Chapter 1 Chitosan and its Derivative Polymers- Recent...

Chapter 1

Chitosan and its Derivative Polymers- Recent Developments in Synthesis, Characterization and Applications

Scope of the Chapter

This chapter focuses an exhaustive literature survey on chitosan and its

derivatives. It begins with a brief back ground of generality on polymers, methods of

polymerization and major divisions of polymers like natural and synthetic ones. A brief

description about some common synthetic and natural polymers widely used in the

biomedical field is also given. The various and diverse methods used for the recovery of

chitosan from its sources, its physico-chemical properties, the various chemical methods

used for the modification of chitosan etc are discussed in detail. The applications of

chitin and chitosan in the area of biomedical, pharmaceutical, cosmetic and industrial

area are detailed. Recent developments in chemistry and applications of chitosan and

chitosan derivatives are surveyed. The scope and objectives of the present study are

described at the end of this chapter.

Chapter Chapter Chapter Chapter 1111

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1.1. Generality on Polymers

Polymers, formed from hydrocarbons, hydrocarbon derivatives, or sometimes

from inorganic elements, are the basis not only for numerous natural materials, but also

for most of the synthetic plastics that one encounters every day. Polymers consist of

extremely large, chain-like molecules that are, in turn, made up of numerous smaller,

repeating units called monomers. Polymers can be composed of more than one type of

monomer, and they can be altered in other ways. When only one species or building

block is used to form a polymer, the product is called a photopolymer. If the polymer

chains are composed of two types of monomer unit, the material is known as a

copolymer; for three monomer units it is called a terpolymer, etc. It is very difficult to

spend a day without encountering a natural polymer but in the twenty-first century, it is

probably even harder to avoid synthetic polymers, which have collectively revolutionized

human existence. The length of the polymer chain is specified by the number of repeat

units in the chain. This is called the degree of polymerization (DP). The molecular weight

of the polymer is the product of the molecular weight of the repeat unit and the DP.

1.2. Synthesis of polymers

Polymerisation

Step growth Addition Insertion

Free radicalCationicAnionic

Copolymerisation

AlternatingRandom

Blocks & Grafts

Scheme1. Types of Polymerisations


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There are number of different methods of synthesising polymers from suitable

monomers, these are step-growth polymerisation, addition polymerization,

copolymerisation and insertion polymerisation.

1. 2.1. Condensation or Step-growth Polymerisation

Step growth polymerisation is usually used for monomers with functional groups

such as -OH, -COOH etc. In polymer formation the condensation takes place between

two polyfunctional molecules to produce one larger polyfunctional molecule. The

reaction leads successively from monomer to dimer, trimer, tetramer, pentamer and so on,

until finally a polymer molecule with large DP is formed. It is usually a succession of

non-catalysed, chemical condensation reactions associated with the elimination of low

molar-mass side-products, eg. water. The reaction continues until almost all of one of the

reagents is used up; equilibrium is established that can be shifted at high temperatures by

controlling the amounts of the reactants and products.

Eg: Hexamethylenediamine and adipic acid reacts to form the polyamide nylon66,

and formation of polyester from terepthalic acid and ethylene glycol.

1.2.2. Addition Polymerisation

Addition or chain reaction polymerization involves chain reactions in which the

chain carrier may be an ion or a reactive substance with one unpaired electron called a

free radical. Usually addition polymerisations involve the polymerisation of olefinic

monomers. A free radical is usually formed by the decomposition of a relatively unstable

material called an initiator. The free radical is capable of reacting to open the double

bond of a vinyl monomer and add to it, with an electron remaining unpaired. In a very

short time (usually a few seconds or less) many more monomers add successively to the

growing chain. The product, then, unlike that obtained from step-growth polymerisation,

has the same chemical composition as the starting monomer. Finally two free radicals

react to annihilate each other’s growth activity and form one or more polymer molecules.


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Eg: Ethylene polymerises to polyethylene, Vinyl chloride polymerises to

poly(vinyl chloride), Vinyl acetate to poly(vinyl acetate) etc.

Cationic polymerizations are those where the active site has a positive charge, i.e.

carbocation. The monomers which contain an electron donating group are most

appropriate for these types of polymerizations.

Eg: Isobutylene, alkylvinyl ethers, vinyl acetals, para-substituted styrenes.

Initiation can be achieved using protonic acids and Lewis acids, with the latter

apparently requiring a co-catalyst such as water or methanol.

An anionic polymerization is one where the active site on the end of a growing

chain is negatively charged, i.e. carbanion. Certain types of ring-opening polymerizations

can also proceed by this mechanism. In protic solvents the chain growth can be stopped

by transfer to solvent. If an inert solvent is used and there are no contaminants (eg: those

containing an active hydrogen), then it is possible to obtain a system where carbanion end

groups are always present, because of the absence of termination reactions. One

immediate consequence of the “living” nature of these polymerizations is that it allows

the synthesis of block copolymers by sequential addition of monomers to the living

polymer anion.

1.2.3. Copolymerisation

Polymers can be synthesized with more than one type of monomers in the chain.

Such polymers are called copolymers. Copolymers can be of different types, depending

on the monomers used and the specific method of synthesis. The copolymer with a

relatively random distribution of the different monomers in its structure is referred to as a

random copolymer. Representing, say, two different monomers by A and B, a random

copolymer can be depicted as

ABBABBBAABBAABAAABBA


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Three other copolymer structures are known: alternating, block and graft

copolymer structures. In the alternating copolymer the two monomers alternate in a

regular fashion along the polymer chain

ABABABABABABABABABAB

A block copolymer is a linear copolymer with one or more long uninterrupted

sequences of each monomer in the chain

AAAAAAAAAABBBBBBBBBB

A graft copolymer, on the otherhand, is a branched copolymer with a backbone of

one type of polymer to which is attached one or more side chains of another polymer.

AAAAAAAAAAAAAAAA

A

A

A

A

A

A

A

Copolymerisation, which may be compared to alloying in metallurgy, is very

useful for synthesizing polymers with the required combination of properties. For

example, polystyrene is brittle, and polybutadiene is flexible; therefore copolymers of

styrene and butadiene should be more flexible than polystyrene but harder than

polybutadiene. The general-purpose rubber GRS (or SBR), the first practical synthetic

rubber, is a copolymer of styrene and butadiene.


