chitosan_a versatile semi-synthetic polymer in biomedical

Progress in Polymer Science 36 (2011) 981–1014

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Progress in Polymer Science

journa l homepage: www.e lsev ier .com/ locate /ppolysc i

Chitosan—A versatile semi-synthetic polymer in biomedicalapplications

M. Dasha, F. Chiellini a, R.M. Ottenbriteb, E. Chiellini a,∗

a Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab), UdR INSTM, Department of Chemistry and IndustrialChemistry, University of Pisa, Pisa, Italyb Department of Chemistry, Virginia Commonwealth University, Richmond, VA, USA

a r t i c l e i n f o

Article history:Received 4 May 2010Received in revised form 21 October 2010Accepted 4 February 2011Available online 22 February 2011

Keywords:ChitosanTissue engineeringDrug deliveryGene therapyBioimaging

a b s t r a c t

This review outlines the new developments on chitosan-based bioapplications. Over thelast decade, functional biomaterials research has developed new drug delivery systemsand improved scaffolds for regenerative medicine that is currently one of the most rapidlygrowing fields in the life sciences. The aim is to restore or replace damaged body parts orlost organs by transplanting supportive scaffolds with appropriate cells that in combinationwith biomolecules generate new tissue. This is a highly interdisciplinary field that encom-passes polymer synthesis and modification, cell culturing, gene therapy, stem cell research,therapeutic cloning and tissue engineering. In this regard, chitosan, as a biopolymer derivedmacromolecular compound, has a major involvement. Chitosan is a polyelectrolyte withreactive functional groups, gel-forming capability, high adsorption capacity and biodegrad-ability. In addition, it is innately biocompatible and non-toxic to living tissues as well ashaving antibacterial, antifungal and antitumor activity. These features highlight the suit-ability and extensive applications that chitosan has in medicine. Micro/nanoparticles andhydrogels are widely used in the design of chitosan-based therapeuticsystems. The chemi-
cal structure and relevant biological properties of chitosan for regenerative medicine havebeen summarized as well as the methods for the preparation of controlled drug releasedevices and their applications.
© 2011 Elsevier Ltd. All rights reserved.

Abbreviations: AL, alginate; ASGPR, asialoglycoprotein receptor; RGD, arginine–glycine–aspartic acid; BAL, bioartificial liver; BMP, bone morphogeneticprotein; CP, calcium phosphate; CPC, calcium phosphate cement; CSF, colony-stimulating factor; DD, degree of deacetylation; DCs, dendritic cells; DTPA,diethyl triamine penta acetic acid; EDC, 1-ethyl-3-[3-imethylaminopropyl]carbodiimide hydrochloride; EGFP, enhanced green fluorescent protein; ECM,extra cellular matrix; FGF-2, fibroblast growth factor-2; FRET, fluorescence resonance energy transfer; FHF, fulminant hepatic failure; Gd, gadolinium;GC, galactosylated chitosan; GDNF, glial cell line-derived nerve growth factor; GP, glycerophosphate; GAGs, glycosamine glycans; GM-CSF, granulocyte-macrophage colony-stimulating factor; GTR, guided tissue regeneration; hGH, human growth hormone; hUCMSCs, human umbilical cord mesenchymalstem cells; HA, hydroxyapetite; HEC, hydroxyethyl cellulose; IBL, implantable bioartificial liver; 131I-NC, 131I-norcholesterol; IL, interleukin; IPN, interpen-etrating network; ILs, ionic liquids; LCST, lower critical solution temperature; MRI, magnetic resonance imaging; MSCs, mesenchymal stem cells; NHS,N-hydroxysuccinimide; NCT, neutron-capture therapy; pDNA, plasmid DNA; PAA, poly(acrylic acid); PEC, polyelectrolyte complex; PEO, polyethylene oxide;PEI, poly(ethylenimine); PVP, poly(vinyl pyrrolidine); PNIPAM, poly(N-isopropylacrylamide); PVA, poly vinyl alcohol; RES, reticuloendothelial system; RII,retrograde intrabiliary infusion; RTILs, room temperature ionic liquids; RWM, round window membrane; SCs, Schwann cells; TPP, sodium tripolyphosphate;SPIOs, super paramagnetic iron oxide; SBF, synthetic body fluids; TCP, tricalcium phosphate; TGF-�1, transforming growth factor �1; TEM, transmissionelectron microscopy; TAA, triamcinolone acetonide; UV, ultra-violet; WSC–LA, water-soluble chitosan–linoleic acid; XRD, X-ray diffraction.

∗ Corresponding author. Tel.: +39 050 2210301/2/3; fax: +39 050 2210332.E-mail address: [email protected] (E. Chiellini).

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982 M. Dash et al. / Progress in Polymer Science 36 (2011) 981–1014

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9822. General aspects of chitosan-structural and functional features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983

2.1. Structure, source and physicochemical properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9832.2. Structure–property relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9832.3. Biodegradability of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985

2.3.1. Chitosan in-vitro biodegradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9852.3.2. Chitosan in vivo biodegradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985

2.4. Biological properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9852.5. Chitosan toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986

2.5.1. In-vitro toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9862.5.2. In vivo toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986

3. Chitosan-based systems for biomedical applications – types and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9873.1. Chitosan micro/nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 987

3.1.1. Emulsion cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9873.1.2. Coacervation/precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9883.1.3. Spray-drying. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9883.1.4. Emulsion-droplet coalescence method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9893.1.5. Ionic gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9893.1.6. Reverse micellar method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9893.1.7. Sieving method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9903.1.8. Chitosan micro/nanoparticles – drug loading and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991

3.2. Chitosan hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9913.2.1. Physical association networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9923.2.2. Cross-linked networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9933.2.3. Chitosan hydrogels – drug loading and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994

4. Biomedical–pharmaceutical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9954.1. Chitosan for tissue engineering applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995

4.1.1. Chitosan in bone tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9954.1.2. Chitosan in cartilage tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9964.1.3. Chitosan in liver tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9974.1.4. Chitosan in nerve tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999

4.2. Chitosan in wound-healing applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10004.3. Chitosan in drug delivery applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000

4.3.1. Chitosan-based systems for the delivery of anti-cancer drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10004.3.2. Chitosan-based systems for the delivery of proteins/peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10004.3.3. Chitosan-based systems for the delivery of growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10014.3.4. Chitosan-based systems for the delivery of antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10014.3.5. Chitosan-based systems for the delivery of anti-infammatory drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10024.3.6. Chitosan-based systems for vaccines delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10024.3.7. Chitosan membranes in drug release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002

4.4. Chitosan in gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
4.5. Chitosan in bioimaging applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
. . . . .

. . . . .

. . . . .

dimensional properties, highly sophisticated functionalityand a wide range of applications in biomedical and otherindustrial areas [3–5]. They have become interesting notonly because they are made from an abundant renew-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006

able resource but because they are very compatible andeffective biomaterials that are used in many applica-tions [6–8]. Chitosan is a linear copolymer of �-(1–4)linked 2-acetamido-2-deoxy-�-d-glucopyranose and 2-amino-2-deoxy-�-d-glycopyranose (Fig. 1). It is obtainedby deacetylation of its parent polymer chitin, a polysaccha-ride widely distributed in nature (e.g. crustaceans, insectsand certain fungi) [9,10]. Due to chitin’s poor solubilityin aqueous solution and organic solvents, it does not findpractical applications whereas chitosan as an artificial vari-ant of chitin is more suitable for useful bioapplications
[11]. The positive facets of excellent biocompatibility and
4.6. Chitosan in green chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . .5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

The history of chitosan dates back to the 19th century,when Rouget [1] discussed the deacetylated forms of theparent chitin natural polymer in 1859. During the past 20years, a substantial amount of work has been reportedon chitosan and its potential use in various bioapplica-tions. Chitosan is derived from naturally occurring sources,which is the exoskeleton of insects, crustaceans and fungithat has been shown to be biocompatible and biodegrad-able [2]. Chitosan polymers are semi-synthetically derivedaminopolysaccharides that have unique structures, multi-

admirable biodegradability with ecological safety and lowtoxicity with versatile biological activities such as antimi-crobial activity and low immunogenicity have providedample opportunities for further development [12–17].

M. Dash et al. / Progress in Polymer Science 36 (2011) 981–1014 983

F l atomsa le � defi

2f

2c

admogidegs2o[t

trifwshama[ouaf(

Chitosans, with the main structural differences beingrepresented by the relative proportions of N-acetyl-d-glucosamine and d-glucosamine residues, provide specificstructural changes. This difference in the structure givesrise to several batches of chitosan that are distinguished

ig. 1. (a) Chitosan structure; (b) chemical structure of chitosan. Individuangles (ϕ, ) defining the main chain conformation and one dihedral ang

. General aspects of chitosan-structural andunctional features

.1. Structure, source and physicochemical properties ofhitosan

Chitosan molecule is a copolymer composed of N-cetyl-d-glucosamine andd-glucosamine units available inifferent grades depending upon the degree of acetylatedoieties [18] (Fig. 1). It is a polycationic polymer that has

ne amino group and two hydroxyl groups in the repeatinglucosidic residue [19] (Fig. 1). The carbohydrate backbones very similar to cellulose, which consists of �-1,4-linked-glucosamine with a variable degree of N-acetylation,xcept that the acetylamino group replaces the hydroxylroup on the C2 position. Thus, chitosan is a copolymer con-isting of N-acetyl-2-amino-2-deoxy-d-glucopyranose and-amino-2-deoxy-d-glucopyranose, where the two typesf repeating units are linked by (1 → 4)-�-glycosidic bonds20]. After refinement, chitosan has a rigid crystalline struc-ure through inter- and intra-molecular hydrogen bonding.

The source of chitosan is a naturally occuring polymer,he chitin that is the second most abundant polysaccha-ide in nature, cellulose being the most abundant. Chitins found in the exoskeleton of crustacea, insects, and someungi. The main commercial sources of chitin are the shellaste of shrimps, lobsters, krills and crabs. In the world

everal millions tons of chitin are harvested annually andence this biopolymer represents a cheap and readilyvailable source [20–22]. Chitosan is obtained by the ther-ochemical deacetylation of chitin in the presence of alkali

nd naturally it occurs only in certain fungi (Mucoraceae)23]. Several alkaline methods have been proposed, most
f them involving the hydrolysis of the acetated positionsing sodium or potassium hydroxide solutions as wells a mixture of anhydrous hydrazine and hydrazine sul-ate [24]. The treatment of chitin with an aqueous 40–45%w/v) NaOH solution at 90–120 ◦C for 4–5 h results in
are numbered. Dashed lines denote O3–O5 hydrogen bonds. Two dihedralning the O6 orientation are indicated [19]. Copyright 2009, Elsevier Ltd.

N-deacetylation of chitin. The conditions used for deacety-lation determines the polymer molecular weight and thedegree of deacetylation (DD).

The active primary amino groups on the molecule beingreactive provide sites for a variety of side group attach-ment employing mild reaction conditions (Fig. 2). The facilederivatization makes chitosan an ideal candidate for bio-fabrication [17]. In addition, the characteristic features ofchitosan such as being cationic, hemostatic and insolubleat high pH, can be reversed by sulfating the amine whichmakes the molecule anionic and water-soluble, with theintroduction of anticoagulant properties [25]. The attachedside groups on chitosan provide versatile materials withspecific functionality, alter biological properties or modifyphysical properties.

2.2. Structure–property relationship

Fig. 2. Schematic illustration of chitosan’s versatility. At low pH (less thanabout 6), chitosan’s amine groups are protonated conferring polycationicbehavior to chitosan. At higher pH (above about 6.5), chitosan’s aminesare deprontonated and reactive.

olymer S
984 M. Dash et al. / Progress in P
on the basis of their DD and molecular weight. DD andmolecular weight directly affect the chemical and bio-logical properties of the polymer (Table 1). Commercialchitosan as sold by Sigma–Aldrich is available in two gradesof high and low molecular weight. Low molecular weightchitosan grade is characterized by molecular weight com-prised between 20 kDa and 190 kDa with DD < 75%. Thehigh molecular weight chitosan grade is generally charac-terized by molecular weight comprised between 190 kDaand 375 kDa with DD > 75%.

The parent chitin is insoluble in most organic solvents,chitosan is readily soluble in dilute acidic solutions belowpH 6.0 due to the quaternisation of the amine groups thathave a pKa value of 6.3 making chitosan a water-solublecationic polyelectrolyte. The presence of the amino groupsindicates that pH substantially alters the charged state andproperties of chitosan [17]. At low pH, these amines getprotonated and become positively charged and that makeschitosan a water-soluble cationic polyelectrolyte. On theother hand, as the pH increases above 6, chitosan’s aminesbecome deprotonated and the polymer loses its chargeand becomes insoluble. The soluble–insoluble transitionoccurs at its pKa value around pH between 6 and 6.5. Asthe pKa value is highly dependent on the degree of N-deacetylation, the solubility of chitosan is dependent onthe DD and the method of deacetylation used [26]. Apartfrom the DD, the molecular weight is also an importantparameter that significantly affects the solubility and otherproperties [27–31].

The choice of chitin and its isolation process are also fac-tors that affect chitosan quality [32] in a significant way.There are three forms of chitin known �, �, �. Depend-ing on the origin of the polymer and its treatment duringthe extraction process, chitosan shows crystallinity andpolymorphism. Crystallinity is maximum for chitin (i.e.0% deacetylated) and fully deactylated chitosan (i.e. 100%deacetylated) [33]. A linear unbranched structure and highmolecular chitosan is an excellent viscosity enhancingagent in acidic environments and behaves as a pseudo-plastic material demonstrating a decrease in viscosity with
increasing rates of shear. The viscosity of chitosan solu-tion increases with an increase in the concentration ofchitosan, decrease in temperature and with increasingDD. Viscosity also influences biological properties such as
Table 1Relationship between structural parameters and properties.

Property Structural characterist

Solubility ↑ DDCrystallinity ↓ DDBiodegradability ↓ DD, ↓ Molecular weiViscosity ↑ DDBiocompatibility ↑ DD

BiologicalMucoadhesion ↑ DD, ↑ Molecular weiAnalgesic ↑ DDAntimicrobial ↑ DD, Molecular weighPermeation enhancing effect ↑ DDAntioxidant ↑ DD, ↓ Molecular weiHemostatic ↑ DD

a ↑ – Directly propotional to property; ↓ – inversely propotional to property.

cience 36 (2011) 981–1014

wound-healing properties and osteogenesis enhancementas well as biodegradation by lysozyme [34].

