Journal of Controlled Release 186 (2014) 5487

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review

The implications of recent advances in carboxymethyl chitosan based

targeted drug delivery and tissue engineering applications
Laxmi Upadhyaya a,, Jay Singh b,, Vishnu Agarwal c, Ravi Prakash Tewari a
a
Department of Applied Mechanics, MotiLal Nehru National Institute of Technology, Allahabad 211004, India
b
Department of Applied Chemistry & Polymer Technology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, New Delhi 110042, India
c
Department of Applied Mechanics (Biotechnology), MotiLal Nehru National Institute of Technology, Allahabad 211004, India

a r t i c l e i n f o a b s t r a c t

Article history: Over the last decade carboxymethyl chitosan (CMCS) has emerged as a promising biopolymer for the develop-
Received 10 February 2014 ment of new drug delivery systems and improved scaffolds along with other tissue engineering devices for regen-
Accepted 23 April 2014 erative medicine that is currently one of the most rapidly growing elds in the life sciences. CMCS is amphiprotic
Available online 5 May 2014
ether, derived from chitosan, exhibiting enhanced aqueous solubility, excellent biocompatibility, controllable
biodegradability, osteogenesis ability and numerous other outstanding physicochemical and biological proper-
Keywords:
Carboxymethyl chitosan
ties. More strikingly, it can load hydrophobic drugs and displays strong bioactivity which highlight its suitability
Drug delivery and extensive usage for preparing different drug delivery and tissue engineering formulations respectively. This
Tissue engineering review provides a comprehensive introduction to various types of CMCS based formulations for delivery of ther-
Formulation of drug apeutic agents and tissue regeneration and further describes their preparation procedures and applications in dif-
ferent tissues/organs. Detailed information of CMCS based nano/micro systems for targeted delivery of drugs
with emphasis on cancer specic and organ specic drug delivery have been described. Further, we have
discussed various CMCS based tissue engineering biomaterials along with their preparation procedures and ap-
plications in different tissues/organs. The article then, gives a brief account of therapy combining drug delivery
and tissue engineering. Finally, identication of major challenges and opportunities for current and ongoing ap-
plication of CMCS based systems in the eld are summarised.
2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2. Scope of the present review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3. Limitations with the use of chitosan in drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4. Biopharmaceutical and toxicological prole of CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5. Synthesis of various carboxymethyl chitosans (CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.1. Synthesis of O-carboxymethyl chitosans (O-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.2. Synthesis of N-carboxymethyl chitosans (N-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.3. Synthesis of N,O-carboxymethyl chitosans (N,O-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
5.4. Synthesis of N,N-dicarboxymethyl chitosans (N,N-di-CMCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
6. Preparation techniques of CMCS based formulations for drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Abbreviations: ADR, adriamycin; AQCOM, alginate-Q-CMCS-organic montmorillonite; bFGF, basic broblast growth gactor; BSA, bovine serum albumin; BMSCs, bone marrow stromal
cells; CPT, camptothecin; CMC, critical micelle concentration; CMCS, carboxymethyl chitosan; O-CMCS, O-carboxymethyl chitosans; N-CMCS, N-carboxymethyl chitosans; N,O-CMCS, N,O-
carboxymethyl chitosans; N,N-di-CMCS, N,N-di-carboxymethyl chitosans; CMHC, carboxymethylhexanoyl chitosan; CMCPEG, methoxy poly(ethylene glycol)-grafted carboxymethyl chi-
tosan; CMCS-g-D-GA, CMCS-graft-D-glucuronic acid; CD, cyclodextrin; DD, degree of deacetylation; DS, degree of substitution; DA, deoxycholic acid; DOX, doxorubicin; EPR, enhanced per-
meability and retention; EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride; FA, folic acid; 5-FU, 5-uorouracil; GA, glutaraldehyde; GL, glycyrrhizin; GFLX, gatioxacin;
HAP, hydroxyapatite; IPN, interpenetrating; iPSCs, induced pluripotent stem cells; LA, linoleic acid; mPEG-g-CMC, methoxy poly (ethylene glycol) grafted carboxymethyl chitosan; MW,
molecular weight; MTA, mineral trioxide aggregate; MIC, minimum inhibitory concentration; MMA, methyl methacrylate; N-CECS/nano-HAP, N-carboxyethyl chitosan/
nanohydroxyapatite; OCT, octreotide; OCC, N-octyl-O,N-carboxymethyl chitosan; OD, ornidazole; OMMT, organic montmorillonite; PTA, Cis-3-(9H-purin-6-ylthio)-acrylic acid; PTX, pac-
litaxel; PNIPAM, poly(N-isopropylmethacrylamide); PBS, phosphate buffer saline; PEG, polyethylene glycol; PAMAM, poly(amidoamine); PVA, poly-vinyl alcohol; QCMCS, quaternised
carboxymethyl chitosan; SA, stearic acid; SMCs, smooth muscle cells; TLAC, thiolated lactosaminated; VEGF, vascular endothelial growth factor.
Corresponding authors. Tel.: +91 11127871045 (ofce), +91 9871765453 (mobile).
E-mail addresses: laxmi4.phd@gmail.com (L. Upadhyaya), jay_singh143@yahoo.co.in (J. Singh).

http://dx.doi.org/10.1016/j.jconrel.2014.04.043
0168-3659/ 2014 Elsevier B.V. All rights reserved.
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 55

7. CMCS formulations for drug delivery applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

7.1. CMCS hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
7.2. CMCS microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
7.3. CMCS micelles/aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
7.4. CMCS nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
7.5. CMCS lms and bers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
7.6. CMCS composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8. CMCS based targeted drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8.1. Cancer-specic drug delivery based on CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
8.2. Organ-specic drug delivery based on CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.2.1. Colon-specic drug delivery using CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.2.2. Liver targeted drug delivery using CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
8.2.3. Ocular drug delivery using CMCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
8.2.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
9. Tissue engineering: origin and strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
10. Preparation techniques of CMCS based biomaterials for tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
11. CMCS based biomaterials for tissue engineering and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
11.1. CMCS scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
11.2. CMCS hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
11.3. CMCS composites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
11.4. CMCS injectable gels, lms and membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12. CMCS applications in different tissues/organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12.1. CMCS in bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12.2. CMCS in cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
12.3. CMCS in nerve tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
12.4. CMCS in wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13. Tissue engineering application is often combined with drug delivery strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
14. CMCS based systems: current challenges and opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
15. Summary and future remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

1. Introduction
Natural polysaccharides, due to their non-toxicity, biocompatibility
and biodegradability, are widely being studied as biomaterial for drug
delivery and tissue engineering applications. Among them, chitosan,
which is known to be biocompatible, biodegradable, non-toxic,
mucoadhesive and antimicrobial, has been exhaustively exploited for de-
veloping different formulations for controlled delivery of biotherapeutics
and in regenerative medicine. But limited solubility of chitosan in water
and other organic solvents has prevented its full exploitation in drug de-
livery and tissue repair and reconstruction [1,2]. In addition to this, the
limited colloidal stability of chitosan based particulate drug delivery sys-
tems are known to exhibit immunogenicity [3] and degradability of chi-
tosan based formulations in tissue regeneration and drug delivery is
uncontrollable [4]. Therefore derivatisation of chitosan seems a promis-
ing way to get rid of these limitations of chitosan and widening range
of drug delivery and tissue engineering applications. In fact, life sciences
and bio-technologies is the realm where chitosan and chitosan deriva-
tives have raised greater scientic interest because of their remarkable
structural and functional properties. Among them, carboxymethyl chito-
san (CMCS), a water soluble derivative of chitosan, has attracted boom-
ing interests in several elds such as in vitro diagnostics [57],
theranostics [8] bioimaging [9], biosensors [10,11], wound healing [12],
gene therapy [1316] and food technology [17,18] but its greatest impact
has been in the area of drug delivery and tissue engineering. CMCS is
potentially biologically compatible material that is chemically versatile
(NH2 and COOH) groups and various molecular weight, (MW). The
positive facets of increased water solubility, excellent biocompatibility
[19], admirable biodegradability, high moisture retention ability [20],
improved antioxidant property [21], enhanced antibacterial [22] and
antifungal [23] activity and non-toxicity as compared to chitosan has
provided ample opportunities to the drug delivery and tissue engineer-
ing scientists to create a plethora of formulations and scaffolds. In addi-
tion, it is also known to be more bioactive [24], promotes osteogenesis
[25] and its safety evaluation on compounds [26], in vitro models,
blood systems [27] and tumor application [28] has been well established.
All these favorable physical, chemical and biological properties of CMCS
make it a promising biomedical material for drug delivery and tissue en-
gineering applications in several formulations. Recently numerous ex-
perimental results have been reported on the potential therapeutic
applications of CMCS in reduction in post surgical adhesion formation
[29], antibacterial biomaterial [30] and accelerated wound healing [31].
The most exhaustively investigated CMCS based drug delivery formula-
tions include hydrogels [32], microspheres [26], beads [33], micelles
[34], aggregates [35], nanoparticles [36,37], lms [38] and membranes
[39]. Similarly, repairing and reconstruction of tissues like bone, cartilage,
and nerve by CMCS based tissue engineering devices like scaffolds [40],
injectable gels [2], and biocomposites has been reported by various
researchers in recent years.
2. Scope of the present review
In consideration of the above, the scope of the present review is to
identify the lines of applied research that are now consolidating major
advances made with the CMCS during the last decade in the eld of
drug delivery and tissue engineering. The novelty of these facts is
underlined by the fact that a previous attempt to review the literature
on CMCS has focused primarily on the general biomedical applications
of CMCS with only a minor part dealing with drug delivery and tissue
engineering applications [41]. While another review article authored
by Mourya et al. [42] mainly covers literature regarding the synthesis
and characterisation of CMCS along with its pharmaceutical applica-
tions. CMCS being inherently bestowed with astonishing physical,
chemical and biological features, emerging trends show that it is highly
suitable for the delivery of numerous bioactive and therapeutic com-
pounds and for the repair and reconstruction of damaged and/or dis-
eases tissues. Excellent biocompatibility, improved biodegradability,
enhanced antimicrobial activity, better chelating ability, moisture
56 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
absorption and retention capacity, increased antioxidant property, and
higher bioactivity of CMCS compared to its parent polymer chitosan
has contributed to the exhaustive usage of this biopolymer in drug de-
livery and tissue engineering applications both in vitro and in vivo.
This review therefore intends to convey to the reader the detailed infor-
mation on various CMCS based formulations for drug delivery and tissue
engineering and their preparative techniques along with applications in
tissues like bone, cartilage and nerves in last few years. The review gives
an overview of CMCS based formulations for targeted delivery focusing
cancer specic and organ specic drug delivery. The article also gives a
brief discussion about the fact that tissue engineering applications are
often combined with drug delivery strategy. In this regard, it is very im-
portant to understand the basic anatomy of a drug delivery vehicle
which is elucidated in the section discussed later.
3. Limitations with the use of chitosan in drug delivery
The drug absorption enhancing property of chitosan due to its bio/
mucoadhesive nature (i.e. delayed clearance of the formulation from
the absorption site) and the transient opening of the tight junctions be-
tween the epithelial cells of the mucosal membrane are well experi-
mentally demonstrated and reported in literature. But the major
limitation associated with the use of chitosan in efcaciously increasing
mucosal drug absorption is its poor solubility at pH higher than its pKa
(pH 5.56.5). Chitosan is soluble in aqueous dilute acid such as hydro-
chloric acid and aqueous organic acid such as formic, acetic, oxalic and
lactic acids when the degree of deacetylation (DD) of chitin reaches
about 50%. While it remains insoluble at neutral and alkaline pH values.
The main mechanism involved in the solubilisation of chitosan is pro-
tonation of the NH2 function on the C-2 position of the D-
glucosamine monomeric unit of chitosan, whereby the polysaccharide
is converted to a polyelectrolyte in acidic media. The main factors
governing the solubility of chitosan are DD, the ionic concentration,
the pH, the MW, the nature of the acid used for protonation, the distri-
bution of acetyl groups along the chain and the conditions of the meth-
od of isolation and drying of the polysaccharide. Highly deacetylated
chitosans (N85%) are soluble only up to a pH of 6.5. The dependence
of the degree of ionisation on the pH and the pKa of the acid have
been experimentally displayed by examination of the role of proton-
ation in the presence of acetic acid and hydrochloric acid on solubility
of chitosan [43]. The decrement of intermolecular interactions and the
lower crystallinity causing change in the microstructure of chitosan,
which facilitate the permeation of water, LiOH hydrates and urea hy-
drates, thereby enhancing chitosan dissolution in an aqueous solution
of LiOH/urea [44].
It is a well known fact that the pH of the small intestines increases
from the duodenum to the terminal ileum from pH about 4.5 to 7.4.
Therefore, in the lower part of intestine, chitosan will not be soluble.
And in order to achieve higher oral bioavailability of drugs through
the use of chitosan as an absorption enhancing delivery system, dissolu-
tion of chitosan at the pH values present in the lumen of small and
large intestines is must. In consideration of the above facts, several at-
tempts have been made and a large body of research exists on
chemical modication of chitosan through derivatisation of the amine
and/or hydroxyl groups to enhance the water solubility of chitosan.
These derivatives mainly include sulfonation [45], quaternarisation
[46], carboxymethylation [47], N- and O-hydroxyalkylation [48] and dif-
ferent grafted copolymers of chitosan [4951]. Among these, chitosan
derivatives, CMCS has drawn signicant attention of the researchers
for drug delivery applications due to its outstandingly enhanced water
solubility, superior bio/mucoadhesive property, ease of preparation
and numerous other admirable physicochemical and biological charac-
teristics that are elaborated in Fig. 1 which make it highly suitable for
fabricating different drug delivery formulations and tissue engineering
biomaterials.
4. Biopharmaceutical and toxicological prole of CMCS
As a result of the research undertaken over the last decade there is
an acceptable understanding of suitability of CMCS as a versatile func-
tional biomaterial for delivering different therapeutic moieties and tis-
sue engineering applications. Among the water soluble chitosan
derivative, CMCS is an amphiprotic ether derivative containing carboxyl
and amine groups in the molecule. Chemically, chitosan is poly-1,4-
linked -D-glucosamine, a cationic polysaccharide prepared by alkaline
N-deacetylation of chitin. The inherent properties of chitosan itself like
biocompatibility, non-toxicity, biodegradability and antimicrobial char-
acteristic makes it suitable for various drug delivery [52,53] and tissue
engineering applications [54,55]. But the limited solubility of chitosan
in common solvents including water hinders its full potential exploita-
tion in biomedical and pharmaceutical industry. Therefore introducing
carboxymethyl group into the chitosan polymer chain endows it with
some outstanding physical, chemical and biological properties suitable
for delivery of therapeutic compounds and tissue repair and reconstruc-
tion purpose. The water solubility property of CMCS at various pH and
different preparation conditions has been experimentally demonstrat-
ed. The study showed that degree of carboxymethylation has critical ef-
fect on aqueous solubility of CMCS. The study demonstrated that the
water insolubility of CMCS at different pHs varied with the degree of
carboxymethylation. The experiment also showed that the increase in
the reaction temperature increased the fraction of carboxymethylation
and thereby increased the insolubility at lower pHs. Also the decrease
in the fraction of carboxymehylation increased the insolubility at higher
pHs [56]. Similarly, DD and degree of substitution (DS) are known to
have critical effect on the moisture-absorption and moisture-retention
abilities of CMCS. The study revealed that under conditions of high rel-
ative humidity, the maximum moisture absorption and moisture reten-
tion were obtained at DD values of about 50%. Also when the DD values
deviated from 50% moisture absorption and moisture retention de-
creased. While both moisture absorption and moisture retention in-
creased with the increase in DS value [57]. Currently, a quantitative
study of the acid base equilibrium of CMCS has been carried out by a
group of researchers which can be useful for many biomedical applica-
tions. The study investigated the effect of metal ion properties on the
stability of the complexes. The results showed that the study of com-
plexes can be ordered as Mn(N,O-CMCS) b Co(N,O-CMCS),Ni(N,O-
CMCS) b Cu(N,O-CMCS) b Zn(N,O-CMCS) [58]. Apart from these, the
antibacterial characteristics of CMCS [59], its fungistatic activity [60], an-
timicrobial properties of modied forms of CMCS [6163] and CMCS
based composites [64,65] have been well reported in literature.
CMCS has been known to be highly biocompatible and is also known
to promote the proliferation of the normal skin broblast signicantly
but inhibited the proliferation of keloid broblast [19]. Tao et al. pre-
pared CMCS characterised with different sulfate content and concentra-
tion. MTT method was applied to evaluate effect of CMCS on broblast
proliferation. The results of the study demonstrated that CMCS with sul-
fate content 26.26% at the concentration of 100 g/mL shows best po-
tential for skin broblast proliferation [66]. Currently, it is also shown
that oligo-chitosan, N,O-CMCS and N-CMCS in sheets and pastes are
cytocompatible with potential of wound healing when cytotoxicity
was evaluated using primary normal human dermal broblast cultures
and hypertrophic scars [67]. The biological safety of CMCS in blood sys-
tems of rats has been experimentally established [27]. Moreover, CMCS
is known to be safe in vivo and slightly inhibited growth of sarcoma 180
and enhanced body immunity via elevation of serum IL-2 and TNF-
level in treated mice [28]. Apart from this, the excellent biodegradability
of CMCS by in vitro and in vivo evaluation has been experimentally dem-
onstrated in rats. This study revealed that liver played central role in
biodegradation of CMCS [68]. The better biodegradability of 1-ethyl-
3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross
linked CMCS lms in lysozyme solution (pH 7.4, 37 C) has been report-
ed. Also, these lms enhanced the spread of Neuro-2a cells and provided
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 1. Represent desirable property of CMCS and which makes it highly suitable for fabricating different drug delivery formulations and tissue engineering biomaterials nanosystem.
a good proliferation substratum for Neuro-2a cells, as compared to chi-
tosan lms [69]. The biodegradation study on CMCS-g-medium chain
length polyhydoxyalkanoates by Bhatt and coworkers showed that de-
pends on several factors such as temperature, pH, water potential, oxy-
gen content, stereo regularity and crystallinity of the polymer, and its
material processing [70]. The spontaneous degradation of CMCS in soil
and by enzymes with satisfactory results has been earlier reported in lit-
erature [71]. In this view, controlling the degradation of covalently
cross-linked CMCS utilizing bimodal MW distribution as reported by
Guangyuan and coworkers is worth mentioning [72]. Recently, a study
demonstrating the signicant effect of MW of CMCS on its uptake
from the lumen of abdomen and blood vessels to peripheral tissues,
the distribution of this chemical and urinary excretion after intraperito-
neal administration has been carried out [73].
The earliest study establishing the low toxicity of CMCS dates back to
early 1990s when Tokura et al. [74] studied the biological activities of dif-
ferent biodegradable polysaccharides. With regard to the in vivo toxicity,
no acute toxicity was detected in blood systems of rats after CMCS was
absorbed in the abdominal cavity and degraded gradually in the blood
[75]. Finally a recent study exploring the in vitro cytotoxicity prole of
chitosan, O-CMCS and N,O-CMCS nanoparticles to breast cancer cells-
MCF-7 showed less toxicity (almost 98% viability was found) [76]. In
this context, the cytocompatibility of some of the modied CMCS formu-
lations like CMCS-2, 2 ethylenedioxy bisethylamine-folate [77], CMCS-
Polyamidoamine dendrimer nanoparticles [78] and N-octyl-N,O-CMCS
[79] demonstrated currently is worth mentioning. In addition to the tox-
icological prole of CMCS in different forms, the ability of CMCS in pre-
vention and reduction of postsurgical adhesion formation [80,81,29]
and in vivo wound healing ability [82] have been experimentally demon-
strated. Finally, the inherent excellent mucoadhesive and absorption en-
hancing property of chitosan are retained in CMCS which is favorable for
drug delivery applications. Therefore, this interesting biopharmaceutical
and toxicological prole of CMCS has encouraged its application as drug
delivery and tissue engineering biomaterial. Fig. 2 illustrates different
outstanding and astonishing physicochemical and biological properties
of CMCS that contribute for its suitability in numerous drug delivery
and tissue engineering applications.
57
5. Synthesis of various carboxymethyl chitosans (CMCS)
Synthesis of different types of CMCS by different chemical routes and
effects of DD and DS along with relationship between molecular struc-
ture and moisture absorption and moisture retention abilities of CMCS
has been well described earlier by several researchers [8387]. The ef-
fect of various parameters like acid, pH, ionic concentration on the ag-
gregation behavior of the polymer in aqueous systems has also been
reported [88,89]. In fact, recently, Kong and coworkers developed a
novel method for simultaneously determining DD, DS, and the distribu-
tion factor of COONa or COOH in CMCS by potentiometric titration
[90]. Fig. 3 shows the preparative methods of different types of CMCS
5.1. Synthesis of O-carboxymethyl chitosans (O-CMCS)
Among the water soluble derivatives of CMCS, O-CMCS is known to
be an amphiprotic ether derivative which contains COOH groups and
NH2 groups in the molecule. The reaction medium used for preparation
of O-CMCS is strongly alkaline. O-CMCS is prepared by suspending chi-
tosan, sodium hydroxide and solvent isopropanol into ask and stirring
the alkaline slurry at room temperature for 1 h. Subsequently,
monochloroacetic acid dissolved in isopropanol is added to reaction
mixture drop wise within 30 min. The whole reaction mixture is reacted
for 4 h at 55 C. Finally, the solid is ltered and washed with ethyl alco-
hol and dried in vacuum. The preparation conditions and degree of
carboxymethylation govern water solubility of O-CMCS as experimen-
tally demonstrated by Chen at al. [57].
5.2. Synthesis of N-carboxymethyl chitosans (N-CMCS)
Synthesis of N-CMCS takes place in slightly acidic medium. To pre-
pare N-CMCS, free amino group of chitosan is reacted with glyoxylic
acid to give soluble aldimine and then aldimine is reduced with reduc-
ing agent sodium cyanoborohydride. The reaction neither requires
warming, nor cooling. The proportions of acetyl, carboxymethyl and
free amino groups are determined by DA and MW of the chitosan
used and the quantity of glyoxylic acid used [41].
58 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Fig. 2. Illustrates the different outstanding and astonishing physicochemical and biological properties of CMCS that contribute for its suitability in numerous drug delivery and tissue en-
gineering applications.