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1.2.4. Insertion Polymerisation (Coordination polymerization) Polymerisation reactions, especially of olefins and dienes, catalysed by organo-

metallic compounds, fall under the category of coordination polymerization. The

monomer is inserted in between the metal ion and the carbanion with the result that the

polymer chain formed is pushed out from the solid catalyst surface. For this reason,

coordination polymerization is also known as insertion polymerization.

Usually addition polymerisations involve the polymerisation of olefinic

monomers. Polymerisation occurs via an insertion of a monomer at the end of the

growing chain, mediated by a catalyst. The catalyst stays at the end of the growing chain.

Polymers synthesised by insertion polymerisation are typically characterised by a very

high stereo regularity. An example for such a polymerisaiton technique is the Ziegler-

Natta polymerisation. Ziegler-Natta is especially useful, because it can make polymers

that can't be made any other way, such as linear unbranched polyethylene and isotactic

polypropylene. Free radical vinyl polymerization can only give branched polyethylene,

and propylene won't polymerize at all by free radical polymerization.

1.3. Classification of Polymers

Polymers

NaturalSynthetic

ProteinsPolysachharides

Elastomers

ThermoplasticsThermosets

FibersNucleic acids

HydroxyapatitesPolyesters

Scheme 2. Classification of Polymers


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1.3.1. Synthetic Polymers

1.3.1. 1. Thermoplastics Polymers Some polymers soften on heating and can be converted into any shape that they

can retain on cooling. The processes of heating, reshaping and retaining the same on

cooling can be repeated several times. Such polymers, that soften on heating and stiffen

on cooling, are termed ‘thermoplastics’. Most thermoplastics are high molecular weight

polymers whose chains associate through weak van der Waals forces (e.g. polyethylene);

stronger dipole-dipole interactions and hydrogen bonding (e.g. nylon); or even stacking

of aromatic rings (e.g. polystyrene). Thermoplastics are elastomeric and flexible above

the glass transition temperature. Some thermoplastics normally do not crystallize: they

are termed "amorphous" plastics and are useful at temperatures below the Tg. They are

frequently used in applications where clarity is important. Some typical examples of

amorphous thermoplastics are PMMA, PS and PC. Generally, amorphous thermoplastics

are less chemically resistant and can be subjected to stress cracking. Most other

thermoplastics will crystallize to a certain extent and are called "semi-crystalline" for this

reason. Typical semi-crystalline thermoplastics are PE, PP, PBT and PET. The speed and

extent to which crystallization can occur depends, in part, on the flexibility of the

polymer chain. Semi-crystalline thermoplastics are more resistant to solvents and other

chemicals. If the crystallite size is larger than the wavelength of light, the thermoplastic is

hazy or opaque. Polyethylene, poly(vinyl chloride), polyacrylonitrile polycarbonate,

polyacetal, nylon and sealing wax are examples of thermoplastic polymers.

1.3.1.2. Thermosetting Polymers Some polymers on the other hand, undergo some chemical change on heating and

convert themselves into an infusible mass. Once set, they can not be reshaped. Such

polymers that become an infusible and insoluble mass on heating are called

‘thermosetting’ polymers.


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Thermoset materials are usually liquid, powder, or malleable prior to curing, and

designed to be molded into their final form, or used as adhesives. The curing process

transforms the resin into a plastic or rubber by a cross-linking process. Energy and/or

catalysts are added that cause the molecular chains to react at chemically active sites

(unsaturated or epoxy sites, for example), linking into a rigid, 3-D structure. The cross-

linking process forms a molecule with a larger molecular weight, resulting in a material

with a higher melting point. During the reaction, when the molecular weight has

increased to a point so that the melting point is higher than the surrounding ambient

temperature, the material forms into a solid material. Subsequent uncontrolled reheating

of the material results in reaching the decomposition temperature before the melting point

is attained. A thermoset material cannot be melted and re-shaped after it is cured.

Thermoset materials are generally stronger than thermoplastic materials due to this 3-D

network of bonds, and are also better suited to high-temperature applications up to the

decomposition temperature of the material. They do not lend themselves to recycling like

thermoplastics, which can be melted and re-molded. Examples are vulcanized rubber,

phenol formaldehyde resin, urea-formaldehyde resin (used in plywood, particle board and

medium-density fibre board), melamine (used on worktop surfaces), polyester resin (used

in glass-reinforced plastics/fibreglass (GRP)), epoxy resin (as an adhesive, in fibre

reinforced plastics such as glass reinforced plastic and graphite-reinforced plastic) etc.

1.3.1.3. Elastomers Elastomers are the group of polymers that can easily undergo very large,

reversible elongations (< 500-1000%) at relatively low stresses. This requires that the

polymer be completely or almost completely amorphous with a low glass transition

temperature and low secondary forces so as to obtain high polymer chain mobility. Some

degree of cross-linking is needed so that the deformation is rapidly and completely

reversible (elastic). Most elastomers obtain the needed strength via cross-linking and the

incorporation of reinforcing inorganic fillers (eg:carbon black, silica). Some elastomers

undergo a small amount of crystallization during elongation, especially at very high

elongations, and this acts as an additional strengthening mechanism. The Tm of the


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crystalline regions must be below or not significantly above the use temperature of the

elastomer in order that the crystals melt and deformation be reversible when the stress is

removed. Polyisoprene (natural rubber) is a typical elastomer, it is amorphous, is easily

crosslinked, has a low Tg (-73ºC), and has a low Tm (28ºC). Cross linked polyisoprene

has a modulus that is initially less than 70 Ncm-2, however its strength increases to about

1500 Ncm-2 at 400% elongation and about 2000 Ncm-2 at 500% elongation. Its elongation

is reversible over the whole elongation range that is up to just prior to the rupture point.

The extent of crosslinking and the resulting strength and elongation characteristics of an

elastomer cover a considerable range depending on the specific end use. The use of an

elastomer to produce an automobile tire requires much more crosslinking and reinforcing

fillers than does the elastomer used for producing rubber bands. The former application

requires a stronger rubber with less tendency to elongate than the latter application.

Extensive crosslinking of a rubber converts the polymer to a rigid plastic.

