Peptides 32 (2011) 173187

Contents lists available at ScienceDirect

Peptides
journal homepage: www.elsevier.com/locate/peptides

Review

Development of peptide and protein nanotherapeutics by nanoencapsulation and

nanobioconjugation
Subhash Chandra Yadav , Avnesh Kumari, Ramdhan Yadav
Nanobiology Lab, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientic and Industrial Research, Palampur 176061 HP India

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

Article history: The targeted delivery of therapeutic peptide by nanocarriers systems requires the knowledge of inter-
Received 15 September 2010 actions of nanomaterials with the biological environment, peptide release, and stability of therapeutic
Received in revised form 2 October 2010 peptides. Therapeutic application of nanoencapsulated peptides are increasing exponentially and >1000
Accepted 3 October 2010
peptides in nanoencapsulated form are in different clinical/trial phase. This review covers current sce-
Available online 8 October 2010
nario of therapeutic protein and peptides encapsulation on polymer to metallic nanocarriers including
methods of protein encapsulation, peptide bioconjugation on nanoparticles, stability enhancement of
Keywords:
encapsulated proteins and its biomedical applications.
Peptide therapeutics
Peptide nanomedicines 2010 Elsevier Inc. All rights reserved.
Nanoencapsulation
Nanocarriers
Protein nanoparticles interaction
Bioconjugation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2. Therapeutic use of peptide and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
3. Methodology of peptide and protein nanoencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
3.1. Emulsicationpolymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.2. Interfacial polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.3. Solvent evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.4. Salting out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.5. Coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
3.6. Emulsication/solvent diffusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
3.7. Surface functionalization of gold NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
3.8. Taylor cone jet methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
4. Nanocarriers of peptide and protein encapsulant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
4.1. Metallic nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
4.2. Polymeric nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.2.1. Natural nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
4.2.2. Synthetic nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4.3. Protein itself as nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
5. Bioconjugation of peptide and protein on nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
5.1. Maleimide reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
5.2. EDCNHS bioconjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.3. Afnity interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.4. Click chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.5. Streptavidinbiotin reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
6. Nanoencapsulation improves the therapeutic importance of proteins and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

Corresponding author. Tel.: +91 9418096177; fax: +91 1894230428.

E-mail address: subhash@ihbt.res.in (S.C. Yadav).

0196-9781/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2010.10.003
174 S.C. Yadav et al. / Peptides 32 (2011) 173187

7. The stability of nanoencapsulated peptide and protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

8. Therapeutic use of the nanoprotein molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

1. Introduction to repel the absorption of opsonin proteins via steric repulsion [83].
Advances in protein engineering and materials science have con-
Proteinaceous biomolecules such as antibodies, antigens, tributed to novel nanoscale targeting approaches that may bring
growth factors, and bioactive peptides are well known for their new hope to develop a new therapeutic nanobiomolecules [89].
therapeutic potential in various diseases. These therapeutic pro- In this context, this review will address the recent advancement
teins and peptides are potential target for development of of protein and peptide nanoparticles conjugates and their specic
therapeutic nanoprotein and peptides because of their nanoscale application as nanomedicines. Our aim is to introduce some facets
dimension, highly specic therapeutic importance, and numerous of protein nanobiotechnology and therapeutics to highlight the
other specic enzymatic activities. Encapsulation or bioconjugation broadness and perspective of the topic.
of these proteins/peptides on suitable nanocarriers may improve
the potential of these therapeutic proteins to create a novel com- 2. Therapeutic use of peptide and proteins
ponent for therapeutics, disease diagnosis, uorescent microscopy,
imaging, and other life science devices [6]. The protein and peptides Insulin was the rst peptide to be used as therapeutics and
nanoparticles are developed by adsorption on the nanocarri- since then many therapeutic proteins and peptides have been
ers surfaces, encapsulation in nanoparticles, bioconjugation on reported in almost every eld of medicine [60]. The protein
nanoparticles and by molecular self assembly of small peptides into therapeutics summarizes more than 130 currently used protein
nanoparticles size. The encapsulated protein on nanoparticles has therapeutics (approved for clinical use by the US Food and Drug
potential to enhance therapeutic activity of peptides by sustained Administration FDA) and 1000 proteins/peptides are in different
and targeted delivery, improve stability, better bioavailability, and clinical/trial/approval phase [60]. This therapeutics are currently
assemblies of nanocomposites [44]. The properties of protein to preferred due to its high specicity and complex set of functions;
interact with each other (antigenantibody interaction, subunit less interference to normal biological processes; less likely to elicit
association, disulde linked dimerization) make them an excel- immune responses; effective replacement treatment in mutated or
lent candidate to serve as a linker to form an ordered nanoparticles deleted normal protein and faster clinical development as well as
structure [31]. These ordered nanoparticles improve the therapeu- FDA approval time [96].
tic potential of encapsulated protein and peptides. Today there are more than 300 reports of plant-based pro-
Most of the therapeutic proteins and peptides are quite duction of therapeutic proteins (antibodies, single chain variable
unstable, prone to denaturation compare to conventional ther- fragments (scFv), antigens, vaccines, hormones, growth factors
apeutic small molecules. The therapeutic activities of peptides and human blood proteins). Various plant isolated cyclic peptides
are simply lost due to aggregation, degradation and unfold- (cyclotides), cysteine proteinases, defensins, plant-made vaccines
ing [64]. Apart from this, most of the protein lost its activity are well known for its therapeutic importance. These peptides and
and structural organization during encapsulation, or bioconju- other therapeutic protein from animal, bacterial, fungal and syn-
gation on the nanoparticles e.g. glucose oxidase encapsulation thetic are under various stages of research and development for
[64]. The retention of activity, structural identity, and stability better therapeutic applications [39]. A detail description of FDA
of protein and peptides after encapsulation on suitable nanode- approved commercial protein and peptides are reviewed by Leader
vices are basic concern in development of protein and peptides et al. [60]. They have classied the therapeutic protein in four major
nanomedicine. A variety of nanoparticulate systems like polymeric groups (Group IIV) based on mechanism and pharmacological
microspheres/nanoparticles, liposomes and solid lipid nanoparti- actions [60]. Recently, transgenic tobacco cell cultures produced
cles (SLNs) etc are being used for the nanoencapsulation protein recombinant animal vaccine against Newcastle disease virus (NDV)
and peptides to improve protein drug accumulation inside tar- was approved by FDA (February 2006) [57]. Insulin, angiotensin
get cells due to easy and efcient cellular internalization [8]. I-converting enzyme, inhibitory 1.3 kDa peptide (Met-Ile-Phe-Pro-
Further, nanoencapsulation of protein to develop nanomedicines Gly-Ala-Gly-Gly-Pro-Glu-Leu) [56], 15 new antimicrobial peptides
require the complete information about the changes in cell recep- form the skin of various amphibians [97], peptide venom of Both-
tors that occur with progression of disease, mechanism and site rops marajoensis snakes [22], synthetic peptides containing amino
of action, retention of drugs, multiple administration, molecular acid residue 161173 of HIV-1 integrase protein (IN) [5] are
mechanisms, stability and therapeutic activity of protein in vivo reported to have the great therapeutic importance.
and pathobiology of the disease. The opsonization and clearance
of these particles (macrophage action) from the body are impor- 3. Methodology of peptide and protein nanoencapsulation
tant point of consideration during the encapsulation. However,
these can be minimized by suitable and balanced surface modica- Protein and peptides encapsulation or adsorption on nanocar-
tion of nanocarrier without affecting the drug loading and release riers have been achieved by various methods like emulsion
mechanisms [43]. Several methods have been developed to mask polymerization, interfacial polymerization, solvent evaporation,
nanoparticles from the mononuclear phagocytic system (MPS). salting out, coacervation, combination of sonication and layer by
The most preferred method is the adsorption or grafting of poly layer technology [3], solvent displacement/solvent diffusion etc.
(ethylene glycol) (PEG) to the surface of nanoparticles. Addition of Each methods of protein encapsulation have their own advan-
PEG and PEG-containing copolymers to the surface of nanoparti- tage and disadvantage. Since each protein and peptides requires its
cles results in an increase in the blood circulation half-life of the own specic condition for stability, solubilization, control releases
particles by several orders of magnitude. This method creates a immune elimination. The method of encapsulation is entirely based
hydrophilic protective layer around the nanoparticles that is able on the physicochemical activity of protein and its application. A
Table 1
Nanocarriers for the protein and peptide encapsulation.

