Chitosan-Modifications and Applications: Opportunities Galore PDF

Reactive & Functional Polymers 68 (2008) 1013–1051 Contents lists available at ScienceDirect Reactive & Functional Polymers j...

Reactive & Functional Polymers 68 (2008) 1013–1051 Contents lists available at ScienceDirect Reactive & Functional Polymers journal homepage: www.elsevier.com/locate/react Review Chitosan-modifications and applications: Opportunities galore V.K. Mourya *, Nazma N. Inamdar Department of Pharmaceutics, Government College of Pharmacy, Vedanta Hotel Road, Usmanpura, Aurangabad 431 001, India a r t i c l e i n f o a b s t r a c t Article history: Of late, the most bountiful natural biopolymer chitin and chitosan have become cynosure Received 19 January 2008 of all party because of an unusual combination of biological activities plus mechanical and Received in revised form 5 March 2008 physical properties. However applications of chitin are limited due to its inherent insoluble Accepted 6 March 2008 and intractable nature. Chitosan, alkaline hydrolytic derivative of chitin has better solubil- Available online 13 March 2008 ity profile, less crystallinity and is amenable to chemical modifications due to presence of functional groups as hydroxyl, acetamido, and amine. The chemical modification of chito- san is of interest because the modification would not change the fundamental skeleton of Keywords: Chitin chitosan, would keep the original physicochemical and biochemical properties and finally Chitosan would bring new or improved properties. In view of rapidly growing interest in chitosan its Chemical modification chemical aspects and chemical modification studies is reviewed. The several chemical Grafting modifications such as oligomerization, alkylation, acylation, quternization, hydroxyalkyla- Drug delivery tion, carboxyalkylation, thiolation, sulfation, phosphorylation, enzymatic modifications and graft copolymerization along with many assorted modifications have been carried out. The chemical modification affords a wide range of derivatives with modified proper- ties for specific end use applications in diversified areas mainly of pharmaceutical, biomed- ical and biotechnological fields. Assorted modifications including chitosan hybrids with sugars, cyclodextrin, dendrimers, and crown ethers have also emerged as interesting mul- tifunctional macromolecules. The versatility in possible modifications and applications of chitosan derivatives presents a great challenge to scientific community and to industry. The successful acceptance of this challenge will change the role of chitosan from being a molecule in waiting to a lead player. Ó 2008 Elsevier Ltd. All rights reserved. Contents 1. Introduction........................................................................................... 1014 2. Chemistry............................................................................................. 1014 3. Chemical characteristics................................................................................. 1015 4. Chemical modifications of chitin........................................................................... 1015 5. Chitosan and chemical modifications of chitosan............................................................. 1016 6. Chitosan oligomers...................................................................................... 1016 7. Chitosan derivatives of importance......................................................................... 1017 7.1. Quaternized chitosan and N-alkyl chitosan............................................................ 1017 7.2. Highly cationic derivatives......................................................................... 1019 7.3. Hydroxyalkyl chitosans............................................................................ 1020 7.4. Carboxyalkyl chitosans............................................................................ 1020 7.5. Sugar-modified chitosan........................................................................... 1022 * Corresponding author. Tel.: +91 95240 2346820; fax: +91 95240 2321130. E-mail address: [email protected] (V.K. Mourya). 1381-5148/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2008.03.002 1014 V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 7.6. Cyclodextrin linked chitosan........................................................................ 1024 7.7. N-Acyl chitosan.................................................................................. 1025 7.8. O-Acyl chitosan.................................................................................. 1027 7.9. Thiolated chitosan................................................................................ 1028 7.10. Sulfated chitosan................................................................................. 1034 7.11. Miscellaneous derivatives.......................................................................... 