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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 tion, giving rise to a polymer rich dense phase. This method has
based on the separation of a water miscible solvent from aqueous been classied into simple and complex processes depending on
solution by adding salting out agent like magnesium chloride, cal- the number of participating macromolecules. In simple polyelec-
cium chloride, etc. The main advantage of salting out procedure trolyte coacervation, addition of salt or alcohol normally promotes
is that it minimizes unfolding stress to protein encapsulates. Var- coacervation. In complex coacervation, two oppositely charged
ious peptides are nanoencapsulated on different nanoparticulate macromolecules (or a polyelectrolyte and an oppositely charged
system by using this technique [74]. colloid) can undergo coacervation through associative interactions
(Fig. 1B). The charges on the polyelectrolytes must be sufciently
3.5. Coacervation large to cause signicant electrostatic interactions but not so large
to cause precipitation. The dilute liquid phase, (usually super-
Coacervation is a process during which a homogeneous solution natant), remains in equilibrium with the coacervate phase. These
of charged macromolecules undergoes liquidliquid phase separa- two liquid phases are incompatible and immiscible. BSA has been
S.C. Yadav et al. / Peptides 32 (2011) 173187 177

successfully encapsulated on PLA and PLGA by using coacervation of peptides, proteins, and genes due to their ability to protect pro-
(Table 1) [113]. tein and peptides from degradation in the gastrointestinal track and
also in blood circulation. These protein encapsulated carriers have
3.6. Emulsication/solvent diffusion many advantages, such as a potential for selective targeting, con-
trolled release and increase persistence of proteins therapeutics in
The emulsication/solvent diffusion (ESD) method is very the body. The protein therapeutic agents have short half-life due to
efcient technique for encapsulation of protein on polymeric proteolysis, rapid clearance from the blood stream and repeated
nanoparticles. This technique provides the maximum encapsu- administration. Encapsulation of protein drugs on nanoparticles
lation efciency without homogenization, high batch to batch provides sustained release and protects the non-released protein
reproducibility, ease of scale up, simplicity and narrow size dis- from degradation [10]. The therapeutic protein molecules inter-
tribution of protein nanoencapsulate [100]. In these methods, act with nanocarriers by forming a coat or adsorption on the
the polymers with proteins are dissolved in a partially water- surface, or by bioconjugation (direct or using cross linkers). Var-
soluble solvent and saturated with water. Subsequently, the ious methods and nanocarriers systems are used to overcome
polymerwater saturated solvent phase is emulsied in an aque- the problems associated with functional native protein encap-
ous solution containing stabilizer, leading to solvent diffusion to sulation on nanoparticles. The choices of encapsulation methods
the external phase and the formation of the nanoparticles. Bovine and nanocarriers system entirely depends on the application and
serum albumin (BSA) and immuno--globulin (IgG) encapsulated nature of protein encapsulate. Some proteins, enzymes, and DNA
on (PEOPLGA) using this method has high encapsulation ef- plasmids are encapsulated in biodegradable polymeric nanobers
ciency (58.9%) and slow in vitro release rate. The problem of protein by electro-spinning technique to retain their bioactivity [72]. The
hydration, pH reduction derived from polymer degradation and strength and selectivity of proteinprotein interactions make the
presence of the hydrophobic interfaces in poly lactic-co-glycolic protein therapeutics as excellent candidates to serve as a linker
acid (PLGA)-based delivery devices lead to protein inactivation or between nanoparticles ordered structures. In addition, the pro-
irreversible aggregation inside PLGA or PLA nanoparticles [100]. teins therapeutic molecules are encapsulated on various kinds of
These problems are prevented by incorporation of stabilizers such nanoparticles like metallic, polymeric (natural and synthetic), or
as polyethylene oxide (PEO) and its derivatives (Table 1). 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).
3.7. Surface functionalization of gold NPs
4.1. Metallic nanocarriers
Bayraktar et al. [12] demonstrated the ability to disrupt
proteinprotein interactions using surface-functionalized gold
Semiconductor and metallic NPs encapsulated protein and pep-
nanoparticles. They have designed nanoparticles that bound selec-
tides therapeutics illustrate unique electronic, optical and catalytic
tively to cytochrome c (Cyt c) or cytochrome c peroxidase
properties (Fig. 2). Proteins and peptides encapsulated on nanopar-
(CCP), thereby inhibiting enzyme turnover. Surface-functionalized
ticles (NP) or quantum dot (QD) has yielded composite material
nanoparticles with gold cores (2 nm) are prepared using thiolates
with new functionality. Gold NP has been used as nanoelectrode to
with oligo(ethylene glycol) groups terminated in carboxylate (Au-
attach proteins and peptides by dithiol bridge due to inactivation
TCOOH) and trimethylamine (Au-TTMA) functionalities. Au-TTMA
of active site on encapsulation [65]. Horse spleen apoferritin was
nanoparticles bind selectively to a negative patch on CCP sur-
attached on gold nanoparticles by applying this technique. This
face, whereas Au-TCOOH nanoparticles bind selectively to basic
enzyme forms a covering layer that stabilizes the gold nanoparticles
amino acid rich surface of Cyt c. Tseng et al. [118] fabricated
in solution [65]. Cyt c, hemoglobin and myoglobin are also biocon-
amino-acid-functionalized gold nanoparticles that modulate the
jugated on silver and gold nanoparticles by using this technique.
catalytic activity of R-chymotrypsin. They proposed that the amino
The spectral features of protein encapsulated on nanoparticles are
acid monolayer on the nanoparticles controls both the capture of
broadened and red shifted due to surface binding of these proteins
substrate by the active site and the release of product through elec-
[80]. Similarly, CD11b interleukins conjugated gold nanoparticles
trostatic interactions (Table 1).
