 nanoparticles are frequently termed “ferrofluid” because of their
good dispersibility in water [73]. The complete precipitation of Fe3O4 according to equation
(1) is achieved in the pH range from 9 to 14 at Fe3+/ Fe2+ = 2/1 (w/w) and in oxygen-free
reaction medium. However, because of their hydrophobicity and high surface-to-volume ratio
as well as because of the occurrence of Van der Waals and magnetic dipole-dipole
interactions the obtained magnetic nanoparticles rapidly agglomerate. It is known that this
problem can be avoided by modifying or coating the surface of the magnetic nanoparticles
with an appropriate metal, oxide or stabilizer [64,65,74,75]. Most frequently, however, the
dispersibility in aqueous media is improved by adding surfactants (e.g., oleic acid, sodium
oleate, dodecylamine, sodium carboxymethylcellulose, cetyltrimethylammonium bromide)
and/or steric stabilizers: polymers [e.g. poly(ethylene glycol) (PEG), poly(acrylic acid)
(PAA), PVA, polysaccharides) [64,76,77]. In addition, polyelectrolytes with appropriate
functional groups (carboxyl, sulfate, phosphate) can bind to the magnetite surface through
chemisorption and prevent further nanoparticle growth and agglomeration by electrostatic
repulsion [77-83].
Two main models have been applied to explain the structure of the composite materials
that are formed upon interaction of natural polysaccharides with iron ions. The first one, the
site binding model, supposes that iron (III) is bound through the binding sites of the
saccharide residues (e.g. carboxylate, sulfonate, alcoholic hydroxylate or amine) and forms
spatially separated iron (III) centers along the polymer chain [84-87]. The second one
assumes that iron (III) forms a FeOOH precipitate which is coated by the polysaccharide and
thus the aggregation of subcolloidal FeOOH particles is inhibited [88,89]. Although the
interaction between chitosan and iron (III) has not yet been completely elucidated, the ability
of chitosan to form complexes with the iron ions is supposed to play a significant role (Figure
19) [90].
Figure 19. Schematic representation of complex formation between chitosan and Fе3+ at pH = 2 (A, B)
and pH = 6 (C, D). Reproduced from Hernandez et al. [90] by permission of Elsevier.
Figure 20. A photograph of FF(Fe3O4) (A) and FF(Fe3O4×CECh) (B) magnetic suspensions after 40-
day storage; impact of a permanent magnet on FF(Fe3O4×CECh).
The presence of polyelectrolytes as stabilizing agents of Fe3O4 nanoparticle suspensions is

particularly effective when the polyelectrolytes participate in the sol-gel process of inorganic
phase formation [78]. In this case the carboxyl and/or amino groups in CECh macromolecule
initially chelate the iron ions similarly to chitosan [77,89,90], and impede their aggregation by
steric and/or electrostatic repulsion. The stabilizing role of CECh added to the medium during
magnetite nanoparticle formation has been confirmed by the studies performed after a
prolonged storage of the magnetic suspensions. It has been found that the magnetite
dispersions, synthesized in the presence of CECh, further denoted as FF(Fe3O4×CECh)
(Figure 20B), remain stable for more than 40 days and no precipitate formation is observed.
In contrast to them, the magnetite dispersion in the absence of a stabilizer, further denoted as
FF(Fe3O4) (Figure 20A), preserves its stability only within 1-2 days.
The presen nce of magnetite phase onlyo has beenn confirmed by the perfoomed XRD
annalyses of FF F(Fe3O4) andd FF(Fe3O4×C CECh) powdeers. The iron oxide phasee has been
iddentified by thhe peak positiions at 30.0° (220), 35.5° (311),
( 43.1° (400), 53.4° (4422), 57.0°
(5511) and 63° (440),
( which are
a in accordannce with magnnetite standardd data. In the presence
p of
C
CECh broadeniing of the difffraction peaks is observed. This
T can be exxplained by a decrease
d in
thhe particle sizee [91] which is confirmed by
b the values ofo the mean paarticle size, caalculated by
thhe Scherer’s equation. The results
r reveal that in the preesence of CEC Ch particles with
w a mean
diiameter of 10 0 nm are obtaained while inn the absencee of CECh thhe nanoparticlle diameter
vaaries from 20 to 45 nm [78]. The determ mined values have been conffirmed by TEM M analyses
(FFigure 21). Th he typical spheerical shape off the magneticc particles in the
t obtained suspensions
s
ass well as theirr tendency to align in the form
fo of stringss, similarly too biogenic maggnetite, are
cllearly seen it the
t TEM micrographs. It iss noteworthy that in the absence of CEC Ch particles
w broad sizee distribution and
with a agglomeraates are formedd [78].
The magneetization curvves (Figure 222) show minim mal hysteresiss and low vallues of the
reemnant magneetization and coercivity im mplying superpparamagnetic properties annd minimal
aggglomeration of the obtainned nanopartiicles. In addittion, the satuuration magneetization of
FF F(Fe3O4×CEC Ch) is 16.6 emu/g
e and thhat of FF(Fe3O4) is 47.7 emu/g. The performed
M
Mössbauer meaasurements haave confirmedd that superparramagnetism is i most clearlyy displayed
inn the case of FF(Fe
F 3O4×CECh) and at 30 00 К this compponent is abouut four times larger than
thhe one determined for FF(F Fe3O4) (Figure 23). The obtaained results are a consistent with those
obbtained from the XRD andd TEM analyyses and are inn conformity with the dataa for other
sttabilized Fe3O4 nanoparticlees [78].
Fiigure 21. TEM micrographs off FF(Fe3O4) (A)) and FF(Fe3O4×CECh)

× (B). Addapted from Miincheva et
all. [78] by permiission of Elsevieer.
60
40
20
M, emu/g
-20
-40
-60
-8 -6 -4 -2 0 2 4 6 8
H, kOe
Figure 22. Magnetization curves at 300 K for powders of FF(Fe3O4) (S) and FF(Fe3O4×CECh)
(z).Adapted from Mincheva et al. [78] by permission of Elsevier.
These findings reveal that CECh is particularly effective in the stabilization of Fe3O4
nanoparticle aqueous suspensions, especially when participating in the sol-gel process of their
formation.
The nanoscale dimensions of these particles and the stability of the suspensions allow the
fabrication of biomimetic hybrid nanocomposite fibers with magnetic properties by means of
electrospinning. Up to date electrospinning has been applied for fabrication of
organic/inorganic hybrid nanofibers with magnetic properties using polymers such as PVA,
poly(ethylene oxide) (PEO), poly(vinyl pyrrolidone), poly(L-lactide) (PLLA), poly(ε-
caprolactone) [92-95]. As discussed in Section 2.1. of the present Chapter, it is not possible to
electrospin CECh alone [15]. Similarly, electrospinning of CECh in the presence of Fe3O4
nanoparticle magnetic suspensions is also not feasible and leads only to the formation of
micro- and nanosized „tailed” beads [78]. Based on the successful electrospinning of CECh in
the presence of the non-ionogenic water-soluble polymers PAAm and PVA [15,52] the
optimal conditions for fabrication of magnetically sensitive non-woven mats have been found.
In the case of the system consisting of CECh, PAAm, and ferrofluid this has been achieved
using a spinning solution at CECh/PAAm = 1/4 (w/w) and total polymer concentration of 3
wt%.
Fiigure 23. Mössb

bauer spectrum of FF(Fe3O4) (A A) and FF(Fe3O4×CECh) (B) recorded
r at 3000 K.
A
Adapted from Mincheva
M et al. [778] by permissiion of Elsevier.
The electrospin nning of the system com mposed of CE ECh, PVA, and a ferrofluidd has been
prreformed at CECh/PVA
C = 1/8 (w/w) anda total polyymer concenttration of 14 wt%. The
diifference in th
he total polymeer concentrations has an im mpact on the main
m characteriistics of the
sppinning suspen nsions of CEC Ch/ferrofluid/P
PAAm and CE ECh/ferrofluidd/PVA, as welll as on the
chharacteristics of the obtaineed fibers (Tablle 1). The low
w contrast of thhe polymers hash favored
thhe visualizatio
on of the maggnetite particlees incorporateed in the fibeers and their distribution
d
allong the fiberrs by TEM (Figure 24). It has h been founnd that in the absence of a stabilizing
poolymer the maagnetic particlles accumulatee only in certaain parts of thee fibers. The presence
p of
C
CECh in the magnetic
m suspeensions has leed to consideraably more uniiform distribuution of the
m
magnetic particcles along the fibers.
N-Carbboxyethylchitoosan-Based Poolymer Materials 289
Table 1. Chharacteristicss of the CECh

h/ferrofluid/P
PAAm and CE ECh/ferrofluiid/PVA
systems, the applied field strength and
d the electrosp
pun hybrid nanocomposit
n e fibers*
S
System η, cP σ, mS//cm AFS, kV/cm
k d , nm
n SD
1.0 160 38
а
C
CECh/FF(Fe3O4)/PAAm 2885 2.57 1.2 180 34
1.4 230 68
1.0 150 17
C
CECh/FF(Fe Amа
3O4×CECh)/PAA 4997 6.43 1.2 180 48
1.4 110 22
1.3 250 74
b
C
CECh/FF(Fe3O4)/PVA 16650 4.33 1.6 220 65
2.2 230 38
1.3 470 63
C
CECh/FF(Fe Ab
3O4×CECh)/PVA 38800 6.98 1.6 420 73
2.2 480 70
* dynamic viscossity (η); conducctivity (σ); meann fiber diameterr ( d ); standardd deviation (SD)).
а b
t
total polymer co
oncentration 3 wt%,
w total pollymer concentraation 14 wt%.
A
Adapted from Mincheva
M et al. [778] by permissiion of Elsevier.
Fiigure 24. TEM micrographs off CECh/FF(Fe3O4×CECh)/PAA ECh/FF(Fe3O4×C

Am (A) and CE CECh)/PVA
(B
B). Adapted from
m Mincheva et al. [78] by perm
mission of Elsevvier.
This is additionnal evidence of the stabilizzing role of CECh

C in the preparation ofo magnetic
suuspensions. String-like aliggnment of thhe magnetic particles
p paralllel to the fibber axis is
obbserved (Figuure 24), whichh mimics the alignment off magnetite inn magnetotacttic bacteria
[996]. This can be explainedd by the tendeency of the dipole-dipole
d i
interactions too align the
C
CECh-stabilize ed particles duuring the electrospinning process, as weell as by the interactions
i
beetween the polymers and between the polymers annd magnetic particles. p Thee discussed
reesults reveal that
t electrosppinning is an effective toool for fabricattion of hybridd magnetic
m
materials whichh mimic the naaturally occurrring ones.
2..2.3. Electrosp
spun Hybrid Nanofibers
N C
Composed of CECh/AgNP Ps
AgNPs aree of particular interest due too their inherennt antimicrobiaal activity agaainst a wide
raange of pathoogenic microoorganisms andd can be empployed in the design of noovel hybrid
materials for wound-healing applications or in the fabrication of antibacterial filters. High

surface area-to-volume and aspect ratios are of significant importance for hybrid polymer-
AgNPs systems that are required to possess antimicrobial activity. Such characteristics are
favorable for the efficient contact between the hybrid material and the cell membranes of
pathogenic microorganisms, i.e. for the high biocidal activity. For this reason, it is of interest
to apply electrospinning for preparation of AgNPs-containing hybrid materials.
Polyacrylonitrile [97], cellulose acetate [98,99], PVA [100], and poly(vinyl pyrrolidone)
[101] have already been used as polymer partners. During the last two years the preparation
of hybrid materials from AgNPs and chitosan has attracted increasing attention [102-105].
Recently it has been demonstrated that hybrid nanoparticles consisting of chitosan and
AgNPs, display significantly higher antibacterial activity as compared to that exhibited by the
two partners alone [103]. As outlined in Section 1.3., the modification of chitosan to CECh
leads to loss of the antibacterial properties of the natural polymer. That is why for certain
applications the incorporation of antibacterial agents, such as AgNPs, in CECh-based
polymer materials is appropriate. The reduction of AgNO3 into elemental silver under the
action of concentrated НСООН [107] proceeds according to the reaction (2):
_
2 AgNO 3 + HCOOH 2 Ag(0) + CO 2 + 2 NO 3 + 2 H+
(2)
Penchev et al. [106] have demonstrated that AgNO3 is also reduced in CECh or chitosan
solutions in 85% НСООН to AgNPs. Since CECh and chitosan are soluble under
physiological conditions the authors have aimed at the preparation of AgNPs-containing
nanofibrous materials from the natural polymers crosslinked with GA.
Figure 25. SEM micrograph of net-CECh-sipn-PEO/AgNPs nanofibers; CECh/PEO = 1/1 (w/w); total
polymer concentration - 3.4 wt. %; 0.02 mol/l AgNO3; magnification: × 10000. Penchev et al. [106],
Electrospun hybrid nanofibers based on chitosan or N-carboxyethylchitosan and silver nanoparticles,
Macromol. Biosci., 2009, 9, 884–894. Copyright Wiley-VCH Verlag GmbH and Co. KGaA.
CECh in 85% HCOOH
Ag- Ag-
Ag-
Ag-
Ag-
Ag- Ag-
AgNO3
reduction 30 min
PEO in 85% HCOOH

CECh/AgNPs
dispersion
50% GA aq. solution
electrospinning
CECh in 85% HCOOH PEO in 85% HCOOH
Ag- Ag-
Ag-
Ag-
Ag-
Ag- Ag-
AgNO3
reduction 30 min
1st step electrospinning
cross-linking
2nd step
GA vapors B
Scheme 3. Representation of one- (A) and two-step (B) approaches for prepraration of water-insoluble
net-CECh-sipn-PEO/AgNPs nanofiberous materials.
Fiigure 26. A pho

otograph of yarnn formation durring the electrosspinning of CECCh/PEO/AgNO3 solution in
855 % НСООН; CECh/PEO
C = 1//1 (w/w); total polymer
p concenntration: 3.4 wt.. %; 0.02 mol/l AgNO3.
They have prop posed two appproaches whichh are schematiically presenteed in Scheme 3. The first
appproach consists in reactivee electrospinniing of CECh/P PEO/AgNO3 system
s in the presence
p of
G and aims at one-step preparation
GA p off crosslinked fibrous materrials (Schemee 3A). The
seecond approacch comprises two steps: innitial electrosppinning of CE ECh/PEO/AgN NO3 system
foollowed by crosslinking of the mats withh GA vapors (Scheme 3B). A SEM miccrograph of
neet-CECh-sipn--PEO/AgNPs fibers obtaiined using thhe first apprroach (reactivve electro-
sppinning) is shoown in Figure 25.
The perforrmed studies on o the stabilityy of the nanoffibrous materiials in aqueouus medium,
hoowever, have shown that thhe mats lose thheir integrity and fibrous sttructure after a 24-h stay
inn aqueous solu ution, i.e. thee crosslinking of CECh witth GA has noot proceeded effectively.
e
This result has been attributeed to participattion of GA in the reductionn process of sillver ions to
A
AgNPs, similarrly to the action of formalddehyde [108]. The two-stepp approach (Scheme 3B)
alllows crosslin nked water-innsoluble fibroous materials from both CECh/PEO/Ag
C gNO3, and
chhitosan/PEO/A AgNO3 system ms to be prepaared, as evideenced by the performed
p studdies on the
sttability of the mats after a 244-h contact with aqueous medium
m of рН 4 or saline (рН Н 7). Thus,
thhe crosslinking with GA vapors is an efficient route for preparation of net-chitosan-sipn-
PE EO/AgNPs an nd net-CEChh-sipn-PEO/AggNPs nanofibbrous mats thhat retain theiir integrity
unnder condition ns close to thhe physiologiccal ones. The presence of AgNPs in the chitosan/
PE EO/AgNPs an nd CECh/PEO O/AgNPs nanoofibers has beeen detected byy energy disperrsive X-ray
m
mapping of Ag g. It has beeen found that the nanofibeers contain hoomogeneouslyy dispersed
A
AgNPs. In adddition, the perfformed analysses by means of X-ray phootoelectron sppectroscopy
(XXPS) have rev vealed that 15 % of the incorrporated AgN NPs are on the fibrous materiial surface.
Figure 27. ТЕМ micrograph of a single CECh/PEO/AgNPs fiber; 85% НСООН; CECh/PEO = 1/1
(w/w); total polymer concentration - 3.4 wt. %; 0.02 mol/l AgNO3.
It should be noted that during the electrospinning of CECh/PEO/AgNO3 system at

concentration of the low molecular weight salt 0.02 mol/l, the following phenomenon has
been observed: the initially formed fibers grow in height from the collector to the capillary
tip, which is accompanied by an intensive process of self-bundling of the fibers (Figure 26).
This self-assembly leads to yarn formation. This phenomenon has been observed during the
electrospinning of a non-ionogenic polymer in the presence of a low molecular weight
conductive salt [109]. It has been found that the self-assembling of the respective fibers in
bundles is enabled when the conductivity of the spinning solution is higher than 400 μS/cm.
In the case of CECh/PEO/AgNO3 system the electrical conductivity of the spinning solution
is 22.1 mS/cm.
Apart from the observed self-bundling of the fibers, the performed TEM analyses of
single fibers have shown that the presence of AgNO3 in the spinning solution has an impact
on the fiber morphology, as well (Figure 27).
As seen, on the surface of a CECh/PEO/AgNPs nanofiber extremely fine dendrite-like
structures (mean base diameter of the dendrites emerging from the fiber - 2.7 nm; mean
diameter of the dendritic branches - 1.5 nm) with a length of 65 nm are observed. Structures
of this type are not observed in the case when the fibers have been obtained by
electrospinning of CECh/PEO system in the absence of a low molecular weight salt. The
formation of these fine tendrils on the fiber surface can be attributed to ionic imbalance
induced by the low molecular weight salt on the surface of the spinning jet during the
electrospinning process, similarly to the recently reported formation of a spider-net binding
the main nanofibers in the case of electrospinning solutions of non-ionogenic polymers
containing a low molecular weight salt [110]. In accordance with the results from energy
dispersive X-ray mapping of Ag the AgNPs are uniformly distributed along the fiber length
having a mean size of 3 nm. Thus, the developed approach for incorporation of AgNPs into
CECh-based nanofibrous materials is a highly promising one, and can be used for preparation
of antibacterial wound healing dressings.
3. MATERIALS BASED ON POLYELECTROLYTE

COMPLEXES OF CECh
PEC-based polymer materials are of particular relevance because they combine the
physicochemical and biological properties of different types of polyelectrolytes. In addition,
these materials possess new, specific properties which differ from those of the components
they are composed of. PECs are formed as a result of cooperative interactions between
oppositely charged macromolecules in solution [111]. Depending on the reaction conditions
and the nature of the polymer partners complexes can be obtained in the form of coacervates
[112], highly swollen hydrogels [113], amorphous precipitates [114], colloidal aggregates
[115] and nanoparticles [116,117]. These polymer materials can find application as drug
delivery systems [118,119], gene transfer agents [120-123], dialysis and ultrafiltration
membranes [111], medical implants [124], carriers for enzyme immobilization [125], coatings
of films and fibrous materials [126-128].
Recently, PECs based on natural polysaccharides and their derivatives have attracted
great attention [129]. One of the reasons for this is their envisaged pharmaceutical and
biomedical application. Complex formation with suitable natural or synthetic polyelectrolyte
partners represents a feasible route for targeted modification of the properties of a given
polyelectrolyte. The present Section is focused on PECs between CECh and synthetic
polymers as well as on the possibilities for preparation of novel materials (рН-sensitive
hydrogels, nanoparticles and fibrous materials with targeted surface properties).
Figure 28. pH range of complex formation between chitosan and (co)polymers of AA [130] and AMPS
[131,132]; or between CECh and PAA or PAMPS [8], PEI [8] or (quaternized) PDMAEMA [9].
3.1. Polyelectrolyte Complexes of CECh with Polyacids and Polybases;

Nanoparticles from PECs Based on CECh
As discussed in Section 1 of the present Chapter, depending on the medium рН value

CECh can form water-insoluble PECs with both polyanions (in acidic medium) and
polycations (in alkaline medium). The CECh/synthetic polymer pairs which have so far been
shown to form PECs are presented in Figure 28. For the sake of comparison, the рН range in
which chitosan (the precursor of CECh) is able to form PECs with polyacids is outlined, as
well. Mincheva et al. [8] have demonstrated for the first time the possibility of obtaining
water-insoluble PECs between CECh and the synthetic polyacids - poly(2-acrylamido-2-
methylpropanesulfonic acid) (PAMPS) and PAA. PAMPS is a strong polyacid and in aqueous
solutions the amount of its ionized sulfo groups does not depend on medium рН value. It has
been found that water-insoluble CECh/PAMPS complex is formed in the рН range from 1.2
to 6.0.
CH2 CH n CH
3
O C NH C CH2 SO3H
CH3
Poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS).
CH2 CH n
COOH
Poly(acrylic acid) (PAA).
At pH 1.2 CECh/PAMPS complex of maximum yield, similarly to chitosan/PAMPS complex

[131], is formed at an equimolar ratio between the polymer partners (Figure 29). At рН
values from 4.8 to 6.0 CECh contains betaine-type structures which hamper the complex
formation. For this reason at these pH values maximum yield of the complex is obtained at
CECh excess. The behavior of CECh with respect to the weak polyacid PAA (pKa = 4.8;
[133]) is also similar to that of chitosan. It has been established that CECh/PAA complex is
formed in a narrow рН range: from 4.8 to 6.0, and the higher the medium рН value, the
greater excess of CECh is necessary for the formation of maximum complex yield. The
possibilities of complex formation between CECh and the weak polybase poly(ethylene
imine) (PEI, pKa = 8.8 [134]) [8] have been examined, as well. It has been demonstrated that
a water-insoluble CECh/PEI complex is obtained in the pH range from 5.4 to 7.0.
The complexes are formed at a considerable excess of PEI which is attributed to the fact
that the macromolecule of PEI is highly branched and its protonated amino and imino groups
are hardly accessible for interactions with the ionized carboxyl groups of CECh. PAMPS
possesses anticoagulant activity commensurable with that of the natural anticoagulant heparin
[132]. The complex formation between CECh and PAMPS has been used successfully by
Mincheva et al. [8] as a route for preparation of polymer materials with improved
hemocompatibility.
Yancheva et al. have performed a systematic study on the complex formation between
CECh and synthetic polycations in neutral and alkaline media [9,30]. Poly[2-
(dimethylamino)ethyl methacrylate] (PDMAEMA) and its 50 and 100% quaternized
derivatives: PDMAEMAQ50 and PDMAEMAQ100, have been used as partners of CECh
(Figure 30).
100 1.00
Yield of complex, %
Specific viscosity
75 0.75
50 0.50
25 0.25
0 0.00
0.0 0.2 0.4 0.6 0.8 1.0
Mole fraction of CECh-units
Figure 29. Dependence of the yield of the complex CECh/PAMPS (■) and supernatant specific
viscosity (▲) on the mole fraction of CECh-units (0.2% w/w); pH 1.2, 25°C, I = 0.1. Reproduced from
Mincheva et al. [8] by permission of Elsevier.
Figure 30. Fragments of the macrochains of PDMAEMA (a), PDMAEMAQ50 (b), and
PDMAEMAQ100 (c); R = C2H5OOC(CH3)2; X = Br; A = I. Reproduced from Yancheva et al. [9] by
permission of ACS.
N-Carbboxyethylchitoosan-Based Poolymer Materials 297
Fiigure 31. Depen ndence of the eqquilibrium wateer content (αeq) of CECh/PDMA AEMA (FDMAEM MA = 0.64),
CECh/PDMAEM MAQ50 (FDMAEM MA = 0.53), and CECh/PDMAE EMAQ100 (F DM
MAEMA = 0.47) c
complexes
onn the nature of DMAEMA-bas
D sed polymers (ppH = 7.0 and 9.00, I = 0.1, 25 ºC
C). Yancheva et al.[30],
Poolyelectrolyte complexes
c basedd on (quaternizeed) poly[(2-dim
methylamino)ethhyl methacrylatte]: behavior
inn contact with blood, Macromool. Biosci., 20077, 7, 940-954, Copyright
C Wileyy-VCH Verlag GmbH
G and
Co. KGaA. Reprroduced with peermission.
PDMAEM MA has been synthesized in i a controlleed manner byy atom transsfer radical
poolymerization (ATRP) as described
d prevviously [135], and the quaaternization reeaction has
beeen carried out subsequeently using methyl iodidde as a quaaternizing aggent [136].
(QQuaternized) DMAEMA-based (co)polyymers possess intrinsic biiological activvity which
m
makes them particularly
p atttractive partnners for compplex formatioon with polyaanions. As
poolycations, th hey display antimicrobiall activity against a widee range of pathogenic
m
microorganism s [137,138]. It is also known
k that DMAEMA-ba
D ased (co)polym mers have
innherent hemosstatic propertiees [122,139]. PDMAEMA is a weak polyybase (pKa = 7.0, [140])
thhat is soluble in
i aqueous meedium because of protonatiion of its tertiaary amino grooups. It has
beeen demonstraated that a watter-insoluble complex
c betw
ween CECh andd PDMAEMA A is formed
inn an extremelly narrow рН Н range (arounnd 7.0), and a maximum yield y of the complex
c is
obbtained at a sllight excess off DMAEMA-uunits (FDMAEM MA = 0.64, whe ere FDMAEMA iss the molar
frraction of DMMAEMA-units)) [9]. The neccessity of PDM MAEMA exceess for the preparation of
m
maximum amount of CECh//PDMAEMA complex has been b attributed by Yanchevva et al. [9]
too the fact that at рН values close to the pKp a value of PDMAEMA
P thhe amount of protonated
teertiary amino groups is reeduced. The partialp quaternnization of PDMAEMA
P e
enables the
coomplex formaation with CEC Ch in both neeutral and alkaaline media. ItI has been foound that at
рН Н 7.0 a maxim mum amount of CECh/PDM MAEMAQ50 complex is obtainedo at ann equimolar
raatio between the
t partners (F
( DMAEMA = 0.53),
0 р 9.0 an exccess of PDMA
and at рН AEMAQ50
(F
FDMAEMA = 0.663) is requiredd for the prepaaration of a maximum
m amouunt of complex. This has
beeen ascribed to the fact that
t at рН 9..0 a considerrable amount (ca. 99%) of the non-
quaternized tertiary amino groups in the structure of PDMAEMAQ50 are not protonated and
they cannot participate in complex formation. The quaternization of all of the amino groups in
the case of PDMAEMAQ100 allows the preparation of complexes with a stoichiometry close
to the equimolar one in both neutral (FDMAEMA = 0.47) and alkaline (FDMAEMA = 0.57) media.
Yancheva et al. [30] have examined the dependence of the equilibrium water content
(αeq) of the complexes they have obtained on the nature of the DMAEMA-based polymers at
рН 7.0 and 9.0 (Figure 31). No significant difference between αeq values of
CECh/(quaternized) PDMAEMA complexes has been detected at pH 7.0. This result has been
attributed to the fact that at this pH value the amount of the ionized free groups of both
polymer partners is low; almost all of the carboxyl groups of CECh and the (quaternized)
tertiary amino groups of PDMAEMA are engaged in ionic bond formation in the respective
complexes. At рН 9.0, the CECh/PDMAEMA complex dissolves completely which renders
the determination of the αeq value for this complex impossible at this pH value. As seen in
Figure 31, CECh/PDMAEMAQ50 complex is characterized by the highest αeq value at рН
9.0. This observation has been explained by the occurrence of two processes. On the one
hand, the non-quaternized amino groups of PDMAEMAQ50 at this рН value are in the non-
protonated form and the ionic bonds between these groups and the carboxylate anions of
CECh are disrupted. On the other hand, at pH 9.0 CECh does not exist in the form of betaine-
type structures. This leads to an increased amount of free hydrophilic groups and enhanced
complex swellability. The effect of the carboxyl groups liberated from the betaine-type
structures of CECh on the swelling ability of the complexes has been confirmed in the case of
CECh/PDMAEMAQ100 complex. The αeq value of this complex is higher at pH 9.0 than at
pH 7.0.
Yancheva et al. [9] have prepared films of crosslinked CECh by a reaction with GA at
[NH2]/[CHO] = 1/1 (mol/mol). The obtained films are insoluble in aqueous media over the
entire pH range. It has been demonstrated that the crosslinking of CECh using GA does not
interfere with CECh ability to form complexes with (quaternized) PDMAEMA, since GA
interacts with the N-containing groups of CECh while preserving its carboxyl groups free.
Similarly to CECh/(quaternized)PDMAEMA PECs a complex between crosslinked CECh
and PDMAEMA is formed in a narrow рН range around 7.0, while complexes between CECh
and PDMAEMAQ50 or PDMAEMAQ100 are formed in both neutral and alkaline media.
The engagement of the N-containing groups of (quaternized) PDMAEMA in complex
formation with CECh should lead to changes in the polycation behavior upon contact with
pathogenic microorganisms and blood cells. Yancheva еt al. [9] have demonstrated that the
behavior of crosslinked CECh/(quaternized)PDMAEMA complexes with respect to E. coli
depends on the polycation nature. The CECh/PDMAEMA complex induces complete
inhibition of the bacterial growth of E. coli. Using PDMAEMAQ50 as a polymer partner
yields complexes which possess feeble antibacterial activity and in the case of fully
quaternized PDMAEMA (PDMAEMAQ100) the complex formation with CECh leads to loss
of the polycation antibacterial activity. The different behavior of the complexes is due to the
different amount of free N-containing groups which are responsible for the antibacterial
activity of (quaternized) PDMAEMA. The performed studies have shown that in the case of
the CECh/PDMAEMAQ100 complex all of the quaternized amino groups are engaged in
ionic bonds with the carboxyl groups of CECh and that accounts for the lack of antibacterial
activity of this complex.
Figure 32. Effect of PDMAEMA (A), PDMAEMAQ50 (B), PDMAEMAQ100 (C), (crosslinked)
CECh, and their complexes (A–C) on the counts of the white blood cells, red blood cells and platelets;
concentration of the polymers 5 mg/ml, concentration of the (crosslinked) CECh/(quaternized)
PDMAEMA complexes 10 mg/ml; duration of the contact with whole blood 30 min. Yancheva et al.
[30], Polyelectrolyte complexes based on (quaternized) poly[(2-dimethylamino)ethyl methacrylate]:
behavior in contact with blood, Macromol. Biosci., 2007, 7, 940-954. Copyright Wiley-VCH Verlag
GmbH and Co. KGaA. Reproduced with permission.
Yancheva еt al. [30] have demonstrated that the behavior of the complexes in contact
with blood cells also depends significantly on the polycation nature, as well as on the type of
CECh: non-crosslinked or crosslinked. The results from the blood cell counting tests
performed after contact of the complexes with whole blood are presented in Figure 32. The
complex formation between (crosslinked) CECh and (quaternized) PDMAEMA leads to a
substantial decrease in the polymer partners toxicity in respect to white and red blood cells;
the counts of the latter remain almost unchanged after a 30-min contact with the complexes.
The platelet counts, however, are reduced. An explanation of this fact has been found in the
structure of the complexes. CECh/PDMAEMA and CECh/PDMAEMAQ50 complexes are
highly porous, as evidenced by SEM observations. Platelets, due to their small size (2 - 4
μm), can penetrate into the pores of the complexes and be physically entrapped in the bulk of
the complexes. After their penetration the platelets may interact with the free positively
charged N-containing groups located in the bulk of the complexes. This can lead to platelet
activation and aggregation resulting in a reduction in the number of platelets in the blood
sample. Using crosslinked CECh as a partner of (quaternized) PDMAEMA in PEC formation
allows the preparation of materials which are less harmful in respect to platelets (Figure 32).
The presented results indicate that CECh is a suitable partner for the preparation of PECs
which display interesting behavior upon contact with pathogenic microorganisms and blood
cells. The systematic investigations conducted by Mincheva et al. and Yancheva et al.
[8,9,30] have paved the way for finding new solutions for preparation of stable aqueous
dispersions of nanoparticles consisting of PECs between CECh and copolymers of
PDMAEMA or PAMPS, as well as for preparation of PEC-based coatings on the surface of
electrospun fibrous materials. The obtained results are briefly described below.
Since the first reports on PECs as colloidal dispersions [116,141,142], this field has
attracted significant attention as the obtained materials demonstrate effectiveness in
flocculation [143], surface modification of different substrates [144, 145], and are very
promising in ecology, drug delivery and controlled release systems [146,147]. The driving
forces are the same as for the preparation of macroscopic PECs: electrostatic interactions
based on Coulombic attraction and entropy gain through counterion release. Depending on
the chemistry of the different components additional interactions, such as hydrogen bond
formation and Van der Waals forces or dipole-charge transfer might also contribute. Up to
now, numerous ionogenic homopolymers, neutral-ionic block, graft, or random copolymers
have been complexed with oppositely charged natural polymers or synthetic (co)polymers of
various architectures [117]. To describe the formed structures four different terms are used:
(i) polyion complex micelles (PICs) [116]; (ii) (inter)polyelectrolyte complexes (I)PECs
[148,149]; (iii) block ionomer complexes (BICs) [141] and (iv) complex coacervate core
micelles (C3Ms) [144]. Typically, when polyelectrolytes of significantly different molecular
weights are mixed in non-stoichiometric ratios nanoparticles consisting of a long host
macromolecule complexed with shorter molecules of an oppositely charged guest
polyelectrolyte are formed [141, 150-152]. However, such nanosized PEC-based systems are
only obtained far from charge stoichiometry as secondary aggregation leading to precipitation
upon approaching charge unity. Secondary aggregation occurs also in solutions containing
low molecular weight salts, which is a disadvantage when potential biomedical applications
requiring contact with body fluids are targeted. To avoid these drawbacks, neutral-ionic block
copolymers are used resulting in nanoparticles with a stimuli-responsive complex coacervate
core and neutral (usually, PEO) shell, impeding aggregation through steric stabilization
[117,144,146,151,153]. The size and shape of such PEC nanoparticles depend significantly on
the length ratio between the neutral and the ionic blocks, medium conditions and molar ratio
between the oppositely charged partners [117].
PEC nanoparticles from natural polymers, such as D(R)NA, oligonucleotides, proteins,
peptides, enzymes and polysaccharides (like chitosan) evoke special attention [117, 154-162].
Such nanoparticles are considered very promising for the development of novel materials
with improved in vivo distribution and preserved or even enhanced stability and activity.
Nanoparticles from chitosan and chitosan derivatives are prepared by PEC formation with
diverse synthetic and natural polyions.
Mincheva et al. [163] have studied the possibility of obtaining nanoparticles consisting of
PECs between CECh and DMAEMA-based (co)polymers. When using PDMAEMA as a
polymer partner, it has been found that regardless of the polymer concentration (0.01, 0.05, or
0.1 mg/ml) and the medium pH value (pH 6.5, 7.0, or 7.5) at ionic strength (I) of 0.1 and
FDMAEMA = 0.6 (mixing ratio between the oppositely charged groups equal to 1/1) the
complex formation with CECh leads to the occurrence of macroscopic phase separation in the
system. Precipitation has been avoided by lowering the ionic strength value up to 0.01,
however, large nanoparticles bimodal in distribution with apparent hydrodynamic diameters
(Dh,app) of 800–900 nm and 50–100 nm, have been detected by DLS. These observations are
indicative of very heterogeneous populations of nanoparticles, consisting of a long host
molecule complexed with shorter guest polyions carrying oppositely charged groups
[141,164]. Another explanation is the appearance of diffusion/shielding effects during the fast
PEC formation, resulting in the preparation of particles with a core consisting of the high
molecular weight polymer where parts of the chains are inaccessible for the low molecular
weight polymer, and a shell of insoluble PEC between the partners [165].
O CH3 CH3 O CH3 CH3

CH3O CH2CH2O C C CH2 C n Br CH3O CH2CH2O C C CH2 C n Br
114 114
CH3 C O CH3 C O
O O
(CH2)2 (CH2)2
(1) (2) H3C + _
N N ,I
H3C CH3 H3C
CH3
O CH3
CH3O CH2CH2O C C CH2 CH m Br
114
CH3 C O
NH
H3C _ +
(3) C CH2 SO3, Na
H3C
Figure 33. Chemical structures of: PЕO114-b-PDMAEMAn (1, n = 41, 70 or 119); PЕO114-b-
PDMAEMAnQ100 (2, n = 41, 70 or 119), and PЕO114-b-PAMPSNam (3, m = 41, 66 or 150).
The replacement of PDM MAEMA withh the double hydrophilic dibblock copolym mer PЕO-b-
PDDMAEMA has h allowed the t preparatioon of aqueouus dispersionss of PEC naanoparticles
w
without occurreence of macroscopic phase separation.
s
The approaach based on using
u double hydrophilic
h mers has been applied by
dibblock copolym
M
Mincheva et all. [163,166] for
fo the preparaation of nanopparticles conssisting of PEC Cs between
C
CECh and copo olymers contaaining a weak polycationic block (PЕO-bb-PDMAEMA A), a strong
poolycationic block (PЕO-b-PDMAEMAQ100), or a strong polyyanionic blockk (PЕO-b-
PAAMPSNa). Th he chemical structures
s of the
t synthetic polyions
p are given
g in Figuure 33. The
PEEC-based nan noparticle disppersions havee been evaluaated with resppect to CECh molecular
w
weight, polyelectrolyte blocck length in the diblock copolymers, polymer conncentration,
paartner molar ratio,
r pH and ionic strengthh value. Two CECh samplees, having M w of 9.5 ×
5
100 g/mol (HM MMCECh) andd 7.1 × 103 g/m mol (LMMCE ECh) have beeen prepared acccording to
thhe procedure described
d by Sashiwa
S et al. [2]. The doubble hydrophilicc block copolyymers have
beeen synthesizzed in a conttrolled manneer by water-bbased ATRP using α-methhoxy-ω-(2-
m
methylbromois obutyrate) PE
EO as a macrooinitiator [1677,168]. The leength of the ionic
i block
haas been varied
d while keepinng the PEO bllock length coonstant (degree of polymeriization, n =
114; M n = 500 00 g/mol). It has
h been demoonstrated that in
i the case of the HMMCEC Ch/PЕO-b-
PDDMAEMA pair p in contraast to HMMCECh/PDMA AEMA at mixxing ratio beetween the
opppositely charrged groups eqqual to 1/1 no flocculation occurs
o at polym
mer concentraations up to
0..05 mg/ml an nd I = 0.1. Thhe dependencce of Dh,app off HMMCEChh/PEO114-b-PD DMAEMAn
paarticles on thee molar fractioon of DMAEMMA-units is shoown in Figuree 34.
Fiigure 34. Depen ndence of the appparent hydrodyynamic diameteer (Dh,app) on thee mole fraction of
D
DMAEMA unitss for HMMCEC Ch/PEO114-b-PD DMAEMAn nannoparticles (n = 45, 55, and 1344) as
obbtained at initiaal polymer concentration 0.05 mg/ml;
m pH 7.0; I = 0.1; 25 ± 0..1 ºC. Minchevaa et al.
[1163], Polyelectrrolyte complex nanoparticles
n frrom N-carboxyeethylchitosan annd polycationicc double
hyydrophilic diblo ock copolymerss, J. Polym. Sci. Part A: Polym. Chem., 2009, 47, 2105-2117.. Copyright
W
Wiley-VCH Verllag GmbH and Co. KGaA. Repproduced with permission.
p
Figure 35. Cryo-TEM micrographs of HMMCECh/PEO114-b-PDMAEMA45 (A) and

LMMCECh/PEO114-b-PDMAEMA45 (B) nanoparticles as obtained upon mixing polymer solutions with
concentration 0.01 mg/ml. pH 7.0; I = 0.1; FDMAEMA = 0.6. Mincheva et al. [163], Polyelectrolyte
complex nanoparticles from N-carboxyethylchitosan and polycationic double hydrophilic diblock
copolymers, J. Polym. Sci. Part A: Polym. Chem., 2009, 47, 2105-2117. Copyright Wiley-VCH Verlag
GmbH and Co. KGaA. Reproduced with permission.
As seen, the size of the obtained nanoparticles increases on increasing the PDMAEMA block
length. The obtained results are in agreement with literature data showing that the size of PEC
nanoparticles increases on increasing the ionogenic block length [144]. Regardless of the fact
that no flocculation has been observed under the selected conditions, DLS results have shown
that when PEO114-b-PDMAEMAn (n = 55 or 134) is used the particle size distribution
becomes bimodal on approaching mixing ratio between the oppositely charged groups equal
to 1/1. Thus, nanoparticles with well-defined PEC core-PEO shell structure are not obtained
from HMMCECh/PEO114-b-PDMAEMAn (n = 55 or 134). When using these polycations it
has also been found that upon increasing the polymer concentration up to 0.1 mg/ml aqueous
dispersions of PEC nanoparticles without flocculation are obtained only at FDMAEMA < 0.3 or
FDMAEMA > 0.8. Using the copolymer with the shortest ionogenic block (PEO114-b-
PDMAEMA45) allows the preparation of aqueous dispersions without occurrence of
flocculation in the entire range of concentrations (from 0.01 mg/ml to 0.1 mg/ml) irrespective
of FDMAEMA value. This is attributed to the fact that the ratio between the neutral and the
polyelectrolyte block is 3, which is often considered as the minimum ratio required for
stabilization of PEC nanoparticles near charge stoichiometry [141,144]. The conducted DLS,
AFM and cryo-TEM analyses have shown that in the case of the HMMCECh/PEO114-b-
PDMAEMA45 pair core-shell nanoparticles are formed only at ionic strength of the medium
equal to 0.01. At ionic strength value close to the physiological one (I = 0.1) secondary
aggregation occurs (Figure 35A).
It is known that unmatched pairs (the molecular weight of HMMCECh is about 130 times
higher than that of the PDMAEMA block in PEO114-b-PDMAEMA45) cannot form
nanoparticles without mixing of the shell and the PEC forming phases [141,144]. The
alignment of the molecular junctions between the ionogenic and PEO segments at the core-
shell interface is a prerequisite for well-defined phase separation between the core and the
shell in the nanoparticles. When one of the partners is significantly longer, important
constraints exist because charge neutralization and uniform distribution of the ionogenic
segments in the core have to be respected. That is why, in the case of HMMCECh/PEO114-b-
PDMAEMA45, mixing of the core- and shell-forming phases is possible thus leading to large
particle formation (Figures 35A and 36A). According to Kabanov et al., the simplest way to
describe the formed structures is as micelle-like aggregates consisting of neutralized polyions
surrounded by a PEO shell [141]. According to van der Burgh et al., particles with deformed
core are obtained on increasing the chain length of the homopolymer [144]. For LMMCECh (
M w = 7.1 × 103 g/mol) the length of the chain (n = 30) is commensurable with the length of
the PDMAEMA block in PEO114-b-PDMAEMA45. In the case when the lengths of the
ionogenic segments of the polymer partners do not differ significantly, the alignment of the
molecular junctions between the ionogenic and PEO segments at the core-shell interface is
allowed without occurrence of phase mixing. Besides, nanoparticles with strictly separated
PEC-core and PEO-shell are formed (Figures 35B and 36B). The core-shell structure of
LMMCECh/PEO114-b-PDMAEMA45 nanoparticles has been evidenced by the performed
XPS analyses, revealing no nitrogen on their surface. Thus, it has been concluded that these
particles consist of a coacervate core from LMMCECh/PDMAEMA complex surrounded by
a PEO shell.
While PEO-b-PDMAEMA copolymers allow the formation of PEC nanoparticles with
CECh in a rather narrow pH range (ca. 7.0) [163], the quaternized PDMAEMAs enable as
discussed above the preparation of complexes with CECh in a broader pH range from 6.5 up
to 14.0. Therefore, the peculiarities of the preparation of core-shell nanoparticles from the
CECh/PEO-b-PDMAEMAQ100 pair have been studied [166]. At first, experiments have
been performed using HMMCECh at conditions at which PEC nanoparticles from the
CECh/PEO-b-PDMAEMA pair have been obtained.
Figure 36. Schematic representation of HMMCECh/PEO114-b-PDMAEMA45 (A) and

LMMCECh/PEO114-b-PDMAEMA45 (B) nanoparticles as obtained at pH 7.0, I = 0.1 and FDMAEMA =
0.6. Mincheva et al. [163], Polyelectrolyte complex nanoparticles from N-carboxyethylchitosan and
polycationic double hydrophilic diblock copolymers, J. Polym. Sci. Part A: Polym. Chem., 2009, 47,
2105-2117. Copyright Wiley-VCH Verlag GmbH and Co. KGaA. Reproduced with permission.
Figure 37. DLS histograms of the LMMCECh/PEO114-b-PDMAEMA45Q structures obtained at

different polymer concentrations (A), and dependence of the diffusion coefficient (D) on the scattering
vector (q2) for nanoparticles prepared at polymer concentration of 0.01 mg/ml (B); FDMAEMAQ = 0.6; pH
7.5; I = 0.01; 25 ± 0.1 ºC. Reproduced from Mincheva et al. [166] by permission of ACS.
In sharp contrast, macroscopic flocculation has occurred in all studied systems from the
HMMCECh/PEO-b-PDMAEMAnQ100 pair at pH 7.5, 8.0, and 9.0 even at polymer
concentration as low as 0.005 mg/ml. The failure to prepare aqueous dispersions has been
attributed to the high ionic strength value used in the experiments (I = 0.1). Taking into
account the obtained results experiments at 10 times lower ionic strength values (down to
0.01) have been conducted. However, even under these conditions a PEC nanoparticles-
containing dispersion without macroscopic phase separation at charge stoichiometry has only
been obtained using the copolymer with the shortest quaternized PDMAEMA block and at
low polymer concentration of 0.005 mg/ml. The Dh,app values depend strongly on
FDMAEMAQ100 value, and at mixing ratio between the cationic and anionic groups close to 1/1
two populations of nanoparticles have been detected, which is indicative for the occurrence of
secondary aggregation. Thus, the presence of a PEO block cannot prevent the aggregation of
the particles, at least when its degree of polymerization is 144. To evaluate the effect of the
chain lengths of the polymer partners, HMMCECh has been replaced by its low molecular
weight analogue (LMMCECh). This has enabled the preparation of nanoparticles at polymer
concentration as high as 0.1 mg/ml, without occurrence of macroscopic phase separation,
irrespective of FDMAEMAQ100 value. At polymer concentration equal to 0.01 mg/ml Dh,app (240-
290 nm) does not depend significantly on FDMAEMAQ100, while at 0.1 mg/ml it increases on
increasing FDMAEMAQ100 and passes through a maximum. Similarly to the LMMCECh/PEO114-
b-PDMAEMA45 pair, the size of the particles increases on increasing the polymer
concentration. The DLS histograms of the LMMCECh/PEO114-b-PDMAEMA45Q100
nanoparticles in the studied concentration range are monomodal even at stoichiometric
conditions (Figure 37). As evidenced by AFM and XPS analyses, core-shell nanoparticles are
obtained only at polymer concentration of 0.01 mg/ml (the detailed conditions are given in
the figure caption of Figure 37).
The possibility of preparing PEC nanoparticles from CECh/PEO-b-PAMPSNa in acidic
medium has been evaluated, as well [166]. Similarly to the previous cases of
CECh/polycationic block copolymer pairs, at first the possibility of obtaining nanoparticles
from HMMCECh/PEO-b-PAMPSNa pair at ionic strength 0.1 at different polymer
concentrations and pH values of 1.2, 4.8 and 6.0 has been examined. Again, macroscopic
flocculation has been observed after mixing the polymer solutions. The decrease of the ionic
strength to 0.01 while using HMMCECh has allowed the formation of nanosized structures
only with the shortest polyanionic block, but in excess of CECh macroscopic flocculation has
occurred even at polymer concentration of 0.005 mg/ml. Thus, in contrast to the previously
discussed cases, the lower ionic strength does not prevent the pronounced aggregation of the
particles. Aqueous dispersions of nanoparticles have only been obtained at ionic strength of
0.01 when LMMCECh and PEO114-b-PAMPSNa41 have been paired similarly to
CECh/PEO114-b-PDMAEMA41Q100. As evidenced by DLS analyses, the Dh,app values for the
LMMCECh/PEO114-b-PAMPSNa41 nanoparticles depend strongly on pH. It has been found
that the Dh,app values increase on increasing pH from 1.2 to 6.0 and the maxima of the curves
are shifted to higher FCECh. This is ascribed to the existence of intermacromolecular dipole-
dipole interactions in CECh at pH values higher than 4.8, as discussed above for
CECh/PAMPS complex formation. Moreover, hydrogen bonds cannot be precluded. Such
interactions can accompany the PEC formation with participation of PAMPS sulfo groups
and the protonated N-containing groups of CECh, thus resulting in highly aggregated
systems.
It is evident that the preparation of nanoparticles consisting of CECh-based PECs
depends to a great extent on the oppositely charged polyelectrolyte nature as well as on the
medium conditions (рН and ionic strength values).
3.2. Preparation of Non-Woven Textiles by Combining PEC Formation and

Electrospinning
The preparation of PEC-coated fibrous materials by consecutive deposition of oppositely

charged polyelectrolytes is a promising trend in the preparation of novel materials by means
of electrospinning [128,169,170]. In this manner targeted modification of the biological
behavior of the fibrous materials can be achieved. Recently Yancheva et al. [171] have
demonstrated the formation of a coating based on a PEC between CECh and PEO-b-
PDMAEMAQ100 on the surface of PLLA and PLLA/PEG electrospun materials.
Figure 38. Photographs showing the wettability of PLLA and PLLA/PEG mats on contact with a drop
of water or a drop of water/ethanol mixed solvent (1/1 v/v). Reproduced from Yancheva et al. [171] by
permission of ACS.
Figure 39. SEM micrographs of PLLA fibers triple-coated with CECh (cross-linked): × 1000 (A) and ×
10 000 (B), and coated with CECh (cross-linked)/PEO-b-PDMAEMAQ100 complex: × 1000 (C) and ×
10 000 (D). Reproduced from Yancheva et al. [171] by permission of ACS.
Figure 40. High resolution C 1s spectra of PLLA mats: pristine (A), triple-coated with CECh(cross-
linked) (B), and coated with CECh(crosslinked)/PEO-b-PDMAEMAQ100 complex (C). For the sake of
comparison, high resolution C 1s spectra of a film of CECh(cross-linked) (D) and PEO-b-
PDMAEMAQ100 powder (E) are presented. Reproduced from Yancheva et al. [171] by permission of
ACS.
It has been found that in contrast to PLLA/PEG mats, which are hydrophilic, the surface of
PLLA mats is superhydrophobic (the determined water contact angle is 142.3 ± 2.2°). Thus,
aqueous solutions of the polyelectrolyte partners are not suitable for preparation of coatings
of CECh or PEC because of the poor water wettability of PLLA mats. It has been suggested
that replacing part of the water with ethanol can improve PLLA mats wettability. Photographs
showing the wettability of PLLA and PLLA/PEG mats on contact with a drop of water and
with a drop of water/ethanol mixed solvent (1/1 v/v) are presented in Figure 38. As seen, the
use of a mixed water/ethanol solvent leads to improved wettability of the PLLA mats by
aqueous solutions. Therefore, this solvent has been used to prepare solutions of CECh and
PEO-b-PDMAEMAQ100 for consecutive deposition of the polymer partners on the surface
of the PLLA and PLLA/PEG mats. The PLLA mats have been coated by immersion in the
CECh solution followed by drying; the coating/drying procedure has been repeated three
times. In order to prevent dissolution of the coating in the course of depositing a layer of the
oppositely charged polyelectrolyte CECh has been crosslinked in an atmosphere enriched
with GA vapors. Subsequently, deposition of PEO-b-PDMAEMAQ100 has been carried out.
SEM micrographs of CECh(crosslinked)- and PEC-coated fibers are shown in Figure 39. As
seen, the triple coating with CECh leads to film formation between some of the fibers. In
addition, a 2-fold decrease of the mean pore number per unit area is observed (from 48
pores/9 μm2 to 22 pores/9 μm2). Moreover, the mean length and width of the pores decrease,
respectively, from 370 ± 70 nm to 240 ± 50 nm and from 160 ± 40 nm to 120 ± 30 nm
(Figure 39B). The complex formation between CECh(crosslinked) and PEO-b-
PDMAEMAQ100 leads to additional reduction of the pores number (up to 15 pores/9 μm2),
as well as of their size (up to a mean length of 120 ± 30 nm and a mean width of 100 ± 23
nm, respectively) (Figure 39D). The observation of a film between some of the fibers as well
as the reduction of the mean size and number of the pores along PLLA fibers indicate that a
coating of CECh(crosslinked) or CECh(crosslinked)/PEO-b-PDMAEMAQ100 complex is
formed on the surface of the fibers.
The successful coating of the fibers with CECh or PEC, respectively, has been verified by
XPS analyses. In the survey spectra a new peak characteristic of nitrogen has been recorded at
binding energies of 396-403 eV (0.15 atomic % for PLLA mats coated with CECh
(crosslinked) and 0.84 atomic % for PEC-coated mats). In addition, the appearance of a new
peak corresponding to carbon atoms participating in C-N bond is observed in the C 1s spectra
of the coated fibers at 285.79 eV (Figures 40B,C). Carbon atoms of this type are present in
the structure of CECh and PEO-b-PDMAEMAQ100 (Figure 40D,E). The area of the peak at
285.79 eV increases upon coating with PEC (Figure 40C). The high resolution N 1s spectra of
the (coated) PLLA fibrous materials provide additional evidence of the successful coating
with CECh or PEC. In the spectrum of PLLA mats coated with CECh(crosslinked), two types
of nitrogen atoms have been recorded, which are assigned to nitrogen from NHCOCH3
groups (at 399.83 eV) and nonsubstituted, mono-, and disubstituted amino groups (at 398.39
eV) which are present in CECh structure. In the high resolution N 1s spectrum of PEC-coated
mats, two peaks are observed at 402.77 and 397.19 eV, respectively, and the ratio between the
peak areas is 60/40. The first peak is attributed to the presence of nitrogen from quaternary
ammonium groups from the quaternized PDMAEMA block in the copolymer structure, and
the second one is assigned to the presence of nitrogen from nonsubstituted, mono-, and
disubstituted amino groups from CECh structure. It is noteworthy that the atomic percentages
of nitrogen atoms on the surface of CECh-coated PLLA and PLLA/PEG mats obtained from
the survey spectra are very close (Table 2).
Table 2. Elemental composition of pristine, CECh(cross-linked)-coated or CECh(cross-

linked)/PEO-b-PDMAEMAQ100 complex-coated PLLA and PLLA/PEG matsa
Elemental composition, atomic %

Composition
C O N I
PLLA 64.98 35.02 - -
PLLA/CECh 65.48 34.37 0.15 -
PLLA/CECh/
69.44 29.49 0.84 0.23
PEO-b-PDMAEMAQ100
PLLA/PEG 68.05 31.95 - -
PLLA/PEG/CECh 62.65 37.25 0.10 -
PLLA/PEG/CECh/
71.19 27.83 0.73 0.24
PEO-b-PDMAEMAQ100
a
Determined by XPS analysis.
Figure 41. Effect of triple-coating with CECh(cross-linked) (net-CECh) and of the formation of a
CECh(cross-linked)/PEO-b-PDMAEMAQ100 complex on the behavior of PLLA mats in contact with
blood cells; duration of the contact with whole blood: 30 min; **p < 0.01; ***p < 0.001. Reproduced
from Yancheva et al. [171] by permission of ACS.
A similar result has been obtained for PEC-coated PLLA and PLLA/PEG mats. This is an
indication that when a mixed water/ethanol solvent (1/1 v/v) is used the chemical composition
and surface topography of the fibrous material do not have any significant impact on the
surface composition of CECh- or complex-coated mats.
As pointed out in Subsections 1.3. and 3.1., CECh and DMAEMA-based (co)polymers
are characterized by intrinsic biological activity. The formation of a CECh- or complex-based
coating on the surface of PLLA and PLLA/PEG fibrous materials is expected to alter their
behavior in respect to blood cells and pathogenic microorganisms. For this reason Yancheva
et al. [171] have conducted ex vivo studies which have demonstrated that the approach
consisting in coating PLLA and PLLA/PEG fibrous materials with CECh/PEO-b-

PDMAEMAQ100 PEC can be used for imparting hemostatic and antibacterial activity to the
surface of the electrospun mats. The results obtained from the blood cell counting tests
performed with (coated) PLLA mats are presented in Figure 41. In accordance with
previously reported results [172], it has been found that PLLA-based materials do not exert
any effect on the blood cell counts. The triple coating with CECh(crosslinked) leads to a
slight reduction in the blood cell counts. The formation of a complex between
CECh(crosslinked) and PEO-b-PDMAEMAQ100 on the surface of PLLA mats allows the
preparation of materials that trigger a considerable decrease in the platelet number and
possess hemostatic activity. The results from the blood cell counting tests have been testified
by the conducted SEM analyses (Figure 42). Single red blood cells with unaltered
morphology are observed on the surface of the pristine PLLA mats (Figure 42А) and the
PLLA mats triple-coated with CECh(crosslinked) (Figure 42B), i.e. the pristine and
CECh(crosslinked)-coated PLLA mats do not display hemostatic activity.
Figure 42. SEM micrographs of PLLA mats after contact with blood: pristine (А, × 503 – large SEM
micrograph, × 2500 – small SEM micrograph), triple-coated with CECh (cross-linked) (B, × 503) and
coated with a CECh(cross-linked) /PEO-b-PDMAEMAQ100 complex (C, × 503 and D, × 2500).
Figure 43. SEM micrographs of S. aureus cells adhered to the surface of mats incubated in cell culture
(2 × 107 cells/ml) for 24 h at 37°С. PLLA mats: pristine (А, ×500), triple-coated with CECh(cross-
linked) (B, ×500), and coated with a CECh(cross-linked)/PEO-b-PDMAEMAQ100 complex (C, ×500).
Figure 44. SEM micrographs of S. aureus cells adhered to the surface of mats incubated in cell culture
(2 × 107 cells/ml) for 24 h at 37°С. PLLA/PEG mats: pristine (А, ×500), triple-coated with CECh(cross-
linked) (B, ×500), and coated with a CECh(cross-linked)/PEO-b-PDMAEMAQ100 complex (C, ×500).
In the case of PLLA mats coated with a CECh(crosslinked)/PEO-b-PDMAEMAQ100

complex a greater number of adhered red blood cells are observed (Figure 42C), as well as
some aggregates consisting of red blood cells and platelets (Figure 42D). The obtained results
are consistent with the results obtained from the blood cell counting tests that attest for
preservation of the red blood cells number, that is, for absence of hemolysis and for a
reduction in the platelets number. Similarly to the PLLA mats, on the surface of the
PLLA/PEG mats with a triple-coating of CECh(crosslinked) single red blood cells with
unaltered morphology are observed whereas the complex-coated mats exhibit hemostatic
properties (on the surface of the latter adhered and aggregated red blood cells and platelets are
observed).
SEM micrographs of S. aureus cells adhered to the surface of mats incubated in cell
culture of S. aureus for 24 h are presented in Figure 43. As seen, a great number of adhered
cells are observed on the surface of the PLLA fibrous materials (ca. 120 cells/400 µm2). The
considerable adhesion of bacterial cells is related to the hydrophobicity of the surface of
PLLA materials [172].
The preparation of a coating from CECh(crosslinked) or CECh(crosslinked)/PEO-b-

PDMAEMAQ100 complex leads to a considerable decrease in the number of cells adhered to
the surface of the mats, up to about 20 cells/400 μm2 and 15 cells/400 μm2, respectively
(Figure 43B,C). As discussed in Subsection 1.3 of the present Chapter, CECh does not exhibit
antibacterial activity. The reduced adhesion of S. aureus cells to CECh(crosslinked)-coated
mats is explained by the presence of negatively charged carboxylate anions on the surface of
the mats, and their repellent activity against the pathogenic microorganism. The reduced
number of S. aureus cells adhered to PEC-coated mats is attributed to the presence of
quaternized tertiary amino groups from the copolymer on the surface of the mats. The
obtained results indicate that the preparation of a PEC coating considerably reduces the
adhesion of the pathogenic microorganism to the surface of PLLA fibrous materials. The
incorporation of PEG in PLLA fibrous materials leads to a decrease in the adhered bacterial
cells number (from 120 cells/400 µm2 in the case of PLLA mats to 90 cells/400 µm2 in the
case of PLLA/PEG mats) (Figure 44А). The reduced bacterial cell adhesion is due to the
greater hydrophilicity of the PEG-containing fibrous materials. The formation of a coating of
CECh(crosslinked) or CECh(crosslinked)/PEO-b-PDMAEMAQ100 complex results in an
additional reduction in the number of the adhered cells - 65 and 35 cells/400 µm2,
respectively (Figure 44B,C).
These findings indicate that coating with CECh(crosslinked) leads to the preparation of
novel materials, which are characterized by good compatibility with blood cells and reduce
the adhesion of pathogenic microorganisms. These materials can find potential applications in
the tissue regeneration field. The preparation of a CECh(crosslinked)/PEO-b-
PDMAEMAQ100 complex coating imparts hemostatic properties to the PLLA and
PLLA/PEG mats. In addition, the PEC coating reduces the adhesion of pathogenic
microorganisms. Thus, the PEC-coated PLLA and PLLA/PEG mats are potential candidates
for wound healing applications.
4. CONCLUSIONS
As evident from the above considered studies on N-carboxyethylchitosan, this chitosan
derivative is a promising candidate for diverse applications because of the easily feasible and
environmentally friendly route of its synthesis and the particular physicochemical behavior of
this polyampholyte-polyzwitterion. In addition, the data available on CECh biological
behavior indicate that it is a biocompatible and biodegradable polymer. This is of great
importance regarding especially biomedical applications. The found conditions for fabrication
of core-shell nanoparticles with a CECh core and of CECh-based non-woven textile by
electrospinning pave the way to design of new generation drug delivery systems and wound
healing dressings. Moreover, the ability of CECh to stabilize metal nanoparticles such as
superparamagnetic iron oxide nanoparticles and silver nanoparticles is propitious for
successful preparation of hybrid organic/inorganic dispersions as well as of hybrid
nanofibrous materials. The formation of polyelectrolyte complexes is an efficient route for
preparation of various CECh-based materials with tunable surface properties. It may be
concluded that amongst the polymers obtained from renewable sources, N-
carboxyethylchitosan is of particular interest due to its numerous beneficial properties and its
versatility.
ACKNOWLEDGMENTS
Financial support from the National Science Fund of Bulgaria (Grant DO-02-237/2008)
is gratefully acknowledged. The authors thank the bilateral cooperation between the
Bulgarian Academy of Sciences and Wallonie-Bruxelles International (WBI, ex-CGRI,
Brussels).
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Chapter 6
CHITOSAN NANOPARTICLES FOR

BIOMEDICAL APPLICATIONS
Paula Pereira, Vera Carvalho, Reinaldo Ramos

and Miguel Gama∗
IBB-Institute for Biotechnology and Bioengineering,
Centre for Biological Engineering, Minho University,
Campus de Gualtar, Braga, Portugal
ABSTRACT
Chitosan is a rather abundant material with exquisite properties, which may be
processed into a variety of materials including hydrogels, fibres, membranes, etc. The
production of chitosan-based nanogels, also known as macromolecular miceles, has been
successfully achieved using different techniques, which will be reviewed. This chapter
covers the properties and applications of chitosan nanogels in the biomedical field,
namely as a drug delivery vehicle for biopharmaceuticals. The main achievements and
recent developments will be addressed.
1. INTRODUCTION
Chitosan (poly[β-(1-4)-2-amino-2-deoxy-D-glucopyranose]) is a natural, non-toxic and
biodegradable linear polysaccharide, composed by β-(1-4)-linked N-glucosamine and N-
acetyl-glucosamine residues [1]. Chitosan (CS) is obtained upon partial alkaline deacetylation
of chitin, a structural element in the exoskeleton of crustaceans and insects, which is the
second-most abundant polysaccharide next to cellulose [2]. Nevertheless, being insoluble in
water and chemically inert, applications of chitin are limited. In turn, having hydroxyl and
amine reactive groups, CS is susceptible to structural modifications.
∗
Corresponding author.
322 Paula Pereira, Vera Carvalho, Reinaldo Ramos et al.
Table 1. Biomedical applications of CS
Biomedical application Remarks Ref.