The understanding and control of the degradation rateof chitin and chitosan-based devices is of great interestsince degradation is essential in many small and largemolecule release applications and in functional tissueregeneration applications. Degradation has been shown toincrease as DD decreases [35–37]. It has also been observedthat the degradation kinetics seem to be inversely relatedto the degree of crystallinity which is controlled mainlyby the DD. The distribution of acetyl groups also seem toaffect biodegradability, the arrangement of acetyl groupsand their homogeneous distribution (random rather thanblock) results in very low rates of enzymatic degrada-tion [25,38]. However, several studies reported that thedegradation rate is affected by the length of the chains(Mw) as well [28,39,40]. Kofuji et al. investigated the enzy-matic behaviors of various chitosans by observing changesin the viscosity of chitosan solution in the presence oflysozyme and found that chitosan with a low DD tend tobe degraded more rapidly [41]. Differences in degradationdue to variations in the distribution of acetamide groups inthe chitosan molecule have been observed [38,42]. Appar-ently, different deacetylation conditions can influence theviscosity of the chitosan solution by changing the inter- andintramolecular repulsion forces [37]. Very fast degradationrates of chitosan cause an accumulation of amino saccha-ride that can produce an inflammatory response. Whilethe lower DD chitosans only induce an acute inflamma-tory response; the higher DD produces a minimal responsedue to the lower degradation rate. More specific infor-mation on the biodegradability of chitosan is providedlater in this review. The cytocompatibility of chitosanhas been observed in vitro with myocardial, endothelialand epithellial cells, fibroblast, hepatocytes, condrocytesand keratinocytes [29]. As the degree of deacetylation(DD) of the polymer increases, the interactions betweenchitosan and the cells increase due to the presence offree amino groups, consequently, cell adhesion and pro-liferation, as well as cell type, depend on DD. Other
biological properties, such as analgesic, antitumor, haemo-static, hypocholesterolemic, antimicrobial, and antioxidantproperties are also affected by the physical properties ofchitosan [6,43,44]. The dependence of the various poly-
icsa References

[17,26–31][33]

ght [25,28,35–42][41][29,45]

ght [39,46–49][50–53]

t [54,55][45,56–60]

ght [61–63][64–68]

olymer S

mT

2

faihsftteatEtagtdu

distetodiad

2

tmufTottbPcwwNsmcfiweem

M. Dash et al. / Progress in P

er properties on the structural parameters are listed inable 1.

.3. Biodegradability of chitosan

Biodegradation plays a major role in the metabolicate of chitosan in the body and is important to respectll polymers used in drug delivery systems and scaffoldsn tissue engineering. In case of systemic absorption ofydrophilic polymers, such as chitosan. For example, auitable molecular weight is required for renal clearancerom 30,000 to 40,000, depending on the polymer used. Ifhe administered polymer’s size is larger than this, thenhe polymer must undergo degradation. Both chemical ornzymatic biodegradation would provide fragments suit-ble for renal clearance. Chemical degradation is referredo acid catalyzed degradation such as in the stomach.nzymatically, chitosan can be degraded by enzymes ableo hydrolyse glucosamine–glucosamine, glucosamine–N-cetyl-glucosamine and N-acetyl-glucosamine–N-acetyl-lucosamine linkages [69]. Even though depolymerisationhrough oxidation–reduction reaction [70] and free radicalegradation [71] of chitosan have been reported these arenlikely to play a significant role in the in vivo degradation.

Chitosan is known to be degraded in vertebrates pre-ominantly by lysozyme and by certain bacterial enzymes

n the colon [72]. Eight human chitinases (in the glyco-ide hydrolase 18 families) have so far been identified,hree of which have shown enzymatic activity [73]. A vari-ty of microorganisms synthesises and/or degrades chitin,he biological precursor of chitosan. Both rate and extentf chitosan biodegradability in living organisms are DDependent, with a decrease in the degradation rate with

ncreasing DD [74,75]. Basically, given adequate time andppropriate conditions, chitosans, in most cases wouldegrade sufficiently to be excreted [69].

.3.1. Chitosan in-vitro biodegradationViscometry and/or gel permeation chromatography are

he commonly used assays to determine the decrease inolecular weight [76]. Following an in vitro incubation

sing lysozyme at pH 5.5 in a phosphate buffer at 37 ◦Cor 4 h, 50% acetylated chitosan lost 66% in viscosity [76].his degradation is DD dependent with the degradationf the higher acetylated chitosan behaving more like chi-osan [77,78]. In addition, a range of proteases was foundo degrade chitosan films, with leucine amino-peptidaseeing the most effective with 38% over 30 days [65].ectinase isozyme from Aspergilus niger has also indicatedhitosan digestion at low pH releasing lower moleculareight fragments [79,80]. From the digestion of chitosanith rat cecal and colonic bacterial enzymes, Zhang andeau observed that extracellular enzymes were respon-

ible for degradation that was related to both DD andolecular weight [78]. McConnell et al. used human fae-

al preparations and studied the degradation of chitosan
lms, cross-linked by glutaraldehyde and tripolyphosphateith interesting results [81]. The rate of porcine pancreatic
nzymes to degrade chitosan films degradation was influ-nced cross-linker; for example, glutaraldehyde degradedore readily than tripolyphosphate.

cience 36 (2011) 981–1014 985

2.3.2. Chitosan in vivo biodegradationVery little has been reported on the in vivo degrada-

tion of chitosan. The mechanism of degradation is currentlyunclear, especially after intravenous injection. However,studies do indicate that distribution, degradation and elim-ination processes are strongly dependent on molecularweight. The liver and kidney were found to be the pos-sible sites of degradation inferred due to the localizationof chitosan. In rabbits, when injected intravenously, chi-tosan oligosaccharides enhanced lysozyme activity in theblood [82]. Oral administration of chitosan has shown somedegradation in the gastrointestinal tract. The digestion ofchitosan, occurring predominantly in the gut, was found tobe species dependent with hens and broilers being moreefficient digesters (67–98% degradation after oral inges-tion) than rabbits (39–83% degradation) [83]. In anotherstudy, of subcutaneous implantation of chitosan, a pro-posed skin substitute such as glutaraldehyde cross-linkedchitosan/collagen was found to be relatively stable overtime compared to collagen alone when implanted subcu-taneously in rabbits [84].

2.4. Biological properties of chitosan

Chitosan has proved to be a safe excipient in drug formu-lations over the last decades [85]. It has attracted attentionas an excellent mucoadhesive in its swollen state and a nat-ural bioadhesive polymer that can adhere to hard and softtissues. Good adhesion was found in epithelial tissues andin the mucus coat present on the surface of the tissues.A number of colloidal delivery systems based on chitosanhave been presented in the literature for the mucosal deliv-ery of polar drugs, peptides, proteins, vaccines and DNA.Clinical tests carried out using chitosan-based biomaterialsdo not report any inflammatory or allergic reactions follow-ing implantation, injection, topical application or ingestionin the human body [29]. The in vitro and in vivo cytocompat-ibility of chitosan films with keratinocytes and fibroblastshas demonstrated that DD has no significant influence. Thechitosan films with a low DD are very good biomaterials forsuperficial wound-healing [29]. Once placed on the wound,they adhere to fibroblasts and favor the proliferation ofkeratinocytes and thereby epidermal regeneration.

One of the upcoming areas of study related to chitosanis its biodistribution, especially using methods other thanintravenous administration. The distribution of chitosanformulates in the body is related to all aspects of the chi-tosan formulation from the molecular weight and DD tothe size of the delivery vehicle. For instance, in case of ananoparticulate formulations, the kinetics and biodistribu-tion will initially be controlled by the size and charge ofthe nanoparticles and not by chitosan structural features.However, after particle decomposition to chitosan and freedrug, inside the cells or target tissues, free chitosan will dis-tribute in the body and eliminate accordingly. Elimination
processes may be preceded by biodegradation. To under-stand chitosan biodistribution the kinetics of its labeled(radio or fluorescent) modifications should be followed,assuming that the label is neither labile nor affecting thephysicochemical properties of the chitosan [86].

olymer S
Banerjee et al. investigated the distribution of intra-venously injected 99mTc labeled nanoparticles (<100 nm)in Swiss albino mice. Radio-label stability of the nanoparti-cles was tested and 80% of the radioactivity was associatedwith the particles after 3 h. Nanoparticles were admin-istered in mice and an apparent scavenging played ofthe reticuloendothelial system (RES) was suggested asradioactivity decreased in organs of this system butremained stable in the blood after 1 h [87]. Unfortunately,the nanoparticles were not sufficiently stable to look atlong-term distributions. In a later study, by the sameauthor sodium borohydride was used in the preparationconditions. The modified method prevented aggregation,improved the kinetics of the nanoparticles and resulted inboth less blood clearance and liver accumulation i.e. avoid-ance of the RES. The radioactivity in blood was present forup to 10% even after 2 h [88].

In most of the studies liver was found to be a signifi-cant site of accumulation due to the action of Kepfer cells;this could be due to this organ being a primary site ofmetabolism as seen with radio-labeled dextran [89].

A suggestion was made by Kean and Thanou who pro-posed that a potential method to study native chitosanwithout significant modification would be to use 14C as alabel e.g. in the food source for the animal/fungi produc-ing the chitin so that the saccharide backbone is labeled, asdetection of native chitosan is appearing to be a challenge[69].

To study the intraperitoneal administration of chitosan,FITC-labeled chitosan (50% DD, 100 kDa) was prepared byFITC coupling. It was observed that this labeled chitosanwas completely absorbed form the peritoneal cavity (noevidence in abdominal fluid after 14 h). FITC-chitosan wasfound to be predominantly localised in the kidney at 1 h ina mouse model. There was a rapid renal excretion rate (25%at 1 h, 100% in 14 h) with evidence of degradation due to adecrease in the molecular weight [76].

Some studies suggest that chitosan binds fat andreduces cholesterol but the mechanism, is still some-what questionable [43,90]. Apart from the effect thatchitosan may have on bile salts and gastrointestinalmilieu, the uptake of chitosan into the bloodstream isgenerally not investigated in oral administration studies.Molecular weight has been a parameter that largely influ-ences chitosan’s systemic absorption and distribution fromthis route of delivery. It has been seen in some casesthat oligomers showed some absorption whereas largermolecular weight chitosans were excreted without beingabsorbed. This effect was investigated using FITC-labeledchitosans with 3.8 kDa (88.4% DD) which was shown toresult into the greatest plasma concentration after oraladministration compared to 230 kDa (84.9% DD) for whichnearly no uptake was detected. In one of the studies aimedat investigating plasma concentration after oral admin-istration, the increasing molecular weight was seen todecrease the plasma concentration [91].

Although intracellular uptake and distribution of nativechitosan have not been investigated, but chitosan/DNAcomplexes have been studied in vitro [92–94]. Chitosanpolyplex uptake at 37 ◦C was 3-fold higher than at 4 ◦Cwhich could be due to increased interaction and not an

cience 36 (2011) 981–1014

ATP dependent endocytotic mechanism [92]. The authorssuggested nuclear localization and they also stated littledissociation of the DNA from the chitosan. In a more exten-sive study, Leong et al. stained lysosomes and found someco-localization with chitosan DNA nanoparticles. However,the majority of the polyplexes were found in the cytosol[93]. A complex of doxorubicin with chitosan was studied;where is was observed that complexes enter cells throughan endocytotic mechanisms which was not further eluci-dated [95].

2.5. Chitosan toxicity

Chitosan is considered as being a non-toxic, biologi-cally compatible polymer [96]. It is approved for dietaryapplications in Japan, Italy and Finland [97] and it has beenapproved by the FDA for use in wound dressings [98]. How-ever, certain modifications implemented on chitosan couldmake it more or less toxic and any residual reactants shouldbe removed carefully.

2.5.1. In-vitro toxicityIn a series of studies, Schipper et al. observed the effects

of chitosan samples characterized by different molecularweight and DD on CaCo-2 cells, HT29-H and in situ ratjejunum. Toxicity was found to be dependent on DD andmolecular weight. At high DD the toxicity is related to themolecular weight and the concentration, at lower DD tox-icity is less pronounced and less related to the molecularweight. Nevertheless, most of the chitosans tested did notincrease dehydrogenase activity significantly in the con-centration range tested (1–500 �g/ml) on Caco-2 cells. Thein situ rat jejunum study showed no increase in LDH activitywith any of the chitosan samples tested (50 �g/ml) [45,99].Red blood cell haemolysis assay is a study that revealssafety of materials. No haemolysis was observed (<10%)over 1 h and 5 h with chitosans of <5 kDa, 5–10 kDa and>10 kDa at concentrations of up to 5 mg/ml [100]. Further-more, no red blood cell lysis was observed with paclitaxelchitosan micelles at 0.025 mg/ml [101].

Interestingly, chitosan and its derivatives seem to betoxic to several bacteria [102], fungi [82] and parasites[103]. This pathogen related toxicity is an effect whichcould be beneficial in the control of infectious disease. Bac-terial inhibition took place in acidic solutions pH 5–5.3, anda 87 kDa 92% DD chitosan was more effective than a 532 kDa73% DD chitosan against both Pseudomonas aeruginosa andStaphylococcus aureus. Antimycotic effect against Candidaalbicans and Aspergillus niger was observed in a lipid emul-sion of the same chitosans [102]. However, none of thesestudies proposed a mechanism of action for the observedinhibitory effect.

2.5.2. In vivo toxicityIn a study by Hirano et al. which was relatively

long (65 days), no detrimental effect on body weight
was found when chitosan oligosaccharides were injected(7.1–8.6 mg/kg over 5 days). An increase in lysozyme activ-ity was apparent on the first day post injections [104].
Rao et al. stated no “significant toxic effects” of chitosanin acute toxicity tests in mice, no eye or skin irritation in

olymer Science 36 (2011) 981–1014 987

rtn[siighfnaAsotnccaa

3a

tdkotmiac

3

itpvsSo

Table 2Chitosan-based drug delivery systems prepared by different methods.