Nanocarriers systems Encap. protein Encapsulation Type of encapsulation Size (nm) EE (%) Release mechn. Advantage Stability Ref.
methods
Metallic/Semiconductors
Iron BSA Adsorption 15 93 Desorption in pH > 7 Max. adsorption at Increases [66]
isoelectric pH of BSA
Bombesin peptide Click chemistry Bioconjugation 15 80 Targeted delivery Activity retained [75]
F3-peptide Bioconjugation 50.3 Targeted delivery [135]
Chymo-trypsin EDCNHS chemistry Bioconjugation 31 69 Thermal stability was 88.7% Res act at [51]
improved 85 C
Gold E. coli Ab 0157 EDCNHS chemistry Bioconjugation 10 Biological activity retained [86]
Lipase Click Chem. Bioconjugation Activity retained [14]
Ni His-Tag proteins Micro emulsion Bioconjugation 145 80 Enhanced IgG and IgG2a Stable at 37 C, pH [88]
responses 7.4
CaF2 -Chitosan BSA Microemulsion Bioconjugation 20 Stable [114]
Quantum Dots UWrB SMCC cross-linker Bioconjugation 1520 Serve as a molecular [124]
marker
CNTs Tumor lysate EDCNHS chemistry Bioconjugation 2030 66 Activity enhanced Stable [77]

Polymeric
Chitosan Insulin Ionotropic gelation Encapsulation 750 70 Pharmacological activity Increased [103]

S.C. Yadav et al. / Peptides 32 (2011) 173187

enhanced
Cyclosporin A Ionotropic gelation Encapsulation 293 73 Dissolution in media Bioavailability increased Similar [27]
BSA Taylor cone Jet Encapsulation 20,000 76 Dissolution Retained >80% of the BSA [129]
bioactivity
BSA Coacervation Encapsulation 200580 Desorption and diffusion Biological activity retained Stable [40]
TMC BSA Ionic gelation method Encapsulation 301 95 Diffusion Activity retained [17]
BHb Encapsulation 349 95 Diffusion 5 days stable [17]
Kon-jac-glu. chitosan (CKGMSCS) BSA Ionotropic gelation Encapsulation 330900 20 Diffusion pH sensitive [30]
Gelatin BSA Emulsion Encapsulation 840 Diffusion Activity retained [62]
bFGF Encapsulation 500018000 80.5 Diffusion Denaturation prevented [63]
Albumin BMP-2 Coacervation Encapsulation 90 Diffusion Stable [134]
Ganciclovir Coacervation Encapsulation 200400 Diffusion Increased release acidic Stable [78]
and basic pH
PLA hGF2 Double emulsion Encapsulation 180 77 Activity retained Increased [87]
solvent evaporation
Protein C Encapsulation 205 65 Diffusion Activity retained Increased [132]
Tetanus toxoid Encapsulation 192 36.7 Diffusion Bioavailability enhanced Increased [121]
Neurotoxin Encapsulation 65 35.5 Bioavailability enhanced Increased [18]
Insulin Nebulisation 400600 Biological activity retained Similar [37]
techniques
BSA Double emulsion Encapsulation 377 71.6 Biodegradation NPs Aggregation after 50 days Stable up to 28 [42]
days
PLA-TPGS BSA Encapsulation 362 75.6 Diffusion Structure retained 35 days [61]
PLGA hGF2 Encapsulation 177 77 Activity retained Similar [87]
BSA Encapsulation 100 77 Activity retained Similar [31]
Cycl. A Solvent displ. Encapsulation 170 Activity retained Similar [99]
BSA Taylorcone jet Encapsulation 20,000 76 Diffusion Activity retained 38 days [129]
PCL Insulin Multiemulsion Encapsulation 358 96 Activity preserved Increased [26]
Cyclodextrin-PLA BSA Double emulsion Encapsulation 377 71.6 Degradation Activity retained Similar [41]
BSA Encapsulation 83.5 Degradation Activity retained Similar [42]
Dex-PCL BSA Double emulsion 188 32 Dissolution Retained activity 4 C/15days [98]
method
Lectin Encapsulation 128 80 Dissolution Hemogglutinating activity 4 C/20 days [98]
conserved
Polymethylmethacrylate Lysozyme Emulsion Encapsulation 220 20 Activity preserved and Increased [122]
polymerization shelf life increased
tert-butyl acrylate BSA Maleimide coupling Bioconjugation 30 5060 Biological activity retained Similar [82]

175
176 S.C. Yadav et al. / Peptides 32 (2011) 173187

brief description about the each reported protein encapsulated

methods along with detail characteristics are provided in subsec-
tions (Table 1).

3.1. Emulsicationpolymerization

The emulsicationpolymerization method is classied in two

categories, based on the use of organic or aqueous continuous
phase. In the continuous aqueous phase, polymers and protein
drugs are dissolved in aqueous solvent without surfactants or
emulsier by using anionic polymerization mechanism with high
energy radiation. For example, insulin and cyclosporin A are encap-
sulated on poly(isobutylcyanoacrylate) nanoparticles of particle
size <500 nm and 120 nm respectively [93]. In the continuous
organic phase, polymers are dissolved in organic non-solvent by
dispersion via surfactants in to solvent. Various enzymes (e.g. calci-
tonin) are encapsulated with polyacrylamide nano/microparticles
of <1000 nm size nanoparticles (Table 1) [125].

3.2. Interfacial polymerization

In interfacial polymerization, the cyanoacrylate monomer and

proteins drug are dissolved in a mixture of an oil and absolute
ethanol. Oils have positive inuence to reduce the unfolding of
protein by ethanol. This mixture is then slowly extruded through
a needle into a well stirred aqueous solution, with or without
some ethanol containing surfactant. An advantage of interfacial
polymerization technique is high efciency drug encapsulation
[23]. Insulin was encapsulated on poly(ethylcyanoacrylate) and
poly(isobutylcyanoacrylate) nanoparticles of particle size 151 nm
and 150300 nm respectively by using this method [26].

3.3. Solvent evaporation

In solvent evaporation method, the polymers along with pro-

teins are dissolved in volatile organic solvent (DCM, acetone, CHCl3 ,
EtAc, etc.) and poured into continuously stirring aqueous phase
with or without emulsier/stabilizer and sonicated. Most of the
proteins are likely to denature after the sonication. Thus, a slow
and intermittent sonication at low temperature is effective to retain
the secondary and tertiary structure of protein drugs (Fig. 1A).
Albumin, and tetanus toxoid are successfully encapsulated on poly-
lactic acid (PLA) nanoparticles of size 100120 nm and 150 nm by
these methods [111]. Solvent displacement method is similar to
solvent evaporation that is based on spontaneous emulsication of
the organic internal phase containing partially dissolved polymer
along with protein into the aqueous external phase. Insulin on PLA
Fig. 1. Methods for proteins nanoencapsulation (A) Solvent evaporation: Polymer is
nanoparticles of size 105170 nm was nanoencapsulated by this
dissolved in organic solvent, emulsied in aqueous solution. The emulsion is dried
methods (Table 1) [9]. under pressure or continues stirring or increasing temperature. (B) Coacervation
Method: Alcoholic solution of desolvating agent is added to aqueous solution of
3.4. Salting out albumin. Associative phase separation of two polymers in water occurs if there is
an electrostatic attraction.