1034 7.11.1. Azidated chitosan....................................................................... 1034 7.11.2. Phosphorylated chitosan.................................................................. 1035 7.11.3. EDTA–chitosan......................................................................... 1036 7.11.4. Thiourea derivatives..................................................................... 1037 8. Enzymatic modification of chitosan....................................................................... 1037 9. Graft copolymers of chitosan............................................................................. 1038 10. Chitosan–dendrimer hybrid.............................................................................. 1042 11. Cyclic-host bound chitosan.............................................................................. 1045 12. Conclusion........................................................................................... 1045 References............................................................................................ 1046 1. Introduction O Chitin and chitosan are aminoglucopyrans composed OH NH2 OH NH of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) O HO O HO O O O O residues. These polysaccharides are renewable resources O HO NH O HO O which are currently being explored intensively for their NH O O OH applications in pharmaceutical, cosmetics, biomedical, OH biotechnological, agricultural, food, and non-food indus- Fig. 1. Structure of chitin. tries as well (water treatment, paper, and textile). These unique polymers have emerged as a new class of physiological materials of highly sophisticated functions due to their versatile biological activity, excellent bio- compatibility, and complete biodegradability in combina- OH OH NH2 NH2 tion with low toxicity [2–4]. To exploit the unique O HO O HO O O O O properties and to realize full potential of these versatile O HO NH O HO O polysaccharides, attempts are being made to derivatize NH2 O OH OH them. Fig. 2. Structure of chitosan. 2. Chemistry Chitin is the second most abundant natural biopolymer derived from exoskeletons of crustaceans and also from the packing and polarities of adjacent chains in successive cell walls of fungi and insect. Chitin is a linear cationic sheets. Generally, the individual chains assume an essen- heteropolymer of randomly distributed GlcNAc and GlcN tially linear structure, which undergoes one full twist, residues with b-1,4-linkage. Chitobiose, 4-O-(2-amino- every 10.1–10.5 Å´ along the chain axis. Because each glyco- 2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-2-amino-2-deoxy-D- sidic unit in the chain is chiral, and all units are connected glucose, is the structural unit of native chitin. Bound by an oxygen atom that links C-1 of one glycosidic unit to water is also a part of the structure. The degree of deacety- C-4 of an adjacent unit, a distinct ‘‘left” and ‘‘right” lation in chitin can be as low as 10 R O O O O O O O O HO NH2 HO NH2 O OH HOOC COOH O O O HO NH OH HOOC COOH Fig. 10. Reactions of chitosan with epoxide. Under certain conditions, substitution degrees higher than 2 may occur. in water, but has unique chemical, physical and biological Sashiwa et al. applied Michael reaction of various acryl properties such as high viscosity, large hydrodynamic vo- reagents with chitosan. With application of water- lume and film, gel-forming capabilities also, all of which soluble acryl reagents for this reaction, novel types of func- make it an attractive option in connection with its use tional groups were introduced by a simple procedure. The in food products and cosmetics [24,141]. Carboxymethyl reagents tried are hydroxyethyl acrylate, hydroxypropyl chitosan is used in development of different protein drug acrylate, acrylamide, acrylonitrile, PEG-acrylate. Reaction delivery systems as super porous hydrogels, pH-sensitive of chitosan with acrylonitrile gives cyanoethyl chitosan hydrogels, cross-linked hydrogels [142–145]. N,N-Dicar- whereas reaction of chitosan with ethyl acrylate in aqu- boxymethyl chitosan has shown to possess good chelat- eous acidic medium gives N-carboxyethyl ester intermedi- ing abilities and its chelate with calcium phosphate ate which can easily be hydrolyzed to free acid or used as favoured osteogenesis while promoting bone mineraliza- an intermediate to substitute with various hydrophilic tion. O-Carboxymethyl chitosan exhibits antibacter- amines, without requiring protecting groups. ial activity and modified adhesive properties for instance, The carboxyl bearing aromatic substitution can be done surface modification of tissue scaffolds of poly(lactide-co- with aromatic aldehydes. Lin et al. synthesized N-carboxy- glycolide acid) with O-carboxymethylchitosan enhances benzyl chitosan by reductive amination sequence with chondrocyte adhesion; surface modification of Dacron 2-carboxy benzaldehyde and cross-linked with glutaralde- vascular grafts enhances the blood compatibility. hyde