Artificial skin and wound Structural similarity with glycosaminoglycans makes [10-13]
healing CS and its derivatives eligible for skin replacement.
CS and its derivatives support blood coagulation,
prevent abnormal fibroblastic reactivity, and act as a
bactericide and wound-healing accelerators.
Ophthalmology CS possesses all the characteristics required for [14]

making an ideal contact lens: optical clarity,
mechanical stability, satisfactory optical correction,
gas permeability and wettability.
Blood anticoagulants Sulfonated derivatives act as blood-thinner and [15]
lipoprotein lipase (LPL)-releasing agents
Orthopedic/periodontal Calcium-based compounds with chitin and CS as [16-19]

applications bone substitutes
Tissue engineering Porous biodegradable matrixes for cell seeding [19-23]
Antimicrobial Inhibition of microorganisms growth [24, 25]
applications
CS is insoluble in water and organic solvents, but soluble in dilute aqueous acidic
solutions (pH < 6.5), due to the protonation of the glucosamine residues into the soluble form
R-NH3+ [3]. Commercial CS has an average molecular weight (Mw) ranging from 3800 to 2
000 000 Daltons (Da) and a deacetylation degree - the proportion of glucosamine residues - of
66 to 100% [4]. The deacetylation degree (DD) has an important influence on various
properties including solubility, biodegradability, toxicity, antimicrobial activity, etc. [5].
Furthermore, CS is non-toxic, stable, biodegradable and sterilizable. It is mucoadhesive, due
to electrostatic interactions with mucosal surfaces [6, 7]. The reactivity and polycationic
character allow the production of a variety of formulations with different properties, ranging
from hydrogels, rods and fibres to nano/microparticles and membranes [8]. Altogether, these
properties make of medical grade CS a versatile material with extensive application in the
biomedical and biotechnological fields [9]. Table 1 summarizes some relevant biomedical
applications of CS.
One of the most well-known biomedical applications is the development of drug delivery
systems. In fact, papers related to drug delivery systems using CS are available by the
hundreds [26-39]. Different types of CS-made drug carriers have been conceived for various
administration routes, such as oral, sub-lingual, nasal, transdermal, parenteral, vaginal,
cervical, intrauterine and rectal.
Chitosan Nanoparticles for Biomedical Applications 323
2. PRODUCTION OF CHITOSAN-BASED NANOPARTICLES

CS microspheres and nanoparticles (NPs) are widely studied drug delivery systems. The
use of NPs offers many advantages, providing targeted delivery of drugs, improving the
bioavailability and stability of the therapeutic agents against chemical/enzymatic degradation
[40]. Micro/nanoparticles may hold drug molecules attached to the matrix, dissolved,
encapsulated or entrapped [41].
Different methods have been used to prepare CS particulate systems. The selection of one
of these methods depends upon the specificities and requirements associated to each
application, namely the physicochemical properties and thermal-chemical stability of the
active agent, the envisaged release kinetic profile, biodistribution, cellular uptake efficiency
and intracellular fate of the nanoparticles, etc.
Emulsion Crosslinking
This technique is based on the reaction between the primary amines and a multifunctional
crosslinking agent bearing aldehyde groups. In this process, a CS solution in acetic acid is
emulsified in liquid paraffin (w/o emulsion). The aqueous droplets are stabilized using a
suitable surfactant. The emulsion is then reticulated with an appropriate crosslinking agent
such as glutaraldehyde, to stabilize the polysaccharide droplets (Figure 1). The amount of
crosslinking agent varies according to the crosslinking density required. The nanospheres are
then washed and dried [42-44]. The incorporation of drugs is achieved by dispersion in the
CS solution, in the beginning of the process, becoming entrapped during the crosslinking
reaction. The entrapment efficiency may be improved by performing multiple emulsions [45].
Major drawbacks of this method are associated with the use of organic solvents and
crosslinking agents, that may adversely affect the stability of proteins [46]. The complete
removal of the unreacted - often toxic - crosslinker is difficult to achieve. Moreover,
glutaraldehyde crosslinked NPs present negative effects on cell viability.
Figure 1. Emulsion crosslinking methodology for the preparation of CS-NPs.

Coacervation/Precipitation
This method takes advantage of the physicochemical properties of CS, specifically its
insolubility in alkaline pH. CS is dissolved in acidic solution and precipitates/coacervates in
contact with an alkaline solution. Spraying the CS solution into sodium hydroxide, NaOH-
methanol or ethanediamine alkaline solutions, using compressed air, originates coacervated
droplets, forming the NPs [47]. Separation and purification of the particles is finally achieved
by centrifugation, followed by successive washing steps with hot and cold water. The size of
the NPs can be controlled by varying the diameter of the compressed air nozzle. Berthold et
al. [48] described a different approach: the formation of the particles is obtained after addition
of sodium sulfate solution to a CS solution, under mild agitation and continuous sonication
for 30 min. However, a crosslinking agent is frequently used to harden the NPs.
Emulsion-Droplet Coalescence Method
This technique was developed by Tokumitsu et al. [49]. In this approach, precipitation is
induced by allowing CS droplets to combine with NaOH droplets. This method involves both
emulsion crosslinking and precipitation. A stable emulsion containing the aqueous CS
solution along with the drug in liquid paraffin oil is produced; a second emulsion, containing
a NaOH solution, is produced in a similar way. When both emulsions are mixed under high-
speed stir, droplets of each emulsion collide at random, coalesce, and finally precipitate as
small size particles. NPs are obtained within the emulsion-droplets. The method is
schematically shown in Figure 2. The particle size varies inversely with the DD of CS, the
larger NPs containing lower amounts of drug.[49]
Figure 2. Production of CS-NPs using the emulsion-droplet coalescence technique.

Figure 3. Preparation of CS-NPs by the ionotropic gelation method.
Figure 4. Reverse micelles method of preparation of CS-NPs

Ionotropic Gelation
This method is based on the electrostatic interactions between the CS amine group and a
polyanion such as tripolyphosphate (TPP) [50-52]. TPP, a non-toxic multivalent anion, is
often used to prepare CS-NPs [53]. The complexation between oppositely charged
macromolecules as a way to prepare CS-NPs has attracted much attention, because the
process is very simple and mild: the acidic solution of CS is added dropwise, under constant
stirring, to the polyanionic TPP solution. Due to the interaction between oppositely charged
species, ionic gelation occurs, giving rise to spherical particles (Figure 3). The particles size
and surface charge can be modified by varying the ratio of CS and stabilizer. The efficiency
of the method is dependent upon the deacetylation of CS. In fact, the gelation process occurs
through the interaction of the protonated amino groups of CS. One of the major drawbacks of
this technique is the poor stability and mechanical properties of the NPs, thus limiting their
usage in drug delivery. Furthermore, the separation and redispersion processes are difficult to
achieve. The negatively charged DNA can also form polyelectrolyte complexes with
cationically charged CS, through ionic gelation, as demonstrated by Mansouri et al. [54]. The
CS properties, namely the Mw, DD and concentration used, have a significant impact on
those of the NPs and on its performance as drug carriers [55-57].
Reverse Micelles Method
Reverse micelles are thermodynamically stable liquid mixtures of water, oil and
surfactant. Macroscopically, the emulsion is homogeneous and isotropic, structured on a
microscopic scale, with the aqueous and oil microdomains separated by surfactant-rich films.
The aqueous core of the reverse micellar droplets can be used as a nanoreactor to prepare
nanoparticles. Since the size of the obtained reverse micellar droplets usually lies between 1
and 10 nm [58], the preparation of drug-loaded nanoparticles will produce extremely fine
particles with a narrow size distribution. In this technique, the reverse micelles are formed by
dissolving a surfactant into an organic solvent, giving rise to a water-in-oil micellar system
(Figure 4). The aqueous phase containing the CS and the drug are added to this emulsion with
constant vortexing and the NPs forms in the core of the reverse micelles. An additional
amount of water may be added to produce larger sized NPs. The maximum amount of drug
that can be dissolved in reverse micelles has to be determined for each case, by gradually
increasing the amount of drug, until the clear microemulsion is transformed into a translucent
solution. To this transparent solution, a cross-linking agent is added with constant overnight
stirring. The organic solvent is then evaporated. The material is redispersed in water with
sonication and the surfactant is salted out. The mixture is finally centrifuged and the
supernatant solution, which contains the drug-loaded NPs, is dialyzed for about 1 h and
lyophilized to dry powder [59].
Template Polymerization
In this technique, the NPs are obtained upon template polymerization of an acrylic
monomer next to the chitosan backbone. CS is firstly dissolved in an acrylic monomer
solution under magnetic stirring. Due to the electrostatic interaction, the negatively charged
acrylic monomers align along the chitosan molecules. After complete dissolution of CS, the
polymerization is started by adding the initiator (K2S2O8) under stirring at 70ºC. The
complete polymerization leads to the appearance of an opalescent solution, indicating the NPs
formation. The NPs solution are then filtered and dialysed to remove the residual monomers
and initiator. The obtained NPs are positively charged and present a size in the range of 50 to
400 nm [60, 61].
Self-Assembled Nanogels
The self-assembly process, defined as the autonomous organization of components into

structurally well-defined aggregates, is characterized by numerous beneficial attributes; it is
cost-effective, versatile and facile; the process occurs towards the system’s thermodynamic
minima, resulting in stable and robust structures. Molecular self-assembly is characterized by
diffusion followed by specific association of molecules through non-covalent interactions,
including electrostatic and/or hydrophobic associations. Individually, such interactions are
weak, but dominate the structural and conformational behavior of the assembly due to the
large number of interactions involved. While oppositely charged polysaccharides associate
readily as a result of electrostatic attractions, interactions among neutral polysaccharides tend
to be weaker, or nonexistent, a modification with chemical entities able to trigger assembly
being necessary [62]. Hydrophobically modified chitosan is an interesting strategy, consisting
on chemical linkage of hydrophobic grafts on hydrophilic backbone, to induce the formation
of nanoparticles via hydrophobic interactions [63-67]. Upon contact with an aqueous
environment, hydrophobically modified chitosan spontaneously form self-aggregated
nanoparticles, via intra- or intermolecular associations between the hydrophobic moieties,
primarily to minimize the interfacial free energy.
3. BIOMEDICAL APPLICATIONS
The biomedical application for CS-NPs is particularly relevant concerning the
development of delivery systems for biopharmaceuticals, although many papers describe also
its use as carriers for low molecular weight drugs. The recent progresses in these biomedical
applications are reviewed in this section.
3.1. Protein, Peptide and Oligosaccharide Delivery
Therapeutic proteins are becoming available for the treatment of a wide range of diseases,
such as cancer, autoimmune diseases and metabolic disorders. A main problem limiting the
efficiency of protein therapeutics is the reduced stability and short circulation half-lives after
parenteral administration (i.e. intravenous, intramuscular, or subcutaneous) [68]. As a result
of the invasive nature, injectable formulations are frequently faced with patient discomfort
and noncompliance. In the case of proteins, susceptibility to proteolysis and colloidal
instability are additional difficulties. Consequently, a high drug concentration or a high

dosing frequency becomes necessary, which may lead to adverse side effects [68-71]. Thus,
drug delivery systems (DDS) are urgently needed, for the enhancement of the
biopharmaceuticals bioavailability and selectivity, enabling a targeted controlled release
profile.
Aiming at achieving an effective protein delivery, carriers such as liposomes, polymer
micelles, and micro- or NPs have been developed [68, 70, 72-76]. Among them, nanometer-
sized (<100 nm) polymer hydrogels have attracted growing interest. By trapping proteins in a
hydrated polymer-network, these nanogels minimize denaturation, simultaneously allowing a
slow, continuous and controlled release of the protein, maintaining an effective concentration
for the necessary period of time [73, 77-79]. Nanocarriers enable localized and specific
targeting to their intended tissues or cells, thereby decreasing drug doses and improving
patient compliance [72]. Among nanosized systems, CS nanoparticles have been tested as
carriers for proteins, peptides and oligosaccharides.
Usually, proteins and vaccines are delivered via parenteral routes, due to their low
bioavailability and/or poor immunogenicity when administered via non-parenteral routes [80].
In recent years, substantial progresses have been made on the use of non-invasive routes, such
as mucosal (oral, nasal, pulmonary and colon) and transdermal, for the delivery of proteins
and vaccines, yielding better patient compliance [80-83].
Oral Delivery
The oral route is considered the most convenient and comfortable means of drug
administration, because of its non-invasive nature. It reduces the risk of infection, and do not
require trained personnel. Drug molecules may cross the intestinal epithelium by transcellular
or paracellular pathways. However, the bioavailability of orally administered proteins is
usually poor, because of the hostile gastric and intestinal environments, and also the limited
gastrointestinal (GI) mucosal permeability. The intestinal epithelium is a major barrier to the
absorption of hydrophilic macromolecules, because they cannot diffuse across the lipid
bilayer of the cell membranes, given the large Mw and hydrophilicity [84, 85]. In order to
facilitate the paracellular transport of hydrophilic macromolecules, efforts have been made to
induce transient opening of intercellular tight junctions [86, 87]. It has been reported that CS
is able to enhance absorption in the intestinal lumen. A combination of mucoadhesion and a
transient opening of the tight junctions in the mucosal membrane explain the enhanced
absorption ability of CS. The mucoadhesive properties are attributed to an interaction
between the positively charged CS and the negatively charged sialic-acid groups in mucins,
which provides a prolonged contact time between the protein and the mucosal surface,
thereby promoting its absorption [88, 89]. It has also been suggested that interactions of the
CS positively charged amino groups, with the negatively charged cell surfaces and tight
junctions, induce a redistribution of F-actin and tight junction’s protein ZO-1, which triggers
the increased paracellular permeability.
Lin et al. [90] reported the production, by ionic gelation, of NPs composed of CS and
poly-γ-glutamic acid (γ-PGA) for insulin delivery. In the GI tract, the pH varies from acidic in
the stomach to slightly alkaline in the small intestine. The fasting pH of the stomach is about
2.5 to 3.7, but in the presence of food, the pH drops to about 1.0 to 2.0, due to hydrochloric
acid secreted by parietal cells. The proximal part of the small intestine (duodenum) has a pH
value of about 6.0–6.6 due to neutralization of the acid by bicarbonates secreted by the
duodenal mucosa and pancreas. The jejunum and ileum are the middle portion and terminal
part of the small intestine, respectively, and their pH values are about 7.4. Therefore,
characterization of NPs at distinct pH values, simulating the environments of the GI tract, was
investigated. The stability and functionality of NPs in vitro, using Caco-2 cell monolayers,
and in vivo, in a rat model were studied. The pKa values of CS and γ-PGA are 6.5 and 2.9,
respectively. In the range of pH 2.5–6.6, CS and γ-PGA are ionized. The ionized CS and γ-
PGA can form polyelectrolyte complexes, which results in a matrix structure with a spherical
shape. Outside of this pH range, the NPs become unstable and subsequently broken apart.
This is because, at pH 1.2, γ-PGA is not charged. Therefore, the little electrostatic interaction
between the positively charged CS and the neutral γ-PGA causes the instability of NPs.
Similarly, at pH 7.4, CS is neutral and thus NPs collapse. The authors observed that the CS-
NPs could transiently and reversibly open the tight junctions between Caco-2 cells, thus
enhancing the paracellular permeability. However, the CS-NPs at pH 7.4 appear to be less
effective in opening tight junctions than at pH 6.6, due to the less positively charged CS. It
was suggested that the electrostatic interaction between the positively charged CS and the
negatively charged sites of ZO-1 proteins on cell surfaces induces a redistribution of cellular
F-actin, leading to an increase in paracellular permeability.
The pH sensitivity and functionality of the CS-NPs were confirmed in an animal study.
At the duodenum (pH 6.0–6.6), while adhering and infiltrating into the mucus layer, the orally
administered NPs may be degraded due to the presence of digestive enzymes in the intestinal
fluids. Additionally, the pH environment may be changed (becoming neutral) while the NPs
were infiltrating into the mucosa layer and approaching the intestinal epithelial cells. This
may further lead to the collapse of NPs due to the change in the exposed pH environment.
The dissociated CS from the degraded/collapsed NPs is then able to interact and modulate the
function of ZO-1 proteins between epithelial cells. ZO-1 proteins are thought to link the
occludin and actin cytoskeleton, playing important roles in the rearrangement of cell–cell
contacts. Oral intake of NPs/insulin demonstrated a sustained decrease of the blood glucose
level over a long period of time, at least 10 h. In a further development of this work, Sonaje et
al. [91] prepared self-assembled NPs, by mixing γ-PGA with CS in the presence of MgSO4
and TPP. The introduction of MgSO4 in the preparation of CS-NPs improved the stability in a
broader pH range. The intestinal paracellular transport of insulin was investigated using
Caco-2 cell monolayers. Additionally, the efficacy of NPs for oral delivery and intestinal
absorption of insulin was investigated, in a diabetic rat model. The in vitro results showed
that the mucoadhesive properties of CS-NPs are affected by the pH and additionally, the
transport of insulin across Caco-2 cell monolayers is pH-dependent: with increasing pH, the
amount of insulin transported decreased significantly, due to the lower positive surface charge
of the NPs, hence lower mucoadhesive and absorption enhancement ability. In addition, oral
administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at
least 10 hours, in diabetic rats.
Based on the work of Lin et al. [90] and Sonaje et al. [91], a mechanism for the
paracellular delivery of insulin through the GI tract using CS-NPs was proposed (Figure 5).
Figure 5. Schematic illustration of the hypothetic mechanism of the paracellular transport of insulin
released from CS-NPs via oral administration. NPs adhere and infiltrate into the mucus layer of
intestinal epithelium. The infiltrated NPs disintegrate due the near neutral pH and release the loaded
insulin; simultaneously, the CS opens the tight junctions allowing the insulin permeation through the
paracellular pathway.
Despite some encouraging results, the poor solubility at physiological pH is a limitation

for a more effective use of CS based NPs. Indeed, it has been shown that only protonated –
soluble - CS can trigger the opening of the tight junctions, facilitating the paracellular
transport of hydrophilic compounds [92]. The pH of the intestinal lumen is higher than the
pKa of CS (6.5), limiting its efficiency as an absorption enhancer and suitability for protein
delivery in neutral and physiological environments.
Several strategies have been attempted to overcome these drawbacks. Qian et al. [93]
prepared hydrophilic CS-NPs, by free radical polymerization of methyl methacrylate (MMA),
N-dimethylaminoethyl methacrylate hydrochloride (DMAEMC), or N-trimethylaminoathyl
methacrylate chloride (TMAEMC), which show a higher solubility in a broader pH range.
These graft copolymer NPs enhanced the absorption and improved the bioavailability of
insulin via the GI tract of normal male Sprague-Dawley rats. Trimethyl chitosan (TMC) NPs
were also developed by other authors for insulin delivery ([94, 95]). In an attempt to combine
the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers, Yin
et al. [96] synthetized a TMC-cysteine (TMC-Cys) conjugate. Thiolated polymers have been
developed as a category of mucoadhesive polymers with reactive thiol groups immobilized on
the polymeric structure. They can tightly and long lasting adhere to the intestinal mucus layer,
through covalent bonding with mucin glycoproteins ,via thiol-disulfide exchange reactions,
hence providing a steep drug concentration gradient at the absorption sites and exerting an
additional permeation enhancing effect. However, thiolated CS is also insoluble at
physiological pH, which again restricts its application. TMC-Cys/insulin NPs were prepared
through self-assembly driven by the electrostatic interaction between oppositely charged
TMC-Cys and insulin. The authors [96] confirmed that, when reaching the small intestine, the
positively charged TMC-Cys NPs are directed to the mucus layer, through electrostatic
interaction with negatively charged sialic acid residues on mucin glycoproteins. Meanwhile,
the free thiol groups on TMC-Cys are oxidized at neutral pH and disulfide bonds allowed to
form between TMC-Cys and cysteine-rich mucin, contributing to the notably higher amount
of TMC-Cys NPs immobilized in the mucus layer. So, mucoadhesion and permeation
enhancing effects are significantly enhanced by the presence of cystein conjugates. Besides
being biocompatible, TMC-Cys NPs showed a more potent hypoglycemic effect following
both oral and ileal administration in normal rats than TMC NPs.
Nasal Delivery
The nasal mucosa is an attractive route for the delivery of vaccines because it has a
relatively large absorptive surface and low proteolytic activity [82, 97-101]. Importantly,
nasally administered vaccines can induce both local and systemic immune responses.
However, most proteins are not well absorbed from the nasal cavity when administered as
simple solutions. The major factors limiting the absorption of nasally administered proteins
are the poor ability to cross the nasal epithelia, and the mucociliary clearance, which rapidly
removes protein solutions from the absorption site [82, 83, 97]. Mucoadhesive, hydrophilic
NPs have received much attention to overcome these obstacles and deliver protein antigens
via the nasal route, because they strongly attach the mucosa increasing mucin viscosity [83,
97, 99, 100]. By this means, mucoadhesive NPs are able to decrease the nasal mucociliary
clearance rate and thus increase the residence time of the formulation in the nasal cavity [82,
97].
Amidi and colleagues [102] prepared and characterized protein loaded TMC NPs as a
nasal delivery system, by ionic crosslinking a TMC solution (with or without ovalbumin, the
model protein studied) with TPP. It was observed that TMC NPs have a high loading
efficiency (fraction of protein loaded) and capacity (amount of protein loaded per NPs dry
weight) up to 50% (w/w). The integrity of the entrapped ovalbumin was preserved and release
studies showed that more than 70% of the protein remained associated with the NPs for at
least 3h of incubation in PBS (pH 7.4), at 37ºC. Regarding biocompatibility, the NPs were
non-cytotoxic, whereas a partially reversible cilio-inhibiting effect on the chicken trachea was
observed. In vivo uptake studies indicated the transport of the protein across the nasal mucosa.
Altogether, the authors concluded that TMC NPs are a promising nasal delivery system for
proteins.
Other authors tested CS-NPs as a nasal delivery system for insulin. Zhang et al. [103]
used polyethylene glycol-grafted CS-NPs to improve the systemic absorption of insulin,
following nasal administration. The NPs were prepared by ionotropic gelation using TPP ions
as the crosslinking agent. In vitro release studies showed an initial burst, followed by a slow
release of insulin. Intranasal administration of the NPs in rabbits enhanced the absorption of
insulin to a greater extent than the free protein. The nasal delivery of insulin using chitosan-
acetyl-L-cysteine (CS-NAC) NPs was proposed by Wang et al. [104]. These authors observed
that intranasal administration of CS-NAC NPs in rats enhanced the absorption of insulin by
the nasal mucosa, as compared with unmodified CS-NPs and control free insulin solution.
Mucosal vaccine strategies have emerged as a viable and attractive alternative to
parenteral immunization. Advantages associated with mucosal vaccination are numerous:
easy and low cost of administration, patient compliance, avoidance of the hepatic first pass
metabolism and ability to induce mucosal as well as systemic immunity. Furthermore, the
immune response generated at one mucosal site is able to induce a strong immune response at
distal mucosal surfaces [105]. Westerink et al. [106] examined the effect of mucosal
administration of tetanus toxoid (TT) in the presence of a non-ionic copolymer, Pluronic®
F127 (F127) with CS or lysophosphatidylcholine (LPC), on the systemic and mucosal
immune response. The results suggest that the two components of F127/CS appear to exert an
additive or synergistic effect on the immune response.
Pulmonary Delivery
Pulmonary drug delivery for both local and systemic treatments has many advantages
over other delivery routes. The lungs have a large surface area (43 to 102 m2) [107]. In
addition, mucociliary clearance is slower at the alveoli of the lungs than in the airways.
Furthermore, the epithelia is thinner and more permeable, making possible the systemic
absorption of peptides and proteins. Indeed, a number of high Mw drugs were demonstrated
to be absorbed successfully through the lungs [107-109]. The successful delivery of the
inhaled particles depends mostly on their size and density, and hence, on the aerodynamic
diameter. The respirable fraction of these powders, generally the fraction of particles with an
aerodynamic diameter ranging from 1 to 5 μm, should be as high as possible, as to guarantee
a maximum deposition in the deep lung [110]. Independently of the method used to produce
the aerosol, before reaching the deep lung, inhaled particles must overcome certain obstacles
and lung defense mechanisms, essentially the effect of the branched airways structure and the
mucus layer, which protects the epithelium in the tracheobronchial region. Particles targeted
to the deep lung should be small enough to pass through the mouth, throat and conducting
airways; however, if too small, they mail fail to deposit, being breathed out again. Therefore,
they should have an aerodynamic diameter between 1 and 5 μm. Even so, a certain number of
particles will be transported away from the lung by mucociliary clearance [111]. Once in the
deep lung, particles will have to face at least two other defense mechanisms: the alveolar
macrophages, and the enzymatic activity. The alveolar surface is covered by a thin layer of
fluid with suspended macrophages, which play an important role in the lung defense. With the
capacity of moving freely in the surface, they are able to engulf “foreign” substances from the
airway surface, eliminating potentially damaging agents [111]. There is no consensus
concerning the ideal size range to avoid or delay phagocytosis; however, it has been reported
that the phagocytic activity is maximum for particles of 1–2 μm, decreasing for both smaller
and larger particles out of this range [112]. Generally, authors agree that for particles in the
micrometer range, the smaller the particle size, the higher is the probability of being captured
[112]. Concerning the second defense mechanism (enzymatic activity), it is known that the
lung presents a lower enzymatic activity when compared to other mucosal surfaces, such as
the gastric. However, some enzymes have already been identified, as protease inhibitors,
isozymes of the cytochrome P-450 family and lysozyme [113].
Grenha et al. [114] reported the preparation and characterization of dry powders
containing protein-loaded TPP-CS-NPs, using aerosol excipients. Bovine insulin was chosen
as model protein, with mannitol and lactose as excipients. The results showed that the
obtained microspheres are mostly spherical and possess appropriate aerodynamic properties
for pulmonary delivery. These NPs showed a good protein loading capacity, providing the in
vitro release of 75–80% insulin within 15 min, and could be easily recovered from
microspheres after contact with an aqueous medium, with no significant changes in their size
and zeta potential values. Therefore, protein-loaded CS-NPs could be successfully
incorporated into microspheres with adequate characteristics as to reach the deep lung; in
contact with the aqueous environment, the microspheres were able to release the NPs and
then the therapeutic macromolecule.
Colon Delivery
Colon targeted drug delivery is useful in improving the absorption of peptide drugs via
the GI tract. Site specific drug delivery to the colon is of special interest for drugs instable in
the upper part of the GI tract, because of the peptidase activity in the small intestine. The
colon is thought to have lower enzymatic activity than other regions of the GI, hence a greater
absorption efficiency in this region would be expected, as long as the proteins/peptides are
released locally [115]. Due to negligible activity of brush-border membrane and much less
activity of peptidases and pancreatic enzymes, the colon has been considered suitable for the
delivery of peptides and proteins.
Bayat et al. [116] developed a nanoparticulate system using two new quarternized
derivatives of CS, triethylchitosan (TEC) and dimethylethylchitosan (DMEC), for insulin
colon delivery. Insulin-NPs (CS, TEC or DMEC NPs) were prepared by the polyelectrolyte
complexation method. The three kinds of NPs showed a positive charge that could facilitate
insulin uptake, allowing a low bursting effect and a steady release of insulin in vitro. DMEC
NPs and TEC NPs had smaller particle size, higher insulin loading capacity and improved
transport and absorption of insulin in GI tract, as compared with CS-NPs. The blood glucose
lowering effect of TEC NPs and DMEC NPs, after injection into ascending colon, was
superior to that obtained with free insulin or CS-NPs. This study indicated that NPs prepared
from quaternized derivatives of CS might be a promising vehicle of administration of proteins
and peptides via colon absorption.
Numerous studies highlight the importance of CS-NPs for protein, peptide and
oligosaccharide delivery, as summarized in table 2.
Table 2. Representative examples of the use CS-NPs for proteins, peptides, and
oligosaccharide delivery
Route of
administration Method of preparation Remarks Ref.
Therapeutic agent / Associated disease: Insulin / Diabetes Mellitus
Oral delivery NPs, composed of hydrophilic Oral administration of insulin loaded NPs in [90]
CS and γ-PGA. diabetic rats demonstrated a sustained effect of
decreasing the blood glucose level.
Insulin-loaded CS-NPs prepared by CS-NPs enhanced the intestinal absorption of [117]
ionotropic gelation with TPP. insulin.
Pulmonary Low-molecular-weight CS-NPs In vivo administration of CS-NPs containing insulin [118]
delivery prepared by solvent evaporation showed hypoglycemic activity.
method.
Therapeutic agent/Associated disease: Calcitonin / Osteoporosis
Oral delivery CS–PEG nanocapsules obtained by the In vivo studies showed capacity of CS–PEG [119]
solvent displacement technique. nanocapsules to enhance and prolong the intestinal
absorption of salmon calcitonin.
Pulmonary Surface-modified DL-lactide/glycolide CS-modified PLGA NPs loaded with elcatonin [120]
delivery copolymer (PLGA) NPs with CS reduced blood calcium levels to 80% of the initial
prepared by the emulsion solvent concentration and prolonged the pharmacological
diffusion method. action to 24 h.
Table 2. Continued
Theraputic Agent / Associated Disease: Insulin/ Diabetes Mellitus

Oral delivery NPs prepared by ionic gelation method No significant anticoagulant activity was detected [121]
without chemically modifying heparin. after administration of the free heparin solution in a
rat model, while administration of NPs was
effective in the delivery of heparin into the blood
stream
Pulmonary CS and mixtures of hyaluronic acid Fluorescent heparin-loaded CS–HA NPs were [122]
delivery (HA) with heparin combined to form effectively internalized by rat mast cells. Ex vivo
NPs by the ionotropic gelation experiments evaluated the capacity of heparin to
technique. prevent histamine release in rat mast cells indicating
that the free or encapsulated drug exhibited the
same dose–response behavior.
Therapeutic agent / Effect: Cyclosporin-A (CyA) / Immunosuppressant and extraocular disorders
Ocular delivery CyA-loaded CS-NPs obtained using In vivo experiments showed that, following topical [123]
the ionic gelation technique. instillation of CyA loaded CS-NPs to rabbits, it was
possible to achieve therapeutic concentrations in the
external ocular tissues (i.e., cornea and conjunctiva)
while maintaining negligible or undetectable CyA
levels in the inner ocular structures.
Therapeutic agent/Associated disease: Prolidase / Prolidase deficiency (PD)
Parenteral CS-NPs prepared by combining Ex vivo experiments performed by incubating [124]
administration ionotropic gelation, with TPP, and different amounts of prolidase loaded CS-NPs with
ultrasonication treatment. skin human fibroblasts from PD patients for
scheduled times.
CS-NPs loaded with PEGylated The ex vivo evaluation of prolidase activity on PD [125]
prolidase,obtained by combining fibroblasts indicated a good level of prolidase
ionotropic gelation and ultrasonication activity replacement up to 10 days.
treatment.
Therapeutic agent / Effect: RGD / Anti-carcinogenic
Intratumural Hydrophobically modified glycol CS Intratumoral administration of RGD-HGC [126]
administration (HGC) NPs containing a cyclic RGD significantly decreased tumor growth and
peptide (RGD-HGC) prepared by a microvessel density compared to native RGD
solvent evaporation method. peptide injected either intravenously or
intratumorally.
Therapeutic agent / Purpose: Tetanus toxoid / Tetanus vaccination
Nasal PEG-coated poly(lactic acid) (PLA) The coating of PLGA NPs with the mucoadhesive [127]
administration NPs, CS-coated poly(lactic acid– polymer CS improved the stability of the particles
glycolic acid (PLGA) NPs, and CS- in the presence of lysozyme. Moreover, these
NPs prepared by ionotropic gelation particles were very efficient in improving the local
with TPP. and systemic immune responses to tetanus toxoid.
Therapeutic agent / Associated disease: Anti-neuroexcitation peptide (ANEP) / Neuroexcitation associated diseases
Brain-targeting ANEP-loaded TMC NPs prepared by The results showed that the targetability of ANEP to [128]
delivery ionic crosslinking of TMC with TPP. brain was significantly increased by TMC NPs.
Absorptive-mediated transcytosis was believed to
be the main pathway for the brain-targeting of
ANEP-TMC/NPs.
3.2. Gene Delivery
Gene therapy is an emerging field in medical and pharmaceutical sciences, a very

promising one due its potential for the treatment of a wide range of diseases, both inborn and
acquired, by replacing defective genes, substituting missing ones, or silencing unwanted gene
expression. However, naked therapeutic genes are rapidly degraded by nucleases, showing
poor and non-specific cellular uptake and also low transfection efficiency [129]. Hence, the
development of safe and efficient gene carriers is primordial for the success of gene therapy.
Gene delivery systems include viral vectors, cationic liposomes and polycationic complexes.
In spite of high transfection efficiency, the immune and oncogenic responses associated to
viral vectors limit their therapeutic applications in vivo. To overcome these limitations, non-
viral delivery systems (cationic liposomes and cationic polymers) have been proposed as a
safe alternative. Besides being easily synthesized in large-scale, these nanoparticulate systems
are targetable, have low immune response and unlimited DNA packaging capacity [130].
Non-viral systems, based on cationic polymers bearing amine groups in their backbone, are
now extensively used as gene carriers; they form stable complexes with DNA, keeping it safe
from nuclease degradation, and readily interact with cells membrane. Among non-viral
vectors, CS and its derivatives are good delivery systems for DNA, antisense
oligodeoxynucleotides and siRNA. CS gathers a number of desirable characteristics: cationic
charge, biodegradability, biocompatibility, low toxicity, mucoadhesivness and functional
groups with targeting ability. On the other hand, its transfection efficiency is relatively low,
compromising potential clinical application. Several studies have been carried out to elucidate
the influence of the CS-based formulation parameters on the gene expression [129]. In this
section, we review the state of the art of DNA and siRNA CS-based delivery systems [131].
3.2.1. Chitosan Features Influencing Transfection

Many reports highlight the potential of CS as an efficient polymeric gene carrier. At
acidic pH, below the pKa, the primary amines in the CS backbone are positively charged and
interact with negatively charged DNA leading to the spontaneous formation of nanosized
complexes. Under neutral or alkaline conditions, CS is only slightly charged, thus the CS and
DNA binding is stabilized mainly by hydrogen bonding and hydrophobic interactions [132].
DNA can be carried entrapped into the NPs by hydrophobic [63] or ionic interactions [133].
Numerous factors influence the stability and transfection efficiency of CS-based systems,
including Mw, DD, charge ratio of CS to DNA (N/P ratio), pH, serum and additives, which
will be discussed bellow.
The CS Mw has a major influence on the size of the nanoparticles, the CS/DNA complex
stability [134], the unpacking of DNA after endocytosis and thus, overall, on the transfection
efficiency [135]. Huang et al. [136] reported that CS with low Mw (<20kDa) only poorly
retains the DNA upon dilution, consequentially being less capable of protecting it from
degradation by DNase and serum components, resulting in low transfection efficiencies. On
the other hand, Köpping-Höggard et al. [137] developed highly effective non-viral gene
delivery systems using CS oligomers (1.2 to 10 kDa). The easier dissociation of the
polyplexes was reflected in a greater gene expression, when compared to the more stable
high-molecular-weight ultrapure CS-DNA polyplexes. Consequently, an intermediate
stability must be achieved: the optimal CS Mw that allow extracellular DNA protection
(favored by high Mw) versus efficient intracellular DNA release (favored by low Mw), in
order to optimize the levels of transfection [129].
Higher DD results in increased positive charge, hence greater DNA binding capacity and
cellular uptake [129]. According to Köpping-Höggard et al. [138], only CS with high charge
density form stable complexes with pDNA. Similarly, Huang et al. [136] observed that CS
with low Mw or DD (46 kDa and 61%, respectively) did not retain DNA efficiently and
showed poor cellular uptake. Also, Kiang et al. [139] concluded that the DNA binding
efficacy decrease for DD <70%, resulting in low luciferase expression, due to the particle
destabilization caused by the bulky acetyl groups in the polymer chains. Lavertu et al. [140]
reported as well high levels of luciferase expression, equivalent to those obtained with
positive controls (Lipofectamine TM and FuGENE 6), using CS formulations with DD>80%
and low Mw (10kDa), at pH 6.5.
The N/P ratio is given by the stoichiometry of CS’s nitrogen and DNA’s phosphate. The
surface charge of the polyplexes depends on the N/P ratio, which influences the particles
ability to interact with the negatively charged cell membrane [141]. Ishii et al. [142] reported
that the transfection efficiency increases for charge ratios of 3 and 5, decreasing for further
higher values. Another study, developed by Kim et al. using galactosylated CS/DNA [143],
showed that complete shielding of DNA occurs at charge ratio of 5, with no significant
improvement in the range 5–20. Galactosylated CS/DNA complexes with charge ratio above
5 (slightly positive zeta potential) were suitable for effective gene transfer. The most
enhanced stability was obtained at charge ratio 10, due to the prevention of self-aggregation.
It has been suggested that strong interactions between CS and DNA results in highly
stable particles, thereby preventing dissociation within the cell and leading to the absence of
DNA translation. Attempting to reduce the CS-DNA interaction, Douglas et al. [144]
associated an anionic biopolymer (alginate, 12–80 kDa) with low Mw CS (10 kDa, 90% DD).
The presence of alginate in the complexes improved the yield of cell transfection. With the
same aim, Duceppe et al. [135] used NPs made of ultra low Mw chitosan (ULMWCh, <10
kDa)/Hyaluronic acid (HA), as a novel potential carrier for gene delivery. Addition of HA to
the NP formulation improved transfection rate from 0,7 to 25%. Peng and colleagues [145]
demonstrated that mixtures of CS, DNA and poly(gamma-glutamic acid) (g-PGA) in aqueous
media lead to the formation of ‘‘compounded NPs’’, containing domains of CS/DNA and
CS/g-PGA. With this internal structure, the compounded NPs might disintegrate into a
number of even smaller sub-particles, after cellular internalization, improving the dissociation
capacity of CS and DNA. Consequently, an improved transfection was obtained.
Figure 6. Biological barriers and gene delivery. Firstly, the polymeric complex should (I) be stable in
the systemic circulation for a fairly long period of time, (II) able to access to the target cells and be
internalized, (III) escape the endosomes to avoid degradation, (IV) reach the perinuclear space and
allow unpacking of the DNA complexes and finally (V) translocation to the nucleus.
The transfection efficiency of the CS complexes is also dependent on the pH of the

culture medium. A pH slightly below 7 is optimal to achieve a good balance between DNA
association and dissociation [129]. A recent study highlights the importance of the polyplex
stability under different pH [146]. At pH 5.5, unmodified CS was most efficient in the DNA
condensation. Conversely, when the pH was adjusted to 7.4, in the presence of high ionic
strength, the condensation was strongly compromised, due to the reduction of both the degree
of ionization and solubility. The condensation of DNA with TMC (50kDa) and TMC grafted
with polyethylenoglycol (PEG) is less sensitive to pH and, at neutral pH, to the ionic strength.
Likewise, Kadyala et al. [147] reported that the higher rate of transport of CS-based NP in
Caco-2 cell layers occur at pH 5.5. Increasing the pH decreases the transport efficiency by 3
and 10-folds, for pH 6.4 and 7.4 respectively, owing to the NPs aggregation.
A mandatory requirement for the in vivo therapeutic application of gene delivery systems
is the stability in serum. According to Ishii et al. [148] the presence of 10–20% serum
enhances transfection, higher concentrations of serum yielding poorer results. Probably, the
optimal serum concentration is determined by the overall effect on cells.
3.2.2. Biological Barriers in Cell Transfection

The body defense barriers at the humoral and cellular level have evolved to efficiently
prevent intrusion of exogenous entities. Thus, the transfer of foreign genetic material to cells
is a most challenging area, implying a safe and efficient method to deliver therapeutic genes
to target cells. The gene carriers must meet a number of physical-chemical requirements;
namely, the nanocarrier should have the following properties: stability in biological fluids,
access to the target cells and cellular uptake, endosomal escape ability, appropriate
intracellular trafficking and unpacking of polyplexes, and nuclear transport (Figure 6) [149].
The cationic surfaces increase the solubility of the complexes in aqueous medium and
facilitate its interaction with cells. However, when systemically administered, they readily
interact with serum components, such as negatively charged serum albumin and other
opsonins, originating the aggregation of particles in the blood stream through reduction of the
zeta potential, thereby decreasing the ionic repulsion between particles. Opsonization results
in the rapid clearance by cells of the mononuclear phagocytic system (MPS), specially
macrophages in the liver (the Kupffer cells), spleen and bone marrow. In order to minimize
the interactions with serum proteins, augmenting DNA survival in the bloodstream for the
period of time necessary to reach the target tissue, hydrophilic polymers can be conjugated
with CS (Figure 6, step I) [131]. PEGlyation of CS increases the systemic circulation time
after intravenous administration, possibly by sterically avoiding the non-specific interactions
between the serum-driven components and polyplexes [149]. Jiang et al. [150] synthesized a
galactosylated poly(ethylene glycol)-chitosan-graft-polyethylenimine (Gal-PEG-CHI-g-PEI)
as a potential hepatocyte-targeting gene carrier. After intravenous injection, PEI/DNA
complexes rapidly accumulated in the lungs, whereas Gal-PEG-CHI-g-PEI/DNA complexes
accumulated in the lungs, heart and liver, indicating that complexes had increased circulation
time in vivo due to the hydrophilic group. Park and colleagues [151] introduced another
hydrophilic group, poly(vinyl pyrrolidone) (PVP), into galactosylated CS to prevent
aggregation of the complexes and the interaction with plasma proteins. The PVP surface
effectively prevented the complex from interacting with albumin.
The polyplexes should reach the target cells without loss of integrity (Figure 6, step II).
The positively charged particles readily attach to the cell surface via ionic interactions,
thereby facilitating internalization by different endocytic mechanisms [152]. The cellular
uptake of the polymeric polyplexes mostly occurs via non-specific adsorptive endocytosis.
However, to improve cellular uptake efficiency and specificity, CS can be decorated with
specific ligands (Table 3), which specifically recognize and bind receptors of the target cells
(receptor-mediated endocytosis), improving the transfection efficiency [131].
Table 3. Representative examples of the CS and CS derivatives modified with cell

specific ligands to improve the specificity to target cells
Targeting
CS derivatives ligands Target cells Remarks Ref.
Folate-N- Folate Cancer cells (KB Folate conjugation increased the [153]
trimethyl CS and SKOV3) - cellular uptake of the complex in
(folate-TMC) folate receptor KB cells and SKOV3 cells via
over-expressing) folate receptor. The intracellular
Lung cells (A549) trafficking of the folate-
and fibrosblast TMC/pDNA was faster than
(NIH/3T3) - folate TMC/pDNA due to the use of
receptor deficient) different trafficking pathways.
KNOB-CS KNOB Kidney cells KNOB conjugated NPs improved [154]
protein (HEK293) gene expression level in HeLa and
Cancer cells HEK293 cells by 130 and 7-folds,
(HeLa) respectively.
Transferrin-High Transferrin Cancer cells CS-NPs decorated with transferrin [147]
Mw (HMW) CS (CaCo-2) enhanced the transport of NPs
trough cell layers by 3- to 5-fold
and led to higher stability of the
NPs at higher pH.
Hyaluronic Hyaluronic Corneal (HCE) and The endocytic process was [155]
acid(HA)-CS acid conjuctival (IOBA- mediated by hyaluronan receptor
NHC) cells CD44
Mannosylated Mannose Antigen presenting The transfection efficiency of Man- [156]
chitosan-graft- cells (APCs) CHI-g-PEI/DNA complexes into
polyethylenimine macrophage cell line, which has
(Man-CHI-g- mannose receptors, was higher than
PEI) CHI-g-PEI as well as PEI.
Galactosylated Galactose Hepatocytes Gal-PEG-CHI-g-PEI/DNA [150]
poly(ethylene complexes transfected liver cells
glycol)-chitosan- more effectively than PEI. Gal-CS
graft- is reported as hepatocyte-targeting
polyethylenimine gene carrier due to specific ligand–
(Gal-PEG-CHI- receptor interactions between
g-PEI) galactose-moieties and
asialoglycoprotein receptors
(ASGPRs).
The introduction of the hydrophobic units in the CS-based complexes is also expected to
increase transfection efficiency by modulating the complex interactions with cells, such as
adsorption on cell surfaces and cellular uptake [131]. In addition, hydrophobic units in the
polymeric carriers may assist in the dissociation of polymer/DNA complexes, facilitating the
release of DNA which otherwise would be strongly bound through ionic interactions. Lee et
al. [157] described the potential of thiolated CS for enhanced gene transfer. Indeed, the
thiolated CS/pDNA nanocomplexes exhibited a gradual increase in mucin adsorption,
probably due to the formation of covalent bonds between thiolated CS and cysteine-rich
subdomains of mucin. In vitro and in vivo studies confirmed that thiolation of CS increases
the transfection efficiency and sustained gene expression. The introduction of a
trimethyltriazole group in C-6 position of CS allowed an increase in DNA binding ability,
serum stability and significantly increase of cellular uptake, as compared to unmodified CS
[158]. This effect was assigned to the ability of the trymethylammonium groups to open the
tight junctions, leading to increased paracellular permeability and consequently higher
transfection efficiency.
Once taken up by cells, via either adsorptive or receptor-mediated endocytosis,
polyplexes are localized within the endosomal compartments, where pH rapidly drops to
about 5 by the action of membrane bound ATP-driven proton pumps. The endosomes mature
to lysosomes, where the arrested polyplexes disassemble due to the low pH and the released
DNA may degrade. Therefore, the escape of polyplexes from the endosome is a critical step
in the process (Figure 6, step III). Partially protonated polymers retain a substantial buffering
capacity, which can lead to the protection of DNA from degradation. Since protons are
diverted, the acidification of endosome is prevented; the continued action of the proton pump
leads to the retention of chloride ions and therefore osmotic swelling occurs leading to
subsequent endosome disruption [142]. Unfortunately, the buffering capacity of CS
(pKa=6,2) is weak compared with PEI (pKa=8,7). Hence, CS have been frequently
conjugated with PEI to take advantage of its proton sponge effect [159]. The combination of
PEI with CS/DNA complex dramatically increased the luciferase expression in various cell
lines, and the synergistic effect was proved to be induced by proton sponge effect of PEI
[160]. However, CS-graft-PEI/DNA complexes frequently display higher transfection
efficiency than PEI/DNA [161]. Köpping-Höggard et al. [138] studied in detail the effect of
the addition of PEI to CS in the transfection efficiency. In this study, in contrast to PEI,
ultrapure CS (UPC) displayed no buffering capacity at the acid endosomal pH-interval of 4.5–
5.5, and thus the authors suggested the enzymatic degradation as a more likely mechanism for
the endosomal escape of UPC polyplexes. Indeed, the enzymatic degradation products (oligo-
and monosaccharides) may increase the osmolarity, followed by water influx, subsequent
swelling and rupture of the vesicular membranes. It was stated that, whatever the mechanism,
the efficiency of the PEI and UPC polyplexes depend on the rates at which the two polymers
escape the endo/lysosomal compartment.
Several modifications of CS have been attempted to improve this effect. Imidazole-
containing polymers have also been reported to act as a proton sponge, consequently
enhancing the release of the complex into the cytoplasm following endocytosis. Kim et al.
[162] used water soluble CS (WSC) conjugated with urocanic acid (UA) bearing an imidazole
ring. The transfection efficiency was enhanced by grafting CS with UA, an effect that
increases with the UA contents. Hu et al. [67] grafted hydrophobic moieties, stearic acid
(SA), with CS oligosaccharide (CSO) (CSO–SA). Transfection using the CSO–SA/DNA
complexes reached an efficiency of 15%, slightly below the figures obtained with
LipofectamineTM (about 20%). The high transfection of CSO–SA/DNA complexes – as
compared with CSO/DNA - is believed to rely on the chain of SA, which may favor the
escape of CSO–SA/DNA complex from endosome. When the CSO–SA was used as a
transfection carrier of pEGFP-C1, the fluorescence intensity increased gradually with the
post-transfection time (until 76 h), and during this period cellular growth was observed.
Conversely, a sharp increase on the transfection was detected with LipofectamineTM/DNA in
24h post-transfection, though after this period the DNA expression decreased rapidly,
possibly due to the cytotoxicity of the formulation. The continuous, yet with relatively low
efficiency, transfection of CSO–SA may be related to the slow rate of release of DNA. The
pH sensitivity of the poly(propyl acrylic acid) can also be used to enhance the release of
endocytosed drugs into the cytoplasmic compartment, because it exhibits maximal membrane
disruption ability at pH 6. By incorporating this polymer in a CS gene carrier, Kiang et al.
[163] confirmed the enhancement of the pDNA release from the endosomal compartment and
improved gene expression.
After escaping from endosome, the complex should be able to unpack quickly, allowing
the DNA to move towards the perinuclear space, where nuclear translocation of DNA takes
place (Figure 6, step IV). While highly stable polyplexes may provide robust protection of
DNA from extra- and intra-cellular nuclease attack, maximum transfection efficiency may not
be achieved due to restriction in transcription. In contrast, polyplexes with lower stability may
go through rapid uncoupling, causing premature degradation of plasmid DNA in the cytosol.
Therefore, maximum transfection efficiency may be achieved using a polymer with
intermediate stability. The unpacking can be carried out within the endosome [164] or
cytoplasm [165]. In the following step, the DNA or complex should move to the peri-nuclear
space. To better understand the intracellular trafficking of pDNA/lactosylated-CS complexes,
Hashimoto et al. [165] examined the effect of the endocytosis inhibitors on the transfection
efficiency. Bafilomycin A1, a proton pump inhibitor, greatly depressed the luciferase activity
of both pDNA/CS and pDNA/lactosylated-CS complexes. Monensin, an inhibitor of
endosomal acidification, significantly decreased the gene expression of the pDNA/CS and
pDNA/lactosylated-CS complexes. Nocodazole, which blocks transport from the early to late
endosomes, resulted in the accumulation of cargo in the endosome compartment and
improved transfection efficiency of the pDNA/CS complex, by about 2-3-fold. In contrast, in
the case of pDNA/lactosylated-CS complexes, the transfection efficiency was decreased by
nocodazole to 60% for HepG2 cells. Thus, the entrapment of pDNA/lactosylated-CS
complexes in early endosome resulted in the obstruction of the release from the endosome.
Although the transport of pDNA complexes to the late endosome/lysosome would raise the
risk of the hydrolysis of DNA, the pDNA/lactosylated-CS complex showed high transfection
efficiency, taking advantage of the release in perinuclear region.
Efficient nuclear localization of DNA is considered the final destination of gene delivery,
since eukaryotic transcription is an essential intermediate step to convert genetic information
into protein and is performed in the nucleus (Figure 6, step V). However, the mechanism of
nuclear translocation of DNA from CS/DNA complexes is not yet understood [149].
3.2.3. DNA Therapy

Recent advances in gene delivery emphasize the application of CS-based NPs as gene
carriers in cancer, rheumatoid arthritis, atherosclerosis, allergic asthma, tuberculosis,
hemophilia A, hepatitis B, coxsackievirus B3 and respiratory syncytial virus (RSV) infections

(Table 4), among others.
Table 4. Representative examples of the use CS/DNA NPs for gene therapy
Therapeu
Disease tic agent Administration Remarks Ref.
Colon adeno- IL-12 Intratumoral Mannosylated CS/pmIL-12 complexes administered in [166]
carcinoma gene BALB/c mice bearing CT-26 tumor cells resulted in high
expression levels of IL-12 and IFN-γ, suggesting that tumor
growth was retarded due to the higher production of both
cytokines. The IL-12 down-regulated angiogenesis and
together with IFN-γ promotes apoptosis and cell cycle
arrest.
Rheumatoid IL-1 Ra Intravenous The human IL-1Ra remained in the serum of rats for 10 [167]
arthritis gene days and reverted the alterations in bone turnover (bone
resorption versus formation) observed in arthritic animals.
Atherosclerosis pCR-X8- Intranasal Significant serum anti-CETP (cholesteryl ester transfer [168]
HBc- protein) IgG were detected and lasted for 21 weeks. The
CETP aortic lesions in the rabbits with NPs were lower than those
(pCETP) treated with saline control. CS/pCETP NPs could
significantly attenuate the progression of atherosclerosis.
Asthma IFN-γ Intranasal CS/IFN-γ pDNA NPs (CIN) led to the normalization of [169]
pDNA airway inflammation and hyperresponsiveness (AHR), and
return to normal lung morphology from the hyper-
inflammatory condition induced by Ovalbumin (OVA)
sensitization.
Tuberculosis pDNA Pulmonary [170]
T-cell
epitopes CS/DNA was able to induce the maturation of dendritic
from cells (DCs). pDNA incorporated in CS-NPs induced
Mycobac increased levels of IFN-γ secretion compared to pDNA in
terium solution.
tubercul
osis
Hemofilia A FVIII Oral DNA polyplexes were detected in gastrointestinal tissues as [171]
pDNA well as in liver, spleen and additional systemic tissues in the
hemophilia A mice. Functional Factor VIII protein was
found in plasma reaching a level of 2-4% FVIII at day 22
after delivery.
Hepatitis B pRc/CM Nasal pRc/CMV-HBs loaded CS-NPs resulted in serum anti- [172]
virus infection V-HBs HBsAg and sIgA titers in the mucosal secretions. CS-NPs
were able to induce humoral mucosal and cellular immune
responses.
Coxsackievirus pcDNA3 Intranasal [173]
B3 infection -VP1
(encodin
Mice immunized with CS/pcDNA3-VP1 produced higher
g VP1,
levels of IgG and IgA. CS/DNA vaccine induced CVB3-
major
specific systemic immunity (humoral and cellular) and
structural
protected mice from lethal CVB3 challenge.
protein
of
CVB3)
Respiratory pDNA Intranasal Immunization with pDNA conjugated with CS induced in [174]
syncytial virus encoding vivo peptide- and virus-specific CTL responses. In CS/DNA
(RSV) infection a immunized mice a significant reduction in virus loaded in
cytotoxic the lungs was observed.
T-
lymphoc
ytes
(CTL)
epitope
from M2
protein
of RSV
3.2.4. siRNA Delivery
RNA-interference (RNAi) mediates knockdown of harmful or unwanted genes. In the
RNAi process, double-stranded small interfering RNA (siRNA) with 21–23 nucleotides,
endogenously produced or exogenously introduced, associates with a nucleic acid-protein
complex called RNA-induced silencing complex (RISC). One of the strands is used to target a
specific sequence in a particular messenger RNA (mRNA), leading to its degradation. Hence,
the synthesis of the protein encoded by that mRNA molecule is prevented [175, 176]. The
successful application of siRNA is largely dependent on the development of the delivery
vehicle, due to its rapid degradation and poor cellular uptake in vitro and in vivo [177]. So, an
ideal carrier for siRNA should be able to bind and condense siRNA, provide protection
against degradation, specifically direct the siRNA to target cells, facilitate its intracellular
uptake and escape from the endosome/lysosome into cytosol, and finally promote efficient
gene silencing. Recently, Katas and Alper [178] were the first to explore the use of CS as
polymeric carrier for siRNA delivery.
In comparison to usual DNA-based gene delivery, the extra vulnerability of RNA to
enzymatic degradation represent additional hurdles to CS-mediated gene transfer, and make it
even more challenging than conventional pDNA delivery. As the structure and size of siRNA
are quite different from those of pDNA, the influence of the N/P ratio, serum and CS Mw, is
also different; however, the effect of DD and pH is similar. Therefore, all parameters must be
optimized specifically for the CS/siRNA complexes. NPs stability is required for extracellular
siRNA protection, but disassembly is needed to allow RNA-mediated gene silencing through
interaction with the intracellular RISC. An appropriate balance between protection and
release of siRNA needs to be achieved, using a CS with the convenient Mw. Liu and
colleagues [175] verified that CS with high Mw and DD result in the formation of discrete
and stable NPs, 200 nm in size. CS/siRNA formulations prepared with low Mw CS (~10 kDa)
showed almost no knockdown, whereas highest gene silencing efficiency (80%) was achieved
using CS/siRNA NPs at N:P 150, with high Mw CS (114 and 170 kDa) and DD of 84%
[137]. The influence of N/P ratio, here defined as the ratio of CS amino groups (N) to RNA
phosphate groups (P), in the size of nanoparticle was described by Howard et al. [179], using
a CS with Mw of 114 kDa. The nanoparticle hydrodynamic radius increased along with
decreasing the N/P ratio. This suggests siRNA bridges the CS chains, higher concentrations
leading to greater CS incorporation and possible interparticle aggregation. Liu et al. [175]
investigated the influence of N/P ratio on the gene knockdown efficiency using
chitosan/siRNA nanoparticles (CS with 170 kDa and DD of 84%) in H1299 human lung
carcinoma cells. It was found that the level of EGFP knockdown increased at higher N/P
ratios (50 and 150), in comparison to low N/P (2 and 10) formulations; the NPs formed at N/P
150 showed the greatest level (80%) of EGFP knockdown. This result was explained by the
increased nanoparticle stability at high N:P. Indeed, removal of excess polycations prior to
transfection resulted in virtually no cellular knockdown, suggesting a possible role of a CS
excess in the cellular permeation. Inside cells, the siRNA must be resistant to digestion by
nucleases. Katas and Alper [178] studied the effect of serum in the free siRNA and CS–
siRNA NPs stability and protection from nuclease degradation. After incubation with 5%
FBS, free siRNA was intact only up to 30 min, being fully degraded after 48 h; on the other
hand, siRNA in CS–TPP NPs started to degrade after 24 h incubation and full degradation
was only observed after 72 h incubation. Interestingly, the siRNA recovered from CS–siRNA
NPs was intact up to 7 h and fully degraded after 48 h incubation in 50% serum, while
complete degradation of free siRNA was observed from the very first moments of incubation.
Indeed, CS–siRNA NPs significantly protected siRNA from nuclease activity.
In order to improve the efficiency of RNA transfer using CS, several attempts on the
vector improvement have been made over the past years. Katas and Alpar [178] synthesized
CS-NPs by ionic gelation using CS salts (CS hydrochloride and glutamate) and sodium TPP.
Compared with standard chitosan, these NPs showed efficient siRNA transfer, which may be
related to the higher RNA binding capacity and loading efficiency. Later, another group
conjugated CS with thiamine pyrophosphate [180], a thiamine derivative. The CS-thiamine
diphosphate-mediated siRNA silencing of endogenous EGFP gene occurred at best with 70–
73% efficiency. This efficiency was associated with the increased nucleic acid binding ability
and improved water solubility of the vector, due to the addition of extra amine groups from
thiamine diphosphate and to the salt formation between the phosphate group of thiamine
diphosphate and the amine group of CS. Lee et al. [181] synthesized CS-NPs, by
coacervation, to encapsulate siRNA in the presence of polyguluronate (PG), a block of
guluronic acid residues present in the alginate backbone. The ability of PG to form stable NPs
with CS was hypothesized, given its low Mw and ionic interactions with cations.
CS/(PG+siRNA) NPs were the most efficient formulation to deliver siRNA into HEK 293FT
and HeLa cells, as compared with NPs without PG or with alginate replacing PG. Ji and
colleagues [182] developed CS/FHL2 siRNA NPs with a hydrodynamic radius of about 148
nm, which knock down about 67% FHL2 gene expression (over-expressed human colorectal
cancer Lovo cells), very similar to the 69% reduced gene expression when siRNA was
transfected with liposome LipofectamineTM. CS surface-modified poly(D,L-lactide-co-
glycolide) (PLGA) nanospheres for siRNA delivery were prepared by Tahara et al. [183]. CS-
PLGA nanospheres exhibited much higher encapsulation efficiency and were more
effectively taken up by the cells than unmodified PLGA NPs, possibly due to electrostatics
interactions with cell membrane. Consequently, the gene silencing efficiency of CS-PLGA
nanospheres was higher and more prolonged.
3.2.5. Gene Silencing In Vivo

Only a few studies report the use of CS/siRNA for in vivo therapy. Some works described
ahead in this review show the success of CS/siRNA NPs as a promising approach for the
inhibition of gene expression in vitro and in vivo, and its therapeutic potential for the
treatment of infections, allergic and inflammatory chronic diseases and cancer (Table 5).
3.3. Delivery of Low Molecular Weight Drugs
Currently, the research on nanoparticles based drug delivery systems focus on the
selection of nanoparticulate carriers for suitable drug release profiles and also on its surface
decoration, aiming at improving the targeting ability and in vivo biodistribution [188].
Table 5. Examples of CS/siRNA complexes used in knockdown of gene expression
Disease Therapeutic agent Administration Remarks Ref.