Type of system Method of preparation

Tablets Matrix coatingCapsules Capsule shellMicrospheres Emulsion cross-linking

Coacervation/precipitationSpray-dryingIonic gelationSieving method

Nanoparticles Emulsion-droplet coalescenceCoacervation/precipitation


abbits and guinea pigs respectively. He also concluded inhe same study that chitosan was not pyrogenic. However,o concentration or DD of the chitosan used was noted65]. No detrimental effects were also noted by Richard-on et al., even though there was mention of dose effectn his work [100]. The LD50 of paclitaxel chitosan micellesn mice was 72.2 mg/kg, no anaphylaxis was observed inuinea pigs and no intravenous irritation was observedistopathologically in rabbits at 6 mg/kg [101]. In a study on

at chelation, 4.5 g/day chitosan (molecular weight and DDot specified) in humans was not reported not to be toxic,lthough no significant reduction in fat was found [105].rai et al. found that chitosan has an LD50 comparable toucrose of >16 g/kg in oral administration to mice [106]. Noral toxicity was found in mice treated with 100 mg/kg chi-osan nanoparticles (80 kDa, 80% DD) [107]. Exposure of ratasal mucosa to chitosan solutions at 0.5% (w/v) over 1 haused no significant changes in mucosal cell morphologyompared to control [108]. From most studies reported itppears that chitosan shows minimal toxic effects and thispproves its adoption as a safe material in drug delivery.

. Chitosan-based systems for biomedicalpplications – types and methods

Regenerative medicine offers the opportunity to dras-ically improve our ability to fight disease and to repairamaged organs of the human body. It combines thenowledge and skills of several disciplines towards the aimf addressing impaired function in the body. It is impor-ant to note that the goal is not just to replace what is

alfunctioning, but to provide the elements required forn vivo repair, to devise replacements that seamlessly inter-ct with the living body and to stimulate the body’s intrinsicapacities to regenerate [109].

.1. Chitosan micro/nanoparticles

Chitosan offers several advantages, and these includets ability to control the release of active agents and avoidhe use of hazardous organic solvents while fabricating
articles since it is soluble in aqueous acidic solution. Iniew of the above-mentioned properties, chitosan is exten-ively used in developing drug delivery systems [110–117].pecifically, chitosan has been used in the preparationf mucoadhesive formulations [46,108,118,119], improv-
Fig. 3. Schematic representation of preparation of chitosan pa

Beads Coacervation/precipitationFilms Solution castingGel Cross-linking

ing the dissolution rate of the poorly soluble drugs[113,120,121], drug targeting [122,123] and enhancementof peptide absorption [108,118,124]. Different types ofchitosan-based drug delivery systems are summarized inTable 2. In this section we have addressed the trends in thearea of micro/nanoparticulate chitosan-based drug deliv-ery systems. Literature form past decade has been reviewedand results are evaluated.

Different methods have been employed to prepare chi-tosan particulate systems. Selection of any of the methodsshould take into consideration factors such as particle sizerequirement, thermal and chemical stability of the activeagents, reproducibility of the release kinetic profiles, sta-bility of the final product, residual toxicity associated withthe final products, the nature of the active molecule as wellas the type of the delivery device [125].

3.1.1. Emulsion cross-linkingThis method exploits the reactive functional amine

group of chitosan to cross-link with the available reac-tive groups of the cross-linking agent. In this method, awater-in-oil (w/o) emulsion is prepared by emulsifying thechitosan aqueous solution in the oil phase. A suitable sur-factant is used to stabilize the aqueous droplets. Thereafterstable emulsion is cross-linked by using an appropriatecross-linking agent to harden the droplets. Microspheres
are filtered and washed repeatedly with alcohol and thendried [126]. This method, is helpful in controlling the sizeof the particles by controlling the size of aqueous droplets.However, the particle size of final product is dependenton the extent of cross-linking agent used while hardening
rticulate systems by emulsion cross-linking method.


san par

leading to the formation of free flowing particles [129].Various process parameters should be controlled to get thedesired size of particles such as the size of nozzle, sprayflow rate, atomization pressure, inlet air temperature and

Fig. 4. Schematic representation of preparation of chito

along with the speed of stirring. This method is schemat-ically represented in Fig. 3. The emulsion cross-linkingmethod involves a few drawbacks. Besides being tediousit uses harsh cross-linking agents, which might possiblyinduce chemical reactions with the active agent. Moreover,complete removal of the unreacted cross-linking agent maybe a challenge.

Sankar et al. used this method to prepare the chitosan-based pentazocine microspheres for intranasal delivery.Formulation parameters such as drug loading, polymerconcentration, stirring speed during cross-linking and oilphase were modified to develop microspheres having goodin vivo performance. In vivo studies indicated a significantlyimproved bioavailability of pentazocine. In-vitro releasekinetic models indicated that these systems followed thediffusion controlled release kinetics [127].

3.1.2. Coacervation/precipitationThe physicochemical property of chitosan is utilized in

this method since it is insoluble in alkaline pH medium,but precipitates/coacervates when it comes in contactwith alkaline solution. Chitosan solution is blown intoan alkali solution like sodium hydroxide, NaOH–methanolor ethanediamine using a compressed air nozzle to formcoacervate droplets. Separation and purification of parti-cles are performed by filtration/centrifugation followed bysuccessive washing with hot and cold water. The methodis schematically represented in Fig. 4. Variation in com-pressed air pressure or spray-nozzle diameter can be doneto control the size of the particles. The drug release can becontrolled by using appropriate cross-linking agent.

This technique has been used to prepare chitosan–DNAnanoparticles [128]. Processing parameters such as con-centrations of DNA, chitosan, sodium sulfate, temperature,pH of the buffer and molecular weights of chitosan and DNAhave been investigated. The particle size was successfullyoptimized to 100–250 nm with a narrow distribution bykeeping the amino to phosphate group ratio between 3 and8 and chitosan concentration of 100 �g/ml. Surface charge
of these particles was slightly positive with a zeta poten-tial of 112–118 mV at pH lower than 6.0, and became nearlyneutral at pH 7.2. Results indicated that the nanoparticlescould partially protect the encapsulated plasmid DNA fromnuclease degradation.
ticulate systems by coacervation/precipitation method.

3.1.3. Spray-dryingSpray-drying is a popular method to produce powders,

granules or agglomerates from the mixture of drug andexcipient solutions as well as suspensions. The method isbased on drying of atomized droplets in a stream of hotair. Briefly, chitosan is dissolved in aqueous acetic acidsolution, drug is then dissolved or dispersed in the solu-tion followed by the addition of a suitable cross-linkingagent (Fig. 5). This solution or dispersion is then atomizedin a stream of hot air that leads to the formation of smalldroplets, from which solvent evaporates instantaneously

Fig. 5. Schematic representation of preparation of chitosan particulatesystems by spray-drying method.


Fs

emn

pgmmssi((Igs

3

becbdtpandl6oComaa

ig. 6. Schematic representation of preparation of chitosan particulateystems by emulsion-droplet coalescence method.

xtent of cross-linking. This method is however more com-only used for the preparation of microparticles than for

anoparticles.Huang et al. prepared betamethasone disodium phos-

hate chitosan microspheres by this method using type-Aelatin and ethylene oxide–propylene oxide block copoly-er poloxamer as modifiers. Investigation of the surfaceorphology and surface charges of the prepared micro-

pheres were done which indicated that shape, size andurface morphology of the microspheres were significantlynfluenced by gelatin concentration. A good drug stabilityless 1% hydrolysis product), high entrapment efficiency95%) and positive surface charge (37.5 mV) was observed.n-vitro drug release from the microspheres was related toelatin content. The gelatin/chitosan ratio of 0.4–0.6 (w/w)howed a fairly prolonged release up to 12 h [130].

.1.4. Emulsion-droplet coalescence methodThis emulsion-droplet coalescence method, developed

y Tokumitsu et al., which combines the principles of bothmulsion cross-linking and precipitation [131]. Instead ofross-linking the stable droplets, precipitation is inducedy allowing coalescence of chitosan droplets with NaOHroplets. Two separate emulsions are prepared, one con-aining aqueous solution of chitosan along with drug isroduced in liquid paraffin oil, another containing chitosanqueous solution of NaOH is produced in the same man-er. The emulsions are mixed under high-speed stirring,roplets of each emulsion collide at random and coa-

esce, forming small sized particles that precipitate (Fig.). The particle size increased with the decrease in DDf chitosan which in turn decreased the drug content.
ompletely deacetylated chitosan produced particle sizef 452 nm with 45% drug loading. The efficiency of thisethod depends on the electrostatic interactions with the
mino groups of chitosan, which could not have occurred ifcross-linking agent was used that blocked the free amino

Fig. 7. Schematic representation of preparation of chitosan particulatesystems by ionic gelation method.

groups of chitosan. Thus, it was possible to achieve highergadopentetic acid loading by using the emulsion-dropletcoalescence method that did not involve the use of anycross-linking agent.

3.1.5. Ionic gelationFor the most part, complexation to prepare chitosan

microspheres has attracted much attention since the pro-cess is very simple and mild [132,133]. The reversiblephysical cross-linking by electrostatic interaction, insteadof chemical cross-linking, decreases the potential tox-icity impact of reagents and other undesirable effects.For example, the polyanion, tripolyphosphate (TPP) isa polyanion, which interacts electrostatically with thecationic chitosan [114,134]. After Bodmeier et al. [135]reported the preparation of TPP–chitosan complex bydropping chitosan droplets into a TPP solution, manyresearchers have explored its potential pharmaceuticalusage [117,136–140]. For ionic gelation, chitosan is dis-solved in aqueous acidic solution which quaternizes thechitosan amino groups making it soluble; this solution isthen added dropwise under constant stirring to polyan-ionic TPP solution. The complexation between oppositelycharged species, causes the chitosan to undergo ionic gela-tion and precipitate as spherical particles (Fig. 7).

Various formulations of chitosan nanoparticles pro-duced by the ionic gelation of TPP and chitosan werestudied by Xu and Du [141]. The spherical shaped parti-cles, 20 nm and 200 nm in size, were observed by TEM. Thefactors that affected the release of bovine serum albumin(BSA) as a model protein have been studied which includemolecular weight, DD and concentrations of chitosan andBSA, as well as the presence of polyethylene glycol (PEG)in the encapsulation medium.

3.1.6. Reverse micellar methodReverse micelles are thermodynamically stable liquid

mixtures of water, oil and surfactant. These homoge-neous and isotropic structured on microscopic scaleinto aqueous and oil microdomains are separated by asurfactant-rich film. The dynamic behavior of these reversemicelle systems provides very important characteristics.


chitosa
Fig. 8. Schematic representation of preparation of
The nanoparticles prepared by conventional emulsionpolymerization methods are usually large (>200 nm), witha broad size range. Ultrafine polymeric nanoparticles withnarrow size distribution could be achieved by using reversemicellar medium [142]. Due to the Brownian motion ofthe micellar droplets, they undergo continuous coales-cence followed by re-separation on a time scale thatvaries between millisecond and microsecond [143]. A rapiddynamic equilibrium maintains the size, polydispersityand thermodynamic stability of these droplets. To prepare
reverse micelles, the surfactant is dissolved in an organicsolvent followed by the addition of chitosan and drug underconstant vortexing. To the transparent obtained solution, across-linking agent is added with constant stirring, contin-ued overnight. The maximum amount of drug that can be
Fig. 9. Schematic representation of preparation of chi

n particulate systems by reverse micellar method.

dissolved in reverse micelles varies from drug to drug andhas to be determined by gradually increasing the amountof drug until the clear microemulsion is transformed intoa translucent solution. This method is schematically rep-resented in Fig. 8. Chitosan nanoparticles encapsulatingdoxorubicin–dextran conjugate was prepared by reversemicellar method [144]. The surfactant sodium bis(2-ethylhexyl) sulfosuccinate (AOT), was dissolved in n-hexane.

3.1.7. Sieving method
A simple, novel method to produce chitosan micropar-
ticles has been developed by Agnihotri and Aminabhavi[145]. This method, is devoid of tedious procedures andcan be scaled up easily, microparticles are prepared bycross-linking a 4% acetic acid chitosan solution to form

tosan particulate systems by seiving method.

olymer S

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atrIofmlaedscwmfit

wflid


lassy hydrogels that are passed through a sieve (Fig. 9).lozapine (C18H19ClN4) was incorporated into chitosan gelefore cross-linking with 99% efficiency. Irregular shapedicroparticles, ∼540–700 �m, were formed on seiving and

n vivo studies indicated a slow release of clozapine.

.1.8. Chitosan micro/nanoparticles – drug loading andelease

Usually, drug loading in micro/nanoparticulate sys-ems is achieved using one of the two following methods;a) incorporating the drug during the preparation of thearticles and (b) after the formation of the particles by

ncubation the drug with them. In both systems, therug is physically embedded into the matrix as well asdsorbed onto the surface. Both water-soluble and water-nsoluble drugs can be loaded by these techniques. The

ater-soluble drugs are generally incorporated by mixingith an aqueous chitosan solution to form a homogeneousixture, followed by particles production as described.ater-insoluble drugs and drugs that precipitate in acidic

olutions are usually loaded by incubation which involvesoaking the pre-formed particles in a saturated solutionf drug [146]. Chitosan microspheres were loaded usingwo different methods by Hejazi and Amiji [147]. In therst method, tetracycline was mixed with chitosan solutionnd then simultaneously cross-linked and precipitated thearticles. In the second method, pre-formed microspheresere incubated with the drug for 48 h. The accumulated

mount of tetracycline released and its stability was exam-ned in different pH media at 37 ◦C. When drug was addedo chitosan solution before cross-linking and precipita-ion, only 8% (w/w) was optimally incorporated into the

icrosphere. But when the drug was incubated with there-formed microspheres, 69% (w/w) could be loaded.bout 30% of tetracycline in solution or when released from

he microspheres was found to degrade at pH 1.2 in 12 h147].

The drug release from chitosan particulate systems usu-lly follows three different, mechanisms: (a) release fromhe surface of particles, (b) diffusion through the swollenubbery matrix and (c) release due to surface erosion.n most cases, drug release follows more than one typef mechanism. In case of release from the particle sur-ace, adsorbed drug dissolves on contact with the release

edium; similarly, the drug entrapped onto the surfaceayer of particles also follows this mechanism but perhapslittle slower. These types of drug release lead to a burst

ffect which is encountered in all delivery systems wherebyrug onto the surface is rapidly taken up followed by alower diffusion release from the inner matrix of the parti-le. Diffusion controlled drug release occurs in three steps; (a)ater penetrates into the particulate system, (b) the glassyatrix becomes rubbery and swells, and (c) the drug dif-

uses from the swollen matrix. Hence, this type of release isnitially slow but becomes faster as the drug dissolves andhe matrix swells.

The Higuchi equation is one of the traditional methodshich was used to describe the release of a solute from aat surface [148], but not from a sphere [149]. A good fit

ndicates that the release rate is dependant on the rate ofiffusion through the matrix. To describe the diffusion from

cience 36 (2011) 981–1014 991

a sphere, the release mode is determined using equationsdeveloped by Guy et al. [150].