to develop pH-sensitive hydrogel for colon specific Carboxymethyl chitosan and modified carboxymethyl drug delivery of 5-flurouracil. a-Keto acids such as chitosan at amino function with haexanoic, linoleic acid pyruvic acid (and its derivatives as b-hydroxypyruvic acid, have been employed as a carrier for delivering drugs as phenylpyruvic acid, 4-hydroxyphenylpyruvic acid), a-keto- gatifloxacin, camptothecin, ibuprofen, and adriamycin glutaric acid, levulinic acid are some of the other carbox- [148–151]. Ge and Luo reported preparation of carboxy- yaldehydes being employed for carboxyalkylation of methyl chitosan in aqueous solution under microwave ir- chitosan. Stable and self-sustaining gels are obtained from radiation. A higher homolog of carboxymethyl 4-hydroxyphenylpyruvic acid modified chitosan, i.e. tyro- chitosan, i.e. N-(2-carboxyethyl) chitosan was obtained sine glucan in the presence of tyrosinase. Similar gels are by reaction of chitosan and 3-halopropionic acids under obtained from 3-hydroxybenzaldehyde, 4-hydroxybenzal- mild alkaline condition and ambient temperature where dehyde, and 3,4-dihydroxybenzaldehyde: all of them are alkylation proceeds exclusively at the amino groups hydrolyzed by lysozyme, lipase, and papain. No cross-. This N-carboxyalkyl derivative was tested for anti- linking is observed for chitosan derivatives of vanillin, oxidant and antimutagenic activity. syringaldehyde, and salicylaldehyde. 1022 V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 O OH OH OH H O O O O O NaBH3CN O O HO N HO 1%AcOH NH COOH COOH Schiff base N-Carboxymethyl chitosan COOH OH O O 40% NaOH O O O HO NH2 ClCH2COOH O O 0-30 0C HO O-Carboxymethyl chitosan n NH2 Chitosan OH ClCH2COOH O O O N-Carboxymethyl chitosan methanol HO NH COOH O O X= ONa NH2 OH O NMe2 O N O + X aq.NaHCO3 Y- O O HO O O H2O,AcOH NH n OH O O OH X O O O 7 H OH OH O O COOEt O O O AcOH, H2O, EtOH HO HO NH N R= H, Et COOR COOR COOR Major Minor R'NH2, H20,EtOH OH OH O O O Z= OH, OEt, -NHR' O O HO HO N NH COZ COZ COZ Fig. 11. Carboxylation of chitosan depending on reaction conditions O-carboxylated, N-carboxylated, or N,O-carboxylated chitosan can be obtained. Ding et al. effectively modified chitosan into chitosan proach [161,162]. They synthesized sugar-bound chitosan a-ketoglutaric acid and hydroxamated chitosan a-ketoglu- by reductive N-alkylation using sodium cyanoborohydride taric acid. The modified chitosan were employed in the and unmodified sugar or sugar-aldehyde derivative. Sashi- formation of theophylline-loaded, iron(III)-cross-linked wa and Shigemasa reported N-alkylation of chitosan per- polymeric beads proven to be successful in prolonging formed in aqueous methanol with various aldehydes, drug release as well as in augmenting adsorption proper- monosaccharides, and disaccharides (glycolaldehyde, DL- ties [159,160]. glyceraldehyde, D-ribose, D-arabinose, D-xylose, 2-deoxy- D-ribose, D-gulcose, 2-deoxy-D-glucose, 3-O-Me-D-glucose, 7.5. Sugar-modified chitosan D-galactose, D-mannose, L-fucose, L-rhamnose, GlcNAc). Initially, the sugar-bound chitosans had been investi- Reductive N-alkylation is a valuable process in chitosan gated mainly for rheological studies; but since the specific chemistry (Figs. 12 and 13). Hall and Yalpani were the first recognition of cells, viruses, and bacteria by sugars was to report sugar-modified chitosan derivatives by this ap- discovered, this type of modification has usually been used  V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 1023 Lactose OH OH OH OH O O O O O OH HO O HO HO OH OH NH 1. OH OH OH OH O O HO OH HO 2. NaCNBH3 OH OH OH OH O O O O OH O CHO HO OH HO 1. OH NH O O n O O O OH OH HO HO O NH NH 2. NaCNBH3 O HO OH n Lactobionic acid OH OH OH OH OH O COOH O O O O HO HO OH HO OH HN OH EDC, NHS, RT, 72 hrs OH OH OH O O O HO HO OH OH Fig. 12. Synthesis of sugar linked chitosans. to introduce cell-specific sugars into chitosan. Morimoto et receptors can not only bind galactose-bearing ligands, al. reported the synthesis of sugarbound chitosans, such as but can internalize them within membrane bound vesicles those with D- and L-fucose, and their specific interactions or endosomes. Akin specificity is observed for synthesized with lectin and cells [163–166]. Stredanska and co-work- mannosyl-chitosan for antigen presenting cells as macro- ers synthesized lactose-modified chitosan for a potential phages and dendritic cells. Sashiwa et al. prepared application in the repair of the articular cartilage by the sialic acid bound chitosan as a new family of sialic acid same mode. Kato et al. also prepared lactosaminated containing polymers using p-formylphenyl-a-sialoside N-succinyl-chitosan and its fluorescein thiocarbanyl deri- by reductive N-alkylation. Since sialic acid vative as a liver-specific drug carrier in mice through asia- bound chitosan was insoluble in water, successive N-succi- loglycoprotein receptor. Moreover, lactosaminated nylations were carried out to obtain the water-soluble