5.3. Synthesis of N,O-carboxymethyl chitosans (N,O-CMCS) sites of the glucosamine units of the chitosan structure. The main attrac-
tive features of N,O-CMCS are moisture retention, gel formation and
N,O-CMCS is hydrophilic and amphoteric polyelectrolyte which bears good biocompatibility along with increased water solubility and en-
carboxymethyl substituents at some of the amino and primary hydroxyl hanced antibacterial property that makes this polymer derivative highly

Fig. 3. Preparative methods of different types of CMCS.

 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
suitable for wide variety of biomedical applications. N,O-CMCS can be pre-
pared by using chitosan, sodium hydroxide, isopropanol and chloroacetic
acid [91,92].
5.4. Synthesis of N,N-dicarboxymethyl chitosans (N,N-di-CMCS)
The most crucial parameters for preparing N,N-di-CMCS are the con-
centration of chitosan, water, glacial acetic acid, glyoxylic acid, and sodi-
um borohydride. In order to synthesise N,N-di-CMCS, to a xed
concentration of chitosan (30 g) suspended in demineralised water
(3 l), 27 g of glacial acetic acid is added and stirred for 20 min. After
this glyoxylic acid is added (178 mL 50% v/v corresponding to 119 g of
glyoxylic acid) and the molar ratio of amine/glyoxylic acid is set to
1:9 at pH 23. Finally, with the help of peristaltic pump (1.2 ml min1),
sodium borohydride (90 g) in water (2.5 l) is delivered as a 3.6% solution
to the reaction vessel. The N,N-di-CMCS prepared by this method exhibits
good lm forming ability, good capacity to chelate metal ions and also
possesses excellent osteoinductive properties with calcium phosphate
[87].
6. Preparation techniques of CMCS based formulations for drug
delivery
CMCS based formulations for drug delivery applications have been
prepared by different techniques and methods by various researchers
as per the requirement of the host tissue/organ and the type of drug
being delivered. CMCS based hydrogels are one of the major formula-
tions for drug delivery applications and can be prepared by different
methods. In each process, CMCS is either physically associated or chem-
ically cross-linked. The major schemes of physical interactions that lead
to gelation of CMCS solution in hydrogels are ionic interaction, polyelec-
trolyte complex; inter polymer complex, and hydrophobic associations.
Moreover the physically cross linked hydrogels have the advantage of
gel formation without the use of cross-linking moieties. But they have
certain limitations like difculty in accurately controlling the physical
gel pore size, chemical functionalisation, dissolution and degradation,
thereby providing inconsistent in vivo performance. Cross linked
hydrogels often possess better mechanical properties where cross
links can be incorporated either chemically or through irradiation. But
chemical cross linking method for preparation of hydrogels suffers
from disadvantage of having toxic residues of cross linking chemicals.
On the other hand preparation of microspheres, nanoparticles and mi-
celles/aggregates can be carried out by different methods like emulsion
cross linking, sonication, coacervation/precipitation, spray drying, ionic
gelation, sieving, emulsion droplet-coalescence and reverse micellar
methods. Each method has its own advantage and certain limitations
and therefore the choice of preparative technique depends on the type
of particulate being synthesised, its size range, composition, the drug
being loaded or encapsulated and the target tissue/organ where drug
is to be delivered. Similarly, layer-by-layer assembly and cross linking
are the main preparative methods for the synthesis of composites and
blending, casting and drying methods are the most popular preparative
techniques for lms and bers for the purpose of delivery of different
drugs. Table 1 shows preparation techniques of CMCS based formula-
tions for drug delivery.
7. CMCS formulations for drug delivery applications
7.1. CMCS hydrogels
In the recent few decades, the pharmaceutical industries have
witnessed the emergence of drug delivery technologies as a powerful
and efcient tool in order to effectively use the existing drugs and devel-
op new drug candidates successfully [128]. A hydrogel can be dened as
cross linked network formed from a macromolecular hydrophilic poly-
mer capable of absorbing large amount of water. The volume of water
59
absorbed by hydrogels may range from 10% to thousands of times of
its own volume. The high water content makes hydrogels biocompatible
while coherence of their mechanical properties with the soft tissues as-
sists and enhances healing phenomenon. The compatibility of mechan-
ical properties of hydrogels with the soft tissues also mimics
morphological and functional characteristics of organ tissue. The visco-
elastic property of hydrogels causes minimised damaged to the sur-
rounding tissues in the host. Natural polymer based hydrogels have
also attracted the attention of scientists and researchers because of
their improved biocompatibility, biodegradability and notable capabili-
ty of controlled delivery of bioactive molecules [129131].
As far as covalent cross linking is concerned, it leads to the formation
of hydrogels with permanent network structure because irreversible
chemical links are formed. Therefore, this type of linkage not only al-
lows absorption of water and/or bioactive compounds without dissolu-
tion but also permits drug release by diffusion. Broadly covalently
crosslinked chitosan hydrogels can be divided into three categories: chi-
tosan cross linked with itself, hybrid polymer network (HPN) and semi
or full interpenetrating networks (IPN). In chitosan cross linked with it-
self, crosslinking involves two structural units that may or may not be-
long to the same chitosan polymeric chain [132]. The overall nal
structure of this type of hydrogel can be considered as a crosslinked
gel network dissolved in a second entangled network formed by chito-
san chains with restricted mobility [133]. In hybrid polymer networks
(HPN) based hydrogels the crosslinking reaction occurs between a
structural unit of chitosan chain and a structural unit of polymeric
chain of another type. Although crosslinking of two structural units of
the same type and/or belonging to the same polymeric chain cannot
be excluded. Finally semi or full IPNs contain a non reacting polymer
which is added to the chitosan solution before crosslinking. Thus cross
linked polymer network in which the non reacting polymer is
entrapped (semi-IPN) are thus formed. This additional polymer can fur-
ther be crosslinked in order to have two entangled crosslinked net-
works forming a full IPN whose microstructure and properties can be
quite different from its corresponding semi-IPN [134]. Ionically cross
linked hydrogels leads to the formation of non permanent network
structure since reversible links are formed [135]. Ionically cross linked
hydrogels show higher swelling sensitivity to pH changes as compared
to hydrogels having covalent cross linking. The entities reacting with
chitosan are ions or ionic molecules with well dened MW in ionically
crosslinking. It is different from polyelectrolyte complexation as the en-
tities reacting with chitosan are polymer with a broad MW distribution
[136]. In contrast to covalent crosslining ionic cross linking is a simple
and mild procedure as no auxillary molecules such as catalyst are re-
quired [137]. Ionic cross linking can be assured by the classical method
of preparing a cross linked network namely by the addition of
crosslinker either solubilised [138] or dispersed [139,140] to the chito-
san solution.
Superporous hydrogels containing poly (acrylic acid-co-acrylamide)/
O-CMCS IPNs were prepared by cross-linking O-CMCS with glutaralde-
hyde (GA) after superporous hydrogel was synthesised by Yin et al. An
enhanced capacity of loading insulin was reported for the superporous
hydrogels as compared to the non-porous hydrogels. Due to improved
mechanical properties, in vitro muco-adhesive force and loading capaci-
ties, these IPNs showed potential of muco-adhesive system for peroral
delivery of peptide and protein drugs [95]. Similarly, Chen et al. prepared
a novel type of IPN hydrogel membrane of poly (N isopropylacrylamide)/
CMCS and systematically studied the effects of the feed ratio of compo-
nents, swelling medium and irradiation dose on the swelling and
deswelling properties of the hydrogel. The results showed that a combi-
nation of pH and temperature can be coupled to control the responsive
behavior of poly(N-isopropylmethacrylamide (PNIPAAM)/CMCS
hydrogels [93]. There are several CMCS based semi-IPNs reported in lit-
erature prepared by crosslinking with genipin [20], GA or cross-linked/
grafted with ethylene glycol diglycidyl ether [99], and N,N-
methylenebisacrylamide [97]. Min Wang and coworkers synthesised
60 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Table. 1
Preparation techniques of CMCS based formulations for drug delivery.

CMCS based formulation Composition of formulation Method of preparation Refs.