The most commonly used equation for diffusion con-trolled matrix system is an empirical equation used byRitger and Peppas [151], in which the early time releasedata can be fitted to obtain the diffusion parameters,

MtM∞

= ktn (1)

where, Mt/M∞ is the fractional drug released at time t, k isa constant characteristic of the drug–polymer interactionand n is an empirical parameter characterizing the releasemechanism.

The drug transport can be classified as Fickian behavior(n = 0.5), Case II transport (n = 1), non-Fickian or anomalousbehavior (0.5 < n < 1) and super Case II (n > 1), based on thediffusion exponent [152].

The dynamic swelling data for chitosan microparti-cles was analysed by Agnihotri and Aminabhavi usingequation (1) to predict drug release from microparticlescross-linked with 5.0, 7.5 and 10.0 × 10−4 ml of glutaralde-hyde/mg of chitosan [145]. It was found that the swellingof chitosan microparticles decreased as the cross-linkingincreased. And the values of “n” < 0.5 were due to the irreg-ular shaped particles and that “n” decreased systematicallywith increasing cross-linking.

A good correlation for the cumulative drug releasedvs. the square root of time was obtained by Jameela etal. [153]; this indicated that drug release from the micro-sphere matrix is diffusion controlled and obeys the Higuchiequation [148]. In addition, the smaller sized microspheresreleased drugs faster than the large microspheres due totheir relative greater surface area and that the duffusionpath length was shorter from the smaller particles into thedissolution medium.

As stated earlier, the release of a specific drug fromchitosan-based particulate systems depends upon severalfactors, such as the cross-linking, morphology, size anddensity of the particles used. Other considerations involvethe physicochemical properties of the drug as well as thepresence of adjuvants. In-vitro release also depends onpH, polarity and presence of enzymes in the dissolutionmedia.

3.2. Chitosan hydrogels

Hydrogels are composed of three-dimensional polymernetworks that can absorb large quantities of water. Conse-quently, they are soft, pliable, wet materials with a widerange of potential biomedical applications. Hydrogels arewidely used in bioapplications and play a crucial role incurrent strategies to remedy malfunctions in and injuriesto living systems. The high water content of hydrogels ren-ders them compatible with most living tissue and theirviscoelastic nature minimizes damage to the surround-ing tissue when implanted in the host. In addition, their
mechanical properties parallel those of soft tissue, makingthem particularly appealing with the host tissues, assistingand improving the healing process, and mimicking func-tional and morphological characteristics of organ tissue.Chitosan hydrogels can be prepared with a variety of ways.

992 M. Dash et al. / Progress in Polymer S

Fig. 10. Schematic representation of chitosan hydrogel networks derived
by physical associations: networks formed with ionic molecules, poly-electrolyte polymer and neutral polymers [161]. Copyright 2009, ElsevierLtd.
In each process, chitosan is either physically associated orchemically cross-linked networks to form hydrogel.

3.2.1. Physical association networksTo construct a stable hydrogel, the chitosan polymer

network must satisfy two conditions: (a) interchain inter-actions must be strong enough to form semi-permanentjunction points in the molecular network and (b) the net-work must be capable of exchanging water molecules withthe hydrated polymer network. The chitosan hydrogelsthat meet these basic requirements may be prepared bynon-covalent strategies that take advantage on electro-static, hydrophobic, and hydrogen bonding forces betweenpolymer chains [154,155]. The schemes of four majorphysical interactions ionic, polyelectrolyte, interpolymercomplex, and hydrophobic associations that lead to thegelation of a chitosan solution are in Fig. 10. Since all ofthese are purely physical interactions, gel formation can bereversed. Swelling behavior can be tuned by adjusting theconcentration and nature of the component(s) used duringthe fabrication process. However, the number of appli-cations of chitosan hydrogels is limited due to the weakmechanical strength and uncontrolled dissolution [156].

3.2.1.1. Ionic complexes. Ionic complexes are readilyformed by ionic interactions between the cationic chi-tosan and negatively charged molecules, such as sulfates,citrates, and phosphates ions [157,158] and anionic metalsPt(II), Pd(II), and Mo(VI) [159,160]. These interactions canbe manipulated to form hydrogels with varying structuralproperties that depend upon the charge density andsize of the anionic agents, as well as chitosan DD andconcentration [161].

Anions and small molecules bind chitosan via itsprotonated amino groups, whereas metal ions formcoordinate–covalent bonds with the polymer instead ofelectrostatic interactions [159,160]. Ionic complexation isusually accompanied by other secondary interchain inter-

cience 36 (2011) 981–1014

actions including hydrogen bonding between chitosan’shydroxyl groups and the ionic molecules, or interactionsbetween deacetylated chitosan chains after neutralizationof their cationic charge [159,162]. These interactions oftenenhance certain physical properties of the hydrogel, whichcan be modulated to express unique material properties,such as pH sensitivity.

3.2.1.2. Polyelectrolyte complexes (PEC). Chitosan electro-static interactions with polyelectrolytes, are different fromthe ionic complexes in that they are larger molecules. Theinteractions between the chitosan and polyelectrolytes arestronger than other secondary binding interactions likehydrogen bonding. This type of complexes avoids the useof organic precursors, catalysts, or reactive agents, alle-viating the concern about toxicity or reactions with atherapeutic payload. Since, PEC only consist of chitosanand the polyelectrolyte, their complexation is generallystraightforward and reversible [163]. Chitosan-based PECnetworks have been produced with DNA, anionic polysac-charides such as alginate, glucosamineglycans, chondroitinsulfate, hyaluronic acid, heparin, carboxymethyl cellulose,pectin, dextran sulfate, xanthan [164–166], proteins suchas gelatin, albumin, fibroin, keratin, and collagen [167–169]and synthetic anionic polymers such as (polyacrylic acid)[170]. The stability of these complexes is dependent oncharge density, solvent, ionic strength, pH, and temper-ature [171]. The charge of the anionic molecule underphysiological conditions is an important factor for PEC tooccur; since the pH of the hydrogel environment modifiesthe ionic interactions, in vivo environment must be consid-ered when using PEC for hydrogels.

3.2.1.3. Physical mixtures and secondary bonding. In addi-tion, hydrogels are formed by polymer blends betweenchitosan and other water-soluble nonionic polymers, suchas poly(vinly alcohol) (PVA). After lyophilization or a seriesof freeze–thaw cycles, these polymer mixtures form junc-tion points in the form of crystallites and interpoymercomplexation [154,172]. The chain–chain interactions per-form as cross-linking sites of the hydrogel formation. Inthe case of chitosan–PVA polymer blends, increasing thechitosan content negatively affects the formation of PVAcrystallites, leading to the formation of poorer hydrogelstructures. Chitosan is also capable of forming hydrogels byitself without the use of any polymer or complexing agent.A chitosan hydrogel using a hydro-alcohol gel formationprocess based on the neutralization of chitosan’s aminogroups using a sodium hydroxide solution was preparedby Ladet et al. [173]. This process inhibited ionic repulsionbetween the polymer chains that allowed the formationof hydrogen bonds, hydrophobic interactions, and chitosancrystallites to form the necessary cross-links for hydrogelformation (Fig. 11). An interrupted gelation method led tomultilayered “onion-like” hydrogels (Fig. 11); this uniquefeature is being explored to encapsulate drugs to co-deliver
multiple therapeutics and for pulse-like delivery of a givenpayload [174,175].
3.2.1.4. Thermoreversible hydrogels and hydrophobic asso-ciations. Thermoreversible hydrogels and hydrophobic


Fig. 11. (a) Multi-membrane biomaterial with ‘onion-like’ structure based on chitosan hydrogel. (b) Schematic diagram of the multi-membrane onion-l n as a fc .5 wt.% ia blishin

atrbmslupaautaagtaTcpmscbtb[

ike structures. (c) Variation of hydrogel shrinkage during neutralizatiooncentration in the non-neutralized alcohol gel is constant and close to 4s a function of the NaOH neutralization [173]. Copyright 2008, Nature Pu

ssociation form an interesting class of hydrogels that areransient gels or in liquid states depending upon the envi-onmental temperature. These hydrophobic and secondaryonding interactions develop junctions between the poly-er chains that form semi-rigid gels from a flowable liquid

olution. When temperature of the system is below theower critical solution temperature (LCST), the materialndergoes a hydrophilic–hydrophobic transition. Thus, aolymer solution that has a low viscosity at room temper-ture, can form a gel above the LCST; this is very significants these materials can be injected into the body as a liq-id below LCST and then it forms a gel in situ at bodyemperature which is above the LCST. This carrier matrixpplication is being used in a wide range of biomedicalnd pharmaceutical applications [176,177]. The injectableelling systems can be introduced into the body withouthe need for invasive surgery and deliver the bioactivegents to the defect site without significant side effects.he hydrogels prepared by aggregation of chitosan-basedopolymers or by neutralization with polyol salts showromising thermoreversible gelation properties in aqueousedia [178–182]. The latter strategy uses the temperature-

ensitivity of glycerol phosphate disodium salt (GP) and
hitosan physical mixture. The phosphates of the GP salt areelieved to neutralize the ammonium groups of chitosan,hus increasing the hydrophobic and hydrogen bondingetween the chitosan chains at elevated temperatures182].
unction of the concentration of sodium hydroxide (the initial polymern each case). (d) Evolution of the chitosan mass fraction in the gel (WCH)

g Group.

3.2.2. Cross-linked networksAlthough physically bonded hydrogels have the advan-

tage of gel formation without the use of cross-linkingmoieties, they have certain limitations. For example, it isdifficult to accurately control the physical gel pore size,chemical functionalizations, dissolution and degradation.Consequently they often provide inconsistent in vivo per-formance. Better chitosan hydrogel mechanical propertiescan be produced using irreversible networks. To formthe hydrogel, the polymer chains are covalently bondedtogether by small cross-linker molecules, secondarypolymerizations, or irradiation. Covalently cross-linkedhydrogels are also obtained by attaching photo-reactiveor enzyme-sensitive molecules on the chitosan, followedby their subsequent exposure to UV or sensitive enzymes,respectively. The properties of cross-linked hydrogelsdepend mainly on their cross-linking density and the ratiocross-linker molecules to the moles of polymer repeatingunits [183]. The different method to make irreversible chi-tosan hydrogels is described below.

3.2.2.1. Chemical cross-linking. Chemical cross-linking isthe most straightforward method to produce permanent
hydrogel networks using covalent bonding between thepolymer chains. The available –NH2 and –OH groups onchitosan are active sites capable of forming a numberof linkage, including amide and ester bonding as wellas Schiff base formation [162,184,185]. These networks

olymer S
can be formed by using small molecule cross-linkers,polymer–polymer reactions between activated functionalgroups, photosensitive agents and enzyme-catalyzed reac-tions.

The new cross-linking agent, genipin (C11H14O5), is anaturally derived chemical that can bind biological tissuesand biopolymers through covalent coupling [185–187].Genipin is particularly effective for cross-linking polymerscontaining amino groups. In addition it is less toxic anddegrades slower than agents like glutraldehyde [188,189].Genipin also provides extended drug release by chitosanhydrogels cross-linked in situ [179,190]. Although, genipinhas good biocompatibility, it is susceptible to interact withthe encapsulated drugs; this can be a problem for gelationin the presence of a therapeutic agent [161].

A thermo-sensitive, chitosan–pluronic hydrogel wasalso produced using ultraviolet (UV) photo-cross-linking[191]. The UV exposure cross-links chitosan and pluronicgroups functionalized with photosensitive acrylate groups.These systems form physical networks at temper-atures above the LCST. Thermo-sensitive hydrogelshave been used for sustained release of encapsulatedhuman growth hormone (hGH) and plasmid DNA andexhibit potential application for different types of drugs[191,192].

3.2.2.2. Interpenetrating networks (IPN). Entangled poly-mer networks within the cross-linked networks can bestrengthened by interlacing them with secondary poly-mers. In this case, a cross-linked chitosan network isallowed to swell in an aqueous solution of monomers.When these monomers are polymerized, a physicallyentangled polymer mesh is formed called an inter-penetrating (IPN) network. A semi-IPN involves onecross-linked polymer network with another polymerin the linear states. There are several chitosan-basedsemi-IPNs reported in literature prepared with polyether[193,194], silk [195], polyethylene oxide (PEO) [196], andpolyvinylpyrrolidine (PVP) [197] and full-IPNs (preparedwith poly(N-isopropylacrylamide) (PNIPAM) [198]).

This technique has the advantage of specifically select-ing polymers that can complement the deficiencies of oneanother. Based on the target application, cross-linking den-sity, hydrogel porosity, and gel stiffness can be adjustedin IPN-based hydrogels; however it is more difficult toencapsulate a wide variety of therapeutic agents, espe-cially sensitive biomolecules. IPN preparations generallyinvolve the use of toxic agents to initiate or catalyzethe polymerization or the cross-linking; their completeremoval is very difficult, making the clinical application aconcern.

3.2.3. Chitosan hydrogels – drug loading and releaseThe drug loading in a hydrogel depends upon the phys-

ical and chemical properties of the gel as well as from thestructural features of the therapeutic agent. Three major
approaches to drug loading can be summarized as: diffu-sion, entrapment, and tethering [199–203]; each methodhas advantages and disadvantages. The easiest drug loadingmethod is to place the fully formed hydrogel into a mediumsaturated with the therapeutic agent [204,205]. The drug
cience 36 (2011) 981–1014

slowly diffuses into the gel depending upon the poros-ity of the hydrogel, the size of the drug and the chemicalproperties of each, such as hydrophobicity/hydrophilicity.When placed in vivo, the drug diffuses out of the hydrogelinto the neighboring tissue. This technique has proved tobe effective in loading small molecules, but larger thera-peutics peptides and proteins, in particular, do not readilyable to migrate through the small pores of the hydrogel[161]. However, this process is also very time consumingand, therefore, it is not recommended for manufacturingpurposes. Therefore, in the case of large sized drugs andbioligands, it is preferable to have the drug entrappedduring the gelation process. Usually, the drug is mixedin a polymer solution, and a cross-linking or complexa-tion agent is added. The chemistry of the drug moleculemust be considered to prevent unwanted cross-linking ordeactivation of the therapeutic agent during gelation. Freemovement of the therapeutic agent out of the hydrogelnetwork is takes place in both diffusion and entrapmentsystems. This process often leads to an initial burst releaseafter implantation of the hydrogel in vivo due to theconcentration gradient formed between the gel and thesurrounding environment.