show selective binding to macrophage cells [92]. This selective
destruction of the target cell line is possible by laser irradiation
3.8. Taylor cone jet methods of plasmon resonant frequency of protein encapsulated on gold
nanoparticles [92]. Silver nanoparticles interact with the HIV-1
The secondary structure of adsorbed proteins depends on both virus via preferential binding to the gp120 glycoprotein knobs. Due
the identity of the adsorbed protein and the mode of presentation to this interaction, silver nanoparticles inhibit the virus from bind-
of chemical functionalities. Taylor Cone Jet Mode is a new technique ing to host cells [35]. Some enzymatic proteins molecules lose their
for protein encapsulation on nanoparticles to protect their struc- proper function and structural organization during encapsulation
ture and function. In this technique, protein solution is dispersed on nanoparticles. Small perturbation occurs in the native structure
in organic phase (with DCM) dissolved PLGA by controlled soni- of chymotrypsin on conjugation to silver nanocluster [81]. Ni-NTA
cation processes. Further, electrospray with well dened potential modied Fe3 O4 NH+ nanoparticles have high efciency and speci-
difference is created between the nozzle and the ring by applying city to His-tagged proteins [118]. These modied nanoparticles
high voltage on the nozzle and a lower high voltage in the ring. The are used more efciently for the purication of His-tagged pro-
emulsion solution was pumped through nozzle to form liquid cone. teins. Gold and silver nanoparticles produced by citrate reduction
This creates a thin jet from the apex to break up into monodispersed have been functionalized with immunoglobulin (IgG) molecules
droplets (Table 1) [128]. at pH slightly above the isoelectric point of the citrate ligand. This
allows effective binding between the positively charged amino
4. Nanocarriers of peptide and protein encapsulant acid side chains of the protein and the negatively charged citrate
groups of the colloids [110]. Other examples of protein coating
Nanocarriers are one of the useful tools for achieving the main through electrostatic interactions include the direct adsorption
objective of protein therapeutics and its targeted delivery. A vari- of heme-containing redox enzymes at citrate-stabilized silver
ety of nanocarriers have received much attention for the delivery 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- mation [115]. Chitosan nanoparticles have low toxicity and high
cles. These are some examples of well known nanoencapsulates of susceptibility to biodegradation, mucoadhesive properties and
protein and peptides on metallic nanoparticles (Table 1). has an important capacity to enhance protein drug permeabil-
ity/absorption at mucosal sites [115]. More importantly, chitosan
4.2. Polymeric nanocarriers micro/nanoparticles can be spontaneously formed through ionic
gelation using tripolyphosphate as the precipitating agent. This
The major advantage of colloidal drug carrier system in pro- reduces the use of harmful organic solvents during preparation
tein and peptide therapeutics are the controlled drug targeting, and loading of protein therapeutics [119]. Chitosan solubility is
modied body distribution and enhancement of cellular uptake. poor above pH 6.0 which is a major drawback of this system. At
The polymeric nanocarriers are very promising since they are physiological pH, chitosan is known to lose its capacity to enhance
biodegradable, non-antigenic, relatively easy to prepare and full drug permeability and absorption, which can only be achieved in its
control on size distribution. A variety of polymeric nanoparti- protonated form in acidic environments. In contrast, quaternized
cles (natural and synthetic) can be synthesized in laboratory and chitosan derivative, N-trimethyl chitosan chloride (TMC) shows
are also commercially available. Polymeric materials used for the perfect solubility in water over a wide range of pH [29]. In addition,
formulation of nanoparticles include synthetic (poly(lactic acids) these chitosan derivatized nanoparticles have bio-adhesive prop-
(PLA), poly(lactic-coglycolic acids) (PLGA), poly(-caprolactone) erties. Thus, it is used for enhancement of permeability and absorp-
(PCL), poly(methyl methacrylates), and poly(alkyl cyanoacrylates)) tion of diverse protein drugs in neutral and basic-pH condition. N-
or natural polymers (albumin, gelatin, alginate, collagen or chi- trimethyl chitosan chloride (TMC) nanoparticles to carry proteins
tosan) (Fig. 2) [59]. were prepared by ionic cross linking of TMC with tripolyphosphate
(TPP) [47]. The results indicate that different degree of quaterniza-
4.2.1. Natural nanocarriers tion (1/ particle size) of TMC has inuenced the physicochemical
Chitosan is a modied natural carbohydrate polymer pre- properties, release prole and degree of loading of different pro-
pared by the partial N-deacetylation of crustacean derived natural teins. Thus, the particle size may depend upon the nature and
biopolymer chitin (Fig. 2). It is the second most abundant polysac- concentration of the loading proteins [47]. Insulin was observed to
charide in nature, and has attracted particular interest as a be directly internalized by enterocytes in contact with intestine and
biodegradable material for mucosal delivery systems (Fig. 2). There retention of drugs at their absorptive sites by mucoadhesive carri-
are at least four methods reported [115] for the preparation ers [103]. Insulin loaded chitosan nanoparticles markedly enhanced
of chitosan nanoparticles as ionotropic gelation, microemulsion, intestinal absorption of insulin following oral administration. The
emulsication solvent diffusion and polyelectrolyte complex for- hypoglycemia effect and insulinemia levels were signicantly
S.C. Yadav et al. / Peptides 32 (2011) 173187 179

higher than that obtained from insulin solution and physical mix- lite monomers, lactic acid and glycolic acid (Fig. 2) [59]. Since
ture of oral insulin and empty nanoparticles. The mechanism of the body effectively deals with these two monomers, there is
insulin absorption seems to be a combination of both insulin inter- very minimal systemic toxicity associated by using PLGA for drug
nalization, probably through vesicular structures in enterocytes delivery or biomedical applications. Insulin was encapsulated in
and insulin loaded nanoparticles uptake by Payers patches cells a blend of poly (fumaric anhydride) (poly(FA) and poly(lactide-
[103]. Chitosan nanoparticles are also explored for their efcacy co-glycolide) (PLGA) at a 50:50 ratio (poly(FA:PLGA)) using the
to increase systemic absorption of hydrophobic peptides such as inversion phase method [16]. Animals feeding the poly (FA: PLGA)
cyclosporin A [34]. The relative bioavailability of cyclosporin A encapsulated insulin preparation showed a better ability to regu-
encapsulated chitosan nanoparticles was increased by about 73%. late glucose load than the controls. This gave an indication that the
This formulation provides the highest Cmax (2762.8 ng/ml) of Cy-A insulin has crossed the intestinal barrier and was released from
after 2.17 h. Chitosan nanoparticles administered orally to beagle the microspheres in a biologically active form [16]. Kawashima
dogs provide an improved absorption compared to the currently et al. evaluated the effectiveness of mucoadhesive polymeric
available cyclosporin A microemulsion (Neoral ) [103]. It has been nanospheres in the absorption of calcitonin on chitosan coated
shown that ovalbumin loaded chitosan microparticles are taken up elcatonin-loaded PLGA nanospheres [25]. BSA and immuno--
by the Peyers patches of the gut associated lymphoid tissue (GALT). globulin (IgG) have been encapsulated on PEOPLGA nanoparticles.