Respiratory RSV-NS1gene Intranasal Treatment of rats with siNS1 prior to [184]
syncytial virus siRNA (siNS1) RSV exposure reduced virus titers in
(RSV) infection the lung and prevented the
inflammation and airway
hyperresponsiveness (AHR)
associated with the infection and
development of asthma.
Asthma Imiquimod and Transdermal Imiquimod cream containing siNPRA [185]
natriuretic peptide CS-NPs showed significantly reduced
receptor A siRNA AHR, eosinophilia, lung
(siNPRA) histopathology and pro-inflammatory
cytokines IL-4 and IL-5 in lung
homogenates compared to controls.
Rheumatoid anti-TNF-α Dicer- Intraperitoneal CS-NPs containing siRNA mediated [186]
arthritis substrate siRNA TNF-α knockdown in primary
(DsiRNA) peritoneal macrophages. Histological
analysis of joints revealed minimal
cartilage destruction and
inflammatory cell infiltration in anti-
TNF-α-treated mice.
Lung cancer onco-protein Akt1 Nasal CS-graft-polyethylenimine carrier [187]
siRNA (siAkt) efficiently delivered siAkt and
silenced onco-protein Akt1.
These are crucial goals, since drugs often fail to get favorable clinical outcomes, due to
instability and reduced bioavailability. Furthermore, drugs need to be protected from
degradation in the biological environment. The bioactivity is often limited by the inability to
cross biological barriers and to reach the target site, specially when intracellular or
intranuclear sites of action must be reached [124]. In addition, significant amounts of the
administrated drug may distribute over the healthy tissues or organs, often leading to severe
side effects. Among other drug delivery strategies, a great deal of attention has been directed
to CS-NPs as promising systems able to improve drug bioavailability, modify
pharmacokinetics and/or protect the encapsulated drug [189]. In fact, CS-NPs improve
transmucosal permeability, enhance transport through the paracellular pathway, due to the
good bio- and mucoadhesive properties, and induce structural reorganization of tight junction
[190]. CS derivatives have been designed to improve the properties of native CS. Chemical
modifications of CS originate amphiphilicity, an important characteristic for the formation of
self-assembled NPs, potentially suited for drug delivery applications. The hydrophobic cores
of the NPs may act as reservoirs for a variety of bioactive substances [191]. CS-based NPs as
delivery systems of low molecular weight drugs have attracted attention for cancer and organ-
specific therapy.
3.3.1. Cancer-Targeted Drug Delivery

Most anticancer agents do not specifically target cancer cells, but also normal tissues,
leading to adverse effects following systemic administration. In addition, anticancer drugs are
often poorly soluble in water; thus, organic solvents or detergents are necessary for clinical
applications, resulting in undesirable side effects such as venous irritation and respiratory
distress. Therefore, in an attempt to circumvent these limitations, research efforts have been
concentrated on the development of new nanoparticulate drug delivery systems able to
encapsulate a large quantity of drugs. Targeted drug delivery using long-circulating
particulate drug carriers, such as polymeric NPs of controlled size, has high potential to
improve the cancer therapy, providing a selective effect owing to the concentration of drugs
at the tumor site, through enhanced permeability and retention (EPR) effect (Figure 7) and
allowing lower distribution in healthy tissues [192] (Figure 7).
Figure 7. Schematic representation of anatomical differences between normal and tumor tissues. The
defective tumor vasculature with disorganized endothelium allows passive targeting of nanoparticle
carrier due EPR effect.
The use of polymeric NPs has been recognized as an effective strategy for passive tumor
targeting, because its prolonged circulation time allows the accumulation and extravasation
into the tumor tissue. The NPs diffuse trough the anatomical and pathophysiological
abnormalities of the tumor vasculature, utilizing the principle of EPR effect [193]. The size
and surface of nanoparticle carriers play a crucial role in this process. Particles smaller than
200 nm and with hydrophilic surfaces tend to exhibit improved EPR effect, due to the
increased residence time in the blood stream, which can only be possible avoiding
opsonization.
The encapsulation of drugs into CS-based NPs can be physically achieved by

hydrophobic interactions [194] [195] or covalently binding to the polymer via a
biodegradable spacer [196] [197] or using chemical cross-linkers [198] [199]. CS-NPs have
been investigated as carriers for diverse small molecular drugs, in recent years, as referred in
Table 6.
Table 6. Examples of CS-based NPs systems for the delivery
of low molecular weight drugs
Drug CS derivative Remarks Ref.

Camptothecin Hydrophobically (5β- The CPT-HCG NPs intravenous injected exhibited [200]
(CPT) cholanic acid) significant antitumor effect and high tumor targeting ability
modified glycol towards human breast cancer xenografts, owing prolonged
chitosan (HGC) blood circulation time and high accumulation in tumors.
poly(N- NIPAAm/CS NPs were sensitive to pH, which can be [201]
isopropylacrylamide) advantageous to target tumor cells. The CAMP loaded NPs
(NIPAAm)/CS drastically enhanced the cytotoxicity at pH 6.8
Doxorubicin CS-poly(acrylic acid) The DOX-loaded CS-PAA nanospheres remained in the [202]
(DOX) (PAA) systemic circulation for a longer period, compared with
DOX solution. In mice liver, the DOX delivered from
nanospheres was maintained constant at relatively high level
Glycol CS DOX loaded self-aggregates exhibited lower systemic [203]
toxicity but equivalent anti-tumor activity, compared to free
DOX. The increased blood circulation time and sustained
released resulted in the suppression of the tumor growth.
Paclitaxel (PTX) Linoleic acid (LA) The PTX-LMC1 was more effective in tumor suppression [204]
and poly(β-malic than free PTX, because the hydrophilic PLMA on the
acid) (PMLA) grafted surface of LMC NPs decreased the uptake by the
CS (LMCs) reticuloendothelial system.
Hydrotropic HO-GC-PTX NPs presented a sustained drug release profile, [205]
oligomer-conjugated rapid cellular uptake, and lower cytotoxicity. The PTX-
glycol CS (HO-GC) encapsulated NPs were predominantly accumulated in the
tumor tissue.
Cisplatin HGC CDDP-HGC NPs were successfully accumulated by tumor [206]
(CDDP) tissues in tumor-bearing mice, due to the prolonged
circulation, enhanced permeability and EPR effect.
Mitomycin C poly-ε-caprolactone CS-PCL NPs were selectively incorporated by bladder [207]
(MMC) coated with CS (CS- cancer cell line and MMC loaded NPs exhibited higher
PCL) toxicity than free MMC.
5-fluorouracil CS-polyaspartic acid The Bcl-2 gene family (regulatory factor group in apoptosis) [208]
(5-FU) (CTS-PASP) was regulated by CTS-Pasp-5Fu and 5-Fu. The regulation
effect of CTS-Pasp-5Fu NPs was more effective, enhancing
the inhibition and inducing apoptosis of the gastric
carcinoma.
Norcantharidin Galactosylated CS NCTD-GC NPs displayed tumor inhibition effect in mice [209]
(NCTD) (GC)
protophorphyrin HGC The released PpIX from NPs became highly phototoxic [210]
IX (PpIX) upon visible irradiation and in SCC7 tumor-bearing mice
exhibited enhanced tumor specificity and increased
therapeutic efficacy compared to free PpIX.
All- trans Methoxy In vitro results suggested that tumor cell migration was most [211]
retinoic acid poly(ethylene glycol) effectively inhibited by the polyion complex micelles
(ATRA) (mPEG)-grafted (ATRA/CP) than ATRA free.
CS (CP)
Docetaxel HGC DTX-HGC NPs presented deformability, passing through [64]
(DTX) the smaller pore size, owing their highly flexible features,
prolonged circulation time, tumor targeting ability and
higher antitumor efficacy.
The surface decoration of CS-NPs with poly(ethylene glycol) (PEG) has attracted
attention since it increases the physical stability of NPs, prolonging the circulation time in
blood by avoiding the removal by the reticuloendothelial system and decreasing the positive
charge of the particle surface. Hu et al. [212] verified that the PEGylation of stearic acid-
grafted CS oligosaccharide (CSO-SA) micelles reduce significantly the cellular uptake by
macrophages, not affecting the internalization by normal and tumor liver cells. A similar
effect was observed by Qu et al. [213] using paclitaxel loaded mPEGOSC micelles (CS
derivative with hydrophobic moieties of octyl and hydrophilic moieties of sulfate and
polyethylene glycol monomethyl ether (mPEG) groups).
However, the accumulation of drugs in the tumor tissue is not always a guarantee of a
successful therapy if the drug does misses the target site within the tumor cell, such as the cell
membrane, cytosol, or nucleus. Park et al. [214] synthesized self-assembled NPs made of N-
acetyl histidine-conjugated glycol CS (NAcHis-GC), a promising system for intracytoplasmic
delivery of paclitaxel. Cellular uptake of NAcHis-GC NPs occurred by adsorptive
endocytosis initiated by nonspecific interactions between NPs and cell membranes. Then, the
NPs were exocytosed or localized in endosomes. In the slightly acidic environment of the
endosomes, the drug-loaded NPs were disassembled due to breakdown of the
hydrophilic/hydrophobic balance by the protonation of the imidazole group of NAcHis,
providing a drug release into the cytosol. Therein, paclitaxel was effective in inducing arrest
of cell growth. You et al. [65] developed another strategy for paclitaxel delivery. Micelles
made of stearic acid and CS hold multiple hydrophobic “minor cores” near the surface, which
improved the micelles internalization into cancer cells and accumulation of the drug in the
cytoplasm. For antitumor drugs acting on the nucleus, effective internalization and nucleus
accumulation is mandatory. Although nuclear import of many nuclear proteins is based on the
presence of a peptidic nuclear localization signals (NLS), other non-peptidic NLS exist,
namely sugar molecules. You et al. [66] observed that the CS-g-stearic acid micelles loaded
DOX presented an enhanced nuclear location comparing to free DOX, possibly due to the
import of the micelles loaded drug occurring via a sugar-dependent manner.
Cancer cells often over-express specific antigens or receptors on the cell surface that can
be used for active targeting. Chemical modification of the drug carrier using targeting
moieties can precisely direct NPs to receptors on the tumor tissue [191]. For successful active
targeting, the specific receptors should be expressed exclusively on the cancer cells. The
targeting moieties most used are galactose, transferrin (Tf), folic and hyaluronic acids (HA).
Glycotargeting takes advantage of a highly specific interaction between the carbohydrate
ligands conjugated on macromolecules and the endogenous lectins present on the targeted
cells. Because of their high density on the surface of hepatoma cells in the liver cancers, the
asialoglycoprotein (ASGP) receptors are a particularly attractive site for glycotargeting.
Among the glycoconjugated macromolecules, galactosylated CS was found to be a suitable
material for liver-targeting drug/gene delivery [215]. Mi et al. [215] confirmed that the
galactosylated CS-NPs had higher specific interaction with hepatoma cells than CS-NPs, via
the ligand-receptor (ASPG)-mediated recognition, leading to a high affinity to HepG2 cells.
The Tf is also over-expressed in tumor tissues, hence it can be used as a ligand for tumor
targeting. Tf was covalently bound to the Dox-loaded palmitoylated glycol CS (GCP) vesicles
by Dufes et al. [216]. The Tf decorated vesicles were taken up faster (after 1–2 h) and DOX
reached the nucleus after 60–90 min, leading to higher cytotoxicity than GCP DOX in vitro,
although this good in vitro performance did not translate into a therapeutic advantage in vivo.
All vesicles reduced the tumor size on day 2, but were, overall, less active than the free drug.
Folate receptors (FRs) are also frequently over-expressed in human epithelial cancerous cells.
Therefore, folate-conjugated drugs or carriers can be rapidly internalized into cancer cells via
receptor-mediated endocytosis. Folate-conjugated stearic acid grafted CS (54 kDa) NPs,
produced by You et al. [217], were rapidly took up by Hela cells (over-expressed FRs) as
compared to A549 cells (deficient FRs cell line). PTX was encapsulated into these micelles;
the lethal half dose of taxol (a clinical formulation containing PTX) on A549 and Hela cells is
7.0 and 11.0 µg ml−1, respectively while for PTX-loaded micelles these values were reduced
to 0.32 µg ml−1 and 0.268 µg ml−1. These results were attributed to the increased intracellular
delivery of the drug. Most malignant solid tumors and their surrounding stromal tissue
contain elevated levels of HA, which can provide a matrix that facilitates invasion [218]. HA
receptors, such CD44, are also over-expressed in tumor cells; indeed, cells with metastatic
potential often show enhanced binding and internalization of HA. Jain and Jain [218]
explored the utilization of HA grafted CS-NPs for the effective delivery of 5-FU to colon
tumors. HA-CS-NPs showed significantly higher uptake by cancer cells, about 7.9 fold as
compared to uncoupled NPs, which clearly indicate that the uptake of HA coupled NPs
occurred via CD44 receptors of HT-29 cancer cells.
3.3.2. Organ-Specific Drug Delivery

NPs constitute a versatile drug delivery system, with the ability to overcome
physiological barriers, guiding drugs to specific cells or intracellular compartments, either by
passive or receptor-mediated targeting mechanisms. The NPs can be targeted to organs such
as the eye, liver, spleen, lung and lymph; because of their very small size, they can pass
through the narrowest capillaries [219].
Ocular Delivery
Efforts in ocular drug delivery have been made to improve the bioavailability and to
prolong the residence time of drugs applied topically onto the eye [220]. Campus et al. [221]
concluded that CS-coated poly-ε-caprolactone nanocapsules enhanced the penetration of an
encapsulated dye through the cornea, probably due to the extended adhesion of the
nanocapsules at the superficial layers of the epithelium. In vivo studies showed that the
amounts of fluorescent CS in cornea and conjunctiva were significantly higher for fluorescent
CS-NPs than for a control fluorescent CS solution, these amounts being fairly constant for up
to 24 h [222]. More recently, Motwani et al. [223] used mucoadhesive CS-sodium alginate
(ALG) NPs as a new vehicle for the prolonged topical ophthalmic delivery of antibiotic
gatifloxacin. Unfortunately, no evidence of the in vitro or in vivo behavior of this formulation
has been reported. Badawi et al. [224] observed that, following topical instillation of a CS-
nanocarrier loaded with indomethacin (IM) to rabbits, it was possible to achieve therapeutic
concentration of IM in the cornea and fairly high IM level in inner ocular structure. These
levels were significantly higher than those obtained following instillation of IM solution. IM
concentration delivered from nanocarriers in the cornea was sufficiently high to adequately
suppress inflammatory process.
Liver-Target Drug Delivery

The diammonium glycyrrhizinate (DG) is used for the treatment of chronic hepatitis.
Yang et al. [225] produced lactose-conjugated PEG-grafted-CS (Lac-PEG-g-CS) to promote
liver-targeted delivery of DG, because lactose can be recognized by asialoglycoprotein
receptor (ASGP-R) on the cell surface of liver. Indeed, the Lac-PEG-g-CS delivered DG
more effectively to the liver than the PEG-g-CS micelles. Although reported as a therapeutic
agent, the glycyrrhizin was also used for the surface decoration of the CS-NPs (CS-NPs-GL),
a novel hepatocyte-targeted delivery system [226]. CS-NPs-GL NPs were preferentially
accumulated into hepatocytes and the cellular uptake was dependent on incubation time and
dose of NPs, which indicated that the internalization of the NPs into hepatocytes was mostly
mediated by a ligand-receptor interaction. Lin et al. [227] also described the conjugation of
glycyrrhizin with N-caproyl CS.
Brain-Target Drug Delivery

The Alzheimer disease (AD) is a chronic neurodegenerative disorder accompanied by the
gradual and progressive loss of functional and psychomotor abilities. The female sex
hormone, 17β-estradiol, is involved in the regulation of brain development. Long-term
oestrogen replacement has proved to be beneficial in the prevention and treatment of
Alzheimer’s disease [228]. Considering the therapeutic potential of E2, Wang et al. [228]
studied the levels of estradiol (E2) in blood and the cerebrospinal fluid in rats following
intranasal administration of E2-loaded CS-NPs. The E2 was directly transported from the
nasal cavity into the cerebrospinal fluid, in rats; the CS-NPs are thus able to significantly
improve the E2 transport to central nervous system. Later, the same group synthesized TMC
surface-modified poly(D,L-lactide-co-glycolide) (PLGA) NPs (TMC/PLGA–NP) [229]. As a
cationic ligand, TMC can facilitate the active transport of NPs via absorptive-mediated
transcytosis (AMT) across the cerebral endothelium and, so TMC-modified NPs could be
used as a drug carrier for brain delivery, overcoming the blood–brain barrier (BBB). PLGA–
NP and TMC/PLGA–NP loaded 6-coumarin, as a probe, were injected into the caudal vein of
mice; higher accumulation of TMC/PLGA–NP in the cortex and third ventricle was observed,
as compared to PLGA–NP, demonstrating that the TMC/PLGA NPs pass through the
endothelial cells of the BBB, reaching the brain parenchyma. This effect was confirmed by
the behavior tests in AD transgenic mice, in which the neuroprotective effects of
TMC/PLGA–NP loaded with coenzyme Q10 (Co Q10) were superior to the PLGA–NP and
solution, and markedly improved the spatial memory.
Lung-Target Drug Delivery

In general, nanoparticle delivery to the lungs is an attractive concept because retention of
the particles in the lungs, accompanied with a prolonged drug release, can be achieved using
large porous nanoparticle matrices. On the other hand, it has been shown that NPs uptake by
alveolar macrophages can be reduced using particles smaller than 260 nm. Both effects
combined might improve local pulmonary drug therapy [230]. Asthma is a chronic
inflammatory disease of the airway characterized by the infiltration of eosinophils, epithelial
hyperplasia leading to hypersecretion of mucus and the presence of airway
hyperresponsiveness to a variety of stimuli. Theophylline was used for the treatment of
asthma but side effects limit its application. Thus, Lee et al. [231] hypothesized that the
absorption of theophylline through bronchial mucosa could be enhanced by administration

with thiolated CS-NPs (TCNs), because of their greater mucoadhesiveness and permeability
properties. In an allergic asthma mouse model, intranasal delivery of theophylline complexed
with TCNs augmented the anti-inflammatory effects of the drug compared to theophylline
administered alone, or loaded into unmodified CS-NPs.
4. CONCLUSIONS
The application of CS nanoparticulate systems in drug delivery has great potential.
Exciting concepts and sophisticated formulations have been produced using CS and its
derivatives. However, several issues must be addressed such that these possibilities can be
fully exploited and reach clinical application. A wider choice of pure, medical grade chitosan
and its derivatives is needed. A better control over the stability of the NPs is necessary, in
particular at physiological pH. In many cases, in vitro results are not reproduced in vivo,
hence more knowledge on the fate of the CS-NPs in vivo is mandatory. The interaction of
NPs with serum proteins (in the blood), the biodistibution and intracellular trafficking must
be more comprehensively characterized, as well as toxicological issues.
ACKNOWLEDGMENTS
Paula Pereira, Vera Carvalho and Reinaldo Ramos were supported respectively by the
grant SFRH/BD/64977/2009, SFRH/BD/27359/2006, SFRH/BD/27404 / 2006, from
Fundação para a Ciência e Tecnologia (FCT), Portugal. This review was also supported by
FCT through the project PTDC/BIO/67160/2006.
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Chapter 7
CHITOSAN-BASED NANOCARRIERS:
EFFECTIVE VEHICLES FOR MUCOSAL
PROTEIN DELIVERY
Luis Braz1,2, Marita Dionísio3 and Ana Grenha3∗

1
CIQA – Algarve Research Centre of Chemistry,
University of Algarve, Campus de Gambelas, Faro, Portugal
2
School of Health, University of Algarve,
Av. Dr. Adelino da Palma Carlos, Faro, Portugal
3
CBME - Centre for Molecular and Structural Biomedicine,
IBB – Institute for Biotechnology and Bioengineering,
University of Algarve, Campus de Gambelas,
Faro, Portugal
ABSTRACT
Many newly designed therapeutic biomacromolecules, including protein-based
drugs, are characterised by reduced capacity to permeate biological membranes and/or
low stability in physiological environments. Over recent years, a major challenge of the
pharmaceutical industry has concerned the necessary development of suitable non-
injectable drug carriers that permit overcoming these limitations, opening the possibilities
for the administration of the referred molecules through routes which are alternative to
the parenteral. In this regard, nanocarriers have emerged as one of the most exciting tools
to circumvent these drawbacks, mainly owing to their increased surface-to-volume ratio,
which results in improved interaction with epithelial surfaces and, in some cases, in the
ability to cross epithelial barriers. Moreover, nanoparticles further enable the protection
of the encapsulated molecules, which retain their biological activity, permitting their
administration through routes that were previously unviable. The compelling need to
design biocompatible, biodegradable and non-toxic vehicles has turned polysaccharides
into a very attractive class of materials. In this group, chitosan has reached a position of
evidence, due to its interesting physicochemical and biopharmaceutical properties.
∗
Corresponding author: Phone: 00351 289800100-7556, Fax: 00351 289819419, E-mail: amgrenha@ualg.pt.
366 Luis Braz, Marita Dionísio and Ana Grenha
Chitosan nanoparticles have in fact proven to be very effective vehicles for systemic
mucosal protein delivery, demonstrating high capacity for protein association, enabling
their protection from harsh environments and, owing to chitosan mucoadhesive character,
improving the proteins’ residence time in contact with the absorptive epithelia. Overall,
chitosan nanocarriers have been demonstrating the ability to increase the bioavailability
of encapsulated drugs. In this chapter, we present chitosan-based nanoparticles that have
been proposed for the administration of proteins through mucosal routes such as the oral,
buccal, nasal and pulmonary. More specifically, the various techniques and materials
applied on the elaboration of the carriers, along with chitosan, are analysed and the
efficacy of these carriers is discussed, analysing the various mechanisms proposed for
their effectiveness.
INTRODUCTION
Many protein-based drugs resulting from the work of the biotechnology industry, have
been identified as potential therapeutic agents. However, proteins are very instable
compounds and their administration is extremely challenging, due to inherent
physicochemical and biopharmaceutical properties [1], making parenteral delivery usually the
unique possibility. Parenteral administration is invasive and painful, frequently leading to
therapeutic incompliance [2] and, thus, it is urgent to find adequate alternatives. In
comparison, mucosal administration is advantageous as systemic pathway, because it is non-
invasive. In this manner, the development of non-injectable delivery systems for mucosal
administration could enhance significantly patient’s compliance, thereby leading to increased
therapeutic benefits [3]. The therapeutic action of proteins is not only limited by the potential
degradation in biological environments, but is also compromised by their low ability to reach
the therapeutic site of action [4]. In fact, these limitations are related with the presence of a
vast number of functional groups susceptible of chemical degradation and, also, with the high
hydrophilic character of the proteins, which results in poor permeability [5]. In addition, drug
delivery via mucosal routes faces other major restrictions, including the immune reactions at
the delivery site and, generally, limitations in the surface area available for absorption [6]. As
such, a meaningful challenge for pharmaceutical scientists nowadays has been the need to
develop suitable vehicles that permit delivering macromolecules through alternative routes of
administration, while providing for their protection. Apart from these features, in most cases
the carriers are expected to provide a controlled release pattern and, ideally, they should
enable the molecules to cross the mucosal barriers, in order to reach the specific sites of
action. In such a task, there is a consensus in that the materials and methods used to prepare
the referred vehicles play relevant roles [7].
Addressing the research efforts towards the development of adequate vehicles for the
purpose of drug delivery through distinct routes of administration, resulted in the appearance
of several innovative drug delivery systems like nanoparticles, liposomes and microspheres.
Nanoparticulate-based technologies have reached a position of evidence, because they display
an increased surface-to-volume ratio and surface functionality, thus offering high potential for
macromolecules association [8]. Furthermore, they have been reported to increase drug
absorption by reducing the resistance of the epithelium to drug transport in a localised area or
by carrying the drug across the epithelium [9]. In this regard, transport has been described as
more favorable for nanoparticles than for microparticles [1;10;11]. In addition, these colloidal
Chitosan-Based Nanocarriers: Effective Vehicles for Mucosal Protein Delivery 367
carriers are reported to have the capacity to interact with mucosal epithelial membranes, with
maximum interaction within 50 and 500 nm [5], and they have shown several times the ability
to control the drug release profile of encapsulated molecules [12]. Table 1 summarises the
advantages and drawbacks of nanoparticles for drug delivery applications.
Table 1. Advantages and limitations of nanoparticles for drug delivery applications
Advantages Limitations
High surface/volume ratio Undefined physical shape
Limited capacity to co-associate other
Ease of surface modification
functional molecules
Maximised contact with mucosae Unknown toxicity profile
Lack of suitable large-scale production
High drug concentration in desired site
methods
Ability to enter cells Low stability in some biological fluids
Protection of encapsulated molecules Tendency for aggregation
Limited loading capacity (unsuitable for less
Possibility to provide controlled release
potent drugs)
Small size can provide access to unintended
Possibility of targeted delivery
environments
Table 2. Secondary materials used in combination with chitosan to prepare chitosan-

based nanoparticles proposed for mucosal (oral, buccal, nasal and pulmonary) delivery
and associated molecules
Secondary material
Arabic gum, cyclodextrin, dextran sulfate, glucomannan,
Natural polymers
hyaluronic acid, polyglutamic acid, sodium alginate
Acrylic acid, methacrylic acid, polyacrylic acid,
Synthetic excipients
polyethylene glycol
Small anionic molecules Tripolyphosphate
Metals Gold
Surfactants Lecithin, poloxamer, span
Salts Magnesium sulfate, sodium sulfate
Associated molecules
Bovine serum albumin, cyclosporin-A, CpG
Proteins/peptides oligodeoxynucleotide, estradiol, FITC-BSA, FITC-
ovalbumin, heparin, insulin, ovalbumin, silk peptide
Antigens Cholera toxoid, HBsAg, Tat toxoid, Tetanus toxoid
Markers FD4, FD40
FD4: fluorescein isothiocyanate dextran 4 kDa; FD40: fluorescein isothiocyanate dextran 40 kDa;
FITC-BSA: fluorescein isothiocyanate labelled bovine serum albumin; HBsAg: hepatitis B surface
antigen.
Many materials have been applied so far in the production of these structures but,
concerning mucosal administration, one of the most promising strategies has been the
incorporation of mucoadhesive polymers, since this feature is known to prolong the residence
time of drug carriers at the absorption sites, thus resulting in higher permeation [7]. In this
regard, the application of chitosan has come to be particularly interesting for the association
and mucosal delivery of protein-based compounds, given its demonstrated strong
mucoadhesive character [13]. Chitosan further displays other very attractive properties for
drug delivery applications, such as its cationic character and solubility in aqueous solutions,
which enable the interaction with oppositely charged polymers, macromolecules and small
anions present in aqueous media. In occasions, the contact with specific polyanions results in
the formation of gels, a process derived from inter- and intramolecular crosslinkages
mediated by the anionic species [4].
Furthermore, it has demonstrated the ability to enhance the permeation of
macromolecules through well-organised epithelia, by means of transiently opening epithelial
tight junctions [14-16]. In addition, chitosan is known to be biocompatible and to exhibit very
low toxicity [4;17], both mandatory requisites for drug delivery applications. The first
approach to produce chitosan-based nanoparticles was reported in 1994 by Ohya et al. [18]
for the delivery of an anticancer drug and, since that date, these novel systems have been
extensively studied for drug delivery purposes. Table 2 displays an array of molecules already
associated to chitosan-based nanoparticles. The original formulation of chitosan nanoparticles
was then adopted by other scientists who found different applications, for instance in the
delivery of toothpaste actives [19], while others directed their work to the development of
new production methods [20-22] or to the diversification of the applied secondary materials
[23-26], obtaining rather different chitosan-based formulations, in many cases with
outstanding results. As can be seen in Table 2, a huge amount of secondary materials were
already combined with chitosan to provide the formation of chitosan-based nanoparticles.
Some formulations have proven specifically efficient for the association of peptides and
proteins, garnering excellent results for the systemic administration of these macromolecules
through the oral [27], nasal [28] and pulmonary [29] routes, demonstrating the nanoparticles
ability to act as permeation enhancers.
Research on chitosan is not limited to its application as obtained from the parent
molecule chitin. In fact, the presence of several reactive functional groups, as well as the
appearance of new synthetic routes and reaction media that permit its chemical modification,
has prompted the possibility of modifying its surface chemistry with the aim of promoting
new biological activities or achieving cell targeting [30;31]. In this manner, several
approaches have been conceived to chemically modify the chitosan molecule in order to
improve its performance as a drug delivery material. These include the synthesis of various
chitosan derivatives, which showed promising results when formulated into nanocarriers, as
reviewed elsewhere [31-33]. Chitosan has also been used as coating material to modify the
surface characteristics of previously formed carriers, contributing for enhanced mucoadhesion
and, thus, therapeutic effect of the encapsulated drugs [7;34;35]. Nevertheless, both the
referred strategies are beyond the scope of this review.
A few general reviews on the potential of chitosan for pharmaceutical applications have
been recently published [36-38]. We are aware of the promising results obtained for gene
delivery using chitosan nanoparticles as carriers. However, this subject was exhaustively
reviewed very recently [38] and, therefore, this chapter covers the application of chitosan in
nanoparticulate form for the systemic administration of protein-based drugs, namely, proteins,
peptides and antigens. The primary objective of this chapter is to provide an insight into the
relevance of chitosan nanoparticles as protein carriers for systemic administration through
mucosal routes, focusing the oral, buccal, nasal and pulmonary administration. The oral route
is always the first choice for the patients, because of its convenience and easy access [39];
while the buccal mucosa is readily accessible and permits avoiding the first-pass effect [40].
In turn, the nasal and pulmonary routes are more permeable and highly vascularised sites, also
avoiding the hepatic first-pass metabolism [41]. In vitro characterisation of carriers and in
vivo outcomes will be discussed, and special emphasis will be placed on the methods applied
for the carriers’ production.
METHODS FOR THE PREPARATION

OF CHITOSAN NANOPARTICLES
As mentioned in the introduction, the preparation of chitosan nanoparticles was reported

for the first time in 1994 by Ohya and co-workers [18]. In this case, chitosan nanoparticles
were obtained by application of a technique of emulsification, with a subsequent step of
cross-linking with glutaraldehyde. Although these pioneer studies demonstrated the feasibility
of synthesizing stable chitosan nanoparticles with capacity for drug encapsulation, the
detrimental effects later attributed to glutaraldehyde shifted the scientific interest to less
aggressive products, demanding the development of different technologies for the production
of chitosan-based nanoparticles. In this manner, several other techniques have been
developed, such as ionotropic gelation [42], emulsion-droplet coalescence [43], reverse
micellar method [44], emulsion solvent diffusion [20], polyelectrolyte complexation [24] and
desolvation [45]. Each of these methods has particularities affecting the resultant particles
but, in accordance with the objective of this chapter, in this section we will only review in
more detail the techniques applied in the production of chitosan nanoparticles with an
application described herein.
Ionic Gelation and Polyelectrolyte Complexation
The technique of ionotropic gelation of chitosan (CS) was first reported by Bodmeier et
al. [46] to produce chitosan beads, being adapted later on by Calvo et al. [42] for the
production of nanoparticles, in an attempt to produce these nanostructures in a complete
hydrophilic environment. This procedure explores the cationic character of chitosan and is
based on the interaction between positively charged amino groups present in chitosan’s
backbone and the polyanion tripolyphosphate (TPP), which acts as chitosan cross-linker [42].
In this case, nanoparticles are formed immediately after dropping the TPP solution into
chitosan solution, under mild stirring, at room temperature (Figure 1). After approximately 10
minutes of stirring, nanoparticles suspension is centrifuged, the supernatant discarded and the
resultant pellet of nanoparticles resuspended in water. Ionic gelation should be referred
anytime that chitosan gelation is induced by a complexation with small anionic molecules,
such as phosphates, citrates, sulfates, etc. When negatively charged macromolecules are used
instead of small molecules, a polyelectrolyte complexation occurs [47], a technique often

termed complex coacervation or interfacial coacervation [48;49]. Materials applied in the
production of such chitosan nanoparticles include several polymers, such as dextran sulfate
[23], sodium alginate [24], carrageenan [25], poly(acrylic acid) [50], arabic gum [51] and
poly-γ-glutamic acid [52;53], and also protein-based substances, like insulin [54] or even
DNA [21].
The setting up of the protocol for the preparation of chitosan nanoparticles by ionic
gelation demonstrated the relevance of controlling several parameters considered crucial to
succeed on their elaboration. These are mainly related with both the type of chitosan that is
used and the specific preparation conditions. Chitosan exists under many different chemical
structures, being available either as a base or a salt (there are different types of salt, such as
chloride, lactate, glutamate, etc.), and with distinct molecular weights and deacetylation
degrees. Several studies have been performed regarding the influence of these properties in
the characteristics of the obtained chitosan nanoparticles [55-57], revealing that chitosan
structure determines the nanoparticles preparation variables as well as their final
characteristics, namely size and zeta potential. The influence of these features in the polymer
performance in mucosal and transmucosal drug delivery was also extensively reviewed
recently [37]. Concerning the experimental conditions, the stirring pattern (speed and type of
vial, most importantly) used for the nanoparticles’ assembly and the conditions of
centrifugation (speed and duration) are the most important to obtain suitable nanocarriers
[58]. In the specific case of CS/TPP nanoparticles it is very important to optimise variables
such as chitosan and TPP concentrations, mass ratio between the materials, time and speed of
centrifugation and volume of resuspension [42;58]. However, these are the parameters that
should be optimised in a general manner, independently of secondary nanoparticles-forming
materials.
Figure 1. Schematic representation of chitosan nanoparticles preparation by the method of ionic

gelation.
Figure 2. Schematic representation of chitosan nanoparticles preparation by the method of modified

ionic gelation with radical polymerisation.
These techniques have been claiming important advantages, amongst which the complete
hydrophilic environment and the mild preparation conditions, which only involve gentle
stirring [34], should be highlighted. In fact, owing to the absence of organic solvents or high
shear forces, it is possible to encapsulate labile drugs such as proteins [59] and, possibly
because of that, these techniques have been the most applied in the production of chitosan
nanoparticles, as is reflected in this chapter, where they cover more than 60% of the described
works. The development of these methods to produce chitosan nanoparticles enabled an
important achievement, since using a physical cross-linking mediated by an electrostatic
interaction, instead of the chemical and covalent cross-linking with glutaraldehyde, provided
the possibility to ensure cell viability and drug integrity [4;34].
Modified Ionic Gelation with Radical Polymerisation
This process involves a modification of the above described ionic gelation method,
because chitosan gelation occurs concomitantly with the polymerisation of acrylic acid
monomers. In a brief description, an aqueous solution containing monomers of acrylic or
methacrylic acid is mixed, under stirring at room temperature, with an aqueous solution of
chitosan. In occasions, polyethylene glycol (PEG) or polyether (polethylene glycol-
polypropylene glycol-polyethylene glycol), have been also added to the reaction medium.
These polymers can be either previously mixed with chitosan or they can be added separately
into the monomers’ solution. As depicted in Figure 2, the opposite charges of chitosan and
acrylic acid enable the occurrence of an ionic interaction, leading to chitosan gelation.
Concomitantly, radical polymerisation is started by the addition of potassium persulfate and
the reaction takes place under a nitrogen stream. Temperature is usually raised to 60 - 70 ºC
and polymerisation lasts for approximately 6 h, after which the formed suspension of
nanoparticles is allowed to settle overnight. In order to remove residual monomers that have
not reacted, subsequent washes of the formed particles are performed with distilled water or
by application of a dialysis procedure [50;60;61].
Figure 3. Schematic representation of chitosan nanoparticles preparation by the method of desolvation

with sodium sulfate.
Parameters such as chitosan/acrylic monomers ratio and polymers concentration have

been found to strongly affect the nanoparticles’ properties, namely in which concerns their
physicochemical characteristics.
Desolvation
The process of desolvation is also frequently referred to as simple coacervation or phase

separation. It involves a macromolecular aggregation brought about by partial desolvation of
fully solvated molecules [48]. The use of desolvating agents to produce chitosan particles was
reported for the first time for the preparation of microparticles [62] but, nowadays, this
procedure is frequently applied to produce chitosan nanoparticles. Substances such as sodium
sulfate and non-solvents miscible with water (acetone, ethanol, isopropanol) have been
proposed as precipitating agents, the former being the most used. The formation of chitosan
nanoparticles by this technique is very simple, involving the dropwise addition of a solvent
competing agent of greater hydrophilicity (sodium sulfate for instance) into a chitosan
solution (Figure 3). When the salt is added to chitosan solution, it induces the elimination of
solvation water disposed around chitosan, given its higher affinity for water, thus leading to
the insolubilisation of the polymer and its consequent precipitation [4;49;63]. In fact, a phase
separation is occurring during the particles formation, because water-salt interactions are
more favourable than water-polymer, inducing the partial desolvation of chitosan. This, in
turn, leads to increased interactions between chitosan molecules, forming the referred
nanoparticles [48].
Chitosan solution usually contains a stabiliser, such as polissorbate 80, to stabilise the
formed nanoparticles suspension. A subsequent process of cross-linking, for instance with
glutaraldehyde, has been described, in order to harden the nanoparticles [63]. Factors such as
chitosan molecular weight, chitosan concentration and stirring rate have been found to
strongly affect final characteristics of nanoparticles, thus, being required to undergo

optimisation.
Emulsion Solvent Diffusion
The technique of emulsion solvent diffusion as a method to prepare chitosan

nanoparticles was adapted from the original procedure developed to produce PLGA
nanoparticles [20]. This method is based on the partial miscibility of an organic solvent with
water and it involves the addition of an organic phase (for instance, comprised of methylene
chloride and acetone) which contains the hydrophobic drug, to an aqueous solution containing
chitosan and a stabiliser (poloxamer, for example), under stirring (Figure 4). An O/W
emulsion is then formed and a subsequent high pressure homogenisation takes place.
Methylene chloride is afterwards removed under reduced pressure at room temperature. At
this time, acetone diffuses to the aqueous phase, decreasing the solubility of chitosan and,
thus, leading to the polymer precipitation to form nanoparticles. An additional amount of
water is usually added at this stage, in order to permit the complete diffusion of acetone.
Finally, the nanoparticles are isolated by centrifugation.
This method has demonstrated to be suitable for encapsulating hydrophobic drugs,
achieving high encapsulation efficiencies [20;59]. However, it is important to highlight the
need of aggressive preparation conditions, such as organic solvents and high shear forces,
which can be avoided by some of the previously described methods.
Although few studies are available applying the technique, parameters such as type of
chitosan, homogenisation rate and reaction time (period of evaporation and diffusion), should
affect the final outcome of the procedure and, consequently, nanoparticles characteristics.
Figure 4. Schematic representation of chitosan nanoparticles preparation by the method of emulsion

solvent diffusion.
Table 3. Potential advantages and disadvantages of oral administration
Advantages Disadvantages
Readily accessible and non-invasive Variability
Large surface area available for drug Drug diffusion limited by mucus barrier and
absorption (200 m2) intestinal motility
Rich blood supply Acidic gastric environment
Patient acceptability and compliance High metabolic activity
Ease of administration Low permeability of epithelium
APPLICATIONS OF CHITOSAN NANOPARTICLES IN MUCOSAL

DELIVERY OF PROTEINS
Oral Delivery
The oral route is the most common of the existing administration routes to achieve
systemic drug delivery, because of its convenience and high patient acceptance [1]. Under a
general point of view, oral drug delivery is simple, safe, non-invasive and highly cost-
effective, thus being the preferential route of administration in drug delivery research [39].
These are also the factors mostly contributing for the high therapeutic compliance of orally
administered drugs. Nevertheless, other relevant characteristics related with more scientific
considerations should be mentioned, such as the large intestinal absorptive surface area
(approximately 200 m2) and the high vascularisation, both strongly contributing for effective
drug absorption. Successful oral delivery is however compromised by several limitations,
which are particularly severe for labile drugs, such as protein-based molecules [1]. In this
context, drawbacks related to drug degradation, permeation and bioavailability in general are
still to overcome [5]. Several other parameters are known to affect oral drug delivery, which
were extensively reviewed in Lee and Yang [39]. A summary of the advantages and
disadvantages of drug delivery through the oral route is available in Table 3.
Overcoming the inherent limitations is only possible with the design of carriers that
respond to the challenge of maintaining protein functionality, while increasing their
bioavailability. To accomplish these requests, carriers are required to provide protein
protection from harsh gastric environment and should preferably permit an intimate
interaction with the epithelium to facilitate drug transport, enabling biomolecules to cross the
intestinal barrier [64]. Amongst the approaches developed for this end, nanoparticulate
chitosan systems have shown great potential, with much emphasis attributed to the recognised
mucoadhesive properties. Chitosan nanoparticles have been proposed to target specific
regions of the gastrointestinal tract by acting as a pH sensitive system [53;61;65;66].
Furthermore, they have demonstrated promising results in oral vaccination [67], taking
benefit of the fact that intestinal epithelium possesses specialised antigen presenting cells (M
cells) located at the Peyer’s patches [39]. Mucosal vaccination would offer several advantages
over parenteral immunisation, such as the avoidance of injection and the induction of local
immune responses, blocking pathogens at their portal of entry before their establishment [68].
Table 4. Description of in vitro studies performed with chitosan nanoparticles for oral drug delivery
Materials Associated Preparation Performed studies Major findings Ref.

molecule method
CS, TPP Insulin Ionic gelation Characterisation Nanoparticles (230-330 nm) evidenced a pH-dependent association efficiency (2%-85%). [69]
Drug release Certain pH values of nanoparticles preparation media rendered insulin more susceptible to
acid and enzymatic hydrolises, as compared to free peptide
CS, TPP FITC-BSA Ionic gelation Characterisation No alterations in tight junction functionality or membrane integrity were observed upon [72]
Cell culture (Caco-2 nanoparticles (290 nm) incubation with cells, thus evidencing absence of cytotoxicity.
and MTX-E12) Nanocarriers demonstrated ability to penetrate the mucus layer
CS, TPP, Insulin Ionic gelation Characterisation Nanoparticles size (260-390 nm) varied with poloxamer concentration and insulin was [27]
Poloxamer 188 associated with efficiency of 87%
CS, TPP Polypeptide Ionic gelation Characterisation Nanocarriers (170-300 nm) surface charge shifted from positive (> +30 mV) to negative (- [73]
GM mixture * NP stability 18 mV) upon coating with glucomannan. Glucomannan preserves nanoparticles structure
during freeze-drying, increasing their stability in PBS pH 7.4
CS, γ-PGA Insulin Polyelectrolyte Characterisation Insulin-loaded nanoparticles (100-200 nm; 57% association efficiency), have pH- [52]
complexation Drug release dependent release, with a burst release at pH 7.4 and slower release for lower pH values.
Cell culture Caco-2) All positive charged particles increased paracellular permeability
CS, γ-PGA, Insulin Ionic gelation Characterisation Nanoparticles of 218 nm associated insulin efficiently (68% - 74%). TPP and magnesium [53]
TPP, MgSO4 Drug release sulfate permitted a higher control over release, at pH 7.0 releasing 35% of insulin in 6 h,
Cell culture (Caco-2) in contrast with 70% release from formulation without these components
CS, Arabic Insulin Polyelectrolyte Characterisation Insulin was efficiently associated (38%) to nanocarriers, which displayed a size of 180 [51]
gum complexation Drug release nm. At pH 1.2, 30-50% of peptide was released in 15 minutes. At pH 6.5 and 7.2, 15%
and 40% cumulative release was observed, respectively, in 1h. Above pH 6.5, arabic gum
swells, resulting in more porous nanoparticles
CS, PAA Silk peptide Ionic gelation/ Characterisation Nanoparticles (200-300 nm) encapsulated silk peptide with efficiency of 82%. At pH 2 – 3 [50]
AA monomers radical Drug release the peptide released rapidly, but at pH values of 4.5 and 7.4 an initial burst, followed by
polymerisation extended release for up to 10 days was observed
CS, MA Insulin Ionic gelation/ Characterisation Insulin was associated efficiently (85%) to nanocarriers (500-800 nm). Insulin [60]
monomers Insulin-β-CD radical Drug release complexation with β-CD did not affect its pH-dependent release profile. In 2 h, 20% of
polymerisation Mucoadhesion insulin was released at pH 1.2, in contrast with 80% release at pH 7.4. Nanoparticles
preserve insulin biological activity and are mucoadhesive
CS, MA BSA Iionic gelation/ Drug release Nanoparticles revealed a controlled release of protein at pH 1.2 (less than 15% in 2 h), but [61]
monomers, radical at pH 7.4 evidenced complete release in 3 h
PEG polymerisation
Table 4. Description of in vitro studies performed with chitosan nanoparticles for oral drug delivery (Continued)
Materials Associated Preparation method Performed studies Major findings Ref.

molecule
CS, DS Insulin Polyelectrolyte Characterisation Nanoparticles (490 nm, -14 mV) efficiently associated insulin (85%) and displayed pH- [23]
complexation Drug release sensitive behaviour. No insulin was released at pH 1.2, 4.5 and 5.2, while sustained
release is obtained at pH 6.8 (40% release in 15 minutes, followed by extended release
until 6 h)
CS, ALG Insulin Polyelectrolyte Characterisation Insulin was associated with efficacy of 66%. Rapid disintegration of nanoparticles occurs [24]
complexation Drug release at pH 1.2 and 6.8, leading to immediate insulin release
CS, CK-GM BSA Polyelectrolyte Characterisation Positively charged particles were obtained in a broad size interval (50 nm – 1200 nm), [26, 74]
complexation Drug release which efficiently associated BSA (30-45%). Protein displays pH-dependent release,
releasing 20% at pH 1.2 and 50% at pH 7.4 after 2 h
CS, TPP Insulin Polyelectrolyte Characterisation Polyelectrolyte complexation permitted higher peptide association (90%) than ionic [75]
complexation Drug release gelation (50%), but release profile is similar for both methods. Strong burst release was
Ionic gelation observed at pH 1.5 (80% at 5 minutes), which was much less pronounced at pH 3.0 and
6.8 (around 20%)
CS Insulin Polyelectrolyte Characterisation Nanoparticles presented size of 270 nm and displayed sustained insulin release at pH 6.8, [54]
complexation Drug release with only 10% of insulin released after 1 h
CS, oleic acid, Insulin Polyelectrolyte Characterisation The oily based system (oleic acid and surfactant mixture) protects insulin from proteolysis [76]
surfactant complexation Drug release (90% intact insulin recovery) in simulated gastric fluid containing pepsin. Presence of
mixture surfactant is responsible for reduction of nanoparticles size from 2000 nm to 100 nm
CS, lecithin, Cyclosporin- Emulsion solvent Characterisation Nanoparticles presented a size of 150 nm and encapsulated cyclosporine-A with 94% [20]
poloxamer A diffusion efficiency
188
CS, Gold Insulin Reduction of Characterisation Nanoparticles display sizes between 10 and 100 nm and those formed with CS [77]
chloroauric acid concentration > 0.1% (maximum of 1%) did not aggregate for up to 6 months. Insulin was
associated with 53% efficacy
CS, TPP Ovalbumin Ionic gelation Characterisation Ovalbumin was associated with 65% efficiency to nanoparticles (300 nm). The [67]
Cell culture (Caco-2) nanocarriers evidenced absence of cytotoxicity
CS Ovalbumin Desolvation Characterisation Nanoparticles (640 nm) adsorbed ovalbumin with 40%-60% efficacy. Alginate coating [79]
ALG Drug release inverted zeta potential from +41 mV to -35 mV, and increased nanoparticles stability in
SIF (pH 6.8). However, in SGF the coating did not avoid a total loss of nanoparticles
integrity
CS Ovalbumin Desolvation Drug release Alginate coating provided controlled release of ovalbumin in PBS pH 7.4, with ± 45% [80]
ALG Cell culture released in 60 min and a total of 70% in 16 h. No evidence of nanoparticles cytotoxicity
(Splenocytes) was observed
molecule
CS HBsAg Desolvation Characterisation HBsAg and CpG ODN were associated with 86% and 99% efficacy to nanoparticles, [81]
CpG ODN respectively, and the antigen maintained its integrity
CS, Na2SO4, BSA Desolvation Characterisation BSA-loaded nanoparticles (220 nm, 95% association efficiency) encapsulated in lipidic [82]
soya lecithin, Drug release vesicles prevented almost 2-fold more the loss of antigen at pH 1.2. Controlled release
Span 60 was observed at pH 7.4 (40% at 10 h and 50% at 24 h)
AA: acrylic acid; ALG: sodium alginate; BSA: bovin serum albumin; β-CD: β-cyclodextrin; CpG ODN: CpG oligodeoxynucleotide; CS: chitosan; CK-GM:
carboxymethyl Konjac glucomannan; DS: dextran sulfate; GM: glucomannan; HBsAg: hepatitis B surface antigen; MA: methacrylic acid; MgSO4: magnesium
sulfate; Na2SO4: sodium sulfate; NP: nanoparticles; PAA: poly(acrylic acid); PBS: phosphate buffer saline; PEG: polyethylene glycol; γ-PGA: poli(γ-glutamic
acid); SGF: simulated gastric fluid; SIF: simulated intestinal fluid; TPP: tripolyphosphate; *: mixture of polypeptides isolated from seeds of Ricinus comunis.
Table 5. Description of in vivo studies performed with chitosan nanoparticles for oral drug delivery
Materials Associated Preparation method Animal Major findings Ref.