de- N-succinylchitosan was found to be a good drug carrier rivative N-succinyl-sialic acid bound chitosan. Specific for mitomycin C in treatment of liver metastasis. binding of wheat germ agglutinin with lectin was shown Galactosylated chitosan prepared from lactobionic acid in the presence of N-succinyl-sialic acid bound chitosan. and chitosan with 1-ethyl-3-(3-dimethylaminopropyl)- Water-soluble a-galactosyl chitosan prepared by the same carbodiimide (EDC) and N-hydroxysuccinimide (NHS) strategy as sialic acid showed specific binding against a ga- showed promise as a synthetic extracellular matrix for he- lactosyl specific lectin (Griffonia simplicifolia). The patocyte attachment. Furthermore, graft copolymers different type of spacer has been prepared on sialic acid of galactosylated chitosan with poly(ethylene glycol) or or a-galactosyl epitope bound chitosans. These epi- poly(vinyl pyrrolidone) and dextran were useful as hepato- tope bound chitosans may be useful as potent inhibitors cyte-targeting DNA carriers since PEG reduces particle of influenza viruses or blocking agents for acute rejection sizes of complexes and PVP prevents albumin from interac- [179,181]. tion with complexes [171–173]. The quaternized galaocto- A larger carbohydrate as amylose can be introduced on sylaed chitosan too hold the cellular recognition ability and chitosan by chemoenzymatic method as accomplished by possibility of gene delivery [174,175]. Such selective tar- Kaneko et al.. In this, grafting of maltoheptaose to geting of the substrate delivery to hepatocyte cells is feasi- chitosan by a reductive amination using sodium cyanobor- ble because hepatocytes are the only cells that possess ohydride in a mixed solvent of 1 M aqueous acetic acid– large numbers of high-affinity cell-surface asialoglycopro- methanol at room temperature preceded the phosphory- tein receptors that can bind to asialoglycoproteins. These lase-catalyzed enzymatic polymerization of a-D-glucose 1024 V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 OH OH O O O O O HO HO NH 2 NH 2 OH CHO OH COONa NaCNBH 3 HO O O AcOH, H 2 O, MeOH AcNH RT, 1 day HO OH OH OH OH O O O O O O O O O O HO HO HO HO NH NH2 NH NH OH O COONa OH HO O O Water insoluble COONa O AcNH HO O O O O HO O OH HO 1.AcOH, H2O, MeOH, RT, 1 day. HO HO 2. 0.5M NaOH,RT, 2 hrs HO OH OH O O alpha-galactosyl succinyl chitosan O O O Water soluble HO HO NH NH OH COONa O OH HO O O AcNH HO COONa Sialic acid -succinyl-chitosan Water soluble Fig. 13. Synthesis of sialic acid–chitosan and a-galactosyl chitosan and their N-succinylation. 1-phosphate. The functionality of maltoheptaose to chito- naudo also reported similar synthesis of chitosan bearing san depended on reaction time where as the average de- pendant CD through reductive amination with the studies gree of polymerization of amylose graft chains depended of formation inclusion complexes with 4-tert-butyl benzoic on the feed ratios of a-D-glucose 1-phosphate to maltohep- acid , or of supramolecular assemblies with adaman- taose primers. The amylose-grafted chitosan does not dis- tyl groups linked on the chitosan backbone. The solve in any solvent, e.g., aqueous acetic acid and CD-chitosan derivative prepared similarly with CD mono- dimethyl sulfoxide, which are good solvents for chitosan aldehyde has been evaluated for mucoadhesion by the and amylose, respectively. Further studies on the mechan- same team. Chen and Wang obtained CD- ical properties and applications of the present material are linked chitosan using tosylated b-CD and further evaluated underway. the potential of b-CD for the release of I-131 in vivo and improved solubility. Georgeta et al. allowed the reaction 7.6. Cyclodextrin linked chitosan of chitosan microspheres, obtained through cross-linking with glutaraldehyde with chloroacyl CDs in organic basic Chitosans bearing cyclodextrin (CD) pendant are devel- solvents. The higher amounts of acyl CD are linked to the oped with an aim to combine unique characteristics of microspheres through spacer and C–N bonds with a smal- chitosan with the potential of CD to form non-covalent in- ler cross-linking degree. The inclusion efficiency was clusion complexes with a number of guest molecules alter- checked with nalidixic acid, piroxicam, and p-nitrophenol ing their physicochemical properties for improved drug. The CD-linked chitosan could also be prepared by delivery system, cosmetics, and analytical chemistry the monochlorotriazinyl derivative of CD. Triazinyl moiety [183,184]. There are different means to link cyclodextrin acts as a spacer. This compound was used for decon- to chitosan (Fig. 14). Sakairi and co-workers [184,61,185] tamination of waters containing textile dyes. El-Tahlawy et prepared a-CD-linked chitosan using 2-O-formylmethyl- al. used a novel technique for preparation of b-CD grafted a-CD by reductive N-alkylation and confirmed the host– chitosan of reacting b-CD citrate with chitosan dissolved guest complex of with