1.3.1.4. Fibers Fibers are polymers that have very high resistance to deformation-they undergo

only low elongations (<10-50%) and have very high moduli (>35,000 Ncm-2) and tensile

strengths (>35,000 Ncm-2). A polymer must be highly crystalline and contain polar

chains with strong secondary forces in order to be useful as a fiber. Mechanical stretching

is used to impart very high crystallinity to a fiber. The crystalline melting temperature of

a fiber must be above 200ºC so that it will maintain its physical integrity during the use

temperatures encountered in cleaning and ironing. However, Tm should not be

excessively high-not higher than 300ºC, otherwise fabrication of the fiber by melt

spinning may not be possible. The polymer should be soluble in solvents used for

solution spinning of the fiber but not in dry-cleaning solvents. The glass transition

temperature should have an intermediate value; low aTg would not allow crease retention

in fabrics. Poly(hexamethylene adipamide) is a typical fiber. It is stretched to high

crystallinity, and its amide groups yield strong secondary forces due to hydrogen

bonding; the result is high tensile strength (70,000 Ncm-2), and modulus (500,000 Ncm-2),

and low elongation (< 20%). The Tm and Tg have optimal values of 265°C and 50ºC,


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respectively. The propylene used as a fiber is an exception to the generalization that polar

polymers are required for fiber applications. The propylene used as a fiber has a highly

stereo regular structure and can be mechanically stretched to yield a highly oriented

polymer with the strength characteristics required of a fiber. Some examples of a few

synthetic polymers relevant to biomedical applications are acrylates, vinyl polymers,

vinyl esters, polycarbonates etc.

1.3.2. Synthetic Polymers Relevant to Biomedical Applications

1.3.2.1. Poly(methyl methacrylate)

Methyl methacrylate Poly(methyl methacrylate)

Poly(methyl methacrylate), PMMA, a synthetic resin belonging to the family of

polymeric organic compounds, is manufactured by bulk, solution, suspension, and

emulsion polymerization of MMA monomer. The resin has excellent transparency which

takes a good gloss with the property of weather resistance, high surface hardness and

coloring property. It is used as a replacement for glass. Its application includes the semi-

finished products of automotive parts, electronic parts, illuminated display sheet for

advertising, building materials and LCD (Liquid Crystal Display) plates. It is available

commercially in both pellet and sheet form. Outstanding properties include

weatherability and scratch resistance. The most serious deficiencies are low impact

strength and poor chemical resistance. PMMA is a commodity polymer which forms the

basis of the Perspex, Plexiglass and Lucite families of materials. These have a wide range

of applications many of which exploit the polymer's high optical clarity. The softening

temperature (Tg) is technologically one of the most important properties of the material


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and this is strongly influenced by the tacticity of the polymer, i.e. the placements of the

ester and methyl groups along the polymer backbone. For example, free radical-generated

material is biased towards the syndiotactic form (ca. 67%) affording a Tg of ca. 105°C.

However, as the syndiotacticity is increased, the Tg also rises (to ca. 135°C) for highly

syndiotactic material, and this holds clear advantages for applications where the polymer

is exposed to higher temperature environments.

PMMA has a good degree of compatibility with human tissue, and can be used for

replacement intraocular lenses in the eye when the original lens has been removed in the

treatment of cataracts. Hard contact lenses are frequently made of this material. Soft

contact lenses are often made of a related polymer, where acrylate monomers containing

one or more hydroxyl groups make them hydrophilic. In orthopaedics, PMMA bone

cement is used to affix implants and to remodel lost bone. Bone cement acts like a grout

and not so much like a glue in arthroplasty. Although sticky, it primarily fills the spaces

between the prosthesis and the bone preventing motion. It has a young's modulus

between cancellous bone and cortical bone. Thus it is a load sharing entity in the body

not causing bone resorption. Dentures are often made of PMMA, and can be colour-

matched to the patient's teeth. In cosmetic surgery, tiny PMMA microspheres suspended

in some biological fluid are injected under the skin to reduce wrinkles or scars

permanently.

1.3.2.2. Poly(2-Hydroxyethyl methacrylate) [Poly(HEMA)]

C CCH3H

H C OO

CH2CH2OH

CH2 C

CH3

C OO

CH2CH2OH

n

free radical

vinyl polymerisation

(2-Hydroxyethyl methacrylate) Poly(2-Hydroxyethyl methacrylate)


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Poly(2-Hydroxyethyl methacrylate), or poly(HEMA), is one of the most

important hydrogels in the biomaterial world since it has many advantages over other

hydrogels. These include a water-content similar to living tissue, inertness to biological

processes, resistance to degradation, permeability to metabolites, resistance to absorption

by the body. It can be easily manufactured into many shapes and forms, and be easily

sterilized. The most common example of poly(HEMA) is its use as contact lenses.

Immobilization of proteins can be done in it at low temperature to preserve the

structure and the biological activity of a protein. It is also used in light curing polymer

system and high performance coatings for lasting high gloss against scratching, solvents

and weathering. It is used in paint resins and emulsions, binders for textiles and paper. It

is used as an adhesion promoter for metal coatings.

1.3.2.3. Poly(vinyl acetate)

Vinyl acetate Poly(vinyl acetate)

Poly(vinyl acetate) is an amorphous, flavourless and odourless plastic which is

obtained by radical polimerisation or, on a large scale, by emulsion polymerisation of

vinyl acetate. This polymer is insoluble in water but well soluble in ketones, esters,

ethers, aromatic and chlorinated hydrocarobons. Pure vinyl acetate exists in the form of

emulsions or colourless beads. Because of its low softening temperature it is not suitable

for producing moulding components but instead well compatible with diluents. In the

form of coatings, it can be easily applied on a variety of surfaces. In addition to that,

PVAc is non-fading and weather-resistant.


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Usage: typical binding agent, resource for the fabrication of adhesives laquers,

coatings, impregnating agents, latex colours, wrapping films, coatings for food

packagings (sausages and cheese), polyvinyl alcohols, polyvinyl acetals, additive for

concrete. Poly(vinyl alcohol) is obtained by controlled hydrolysis of poly(vinyl acetate)

1.3.2.4. Polyethylene glycol

condensationpolymerisation

Ethylene glycol Polyethylene glycol

Polyethylene glycol is a condensation polymer of ethylene glycol or addition and

ring polymers of ethylene oxide with the general formula H(OCH2CH2)nOH, where n is

the average number of repeating ox ethylene groups typically from 4 to about 180. One

common feature of PEG appears to be the water-solubility. It is soluble also in many

organic solvents including aromatic hydrocarbons (not aliphatics). They are used to

make emulsifying agents and detergents, and as plasticizers, humectants, and water-

soluble textile lubricants. The wide range of chain lengths provides identical physical and

chemical properties for the proper application selections directly or indirectly in the field

of alkyd and polyester resin preparation to enhance water dispersability and water-based

coatings. It finds use as antidusting agents in agricultural formulations and as cleaners,

detergents and soaps with low volatility and low toxicity properties. Other applications

include coupling agents, humectant, solvent and lubricant in cosmetics and personal care

bases, dimensional stabilizer in wood working operations, dye carrier in paints and inks,

heat transfer fluid formulation and defoamer formulations, low volatile, water soluble,

and noncorrosive lubricant without staining residue in food and package process. It finds

application as plasticizer to increase lubricity and to impart a humectant property in

ceramic mass, adhesives and binders. PEG is a candidate material for softener and

antistatic agents for textiles, soldering fluxes with good spreading property.