The protein and peptides therapeutic are sensitive to unfolding

or inactivation. These problems are minimized by the salting out
methods of protein and peptide encapsulation. This procedure is
based on the separation of a water miscible solvent from aqueous
solution by adding salting out agent like magnesium chloride, cal-
cium chloride, etc. The main advantage of salting out procedure
is that it minimizes unfolding stress to protein encapsulates. Var-
ious peptides are nanoencapsulated on different nanoparticulate
system by using this technique [74].
3.5. Coacervation
Coacervation is a process during which a homogeneous solution
of charged macromolecules undergoes liquidliquid phase separa-
tion, giving rise to a polymer rich dense phase. This method has
been classied into simple and complex processes depending on
the number of participating macromolecules. In simple polyelec-
trolyte coacervation, addition of salt or alcohol normally promotes
coacervation. In complex coacervation, two oppositely charged
macromolecules (or a polyelectrolyte and an oppositely charged
colloid) can undergo coacervation through associative interactions
(Fig. 1B). The charges on the polyelectrolytes must be sufciently
large to cause signicant electrostatic interactions but not so large
to cause precipitation. The dilute liquid phase, (usually super-
natant), remains in equilibrium with the coacervate phase. These
two liquid phases are incompatible and immiscible. BSA has been
 S.C. Yadav et al. / Peptides 32 (2011) 173187
successfully encapsulated on PLA and PLGA by using coacervation
(Table 1) [113].
3.6. Emulsication/solvent diffusion
The emulsication/solvent diffusion (ESD) method is very
efcient technique for encapsulation of protein on polymeric
nanoparticles. This technique provides the maximum encapsu-
lation efciency without homogenization, high batch to batch
reproducibility, ease of scale up, simplicity and narrow size dis-
tribution of protein nanoencapsulate [100]. In these methods,
the polymers with proteins are dissolved in a partially water-
soluble solvent and saturated with water. Subsequently, the
polymerwater saturated solvent phase is emulsied in an aque-
ous solution containing stabilizer, leading to solvent diffusion to
the external phase and the formation of the nanoparticles. Bovine
serum albumin (BSA) and immuno--globulin (IgG) encapsulated
on (PEOPLGA) using this method has high encapsulation ef-
ciency (58.9%) and slow in vitro release rate. The problem of protein
hydration, pH reduction derived from polymer degradation and
presence of the hydrophobic interfaces in poly lactic-co-glycolic
acid (PLGA)-based delivery devices lead to protein inactivation or
irreversible aggregation inside PLGA or PLA nanoparticles [100].
These problems are prevented by incorporation of stabilizers such
as polyethylene oxide (PEO) and its derivatives (Table 1).
3.7. Surface functionalization of gold NPs
Bayraktar et al. [12] demonstrated the ability to disrupt
proteinprotein interactions using surface-functionalized gold
nanoparticles. They have designed nanoparticles that bound selec-
tively to cytochrome c (Cyt c) or cytochrome c peroxidase
(CCP), thereby inhibiting enzyme turnover. Surface-functionalized
nanoparticles with gold cores (2 nm) are prepared using thiolates
with oligo(ethylene glycol) groups terminated in carboxylate (Au-
TCOOH) and trimethylamine (Au-TTMA) functionalities. Au-TTMA
nanoparticles bind selectively to a negative patch on CCP sur-
face, whereas Au-TCOOH nanoparticles bind selectively to basic
amino acid rich surface of Cyt c. Tseng et al. [118] fabricated
amino-acid-functionalized gold nanoparticles that modulate the
catalytic activity of R-chymotrypsin. They proposed that the amino
acid monolayer on the nanoparticles controls both the capture of
substrate by the active site and the release of product through elec-
trostatic interactions (Table 1).
3.8. Taylor cone jet methods
The secondary structure of adsorbed proteins depends on both
the identity of the adsorbed protein and the mode of presentation
of chemical functionalities. Taylor Cone Jet Mode is a new technique
for protein encapsulation on nanoparticles to protect their struc-
ture and function. In this technique, protein solution is dispersed
in organic phase (with DCM) dissolved PLGA by controlled soni-
cation processes. Further, electrospray with well dened potential
difference is created between the nozzle and the ring by applying
high voltage on the nozzle and a lower high voltage in the ring. The
emulsion solution was pumped through nozzle to form liquid cone.
This creates a thin jet from the apex to break up into monodispersed
droplets (Table 1) [128].
4. Nanocarriers of peptide and protein encapsulant
Nanocarriers are one of the useful tools for achieving the main
objective of protein therapeutics and its targeted delivery. A vari-
ety of nanocarriers have received much attention for the delivery
177
of peptides, proteins, and genes due to their ability to protect pro-
tein and peptides from degradation in the gastrointestinal track and
also in blood circulation. These protein encapsulated carriers have
many advantages, such as a potential for selective targeting, con-
trolled release and increase persistence of proteins therapeutics in
the body. The protein therapeutic agents have short half-life due to
proteolysis, rapid clearance from the blood stream and repeated
administration. Encapsulation of protein drugs on nanoparticles
provides sustained release and protects the non-released protein
from degradation [10]. The therapeutic protein molecules inter-
act with nanocarriers by forming a coat or adsorption on the
surface, or by bioconjugation (direct or using cross linkers). Var-
ious methods and nanocarriers systems are used to overcome
the problems associated with functional native protein encap-
sulation on nanoparticles. The choices of encapsulation methods
and nanocarriers system entirely depends on the application and
nature of protein encapsulate. Some proteins, enzymes, and DNA
plasmids are encapsulated in biodegradable polymeric nanobers
by electro-spinning technique to retain their bioactivity [72]. The
strength and selectivity of proteinprotein interactions make the
protein therapeutics as excellent candidates to serve as a linker
between nanoparticles ordered structures. In addition, the pro-
teins therapeutic molecules are encapsulated on various kinds of
nanoparticles like metallic, polymeric (natural and synthetic), or
by self assembly to forms nanoparticles (Fig. 2). The proteins and
peptides encapsulated on these nanocarriers systems are briey
described in following subsections (Table 1).
4.1. Metallic nanocarriers
Semiconductor and metallic NPs encapsulated protein and pep-
tides therapeutics illustrate unique electronic, optical and catalytic
properties (Fig. 2). Proteins and peptides encapsulated on nanopar-
ticles (NP) or quantum dot (QD) has yielded composite material
with new functionality. Gold NP has been used as nanoelectrode to
attach proteins and peptides by dithiol bridge due to inactivation
of active site on encapsulation [65]. Horse spleen apoferritin was
attached on gold nanoparticles by applying this technique. This
enzyme forms a covering layer that stabilizes the gold nanoparticles
in solution [65]. Cyt c, hemoglobin and myoglobin are also biocon-
jugated on silver and gold nanoparticles by using this technique.
The spectral features of protein encapsulated on nanoparticles are
broadened and red shifted due to surface binding of these proteins
[80]. Similarly, CD11b interleukins conjugated gold nanoparticles
show selective binding to macrophage cells [92]. This selective
destruction of the target cell line is possible by laser irradiation
of plasmon resonant frequency of protein encapsulated on gold
nanoparticles [92]. Silver nanoparticles interact with the HIV-1
virus via preferential binding to the gp120 glycoprotein knobs. Due
to this interaction, silver nanoparticles inhibit the virus from bind-
ing to host cells [35]. Some enzymatic proteins molecules lose their
proper function and structural organization during encapsulation
on nanoparticles. Small perturbation occurs in the native structure
of chymotrypsin on conjugation to silver nanocluster [81]. Ni-NTA
modied Fe3 O4 NH+ nanoparticles have high efciency and speci-
city to His-tagged proteins [118]. These modied nanoparticles
are used more efciently for the purication of His-tagged pro-
teins. Gold and silver nanoparticles produced by citrate reduction
have been functionalized with immunoglobulin (IgG) molecules
at pH slightly above the isoelectric point of the citrate ligand. This
allows effective binding between the positively charged amino
acid side chains of the protein and the negatively charged citrate
groups of the colloids [110]. Other examples of protein coating
through electrostatic interactions include the direct adsorption
of heme-containing redox enzymes at citrate-stabilized silver
nanoparticles [73] and the binding of basic leucine zipper proteins
178 S.C. Yadav et al. / Peptides 32 (2011) 173187
Fig. 2. Various protein nanocarriers as pillars of protein nanotechnology. Protein nanotechnology includes various nanocarriers system like metallic, polymeric and self
assembly.
to lipoic acid-stabilized semiconductor CdSe core/ZnS shell parti-
cles. These are some examples of well known nanoencapsulates of
protein and peptides on metallic nanoparticles (Table 1).
4.2. Polymeric nanocarriers
The major advantage of colloidal drug carrier system in pro-
tein and peptide therapeutics are the controlled drug targeting,