p-nitrophenol. Auzely-Velty and Ri- in formic acid solutions and evaluated these polymers as  V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 1025 i) Formylmethylene CD CD Chitosan- Epoxy activated Chitosan-CH2-CH2-CD ii) NaCNBH3 2-hydroxypropyl-CD chitosan (2-OH) Tosylated CD Chitosan-CD (2-OH) Aminated CD Succinyl Chitosan Chitosan-succinic acid-CD chitosan Monochlorotriazinyl CD Chitosan-triazinyl-CD (2,3 or 6-OH) Chloroacyl CD CD-citrate or itaconate Chitosan- Glutaraldehyde Chitosan-citricacid/itaconic acid CD glutarldehyde-CD chitosan Fig. 14. Cyclodextrin linked chitosan: (1) by the reductive amination using formylmethylene CD, (2) by using tosylated CD, (3) by the nucleophilic substitution reaction using monochlorotriazinyl derivative of CD, (4) via epoxy-activated chitosan, (5) by using redox aminated CD (mono-6-amino-mono- 6-deoxy-b-cyclodextrin), (6) by the condensation of CD-citrate or itaconate with chitosan, (7) cross-liking of CD and chitosan by glutaraldehyde. antimicrobial agents. They also reported analogous ble polymers have emerged as a new class of industrially synthesis with b-CD-itaconate and chitosan along with its important macromolecules. Some of these are intended utility as ion exchange resin. The b-CD linked chito- to mimic the endotoxins. The introduction of hydro- san using 1,6-hexamethylene diisocyanate as spacer was phobic branches also endows the polymers with a better also prepared by Sreenivasan [194,195]. This material soluble range than chitosan itself. Zong et al. synthesized interacts with cholesterol and might be useful as an acylchitosan with longer chains by reacting chitosan in adsorbent. The spacer can be 2-hydroxypropyl moiety pyridine/chloroform with hexanoyl, decanoyl, and lauroyl introduced by grafting b-CD onto chitosan using epoxy- chlorides. These acylated chitosans with 4 degree of substi- activated chitosan. The spacer can be a reducing tution per monosaccharide ring (disubstitution at amino sugar derivative. Aime et al. functionalized and monosubtitution each at hydroxyl groups) exhibited CD by means of a maleic spacer, whose free carboxyl group an excellent solubility in organic solvents such as chloro- is subsequently activated with a carbodiimide to form form, benzene, pyridine, and THF. The analyses indicate amide linkages with amino groups of chitosan. The regios- that these polymers form a layered structure in solid state electivity of the coupling could be accurately controlled if and the layer spacing increases linearly with increasing the the 6-monotosyl-CD derivative is used as substrate for nu- length of side chains. The presence of such layered cleophilic substitution with sodium maleate. An insoluble structure was elucidated with N-aliphatic acyl chitosans cross-linked chitosan bearing b-CD was prepared using and N-aliphatic-O-dicinnamoyl-chitosans with acyl as N-succinyl chitosan and aminated-b-CD (mono-6-amino- acetyl, butyryl, octanoyl, lauroyl and stearoyl moieties. mono-6-deoxy-b-cyclodextrin) via amide bond formation None of the polymers belonging to the N-aliphatic acyl ser- in the presence of the water-soluble 1-ethyl-3-(3-dimethy- ies could be dissolved in the solvent systems investigated laminopropyl) carbodiimide (EDC) under homogeneous (CHCl3, CH2Cl2, THF, (Me)2CO, DMAc, DMF, DMSO, DMSO/ conditions. CHCl3). The reason for the striking stability of N-aliphatic acyl against solvents is obviously due to the compact ar- 7.7. N-Acyl chitosan rangement of both the main chains and the side chains of N-aliphatic acyl to form a crystal with strong hydrogen N-Acyl derivatives of chitosan can be easily obtained bond interactions together with strong interactions be- from acyl chlorides and anhydrides (Fig. 15). In a general tween closely packed hydrophobic side chains. On the way, acylation reactions lead frequently in mediums as other hand, the polymers belonging to the series of N-ali- aqueous acetic acid/methanol, pyridine, pyridine/chloro- phatic-O-dicinnamoyl-chitosans displayed solubilities form, trichloroacetic acid/dichloroethane, ethanol/metha- strongly related to the length of the flexible side chains. nol mixture, methanol/formamide or DMA–LiCl. In general, increasing length of the flexible side chains re- Due to fairly different reactivities of the two hydroxyl duced the solubility. and the amino group on the repeating unit of chitosan, acy- N-Acylated chitosans with saturated (e.g. C2–C18) and lation can be controlled at the expected sites, i.e. on either unsaturated acyl groups of different chain length (e.g. oleic, amino [200–202], hydroxyls , or on both groups linoleic, elaidoic, erucoyl) as well as aromatic acyl groups [204–207]. The introduction of hydrophobic branches gen- (e.g. phthaloyl, p-nitrobenzoyl, cinnamoyl) had been suc- erally endows with new physicochemical properties such cessfully synthesized to obtain randomly distributed sub- as the formation of polymeric assemblies, including gels stituents in a controlled amount along the