To reduce the loss of the therapeutic reserve and therisk of toxic exposure, drugs usually covalently or phys-ically are linked to the polymer chains prior to gelation.This technique is termed as tethering which limits tissueexposure to the agent only when the hydrogel breaks downor the molecular tether is broken [184]. Drug loading canbecome complicated by molecules that have the oppositesalvation features or similar charge as the continuous poly-mer matrix [206]. An alternative in such cases is to forma complex with amphiphilic additives before the hydro-gel and drug are blended in solution [207,208]. This hasbeen accomplished by binding paclitaxel (C47H51NO14) toalbumin (Abraxane) or by mixing it in an aqueous cit-ric acid/glyceryl monooleate solution prior to hydrogelloading [208]. Therapeutic agents can also been loadedinto small secondary release vehicles, such as microparti-cles, microgels, liposomes, and micelles, prior to hydrogelencapsulation [209,210].

The release of a therapeutic from a hydrogel canfollow one of three different modes: diffusion, chemi-cal/environmental stimulation, or enzyme-specific stimula-tion [211]. Diffusion is regulated by the movement of thedrug through the polymer matrix or by bulk erosion of thehydrogel carrier as it breaks down in vivo. Environmen-tally responsive hydrogels swell in response to externalconditions, such as pH and temperature that effectivelyopen the pores to enhance diffusion of the entrappedtherapeutic under predetermined conditions [161]. Thistype of controlled release is used to limit drug releaseoutside of the effective range of the diseased tissue. Therelease of a drug payload can also be triggered by localenzymatic action. These biochemically responses occurby tethering drugs to the hydrogel via labile domains
that are susceptible to enzymes or using polymers thatare targeted by enzymes [212]. This method though notwidely used offers selective, sustained release mechanismsis beginning to receive attention from chitosan hydrogelengineers.

olymer S

4

4

tatbblivctmcslt

4

nmtape(mtTsmmaatnricwdsefcbtipsctsttc[


. Biomedical–pharmaceutical applications

.1. Chitosan for tissue engineering applications

Tissue engineering is a highly interdisciplinary fieldhat combines the principles and methods of life sciencesnd engineering to utilize structural and functional rela-ionships in normal and pathological tissue to developiological substitutes to restore, maintain, or improveiofunction [213]. It involves the in vitro seeding and pro-

iferation of relevant cells in a scaffold support. Since its biodegradable, non-toxic and can be formulated in aariety of forms including powders, gels and films for appli-ations, it has a wide range of potential applications inissue engineering. In addition to being implied in the for-

ulation of controlled delivery systems a wide range ofhitosan modifications can be made to improve the celleedings. Many tissue analogs including cartilage, bone,iver, and nerve have been prepared using this engineeringechnology.

.1.1. Chitosan in bone tissue engineeringChitosan has been extensively used in bone tissue engi-

eering, since it was shown to promotes cell growth andineral rich matrix deposition by osteoblasts cells in cul-

ure [214]. The biocompatibility of chitosan minimizesdditional local inflammation, and it can be molded intoorous structures to allow osteoconduction [215]. Sev-ral studies have focused on chitosan–calcium phosphateCP) composites for this purpose [216–218] with a 3D

acroporous CP bioceramic embedded with porous chi-osan sponges being developed by Zhang and Zhang [219].his nested chitosan sponge enhances the mechanicaltrength of the ceramic phase through matrix reinforce-ent and preserves the osteoblast phenotype [220]. Theacroporous chitosan scaffolds incorporating hydroxyap-

tite (HA) or CP glass had an interconnected porosity ofpproximately 100 �m and are used for clinical applica-ions [221]. Hu et al. prepared a chitosan–HA multilayeranocomposite with high strength and bending modulusendering the material suitable for possible application fornternal fixation of long bone fractures [222]. A series ofhitosan-� tricalcium phosphate (TCP) composite scaffoldsere developed for bone tissue engineering using freeze-rying process which provide macroporous compositecaffolds with different pore structures. Compressive prop-rties were improved, especially compressive modulusrom 4 MPa to 11 MPa. The biocompatibility, evaluated sub-utaneously on rabbits indicated that these scaffolds cane utilized in non-loading bone regeneration [223]. Chi-osan is also used as an adjuvant with bone cements toncrease their injectability while keeping the chemico-hysical properties suitable for surgical use with respect toetting time and mechanical properties [224]. The choice ofhitosan for this purpose is based on the property that chi-osan solutions tend to gel in response to a pH change from
lightly acidic to physiological environment. It is importanto note that, the chitosan–CP composites address the needo develop bone fillers that set in response to physiologicalonditions, but not while mixing the components in vitro221].
cience 36 (2011) 981–1014 995

Xu et al. studied the feasibility of creating macroporesin calcium phosphate cement (CPC) using chitosan and/orabsorbable mesh. This injectable, bioabsorbable compositematerial formed interconnected macropores (osteocon-ductive) and provided strength to the implant duringtissue regeneration [225]. Zhao et al. used phase separationtechnique to fabricate biomimetic HA/chitosan–gelatinnetwork composites in the form of 3D-porous scaffoldsthat improved adhesion, proliferation and expression ofrat calvaria osteoblasts on these highly porous scaf-folds [226]. Kim et al. showed the application of thisproperty through composites of chitosan with poly methyl-methacrylate (PMMA). This specially developed compositematerial exhibited lower exothermic curing tempera-tures and possessed higher interconnected porosity witha pore size suitable for osteoconduction with betteranchorage to the surrounding bone. It was observed thatthe pore size of this composite material increased withtime due to biodegradation of the chitosan [227]. Chi-tosan is also used to modify the surface properties ofprosthetic materials for the attachment of osteoblasts[228,229].

Recently, Zhao et al., using a scaffold with CPC andchitosan fibers, harvested human umbilical cord mes-enchymal stem cells (hUCMSCs) without an invasiveprocedure that is commonly required when studying bonemarrow mesenchymal stem cells (MSCs). The objectiveswere to develop CPC scaffolds with improved resistanceto fatigue and fracture, for the delivery of hUCMSC aimedat bone tissue engineering. In “fast fractures”, CPC with15% chitosan and 20% polyglactin [(C2H2O2)m(C3H4O2)n]fibers (CPC–chitosan–fiber scaffold) had flexural strengthof 26 MPa, while CPC control was 10 MPa. The hUCM-SCs showed excellent viability when seeded in CPC andCPC–chitosan–fiber scaffolds, the live cell retention was96–99%. The cell density was about 300 cells/mm2 on day1; and with proliferation ∼700 cells/mm2 at day 4 (Fig. 12).Wst-1 assay showed that the stronger CPC–chitosan–fiberscaffold had hUCMSC viability that matched the CPC control(p > 0.1). This study indicated that chitosan and polyglactinfibers substantially increased the fatigue resistance of CPC,and that hUCMSCs had excellent proliferation and viabilityon the scaffolds [230].

A robotic desktop rapid prototyping (RP) system tofabricate scaffolds for tissue engineering applicationswas designed by Ang et al. The set-up consisted of acomputer-guided desktop robot and a one-componentpneumatic dispenser. The dispensing material, chitosanand chitosan–hydroxyapatite (HA) dissolved in acetic acid,was forced out through a small Teflon lined nozzle into adispensing sodium hydroxide–ethanol medium. Layer-by-layer, the chitosan was fabricated with a pre-programmedlay-down pattern. The attachment between layers allowedthe chitosan matrix to form interconnected channeledarchitectures. The in vitro osteoblast cell culture stud-ies revealed the biocompatibility of the scaffolds with
cells exhibiting healthy morphology and avid prolifera-tion throughout the culture period. The rapid prototypingrobotic dispensing (RPBOD) system is capable of fabricatingthree-dimensional (3D) scaffolds with regular and repro-ducible macropore architecture [231].


Fig. 12. (1) hUCMSCs were cultured on CPC control and CPC–chitosan–fiber for 1, 4, and 8 days: (A) percent of live cells, and (B) live cell attachment(mean ± sd; n = 5). PLive reached 96–99%, not different from each other (p > 0.1). CAttach was less than 300 cells/mm2 at day 1; it more than doubled to

2 ters indre desiga highe

700 cells/mm at day 4, due to hUCMSC proliferation. In (B), dissimilar letattachment on: (A) CPC control, and (B) CPC–chitosan–fiber scaffold. Cells ain (B). Cells developed long, cytoplasmic extensions “E”, shown in (C) atscaffold [230]. Copyright 2010, Elsevier Ltd.

4.1.2. Chitosan in cartilage tissue engineeringThe choice of biomaterial is very critical for the success

of tissue engineering approaches, especially in cartilagerepair [232]. The ideal cell-carrier substance should mimicthe natural environment in the articular cartilage matrix.The cartilage-specific extracellular matrix (ECM) compo-nents such as type II collagen and GAGs, play a criticalrole in regulating expression of the chondrocytic pheno-type and in supporting chondrogenesis in vitro and in vivo[233,234].

Three-dimensional (3D) scaffolds are especially impor-
tant for fabricating articular cartilage. Ideal scaffolds aredesigned to be biocompatible, bioabsorbable and exhibitpredictable porosity and degradation rate. They providea framework that facilitates new tissue in growth; more-over and mechanical characteristics that match those of the
icate values that are significantly different (p < 0.05). (2) SEM of hUCMSCnated as “C”, which anchored to CPC in (A), and to the fibers in the scaffoldr magnification, attaching firmly to the fiber in the CPC–chitosan–fiber

native tissue; this increases the chances that the reparativeprocess will be compatible with the host’s tissue physiology[235,236]. Chitosan was chosen as a scaffolding material inarticular cartilage engineering due to its structural similar-ity with various GAGs found in articular cartilage [25,237].This is of importance since GAGs are considered to playa pivotal role in modulating chondrocytes morphology,differentiation, and function [238]. An alginate–chitosanhybrid based on polymer fibers, that increased cell attach-ment and proliferation in vitro compared to alginate wasreported by Iwasaki et al. [239]. These hybrid polymer
fibers showed increased tensile strength, implying a pos-sible use in developing a 3D load-bearing scaffold forcartilage regeneration. Chondrocytes cultured on chitosansubstrates in vitro maintained a round morphology andcell-specific ECM [237,240]. Chitosan modified PLLA sub-


Fig. 13. (a) Accumulation of matrix in the tissue-engineered cartilage. Histology and immunohistochemistry of chondrocytes cultured in chitosan hydrogels3 weeks. The results showed that the chondrocytes in the chitosan hydrogels accumulated pericellular sulfated GAG-containing matrix. A, H.E. staining;B, type II collagen immunohistochemical staining; C, toluidine blue staining; D, safranin O staining. Star: chitosan hydrogel; arrowhead: cell nucleus;arrow: matrix of the chondrocytes. Bar = 100 �m. (b) Gross observation of the articular cartilage repair at 24 weeks post-operation. A, the defect part ofthe cartilage in the experimental group was covered by consistently smooth, glistening white hyaline tissue nearly indistinguishable from the surroundingn spottedw n the des artilagei s referre

sbewccoaspom

ctpfceionsfrceoyaio[

mdt

ormal cartilage. No clear signs of margin with normal cartilage could beere partially repaired with fiber-like tissue, leaving a small depression i

urface tissue, with obvious defects and cracks surrounding the normal cnterpretation of the references to color in this figure legend, the reader i

trate showed increased cell adhesion, proliferation andiosynthetic activity [241]. Chondrocyte adhesion, prolif-ration, and the synthesis of aggrecan and type II collagenere significantly higher on the hybrid fiber than on

hitosan [242]. Similarly, chitosan–alginate–hyaluronanomplexes increase the cellular adhesiveness with or with-ut covalent attachment with arginine–glycine–asparticcid (RGD) containing protein. When chondrocytes seededcaffolds were implanted into rabbit knee cartilage defects,artial repair was observed after 1 month both in presencer absence of RGD indicating potential of this compositeaterial for cartilage regeneration [243].Chitosan-based scaffolds deliver growth factors in a

ontrolled fashion to promote the in-growth and biosyn-hetic ability of chondrocytes. Lee et al. [244] reportedorous collagen/chitosan/GAG scaffolds loaded with trans-orming growth factor (TGF)-�1. This scaffold exhibitedontrolled release of TGF-�1 and promoted cartilage regen-ration. The addition of chitosan to the collagen scaffoldmproved the mechanical properties [244] and the stabilityf the collagen network by inhibiting the action of collage-ases [245]. Kim et al. used a porous freeze-dried chitosancaffold incorporating TGF-�1-containing microspheres,or the treatment of cartilage defects [246]. The TGF-�1 waseleased in a sustained fashion, and promoted chondro-yte proliferation and matrix formation. In a similar trial, Lut al. studied the effect of intraarticular injection of chitosann regeneration of articular cartilage. An increase in epiph-sis cartilage in the tibial and femoral joints was seen withn activation of chondrocytes proliferation. Similarly, anntra-articular fibrous tissue was observed for the 6 weeksf the experiment, together with residual injected chitosan
247].
A noteworthy accomplishment was achieved by Hoe-ann et al. who showed that microfractured ovine

efects are repaired with more hyaline cartilage whenhe defect is treated with in situ-solidified implants of

on the surface of the regenerated areas; B, the defects in control group 1fect areas; C, the defects in control group 2 detected a thin and irregular. Arrow: the defect; Bar = 0.5 cm [252]. Copyright 2010, Elsevier Ltd.(Ford to the web version of the article.)

chitosan–GP mixed with autologous whole blood, com-pared to microfracture alone in an ovine model at 6 months[248]. Since bleeding has been identified as an initiat-ing event in post-surgical repair, it was hypothesized thatmicrofracture-based repair could be improved by stabi-lizing the clot formed in the lesion with chitosan that isthrombogenic and actively stimulates the wound-healingprocess. These chitosan–GP/blood clots are adhesive andcontract much less than whole blood clots, thereby main-taining a voluminous scaffold [248]. Chitosan–GP/bloodimplants were applied to marrow-stimulated chondraldefects in rabbit cartilage repair models [249], where theyinduced greater fill of chondral defects with repair of tissuecompared to marrow-stimulation alone [248]. In addition,a more cellular and hyaline repair cartilage was producedwith a porous subchondral bone structure [248–251].