Additionally, after co-administering chitosan with antigens in nasal Different amounts of BSA or IgG corresponding to 1%, 2% and 4%
vaccination studies, a strong enhancement of both mucosal and sys- theoretical loadings were encapsulated into PEOPLGA nanopar-
temic immune responses was observed [119]. Van der Lubben et al. ticles of different composition. The type of used PEO derivative
[119] have demonstrated that large amounts of bovine serum albu- and pH of internal aqueous phase are the most important fac-
min (BSA) or tetanus toxoid (TT) vaccine were easily encapsulated tors inuencing BSA protein encapsulation and release kinetics.
in chitosan nanoparticles (Table 1). Recently, Alonsos group has Degradation and release characteristics of polyester particles can be
developed chitosan nanoparticles as carrier systems for transmu- improved by the incorporation of polyoxyethylene derivatives with
cosal delivery. They have shown the enhanced mucosal absorption different hydrophilialipophilia balance [100]. BSA encapsulated
of chitosan NPs on rats and rabbits [120]. The nanoparticles were on PEGPLGA nanoparticles are reported to extend the half-life
in the 350 nm size range, and exhibited a positive electrical charge as 4.5 h than 13.6 min in simple BSA. This extended half-life obvi-
(+40 mV) and high loading efciency (5060%). They have reported ously change the protein bio-distribution in rats compared with
important capacity of chitosan nanoparticles for the association that of BSA loaded on PLGA nanoparticles [65]. Similarly, tetanus
of peptides such as insulin, salmon, calcitonin and tetanus tox- toxoid is also encapsulated on PLA and PLAPEG nanoparticles
oid. Their mechanism of interaction with epithelia was investigated of similar particle size (137156 nm) but differed in their hydr-
using the Caco-2 model cell line. The results showed that chitosan phobicity [108]. PLAPEG nanoparticles led to greater penetration
coated systems caused a concentration-dependent reduction in the of tetanus toxoid into the blood circulation and in lymph nodes
transepithelial resistance of the cell monolayer (Table 1) [120]. than PLA encapsulated tetanus toxoid [121]. The results conrm
Gelatin nanoparticles are extensively used in food and medici- that PLA nanoparticles suffered an immediate aggregation upon
nal purpose. It is an attractive nanomaterial to exploit in controlled incubation with lysozyme, whereas the PEG-coated nanoparticles
release of peptide therapeutics due to its nontoxic, and biodegrad- remained totally stable [109]. The antibody levels elicited following
able nature (Fig. 2). This polymer works as a polyampholyte in administration of PEG-coated nanoparticles were signicantly
consisting both cationic and anionic groups along with hydrophilic higher than those corresponding to PLA nanoparticles [120].
functionality [58]. Due to this nature, gelatin molecules are fre- Insulin encapsulated into poly(isobutylcyanoacrylate) (PIBCA)
quently used for encapsulation of both acidic and basic peptides. nanoparticles by interfacial polymerization methods protect the
Gelatin nanoparticles are prepared by desolvation/coacervation insulin against proteolytic enzymes and promote absorption by
[70] or emulsion method [132]. The addition of natural salt or the intestinal mucosa [26]. There is evidence that PIBCA nanopar-
alcohol normally promotes coacervation and the control of tur- ticles is able to pass from the gut lumen to the blood compartment
bidity/cross linking that resulted in desired nanoparticles [70]. by means of a paracellular pathway [2]. These insulin-containing
Gelatin nanoparticles have been used for encapsulation of BSA. nanocapsules induced a signicant hypoglycemic effect for sev-
These nanoparticles can absorb 5172% of water, thus the release eral days in fasting and fed diabetic rats but were ineffective
of BSA from the gelatin nanoparticulate matrix follows a diffu- in normal rats [26]. Damge et al. developed insulin-loaded poly
sion controlled mechanism (Table 1). The average diameter of (isobutylcyanoacrylate) of 60300 nm nanocapsules that upon oral
the BSA-containing gelatin nanoparticles is approximately 840 nm administration could deliver insulin directly to the blood [26].