chitosan chain , polymeric vesicles , Langmuir–Blodgett films [219–222]. Cyclic acid anhydrides too are used for acyla- [210,211], liquid crystals [212,213], membranes , tion purpose via ring-opening reactions giving N-carboxya- and fibers [215,216]. Hydrophobic associating water-solu- cyl chitosans (e.g. succinic, maleic, glutaric, itaconic, 1026 V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 O O or O OH R O R R Cl O O CH3OH CH3COOH O HO NH R O Acyl chitosan O OH R O OH O O O O Cyclic anhydride O O O HO HO NH NH2 R COOH O Carboxyacyl chitosan OH O O Lactone O HO NH R OH O Hydroxyacyl chitosan OH RCOOH O O Carbodiimide O HO NH O R Acyl chitosan Fig. 15. Acylation of chitosan. phthalic, cis-1,2,3,6-tetrahydrophthalic, 5-norbornyl-endo- amphiphatic hydrogel with excellent water-absorption 2,3-dicarboxylic, cis-1,2-cyclohexyl dicarboxylic, trimellitic and water retention abilities under neutral conditions anhydride, (2-octen-1-yl)succinic, citraconic, trimellitic, and then employed as a carrier for delivering amphiphatic pyromellitic) [39,216]. Addition reactions of lactone with agents. The hexanoyl substitution improves signifi- chitosan offers hydroxyl substituted carboxylchitosan by cantly the water-absorption ability of hydrogel by altering acylation. the number of water-binding sites under low humidity One of the methods of obtaining acylated chitosan deri- and the state of water in fully swollen state, retards water vatives includes the thermolysis of its acylammonium mobility during deswelling, and enhances amphiphatic salts in the solid state. This method was used to pre- drug encapsulation efficiency compared to pristine chito- pare chitosan amides derived from acids, such as acetic, san. The acylated chitosan are being applied for stabiliza- acrylic, methacrylic, trifluoroacetic, and myristic. tion of nanoparticles as iron oxide, and gold [226,227]. Initially the chitosan derivatives were prepared for exam- N-Succinyl-chitosan obtained by introduction of succi- ining physicochemical properties which subsequently nyl groups into N-terminal of the glucosamine units of amended into applications. Mi et al. prepared biodegrad- chitosan could be modified easily with respect to succiny- able N-acylchitosan microspheres by water-in-oil (w/o) lation degree by changing reaction conditions and the mo- interfacial N-acylation method for controlled release of lecular weight using hydrochloric acid [228,229]. Although 6-mercaptopurine using acetic, propionic and n-butyric N-succinyl-chitosan was initially developed as wound anhydrides as reagents for the interfacial N-acylation dressing materials , it is currently also applied as cos- reaction. N-Acylation of chitosan with longer chain metic material. New wound dressings composed of acid (C6–C16) chlorides increased its hydrophobic character N-succinyl-chitosan and gelatin were also developed (hydrophobic self-assembly) and made important changes. N-Succinyl-chitosan has unique characteristics in vi- in its structural features. It was reflected in improved tro and in vivo due to many carboxyl groups. For example, mechanical properties of tablets prepared using these ordinary chitosan can be dissolved in acidic water but not derivatives. The release characteristics of the drug sug- in alkaline, whereas N-succinyl-chitosan with high degree gested that release is controlled by diffusion or by swe- of substitution (degree of succinylation: >0.65) exhibits lling followed by diffusion, depending on both the acyl the opposite behaviour. N-Succinyl-chitosan can chain length and the degree of acylation. Hexanoyl easily react with many kinds of agents due to –NH2 and chitosan with carboxymethylation was developed into –COOH groups in its structure. It is valuable as the drug  V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 1027 carrier to readily prepare its conjugates with various drugs droxyl group (Fig. 16). This approach was used to prepare to avoid vexatious complications. The water-insoluble and N-chloroacyl 6-0-triphenylmethyl chitosan which can be water-soluble drug conjugates could be prepared using a further substituted or quaternized with amines as pyri- water-soluble carbodiimide and mitomycin C or using an dine, imidazole, triethylamine, tributylamine, N-chlorobe- activated ester of glutaric mitomycin [233–235]. N-Succi- tainyl chloride [242–244]. The betaine derivatives have nyl chitosan, which can form self-assembly of well-dis- two major advantages over the parent chitosan: (i) they persed and stable nanospheres in distilled water, shows are water-soluble at physiological pH, and (ii) they have great potential in the drug controlled release delivery a permanent positive charge on the polysaccharide. It was used for preparation of oxymatrine nanopar- backbone. ticles. The use of condensing agent as carbodiimide for N-acy- The succinyl chitosan can be adapted for solubility by lation chitosan has been done with amino acids (lysine, ar- addition of a long