modied body distribution and enhancement of cellular uptake.
The polymeric nanocarriers are very promising since they are
biodegradable, non-antigenic, relatively easy to prepare and full
control on size distribution. A variety of polymeric nanoparti-
cles (natural and synthetic) can be synthesized in laboratory and
are also commercially available. Polymeric materials used for the
formulation of nanoparticles include synthetic (poly(lactic acids)
(PLA), poly(lactic-coglycolic acids) (PLGA), poly(-caprolactone)
(PCL), poly(methyl methacrylates), and poly(alkyl cyanoacrylates))
or natural polymers (albumin, gelatin, alginate, collagen or chi-
tosan) (Fig. 2) [59].
4.2.1. Natural nanocarriers
Chitosan is a modied natural carbohydrate polymer pre-
pared by the partial N-deacetylation of crustacean derived natural
biopolymer chitin (Fig. 2). It is the second most abundant polysac-
charide in nature, and has attracted particular interest as a
biodegradable material for mucosal delivery systems (Fig. 2). There
are at least four methods reported [115] for the preparation
of chitosan nanoparticles as ionotropic gelation, microemulsion,
emulsication solvent diffusion and polyelectrolyte complex for-
mation [115]. Chitosan nanoparticles have low toxicity and high
susceptibility to biodegradation, mucoadhesive properties and
has an important capacity to enhance protein drug permeabil-
ity/absorption at mucosal sites [115]. More importantly, chitosan
micro/nanoparticles can be spontaneously formed through ionic
gelation using tripolyphosphate as the precipitating agent. This
reduces the use of harmful organic solvents during preparation
and loading of protein therapeutics [119]. Chitosan solubility is
poor above pH 6.0 which is a major drawback of this system. At
physiological pH, chitosan is known to lose its capacity to enhance
drug permeability and absorption, which can only be achieved in its
protonated form in acidic environments. In contrast, quaternized
chitosan derivative, N-trimethyl chitosan chloride (TMC) shows
perfect solubility in water over a wide range of pH [29]. In addition,
these chitosan derivatized nanoparticles have bio-adhesive prop-
erties. Thus, it is used for enhancement of permeability and absorp-
tion of diverse protein drugs in neutral and basic-pH condition. N-
trimethyl chitosan chloride (TMC) nanoparticles to carry proteins
were prepared by ionic cross linking of TMC with tripolyphosphate
(TPP) [47]. The results indicate that different degree of quaterniza-
tion (1/ particle size) of TMC has inuenced the physicochemical
properties, release prole and degree of loading of different pro-
teins. Thus, the particle size may depend upon the nature and
concentration of the loading proteins [47]. Insulin was observed to
be directly internalized by enterocytes in contact with intestine and
retention of drugs at their absorptive sites by mucoadhesive carri-
ers [103]. Insulin loaded chitosan nanoparticles markedly enhanced
intestinal absorption of insulin following oral administration. The
hypoglycemia effect and insulinemia levels were signicantly
 S.C. Yadav et al. / Peptides 32 (2011) 173187
higher than that obtained from insulin solution and physical mix-
ture of oral insulin and empty nanoparticles. The mechanism of
insulin absorption seems to be a combination of both insulin inter-
nalization, probably through vesicular structures in enterocytes
and insulin loaded nanoparticles uptake by Payers patches cells
[103]. Chitosan nanoparticles are also explored for their efcacy
to increase systemic absorption of hydrophobic peptides such as
cyclosporin A [34]. The relative bioavailability of cyclosporin A
encapsulated chitosan nanoparticles was increased by about 73%.
This formulation provides the highest Cmax (2762.8 ng/ml) of Cy-A
after 2.17 h. Chitosan nanoparticles administered orally to beagle
dogs provide an improved absorption compared to the currently
available cyclosporin A microemulsion (Neoral ) [103]. It has been
shown that ovalbumin loaded chitosan microparticles are taken up
by the Peyers patches of the gut associated lymphoid tissue (GALT).
Additionally, after co-administering chitosan with antigens in nasal
vaccination studies, a strong enhancement of both mucosal and sys-
temic immune responses was observed [119]. Van der Lubben et al.
[119] have demonstrated that large amounts of bovine serum albu-
min (BSA) or tetanus toxoid (TT) vaccine were easily encapsulated
in chitosan nanoparticles (Table 1). Recently, Alonsos group has
developed chitosan nanoparticles as carrier systems for transmu-
cosal delivery. They have shown the enhanced mucosal absorption
of chitosan NPs on rats and rabbits [120]. The nanoparticles were
in the 350 nm size range, and exhibited a positive electrical charge
(+40 mV) and high loading efciency (5060%). They have reported
important capacity of chitosan nanoparticles for the association
of peptides such as insulin, salmon, calcitonin and tetanus tox-
oid. Their mechanism of interaction with epithelia was investigated
using the Caco-2 model cell line. The results showed that chitosan
coated systems caused a concentration-dependent reduction in the
transepithelial resistance of the cell monolayer (Table 1) [120].
Gelatin nanoparticles are extensively used in food and medici-
nal purpose. It is an attractive nanomaterial to exploit in controlled
release of peptide therapeutics due to its nontoxic, and biodegrad-
able nature (Fig. 2). This polymer works as a polyampholyte
consisting both cationic and anionic groups along with hydrophilic
functionality [58]. Due to this nature, gelatin molecules are fre-
quently used for encapsulation of both acidic and basic peptides.
Gelatin nanoparticles are prepared by desolvation/coacervation
[70] or emulsion method [132]. The addition of natural salt or
alcohol normally promotes coacervation and the control of tur-
bidity/cross linking that resulted in desired nanoparticles [70].
Gelatin nanoparticles have been used for encapsulation of BSA.
These nanoparticles can absorb 5172% of water, thus the release
of BSA from the gelatin nanoparticulate matrix follows a diffu-
sion controlled mechanism (Table 1). The average diameter of
the BSA-containing gelatin nanoparticles is approximately 840 nm
[62]. Basic broblast growth factor (bFGF) has been successfully
loaded on gelatin particles. The average diameter of the gelatin
particle-PLGA microsphere composite was 518 m, and bFGF-
loading efciency was up to 80.5%. The bFGF releasing experiment
indicated that this new composite system could release bFGF con-
tinuously and protect bFGF from denaturation [69].
4.2.2. Synthetic nanocarriers
Natural polymeric nanoparticles provide a relatively quick drug
release. However, synthetic polymers enable extended drug release
over periods from days to several weeks. Poly(dl-glycolide-co-
lactide) (PLGA) [32], poly(d,l-lactide) (PLA) and polycaprolactone
(PCL) are used to encapsulate proteins drug due to its biodegradable
and biocompatible nature (Table 1) [59]. Among these nanopar-
ticles, PLGA (poly-d,l-lactide-co-glycolide) and PLA nanoparticles
are one of the most successfully used biodegradable nanosys-
tem for the development of protein nanomedicines. They undergo
hydrolysis in the body to produce the biodegradable metabo-
179
lite monomers, lactic acid and glycolic acid (Fig. 2) [59]. Since
the body effectively deals with these two monomers, there is
very minimal systemic toxicity associated by using PLGA for drug
delivery or biomedical applications. Insulin was encapsulated in
a blend of poly (fumaric anhydride) (poly(FA) and poly(lactide-
co-glycolide) (PLGA) at a 50:50 ratio (poly(FA:PLGA)) using the
inversion phase method [16]. Animals feeding the poly (FA: PLGA)
encapsulated insulin preparation showed a better ability to regu-
late glucose load than the controls. This gave an indication that the
insulin has crossed the intestinal barrier and was released from
the microspheres in a biologically active form [16]. Kawashima
et al. evaluated the effectiveness of mucoadhesive polymeric
nanospheres in the absorption of calcitonin on chitosan coated
elcatonin-loaded PLGA nanospheres [25]. BSA and immuno--
globulin (IgG) have been encapsulated on PEOPLGA nanoparticles.
Different amounts of BSA or IgG corresponding to 1%, 2% and 4%
theoretical loadings were encapsulated into PEOPLGA nanopar-
ticles of different composition. The type of used PEO derivative
and pH of internal aqueous phase are the most important fac-
tors inuencing BSA protein encapsulation and release kinetics.
Degradation and release characteristics of polyester particles can be
improved by the incorporation of polyoxyethylene derivatives with
different hydrophilialipophilia balance [100]. BSA encapsulated
on PEGPLGA nanoparticles are reported to extend the half-life
as 4.5 h than 13.6 min in simple BSA. This extended half-life obvi-
ously change the protein bio-distribution in rats compared with
that of BSA loaded on PLGA nanoparticles [65]. Similarly, tetanus
toxoid is also encapsulated on PLA and PLAPEG nanoparticles
of similar particle size (137156 nm) but differed in their hydr-
phobicity [108]. PLAPEG nanoparticles led to greater penetration
of tetanus toxoid into the blood circulation and in lymph nodes
than PLA encapsulated tetanus toxoid [121]. The results conrm
that PLA nanoparticles suffered an immediate aggregation upon
incubation with lysozyme, whereas the PEG-coated nanoparticles
remained totally stable [109]. The antibody levels elicited following
in administration of PEG-coated nanoparticles were signicantly
higher than those corresponding to PLA nanoparticles [120].
Insulin encapsulated into poly(isobutylcyanoacrylate) (PIBCA)