molecule
CS, TPP Insulin Ionic gelation Rats Nanoparticles formulated at pH 5.3 have onset of action of 10 h and decreased 40% serum glucose at [70]
16 h; while those formulated at pH 6.1 had onset of action of 2 h but lower efficiency (60-75% of
baseline) for 6-24 h after administration
CS, TPP FITC-BSA Ionic gelation Rats Nanoparticles were internalised by Peyer’s patches and epithelial tissue after intra-duodenal injection [72]
CS, TPP, Insulin Ionic gelation Rats Nanoparticulate system protects entrapped protein from aggressive gastrointestinal conditions. [27]
Poloxamer 188 Nanoparticles induced a long-lasting hypoglycemic effect (at least 10 h)
CS, γ-PGA Insulin Polyelectrolyte Rats Blood glucose levels decreased to 50% 5 h after nanoparticles administration. Hypoglycemic effect [52]
complexation remained for up to 10 h
CS, γ-PGA, Insulin Ionic gelation Rats Daily dose of empty nanoparticles administered for 14 days did not induce toxicity. Nanoparticles [65]
TPP, MgSO4 Mice induced continuous decrease of glucose levels for at least 10 h (50% of basal), while SC injection
provided a minimum peak at 2 h (30% of basal), with subsequent recovery after that
CS, DS Insulin Polyelectrolyte Rats Nanoparticles achieved a 2-3 fold increase in oral insulin relative pharmacological availability, [66]
complexation comparing to an insulin solution
CS, oleic acid, Insulin Polyelectrolyte Rats Nanoparticles dispersed in an oily-based system (oleic acid + surfactant) led to a pharmacological [76]
surfactant complexation responses similar to non-dispersed nanoparticles (40% maximum decrease of blood glucose levels 8 h
mixture post-administration)
CS, lecithin, Cyclosporin-A Emulsion solvent Dogs Nanoparticles have better therapeutic response than commercial microemulsion (Neoral®), with [20]
poloxamer 188 diffusion approximately 73% increase in relative bioavailability of cyclosporine-A
CS, Gold Insulin Reduction of Rats Nanoparticles induced a blood glucose reduction of 30%, in contrast with no hypoglycemic effect [77]
chloroauric acid upon administration of an insulin solution
Table 5. Description of in vivo studies performed with chitosan nanoparticles for oral drug delivery (Continued)

molecule
CS, TPP Ovalbumin Ionic gelation Mice Nanoparticles administration induced a significant enhancement in IgG production (over 1000 fold) [67]
compared to the intraduodenal immunisations with free antigen, due to M-cell specific uptake of the
particulate system
CS Tat toxoid Desolvation Mice Nanoparticles administration elicited both mucosal and systemic immunity [78]
CS, Ovalbumin Desolvation Rats Alginate coated-nanoparticles were internalised by Peyer’s patches, in contrast with a control FITC- [80]
ALG FITC- ovalbumin solution. Negatively-charged hydrophilic nanoparticles can undergo cellular uptake
Ovalbumin
CS HBsAg, Desolvation Mice HBsAg-loaded nanoparticles alone or mixed with CpG ODN (in solution or in nanoparticles) [81]
CpG ODN enhanced mucosal and systemic immune responses
CS, soya BSA Desolvation Rats Nanoparticles encapsulated in vesicles elicited, after 4 weeks, a systemic immune response about 1.5- [82]
lecithin, Span 60 Reverse-phase fold stronger than unmodified nanoparticles. Induced IgA titers were also 2-fold higher
ALG: sodium alginate; AUC: area under the curve; BSA: bovine serum albumin; CpG ODN: CpG oligodeoxynucleotide; CS: chitosan; DS: dextran sulfate; FITC:
fluorescein isothiocyanate; FITC-BSA: isothiocyanate fluorescein-labelled bovine serum albumin; HBsAg: hepatitis B surface antigen; IgA: immunoglobulin
A; IgG: immunoglobulin G; MgSO4: magnesium sulfate; Na2SO4: sodium sulfate; γ-PGA: poli(γ-glutamic acid); TPP: tripolyphosphate.
Many research works have, thus, been developed towards the design of chitosan
nanoparticulates that provide efficient delivery of protein-based drugs through the oral route.
Tables 4 and 5 present a summary of these works, with emphasis on applied materials,
methods and major findings, respectively for in vitro and in vivo studies.
It is noteworthy the observation that a very considerable percentage of the works
comprising the production of chitosan nanoparticles for oral delivery is obtained by a process
of ionic complexation, in many cases mediated by TPP. Ma et al. studied the efficiency and
mechanism of association of insulin to these CS/TPP nanoparticles, in the pH range of 2.3 to
6.3. With sizes ranging approximately between 230 nm and 330 nm, nanoparticles revealed a
pH-dependent association efficiency between 2% and 85% (highest association at pH 6.1).
Curiously, it was observed that at certain pH values (i.e. pH 5.3) insulin association to
nanoparticles rendered the peptide more susceptible to acid and enzymatic hydrolyses, as
compared to free peptide [69]. Based on the consideration that these differences could be
reflected on nanoparticles in vivo behaviour, a subsequent study was performed in diabetic
rats, evaluating formulations produced at pH 5.3 and 6.1. It was then showed that
nanoparticles formulated at pH 5.3 displayed an onset of action of 10 h, inducing a strong
decrease of serum glucose levels 16 h after the administration, reaching 60% of baseline and
maintaining this level at least until 24 h; while those formulated at pH 6.1 had a faster onset
of action (2 h) but lower efficiency (60-75% of baseline) for 6-24 h after administration. It is
worth mentioning that, in spite of the obtained hypoglycemic response, no correspondence
was observed with higher serum insulin concentrations, as compared to the control group
[70]. In this sense, as similar results were obtained for other polymer formulation [71], the
authors ascribed the phenomenon to a local effect of insulin in the intestine [70]. Other
authors used a fluorescent (fluorescein isothiocyanate-labelled bovine serum albumin, FITC-
BSA) formulation of CS/TPP nanoparticles to evaluate the effect of mucus on cellular
association, using Caco-2 and MTX-E12 cells, which are models of intestinal epithelium and
mucus-secreting cells, respectively. Cytotoxicity studies based on TER monitorisation and
lactate dehydrogenase release, revealed that presence of nanoparticles did not alter tight
junction functionality or membrane integrity. It was further demonstrated that nanoparticles
can penetrate the mucus layer, remaining associated to cells. Furthermore, in vivo studies
revealed an extensive internalisation by Peyer’s patches and epithelial cells, after intra-
duodenal injection of the particles [72].
Approaches such as adding absorption enhancers to the formulations or coating chitosan
nanoparticles with other polymers have been attempted to achieve improved pharmacological
responses after oral administration. In this regard, Pan et al. prepared insulin-loaded CS/TPP
nanoparticles containing poloxamer 188, a surfactant that provides enhanced intestinal
absorption by prolonging residence time in the intestinal tract. Nanoparticles size varied with
poloxamer concentration, ranging between 260 nm and 390 nm, and insulin was associated
with efficiency of 87%. In vivo administration showed a maximum decrease of glucose levels
at 10 h (40% reduction), in contrast with 20% reduction obtained upon administration of
insulin-chitosan solution with the same dosis. Moreover, the hypoglycemic effect provided by
the nanocarriers was found to last for at least 10 h. The authors concluded that the
nanoparticulate system provides improved protection of entrapped protein in the harsh
conditions characteristic of gastrointestinal tract and that, including poloxamer 188 in the
formulation, rendered nanoparticles the ability to escape clearance, consequently providing a
longer residence time at the absorption site, thereby leading to prolonged hypoglycemic effect
[27]. However, this latter conclusion should be supported by a direct comparison of in vivo
behaviour between poloxamer-containing nanoparticles and nanoparticles without poloxamer
188, but unfortunately this last formulation was not tested. Other research group has
administered orally CS/TPP nanoparticles (no poloxamer included) and the resultant
hypoglycemic effect was similar to that reported for the poloxamer-containing nanoparticles
[70]. Nevertheless, the administered dose is about 7-fold higher in this case and chitosan
displays different properties, thus not allowing a direct comparison.
Concerning the second mentioned approach, which comprises coating of chitosan
nanoparticles, glucomannan (GM) is a polysaccharide that was used for this end, conjugating
chitosan ability to promote transport of associated molecules and the skill of glucomannan to
interact with mannose receptors expressed in intestinal M-cells. It was argued by the authors
that glucomannan further provides a more rigid structure, which is resistant to the gastric
environment. Nanoparticles were prepared by chitosan ionic gelation with TPP and
subsequent coating with phosphorylated glucomannan, displaying sizes between 170 and 300
nm. Particles surface charge shifted from highly positive (> +30 mV) to negative (-18 mV)
upon coating with glucomanan. The system capacity to associate the model molecule (a
mixture of polypeptides isolated from seeds of Ricinus comunis) was strongly affected by the
presence of salts surrounding the protein environment, varying between 5% and 26%.
Interestingly, CS/GM nanoparticles were found to maintain their physicochemical
characteristics upon freeze-drying without any cryoprotectant agent, in contrast with the
observed for CS/TPP nanoparticles. This suggests that glucomannan plays a role in
preserving nanoparticles structure during freeze-drying. Furthermore, the carbohydrate was
found to increase nanoparticles stability in PBS pH 7.4 [73]. Considering the positive effect
observed in vitro as result of the glucomannan coating of CS/TPP nanoparticles, it would be
very interesting to observe the in vivo outcome, which was not displayed so far.
Several other materials have been conjugated to chitosan, with the final goal of obtaining
nanoparticles. In this context, Lin et al. used poli(γ-glutamic acid) (γ-PGA) to produce
CS/PGA nanoparticles aimed at the oral delivery of insulin. Sizes ranged within 100-200 nm
and zeta potential oscillated from strongly negative (-24 mV) to strongly positive (+33 mV)
as a function of the used CS/PGA mass ratios. Nanoparticles, which associated insulin with
57% efficiency, demonstrated to preserve the peptide integrity and evidenced a pH-dependent
release, showing a burst release at pH 7.4 but enabling slower release for lower pH values
until pH 2.5. In addition, given chitosan characteristics, nanoparticles were found to be
unstable at pH 1.2 and pH 7.4. Transepithelial electrical resistance (TER) measurements and
confocal laser scanning microscopy (CLSM) visualisations demonstrated that all positively
charged particles transiently opened Caco-2 cells tight junctions, increasing paracellular
permeability. In vivo experiments conducted with diabetic rats clearly indicate that these
nanoparticles protect insulin from gastrointestinal environment, considering the long-lasting
decrease of blood glucose levels, with a maximum reduction of 50% at 5 h, which remained
for up to 10 h [52]. With the objective of producing nanoparticles stable in a broader pH
range, the same research group introduced in the formulation of CS/PGA two other materials,
TPP and magnesium sulphate, using a multi-ion-crosslinking to prepare pH-responsive
nanoparticles. Insulin was associated with higher efficiency (68% - 74%) to this new
formulation, but comparable size (220 nm) and zeta potential (+26 mV) were obtained. Multi-
ion crosslinked nanoparticles achieved increased stability, with the inclusion of TPP and
magnesium sulfate in the formulation providing higher control of release behaviour, which
resulted in 35% release of insulin after 6 h in pH 7.0, in contrast with 70% of parent
formulation. Moreover, enhancement of paracellular permeability of insulin increased with
the new nanoparticles [53]. In order to provide the real proof-of-concept of this formulation,
in vivo studies were carried out, evaluating toxicological behaviour and the nanoparticles
potential for oral insulin delivery. No toxicity signs were found in mice treated during 14 days
with a daily dose of empty nanoparticles. It was very curious to compare the behaviour of
insulin-loaded nanoparticles administered orally to rats with that of a subcutaneous (SC)
injection of insulin. Until 7 h-post administration, nanoparticles induced a hypoglycemic
effect similar or inferior to that of SC insulin but, after that time, the profile inverted and,
while the animals tested with SC insulin recovered their blood glucose levels, those receiving
nanoparticles registered a continuous decrease of glucose levels, which achieved 50% of
basal at 10 h. Unfortunately, the authors stopped the experiment at that time and only said that
glucose levels did not return to initial values at 24 h post-dosing, not mentioning what
happened exactly between 10 h and 24 h. Interestingly, plasmatic insulin levels achieved a
peak 5 h post-administration, decreasing afterwards until the end of the experiment (10 h), but
no comment was performed on its relation with the decrease of glycemia until 10 h. Relative
bioavailability of orally administered insulin as compared to SC administration of insulin
solution, was reported to be 15%, an encouraging result considering the numerous difficulties
encountered with the oral absorption of insulin [65].
Arabic gum was also used to prepare chitosan-based nanoparticles, which were produced
according to a 23 factorial design, varying polymer concentrations and amount of associated
insulin. The highest association efficiency (38%) was obtained with the highest polymers
concentration and insulin amount, resulting in nanoparticles of 180 nm and surface charge of
+38 mV. An in vitro burst release effect was observed at pH 1.2, with 30-50% peptide being
released in the first 15 minutes. This was not observed at pH 6.5 and 7.2, which enabled,
respectively, 15% and 40% cumulative release in 1 h. This difference (between pH 6.5 and
7.2) was attributed to the swelling of arabic gum at pH above 6.5, which disturbs
nanoparticles structure, resulting in higher porosity and, thus, increasing insulin release [51].
Hu et al. described the preparation of chitosan/poly(acrylic acid) nanoparticles by simply
mixing solutions of both polymers, which interact ionically. In addition, they reported the
adaptation of this polyelectrolyte complexation technique to permit a concomitant radical
polymerisation of acrylic acid monomers to produce poly(acrylic acid). Protocol details are
available in the section of Methods for the preparation of chitosan nanoparticles.
Nanoparticles produced by polymerization presented smaller size (200 - 300 nm) as compared
to those obtained by simple ionic interaction (350 – 450 nm). Moreover, the first method
resulted in positively charged nanoparticles, while the latter produced nanoparticles with
charge dependent on the polymer mixing order (dropping poly(acrylic acid) over chitosan
results in positively charged nanoparticles and vice-versa). Based on zeta potential results and
transmission electron microscopy (TEM) visualisations, it was concluded that nanoparticles
can be formed with either a chitosan or poly(acrylic acid) core. To evaluate the efficacy of
these nanoparticles as protein delivery systems, silk peptide was loaded in particles produced
by polymerization (encapsulation efficiency of 82%) and release profile was determined. The
peptide released rapidly at pH 2 – 3, but at pH values of 4.5 and 7.4 a profile characterised by
an initial burst, followed by extended release for up to 10 days was observed [50].
Polymethacrylic acid was used in other occasion to produce chitosan-based nanoparticles,
simply performing radical polymerisation of methacrylic acid monomers in presence of
chitosan. Insulin alone and complexed with β-ciclodextrin was used as model protein (85%
association efficiency) and particle size varied between 500 and 800 nm. Insulin
complexation did not affect its release profile which was pH-dependent (in 2 h, 20% of
insulin was released at pH 1.2, in contrast with 80% release at pH 7.4) [60]. In a preliminary
study the authors had encapsulated BSA in similar nanoparticles, this time further added of
polyethylene glycol, and the same pH-dependent release behaviour was described [61].
Nanoparticles further enabled preservation of insulin biological activity and demonstrated to
be mucoadhesive (84% of nanoparticles still retained in intestinal epithelium after 20 minutes
of constant buffer flux) [60]. In general, these nanocarriers seem to be adequate for oral
delivery of proteins, but the lack of in vivo assays and experiments with specialised model
cell lines, do not allow a conclusion on their efficacy, which cannot be drawn only based on
the described in vitro behaviours.
Materials such as dextran sulfate, alginate or glucomannan have also been described to
form nanoparticles by ionic interaction with chitosan, by means of a process of
polyelectrolyte complexation or complex coacervation (for further details please consult the
section of Methods for the preparation of chitosan nanoparticles). Insulin was used as model
protein in all cases, displaying encapsulation efficiencies varying between 65% and 85%.
Nanoparticles produced with chitosan and dextran sulfate (DS) of various mass ratios, had a
minimum size of 490 nm (CS/DS = 1/1.5) and a negative zeta potential of -14 mV, reflecting
the higher amount of dextran sulfate in the particles composition, as compared to chitosan. In
vitro results clearly indicate a pH-sensitive character of nanoparticles. In fact, as observed in
Figure 5, no insulin released from the nanoparticles at pH 1.2, 4.5 and 5.2, while a sustained
release profile is obtained at pH 6.8 (40% release in 15 minutes), followed by an extended
release until 6 h.
Reprinted with permission from [23].
Figure 5. Insulin cumulative release profile from insulin-loaded dextran sulfate/chitosan nanoparticles
produced with dextran sulfate:chitosan mass ratio of 1.5:1 at final pH of 4.8. Nanoparticles were
submitted to curing time of 15 min and the theorical insulin loading was 2.3% (final insulin 0.006%).
Release assay was performed at pH 1.2 (U), 4.5 ({), 5.2 () and 6.8 (z) (vertical bars represent
standard deviations based on three replicates).
This pH-sensitive behaviour was ascribed to strong electrostatic interactions established

between insulin and dextran sulfate, which contribute to retain insulin in the nanoparticles
network. It was also demonstrated that released insulin did not suffer any degradation as
compared to a fresh sample, thus confirming the nanoparticles protection capacity [23]. In
vivo administration of insulin-loaded nanocarriers to diabetic rats has later evidenced their
efficacy in oral protein delivery. A plasma glucose reduction of approximately 32% was
observed 14 h after administration of nanoparticles, in contrast to a maximum decrease of
20% elicited by an insulin solution at 6 h. As compared to the insulin solution, nanoparticles
enabled a 2-3 fold increase in oral insulin relative pharmacological availability [66].
Nanoparticles were further prepared by chitosan polyelectrolyte complexation with sodium
alginate, but insulin released immediately either at pH 1.2 or 6.8, indicating a rapid
disintegration of nanoparticles, which is devoid of interest for the final aim of oral protein
delivery. This behaviour changes when nanoparticles are prepared by alginate pre-gelation
with Ca2+ followed by chitosan complexation but, this produces a structure based on chitosan
coating, which is beyond the scope of the present chapter [24].
Chitosan complexation with varying concentrations of carboxymethyl Konjac
glucomannan produced positively charged small nanoparticles from 50 nm up to
microspheres of 1.20 µm, which evidenced the ability to efficiently associate BSA (efficacy
from 30% to 45%) [26]. In a subsequent work the release profile was characterised and a
chitosan concentration-dependent release behaviour was described at pH 7.4. Nevertheless,
reporting to the displayed graphic, values are all very similar and the authors do not provide a
statistical analysis, thus not allowing a definitive conclusion on the observation. BSA release
was also pH-dependent, at 2 h releasing 20% in pH 1.2, in contrast with 50% at pH 7.4 [74].
A very interesting study was performed by Sadeghi et al. in which chitosan/insulin
nanoparticles prepared by polyelectrolyte complexation were compared with insulin-loaded
CS/TPP nanoparticles prepared by ionic gelation. Physicochemical characteristic were very
similar, with sizes around 200 nm and positive zeta potential of approximately +20 mV.
Polyelectrolyte complexation permitted higher peptide association (90%) than ionic gelation
(50%), given that in the former method insulin interacts directly with chitosan amino groups,
while in ionic gelation TPP is also competing for these groups. Importantly, no differences
were found in insulin release profile between nanoparticles produced by both methods, a
strong burst release being observed for the most acidic pH tested (pH 1.5, 80% release at 5
minutes), which was much less pronounced in higher pH (3.0 and 6.8), both around 20% [75].
Nevertheless, considering the association efficiency, polyelectrolyte complexation is
apparently the most adequate technique to produce these nanoparticles, although only a
comparison of in vivo pharmacological responses would provide a definitive conclusion.
Bayat et al. produced the same polyelectrolyte complexes (chitosan-insulin) with comparable
properties (270 nm, +18 mV and 84% association efficiency) [54]. It is, then, curious to
observe that in this case a very different release profile was found at pH 6.8. While in the
present study only 10% of insulin released after 1 h [54], in the previous one the level of
insulin raised to approximately 50% in the same period [75]. The works were performed by
the same research group, but none of the papers reported to the other. A possible explanation
for this difference can be related with chitosan characteristics, for instance concerning
deacetylation degree or molecular weight, which were not completely reported.
In a different approach, these chitosan/insulin nanoparticles (79% association efficiency)
were dispersed in a W/O microemulsion (oleic acid and surfactant mixture) in order to reduce
proteolytic degradation and improve absorption. The size of nanoparticles dispersed in the
oily-based system (oleic acid and surfactant mixture) was around 100 nm, in contrast with the
2000 nm obtained upon dispersion in the absence of surfactant, demonstrating the importance
of its presence. In vitro assays in simulated gastric fluid containing pepsin clearly
demonstrated that the oily based system protects nanoparticles against proteolyses, allowing
the recovery of 90% unaltered insulin, while complete degradation occurs in insulin solution
and aqueous suspension of insulin/chitosan nanoparticles. Curiously, this was not supported
by in vivo results, which revealed that, whether dispersed or not in the oily system,
nanoparticles led to similar pharmacological responses when administered to diabetic rats
(40% maximum decrease of blood glucose levels 8 h post-administration) [76]. Chitosan
nanoparticles with approximately 150 nm were further produced by emulsion solvent
diffusion, using lecithin and poloxamer 188 as emulsifiers. Cyclosporin-A was encapsulated
with 94% efficiency. Nanoparticles were tested in vivo in beagle dogs and their behaviour
was compared to that of a commercial cyclosporin-A microemulsion (Neoral®). A better
therapeutic response was reported for nanoparticles as compared to the microemulsion, with
higher Cmax and a 1.8-fold increase in AUC0→24, which led to an increase of approximately
73% in relative bioavailability of nanoparticulate form of cyclosporin-A [20].
A different application of chitosan is its use to produce chitosan-gold nanoparticles aimed
at the oral delivery of insulin. The polymer plays a dual role, acting as reducing agent of
chloroauric acid (HAuCl4) to produce gold nanoparticles and as enhancing agent to in vivo
uptake. Nanoparticles were prepared using several chitosan concentrations (0.01% - 1%) and
displayed sizes between 10 nm and 100 nm. Nanoparticles associated insulin efficiently
(53%) and their oral delivery to diabetic rats led to 30% reduction of blood glucose levels, in
contrast with no hypoglycemic effect upon administration of an insulin solution. The authors
refer that studies on excretion, accumulation and toxicity of nanoparticles after chronic
administration are being conducted, which could reinforce their potential [77].
Adapted with permission from [67].
Figure 6. Ovalbumin-specific IgG titres in serum of Balb/c mice intraduodenally fed with Ovalbumin
solution (OVA), Ovalbumin-loaded chitosan nanoparticles (CS/TTT/OVA), as compared to
intramuscular injection with Ovalbumin solution (OVA (IM)). a) Total IgG titres 14 days after priming.
b) Total IgG titres 42 days after priming (28 days after boosting). ** Ovalbumin-loaded chitosan
nanoparticles induced higher IgG titres than Ovalbumin solution (p<0.01).
As referred previously, one of the most promising applications of chitosan nanoparticles

is oral vaccination. Slütter et al. have studied CS/TPP nanoparticles (300 nm) in order to
understand the mechanisms by which oral administration of antigen-loaded nanoparticles
induces an immune response. To do so, ovalbumin was used as a model antigen, being
associated with 65% efficiency. Studies on Caco-2 cells indicated an absence of significant
toxicity induced by nanoparticles. As demonstrated in Figure 6, oral in vivo administration of
ovalbumin-loaded nanoparticles to mice induced a strong and significant enhancement in IgG
production (over 1000 fold) compared to the intraduodenal immunisation with free
ovalbumin.
The authors concluded that this immune-enhancing effect of chitosan nanoparticles
should not be attributed to their ability to modify tight junctions, but to the M-cell specific
uptake of the particulate system [67]. Several other studies addressed the potential of chitosan
nanoparticles for oral immunisation, all describing nanoparticles production by desolvation
with sodium sulfate. As far as we concern, the first particles produced by this method for oral
vaccination were reported by Le Buanec et al., who associated Tat toxoid, a protein with an
essential role in HIV-1 replication, which also participates in T-cell immunosuppression, with
the objective of producing anti-Tat mucosal immunity. Nanoparticles characterisation was not
reported and only results of in vivo administration were shown, demonstrating the
achievement of both systemic and mucosal immunity, although no control (plain particles
and/or toxoid alone) were used to allow a comparison [78].
Coating chitosan nanoparticles with alginate was reported as an attempt to improve
immune pharmacological response. The used antigen, ovalbumin, was adsorbed to chitosan
nanoparticles with efficacy between 40% and 60%, and alginate coating was expected to
circumvent enzymatic or acid degradation. Nanoparticles had size around 640 nm and a
positive net surface charge, which undergone a complete inversion from +41 mV to -35 mV
upon alginate coating. Release studies in simulated intestinal fluid (pH 6.8) revealed the
capacity of the alginate coating to increase nanoparticles stability, preventing the complete
release of ovalbumin that is observed from uncoated chitosan nanoparticles. However, the
coating was not efficient in avoiding a total loss of nanoparticles integrity in simulated gastric
fluid, with consequent release of all drug-load. It was further demonstrated that ovalbumin
maintains its structural integrity after entrapment in the nanoparticles [79]. In a subsequent
work, the authors studied the release behaviour in a phosphate buffer of pH 7.4, observing
that coated-nanoparticles provided a controlled release of ovalbumin, with approximately
45% released in 60 min and a total of 70% in 16 h. As mentioned before, coated nanoparticles
released almost all associated ovalbumin at pH 1.2. Although this was not discussed by the
authors, it is probably the reason why in vivo experiments comprised a direct administration
of formulations in duodenum, this way avoiding nanoparticles contact with gastric medium.
Cytotoxicity pattern of these nanoparticles was assessed in splenocytes, a sensitive model of
different mucosal immune cells. No evidence of nanoparticles cytotoxicity was found by an
array of tests (MTT, trypan blue and propidium iodide). Coated-nanoparticles were loaded
with FITC-labelled ovalbumin and administered in vivo to rats directly in the duodenum. In
contrast with the observed for a control FITC-ovalbumin solution, coated-nanoparticles were
shown to be internalised by Peyer’s patches, which is a good indication of the possibility of
inducing an immune response. Moreover, this finding has demonstrated that, not only positive
or hydrophobic surfaces, but also negatively-charged hydrophilic particles, can undergo
cellular uptake [80]. Notwithstanding these promising results, it is important to notice that the
contact of nanoparticles with gastric acidic medium was avoided, thus not permitting a real
evaluation of their potential. Nevertheless, the authors used this alginate-coated chitosan
nanoparticulate system to encapsulate and deliver hepatitis B antigen (HBsAg), in order to
obtain local and systemic immunisation after oral vaccination. In addition, an
immunopotentiator (CpG oligodeoxynucleotide – CpG ODN) was also encapsulated (in
nanoparticles without HBsAg) to evaluate its adjuvant capacity. Although the antigen is not
the same used in the previous works, nanoparticles physicochemical properties were assumed
similar. HBsAg and CpG ODN were associated with 86% and 99% efficacy, respectively, this
difference being justified by the known strong interaction of oligodeoxynucleotides with
chitosan. The antigen was shown to maintain its integrity after entrapment in nanoparticles.
Oral administration to mice showed enhanced mucosal and systemic immune responses in the
groups vaccinated with HBsAg-loaded nanoparticles and with a mixture of these
nanoparticles and those containing CpG ODN [81]. Nevertheless, it is important to mention
that an increase of gastric pH was induced prior to mice immunisation by administration of
sodium bicarbonate, in order to avoid nanoparticles disintegration, as predicted by the release
studies described above [80].
In a different approach, Jain et al. associated BSA (95% association efficiency) to
chitosan nanoparticles of approximately 220 nm and performed a subsequent encapsulation of
these in lipidic vesicles (liposomes and niosomes), by a technique of reverse-phase, in order
to overcome the drawback of complete drug release in acidic media. Although the protocol
involves the use of glutaraldehyde as cross-linking agent, which is known for its cytotoxicity
[4], results were very promising. A stability study performed in simulated gastric and
intestinal fluids compared the loss of antigen from unmodified nanoparticles and
nanoparticle-loaded vesicles. It was then demonstrated that, at pH 1.2, vesicular encapsulation
prevented almost 2-fold more the loss of antigen. Nevertheless, in intestinal media the
vesicular encapsulation provided worst results, due to vesicles’ destabilization/dissolution by
pancreatin lipase and bile salts. At pH 7.4, BSA released in vitro according to a sustained
release profile, with 40% of antigen releasing at 10 h and 50% at 24 h. The vesicular system
was administered in vivo to rats eliciting, after 4 weeks, a systemic immune response about
1.5-fold stronger than unmodified nanoparticles. Moreover, after the same period of time, IgA
titers were 2-fold higher upon administration of the vesicular system, thus demonstrating the
potential of this system not only to induce systemic immunisation but also to provide a local
mucosal response, which can be advantageous regarding antigen neutralisation at the first
exposure [82].
It is noticeable the great amount of works using chitosan-based nanoparticles to address
the issue of oral protein administration. This reveals the potential of the polysaccharide as
nanoparticle-formation material and the efforts directed to the design of efficient formulations
that provide improved therapeutic responses.
Buccal Delivery
The buccal route offers a number of advantages for drug administration, which include
the avoidance of hepatic first-pass metabolism and of gastrointestinal degradation, two major
reasons for the poor bioavailability of some compounds when administered orally. Moreover,
there are few proteolytic enzymes as compared to the gastrointestinal environment and the
buccal mucosa is highly vascularised, robust and easily accessible, ensuring that a dosage
form intended to have a systemic effect can be applied directly to the required absorption site
[83]. Other features affecting buccal delivery of drugs have been extensively addressed in
some valuable reviews [84-86]. A more detailed consideration on the advantages and
limitations of buccal delivery may be found in Table 6. As can be seen, several limitations are
still to overcome to achieve successful delivery through this route. In fact, adequate buccal
absorption requires that a drug administered in a solid dosage form must exhibit biphasic
solubility, which means that it is soluble in saliva and in the lipid membranes of the buccal
cavity [87]. On the other hand, a major limitation in developing a buccal drug delivery device
is the relatively low permeability of the buccal mucosa, although this might be altered by
using of penetration enhancers [40]. Due to these issues, buccal administration is often ranked
as less effective when compared to other alternative routes, such as the nasal [88], and these
are probably the reasons why there are only a limited number of papers dealing with the
development of drug delivery systems for drug administration through this route.
Nevertheless, a successful approach for buccal mucosal delivery has been the development of
bioadhesive formulations that permit an intimate and increased contact with the mucosa, thus
facilitating high drug concentration and, consequently, increased bioavailability [40;88]. It is
in this context that chitosan has been mentioned as a promising vehicle for buccal delivery
applications. In fact, chitosan solutions of varying characteristics (different chitosan salts and
molecular weights) were tested in a model cell line of the buccal epithelium (TR146 cells),
evidencing a clear ability to increase the permeability of substances such as mannitol and
fluorescein isothiocyanate labeled dextrans [89]. Curiously, the application of chitosan in the
nanoparticulate form is really scarce for this end and only two works are available in the
literature reporting this specific application [90;91]. From these, only the work from Teijeiro-
Osorio and co-workers reported the delivery of a protein, FITC-BSA, which was used as a
model. This work has focused on the evaluation of chitosan nanoparticles ability to increase
macromolecule permeation across the buccal mucosa, as well as on the assessment of the
cytotoxicity profile. Tested nanoparticles, prepared by ionic gelation of chitosan (CS) with
tripolyphosphate (TPP), were formulated using chitosan of different molecular weights (MW)
and deacetylation degrees (DD). Results revealed that nanoparticles promote the absorption of
the model protein across TR146 cells, demonstrating a clear effect of chitosan DD on the
absorption enhancing effect, with highly deacetylated chitosan showing the highest
enhancement ratio.
Table 6. Potential advantages and disadvantages of buccal administration
Relatively small area available for absorption
Avoids first-pass metabolism
(50 cm2)
Low metabolic activity (compared to oral Low permeability to high molecular weight
route) and hydrophilic compounds
High vascularisation Limited to potent molecules
Short recovery time of mucosa after stress
Variable saliva pH (5.5-7) and flow rate,
or damage
Readily accessible and non-invasive Continuous dilution of drug
High patient compliance, no pain Involuntary swallowing of drug
However, no improvement has been reported on permeation when comparing to a

corresponding chitosan solution (same DD), although nanoparticles demonstrated an
important decrease in toxicity as compared to the solution (8-fold increase in IC50) [91],
which is per se an outstanding achievement. Another study on the mechanism of
macromolecules penetration enhancement induced by chitosan nanoparticles, suggested that
this effect is due to repackaging of the epithelial cells up to the basal membrane and a partial
disarrangement of desmossomes [90].
Regardless of the limited work developed so far on chitosan nanoparticles with
application in buccal delivery of proteins, it is worth to mention that there are at least three
reasons to support the expected success of chitosan nanoparticles in buccal drug delivery.
First, it has been extensively reported that the administration of bioadhesive nanoparticles that
adhere to the epithelia, might improve significantly the residence time and, thus, the
possibility for drug release and absorption [42]; secondly, it is also known that the
nanoparticles and/or their components can promote the permeation of macromolecules across
different mucosae, such as the intestinal, ocular or nasal [27;28;92]; and finally, drug
encapsulation into nanoparticles protects active compounds from degradation [93;94].
Undoubtedly, taking into account the potential of the buccal route, research and
development of new nanotechnologies aimed at the successful buccal delivery of high
molecular weight molecules, such as protein-based drugs, encompasses an exciting challenge
for coming years.
Nasal Delivery
So far, nasal delivery has been the most successful of the alternative mucosal routes of
administration (which exclude the oral route, envisaged as the traditional mucosal route), or
simply the most studied, and there are already some systemically acting drugs available in the
market as nasal formulations, including peptides, such as calcitonin and buserelin [95].
Owing to the relatively high surface area available for absorption and the high
vascularisation, nasal administration enables rapid absorption and, therefore, a rapid onset of
therapeutic action [96-98].
Table 7. Potential advantages and disadvantages of nasal administration
Large interspecies variation, leading to
Readily accessible and non-invasive
difficult extrapolations of results
Relatively large surface area available for Drug diffusion limited by the mucus barrier
drug absorption (160 cm2) and mucociliary clearance
High vascularisation Administered volume limited to 25-200 μL
Low enzymatic metabolism, compared to Degradation by the nasal mucosa and secretion
other routes (i.e. oral) enzymes
Avoidance of first-pass metabolism Molecular weight cut-off of ~ 1 kDa
Rapid absorption and onset of action Absorption affected by pathological conditions
Fewer side effects Limited to potent molecules
Ease of administration Lack of reproducibility
Nevertheless, although the permeation of macromolecules across the nasal mucosa is often
superior to that across other mucosal routes, the bioavailability of some protein drugs
following intranasal administration is still low due to both drug metabolism and limited
permeability. Several other factors are identified as influencing systemic nasal drug delivery,
which have been already reviewed extensively [96;98;99]. A brief overview of the major
advantages and limitations of nasal administration is displayed in Table 7.
Attempts to overcome the referred bioavailability problems have mainly focused the
inclusion of absorption enhancers in the developed formulations, but these are often reported
to induce mucosal irritation and ciliary stasis at the site of administration [100;101], thus
demanding caution on their utilisation. Chitosan nanoparticles have appeared in this context
as a potential mucoadhesive alternative, demonstrating to act as important peptide absorption
enhancers, being non-toxic at the same time [28;102]. It is worth saying that the increased
interest in the nasal administration route is also a reflex of the amazing possibility of
immunisation, as well as of the potential to reach the brain in a rapid manner [95;103;104].
The use of the nasal cavity to obtain vaccination, especially against respiratory infections, has
been frequently reported and is related to the fact that most of the pathogens invade their
human hosts at the mucosal surfaces. As mentioned before in the section of oral delivery,
mucosal immunisation is advantageous as compared to parenteral vaccination. Indeed, the
great interest of this route regarding vaccine administration appeared as a consequence of the
demonstration that it can provide not only a systemic immune response, but also a local
mucosal one, providing an additional protection barrier [98;100;103]. Besides, the fact that
nasal immunisation does not require needles, which might be potential infection sources,
provides an outstanding possibility of vaccination for sub-developed countries [103]. The
second mentioned approach of nasal delivery is the recently suggested presence of a direct
pathway from the nasal cavity to the central nervous system (CNS), this way enabling the
targeting of drugs directly to the brain via the olfactory neurons [105]. Studies performed
with some drugs, demonstrated that their concentration in the brain is higher after nasal
instillation than after intravenous administration. This would be especially important for
drugs which are not able to cross the blood-brain barrier [106]. Reddy and Venkateswarlu
present an exhaustive review on drug delivery to the brain, where they describe the results of
some in vivo works with drugs which are potential candidates to be delivered to the brain via
the olfactory pathways. Among the studied drugs are peptides like estradiol [106].
The previous comments suggest that nasal delivery is challenging, though promising, and
that successful nasal systemic administration will rely on the capacity of designing
formulations endowed with such properties that enable overcoming distinct barriers. As
previously mentioned, chitosan nanoparticles have been proposed several times to succeed on
this exigent task. Tables 8 and 9 present summarised aspects of the research works described
herein, respectively concerning in vitro and in vivo studies. More specifically, materials
composing the nanoparticles, preparation methods and major findings of the respective
studies are indicated. As already observed in the section of oral delivery, the great majority of
chitosan nanoparticles with an application in nasal delivery is elaborated by techniques
involving ionic interaction, in this case specifically ionic gelation, representing almost 75% of
the published works.
Table 8. Description of in vitro studies performed with chitosan nanoparticles for nasal drug delivery

molecule
CS, TPP Insulin Ionic gelation Characterisation Insulin-loaded CS/TPP nanoparticles have size between 300 and 400 nm and positive [28,102]
Drug release zeta potential of +25 - +40 mV. Insulin is associated up to 97%, with maximum
loading of 55%. Insulin releases completely in few minutes. Nanoparticles are easily
reconstituted without alterations upon freeze-drying using trehalose or sucrose as
cryoprotective agents
CS, CD, Insulin Ionic gelation Characterisation SBE-β-CD produced nanoparticles with slightly smaller size (200-300 nm) than CM- [107]
TPP Cell culture (Calu-3) β-CD (300-400 nm), due to stronger negative charge. Nanoparticles induce reversible
reduction in cells’ TER and are internalised
CS, ALG, Insulin Ionic gelation Characterisation Nanoparticles have size around 300 nm and insulin association efficiency ranges [108]
TPP Drug release between 44-50%, irrespective of ALG MW. Presence of ALG did not modify insulin
Insulin integrity release profile as compared to CS/TPP nanoparticles. Insulin remains stable upon
release
CS, Gold Insulin Reduction of Characterisation Nanoparticles display sizes between 10 and 100 nm and those formed with CS [77]
chloroauric acid concentration > 0.1% (maximum of 1%) did not aggregate for up to 6 months. Insulin
was associated with efficiency of 53%
CS, TPP Tetanus toxoid Ionic gelation Characterisation Nanoparticles of approximately 350 nm associated tetanus toxoid with 55% [116]
NP stability efficiency. The colloids were stable in presence of lysozyme during 2 h
CS, TPP Tetanus toxoid Ionic gelation Characterisation Chitosan MW did not affect nanoparticles size, which was around 350 nm; displaying [117]
Drug release a positive zeta potential (+ 40 mV). Tetanus toxoid association efficiency ranged
between 44-55% and revealed an initial burst released followed by extended delivery
up to 16 days
CS Ovalbumin Desolvation Characterisation Sonication reduced nanoparticles size from 700 nm to the final 300 nm, which [118]
Drug release increased to 400 nm upon antigen association. Ovalbumin was associated with
efficiency of >80% and no release was observed for 3 h (no data after that)
CS HBsAg Desolvation + Characterisation Nanoparticles of 300-600 nm associated efficiently HBsAg (84%). ALG coating [119]
ALG coating attributed a negative surface charge (- 35 mV)
CS, TPP Estradiol Ionic gelation Characterisation Estradiol is associated with 65% efficiency to CS/TPP nanoparticles of 270 nm, which [120]
display positive zeta potential of + 25 mV
ALG: sodium alginate; CD: cyclodextrin; CM-β-CD: carboximethyl-β-cyclodextrin; CS: chitosan; HBsAg: hepatitis B surface antigen, MW: molecular weight;
Na2SO4: sodium sulfate; NP: nanoparticles; SBE-β-CD: sulfobutylether-β-cyclodextrin; TER: transepithelial electrical resistance; TPP: tripolyphosphate.
Table 9. Description of in vivo studies performed with chitosan nanoparticles for nasal drug delivery

molecule
CS, TPP Insulin Ionic gelation Rabbits Insulin-loaded nanoparticles elicit a stronger hypoglycemic response as compared to [28]
chitosan solution containing insulin (40% reduction versus approximately 17%)
CS, CD, TPP Insulin Ionic gelation Rabbits Nanoparticles have the ability to enter the nasal mucosa. Nanoparticles reduce [107]
plasma glucose levels more than 35% 60 minutes after administration, as compared
to the 14% decrease elicited by a control insulin solution
CS, ALG, TPP Insulin Ionic gelation Rabbits CS/ALG/TPP nanoparticles induce 35% decrease in glucose levels 60 minutes after [108]
administration, a response similar to CS/TPP nanoparticles. Alginate MW affects
duration of hypoglycemic effect; ALG 18 kDa eliciting effect prolonged for 5 h
CS, TPP Insulin Ionic gelation Rats CS solution containing insulin induced a stronger hypoglycemic effect (47% - 60% [115]
Sheep reduction) compared to insulin-loaded nanoparticles (27% - 40%). In sheep,
nanoparticles have a bioavailability of 1.3% and CS solution achieved 3.6%, as
compared to SC injection
CS, Gold Insulin Reduction of Diabetic rats Nanoparticles induced a blood glucose reduction of 34% 3 h after administration, as [77]
chloroauric acid compared to a decrease of 12% induced by a chitosan solution containing insulin
CS Tat toxoid Desolvation Mice Nanoparticles have the ability to induce systemic and mucosal immune response [78]
CS, TPP Tetanus toxoid Ionic gelation Mice Nanoparticles induced higher systemic and mucosal responses as compared to the [116]
fluid vaccine. IgA was detected for up to 6 months post-administration
CS, TPP Tetanus toxoid Ionic gelation Mice Chitosan amount and MW do not affect the immune response elicited by [117]
FITC-BSA nanoparticles. The carriers evidence the ability to cross the nasal mucosa
CS Cholera toxin Desolvation Rats IgA titers induced by nanoparticles administration were significantly higher than [118]
Ovalbumin those elicited by fluid vaccine and IgG levels were comparable to those obtained by
intraperitoneal injection
CS HBsAg Desolvation + Mice Nanoparticles added of an immunopotentiator (CpG ODN) induce both systemic [119]
ALG coating and mucosal immune response; while only mucosal response is observed in the
absence of the potentiator
CS, TPP Estradiol Ionic gelation Rats Estradiol concentration in cerebrospinal fluid is significantly higher (more than 2.5- [120]
fold) upon nasal administration as compared to an IV injection
ALG: sodium alginate; CS: chitosan; CpG ODN: synthetic oligodeoxynucleotide; CD: cyclodextrin; FITC-BSA: isothiocyanate fluorescein-labelled bovine serum
albumin; HBsAg: hepatitis B surface antigen, IgA: immunoglobulin A; IgG: immunoglobulin G; IV: intravenous; MW: molecular weight; Na2SO4: sodium
sulfate; SC: subcutaneous; TPP: tripolyphosphate.
Figure 7. Effect of CS/CD nanoparticles (40 μg/cm2) on the TEER of Calu-3 cell monolayers at pH 6.4.
Each point represents the mean ± SD (n = 5). Keys: (O) control HBSS pH 7.4; (Δ) control HBSS pH
6.4; (▲) CS/SBE-β-CD/TPP (4/3/0.25) nanoparticles; (♦) CS/CM-β-CD/TPP (4/4/0.25) nanoparticles;
dotted line (----) represents the start of the reversibility experiment.
The utility of chitosan nanoparticles for nasal protein delivery was first investigated in
rabbits, using insulin as a model compound. These nanoparticles were obtained by ionic
gelation of chitosan with TPP, resulting in sizes around 300-400 nm and achieving insulin
association efficiencies of more than 87%. Following intranasal instillation of insulin-loaded
nanoparticles a reduction of 40% in plasma glucose levels was observed, in contrast with the
lower response induced by the administration of a chitosan solution containing the peptide
(approximately 17% reduction of glucose levels). This revealed for the first time the ability of
chitosan nanoparticles to enhance nasal absorption of macromolecules [28;102] and, at the
same time, suggested different mechanisms of action between chitosan nanoparticles and
chitosan solutions. In this regard, it was proposed that the nanoparticulate form could
intensify the contact of insulin with the absorptive mucosa [28]. Insulin was found to be
released very rapidly (few minutes) from CS/TPP nanoparticles [28] and these demonstrated
the capacity to undergo a freeze-drying process in the presence of cryoprotectants, such as
trehalose and sucrose, without compromising their original characteristics [102].
Insulin has been, in fact, the most tested peptide, with several other works focusing on its
administration through the nasal route. The same group that developed these CS/TPP
nanoparticles, proposed a slight modification of the method to include in the formulation
other carbohydrates, such as cyclodextrins and alginate, thus forming hybrid nanoparticles
[107;108]. The rational of including cyclodextrins was based on the previous knowledge that
these cyclic oligosaccharides also promote nasal absorption of peptides [109;110]; while
alginate provides extra bioadhesiveness to the system [111;112], in addition to that provided
by chitosan. This way, it was developed a single system that includes the favorable properties
of chitosan and cyclodextrins or alginate. Chitosan/cyclodextrin/tripolyphosphate
(CS/CD/TPP) nanoparticles were prepared with either sulfobutylether-β-cyclodextrin (SBE-β-
CD) or carboximethyl-β-cyclodextrin (CM-β-CD) and the first derivative induced the
formation of slightly smaller nanoparticles (200-300 nm versus 300-400 nm), due to its
higher density of negative charges. TPP was included as cross-linking agent. The incubation
of CS/CD/TPP nanoparticles with Calu-3 cells, a cell line representative of the nasal mucosa,
revealed their ability to open tight junctions and, consequently, to decrease TER (Figure 7),
thus providing the means for an absorption promotion. Importantly, it was found that this
effect was completely reversible upon removal of the nanoparticles [107]. This demonstrates
that nanostructured chitosan and cyclodextrin maintain the intrinsic permeation properties of
the individual materials.
The enhancement effect of chitosan on permeation has been attributed to the
reorganisation of actin rings, as well as to the translocation of the ZO-1 and occludin tight
junction proteins from the membrane to the cytoskeleton [113]. In turn, the promoting effect
of cyclodextrins has been related to their capacity to disrupt the epithelial membrane by
extraction of phospholipids and/or opening tight junctions [101;114]. A fluorescent
formulation of CS/CD/TPP nanoparticles was administered in vivo to rabbits and CLSM
viewing demonstrated the nanoparticles ability to enter the nasal mucosa, thus evidencing a
transcellular transport of the nanostructures. Furthermore, intranasal administration of insulin-
loaded nanoparticles revealed a significant decrease of plasma glucose levels of more than
35% 60 minutes after the administration, as compared to the 14% reduction elicited by a
control insulin solution [107]. Interestingly, it was observed that this reduction of glucose
levels was obtained with an amount of chitosan of 0.096 mg/kg [107], while for CS/TPP
nanoparticles described before an amount of 0.16 mg/kg was needed to achieve a similar
hypoglycemic response [102]. The release profile of CS/CD/TPP nanoparticles was not
explored by the authors in this work. However, an eventual difference in the pattern probably
would not justify the need of a lower amount of chitosan to obtain the same hypoglycemic
effect. Most likely, in CS/CD/TPP nanoparticles, both chitosan and cyclodextrins have a
contribution on the absorption increase, by means of opening tight junctions or any other
mechanism.
Chitosan/alginate/tripolyphosphate (CS/ALG/TPP) nanoparticles were prepared by the
same method, using alginates of different molecular weights (4 to 32 kDa), displaying sizes
around 300 nm. TPP was still used as cross-linking agent. Insulin was associated with
efficacy between 44 and 50%, irrespective of alginate molecular weight. Insulin release
profile was not different from that observed for the original CS/TPP nanoparticles, which
release the majority of the peptide in a few minutes. An analysis of its conformational
stability using circular dichroism demonstrated that the peptide is released in its original
monomeric form, thus keeping its integrity and, presumably, its bioactivity. The intranasal
administration of insulin-loaded nanoparticles to rabbits demonstrated their ability to enhance
the systemic absorption of insulin, with a strong reduction of 35% on the glucose serum
levels being observed at 60 minutes post-administration, similar to that obtained upon
administration of CS/TPP nanoparticles. However, it is remarkable that the formulation
containing alginate of 18 kDa elicited a prolonged hypoglycemic effect for up to 5 h, although
this was not observed for higher molecular weight. The authors speculated that the affinity of
alginate by cellular Ca2+ could be responsible for this prolonged effect, but the possibility of a
slower release in vivo from this formulation was also proposed as an explanation [108].
It is interesting to notice that the three formulations described above (CS/TPP;
CS/CD/TPP; CS/ALG/TPP) elicit similar in vivo responses for the same insulin dose (5
IU/kg), in which refers to the maximum decrease of glucose levels. This indicates that
different composition of carriers was not determinant in their in vivo behaviour, suggesting a
very important role for the presence of chitosan in nanoparticulate form. Although some
aspects of these new hybrid formulations of chitosan nanoparticles need further studies, it
seems obvious that they represent valuable tools for enhancing the transport of complex
molecules, such as proteins, across the nasal barrier.
Considering all the above described studies, which overall describe a very positive effect
of chitosan-based nanoparticles on the absorption enhancement of insulin, it is interesting to
observe the results reported by Dyer et al. in 2002. These authors concluded that the
administration of a chitosan solution with insulin to both rats and sheep, induced a stronger
hypoglycemic response when compared to CS/TPP nanoparticles, prepared by ionic gelation.
In rats, the response was similar upon the administration of either a complex of insulin and
chitosan, or insulin-loaded CS/TPP nanoparticles. In the sheep model, nanoparticles were
reported to have a bioavailability of 1.3% as compared to a subcutaneous injection, while
chitosan solutions achieved 3.6% bioavailability [115]. The literature does not display any
other similar results and so, maybe some kind of formulation discrepancies could explain the
reported differences, although the method reported for the formulation of nanoparticles is
apparently the same.
A different approach on the use of chitosan to produce nanoparticles for insulin nasal
delivery was based on its application as reducing agent for gold nanoparticles. Nanoparticles
were prepared by simply reducing a solution of chloroauric acid (HAuCl4) by heating in a
chitosan solution, displaying sizes between 10 and 100 nm. Chitosan was used in
concentrations varying within 0.01% and 1% (w/v) and those nanoparticles formed with a
concentration higher than 0.1% were found to be stable, with no signs of aggregation for up to
6 months. This is probably related with the particles zeta potential, because when chitosan
concentration is above 0.1% the surface is highly charged (> +50 mV), thus inducing
electrical repulsion. Nanoparticles associated insulin with an efficiency of 53% and their
nasal administration induced a significant reduction in blood glucose levels of diabetic rats
(20% and 34% reduction at 2 and 3 h, respectively), as compared to the administration of a
chitosan solution containing insulin (12% reduction at 3 h). Moreover, serum insulin levels
determined after nasal instillation were comparable to those obtained after subcutaneous
administration of insulin solution [77]. Taking into account the obtained results, there is an
indication that these nanoparticles are a valuable alternative for nasal insulin administration.
A step forward on the evidence of the potential of chitosan nanoparticles as nasal carriers
of macromolecules was given with their application in nasal immunisation. The first report
for this end was made available in 2001, regarding the administration of Tat toxoid, a protein
with relevant role in HIV-1 replication, to mice. Chitosan nanoparticles were produced by
desolvation of chitosan with sodium sulfate and demonstrated the ability to elicit both
systemic and mucosal immune responses [78]. Unfortunately, the authors did not privilege the
characterisation of the obtained drug delivery systems and parameters such as size, zeta
potential and release profile were not addressed. Moreover, a control of the administration of
free toxoid, or other relevant control, was not provided, not allowing a meaningful
comparison of results and a rigorous perception of the effect of nanoparticles.
A study by Vila et al. was truly the first to provide an insight into the ability of chitosan
nanoparticles to grant effective nasal immunisation. Using tetanus toxoid as a model antigen
(55% association efficiency), it was demonstrated that chitosan nanoparticles (chitosan
molecular weight (MW) = 70 kDa) of approximately 350 nm, produced by ionic gelation with
TPP, were able to elicit higher and long-lasting IgG immune responses, as compared to the
fluid vaccine, upon intranasal administration to conscious mice (Figure 8). Similar responses
were obtained for antitetanus IgA titers in saliva, bronchoalveolar and intestinal lavages 6
months post-administration. This improved immune response was attributed to the facilitated
access that chitosan provides to the antigen to the nasal-associated lymphoid tissue (NALT),
as well as to the antigen presenting cells underlying the nasal epithelium.
Nevertheless, although chitosan has proven before to increase the paracellular transport
of macromolecules by transiently opening tight junctions, the authors suggested that other
mechanisms might be responsible for such a positive effect of chitosan nanoparticles. In this
regard, it was proposed that chitosan itself can display an immunostimulatory effect or that
the nanoparticles might cross the nasal mucosa, transporting the antigen and releasing it in a
sustained manner [116]. After these encouraging results, the authors performed another study
to evaluate the effect of chitosan amount and MW on the ability of nanoparticles to elicit an
immune response in mice. Chitosan’s of 23, 38 and 70 kDa were used and tetanus toxoid was
again the model antigen. Irrespective of chitosan MW, nanoparticles maintained a size around
350 nm and association efficiency between 44 and 55%. The antigen was found to release
according to a biphasic profile, showing an initial burst release followed by an extended
delivery up to 16 days. It was demonstrated that neither the amount nor the molecular weight
of chitosan had a significant effect on the IgG or IgA titers. These CS/TPP nanoparticles were
further labelled with FITC-BSA and, using confocal microscopy, it was observed that they
can cross the nasal mucosa. This way, the antigen is carried to some specific body
compartments, from which there is a prolonged delivery of the antigen [117].
Figure 8. IgG antibody levels following intranasal administration of TT entrapped in CS nanoparticles

(70 kDa) or in PBS solution to mice (mean ± SEM, n=6-9).
Nagamoto et al. also reported the efficacy of chitosan nanoparticles for nasal
immunisation of rats, using cholera toxin and ovalbumin as model antigens. Nanoparticles
were produced by desolvation with sodium sulfate and sonication was subsequently applied
to achieve a final particle size of 300 nm. Model antigens were associated by incubation with
previously formed nanoparticles (> 80% encapsulation efficiency) and the final systems
achieved sizes around 400 nm. The authors refer an absence of release for up to 3 h, but no
information is provided after that time, which should help the interpretation of in vivo data.
Rat immunisation showed that nasal administration of nanoparticles induced IgG levels
comparable to those elicited by intraperitoneal injection and IgA titers significantly higher
than those obtained for the fluid vaccines. These nanoparticles also demonstrated to be more
effective in obtaining mucosal immunisation as compared to larger particles (1 and 3 μm),
inducing higher IgA levels. This was justified by the fact that microparticles are retained only
in the M-cells, whereas nanoparticles can also be uptaken from the nasal-associated lymphoid
tissue (NALT), thus inducing not only mucosal, but also systemic immune response [118].
One of the relevant challenges of mucosal delivery is enzymatic degradation. The coating
of chitosan nanoparticles with alginate was proposed in this regard as a way to protect
antigens adsorbed on the surface of the core chitosan nanoparticles. These were produced by
desolvation with sodium sulfate and the coating was simply obtained by incubation of pre-
formed nanoparticles with an alginate solution. Resultant nanoparticles associated efficiently
(84%) recombinant hepatitis B surface antigen (HBsAg). Although the authors refer that these
nanoparticles were characterised in a previous work, that work concerns ovalbumin-loaded
nanoparticles and not HBsAg-loaded nanoparticles and, therefore, a specific characterisation
of the present system has not been provided. Nevertheless, the authors assumed that the
coated systems displayed sizes within 300 and 600 nm. The intranasal administration of
antigen-loaded nanoparticles to mice generated a mucosal immune response but not a
systemic immune response; although the concomitant vaccination with HBsAg-loaded
nanoparticles and an immunopotentiator (CpG oligodeoxynucleotide) induced both type of
responses. Nevertheless, the induced immune responses were not improved in comparison
with those elicited by a PBS solution containing both the antigen and the immunopotentiator
[119]. A previous work of the same group has demonstrated that the presence of the alginate
coating over chitosan nanoparticles prevents a burst release of ovalbumin, as compared to
uncoated particles [79]. Unfortunately, the authors assume the same release behaviour for the
present formulation containing HBsAg, without showing any result on the subject.
Considering that the interaction between entrapped molecules and polymers comprising
particles plays a role in the molecules release profile, it would be of added value the
performance of such an assay to demonstrate whether the behaviour remains the same.
Finally, it is important to mention one work that takes benefit of the direct pathway
between the nasal cavity and the CNS, which proposes chitosan nanoparticles as vehicles of
estradiol to the CNS, for the treatment of Alzheimer’s disease. CS/TPP nanoparticles
prepared by ionic gelation (270 nm) were loaded with estradiol (65% association efficiency)
and subsequently administered to rats via the nasal route. Results showed that, while the
plasma estradiol levels were lower upon nasal administration as compared to an intravenous
injection (IV), the drug concentration in cerebrospinal fluid was significantly higher when
nasal administration was performed (76.4 ± 14.0 ng/mL; Tmax 28 ± 18 min), as compared to
the same IV control (29.5 ± 7.4 ng/mL; Tmax 60 min) (120).
Table 10. Potential advantages and disadvantages of pulmonary administration
Non-invasive route Lungs are not readily accessible
Large alveolar surface area suitable for drug
Airway structure acts as a filter
absorption (100 m2)
Extensive vascularization Mucociliary clearance
Low thickness epithelial barrier Alveolar macrophages
Low proteolytic activity compared to other Absorption affected by pathological
routes conditions
Avoidance of first-pass metabolism and Requires complex devices and particles
gastrointestinal degradation with specific aerodynamic properties
Rapid absorption and onset of action Many factors affecting reproducibility
Possibility of administering lower doses Particles can be exhaled
Considering all the works presented in this section, it is interesting to observe that the
authorship of 50% of those, belongs to the same research group, which demonstrates well the
variety of approaches permitted by chitosan nanoparticles. In light of all the presented results,
it seems adequate to say that the increasing interest in the application of these nanoparticles
for the systemic delivery of protein-based drugs through the nasal route is really justified by
the promising results achieved so far. However, it is also assumed that there is a long road
ahead, especially concerning the improvement of the absorption pattern of the biomolecules.
Pulmonary Delivery
The unique pulmonary features are opening a wide and precious way towards the
systemic delivery of therapeutic macromolecules. The most attractive properties of the lung
include the large surface area available for absorption, the low thickness of the alveolar
epithelium and the high vascularisation, which could lead to rapid absorption and, thus, rapid
onset of action. Moreover, proteolytic activity is low compared to other routes and hepatic
first-pass metabolism is avoided [2;121]. Table 10 depicts the most relevant advantages and
limitations of the pulmonary route.
The pulmonary bioavailability of some drugs is still poor when compared to parenteral
routes, mainly due to enzymatic degradation and clearance processes [2]. The use of
absorption enhancers has been punctually proposed for improving drug absorption, although
only few studies have been performed on their local toxicity following administration.
However, it was already demonstrated that some of these materials, although efficient
pulmonary absorption enhancers (i.e. n-lauryl β-D-maltopyranoside, laureth-9 and sodium
glycocholate), induce lung damage [122;123], indicating the necessity of a very cautious
utilisation, as previously mentioned concerning the nasal administration. The success of
inhaled aerosol particles depends mostly on their aerodynamic properties. In fact, particles
aimed at a systemic absorption, such as is generally desired for proteins, should be small
enough to pass through the mouth, throat and conducting airways into the alveoli, but not too
small that they fail to deposit and are breathed out again [124-126].
Table 11. Description of in vitro/in vivo studies performed with systems developed for lung drug delivery
Associated Drug delivery Preparation Animal /