A chitosan hydrogel in the form of a scaffold wasprepared for chondrocyte cells to reconstruct tissue-engineered cartilage and repair articular cartilage defectsin the sheep model was reported by Hao et al. [252]. In thisstudy, temperature-responsive chitosan hydrogels wereprepared by combining chitosan, GP and hydroxyethylcellulose (HEC). The in vitro tissue-engineered cartilagereconstructions were made by mixing sheep chondrocyteswith a chitosan hydrogel. Cell survival and matrix accumu-lation analysis were done after 3 weeks in culture (Fig. 13).For in vivo repair, reconstructions cultured for 1 day andtransplanted to the freshly prepared defects of the articu-lar cartilage of sheep. The cultured chondrocytes survivedand retained their ability to secrete. Thus in vivo trans-plantation repaired cartilage defects completely within 24weeks (Fig. 13). This study showcased the success of a new
technique in its ability to repair articular cartilage defects.
4.1.3. Chitosan in liver tissue engineeringDue to the insufficient donor organs for orthotopic

liver transplantation the need for new therapies for


ed chiton cell suard devi

Fig. 14. (A) SEM images of hepatocytes cultured within fructose-modifihepatocytes and cell aggregates; (b) high-magnification of the microvilli owithin different scaffolds. (Each time point represents the mean ± standLtd.

acute and chronic liver disease is critical [253]. Currently,bioartificial liver (BAL) is a promising application of tis-sue engineering for the treatment of fulminant hepaticfailure (FHF). An important issue for BAL devices is theproper choice of cell sources, such as primary hepato-cytes, hepatic cell lines, and liver stem cells [254]. Theprimary hepatocyte of these cells represent the mostdirect approach to BAL devices however hepatocytesare anchor dependent cells and are highly sensitive tothe ECM milieu for the maintenance of their viabilityand differentiation functions [255–257]. The structuralsimilarity of chitosan to GAGs and since GAGs are compo-nents of the liver ECM, chitosan was selected as a scaffoldmaterial for hepatocytes culture [258–261]. Wang et al.prepared chitosan/collagen matrix (CCM) by cross-linking
agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) in N-hydroxysuccinimide (NHS)buffer system [261]. The cross-linked matrix had moder-ate mechanical strength, good hepatocyte compatibilityas well as excellent blood compatibility. However,
san scaffold: (a) low-magnification view showing the higher density ofrface and the cell morphology. (B) Urea synthesis of hepatocytes culturedation of the mean of three experiments) [260]. Copyright 2003, Elsevier

implantable bioartificial liver (IBL) has severe complexi-ties, unlike those for bioartificial skin, bone and cartilage.Since thrombus formation can lead to occlusion anddecrease membrane efficiency, special designs of thecomplex architecture, as well as the anti-thrombogenicextracellular component, were necessary to develop thisblood-contact device [254]. A superior blood compatiblechitosan/collagen/heparin matrix in implantable bioartifi-cial liver (IBL) applications was, subsequently, developedby Wang et al. [262].

Multivalent galactose residues to bind to the asialo-glycoprotein receptor (ASGPR) expressed on the surfaceof hepatocytes is another strategy for liver tissue engi-neering [263,264]. Typical cell–matrix interactions aremediated by an adhesion receptor like integrin which
specifically binds RGD sequence [265]. The ASGPR was thefirst reported mammalian lectin, or carbohydrate-bindingprotein discovered [266,267]. The hepatic ASGPR is a clas-sical system for studying receptor-mediated endocytosis.Chung et al. reported the potential ability to improve hepa-


F be and[

t(s[ci

Acsca[

idfattfcme

4

fioiblpcaom[adaoc

ig. 15. Optical microscope longitudinal view of (a) a chitin hydrogel tu280]. Copyright 2005, Elsevier Ltd.

ocyte attachment to alginate (AL)/galactosylated chitosanGC) scaffolds for short-term culture [268] while Seo et al.,howed enhanced hepatocyte functions in AL/GC scaffolds269]. The study was done for long periods and hepatocytesultured enhanced function through its spheroid formationn co-culture condition with fibroblast cells.

Concurrently, fructose known as a specific ligand ofSGPR in hepatocyte, was conjugated, by Li et al., on poroushitosan scaffolds by forming Schiff’s bases. The chitosanurface modified with fructose induced the formation ofellular aggregates and enhanced liver specific metabolicctivities and cell density to a satisfactory level (Fig. 14)259,260].

Chitosan micro/nanofibers can be fabricated by chem-cal and electrospinning techniques, however, Lee et al.eveloped a microfluidic-based pure chitosan microfibersor liver tissue engineering applications without the use ofny chemical additives [270]. To evaluate the capability ofhe microfibers, hepatoma HepG2 cells were seeded ontohe chitosan microfibers. The HepG2 cells self-aggregatedorming spheroids that had a higher liver function that wasonfirmed by albumin secretion and urea synthesis. Thisethod represents a potentially useful tool for liver tissue

ngineering applications.

.1.4. Chitosan in nerve tissue engineeringOnce the nervous system is impaired, its recovery is dif-

cult and malfunctions in other parts of the body generallyccurs [271]. Nerve injuries complicate successful rehabil-tation since mature neurons like many other cells in theody do not undergo cell division. The goal to repair nerve

esions is to direct the regenerating nerve fibers into theroper endoneurial tubes. The current strategies can belassified into two categories: (a) bridging, using graftingnd tubulization techniques and (b) end-to-end suturingf the nerve stumps. The former technique seems to beore effective, as it avoids tension across the repair site

272]. A wide variety of biocompatible, non-degradable
nd degradable materials have been suggested for the pro-uction of artificial tubes for nerve repair. However, thertificial tubes have been suggested for the productionf artificial tubes for nerve repair. However, the artifi-ial tubes lack sufficient internal surface area for nerve
(b) a chitin gel tube reinforced with a PLGA coils embedded in the wall

fibers and Schwann cells (SCs) to cohere [254]. For arti-ficial tubes to bridge large defects in nerve repair thebiodegradable matrix should provide a cellular, and molec-ular framework for SCs and neurite migration across thenerve gap. Chitosan is suitable for nerve regeneration basedon its biocompatibility and biodegradability. Haipeng et al.reported that neurons cultured on the chitosan membranecan grow well and that chitosan tube can promote repairof the peripheral nervous system [273]. Yuan et al. foundthat chitosan fibers supported the adhesion, migrationand proliferation of SCs, which provide a similar guidefor regenerating axons to Büngner bands in the nervoussystem [274]. A novel biomaterial for nerve regenerationthrough immobilization of laminin peptide in molecu-larly aligned chitosan by covalent bonding was designedby Matsuda et al. [275]. Progestrone delivered from chi-tosan prostheses were reported by, Chavez-Delgado et al.,to provide better facial nerve regenerative response ofthe rabbits than chitosan prostheses without progesterone[276]. An improved attachment, differentiation and growthon the chitosan/poly(l-lysine) composite materials whencompared to cells cultured on chitosan membranes wasfound by Mingyu et al. The improved nerve cell affin-ity on the chitosan/poly(l-lysine) composite materialswas attributed to the increased hydrophilicity by thehydroxyl groups and the positive surface charge of chitosan[277].

Gelatin added by Cheng et al. provided a soft and elasticcomplex that has good nerve cell affinity. The compositefilm exhibited a lower modulus with a higher percentage ofelongation at break compared to chitosan films. In addition,PC12 cells cultured on the composite films differentiatedmore rapidly and with longer neurites than on chitosanfilms [278,279]. Using mold casting techniques Frier et al.developed chitin hydrogel tubes by using no toxic cross-linking agent (Fig. 15). Both chitin and chitosan indicatednerve cell adhesion and neurite outgrowth, indicating thepotential of these materials for scaffolds in neural tissue
engineering [280].
A new method was developed by Yeh et al. forculturing in chitosan microfibers scaffolds; a chitosanlaminar flow by sheath force in a microfluidic chip wasused to produce sodium tripolyphosphate (TPP) chitosan

olymer S
microfibers via the ionic cross-linking reaction. A poly-methyl-methacrylate (PMMA) microfluidic chip with a 45◦

cross-junction microchannel was fabricated using a CO2laser machine to generate the chitosan microfibers. Bychanging the ratios of the core and sheath flow rate, dif-ferent sizes of chitosan microfibers were produced. Themicrofibers were then coated with collagen and Schwanncells and fibroblast cells were cultured on the chitosanmicrofibers. Both cell types adhered to the surface of thechitosan microfibers and began to proliferate after 24 h.After 72 h, the Schwann cells had proliferated linearly whilethe fibroblast cells covered the surface of the chitosanmicrofibers. The chitosan microfibers provide very goodscaffolds for many tissue engineering applications with theadvantages of ease of fabrication, simplicity and cost effec-tiveness [281].

4.2. Chitosan in wound-healing applications

In wound-healing, an ideal dressing should protect thewound from bacterial infection as well as promote heal-ing [282]. Chitosan-based materials, produced in varyingformulations, have been used in a number of wound-healing applications. Chitosan induces wound-healing onits own and produces less scarring [283–285]. It seems toenhance vascularization and the supply of chito-oligomersat the lesion site, which have been implicated in bettercollagen fibril incorporation into the extracellular matrix[65,286]. While different material dressings have beenused to enhance endothelial cell proliferation, the deliveryof growth factors involved in the wound-healing pro-cess can improve that process [287]. In addition to thereparative nature of the chitosan hydrogels they can alsodeliver a therapeutic payload to the local wound, for exam-ple, fibroblast growth factor-2 (FGF-2) which stimulatesangiogenesis by activating capillary endothelial cells andfibroblasts [288,289]. To sustain FGF-2 residence at thewound site, FGF-2 was incorporated into a high molecularweight chitosan hydrogel, formed by UV-initiated cross-linking [290].

A chitosan hydrogel scaffold impregnated with �-FGF-loaded microspheres were developed by Park et al. thataccelerates wound closure in the treatment of chroniculcers [288]. Films of chitosan, in combination [291] werefound to promote accelerated healing of incisional woundsin a rat model [292]. The wounds closed within 14 daysand mature epidermal architecture observed histologicallywith keratinized surface of normal thickness and a sub-sided inflammation in the dermis.

4.3. Chitosan in drug delivery applications

Drug delivery has been a very active area, especially forchitosan as a carrier for various active agents [293–295].Chitosan has been effectively used in drug delivery as a
hydrogel system, drug conjugate, biodegradable releasesystem, and PEC for many components. Chitosan-basedsystems are used for the delivery of proteins/peptides,growth factors, anti-inflammatory drugs, antibiotics, aswell as, in gene therapy and bioimaging applications.
cience 36 (2011) 981–1014

4.3.1. Chitosan-based systems for the delivery ofanti-cancer drugs

Cisplatin loaded chitosan microspheres were preparedusing a w/o emulsion system; the incorporation effi-ciency was ∼30% [296]. The type of oil used was foundto affect release properties of cisplatin, and the initialburst effect. Pharmacokinetics, targeting, embolizationeffects and alteration of liver function using cisplatin chi-tosan microspheres were evaluated after hepatic arterialembolization in dogs. A remarkable decrease in the num-ber of arterioles in liver, necrosis of nodules and hepatic celldegeneration in the embolized region. A chitosan-basedhydrogel with 131I-norcholesterol (131I-NC) was tested in abreast cancer xenograft mouse model by Azab et al. Thishydrogel, cross-linked with glutaraldehyde, reduced theprogression of the tumor and [297,298] prevented 69% oftumor recurrence and metastatic spreading. Most impor-tantly, there was little or no systemic distribution of theradioisotope after hydrogel implantation. Recently, pacli-taxel was effectively delivered from salicylic acid-graftedchitosan oligosaccharide nanoparticle by Wei et al. [299].

4.3.2. Chitosan-based systems for the delivery ofproteins/peptides

Chitosan particles or polyelectrolytes complexes havebeen studied for nasal delivery of therapeutic proteins havebeen done [300–304]. It was found that insulin-loadedchitosan nanoparticles enhanced nasal absorption of pro-teins to a greater extent than relevant chitosan solutions[300,301,304]. Insulin-loaded nanoparticles were made byspray-drying a mannitol/lactose solution to yield ∼1–3 �mmicroparticle powders for alveolar deposition [305–307].The nanoparticles have a good loading capacity (65–80%)and a fast release of insulin [307]. An inhalable chitosan-based powder formulation of salmon calcitonin containingmannitol (as a cryoprotecting agent) was prepared by Yanget al. using spray-drying. The dissolution rate of the proteindecreased when formulated with chitosan, which might bedue to irreversible complex formation between the (aggre-gated) protein and chitosan during the drying process[308]. In a recent study, Ma and Liu prepared protein-loaded chitosan microspheres by a modified ionotropicgelation method combined with a high voltage electrostaticfield [309]. The morphology, particle size, encapsulationefficiency and in vitro release behavior of the preparedmicrospheres were investigated. Microspheres with gooddispersity and spherical shape were obtained from a mix-ture of TPP and ethanol was applied as the coagulationsolution. The encapsulation efficiency depended on theratio of BSA to chitosan with 35% of the BSA being releasedfrom the microspheres cured in 3% coagulation solution,and >50% of the BSA was released from the microspherescured in 1% coagulation solution. The combination of anionotropic gelation method with a high voltage electro-static field seems to be an effective method to fabricatechitosan microspheres for sustained delivery of protein.
Hu et al. polymerized acrylic acid with chitosan to formnanoparticles; the lower molecular weight, the better theyield (∼70%) [310]. The release of silk peptide occurredas an initial burst followed by prolonged release up to 10days. These nanospheres were suggested for pH dependent


F ally crop after so

dbiutcm(cpopTXntcon[

4f

priefoswtgf

ig. 16. (A) TEM images of chitosan–TPP nanocomplexes formed by ionichotos of (a) chitosan–TPP nanoparticles and (b) chitosan–TPP nanofibers

rug release applications in the gastric cavity. Chitosan-ased nanocomplexes were prepared by Zeng et al. by

onically cross-linking with TPP in different acidic mediander mild conditions. The self-assembly and ionic interac-ions of chitosan and TPP were affected by reaction media;hitosan-based nanofibers could be obtained in adipic acidedium while nanoparticles were formed in acetic acid

Fig. 16A). The in vitro drug release of BSA indicated thathitosan-based nanofibers and nanoparticles have similarrolonged release profile. The bioinspired mineralizationf both chitosan-based nanofibers and nanoparticles waserformed by soaking them in synthetic body fluids (SBF).ransmission electron microscopy (TEM) (Fig. 16B) and-ray diffraction (XRD) data indicate that chitosan-basedanofibers induced nanohydroxyapatite formation bet-er than chitosan-based nanoparticles. These biomimetichitosan-based systems have a controlled release capacityf bioactive factors that may be of use in bone tissue engi-eering to enhance the bioactivity and bone inductivity311].