nanoparticles by interfacial polymerization methods protect the
insulin against proteolytic enzymes and promote absorption by
the intestinal mucosa [26]. There is evidence that PIBCA nanopar-
ticles is able to pass from the gut lumen to the blood compartment
by means of a paracellular pathway [2]. These insulin-containing
nanocapsules induced a signicant hypoglycemic effect for sev-
eral days in fasting and fed diabetic rats but were ineffective
in normal rats [26]. Damge et al. developed insulin-loaded poly
(isobutylcyanoacrylate) of 60300 nm nanocapsules that upon oral
administration could deliver insulin directly to the blood [26].
Das and Lin [84] developed double-coated (Tween 80 and PEG
20000) poly(butylcyanoacrylate) nanoparticulate delivery systems
(PBCA) for oral delivery and brain targeting of dalargin. Even if
they did not elucidate the mechanisms of PBCA nanoparticle trans-
port from gastrointestinal tract to brain, they observed a signicant
dalargin-induced analgesia with double-coated PBCA nanoparticles
compared to single-coated PBCA nanoparticles (either Tween 80
or PEG) [84]. Earlier studies described similar approaches to pro-
tect the orally delivered cyclosporin A, LHRH, vasopressin, INF and
peptidomimetics (Table 1).
Polyalkylcyanoacrylate (PACA) nanocapsules are used as
biodegradable polymeric drug carriers for subcutaneous and oral
delivery of octreotide, a long-acting somatostatin analogue. It
has the ability to reduce secretion of insulin or of prolactin in
response to estrogens. Octreotide-loaded nanocapsules reduce
(higher than 72%) prolactin secretion, increased plasma octreotide
level, and prolonged therapeutic effect of a somatostatin analogue
in estrogen-treated rats given orally [26]. Similarly, luteinizing
180 S.C. Yadav et al. / Peptides 32 (2011) 173187
hormone releasing hormone (LHRH), conjugated with hydrox-
ypropylmethacrylamide nanoparticles are effective formulations
for enhancing its stability and improve targeting possibilities [45].
LHRH-loaded copolymerized peptide particle systems (mean size
of 100 nm) administered orally show a half-life of LHRH in blood of
12 h than normal 28 min [45]. Calcitonin, a peptide secreted by the
parathyroid gland of the human body has been encapsulated into
polyacrylamide nanospheres, PIBCA nanocapsules and chitosan
nanoparticles [68]. This encapsulated calcitonin greatly decreases
the level of ionized calcium in blood than simple calcitonin on
oral administration [25]. Cyclosporine was also encapsulated in
PIHCA by interfacial and emulsion polymerization. This thera-
peutic peptide is a cyclic nonribosomal peptide produced by the
fungus Hypocladium inatum, initially isolated from a Norwegian
soil sample. The nanoparticle formulation had a notably increased
bioavailability compared with that of the commercial formulation
[45].
Poly (N-isopropyl acrylamide) (PNIPAm) undergoes a sharp
coil globule transition in water at 32 C. It is being hydrophilic
below this temperature and hydrophobic above it. PEG coat-
ing on encapsulated therapeutic proteins have been shown to
stabilize the protein without losing their biological function
on PNIPAm nanoparticles [90]. Amine functionalized polymeric
nanoparticles is being used to enhance charge directed target-
ing of lysozyme nanoparticles conjugates to bacteria. Activity of
lysozyme conjugated to positively charged PNIPAm nanoparticles
was approximately twice, while lysozyme conjugated to neg-
atively charged PNIPAm nanoparticles showed little detectable
activity [104]. When calcitonin is incorporated in nanoparticles,
oral absorption is enhanced in rats and consequently calcium con-
centration in blood decreases compared to oral administration of
a calcitonin solution [25]. BSA was loaded on PNIPAm gels fabri-
cated with higher encapsulation efciency (40%) in the presence of
lower cross-linker contents. An incomplete release of encapsulated
BSA from the PNIPAm gels was observed in all cases. Enhanced mass
transfer was created by oscillating swellingdeswelling in response
to temperature cycling across the LCST. This reveals that lowering
in vitro release and temperature did not promote BSA release due
to strong BSAPNIPAm gel interactions (Table 1) [127].
4.3. Protein itself as nanoparticles
Peptides serve as excellent building blocks for bionanotech-
nology owing to the ease of their synthesis, small size, relative
high stability and chemical/biological modiability. Understand-
ing the physicochemical determinants that underlie peptide self
assembly is a fundamental step in view of the rational design of
new nano building blocks for biotechnological applications or new
drugs (Fig. 1). A large number of nonpathogenic and pathogenic
peptide have been shown to form ordered brils under particular
solvent, temperature and pH conditions [55]. Native folded struc-
tures of proteins and peptides have capability to self assembles
as protein bres e.g. coilcoil or amylogenic peptides [46] (Fig. 2).
Three classes of protein components as planar crystalline arrays,
engineered proteins pores and molecular motors are frequently
used for development of protein nanodevices. The surface-layer (S-
layer) proteins having square or hexagonal symmetry of 330 nm
unit cells with 510 nm thickness are one of the good examples of
planar protein crystalline arrays. Several therapeutic applications
have been suggested for S-layer as nanoscale immobilization matri-
ces. This includes S-layer streptavidin fusion nanodevice and IgG
binding domain based high density adsorbent for extracorporeal
blood purication. This is reported to have 20 times higher capac-
ity to remove autoantibodies [102]. The protein nanopores such
as -hemolysin (HL) are developed as medical analytes for single
molecule level by stochastic sensing [11]. Various molecular motors
like linear kinesin, myosin, ATP synthase have strong potential to
be used as nanodevices for various cystis brosis diseases [67].
Despite the high sequence diversity, many proteins and peptides
aggregate into a common cross--sheet structure, and the resulting
brils show remarkable ultra-structural and biophysical similarity.
These assemblies belong naturally to the realm of the nanoscale.
The study of their properties and mechanisms of formation might
be a source of inspiration for the development of ordered, rationally
designed nanostructures with potentially interesting applications
in biotechnology and other elds (Fig. 2). Normal proteins are engi-
neered to self assemble into nanodevice and thus self assembly at
nanoscale is important for the fabrication of novel supramolecu-
lar structure, having applications in the eld of nanotechnology
and nanomedicines. Peptides represent the most favorable build-
ing block for the design and synthesis of nanostructures because
they offer a great diversity of chemical and physical properties.
They can be synthesized in large amounts, can be modied and
decorated with functional elements for diverse nanotherapeutic
applications. Self assembly of polypeptide in nature such as amy-
loid brils is associated with human medical disorder. Structural
element as short as dipeptides can form well ordered assemblies at
the nanoscale [95]. Many novel building blocks that are based on
either natural or synthetic amino acids have capability to develop
linear or cyclic congurations. The diversity of the side chains of
amino acids enables the control of both conformation and function-
ality. A number of investigator used antigenantibody interactions
and biotinavidin interactions to study the direct assembly of inter-
acted protein [101].
5. Bioconjugation of peptide and protein on nanoparticles
Most of the therapeutic proteins and peptides are encapsulated
by adsorption on the surface of nanocarriers. However, various
biochemical conjugation reactions are advantageous for the ef-
cient and controlled encapsulation of therapeutic proteins (Fig. 3).
Furthermore, the activity of the protein polymer/nanoparticles
conjugate is closely regulated by altering the response stimulus
of the synthetic polymer and the site of attachment to the pro-
tein and peptides. These chimeric systems may thus be considered
as true molecular-scale devices for the development of delivery
systems. The nanoparticles and specic ligands are activated by
incorporation of functional group (NH2 , SH, COOH,), and block-
ing of specic functional group (NH2 , SH, CHO, COOH,). These
functionalized nanoparticles and its complementary functionalized
ligands are bioconjugated by functional group specic bioconju-
gation reaction. A variety of bioconjugation reaction are used like
maleimide reaction, EDC/NHS chemistry, afnity interactions, click
chemistry, streptavidin biotin reaction etc for the covalent conju-
gation of specic ligands on functionalized nanoparticles (Table 2)
[49].
5.1. Maleimide reactions
The maleimide reaction is well known for the bioconjuga-
tion of protein containing free thiol group on nanoparticles.
Thiol groups of proteins or functionalized nanoparticles (GNPs
etc.) react with maleimide functionality to form thioether bond
(Fig. 3A). In addition, thiol-maleimide based couplings are known
to proceed efciently in solution, with site specic attachment
of maleimide groups on Fc region of antibodies [49]. It is more
benecial to incorporate this functionality on the biomacro-
molecule than the nanoparticles, because nanoparticles require
several steps of purication in aqueous solution by dialysis over
a longer period of time. Maleimide functionalized bovine serum
albumin (BSA) as a model biomacromolecule was bioconjugated
 S.C. Yadav et al. / Peptides 32 (2011) 173187 181