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Polyethylene glycol is non-toxic, odorless, neutral, lubricating, non-volatile and

non-irritating and is used in a variety of pharmaceuticals and in medications as a solvent,

dispensing agent, ointment and suppository bases, vehicle, and tablet excipient.

1.3.2.5. Polyethylene

Polyethylene is the simplest hydrocarbon polymer.

Ethylene Polyethylene

Polyethylene, PE, is created through polymerization of ethene. It can be produced

through radical polymerization, anionic addition polymerization, ion coordination

polymerization or cationic addition polymerization. This is because ethene does not have

any substituent groups which influence the stability of the propagation head of the

polymer. Each of these methods results in a different type of polyethylene. Polyethylene

is classified into several different categories based mostly on its density and branching.

The mechanical properties of PE depend significantly on variables such as the extent and

type of branching, the crystal structure, and the molecular weight. UHMWPE (ultra high

molecular weight PE), HMWPE (high molecular weight polyethylene), HDPE (high

density PE), HDXLPE (high density cross-linked PE), PEX (cross-linked PE), MDPE

(medium density PE), LDPE (low density PE), LLDPE (linear low density PE), VLDPE

(very low density PE) .

1.3.3. Medical Applications of Synthetic Polymers Synthetic polymers can be employed in various fields of application, as shown in

Table 1.1. Polymers which, due to their fairly good intrinsic haemocompatibility

properties, have been largely employed in endocorporeal permanent applications of


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prosthetic type include polyurethanes, silicone rubbers, hydrogels, teflon and some vinyl

polymers or copolymers.

Table1.1. Synthetic polymers in medicine

Type of polymers Application fields

Vinyl polymers Polyethylene (LDPE, HDPE, HMWPE); Polypropylene)

Poly(vinyl chloride)

Vinyl copolymers Fluorinated polymers (Teflon)

Polyamides (Nylon)

Polyestres (Polyethylene-terephthalate)

Silicone rubber

Hydrogels (PMMA, PHEMA, PVP)

Polyurethanes

PMMA

Sutures, ligament prostheses, reconstructive surgery, orthopedics.

Tubing in biomedical applications

Haemodialysis filters General surgery, vascular grafts, sutures

Sutures

Sutures, vascular grafts.

Plastic surgery, orthopedics

Ophthalmics

Blood contact devices

Bone cements, contact lenses, dentistry.

1.3.4. Biodegradable Synthetic Polymers A vast majority of biodegradable polymers studied belongs to the polyester

family, which includes polyglycolides and polylactides. Biodegradable synthetic

polymers such as poly(glycolic acid), poly(lactic acid) and their copolymers, poly(p-

dioxanone), and copolymers of trimethylene carbonate and glycolide have been used in a

number of clinical applications [1-5]. The major applications include resorbable sutures,

drug delivery systems and orthopaedic fixation devices such as pins, rods and screws [6, 7].

Among the families of synthetic polymers, the polyesters have been attractive for these

applications because of their ease of degradation by hydrolysis of ester linkage,

degradation products being resorbed through the metabolic pathways in some cases and

the potential to tailor the structure to alter degradation rates. Polyesters have also been

considered for development of tissue engineering applications [8-13]

1.3. 5. Natural Polymers

Some of the representatives of biological materials are given below.


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1.3.5.1. Polypeptides A mammalian body has different kinds of polypeptide including plasma,

structural and functional proteins. The majority of proteins used as biomaterials originate

from blood plasma and structural skeletons. Functional proteins such as enzymes, cell

growth factors and interleukins are also used but mostly incorporated in biomaterials as

ingredients.

1.3.5.1.1. Plasma Proteins The plasma protein which is present in blood in the largest amount is serum

albumin. This globular protein is unable to form tough film or sponge, but only

microbead through its denaturation or chemical cross-linking. In contrast, fibrinogen is

readily polymerized into hydrogel when partially hydrolysed by thrombin. This fibrin

hydrogel can be further cross-linked chemically by factor XIII.

1.3.5.1.2. Collagen and Gelatin Approximately one-thirds of the proteins present in the human body are collagen,

which forms a family of different kinds from Type 1 to Type XIII. The collagen most

widely used as biomaterial is of Type 1, which is distributed in the skin, bone, tendon,

and intestinal gut. The regenerated collagen obtainable by extraction from animal tissues

under mild conditions keeping triple-helical collagen molecules are employed as

biomaterials. Although collagen films and sheets are insoluble in water of pH 7, they are

quickly degraded and absorbed in the body when implanted. Delay in collagen absorption

can be realized if chemical cross-linking is introduced into collagen molecules.

Gelatin is a denatured form of collagen consisting of random chains without triple

helix. Extraction of animal collageneous tissues with lime or acids yields gelatin. Gelatin

is readily soluble in water over a wide pH range, unless the temperature is lower than 25 ºC.

This sol-gel transition is reversible for aqueous gelatin solution. To avoid very rapid

absorption of gelatin films or sponges implanted in the body, they should be cross-linked


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chemically. It is noteworthy that cross-linked gelatin generally possesses excellent

mechanical properties than the cross-linked collagen.

1.3.5. 2. Polysaccharides In animals, polysaccharides are important as components of extra cellular matrix

while those biomacromolecules, especially cellulose and chitin, are the major elements of

the skeleton of plants, crustacean and insects. Cellulose has the largest production among

the whole natural and synthetic macromolecules existing on the earth, while the

macromolecule of the second largest production is chitin.