Materials Major findings Ref.
molecule system method Cell line
Nanoparticles increase IC50 8-fold as compared to
FITC- Calu-3
CS, TPP Nanoparticles Ionic gelation chitosan solution and cross epithelial barrier, entering [91]
BSA cells
intracellular compartment
Incubation with mast cells for 2 h gathers > 90% viability
CS, HA, and enables cell internalisation. Prevention of histamine
Heparin Nanoparticles Ionic gelation Mast cells [141]
TPP release from mast cells is similar for heparin-loaded
nanoparticles and free heparin
Dry powder with adequate aerodynamic diameter (2-3
Microencapsul Ionic gelation
CS, TPP Calu-3cells µm). Spray-drying has no effect on insulin release from
Insulin ated / Spray- [140, 143]
Mannitol A549 cells nanoparticles. System is biocompatible with Calu-3 and
nanoparticles drying
A549 cells and there is evidence of bioadhesion
Intratracheal administration of nanoparticle-loaded
Microencapsul Ionic gelation
CS, TPP mannitol microspheres induces 70% reduction of glucose
Insulin ated / Spray- Rats [29]
Mannitol levels as compared to 40% reduction obtained by
nanoparticles drying
administration of insulin solution
Complete coating of nanoparticles is achieved using both
Microencapsul
CS, TPP, Ionic gelation DPPC and DMPG, providing a controlled release of
ated
DPPC, / lipid film insulin, as compared to uncoated nanoparticles.
Insulin lipid/chitosan - [144, 145]
DMPG hydration/ Microencapsulated complexes have aerodynamic
nanoparticles
Mannitol Spray-drying diameters of 2-3 µm. Microencapsulation does not affect
complexes
insulin release profile
CS: chitosan; DPPC: dipalmitoylphosphatidylcholine; DMPG: dimiristoylphosphatidylglycerol; HA: hyaluronic acid; IC50: 50% inhibitory concentration; TPP:
tripolyphosphate.
In this manner, it has been reported that particles aimed at reaching the deep lung and,
thus, enabling a systemic absorption, require a size in the range of 1-5 µm; although some
authors narrow this range to 2-3 µm [2;127]. For a broad review on the factors affecting the
pulmonary delivery of drugs, please consult Courrier et al., Taylor and Kellaway and Cryan
[2;128;129].
Colloidal carriers like nanoparticles have been identified as having great potential for
systemic lung delivery, because their small size has been reported to enable avoiding the
mucociliary clearance and phagocytic activity [130;131]. Furthermore, nanoparticles made of
diverse polymers, including chitosan, demonstrated to be efficiently uptaken by alveolar
epithelial cells [132-134]. Chitosan nanoparticles represent an added value, considering the
previously mentioned mucoadhesive capacity. Nevertheless, the utility of nanoparticulate
systems for pulmonary application is severely hindered because of low inertia caused by the
excessively small dimensions and mass, which causes them to escape from lung deposition
and to be predominantly exhaled [127;135;136].
The challenge of developing adequate delivery systems is, therefore, a major concern in
lung delivery and an appreciable amount of new particulate systems is appearing as
alternative. In fact, the combination of some delivery systems to form a single complex
system, like the production of dry powders containing colloidal systems, in order to solve
aerodynamic and stability limitations, was recently proposed by several authors [137-140].
Following in this section, several works are reported with the application of chitosan
nanoparticles in systemic lung delivery and Table 11 displays summarised aspects of the
developed formulations, indicating methods and major findings of the respective studies.
CS/TPP nanoparticles prepared by ionic gelation were evaluated in a bronchial epithelial cell
line (Calu-3) by the group that proposed their formation for the first time, with regard to their
effect on cell viability. It was described that the nanoparticulate form of the polymer induced
a cytotoxicity reduction, increasing the IC50 approximately 8-fold as compared to a chitosan
solution. CS/TPP nanoparticles were further labelled with FITC-BSA and confocal
microscopy revealed their ability to enter the intracellular compartment [91]. This behaviour
was previously described by other authors for a fluorescent formulation of the same
nanoparticles (FITC-labelled CS/TPP nanoparticles) in a study with A549 cells, a cell line
representative of the alveolar epithelia. In this case, nanoparticles uptake by A549 cells was
1.8-fold higher than that of chitosan molecules in solution. Moreover, nanoparticles uptake
was found to be concentration and temperature dependent, increasing with the concentration
and being significantly reduced at 4 ºC comparing to 37 ºC. This led the authors to conclude
that nanoparticles internalisation by cells occurs predominantly by adsorptive endocytosis
initiated by non-specific interactions between nanoparticles and cell membranes, and partially
mediated by clathrin-mediated process [132]. The research group that performed the study
with the Calu-3 cells, described above, proposed a new formulation of hybrid chitosan
nanoparticles, in the sequence of those mentioned for nasal delivery, also formed by ionic
gelation but this time composed of chitosan, hyaluronic acid and TPP (cross-linking agent).
Heparin was chosen as drug in a strategy of antiasthmatic therapy and was used both in the
form of unfractionated heparin and low molecular weight heparin. Nanoparticles displayed
sizes within 150 and 220 nm and heparin was associated with efficiency of approximately
70%. Nanoparticles size demonstrated to remain stable in PBS pH 7.4 for at least 24 h and in
water, at 4 ºC, for up to three months. Nanoparticulate systems loaded with unfractionated
heparin presented a much slower release profile (10% release in 12 h) as compared to low
molecular weight heparin (80% release in 12 h), a result attributed to the smaller molecular
weight of the latter referred molecule, which permits faster diffusion from nanoparticles.
These heparin-loaded nanoparticles were incubated with purified mast cells for 2 h, resulting
in cell viability of more than 90%. Moreover, a confocal microscopy study evidenced the
nanoparticles internalisation after this period of interaction with mast cells. Nevertheless, the
ability of heparin-loaded nanoparticles to prevent histamine release from mast cells was
similar to that found for free heparin, both evidencing a dose-dependent response [141].
However, the in vivo performance of the systems will be dependent on a variety of
parameters, including the formulation and physiological conditions, and not only on the
formulation itself. In this manner, considering the well-known characteristics of the two
polymers, chitosan and hyaluronic acid, and of chitosan nanoparticles, these might represent
an improvement on overcoming physiological barriers such as the mucociliary escalator and
enzymatic metabolism. Moreover, the nanoparticulate formulation might provide a controlled
release of the drug in vivo, which cannot be obtained by administration of the free drug.
Nevertheless, the authors did not address the issue of the administration of the developed
nanoparticles through the lung route, an event of major relevance for the therapeutic success
of the proposed formulation.
As mentioned above, achieving effective pulmonary administration is still a major
challenge and the compelling need of designing adequate carriers is evident. With this idea in
mind, in the recent years several new concepts of drug delivery systems have appeared,
including single systems that are in fact the combination of few others all together. It was in
the later 90s when, to our best knowledge, the encapsulation of nanospheres inside
microspheres was first reported, in an attempt to improve the inhalation efficacy of
nanospheres [138], overcoming aerosolisation and stability problems. Grenha et al. adopted
this strategy for the pulmonary administration of insulin-loaded chitosan nanoparticles, for the
first time focusing efforts in the design of a system fully comprised of hydrophilic polymers
and prepared in an entirely aqueous medium. According to the scheme presented in Figure 9,
CS/TPP nanoparticles of approximately 300 nm (Figure 10a), obtained by ionic gelation,
were successfully encapsulated in mannitol microspheres (Figure 10b) using a spray-drying
technique.
A dry powder with optimal properties for lung delivery was obtained, displaying
aerodynamic diameters varying within 2 and 3 μm [140;142]. Nanoparticles formulation
under the form of a dry powder presents the advantage of increased stability in addition to an
easier handling of the nanoparticles. Insulin therapeutic action requests a systemic absorption,
demanding the microspheres to reach the deep lung, where these are expected to dissolve into
nanoparticles, subsequently enabling the peptide release.
The capacity of microspheres to endow this process was demonstrated in vitro, when
CS/TPP nanoparticles were recovered without alterations on size and zeta potential, upon
microspheres contact with an aqueous medium with pH similar to that of the lung lining fluid
(around 7). The microencapsulation process has further evidenced to not affect the insulin
release profile [140]. This combined system has also demonstrated in vitro biocompatibility
with two pulmonary epithelial cell lines, Calu-3 and A549. In addition, although cell
internalisation of nanoparticles was not observed upon 2 h incubation, bioadhesion was
clearly evident [143], certainly as a result of chitosan presence, thus opening the possibility of
increased residence time and, consequently, increased absorption of the encapsulated
molecule. In fact, the in vivo intratracheal administration of this combined system to rats
revealed a significantly stronger hypoglycemic response (70% reduction of basal glucose) as

compared to that obtained upon administration of an insulin solution of the same dosis (40%
reduction of basal glucose) [29].
Figure 9. Schematic representation of chitosan nanoparticles microencapsulation by the technique of

spray drying.
Figure 10. Microphotographs of a) CS/TPP nanoparticles and b) mannitol microspheres containing

CS/TPP nanoparticles, obtained by transmision electron microscopy and scanning electron microscopy,
respectively.
In the sequence of the design of these microencapsulated chitosan nanoparticles, a novel

chitosan-based delivery system for pulmonary administration was proposed by the same
authors. This comprised a combination of three different systems, consisting in coating
chitosan nanoparticles with a phospholipid layer to obtain lipid/chitosan nanoparticles
complexes, which were then microencapsulated in mannitol microspheres. Different
combinations of lung endogenous phospholipids, namely dipalmitoylphosphatidylcholine
(DPPC) and dimiristoylphosphatidylglycerol (DMPG), were used and the complexes were
prepared by hydration of the lipid film with a suspension of CS/TPP nanoparticles. The
combination of zeta potential measurements with the results of X-ray photoelectron
spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS),
demonstrated that a complete coating of the nanoparticles could be achieved using both
phospholipids [144]. In this case, the lipid coating provided a controlled release of insulin, as
compared to the pattern evidenced by uncoated CS/TPP nanoparticles. Microencapsulated
lipid/chitosan nanoparticles complexes also displayed adequate properties for lung systemic
delivery and the microencapsulation process did not have any effect on insulin release profile
[145]. Considering the apparent promise of this drug delivery system, a real demonstration of
its potential is expected through in vivo studies.
Taking into account the exposed potentialities of the lung for systemic delivery of
proteins and the well-known beneficial characteristics of chitosan nanoparticles, it is
surprising to observe the reduced number of works in the subject. That is most probably a
result of the challenge that represents the pulmonary administration of nanoparticles. It is also
noticeable that the totality of works on chitosan nanoparticles for lung administration of
proteins has been performed by the same research group.
CONCLUSION
Over the years, chitosan has been demonstrating to be a very interesting material for drug
delivery purposes, owing to important properties such as very low toxicity, biocompatibility,
permeation enhancement capacity and relatively low production costs. Chitosan nanoparticles
can be obtained by simple and mild methods, with special emphasis on techniques involving
ionic interaction, and have been showing to be prospective drug delivery carriers for a variety
of drugs, including protein-based molecules. Furthermore, as variable types of chitosan are
available, flexible nanoparticle formulations can be easily obtained. All together, the
promising properties of chitosan and the advantages granted by the reduced size of
nanoparticles, which can inclusively permit overcoming epithelial barriers, encompass
relevant issues to achieve the challenging goal of improving bioavailability. The approaches
presented in this chapter demonstrate that a rational modification of chitosan nanoparticles
composition and structure increases the prospects of their usefulness for mucosal protein
delivery and immunisation. Oral and nasal routes are currently the most frequently envisaged,
although pulmonary and buccal display valuable characteristics that provide the basis for
successful systemic protein delivery. In all cases, encouraging results were obtained both in
vitro and in vivo. Nevertheless, chitosan-based nanoparticles need to provide the final proof-
of-concept through clinical trials.
ACKNOWLEDGMENTS
The authors acknowledge funding from the Portuguese Foundation for Science and
Technology, through the project PTDC/SAU-FCF/100291/2008 and from IBB/CBME, LA,
FEDER/POCI 2010.
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Chapter 8
CHITOSAN: A POTENTIAL BIO-POLYMER

FOR DRUG DELIVERY
Sanjay K Jain∗, Piush Khare, Arvind Gulbake

and Satish Shilpi
Department of Pharmaceutical Sciences,
Dr. Harisingh Gour University, Sagar (M.P.) India
ABSTRACT
Chitosan is a natural biopolymer derived from chitin found in the cell walls of fungi,
molds, yeasts and exoskeletons of crustaceans, mollusks, crabs and shrimps etc. Chitosan
is generally partially deacetylated polymer of N-acetylglucosamine which is second most
abundant polymer on earth after cellulose. Chitosan is biocompatible, biodegradable,
non-toxic and mucoadhesive polymer which has attracted the attention of the scientists
and researchers in the pharmaceutical and biomedical fields. Chitosan has many
properties which make it a suitable candidate to be utilized in various applications.
Besides being biosafe it possesses antimicrobial activity where it is effective against the
gram negative bacteria and also possesses antifungal activities. It has wound healing
property. It also finds application in the cosmetics and biomedical arena. Major impetus
is being laid in the field of pharmaceuticals where the polymer is being utilized for
formulation development and novel drug delivery research. The polymer has been
deployed in the formulation of the extended release tablets and various sustained release
matrix tablets which have been utilized for the treatment of gastrointestinal related
ailments such as amoebiasis. The same has been used to formulate the biofilms which
have been developed for wound dressings and in manifestations such as mucositis.
Chitosan has been engineered to transform into novel drug delivery systems such as
nanoparticles, microspheres, gastrointestinal patches, buccal patches etc. through a
number of reported methods. There are a variety of published reports where the chitosan
microspheres have been utilized for colonic delivery and colon associated malfunctions.
The same have also been used for other gastrointestinal related manifestations such as in
∗ Lead and corresponding author: Sanjay K Jain, Department of Pharmaceutical Sciences, Dr. H. S. Gour
University, Sagar (M.P.) INDIA, 470003. Email: drskjainin@yahoo.com.
414 Sanjay K Jain, Piush Khare, Arvind Gulbake et al.
H. pylori infection, duodenal ulcers etc. Moreover, these have been utilized for targeted
drug delivery for the cure of a particular diseased organ. In continuation to this the
nanoparticles have been utilized for the delivery of the antigen and protein i.e. in vaccine
delivery wherein the immunoadjuvant property of the polymer has been harnessed to
increase the potential of the vaccine since chitosan also improves the transfection
efficiency and hence the absorption of drug or protein is enhanced. The gastrointestinal
patches and other delivery systems have also been developed against a variety of
malfunctions. This chapter is directed towards the elucidation of the use and development
of the chitosan in the drug delivery field and includes a detailed discussion of the same
alongwith a focus on the future prospects of the polymer in drug delivery.
INTRODUCTION
Drug delivery is a fascinating field of pharmaceutical sciences wherein different
strategies of treatment against number of ailments are developed. There are a number of drug
carrier systems and at the same time variety of routes for the administration of drug into the
body. The carrier systems can be broadly categorized into conventional systems viz. tablets,
capsules, ointments, syrups etc and Novel carrier systems viz. nanoparticles, microspheres,
niosomes, microemulsions, Gastrointestinal patches, liposomes etc. All of the systems utilize
a variety additives and constituents apart from the drug. Recently, major impetus is being laid
on different types of the substances which are otherwise known as polymers because of a
number of advantages that they lend alongwith the flexibility of being engineered and
developed into carrier system according to ones need. Scientists linked with the medical and
pharmaceutical field have carried out enormous research on the polymers to harness their
potential in the field of drug delivery and drug carrier systems. The research seems to open
new vistas in the field of drug delivery which is being subsequently utilized for the betterment
of the therapy. Although, there is variety of polymers which are being deployed in the drug
delivery applications but main concern is being given to the polymers which are natural,
biosafe and biodegradable. Chitosan is one of these polymers. A comprehensive discussion
about the role of chitosan in drug delivery is being presented in this chapter.
Chemistry: Chitosan (2-amino-2-deoxy-D-glucose) is a polysaccharide and polycationic
polymer which is derived from chitin (β linked N-acetyl- D-glucosamine) after its partial N-
deacetylation and hydrolysis, primarily from crustacean and insect shells. It consists of
repeating units of glucosamine and N-acetyl-glucosamine, the proportions of which determine
the degree of deacetylation of the polymer. Chitosan has primary and secondary hydroxyl
group along with free amino group. Chemical structure of chitosan and Chitin is shown in
Figure (1). Chitin is in fact the second most abundant polymer on this earth after cellulose
(Muzarrelli 1977).
Sources: It is found naturally in the shells of crustaceans, such as crab, shrimp and
lobster, as well as in the exoskeleton of marine zoo-plankton, including coral and jelly fish.
Insects, such as butterflies and ladybugs, have chitin in their wings. And the cell walls of
yeast, mushrooms and other fungi also contain this natural substance. Chitin may be obtained
from arthropods/ exoskeletons of crustaceans such as crabs, shrimps, lobsters, fungi cell wall
and insect shell (scorpions, spiders, cockroaches and silk worms). Chitin was first found in
mushrooms in 1811 by Professor Henri Braconnot while he was Professor of Natural History
and Director of the Botanical Gardens at the Academy of Sciences in Nancy, France. In the
Chitosan: A Potential Bio-Polymer for Drug Delivery 415
1830's, it was isolated in insects and named chitin. Professor C. Rouget discovered chitosan in
1859, and over the next century, much fundamental research took place on these compounds.
An intense interest in new applications grew in the 1930s and early 1940s, as evidenced by
almost 50 patents; however, the lack of adequate manufacturing facilities and competition
from synthetic polymers hampered commercial development. Renewed interest in the 1970s
was encouraged by the need to better utilize shellfish shells. Scientists worldwide began to
chronicle the more distinct properties of chitin and its derivatives and understand the potential
of these natural polymers.
Properties: Chitosan possess varieyty of physicochemical properties. It has a pKa of
approximately 6.5. Chitosan is insoluble at neutral pH but is soluble and positively charged at
acidic pH (Singla and Chawla 2001; Hejazi and Amiji 2003a). Number of protonatable amine
groups alongwith the degree of deacetylation fundamentally determines the polymer
properties including solubility, hydrophobicity, and its ability to interact electrostatically with
polyanions (Kiang, Wen, et al., 2004; Huang et al., 2005). The solubility of chitosan in
neutral and basic pH can be improved by quaternization which forms trimethyl chitosan
derivatives (Van Der Merwe et al., 2004). The molecular weight of chitosan is also of
fundamental importance and exerts its effect on the properties of the chitosan. Generally,
chitosans having lower molecular weights and lower degrees of deacetylation exhibit greater
solubility and faster degradation than their high-molecular-weight counterparts (Zhang and
Neau 2001, 2002; Köping-Höggård et al., 2004; Mao et al., 2004; Ren et al., 2005).
Figure 1. Chemical structure of Chitin and Chitosan.

Figure 2. Derivatives of Chitosan used for Pharmaceutical applications.
On the other hand positively charged chitosan will bind to cell membranes and is reported
to decrease the trans-epithelial electrical resistance (TEER) of cell monolayers as well as to
increase paracellular permeability (Artursson et al., 1994; Dodane et al., 1999). Chitosan
solutions have been shown to increase trans and para-cellular permeability in a reversible,
dose-dependent manner that also depends on the molecular weight and degree of
deacetylation of the chitosan (Schipper et al., 1996). The mechanism of action, which appears
to be mediated by the positive charges on the chitosan, includes interactions with the tight
junction proteins occludin and ZO-1, redistribution of F-actin, and slight destabilization of the
plasma membrane (Dodane et al., 1999; Fang et al., 2001; Thanou et al., 2001a). Thus, the
ability of chitosan to enhance permeation is influenced by the pH of the environment. As
mentioned above, trimethyl chitosan derivatives are soluble at higher pH than unmodified
chitosan. For example, a trimethyl derivative with 61.2% quaternization was able to decrease
TEER of Caco-2 cells and increase mannitol permeability at pH 7.4, unlike unmodified
chitosan hydrochloride or 12.3% quaternized trimethyl chitosan (Kotzé et al., 1999). Chitosan
is also mucoadhesive (Deacon et al., 2000). Mucus is a blend of molecules including salts,
lysozyme, and mucins, which are highly hydrated glycoproteins primarily responsible for the
viscoelastic properties of mucus. Sialic acid residues on mucin have a pKa of 2.6, making
them negatively charged at physiological pH (Deacon et al., 2000; Wang et al., 2000).
Therefore, the presence of mucus affects free drug permeability as well as the uptake of
particulates by forming both a physical barrier to diffusion as well as by interacting
electrostatically with cationic molecules, such as chitosan. Derivatives of chitosan such as
trimethyl chitosan retain their mucoadhesive properties, although to a lesser extent than
unmodified chitosan (Snyman et al.., 2003). In addition, formation of chitosan into micro-
and nano-particles also preserves mucoadhesion (Behrens et al.., 2002; Kockisch et al.., 2003;
Dhawan et al.., 2004). Chitosan is generally considered nontoxic and biodegradable, with an
oral LD50 in mice of over 16 g/kg (Hirano 1996). Antimicrobial, antifungal, and wound-
healing properties have also been reported (Singla and Chawla 2001). The safety of chitosan,
its ability to prolong residence time in the gastrointestinal tract through mucoadhesion, and its
ability to enhance absorption by increasing cellular permeability have all been major factors
contributing to its widespread evaluation as a component of oral dosage forms.
Chitosan has been modified in variety of forms in accordance with the type of their use or
purpose to be served. Various chitosan deriavatives are given in Figure 2.
Advantages of the Chitosan
• Chitosan is obtained from natural source and is second most abundant polymer on
this earth after celluloe as mentioned above. Hence, it is readily available.
• It is cheap and hence large quantities can be utilized and availed.
• It can be modified in different forms according to need.
• It is natural and non toxic.
• It is biosafe, biocompatible and at the same time it is also non immunogenic
(Mattioli-Belmonte et al.., 1997, Shigemasa and Minami 1995).
• It is biodegradable (Kean and Thanou 2009).
• It increases paracellular permeability across mucosal epithelia and acts as absorption
and permeation enhancer (Thanou et al.., 2001b, Madsen and Peppas 1999).
APPLICATIONS OF CHITOSAN AS A DRUG DELIVERY POLYMER

Chitosan has been modified and evaluated to serve different purposes in a variety of
fields. Various applications of chitosan include:
• As carrier system for the delivery of the therapeutic agents/drugs (Dini et al..,
2003);
• As antibacterial and antimicrobial (Jumaa et al.., 2002, Helander et al.., 2001,
Kim et al.., 2003);
• Used as wound healer and in orthopaedics (Muzzareli and Biagini 1993,
Muzzareli RAA 1997, Vila et al.., 2002 Khan et al.., 2000);
• Utilized in surgical sutures;
• In ophthalmology (Felt et al.., 2000);
• It is use in nutritional field: possesses cholesterol lowering effects and weight
loss effects due to fibrous nature (International commission on Natural Health
products 1995);
• In cosmetics;
• In the formulation of drug delivery systems such as microspheres, nanoparticles,
GIT patches, coated tablets, etc.
As evident from the above discussion chitosan has many applications and possess
potential to be utilized in different roles. On the other hand it is also extensively used in the
drug delivery. In last decade, chitosan has been established as polymer of choice to be
formulated as a drug carrier system because of the already mentioned properties and
advantages. The polymer has been maneuvered and engineered to evolve into array of drug
delivery systems ranging from nanoparticles to matrix tablets on one hand and microspheres
to gastrointestinal patches on the other hand. The delivery systems which deploy chitosan can
briefly be categorized into conventional viz. matrix tablets, capsules and novel drug delivery
systems viz. microspheres, nanoparticles etc. Both the category utilizes chitosan in one or the
other form for a definite purpose. A brief account of chitosan regarding its use is being given
below wherein chitosan as a constituent of diverse drug delivery system is discussed.
Conventional Drug Delivery Systems
Conventional Drug delivery include those delivery systems which have been utilized for
the treatment since the advent of the pharmaceutical sciences or the formulation science
where the prime aim is to release the drug after it is administered into the body. These include
tablets, capsules, pills, ointments, suspensions etc. The chitosan has been deployed in some of
these dosage forms with the aim of adding advancement to the existing system. Some of these
use and attributes are being discussed below:
Tablets: Chitosan has been used in different ways as matrix constituent of the tablets or in
film coating of the tablets. As already discussed that the polymer is mucoadhesive and
possess swelling properties. The viscosity imparting and adhsive nature of the polymer has
been utilized to serve as the binding agent in the tablet formulation. The solution of chitosan
of definite viscosity can be used as binding agent for the preparation of granules for tableting.
Chitosan possess swelling properties and can imbibe lot of water which make it suitable
candidate to be used as disintegrant. Since the disintergrant follow the same path i.e. they
imbibe water in gastric mileu and swell, thereby increasing the volume resulting in the
explosion of the tablet into smaller aggregates releasing drug. The polymer could be utilized
in the bioadhesive systems for prolonged drug release wherein mucoadhesive buccal tablets
can be formulated alongwith the gastroretentive systems for the delivery of the therapeutics to
the gastrointestinal tract. Major drawback of the ordinary tablet can be outlined as altogether
release of the medicament in GIT irrespective of the existence of the absorption window and
extent of the absorption of a particular drug. This drawback can be counteracted by the design
of a bioadhesive system which can release the drug at a controlled rate so that the therapeutic
concentration can be maintained in the body. Since it has also been evaluated as a vehicle for
sustained release of the drugs (Nigalaye et al.., 1990, Adusumili and Bolton 1990) and hence
bioadhesive tablets can be designed for the delivery of medicaments which possess limited
absorption, wherein the tablets will stick to the GIT lining for longer period of time and
simultaneously will release the drugs in controlled way. The polymer is a polysaccharide and
hence the cross linked polymeric composites can also be utilized for colon specific drug
delivery where the polymer will be degraded by the polysaccharidases of the colon and the
drug will be released in the colon. Depending upon the use of these properties the polymer
has been deployed in various forms for different uses discussed ahead. Chitosan has been
used as diluent and as disintegrant also in the preparation of tablets alongwith other diluents
like lactose, microcrystalline cellulose, potato starch etc. (Sawayanagi et al.., 1982). Chitosan
granules with internal cavities and laminated chitosan granules with coating of chitosan for
delivery of prednisolone (Machida et al.., 1989). It was found that these buoyant granules
retained more in stomach than ordinary tablets and resulted in higher drug plasma levels for
longer period of time. Knapczyk (1992) has developed antimycotic buccal and vaginal tablets
utilising chitosan as an auxillary substance for direct tableting. These tablets contained a
mixture of deacetylated chitosans as a slow disintegrating agent which retained their
properties during storage while Ritthidej et al.., (1994) used chitosan as a disintegrating agent
in paracetamol tablets. Chitosan has also been assessed for its role as binder (Upadrashta
et.al. 1992). Nunthanid et al.., (2004) utilised chitosan acetate as a binder for the tablets and
assessed its properties. They suggested that the simple incorporation of spray-dried chitosan
acetate as a tablet binder could exhibit sustained drug release. An oral mucoadhesive tablet
containing diltiazem were developed using chitosan and sodium alginate as diluents
(Miyazaki et al.., 1995). Tominaga et al.., (1998) have prepared a colon specific dosage form
bearing acetaminophen using a double coating system where the core which is composed of
acetaminophen, was first coated with chitosan and thencoated with phytin, a gastric acid
resistant material. The outer coated layer protected the core from the acidic environment in
the stomach which dissolved in the small intestine. The inner coating layer protected the core
in the small intestine and then biodegraded in the colon where the drug was released. In
another study Phaechamud et al.., (2000) utilised chitosan (5% w/w chitosan in citric acid
solution) for film coating of the lactose tablets containing propanolol as drug. It was found
that no color migration appeared and the film coated onto the core tablet was smooth,
homogeneous and well attached to the surface of the tablet. Raghavan et al.., (2002) utilized
chitosan alongwith locust bean gum in different ratios to coat the tablets loaded with
mesalazine, for colon targeted delivery. It was found that the tablets surpassed stomach and
small intestine and released the drug in colonic mileu. Recently, Amrutkar and Gattani (2009)
utilized chitosan alongwith chondroitin sulfate as a diluent in the formulation of the tablets of
indomethacin for colonic delivery. The study confirmed that selective delivery of
indomethacin to the colon can be achieved using cross-linked Chitosan and Chondroitin
sulfate polysaccharides. In another work, chitosan alongwith chondroitin sulfate has been
used for colon specific coating of the tablets containing budesonide. It was observed that
sulfate groups of chondriotin sulfate and amine groups of chitosan interacted to form
interpolymer complex resulting in colon specific coating (Kaur et al.., 2009).
Capsule: Chitosan in a capsule form has been studied for colonic delivery. Muranishi’s
group coated chitosan capsules to deliver 5-ASA (Odoriba et al.., 1997) and insulin (Tozaki
et al.., 1997) to the colonic region using a rat model. These capsules appeared to be stable in
the stomach and in the small intestine; however, they were degraded by microorganisms in
the cecal contents of the colon. The results suggested that colon-specific drug delivery could
be achieved through the use of the chitosan capsules.
Table 1. Chitosan hydrogels in cancer treatments
Cancer type Formulation Drug Reference

Azide-chitosan- lactose (Az- Obara et al..,
Lung cancer Paclitaxel
CH-LA) 2005
Chitosan/β- Ruel-Gariepy et
Breast cancer Paclitaxel
glycerophosphate (C/GP) al.., 2004
Chitosan/β- Berrada et al..,
RIF-1 fibrosarcoma Camptothecin
glycerophosphate (C/GP) 2005
Doxorubicin and
Chitosan/β-
Cervical cancer Vaccinia virus-based Han et al.., 2008
glycerophosphate (C/GP)
vaccine
Mucin-production Chitosan/Glyceryl Jauhari et al..,
Paclitaxel
associated cancers monooleate (C/GMO) 2006
Novel Delivery Systems
Chitosan has been extensively investigated in the formulation of the novel drug delivery
systems. These systems are novel in the sense that these are more advanced than the
conventional dosage forms with respect to drug delivery. These are controlled drug delivery
systems which deliver drug in a controlled way with uniform drug release pattern. Moreover,
these can be targeted to a specific site surpassing the other organs of the body and thus
eventually lower down the side effects alongwith the maintenance of therapeutic
concentration in the body for a longer period of time. These include microspheres,
nanoparticles, gastrointestinal patches, niosomes etc. Some of the delivery systems
formulated with chitosan are being discussed below:
Hydrogels: The cross-linked polymeric networks that have a high number of hydrophilic
groups or domains that can absorb large amount of water or biological fluid and dramatically
increase the volume are known as Hydrogels (Kost and Langer 1987; Graham 1990). The
preparation of hydrogel basically depends on the cross linking of polymers which is either
physical (e.g., ionic interaction, crystallization) or chemical (e.g., chemical reaction, radical
polymerization, enzyme induced) (Hamidi et al.., 2008). Chitosan is one of the most suitable
polymers for the hydrogel due to its biocompatible, biodegradable and hydrophilic nature
alongwith functional amino groups and a net cationic charge. Chitosan hydrogls have been
used for the delivery of macromolecular compounds, such as peptides, proteins, antigens,
oligonucleotides, and genes. The chitosan-based hydrogels have also been used for cancer
therapeutics, subcutaneous release, and oral delivery. Chitosan hydrogel has shown strong
potential for use as a new tissue adhesive in surgical applications and wound dressing (Lu et
al.., 2010). The hydrogel membrane maintained moist environment over the wound bed for
enhancing reepithelialization and also exhibited barrier function, as it was impermeable to
bacteria but permeable to oxygen. These studies on skin revealed that the hydrogel membrane
have neither cytotoxicity nor effect on cell proliferation (Lu et al.., 2010). The pH-responsive
hydrogel beads based on chitosan and sodium alginate were prepared using ionotropic
gelation reaction and have been subsequently characterized for the controlled release study of
protein drugs in the small intestine (El-Sherbiny et al.., 2010). Injectable thermosetting
chitosan hydrogels are attractive carrier systems for drug delivery and tissue engineering to
form in situ gel-like implants (Schuetz et al.., 2008). The chitosan based hydrogels have been
extensively studied by many researchers and established as a potential chemotherapeutic
delivery system. Some of the examples have been tabulated in Table 1.
Chitosan Nanoparticles
Nanoparticles are defined as particulate dispersions or solid particles with a size range of
10-1000nm. The drug is dissolved, entrapped, encapsulated or attached to a nanoparticle
matrix depending upon the method of preparation. Nanocapsules are systems in which the
drug is confined to a cavity surrounded by a unique polymer membrane, while nanospheres
are matrix systems in which the drug is physically and uniformly dispersed. In recent years,
biodegradable polymeric nanoparticles have been used as potential drug delivery devices
because of their ability to circulate for a prolonged period of time. They are also used to target
a particular organ, as carriers of DNA in gene therapy, and due to their ability to deliver
proteins, peptides and genes (Langer 2000, Kommareddy et al.., 2004, Lee and Kim, 2005).
The major goals in designing nanoparticles as a delivery system include control particle size,
surface properties and site specific release of pharmacologically active agents to achieve
maximum therapeutic effect of the drug with minimum side effects. Polymeric nanoparticles
possess advantages with respect to the stability of drugs/proteins and possess useful
controlled release properties (Vila et al.., 2002, Mu and Feng, 2003).
The advantages of using nanoparticles as a drug delivery system include the following:
1. Particle size and surface characteristics of nanoparticles can be easily manipulated to

achieve both passive and active drug targeting;
2. Controlled and sustained release can be attained thereby targeting specific site which
results in increased drug therapeutic efficacy and reduced side effects;
3. Wide range of matrix constituents for choice of reserachers. High drug loading
without any chemical reaction;
4. Site-specific targeting can be achieved by attaching targeting ligands to surface of
particles or use of magnetic guidance;
5. The system can be used for various routes of administration including oral, nasal,
parenteral, intra-ocular etc;
Nanoparticles as a matter of fact also possess pros and cones. For example, their small
size and large surface area can lead to particle-particle aggregation which makes its handling
difficult in liquid and dry forms. In addition, small particles size and large surface area readily
result burst release. Biodegradable nanoparticles are frequently used to improve the
therapeutic value of various water soluble/insoluble medicinal drugs and bioactive molecules
by improving bioavailability, solubility and retention time (Shenoy et al.., 2005). These
nanoparticle–drug formulations reduce the patient expenses, and risks of toxicity as well as it
increases drug efficacy, specificity, tolerability and therapeutic index of entrapped/
encapsulated drugs (Safra et al.. 2000, Schroeder et al.., 1998, Kreutera et al.., 1997, Fassas et
al.., 2003, Jean-Christophe et al.., 1996).
For the cellular internalization of the nanoparticles, surface charge is important in

determining the cluster of the nanoparticles in blood flow or adherence to, or interaction with
oppositely charged cells membrane. Cationic surface charge is desirable as it promotes
interaction of the nanoparticles with the cells and hence increases the rate and extent of
internalization (Feng 2004).
There are many biodegradable polymers available which can be utilized to formulate
their nanopaticulate formulation for drug delivery, in which some polymer are water insoluble
and some are water insoluble. Chitosan is one of the water soluble polymers which possess
cell adhesion and has potential uptake properties due to their positive charge. Chitosan
particles in this respect are most favorable due to their attraction to negatively charged cell
membranes. Due to these properties of the chitosan, it has a valuable role in treatment of solid
tumors. Careful design of Chitosan based delivery systems with respect to target and route of
administration may solve some of the problems faced by new classes of active molecules.
Most of the reported methods are frequently used for the synthesis of biodegradable
nanomedicines. Polymeric nanoparticles have been prepared using various methods according
to needs of its application and type of drugs to be encapsulated. These nanoparticles are
extensively used for the nanoencapsulation of various useful bioactive molecules and
medicinal drugs to develop nanomedicine. Biodegradable polymeric nanoparticles are highly
preferred because they show promise in drug delivery system. Such nanoparticles provide
controlled/sustained release property, subcellular size and biocompatibility with tissue and
cells (Panyam et al.., 2003) Apart from this, these nanomedicines are stable in blood, non-
toxic, nonthrombogenic, nonimmunogenic, noninflammatory, do not activate neutrophils,
biodegradable, avoid reticuloendothelial system and applicable to various molecules such as
drugs, proteins, peptides, or nucleic acids (Rieux, et al.., 2006).The various modes of drug
loading into various nanoparticles are represented in Figure 3. The drug molecules either
bound to surface as nanosphere or encapsulated inside as nanocapsules.
Figure 3. Mode of drug entrapment in nanoparticles.

Preparation of Chitosan Nanoparticles
Chitosan as already mentioned is a natural carbohydrate copolymer of N-acetyl-d-

glucosamine and d-glucosamine units and is one of the polysaccharides most suitable as
carriers for a large number of active compounds. Amino groups arranged along chitosan
backbones allow solubility in dilute organic acid solutions, ionic cross-linking, and chemical
modification of the biopolymer to form gels, beads, films, particles, etc. In addition to its
biodegradability, biocompatibility and non-toxicity, chitosan has received much attention in
the development of micro and nanoencapsulation systems (Ko et al.., 2002, Xu et al.., 2003,
Desai and Park 2006, Kean and Thanou, 2009). In addition, chitosan is known to have various
biological activities including antitumor activities, immune-enhancing effects, antifungal and
antimicrobial activities, the unique cationic character of chitosan nanoparticles could provide
higher affinity with negatively charged biological membranes and site-specific targeting
(Sudarshan et al.., 1992, Tsai et al.., 1999). The nanoparticles of chitosan are prepared using
ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte
complex methods (Tiyaboonchai et al.., 2003)
Ionotropic gelation: Much research has been focused on the preparation of nanoparticles
using biodegradable hydrophilic polymers such as chitosan, gelatin and sodium alginate.
Calvo and co-workers developed a method for preparing hydrophilic chitosan nanoparticles
using ionic gelation technique (Janes et al.., 2001; Pan et al.., 2002). Ionotropic gelation is
based on electrostatic interaction between amine groups of chitosan and negatively charge
groups of polyanion such as tripolyphosphate (Calvo et al.., 1997, Pan et al.., 2002, Janes et
al.., 2001). The method involves a mixture of two aqueous phases, of which one is the
polymer chitosan, a di-block co-polymer ethylene oxide or propylene oxide (PEO-PPO) and
the other is a polyanion sodium tripolyphosphate. In this method, positively charged amino
group of chitosan interacts with negative charged tripolyphosphate to form coacervates with a
size in the range of nanometer. Coacervates are formed as a result of electrostatic interaction
between two aqueous phases, whereas, ionic gelation involves the material undergoing
transition from liquid to gel due to ionic interaction conditions at room temperature.
Polyanion was then added and nanoparticles were spontaneously formed under mechanical
stirring (Bodmeier et al.., 1989). The size and surface charge of particles can be modified by
varying the ratio of chitosan and stabilizer (Calvo et al.., 1997).
Microemulsion Method
Chitosan NP prepared by microemulsion technique was first developed by Maitra et al..,

(1999). This technique is based on formation of chitosan NP in the aqueous core of reverse
micellar droplets and subsequently cross-linked through glutaraldehyde. In this method, a
surfactant is dissolved in N-hexane and chitosan in acetic acid. Then chitosan solution and
glutaraldehyde are added to surfactant/hexane mixture under continuous stirring at room
temperature. Nanoparticles are formed in the presence of surfactant. The system is stirred
overnight to complete the cross-linking process, which the free amine group of chitosan
conjugates with glutaraldehyde. The organic solvent is then removed by evaporation under
low pressure. The obtained cross-linked chitosan nanoparticles contain excess surfactant. The
excess surfactant is then removed by precipitate with CaCl2 and then the precipitant is
removed by centrifugation. The final nanoparticles suspension is dialyzed before

lyophilyzation. This technique offers a narrow size distribution of less than 100 nm and the
particle size can be controlled by varying the amount of glutaraldehyde that alters the degree
of cross-linking. Nevertheless, some disadvantages exist such as the use of organic solvent,
time-consuming process, and complexity in the washing step.
Reverse microemulsion method followed by ionic gelation The ultrafine chitosan particles
are prepared by using reverse microemulsion, in which Triton X-100 is used as surfactant,
octanol as co-surfactant, and cyclohexane as oil phase. Ultrafine chitosan particles are first
prepared by ionic gelation. The preformed microemulsion is then added drop-wise into the
chitosan suspension till pH reached 10 followed by sonication and stirring to precipitate
chitosan particles (Maitra et al.., 1999).The resulting chitosan suspension is further modified
by cross-linking with glutaraldehyde and formaldehyde. To extract the ultrafine chitosan
particles from reverse micelles and purify them from unreacted materials, the ultrafine
particles suspensions are centrifuged at 5000 rpm for 30 min and the precipitate is then
washed with acetone and ethanol for two or three times prior to drying under vacuum for 12 h
to obtain ultrafine chitosan particles.
Emulsification Solvent Diffusion Method
El-Shabouri et al.., (2002) reported an emulsion solvent diffusion method for the
preparation of chitosan nanoparticles, which was originally developed by Niwa et al.., 1993
employing PLGA. This method is based on the partial miscibility of an organic solvent with
water. An o/w emulsion is obtained upon injection an organic phase into chitosan solution
containing a stabilizing agent (i.e. poloxamer) under mechanical stirring, follow by high
pressure homogenization. The emulsion is then diluted with a large amount of water to
overcome organic solvent miscibility in water. Polymer precipitation occurs as a result of the
diffusion of organic solvent into water, leading to the formation of nanoparticles. This method
is suitable for hydrophobic drug and showed high percentage of drug entrapment.
Polyelectrolyte Complex (PEC)
Polyelectrolyte complex or self assemble polyelectrolyte is a term to describe complexes

formed by self-assembly of the cationic charged polymer and plasmid DNA. Mechanism of
PEC formation involves charge neutralization between cationic polymer and DNA having
anionic charge leading to a fall in hydrophilicity as the polyelectrolyte component self
assembly. Several cationic polymers (i.e. gelatin, polyethylenimine) also possess this
property. Generally, this technique offers simple and mild preparation method without
involving harsh conditions. The nanoparticles suddenly formed after addition of DNA
solution into chitosan dissolved in acetic acid solution, under mechanical stirring at room
temperature The complexes size can be varied from 50 nm to 700 nm. (Erbacher, et al..,
(1998).
Preparation of Microspheres: Microspheres are matrix type controlled drug delivery
systems (Ravi Kumar 2000) which can be targeted to a specific site and as well as formulated
in different ways. Recently, microspheres of chitosan have been prepared on large scale for
drug delivery. Microspheres of chitosan can be formulated through various methods

depending upon the properties of the therapeutic agent to be encapsulated and other attributes
of the system to be designed. Sinha et al.., (2004) discussed the potential of chitosan
microspheres as delivery system. There are many methods for the preparation of
microspheres, some of them are described here.
Coacervation: In this method the polymer is solubilized to form a solution followed by
addition of a solute, which results in the precipitation of insoluble polymer derivative. This
process also prevents the use of harsh and toxic chemicals. Berthold et al.., 1996 prepared
prednisolone sodium phosphate loaded chitosan microspheres using sodium sulphate as a
precipitant.
Emulsification method: Thanoo et al.., (1991) reported the preparation of the chitosan
micropsheres using emulsification method. A W/O emulsion was formed by dropping
aqueous chitosan solution in the non –aqueous phase with constant stirring which resulted in
the formation of emulsion and the globules were crosslinked on the surface by the addition of
the crosslinker with eventual hardening and formation of the microspheres. The microspheres
formed are filtered, washed with suitable solvents and dried. The same method has also been
utilized by other researchers (Jameela et al.., 1998; Denkbas et al.., 1999). Different types of
crosslinking agents have been utilized which include glutaraldehyde, tripolyphosphate (Shu
and Zhu 2001), genipin (Mi et al.., 2002a). On the other hand thermal crosslinking has also
been used for the preparation of the microspheres whereby application of heat is utilized (El-
Shafy et al.., 2000). Recently, we utilized the emulsification method for the preparation of
chitosan microspheres where the effect of the viscosity of oil phase on the preparation of the
microspheres was elucidated. It was found that lowering of the viscosity results in the
formation of smaller spherical and free flowing microspheres (Khare and Jain 2009).
Multiple Emulsion method: The method involves formation of (o/w) primary emulsion
(non-aqueous drug solution in chitosan solution) and then addition of primary emulsion to
external oily phase to form o/w/o emulsion followed by either addition of glutaraldehyde
(crosslinking agent) and evaporation of organic solvent (Pavanetto et al.., 1996). Genistein
chitosan microspheres were also prepared by o/w/o multiple emulsion method (Wu and Li
2002).
Solvent evaporation method: In this method the polymer solution is dispersed in a
immiscible solvent to form an emulsion and the polymer solvent is evaporated under stirring
to result in microspheres. Bogataj et al.., (2000) have prepared microsphere by solvent
evaporation method using liquid paraffin and acetone. The drug solution was dispersed in
chitosan solution and this mixture was emulsified in liquid paraffin and stirred resulting in
microspheres.
Spray Drying: This method involves spraying of the polymeric solution and air drying
alongwith the addition of the crosslinking agent. He et al.., (1999) have reported preparation
of microspheres by spray drying of multiple emulsion (o/w/o or w/o/w) with drugs cimetidine
and famotidine. Similarly, Filipovic-Grcic et al.., (2003) have also reported the preparation of
microspheres through spray drying. Various classes of the drugs have been entrapped in the
microspheres for different ailments which are summarized in Table 2.
Table 2. Chitosan Microspheres utilized in different types of delivery
S.No Drug Purpose/ Class of Reference

Drug
1 5-Fluorouracil Anti cancer Delivery Li et al.., 1991, Chandy et al..,
2000
2 Cisplatin Anti Cancer delivery Akbuga and Bergisadi 1999
3 Mitoxantrone Antitumor activity Jameela et al.., 1996
4 Methotrexate Antitumor activity Singh and Udupa 1998
5 Indomethacin Anti-inflammatory Aggarwal et al.., 2001
6 Diclofenac sodium Anti-inflammatory Acikgoz et al.., 1995
7 Prednisolone Anti-inflammatory Mooren et al.., 1998
8 Diltiazem HCl Cardiac agent Bayomi et al..,1998
9 Nifedipine Cardiac agent Filipovic-Grcic et al.., 1996
10 Amoxycillin H.pylori infection Shah et al.., 1999
11 Tetracycline Antibiotic delivery Hejazi and Amiji 2003b
12 Sulfadiazine Antibiotic delivery Bodemier et al.., 1989
13 Antigen New castle disease Mi et al.., 1999
14 Insulin Antidiabetic delivery Huang et al.., 2001
15 DNA Gene Delivery Aral et al.., 2000
16 Furosemide Diuretic Akbuga and Durmaz 1994
17 Pentazocine CNS agents Sankar et al.., 2001
18 Metoclopramide HCl GIT agents Ganza-Gonzalez et al.., 1999
19 Cimetidine H2- antagonist He et al.., 1999
20 Acyclovir Ocular delivery Genta et al.., 1997
APPLICATIONS OF CHITOSAN FOR DIFFERENT DRUG/BIOLOGICAL

PRODUCTS DELIVERY
Several drug delivery systems and dosage forms as discussed earlier have been
investigated for different purposes. The administration of these systems follows one or the
other available routes viz. oral, transdermal, nasal, topical, parenteral, ocular, vaginal etc. On
the basis of the delivery systems and the route of administration, different strategies or
approaches have been designed to achieve a specific goal and achieve maximal therapeutic
effectiveness. Some of the routes and the strategies linked with them with reference to
chitosan delivery system are being discussed below:
Oral: Oral route remains to be the most preferred route for the administration of the
therapeutics or drugs (Ponchel and Irache 1998). There exist many plans which utilize oral
route for the accomplishment of the delivery of the therapeutics. These include delivery of the
drugs to buccal cavity, stomach, small intestine, colon, etc. Oral delivery through buccal
cavity encompasses treatment of the local ailments such as mouth ulcers, stomatitis and other
infections of the cavity alongwith the delivery of the therapeutic agents which suffer first pass
metabolism. Drug absorption from the oral cavity has been investigated and reviewed by
Rathbone and Hadgraft (1991). Chitosan is an example of cationic materials that have been
proposed as mucosal-adhesive polymers (Lehr et al.., 1992). Chitosan and sodium

hyaluronate has been utilized as matrix in some of the buccal adhesive matrix tablet
formulations (Takayama et al.., 1990). As discussed earlier Jan Knapczyk (1992) has utilized
chitosan for the preparation of antimycotic Buccal Tablets. The bioadhesive property of
chitosan makes it a suitable candidate for the formulation of buccal delivery systems which
can adhere to the mucosa and release the contents in the buccal cavity for the treatment of the
local infections. Another fascinating property of this very interesting polymer is that it acts as
permeation enhancer and hence increases the absorption of the molecules across the
epithelium. Illum et al.., (1994) reported that the chitosan is able to promote the transmucosal
absorption of small polar molecules alongwith peptides and protein drugs. The mode of action
was attributed to bioadhesion and transient widening of the tight junctions of the membrane.
Moreover it has been found that Chitosan’s effects on the integrity of the epithelium or the
cell membranes are minimal when compared to the effects of known absorption enhancers
(Thanou et al.., 2001b). Following this Artursson et al.., (1994) reported that chitosan can
increase the paracellular permeability across caco-2 intestinal epithelia. The findings clearly
reflect the permeation enhancement nature of chitosan. This very property can be utilized for
the delivery of the poorly soluble macromolecules by encapsulating these molecules in the
chitosan based delivery systems. In this respect a strategy can be proposed which includes the
formulation of GIT patch with a mucoadhesive matrix (chitosan) containing drug covered
from three sides by impermeable membrane and open from one side. This delivery system
when administered through oral route will stick to the GIT tract from the open side and
release its contents in sustained manner for a longer period of time. Such strategy can be
schematically represented as shown in Fig 4. In similar way, the gastro-retentive systems
which stick to the mucosa and release drug, can also be formulated. Apart from this, delivery
of peptides and proteins can be brought about by delivery systems such as microspheres,
nanoparticles to the lower part of GIT where they are intended to be targeted. Drugs which
have absorption window in small intestine or the antigen/ peptides which are to be targeted to
the GALT (gut associated lymphoid tissue) located mainly in the small intestine, can be
administered through these carriers. As already mentioned that chitosan acts as an
immunoadjuvant and hence immune response can be multiplied by the use of chitosan based
delivery system containing antigen/peptide. Since chitosan is also a wound healer and hence
the drug loaded chitosan carrier can be utilized to cure the localised infection wherin there is
marked inflammation in the GALT tissue viz. peyers patches of the small intestine. Such
cases are observed when a pathogen infects and resides in these tissues. There exists a size
dependent uptake of particles at the site of GALT (Florence, 1997) and this can be utilized to
target the particulate carriers to these tissues. Such type of treatment strategy is represented
schematically in Figure 5. This strategy in particular is infact part of our Ph. D (unpublished)
work. As already discussed, this strategy can be utilized for the delivery of vaccines by
targeting the antigen to the GALT tissues for eliciting immune responses. In a similar way,
colon targeted dosage form and carrier system of chitosan can also be formulated for the
delivery of therapeutics to the colon. There are numerous reports and published data on the
use of chitosan in designing of colon targeted delivery systems via oral route. Recently,
tablets for colonic delivery containing crosslinked chitosan and chondroitin sulfate
polysacharides were reported by Amrutkar and Gattani (2009). Following this, Kaur et al..,
(2009) developed tablets coated with chitosan solution for delivery of budenoside. This era
has witnessed design and development of novel drug delivery systems for different delivery
and colon delivery is not an exception to this and is also one of the investigated areas.
Lorenzo-Lamosa et al.., in 1998 has developed microencapsulated chitosan microspheres for
delivery of diclofenac sodium to colon. Brielfy, they prepared the chitosan microspheres by
spray drying and then coated the microspheres with pH sensitive polymer Eudragit S-100 to
prevent the release of the drug in the gastric environment and release drug in colon.Chitosan
based hydrogels have been utilized for delivery of therapeutics to colon wherein the polymer
is cleaved due to the presence of enzymes and caecal contents resulting in drug release (Jain
et al.., 2007). Tozaki et al.., (1997) have reported the use of chitosan based capsules in colon
specific delivery of insulin. It was observed that the efficient delivery of the insulin resulted
since proteolytic activity is lower in colon as compared to the other parts of gastrointestinal
tract. The release of the therapeutics by carrier systems for colon specific drug delivery has
been depicted in figure 6.
Figure 4. Schematic representation of GIT patch.
Figure 5. Schematic representation of strategy to target microparticles to GALT tissue.