Hydrogel CMCS/poly(N-isopropylacrylamide) (PNIPAAM) Physical cross linking [93]

Hydrogel N,O-CMCS & alginate Chemical cross linking [20]
Hydrogel N,O-CMCS Physical cross linking [94]
Hydrogel CMCS Chemical cross linking [95]
Hydrogel Poly(acrylic acid-co-acrylamide)/O,CMCS Chemical cross linking [96]
Hydrogel CMCS/poly(N-isopropylacrylamide) Chemical cross linking [97]
Hydrogel CMCS Chemical cross linking [98]
Hydrogel 6-O,CMCS/polyurethane Chemical cross linking [39]
Hydrogel CMCS/poly(acrylonitrile) Chemical cross linking [99]
Hydrogel CMCS Radiation cross linking [100]
Hydrogel CM-cellulose/CMCS Radiation cross linking [101]
Hydrogel CMCS Radiation cross linking [102]
Hydrogel CMCS/gelatin Radiation cross linking [103]
Hydrogel Nano-Ag/gelatin/CMCS Radiation cross linking [104]
Hydrogel N-isopropylacrylamide/CMCS Radiation cross linking [105]
Nanoparticles Cross linked lactosaminated CMCS Ionic gelication [106]
Nanoparticles O,CMCS Ionic gelation [107]
Nanoparticles Amphiphilic octadecyl Q-CMCS Microemulsion [108]
Nanoparticles Cholesterol modied O-CMCS Sonication [109]
Nanoparticles CMCS bound Fe3O4 Spraying co-precipitation [110]
Nanoparticles CMCS/poly(amidoamine) Coprecipitation [111]
Nanoparticles O-CMCS Ionic cross-linking [112]
Nanoparticles Oleoyl-CMCS Self-assembly [113]
Nanoparticles Oleoyl-CMCS Oil in water emulsication [114]
Microspheres CaCO3/CMCS Precipitation [115]
Microcapsules and microparticles CMCS & CMCS graft-poly(N,N-diethylacrylamide) Self-assembly [116]
(CMCTS-g-PDEA)
Microspheres O-CMCS bound with iron oxide In situ coprecipitation [117]
and incorporation
Microspheres Alginate/Q-CMCS/clay nanocomposite Ion-crosslinking [118]
Microspheres Alginate & chemically modied CMCS Ionotropically-crosslinked [22]
Microspheres Ambroxol hydrochloride CMCS Emulsion chemical cross-linking [119]
Micelles Linoleic acid modied CMCS Sonication [120]
Micelles PEG-grafted-CMCS Ion complex formation [121]
Composite CMCS/TMCS Direct crosslinking [122]
Composite Folate conjugated CMCS/Fe3O4/CdTe Layer-by-layer assembly technique [123]
Composite O-CMCS/ cyclodextrin Crosslinking method [124]
Composite FACMCSZnS:Mn Ionic crosslinking [125]
Film Poly-vinyl alcohol/CMCS Blending/casting [126]
Fibers Ag nanoparticles/poly vinyl alcohol/CMCS Electrospinning technique [127]

CMCS hydrogels by gamma rays induced cross linking where adsorption
kinetics study showed fast adsorption of Fe (III) ions onto CMCS gels. This
was primarily due to the coordination of Fe (III) ions with amino, hy-
droxyl and carboxyl groups of CMCS molecules [102]. Also an ESR
study on CMCS radicals in a highly concentrated CMCS aqueous solution
which forms hydrogels by ionizing radiation was performed by Seiichi
Saiki et al. [100]. Physically cross linked alginate-N,O-CMCS hydrogels
with calcium for oral delivery of protein drugs was prepared by Lin and
coworkers [94]. Substantial research has been done in chemically
cross-linked hydrogels also. Currently, pH sensitive hydrogels composed
of CMCS, chemically cross linked by GA was prepared and evaluated
in vitro as a potential carrier for colon targeted drug delivery of
ornidazole [98]. In this regard, the synthesis of novel polyampholyte
hydrogels based on CMCS of varying DD and DS cross linked with GA
by Chen et al. [95] is worth mentioning. With increasing DD or DS
value, the hydrogel changed from polyampholyte into polycations or
polyanions, respectively. The release of bovine serum albumin (BSA)
was much quicker at pH 7.4 buffer than pH 1.2 solutions. In recent
years radiation cross linking has proved to be safer, clean and effective
method of hydrogel synthesis. The products formed are free of toxicity
additives as neither initiator nor cross linker is required as in convention-
al chemical routines. The synthesis of CMCS using -rays radiation [101,
102] and ionizing radiation [100] has been reported in the literature.
Yang et al. [141] have in situ synthesised dark bluish coloured 5-
uorouracil (5-FU) loaded CMCS hydrogels using genepin as the cross
linker and it was speculated that the dark bluish colour of the hydrogels
prepared resulted from the cross linking reaction between genipin and
the amino groups of CMCS (Fig. 4A). The possible cross linking
mechanism for the reaction of CMCS with genepin was speculated as il-
lustrated in Fig. 4B [141]. Similarly, Yang et al. [142] prepared hydrogel
beads based on methoxy poly (ethylene glycol) grafted carboxymethyl
chitosan (mPEG-g-CMC) and alginate in order to construct interpen-
etrating polymeric matrix. Fig. 5 [142] illustrates the synthesis of
mPEG-g-CMC copolymer using Schiff's base method. Some of the CMCS
based hydrogels for delivery of different therapeutics are summarised
in Table 2.
7.2. CMCS microspheres
Microspheres are spherical free owing particles having size range
between 50 m and 2 mm with drugs entrapped inside them. While
in matrix type microspheres drug is mainly released by erosion mecha-
nism, release of drug takes place by diffusion and erosion in matrix and
reservoir type microspheres [149]. In general drug release rate depends
upon solubility, diffusion, biodegradability of the matrix, drug loading
efciency, and size of the microspheres. Therefore the drug release
mechanism can be altered by varying polymer employed and its proper-
ties. Microspheres are mainly exploited in controlled drug delivery but
by derivatisation and surface modication they can also be used for
drug targeting purpose. The partition coefcient determines the drug
distribution within the microspheres. CMCS based microspheres have
emerged as efcient drug carriers due to their ability to encapsulate a
variety of drugs, biocompatibility, protection of fragile drugs, high bio-
availability and sustained drug release characteristics. Currently,
carboxymethyl cellulose and CMCS based biodegradable and highly po-
rous microspheres were prepared with an inverse emulsion-cross-
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 61

Fig. 4. Possible representations for (A) in situ synthesis of the drug loaded CMCS hydrogel and its photo and (B) cross linking mechanism between CMCS and genipin (Reproduced with
permission from [141]).

linking method without incorporating any extraneous cross-linking
agents capable of loading anticancer drug doxorubicin (DOX) whose re-
lease prole was adjustable. The in vitro and in vivo studies indicated
that these microspheres can be used as biocompatible and biodegradable
embolic agents for transarterial embolisation [150]. Recently, quaternised
carboxymethyl chitosan (QCMCS) was intercalated into the interlayer of
organic montmorillonite (OMMT) to obtain the QCMCS/OMMT nano-
composites. The cross linked alginate-Q-CMCS-organic montmorillonite
(AQCOM) microspheres were prepared by crosslinking with CaCl2 and
the drug-controlled release was evaluated by using BSA as model drug.
The AQCOM microsphere displayed more excellent encapsulation and
controlled release capacities than the microsphere without OMMT and
in vitro active cutaneous anaphylaxis test on Guinea pigs did not cause
anaphylaxis [118]. Fig. 6 shows the pictures of Guinea pigs after ACA
test of AQCOM-3 microspheres. Signicant anaphylaxis such as swelling
and even ulcerated induced by 2,4-dinitrophenol was observed (as

Fig. 5. Synthesis of the methoxy poly (ethylene glycol) grafted carboxymethyl chitosan (mPEG-g-CMC) copolymer (reproduced with permission from [142]).
62 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Table. 2
Summarizes different CMCS based hydrogels for delivery of different therapeutics in drug delivery applications.

Composition of hydrogel Remark Refs.

Photocrosslinked methacrylated O-CMCS & PEG diacrylate OCMCS-based hydrogels with varying concentrations of carboxy and methacrylate groups are envisaged as [143]
attractive and versatile synthetic extracellular matrices that can effectively sequester, protect and controllably
release basic proteins. Varying the carboxyl and methacrylate groups respectively allows variation of charge and
water content/pore size which ultimately allows control of the absorption and dynamic release, via substrate
biodegradation, of basic proteins.
Covalently crosslinked N,O-CMCS and oxidized alginate Good cytocompatibility against NH3T3 cells by in vitro test and no obvious cytotoxicity for major organs during [144]
hydrogel the period of 21-day intraperitoneal administration was observed by acute toxicity test. The hydrogels developed
also showed good hemocompatibility.
Nanocomposite hydrogel composed of Curcumin, Nano curcumin with improved stability by using methoxy poly(ethylene glycol)-b-poly( -caprolactone) [32]
N,O-CMCS and oxidized alginate copolymer as carrier was slowly released from the hydrogel with the diffusion controllable manner at initial phase
followed by the corrosion manner of hydrogel at terminal phase. In vivo wound healing study showed that the
hydrogel could signicantly enhance there-epithelialisation of epidermis and collagen deposition in the wound
tissue.
CMCS and carbopol 934 hydrogel for delivery of In vitro swelling studies have shown little swelling in acidic pH 432% at the end of 2 h and 1631% in basic pH at the [145]
theophylline end of 12 h. The pH-sensitive hydrogel of CMCS can be used for extended release of theophylline in intestine and
can be highly useful in treating symptoms of nocturnal asthma.
CMCS hydrogel A simple and economical method with no toxic chemicals involved was introduced. The steam-induced [146]
crosslinking of CMCS sodium salt was observed to be quite efcacious. Depending on the harshness of steaming
conditions used, the DS of the hydrogels was found to be up to 36. The increasing temperature and duration of
steam exposure changed the coloration of the samples from light beige to brown.
In situ gelable hydrogel composed of N-CMCS and oxidized The rate of gelation was directly related to the degree of oxidation of Odex and the hydrogels underwent fast mass [147]
dextran (Odex) loss in the rst 2 weeks, followed by a more moderate degradation. The in vivo studies in mice full-thickness
transcutaneous wound models showed its ability of enhanced wound healing ability.
Physically crosslinked hydrogels of CMCS with cellulose With the increase in the interaction of component polymers, the swelling and drug release rate of hydrogels [148]
ethers including hydroxyethylcellulose decrease. Component polymer ratio controlled the swelling and drug release from hydrogels.
and methylcellulose
5-FU or bevacizumab loaded N,O-CMCS hydrogels The in vitro drug release experiments showed that nearly 100% of 5-FU was released from the drug-loaded [141]
crosslinked with genepin hydrogels within 8 h, but less than 20% bevacizumab was released after 53 h. The hydrogels provided great
opportunity to increase the therapeutic efcacy of glaucoma ltration surgery.