Fig. 3. Biochemical basis of protein nanoparticles interactions. (A) Maleimide reactions; in this method, peptide and protein containing free thiol group react with
maleimide functionality to form thioether bond with the nanoparticles. (B) EDCNHS bioconjugation; carboxyl groups of nanoparticles are activated by 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride)) (N-hydroxysulfosuccinimide) (EDC/NHS) to form reactive sulfo-NHS ester which reacts with amine functionality of
proteins via formation of amide bond. (C) Afnity interactions; bioconjugation of recombinant proteins on Ni-NTA coated nanoparticles. (D) Click chemistry; this reaction is
based on the reaction between alkyne moiety and azide moiety to from triazole ring. (E) Streptavidin biotin interactions; specicity of biotin binding to streptavidin provides
the basis for developing a bioassay system to detect or quantify analytes.

with thiol-terminated poly(ethylene glycol) (PEG) chains on shell
cross linked knedel-like (SCK) nanoparticles [82]. The particle
size was increased from 20 nm to 30 nm after encapsulation of
BSA on SCK nanoparticles with >5060% encapsulation efciency.
Pioneering work has been carried out by Hoffman, Stayton and
co-workers for the bioconjugation of peptide with nanoparticles
[15]. They engineered single cysteine via site-directed mutagene-
sis a mutant of cytochrome b5. This is accessible for reaction with
maleimide end-functionalized poly-N-isopropylacrylamide (PNI-
PAm). Since the native cytochrome b5 does not contain any cysteine
residues this substitution provided a unique attachment point for
the polymer. The resultant polymerprotein conjugate displayed
low critical solution temperature (LCST) behavior and could be
reversibly precipitated from solution by variation in temperature.
This approach has proved to be very versatile and a large num-
ber of nanopolymer protein conjugates incorporating biological
components like antibodies, protein A, streptavidin, proteases and
hydrolases have now been prepared [50]. Small changes in envi-
ronmental conditions can then cause large changes in the polymer
conformation, leading to reversible blocking or unblocking of
the proteins active site; such changes also can lead to triggered
release of a bound ligand from the protein binding site. The bio-
182 S.C. Yadav et al. / Peptides 32 (2011) 173187
Table 2
Bioconjugation reaction of protein and peptides to nanoparticles.
Bioconjugation method Proteins
Maleimide coupling BSA
Adsorption BSA
Lysozyme
EDCNHS Chemistry Chymotrypsin
Anti-E. coli antibody
Monoclonal 19
Afnity interactions Horseradish peroxidase
His-Tag proteins
Click chemistry F3-peptide
Lipase
Bombesin peptide
Streptavidinbiotin interactions Biotin
Biotin
Electrostatic interactions BSA
logical functions or activities of these conjugate systems are fully
similar to their native counterparts, but they are switched on or
off as a result of thermally induced polymer phase transitions
[106]. Lactoferrin-conjugated PEGPLA nanoparticles synthesized
by thiolated reaction containing 55 molecules per nanoparticles
showed an improved brain delivery [107]. Lactoferrin engineer-
ing signicantly increased the brain uptake of drugs associated to
nanoparticles following an intravenous administration. Thiol group
is an attractive functionality to have on the surface of nanoparticles
or protein, since reversibly linked bioconjugates can be prepared by
disulde formation, which may allow reductive release of a target-
ing or therapeutic component [13].
5.2. EDCNHS bioconjugation
EDC/NHS bioconjugation is frequently used for the bioconju-
gation of peptide on variety of nanoparticles. Carboxyl groups
present on the surface of nanoparticles are activated by using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride))
(N-hydroxysulfosuccinimide) (EDC/NHS) to form reactive sulfo-
NHS ester. This ester reacts with amine functionality of proteins
via formation of amide bond (Fig. 3B). When working with pro-
teins and peptides, experience indicates that EDC-mediated amide
bond formation effectively occurs between pH 4.5 and 7.5. Beyond
this pH range, however, the coupling reaction occurs more slowly
with lower yields. The presence of both carboxylates and amines
on the molecules to be conjugated with EDC may result in self-
polymerization. This is because the substance can react with
another molecule of its own kind instead of the desired tar-
get. PLGA nanoparticles have been conjugated to Anti-Fas mAb
and IgG using EDC/NHS chemistry [76]. Variations in surface car-
boxyl density permitted up to 48.5 g coupled Ab per mg of NP.
The amount of anti-Fas mAb that could be conjugated to the NP
using two-step carbodiimide activation was dependent on the
ratio of non-end-capped to end capped PLGA types. In addition, it
was observed that anti-Fas conjugation was not affected by drug
loading. The model proteins like BSA, streptavidin or goat anti-
rabbit immunoglobulin G (IgG) are also bioconjugated on magnetic
nanoparticles with carboxylic or aminopropyltrimethoxysilane
(APTS) groups using carbodiimide as a zero-length cross-linker
[48]. The average hydrodynamic diameters of the nanoparticles
were about, 154 nm and 165 nm and the conjugation yield of
proteins were very low. 3-mercaptopropyl acid-stabilized CdTe
quantum dot nanoparticles are conjugated with peptides or pro-
teins (active) mediated by N-hydroxysulfo-succinimide (NHS) or
1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride
(EDC) [137]. The enzyme -chymotrypsin was nanoencapsulated
Nanocarriers Speciality Ref.
t-BA Protein activity retained [82]
Fe Biological activity retained [66]
Au Bioactivity of the lysozyme should be retained [130]
Fe Thermal stability was improved [51]
Au Suitable for antigenantibody interactions [86]
Au Facile identication of cancer tissue [33]
NTA-GNP Specic immobilization of His-tagged proteins [1]
Ni Suitable for purication of His-tag proteins [88]
Fe Good for targeted delivery [135]
Au Enzyme activity was retained [14]
Fe Good for targeted delivery [75]
Fe High afnity for the Cy3-labeled streptavidin [19]
PLGA Specic binding of biotinylated NP to a [126]
NeutrAvidin (NAv)-coated surface
Folate-PEG-TMC Suitable for intracellular transport of [136]
negatively charged therapeutic proteins into
folate receptor over-expressing tumor cells
on polyacrylamide-coated Fe3 O4 nanoparticles synthesized by
photochemical in situ polymerization [53]. Mean particle size
of the immobilized enzyme was 31 nm and super paramagnetic
properties of Fe3 O4 were retained after enzyme immobilization.
The wheat germ agglutinin (WGA)-conjugated PLGA nanoparticles
loaded with paclitaxel and isopropyl myristate has been prepared
by two-step carbodiimide method to use it as an anticancer agents
(Table 2) [31]. WDA-conjugated PLGA nanoparticles had lower
yields and encapsulation efciency because they require more
purication steps.
5.3. Afnity interactions
An alternative approach taking advantage of afnity interactions
has been extensively used for bioconjugation of recombinant pro-
teins on Ni-NTA coated nanoparticles. This approach exploits the
interaction between divalent metal ions such as nickel, copper, or
cobalt and a short sequence of histidine residues (410 repeating
units) added to the N- or C-terminus of the protein (histidine-tags)
(Fig. 3C). These purication methods generally involve immobi-
lizing the metal ion onto the column packing material using a
chelating agent such as nitrilotriacetic acid (NTA). In the case of
Cu2+ and Ni2+ which have six coordination sites, NTA forms a strong
complex with four of the metal sites, leaving two additional sites
for interaction with the His-tag present on the protein. His-tag
HIV-1 Gag p24 protein was coupled to DOGSYNTAYNi nanoparti-
cles utilizing the above approach [88]. The advantages of afnity
interactions between divalent Ni nanoparticles are effective for
enhancing its interaction with His-tag proteins. The stronger afn-
ity of antigen with the Ni-NPs resulted in superior humoral immune
responses in vivo compared to protein adjuvant with alum [88]. Fol-
lowing this method various metallic nanoparticles have been used
for the attachment of protein molecule by polymeric hydrophilic
nanolayer surface modication (Fig. 3C).
5.4. Click chemistry
Click chemistry is one of the most useful reactions for the
bioconjugation of proteins on the nanoparticles. This reaction is
based on the reaction between alkyne moiety and azide moiety
to from triazole ring. Beauty of this reaction is that alkyne moiety