Figure 6. Schematic representation of polymer based colon specific microspheres.
Ocular: Chitosan has also been utilized for the delivery of drugs, proteins, genes,
peptides and other biological products to the ocular tissues (Wadhwa et al.., 2009). The
specific biadhesiveness of Chitosan to the ocular surface was first observed in an ex-vivo
study, in which the activity of radiolabelled Chitosan was measured by scintillation counting
(Henriksen et al.., 1996). The polycationic nature of Chitosan interacts with the polyanionic
surface of ocular mucosa through hydrogen bonding /ionic interactions which results in
mucoadhesivity of Chitosan based formulations. Moreover, Chitosan has penetration-
enhancing properties and has attracted a lot of attention as a potential absorption enhancer
across the mucosal epithelia. This fact has been mainly attributed to the opening of the tight
junctions located between epithelial cells, resulting in an enhancement of the absorption via
the paracellular route (Schipper et al.., 1997, Artursson et al.., 1994, Van Der Merwe et al..,
2004). Chitosan solutions also exhibit pseudoplastic and viscoelastic nature (Mucha et al..,
1997, Wang et al.., 1994), which is very essential for ocular drug delivery, since the main
aspect of the delivery in ophthalmic therapy centers around the retention time of the delivery
system in the eye. Sustained and controlled ocular delivery can be achieved with chitosan
based formulations like chitosan gels, inserts, chitosan coated liposome/niosome and chitosan
nanoparticles. Chitosan based carrier systems deployed in ocular delivery have been
summarized in Table 3.
Table 3. Chitosan based Carrier for Ocular delivery
Carrier System Drug Utilization/Methodology References

Inserts Pilocarpine Hybrid polymeric hydrogels were prepared by the Verestiuc et
reaction of acrylic acid-functionalized chitosan with al.., 2006
either N-isopropylacrylamide or 2-hydroxyethyl
methacrylate monomers, and investigated for
controlled release system for ocular drug delivery
Chitosan Timolol Timolol loaded niosomes and coated with chitosan Aggarwal et
coated prolonged the drug release and also help in lowering al.., 2005
Niosomes of Intra Ocular Pressure with minimum side effects
Insert- Ofloxacin Poly (ethylene oxide) inserts showed prolonged the Di Colo et
microspheres release of ofloxacin and enhanced its transcorneal al.., 2002
permeation when Chitosan microparticles prepared
by spray drying were added into it.
Nanoparticles Fluorescent In-vitro studies revealed the stability of chitosan Di Colo et
fluorescent (Cs-fl) nanoparticles upon incubation al.., 2005
with lysozyme and confocal studies showed high
amount of (Cs-fl) NP in corneal tissues as compared
to Cs-fl solutions with 100% cell viability
Nanoparticles Cyclosporin A Modified ionic gelation. In-vitro studies revealed De Campos
the fast release of CyA loaded nanoparticles (293 et al.., 2001
nm) during the first hour followed by a more
gradual released of drug during a 24 hr period.
Chitosan enhanced the therapeutic index of drug
and in-vivo studies observed, high therapeutic
concentration in external ocular tissues after its
topical instillation
Nanoparticles Fluorescein Ionotropic gelation. Nanoparticles formulated by Salamanca et
isothiocyanate ionic gelation technique are well tolerated and safe al.., 2006
- to use in ocular drug delivery. These chitosan
BSA nanoparticles were internalized by IOBANHC cell
cultures via active transport mechanism and showed
high viability
Nanoparticles Gatifloxacin The gatifloxacin nanoparticles were prepared using Motwani et
modified coacervation method and In-vitro studies al.., 2008
showed fast release during the first hr followed by a
more gradual release during a 24 hr period.
Nanoparticles Indomethacin Chitosan prolonged the delivery of indomethacin Badawi et
Nanoemulsions and enhanced its bioavailability in both external and al.., 2008
internal ocular tissues
Nanoparticles Dorzolamide The nanoparticle were prepared using ionic gelation Papadimitrio
method and In-vitro studies showed that u et al.., 2008
dorzolamide was released in a sustained manner in
PBS (pH 7.4) when delivered in ocular tissues for
the treatment of glaucoma
Nanoparticles (GFP,RFP) PCEP and MNP nanoparticles prepared by Prow et al..,
Gene delivery coacervation method are nontoxic and show high 2008
transfection efficiency while MNP yields good
transfection as compared to PCEP.
Carrier System Drug Utilization/Methodology References

Nanoparticles Gatifloxacin Drug was released in a sustained manner and Mishra et
showed good antimicrobial efficiency al.., 2008
Solution Vancomycin The 0.1% and 0.3% chitosan solutions were used Khangtragool
for the ocular drug delivery of vancomycin. et al.., 2009
Microspheres Rokitamycin The spray dried microspheres of new quaternary Rassu et al..,
ammonium chitosan derivatives were prepared and 2009
showed better characteristics (solubility, penetration
enhancement) compared with chitosan itself. The in
vitro release behaviour and good mucoadhesiveness
are making them more suitable for ocular or nasal
administration.
Thermosensitiv Ofloxacin The in situ thermosensitive ofloxacin liposomal Hosny 2009
e liposomal hydrogel ensures steady and prolonged transcorneal
hydrogel permeation, which improves the ocular
bioavailability, minimizes frequency of
administration and ocular side effect of ofloxacin.
Nanoparticles DNA The chitosan-DNA nanoparticles of NOVAFECT Eytan et al..,
(ultrapure chitosan oligomers) were prepared. In 2010
vitro transfection studies show the ability of
nanoparticles to effectively transfect COS-7 cells.
The formulation injected into the stroma showed
increased luciferase gene expression.
Topical: The chitosan and its derivatives have been utilized topically to heal wounds
where it accelerates the granulation (Ueno et al.., 1999). The chitosan also activates
immunocytes and inflammatory cells such as PMN, macrophage, fibroblasts and
angioendothelial cells (DiPietro 1995; Nishimura et al.., 1986; Ueno et al.., 2001).
Terbinafine HCl gel was prepared using chitosan of different molecular weight for the
treatment of fungal infections. In vitro and ex vivo studies suggested the advantages of the
formulations for topical antifungal therapy against Candida species and filamentous fungi
(Özcan 2009). Wang et al.., (2008) prepared cream containing siNPRA chitosan nanoparticles
and imiquimod and performed in vivo study on BALB/c mice (asthma model) which showed
anti-inflammatory effect and it was proposed that it may provide a potential therapeutic
approach for asthma. Saito et al.., (2006) developed a simple chitosan sheet containing
adriamycin and was inserted into the peritoneal cavity of mice which provoded improved
therapeutic efficacy in topical lesions. The chitosan sponges of norfloxacin were prepared by
a solvent evaporation method. The drug release was found to be swelling controlled initially
and diffusion controlled at later period of time where antibacterial activity was directly
proportional to the release rate (Denkbaş et al.., 2004). A novel bilayer chitosan membrane
was prepared by a combined wet/dry phase inversion method consisting of a dense upper
layer (skin layer) and a sponge-like lower layer (sublayer), for a topical delivery of silver
sulfadiazine (AgSD) for the control of wound infections. In vivo antibacterial study
confirmed that dressing is effective for long-term inhibition of the growth of Pseudomonas
aeruginosa and Staphylococcus aureus at an infected wound site (Mi et al.., 2002b).
Transdermal: As evident from various reports discussed previously about the ability of
chitosan and its derivatives could significantly enhance drug absorption across mucosa
epithelia, extends a potential approach for trandermal delivery of drug/ biological products
(Thanou et al.., 2001a; Hamman et al.., 2002; Jonker et al.., 2002; Sinswat and Tengamnuay,
2003; Di Colo et al.., 2004; Giuseppina et al.., 2005). The chitosan interacts with negative
charges of the skin to improve diffusion of therapeutic actives to deeper layers of skin
(Taveira et al.., 2009). Zhou et al.., (2010) evaluated low molecular weight chitosans for
transdermal delivery of baicalin, for atopic dermatitis, viral hepatitis, and HIV infection. They
have also observed enhanced skin permeation in their in vitro permeation studies. He et al..,
(1999) have investigated the mechanism of transdermal permeation enhancer activity of
chitosan and its derivatives and suggested that it could be due to the effects on the secondary
structure of keratin and water content in stratum corneum, cell membrane potential and
fluidity. Transdermal patches of carvedilol containing soyabean extract-chitosan mixture have
been developed which exhibited better performance in controlling hypertension in
deoxycorticosterone acetate-induced hypertensive rats (Sapra et al.., 2009). Kahlig et al..,
(2009) developed an adhesive matrix of chitosan-glycolic acid for a transdermal application
of progesterone exhibiting excellent skin adhesiveness and permeation properties. Permeation
enhancement alongwith bioadhesive nature of chitosan makes it a preferred polymer for the
designing of the transdermal delivery systems.
Nasal: The cationic nature of chitosan has been providing adhesiveness between the
formulation and the biomembranes. Nasal mucosal layer is no exception to this well known
fact. Chitosan-based formulations can greatly improve the absorption of drugs from the nasal
cavity for the treatment of various diseases. Enhancement in paracellular transport through
cell layes (CaCo-2) as well as in animal models, have demonstrated that chitosan exerts an
effect in modifying paracellular transport (Dodane et al.., 1999). The drug/ biological
products delivery directly from nose-to-brain is a potential approach for by-passing the blood
brain barrier (Illum, 2000). Alhalaweh et al.., (2009) have prepared mucoadhesive dry
powders of the zolmitriptan (antimigraine drug), in combination with chitosan, for nasal
administration using spray drying method. The properties and composition of the chitosan
affects the dispersion and release of the drug. Khan et al.., (2009) have prepared formulation
of chitosan with hydroxypropyl beta-cyclodextrin containing buspirone hydrochloride for
bioavailability enhancement and directly transport the drug from nose to brain. The result
showed increased access of buspirone to the blood and brain from intranasal solution
designed with chitosan. Wang et al.., (2008) have prepared estradiol-loaded chitosan
nanoparticles and their in vivo studies on male Wister rats revealed that the drug can be
directly transported from the nasal cavity into the cerebrospinal fluid in rats with an improved
drug delivery to central nervous system (CNS) using chitosan nanoparticles. Jain et al..,
(2007) have prepared multivesicular liposomes coated with chitosan for intranasal delivery of
insulin and performed in vivo studies on Streptozotocin induced diabetic rats. Chitosan coated
formulation showed a better hypoglycemic profile as compared to plain insulin solution.
The nasal route is promising immunization site, and chitosan-based nasal delivery
systems for various antigens have now been developed and studied by some researchers due
to bioadhesion and paracellular transport effects (McNeela et al.., 2000; Bacon et al.., 2000;
Jabbal-Gill et al.., 1998). The influence of N,N,N-trimethylchitosan on the intra nasal
delivery of whole inactivated influenza virus was studied in mice for nasal residence time and
the specific location in the nasal cavity of formulation (Hagenaars et al.., 2010).
Vaginal: The anatomical position, rich blood supply and huge surface area of the vagina
makes it potential route for locally and systemic drug delivery, uterine targeting or even
vaccination (Benzinger and Edelson 1983; Muranishi et al.., 1993; Bourin et al.., 1983). The
vagina might serve as a better route compared to the oral cavity, for the delivery of hormonal
contraceptives owing to the lack of drug interactions observed in the gastrointestinal tract.
Perioli et al.., (2009) have prepared vaginal mucoadhesive tablets using chitosan (FG90C),
polyvinylpyrrolidone (PVPK90) and polycarbophil (PCPAA1). The topical administration of
metronidazole for bacterial vaginosis (BV) shows better mucoadhesiveness and antimicrobial
activity, and representing a good alternative to traditional dosage forms for vaginal
administration. Gels have been prepared using hydroxyethylcellulose, chitosan and 5-
methylpyrrolidinone-chitosan loaded with the antibacterial metronidazole. The 5-
methylpyrrolidinone derivative of chitosan shows improved gel characteristics in terms of
mucoadhesive force, rheological behaviour and drug release, and potential for vaginal
delivery (Perioli et al.., 2008). The properties of chitosan viz. mucoadhesivity, anti bacterial
nature and permeation enhancement make it a promising candidate for the development of the
delivery system acting through vaginal route.
Gene Delivery: Viruses can efficiently transfer genes into cells but concerns such as host
immune response, residual pathogenicity, and potential induction of neoplastic growth
following insertional mutagenesis limit the use of viral vectors in the same regard. This has
led to the exploration of non-viral gene transfer systems (Chong and Vile, 1996; Otto et al..,
1994). These non-viral delivery systems are generally considered to be safer since they are
typically less immunogenic and lack mutational potential. But for successful gene delivery
there are usually five primary barriers that must be overcome by delivery vehicle that are in
vivo stability, cell entry, endosome escape, intracellular trafficking and nuclear entry.
Cationic polymers and lipids both have potential as gene delivery agents because both are
cationic in nature which when interact with DNA produces particles that reduce one or more
of these barriers. Particle formed after interaction of DNA and polymer reduce negative
charge of DNA which result in increased positive charge which may facilitate the binding to
the cell surface and enhanced endocytosis (Boussif et al.., 1995; Labat-Moleur et al.., 1996;
Mislick and Baldeschwieler, 1996). In many cases, cationic polymers seem to produce more
stable complexes thus offering more protection during cellular trafficking than cationic lipids
(Hwang and Davis, 2001; Pollard et al.., 1998). Among cationic polymers such as PEI, poly-
L-lysine and chitosan, PEI and poly- l-lysine are particularly promising as a vector and give
relatively high level of transfection efficiency due to their high charge density. But their
polycationic nature cause marked toxicity, this toxicity has severely limited the use as a gene
delivery vector in vivo.
On the contrary, chitosan is a cationic polymer with significantly lower toxicity than
poly-L-lysine and PEI and it also enhances the transport of drug across cell membrane. The
transfection efficiency showed to be lower than that of other cationic polymer vehicles such
as polyethylenimine (MacLaughlin et al.., 1998) and it is depends on the cell type, serum
concentration, pH and molecular weight of chitosan (Ishii et al.., 2001; Sato et al.., 2001). It
is found the transfection efficiency to be higher at pH 6.9 than that at pH 7.6. Transfection
efficiency meditated by chitosan of high molecular weight, >100 kDa, is less than that of low
molecular weight, 15 and 52 kDa. To increase their transfection efficiency, two approaches
have been developed. First, by increasing chitosan solubility and this can achieved by using
their derivative such as trimethyl chitosan (TMC), quaterinzed chitosan have better solubility
and transfection efficiency than chitosan. It is necessary that the derivatives should be
protonated because only protonated nature of chitosan can cause transient opening of tight
junctions. Another approach is attachment of cell targeting ligands to the chitosan particles.
Park et al.., (2000) developed liver targeted delivery system by preparing galactosylated-
chitosan-graft-dextran DNA complexes, as galactose is known as liver targeting ligand.
Similarly, Mao et al.., (2001) have prepared transferrin-chitosan-DNA nanoparticles as a
targeted drug delivery. However, when KNOB (C-terminal globular domain of fiber protein)
conjugated to the chitosan, the transfection efficiency in Hela cells can be improved by 130
fold. All this indicates towards the use of chitosan as promising non viral vector for gene
delivery which comes with a bonus pack of reduced toxicity, biodegradability and non
immunogenecity.
Table 4. Bioconjugates of Chitosan and their role in therapeutics
Conjugate Delivery system Purpose/ target cell Activity Reference

Chitosan– thermo- sustained-release profiles anti-cancer activity Cho et
doxorubicin responsive and -lung adenocarcinoma al.., 2009
photo-
crosslinkable
hydrogels
N,N,N- gene delivery galactosidase Murata et
trimethyl(TM)- -HepG2 human activity al.., 1997
chitosan/tetragaIa hepatoma cells
ctose antenna
conjugate (TC-
Ga14A20),
chitosan-amino chitosan beads increase the adsorption removal of heavy Ishii et
acid capacity metals (Cu, Ni, Co al.., 1995
and Mn)
chitosan- solution evaluate in vivo antitumor effects Lee et al..,
conjugated antitumor efficacy and 2009
docetaxel subacute toxicity of
docetaxel
- human non-small cell
lung carcinoma
(NCIH358) and
glioblastoma (U87MG)
Eudragit-coated microspheres Gastrointestinal treatment of Oosegi et
chitosan– distribution and inflammatory bowel al.., 2008
prednisolone absorption behavior, disease
conjugate reducing toxicity and
inflammation
cholesterol- epirubicin Synthesis of conjugate Drug loading and Wang et
modified loaded sustained release al.., 2007
chitosan nanoparticles
conjugate
PEG- chitosan Water soluble Solubility enhancement Suitable carrier for Hiroshi et
dispersion of chitosan DNA, protein, al.., 1997
anionic drug
lipase-chitosan Particulate To improve lipase Increase activity of Kwon et
conjugate system activity lipase From simple al.., 2007
lipase (40%) to
chitosan conjugate
(93 %)
Bioconjugates: Chitosan has been found to be used as a support material for gene
delivery, cell culture, and tissue engineering. However, practical use of chitosan has been
mainly confined to the unmodified forms. It has been found that chitosan itself has no
recognizable moiety in respect to cellular surface or biological fluid (Jun-ichi Murata et al..,
1997). The introduction of new moiety or make the conjugate with the chitosan molecule
increases the recognization potential of chitosan. Chemical modification of chitosan is useful
for the association of bioactive molecules to polymer and controlling the drug release profile.
Advance utilization of chitosan, especially in the field of controlled drug delivery, graft
copolymerization onto chitosan can function as a key point in the delivery. The
bioconjugation approaches and strategies may transform into enhanced specificity,
reproducibility, targetabilty and sustained release profile and may be used for the treatment of
different diseases. This introduces desired properties to the therapeutic molecules and
enlarges the field of the potential applications of chitosan by choosing various types of side
chains and subsequently is helpful to conjugate many drugs, bioactive macromolecules such
as antibody, ligonucleotides, interleukins, and interferons, trnsferrin, folic acid etc., enzymes,
glycoproteins, and some polymer such as PEG, poly-l-lysine and related polymers. Some
important examples of such bioconjugates have been presented in Table 4 for a brief
overview.
CONCLUSION
Chitosan possesses a variety of physicochemical properties which contribute it to be a
vast resource with enormous pharmaceutical and medical potential. More than thousand
articles related to chitosan indexed in PubMed in 2008-09 are indicative of the enormous
research that is being carried out on chitosan. Chitosan has found to be biocompatible,
biodegradable, non toxic and mucoadhesive as a result of many studies. Some of the studies
also point towards its anti-microbial and immunoadjuvant property. All these properties either
collletively or alone have been utilized for accomplishment of different goals by many
researchers. Promising results have witnessed the potential of chitosan as a polymer for its
use in the field of drug delivery. The applications and use of chitosan range from its
utilization in tablets to transdermal patches, from formulated gels to gene delivery conjugates
and from oral to ocular delivery. The polymer finds role in conventional delivery system vis a
vis its widespread use in target specific novel and controlled drug delivery systems. The
polymer has also been found to be a wound healer and a permeation enhancer which has
equipped the scientists with an option of its use in enhancing the absorption of the poorly
permeated drugs either through oral or topical route. More fascinating fact about chitosan is
that it lends itself to modification in chemical way which results in variety of derivatives of
chitosan with modified properties according to ones need. This can be utilized for different
purposes. Several strategies have been chalked out and worked upon for delivery of
therapeutics to different sites of the body. Some of them have been discussed in the chapter
schematically. Chitosan has been utilized in almost every facet of drug delivery and several
reported work show huge potential of this polymer in drug delivery. Although there exists a
vast published data on chitosan and lot of work has been done but yet there remains a lot to be
explored, for example use of this polymer in the field of vaccination and vaccine development
can open new vistas in immunology. Different properties the polymer should be combined
with that of the drug for development of effiecient therapeutics with dual line of action. The
immunoadjuvant property of chitosan can be utilized in the vaccine development wherein the
polymer will act along with the antigen/peptide to result in eliciting a marked and increased
immunogenic response. Chitosan has made its presence in the medical, pharmaceutical and
biomedical applications but efforts must be laid and entire scientific fraternity must strive
towards the launch of polymeric products in the market so that mankind as a whole can be
benefitted and this has been the ltimate goal of science from time unknown.
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Chapter 9
CURRENT STATUS OF CHITOSAN

ON DERMAL/TRANSDERMAL DRUG
DELIVERY SYSTEMS
Ipek Ozcan∗, Taner Senyigit, Evren Homan Gokce

and Ozgen Ozer
Ege University, Faculty of Pharmacy,
Department of Pharmaceutical Technology, Izmir, Turkey
ABSTRACT
In case of targeting the drug to the desired part of the skin, vehicles play an
important role, beside the characteristics of the drug. Many natural and synthetic vehicles
have been used for various topical dermal/transdermal preparations. However, chitosan
has been standing out with its many advantages based mainly on its biological and
physicochemical properties. Chitosan is a unique hydrophilic biopolymer obtained by
partial deacetylation of chitin, which is one of the most abundant polysaccharide. It is a
natural product widely found in crustacean shells, fungal cell walls, insect exosceletons,
and mollusks. Chitosan is a linear glycosaminoglycan made up of N-acetyl-D-
glucosamine units.
Characteristics of chitosan, such as the molecular weight, viscosity and the degree of
deacetylation, greatly influence the properties of formulations. The by-products formed
after the biodegradation of the polymer does not cause immune responses making it
biocompatible. Due to the specific cationic glucosamine groups of chitosan, it can be
interacted with anionic proteins in the skin providing the bioadhesive characteristics.
These properties result in improved efficacy, enhanced bioavailability and reduced
toxicity -generally recognized as safe (GRAS). Furthermore, the antimicrobial/
antibacterial and skin hydrating effects of chitosan have been received considerable
attention for dermal/transdermal applications. It plays an important role in the cell
∗
Corresponding Author: Ipek Ozcan. Ege University, Faculty of Pharmacy, Department of Pharmaceutical
Technology, 35100, Bornova, Izmir-Turkey. Tel: +90 232 3884000 / 1368 Fax: +90 232 3885258. E-mail
address: ipek.ozcan@ege.edu.tr.
450 Ipek Ozcan, Taner Senyigit, Evren Homan Gokce et al.
regulation, tissue regeneration and collagen production. Chitosan and some of its
complexes were approved by FDA for use in wound dressing products.
Chitosan also provides the controlled release of numerous active agents used for the
treatment of skin diseases such as corticosteroids, antifungal agents, nonsteroidal anti-
inflammatory drugs, hormones, local anesthetics, antiviral and antiseptic agents, etc.
Regarding to the good bioadhesive property of chitosan and its ability to sustain the
release of the active compounds, it has found many practices in the formulation of gels,
dermal/transdermal patches, sponges, micro- and nanoparticulate systems as drug
carriers. Particularly, chitosan has been used in the preparation of mucoadhesive
formulations, for improving the dissolution rate of the poorly soluble drugs, drug
targeting and enhancement of peptide absorption.
This paper is focused on the use of chitosan for dermal/ transdermal drug delivery
systems following a general overview of chitosan. This natural polymer is a promising
carrier or excipient as a delivery system and remarkable advances have been made about
its potential applications in skin delivery.
INTRODUCTION
Skin is the largest body organ, weighing approximately 5 kg with a surface area of about
two square meters in adult humans. This multilayered organ is an effective barrier protecting
the body from the surrounding environment, thus being an efficient permeation obstacle for
exogenous molecules [1]. When this barrier function is compromised for any reason, it
becomes a port of entry for microorganisms into the body as seen in burns patients and
various dermatological conditions [2].
The skin is composed of the outermost epidermis, and the dermis -vascular structure
containing free nerve endings. The superficial layer of epidermis is the stratum corneum and
it is almost impermeable and responsible for the barrier function of the skin. This highly
hydrophobic layer is composed of differentiated non-nucleated cells, corneocytes, which are
filled with keratins and embedded in the lipid domain. It plays a vital role in the absorption of
drugs. Stratum corneum therefore, provides the rate-limiting step in the penetration process
[3].
The penetration of drugs through the skin involves diffusion via i) transcellular pathways
ii) intercellular pathways iii) hair follicles and sweat glands. The permeation through the
appendages allows diffusional leakage into the epidermis and penetration directly into the
dermis. This is supposedly the fastest route for hydrophilic molecules. However, the major
transport pathway is through the intercellular lipid domains [1, 4].
Dermal (topical) delivery term is used to define a targeting within the skin, which
involves ensuring minimal systemic absorption. This type of drug localization is important in
the treatment of dermatological conditions such as skin cancer, psoriasis, eczema, and
microbial infections, where the seat of the disease is located in the skin [5]. Dermal
administration of drugs is performed for various destinations. Going from outside to inside
the skin, the drug has to survive from the cleaning and protecting properties on the surface.
Keratolytic and moisturizing effects, the stratum corneum and drug interactions with
pharmacological targets localized in the viable epidermis and dermis are limiting factors for
drug delivery. Systemic bioavailability is the aim of transdermal delivery [6].
Current Status of Chitosan on Dermal/Transdermal Drug Delivery Systems 451
Transdermal delivery is a term describing the situation in which a solute diffuses through
the various layers of the skin and reaches the systemic circulation for a therapeutic effect [7,
8]. Transdermal drug delivery offers a number of potential advantages over conventional
methods, such as pills and injections: i) no degradation due to stomach, intestine, or first pass
of the liver, ii) probable improved patient compliance because of a user-friendly method, and
iii) potential for controlled delivery. Nevertheless, very few drugs can be administered
transdermally at therapeutic levels, due to the low permeability and lipophilic nature of
human skin [6, 9].
To target the drug to the desired part of the skin, vehicles play an important role, beside
the characteristics of the drug such as solubility, partition coefficient, particle size, charge and
molecular weight etc. Many natural and synthetic vehicles have been used for various topical
dermal/transdermal preparations. However, chitosan has been standing out with its many
advantages based mainly on its biological and physicochemical properties [10].
Chitosan is a linear glycosaminoglycan made up of N-acetyl-D-glucosamine units
derived from chitin by deacetylation. It is a natural product widely found in crustacean shells
(crab, shrimp, crayfish), fungal cell walls, insect exosceletons, and molluscs. [11-13].
Since chitosan exhibits a variety of physicochemical and biological properties, it has
found numerous applications in various fields such as environmental protection, agriculture,
fabric and textiles, cosmetics, nutritional enhancement, and food processing [14].
In biomedical applications, chitosan and some of its complexes have been employed in
wound dressings, drug delivery systems and space-filling implants [11]. Particularly, chitosan
has been used in the preparation of mucoadhesive formulations [15, 16], for improving the
dissolution rate of the poorly soluble drugs [17, 18], drug targeting [19] and enhancement of
peptide absorption [10, 20, 21].
Chitosan has a very low toxicity and has been of GRAS status (generally recognized as
safe) [15, 22], thus has found many practices in the formulation of gels, dermal/transdermal
patches, micro- and nanoparticulate systems as drug carriers.
The aim of this chapter is to focus on the chitosan based formulations for dermal and
transdermal applications by analyzing the physicochemical and biological properties of this
natural polymer.
PHYSICOCHEMICAL AND BIOLOGICAL PROPERTIES

OF CHITOSAN
Chitosan can be produced in a variety of ways including thermal deacetylation, isolation

from raw material and bioconversion methods [12]. Acetamide group of chitin can be
converted into amino group to give chitosan, which is carried out by treating chitin with
concentrated alkali solution [10]. Because of the presence of functional groups (amine and
hydroxyl) various chemical chitosan derivatives have been synthesized and studied for
different applications such as; thiolated chitosans, mono-N-carboxymethyl chitosan,
quaternary chitosan and partially quaternized chitosan (N-trimethyl chitosan) [23].
Chitin and chitosan represent long-chain polymers having molecular mass up to several
million Daltons [10]. Commercially available chitosan is in a range of molecular weights
between 3800 and 20000 Daltons and has 66-95% degrees of deacetylation with different
types of salts such as glutamate, hydrochloride and lactate [24]. Due to high molecular weight
and a linear unbranched structure, chitosan is an excellent viscosity enhancing agent in an
acidic environment. It behaves as a pseudoplastic material exhibiting a decrease in viscosity
with increasing rates of shear [25].
The viscosity of chitosan solution is affected by its molecular weight, ionic strength, pH
and temperature of solution. Low molecular weight chitosan oligomers provide a low solution
viscosity [26]. The viscosity of chitosan solution is directly proportional to chitosan
concentration and degree of deacetylation but inversely proportional to solution temperature
and pH [25].
Chitosan with a low degree of deacetylation (of about 40%) is soluble up to pH 9.0,
whereas with a high degree (of about 85%) is soluble only up to pH 6.5. Solubility is also
greatly infuenced by the addition of salt to the solution. The higher is the ionic strength, the
lower is the solubility [27].
The potential of chitosan stems from its cationic nature in neutral or basic pH conditions
and high charge density in solution [28]. This polymer is distinct from other commonly
available polysaccharides due to the presence of nitrogen in its molecular structure, its
cationicity, and its capacity to form polyelectrolyte complexes. The cationic nature of the
polymer allows it to become water-soluble after the formation of carboxylate salts, such as
formate, acetate, lactate, malate, citrate, glyoxylate, pyruvate, glycolate, and ascorbate and
show strong adhesive properties [29]. When adhesion is allowed to develop, substantial
amounts of drug is delivered until a limit imposed by the drug partition coefficient [30, 31].
Adhesion to specific sites increases the bioavailability by optimum contact due to the
extended time of residence [32, 33].
The mechanism of adhesion is predominantly explained by primary electrostatic
interaction followed by secondary hydrogen bonding which is highly dependent on the ionic
strength and chitosan possesses -OH and -NH2 groups that can give rise to this confirmation
[16, 34].
The strong mucoadhesive characteristic displayed by chitosan is the electrostatic
interaction between the positively charged amino groups in chitosan and the negatively
charged sialic acid residues present in mucins [28].
Chitosan salts as well as trimethylchitosan are able to enhance the paracellular
permeability of intestinal, nasal and buccal mucosal epithelia by transiently opening the tight
junctions, thereby increasing the paracellular absorption of hydrophilic and macromolecular
drugs. In the last years it has been proven that tight junctions occur also on skin and have
been characterised in the granular cell layer of human epidermis [35, 36]. The positive charge
of chitosan salts and trimethyl chitosan might help them to be used as penetration enhancers
due to the ionic interaction with negatively charged groups of glycocalyx [37, 38].
Chitosan and hydroxypropyl chitosan were found to be enzymatically degraded so that
they can be used for biodegradable controlled-release dosage forms [12]. Chitosan breaks
down slowly to harmless products (amino sugars), which are completely absorbed by the
human body and these by-products do not cause allergic reactions/rejection and exert
moderate immunostimulating effects [39, 40]. This attractive natural polysaccharide shares
the benefits of other natural polymers (lysozomal degradation, etc.), but does not induce an
immune response [29]. It is biocompatible with living tissues. In-vivo toxicity studies show
chitosan to be inert, non-toxic and easily removable from the organism without causing
concurrent side reactions. The LD50 (lethal dose 50%) of chitosan in mice was determined to
be greater than 16 g kg−1 which is close to sugar or salt [10, 25].
Table 1. The summary of chitosan usage in dermal/ transdermal delivery systems
The role of Reference

Aim Formulation Active agent
chitosan number
Berberine [65]
Tiaprofenic acid [66]
Terbinafine HCl [67]
Gels Nonivamide [68]
Propranolol HCl [69]

Clobetasol propionate and
[70]
mometasone furoate
Enhanced drug release Testosteron [75]

and/or
Patches Nifedipine [88]
Skin absorption
Sponges Paracetamol [93]
Microparticles Retinoic acid [133]
Aciclovir [144]
Nanoparticles
Gene-DNA [149]
Cyproterone acetate [156]
17-β-estradiol, progesterone,
[157]
Coated cyproterone acetate and finasteride
Network Liposomes
Aciclovir and minoxidil [164]
Matrix
Carrier system
Lidocaine HCl [176]
Microneedles Calcein and bovine serum albumine [238]
Gels Fibroblast growth factor [74]
Lidocaine hydrochloride [83]

Patches
Etoricoxib [87]
Paclitaxel [89]
Sponges Curcumin [94]
Norfloxacin [95]
Controlled
Diltiazem hydrochloride [110]
and/or
Microparticles
Localized drug delivery Fibroblast growth factor [131]
Artocarpin [132]
Gene-RNA [136]
Clobetasol propionate [137]
Nanoparticles
Retinol [145]
Prolidase [146]
Gene-DNA [150]
Table 1. The summary of chitosan usage in dermal/

transdermal delivery systems (Continued)
The role of Reference

Aim Formulation Active agent
chitosan number
Oligonucleotide [151]
Quinacrine [152]
Coated
Glycolic acid [161]
Liposomes
Superoxide dismutase [183]
Gels No drug incorporated [43]
Fibroblast growth factor [74]

Wound healing
Patches Silver sulfadiazine [85]
and/or
Improved regeneration
Sponges Fibroblast growth factor [96]
and re-epithelization
Microparticles Fucoidan [106]
Ampicillin [130]
Chitosan’s monomeric unit, N-acetylglucosamine, occurs in hyaluronic acid, an

extracelular macromolecule that is important in wound repair [41]. There are two stages of
dermal healing; the inflammatory phase and the new tissue formation phase. During the
inflammatory phase, infiltrating neutrophils aid in the removal of foreign agents in the area.
When the new tissue is formed, fibroplasia begins by the formation of granulation tissue
within the wound space. It was found that chitin and chitosan could accelerate the infiltration
of inflammatory cells, consequently accelerating wound cleaning. Therefore chitosan has
been used as a wound dressing for proliferation and activation of inflammatory cells in
granulation tissue [42, 43].
The N-acetylglucosamine moiety in chitosan is structurally similar to
glycosaminoglycans (GAGs), heparin and chondroitin sulphate beside hyaluronic acid. These
compounds hold the specific interactions with various growth factors, receptors and adhesion
proteins. Therefore, the analogous structure in chitosan aid to exert similar bioactivity and
biocompatibility [44, 45].
Chitosan inhibits the growth of bacteria and this effect is reported to be dependent on the
molecular weight [46]. The variety of chitosan salts markedly inhibited the growth of most
bacteria tested. Their inhibitory effects differed with molecular weight and the particular
bacterium. It was shown that chitosan had stronger bactericidal effects for gram-positive
bacteria than gram-negative bacteria [47].
Not only due to its multitude of applications but due to increasing environmental
awareness, the ecological production of chitosan with its low cost makes this natural polymer
superior to other synthetic compounds [48].
In view of the above-mentioned properties, chitosan is extensively used in developing
drug delivery systems for dermal and transdermal applications. Table 1 represents the
chitosan usage in various dermal/ transdermal delivery systems.
CHITOSAN BASED FORMULATIONS

Gels
Gels are semi-solid dispersions of small or large molecules in an aqueous vehicle with a
gelling agent. In other words, as stated by Peppas [49] they are macromolecular networks
swollen in water or biological fluids. Gel formulations are suitable for topical delivery of
drugs for treatment of diseases involving skin lesions due to lack of irritating components
[50].
The development of hydrogels from a variety of synthetic materials has provided a great
deal of flexibility in engineering the characteristics of the fabricated drug delivery systems.
Polyethylene glycol (PEG), polyvinyl alcohol (PVA) and methacrylate derivatives have all
been used to form hydrogels with variable mechanical strengths and biological responses
[29]. However after many researches, chitosan gels were found to be advantageous and still
preferable in comparison with other gel systems for dermal/transdermal drug delivery due to
the favorable biological aspects. Especially its high capacity to adhere negatively charged
membranes, causes enhanced muco/bioadhesion and improves wound and burn healing with
low toxicity [25, 51, 52].
Hydrogels are often divided into three classes depending on the nature of their network, i)
entangled networks, ii) covalently crosslinked networks and iii) networks formed by
secondary interactions. However, with respect to chitosan hydrogels, this classification is not
entirely suitable. Since the boundaries were not so district between these classes, a modified
classification for chitosan hydrogels was offered as chemical and physical hydrogels.
Chemical hydrogels are formed by irreversible covalent links, as in covalently crosslinked
chitosan hydrogels. On the contrary, physical hydrogels are formed by various reversible
links. These can be ionic interactions as in ionically crosslinked hydrogels and polyelectrolyte
complexes, or secondary interactions as in chitosan/PVA complexed hydrogels, grafted
chitosan hydrogels and entangled hydrogels [29, 49, 53-55].
The easiest way to prepare the chitosan gel is to solubilize chitosan in acidic aqueous
media. The solubility of chitosan is the most crucial point for the preparation of gels.
Chitosan is insoluble at alkaline and neutral pH values but is soluble in acidic media. The
solubility of chitosan in acidic media is enhanced when its degree of deacetylation reaches
50% or more. In addition, the solubility of chitosan in inorganic acids is limited when
compared with its solubility in common organic acids. Chitosan has a low solubility at
physiological pH of 7.4 or higher pH as it is a weak base with pKa values ranging from 6.2 to
7 [56]. This type of chitosan hydrogels are limited by their lack of mechanical strength and
tendency to dissolve. Moreover, they do not exhibit characteristics that allow drug delivery to
be efficiently controlled [48].
Another way for the preparation of chitosan gel is the addition of covalent or ionic
crosslinking agent [29, 48]. In crosslinked hydrogels, polymeric chains are interconnected by
crosslinkers, leading to the formation of a 3D network. Crosslinkers are molecules of
molecular weight much smaller than the molecular weight of the chains between two
consecutive crosslinks. The properties of crosslinked hydrogels depend mainly on their
crosslinking density; the ratio of moles of crosslinking agent to the moles of polymer
repeating units [49].
The preparation of a hydrogel containing covalently crosslinked chitosan requires

minimum amount of chitosan and crosslinker in an appropriate solvent, especially water. To
date, the most common crosslinkers used with chitosan are dialdehydes such as glyoxal and in
particular glutaraldehyde. However, the main disadvantage of these dialdehydes is that they
are generally considered to be toxic [57]. Besides dialdehydes, epichlorohydrin, genipin,
diioscyanate and oxalic acid are alternative crosslinkers. Among these alternatives, genipine
is promising due to being naturally occurring and biocompatible [58]. Covalently crosslinked
hydrogels are the only systems that are characterised by a permanent network, due to their
irreversible chemical links. Therefore, they exhibit good mechanical properties and can
overcome dissolution, even in extreme pH conditions [48].
In order to prepare ionically croslinked chitosan hydrogels, a charged ionic crosslinker
and chitosan dispersed in a solvent (commonly water) are needed. Chitosan hydrogel could be
obtained through treating the polymer with multivalent anions such as glycerol phosphate,
oxalic acid, pyrophosphate, tripolyphosphate, tetrapolyphosphate, octapolyphosphate, and
hexametaphosphate [56]. Ionic crosslinking is an extremely simple and mild procedure. In
contrast to covalent crosslinking, no auxiliary molecules such as catalysts are required [59].
Ionically crosslinked chitosan hydrogels offer more possibilities as drug delivery systems
compared to covalently crosslinked hydrogels. They can be formulated for controlled release
not only in acidic but also in basic media and used for rapid release. However, their main
disadvantages are the possible lack of mechanical stability and the risk of dissolution of the
system, due to highly pH-sensitive swelling [48].
The pH of these systems has a substantial role in the swelling behaviour of chitosan gels.
When pH decreases, the crosslinking decreases due to the reducement in crosslinker’s charge
density and swelling occurs. If the decrement in pH is too much, dissociation of ionic
linkages and dissolution of the network can occur [59], leading to a fast drug release [60].
When pH increases the protonation of chitosan decreases and the crosslinking density
declines thus blocks swelling. The protonation and repulsion of chitosan free ammonium
groups helps swelling. If the pH becomes too high, amino groups of chitosan are neutralised
and ionic crosslinking is inhibited. If the crosslinking density becomes too small, interactions
are no longer strong enough to avoid dissolution and the ionic crosslinker is then released [61,
62].
An effective approach for developing a clinically applicable chitosan is to modify the
surface of the material that has biofunctionality. Blending technologies with various additives
may cause cytotoxicity. Hence, the modified biomedical-grade of chitosan with various
derivatives must be submitted for biocompatibility testing [28, 63].
The technical properties and microbial stability of chitosan-EDTA were compared with
well established gelatinizing agents such as carmellose sodium, hydroxypropylmethyl-
cellulose, polycarbophil and carbopol 980. Chitosan-EDTA gels were shown to have the
advantage of compatability with higher concentration of ethanol leading to excellent swelling
and stability against microorganisms [37, 64].
Many drugs have been incorporated in chitosan gels in terms of dermal and transdermal
drug delivery. The penetration of berberine into and through the rat skin was found in
significant amounts after co-administration with absorption enhancers from chitosan gels
[65]. Tiaprofenic acid loaded chitosan gel was compared to two types of emulsion (w/o and
o/w) and hydrophilic petrolatum based ointment by means of in vitro release from cellophane
membrane. The diffusion coefficient of the drug from chitosan gel was found to be the
highest compared to all other vehicles [66].
In a study of Ozcan et al. [67], terbinafine hydrochloride loaded chitosan gels were
prepared using different types of chitosan at different molecular weight, and the antifungal
inhibitory activity was evaluated and compared to a marketed ointment product of the drug.
Low molecular weight chitosan gel performed significant increament in antifungal activity
caused by higher drug release resulting in larger zone of inhibition among other tested
formulations prepared with medium and high molecular weight chitosan.
In vitro percutaneous absorption and in vivo pharmacodynamic responses of nonivamide
(synthetic capsaicin) incorporated into chitosan, Pluronic F-127, carboxymethylcellulose gels
and cream formulation were compared after topical administration to rats. Chitosan gels
produced the highest nonivamide permeation across and the greatest cumulative amount
trapped in the skin [68]. Propranolol hydrochloride was incorporated into physically cross-
linked chitosan hydrogels with lauric, myristic, palmitic or stearic acid by freeze-drying for
transdermal use [69]. The chitosan hydrogels provided more transcutaneous permeation of
propranolol hydrochloride than the corresponding solution of the drug due to the possible
enhancement of the drug solubility in the skin, probably produced by the interaction of the
polymer with the stratum corneum.
Clobetasol propionate and mometasone furoate, as model drugs of topically applied
corticosteroids, were loaded into medium molecular weight chitosan gels. Skin permeation
was improved in comparison with commercial cream [70].
The release of cationic (lidocaine hydrochloride), anionic (benzoic acid) and neutral
(hydrocortisone) model drug molecules from chitosan gel were studied for electrically-
modulated drug delivery systems. The behavior of gels in an electric field resulted as changes
in their surface pH during electrification and in the electrically modulated release of drug
from the gel formulations. The results indicated that the release of the model drugs from the
gel matrix was ranked in order as benzoic acid > hydrocortisone > lidocaine, which is
consistent with the electrokinetically competing forces that are involved in these gels.
Overall, effective and reliable electrically modulated drug delivery systems could be prepared
using chitosan gels with adequate characterization of electrical effects on formulation
matrices [71].
Taveira et al. [72] investigated the effect of iontophoresis on skin penetration of
doxorubicin from chitosan gels. The results indicated that chitosan appeared to interact with
negative charges in the skin. Hence, chitosan gel was reported to not only reduce
electroosmotic flow, but also improve doxorubicin diffusion throughout the deeper layers of
the skin.
In the area of wound healing, chitosan-based materials have been used in varying
formulations. Chitosan itself can induce faster wound healing and produce smoother scarring
[43, 73]. Chitosan hydrogels had an additional advantage for delivering a therapeutic agent to
the local wound because of the reparative nature of the polymer. When chitosan hydrogels
were tested as a carrier material for controlled release of fibroblast growth factor-2 (FGF-2)
and as a wound healing acceleration property, a significant accelerated wound closure was
observed and shortened healing process in mice [74].
Testosterone transdermal permeation enhancement from N-trimethyl chitosan gels with
different degrees of quaternization were evaluated. Both in vitro and in vivo rabbit skin
permeation studies suggested that transdermal permeation ascended with N-trimethyl chitosan
formulations compared to the testosterone gel without enhancer [75].
Chitosan hydrogels have also been prepared as the carrier systems for a variety of
different shapes, geometries, and formulations that include liquid gels, powders, beads, films,
tablets, capsules, microspheres, microparticles, sponges, nanofibrils, textile fibers, and
inorganic composites [29].
Patches/Films/Membranes
Patches are drug delivery systems intended for skin application in view of achieving local
or systemic effect. In some cases, they might be of hydrocolloid characteristic and this helps
them to serve as an occlusive and adhesive wafer dressing containing gel-forming agents.
Patches provide the administration of effective and known drug amount to the skin [76].
The structure of the membrane depends mainly on the polymer type: such as molecular
weight, deacetylation degree and also the polymer concentration in the film-forming solution
[77, 78].
Lamination and casting techniques are well-known methods for the preparation of patch
formulations and both of them could be implementated to the chitosan patches. Similar to gel
formulations, solubilizing of chitosan in appropriorate solvent is substantial for preparing
patches. Lactic acid is generally preferred to solubilize chitosan because it has been proven to
be non-irritating compared to other alternatives, such as acetic acid. Besides, lactic acid has
the ability to improve the flexibility of the film due to a plasticizing action [79, 80]. Larena et
al. [81] prepared several chitosan films using glycerine as a plasticizer alternatively.
Another important point for the preparation of chitosan patches is the choice of the
chitosan type. In general, low molecular weight chitosan is preferable with greater patch
properties such as being thin, transparent and less permeable to water vapor compared to
patches prepared with high molecular weight chitosan [82].
A transdermal lidocaine hydrochloride chitosan patch was developed for rate control and
a chitosan hydrogel as a drug reservoir. Drug release was found to be slower through chitosan
membranes produced by a high degree of deacetylation with a larger thickness. The
functionality of this transdermal patch was studied on the forearm of human volunteers.
Patches prepared with a 95% degree of deacetylation, prolonged the anesthetic effect [83].
The degree of acetylation also plays a key role on some biological properties of chitosan
gels. Chatelet et al. [84] demonstrated that the higher degree of deacetylation caused lower
cell adhesion and proliferation.
The suitability of chitosan films (prepared with two different solvents: acetic acid and
lactic acid) for wound dressing were investigated and compared with a commercial,
polyurethane membrane supported gel, namely Omiderm®. In addition to physical
characterizations (mechanical properties, in vitro bioadhesive strength and vapour
permeability), the biological evaluations were also performed via primary skin irritation,
intracutaneous and systemic injection tests. It was stated that chitosan films showed
significantly better mechanical and bioadhesive strength properties from Omiderm®. Chitosan
films prepared with lactic acid were found to be softer, more flexible and more bioadhesive
than other tested films and did not cause erythema, edema or systemic toxicity [79]. Chitosan
membrane loaded with silver sulfadiazine (AgSD) showed prolonged antibacterial activity
and decreased potential silver toxicity as a result of the agar plate bacteria-cultures
(Pseudomonas aeruginosa and Staphylococcus aureus) and cell-culture (3T3 fibroblasts)
assays. The new formulation was less cytotoxic than the traditionally used AgSD cream, and
was very effective for long-term inhibition of the growth of bacterium on an infected wound
[85].
A series of chitosan based polyelectrolyte complexes were prepared in order to obtain
films possessing the optimal functional properties (flexibility, resistance, water vapour
transmission rate and bioadhesion) to be applied on the skin by the combination of chitosan
and two polyacrylic acid polymers with different crosslinkers. The optimized film had shown
appropriate properties for skin application and represented a promising formulation for
further incorporation of drugs for topical and transdermal administration [86].
Etoricoxib transdermal patches were prepared using chitosan, its modified derivatives
(with acetaldehyde and propioaldehyde) and chitosan-hydroxy propyl methyl cellulose
(HPMC) blend with glycerol as plasticizer. The drug loaded films were cross-linked with
sodium citrate and investigated for their permeation characteristics across dialysis membrane
and rat skin. It has been observed that diffusion is the dominant mechanism for drug release
following non-Fickian type of diffusion and the film prepared with acethaldehyde modified
chitosan showed sustained release of drug [87].
Propranolol and nifedipin were evaluated for their transdermal delivery potentials using
chitosan as a release controlling membrane. The membranes were permeable to both
lipophilic and hydrophilic drugs. The drug release was significantly reduced when crosslinked
chitosan membranes were used [88].
Drug-loaded chitosan films are emerging as alternative drug delivery systems and films
appear to have potential for local sustained delivery of cancer chemotherapeutic agents such
as paclitaxel [89].
Sponges
Sponges may be defined as dispersions of gas (usually air) in a solid matrix. There has
been considerable recent interest in the use of sponges particularly as matrices for controlled
drug delivery, as wound dressings and as matrices for cell growth within the tissue
engineering field [90].
Sponges based on polysaccharides such as alginate and chitosan have been studied due to
their low toxicity, favourable mechanical properties and capacity for bioresorption of the
constituent materials [25, 91, 92].
As mentioned before the use of chitosan with different polymers is a general approach to
modify the required properties of a formulation. The addition of alginate to chitosan were
shown to accelarate the release of the drug (paracetamol) from the sponge. The use of
chitosan and alginates together, was suggested as a strategy to manipulate both the
mechanical properties and the drug release [93].
A biodegradable sponge, composed of chitosan and sodium alginate was investigated and
it was seen that the gel’s swelling ability was directly proportional to chitosan concentration
in the sponge. The release of curcumin from the sponges have been extended for a period up
to 20 days and could be controlled by the crosslinking degree [94]. In a similar study chitosan
sponges including norfloxacin, the most effective parameter was found to be the degree of
neutralization. It was also observed that the equilibrium swelling ratio decreased with
increasing cross-linking density. The norfloxacin release was found to be swelling controlled
initially and diffusion controlled at the extended release periods. It was also found that the
antibacterial activity was directly proportional to the release rate [95].
Chitosan provided several advantages required for wound dressing. Wang et al. [96]
assessed the biochemical and biophysical improvement of the chitosan crosslinked collagen
sponge containing recombinant human fibroblast growth factor as a new wound dressing for
the diabetic rats. The diabetic wound healing was found to be significantly improved by this
formulation, as compared to collagen sponge alone and growth factor alone.
Particulate Systems
Chitosan has many advantages, particularly for developing micro/nanoparticles. These

include: i) the ability to control the release of active agents, ii) avoiding the use of organic
solvents in the preparation step since it is soluble in aqueous acidic solution, iii) the ability to
crosslinking due to free amine groups, iv) easy ionic-crosslinking property with multivalent
anions since cationic nature and v) increasing residual time at the site of absorption due to its
mucoadhesive character [10].
Microparticles
Since the introduction of microcapsules by Green et al. in the 1950s, the term
microcapsule is defined as a spherical particle with size varying from 50 nm to 2 mm and
containing a core substance. The terms microcapsules and microspheres are often used
synonymously and, microbeads / beads are used alternatively. Spheres and spherical particles
indicates a large size and rigid morphology [73].
Microspheres are composed of artificial or natural polymers that can be modified to
speed up or slow down the degradation of the polymer reservoirs (and, therefore, modify drug
release kinetics) [97].
Chitosan-based particles can be prepared by both chemical and physical methods as
listed; spray drying [98, 99], solvent evaporation [100], precipitation – chemical cross linking
[20, 101], multiple emulsion method [102], thermal cross-linking [103], complex
coacervation [104], ionotropic gelation [105] and wet inversion [23, 62].
Reverse micellar and sieving methods has been suggested as new methods for preparation
of micro- and nanoparticles of chitosan [10]. Supercritical fluid drying has been recently
investigated as an alternative process for producing powder formulations. Critical properties
of microcarriers have to be elaborated after the production by means of size and morphology,
permeability, mechanical integrity, and biocompatibility [11].
Pharmaceutical applications of chitosan in the form of beads, microspheres and
microcapsules were developed in the early of 1990's. Large chitosan microspheres and beads
(up to thousands of microns) have typically been used for the prolonged release of drugs [106,
107] and proteins [20]. Small particle size (<5 μm) chitosan microspheres, containing
anticancer agents such as 5-fluorouracil (5-FU) [104], and magnetic microspheres [108] have
been described for site specific delivery. Cardiac agents [109, 110], anticancer drugs [111-
114], anti-inflammatory drugs [115-120], steroidal drugs [20], antigens [121], insulin [122],
growth factor [123], genes/DNA [26, 124, 125] and antibiotics [126-128] are the most studied
groups for chitosan microspheres.
The strong interaction between chitosan microparticles and mucin was suggested to
increase adsorption in biological surroundings by many studies [16, 100, 129].
The microsphere system based on polyion complexation of fucoidan with chitosan, was
applied to treat dermal burns and the treatment period was shortened by improving
regeneration and re-epithelization [106]. Similarly both of blank and ampicillin-loaded
chitosan microspheres were shown to perform good wound healing properties in vivo [130].
Not only chitosan itself but also chitosan treated gelatin and alginate microspheres has
found many applications in biomedical field. To provide a prolonged, site-specific delivery of
basic fibroblast growth factor (bFGF) to the grafted skin in a convenient manner,
biodegradable chitosan-gelatin microspheres were fabricated and incorporated into a porous
chitosan-gelatin scaffold. The release kinetics of bFGF showed a fast release at the initial
phase in the first 2 days, and the ultimate accumulated release was approximately 71.8% by
day 14, indicating an extended time course for complete release [131]. In another study
artocarpin (Artocarpus incisus extract) were loaded into alginate/chitosan microparticles for
targeted transfollicular delivery. The efficiency of the loaded microparticles was shown to be
comparable to the same dose of applied as solution. Moreover no systemic action was seen
for microparticles as desired in a dermal application [132].
Chitosan treated alginate microparticles were prepared with the purpose of incorporating
all-trans retinoic acid (ATRA) and microparticle characterization, drug–polymer interaction,
release profiles and in vitro skin retention were investigated. The drug content of these
microparticles was reported to be affected by ATRA concentration and by the solvent used
and it was more weakly affected by chitosan concentration. The release of ATRA was also
affected by chitosan concentration. In vitro retention studies indicated that maintenance of
these microparticles in stratum corneum, improved the ATRA concentration in the deeper
skin layers [133].
A transdermal drug delivery system of diltiazem hydrochloride was developed to obtain a
prolonged controlled release with both matrix diffusion controlled and membrane permeation
controlled systems. It was seen that zero order was fitted for the release of drug from
membrane system, whereas non-Fickian pattern was fitted for matrix system. Membrane
controlled delivery sustained the drug release more and exhibited a more steady state plasma
concentration [110].
Nanoparticles
Polymeric nanoparticles are small colloidal particles which are made of non-
biodegradable and biodegradable polymers with a diameter generally around 200 nm. These
structures can enhance dermal uptake or improve tolerability of active substances and allow
drug targeting to the different layers of skin [134, 135]. In the preparation stage, the choice of
the polymeric material is a crucial step for developing pharmaceutical strategy. The
percutaneous delivery of difficult-to-uptake substances might be facilitated by the selection of
an appropriate polymer [134].
From a literature survey, it can be realized that the number of researches on chitosan
nanoparticulate systems containing different active substances have been ascended due to its
many advantages for developing nanoparticles.
Chitosan nanoparticles have been extensively investigated for site-specific drug and gene
delivery systems. Chitosan nanoparticles as topical vehicles, have been proven to prolong the
residence time and to sustain the release of active substance with respect to the skin [136,
137].
Considering the preparation method of chitosan particles, different techniques have been
employed such as emulsion cross-linking [20], coacervation/precipitation [138], spray drying
[139], emulsion-droplet coalesence [140], ionic gelation [141] and reverse micellar methods
[10, 142, 143]. Screening of these methods depends upon several factors such as particle size
requirement, stability of the active agent, stability of the final product and residual toxicity
associated with the final product.
Many studies have been reported to assess the suitability of chitosan nanoparticles for
dermal/transdermal drug delivery. The potential of chitosan-tripolyphosphate nanoparticles
for delivery of aciclovir to skin was evaluated. The chitosan-tripolyphosphate nanoparticles
loaded with aciclovir improved the chemical stability of the drug significantly and the
permeation to skin was ameliorated especially from nanoparticles with higher chitosan
content [144].
Beside the improvement in stability, the solubility of the drugs might also be increased by
chitosan nanoparticles. The solubility of retinol was found to be more than 1600-fold by
encapsulation into chitosan nanoparticles [145].
In case of dermal delivery, the challenge is the control of the drug localization in the
desired layers of skin. Chitosan nanoparticles seem to facilitate the formulators to overcome
this difficulty. Senyigit et al. [137] studied clobetasol propionate loaded lecithin/chitosan
nanoparticles giving special attention to the transport across the skin by comparing the
chitosan gel and commercially available cream of the drug. The accumulation in the
epidermis was achieved without any significant permeation across the skin. In the light of this
finding, a dose damping to 10% was offered for the reduction of the possible side effects of
clobetasol propionate.
The potential toxicity of the applied nanoparticle is another serious matter to be dealt
with for a researcher. Chitosan differs with its strong biocompatible nature among other
polymers. Moreover longer and effective drug delivery can be obtained by these
nanoparticles. Prolidase loaded chitosan nanoparticles suggested for prolidase deficiency
(PD) were evaluated in terms of growth and the viability of cultured skin fibroblasts in order
to verify the compatibility of the chitosan nanoparticles with cells. The results indicated good
biocompatibility and further ex vivo experiments showed that prolidase loaded chitosan
nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period
of time up to 8 days [146]. In another study the immunogenicity of antigen containing N-
trimethyl chitosan nanoparticles were proved to induce dendritic cell maturation and to
enhance immune responses [147].
Chitosan nanoparticles are shown to be good candidates for the delivery of DNA to skin.
The topical application of chitosan-based nanoparticles containing plasmid DNA to the mice
skin resulted in detectable levels of luciferase expression in skin after 24 h, and significant
antigen-specific IgG titer expressed β-galactosidase 28 days after the first application [148].
Likewise, chitosan and poly-γ-glutamic nanoparticles for transdermal delivery were able to
effectively retain the encapsulated DNA and also protect it from nuclease degradation.
Nanoencapsulation improved penetration depth into the mouse skin and enhanced gene
expression [149].
A multifunctional core-shell polymeric nanoparticles system composed of hydrophobic

poly (D, L-lactic-co-glycolicacid) core and a positively-charged glycol chitosan shell were
developed for transdermal DNA delivery and epidermal langerhans cells tracking. The core of
the nanoparticles was used to load fluorescent quantum dots for ultrasensitive detection of
langerhans cell migration following transdermal delivery. The results of animal studies
indicated that bombardment of nanoparticles transfected DNA directly into langerhans cells
present in the epidermis; the transfected langerhans cells then migrated and expressed the
encoded gene products in the skin draining lymph nodes. This developed nanoparticle system
would help to monitor and fine-tune important functional aspects of the immune system and
thus offered as a potential for use in immunotherapy and vaccine development [150].
Ozbas-Turan et al. [151] prepared antisense oligonucleotide loaded chitosan
nanoparticles and applied topically to rats for measurement of beta-galactosidase expression.
The results indicated that free antisense oligonucleotides exhibited 35% of inhibition while
nanoencapsulated antisense oligonucleotides inhibited approximately 82-85% of beta-
galactosidase expression.
Liposomes
Liposomes are made from natural biodegradable and non-toxic phospholipid molecules
which can entrap or bind different kinds of drugs into or on the lipid membrane [152, 153].
Numerous studies report that the application of liposomes on the skin surface is able to
improve permeability for various entrapped drugs through the major barrier the stratum
corneum [154-157]. As a result of liposome encapsulation, the drug was accumulated in the
epidermis and dermis, therefore the systemic drug concentration was generally lower than
that produced by the control preparations (ointment, cream, gel or lotion) [158]. Drug transfer
mechanism from the liposomes is probably due to the similarity in the composition of the
vesicle bilayer and the skin lipids, thus fusion of vesicles in the intercellular space of the skin
occurs [159-161].
Liquid liposomes can be applied directly to skin. Generally it is impossible to incorporate
liposomes in creams because of interactions between the surface active agents and the
liposomal layers [162]. Due to the formation of a highly hydrated network of the hydrophilic
polymers, liposomes are immobilized within the gel network and thus mechanically stabilized
[163]. Hydrophilic polymers like chitosan are suitable thickening agents for topical
application of liposomes in gel form. However, the type and concentration of the polymer,
which forms the gel matrix, could influence the stability as well as the release rate of the
incorporated drug.
The major drawback of liposomes is their instability during storage or in biological
media, which is related to surface properties. They generally adhere to each other and after a
certain time fuse to form larger vesicles. Biocompatible polymers have been used for the
external surface modification of liposomes, obtaining a stable system for different way of
applications [164-168]. Different types of biocompatible polymers such as chitosan can be
employed to improve the efficiency of conventional liposomal systems [161, 169]. The
biological properties of chitosan makes it a good candidate to combine to liposomes and to
render them stable and bioadhesive structures [170]. Liposomes in a chitosan gel formulation
can protect the encapsulated drugs against the degradation and provide their controlled and
sustained release [171, 172]. Glavas-Dodov et al. prepared topical formulation by

incorporation of liyophilized liposomes into the chitosan gel base. Liposomes embedded into
a structured vehicle of chitosan gels sustained the release more than hydrogels [173, 174].
By combining the advantages of chitosan and liposomal characteristics, specific,
prolonged, and controlled release may be achieved. Chitosan-coated liposomes were formed
via ionic interaction between the positively charged chitosan and negatively charged diacetyl
phosphate or phosphatidylcholine [175-179]. Several authors have used chitosan as a
liposome coating to increase the stability of drug release [167, 169, 172, 180] and for
targeting purposes [161, 177, 179, 181, 182].
Positively, neutrally and negatively charged liposomes were coated with two types of low
and medium molecular weight chitosans. Both types of chitosan coating increased the
mucoadhesive characteristics of all three types of vesicles. Using chitosan coating, high
efficient superoxide dismutase-loaded vesicles for drug targeting on mucosal tissues could be
produced [183].
Stability of aciclovir and minoxidil encapsulated liposomes and skin permeation were
improved by coating with chitosan. The coating of liposomes led to higher skin diffusion for
both drugs, which was explained as an effect of positively charged liposomes to interact
stronger with skin’s negatively charged surface and their possible interactions with structures
below the stratum corneum. This interaction might result in transfering to deeper layers,
disrupting the tight junctions in lower epidermis [164].
There are number of factors that can influence a polymer/colloid system such as
chitosan/liposome ratio. A small change in one factor may seriously alter the stability as well
as other characteristics of the system. Lidocaine hydrochloride chitosomes (liposome-chitosan
combination) were prepared by coating multilamellar liposomes with chitosan to achieve an
increment in the positive charge on the liposome surface. A high chitosan concentration was
required at a low stirring range for production of the systems [176].
Ruel-Gariépy et al. [184] reported that negatively-charged liposomes were able to interact
with thermosensitive chitosan hydrochloride (Protasan®) -based hydrogel to decrease slightly
the gelation rate and the gel strength thus they could be useful for delivery of hydrophilic
molecules.
By taking advantage of the biodegradable and biocompatible properties of chitosan,
Chung et al. [152] developed the fibrin encapsulated liposome-in-chitosan matrix (FLCM) for
sustained release of water soluble, low molecular weighed drugs. FLCM was offered for
developing depot delivery system with a high volume capacity of reservoir.
Skin Tissue Engineering
Tissue engineering is a rapidly growing area that seeks to create, repair and/or replace
tissues by using combinations of cells seeded on a scaffold, biomaterials, and/or biologically
active molecules [185-187]. Skin tissue engineering offers exciting opportunities in the
treatment of acute wounds (burn and skin excision), chronic wounds (diabetic ulcer, pressure
ulcer), vitiligo and the scar revision surgery. Fibroblasts, the main component of dermal cells,
can be easily isolated and cultured in monolayers by conventional cell culture techniques and
they may enhance cellular motility in the wound. These cells play an important role in
epithelial morphogenesis, keratinocyte adhesion, and the formation of the complex dermal-
epithelial junction [188, 189]. The development of human skin models that have the same
properties as genuine human skin is of particular significance [190, 191]. Protein-based
polymers including collagen, gelatin, fibrin and other natural polymers such as alginate and
chitosan are widely employed for tissue engineering. These polymers can provide not only
physical support for tissue regeneration but also serves as biomimetic matrices with biological
functions to actively induce tissue regeneration [192, 193].
The pioneers of skin tissue engineering focused on the application of collagen matrix as a
cell carrier [194]. Similarly, chitosan was incorporated in tissue engineering because of its
excellent biocompatibility and biodregadability. Chitosan could be easily formed into
structures under mild processing conditions and chemically modified. Furthermore it has
excellent wound healing properties as mentioned previously and an ability to be processed
into porous structures for use in tissue regeneration [21, 195-201]. Table 2 summarizes
applications and approaches of chitosan usage in dermal healing.
Physicochemical properties of chitosan influence fibroblast proliferation. Howling et al.
compared the ability of 37, 58 and 89% deacetylated chitosan to modulate fibroblast
mitogenesis in vitro. Results revealed that highly deacetylated chitosan was generally more
capable of stimulating fibroblast mitogenesis [202].
Table 2. Different applications and approaches of chitosan usage in dermal healing
Reference
Aim Formulation Content of structure
number
Chitosan-fibroblast growth factor [21]
Chitosan-minocycline [195]
Chitosan-hyaluronic acid-
[199]
chlorhexidine diacetate
Chitosan-peptide [201]
Chitosan-plasenta [222]
Chitosan-fibrinojen [222]
Wound healing Wound dressing
[96], [205] [206],
[208] [209], [210]
Chitosan-collagen
[211], [214]
[215]
[216], [218]
Chitosan-gelatine
[219], [220]
[188], [190]
Chitosan-glycosaminoglycan-collagen
[212], [213]
Suture Chitosan - poly (ε-caprolactone) [232]
Wound dressing Chitosan-collagen [204]
Hemostatic
Bandage Chitosan acetate [228]
activity
Suture Chitosan-poly(L-lactic acid) [235]
Antimicrobial
Bandage Chitosan acetate [229]
activity
In the inflammatory phase, chitosan has unique hemostatic properties that are
independent of the normal clotting cascades [203]. The incorporation of collagen to chitosan
was proven to enhance the cell attachement ability when compared to solely chitosan [204].
Because of the reasons cited above, a number of researchers have studied collagen-chitosan
scaffolds as dermal equivalent in various tissue engineering applications [205-210].
Generally, composite scaffolds were prepared by freezing and lyophilizing of the collagen-
chitosan solution. The effect of physical interaction between collagen and chitosan on
biological properties of scaffolds was described by Tangsadthakun et al. [211]. The fibroblast
proliferation was enhaced by chitosan containing scaffolds compared to pure collagen
assayed by cell culture studies.
A skin equivalent based on a collagen-glycosaminoglycan-chitosan dermal substrate has
been developed to meet the growing demand in tissue engineered skin equivalents [190].
Damour et al. [212] co-cultured fibroblasts and keratinocytes on a substrate composed of
collagen-glycosaminoglycan-chitosan to treat a large skin defect. Skin resections were treated
with a tissue-engineered graft with good wound regeneration. Further, the papers were
published highlighting the clinical potential of this matrix and healing of dermal, epidermal
lesion and burn [188, 213].
A biodegradable chitosan- nanosized collagen membrane as a novel skin substitute was
developed and evaluated by animal studies. The membrane seeded with fibroblasts was found
more effective than both gauze and commercial wound dressing, in healing wounds. An in
vivo histological assessment indicated that covering the wound with the membrane resulted in
its epithelialization and reconstruction [214]. Furthermore, the glutaraldehyde-treated
collagen/chitosan scaffold was also pointed as a potential candidate for dermal equivalent
with enhanced biostability and good biocompatibility [205, 215].
Chitosan-gelatin scaffolds have also been investigated for skin tissue engineering. Gelatin
is a partially denatured derivative of the fibrous protein collagen and it can be completely
resorbable in vivo. The chitosan-gelatin scaffolds were found more wettable than chitosan
alone. The artificial skin obtained with chitosan-gelatin had suitable flexibility and
mechanical properties for skin tissue engineering. Moreover the results indicated an inhibition
of scar formation [216, 217]. Similarly, the researchers have proved that chitosan scaffolds
containing basic fibroblast growth factor loaded gelatin microparticles were effective in
accelerating wound closure of pressure ulcers [218]. To further enhance the properties of
chitosan-gelatin scaffolds for skin tissue engineering, hyaluronic acid was introduced to
chitosan-gelatin complex by Liu et al. to provide a new scaffold for skin tissue engineering
[219, 220].
An increament in cell or DNA damage is associated with high toxicity therefore the
newly developed chitosan derivative in the form of porous skin regenerating templates were
tested for biocompatibility with human epidermal keratinocyte cultures and assessed in terms
of cytotoxicity, genotoxicity and inflammation. Chitosan templates were found to be
cytocompatible, non-toxic and capable of stimulating cell proliferation [221].
ChorioChit (Chorion/placenta + Chitosan) was offered as a biological wound dressing
based on chitosan, combined with a human placenta extract. The dressing was found effective
in the treatment of hard-healing wounds. ChitoFib (Chitosan + Fibrinogen) was another
surgical biological dressing that combined the biopolymer chitosan and tissue glue developed
on the basis of fibrinogen. The dressing enabled surgeons to replace wound stitches with glue
[222].
Many of the chitosan based commercial product available in the market are basicly for
wound healing as dressings and hemostatic products as patches, pads and granules (Table 3).
Table 3. Chitosan-based commercial products available in the market
Product Formulation type Medical application