1.3.5. 2. 1. Cellulose Cellulose is not used as implanted biomaterials at all but is a very important

material for production of hollow fibers used for haemodialysers [14]. They are

fabricated also from cellulose diacetate and cellulose triacetate which activate the

complement system to a lesser extent than cellulose. The cellulose hollow fibers for

haemodialysis are regenerated from cuprammonium solution of refined cotton linters

with high purity, which has higher molecular weight than pulp, leading to higher tenacity

fibers. The superiority of regenerated cellulose is due to 1) high chemical stability, 2) low

price, 3) high tenacity in the wet state, allowing the preparation of a thin membrane, 4)

ease of the control of pore size, ranging from 1 to 100 nm in diameter and 5) controllable

porosity.

Cellulose can be oxidized with NO2 in the fibrous state without altering the

original shape [15]. The oxidized cellulose is absorbed into the body upon implantation

as a result of physical dissolution (not biodegradation).

1.3.5.2.2. Starch Starch is a mixture of amylase and amylopectin, both of which undergo enzymatic

degradation in the body. To delay the rapid clearance of starch from the body, it is

derivatised, for instance, by hydroxyethylation and chemical cross-linking. Hydroxyethyl


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starch was widely used previously as a plasma expander, but derivatised starches are

currently used in most cases as drug carriers.

In contrast to starch, dextran is practically not biodegraded although it is readily

soluble in water over a wide pH range. The major medical application of dextran is its

use as a plasma expander. Once dextran is chemically cross-linked, it no longer becomes

absorbable in the body. This is the reason why cross-linking is not required for medical

application of dextran.

1.3.5.2.3. Hyaluronate The characteristics of hyaluronate, another polysaccharide, as a biomaterial are

high biodegradability and extremely high viscosity of its dilute aqueous solution.

Therefore, this polysaccharide is used clinically in a dilute viscous solution as a

viscoelastic material in ophthalmology. Dilute solutions of hyaluronate have also been

used to prevent tissue adhesion by covering the tissue with the solution for its protection.

It is evident that cross-linking greatly reduces the water content of the hyaluronate film.

Biodegradation of hyaluronate could be also reduced by cross-linking [16]. Films

prepared from derivatised hyaluronates are used clinically to prevent tissue adhesion.

1.3.5.2.4. Chitin and Chitosan Biopolymer chitin, the natural amino polysaccharide, and its most important

derivative, chitosan, are currently considered as the subjects of extensive worldwide

academic and industrial research. All the polysaccharides described above are

hydrophilic and noncrystallisable except for cellulose and its acetates. On the contrary,

chitin, which is an entirely naturally-occurring polymer, is hydrophobic and crystallizable

[17]. Therefore, chitin films and fibers fabricated from organic solutions of chitin have

mechanical properties typical of synthetic fibers and films. In addition, chitin undergoes

enzymatic degradation in rat [16]. These unique properties of chitin appear to provide

high potential for medical applications. A comprehensive review of chitin and chitosan is

given in the following section.


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1.3.5. 3. Nucleic Acids Nucleic acids are some group of organic substances found in the chromosomes of

living cells and viruses that play a central role in the storage and replication of hereditary

information and in the expression of this information through protein synthesis. In most

organisms, nucleic acids occur in combination with proteins; the combined substances are

called nucleoproteins. Nucleic acid molecules are complex chains of varying length. The

two chief types of nucleic acids are DNA (deoxyribonucleic acid), which carries the

hereditary information from generation to generation, and RNA (ribonucleic acid), which

delivers the instructions coded in this information to the cell's protein manufacturing

sites.

1.3.5. 3.1. DNA The chemical and physical properties of DNA suit it for both replication and

transfer of information. Each DNA molecule is a long two-stranded chain. The strands

are made up of subunits called nucleotides, each containing a sugar (deoxyribose), a

phosphate group, and one of four nitrogenous bases, adenine, guanine, thymine, and

cytosine, denoted A, G, T, and C, respectively. A given strand contains nucleotides bearing each of

these four. The information carried by a given gene is coded in the sequence in which the

nucleotides bearing different bases occur along the strand. These nucleotide sequences determine

the sequences of amino acids in the polypeptide chain of the protein specified by that gene.

In 1953 the molecular biologists J. D. Watson, an American, and F. H. Crick, an

Englishman, proposed that the two DNA strands were coiled in a double helix. In this

model each nucleotide subunit along one strand is bound to a nucleotide subunit on the

other strand by hydrogen bonds between the base portions of the nucleotides. The fact

that adenine bonds only with thymine (A—T) and guanine bonds only with cytosine

(G—C) determines that the strands will be complementary, i.e., that for every adenine on

one strand there will be a thymine on the other strand. It is the property of


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complementarity between strands that insures that DNA can be replicated, i.e., that

identical copy can be made in order to be transmitted to the next generation.

1.3.5. 3.2. RNA In order to be expressed as protein, the genetic information must be carried to the

protein-synthesizing machinery of the cell, which is in the cell's cytoplasm. One form of

RNA mediates this process. RNA is similar to DNA, but contains the sugar ribose instead

of deoxyribose and the base uracil (U) instead of thymine. To initiate the process of

information transfer, one strand of the double-stranded DNA chain serves as a template

for the synthesis of a single strand of RNA that is complementary to the DNA strand

(e.g., the DNA sequence AGTC... will specify an RNA sequence UCAG...). This process

is called transcription and is mediated by enzymes.

1.3.5.4. Hydroxyapatites Hydroxyapatite, Ca10(PO4)(OH)2 is the most well known and studied calcium

phosphate material obtained from natural sources (coralline hydroxyapatite and bovine

bone) or synthesized by precipitation using chemical reagents [18, 19]. The major

component of the skeleton of mammals comprises hydroxyapatites, a calcium phosphate,

while calcium carbonate is the main structural body of corals. Both can be used as

biomaterials, but synthetic hydroxyapatite and calcium carbonate are also commercially

available and used in medicine much more than the natural biomaterials. Synthetic

hydroxyapatite (HA) is of importance as a biomaterial as it is chemically similar to the

mineral component of mammalian bone. It is one of few materials that are classed as

bioactive, meaning that it will form strong chemical bonds with surrounding bone, unlike

other materials such as alumina and zirconia, which are identified as foreign materials

and become encapsulated by fibrous tissue. As such the human body is quite happy to

integrate it into it. Coatings of hydroxyapatite are often applied to metallic implants (most

commonly titanium/titanium alloys and stainless steels) to alter the surface properties.