Fig. 6. Photos of Guinea pigs by ACA test: (a) experimental procedure, (b) the rst day after treating by AQCOM-3 microsphere, (c) the seventh day after treating by AQCOM-3 microsphere,
(d) the eighth day after treating by AQCOM-3 microsphere, (e) the eighth day after negative control, and (f) the eighth day after positive control (reproduced with permission from [118]).
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
shown in Fig. 6f). Again the induce contact and even challenge exposure
on Guinea pig treated by AQCOM-3 microspheres did not induce the ana-
phylaxis (bd) which illustrated that AQCOM-3 microspheres did not in-
duce the anaphylaxis [118]. Similarly, clozapine loaded spherical
microspheres of chitosan and CMCS within size range of 100 0.25 m
to 140 0.25 m were prepared by spray drying technique. The in vitro
drug release study showed that CMCS microspheres released most part
of the drug in the intestine compared to chitosan microspheres hence
controlled drug release was achieved [151]. Ma et al. [116] developed a
simple method to fabricate BSA loaded self-assembled CMCS and CMCS-
graft-poly (N,N-diethylacrylamide) microcapsules and microparticles
whose in vitro drug release rate and encapsulation efciency depended
on pH value. The microspheres were found to be non-cytotoxic against
L02 human hepatic natural cell and release of BSA could be sustained.
Also sustained release of BSA from microspheres based on mixtures of
ionotropically cross linked sodium alginate and chemically modied
CMCS and coated through polyelectrolyte complexation with chitosan
grafted with poly (ethylene glycol) has been reported in literature. Fig. 7
[26] illustrates the carboxymethylation of chitosan and synthesis of
CMCS grafted sodium acrylate copolymer. Again, high encapsulation ef-
ciency and sustained release of model anticancer drug DOX has been
achieved by preparing CMCS-CaCO3 microspheres by Wang and co-
workers [115]. There is also information regarding preparation of two
kinds of O-CMCS bounded with iron oxide particles by in situ co-
precipitation and incorporation methods reported in literature. The mag-
netic properties of these microspheres were studied to evaluate its poten-
tial in drug delivery applications [117].
7.3. CMCS micelles/aggregates
When polar or non-polar ends of amphiphilic polymer self assemble
by hydrophobic or ion-pair interaction in a monophasic or biphasic liq-
uid, nanosised (200 nm to 0.5 m) structures are formed which are re-
ferred to as micelles. In an aqueous environment, the amphiphilic
polymers form hydrophobic core by assembly of lipophilic parts and
vice versa. The repositioning of hydrophobic and hydrophilic drug with-
in the hydrophobic or hydrophilic core of micelles improves the solubil-
ity of drugs in monophasic non-solvent medium often referred to as
micellar solubilisation. In biphasic medium, polymeric segments distri-
bution takes place according to their physicochemical characteristics.
In general, the hydrophobicity of the drug and the type of polymer
determines the preparative method of micelle. In fact the experimental
evidences have led to the conclusion that polymeric micelles exhibit nu-
merous promising properties for oral delivery of lipophilic drugs [152].
Fig. 7. Scheme of carboxymethylation of chitosan and synthesis of CMCS grafted sodium acrylate copolymer (reproduced with permission from [26]).
63
Nano precipitation technique, dialysis method and sonication are some
of the preparative methods of micellar structures. Formation of micelles
by self-assembly is the most popular preparative methodology in case of
CMCS based or modied CMCS micelles designed and developed for
drug delivery purpose. The nanoscopic dimension, segregated core/
shell structure, sheltering effect of hydrophobic core on drugs
entrapped and stealthy characteristics by their hydrophilic shells
makes polymeric micelles suitable for tumor targeting [153,154].
CMCS has been extensively exploited by several researchers for devel-
oping micelle based nanoformulations for model anticancer drug DOX
[155,30]. Aifeng et al. synthesised and characterised DOX loaded
octreotide-modied N-octyl-O,N-CMCS micelles which proved to be
promising carrier for efcient intracellular targeting of antitumor
drugs [156]. Similarly self-aggregated nanoparticles from linoleic acid
(LA) modied CMCS were prepared by Yu-long Tan and coworkers for
delivery of hydrophobic anticancer drug adriamycin (ADR). The critical
aggregation concentration determined by measuring the uorescence
intensity of the pyrene as a uorescent probe, was in the range of
0.0610.081 mg/mL [120]. Gong and coworkers grafted Cis-3-(9H-
purin-6-ylthio)-acrylic acid (PTA) on primary amino of CMCS, under
the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride EDCHCl and N-Hydroxysuccinimide (NHS) i.e. the reaction
took following two steps: synthesis of PTA (Fig. 8A) and synthesis of PTA
grafted CMCS (PTA-g-CMCS) Fig. 8B [34]. Amphiphilic polymeric pro-
drug PTA-g-CMCS designed and synthesised could self-assemble into
spherical micelles with a size ranging from 104 to 285 nm with 6-
mercaptopurine loaded into it. The result demonstrated that
nanocarrier was found suitable for controlled drug delivery. Fig. 8C
shows 6-methoxy propyl release from PTA-g-CMCS in the presence of
glutathione, which follows a Michael additionelimination reaction [34].
Jeong et al. [121] synthesised methoxy poly(ethylene glycol)-grafted
carboxymethyl chitosan (CMCPEG) copolymer using a water-soluble
carbodiimide. In addition, DOX-incorporated nanoparticles using
CMCPEG were prepared by ion complex formation between the amine
groups of DOX and the carboxyl group of CMCPEG. The self assembled
N-phthaloyl-CMCS based micelles for drug delivery of levooxacine hy-
drochloride and BSA have also been earlier prepared by Peng et al.
[157]. Another technique of nano-precipitation was employed for the
preparation of copolymer polylactide-polyethylene glycol succinate,
1,3-beta-glucan (Glu), O-CMCS and folate-conjugated O-CMCS micelles
for efcient encapsulation of herbal anticancer drug curcumin. The nano-
formulation displayed better aqueous solubility and biodegradation
along with signicantly enhanced the cellular uptake by cancer cell
HT29 and HeLa. The anti-tumor-promoting effect of the curcumin
64 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 8. (A) Synthesis of PTA, (B) synthesis of PTA-g-CMCS, (C) 6-MP release from PTA-g CMCS in the presence of glutathione (GSH), which follows a Michael additionelimination reaction:
GSH attacks the -C of the unsaturated bonds, leading to the cleavage of the unsaturated bonds (reproduced with permission from [34]).
encapsulated by copolymer in Hep-G2 cell lines after two weeks of cell
growth is shown in (Fig. 9). The sudy showed that in the cytotoxicity
assay there were no distinct differences in the cell survival. Also in the
anti-tumor promoting assay, the ration of tumor promotion with the
Cur, Glu, PLA-TPGS alone was comparable to the control. But contrary
to this clear changes in the size and morphology of tumor between the
control and all the tested samples particularly curcumin encapsulated
with glucan copolymer. It was observed that in comparison to the
tumor on the tested wells, in control the tumor size was much larger
and their surface was very rough (Fig. 9). [158]. Wang and coworkers
prepared CaP/CMCS/KALA nanoparticles by self assembly via electro-
static interaction between positively charged peptide KALA and nega-
tively charged CaP/CMC nanoparticles in an aqueous solution after
loading anticancer drug DOXHCl onto the CaP/CMCS hybrid nanopar-
ticles. The investigation results showed that DOXHCl could be encapsu-
lated with high efciency and the presence of KALA peptide signicantly
enhanced the cell inhibition effect [159]. Dialysis method was employed
for the preparation of methotrexate conjugated O-CMCS micelles where
methotrexate exhibited signicant sustained release behavior in PBS so-
lutions (pH 4.0, 7.2 and 9.0). The critical micelle concentration (CMC) of
O-CMCSmethotrexate conjugates determined in aqueous media was in
the range of 0.00840.0424 mg/mL [160]. In addition, sonication method
has also been utilised for the designing and development of CMCS based
nanocarriers for delivery of anticancer drugs like DOX [161] and paclitax-
el (PTX) [162]. Table 3 shows different CMCS based self aggregates and
the drugs delivered by these fabrications.
7.4. CMCS nanoparticles
The emerging trends and recent advances in nanotechnology has
made a signicant impact on the development of drug delivery systems,
after the liposomes were rst described in the 1960s as carriers of pro-
teins and drugs for disease treatment [167]. Most of the front-line
drugs are toxic entities that act in unspecic fashion being untargeted,
often eliciting unwanted, dose limiting and debilitating side effects. Com-
pared to conventional drug delivery systems, nanoscale drug delivery ve-
hicles are capable of enhancing therapeutic activity by prolonging drug
half-life, improving solubility of hydrophobic drugs, reducing potential
immunogenicity, and/or releasing drugs in a sustained or stimuli-
triggered fashion. Thus, the toxic side effects and administration fre-
quency of the drugs can be reduced [168]. In addition, nanoscale particles
can passively accumulate in specic tissues (e.g., tumors) through the
enhanced permeability and retention (EPR) effect [169]. Polymers like
CMCS have emerged as a promising drug delivery nanovehicle due to in-
herent increased water solubility, biological functionality, compatibility,
safety, biodegradability, and antimicrobial nature. In the context of
drug encapsulated CMCS based nanoparticles, nanoprecipitation, dialy-
sis, micro emulsion, emulsion-solvent diffusion technique, solvent evap-
oration and ionic gelation methods have been employed so far. The
preparation of self-aggregated cholesterol modied O-CMCS nanoparti-
cles by sonication method were prepared to be used as a novel carrier
for PTX has been reported by Wang et al. [170]. These results suggest
that cholesterol modied O-CMCS self-assembled nanoparticles can ef-
fectively solubilise PTX and modify its tissue bio-distribution, which
may be advantageous in enhancing the therapeutic index and reducing
the toxicity of PTX. Similarly Zhang et al. [110] reported synthesis of
magnetic Fe3O4 nanoparticles functionalised with CMCS by spraying
co-precipitation method and these magnetic nanoparticles are suitable
for use as nanomagnetic carriers of drugs formulation. In this context,
the preparation of vincristine loaded polymeric ethosomes; formed
from amphiphilic octadecyl Q-CMCS with different DS by micro emul-
sion method is worth mentioning [171]. Biocompatible ciprooxacin-
loaded CMCS nanoparticles by ionic cross-linking method and optimised
by using BoxBehnken response surface method by Zhao et al., demon-
strated, stronger antibacterial activity against Escherichia coli than the
free ciprooxacin because they can easily be uptaken by cells. Still fabri-
cation of CMCS based nanostructure delivery vehicles by ionic gelation is
the most popular method employed till date [105,106].
A number of CMCS based nanoscale carriers have been constructed
in recent years for the efcient delivery of different anticancer, anti-
inammatory drugs, antibiotics, proteins, peptides and vaccines.
Model anticancer drug DOX has been encapsulated into a number of
modied CMCS nanosystems like FA modied CMCS nanoparticles
[172] amphiphatic carboxymethylhexanoyl chitosan nanocapsules
[155], and acylated CMCS nanoaggregates [35]. Also, liposomes modi-
ed with CMCS [173] and CMCS capped magnetic nanoparticles [164]
have also been exploited for DOX delivery. The drug release prole of
DOX loaded-CMCS-capped-MNP/MMT was tested at pH 7.4 and
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 9. Anti-tumor-promoting effects of the curcumin encapsulated by copolymer in Hep-
G2 cell lines after two weeks of cell growth on agar: control (a), Cur (b) and Cur-PLA-TPGS
(c) under inverted microscope 100 (reproduced with permission from [158]).
pH 5.0 respectively and the released DOX concentration was deter-
mined by UVvis measurement. From the drug release curve (Fig. 10)
it is clear that the release of DOX from DOX loaded-CMCS-capped-
MNP/MMT is pH triggered and the release rate of DOX was higher at
pH 5.0 than pH 7.4. It is well known fact that the pH of tumor tissue is
lower than normal cells. Thus this delivery system will exhibit smaller
toxicity towards normal cells as compared to tumor cells thereby illus-
trating its advantage of safety [174]. Recently, researchers have also de-
veloped O-CMCS [175] and folate conjugated O-CMCS nanosystems
[158] for efcient delivery of another anticancer compound: curcumin.
The cellular uptake study of curcumin-O,CMCS NPs by normal L929
cells and cancer cell line MCF-7 cells using ow cytometry showed
(Fig. 11) that the nanoformulation uptake was concentration dependent
and non specic. This meant that both the cell line showed increasing
uptake of the nanoformulation with the increase in the concentration
of the curcumin-O-CMCS nanoformulation. Also there is not much
65
signicant difference in the cellular uptake of curcumin-O,CMCS NPs
comparing normal and cancer cell lines [175] Another multifunctional
nanodrug system containing Fe3O4, O-CMCS and curcumin has been re-
cently fabricated by Thu et al. [176]. Similarly, Shen et al. [123] prepared
CMCS-ferroferric oxide doped cadmium telluride quantum dot nano-
particles with folate receptors afnity, magnetic responsiveness and lu-
minescent property via layer-by-layer assembly technique in situ
surface as shown in Fig. 12. The results indicated that the novel multi-
functional folate conjugated carboxymethyl chitosan-ferroferric oxide
doped cadmium telluride quantum dot nanoparticles synthesised ex-
hibit a high drug loading efciency, minimised cytotoxicity and desir-
able cell compatibility, and are potential candidates for CMCS-based
targeted drug delivery and cellular imaging. In addition to this,
nanoformulations of other anticancer drugs like vincristine,
camptothecin, PTX, and ADR have been developed by different modi-
cations of CMCS such as amphiphilic carboxymethylhexanoyl chitosan
[177], PEGylated O-CMCS nanoparticles grafted with cyclic Arg-Gly-Asp
(RGD) peptide [178] and LA modied CMCS nanoparticles [162] respec-
tively. Apart from these drugs, CMCS based nanoparticles mediated ve-
hicles have been prepared for delivery of anti-inammatory compounds
[179], proteins/peptides [180] and vaccines [181] along with poorly
water soluble drugs like triamcinolone [182] and vitamin D3 [183].
Table 4 describes various nanoformulations for delivery of different
drugs along with brief method and their outcome.
7.5. CMCS lms and bers
In the recent years, drugs and polymers are manufactured into vari-
ous lm congurations, like coating of some precarious drugs or pro-
teins by thin polymer lms or generating drugpolymer matrix lms.
These different matrices are suitable for facilitating diffusion mediated
drug delivery or adaptable for accomplishing transdermal drug delivery.
The parameters that determine the efcacy of polymeric lms in deliv-
ery of therapeutic compounds are thickness, surface morphology, de-
gree of swelling, degradation behavior, drug release and therapeutic
effect. The drug release pattern and rate of drug release from the lm
coating systems depend on the coating material, thickness of the lm
and circumstance of the applied site. Polymeric lms have also gained
importance as promising drug delivery formulation as its thin layer
resists change in crystallinity and segmental motions. As CMCS shows
inherent excellent biocompatibility, improved biodegradability, antimi-
crobial strength and easily lm forming ability, it has emerged as a suit-
able biopolymer for lms fabrication for drug delivery. The ornidazole
(OD) loaded poly-vinyl alcohol PVA/CMCS lms prepared by blending/
casting method demonstrated excellent antibacterial and biocompati-
bility characteristics which were enhanced with increasing CMCS con-
tent in the lms. The process of preparing the placebo lms and their
corresponding drug OD loaded lms (loaded OD) via blending/casting
is shown in Fig. 13 [38]. The results from in vitro and in vivo study dem-
onstrated the blend lms as an excellent candidate for local drug deliv-
ery system. A similar study was carried out where PVA/CMCS blend
lms prepared by casting and drying method were evaluated as coating
material for site specic drug delivery. The drug release kinetics study
by salicylic acid, theophyline, and OD also revealed that a desired rate
of drug release could be obtained by controlling the content of CMCS
in the blend lm, type and MW of drugs, pH of the medium and thick-
ness of the lm. As pH of the buffer increased the permeability of OD
with a maximum at pH 7 as shown in Fig. 14. The reason for this phe-
nomenon can be attributed to the fact that as pH increased, deproton-
ation of the charge groups of CMCS takes place. CMCS molecules
became uncoiled and assumed to be elongated thereby causing the ex-
pansion of the bulk of lms. It caused the enhanced water absorption
ability and lead to the increase of permeability [126]. A study on the
BSA and bovine brinogen equilibrium adsorption amount on the
PVA/CMCS lms prepared by mechanical blending showed the inu-
ence of CMCS content and pH and ionic strength of protein solutions
66 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Table. 3
Different CMCS based self aggregates.

CMCS based formulation Size range of Brief method and outcome Refs.
self-aggregates

O-CMCS aggregates
Cholesterol modied O-CMCS aggregates
LA-modied CMCS self-aggregates
LA-modied CMCS aggregates
Deoxycholic acid-O-CMCS-folic acid conjugates
CMCS nanoclusters
Not only the aggregates but also the unimers of O-CMCS enhanced the solubility [163]
of camptothecin. The aggregates showed good drug loading capacity and sustained
drug release demonstrating potential for localised drug delivery.
234.9100.1 nm A series of cholesterol modied O-CMCS conjugates with different DS of cholesterol [109]
were synthesised and self-aggregates were prepared by probe sonication in water.
The relationships between the chemical structure, the amphiphilic property and the
morphological characteristics of O-CMCS self-aggregated nanoparticles were
investigated in this study.
222.81028 nm Covalently conjugated LA to CMCS via EDC mediated reaction to generate self-aggregated [164]
nanoparticles by sonication method. Physically entrapped ADR showed slow release which
can be adjusted by change in medium pH.
417.8 17.8 nm The LA modied CMCS self aggregates prepared by sonication method exhibited increased [120]
loading capacity and loading efciency and decreased sustained release for ADR with
increasing DS of LA in CMCS with critical aggregation concentration values in the range
of 0.0610.081 mg/mL.
179212 nm Deoxycholic acid and FA modied O-CMCS self-aggregates were prepared by sonication [165]
method and the mean diameter and critical aggregation concentration value changed with
change in DS and pH values.
301 nm The results showed that demonstrated that BoxBehnken design methodology was an [166]
effective way to obtain the optimal formulation of tea polyphenols-loaded chitosan nanoclusters,
and the nanoclusters complexation synthesizing through ionic gelation
between CMCS and chitosan hydrochloride was good biomaterials, which could
be successfully used to encapsulate tea polyphenols

on the adsorption amount of these plasma proteins [195]. Drug loaded
biodegradable polymer based nanobers intended to deliver drugs at
the targeted site in a sustained fashion avoiding burst release have re-
ceived attention by the researchers in the past decade. As far as CMCS
based nanobers are concerned, electrospinning technique has been
most widely accepted method. It is relatively a simple approach to con-
trol the morphology of ultrane bers with outstanding advantages of
very large surface-to-volume ration and high porosity with a small
pore size [196]. The silver nanoparticles/PVA/CMCS nanobers of uni-
form diameter of 295 to 343 nm with 4 to 14 nm sized Ag nanoparticles
were synthesised via electrospinning technique which proved suitable
as antibacterial biomaterial [197].
attenuate the degradation or release behavior of the incorporated drugs
or to enable targeted delivery by magnetic or surface modication. The al-
ginate/chitosan/CMCS composite microcapsules prepared by extrusion
method encapsulating Lactobacillus casei ATCC 393 proved to be useful
for the delivery of probiotic cultures to the human gastro-intestinal
tract [198]. Geisberger et al. [122] prepared polyoxometalates-trimethyl
chitosan nanoparticles which are obtained from the direct electrostatic
interaction of positively charged trimethyl chitosan with negatively
charged polyoxometalates as shown in Fig. 15. Table 5 shows different
CMCS based composites synthesised for delivery of different drugs.
8. CMCS based targeted drug delivery

Discovery and development of a new drug is a highly challenging,

7.6. CMCS composites
Composites are biomaterials that are composed of more than one con-
stituent of different physical and chemical properties that are blended to
form macroscopic, microscopic or nano structure. In case of composites
for delivery of drugs, usually the polymers are modied either to
Fig. 10. Release proles of DOX from CMCS-capped-MNP/MMT at different pH. The lines
are based on the tting with the empirical Peppas's model (reproduced with permission
from [174]).
labor intensive and expensive process. On an average development pro-
cess of each new drug takes approximately 15 years with an estimated
cost of about US$802 million. And, this estimated cost has been report-
ed to markedly increase at an annual rate of 7.4% above general price in-
ation [199]. Due to their inability to reach the target site of action, most
of the drugs in the clinical phase fail to achieve desired clinical out-
comes. In fact a considerable amount of drug administered gets distrib-
uted over normal tissues or organs, which are not involved in the
pathological processes causing severe side effects. An effective approach
to overcome this critical problem is the development of targeted drug
delivery system that can release bioactive compound at the desired
site of action. The main advantages of targeted drug delivery include
the accumulation of drug in the action site, increase in therapeutic ef-
cacy, reduction of therapeutic dose and toxicity, etc. [200].
The concept of developing a drug that could selectively destroy dis-
eased cells without harming healthy cells was proposed by Paul Ehrlich
almost a century ago. He gave this hypothetical drug name of the magic
bullet [201]. Since then, out of several polymers that have been
exploited in targeted drug delivery till date, CMCS has received signi-
cant attention due to its active functional groups which can easily attach
targeting ligands like FA. It can protect therapeutic agents from hostile
conditions in body and can release the entrapped agents selectively at
desired site in a controlled fashion. In addition to it, the inherent phar-
macological properties and excellent biological properties of CMCS
like biocompatibility, biodegradability, non-toxicity, increased water
solubility and unique mucoadhesivity also facilitate site specic drug
delivery. Fig. 16 illustrates the route followed by different CMCS based
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 67

Fig. 11. (A) Cellular uptake of curcumin-O-CMC Nps by FACS (a) control L929 cells alone (b) and (c) L929 exposed to 1 and 5 mg/ml curcumin-O-CMC Nps (d) control MCF-7 cells alone (e)
and (f) exposed to 1 and 5 mg/ml curcumin-O-CMC Nps (B) Apoptosis assay by FACS (a) control L929 cells exposed to O-CMC Nps (b) and (c) L929 exposed to 1 and 5 mg/ml curcumin-O-
CMC Nps (d) control MCF-7 cells exposed to O-CMC Nps (e) and (f) exposed to 1 and 5 mg/ml curcumin-O-CMC Nps (reproduced with permission from [175]).