[62]. Basic broblast growth factor (bFGF) has been successfully Das and Lin [84] developed double-coated (Tween 80 and PEG
loaded on gelatin particles. The average diameter of the gelatin 20000) poly(butylcyanoacrylate) nanoparticulate delivery systems
particle-PLGA microsphere composite was 518 m, and bFGF- (PBCA) for oral delivery and brain targeting of dalargin. Even if
loading efciency was up to 80.5%. The bFGF releasing experiment they did not elucidate the mechanisms of PBCA nanoparticle trans-
indicated that this new composite system could release bFGF con- port from gastrointestinal tract to brain, they observed a signicant
tinuously and protect bFGF from denaturation [69]. dalargin-induced analgesia with double-coated PBCA nanoparticles
compared to single-coated PBCA nanoparticles (either Tween 80
4.2.2. Synthetic nanocarriers or PEG) [84]. Earlier studies described similar approaches to pro-
Natural polymeric nanoparticles provide a relatively quick drug tect the orally delivered cyclosporin A, LHRH, vasopressin, INF and
release. However, synthetic polymers enable extended drug release peptidomimetics (Table 1).
over periods from days to several weeks. Poly(dl-glycolide-co- Polyalkylcyanoacrylate (PACA) nanocapsules are used as
lactide) (PLGA) [32], poly(d,l-lactide) (PLA) and polycaprolactone biodegradable polymeric drug carriers for subcutaneous and oral
(PCL) are used to encapsulate proteins drug due to its biodegradable delivery of octreotide, a long-acting somatostatin analogue. It
and biocompatible nature (Table 1) [59]. Among these nanopar- has the ability to reduce secretion of insulin or of prolactin in
ticles, PLGA (poly-d,l-lactide-co-glycolide) and PLA nanoparticles response to estrogens. Octreotide-loaded nanocapsules reduce
are one of the most successfully used biodegradable nanosys- (higher than 72%) prolactin secretion, increased plasma octreotide
tem for the development of protein nanomedicines. They undergo level, and prolonged therapeutic effect of a somatostatin analogue
hydrolysis in the body to produce the biodegradable metabo- 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- like linear kinesin, myosin, ATP synthase have strong potential to
ypropylmethacrylamide nanoparticles are effective formulations be used as nanodevices for various cystis brosis diseases [67].
for enhancing its stability and improve targeting possibilities [45]. Despite the high sequence diversity, many proteins and peptides
LHRH-loaded copolymerized peptide particle systems (mean size aggregate into a common cross--sheet structure, and the resulting
of 100 nm) administered orally show a half-life of LHRH in blood of brils show remarkable ultra-structural and biophysical similarity.
12 h than normal 28 min [45]. Calcitonin, a peptide secreted by the These assemblies belong naturally to the realm of the nanoscale.
parathyroid gland of the human body has been encapsulated into The study of their properties and mechanisms of formation might
polyacrylamide nanospheres, PIBCA nanocapsules and chitosan be a source of inspiration for the development of ordered, rationally
nanoparticles [68]. This encapsulated calcitonin greatly decreases designed nanostructures with potentially interesting applications
the level of ionized calcium in blood than simple calcitonin on in biotechnology and other elds (Fig. 2). Normal proteins are engi-
oral administration [25]. Cyclosporine was also encapsulated in neered to self assemble into nanodevice and thus self assembly at
PIHCA by interfacial and emulsion polymerization. This thera- nanoscale is important for the fabrication of novel supramolecu-
peutic peptide is a cyclic nonribosomal peptide produced by the lar structure, having applications in the eld of nanotechnology
fungus Hypocladium inatum, initially isolated from a Norwegian and nanomedicines. Peptides represent the most favorable build-
soil sample. The nanoparticle formulation had a notably increased ing block for the design and synthesis of nanostructures because
bioavailability compared with that of the commercial formulation they offer a great diversity of chemical and physical properties.
[45]. They can be synthesized in large amounts, can be modied and
Poly (N-isopropyl acrylamide) (PNIPAm) undergoes a sharp decorated with functional elements for diverse nanotherapeutic
coil globule transition in water at 32 C. It is being hydrophilic applications. Self assembly of polypeptide in nature such as amy-
below this temperature and hydrophobic above it. PEG coat- loid brils is associated with human medical disorder. Structural
ing on encapsulated therapeutic proteins have been shown to element as short as dipeptides can form well ordered assemblies at
stabilize the protein without losing their biological function the nanoscale [95]. Many novel building blocks that are based on
on PNIPAm nanoparticles [90]. Amine functionalized polymeric either natural or synthetic amino acids have capability to develop
nanoparticles is being used to enhance charge directed target- linear or cyclic congurations. The diversity of the side chains of
ing of lysozyme nanoparticles conjugates to bacteria. Activity of amino acids enables the control of both conformation and function-
lysozyme conjugated to positively charged PNIPAm nanoparticles ality. A number of investigator used antigenantibody interactions
was approximately twice, while lysozyme conjugated to neg- and biotinavidin interactions to study the direct assembly of inter-
atively charged PNIPAm nanoparticles showed little detectable acted protein [101].
activity [104]. When calcitonin is incorporated in nanoparticles,
oral absorption is enhanced in rats and consequently calcium con-
5. Bioconjugation of peptide and protein on nanoparticles
centration in blood decreases compared to oral administration of
a calcitonin solution [25]. BSA was loaded on PNIPAm gels fabri-
Most of the therapeutic proteins and peptides are encapsulated
cated with higher encapsulation efciency (40%) in the presence of
by adsorption on the surface of nanocarriers. However, various
lower cross-linker contents. An incomplete release of encapsulated
biochemical conjugation reactions are advantageous for the ef-
BSA from the PNIPAm gels was observed in all cases. Enhanced mass
cient and controlled encapsulation of therapeutic proteins (Fig. 3).