Hydroxyapatite may be employed in forms such as powders, porous blocks or beads to


21

fill bone defects or voids. These may arise when large sections of bone have had to be

removed (e.g. bone cancers) or when bone augmentations are required (e.g maxillofacial

reconstructions or dental applications). The bone filler will provide a scaffold and

encourage the rapid filling of the void by naturally forming bone and provides an

alternative to bone grafts. It will also become part of the bone structure and will reduce

healing times compared to the situation, if no bone filler was used.

1.4. Chitin and Chitosan in Detail

1.4.1. Source and Composition Chitosan is a fiber-like substance derived from chitin, a homopolymer of ß-

(1→4)-linked N-acetyl-D-glucosamine. It is the second most abundant organic compound

in nature after cellulose [20]. Chitin is widely distributed in marine invertebrates, insects,

fungi, and yeast [21]. However, it is not present in higher plants and higher animals.

Generally, the shell of selected crustacean was reported [22] to consist of 30-40%

protein, 30-50% calcium carbonate and calcium phosphate, and 20-30% chitin. Chitin is

widely available from a variety of source among which, the principal source is shellfish

waste such as shrimps, crabs, and crawfish [23]. It also exists naturally in a few species

of fungi.

In terms of its structure, chitin is associated with proteins and, therefore, high in

protein contents. Chitin fibrils are embedded in a matrix of calcium carbonate and

phosphate that also contains protein. The matrix is proteinaceous, where the protein is

hardened by a tanning process [24]. Studies of Asford and co-workers demonstrated that

chitin represents 14-27% and 13-15% of the dry weight of shrimp and crab processing

wastes, respectively [25]. As seen depicted in schemes (3a to 3c), the only difference

between chitosan and cellulose is the amine (-NH2) group in the position C-2 of chitosan

instead of the hydroxyl (-OH) group found in cellulose. However, unlike plant fiber,

chitosan possesses positive ionic charges, which give it the ability to chemically bind

with negatively charged fats, lipids, cholesterol, metal ions, proteins, and

macromolecules [26]. In this respect, chitin and chitosan have attained increasing


22

commercial interest as suitable resource materials due to their excellent properties

including biocompatibility, biodegradability, adsorption, and ability to form films, and to

chelate metal ions [27].

.

Scheme 3a. Chitin

Scheme 3b. Chitosan

Scheme 3c. Cellulose

The actual difference between chitin and chitosan is the acetyl content of the

polymer. Chitin is made up of a linear chain of acetylglucosamine groups while chitosan

is obtained by removing enough acetyl groups (CH3-CO) for the molecule to be soluble


23

in most diluted acids [28]. When the number of N-acetyl-glucosamine units is higher than

50%, the biopolymer is termed chitin. Conversely, when the number of N-glucosamine

units is higher, the term chitosan is used [29]. Chitosan having a free amino group is the

most useful derivative of chitin [28].

1.4.2. Production of Chitin

Shell

Wash and crush

Demineralize with HCl

Deproteinate with NaOH

Raw chitin

Fungi

Harvest, wash and dry

Pulverize and treat with NaOH

Extract with LiCl/DMAc

Precipitate in water,

collect chitin and dry

Scheme 4. Preparation of chitin

Cellulose and chitin are the most abundant organic compounds in nature and

estimated to be at levels approaching 1011 tons annually. The total amount of chitin

harvestable without imbalancing the marine ecosystem is estimated to be 1.5x108 kg/year

mostly from the shells of crustaceans such as crab, shrimp and krill [30, 31].

Chitosan as mentioned before is extracted from crustacean shell waste such as

crab, shrimp, lobster, and crawfish. The protein and mineral content vary with crustacean


24

species and seasons [32, 33]. The isolation of chitin specifically consists of two steps:

demineralization (DM) and deproteinization (DP), which involves the dissolution of

calcium carbonate with 1.0 N HCl and the removal of proteins with 3% NaOH,

respectively. The sequence of demineralization and deproteinization steps can be

reversed.

1.4.2.1. Deproteinization Chitin occurs naturally in association with protein (Chitinoprotein). Some of this

protein can be extracted by mild methods, but other portion is not readily extracted,

suggesting strong covalent bonding to chitin [34]. With regards to chemical structure,

protein is bound by covalent bonds to the chitin through aspartyl or histidyl residues, or

both, thus forming stable complexes such as glycoproteins. Crustacean shell waste is

usually ground and treated with dilute sodium hydroxide solution (1-10%) at elevated

temperature (65-100ºC) to dissolve the proteins present. Reaction time usually ranges

from 0.5 to 12 h depending on preparation methods. Prolonged alkaline treatment under

severe conditions causes depolymerization and deacetylation. To obtain uniformity in

reaction, it is recommended to use relatively high ratios of solid to alkali solution of 1:10

or 1:15-20 with proper agitation because a minimum ratio of 1:4 (w/v) of shell weight to

KOH solution, had only a minor effect on the DP efficiency of shells. Deproteinization is

conventionally accomplished by extraction with dilute sodium hydroxide solution (1-

10%) at elevated temperature (65-100 ºC) for 1-6 hr [35]. Bough et al extracted protein

from shrimp shells with 3% NaOH at 100ºC for 1 h and No et al similarly treated

crawfish shell waste with 3.5% NaOH at 65ºC for 2 h [36, 37]. Optimal deproteinization

can be accomplished by treatment with dilute potassium hydroxide solution [38]. Cosio et

al. and Chen et al. in their various studies accomplished deproteinization for shrimp shell

at pH 11.5 at 30ºC, and for prawn in 5N NaOH for 1 h at 100ºC, respectively [39, 40].