Tegasorb® Hydrocolloid dressing Partial and full
thickness dermal ulcers, superficial wounds, abrasions,
burns
Tegaderm® Transparent film Leg ulcers, sacral wounds, chronic wounds

dressing
Beschitin® Patch Dress burns and other wounds
has an analgesic efect and favours early granulation, no
retractive scar formation. For traumatic wounds, surgical
tissue defects.
Syvek patch® Patch Hemostatic, achieving hemostasis.
Vulnosorb® Sponge Wound healing
Chitipack S® Sponge like chitin For traumatic wounds and surgical tissue defects.
Favours early granulation, no retractive scar formation.
Chitopack C® Cotton-like chitosan Complete reconstruction of body tissue, re-building of

normal subcutaneous tissue, and regular regeneration of
skin.
Chitipack P® Chitosan layer For the treatment of large skin defects. Favours early
granulation. Suitable for defects difficult to suture. After
skin tumor surgery
HemCon Bandage For emergency use to stop bleeding.

Bandage® Antibacterial, biocompatible wound dressing designed to
be stuffed into a wound track to control moderate to
severe bleeding.
Chitoseal® Pad Hemostatic in bleeding wounds
Clo-Sur® Pad Antimicrobial barrier, hemostatic wound dressing
Aquanova® Pad Leg ulcers, diabetic ulcers, surgical or traumatic wounds,

minor and other burns, superficial cuts, lacerations and
abrasions, and minor irritations of the skin.
Celox® Granules Emergency haemostat, minor cuts and bleeding
Chitodine® Chitosan powder with For the disinfection and cleaning of wounded skin and
adsorbed elementary for surgical dressing.
iodine
Chitopoly® Chitosan and Polynosic Antimicrobial wears, suitable to prevent dermatitis
Junlon poly(acrylate)
Cosmetic applications
Chitosan is an excellent cosmetic excipient that is remarkably well tolerated by the skin.
Since it is an efficient trapper of heavy metals that are responsible for many contact allergies;
its usage in cosmetics may avoid skin allergies. Chitosan has a wide application as
moisturizing component in different cosmetic investigations. Chitosan forms a protective
tensor film on the skin's surface allowing active principles to be placed in close contact with
the skin. Thus other hydrating agents, solar filters, organic acids or other active principles can
be combined with chitosan to facilitate their effects [203, 223-226].
Therefore cosmetic compositions based upon chitosan is promising upon variety of
applications for treatment of hair or skin. Moreover chitosan may be an essential component
in cosmetics due to its antibacterial properties. Chitosan based products can be provided as
hair care systems (hairspray, setting lotion, colouring products, shampoo), creams, lotions,
cleansing products (cleansing milk, face peel, facial toner, soap) and as cosmetic agents for
the care, protection, or cleaning of skin [227].
Other Applications
Chitosan has found many applications in different tools such as bandage, suture and
microneedle because of its versatility and beneficial physicochemical and biological
properties.
Bandage
Chitosan’s properties allow to rapidly clot blood, and has recently gained approval for
use in bandages from FDA. Chitosan is hypoallergenic, and has natural anti-bacterial
properties, further supporting its use in field bandages. Chitosan acetate bandage was
developed as a hemostatic, antimicrobial topical dressing. This product performed superior
properties when compared with gauze and alginate bandage [228]. Dai et al. [229] have
demonstrated that the chitosan acetate bandage performed better than the clinically approved
nanocrystalline silver bandage when it is topically applied to third-degree burns that are
heavily contaminated with pathogenic bacteria.
Pads
Mechanical closure devices are not available to achieve fast and secure hemostasis. As an
alternative chitosan pads were investigated and found to be useful for improvement in
hemostasis. In a study, the use of chitosan pads significantly decreased the bleeding time in
the first three minutes after manual compression time (p < 0.01) [230].
Suture
The application of chitosan fibers as sutures is remarkable due to its low immunogenicity
[231]. The acceleration in wound healing and absorbtion by human body, favors its use since
no second operation is required. It was reported that the chitin suture was absorbed in about 4
months in rat muscles. Moreover, application in patients proved satisfactory results in terms
of tissue reaction and good healing. Toxicity tests, including acute toxicity, pyrogenicity, and
mutagenicity were found negative [232]. It is known that successful wound healing depends
on an appropriate tensile strength provided by the suture materials. In a study, with regard to
wound healing in subcutaneous tissue, chitin administration significantly increased the tensile
strength of skin sutures within 5 days, compared with the control [233]. Standard silk and
catgut sutures coated with regenerated chitin or chitosan showed good wound-healing
activities [234]. Chitosan coated poly(L-lactic acid) wires showed better hemostatic activity
than wires without chitosan [235].
Microneedle
Transdermal drug delivery systems have been limited because of the strong barrier
function of the skin. The use of micron-scale needles showed a dramatical increase of the skin
permeability of drugs [236, 237]. It was suggested that the micro-needle patch of chitin and/or
chitosan made insertion of the microneedles into a living body easier. Chitosan was pointed
as a promising polymer for microneedle-mediated release of drugs through skin for enhancing
transdermal drug delivery [238].
Future Directions
Drug delivery systems have changed to meet the needs in the area of dermal and
transdermal therapy to obtain a sufficient efficacy with low toxicity. The demand for
developing new formulations, coupled with the shortcomings of synthetic polymer based
systems, caused the progress of less toxic and natural polymer based systems.
Numerous arrays of choices for these natural polymers as drug carriers are available,
which offer different perspectives of formulation design.
As being a biocompatible, antimicrobial and cationic natural polymer, chitosan stands out
for applications of dermal and transdermal drug delivery. The cabability of building up a
matrix for different dosage forms such as hydrogel, patch, micro and nanoparticles and the
ability of enhancing the adhesion properties of the formulations with its unique cationic
charge, makes chitosan one of the first choice natural polymers.
The accelarated wound and burn healing observed with the chitosan added formulations
or engineered skin tissues is another promising field in the area of dermal treatment.
As stated by many scientists, potential applications of chitosan in medicine can only be
exploited if its usable forms are properly developed and prepared. Drug products for human
use should be safe, efficacious and of an acceptable quality. Evidently, there is a lot to drive
benefit from chitosan in dermal/transdermal treatments by these means. Interdisciplinary
collaboration, especially the collaboration of clinicians with formulators would facilitate
emphasizing the advantages and disadvantages of chitosan, to indicate where the future
attempt should be set to. As such industry will be more willing to provide leadership in the
area to further advance applications of the technology and the approval and marketing of
chitosan based systems will ascend.
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INDEX
additional dissociation, 231

# additives, 65, 283, 335, 414, 456
adenocarcinoma, 434
21st century, 185
adhesion, 29, 56, 70, 75, 86, 101, 105, 134, 145, 146,
155, 162, 175, 179, 184, 213, 222, 224, 226, 228,
A 234, 237, 244, 255, 312, 313, 348, 404, 422, 452,
454, 458, 464, 469, 471
accelerator, 119, 143, 189, 192 adhesion properties, 469
access, 120, 276, 336, 337, 367, 369, 395 adhesive interaction, 233
accounting, 7 adhesive properties, 75, 86, 88, 452
acetaldehyde, 459 adsorption, viii, 2, 30, 32, 34, 36, 38, 47, 48, 50, 55,
acetaminophen, 419 58, 66, 67, 69, 87, 90, 92, 94, 105, 111, 131, 149,
acetic acid, 4, 8, 11, 13, 18, 19, 30, 37, 56, 73, 92, 191, 225, 254, 338, 407, 434, 436, 461
96, 98, 102, 104, 130, 135, 137, 140, 143, 150, advancement, 418
151, 156, 158, 159, 168, 170, 171, 173, 176, 179, adverse effects, 109, 344
202, 229, 238, 240, 323, 423, 424, 458 aerosols, 411
acetone, 137, 139, 140, 176, 227, 372, 373, 424, 425 AFM, 201, 233, 262, 271, 272, 273, 303, 306
acetylation, viii, 8, 13, 61, 70, 71, 125, 138, 139, agglutination, 163
140, 141, 142, 143, 147, 152, 154, 155, 156, 158, aggregation, 27, 108, 115, 116, 130, 142, 143, 159,
159, 164, 165, 167, 168, 170, 173, 177, 188, 189, 160, 165, 218, 221, 232, 233, 245, 254, 273, 284,
190, 191, 197, 199, 202, 203, 205, 212, 225, 232, 285, 300, 303, 305, 306, 336, 337, 342, 351, 367,
245, 247, 264, 403, 445, 446, 458, 471, 474 372, 394, 421, 472
acidic, 11, 12, 13, 18, 22, 33, 34, 73, 79, 84, 85, 87, agriculture, 220, 241, 451
99, 102, 108, 115, 142, 156, 157, 178, 214, 222, airway epithelial cells, 356
223, 225, 233, 236, 237, 239, 248, 263, 268, 270, airway hyperresponsiveness, 344, 349
295, 306, 322, 324, 326, 328, 335, 347, 383, 386, airway inflammation, 341, 360, 448
415, 419, 452, 455, 456, 460, 475 airways, 332, 397, 411
acrylate, 87, 101, 113, 116, 120, 130, 266, 467 albumin, 19, 238, 251, 337, 378, 407
acrylic acid, 63, 101, 104, 107, 116, 130, 148, 163, aldehydes, viii, 71, 77, 78, 155, 210
210, 225, 240, 248, 253, 256, 257, 262, 264, 284, algae, 136
295, 340, 346, 353, 359, 361, 370, 371, 377, 381, alimentary canal, 238
406, 430, 471 alkaline hydrolysis, 139, 144, 161, 163, 206
acrylonitrile, 101, 103, 121, 126 alkaline media, 237, 239, 263, 268, 296, 297, 298
activated carbon, 32, 39, 55, 67 alkaline pH, viii, 71, 324
active compound, viii, xi, 217, 388, 423, 450 alkyl, viii, 71, 77, 78, 83, 85, 99, 110, 120, 130, 160,
active site, 112, 140 234
active transport, 349, 430 alkylation, 78, 82, 83, 91, 145, 155, 264, 265, 266,
acylation, 82, 95, 96, 98, 99, 115, 116, 129, 143, 267, 268
145, 212, 224 allergic asthma, 340, 349
adaptation, 10, 381 allergic reaction, 452
486 Index
ALT, 395, 396 aquatic systems, 32

alters, 156, 424 aqueous solutions, ix, 28, 39, 188, 205, 219, 225,
alveolar macrophage, 332, 349, 411 226, 234, 261, 263, 267, 268, 269, 270, 271, 272,
alveoli, 332, 397, 411 277, 278, 283, 284, 295, 309, 368
amine, viii, 71, 88, 89, 102, 109, 140, 141, 145, 170, aqueous suspension, 287, 384
171, 284, 321, 326, 335, 343, 415, 419, 423, 451, arrest, 341, 347
460 arthritis, 103, 169, 211, 341, 344, 359, 360
amine group, 88, 109, 140, 141, 145, 170, 171, 326, arthropods, 2, 135, 144, 152, 414
335, 343, 415, 419, 423, 460 articular cartilage, 29, 64
amines, 74, 78, 87, 141, 156, 323, 335 ascending colon, 333
amino, viii, ix, 13, 14, 16, 18, 33, 34, 36, 37, 40, 41, ascites, 440, 446, 476
42, 45, 48, 50, 67, 71, 72, 73, 74, 76, 77, 80, 82, ascorbic acid, 31, 66
83, 84, 85, 86, 88, 90, 94, 98, 99, 101, 102, 110, Asian countries, 3
133, 138, 141, 142, 147, 149, 154, 155, 156, 157, aspartic acid, 100, 241, 257, 352
165, 177, 218, 222, 225, 229, 232, 234, 237, 244, assessment, 64, 72, 115, 207, 249, 387, 447, 466
261, 263, 264, 265, 266, 267, 268, 270, 274, 275, asthma, 344, 349, 357, 411, 431
280, 285, 295, 297, 298, 309, 313, 321, 326, 328, atherosclerosis, 340, 341, 359
342, 369, 383, 414, 420, 423, 434, 440, 451, 452, atmosphere, 309
456 atomic force, 225, 237, 254, 262, 271
amino acid, 67, 100, 149, 237, 244, 434, 440 atomic force microscope, 225
amino groups, viii, 13, 16, 18, 33, 34, 35, 37, 40, 42, atoms, viii, 17, 71, 155, 309
45, 48, 50, 71, 73, 74, 76, 80, 84, 85, 86, 88, 90, atopic dermatitis, 432
101, 102, 110, 141, 154, 156, 157, 165, 177, 218, ATP, 38, 339
222, 229, 232, 234, 263, 264, 265, 266, 267, 268, attachment, 30, 48, 50, 105, 232, 237, 283, 433
270, 274, 275, 285, 297, 298, 309, 313, 326, 328, authorities, 24
342, 369, 383, 420, 452, 456 autoimmune diseases, 327
aminopolysaccharides, viii, 133, 185 auxins, 8
ammonia, 240, 257 avoidance, 331, 374, 386
ammonium, 83, 84, 95, 100, 102, 117, 129, 166, 456
ammonium persulphate, 100
ammonium salts, 95 B
amoebiasis, x, 413
amorphous precipitate, 294 BAC, 243
amylase, 141, 164, 249 Bacillus subtilis, 188
analgesic, 442, 467 bacteria, x, 8, 69, 83, 84, 104, 108, 136, 177, 227,
anionic polyelectrolytes, vii, 218, 245 230, 231, 242, 263, 276, 282, 289, 413, 420, 439,
antibiotic, 2, 6, 26, 29, 231, 348, 351, 443 454, 459, 468
antibody, 30, 232, 395, 435, 436, 440, 443 bactericides, 25
anti-cancer, 434, 437 bacteriophage, 231
anticancer drug, 201, 344, 368, 443, 460 bacteriostatic, viii, 71, 73, 104, 225
anticoagulant, 125, 222, 229, 230, 295, 334, 355 bacterium, 39, 68, 454, 459
antigen, x, 28, 30, 65, 104, 112, 232, 354, 367, 374, Bahrain, 39
377, 378, 385, 386, 390, 391, 395, 396, 408, 410, barriers, ix, 336, 337, 344, 348, 355, 365, 366, 389,
414, 427, 436, 443, 462 400, 402, 403, 433, 440
antigenicity, 236 base, ix, 16, 32, 64, 73, 77, 90, 113, 122, 124, 155,
anti-inflammatory drugs, xi, 353, 406, 437, 450, 460 156, 177, 178, 204, 205, 209, 210, 213, 235, 245,
antimicrobial activity, viii, x, 88, 104, 121, 124, 128, 259, 293, 354, 357, 365, 366, 368, 369, 370, 374,
133, 134, 190, 192, 275, 289, 297, 322, 413, 433 379, 388, 397, 402, 404, 406, 432, 438, 439, 455,
antioxidant, viii, 30, 65, 71, 88, 180, 276 457, 460, 464, 467, 471, 473, 475
antisense, 28, 75, 247, 335, 358, 463 base pair, 235
antisense oligonucleotides, 28, 75, 358, 463 basicity, 79
antitumor, 84, 126, 346, 347, 354, 423, 434, 439, 442 batteries, 27, 179
apoptosis, 247, 341, 346 BBB, 349
behaviors, 118, 168, 255, 256, 258
Index 487
benefits, 218, 452 biopolymer, x, 2, 36, 63, 70, 72, 133, 135, 144, 152,
beverages, 134 218, 263, 336, 413, 423, 449, 466
bicarbonate, 140, 386 biopolymers, 76, 110, 133, 152, 186, 197, 198, 237,
bile, 135, 151, 166, 174, 178, 386 249, 436
bile acids, 178 biosensors, 29, 31
binding energies, 309 biosphere, 2, 152
bioactive agents, 248 biotechnological applications, 245
bioavailability, ix, xi, 102, 120, 228, 323, 328, 330, biotechnology, vii, viii, 1, 133, 188, 200, 217, 220,
344, 348, 357, 366, 374, 377, 381, 384, 386, 389, 232, 235, 241, 243, 245, 284, 366, 439
391, 394, 397, 402, 404, 407, 421, 430, 431, 432, bisphenol, 38, 39, 68, 111
438, 449, 450, 452 bladder cancer, 346
bio-catalysts, vii, 1 bleaching, 137
biochemistry, 134, 203, 256 bleeding, 467, 468
biocompatibility, viii, 29, 31, 55, 56, 71, 72, 89, 102, bleeding time, 468
109, 110, 112, 122, 133, 134, 144, 145, 146, 149, blends, 164, 166, 167, 170, 173, 175, 177, 240
150, 151, 155, 169, 175, 179, 192, 193, 194, 196, blood, viii, 56, 86, 88, 99, 105, 108, 110, 120, 121,
208, 211, 212, 214, 215, 218, 225, 249, 261, 284, 122, 126, 145, 147, 163, 169, 193, 198, 208, 217,
331, 335, 400, 402, 404, 422, 423, 443, 454, 456, 220, 222, 228, 229, 230, 237, 239, 244, 249, 255,
460, 462, 465, 466, 473, 484 258, 274, 275, 284, 297, 298, 299, 300, 310, 311,
biocompatibility test, 456 312, 313, 322, 329, 333, 334, 337, 345, 346, 349,
biocompatible materials, 105, 228 350, 374, 377, 380, 384, 389, 391, 394, 422, 432,
bioconstruction materials, viii, 217 440, 442, 446, 468
bioconversion, 451 blood circulation, 108, 346
biodegradability, vii, viii, 55, 71, 72, 89, 104, 116, blood flow, 422
129, 133, 134, 138, 144, 146, 147, 149, 150, 151, blood plasma, 230, 275
155, 166, 169, 175, 192, 194, 195, 196, 213, 214, blood stream, 334, 337, 345
261, 274, 322, 335, 423, 434 blood supply, 374, 432
biodegradable materials, 210 blood vessels, viii, 217, 237, 244
biodegradation, x, 113, 146, 147, 148, 149, 150, 169, blood-brain barrier, 389
175, 188, 195, 196, 213, 252, 449 bloodstream, 337
biofilms, x, 413 body fluid, 56, 300
biological activities, 84, 102, 134, 222, 368, 423 body weight, 108, 144, 169
biological activity, vii, ix, 155, 201, 297, 310, 365, bonding, 154, 155, 165, 170, 218, 219, 221, 236,
375, 382, 405 239, 245, 257, 268, 272, 335, 429, 452
biological behavior, viii, 261, 263, 264, 274, 306, bonds, 17, 74, 75, 76, 90, 120, 153, 180, 218, 219,
313 220, 223, 224, 225, 226, 229, 237, 240, 242, 276,
biological fluids, 337, 367, 455 298, 330
biological media, 220, 463 bone, viii, 24, 28, 32, 55, 56, 62, 64, 70, 73, 86, 88,
biological responses, 455 103, 112, 116, 135, 139, 167, 171, 184, 210, 212,
biological samples, 31 217, 236, 244, 250, 322, 337, 341, 351
biological sciences, 62 bone form, 70, 73
biologically active compounds, 244 bone marrow, 337
biomass, 2, 4, 6, 11, 12, 33, 37 bone resorption, 341
biomaterials, 103, 117, 169, 185, 190, 191, 193, 194, bone tissue, viii, 28, 55, 62, 64, 184, 212, 217, 244
195, 199, 201, 207, 208, 209, 211, 222, 224, 236, bones, 55, 179
245, 255, 259, 354, 446, 464, 472 brain, 65, 334, 349, 357, 363, 389, 409, 410, 432,
biomedical applications, 98, 105, 135, 144, 166, 191, 442, 446, 448
193, 245, 255, 277, 281, 300, 313, 322, 327, 436, branching, 10, 155, 240
442, 451, 472 Brazil, 68
biomedical field, vii, ix, x, 282, 321, 413, 461 breakdown, 347
biomimetic coatings, viii, 2, 56 breast cancer, 346, 446
biomolecules, 56, 222, 374, 397 bridge model, 33
biopharmaceuticals, ix, 321, 327, 328, 354 buccal epithelium, 387, 408
488 Index
buccal mucosa, 369, 387, 408, 452 catalyst, 84, 97, 100, 101, 114, 164
building blocks, 88 catalytic activity, 230
Bulgaria, 261, 314 cation, 36, 443
burn, 164, 455, 464, 466, 469 cationic character, viii, 71, 83, 84, 142, 368, 369,
by-products, x, 2, 6, 7, 33, 237, 449, 452 423
C-C, 473
CDC, 90, 94
C CECh molecular weight, ix, 261, 272, 273, 302
cell culture, 72, 122, 124, 134, 146, 179, 312, 408,
Ca2+, 383, 394 430, 435, 464, 466
calcification, 69 cell cycle, 8, 341
calcitonin, 115, 333, 357, 388, 410, 446, 475 cell differentiation, 56
calcium, 11, 28, 29, 64, 66, 88, 94, 115, 120, 121, cell line, 109, 127, 232, 338, 339, 346, 348, 382,
123, 136, 137, 152, 171, 227, 249, 333, 351, 442 387, 393, 399, 400, 407, 436, 437
calcium carbonate, 11, 66, 120, 136, 137 cell lines, 127, 232, 339, 382, 400, 407, 436
calibration, 19 cell membranes, 32, 241, 274, 290, 328, 347, 399,
cancer, 30, 130, 201, 223, 327, 340, 343, 344, 346, 416, 422, 427
347, 353, 357, 359, 360, 361, 362, 405, 420, 426, cell surface, 79, 142, 328, 329, 338, 347, 348, 433
434, 437, 439, 446, 450, 459, 478 cellulose, x, 2, 11, 13, 22, 29, 62, 115, 116, 133, 135,
cancer cells, 344, 347, 362 141, 148, 152, 153, 155, 156, 165, 166, 167, 168,
cancerous cells, 95, 100, 347 171, 173, 177, 178, 185, 189, 190, 201, 202, 208,
candidates, 39, 103, 178, 313, 389, 462 210, 222, 247, 251, 256, 263, 277, 290, 321, 413,
capillary, 18, 88, 114, 293 414, 456
capsule, 419 cellulose xanthate, 167
carbohydrate, 202, 237, 256, 347, 380, 423 central nervous system, 349, 389, 409, 432, 440
carbohydrates, 11, 237, 392 ceramic, 70, 112
carbon, vii, 1, 7, 27, 29, 30, 31, 32, 39, 63, 65, 146, cerebrospinal fluid, 391, 396, 432
149, 150, 184, 274, 309 cervix, 356
carbon atoms, 309 challenges, 396
carbon dioxide, 39, 149 charcoal, 52, 69
carbon nanotubes, 27, 30, 32, 63, 65 charge density, 73, 168, 173, 223, 226, 227, 238,
carbonyl groups, 86 245, 249, 259, 270, 335, 433, 452, 456
carboxyl, ix, 85, 88, 89, 90, 99, 222, 231, 239, 261, chemical characteristics, 14, 254
263, 266, 268, 274, 280, 284, 285, 295, 298 chemical degradation, 366
carboxylic acid, 74, 96, 104, 115 chemical modifications, viii, 34, 133, 145, 147, 155,
carboxylic groups, 85, 157, 223, 230, 242 190, 201, 405
carboxymethyl cellulose, 248, 251 chemical properties, 11, 13, 21, 39, 40, 148, 161, 176
carboxymethyl chitosan, 28, 64, 72, 79, 87, 88, 100, chemical reactions, 140, 155
113, 114, 115, 117, 120, 121, 124, 127, 130, 131, chemical stability, 11, 323, 462
198, 361, 451, 480 chemical structures, 302, 370
carboxymethyl groups, viii, 71, 89, 224 chemicals, 2, 7, 33, 68, 171, 198, 213, 425
carboxymethylation, 89, 99, 131 chemisorption, 284
carcinoembryonic antigen, 65 chemotherapeutic agent, 459
carcinoma, 30, 341, 342, 346, 359, 361, 434, 440, chemotherapy, 437, 438
446, 476 chicken, 127, 331
cardiovascular, 105, 222 China, 3, 24, 25, 113, 135, 213, 317, 353
cardiovascular disease, 222 Chinese medicine, 177
cardiovascular diseases, 222 chitinase, 141, 168
carrageenans, 226 chitosan production, vii, 1, 2, 3, 4, 7, 8, 9, 22, 58, 59,
cartilage, 29, 64, 178, 214, 224, 259, 344, 472 60, 138, 187
casein, 204, 436 chitosan salts, 74, 79, 80, 84, 119, 123, 203, 355,
casting, 164, 178, 204, 225, 239, 458 387, 452, 454
catabolized, 184 chitosan-based hydrogels, 194, 420
catalysis, 241
Index 489
chloral, 158 complex interactions, 338

chlorinated hydrocarbons, 158 compliance, 328, 331, 366, 374, 387, 451
chlorine, 39, 69 composite materials, vii, 2, 26, 145, 210, 284
chloroform, 96, 98 composites, 27, 29, 56, 63, 64, 150, 167, 170, 179,
chloroplast, 276 193, 196, 210, 212, 215, 458
cholera, 396 composition, 13, 55, 58, 59, 94, 136, 137, 186, 187,
cholesterol, 24, 31, 66, 90, 91, 417, 434, 448 191, 203, 204, 206, 219, 222, 225, 228, 230, 238,
choline, 31, 66 239, 240, 277, 278, 279, 281, 310, 382, 394, 402,
chondrocyte, 86 432, 463, 474
chondroitin sulfate, 222, 223, 224, 247, 419, 427 compost, 147
chromatography, 14, 19, 60, 143, 148, 159, 258 compounds, 11, 39, 45, 69, 75, 79, 82, 94, 103, 115,
chronic diseases, 343 127, 151, 221, 231, 242, 322, 330, 366, 368, 386,
circulation, 53, 102, 327, 336, 337, 345, 346, 451 387, 409, 415, 420, 454
clarity, 322 compression, 468
classes, 422, 425, 455 computer, 62
classification, 219, 455 condensation, 90, 101, 254, 337
cleaning, 30, 55, 450, 454, 467, 468 conductivity, 16, 27, 129, 228, 240, 277, 282, 289,
cleavage, 141, 224, 232, 241 293
clinical application, 111, 335, 344, 350, 471 conference, 134, 202
clinical trials, 110, 402 configuration, 30, 45, 73, 162, 282
closure, 457, 466, 468 conformational analysis, 202
clothing, 276 conjugation, 102, 338, 349
CMC, 88, 89, 177, 226 conjunctiva, 334, 348
C-N, 17, 309 connective tissue, 55, 175, 222, 236
CNS, 158, 389, 396, 426, 432 consensus, 332, 366
CO, 175 consolidation, 151
coatings, viii, 2, 14, 56, 70, 88, 163, 190, 220, 227, constituent materials, 459
244, 250, 258, 294, 300, 309 constituents, 98, 414, 421
collaboration, 469 construction, 235
collagen, xi, 55, 104, 117, 119, 135, 145, 149, 152, consumption, 109
166, 169, 175, 210, 213, 236, 237, 247, 248, 254, contact time, 43, 51, 53, 75, 328
255, 277, 351, 450, 460, 465, 466, 475, 477 contaminated water, 33
collagen sponges, 236 contamination, 39
colon, x, 74, 105, 106, 118, 126, 223, 228, 250, 328, contraceptives, 38, 433
333, 348, 352, 356, 362, 413, 419, 426, 429, 436, control group, 80, 82, 379
440, 445, 447 COOH, 99, 268, 281
colon associated malfunctions, x, 413 cooling, 12, 19, 21
colonic delivery, x, 123, 413, 419, 427 cooperation, 314
color, 139, 276, 419 coordination, 33, 218
colorectal cancer, 30, 343 copolymer, 83, 94, 102, 103, 109, 118, 119, 125,
combined effect, 277 130, 142, 148, 154, 195, 202, 214, 221, 227, 242,
commercial, vii, 1, 7, 14, 18, 23, 26, 136, 138, 141, 243, 244, 247, 258, 302, 303, 305, 306, 309, 313,
150, 154, 173, 177, 377, 384, 415, 448, 457, 458, 330, 331, 333, 353, 355, 404, 423, 442, 443, 476
466, 467 copolymers, 105, 114, 116, 117, 122, 125, 130, 148,
communication, 56 175, 201, 221, 223, 242, 244, 245, 246, 300, 302,
compaction, 232, 239, 259 303, 304, 358, 442
compatibility, 86, 88, 100, 101, 116, 120, 121, 122, copper, viii, 2, 30, 31, 32, 33, 34, 35, 36, 37, 58, 65,
126, 145, 192, 193, 194, 213, 228, 249, 274, 275, 67
313, 444, 462 cornea, 145, 334, 348
compensation, 271, 272 correlation, 18, 45, 46, 48, 471
competition, 415 correlation coefficient, 45, 46, 48
complementarity, 225 correlations, 141
complex carbohydrates, 199 cortical bone, 167
490 Index
corticosteroids, xi, 450, 457, 474 cytochrome, 332

cosmetic, 22, 25, 99, 180, 187, 468 cytocompatibility, 214, 241, 257
cosmetics, vii, x, 25, 84, 86, 88, 89, 133, 151, 184, cytokines, 224, 341, 344
276, 413, 418, 451, 468 cytokinins, 8
cosmetics industry, vii cytoplasm, 339, 340, 347
cost, 42, 58, 170, 327, 331, 374, 454 cytoskeleton, 329, 393
cotton, 28, 64, 135, 166, 177, 214 cytotoxicity, 88, 94, 108, 109, 110, 118, 122, 123,
Coulomb interaction, 221, 235 124, 145, 149, 163, 164, 178, 192, 229, 234, 340,
covalent binding, viii, 71 346, 347, 354, 361, 362, 375, 376, 385, 386, 387,
covalent bond, 75, 177, 275, 330, 339 399, 404, 406, 408, 420, 447, 456, 466, 474
covalent bonding, 330
covering, 151, 466
coverlets, viii, 217, 244 D
coxsackievirus, 340
CPT, 346 deaths, 109
crab shells, vii, 1, 61 decay, 69
crabs, x, 136, 154, 413, 414 decomposition, 141, 221
creatine, 32, 66 decontamination, 67, 90, 91
cristallinity, 226 decoration, 343, 346, 349
critical value, 221, 272 defects, 29, 64, 116, 163, 277, 278, 279, 280, 282,
Croatia, 206 467
cross-linking reaction, 210 defense mechanisms, 332
crown, 221 deficiency, 221, 334, 462
crustacean shells, vii, x, 1, 11, 13, 17, 136, 137, 154, deformability, 346
449, 451 deformation, 167
crustaceans, vii, x, 2, 4, 6, 13, 72, 135, 136, 152, degradation, 29, 38, 39, 70, 72, 80, 89, 101, 103,
186, 197, 263, 321, 413, 414 112, 115, 141, 147, 148, 149, 159, 164, 165, 169,
crystal growth, 70 174, 175, 179, 189, 192, 193, 194, 196, 211, 213,
crystal structure, 14, 143, 157, 163, 189, 199, 200, 215, 228, 233, 236, 237, 240, 268, 323, 335, 336,
206 339, 340, 342, 344, 366, 374, 383, 384, 385, 386,
crystalline, vii, 1, 13, 20, 94, 102, 135, 140, 143, 388, 396, 397, 415, 445, 448, 451, 452, 460, 462,
144, 152, 153, 155, 162, 168, 170, 172, 176, 185, 463
200, 205, 210, 234, 235, 245, 254, 268 degradation rate, 29, 89, 104, 149, 194, 211, 236
crystalline structure, vii, 1, 13, 94, 152, 153, 155, degree of crystallinity, 62, 161, 162, 163, 170, 172
170, 173, 205 degree of deacetylation, vii, viii, x, 1, 11, 13, 14, 15,
crystallinity, 14, 20, 27, 34, 78, 83, 84, 143, 155, 16, 17, 18, 33, 34, 57, 71, 108, 117, 136, 138,
157, 161, 162, 163, 164, 165, 168, 170, 173, 210, 143, 145, 146, 148, 150, 154, 156, 157, 165, 171,
268 189, 190, 191, 199, 200, 203, 208, 218, 253, 258,
crystallites, 143, 190 262, 267, 357, 358, 406, 414, 415, 416, 440, 448,
crystallization, 242, 420 449, 452, 455, 458
crystals, 56 dehydration, 225, 240, 242, 281
cultivation, vii, 2, 5, 6, 7, 10, 29, 57, 59, 145, 236 denaturation, 328
cultivation techniques, vii, 2 dendrites, 293
culture, 10, 60, 113, 119, 169, 171, 172, 175, 212, dendritic cell, 104, 341, 462
234, 283, 337, 375, 376, 390, 437, 459 denitrifying, 68
culture medium, 60, 234, 337 dentin, 88
curcumin, 459, 475 dephosphorylation, 79
cure, x, 414, 427 depolymerization, 11, 109, 138, 141, 157, 164, 208,
cuticle, 135, 136, 144, 199 442
cycles, 225 deposition, 225, 246, 306, 309, 332, 399
cyclodextrins, 92, 392, 393, 446 derivatization, viii, 71, 77, 79, 100
cyclosporine, 376, 377 dermatitis, 467
cysteine, 74, 75, 76, 112, 330, 331, 339 dermis, 450, 463
desiccation, 21
Index 491
desorption, 48, 55 dosage, viii, 39, 71, 110, 111, 115, 169, 387, 417,
destruction, 165, 239, 344 418, 419, 420, 426, 427, 433, 452, 469
detectable, 164, 278, 462 dosing, 328, 381
detection, 30, 31, 32, 52, 65, 66, 463 double helix, 226
detergents, 344 drawing, 276
developed countries, 389 dressing material, 99, 112, 164, 169, 198, 207, 208,
deviation, 222 211, 244, 282, 437, 475
dialysis, 105, 294, 371, 459 dressings, x, 99, 107, 166, 169, 170, 171, 174, 179,
diarrhea, 65 197, 207, 208, 212, 213, 263, 283, 293, 313, 413,
diatoms, 136 451, 459, 466
dichloroethane, 98, 158 drinking water, 32, 39, 45, 151
dielectric constant, 155 drug carriers, ix, xi, 98, 116, 118, 223, 322, 326, 345,
diffraction, 60, 162, 268, 286 361, 365, 368, 437, 450, 451, 469
diffusion, 52, 70, 75, 95, 99, 229, 242, 262, 273, 301, drug interaction, 433, 450
305, 327, 333, 369, 373, 374, 376, 377, 384, 388, drug metabolism, 389
400, 416, 423, 424, 431, 432, 443, 450, 457, 459, drug release, viii, 71, 72, 78, 79, 87, 88, 94, 101,
460, 461, 464 103, 115, 124, 126, 128, 191, 196, 240, 252, 343,
digestibility, 189, 208 346, 347, 349, 362, 367, 386, 388, 418, 420, 428,
digestion, 342 430, 431, 433, 435, 443, 446, 453, 456, 457, 459,
digestive enzymes, 329 460, 461, 464, 473, 475
diisocyanates, 84 drug therapy, 349
diluent, 419 drying, 20, 22, 37, 67, 124, 138, 164, 170, 171, 176,
dilute acids, viii, 71, 137, 170 214, 224, 251, 309, 375, 380, 390, 392, 398, 400,
dimethylformamide, 157, 161, 163 401, 424, 425, 428, 430, 432, 436, 438, 439, 457,
diphenhydramine, 146 460, 462
discomfort, 327 ductility, 164
diseases, 30, 62, 105, 232, 327, 334, 432, 435, 455 duodenal ulcer, x, 413
disinfection, 39, 467 duodenal ulcers, x, 413
disorder, 349 duodenum, 328, 329, 385
dispersion, 52, 235, 285, 305, 323, 384, 411, 432, dyes, 28, 90, 91, 126, 171, 191, 212, 444
434, 441 dynamic viscosity, 18, 289
displacement, 333
dissociation, 40, 218, 231, 233, 335, 336, 337, 339,
456 E
distilled water, 51, 371
distress, 345 Eastern Europe, 161, 162, 195, 196, 206, 207
distribution, 13, 14, 18, 40, 60, 61, 107, 115, 142, ecology, 300
143, 147, 156, 157, 169, 175, 191, 194, 211, 213, economic values, vii, 2
218, 219, 233, 238, 240, 247, 277, 282, 286, 288, editors, 60, 186, 190, 191, 192, 197, 199, 200, 202,
301, 303, 304, 326, 345, 424, 434, 439, 444, 478 203, 205, 206, 207, 208, 209, 211, 212, 405, 406,
diversification, 368 407, 408, 409, 410
DNA, 27, 30, 32, 37, 63, 65, 78, 100, 107, 113, 118, elaboration, x, 225, 366, 370
121, 122, 178, 201, 232, 233, 234, 235, 241, 244, elastomers, 197
253, 254, 259, 276, 326, 335, 336, 337, 338, 339, electric field, 224, 276, 457
340, 341, 342, 353, 354, 357, 358, 359, 360, 370, electrical conductivity, 27, 293
404, 405, 421, 424, 426, 431, 433, 434, 436, 438, electrical resistance, 380, 390, 416
442, 444, 453, 461, 462, 463, 466, 471, 477 electrochemistry, 66
DNA damage, 65, 466 electrodes, 28, 29, 277
DNase, 335 electrolyte, 43, 63, 249
docetaxel, 354, 434, 442 electromagnetic, 15
dopamine, 31, 32, 66 electromagnetic waves, 15
doping, 257 electron, 28, 31, 32, 56, 177, 231, 401, 442
electron microscopy, 56, 177, 401, 442
electrons, 15
492 Index
electrophoresis, 19, 225 epithelial cells, 80, 115, 118, 119, 329, 355, 379,
electrospinning, ix, 114, 116, 161, 175, 179, 185, 388, 399, 403, 411, 429, 436, 442
207, 211, 215, 261, 263, 276, 277, 280, 284, 287, epithelial surfaces, ix, 365
288, 289, 290, 292, 293, 306, 313 epithelium, 75, 83, 84, 328, 330, 332, 348, 366, 374,
elongation, 8, 161, 163, 165, 166, 167, 171, 173, 379, 382, 395, 397, 409, 411, 427, 445
174, 236 EPR, 345, 346, 361
emulsion polymerization, 102 equilibrium, 32, 34, 36, 37, 38, 42, 43, 44, 48, 50,
emulsions, 102, 108, 236, 323, 324, 410 55, 66, 102, 220, 221, 262, 297, 298, 460
enantiomers, 241 equipment, 19
encapsulation, 94, 99, 101, 103, 121, 224, 343, 345, erosion, 220, 252, 258, 476
354, 369, 373, 381, 382, 386, 388, 396, 400, 462, erythropoietin, 88
463, 476 ester, 87, 97, 168, 185, 205, 266, 341
encoding, 341, 359 esterification, 89, 104, 155, 161, 163, 281
endocrine, 39, 68, 69 estriol, 38
endocrine-disrupting chemicals, 68 estrogen, 37
endothelial cells, 101, 255, 349 ethanol, 7, 19, 94, 98, 137, 167, 206, 256, 307, 309,
endothelium, 345, 349 310, 372, 424, 456
endothermic, 149, 164 etherification, 155
endotoxins, 98, 231, 232, 253 ethyl alcohol, 161, 206
energy, 6, 7, 15, 17, 27, 39, 56, 155, 179, 180, 235, ethylene, 8, 27, 28, 83, 84, 85, 94, 104, 105, 107,
292, 293, 359 115, 116, 117, 122, 125, 129, 130, 164, 179, 198,
energy density, 155 208, 242, 243, 244, 246, 247, 262, 284, 287, 295,
energy input, 7 337, 338, 346, 353, 358, 359, 362, 423, 430, 438,
energy transfer, 359 446, 476
engineering, vii, viii, ix, 1, 2, 21, 28, 29, 56, 62, 63, ethylene glycol, 28, 83, 94, 105, 107, 115, 116, 117,
64, 70, 79, 88, 89, 103, 110, 123, 131, 134, 171, 122, 125, 129, 130, 198, 243, 246, 247, 262, 284,
175, 178, 179, 184, 197, 212, 213, 214, 224, 228, 337, 338, 346, 358, 359, 362
232, 236, 247, 248, 255, 259, 261, 263, 282, 322, ethylene oxide, 84, 85, 105, 164, 208, 242, 247, 262,
351, 354, 358, 420, 435, 442, 455, 459, 464, 465, 287, 353, 423, 430, 438, 446, 476
466, 472 Euglena gracilis, 276
entrapment, 28, 131, 257, 323, 340, 385, 408, 422, eukaryotic, 340
424 Europe, 55, 442
environment, 11, 28, 31, 38, 79, 142, 150, 196, 231, European Commission, 169
240, 327, 329, 332, 344, 347, 369, 371, 374, 380, European market, 3
386, 416, 419, 420, 428, 450, 452 evaporation, 138, 156, 242, 277, 333, 334, 373, 423,
environmental awareness, 454 425, 431, 460
environmental conditions, 474 evidence, ix, 33, 120, 143, 159, 175, 215, 289, 309,
environmental protection, 225, 284, 451 348, 365, 366, 376, 385, 391, 394, 398
enzymatic activity, 242, 332, 333 exclusion, 19, 60, 143, 159, 234
enzyme, 7, 31, 32, 39, 66, 68, 79, 83, 84, 97, 148, excretion, 38, 147, 169, 175, 384
220, 230, 242, 252, 257, 294, 357, 420, 448, 478 exocytosis, 362
enzyme immobilization, 252, 294 exoskeleton, 6, 135, 136, 144, 152, 199, 263, 321,
enzymes, 12, 21, 32, 102, 141, 146, 180, 184, 202, 414
224, 228, 230, 233, 241, 259, 274, 301, 332, 333, experimental condition, 36, 370
388, 428, 435, 448 experimental design, 7
eosinophilia, 344 export market, 136
eosinophils, 349 exposure, 32, 38, 80, 84, 124, 147, 164, 344, 386
epidermis, 164, 450, 452, 462, 463, 464 external environment, 180
epinephrine, 31 extracellular matrix, ix, 64, 69, 105, 110, 170, 261
epithelia, ix, 119, 127, 128, 331, 332, 366, 368, 388, extraction, viii, 2, 3, 6, 7, 11, 12, 13, 34, 57, 138,
399, 417, 427, 429, 431, 439, 452 141, 154, 393
epithelial barriers, ix, 365, 402 extracts, 11, 19, 197
extravasation, 345
Index 493
fluorescence, 50, 51, 75, 162, 238, 271, 273, 340,

F 359
foils, 22
fabrication, ix, 63, 77, 124, 166, 179, 190, 242, 255,
folate, 78, 121, 131, 201, 338, 347, 353, 358, 359
261, 263, 276, 283, 284, 287, 289, 290, 313, 315,
folic acid, 95, 201, 435
351, 353, 405, 475
food, vii, 1, 25, 32, 86, 88, 109, 134, 136, 137, 144,
factories, 3
151, 154, 169, 218, 226, 237, 239, 245, 254, 263,
fat, 3, 24, 57, 151
328, 451
fatty acids, 241
food chain, 32
FDA, xi, 24, 107, 192, 211, 450, 468
food industry, 136, 154, 218, 239
female rat, 109
food processing industries, vii, 1
fermentation, 13, 59, 188
food products, 86, 88
ferrous ion, 100
force, 17, 220, 233, 254, 276, 433, 437
fiber, 89, 104, 115, 121, 149, 150, 151, 159, 166,
foreign exchange, 136
167, 168, 170, 171, 176, 177, 179, 193, 196, 198,
formaldehyde, 292, 424
205, 209, 210, 211, 212, 213, 214, 215, 228, 240,
formamide, 98, 161, 164
277, 279, 281, 282, 289, 293, 434, 474
France, 135, 169, 205, 212, 414
fiber membranes, 214, 474
free amine functionalities, viii, 71
fibers, 89, 98, 102, 104, 117, 145, 149, 150, 151,
free energy, 220, 327
159, 161, 163, 167, 170, 171, 172, 173, 174, 175,
free radicals, 32, 38, 249
176, 177, 178, 181, 196, 197, 198, 204, 205, 206,
freezing, 158, 224, 236, 466
208, 209, 210, 212, 213, 214, 227, 249, 276, 277,
FTIR, 17, 56, 148, 164, 170, 171, 176, 179, 223,
278, 281, 287, 288, 289, 292, 293, 307, 309, 458,
240, 242, 249
468
fuel cell, 27, 61, 63, 239, 240, 257
fibrin, 108, 464, 465
functionalization, 193, 200
fibrinogen, 275, 277, 466
funding, 403
fibroblast growth factor, 105, 121, 122, 142, 457,
fungal infection, 431
460, 461, 465, 466, 474, 475, 477
fungi, vii, x, 1, 2, 3, 4, 6, 7, 8, 11, 12, 13, 15, 17, 21,
fibroblast proliferation, 218, 465, 466
34, 57, 58, 69, 72, 108, 135, 136, 139, 144, 147,
fibroblasts, 27, 88, 89, 105, 145, 164, 171, 175, 179,
149, 152, 169, 187, 263, 413, 414, 431
192, 212, 222, 237, 248, 274, 283, 334, 351, 431,
fungus, 241, 282
459, 462, 466, 477, 481, 482
fusion, 103, 112, 463
fibrosarcoma, 420
fibrosis, 104
filament, 166, 168, 176 G
film formation, 227, 309
films, 37, 56, 70, 88, 89, 98, 101, 102, 124, 126, 145, gadolinium, 353, 405, 478
147, 149, 150, 158, 159, 164, 169, 171, 180, 181, gamma globulin, 19
189, 191, 195, 196, 203, 204, 205, 209, 211, 213, gamma rays, 141
224, 225, 227, 237, 239, 242, 245, 247, 248, 249, gas diffusion, 201
250, 251, 258, 268, 275, 294, 298, 326, 352, 423, gastrointestinal related ailments, x, 413
441, 458, 459, 474, 475 gastrointestinal tract, 105, 111, 374, 379, 417, 418,
filters, 21, 38, 162, 276, 290, 468 428, 433, 445
filtration, 11, 32, 38, 39, 48 gel, 19, 31, 32, 36, 37, 67, 86, 88, 98, 103, 108, 123,
fish, 38, 136, 191, 414 143, 149, 160, 178, 182, 226, 227, 229, 236, 249,
fishery industries, vii, 1 252, 404, 421, 423, 431, 433, 440, 447, 455, 456,
flexibility, 80, 230, 232, 237, 414, 455, 458, 459, 457, 458, 459, 462, 463, 464, 474, 476
466 gel formation, 236
flocculation, 223, 225, 300, 302, 303, 305, 306 gelation, 83, 84, 94, 98, 157, 195, 204, 242, 325,
flora, 32, 105 326, 328, 331, 333, 334, 343, 369, 370, 371, 375,
Flory-Huggins, 155 376, 377, 378, 380, 383, 387, 389, 390, 391, 392,
fluid, 29, 94, 158, 209, 332, 376, 377, 384, 385, 391, 394, 395, 396, 398, 399, 400, 406, 407, 420, 423,
395, 396, 400, 420, 435, 460 424, 430, 446, 460, 462, 464, 476
494 Index
gene expression, 234, 334, 335, 338, 339, 340, 343, 274, 284, 298, 322, 340, 347, 359, 403, 431, 433,
344, 359, 431, 462 453, 454, 459, 460, 461, 462
gene silencing, 342, 343, 360 growth factor, 38, 104, 105, 110, 222, 224, 403, 453,
gene therapy, 103, 110, 121, 131, 178, 201, 232, 233, 454, 460, 461
236, 334, 341, 353, 358, 359, 421, 437 growth hormone, 8, 13
gene transfer, 253, 294, 336, 339, 342, 358, 441, growth rate, 3, 237
442, 443 guidance, 421
generally recognized as safe (GRAS), xi, 449 guidelines, 63
genes, 178, 334, 337, 342, 420, 421, 429, 433, 461
genetic engineering techniques, vii, 1
genetic information, viii, 217, 232, 244, 340 H
genome, 276
glass transition, 27, 164 H. pylori, x, 413, 439
glass transition temperature, 27, 164 H. pylori infection, x, 413
glaucoma, 430 habitat, 136
glioblastoma, 434 hair, 84, 135, 166, 450, 468
global demand, 2 hair follicle, 450
glow discharge, 104 halogen, 60, 265
glucoamylase, 230 harvesting, 11, 60
glucose, 7, 28, 31, 65, 111, 147, 152, 184, 230, 242, hazardous substances, 151
329, 333, 377, 379, 380, 383, 384, 391, 392, 393, H-bonding, viii, 71, 155, 223
394, 398, 401, 414, 440 healing, ix, x, 55, 56, 88, 104, 105, 108, 110, 111,
glucose oxidase, 31 119, 121, 122, 135, 142, 143, 145, 151, 161, 164,
glue, 108, 466 169, 173, 175, 189, 191, 192, 193, 197, 198, 207,
glutamate, 74, 80, 106, 252, 343, 370, 452, 472 208, 212, 214, 224, 228, 236, 248, 249, 261, 263,
glutamic acid, 166, 233, 241, 244, 253, 328, 336, 290, 293, 313, 322, 413, 417, 438, 442, 447, 454,
355, 358, 370, 377, 378, 380 455, 457, 460, 461, 465, 466, 467, 468, 469, 471,
glutathione, 79, 98, 113, 114 472, 474, 475, 477
glycerol, 27, 146, 191, 204, 241, 456, 459 health, 24, 62, 156, 186, 208
glycine, 30, 78 health care, 24, 208
glycogen, 222 heat transfer, 236
glycol, 28, 85, 103, 118, 122, 123, 125, 128, 130, heavy metals, 28, 32, 33, 48, 151, 434, 468
140, 165, 222, 224, 227, 242, 247, 250, 331, 334, height, 20, 272, 293
346, 347, 353, 354, 357, 361, 362, 367, 371, 377, helical conformation, 129
382, 406, 442, 455, 463 Helicobacter pylori, 446
glycoproteins, 75, 76, 120, 237, 330, 416, 435 hemocompatibility, 296
glycosaminoglycans, 167, 169, 222, 247, 248, 322, hemodialysis, 224
454 hemophilia, 340, 341, 359
gold nanoparticles, 65, 124, 127, 384, 394, 407 hemostasis, 467, 468
GPC, 19, 148 hemostatic properties, 297, 312, 313, 465
gracilis, 276 hepatitis, 28, 64, 340, 348, 359, 367, 377, 378, 386,
graft copolymerization, 72, 100, 103, 104, 113, 114, 390, 391, 396, 408, 410, 432
116, 117, 120, 121, 130, 155, 185, 200, 435 hepatocellular carcinoma, 30
grafting, viii, 71, 90, 100, 101, 102, 104, 113, 117, hepatocytes, 234, 349, 359
119, 120, 121, 122, 124, 131, 148, 155, 170, 255, hepatoma, 347, 434
339 herbal medicine, 63
Gram-negative bacteria, 69 heterogeneity, 43
granules, 418, 466 hexane, 423
grass, 135, 166 high chitosan-yielding fungi, vii, 1
Greece, 211 high strength, 168
growth, vii, 1, 3, 7, 8, 13, 22, 29, 38, 39, 59, 60, 89, hip arthroplasty, 116
104, 105, 110, 166, 179, 222, 224, 225, 237, 242, histamine, 334, 398, 400
histidine, 186, 347, 362
histogram, 305, 306
Index 495
history, 151, 220