formulations during their delivery to the targeted site of action in
human body. Sahu et al. investigated the use of hydrophobically modi-
ed CMCS nanoparticles for the delivery of anticancer drug PTX. The re-
sults showed that the nanoparticles exhibit a signicant inhibitory
effect on the folate receptor over expressing tumor cells like HeLa cells
[162]. Similarly evaluation of FA modied CMCS nanoparticles loaded
with another anticancer drug DOX has been also reported [202]. The
confocal and ow cytometry study revealed that the nanoparticles
could target the cancerous cells more effectively than the normal cells.
Recently, Wang et al. prepared novel biodegradable deoxycholic acid
(DA)-O, CMCS-FA micelles for the delivery of PTX which showed en-
hanced level of uptake compared to plain micelles in MCF-7 cells
[203]. N-succinyl-O-carboxymethyl chitosan (NSO, CMCS), another
modied derivative of CMCS has been proven to be an excellent disper-
sant to prepare a well-dispersed suspension of superparamagnetic
Fe3O4 nanoparticles due to its amphiphilic polyelectrolyte property. Its
good cytocompatibility and functional carboxyl groups showed its po-
tential for targeted dug delivery [204]. Similarly, magnetic nanoparticles
complexed with CMCS were prepared through spraying co-
precipitation method and their core-shell structure, stability and mag-
netic properties were also investigated. The studies revealed that the
spraying process is a more practical method due to an increased quan-
tity of adsorbed CMCS and a simplied automated preparation proce-
dure for targeted drug delivery [110]. In this context, the preparation
of well-dispersed suspension of superparamagnetic Fe3O4 nanoparticles
stabilised by chitosan and O-CMCS respectively by Zhu et al. is worth
68 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 12. Schematic diagrams of the fabrication procedure for CMCH-based folate/luminescent/magnetic nanoparticles (reproduced with permission from [123]).
mentioning. The adsorption mechanism of chitosan and O-CMCS onto
the surface of Fe3O4 nanoparticles is believed to be the electrostatic
and coordination interactions, respectively and was suitable for site spe-
cic drug delivery [205]. Novel multifunctional FA-CMCS-ZnS:Mn (FA-
CMCS-Zinc Sulphide: Manganese) nanoparticles developed through
simple aqueous route by Mathew et al. [125], have also shown potential
for effective targeted drug delivery and imaging of cancer cells.
8.1. Cancer-specic drug delivery based on CMCS
Despite of tremendous developments in the eld of medical science,
the cure for cancer is still a major challenge. Indiscriminate distribution
of most of the anticancer drugs towards disease and healthy cells fol-
lowing systemic administration ultimately leading to high toxicity re-
mains the critical bottleneck of conventional chemotherapeutics. In
addition to this, the poor solubility of most of the anticancer drugs in
water creates the need to use organic solvents or detergents for clinical
applications, resulting in undesirable side effects such as venous irrita-
tion and respiratory distress [206]. Therefore, in order to develop a suc-
cessful anticancer therapy, it is important to design a distinct carrier
system that can encapsulate large amount of anticancer drug and deliv-
er it specically to the cancerous cells. Till date a large number of CMCS
based matrixes that include nanoparticles, micelles, microspheres,
nanobers, composites, hydrogels, lms and bers have been designed
by different researchers for delivery of different anticancer drugs like
DOX, 5-FU, methotrexate, PTX, ADR, curcumin, camptothecin, 6-Mer-
captopurine, Vincristine etc. A study of glycol chitosan-carboxymethyl
cyclodextrins drug carrier for efcient targeted delivery of three hydro-
phobic anticancer drugs namely 5-FU, DOX, and vinblastine have been
carried out by Tan et al. [207]. Moreover, the development of multifunc-
tional novel folate conjugated CMCSFe3O4CdTe nanoparticles
exhibiting high drug loading efciency, low cytotoxicity and favorable
cell compatibility for the delivery of another hydrophobic anticancer
drug ADR has been reported by Shen et al. [123]. CMCS based delivery
system of another model anticancer drug has been experimented by
Jin et al. [161], where the group demonstrated the preparation and
in vitro evaluation of amphiphilic DA modied CMCS based pH sensitive
self aggregated nanoparticles. The results of the experiment showed the
greater cellular uptake and enhanced retention of drug loaded nanopar-
ticles in drug-resistant cells thereby conrming its superior efcacy
over free DOX. Maya et al. [208] have prepared O-CMC nanoparticles
by ionic gelation technique where negatively charged carboxyl groups
of O-CMC were cross-linked using CaCl2 and PTX was loaded within
the nanoparticles before the cross-linking step, after allowing the drug
to interact with O-CMC. In order to facilitate the targeted delivery of
PTX, antibody Cetuximab (Cet) was conjugated onto the PTX O-CMC
nanoparticles by EDC coupling chemistry. The complete reaction
scheme for the preparation of Cet-PTX-O-CMC nanoparticles is shown
in Fig. 17 [208]. Zou et al. [209] used octreotidepolyethylene glycol
stearic acid (OCTPhePEGSA) as a targeting molecule for N-octyl-O,
N-carboxymethyl chitosan (OCC) micelles loaded with DOX to prepare
novel active tumor targeting carrier. Fig. 18 [209] illustrates the proce-
dure of self assembly and receptor-mediated cellular internalisation of
OCC-OCT micelles. When OCT conjugated to stearic acid (SA) via poly-
ethylene glycol (PEG) spacer (OCTPhePEGA) was used to modify
OCC micelles. SA could insert into the inner hydrophobic core of OCC
when it self-assembled in the aqueous environment, thereby increasing
the soundness of modication of OCC. On the other hand, PEG, covering
the surface of micelles, could increase the blood circulation time of mi-
celles whereas OCT increased the targeting efciency of the micelles to
tumor cells. The results of the in vitro and in vivo studies indicated that
OCCOCT micelles might be a promising tumor-targeting carrier for
cancer therapy [156]. Table 6 shows different CMCS based matrices for
the delivery of various anticancer drugs.
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 69

Table. 4
Different nanoformulation for delivery of various anti-cancers, anti-inammatory, anti-microbial drugs, proteins/peptides and vaccines along with their brief outcome.

Drug Drug Brief method and outcome Refs.

category used

Anti-cancer Adriamycin Novel CMCS based folate/Fe3O4/CdTe nanoparticles within size range of 170190 nm possessing intense [123]
superparamagnetic effect and photoluminescence property at room temperature exhibiting high drug
loading efciency, low cytotoxicity and favorable cell compatibility were prepared.
Camptothecin A novel organicinorganic hybrid molecule consisting of carboxymethyl-hexanoyl chitosan modied with [177]
(3-aminopropyl) triethoxysilane was synthesised which showed self-assembly behavior into nanoparticles
with a stable polygonal geometry consisting of ordered silane layers of 6 nm in thickness. This formulation
demonstrated excellent cytocompatibility and cellular internalisation capability in ARPE-19 cell line along
with well controlled encapsulation and release proles.
Curcumin Curcumin loaded N,O-CMCS formulation was prepared by simple ionotropic gelation method with size [184]
range of 150 30 nm having high encapsulation efciency of 80% and indicated slow, controlled and
sustained drug release. The nanoformulation was specically toxic to cancer cells and non-toxic to normal
cells
Doxorubicin DOXHCl loaded nanospheres and microspheres were prepared by precipitation method with high [115]
encapsulation efciency which displayed sustained drug release.
Doxorubicin DOX loaded methoxy poly(ethylene glycol)-grafted-CMCS nanoparticles were synthesised by ion [121]
complex formation with size b300 nm. Drug release was faster at acidic pH than neutral or basic pH and
showed promising antitumor activity.
Doxorubicin Amphiphilic deoxycholic acid modied CMCS based pH-sensitive self-aggregated nanoparticles were pre- [161]
pared within size range of 87 to 174 nm
5-Fluorouracil 5-FU encapsulated FACMCZnS:Mn nanoparticles with size range of 130150 nm were prepared [125]
which were found to be suitable for targeting, controlled drug delivery and cancer cell imaging using
Breast cancer cell line MCF-7 and were non-toxic to L929 cells.
5-Fluorouracil 80 20 nm sized 5-FU loaded N,O-CMCS nanoparticles were prepared with drug entrapment ef- [185]
ciency of 65% and were toxic to breast cancer cells showing good blood compatibility.
Methotrexate O-CMCS-methotrexate nanoparticles were prepared by dialysis method with size range of [160]
187.2363.5 nm and zeta potentials ranged from 8.19 mV to 3.08 mV. Drug release behavior was
sustained in PBS solution of (pH 4.0, 7.2 and 9.0).
6-Mercaptopurine Six 6-Mercaptopurine-CMCS were prepared and structurally characterised. 6-Mercaptopurine-CMCS in [116]
pH 7.4 PBS could self-assemble into nanoparticles with mean diameter of 155.8 6.0 nm by Dynamic
Light Scattering and 100 nm by TEM. 6-Mercaptopurine showed release in media containing 2 mM and
10 mM GSH and maximum cumulative release rates were 65.1% and 74.4%, respectively.
Paclitaxel Target oriented nanoparticles based on O-CMCS modied with stearic acid with FA covalently attached [162]
by carbodiimide reaction by sonication method without using surfactants/emulsiers. The nanoparti-
cles exhibited signicant inhibitory effect on Folate Receptor over expressing tumor cells like HeLa cells.
Vincristine Novel multifunctional O-QCMCS/Cholesterol liposomes were constructed with good physical and [186]
thermal stability, excellent solubility in water, and high drug encapsulation efciency (90.1%) and
displayed steady release action over 2 weeks.
Anti-inammatory Indomethacin Magnetic N-benzyl-O-CMCS nanoparticles were synthesised by incorporation and in situ methods and in- [187]
domethacin was incorporated by solvent evaporation method with loading efciency of 60.8% to 74.8%. The
in vitro drug release prole in Simulated Body Fluid (pH 7.4, 37) displayed an initial fast release, which
became slower as time progressed.
Ibuprofen Novel O-CMCS/-cyclodextrin nanoparticles of spherical shape and size with 166 nm were prepared and [124]
Ibuprofen was loaded with entrapment efciency of 93.25 2.89%. The release rate was slower from O-
CMCS/-cyclodextrin than from chitosan/-CD in Simulated Gastric Medium (pH 1.2) while converse was
true for Simulated Intestinal Fluid (pH 6.8).
Ketoprofen Ampiphillic matrices of CMCS-graft-phosphatidylethanolamine were prepared by a EDC-mediated [188]
coupling reaction.
Dexamethasone Novel surface engineered highly branched CMCS/polyamidoamine dendrimer nanoparticles were [189]
synthesised which did not exhibited signicant cytotoxicity in the range of concentration below 1 mg/mL
and displayed high internalisation efciency by both human osteoblast-like cells and rat bone marrow
stromal cells. Interesting physicochemical and biological properties were reported for these macromolec-
ular systems.
Anti-microbial Tetracycline Tetracycline encapsulated O-CMCS nanoparticles were prepared by ionic gelation method which were [190]
biocompatible and 200 nm in size. This nanomedicine was 6-fold more effective in killing intracellular
S. aureus compared to Tetracycline alone.
Gatioxacin Spherical 3070 nm sized GFLX entrapped O-CMCS nanoparticles were developed which displayed a [191]
(GFLX) four-fold lower MIC value against Gram negative bacteria compared to GFLX solution and a similar MIC
value against Gram-positive bacteria as compared to GFLX solution.
Proteins/Peptides/Vaccines Bovine Serum Albumin (BSA) N,O-CMCS modied pristine nanodiamond particles were developed which were biocompatible and [192]
showed no cytotoxicity to cells.
Basic Fibroblast Growth Factor (bFGF) Out of a series of chitosan derivatives synthesised, 2-iminothiolane modied 2-N sulfated 6-O- [193]
carboxymethylchitosan and chitosan complex was found suitable for preparing nanoparticles by poly-
electrolyte self-assembly method which could successfully protect bFGF from inactivation over a 120 h
period as determined by L929 broblast culture tests. The release of bFGF could be successfully controlled.
Tetanus Toxoid 40400 nm sized N-trimethyl chitosan, chitosan and M,N-CMCS nanoparticles with negative surface [194]
(TT) charge for M,N-CMCS and positive surface charge for chitosan and M,N-CMCS nanoparticles were de-
veloped with TT loaded with efciency N90% m/m. Effective uptake of the Fluorescein isothiocyante-BSA
loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells and
enhanced immune response was reported after intranasal application of nanomedicine.
Protein drugs Water soluble oleoyl-CMCS was synthesised through covalent modication of chitosan with oleic acid [113]
and monochloroacetic acid prepared by self-assembled method. The OCMCS nanoparticles showed high
loading efciency and sustained release of extracellular products. The mucoadhesion and internalisation
capability make oleoyl-CMCS nanosystems interesting candidates that could be effective to improve
protein drugs adsorption after oral administration.
70 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 13. Preparation of the PVA/CMCS blank lms and drug lms (reproduced with permission from [38]).
8.2. Organ-specic drug delivery based on CMCS
Organ, or site-specic drug delivery, has several distinct advantages
over other means of delivering drugs. For example, direct infusion into
the target organ or the vasculature surrounding and supplying blood
to the organ ensures that the majority of the drug goes to the site it is
intended to act on. This allows for the use of the more toxic drug agents
(e.g., chemotherapeutics) in high concentrations, because the exposure
of other organs to the compound is limited. Targeting organs or tissues
in vivo can greatly reduce the risk of toxic side effects and signicantly
increase the efcacy of a variety of drugs, including toxic chemothera-
peutic agents, pain medications, and gene therapies. The advantages
of site-specic employing CMCS vehicles include pH-sensitivity,
bioadhesive ability, solubility and absorbability, controllable biodegrad-
ability, nontoxicity of the degradation end products, sustained release
potential and ease of administration [213]. Many researchers have pre-
pared different CMCS based formulations that exclusively deliver vari-
ous pharmacological agents at specic organs for their activity. These
include different organs of body like intestine, liver, pancreas, colon,
eyes, and others which are elaborated below.
Fig. 14. Release of ornidazole through 30% C blend lm at different pH. Results are means 6
standard deviation for n 3 (reproduced with permission from [126]).
8.2.1. Colon-specic drug delivery using CMCS
Colon-specic delivery of drugs has gained much attention in recent
years for the treatment of various diseases such as Crohn's disease, ul-
cerative colitis, and irritable bowel syndrome [214]. The relatively low
proteolytic activities of protein/peptide drugs in the colon and even
for other nonpeptide drugs such as cardiovascular and antiasthmatic
agents has prompted several researchers to focus on colon targeted
drug delivery. The absorption and degradation pathways in the upper
gastrointestinal tract are the major hindrances in delivering drugs to
the colon. To overcome these obstacles, several strategies of colon-
specic drug delivery have been attempted till date by various scien-
tists, that include use of prodrugs which become active at the colon,
drug-eluting system responding to the pH, and microora-activatable
drug delivery systems have gained increasing attention. These strate-
gies basically emphasise on preventing loss of the drug at the stomach
and the small intestine, thereby facilitating quantitative drug delivery
to the colon.
CMCS based systems have been widely studied for colon targeted
drug delivery as colonic microora can degrade its glycosidic linkage
thus facilitating the release of drugs entrapped specically in colon.
Tavakol et al. [215] investigated the release of sulfasalazine drug from
the alginate-N,O-CMCS gel beads prepared by ionic gelation method.
The in vitro studies revealed that the chitosan coated alginate-N,O-
CMCS hydrogel may be used as potential polymeric carrier for colon-
specic delivery of sulfasalazine. Tu et al. [216] studied the sigmoidal
swelling kinetics of a series of CMCS-g-poly (acrylic acid) hydrogels
which were pretreated under acidic buffer media. The swelling kinetics
of 5-aminosalicylic acid at different pH showed its potential for colon-
specic drug delivery. Recently, Vaghani et al. [98] prepared and
characterised pH sensitive hydrogel composed of CMCS cross linked
with GA. The group evaluated in vitro as a promising carrier for the ad-
ministration of colon targeted drug delivery of OD.
8.2.2. Liver targeted drug delivery using CMCS
Liver targeted delivery of drugs has gained increasing attention for
the treatments of a number of chronic hepatic diseases such as hepatitis,
hepatocirrhosis, hepatoma and hepatic carcinoma in the recent years.
Considerable effort has been made to exploit CMCS as liver-specic
drug carrier. This is because nanoparticles prepared from such amphi-
philic derivative via self-assembly have been recognised as a promising
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 71

Fig. 15. Electrostatic interaction of CMCS (a) and trimethyl chitosan (b) biopolymer matrices with encapsulated polyoxometalates (POMs) (reproduced with permission from [122]).

drug carrier, since their hydrophobic domain can serve as a depot for
sparingly soluble drugs [217]. Zheng et al. [218] synthesised glycyrrhizic
acid encapsulated novel thiolated lactosaminated (TLAC)/CMCS nano-
particles through ionic gelication method and characterised these
nanoparticles in vitro studies. The in vivo studies of pharmacokinetic pa-
rameters were evaluated in rabbits and tissue distribution in mice. The
results showed that the TLAC-CMCS may be used as a promising drug
carrier for hepatic targeting and controlled release. Similarly, prepara-
tion, characterisation and tissue distribution studies of lactosaminated
CMCS nanoparticles have been reported by Zheng and coworkers. The
experiment demonstrated that the in vitro release of glycyrrhizic acid
from the nanoparticles exhibited a biphasic pattern, initial burst release
and consequently sustained release. Also, these nanoparticles modify
the tissue distribution prole of the glycyrrhizic acid solution, the kid-
ney excretion rate is reduced and drug accumulation in the liver is in-
creased [105]. Recently, Shi et al. [212] prepared O-CMC-methyl
methacrylate (MMA) copolymers through the graft copolymerizion of
hydrophobic MMA and hydrophilic O-CMC, which spontaneously self-
assembled into coreshell O-CMC nanoparticle in aqueous solution. The
adjacent hydroxyls of glycyrrhizin (GL) were turned into aldehyde
groups through periodate oxidation in order to obtain O-CMC-GL nano-
particle. The preparation procedure of O-CMC nanoparticle, GL modied
CMC-GL, and PTXL loaded CMC-GL nanoparticle (PTXL/CMC-GL) nano-
particles were illustrated in Fig. 19 [212]. In addition, in vitro and
in vivo studies of PTX loaded GL-modied O-CMCS nanoparticles were
also carried out by for hepatocellular carcinoma targeted drug delivery
application. The results established these nanoformulation as promising
drug carrier for hepatic cancer.
8.2.3. Ocular drug delivery using CMCS
The eld of ocular drug delivery is one of the most interesting and
challenging endeavors facing the researchers and scientist community.
From drug point of view, it is very difcult to study the eye as an isolated
organ. This is due to the presence of highly sensitive ocular tissues like

Table. 5
Different CMCS based composites synthesised for delivery of different drugs.