transfer was created by oscillating swellingdeswelling in response
Furthermore, the activity of the protein polymer/nanoparticles
to temperature cycling across the LCST. This reveals that lowering
conjugate is closely regulated by altering the response stimulus
in vitro release and temperature did not promote BSA release due
of the synthetic polymer and the site of attachment to the pro-
to strong BSAPNIPAm gel interactions (Table 1) [127].
tein and peptides. These chimeric systems may thus be considered
as true molecular-scale devices for the development of delivery
4.3. Protein itself as nanoparticles
systems. The nanoparticles and specic ligands are activated by
incorporation of functional group (NH2 , SH, COOH,), and block-
Peptides serve as excellent building blocks for bionanotech-
ing of specic functional group (NH2 , SH, CHO, COOH,). These
nology owing to the ease of their synthesis, small size, relative
functionalized nanoparticles and its complementary functionalized
high stability and chemical/biological modiability. Understand-
ligands are bioconjugated by functional group specic bioconju-
ing the physicochemical determinants that underlie peptide self
gation reaction. A variety of bioconjugation reaction are used like
assembly is a fundamental step in view of the rational design of
maleimide reaction, EDC/NHS chemistry, afnity interactions, click
new nano building blocks for biotechnological applications or new
chemistry, streptavidin biotin reaction etc for the covalent conju-
drugs (Fig. 1). A large number of nonpathogenic and pathogenic
gation of specic ligands on functionalized nanoparticles (Table 2)
peptide have been shown to form ordered brils under particular
[49].
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). 5.1. Maleimide reactions
Three classes of protein components as planar crystalline arrays,
engineered proteins pores and molecular motors are frequently The maleimide reaction is well known for the bioconjuga-
used for development of protein nanodevices. The surface-layer (S- tion of protein containing free thiol group on nanoparticles.
layer) proteins having square or hexagonal symmetry of 330 nm Thiol groups of proteins or functionalized nanoparticles (GNPs
unit cells with 510 nm thickness are one of the good examples of etc.) react with maleimide functionality to form thioether bond
planar protein crystalline arrays. Several therapeutic applications (Fig. 3A). In addition, thiol-maleimide based couplings are known
have been suggested for S-layer as nanoscale immobilization matri- to proceed efciently in solution, with site specic attachment
ces. This includes S-layer streptavidin fusion nanodevice and IgG of maleimide groups on Fc region of antibodies [49]. It is more
binding domain based high density adsorbent for extracorporeal benecial to incorporate this functionality on the biomacro-
blood purication. This is reported to have 20 times higher capac- molecule than the nanoparticles, because nanoparticles require
ity to remove autoantibodies [102]. The protein nanopores such several steps of purication in aqueous solution by dialysis over
as -hemolysin (HL) are developed as medical analytes for single a longer period of time. Maleimide functionalized bovine serum
molecule level by stochastic sensing [11]. Various molecular motors 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 the polymer. The resultant polymerprotein conjugate displayed
cross linked knedel-like (SCK) nanoparticles [82]. The particle low critical solution temperature (LCST) behavior and could be
size was increased from 20 nm to 30 nm after encapsulation of reversibly precipitated from solution by variation in temperature.
BSA on SCK nanoparticles with >5060% encapsulation efciency. This approach has proved to be very versatile and a large num-
Pioneering work has been carried out by Hoffman, Stayton and ber of nanopolymer protein conjugates incorporating biological
co-workers for the bioconjugation of peptide with nanoparticles components like antibodies, protein A, streptavidin, proteases and
[15]. They engineered single cysteine via site-directed mutagene- hydrolases have now been prepared [50]. Small changes in envi-
sis a mutant of cytochrome b5. This is accessible for reaction with ronmental conditions can then cause large changes in the polymer
maleimide end-functionalized poly-N-isopropylacrylamide (PNI- conformation, leading to reversible blocking or unblocking of
PAm). Since the native cytochrome b5 does not contain any cysteine the proteins active site; such changes also can lead to triggered
residues this substitution provided a unique attachment point for 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 Nanocarriers Speciality Ref.

Maleimide coupling BSA t-BA Protein activity retained [82]


Adsorption BSA Fe Biological activity retained [66]
Lysozyme Au Bioactivity of the lysozyme should be retained [130]
EDCNHS Chemistry Chymotrypsin Fe Thermal stability was improved [51]
Anti-E. coli antibody Au Suitable for antigenantibody interactions [86]
Monoclonal 19 Au Facile identication of cancer tissue [33]
Afnity interactions Horseradish peroxidase NTA-GNP Specic immobilization of His-tagged proteins [1]
His-Tag proteins Ni Suitable for purication of His-tag proteins [88]
Click chemistry F3-peptide Fe Good for targeted delivery [135]
Lipase Au Enzyme activity was retained [14]
Bombesin peptide Fe Good for targeted delivery [75]
Streptavidinbiotin interactions Biotin Fe High afnity for the Cy3-labeled streptavidin [19]
Biotin PLGA Specic binding of biotinylated NP to a [126]
NeutrAvidin (NAv)-coated surface
Electrostatic interactions BSA Folate-PEG-TMC Suitable for intracellular transport of [136]
negatively charged therapeutic proteins into
folate receptor over-expressing tumor cells

logical functions or activities of these conjugate systems are fully on polyacrylamide-coated Fe3 O4 nanoparticles synthesized by
similar to their native counterparts, but they are switched on or photochemical in situ polymerization [53]. Mean particle size
off as a result of thermally induced polymer phase transitions of the immobilized enzyme was 31 nm and super paramagnetic
[106]. Lactoferrin-conjugated PEGPLA nanoparticles synthesized properties of Fe3 O4 were retained after enzyme immobilization.