Removal of protein by enzymatic digestion for production of chitin and chitosan was

attempted by several authors in an effort to minimize deacetylation [41-43]. Bough et al

also extracted protein from shrimp shells with Rhyzome-62 concentrate at 60ºC for 6 h at

pH 7 but complete removal of protein was not attained [37]. Extraction of protein for


25

several days was attempted, but prolonged alkaline treatment under severe conditions

caused depolymerization and deacetylation [44, 45]. Shahidi and Synowiecki reported a 2 h

extraction period for removal of all proteins present in the shells [38]. These workers also

observed that relatively high ratios of solid to alkali solution, 1:10 or above are usually

used to obtain uniformity in reaction with proper agitation. Cho and No performed

deproteinization by autoclaving under conditions of 15 psi/121ºC with 3% NaOH for

15min and a solid: solvent ratio of 1:10 [46]. Their results showed that deproteinization

can effectively be achieved by autoclaving conditions because no significant differences

in nitrogen content and bulk density as well as water and fat binding capacity were

observed. Rout investigated the effects of process modification of crawfish chitin and

chitosan production by reversing the first two steps (deproteinization or demineralization)

or reducing the number of steps (deproteinization or discoloration or both) on fat and

water binding capacities of chitin and chitosans; he reported that simplifying the process

affected both water and fat binding capacities. Among crab and crawfish chitosans, the

DMPA (excluding decolouration) had the highest fat binding capacity [27]. The enhanced

removal of proteins as an effect of sonication during chitin extraction was also reported

[47].

1.4.2.2. Demineralization Demineralization is usually accomplished by extraction with dilute hydrochloric

acid (up to 10%) at room temperature with agitation to dissolve calcium carbonate as

calcium chloride [36, 44]. But, reaction time varied with preparation methods from 30

min to over 2 days. However, Synowiecki et al and Chen et al accomplished

demineralization with 22% HCl and 6N HCl, respectively, at room temperature [48, 40].

To avoid modifications such as depolymerization or deacetylation caused by harsh

treatments, Austin et al. suggested the use of mild acids such as ethylenediaminetetra-

acetic acid (EDTA) for decalcification [49]. Prolonged demineralization time for up to 24 h

results in only a very slight drop in the ash content but can cause polymer degradation or

decreased viscosity [50, 51]. Also it is important that the amount of acid be


26

stoichimetrically equal to or greater than all minerals present in the shells to ensure

complete reaction [52, 38].

A wide variation of the demineralization process has been reported in the

literature. The use of HCl acid at higher concentration and also 90% formic acid to

achieve demineralization has been reported. Optimum demineralization is achieved by

constant stirring of the dried ground crawfish shell with 1N HCl for 30 min at ambient

temperature and a solid to solvent ratio of 1:15 (w/v) [36]. The ash content of the

demineralized shell is an indicator of the effectiveness of the demineralization process.

Elimination of the demineralization resulted in products having 31-36% ash.

During the demineralization process excessive undesirable foams are produced

due to the CO2 generation (CaCO3 + 2HCl → CaCl2 + CO2 + H2O). To control or reduce

the foam, No et al. (1995) recommended the use of commercial antifoam comprising of

10% solution of active silicone polymer without an emulsifier [35]. They also

demonstrated that at 1.0 ml of antifoam /L of 1N HCl, the performance of antifoam is

more efficient during demineralization with smaller shell particle size (< 0.425 mm and

under a slightly faster stirring speed at 300 rpm). Furthermore, they recommended that

deproteinization followed by demineralization is a favorable sequence in terms of the

amount of antifoam required to control foaming.

1.4.2.3. Decolouration Acid and alkali treatments alone produce a coloured chitin product. For

commercial acceptability, the chitin produced from crustacean sources, needs to be

decolourized or bleached to yield cream white chitin powder [36]. The pigment in the

crustacean shells forms complexes with chitin. Fox found one 4-keto-and three 4, 4’-

diketo-ß carotene derivatives firmly bound to the exoskeletal chitin of red kelp crab [53].

The level of association of chitin and pigments varies from species to species among

crustacean. Several workers have used reagents to eliminate pigments from crustacean

exoskeleton, usually crab. Hackman obtained a cream-colored lobster chitin by washing

with ethanol and ether [44], and Blumbeg extracted pigments with cold sodium


27

hypochloride solution, containing 0.5% available chlorine, and Kamasastri with absolute

acetone [54, 55]. Anderson, Brine, and Brzeski also accomplished decolouration of chitin

with chloroform, H2O2, and ethyl acetate, respectively [50, 56, 57]. No et al. prepared a

white colored crawfish chitin by acetone extraction, followed by bleaching with 0.32%

sodium hypochlorite solution [36]. However, Moorjani et al. recommends not bleaching

the material at any stage because the bleaching process considerably reduces the viscosity

of the final chitosan product [51].

1.4.3. Isolation of Chitin from Algae and Fungi Shell fish chitin is an exoskeletal component in a complex network containing

proteins and minerals while the main components in the complex network of fungal

chitin are other polysaccharides such as ά- and β-glucan, mannan and cellulose. The

isolation of chitin from fungal cell wall involves deproteinization and lipid extraction.

The mineral content in fungi is minimal and is normally not an issue. This is a defnite

advantage in providing a biomedical grade material in contrast to shellfish sources.

Deproteinization is achieved with NaOH or enzymes as detailed above. Lipid is usually

removed using organic solvents such as acetone. The glucan component can be

hydrolysed enzymatically at an added cost. The solvent 5% LiCl/DMAc is used to extract

the chitin-glucan complex and subsequently precipitated in water to give the chitin rich

complex [58].

1.4.4. Production of Chitosan The characteristics of the final chitosan products differ depending on the

crustacean species from which chitin is isolated, and the production method or sequence

[59-61]. Various procedures have been developed and proposed by many researchers

over the years for chitosan processing [36]. There are numerous reviews on various and

diverse preparation methods for recovery and evaluation of physicochemical properties of

chitosan.


28

Deacetylation is the process to convert chitin to chitosan by removal of acetyl

group. It is generally achieved by treatment with concentrated sodium or potassium

hydroxide solution (40-50%) usually at 100ºC or higher for 30 min or longer to remove

some or all of the acetyl groups from the polymer [36]. The N-acetyl groups cannot be

removed by acidic reagents without hydrolysis of the polysaccharide, thus, alkaline

methods must be employed for N-deacetylation [24]. Depending upon the production

sequence, deacetylation can be achieved by reaction of demineralized shells or crawfish

chitin with 50% NaOH (w/w) solution at 100°C for 30 min in air using a solid to solvent

ratio of 1:10 (w/v) [36]. There are several critical factors that affect the extent of

deacetylation including temperature and time of deacetylation, alkali concentration, prior

treatments applied to chitin isolation, atmosphere (air or nitrogen), ratio of chitin to alkali

solution, density of chitin, and particle size [62].

A new process for treating chitin under high concentrations of sodium hydroxide

with microwave energy was proposed by Peniston and Johnson to accelerate the

deacetylation of chitin within 18 min with 50% NaOH at a mean temperature under 80ºC

[63]. Chitin was deacetylated with concentrated aqueous NaOH in the presence of water-

miscible organic solvents such as 2-propanol, 2-methyl-2-propanol or acetone [64].