attached to the ligands reacts only with azide moiety of nanoparti-
cles avoiding other side products (Fig. 3D). Cyclic tumor-targeting
peptide LyP-1 (CGNKRTRG) containing thiol and amine groups was
bio-conjugated on polymer-coated magnetouorescent nanopar-
ticles by applying click chemistry [123]. Click nanoparticles are
able to stably circulate for hours in vivo following intravenous
 S.C. Yadav et al. / Peptides 32 (2011) 173187
administration (>5 h circulation time), extravasate into tumors,
and penetrate the tumor interstitium to specically bind p32-
expressing cells in tumors. An acetylene-functionalized lipase has
been attached to azide-functionalized water-soluble gold nanopar-
ticles in a copper-catalyzed 1,2,3-triazole cycloaddition with full
enzymatic activity [14]. Seven fully active lipase molecules are
attached to each nanoparticle. Staudinger ligation reaction work
similarly as click chemistry but the reacting moieties are different. It
is used to ligate modied phosphine and an azide to form an amide
bond. The basis of this reaction is the hydrolysis of amidophos-
phonium intermediate in water. The ligand can be readily attached
either before assembly of the complexes (precomplexation) or
to the assembled nanoparticle (postcomplexation) by using this
bioconjugation methods. The PEGylated polyamidoamine DMEDA-
PEG-DMEDA-(MBA-DMEDA)n + 1 -PEG-DMEDA was attached with
integrin-binding peptide ligand via the Staudinger ligation [85].
Postcomplexation strategy led to small and discrete toroidal
nanoparticles whilst the precomplexation particles showed loose
complexes (Table 2).
5.5. Streptavidinbiotin reaction
Streptavidin is a tetrameric protein which binds very tightly
to a small water-soluble molecule biotin. The specicity of biotin
binding to streptavidin provides the basis for developing a bioas-
say system to detect or quantify analytes. More importantly, both
streptavidin and biotin are effectively conjugated to other pro-
teins or labeled with various detection reagents without loss of
their binding afnity (Fig. 3E). The coupling reaction with biotin-
(poly(ethylene glycol))amine and polymeric PLGA nanoparticles
has been used for the conjugation of various protein and peptides
[126]. Biotin binding proteins (avidin, streptavidin, or neutravidin)
have been used as cross linkers to conjugate proteins on biodegrad-
able nanoparticles (PLGAPEGbiotin). Avidin gave the highest
level of overall protein conjugation, whereas neutravidin is known
to minimize non-specic protein binding to the polymer [116].
6. Nanoencapsulation improves the therapeutic
importance of proteins and peptides
The foremost aim of the protein and peptide drug delivery sys-
tems is the interaction or internalization of protein and peptides
drug into target cells. The encapsulation of these drugs on the suit-
able nanocarrier is the rst step in the development of targeted
peptide delivery. The desired feature of these efcient drug delivery
systems is to deliver therapeutic peptides to active site at the right
time in a therapeutically effective concentration and at highest
patient convenience/compliance, with minimal side effects and low
production cost. The isolation, and costly downstream process for
the production of cost effective therapeutic proteins and peptides
are the major obstacle in the development of protein therapeutics.
Various proteases present at potential site of administration, may
inactivate and degrade these proteins therapeutics. The elimination
of these peptide drugs by body defense system is another major
reason for less utilization of protein and peptides therapeutics.
These problems create the decline of bioavailability and increases
potential immune response against the protein therapeutics. The
alternative route of administration of the peptides therapeutics like
pulmonary, transdermal, and nasal may be benecial than conven-
tional route, towards the reduction of above mentioned problems
and increase in their bioavailability [20]. A variety of methods have
been widely proposed to efcient protein delivery in vivo as well
as in vitro for the delivery of protein and peptides therapeutics
[79]. The most efcient non nanotechnological methods for ef-
cient cellular delivery are transcriptional activator of transcription
183
(TAT) from HIVI, third helix of antennapedia homeodomain and
VP22 protein of herpes simplex virus. These methods are used to
improve the internalization of peptide drugs due to their unique
potential to enter the cells in culture when added exogenously [79].
Though these carriers systems have potential to deliver protein into
the cells but, many of them have shown inefcient delivery for the
protein therapeutics to their actual target site. In these strategies,
a number of other problems, such as complex manipulation, cel-
lular toxicity and immunogenicity are reported [79]. However, the
encapsulation of protein and peptides drugs on suitable nanocarri-
ers is one of the emerging techniques with tremendous potential to
reduce the problem (stability, degradation, proteolysis etc.) associ-
ated with protein and peptide delivery. In addition, encapsulation
of proteins and peptides in nanocarriers in physiologically active
form provides resistant to organic solvents, moisture, acidic envi-
ronment, enzymatic degradation, and immunological elimination.
This technique is being widely accepted practical approach for the
development of protein and peptide nanotherapeutics [28].
The retention of protein structure and activity on nanoscale
support are critical for their therapeutic applications. The other
fundamental interest is to understand the effect of size and sur-
face chemistry of nanomaterial on structure, activity, and stability
of nanoparticles conjugated proteins. The study of lysozyme and
human carbonic anhydrase adsorbed on silica nanoparticles reveals
the change in structure, activity and loss of a helical contents
depending upon the size of nanoparticles [71]. This nding sug-
gests that the smaller nanoparticles are advantageous for protein
stability due to higher surface curvature for the protein nanoen-
capsulation. The retention of protein structure on nanoparticles
is also protein dependent. It is also reported that SWCNTs (sin-
gle walled carbon nanotubes) stabilize the protein under harsh
conditions like high temperature, organic solvents etc [7]. The
surface of nanoparticles also provides the control over the struc-
ture and function of adsorbed chymotrypsin [52]. The disruption
of proteinprotein interaction using surface-functionalized gold
nanoparticles is reported with cytochrom c and cytochrome c per-
oxidase [12].
Cellular localization of protein encapsulated on nanoparticles
is signicantly different from those of small-molecule probes and
is extremely sensitive to the surface charge of nanoparticles. The
nanoparticles are localized in lysosome, nucleus, endosome in ani-
mal cells. Oleylated QDs are localized mainly in the cell membrane
[105]. However, coumarin-six loaded chitosan/GMO formulation is
localized in nuclear material in MDA-MB-231 cells [117]. The appli-
cation of carbon-coated magnetic iron nanoparticles in pumpkin
by pulverization induces the distribution of these nanoparticles to
parenchyma, xylem vessels, cortex and epidermis of the plant cells.
This observation provides some highlights in the cellular move-
ment of nanoparticles in vascular plant [21].
7. The stability of nanoencapsulated peptide and protein
The activity, structural composition and stability of protein
nanomaterial interaction depend on the type of nanomaterial and
protein itself. The protein stability may be inuenced/improved
by nanoparticles interaction. The protein stabilization agents, such
as gelatin, BSA and other low-value proteins (for protein-based
stabilization), mannitol (to regulate the osmotic pressure of the
medium), PVP, PEG, Pluronic F-68 etc are used for the improvement
of stability of nanoencapsulated proteins [79]. It is reported that
fabricated amino acid functionalized gold nanoparticles modulates
the activity of the chymotrypsin [7]. Electrostatic nanocomplexes
(100 nm) consisting of -lactoglobulin and pectin showed a very
good colloidal stability [138]. The hydrophilic peptide insulin for-
mulated in to biodegradable nanoparticles have been stabilized
184 S.C. Yadav et al. / Peptides 32 (2011) 173187
Table 3
Nanopolymer protein conjugates based drugs for cancer treatment.
Nanocarriers Therapeutic compounds
Polymerprotein conjugate Styrene maleic anhydride-
neocarz-inostatin (SMAN
CS)
Polymerprotein conjugate PEG- l-asparaginase