HIV, 232, 385, 394, 432 I
HIV-1, 385, 394
ibuprofen, 86, 88
homogeneity, 13, 143, 162, 226
ideal, 7, 51, 52, 53, 165, 240, 322, 332, 342
homopolymers, 300
identification, 180
Hong Kong, 193, 201
immersion, 174, 309
hormone, 8, 37, 38
immobilization, viii, 31, 36, 37, 66, 67, 74, 75, 76,
hormones, xi, 8, 21, 37, 38, 57, 60, 450
104, 125, 217, 252
host, 194, 221, 246, 300, 301, 433
immune reaction, 366
human body, 32, 135, 147, 151, 166, 169, 452, 468
immune response, x, 112, 178, 331, 335, 341, 356,
humidity, 28, 99, 150, 240
374, 385, 391, 394, 395, 396, 408, 410, 427, 433,
Hunter, 215
449, 452, 462
Hyaluronic acid (HA), 336
immune system, 102, 463
hybrid, ix, 30, 38, 63, 65, 98, 120, 124, 126, 178,
immunity, 331, 341, 378, 385, 408
179, 214, 215, 237, 255, 259, 261, 263, 277, 283,
immunization, 28, 64, 331, 359, 408, 432
287, 289, 291, 313, 392, 394, 399, 409, 447, 472
immunogenicity, viii, 133, 134, 151, 328, 354, 359,
hydrocortisone, 457
462, 468
hydrogels, ix, 28, 29, 64, 86, 88, 94, 98, 102, 103,
immunoglobulin, 30, 378, 391
113, 121, 122, 128, 130, 211, 212, 213, 219, 220,
immunostimulatory, 395
226, 228, 240, 245, 248, 250, 251, 252, 256, 257,
immunosuppression, 385
263, 274, 294, 321, 322, 328, 353, 354, 406, 420,
immunotherapy, 463
428, 430, 434, 437, 444, 455, 456, 457, 458, 464,
implants, 115, 135, 151, 166, 224, 294, 351, 421,
471, 472, 473
451
hydrogen, 30, 34, 39, 65, 98, 137, 152, 153, 154,
incompatibility, 29
155, 156, 162, 165, 167, 170, 177, 218, 221, 224,
increased access, 432
225, 236, 238, 239, 245, 268, 270, 272, 300, 306,
incubation period, 150
335, 429, 452
incubation time, 349
hydrogen bonds, 153, 156, 162, 165, 167, 177, 218,
indium, 31
224, 225, 238, 270, 306
induction, 112, 148, 257, 374, 433
hydrogen peroxide, 30, 39, 65, 137
induction time, 148
hydrolysis, 89, 96, 139, 140, 141, 143, 159, 168,
industrial use of fungi, vii, 1
206, 241, 266, 340, 414
industries, vii, 1, 33, 151, 226, 245
hydrophilic materials, 83
industry, vii, ix, 1, 2, 3, 26, 89, 90, 136, 154, 218,
hydrophilicity, 105, 221, 313, 328, 372, 424
220, 239, 365, 366, 406, 469
hydrophobic properties, 99
infection, x, 22, 64, 328, 341, 344, 389, 413, 426,
hydrophobicity, 101, 122, 242, 284, 312, 415
427, 432, 446
hydroxide, 2, 34, 88, 135, 138, 139
inflammation, 146, 194, 231, 344, 427, 434, 466
hydroxyapatite, 28, 55, 56, 64, 70, 171, 212, 226,
inflammatory bowel disease, 434
248, 255, 351
inflammatory cells, 431, 454
hydroxyethyl methacrylate, 105, 114, 120, 126, 430
inflammatory disease, 349
hydroxyl, viii, 33, 40, 41, 71, 72, 73, 83, 84, 85, 98,
influenza, 110, 115, 432, 439
129, 130, 133, 141, 145, 152, 154, 155, 238, 244,
influenza vaccine, 115, 439
321, 414, 451
influenza virus, 110, 432
hydroxyl groups, viii, 33, 40, 41, 71, 72, 73, 85, 98,
infrared spectroscopy, 56, 61, 147
133
infrared spectroscopy (FTIR), 56
hyperplasia, 349
ingredients, 76
hypertension, 26, 432
inhibition, 79, 104, 108, 112, 142, 228, 274, 298,
hyperthermia, 284
343, 346, 431, 442, 457, 459, 463, 466
hypothesis, 45, 46
inhibitor, 27, 63, 117, 340
hysteresis, 286
inhomogeneity, 231
insects, vii, 2, 7, 13, 72, 135, 136, 152, 263, 321, 414
insertion, 164, 469
496 Index
insulin, 28, 64, 82, 94, 114, 119, 125, 224, 237, 255,
328, 329, 330, 331, 332, 333, 353, 355, 356, 367, J
370, 375, 376, 377, 379, 380, 381, 382, 383, 384,
jejunum, 108, 329
390, 391, 392, 393, 394, 398, 400, 402, 404, 406,
joints, 344
407, 408, 409, 410, 419, 428, 432, 440, 444, 447,
Jordan, 446
460, 478
integrity, 104, 135, 152, 292, 331, 338, 371, 375,
376, 377, 379, 380, 385, 390, 393, 427, 460 K
interface, 56, 65, 149, 210, 241, 303, 471
interfacial adhesion, 210 KBr, 17
interference, 63, 342, 360 keratin, 237, 255, 432
interferons, 435 keratinocyte, 464, 466
intermolecular interactions, 28, 218, 240, 268 keratinocytes, 179, 215, 222, 224, 236, 248, 466
internal fixation, 210 ketones, viii, 71, 77, 155
internalised, 377, 378, 385, 390 kidney, 32, 110, 147, 178
internalization, 233, 336, 338, 347, 348, 349, 422 kinetic model, 50
interpolyelectrolyte complexes, viii, 217, 219, 246, kinetic studies, 67
248 kinetics, 34, 47, 66, 107, 114, 460, 461, 476
intestinal tract, 379
intestine, 28, 72, 328, 379, 419, 427, 451
intoxication, 231 L
intramuscular injection, 384
intraocular, 438 labeling, 147, 169
intravenously, 108, 116, 144, 169, 334 lactate dehydrogenase, 145, 164, 379
intrinsic viscosity, 18, 142, 155, 159, 163, 239 lactic acid, 2, 6, 83, 84, 101, 103, 112, 124, 125, 129,
inversion, 265, 385, 460 131, 145, 150, 156, 167, 179, 193, 196, 215, 334,
invertebrates, 32 357, 458, 465, 469, 476
iodine, 113, 467 lactose, 99, 107, 108, 332, 348, 419, 420, 446
ion adsorption, 67 Langerhans cells, 356
ion-exchange, 240, 258 large intestine, 74
ionic strength, ix, 61, 143, 159, 188, 205, 218, 219, L-arginine, 110
223, 224, 228, 229, 231, 234, 235, 238, 240, 245, lead, 32, 46, 274, 276, 298, 300, 328, 329, 336, 339,
253, 254, 261, 262, 270, 271, 301, 302, 303, 305, 397, 421
306, 337, 452 lecithin, 360, 376, 377, 378, 384, 462
ionicity, 220 lesions, 341, 431, 455
ionizable groups, 277 leucine, 112
ionization, 157, 268, 337 liberation, 239, 242
ions, viii, 14, 21, 32, 33, 34, 37, 43, 48, 66, 67, 71, life expectancy, 55
90, 104, 146, 171, 175, 213, 270, 284, 285, 292, ligament, 259
331, 339 ligand, 232, 338, 347, 349, 362, 434
IR spectra, 18, 239, 267, 268 light, 18, 19, 20, 45, 46, 108, 143, 148, 159, 160,
IR spectroscopy, 61, 88, 161, 207, 240 189, 205, 225, 237, 241, 262, 270, 271, 397, 462
iron, ix, 87, 99, 112, 261, 263, 284, 285, 286, 313 light scattering, 18, 19, 20, 143, 159, 160, 189, 205,
irradiation, 101, 116, 146, 211, 251, 346 225, 237, 241, 262, 271
isoelectric point, ix, 40, 236, 242, 261, 262, 263, light transmittance, 148, 270
268, 269, 272, 273 linear dependence, 230
isolation, 13, 59, 135, 137, 152, 157, 239, 242, 244, linear glycosaminoglycan, x, 449, 451
451 linen, 135, 166
isotherms, 34, 43, 44, 45, 46, 47 linoleic acid, 107, 127
isozymes, 332 lipases, 164, 208, 241
issues, 350, 387, 402, 409 Lipases, 241
Italy, 107, 134, 186, 199, 202, 205, 212 lipids, 11, 12, 21, 32, 72, 136, 137, 241, 433, 444,
463
Index 497
lipoproteins, 230 magnetic properties, 64, 287

liposomes, 219, 328, 334, 356, 366, 386, 411, 414, magnetic resonance, 14, 284
432, 440, 463, 464 magnetic resonance imaging, 284
liquid chromatography, 67, 189, 205 magnetic resonance spectroscopy, 14
liquid crystals, 98, 168, 235 magnetization, 284, 286
liquid phase, 16, 43, 48 majority, 110, 219, 221, 222, 231, 389, 393
liquids, 13, 202 mammalian cells, 28, 73, 178, 237, 444
lithium, 126, 140, 158, 189, 202, 210 management, 165, 197, 212
liver, 24, 32, 99, 107, 108, 110, 118, 129, 147, 234, manganese, 113
337, 338, 341, 346, 347, 348, 357, 362, 434, 451 mannitol, 74, 80, 82, 332, 387, 398, 400, 401, 402,
liver cancer, 347 411, 416, 478
liver cells, 234, 338, 347 manufacturing, 14, 34, 138, 161, 164, 191, 204, 206,
local anesthetic, xi, 450 415, 438
localization, 340, 347, 450, 462 mapping, 292, 293
low immunogenicity, viii, 133, 134, 151, 468 marketing, 63, 469
low temperatures, 226 mass, 16, 34, 37, 47, 50, 51, 54, 89, 135, 141, 164,
low-density lipoprotein, 230 191, 219, 221, 226, 227, 229, 232, 233, 234, 237,
luciferase, 232, 234, 335, 339, 340, 431, 462 238, 240, 242, 277, 370, 380, 382, 399, 402, 411
lumbar spine, 112 mass loss, 240
lumen, 328, 330 mass spectrometry, 402, 411
lung cancer, 360 mast cells, 334, 398, 400
lung disease, 363 material surface, 232, 292
Luo, 65, 121, 255, 314, 475 matrix, ix, x, 27, 29, 31, 32, 43, 64, 65, 66, 69, 94,
lutein, 180 101, 103, 105, 110, 112, 113, 119, 122, 124, 126,
lymph, 348, 463 136, 150, 160, 163, 166, 170, 179, 193, 210, 220,
lymph node, 463 240, 248, 252, 261, 323, 329, 348, 413, 418, 421,
lymphocytes, 341 424, 427, 432, 436, 441, 443, 447, 457, 461, 463,
lymphoid, 395, 396, 427 464, 465, 466, 469, 476
lymphoid tissue, 395, 396, 427 matrix tablet formulations, 427
lysine, 100, 149, 195, 243, 244, 433, 435 matrixes, 322
lysis, 163 maximum sorption, 35, 48
lysosome, 340, 342 measurement, 30, 31, 210, 233, 463
lysozyme, 12, 21, 87, 89, 102, 108, 116, 141, 147, measurements, 10, 20, 28, 31, 66, 162, 203, 214,
164, 168, 169, 175, 211, 213, 236, 332, 334, 356, 225, 233, 254, 270, 286, 380, 402
390, 416, 430 mechanical properties, 89, 95, 99, 151, 161, 167,
168, 171, 175, 176, 177, 178, 191, 199, 207, 208,
211, 212, 237, 239, 240, 326, 351, 456, 458, 459,
M 466
mechanical stress, 8
macromolecular chains, 236, 239 media, 6, 10, 13, 68, 72, 76, 78, 88, 94, 227, 229,
macromolecular networks, 455 236, 237, 239, 241, 252, 263, 284, 298, 336, 368,
macromolecules, 71, 94, 98, 105, 122, 162, 178, 218, 375, 386, 441, 455, 456
219, 220, 226, 230, 231, 232, 240, 242, 245, 268, medical, 14, 24, 26, 30, 96, 144, 146, 151, 161, 169,
269, 271, 272, 294, 326, 328, 347, 366, 368, 369, 174, 193, 196, 197, 200, 209, 212, 213, 220, 226,
388, 389, 392, 394, 395, 397, 403, 409, 427, 435 245, 294, 322, 334, 350, 414, 435
macrophages, 72, 104, 105, 218, 242, 332, 337, 344, medical science, 144
347, 356, 360, 397 medicine, viii, 32, 55, 110, 135, 179, 187, 217, 218,
macro-scale applications, vii, 1, 26 220, 235, 239, 241, 243, 245, 276, 284, 469, 470,
magnesium, 104, 375, 377, 378, 380, 437 472
magnet, 151, 285 medium composition, 9
magnetic field, 15, 28, 284 melanoma, 109, 223, 352
magnetic materials, 284, 289 melt, 73, 150, 276
magnetic moment, 15 melting, 73, 166
magnetic particles, 286, 288, 289
498 Index
membranes, ix, 27, 32, 39, 61, 89, 96, 98, 104, 105, microspheres, x, 72, 90, 91, 92, 103, 106, 107, 111,
114, 120, 126, 134, 146, 163, 175, 179, 193, 208, 113, 114, 119, 122, 123, 124, 149, 192, 196, 197,
214, 224, 227, 233, 236, 237, 239, 240, 243, 245, 213, 251, 252, 323, 332, 350, 353, 356, 366, 383,
248, 250, 251, 254, 255, 256, 257, 258, 283, 294, 398, 400, 401, 402, 406, 411, 413, 414, 418, 420,
321, 322, 339, 365, 367, 387, 423, 455, 458, 459, 424, 425, 427, 429, 430, 431, 434, 436, 437, 438,
474, 475 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,
mesenchymal stem cells, 113, 229, 233 458, 460, 461, 470, 471, 473, 475, 476, 477, 478
messenger RNA, 342 microstructure, 157, 224, 241, 351
metabolic disorder, 327 microwave radiation, 101
metabolic disorders, 327 migration, 29, 48, 105, 232, 346, 419, 463
metabolism, 7, 111, 237, 331, 369, 386, 387, 388, mineralization, 69, 86, 88, 139
397, 400, 426 Minneapolis, 69, 186
metabolites, 11, 38 mitochondria, 38
metal ion, 14, 21, 32, 33, 34, 37, 48, 67, 78, 86, 127 Mitoxantrone, 352, 426
metal ions, 14, 21, 32, 33, 34, 37, 48, 67 mixing, 6, 171, 219, 223, 226, 227, 228, 229, 230,
metal nanoparticles, 283, 313 233, 240, 241, 242, 301, 302, 303, 305, 306, 329,
metal oxides, 31, 283 381
metal salts, 240 models, 33, 34, 42, 43, 44, 46, 47, 48, 54, 55, 96,
metals, viii, 28, 32, 33, 71, 73, 155, 202, 240 114, 232, 239, 284, 379, 410, 432, 465, 470
metastasis, 118 modifications, viii, 34, 62, 89, 107, 133, 141, 145,
meter, 16methacrylic acid, 127, 353, 367, 371, 377, 147, 155, 168, 185, 186, 190, 201, 339, 344, 405
381 modulus, 27, 168, 176, 185
methanol, 19, 27, 63, 96, 98, 147, 164, 171, 176, moisture, 20, 25, 151, 170, 171, 176, 199
227, 240, 324 moisture content, 20, 176
methodology, 59, 155, 179, 323, 471 molar ratios, 224, 241
methyl cellulose, 168, 459 molasses, 2, 5, 6, 7
methyl group, 80, 224 mold, 178
methyl groups, 80, 224 molds, vii, x, 1, 413
methyl methacrylate, 119, 330 mole, 243, 296, 302
methylation, 79, 84, 95, 96 molecular biology, 60, 256
methylene blue, 31 molecular mass, 203, 211, 218, 219, 223, 227, 229,
methylene chloride, 158, 163, 373 231, 232, 233, 234, 235, 238, 242, 245, 253, 254,
Miami, 408 358, 446, 451
mice, 68, 108, 110, 118, 122, 147, 149, 150, 169, molecular structure, 34, 134, 229, 230, 353, 406,
175, 194, 211, 213, 227, 249, 258, 341, 344, 346, 448, 452
349, 359, 360, 361, 362, 381, 384, 385, 386, 394, molecular weight distribution, 89, 115, 189, 191,
395, 396, 408, 410, 417, 431, 432, 440, 443, 444, 204, 205
446, 453, 457, 462, 474, 476 molecules, viii, ix, 8, 17, 19, 21, 33, 43, 45, 46, 47,
microcrystalline, 62, 106, 140, 188, 419 48, 50, 71, 82, 84, 89, 94, 101, 110, 114, 122,
microcrystalline cellulose, 106, 419 141, 158, 165, 180, 186, 203, 218, 226, 231, 233,
microemulsion, 326, 377, 383, 423, 424 234, 235, 236, 238, 254, 300, 323, 327, 328, 347,
microgels, 130, 246, 354 365, 366, 367, 368, 369, 372, 374, 380, 387, 388,
micrometer, 332 394, 396, 399, 402, 406, 408, 416, 421, 422, 427,
microorganism, 274, 313 435, 450, 455, 456, 457, 463, 464, 470
microorganisms, viii, 8, 30, 39, 90, 92, 146, 148, mollusks, x, 136, 413, 449
222, 231, 261, 274, 284, 290, 297, 298, 300, 310, momentum, 48
313, 322, 419, 450, 456 monoclonal antibody, 125
micropatterns, 237 monolayer, 43, 45, 47, 120, 146
microphotographs, 162 monomers, 33, 80, 105, 219, 220, 240, 327, 371,
microscope, 57 372, 375, 381, 430
microscopy, 162, 206, 232, 237, 254, 262, 271, 380, monosaccharide, 98, 152, 237
395, 399, 437 Moon, 25, 63, 247
morphine, 127
Index 499
morphogenesis, 464 N-deacetylated chitin, 61

morphology, 29, 56, 89, 136, 164, 176, 184, 208, negative effects, 323
223, 231, 237, 240, 277, 278, 280, 282, 293, 311, neglect, 43
312, 341, 460 neoplasm, 56
mortality, 32 nerve, 29, 64, 121, 178, 214, 351, 450
Moscow, 217, 253 Netherlands, 254
MRI, 111 neurons, 389
mRNA, 37, 342 neuropeptides, 29
mucin, 31, 66, 115, 142, 238, 256, 330, 331, 339, neutral, viii, 13, 39, 40, 71, 73, 74, 78, 79, 84, 85, 95,
416, 437, 461 96, 99, 101, 119, 141, 145, 157, 164, 204, 227,
mucoadhesivity, viii, 71, 80, 110, 128, 429, 433 233, 237, 239, 261, 263, 296, 297, 298, 300, 303,
mucosa, 75, 114, 131, 329, 331, 349, 362, 387, 388, 327, 329, 330, 335, 337, 415, 442, 452, 455, 457
389, 391, 392, 393, 395, 403, 408, 427, 429, 431 neutrons, 15
mucosal routes, ix, 366, 369, 388, 389 neutrophils, 105, 422, 454
mucus, 75, 76, 80, 120, 126, 329, 330, 332, 349, 374, NH2, 33, 40, 99, 138, 157, 267, 281, 298, 452
375, 379, 388, 404, 407, 416, 436 nicotinamide, 32
multilayered structure, 250 nitric oxide, 66
multiplication, 222 nitrogen, 45, 46, 141, 147, 150, 152, 156, 169, 177,
multiwalled carbon nanotubes, 32, 66 233, 304, 309, 336, 371, 452
muscles, 151, 468 Nuclear Magnetic Resonance (NMR), 15, 16, 61,
mushrooms, vii, 1, 2, 3, 4, 57, 135, 136, 414 126, 136, 147, 158, 161, 170, 204, 207, 223, 243,
mutagenesis, 433 264, 265, 266, 267, 353
Muzzarelli and Hirano, vii, 1, 57 noble metals, 33
mycelium, 3, 6, 10, 59 nodes, 158
non-ionogenic polymer, ix, 261, 263, 281, 293
non-viral vectors, viii, 217, 232, 234, 236, 335
N norepinephrine, 31
novel materials, 179, 294, 301, 306, 313
Na2SO4, 168, 377, 378, 390, 391 nuclei, 15
N-acetylglucosamine, x, 15, 109, 120, 154, 237, 413, nucleic acid, viii, 71, 217, 234, 254, 342, 343, 422
454 nucleotides, 92, 129, 342
NaCl, 43, 142, 159, 224, 238, 271 nucleus, 38, 103, 142, 221, 237, 336, 340, 347, 444
NAD, 32 nutrients, 8, 25, 187
NADH, 32, 66 nutrition, 11, 25
nanobelts, 30
Nanocarriers, v, 328, 365, 375
nanocomposites, 31, 64, 65, 189, 210, 351 O
nanofibers, ix, 184, 203, 215, 240, 261, 277, 278,
279, 280, 281, 282, 283, 287, 291, 292, 293 obstacles, 331, 332
nanofibrous materials, ix, 261, 263, 276, 277, 278, obstruction, 340
281, 282, 283, 290, 292, 293, 313 ofloxacin, 276, 430, 431, 438
nanofibrous membranes, 263 OH, 17, 33, 42, 46, 47, 48, 281, 452
nanomedicine, 179, 422 OH-groups, 42
nanometer, 328, 423 oil, 102, 103, 140, 196, 239, 324, 326, 353, 424, 425,
nano-particles, vii, 417 442
nanoparticles preparation, ix, 261, 263, 370, 371, old age, 171
372, 373, 375 oleic acid, 284, 376, 377, 383
nanostructures, 184, 362, 369, 393 oligomers, 109, 127, 141, 246, 265, 335, 357, 431,
nanotechnology, 179, 354 441, 442, 452, 471, 472
nanotube, 27, 31, 63 oligosaccharide, 198, 231, 237, 333, 339, 347, 354,
natural compound, 215 361, 409
natural polymers, viii, 133, 148, 195, 202, 213, 277, olive oil, 227
290, 300, 301, 403, 415, 442, 452, 460, 465, 469 operations, 55, 128, 152
N-carboxyethylchitosan (CECh), viii, 261, 263 opportunities, 186, 202, 211, 464
500 Index
optical activity, 235 393, 400, 403, 405, 406, 408, 409, 410, 427, 436,
optical properties, 235 440, 442, 447, 448, 450, 451, 465, 473
optimization, 196, 249, 252 peptide absorption, xi, 389, 450, 451
oral cavity, 426, 433, 441, 445 peptides, 75, 119, 232, 301, 328, 332, 333, 367, 368,
organ, x, 344, 414, 421, 450 369, 388, 389, 392, 405, 410, 420, 421, 422, 427,
organic compounds, 43 429, 443
organic solvents, 72, 83, 84, 98, 137, 151, 156, 157, perchlorate, 67
189, 322, 323, 344, 371, 373, 460 periodontal, 104, 322
organism, 6, 39, 137, 231, 452 peritoneal cavity, 431
organs, 38, 224, 249, 344, 348, 420 permeability, 105, 108, 114, 118, 119, 127, 128, 149,
oscillation, 17 172, 191, 193, 225, 231, 239, 240, 257, 322, 328,
osmosis, 39 329, 339, 344, 345, 346, 349, 355, 360, 366, 374,
osmotic pressure, 227 375, 380, 387, 389, 403, 416, 417, 427, 436, 438,
Osteogenesis, 123 451, 452, 458, 460, 463, 469, 472
osteogenic cells, 104 permeable membrane, 239
oxalate, 31, 66 permeation, 14, 19, 72, 75, 76, 79, 88, 112, 113, 120,
oxidation, 65, 66, 68, 74, 79, 138, 177, 284 127, 142, 143, 160, 180, 258, 330, 342, 368, 374,
oxidation products, 68 387, 388, 389, 393, 402, 408, 416, 417, 427, 430,
oxidative reaction, 141 431, 432, 433, 435, 440, 445, 448, 450, 457, 459,
oxidative stress, 30 461, 462, 464, 474
oxide nanoparticles, 112, 313, 405, 437 permit, ix, 365, 366, 368, 373, 374, 381, 387, 402
oxygen, 7, 39, 45, 46, 101, 104, 117, 140, 149, 284, peroxidation, 32
420 peroxide, 30, 212
oxygen plasma, 104 pertussis, 440
ozone, 39, 68, 104, 116 PET, 104, 116, 179, 215
phagocytosis, 241, 332, 356
pharmaceutical, vii, ix, x, 1, 14, 24, 57, 63, 110, 116,
P 119, 134, 186, 239, 245, 294, 334, 354, 365, 366,
368, 405, 406, 407, 409, 410, 413, 414, 418, 435,
PAA, 225, 238, 239, 240, 256, 262, 284, 294, 295, 446, 461, 471, 472
346, 375, 377 pharmaceutical industry, vii, ix, 239, 365, 406
Pacific, 3 pharmaceutical research, vii, 1, 14
paclitaxel, 85, 118, 124, 130, 347, 354, 361, 362, pharmaceuticals, x, 68, 69, 74, 413, 447
440, 443, 444, 445, 459, 475 pharmacokinetics, 102, 344, 446, 476
pain, 387, 470 pharmacology, 240
palladium, 125 phase inversion, 431
parallel, 11, 33, 153, 170, 199, 289 PHB, 149
parasites, 108 phenol, 19, 91, 242
parenchyma, 349 phenolic compounds, 90
partial thromboplastin time, 275 phenylalanine, 31, 100
partition, 69, 80, 451, 452 phosphate, 28, 29, 56, 64, 70, 88, 105, 115, 123, 137,
patents, 134, 158, 415 146, 149, 157, 171, 204, 231, 284, 336, 342, 343,
pathogenic microorganisms, viii, 261, 274, 289, 297, 351, 353, 377, 385, 406, 425, 437, 443, 456, 464,
298, 300, 310, 313 476
pathogens, 374, 389 phosphates, 351, 369
pathophysiological, 345 phosphatidylcholine, 464
pathways, 328, 338, 389, 450 phospholipids, 393, 402
pendant model, 33 phosphorus, 233
pepsin, 258, 376, 384 phosphorylation, 79, 127
peptidase, 333 photoelectron spectroscopy, 411
peptide, xi, 28, 74, 75, 76, 79, 115, 116, 119, 122, photographs, 177
127, 232, 247, 333, 334, 341, 344, 355, 356, 357, phthalate, 106, 113, 171, 411
360, 367, 375, 376, 379, 380, 381, 383, 389, 392, physical and mechanical properties, 72, 100
Index 501
physical characteristics, 356 polymer concentration, ix, 156, 218, 226, 227, 261,
physical interaction, 466 272, 273, 274, 278, 287, 288, 289, 291, 292, 293,
physical properties, 141, 154, 164, 203, 210, 214, 301, 302, 303, 305, 306, 381, 458
241, 257 polymer films, 129, 239
physicians, 135 polymer materials, 263, 290, 294, 296
physicochemical characteristics, 60, 187, 190, 194, polymer matrix, 103, 149, 240
225, 254, 263, 264, 372, 380, 475 polymer mixing, 381
physicochemical properties, x, 74, 89, 98, 138, 189, polymer molecule, 80
195, 200, 208, 214, 221, 239, 241, 242, 323, 324, polymer networks, 130, 256, 447
356, 359, 386, 415, 435, 449, 451 polymer properties, 415
physiology, 175, 198 polymer solubility, 155
plankton, 136, 414 polymer solutions, 80, 226, 229, 230, 303, 306
plant growth, 60 polymer structure, 34
plants, 7, 8, 30, 32, 38, 68, 135, 151, 152 polymer systems, 219
plasma levels, 419 polymeric chains, 455
plasma membrane, 71, 416 polymeric composites, 419
plasma proteins, 337 polymeric materials, 167, 244
plasmid, 107, 178, 232, 234, 235, 253, 340, 357, polymeric products, 436
358, 360, 424, 436, 440, 442, 462 polymerization, 34, 100, 101, 102, 105, 138, 140,
plasticity, 104 170, 241, 302, 305, 326, 381
plasticizer, 458, 459 polypeptides, 377, 380
plastics, 125 polypropylene, 105, 161, 171, 173, 213, 371
platelet count, 275, 300 polysaccharide, vii, x, 88, 89, 100, 114, 136, 141,
platelets, 299, 300, 312 193, 196, 200, 203, 218, 223, 225, 226, 231, 232,
Poland, 161, 162, 163, 169, 207 235, 236, 237, 238, 259, 284, 321, 323, 356, 380,
polar, viii, 22, 71, 155, 158, 159, 219, 427 386, 404, 407, 414, 418, 444, 449, 452, 472
polar media, 219 polysaccharide chains, 231, 232
polarity, 73, 155 Polysaccharides, 11, 69, 123, 126, 186, 188, 193,
polarization, 238 198, 200, 222, 350, 360
pollutants, 39, 69 polystyrene, 101, 164, 241, 257, 411
poly(vinyl chloride), 105, 122 polyurethane, 197, 458
polyacrylamide, 19, 103, 117, 256, 262, 277 polyvinyl alcohol, 27, 455
polyanions, viii, 73, 142, 217, 218, 219, 221, 230, population, 55
241, 245, 259, 263, 268, 269, 295, 297, 368, 415 Porifera, 215
polycondensation, 100 porosity, 51, 55, 171, 175, 213, 232, 237, 253, 276,
polydimethylsiloxane, 101 381
polydispersity, 14, 143, 152, 159, 218 port of entry, 450
polyelectrolyte complex, viii, ix, 27, 142, 217, 218, Portugal, 321, 350, 365
219, 220, 223, 224, 225, 226, 227, 229, 230, 233, positive correlation, 75
236, 241, 245, 246, 247, 248, 249, 250, 251, 252, potassium, 2, 17, 100, 135, 139, 177, 371
253, 254, 255, 256, 257, 258, 259, 261, 262, 263, potassium persulfate, 371
268, 300, 313, 326, 329, 333, 369, 370, 381, 382, potato, 4, 5, 6, 62, 419, 446
383, 404, 407, 423, 452, 455, 459, 474 pranlukast, 411
polyelectrolyte complexes (PECs), ix, 261, 268 precipitation, 32, 120, 157, 170, 223, 224, 226, 230,
polyesters, 411 239, 284, 300, 324, 372, 373, 424, 425, 460, 462
polyether, 371 preservation, 25, 312, 382
polyhydroxybutyrate, 164 prevention, 222, 336, 349
polymer blends, 164, 208, 473 priming, 384
polymer chain, 80, 141, 147, 218, 219, 225, 271, principles, 43, 112, 180, 468
284, 335 probability, 50, 332
polymer chains, 141, 225, 271, 335 probe, 31, 177, 271, 273, 349
process control, 219
producers, 6, 58
502 Index
production costs, 6, 402 quantum dot, 463

profitability, 6 quantum dots, 463
progesterone, 432, 441, 453 quartz, 37
pro-inflammatory, 344 quaternary ammonium, 88, 117, 118, 177, 309, 431
project, 169, 185, 350, 403 quinone, 148
proliferation, 29, 68, 104, 105, 145, 146, 164, 175,
192, 212, 222, 224, 226, 232, 237, 255, 283, 351,
420, 454, 458, 466 R
promoter, 155, 240
proposition, 143, 159 radiation, 20, 101, 105, 120, 121, 124, 130, 141, 163
propranolol, 457 Radiation, 101
propylene, 84, 85, 227, 242, 353, 423, 476 radical polymerization, 256, 262, 297, 330, 420
prostheses, 151 radicals, 32, 38, 249
prosthesis, 179 radioactive isotopes, 33
prosthetic device, 151 radius, 158, 342, 343
protease inhibitors, 332 radius of gyration, 158
protection, ix, 72, 121, 135, 152, 335, 339, 340, 342, radula, 136
360, 365, 366, 374, 379, 383, 389, 407, 433, 468 raw materials, vii, 2, 13
protective role, 180 reactant, 231, 238, 244
protein components, 135 reactants, 107, 228
protein kinase C, 472 reaction mechanism, 34
protein synthesis, 8, 142 reaction medium, 225, 284, 371
proteins, viii, ix, x, 11, 12, 14, 21, 32, 38, 66, 69, 75, reaction rate, 227
88, 101, 114, 136, 137, 138, 142, 217, 221, 231, reaction temperature, 84, 100, 101, 170, 231, 267
235, 236, 237, 241, 254, 301, 323, 327, 328, 329, reaction time, 13, 79, 100, 157, 264, 265, 267, 373
331, 332, 333, 337, 347, 350, 353, 354, 366, 368, reactions, viii, 12, 14, 33, 34, 39, 55, 71, 89, 96, 98,
369, 371, 382, 388, 393, 394, 397, 402, 404, 407, 99, 101, 103, 110, 117, 141, 155, 200, 221, 246,
410, 412, 416, 420, 421, 422, 427, 429, 443, 445, 281, 330, 453
449, 454, 460, 476 reactive groups, 34, 35, 102, 321
proteoglycans, 104, 405, 443 reactivity, 264, 265, 322
proteolysis, 327, 376 reagents, 79, 83, 84, 100
proteolytic enzyme, 386, 442 receptors, 38, 231, 234, 338, 347, 380, 454
prothrombin, 275 recognition, 38, 72, 102, 104, 231, 246, 347, 476
protons, 15, 16, 157, 339 recombination, 444
Pseudomonas aeruginosa, 104, 108, 150, 431, 459 recommendations, iv
psoriasis, 450 reconstruction, 179, 215, 237, 466, 467
PTT, 275 red blood cells, 299, 300, 311, 312
publishing, 187 redistribution, 328, 329, 416
pulp, 134, 171, 228, 244, 251 reflexes, 162
pulp cells, 244, 251 regeneration, xi, 29, 64, 103, 105, 121, 149, 161,
purification, 151, 204, 239, 284, 324 163, 164, 169, 178, 206, 214, 228, 244, 251, 283,
purity, 19, 154 313, 450, 454, 461, 465, 466, 467
PVA, 27, 63, 88, 89, 103, 175, 214, 263, 277, 278, regioselectivity, 90, 265
279, 281, 282, 283, 284, 287, 288, 289, 290, 411, regression, 18
455 reinforcement, 144, 167, 171, 178, 212
PVAc, 164 rejection, 452
PVP, 221, 337, 358 relaxation, 229
pyrophosphate, 231, 343, 360, 456 relevance, 7, 185, 212, 294, 369, 370, 400
pyruvate, 452 repackaging, 388
repair, 29, 64, 170, 182, 454, 464
reparation, 121, 180, 200, 239
Q repulsion, 40, 43, 46, 55, 73, 105, 218, 242, 277,
284, 285, 337, 394, 456
quality control, vii, 1, 63 requirements, 10, 144, 174, 177, 185, 323, 337
Index 503
researchers, x, 11, 21, 39, 85, 100, 101, 170, 219, seafood, 2, 3
238, 413, 421, 425, 432, 435, 466 seasonal, vii, 1, 2, 4, 24
residues, 2, 6, 7, 13, 15, 19, 39, 218, 223, 225, 226, seasonal flu, 2, 4
227, 234, 237, 238, 244, 264, 284, 321, 322, 330, second virial coefficient, 158
343, 416, 443, 452 secrete, 29, 138, 140
resistance, 74, 80, 139, 258, 274, 279, 281, 366, 459 secretion, 341, 351, 356, 388
resolution, 89, 308, 309 selectivity, 31, 34, 92, 225, 230, 234, 239, 328
resources, 52, 146 self-assembly, 123, 210, 223, 273, 276, 293, 327,
respiratory syncytial virus, 340, 360 330, 424
response, 7, 29, 31, 32, 59, 88, 145, 146, 147, 150, SEM micrographs, 177, 279, 307, 309, 311, 312
169, 232, 242, 248, 257, 331, 334, 377, 379, 384, semi-empirical method, 278
385, 386, 391, 392, 393, 394, 395, 400, 401, 410, semi-permeable membrane, 239
436, 471 sensing, 32, 240
response time, 31 sensitivity, 28, 30, 31, 51, 65, 102, 113, 117, 228,
restenosis, 476 240, 252, 256, 329, 340
restoration, 161, 163 sensitization, 144, 169, 341
retrovirus, 437, 444 sensors, 29, 30, 31, 32, 127, 224, 239, 240
rings, 103, 162, 222, 393 serine, 237
risk, 24, 212, 328, 340, 456 serotonin, 31
risk assessment, 212 serum, 32, 65, 101, 116, 194, 211, 233, 242, 254,
risks, 421 335, 337, 339, 341, 342, 350, 358, 367, 377, 378,
RNA, 142, 233, 342, 343, 358, 360, 448, 453 379, 384, 391, 393, 394, 433, 446, 453
RNAi, 342 serum albumin, 242, 337, 367, 377, 378, 379, 391,
rodents, 72 453
rods, 322 sewage, 68
room temperature, 15, 90, 101, 102, 137, 139, 158, sex, 136, 349
164, 165, 284, 369, 371, 373, 423, 424 shape, 29, 64, 149, 173, 202, 242, 273, 277, 286,
routes, ix, 263, 322, 328, 332, 365, 366, 368, 369, 301, 329, 367
374, 387, 388, 389, 397, 402, 408, 414, 421, 426, shear, 156, 237, 371, 373, 452
470 sheep, 29, 64, 355, 391, 394
Royal Society, 255 shellfish, 3, 136, 182, 187, 415
rubber, 167, 189 showing, 98, 147, 150, 184, 303, 307, 309, 334, 380,
Russia, 217 387, 395, 396, 402
shrimp, 6, 136, 143, 144, 159, 164, 169, 171, 187,
188, 414, 447, 451
S sialic acid, 237, 238, 330, 452
side chain, 94, 95, 98, 102, 170, 195, 214, 230, 238,
safety, 24, 63, 72, 110, 111, 134, 144, 169, 198, 355, 435
407, 417 side effects, 111, 328, 344, 349, 388, 420, 421, 430,
saliva, 147, 169, 387, 395 462
salmon, 115, 333, 446, 475 signals, 16, 267, 347
salt formation, 343 signs, 109, 381, 394
salts, 14, 60, 73, 74, 79, 80, 84, 119, 123, 152, 156, silica, 32, 36, 37, 67, 179
158, 163, 203, 221, 226, 254, 284, 300, 343, 355, silicon, 227
380, 386, 387, 405, 416, 452, 454 silk, 135, 165, 166, 167, 173, 179, 189, 208, 215,
Samsung, 24 367, 375, 381, 414, 469
saturation, 286 silver, ix, 167, 171, 175, 213, 261, 262, 263, 284,
savings, 7 290, 291, 292, 313, 431, 458, 468
scanning calorimetry, 14, 163 simulation, 10
scanning electron microscopy, 145, 164, 166, 263, siRNA, 122, 233, 254, 335, 342, 343, 344, 357, 360,
278, 401 478
scattering, 18, 20, 233, 263, 273, 305 skeleton, 56, 57, 127, 179
science, 105, 205, 245, 406, 418, 436
scope, 14, 32, 56, 368, 383
504 Index
skin, x, xi, 84, 105, 112, 162, 164, 166, 171, 172, standard deviation, 289, 382
175, 179, 180, 184, 198, 215, 224, 236, 248, 277, starch, 27, 63, 128, 150, 222, 419, 446
283, 322, 334, 351, 420, 431, 432, 441, 447, 448, stasis, 389
449, 450, 451, 452, 455, 456, 457, 458, 459, 461, state, 14, 16, 22, 61, 99, 124, 144, 156, 190, 199,
462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 203, 204, 209, 218, 219, 223, 234, 235, 275, 335,
473, 474, 475, 478 461
skin cancer, 450 sterile, 26, 281, 282
skin diseases, xi, 450 stoichiometry, 231, 232, 233, 234, 252, 298, 300,
sludge, 38, 68, 148, 274 303, 305, 336
small intestine, 328, 330, 333, 419, 420, 426 stomach, 24, 28, 115, 257, 328, 419, 426, 446, 451
smooth muscle, 237 stomatitis, 426
smooth muscle cells, 237 storage, 27, 159, 230, 285, 419, 463
smoothness, 176 streptococci, 222
society, 408 stress, 11, 30, 56, 60, 179, 237, 387
sodium dodecyl sulfate (SDS), 178 stretching, 17, 159, 161, 239
sodium hydroxide, 103, 136, 137, 138, 139, 152, stroma, 431
158, 166, 170, 324 stromal cells, 250
soft lithography, 237 strong interaction, 98, 167, 176, 336, 386, 461
sol-gel, ix, 31, 146, 149, 261, 284, 285, 287 structural defects, 220
solid matrix, 459 structural modifications, 321
solid phase, 32, 43, 47 structural protein, 341
solid state, 59, 61, 95, 98, 116, 131, 156, 161, 207, styrene, 102, 124, 243, 244
247, 278 subacute, 434
solid tumors, 108, 348, 422 subcutaneous injection, 149, 394
solvation, 372 subcutaneous tissue, 467, 469
solvents, 84, 90, 91, 92, 98, 110, 139, 140, 143, 155, subdomains, 339
156, 158, 159, 160, 161, 166, 185, 202, 209, 220, substitutes, 322
225, 242, 372, 425, 458 substitution, 29, 61, 78, 80, 84, 85, 88, 90, 96, 98,
sorption, 28, 33, 34, 36, 37, 40, 42, 43, 44, 45, 46, 99, 109, 113, 114, 141, 157, 161, 207, 224, 262,
47, 48, 49, 50, 51, 52, 53, 54, 55, 69, 101, 122, 264, 265, 267, 275
162, 225, 229, 230, 239, 240 substitution reaction, 85
sorption experiments, 45, 46 substrate, 2, 4, 6, 10, 31, 36, 43, 47, 90, 101, 102,
sorption isotherms, 34, 44 148, 163, 176, 344, 466
sorption process, 28, 33, 34, 36, 40, 42, 50, 52, 55, substrates, vii, 1, 2, 6, 7, 36, 37, 57, 145, 166, 179,
69 237, 246, 300, 481
South Korea, 69 succinate, 106
spatial memory, 349 sucrose, 7, 390, 392
species, 2, 11, 38, 117, 136, 137, 144, 149, 175, 246, sulfate, 100, 106, 107, 124, 130, 166, 222, 223, 224,
326, 368, 431 225, 227, 229, 230, 238, 241, 243, 247, 248, 250,
specifications, 150 252, 257, 284, 324, 347, 362, 367, 370, 372, 375,
spectrophotometry, 61, 148 377, 378, 380, 382, 383, 385, 390, 391, 394, 396,
spectroscopy, 14, 15, 17, 18, 43, 50, 56, 61, 88, 136, 419, 436
161, 207, 233, 240, 254, 271, 273 Sun, 64, 65, 88, 100, 113, 116, 117, 121, 122, 127,
spherulite, 172 130, 199, 201, 210, 215, 247, 251, 254, 255, 318,
spiders, 414 351, 357, 404, 407
spin, 15, 160, 170 suppliers, 3, 23, 24, 26, 57
spleen, 147, 337, 341, 348 suppression, 346
sponge, 339, 351, 431, 459, 460, 475 supramolecular structure, 162, 179, 231
spore, 9, 10 surface area, 32, 276, 290, 332, 366, 374, 388, 397,
Sprague-Dawley rats, 330 421, 432, 450
stabilization, 99, 277, 278, 287, 300, 303 surface chemistry, 368
stabilizers, 284 surface energy, 47
stable complexes, 335, 433 surface layer, 163
Index 505
surface modification, 86, 105, 122, 300, 367, 411, tensile strength, 27, 75, 151, 159, 161, 163, 166, 167,
463 168, 170, 171, 174, 176, 178, 185, 239, 469
surface properties, 294, 313, 411, 421, 463 tension, 103, 158, 276
surface structure, 237 terpenes, 471
surface tension, 155, 163, 165, 276 testing, 24, 173, 178, 240
surfactant, 178, 194, 323, 326, 376, 377, 379, 383, testosterone, 458, 474
423, 424, 442 tetanus, 331, 334, 356, 390, 395
surfactants, 90, 130, 170, 171, 221, 256, 284 Tetanus, 334, 367, 390, 391
survival, 337 textiles, ix, 151, 165, 170, 171, 179, 185, 196, 197,
susceptibility, 147, 192, 194, 211, 327 198, 209, 211, 212, 262, 263, 451
suspensions, 18, 28, 285, 286, 287, 288, 289, 418, Thalassiosira, 136, 153
424 therapeutic agents, 230, 323, 366, 417, 426
sustainability, 2, 7, 33 therapeutic benefits, 366
suture, 135, 151, 166, 171, 174, 179, 187, 213, 467, therapeutic use, iv
468 therapeutics, 130, 327, 354, 360, 418, 420, 426, 434,
swelling, 27, 28, 31, 34, 88, 95, 99, 103, 120, 130, 435, 471
153, 156, 164, 220, 225, 226, 228, 229, 230, 237, therapy, 24, 26, 28, 105, 110, 178, 334, 343, 344,
239, 240, 241, 242, 249, 252, 298, 339, 381, 418, 345, 347, 351, 352, 353, 357, 361, 362, 399, 405,
431, 442, 445, 456, 459 411, 414, 429, 431, 437, 439, 469, 477, 478
symptoms, 144, 169 thermal degradation, 278
synergistic effect, 331, 339 thermal destruction, 228
synthesis, viii, 6, 37, 38, 60, 64, 74, 80, 90, 102, 104, thermal stability, 27, 103, 170, 228, 230, 240, 251
116, 125, 126, 127, 130, 134, 144, 160, 188, 190, thermal treatment, 240, 242, 258
218, 224, 236, 244, 251, 256, 261, 264, 265, 266, thiamin, 360
267, 276, 313, 342, 358, 361, 368, 422, 442, 447, thickening agents, 463
478 thin films, 267
synthetic polymers, 164, 167, 192, 219, 239, 246, thiolated chitosan, 74, 75, 76, 79, 112, 117, 119, 121,
294, 415 355, 451
systemic immune response, 331, 334, 378, 386, 389, thiophenol, 140
396 threonine, 237
thrombin, 275
thromboresistance, 163
T thromboresistant materials, viii, 217, 243, 258
thrombus, 105
tamoxifen, 446, 471 tissue engineering, vii, viii, ix, 2, 21, 29, 56, 63, 64,
tar, 135 79, 88, 89, 103, 110, 123, 134, 171, 175, 179,
target, 4, 6, 7, 40, 74, 100, 231, 233, 234, 336, 337, 213, 214, 224, 228, 232, 236, 247, 248, 255, 259,
338, 342, 344, 346, 347, 354, 374, 421, 422, 427, 261, 263, 282, 351, 354, 358, 420, 435, 442, 459,
428, 434, 435, 451 464, 465, 466, 472, 481, 482, 483
techniques, vii, ix, x, 1, 2, 31, 38, 57, 100, 102, 129, titanate, 27, 31, 63, 66
140, 144, 162, 176, 179, 185, 187, 231, 256, 258, titania, 31
276, 321, 366, 369, 371, 389, 402, 406, 436, 458, titanium, 70, 227, 250
462, 464 TNF, 344, 360
technologies, 366, 369, 456 TNF-alpha, 360
technology, 24, 58, 69, 144, 161, 202, 207, 211, 215, TNF-α, 344
407, 446, 469 total cholesterol, 32
TEM, 56, 177, 263, 278, 279, 286, 288, 289, 293, toxic effect, 32, 108
303, 381 toxic side effect, 180
temperature, 9, 11, 13, 15, 28, 52, 64, 79, 84, 85, 87, toxic substances, 151
88, 100, 101, 103, 139, 146, 148, 149, 156, 157, toxicity, viii, xi, 21, 32, 102, 108, 109, 110, 115,
161, 165, 171, 189, 204, 205, 219, 236, 241, 242, 120, 126, 127, 133, 134, 144, 150, 151, 169, 175,
245, 253, 258, 268, 279, 281, 399, 452 231, 232, 244, 300, 322, 335, 346, 352, 362, 367,
tendon, 135, 166, 259 368, 377, 381, 384, 385, 388, 397, 402, 410, 421,
tendons, 144
506 Index
423, 433, 434, 437, 439, 441, 445, 449, 451, 452, UV radiation, 39
455, 458, 459, 462, 466, 468, 469, 473
toxin, 391, 396, 440, 443
trafficking, 337, 338, 340, 350, 359, 433 V
transcription, 37, 340
transfection, x, 84, 100, 118, 121, 122, 178, 232, vaccine, x, 28, 258, 331, 341, 354, 359, 360, 389,
233, 234, 235, 253, 254, 334, 335, 336, 337, 338, 391, 395, 407, 408, 410, 414, 420, 435, 436, 439,
339, 340, 342, 354, 357, 358, 359, 404, 414, 430, 442, 443, 463
431, 433, 438, 440, 441, 442, 445, 446, 478 vacuum, 140, 424
transferrin, 338, 347, 362, 434 vagina, 68, 356, 432, 437
transformation, 56, 143, 156 valence, 15, 240
transformations, 156 vancomycin, 224, 228, 250, 431
transgene, 444 vapor, 279, 280
transition metal, 61, 67, 104, 167, 177 variables, 82, 100, 101, 191, 204, 370, 436, 477
transition metal ions, 67, 104 variations, 11, 34, 136
transition temperature, 27 varieties, 232
translation, 187, 336 vascularization, 397
translocation, 336, 340, 393 vasculature, 345
transmission, 56, 239, 241, 263, 278, 381, 459 vector, 121, 131, 186, 232, 234, 254, 263, 273, 305,
transmission electron microscopy, 56, 241, 263, 278, 343, 358, 404, 433, 434, 437, 438, 444
381 vehicles, ix, x, 232, 254, 365, 366, 396, 433, 449,
transport, 8, 74, 75, 80, 82, 119, 125, 127, 142, 192, 451, 457, 462
225, 231, 232, 239, 242, 243, 246, 328, 329, 330, vein, 108, 110, 349
331, 333, 337, 338, 340, 344, 349, 366, 374, 380, velocity, 19, 55
393, 394, 395, 404, 407, 432, 433, 436, 441, 443, ventricle, 349
446, 450, 462 versatility, 314, 468
triggers, 328 vessels, viii, 217, 237, 244
tuberculosis, 340, 341, 359 vinyl monomers, 173
tumor, 108, 110, 123, 124, 129, 334, 341, 345, 346, viral gene, 118, 178, 254, 335, 433
347, 361, 362, 437, 348, 441, 444, 467 viral vectors, viii, 178, 217, 232, 234, 236, 244, 334,
turbinates, 192 433
Turkey, 449 virus infection, 341, 360
turnover, 179, 341 viruses, 232, 234
viscoelastic properties, 29, 416
viscose, 166, 167, 170, 171, 177, 214
U viscosity, x, 13, 14, 18, 86, 88, 140, 141, 142, 144,
151, 156, 158, 159, 188, 191, 204, 205, 214, 226,
UK, 136, 188, 193 239, 269, 270, 271, 277, 278, 296, 331, 418, 425,
ulcer, 72, 155, 464 449, 452
ultrasound, 128 visualization, 288
uniform, 43, 50, 179, 227, 288, 304, 420 vitamin C, 438
United, 3, 26, 211 vitamin E, 276, 443
United Kingdom, 211 vitiligo, 464
United States, 3 vulnerability, 342
urea, 19, 158, 204
uric acid, 31
urine, 135, 147, 151, 166, 169, 175 W
USA, 24, 136, 190, 202, 215, 354, 472
USSR, 246, 256 Washington, 189, 197, 200, 204
uterus, 68 waste, vii, 1, 2, 3, 6, 7, 11, 12, 33, 38, 57, 59, 69,
UV, 14, 15, 39, 43, 50, 61, 64, 101, 113, 123, 162, 136, 137, 148, 154, 187, 239
243, 476 waste carbon sources, vii, 1
UV irradiation, 64, 101 waste mycelia, vii, 1, 2, 3, 6, 57
UV light, 101, 162 waste treatment, 239
Index 507
waste water, 38, 69 313, 322, 413, 442, 447, 457, 460, 461, 465, 466,
wastewater, 4, 5, 6, 12, 30, 32, 38, 39, 45, 58, 63, 68, 468, 472, 474, 475, 482, 484
69, 90, 151, 284 wound infection, 351, 431, 443
water absorption, 95, 96
water permeability, 227
water sorption, 101 X
water vapor, 84, 279, 280, 458
water-soluble polymers, 98, 105, 287, 354 xenografts, 346
wave number, 17 XPS, 249, 304, 306, 309, 310
wavelengths, 20 X-ray diffraction, 14, 20, 62, 143, 165, 204, 243,
WAXS, 148, 162 263, 268
weak interaction, 48 X-ray photoelectron spectroscopy (XPS), 292, 402
weight changes, 191 XRD, 164, 167, 170, 176, 268, 286
weight loss, 3, 24, 58, 141, 147, 417
weight management, 3 Y
weight ratio, 252, 278
wellness, 3 yarn, 26, 176, 205, 292, 293
wettability, 232, 307, 309, 322 yeast, 6, 39, 59, 68, 135, 136, 144, 152, 414
wetting, 163, 438 yield, 7, 8, 9, 10, 11, 60, 101, 136, 140, 165, 178,
wheat germ, 164, 244, 411 223, 225, 228, 233, 257, 295, 296, 297, 336
white biotechnology, vii, 1
white blood cells, 275, 299
workers, 158, 184, 369, 387, 423 Z
World Health Organization, 63
wound healing, ix, x, 55, 88, 105, 119, 121, 122, zinc, 76, 213
161, 164, 165, 169, 173, 189, 191, 192, 193, 197, zirconia, 16, 31
198, 207, 208, 212, 224, 228, 249, 261, 263, 293, zwitterions, 89

Chitosan Manufacture Properties and Usage

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Chitosan Manufacture Properties and Usage

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

Nova Science Publishers, Inc.

NOTICE TO THE READER

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Published by Nova Science Publishers, Inc. † New York

Chitosan is a partially deacetylated derivative of chitin, a natural polysaccharide extracted

improved efficacy, enhanced bioavailability and reduced toxicity -generally recognized as

CHITOSAN FROM FUNGI

Mirko Trutnau1,∗, Thomas Bley1 and Jelka Ondruschka2,•

∗ Bergstraße 120, D-01062 Dresden, Germany.

2. OVERVIEW OF CURRENT INDUSTRIAL PRODUCTION AND

3. USABLE FUNGI AND SUBSTRATES

chitosan, being produced by Zygomycetes, Ascomycetes, Basidomycetes, Deuteromycetes

Fungal strains Source Waste product Reference

Furthermore, widely differing extraction protocols were employed, making it difficult to

3.1. Factors Influencing Chitosan Yields

In order to optimise productivity all production stages have to be considered, both

3.1.1. Optimisation of Experimental Conditions by Statistical Experimental Design

3.1.2. Enhancement of Chitosan Content by Using Plant Growth Hormones

3.1.3. Use of Fungal Properties to Enhance the Chitosan Yield

batch (F4.1, 22.25 h)

batch (F7.2, 23.25 h)

Yield of chitosan [%]

batch (F3, 49.25 h)

batch (F7.2, 23.25 h)

batch (F3, 49.25 h)

From Trutnau et al. [28] with permission.

Thus, by recycling stress-adapted cultures in a semi-continuous batch mode, it has been

5.1. General Properties of Chitosan

Chitosan is a mucopolysaccharide and, ideally, a linear polymer of 1,4-ß glycosidically

Chitosan is a weak alkaline polyelectrolyte [52]. A cross-linked unmodified chitosan

5.2. Characterization Methods

Table 3. Methods for determining physico-chemical characteristics of chitosan

Physico-chemical Determinations methods Reference

5.2.1. Commonly Used Protocols in Laboratory Practice

5.2.1.1. Degree of Deacetylation

5.2.1.1.1. First derivative UV-spectroscopy

To analyse samples a 100 mg portion of each sample is dissolved in 20 mL of 85 %

5.2.1.1.2. Nuclear Magnetic Resonance Spectroscopy

(cNaOH ⋅ VNaOH + cHCl ⋅ VHCl )

mSample ⋅ M acetyl - glucosamine ⋅ f ⋅ c ⋅ (VNaOH + VHCl )

5.2.1.1.4. Infrared Spectroscopy

Wave Type of peak Explanation Baseline Calculation of Degree Range

1320 Measurement C-N 1276-1348 A1320 0-100

5..2.1.2. Moleccular Weightt/Mw Distribution

5.2.1.2.3. Gel Permeation Chromatography

5.2.1.3. Protein Determination by Bradford Assays

5.2.1.4. Crystallinity by X-Ray Diffraction

I crystal (2θ = 20°) − I amorph (2θ = 12°30 ')

∫ ⎡⎣ I amorph (2θ ) ⎤⎦ d (2θ )

5.2.1.5. Moisture Content

5.2.1.6. Ash Content

Table 5. Overview of physico-chemical properties of chitosan isolated from the indicated

Annual number of publications

6.1. Summary of Current Commercial Chitosan Applications

Table 6. Overview of current products and suppliers of chitosan products categorized

weight loss/ dietary Fujian Newgift Enterprisies Co., Ltd.

iummune booster Samsung Chitopia Co., Ltd.

cholesterol/fat blocking Shop for you Health Products Ltd.

medical face mask Pannarajintertrade

immune booster Capistone International, Inc.

immune anti-fatigue (advanced Private Label Nutraceuticals Llc.

health care supplement China Apollo Group International.Co., Ltd

slimming coffee KangBo International Biological Development