Composite Drug used Brief method & output Refs.

Folic acid conjugated CMCS-Mn doped
ZnS nanocomposite
O-CMCS/-CD nanocomposite
CMCS/TMCS nanocomposite
Nano-hybrid CMCS-hexanoyl chitosan
modied with (3-aminopropyl)
triethoxysilane
Folate conjugated CMCS/Fe3O4/CdTe
nanocomposites
5-Fluorouracil (5-FU) Novel multi-functional FACMCSZnS:Mn nanocomposites prepared by simple aqueous route showed [125]
high drug encapsulation of 5-FU (92.08%) with controlled release and were found nontoxic to mouse
broblast L929 cells and toxic to MCF-7 cell line.
Ibuprofen (IB) 166 nm sized IB loaded O-CMCS/ cyclodextrin nanocomposites by simple ionic cross-linking method [124]
showed high drug encapsulation efciency of 93.25 2.89%. The release rate was slower from O-CMCS/
CD than chitosan/ CD in simulated gastric medium while converse was true for simulated intestinal
medium.
Bioactive 5090 nm sized nanocomposites were prepared by direct cross-linking approach. POMCMCS com- [122]
Polyoxometalates posites display negative zeta potentials and larger particle sizes than the positively charged POM
(POMs) Trimethyl Chitosan composites
Camptothecin (CPT) Highly ordered appx. 6 nm thick silane layer formations upon self-assembly of the hybrid molecule [177]
leads to sustained release of CPT. The hybrid molecule showed excellent cytocompatibility and efcient
cellular internalisation with tp ARPE-19 cell line.
Adriamycin (ADR) 170190 nm sized novel folate conjugated CMCS/Fe3O4/CdTe NCs were prepared by layer-by-layer [123]
assembly technique. Initial CMCS concentration, medium pH and reaction time strongly inuenced
coating amount and binding mode of CMCS. The multifunctional nanocomposites exhibited high drug
loading efciency, low cytotoxicity and favorable cell
Compatibility.
72 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487

Fig. 16. Illustrates the route followed by different CMCS based formulations during their delivery to the targeted site of action in human body.

the uveal tract and retina. In addition to this, the presence of tissue bar-
riers to drug penetration which include the lipophilic corneal epitheli-
um, the hydrophilic corneal and scleral stroma, the conjunctival
lymphatics, choroidal vasculature, and the blood-ocular barriers
makes ocular drug delivery even more challenging [219]. The choice
of chitosan for ocular drug delivery has been justied due to its out-
standing mucoadhesive and penetration enhancing properties, as well
as by its good biocompatibility with the ocular structures [220]. Applica-
tion of chitosan based nanostructures in delivery of ocular therapeutics
has been well reviewed by Fuente et al. [221]. CMCS has attracted much
attention in ophthalmic drug delivery due to its inherent low toxicity,
biocompatibility, biodegradability and ability to prolong ophthalmic
drug retention time [222]. In fact the ability of CMCS to prolong the
precorneal drug retention, by virtue of its viscosity-increasing affect, of
its ability to bind ooxacin and, probably, of its mucoadhesive proper-
ties in comparison to chitosan has been experimentally demonstrated
by Colo et al. [222]. An in vitro study of gatioxacin (GFLX) from novel
O-CMCS formulation showed that the release was slower than that
from GFLX solution and the MIC of OCMCS formulation against Gram-
negative bacteria is fourfold lower than the system without OCMCS.
The GFLX is a fourth-generation uoroguinolone and in vitro studies
on isolates from bacterial infections of the eye have shown an

Fig. 17. Schematic illustration depicting preparation of PTXL-O-CMC nanoparticles and bioconjugation of Cet on PTXL-O-CMC Nps through EDC activation chemistry. EDC activated the
carboxyl functionality and subsequently linked to Cet through covalent linkage (reproduced with permission from [208]).
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 73

Fig. 18. Schematic illustration of the self-assembly and receptor-meditated cellular internalisation of OCCOCT micelles (reproduced with permission from [209]).

Table 6
Different CMCS based matrices for the delivery of various anticancer drugs.

Anticancer drug CMCS based matrix Matrix composition Remarks Refs.

structure

Adriamycin Self aggregated LA-modied CMCS Self-aggregated nanoparticles exhibited [120]

nanoparticles an increased Loading Capacity and Loading Efciency, decreased sustained release with an increasing
ratio of the hydrophobic LA
to hydrophilic CMCS
Camptothecin Aggregates O-CMCS Not only the aggregates but also the unimers of OCMCS can help to enhance the solubility of CPT. In vitro [163]
cancer antiproliferative activity test further conrms the
slow release of CPT from OCMCS-drug system.
Curcumin Nanoparticles O-CMCS Spherical 150 30 nm sized curcumin loaded O-CMCS nanoparticles were prepared with entrapment [175]
efciency of 87%. The nanoparticles were toxic to cancer cells and non-toxic to normal cells.
Doxorubicin Nanospheres and CaCO3CMCS The water soluble DOXHCl could be effectively loaded in the hybrid [115]
microspheres microparticles and nanospheres with a high encapsulation
efciency, and the drug release could be effectively sustained,
indicating the hybrid microspheres and nanospheres were
suitable for delivery of water-soluble drugs
Self-assemblied Folic acid (FA) modied 267.8 nm sized nanoparticles were prepared by sonication method. The cellular uptake of Folate [162]
nanoparticles CMCS modied CMCS nanoparticles
was found to be higher than that of nanoparticles
based on LA modied CMCS.
Self assembled Carboxymethyl-hexanoyl The model anticancer drug DOX was entrapped with an efciency of 46.8%, and a corresponding drug [155]
hollow chitosan release from the nanocapsules for a time period exceeded 7 days can be achieved in vitro.
nanocapsule
Nanoparticles Methoxy poly(ethylene The nanoparticles showed increased cytotoxicity compared to DOX alone. [121]
glycol)-grafted-CMCS
Self assembled Acylated CMCS The nanoaggregates exhibited an excellent colloidal and structural stability in aqueous medium. [35]
nanoaggregates
5-Fluorouracil Nanoparticles Folate conjugated CMCS- 130150 nm sized novel nanoparticles loaded with 5-FU exhibited non-toxicity to L929 cells. The [125]
Mn doped zinc sulphide nanoparticles could be used for controlled drug delivery and imaging of cancer cells.
Methotrexate Nanoparticles FA conjugated CMCS The encapsulation efciency and loading capacity of Methotrexate in the FA-O-CMCS nanoparticles [210]
were higher, and the particle size of the FA-O-CMCS nanoparticles was also smaller than those in the FA-
CS nanoparticles.
Self assembled Methotrexate conjugated Spherical shaped nanoparticles within size range of 187.2363.5 nm were prepared by dialysis method. [160]
nanoparticles O-CMCS In vitro drug release study by the dynamic dialysis method showed that Methotrexate exhibited
signicant sustained-release behaviors
in PBS buffer solutions (pH 4.0, 7.2 and 9.0), indicating that these nanoparticles had good in vitro sta-
bility
and the potential to be used as a novel drug carrier system
6- Nanoparticles 6-Mercaptopurine-CMCS The 6-Mercaptopurine release from 6-MP-CMC showed dependence on glutathione concentration. In [211]
Mercaptopu- aqueous solution 6-MP-CMC could self-assemble into the nanoparticles through the intra- and inter-
rine molecular hydrophobic interactions between 6-MP groups
Paclitaxel Nanoparticles Glycyrrhizin (GL)-modied 100205 nm sized PTX loaded O-CMCS nanoparticles were prepared with encapsulation efciency of [212]
O-CMCS 83.7% and performed a biphasic release.
Nanoparticles FA conjugated CMCS modi- The PTX loaded nanoparticles exhibited many desirable properties like pH-sensitive dissolution, low [162]
ed with stearic acid cytotoxicity, and high amount drug encapsulation and signicant inhibitory effect on the FR over ex-
pressing tumor cells like HeLa cells
Vincristine Liposomes O-Q-CMCS-Cholesterol Vincristine was encapsulated in polymeric liposomes with high entrapment efciency (90.1%). The [186]
formulation was stable in aqueous solution and exhibited slow, steady release action over 2 weeks
under physiologic pH (7.4).
74 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 19. Synthetic route of O-CMC-methyl methacrylate (MMA) copolymers and glycyrrhizin (GL) and schematic representation depicting the formation of O-CMC nanoparticles, O-CMC-
GL nanoparticles, and PTXL loaded O-CMC-GL nanoparticle (PTXL/CMC-GL) (reproduced with permission from [212]).
encouraging response to it [191]. Currently, Yang et al. investigated both
in vitro and in vivo, the ability of 5-FU or bevacizumab loaded N,O-CMCS
hydrogels to modulate wound healing after glaucoma ltration surgery.
On one hand the in vitro study showed that the nearly 100% of 5-FU was
released from the drug-loaded hydrogels within 8 h, but less than 20%
bevacizumab was released after 53 h. On the other hand, the in vivo
study carried out in rabbits showed that the CMCS hydrogels were non-
toxic to the cornea and were gradually biodegraded in the eyes [141]. Re-
cently, to investigate the availability of induced pluripotent stem cells
iPSCs as bioengineered substitutes in corneal repair Chien et al. [223] de-
veloped a thermo-gelling injectable amphiphatic carboxymethylhexanoyl
chitosan (CMHC) nanoscale hydrogel and found that such gel increased
the viability and CD44 proportion of iPSCs, and maintained their
stem-cell like gene expression, in the presence of culture media. The
study demonstrated that human keratocyte-reprogrammed iPSCs, when
combined with CMHC hydrogel, can be used as a rapid delivery system
to efciently enhance corneal wound healing.
8.2.4. Others
Apart from these, there are a number of other organs for which
CMCS based drug delivery systems have been designed by different sci-
entists in the recent times. This include synthesis and in vitro study of
microencapsulated beads composed of alginate-N,O-CMCS for the deliv-
ery of model protein BSA to different regions of the intestinal tract. The
results of the study demonstrated excellent pH sensitivity and proved to
be suitable polymeric carrier for site specic delivery of bioactive pro-
tein in the intestine. Also the main advantage was that the bioactivity
of the protein drug was preserved due to the preparation of drug loaded
beads in the aqueous medium at neutral environment [94]. Another
similar work for the in vitro and in vivo evaluation of pH sensitive
CMCS based hydrogels for intestinal delivery of drug theophylline has
also been reported recently by [145]. While the in vitro results showed
that formulation I containing CMC and carbopol in 1:1 ratio showed
sustained release, better prolonged action from the prepared hydrogel
formulation when compared to (standard) marketed sustained release
formulation was reported from the in vivo study. Currently, synthesis,
characterisation and in vitro evaluation of anticancer drug 5-FU loaded
CMCS nanoparticles have been reported by Anitha et al. [184] The toxic-
ity of the drug loaded CMCS nanoparticles was showed by MTT, apopto-
sis and caspase 3 assays thereby conrming the potential of 5-FU loaded
N,O-CMC nanoparticles in breast cancer chemotherapy in which the
side effects of conventional chemo treatment could be reduced. Recent-
ly, preparation of metformin loaded O-CMCS nanoparticles prepared by
ionic gelation method for delivery to pancreatic cancer cells has showed
pH sensitive release of the drug in vitro. While the cytotoxicity study
showed the preferential toxicity of the drug loaded nanoparticles on
pancreatic cancer cells (MiaPaCa-2) compared to normal cells (L929),
the nanoparticles exhibited nonspecic internalisation by normal and
pancreatic cancer cells [106].
9. Tissue engineering: origin and strategies
Tissue engineering, by denition, is a highly interdisciplinary eld
that combines the principles and methods of life sciences and engineer-
ing to utilise structural and functional relationships in normal and path-
ological tissue to develop biological substitutes to restore, maintain, or
improve biofunction [224]. The term tissue engineering was formally
coined in 1987 [225] and since then, it has emerged as a scientic eld
distinct from medical eld by providing numerous promising tech-
niques with practical clinical applications. The popular theory of the
medical eld that the human body possesses an inherent capacity to
heal itself has been the fundamental principal that has been exploited
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 75

in the eld of tissue engineering. In fact, the specic tissue or the organ
determines the level of ability to self-repair and regenerate from dis-
ease, damage or an injury. This capacity is limited by the degree of dam-
age, loss of function and involvement of multiple tissues [226]. The
recent developments in tissue engineering like xeno-transplantation
of tissues, new prosthetics and localised manipulation of lesion sites at
the cellular and molecular level have made signicant advancements
in reconstructive surgery in comparison to the conventional approaches
like tissue auto- and allo-grafting [226,227].
The main objective of tissue engineering is to overcome the lack of
tissue donors and the immune repulsion between receptors and donors.
In fact, by laying emphasis on tissue and cell-based therapy, tissue engi-
neering and clinical practices strive to achieve same goals. The tissue en-
gineering strategy involves the in vitro seeding and proliferation of
relevant cells and/or signaling molecules in an appropriately designed
tissue engineering biomaterial like scaffold to form a natural tissue.
This tissue is implanted into the defect in the patients. While in some
cases, the scaffold or scaffold with cells is directly implanted in vivo
where the host body functions as a bioreactor to construct new tissues.
Thus, these transplantable constructs enable regeneration of functional
tissue in the host providing an alternative to conventional organ
transplantation and tissue reconstruction methods. Over the past
40 years, tissue engineering has shown signicant most tissue types, es-
pecially those damaged or lost following debilitating health problems
such as cancer and degenerative disorders [228].
10. Preparation techniques of CMCS based biomaterials for tissue
engineering
CMCS is biocompatible, biodegradable, and more bioactive than chi-
tosan with enhanced osteogenesis property and it can be easily formu-
lated in a variety of forms like hydrogels, scaffolds, composites, i-gels,
lms and membranes which have wide range of potential applications
in tissue engineering. The techniques that are suitable for preparation
of different CMCS based hydrogels for tissue regeneration purpose
have already been discussed in Section 8. As in hydrogels, the cross
links can be incorporated either by chemical cross linking method or
by radiation cross linking in the scaffolds also as per the requirement
of the host tissue and the tissue engineering biomaterial being fabricat-
ed. Nowadays, enzymatic cross linking has also gained attention due to
its own benets. Selecting the scaffolding approach for tissue engineer-
ing is tissue and application specic. The conventional scaffold

Table. 7
Preparation techniques of CMCS based biomaterials for tissue engineering.

Composition of matrix Technique/method Comment Refs.