by thiolated reaction containing 55 molecules per nanoparticles The wheat germ agglutinin (WGA)-conjugated PLGA nanoparticles
showed an improved brain delivery [107]. Lactoferrin engineer- loaded with paclitaxel and isopropyl myristate has been prepared
ing signicantly increased the brain uptake of drugs associated to by two-step carbodiimide method to use it as an anticancer agents
nanoparticles following an intravenous administration. Thiol group (Table 2) [31]. WDA-conjugated PLGA nanoparticles had lower
is an attractive functionality to have on the surface of nanoparticles yields and encapsulation efciency because they require more
or protein, since reversibly linked bioconjugates can be prepared by purication steps.
disulde formation, which may allow reductive release of a target-
ing or therapeutic component [13]. 5.3. Afnity interactions

5.2. EDCNHS bioconjugation An alternative approach taking advantage of afnity interactions


has been extensively used for bioconjugation of recombinant pro-
EDC/NHS bioconjugation is frequently used for the bioconju- teins on Ni-NTA coated nanoparticles. This approach exploits the
gation of peptide on variety of nanoparticles. Carboxyl groups interaction between divalent metal ions such as nickel, copper, or
present on the surface of nanoparticles are activated by using cobalt and a short sequence of histidine residues (410 repeating
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)) units) added to the N- or C-terminus of the protein (histidine-tags)
(N-hydroxysulfosuccinimide) (EDC/NHS) to form reactive sulfo- (Fig. 3C). These purication methods generally involve immobi-
NHS ester. This ester reacts with amine functionality of proteins lizing the metal ion onto the column packing material using a
via formation of amide bond (Fig. 3B). When working with pro- chelating agent such as nitrilotriacetic acid (NTA). In the case of
teins and peptides, experience indicates that EDC-mediated amide Cu2+ and Ni2+ which have six coordination sites, NTA forms a strong
bond formation effectively occurs between pH 4.5 and 7.5. Beyond complex with four of the metal sites, leaving two additional sites
this pH range, however, the coupling reaction occurs more slowly for interaction with the His-tag present on the protein. His-tag
with lower yields. The presence of both carboxylates and amines HIV-1 Gag p24 protein was coupled to DOGSYNTAYNi nanoparti-
on the molecules to be conjugated with EDC may result in self- cles utilizing the above approach [88]. The advantages of afnity
polymerization. This is because the substance can react with interactions between divalent Ni nanoparticles are effective for
another molecule of its own kind instead of the desired tar- enhancing its interaction with His-tag proteins. The stronger afn-
get. PLGA nanoparticles have been conjugated to Anti-Fas mAb ity of antigen with the Ni-NPs resulted in superior humoral immune
and IgG using EDC/NHS chemistry [76]. Variations in surface car- responses in vivo compared to protein adjuvant with alum [88]. Fol-
boxyl density permitted up to 48.5 g coupled Ab per mg of NP. lowing this method various metallic nanoparticles have been used
The amount of anti-Fas mAb that could be conjugated to the NP for the attachment of protein molecule by polymeric hydrophilic
using two-step carbodiimide activation was dependent on the nanolayer surface modication (Fig. 3C).
ratio of non-end-capped to end capped PLGA types. In addition, it
was observed that anti-Fas conjugation was not affected by drug 5.4. Click chemistry
loading. The model proteins like BSA, streptavidin or goat anti-
rabbit immunoglobulin G (IgG) are also bioconjugated on magnetic Click chemistry is one of the most useful reactions for the
nanoparticles with carboxylic or aminopropyltrimethoxysilane bioconjugation of proteins on the nanoparticles. This reaction is
(APTS) groups using carbodiimide as a zero-length cross-linker based on the reaction between alkyne moiety and azide moiety
[48]. The average hydrodynamic diameters of the nanoparticles to from triazole ring. Beauty of this reaction is that alkyne moiety
were about, 154 nm and 165 nm and the conjugation yield of attached to the ligands reacts only with azide moiety of nanoparti-
proteins were very low. 3-mercaptopropyl acid-stabilized CdTe cles avoiding other side products (Fig. 3D). Cyclic tumor-targeting
quantum dot nanoparticles are conjugated with peptides or pro- peptide LyP-1 (CGNKRTRG) containing thiol and amine groups was
teins (active) mediated by N-hydroxysulfo-succinimide (NHS) or bio-conjugated on polymer-coated magnetouorescent nanopar-
1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride ticles by applying click chemistry [123]. Click nanoparticles are
(EDC) [137]. The enzyme -chymotrypsin was nanoencapsulated able to stably circulate for hours in vivo following intravenous
S.C. Yadav et al. / Peptides 32 (2011) 173187 183

administration (>5 h circulation time), extravasate into tumors, (TAT) from HIVI, third helix of antennapedia homeodomain and
and penetrate the tumor interstitium to specically bind p32- VP22 protein of herpes simplex virus. These methods are used to
expressing cells in tumors. An acetylene-functionalized lipase has improve the internalization of peptide drugs due to their unique
been attached to azide-functionalized water-soluble gold nanopar- potential to enter the cells in culture when added exogenously [79].