alkyl moiety as hydrophobic function ginine, aspartic acid, phenylalanine). The amino acid to the amino group given that succinyl moiety provides hy- functionalized chitosan moieties were subsequently en- drophilic and alkyl moiety provides the hydrophobic prop- trapped onto PLA surfaces which demonstrated good erties. The adapted derivatives can form micelles in cyto-compatibility to chondrocytes. aqueous media, and was used as carriers for the anticancer When chitosan and glycol chitosan were acylated with drug doxorubicin. The introduction of lactose to N- deoxycholic acid and 5b-cholanic acid by this method, succinyl chitosan by reductive amination conferred a li- self-assembling hydrophobic macromolecules were ver-targeting ability in normal or tumour-bearing mice obtained which complexed with DNA with enhanced since the liver parenchymal cells have asialoglycoprotein transfection efficiency due to increase of cell membrane– receptors, which specifically recognize the galactose carrier interactions and/or destabilization of cell [169,168]. A series of hydrophobically modified chitosans membrane [246,247]. DNA delivery can be targeted to N-/2(3)-(dodec-2-enyl)succinoyl/chitosans were prepared cancerous cells with use of chitosan acylated by folic acid by reacting chitosan with 2-(dodecen-1-yl) succinic anhy- [248,249]. The reason for such specificity is that folic acid dride (SIGMA) as a new class of potential non-viral vectors being a ligand for folic acid receptors on binding undergoes for gene delivery. edocytosis moreover these receptors are over-expressed Hu et al. prepared N-acylated chitosan as N-acetyl, N- on many human cancer cell surfaces. Similar effects propionyl and N-hexanoyl with different degrees of substi- are seen with chitosan acylated by uraconic acid due to tution and evaluated in vitro for antibacterial activity. The facilitation of endocytic uptake. results showed that intermolecular aggregation character- istic of N-acetylated chitosans with low DD may help in 7.8. O-Acyl chitosan forming bridge to interact with bacterial cell. Chito- san was N-acylated with butanoic, hexanoic and benzoic Introducing a hydrophobic moiety with an ester linkage anhydride under homogeneous conditions in the presence into chitosan has two benefits: (i) hydrophobic groups con- of methanol. The nanoparticles prepared from N-acyl chit- tribute organosolubility; (ii) the ester linkage is hydrolyzed osan were blood compatible. by enzyme like lipase, etc. (Moreover, the glycoside linkage The acylation can be achieved regioselectively at amino of chitosan derivatives is also degraded by glycosidases.) group by using protection as trityl group at the primary hy- Therefore, chitosan derivatives with O-acyl groups are OH OH O Phthalic O O anhydride O O O O Trityl chloride O NH2 NH2. H2O O O HO HO HO N DMF/H 2O, 120 o C N Pyridine, 90 o C NH2 O O O O N-phthaloyl chitosan N-phtaloyl-O-triphenylmetyl chitosan OH O O O RCOCl, Pyridine, RT Aq. HCl,RT O O O O O O O HO O N-Acylation HO HO NH NH NH2 O R O R O-Triphenylmethyl chitosan N-acyl chitosan N-acyl O- triphenylmethyl chitosan Fig. 16. Regioselective acylation of chitosan. 1028 V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 designed as biodegradable coating materials. The success- by suitable method as by using hydrazine hydrate. Re- ful preparation of N,O-acyl chitosan in methanesulfonic cently a one-pot synthesis for the O-acylation of chitosan acid MeSO3H as solvent was performed (Fig. 17) in MeSO3H is also reported. [203,251]. Although the selective O-acylation of chitosan in MeSO3H owing to the salt formation of primary amino 7.9. Thiolated chitosan group with MeSO3H was partly reported, the detailed che- mical structure and the protecting effect of MeSO3H on The derivatization of the primary amino groups of chit- amino group are not clear yet. The preparation of osan with coupling reagents bearing thiol functions leads O,O-didecanoylchitosan, O-succinyl chitosan was also re- to the formation of thiolated chitosans (thiolated polymers ported through protected N-phthaloylchitosan as an inter- as chitosan, polycarbophil or so-called thiomers are hydro- mediate [252–254]. This method, however, needs several philic macromolecules exhibiting free thiol groups on the steps as the protection of the amino group by phthaloyla- polymeric backbone). So far, four types of thiolated chito- tion, O-acylation, and finally removal of protecting group sans have been generated: conjugates as chitosan–cy- Major OR OH OH R'COCl, MeSO3 H NaHCO3 O O O O O O O O O HO RT,5hrs HO RO NHR NH2 NH3+ R= H, COR', MeSO3 Minor Minor Chitosan Acyl chitosan Fig. 17. O-Acylation of chitosan. OH O O NH 2 O O HO NH HO SH HS Chitosan-Cysteine conjugate EDC O NH 2 OH O O O O HO SH HO NH Chitosan-Thioglycolic acid conjugate OH HS EDC O O O O HO NH 2 OH O O O S NH HO Chitosan-4-Thiobutylamidine conjugate NH HS NH 2+Cl- O NH.HCl OH S O O O O HO Chitosan-Thioethylamidine conjugate NH HS NH 2+Cl- Fig. 18. Synthesis of thiolated chitosan.  