Gelatin/CMCS/-tricalcium
phosphate composite scaffold
Hydroxyapatite coated CMCS scaffolds
HAP/CMCS composite scaffold
CMCS/HAP
n-HAP/CMCS biocomposite
scaffold
N-isopropylacrylamide and CMCS
hydrogels
Gelatin & CMCS hybrid hydrogels
CMCS/n-HAP composite
n-HAP/CMCS composite
CMCS/gelatin/n-HAP i-gel
CMCS lm
CMCS-graft-D-glucuronic acid
membranes
Radiation crosslinking Series of biodegradable scaffolds were prepared where ultrasonic [127]
and lyophilizing treatment on the polymer solutions affected the -TCP particles
distribution. Excellent biocompatibility and ability of bone
regeneration was revealed by in vivo implantation in mandible
of beagle dog.
Freeze drying technique Coating of scaffolds with HAP substantially enhanced the viability, [40]
attachment, proliferation, and differentiation of the osteoblast and
directed stem cell differentiation to osteoblast
Coprecipitation method The scaffolds exhibited 20500 m sized pores with regular interconnection [229]
with appx. 58.9% of porosity determined from microcomputed tomography
analysis. Average scaffolds consisted of 24% HA and 76% CMCS determined
from 2D morphometric analysis.
Electrospinning The SEM showed that nano/micro particles formed on the surface of the [230]
nano-nonwoven CMCS brous scaffold. FTIR and XRD conrmed that the
nano/micro particles were hydroxyapatite crystalline. HAP particles
appeared to have a great effect on the late stages of osteoblast behavior
(alkaline phosphatase).
Freeze drying technique The FTIR and XRD results of genipin cross linked n-HAP/CMCS scaffolds [231]
revealed that CMCS's hydroxyl, amine and amide groups determined
nano homogenous distribution of n-HAP and provided nano topographical
features for nanohybrid scaffolds. The scaffolds had pore size of 150 m sized
pores with less toxicity and more facility for adhesion and proliferation of cells.
Chemical cross linking & Thermo responsive & core-shell microgels were prepared having phase [232]
seed emulsion polymerisation transition temperature nearer to that of body compared to pure PNIPAM
Radiation induced cross linking Due to the high water absorption capacity, a similar [103]
compressive modulus with soft tissue, controllable biodegradation,
and excellent biocompatibility, the hydrogels have potential as skin
scaffolds and wound healing materials.
Porogen leaching method The porosity ratio of CMCS/n-HA is about 75% and the compressive [233]
strength can exceed 21 MPa with circular pores of diameter ranging
from several m to six hundred m. The in vivo experiments showed
no inammatory reaction and bone putrescence and toxicity of liver and kidney.
Particle ltration and lyophilisation The composite scaffold with (VEGF)-transfected bone marrow stromal [234]
followed by genipin crosslinking cells (BMSCs) was studied in a rabbit radial defect model. The scaffold is
biocompatible, nontoxic, promotes the inltration and formation of the
microcirculation, and stimulates bone defect repair with degradation
rate matching the growing rate of bone.
Enzymatic crosslinking The i-gels prepared susceptible to tyrosinase/p-cresol mediated in situ [2]
gelling at physiological temperature that may be used in treating irregular
small bone defects with minimal clinical invasion as well as for bone cell delivery
Covalently crosslinked Controlling the molecular weight distribution of properly tailored chitosan [72]
allows one to regulate the mechanical properties and degradation of
chitosan in a sophisticated manner, while maintaining favorable cell
interactions.
Grafting D-GA onto CMCS in The membranes showed bioactivity which demonstrated its potential for [235]
the presence of 1-ethyl-3-(3- tissue engineering applications.
dimethylaminopropyl)
carbodiimide (EDC)
76 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
fabrication techniques include solvent-casting particulate-leaching
method, gas foaming, bre meshes/bre bonding, phase separation,
melt moulding, emulsion freeze drying, solution casting, and
lyophilisation (freeze drying) method. The particle leaching method ex-
hibits advantages of creation of scaffolds with big pores, wellcontrolled
high interconnected porosity and pore morphology. The benets of gas
foaming technique include lack of solvent, eliminating the risk of re-
maining residues, and the low processing temperatures preventing deg-
radation of the polymer during processing. Emulsion freezedrying is
attractive for creation of scaffolds that are relatively thick having large
pores. Despite of several positive facets of these traditional methods,
the incapability of precisely controlling pore size, pore geometry, spatial
distribution of pores and construction of internal channels within the
scaffold, presence of residual organic solvent and poor mechanical in-
tegrity are some of the most signicant problem facing these conven-
tional techniques due to the risks of toxicity and carcinogenicity it
poses to cells. Therefore, new techniques like self-assembly systems,
solid free-form fabrication and electrospinning technique have
attracted the attention of the researchers as these methods can over-
come many of the demerits of the conventional scaffolding approaches.
Sintering method for scaffold design has also received attention and is
usually applied in case of ceramics powders, metals, glasses and certain
polymers as well as composites. But most of the composites for tissue
repair are prepared by coprecipitation, porogen leaching and freeze dry-
ing (lyophilisation) method. In addition to this, injectable-gels, and
membranes can be prepared by enzymatic and covalent cross linking
and grafting method respectively. CMCS based lms are most common-
ly prepared by blending/casting method that may include covalent cross
linking or other linkages. Table 7 shows preparation techniques of CMCS
based biomaterials for tissue engineering.
11. CMCS based biomaterials for tissue engineering and regeneration
An ideal biomaterial for tissue engineering is expected to meet some
important criteria. It must be biocompatible, easily biodegradable in ap-
propriate time window, its degradation products should be non-toxic,
and it must support cell adhesion and growth and should exhibit me-
chanical strength comparable with the host tissue [236]. The applica-
tions of chitosan and its derivatives in the eld of tissue engineering
have been earlier reviewed [237]. CMCS has attracted considerable at-
tention for tissue engineering application due to its inherent increased
Fig. 20. SEM images of gelatin/CM-chitosan/-TCP composite scaffolds with -TCP fraction of (A) 0%, (B) 5%, (C) 10%, (D) 20%, (E) 30%, and (F) 40%. The ultrasonic time was 20 min
(reproduced with permission from [127]).
bioactivity as compared to chitosan and its ability to promote osteogen-
esis [25]. Also the capacity of CMCS to chelate calcium from mineralizing
solution containing calcium and phosphate to induce calcium phos-
phate or hydroxyapatite (HAP) formation has made it suitable biopoly-
mer for tissue engineering [238]. Apart from this, it is well known to
exhibit excellent biocompatibility, better biodegradability, non-
toxicity, and ability to promote cell adhesion which is desirable in the
eld of tissue engineering and regenerative medicine. In the past few
years, several researchers have utilised CMCS in fabrication of different
tissue engineering biomaterials which include scaffolds, hydrogels,
composites, nanobers, nanoparticles, dendrimer, and membranes
and for implant functionalisation [239241] due to easy processibility
of CMCS into these constructs.
11.1. CMCS scaffolds
Scaffolds are unique tissue engineering biomaterial as they are able to
establish three-dimensional environments for propagated cells and spe-
cic signalling molecules that can mimic native tissues environments.
Tissue engineering scaffolds can be of natural, synthetic or a hybrid of
both. CMCS has emerged as a promising scaffolding polymer due to its
inherent excellent biocompatibility, ability to promote cell adhesion
and increased bioactivity as compared to chitosan. Also in comparison
to chitosan that has relatively slow and uncontrollable degradability
[242]. CMCS shows accelerated degradation rate which can be regulated
through different cross-linking extents by EDC, while retaining excellent
mechanical properties. However, EDC cross-linked CMCS porous tubular
scaffolds were fabricated for nerve regeneration which showed de-
creased hydrophilicity and elastic modulus which is desirable for nerve
repair [60]. Nanobrous collagen-coated porous CMCS microcarriers
were successfully fabricated by a simple modied phase separation
method and thereafter collagen anchoring-assembling. In vitro chondro-
cyte culture revealed better cell attachment, proliferation, and differenti-
ation on the CMC-MCs immobilised with self-assembled collagen
nanobers. Cells were observed to grow into a tissue-like structure
after 7 days of culture. Thus the scaffolds prepared showed potential
for application as injectable scaffolds for cell delivery in cartilage tissue
engineering [72]. Gelatin/CM-chitosan/-tricalcium phosphate compos-
ite scaffolds were prepared using a green fabrication method, i.e.
radiation-induced cross linking. Considering their excellent and adjust-
able water retention capacity, highly interconnected porous network
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 77

Fig. 21. Schiff-base formation between amino groups of CMCS and aldehyde groups of oxidized gellan gum (A). In gellan chains, cis-dihydroxyl of rhamnose was oxidized to dialdehyde,
the addition of Ca2+ introduced ionic bonds between the carboxyl groups of gellan via electrostatic interaction, subsequently aldehyde groups and amino groups of CM-chitosan formed
the second network via the Schiff-base reaction. The cross linking mechanism of complex hydrogel (B). Gellan gum chains formed double helix conformations with Ca2+, and then CMCS
chains link the aldehyde zones to the formation of a three dimensional network, that created the gel (reproduced with permission from [249]).

structure, proper compressive strength and high porosity, the scaffolds
met the criteria for bone tissue regeneration. Fig. 20 shows the SEM mor-
phology of scaffolds with different fractions of -TCP when the ultrasonic
time was set to 20 min. From the Fig it is clear that similar pore size
(about 350 m) were observed in the composite scaffold. Although
some agglomeration appeared in the walls of scaffolds containing higher
fractions of -TCP which is in agreement with the literature [127]. The
bioactivity of a novel CMCS scaffolds with and without incorporating
mineral trioxide aggregate (MTA) in a tooth model was characterised.
The deposition of HAP was signicantly higher (P b 0.05) on MTA-
coated CaC (CaMT) scaffold than that on Cross-linked CMCS scaffold
(CaC). Therefore, it can be concluded that the bioactivity of the CMCS
scaffold can be enhanced by incorporating MTA [238].
11.2. CMCS hydrogels
In the past decade, hydrogels have made signicant progress in the
development of tissue engineering scaffold [243]. A number of synthetic
and natural polymers have been exploited in the last few years as hy-
drogel biomaterials that include alginate [244], chondroitin sulfate
[245], hyaluronic acid [246] and collagen [247]. The insufcient me-
chanical performance and relative harsh gelation conditions for cell en-
capsulation are the major limitations of hydrogels in tissue engineering
application [248]. A double-network complex hydrogel with signicant-
ly improved gelation temperature and mechanical properties composed
of oxidised gellan gum and CMCS by Ca2+ cross linking and Schiff reac-
tion was prepared. Firstly, polymer chains were cleaved into smaller
segments by oxidation reaction, which lead to the decrease of polymer
molecular weight and the increase of cross linking aldehyde groups.
This allowed for the formation of two entangled networks of different
cross linked polymers as described in Fig. 21A [249]. In the second
step, the chemical cross linking reaction (Schiff-base formation) be-
tween the pendant amino groups of CMCS and the aldehyde group
lead to the formation of a complex gel in the oxidised gellan as shown
in Fig. 21B [249]. The results showed that the gelation temperature
was lowered from 42 C to below physiological temperature by
oxidation, and further reduced by complexing with CMCS. Also the com-
plex hydrogel showed an increased compressive modulus of 278 kPa,
and an ability to return to the original shape after release of the com-
pressive load. Thus the hydrogels were promising biomaterials for carti-
lage tissue engineering. A study on the synthesis and characterisation of
UV cross-linked hydrogels derived from novel water soluble
methacrylated O-CMCS and polyethylene glycol diacrylate. The hydro-
gel substrates similarly supported attachment and proliferation of
Smooth Muscle Cells (SMCs). The results from the study demonstrated
these hydrogels to be promising biomaterials for tissue regeneration
[143]. A novel microgel class consisting of biocompatible CMCS and
temperature-sensitive PNIPAM was designed and synthesised by seed-
ed emulsion polymerisation. The presence of PNIPAM in microgels con-
tributed to the thermoresponsive property to CMCS while CMCS added
to the improved biocompatibility to the microgels which made these
microgels suitable for tissue regeneration purpose [232]. Yang et al.
[103] fabricated CMCS/gelatin hydrogels by green method i.e. radiation
cross linking method with excellent and adjustable water retention ca-
pacity (10700 g/g dry gel) and a similar compressive modulus with
that of soft tissue (10200 kPa). These hybrid hydrogels have improved
exibility, antimicrobial and water absorption capacity than gelatin
hydrogels and superior handle ability and mechanical properties than
CM-chitosan hydrogels. Apart from these, they exhibited excellent and
controllable degradability and good cytocompatibility which suggested
their potential application in tissue engineering and wound healing.
11.3. CMCS composites
In the recent years, composites have drawn attention of researchers
towards development of biocompatible and biodegradable composites
for tissue regeneration. In this context, preparation of porous
biocomposites of nano-hydroxyapatite (n-HAP) and CMCS by porogen
leaching method and their characterisation by IR, XRD, SEM and com-
pressive strength has shown potential application for bone tissue engi-
neering. The composites displayed porosity where the pores were
interconnected and the compressive strength can exceed 21 MPa
78 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 22. Synthesis of CMCS-graft-D-glucuronic acid (reproduced with permission from [235]).
which should be desirable for bone tissue engineering [233]. Similarly,
Shi fabricated N-carboxyethyl chitosan/nanohydroxyapatite N-CECS/n-
HAP composites for tissue-engineered trachea and investigate its
biomechanical and biocompatible properties. The N-CECS/n-HAP com-
posites exhibited satisfactory tensile strength and Youngs modulus
values thereby conrming their potential for tissue-engineered trachea
[250]. Also, recently, the effect of the n-HAP/CMCS composite with vas-
cular endothelial growth factor (VEGF)-transfected bone marrow stro-
mal cells (BMSCs) in a rabbit radial defect model was studied [234].
This composite was biocompatible, nontoxic, promotes the inltration
and formation of the microcirculation, stimulates bone defect repair
and the degradation rate of the composite matched that of growing
bone thus demonstrating its potential for bone defect repair.
11.4. CMCS injectable gels, lms and membranes
Apart from popular tissue engineering devices like scaffolds,
hydrogels and composites, injectable gels, lms and membranes have
also been developed by different researchers for tissue repair and regen-
eration purpose. Development of gelatin and CMCS gels in situ in the
presence of tyrosinase and p-cresol where presence of n-HAP does not
hamper in situ gelation of the polymers in physiological pH and temper-
ature has been reported by Mishra and coworkers [2]. The results clearly
indicate the potential of tyrosinase/p-cresol crosslinked CMCSgelatin
gel as injectable gel matrix for cell based bone tissue engineering.
Guangyuan et al. [72] prepared CMCS lms and carboxymethylation
and bimodal MW distribution were successfully combined to regulate
rate of degradation of the lms formed. The results displayed that
these CMCS lms with tunable degradation rates provide a powerful
material system for tissue engineering. In this view, the development
of CMCS-graft-D-glucuronic acid (CMCS-g-D-GA) membranes prepared
by grafting CMCS with D-glucuronic acid by using EDC catalyst in
water by Jayakumar and coworkers is worth mentioning. The synthesis
method of the CMCS-g-D-GA membranes is shown in Fig. 22 [235]. The
results of this investigation indicated that as the membranes were capa-
ble of having bioactivity, they were expected to be suitable for tissue en-
gineering purposes.
12. CMCS applications in different tissues/organs
As the inherent ability of body to self-repair from disease or injury
and to regenerate depends on the specic tissue or organ system, it
seems obvious that the choice of tissue engineering device or formula-
tion largely depends on the tissue or organ being repaired, reconstruct-
ed and/or regenerated [225]. In context of CMCS, its fabrication depends
greatly on the target tissue for which the restorative device is being
made which may include different organs like bone [251], nerve, carti-
lage [72], vascular tissue [239], tooth [238] and even trachea [250].
12.1. CMCS in bone tissue engineering
In recent years, CMCS based scaffolds, hydrogels and biocomposites
have gained importance in orthopedic research because of their poten-
tial to minimise surgical invasiveness. When engineering bone tissue,
the tissue engineering device must meet a number of requirements
like being biocompatible, biodegradable in suitable time window, its
degradation products should be non toxic and capable of being easily
eliminated by the metabolic pathways, must support cell adhesion
and growth and exhibit adequate mechanical stability [252,253]. In ad-
dition to this, the biomaterial designed should allow new bone in-
growths (osteoconductive) [254] and angiogenesis to supply the
newly formed tissue with nutrients while inducing bone formation
(osteoinductive) [255]. In this view, the synthesis, characterisation
and in vivo study of an enzymatically cross linked CMCS/gelatin/n-
HAP injectable in mice reported by Mishra and coworkers have been
demonstrated as promising biomaterials that may be used in treating ir-
regular small bone defects with minimal clinical invasion as well as for
bone cell delivery. The in vivo injectability study of the i-Gels in murine
models (Fig. 23) showed that the injected i-Gels were successfully re-
trieved from the exact position of euthanised mice. At the site of implan-
tation no apparent sign tof inammation (redness or edema) was
observed which illustrated that the i-Gels were nonimmunogenic in na-
ture. Also the yellowish colour instead of purple colour of the retrieved
i-Gels can be attributed to the limitation of ample molecular oxygen in-
side the human body. The in vivo study also demonstrated that the i-
Gels will have lesser gel strength in vivo as compared to in vitro situation
[2]. Oliveira et al. reported high efciency of DOX-loaded CMCS/PAMAM
Fig. 23. In vivo gel stability study. Representative macroscopic image of post-mortal mouse
showing the location and texture of iGel 24 h post-implantation (reproduced with permis-
sion from [2]).
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487 79