ticles in a copper-catalyzed 1,2,3-triazole cycloaddition with full Though these carriers systems have potential to deliver protein into
enzymatic activity [14]. Seven fully active lipase molecules are the cells but, many of them have shown inefcient delivery for the
attached to each nanoparticle. Staudinger ligation reaction work protein therapeutics to their actual target site. In these strategies,
similarly as click chemistry but the reacting moieties are different. It a number of other problems, such as complex manipulation, cel-
is used to ligate modied phosphine and an azide to form an amide lular toxicity and immunogenicity are reported [79]. However, the
bond. The basis of this reaction is the hydrolysis of amidophos- encapsulation of protein and peptides drugs on suitable nanocarri-
phonium intermediate in water. The ligand can be readily attached ers is one of the emerging techniques with tremendous potential to
either before assembly of the complexes (precomplexation) or reduce the problem (stability, degradation, proteolysis etc.) associ-
to the assembled nanoparticle (postcomplexation) by using this ated with protein and peptide delivery. In addition, encapsulation
bioconjugation methods. The PEGylated polyamidoamine DMEDA- of proteins and peptides in nanocarriers in physiologically active
PEG-DMEDA-(MBA-DMEDA)n + 1 -PEG-DMEDA was attached with form provides resistant to organic solvents, moisture, acidic envi-
integrin-binding peptide ligand via the Staudinger ligation [85]. ronment, enzymatic degradation, and immunological elimination.
Postcomplexation strategy led to small and discrete toroidal This technique is being widely accepted practical approach for the
nanoparticles whilst the precomplexation particles showed loose development of protein and peptide nanotherapeutics [28].
complexes (Table 2). 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-
5.5. Streptavidinbiotin reaction
face chemistry of nanomaterial on structure, activity, and stability
of nanoparticles conjugated proteins. The study of lysozyme and
Streptavidin is a tetrameric protein which binds very tightly
human carbonic anhydrase adsorbed on silica nanoparticles reveals
to a small water-soluble molecule biotin. The specicity of biotin
the change in structure, activity and loss of a helical contents
binding to streptavidin provides the basis for developing a bioas-
depending upon the size of nanoparticles [71]. This nding sug-
say system to detect or quantify analytes. More importantly, both
gests that the smaller nanoparticles are advantageous for protein
streptavidin and biotin are effectively conjugated to other pro-
stability due to higher surface curvature for the protein nanoen-
teins or labeled with various detection reagents without loss of
capsulation. The retention of protein structure on nanoparticles
their binding afnity (Fig. 3E). The coupling reaction with biotin-
is also protein dependent. It is also reported that SWCNTs (sin-
(poly(ethylene glycol))amine and polymeric PLGA nanoparticles
gle walled carbon nanotubes) stabilize the protein under harsh
has been used for the conjugation of various protein and peptides
conditions like high temperature, organic solvents etc [7]. The
[126]. Biotin binding proteins (avidin, streptavidin, or neutravidin)
surface of nanoparticles also provides the control over the struc-
have been used as cross linkers to conjugate proteins on biodegrad-
ture and function of adsorbed chymotrypsin [52]. The disruption
able nanoparticles (PLGAPEGbiotin). Avidin gave the highest
of proteinprotein interaction using surface-functionalized gold
level of overall protein conjugation, whereas neutravidin is known
nanoparticles is reported with cytochrom c and cytochrome c per-
to minimize non-specic protein binding to the polymer [116].
oxidase [12].
Cellular localization of protein encapsulated on nanoparticles
6. Nanoencapsulation improves the therapeutic is signicantly different from those of small-molecule probes and
importance of proteins and peptides is extremely sensitive to the surface charge of nanoparticles. The
nanoparticles are localized in lysosome, nucleus, endosome in ani-
The foremost aim of the protein and peptide drug delivery sys- mal cells. Oleylated QDs are localized mainly in the cell membrane
tems is the interaction or internalization of protein and peptides [105]. However, coumarin-six loaded chitosan/GMO formulation is
drug into target cells. The encapsulation of these drugs on the suit- localized in nuclear material in MDA-MB-231 cells [117]. The appli-
able nanocarrier is the rst step in the development of targeted cation of carbon-coated magnetic iron nanoparticles in pumpkin
peptide delivery. The desired feature of these efcient drug delivery by pulverization induces the distribution of these nanoparticles to
systems is to deliver therapeutic peptides to active site at the right parenchyma, xylem vessels, cortex and epidermis of the plant cells.
time in a therapeutically effective concentration and at highest This observation provides some highlights in the cellular move-
patient convenience/compliance, with minimal side effects and low ment of nanoparticles in vascular plant [21].