.3.3. Chitosan-based systems for the delivery of growthactors

Chitosan hydrogels coupled with bone morphogeneticrotein (BMP)-7 have shown the ability to enhance lesionepair [312]. Chondroitin sulfate, a GAG molecule foundn cartilage, was immobilized in chitosan hydrogels tonhance cartilage formation [25]. A platelet derived growthactor has also been loaded into chitosan gels to enhancesteoinduction as the hydrogel degraded at the defect
ite [313,314] while chitosan–alginate hydrogels loadedith BMP-2 and MSCs were shown to induce subcu-
aneous bone formation [315]. Chitosan–laminin nerveuides loaded with glial cell line-derived nerve growthactor (GDNF) enhanced both the functional and sensory

ss-linking in (a) adipic acid medium and (b) acetic acid medium. (B) TEMaking in SBF at 37.0 ± 0.5 ◦C for 7 days [311]. Copyright 2009, Elsevier Ltd.

nerve recovery by releasing GDNF in the early stages ofimplantation [316]. Growth factors that have short thera-peutic half-lives, such as endothelial growth factor, requirefrequent administration to maintain an effective concen-tration [161]. Chitosan–albumin hydrogel microsphereshave shown continuous release for over 3 weeks after sub-cutaneous implantation in rats, indicating possible successfor in vivo applications [317].

4.3.4. Chitosan-based systems for the delivery ofantibiotics

The gastric residence time of tetracycline loaded chi-tosan microspheres (prepared by ionic cross-linking andprecipitation method) was examined following oral admin-istration in gerbils by Hejazi and Amiji [318]. The gastricretention was determined by administering radioiodinated[125I] chitosan microsphere in nonacid-suppressed andacid-suppressed states and then measuring the buildupof radioactivity in specific tissues and fluids. The tetra-cycline concentration profile in the stomach followingadministration of microsphere formulation was similar tothat of aqueous solution. The chitosan microspheres didnot prolong the residence time in the fasted gerbil stom-ach. Magnetic chitosan nanoparticles, as multifunctionalnanocarriers, were loaded with bleomycin and proved tobe a very effective as targeting system by Kavaz et al. [319].

In another study, the use of chitosan drug delivery to theinner ear across the “round window membrane” (RWM)was examined by Saber et al. [294]. Three structurallydifferent chitosans loaded with a tracer drug, neomycin
(C23H46N6O13), were injected into the middle ear cavity ofalbino guinea pigs (n = 35). After 7 days, the hearing organwas examined for hair cell loss and the RWM evaluatedin term of thickness. All chitosan formulations success-fully released the loaded neomycin which diffused across

olymer S
the RWM, and exerted a concentration dependent ototoxiceffect on the cochlear hair cells. Chitosans had no harmfuleffect on the cochlear hair cells and were determined to besafe and effective carriers for inner ear therapy.

4.3.5. Chitosan-based systems for the delivery ofanti-infammatory drugs

The loading and release study of triamcinoloneacetonide (TAA) [C24H31FO6], a drug used to reduceinflammation in the treatment of mouth ulcers, inchitosan–poly(acrylic acid) (PAA) membrane was inves-tigated by Ahn et al. [320]. The TAA in this membranewas found to be effective as a transmucosal drug deliv-ery system that responded to pH changes. Indomethacin(C19H16ClNO4) loaded chitosan microspheres were pre-pared as polyelectrolyte complexation of TPP and chitosanby Shiraishi et al. [117]. After oral administration ofchitosan gel beads to beagle dogs, the plasma exhib-ited a sustained release pattern for indomethacin. Asignificant correlation was observed between the molec-ular weight of chitosan and dissolution rate constantor the mean absorption time and the area under theplasma concentration–time curve. Another study reported,the preparation of indomethacin loaded chitosan micro-spheres using only aqueous solvents [321]. The influenceof formulation variables on indomethacin content in themicrospheres and time for release of indomethacin fromthe microspheres was assessed. Huang et al. observed thatbetamethasone disodium phosphate (C22H28FNa2O8P)loaded microspheres had good drug stability (<1% hydroly-sis product), high entrapment efficiency (95%) and positivesurface charge (37.5 mV) [322]. The results also indi-cated that yield and size of particles was increased withincreasing betamethasone amount but both zeta poten-tial and density of the particles decreased with increasingbetamethasone loaded amount. The in vitro release had adose-dependent burst, followed by a slower release thatwas drug loading between 5% and 30% (w/w) [323].

A novel inorganic–organic pH-sensitive membranebased on an interpenetrating network utilizing inorganicsilicate and organic chitosan was proposed by Park et al.[324]. Percolation of lidocaine-HCl (C14H22N2O), sodiumsalicylate (C7H5NaO3) and 4-acetomidophenol (C8H9NO2)into this membrane was studied and found to be sensitiveto the external pH as well as the ionic interactions of drugswith chitosan. The membrane was also sensitive to otherstimuli such as temperature and light that could serve asalternate routes for drug loading. In a similar approach, thein situ light initiated polymerization of acrylic acid in thepresence of chitosan was used to derive a novel mucoad-hesive membrane [320]. The interactions between the twopolymers were determined to be based on hydrogen bond-ing and the strong adhesive property of this membranerendered it suitable for transmucosal drug delivery appli-cations.

4.3.6. Chitosan-based systems for vaccines deliveryChitosan-based powders and micro/nanoparticles for

parenteral and mucosal delivery of antigen vaccines havebeen studied [325–344]. Immunizations with variousantigens co-administered with chitosan produced both

cience 36 (2011) 981–1014

systemic and local immune responses. In a phase I clini-cal study, intranasal immunization with influenza vaccineformulated with soluble chitosan glutamate showed posi-tive effects [345]. After intraperitoneal administration inmice and guinea pigs, the adjuvant activity of chitosanand its precursor chitin, were investigated in terms ofinduction of cytokines, long-lasting circulating antibodiesand cell-based immunity against bacterial alpha-amylaseand Escherichia coli infection [346,347]. In another study,chitosan induced cytokines, interleukin (IL)-1 and colony-stimulating factor (CSF) in macrophages in vitro [348].Zaharoff et al. found that chitosan dissolved in buffer at pH6.2 enhanced the immunoadjuvant properties of cytokinessuch as granulocyte-macrophage colony-stimulating fac-tor (GM-CSF), when co-administered subcutaneously.Chitosan seems to slow the dissemination of GM-CSF atthe site of injection which prolongs its exposure andenhances the immunoadjuvant properties of GM-CSF. Aftera single subcutaneous injection of GM-CSF/chitosan solu-tion, the cytokine expanded lymph nodes were about5-fold greater than GM-CSF injected four times alone.The chitosan may also be enhancing the antigen capa-bility to present dendritic cells (DCs) and induce greaterallogeneic T-cell proliferation [349]. It was found thatsoluble chitosan substantially increased antigen-specificantibody titers and antigen-specific CD4+ proliferationupon subcutenous administration of an aqueous solu-tion of �-galactosidase, as a mode antigen and chitosan.The authors suggested that the ability of soluble chi-tosan to enhance humoral and cell-mediated immunityis related to it’s physicochemical characteristics thatincreases the retention of formulations at the injectionsites and to induce transient cellular expansion in drain-ing lymph nodes [350]. Ghendon et al. demontrated thatintramuscular administration of soluble chitosan withmonovalent and trivalent split inactivated influenza vac-cine gave strong humoral and cell-immunity responsesagainst different variants of A- and B-type human influenzaviruses [351,352]. The soluble chitosan mixed with inac-tivated influenza vaccine increased cytotoxic activity ofthe splenic NK T-lymphocytes and enhanced the prolifer-ative activity of mononuclear lymphocytes in the spleen.Moreover, it was shown that the number of CD3, C3/NKand CD25 T-cells also increased. The chitosan may beactivating cell immunity because of its proliferation activ-ity, initiated through the receptor complex TCR–CD3 aswell as activation signals linked with lectin receptors[352].

4.3.7. Chitosan membranes in drug releaseThe interaction of oxidized glucose dialdehyde with chi-

tosan is another method of generating chitosan membranes[353]. N-alkyl groups of varying chain lengths were used tomodify the hydrophobicity of these chitosan membranes;the longer the alkyl chain, the more hydrophobic the chi-tosan membrane which affected the drug release rate. The
permeation and diffusion of B2 vitamin as the drug modeldecreased with increasing pH 8; above pH 8 the hydropho-bicity of the chitosan increased. In-vitro studies showed notoxic effects for this membrane system. Chitosan–gelatinfilms entrapped danshen, an herbal extract, for delivery in

olymer S

t[

otdsmtTtpaete

4

tngfpccl

aTttioawbfociptS3bwsnolstttwedwa


he abdominal cavity without the need for cross-linking354].

Recently asymmetric chitosan membranes were devel-ped for the guided tissue regeneration (GTR) by usinghe two-step phase separation [355]. The liquid–liquidemixing by non-solvent induction formed bicontinuoustrucutures and the pore size ranged from 0.5 to 2 �m. Theembrane showed good biocompatibility, tissue integra-

ion, cell occlusivity and osteoconduction, for 3 months.he chitosan membrane prevented bacterial proliferation,hat was significantly superior to current commercial GTRroducts for now. The asymmetric structure would beppropriate for the release of complex drugs with differentffective time periods. These asymmetric structures appearo be capable of releasing complex drugs with differentffective time periods.

.4. Chitosan in gene therapy

Many nucleic acid delivery vehicles have been inves-igated due to the low transfection efficiency of nakeducleic acid injection in vitro and in vivo. Chitosan-basedene delivery systems have been proven to be effectiveor non-viral gene therapy [125]. The chitosan–DNA com-lexes are very easy to synthesise and are more effectiveompared to the commonly used polygalactosamine–DNAomplexes, however, their use is limited because of theower transfection efficiency [356].

Various factors affecting chitosan delivery of nucleiccids were studied by Mao et al. [357] and Sato et al. [358].he molecular weight of chitosan, the charge ratio betweenhe luciferase plasmid to chitosan and the pH of the cul-ure media were found to be the factors related to then vitro transfection efficiency. Chitosan enhanced aden-virus infectivity to mammalian cells in gene therapy [359]nd low concentration and low molecular weight chitosansere better in enhancing adenovirus activity [360]. The sta-

ility of the DNA–chitosan complexes depends on severalactors such as the chitosan chain length and the amountf chitosan. Increasing the chitosan molecular weight andhitosan concentration yielded more stable complexes,ndicating that varying the chitosan chain length mayrovide a tool for controlling the ability of the polyplexo deliver therapeutic gene vectors to cells [361,362].pherical chitosan/DNA nanoparticles with an average8 nm diameter were prepared by using a simple osmosis-ased process [363]. About 30% DNA was incorporatedith prolonged release time. By varying the solvent/non-

olvent couple, temperature and membrane cut-off, severalanostructured systems of different size and shape werebtained and used for several biomedical and biotechno-ogical applications. Using a novel technique, Dai et al.tudied the gene delivery by chitosan–DNA nanoparticleshrough retrograde intrabiliary infusion (RII) and examinedhe efficacy of liver specific targeting [364]. The transfec-ion efficiency of chitosan–DNA nanoparticles, compared
ith poly(ethylenimine) (PEI)–DNA nanoparticles, was
valuated in Wistar rats by infusing into the common bileuct, portal vein, or the tail vein. Luciferase expressionas not detected when chitosan–DNA nanoparticles were

dministrated through the portal vein or tail vein, however,

cience 36 (2011) 981–1014 1003

the rats that received chitosan–DNA nanoparticles had 500times higher luciferase expression in the liver 3 days afterRII; and transgene expression levels decreased graduallyover 14 days. Luciferase expression in the kidney, lung,spleen, and heart was negligible compared with that in theliver. RII of chitosan–DNA nanoparticles did not have anysignificant toxicity or damage to the liver and biliary tree.Luciferase expression by RII of PEI–DNA nanoparticles was17-fold lower than that of chitosan–DNA nanoparticles onday 3, but then it increased slightly over time. These resultssuggest that gene delivery by chitosan–DNA nanoparticlesthrough RII is a possible routine to achieve liver-targetedgene delivery; both gene carrier characteristics and modeof administration significantly influence gene delivery effi-ciency.

In an attempt, to track the efficiency of DNA deliv-ery, Lee et al. employed fluorescence resonance energytransfer (FRET) to monitor the molecular dissociation ofa chitosan/DNA complex with chitosan characterized bydifferent molecular weights [365]. Plasmid DNA and chi-tosan were labeled with Quantum Dots and Texas Red,respectively, and confocal microscopy and fluorescencespectroscopy were used to monitor the dissociation ofthe complexes. As the chitosan molecular weight in thechitosan/DNA complex increased the Texas Red-labeledchitosan gradually lost FRET-induced fluorescence light.This observation was also observed when HEK293 cellswere incubated with chitosan/DNA complex. This indicatedthat the dissociation of the chitosan/DNA complex wasgreater with the higher molecular weight chitosan/DNAcomplex. Fluorescence spectroscopy analysis confirmedthat the molecular dissociation of the chitosan/DNA com-plex at pH 7.4 and 5.0 and confirmed that the dissociationoccurred in acidic environments. Therefore, it appears thatthe high molecular weight chitosan/DNA complexes disso-ciate better in lysosomes than the low molecular weightcomplexes. Furthermore, the high molecular weightchitosan/DNA complex showed superior transfection effi-ciency in relation to the low molecular weight complex.It was concluded that the dissociation of the chitosan/DNAcomplex is a critical event in obtaining the high transfectionefficiency of the gene carrier/DNA complex [365].

Chitosan/pluronic hydrogels as injectable systems forgene therapy to enhance local transgene expression atthe site of injection were prepared by Lee, Kim andYoo. Transfection studies employing HEK293 cells showedthat released fractions from chitosan/pluronic hydrogelsshowed better transfection efficiency than those frompluronic hydrogels [192].

In another study, Khatri et al. investigated the prepa-ration and in vivo efficacy of plasmid DNA(pDNA) loadedchitosan nanoparticles for nasal mucosal immunizationagainst hepatitis B. Chitosan/pDNA nanoparticles were pre-pared using a complex coacervation process [366]. Thechitosan nanoparticles produced humoral (both systemicand mucosal) and cellular immune responses upon nasal
administration. This study signified the potential of chi-tosan nanoparticles as DNA vaccine carrier and adjuvant foreffective immunization through non-invasive nasal route.Even though, the conventional high molecular chitosanshave a few drawbacks such as aggregation, low solubility


er (B–C)Copyrig

Fig. 17. MR images of the central region of mouse liver before (A) and aftat (B) 30 min and (C) 1 h after injection of the nanoparticles. L = left [369].

at neutral pH, and high viscosity at concentrations used forin vivo delivery and a slow onset of action [31], the non-viralgene delivery systems based on chitosan are still regardedas one of the most efficient system for DNA vaccine deliv-ery.