V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 1029 steine, chitosan–thioglycolic acid, chitosan–4-thiobutyla- sitively charged amidine substructure. The thiol midine, and chitosan–thioethylamidine conjugate (Fig. 18). group of the reagent is protected towards oxidation be- The sulfhydryl bearing agents as cysteine, and thiogly- cause of the chemical structure of the reagent. However, colic acid can be covalently attached via the amide bond storage stability studies under nitrogen showed an insuffi- formation between carboxylic acid group of the agent to cient stability of thiomer, which resulted in a decrease of the primary amino group of chitosan mediated by a free thiol moieties. This might be due to the formation of water-soluble carbodiimide [256–258]. N-chitosanyl-substituted 2-iminothiolane structures. This An unintended oxidation of thiol groups during synth- undesired side-reaction occurs after the derivatization of esis can be avoided by performing the reaction under inert different amines with 2-iminothiolane. It involves the loss conditions. Alternatively the synthesis can be performed at of ammonia and yields recyclized N-substituted 2-imi- a pH below 5. At this pH-range the concentration of thio- nothiolane (Fig. 19). late anions, representing the reactive form for oxidation In order to achieve the same properties as chitosan–4- of thiol groups, is low, and the formation of disulfide bonds thiobutyl-amidine and to overcome at the same time its in- can be almost excluded. The modifying reagent for chito- sufficient stability, chemical modification of chitosan can san–4-thiobutylamidine conjugate is 2-iminothiolane (a be done with isopropyl-S-acetylthioacetimidate HCl (i-PA- cyclic thioimidester or thioimidate), which reacts with TAI) resulting in chitosan–thioethylamidine conjugate amino groups and introduces a sulfhydryl residue via a po-. The nucleophilicity of amino groups is dictated by NH2+ RNH2 (Chitosan) HS -NH3 S NH2+Cl- S NR HN R 2-iminothiolane Chitosan thiomer N-substituted iminothiolane Fig. 19. Unstability of the chitosan–4-thiobutylamidine conjugate. OSO3 H Major Sulfating agents O O O Heat HO 3 SO NHSO3 H Minor Minor OH OH O 1.6-O-tritylation O O 2. N-Acetylation O HO 3. Sulfating agent, Heat O NH O 4. Deprotection HO3 SO NH2 Chitosan 1.6-O-tritylation OH 2. Sulfating agent, Heat O 3. Deprotection O O HO3 SO NHSO3 H OH 2HCHO OH O O O O CS2 O O RNH2 HO HO N N N R KOH H S SK S S CH3 Cl R= H2 N SO2 Fig. 20. Synthesis of sulfated chitosan. 1030 V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 the protonation state making the reaction pH dependent. of yielding cyclic non-thiol products. Various properties of The reactions can be carried out at pH 6.5–7 at which pH chitosan are improved by this immobilization of thiol value the oxidation process of thiol groups is decreased groups allocating it to a promising new category of thio- and chitosan is soluble as well. This imidoester reacts mers used in particular for the non-invasive administra- rapidly with an amine—maximum for 1.5 h in comparison tion of hydrophilic macromolecules. to the reaction with 2-iminothiolane which ends after 24 h (I) Mucoadhesive properties: Chitosans offers mucoadhe- under continuous stirring at room temperature. The sive properties due to ionic interactions between the posi- short chain of i-PATAI excludes theoretically the possibility tive charged primary amino groups on the polymer and OH OH OH HOOC N3 O O O O O O O HO O Lactobionic acid HO NH HO NH NH2 Lactose N3 O O Chitosan Azidated lactose modified chitosan Fig. 21. Synthesis of azidated chitosan. The substitution occurs randomly. OH OH O O O O 1.H3PO3, O HO HO NHCH2PO3H2 N(CH2PO3H2)2 2. HCHO N-Methylenephosphonic chitosan O O H P OR P OR OH H O OR O O O O HCHO O HO HO NH NH2 R= H, OEt OH OR P O OR O O O HO NH2 N-Methylenephosphonic chitosan P2O5, MeSO3H Phosphorylated chitosan O O OPC P O O O Cl O + O O P PC= N PCO O Trimethylamine N PC PC Phosphorylcholine chitosan O CH3 + O P N CH3 O XH2C O O + O O P N O CH2 O O R(N)n-2 * O O n Fig. 22. Synthesis of phosphorylated chitosan. The substitution occurs randomly.  V.K. Mourya, N.N. Inamdar / Reactive & Functional Polymers 68 (2008) 1013–1051 1031 negatively charged sialic acid and sulfonic acid substruc- chitosan. Chitosan possess the permeation-enhancing tures of the mucus. These mucoadhesive properties capabilities with increase in the paracellular route of ab- of chitosans can be significantly further improved by the sorption, which is important for the transport of hydrophi- immobilization of thiol groups on the polymer. Orientating lic compounds such as therapeutic peptides and antisense studies with all these thiolated chitosans showed that a oligonucleotides across the membrane. The mechanism degree of modification of 25–250 mmol thiol groups per underlying this permeation-enhancing effect seems to be gram chitosan leads to the highest improvement in the based on t