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. 7. The stability of nanoencapsulated peptide and protein
Various proteases present at potential site of administration, may
inactivate and degrade these proteins therapeutics. The elimination The activity, structural composition and stability of protein
of these peptide drugs by body defense system is another major nanomaterial interaction depend on the type of nanomaterial and
reason for less utilization of protein and peptides therapeutics. protein itself. The protein stability may be inuenced/improved
These problems create the decline of bioavailability and increases by nanoparticles interaction. The protein stabilization agents, such
potential immune response against the protein therapeutics. The as gelatin, BSA and other low-value proteins (for protein-based
alternative route of administration of the peptides therapeutics like stabilization), mannitol (to regulate the osmotic pressure of the
pulmonary, transdermal, and nasal may be benecial than conven- medium), PVP, PEG, Pluronic F-68 etc are used for the improvement
tional route, towards the reduction of above mentioned problems of stability of nanoencapsulated proteins [79]. It is reported that
and increase in their bioavailability [20]. A variety of methods have fabricated amino acid functionalized gold nanoparticles modulates
been widely proposed to efcient protein delivery in vivo as well the activity of the chymotrypsin [7]. Electrostatic nanocomplexes
as in vitro for the delivery of protein and peptides therapeutics (100 nm) consisting of -lactoglobulin and pectin showed a very
[79]. The most efcient non nanotechnological methods for ef- good colloidal stability [138]. The hydrophilic peptide insulin for-
cient cellular delivery are transcriptional activator of transcription 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 Targeted cancer Commercial name

Polymerprotein conjugate Styrene maleic anhydride- Hepatocellular carcinoma Zinostatin/Stimalmer


neocarz-inostatin (SMAN
CS)
Polymerprotein conjugate PEG- l-asparaginase Acute lymphoblastic leukemia Oncaspar
Polymerprotein conjugate PEG-granulocyte Prevention of Neulasta/PEG lgrastim
colony-stimula-ting factor chemotherapy-associated
(G-CSF) neutropenia
Immunotoxin (fusion protein) IL 2 fused to diphtheria Cutaneous T-cell lymphoma Ontak (Denile-lukin diftitox)
toxin
Chemo-immunoconjugate Anti-CD33 antibody Acute myelogenous leukemia Mylotarg
conjugated to
calicheamicin
Radio-immunoconjugate Anti-CD20 conjugated to Relapsed or refractory, low-grade, Zevalin
yttrium-90 or indium-111 follicular, or transformed
non-Hodgkins lymphoma
Anti-CD20 conjugated to Bexxar
iodine-131
Immunotoxins, immunopolymers Various drugs and toxins Various type of cancer Clinical trials phage III
and fusion proteins (315 nm)

by the formation of a phospholipid complex [25]. Inuenza HA drainage in tumors provides the permeability of <200 nm nanopar-
loaded PLGA nanoparticles reveals that the encapsulated proteins ticles in the tumor cells [24]. Targeting cancer with mAb and
are stable inside the nanoparticles and does not degrade dur- its feasibility has been clinically demonstrated. Over 17 different
ing the production process as well as on long term storage [94]. mAb are approved by the US Food and Drug Administration (FDA)
Zhu et al., recently reported that combined physical and chem- for cancer treatment. The rituximab (rituxan) and trastuzumab
ical immobilization of glucose oxidase in alginate microspheres (herceptin binds to ErbB2 receptor) mAb was used for non-
improves stability of encapsulation and activity [137]. The reten- Hodgkins lymphoma and breast cancer treatment respectively [4].
tion of alcohol dehydrogenases (ADH) activity was dependent An antiVEGFmAb as angiogenesis inhibitor for treatment of colorec-
on the concentration of ADH in the feed solution. However, the tal cancer Bevacizumab (Herceptin) was approved in 2004 [38].
addition of other proteins, such as bovine serum albumin and Over 200 delivery systems based on antibodies encapsulated on
-lactoglobulin, exhibited an additional improvement of the reten- nanoparticles or their fragments are in preclinical and clinical tri-
tion of ADH activity. The inlet and outlet temperature of the drying als [112]. The tissue rejection could be minimized by using the
air was another key factor for the enzyme activity on the spray specic protein coated nanoparticles. This nanoparticle reduces
drying method of protein encapsulation [131]. Biomimetic silica the chances of rejection as well as to stimulate the production
entrapment of chemically derivatized horseradish peroxidase for of osteoblasts. One of the most exciting applications of protein
amperometric sensing applications shows very high stability in nanoparticles in tissue engineering and regeneration are the abil-
comparison to nanoencapsulated native enzymes [133]. Itoh et al., ity of these nanostructures to provide a permissive environment
group has reported the similar activity and much higher stability for axonal regeneration in the central nervous system (CNS) after
(four times) of catalase upon encapsulation in mesoporous silica injury. When the peptide scaffold was applied to damage optic
synthesized in the pores of an alumina membrane (Table 1) [54]. tracts in hamsters, regenerated axons could reconnect, and target
tissues with sufcient density as to enable the functional return of
vision [36].
8. Therapeutic use of the nanoprotein molecules

Proteomic technology to choose appropriate targets is an active 9. Conclusion


area of research. However, till date no clinically effective targets
have been identied. The fusion proteins, engineered antibody, The therapeutic peptides have more potential for the dis-
dimerization, detection of specic conformation of target receptors ease diagnosis, cure and have minimal side effect than the
together with nanocarriers encapsulation provides novel strategies small-molecule drugs. Various peptides of therapeutic poten-
to cure target cells. The use of peptide as targeting agents results tial are well known and characterized but their therapeutic
in increased intracellular drug delivery in different murine tumor use is restricted due to less functional and structural stability
models (Table 3). Likewise, several protein and peptide encap- and susceptibility to proteolysis. The denaturation and rst pass
sulated nanosystems with different compositions and biological metabolism before reaching the target are other major prob-
properties have been extensively investigated for drug and gene lems prior use as therapeutic medicines. The eld of protein
delivery applications [91]. therapeutics has achieved the exponential growth along with
The small size of nanoparticles was used to spy at the cellu- nanotechnology. Insulin provides the major attention for the
lar machinery level with least interference in function. Protein future development of peptides as future medicine. FDA has
nanotechnology have potential to use in the study of immuno- approved >130 peptide for therapeutic and diagnostic applications.
complex (antigenantibody) formation, drug and gene delivery, At present, more than 800 different antimicrobial peptides (list
biodetection of pathogen, detection of proteins, probing of DNA at http://www.bbcm.units.ittossi/pag1.htm) have been described
structure, tissue engineering, separation and purication of bio- from invertebrates, plant and animal species). The suitable
logical molecules and cells etc. These peptide nanomedicines nanoencapsulation of various potential therapeutic peptides on
reduce the frequency of drug administration, and improved patient biodegradable and biocompatible nanocarriers eliminates the pos-
compliance. This would offer the protection and improved phar- sibilities of degradation, structural and functional deformities and
macokinetics of easily degradable, short half-life of protein in vivo metabolism before reaching the target sites. The nanoencapsula-
and in vitro conditions. Leaky blood vessels and poor lymphatic tion of these peptides are the rst step towards the development
S.C. Yadav et al. / Peptides 32 (2011) 173187 185

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