To develop chitosan nanoparticles for siRNA deliv-ery, Katas and Alpar, prepared chitosan nanoparticles bytwo methods of ionic cross-linking, simple complexationand ionic gelatin using TPP. In-vitro studies using twotypes of cells lines, CHO K1 and HEK 293, revealed thatthe preparation method of siRNA association to the chi-tosan plays an important role on the silencing effect.Chitosan–TPP nanoparticles with entrapped siRNA hadbetter vectors than chitosan–siRNA complexes; this maybe due to their high binding capacity and loading effi-ciency the chitosan–TPP nanoparticles appear to have goodpotential as viable vector candidates with safer and cost-effective siRNA delivery [367]. Exploring the efficiency ofchitosan/siRNA nanoparticles as a therapeutic agent, Liuet al. reported that the physicochemical properties andin vitro gene silencing of chitosan/siRNA nanoparticles arestrongly dependent on chitosan molecular weight and DD[368]. Chitosan with high molecular weight and high levelof DD resulted in the formation of discrete stable nanopar-ticles of 200 nm in size. Chitosan/siRNA formulations (N/P:50) prepared with low molecular weight chitosan (10 kDa)showed almost no knockdown of endogenous enhancedgreen fluorescent protein (EGFP) in H1299 human lungcarcinoma cells, whereas those prepared from highermolecular weight (65–170 kDa) and DD (80%) showedgreater gene silencing ranging between 45% and 65%.The highest gene silencing efficiency (80%) was achievedusing chitosan/siRNA nanoparticles at N/P 150 using highermolecular weight (114 kDa and 170 kDa) and DD (84%)that correlated with formation of stable nanoparticles of200 nm. From their studies it can be concluded that thereis still scope for improvement and for the optimizationof gene silencing using chitosan/siRNA nanoparticles andcertain improvements in the polymeric properties wouldmake significant differences.

4.5. Chitosan in bioimaging applications

Chitosan appears to be an exemplary polymer inbiological applications owing to its biocompatible prop-

injection of SPION-loaded WSC–LA nanoparticles. Images were obtainedht 2009, Elsevier Ltd.

erties. In this context, its use in bioimaging applicationsis also gaining rapid attention. The incorporation ofimaging agents is enabling its use for bioimaging, forexample, the incorporation of imaging agents such asFe3O4 for Magnetic Resonance Imaging (MRI) into theself-assembled nanoparticles could enhance hepatocyte-targeted imaging [369] and the particle could serve asMR molecular imaging agent. Several inorganic materialsincluding metals are being incorporated into the chitosancomposite preparations and their combined characteris-tics are proving beneficial for biomedical applications [19].Chitosan polyion complex composites can be preparedby interactions of chitosan with natural and syntheticpolyanion molecules [370]. PAA (Carbopol), an anionicsynthetic polymer having mucoadhesive properties, isextensively used with chitosan to form polymer compos-ites, which have longer circulation times in vivo, resultingin higher bioavailability of incorporated therapeutic agents[243,370–372]. These composite systems are being widelyinvestigated by incorporating contrast agents for imag-ing purposes. Preparing fluorescent chitosan quantum dotcomposites enables the combination of targeted drug andgene delivery with optical imaging [373,374]. Lee et al.have developed novel self-assembling nanoparticles com-posed of amphipathic water-soluble chitosan–linoleic acid(WSC–LA) conjugates for encapsulation of super param-agnetic iron oxide (SPIOs) as a contrast agent to targethepatocytes [369]. The WSC–LA conjugates self-assembledinto core–shell structures in aqueous solution. Since itsincorporation in nanoparticles, its potential for in vivomolecular imaging applications has increased tremen-dously (Fig. 17).

Chitosan-based gadolinium (Gd)-nanoparticles wereprepared by incorporating Gd complexed with diethyl tri-amine penta acetic acid (DTPA) using the emulsion-dropletcoalescence technique [6]. Their release properties andtheir ability for long-term retention of Gd-DTPA in thetumor indicated that these Gd nanoparticles might be use-ful as an intratracheal injectable device for gadoliniumneutron-capture therapy (Gd-NCT) [131]. The Gd loading
depended on the deacetylation of the chitosan used. Thehighest Gd load was achieved with 100% deacetylated chi-tosan in 15% Gd-DTPA aqueous solution, and the particlesize was 452 nm, whereas chitosan with lower deacetyla-tion level produced much larger particles with decreased

olymer S

Gtrdas

4

tgplinfmps[

ispsssIcofsabf[vTabItn

icftaaoocaatpsan


d-DTPA content. After an in vivo intratumoral injectionhe Gd-DTPA-chitosan nanoparticles displayed prolongedetention in the tumor tissue [375,376]. Kumar et al. alsoescribed the chemistry and preparations of Holmium-166nd Samarium-153 chitosan complexes, that are mainlyuited for radiopharmaceutical applications [6].

.6. Chitosan in green chemistry

Ionic liquids (ILs), have recently received much atten-ion as green solvents as a result of the development ofreen chemistry and the requirement for environmentalrotection [377]. The acceptance of ILs has come as a revo-

ution that has excited both the academia and the chemicalndustries. The terms room temperature ionic liquid (RTIL),onaqueous ionic liquid, molten salt, liquid organic salt and

used salt have all been used to describe ILs (salts in theolten state) [378]. The ILs have a unique physicochemical

roperties that are very useful when conventional organicolvents are not sufficiently effective or not applicable379].

Based on the combination of their unique properties,onic liquids have a number of advantages for synthe-is and extractions. These liquids have negligible vaporressure, a liquid range of up to more than 400 K and den-ities greater than water. Ionic liquids are miscible withubstances within a wide range of polarities, as well as,imultaneously dissolve organic and inorganic substances.n many cases, some processes would be impossible withonventional solvents because of their limited liquid ranger miscibility. Even greater potential is the use of RTILsor chemical synthesis because the charged nature of theseolvents can influence the synthesis itself. The most novelnd striking feature of RTILs as solvents is the possi-ility to design one with specialized properties neededor a specific application, known as “designer solvents”380]. Ionic liquids have been applied as alternative sol-ents in many catalytic organic transformations [378,381].hey efficiently dissolve biological macromolecules thatre linked by intermolecular hydrogen bonds, such as car-ohydrates, cellulose, wool keratin and silk fibroin [382]Ls are also being used to dissolve chitosan for waste waterreatment, cosmetics, heavy metal chelation, heteroge-eous catalysts.

Chitosan has one amino group and two hydroxyl groupsn the repeating hexosaminide residue (Fig. 1). During thehemical modification of any biopolymer, it is essential toorm a stable homogeneous solution in order to improvehe efficiency of modification. However, the strong inter-nd intramolecular hydrogen bonding between the chitinnd chitosan chains decreases their solubility in manyrganic solvents. Therefore, dilute aqueous solutions ofrganic and mineral acids solutions are used to dissolvehitosan. However, these solvents are corrosive and requiren alkaline solution treatment process to remove the acidfter the process. Furthermore, in certain applications like
ransition metal sorbents [383] and drug carriers [384], theolyelectrolyte solutions formed have limited applicationince bioactive agents may be affected by the presence ofcetic acid used for dissolving chitosan. Consequently, aew processing strategy for developing potential applica-
cience 36 (2011) 981–1014 1005

tions of these biorenewable resources is desirable [377].Xie et al., utilized 1-butyl-3-methyl-imidazolium chlo-

ride as a solvent for chitin and chitosan and used themas substitutes for amino-functionalized synthetic poly-mers for capturing and releasing CO2. To have efficientCO2 recovery methods from industrial waste gases isvery important to both reutilization of the CO2 as a car-bon resource as well as environmental issues related togreenhouse effects. Traditionally, the most commonly usedprocess for CO2 recovery is chemically reversible CO2 fixa-tion with amines at room temperature to form ammoniumcarbamates; the CO2 is then released from the ammoniumcarbamates by heating [385]. Amino-functionalized syn-thetic polymers and ionic liquids have also been developedto fixate CO2 based on this principle [386]. Chitosan is a nat-ural polyamine, and hence, could be a suitable alternativeamino-functionalized polymer for CO2 fixation. Althoughthe ionic liquids do not completely disrupt the crystallinedomains of chitosan and only a partially dissolved solutionis obtained, it apparently does not affect their utilization.Both the chitin/IL and chitosan/IL were effective for thereversible fixation of carbon dioxide. This environmentallyfriendly process has the potential not only for recoveringCO2 from industrial exhaust but also for CO2 sensing [377].

A novel polymer/RTIL composite material based onchitosan and 1-butyl-3-methyl-imidazolium tetrafluorob-orate (BMIM.BF4) was developed by Lu et al. The compositesystem could be readily used as an immobilization matrixto entrap proteins and enzymes. Hemoglobin (Hb) waschosen as a model protein to investigate the compositesystem. The bioactive composite film-modified glassy car-bon (GC) electrode was prepared by direct electron transferbetween the protein and the GC electrode. The film elec-trode exhibited dramatically enhanced bioelectrocatalyticactivity towards oxygen and trichloroacetic acid alongwith good stability in solution. Two well-defined quasire-versible redox peaks for hemoglobin were observed at thecomposite film-modified GC electrode. Thermogravimetricanalysis (TGA) indicates that the chitosan–BMIM.BF4–Hbcomposite had higher thermal stability than chitosan–Hbitself. These unique composite material could provide agood electrochemical sensing platform for redox proteinsand enzymes, and thus have potential applications in directelectrochemistry, biosensors, and biocatalysis by directelectron transfer [387].

A sensitive glucose biosensor, using an electrodeposit-ing chitosan–ionic liquid–glucose oxidase biocompositeonto nano-gold electrode, was fabricated by Zeng et al. Anano-gold electrode was constructed by electrochemicallydepositing gold nanoparticles onto a flat gold electrodesurface followed by immersing into a bath containing p-benzoquinone (BQ), chitosan, glucose oxidase (GOD) and ILfor electrodeposition of enzymatic electrode. This biosen-sor exhibits a fast amperometric response (<5 s) to glucosewith a high current sensitivity, that is 2.8 times better than abiosensor prepared by electrodepositing chitosan–IL–GOD
biocomposite on a flat gold electrode. The detection limitfor glucose was 20-fold better compared to the biosensorprepared on the flat gold electrode. The biosensor has highreproducibility, long-time storage stability and satisfactoryanti-interference ability [388].

olymer S
Chitosan/cellulose composites, as biodegradablebiosorbents, were prepared based on ionic liquids; nocross-linking agent and adsorption for heavy metal ionsby Sun et al. The freeze-dried chitosan/cellulose biosor-bent was indicated to possess higher adsorption capacitytogether with better stability. The interaction of the twocomponents and the resulting material’s adsorption capac-ity to Ni(II) were confirmed by IR and XPS. In addition,other heavy metal ions, were effectively adsorbed withthe following capacity Cu(II) > Zn(II) > Cr(VI) > Ni(II) > Pb(II)[389].

An ingenious approach for the fabrication of a promisingglucose sensor, GOx/C60-Fc-CS-IL, that exploits the syner-gistic benefits of fullerene (C60), ferrocene (Fc), chitosanand ionic liquid (IL) for glucose oxidase (GOx), was devel-oped by Zhilei et al. The electrocatalytic activity of C60and Fc remarkably improved the electron relays whichactivated the oxidation of the glucose and accelerated theelectrochemical reaction while the chitosan–IL networkprovided a favorable microenvironment that maintainedthe bioactivity of GOx. The biosensor exhibited a very highsensitivity, low detection limit, fast response time, widecalibration range and excellent long-term stability up to30 weeks [390].

Although chitosan is relatively non-toxic, biocompati-ble material, care must be taken to ensure that it is pureand free from contaminations, such as protein, metal orother contaminants that could potentially cause manydeleterious effects both in cross-linking approaches andin dosage forms. After each chemical reaction, care mustbe taken to thoroughly remove the unreacted reagents,particularity those that are cytotoxic, to prevent confusingresults.

5. Conclusions

Regenerative medicine has witnessed a significantprogress with the development of modern science andtechnology. Chitosan, with its exiciting properties, is oneof the most promising bio-based polymers for drug deliv-ery, tissue engineering, gene therapy and theranostics. Itis almost the only cationic polysaccharide in nature withsuch great innate medical potential. The various prepara-tion techniques presented herein can be helpful in decidingthe context of using chitosan in selectively capturing a ther-apeutic payload and control release in a target site as wellas for tissue engineering purposes. It has been documentedthat chitosan particle/hydrogels can be used as carriers forencapsulation and controlled release. There is a windowfor the preparative conditions to systematically manipulatethe incorporation process and to control the basic proper-ties, such as size and surface charge density, to develop asuccessful system.

Chitosan has been shown to improve the dissolutionrate of poorly soluble drugs, and thus, can be exploitedfor bioavailability enhancement of drugs and their deliv-
ery. Various therapeutic agents, such as anticancer,anti-inflammatory, antibiotics, antithrombotic, steroids,proteins, amino acids, antidiabetic and diuretics have beenincorporated in chitosan-based systems to achieve con-trolled release. A new application related to chitosan is
cience 36 (2011) 981–1014

based on the fact that chitosan particles provide an excel-lent template for bioimaging.

Chitosan’s tissue engineering potential as a biomate-rial to generate structures with predictable pore sizes anddegradation rates makes it particularly suitable for boneand cartilage regeneration. However, efforts to improve themechanical properties of chitosan-based composite bio-materials are essential for this type of application. Anothervery significant chitosan property is its capability to bindanionic molecules, such as growth factors, glucosamineglycans and DNA. The ability to link chitosan to DNAmolecules as a substrate for gene activated matrices makesthis material a good candidate in gene therapy applications.In fact, the combination of chitosan’s good biocompatibil-ity, intrinsic antibacterial activity, ability to bind growthfactors and to be processed into a variety forms, makesit an appropriate candidate as scaffold material for tissueengineering. Chitosan is also contributing to green chem-istry by being actively used with liquid solvents. The chiefapplication of this combination is in waste water treatment,heavy metal chelation, and biosensing. Although some ofthe parameters, such as molecular weight and viscosityhave to be considered in order to use chitosan at its fullpotential, the benefits are significant enough to make theeffort highly rewarding.

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