Polymerprotein conjugate PEG-granulocyte
colony-stimula-ting factor
(G-CSF)
Immunotoxin (fusion protein) IL 2 fused to diphtheria
toxin
Chemo-immunoconjugate Anti-CD33 antibody
conjugated to
calicheamicin
Radio-immunoconjugate Anti-CD20 conjugated to
yttrium-90 or indium-111
Anti-CD20 conjugated to
iodine-131
Immunotoxins, immunopolymers Various drugs and toxins
and fusion proteins (315 nm)
by the formation of a phospholipid complex [25]. Inuenza HA
loaded PLGA nanoparticles reveals that the encapsulated proteins
are stable inside the nanoparticles and does not degrade dur-
ing the production process as well as on long term storage [94].
Zhu et al., recently reported that combined physical and chem-
ical immobilization of glucose oxidase in alginate microspheres
improves stability of encapsulation and activity [137]. The reten-
tion of alcohol dehydrogenases (ADH) activity was dependent
on the concentration of ADH in the feed solution. However, the
addition of other proteins, such as bovine serum albumin and
-lactoglobulin, exhibited an additional improvement of the reten-
tion of ADH activity. The inlet and outlet temperature of the drying
air was another key factor for the enzyme activity on the spray
drying method of protein encapsulation [131]. Biomimetic silica
entrapment of chemically derivatized horseradish peroxidase for
amperometric sensing applications shows very high stability in
comparison to nanoencapsulated native enzymes [133]. Itoh et al.,
group has reported the similar activity and much higher stability
(four times) of catalase upon encapsulation in mesoporous silica
synthesized in the pores of an alumina membrane (Table 1) [54].
8. Therapeutic use of the nanoprotein molecules
Proteomic technology to choose appropriate targets is an active
area of research. However, till date no clinically effective targets
have been identied. The fusion proteins, engineered antibody,
dimerization, detection of specic conformation of target receptors
together with nanocarriers encapsulation provides novel strategies
to cure target cells. The use of peptide as targeting agents results
in increased intracellular drug delivery in different murine tumor
models (Table 3). Likewise, several protein and peptide encap-
sulated nanosystems with different compositions and biological
properties have been extensively investigated for drug and gene
delivery applications [91].
The small size of nanoparticles was used to spy at the cellu-
lar machinery level with least interference in function. Protein
nanotechnology have potential to use in the study of immuno-
complex (antigenantibody) formation, drug and gene delivery,
biodetection of pathogen, detection of proteins, probing of DNA
structure, tissue engineering, separation and purication of bio-
logical molecules and cells etc. These peptide nanomedicines
reduce the frequency of drug administration, and improved patient
compliance. This would offer the protection and improved phar-
macokinetics of easily degradable, short half-life of protein in vivo
and in vitro conditions. Leaky blood vessels and poor lymphatic
Targeted cancer Commercial name
Hepatocellular carcinoma Zinostatin/Stimalmer
Acute lymphoblastic leukemia Oncaspar
Prevention of Neulasta/PEG lgrastim
chemotherapy-associated
neutropenia
Cutaneous T-cell lymphoma Ontak (Denile-lukin diftitox)
Acute myelogenous leukemia Mylotarg
Relapsed or refractory, low-grade, Zevalin
follicular, or transformed
non-Hodgkins lymphoma
Bexxar
Various type of cancer Clinical trials phage III
drainage in tumors provides the permeability of <200 nm nanopar-
ticles in the tumor cells [24]. Targeting cancer with mAb and
its feasibility has been clinically demonstrated. Over 17 different
mAb are approved by the US Food and Drug Administration (FDA)
for cancer treatment. The rituximab (rituxan) and trastuzumab
(herceptin binds to ErbB2 receptor) mAb was used for non-
Hodgkins lymphoma and breast cancer treatment respectively [4].
An antiVEGFmAb as angiogenesis inhibitor for treatment of colorec-
tal cancer Bevacizumab (Herceptin) was approved in 2004 [38].
Over 200 delivery systems based on antibodies encapsulated on
nanoparticles or their fragments are in preclinical and clinical tri-
als [112]. The tissue rejection could be minimized by using the
specic protein coated nanoparticles. This nanoparticle reduces
the chances of rejection as well as to stimulate the production
of osteoblasts. One of the most exciting applications of protein
nanoparticles in tissue engineering and regeneration are the abil-
ity of these nanostructures to provide a permissive environment
for axonal regeneration in the central nervous system (CNS) after
injury. When the peptide scaffold was applied to damage optic
tracts in hamsters, regenerated axons could reconnect, and target
tissues with sufcient density as to enable the functional return of
vision [36].
9. Conclusion
The therapeutic peptides have more potential for the dis-
ease diagnosis, cure and have minimal side effect than the
small-molecule drugs. Various peptides of therapeutic poten-
tial are well known and characterized but their therapeutic
use is restricted due to less functional and structural stability
and susceptibility to proteolysis. The denaturation and rst pass
metabolism before reaching the target are other major prob-
lems prior use as therapeutic medicines. The eld of protein
therapeutics has achieved the exponential growth along with
nanotechnology. Insulin provides the major attention for the
future development of peptides as future medicine. FDA has
approved >130 peptide for therapeutic and diagnostic applications.
At present, more than 800 different antimicrobial peptides (list
at http://www.bbcm.units.ittossi/pag1.htm) have been described
from invertebrates, plant and animal species). The suitable
nanoencapsulation of various potential therapeutic peptides on
biodegradable and biocompatible nanocarriers eliminates the pos-
sibilities of degradation, structural and functional deformities and
metabolism before reaching the target sites. The nanoencapsula-
tion of these peptides are the rst step towards the development
 S.C. Yadav et al. / Peptides 32 (2011) 173187
of peptides nanomedicines. The others foundation towards the
development of the peptides nanomedicines are understanding,
controlling and applying biomoleculenanomaterial interaction for
sensing; enhance therapeutic activity; delivery and the assem-
blies of nanocomposites. The development and approval of
peptide nanomedicines require the complete information about
the changes in cell receptors that occur with progression of disease,
mechanism and site of action, retention, multiple administration,
molecular mechanisms, stability and therapeutic activity of protein
in vivo and pathobiology of the disease. The binding of plasma pro-
teins (opsonization) is also equally important in determining the in
vivo organ distribution and clearance of certain nanotechnology-
derived protein carriers. Thus, a lot of research in the above
mentioned eld is essential and several groups including ours are
involved in this area. The suitable and specic nanoencapsulation
of therapeutic protein and peptides is the key steps in the direc-
tion of step up of peptide nanomedicine. This review could open
up the opportunity on the way to speed up the research for the
development of peptide nanomedicines.
Remarkable progress has been achieved in the development of
protein-based component for nanodevices. The mean to assemble
the component and ultimately the nanodevices is of crucial impor-
tance. The major issue to control the nanodevices has been greatly
solved by protein engineering. Sophisticated application of protein
containing nanodevices is far off, but several can be envisaged. A
relatively simple device also includes sensors, which would have
added value in, say, and medical diagnostic if they were self pow-
ered by biochemical fuels. To reduce the multiple drug resistance
(MDR) the nanocarriers has been programmed by attaching cancer
cell specic ligand protein by conjugation chemistry. This nanopar-
ticle binds to specic receptor and internalized before the drug is
released.
Acknowledgements
We are grateful to Dr. P.S. Ahuja Director, IHBT for providing nec-
essary facilities for carrying out this work. The IHBT communication
number of this article is 2020. Financial assistance from Council of
Scientic and Industrial Research (CSIR) and Department of Science
and Technology (DST), Government of India is truly acknowledged.
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