Fig. 24. Optical microscopy images of Alizarin Red stained osteoblasts after culturing for 14 days on (a) Ti, (b) Ti-CMCS, (c) Ti-CMCS-VEGF, (d) Ti-HAC and (e) Ti-HAC-VEGF. Initial seeding
was carried out with 3104 cells/cm2. (f) shows the Ti-CMCS substrate which had been placed in cell culture medium for 14 days without cell seeding after Alizarin Red staining. Scale bar
200 mm (reproduced with permission from [240]).

dendrimer nanoparticles on being internalised by different cell types,
in vitro, promoting the osteogenic differentiation of rat bone marrow
stromal cells in tissue culture polystyrene dishes [256]. Similarly, the
low generation poly(amidoamine) (PAMAM) dendrimers with CMCS,
CMCS/PAMAM dendrimer nanoparticles were surface engineered and
loaded with DOX with the expectation of exhibiting high drug loading
efciency and non cytotoxicity compared to amine-terminated
PAMAM dendrimers of high generation [257]. Xuefeng Hu carried out
a study on an in vitro assessment of titanium functionalised with dopa-
mine followed by CMCS or hyaluronic acid catechol (HAC) conjugated
with vascular endothelial growth factor (VEGF). It proved to be promis-
ing alternative for enhanced osteointegration and inhibition of bacterial
inhibition. Fig. 24 displays the results of mineralisation of cells after two
weeks culture on different substrates which was assessed by staining by
Alizarin Red. The degree of staining on the Ti-CMCS substrates (Fig. 24b)
and Ti-HAC substrates (Fig. 24d) is not substantially higher than pris-
tine Ti (a). Figure shows an absence of purplish red stains which was
control experiment carried out with Ti-CMCS in cell culture medium
without cells. As shown by the dense coverage of calcium deposits on
the Ti-CMCS-VEGF (Fig) and Ti-CMCS-HAC (Fig) substrates
mineralisation is greatly enhanced which can be attributed to the pres-
ence of immobilised VEGF [240]. Also the development of hydroxyapa-
tite scaffolds with macroporous structure and non-cytotoxicity which
could efciently support the adhesion, proliferation and osteogenic
differentiation of rat bone marrow stromal cells in the presence of
0.01 mg ml 1 DOX-loaded CMCS/PAMAM dendrimer nanoparticles,
in vitro has been reported by several research groups [257,258].
12.2. CMCS in cartilage tissue engineering
The choice of biomaterial is very critical for the success of tissue en-
gineering approaches, particularly in cartilage repair [259]. The struc-
tural similarity of chitosan with various glycosaminoglycans found in
articular cartilage has made it a suitable scaffolding biomaterial in artic-
ular cartilage engineering [260,261]. Therefore, CMCS has been now
experimented for different cartilage tissue engineering applications.
Earlier, N,N-di-CMCS as delivery agent for bone morphogenetic protein
in the repair of articular cartilage has already been experimented [262].
Guangyuan et al. [72] successfully fabricated nanobrous collagen-
coated porous CMCS microcarriers for cultivating cells and for applica-
tion in cartilage tissue engineering as injectable scaffolds for cell deliv-
ery. Recently, double-network complex hydrogel with signicantly
improved gelation temperature and mechanical properties have been
prepared by mixing oxidised gellan gum with CMCS which was found
to be promising material for cartilage tissue engineering [249]. Apart
from these research studies, the ability of chitosan and CMCS to protect
chondrocytes from apoptosis, signicantly suppress the degeneration of
80 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
cartilage in osteoarthritis and to protect chondrocytes from the IL-1beta
induced catabolism have also been reported in literature [263,264]
12.3. CMCS in nerve tissue engineering
Researchers have investigated different biodegradable biomaterials,
including collagen, polyglycolic acid, polylactic acid and co-polymers of
L-lactide and -caprolactone, for nerve repair [265,266]. A research study
on the degradation of covalently cross-linked CMCS in vitro for the rst
time and its potential application for peripheral nerve regeneration has
been carried out by Lu et al. [69]. The results revealed that the faster
degradation of EDC-crosslinked CMCS than chitosan and decrease in
the hydrophilicity and elastic modulus of CMCS lms are benecial for
application of CMCS in nerve repair. The study also suggested that the
EDC cross-linked CMCS lms enhanced the spread and provided a
good proliferation substratum of Neuro-2a cells. Similarly the tunable
degradation rates of CMCS have been experimentally demonstrated re-
cently by Guangyuan et al. [72]. He described the ability of Neuro-2a
cells to adhere and proliferate when cultured on binary and unary
CMCS lms was found to be comparable to those on non-modied
unary chitosan lms which established CMCS as a promising material
for neural tissue engineering. Recently, ability of CMCS to stimulate pro-
liferation of Schwann cells in vitro by activating the intracellular signal-
ing cascades of extracellular signal-regulated kinase (ERK1/2) and
phosphatidylinositil-3 kinase (PI3K/Akt) has been reported by Bin He
and coworkers [267].
12.4. CMCS in wound healing
CMCS-based materials, produced in varying formulations, have been
used in a number of wound healing applications. The inherent acceler-
ative wound healing effects of CMCS on second degree burn models per-
formed in rats was demonstrated in vitro and in vivo by Peng et al. [268].
In another experimental study, N,CMCS was used as biomaterial to heal
deep second-degree burn wounds. The results demonstrated that the N,
CMCS was efcient in accelerating wound healing via activating
transforming growth factor-1/Smad3 signaling pathway [269]. The im-
proved wound healing ability of CMCS of different MW has earlier been
experimentally investigated by Chen et al. [20] by using a cell culture
which showed positive results. The wound dressings of CMCS devel-
oped by Qin and coworkers evaluated for wound healing ability
in vitro also displayed promising results [270]. CMCS, not only in its in-
herent form, but also in many modied forms or in combination with
Fig. 25. In vitro Ag release curves of AgNPs/PVA/CM-chitosan nanobers in PBS
(reproduced with permission from [197]).
other biomaterials has demonstrated potential of enhanced wound
healing property. In fact, oligo-chitosan, N,O-CMCS and N,CMCS in
sheet and paste forms were evaluated in vitro for possible utilisation
in wound dressing applications for wound healing [67]. Similarly, N,O-
CMCS/Collagen matrixes containing chondroitin sulfate or an acellular
dermal matrix developed by Chen et al. [82] demonstrated potential
as wound dressings for clinical applications. An in vivo experiment to
evaluate the wound healing effect of water soluble chitosan/heparin
complex on the full thickness skin excision performed on the backs of
the rat displayed that the complex is most effective in wound healing
[271]. Also, in an in vivo animal experiment using a burn wound
model water-soluble O-CMCS derivatives modied with furfuryl glyc-
idyl ether (O-CMCS/FGE) displayed wound healing effects. The results
of the study showed that O-CMCS/FGE would be a promising candidate
as an anti-adhesion material for biomedical applications [31]. A study on
subcutaneous implantation of the ornidazole loaded (PVA)/CMCS lms
prepared by blending/casting method in the surgical wound did not
promote any adverse effect. Over a long period of time, it is expected
that these drug lm would be absorbed and the wound would be
cicatrised by new forming tissues eventually [34]. Angiogenesis of full-
thickness burn wounds repaired with collagen-sulfonated CMCS porous
scaffold encoding VEGF, DNA plasmids has been reported by Teng et al.
[272]. Recently, Ag nanoparticles/(PVA)/CMCS nanobers prepared by
electrospinning technique by Zhao et al. showed potential for wound
dressing biomaterial. Fig. 25 shows the in vitro Ag released amount of
crosslinks AgNPs/PVA/CMCS nanobers as a function of time. At the
third hour the release of Ag in both the samples was fast and after
that became relatively slow. Finally in both the samples after 24 h the
concentration of Ag+ reached equilibrium [197]. Apart from enhanced
wound healing capacity, CMCS has also been experimentally proven ef-
cacious in preventing as well as reducing post-operative surgical adhe-
sions by several researchers [80,81,273].
13. Tissue engineering application is often combined with drug
delivery strategy
In the area of regenerative medicine and reconstructive surgery sub-
stantial input has been made from developments in tissue engineering
and drug delivery technologies. The regeneration of functional tissue re-
quires an appropriate microenvironment that closely mimics the host
site for desired cellular responses which is typically provided by 3-D tis-
sue engineering scaffold that acts as an architectural template [274]. But
repair and reconstruction of diseased and/or damaged tissues/organs
demands therapy that cannot only provide mechanical and structural
integrity to the tissue but also maintains sustained/controlled delivery
of therapeutics and/or growth factors in order to enhance the healing
and regeneration process. While scaffold provides structural support,
diffusivity to enable cellular inltration and acts as substrate for tissue
differentiation and organisation, the drugs/bioactive molecules embed-
ded in it, cues for the surrounding tissues to heal and regenerate. Recent
advances in the eld of tissue engineering and drug delivery have en-
abled the design and fabrication of scaffolds that can deliver growth fac-
tors/therapeutic agents in a more controlled fashion over a dened
period of time. In fact the control over the regenerative and repairing
potential of tissue engineering scaffolds has dramatically improved in
recent years, mainly by using drug releasing scaffolds or by incorpora-
tion of drug delivery devices in the tissue engineering scaffolds
[275277]. While previous approaches of developing drug delivery for-
mulations mainly focused on the encapsulation or embedment of drugs
within the bulk phase and targeted delivery, recent strategies of tissue
engineering open up the new possibility of constructing scaffolds that
can provide the control over the sequestration and delivery of specic
bioactive factors to enhance and guide the regeneration process [278,
279]. Fig. 26 shows the more efcient and effective approach of combin-
ing tissue engineering applications with drug delivery strategy for
 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
Fig. 26. Shows the more efcient and effective schematic approach of combining tissue engineering applications with drug delivery strategy for enhanced repairing and regeneration of
damaged and/or diseased tissues/organs.
enhanced repairing and regeneration of damaged and/or diseased tis-
sues/organs.
Extensive overviews of recent research studies on development and
applications of three dimensional scaffolds with potential capabilities
for the controlled delivery of therapeutic drugs particularly osteogenic
drugs for bone regeneration are reported in literature [280,281]. In
fact, this paradigm shift that has taken place to utilise the tissue engi-
neering and drug delivery approaches towards the regeneration of den-
tal, oral and craniofacial structures using matrices and scaffolds capable
of controlled drug release has earlier been exhaustively reviewed [282].
In this context, the research work for development of scaffolds with po-
tential of drug delivery for neural [283] and bone [284,285] tissue engi-
neering is worth mentioning. As far as CMCS is considered, not much
research work has been done in 3D scaffolding delivery system. But
CMCS/gelatin/n-HAP i-gels susceptible to tyrosinase/p-cresol mediated
in situ gelling at physiological temperature capable of treating irregular
small bone defects have been investigated to exhibit potential of bone
cell delivery [2]. Reves et al. [286] prepared microspheres cross linked
by two different methods which were incorporated successfully into
the composite scaffolds. The X-CMCS beads (obtained by carbodiimide
chemistry cross linking) displayed good potential for use in bone tissue
engineering applications in which degradation and local drug delivery
are desired as compared to Gen-X CMCS (cross linked by genepin)
which showed poor degradation and drug release proles.
14. CMCS based systems: current challenges and opportunities
CMCS based nanocarriers have become one of the most extensively
studied nanometric drug delivery platforms. But these trials are still lim-
ited to experimental purposes and are not implicated widely as
marketed formulation. Despite of its increased aqueous solubility as
compared to native molecule chitosan, its signicant hydrophobicity re-
mains the major drawback responsible for its limited use in biomedical
eld. A drug requires compatible physicochemical properties of the ma-
trix polymer for developing a formulation successfully. CMCS alone or in
combination with other polymers, metals, and metal oxides has been
81
used either as blends, co-polymers or composites to alter the physico-
chemical characteristics and degradation behavior. CMCS has been
trialled in almost all novel drug delivery systems of current drug deliv-
ery approach due to its compatibility with wide range of polymers and
its suitability with drugs of different properties. A number of research
works have been reported where same drug being encapsulated in dif-
ferent CMCS formulations. Therefore, during the process of formulation
development for a particular drug, such information can be used for
comparative efcacy improvement studies between two or more for-
mulations. Also, owing to its enhanced bioactivity and its ability to pro-
mote osteogenesis, CMCS is being studied at preclinical and clinical level
ensuring its potentiality in tissue engineering. CMCS can chelate calcium
from mineralizing solution containing calcium and phosphate and in-
duce calcium phosphate or HAP formation thereby making it suitable
for bone and cartilage regeneration. But being polymer, the mechanical
properties of CMCS are not compatible enough with the host tissues
particularly with bone which restricts its usage as such in tissue engi-
neering applications. Thus, efforts to improve the mechanical properties
of CMCS based formulations are essential for this type of application.
Therefore, CMCS has often been exploited in the form of either compos-
ites with ceramics or HAP or in combination with some other polymers
with better mechanical strength in order to make it suitable for tissue
engineering. CMCS based 3D scaffolds mimic the extracellular matrix
and have proved to be highly useful formulations for repair and recon-
struction of damaged organs in general and tissues in particular. But de-
spite of its versatility, CMCS based formulations are not commercialised
widely in clinical drug delivery and tissue engineering practice. Also,
there is a paucity of studies regarding the development of technological
strategies to integrate and position drug delivery devices with a
submicrometric spatial resolution within the scaffolds. Nevertheless,
numerous studies involving study of CMCS as drug delivery carriers
and tissue engineering devices are being patented and some are under
preclinical or clinical investigation. The main goals are to improve
their stability in the biological environment, to mediate the bio distribu-
tion of active compounds, enhanced drug loading, targeting, transport,
release, and interaction with biological barriers. The cytotoxicity of
82 L. Upadhyaya et al. / Journal of Controlled Release 186 (2014) 5487
nanoparticles or their degradation products remains a major problem,
and improvements in biocompatibility obviously are a major concern
of future research in CMCS based drug delivery.
15. Summary and future remarks
CMCS based drug delivery and tissue engineering formulations re-
search has reached signicant maturity in the last few years. There are
numerous compelling evidences for the potential of CMCS as biomateri-
al for many novel challenging drug delivery and tissue engineering ap-
plications. CMCS has been shown to improve the dissolution rate of
many otherwise poorly soluble drugs, and thus, can be exploited for bio-
availability improvement of drugs. Various therapeutic agents, such as
anticancer, anti-inammatory, antibiotics, antithrombotic, proteins,
and amino acids have been effectively incorporated in CMCS-based sys-
tems to increase bioavailability and to achieve targeted and/or con-
trolled release. While this concept is still mostly within the academic
frame, results from several laboratories studies conrm enhancements
in the bioavailability of these macromolecules to a level that might suf-
ce for industrial development. Various CMCS based formulations pre-
sented herein this review like hydrogels, microspheres, nanoparticles,
lms, bers, composites and scaffolds can be helpful in deciding the
context of using CMCS in selectively capturing a therapeutic payload
and control release in a target site as well as for tissue engineering pur-
poses. CMCS's innate tissue engineering potential as a biopolymer to
generate structures with predictable pore sizes and controllable degra-
dation rates makes it particularly suitable for bone and cartilage regen-
eration. In concluding remarks, CMCS, the polymer with intact or
derived properties makes it suitable to use and prepare all kinds of
novel drug delivery and tissue engineering formulations. From the
above investigations it may be concluded that CMCS is indeed a versatile
biodegradable polymer derivative having tremendous potential in
pharmaceutical drug delivery and tissue engineering application in
near future.
Acknowledgement
The authors would like to thank Ministry of Human Resource Devel-
opment, Govt. of India for providing institute (MNNIT) fellowship for re-
search. The authors also want to acknowledge the Director, Motilal
Nehru National Institute of Technology, Allahabad, India for providing
other necessary facilities for research work. JS is kindly acknowledged
to Department of Science and Technology, New Delhi, India for
awarding the INSPIRE FACULTY AWARD [IFA-13-CH-105].
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