Although it is difficult to prepare chitosan with a degree of deacetylation greater than

90% without chain degradation, Mima et al developed a method for preparation of

chitosan having a desired degree of deacetylation of up to 100%, without serious

degradation of the molecular chain [65]. This was achieved by intermittently washing the

intermediate product in water two or more times during the alkali treatment for less than

5 h in 47% NaOH at 110ºC. A simple and inexpensive technique for deacetylation of

chitin has been developed in which Alimuniar and Zainuddin produced chitosan by

treatment of prawn chitin with strong sodium hydroxide at ambient temperature (30ºC)

without heating, in an inert atmosphere or without the addition of other additives to

control the reaction [66]. With 50% NaOH, the acid soluble chitosan with 87% degree of

deacetylation could be formed in a single day using 560 ml of the solution for 10 g of

chitin, two days using 420 ml, three days using 280 ml and six days using 140 ml. For a


29

large-scale preparation of chitosan, the process of deacetylation needs to be optimized.

No et al. used autoclaving conditions (15 psi/121ºC) to deacetylate chitin to prepare

chitosan under different NaOH concentration and reaction times [67]. Effective

deacetylation was achieved by treatment of chitin under an elevated temperature and

pressure with 45% NaOH for 30 min with a solid: solvent ratio of 1:15. Treated chitosan

showed similar nitrogen content, degree of deacetylation, and molecular weight, but

significantly higher viscosity value than those of commercial chitosan.

The bioconversion of chitin to chitosan was reported by Dimitris et al. Chitin

deacetylase from Mucor rouxii, the enzyme that catalizes the hydrolysis of acetamido

groups of N-acetyl glucosamine in chitin, is active on several chitinous substrated and

chitin derivatives

1.4.5. Degree of Deacetylation (DD) and its Significance The process of deacetylation involves the removal of acetyl groups from the

molecular chain of chitin, leaving behind a compound (chitosan) with a high degree

chemical reactive amino group (-NH2). This makes the degree of deacetylation (DD) an

important property in chitosan production as it affects the physicochemical properties,

hence determines its appropriate applications [21]. Deacetylation also affects the

biodegradability and immunological activity. The degree of deacetylation can be

employed to differentiate between chitin and chitosan because it determines the content

of free amino groups in the polysaccharides. In fact, there are two advantages of chitosan

over chitin. In order to dissolve chitin, highly toxic solvents such as lithium chloride and

dimethylacetamide are used whereas chitosan is readily dissolved in diluted acetic acid.

The second advantage is that chitosan possesses free amine groups which are an active

site in many chemical reactions [68].

Various methods have been reported for the determination of the degree of

deacetylation of chitosan. These included ninhydrin test, linear potentiometric titration,

near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, hydrogen bromide


30

titrimetry, infrared spectroscopy, and first derivative UV-spectrophotometry [69]. The IR

spectroscopy method, which was first proposed by Moore and Roberts, is commonly used

for the estimation of chitosan DD values [70]. This method has a number of advantages

and disadvantages. First, it is relatively fast and unlike other spectroscopic methods, does

not require purity of the sample to be tested nor require dissolution of the chitosan sample

in an aqueous solvent [71]. For IR spectroscopic determination, the absorbance ratio of

A1550/A2878 was first suggested but several other ratios, [A1320/A1420] were also reported

[72, 73].

ASTM F2260-03 covers the determination of the degree of deacetylation in

chitosan and chitosan salts intended for use in biomedical and pharmaceutical

applications as well as in Tissue Engineered Medical Products (TEMPs) by high-

resolution proton NMR (1H NMR) [74]. Chitosan with higher degree of deacetylation and

molecular weight is more suitable for tissue engineering applications. The DD values of

chitosan are highly affected by the analytical methods employed. Hence, the

quantification method for DD should also be stated when reporting the DD value of

chitosan sample [75]. Hsu et al investigated in vitro the role of the degree of deacetylation

and molecular weight on some biological properties of chitosan films [76]. The results

showed that the degree of deacetylation affected the hydrophilicity and biocompatibility

of the chitosan films. The molecular weight, on the other hand, affected the rate of

degradation and the mechanical properties. Chitosan with higher degree of deacetylation

and molecular weight is more suitable for tissue engineering applications. According to

Piotrowska et al it is not possible to obtain products with a degree of deacetylation higher

than 84% from chitosan with an initial degree of deacetylation of 68% when a 1.2%

solution is used as a substrate for the pre-purified deacetylase from Mucor rouxii

mycelium. The incomplete deacetylation of chitosan under the applied conditions of

enzymatic reactions was not the result of the thermal denaturation of deacetylase during

the relatively long enzymatic reaction. It was shown that enzymatic deacetylation could

be partially inhibited by acetic acid released during deacetylation [77]. Deacetylation of

chitin under homogeneous conditions was optimized by Nemtsev et al [78]. Effect of


31

molecular weight and degree of deacetylation of chitosan on urea adsorption properties of

copper chitosan was studied by Zhou et al [79]. Experimental results showed that the

adsorption capacity for urea of copper chitosan increased with an increasing degree of

deacetylation and decreasing molecular weight of chitosan. Nunthanid et al studied the

physical properties and molecular behavior of chitosan films and found that the increase

in molecular weight of chitosan would increase the tensile strength and elongation as well

as moisture absorption of the films, whereas the increase in degree of deacetylation of

chitosan would either increase or decrease the tensile strength of the films depending on

its molecular weight. Moreover, the higher the degree of deacetylation of chitosan, the

more brittle and the less moisture absorbing the films are [80].

1.4.6. Properties Chitin and chitosan are highly basic polysaccharides while most of the naturally

occurring polysaccharides e.g. cellulose, dextrin, pectin, alginic acid, agar, agarose and

carragenous are neutral or acidic in nature. Their unique properties include polyoxy salt

formation, ability to form films, metal chelations, optical and structural characteristics.

Although the β (1-4) anhydroglucosidic bond of chitin is also present in cellulose, the

characteristic properties of chitin/chitosan are not shared by cellulose.

1.4.6.1. Molecular Weight Chitosan is a biopolymer of high molecular weight. Like its composition, the

molecular weight of chitosan varies with the raw material sources and the method of

preparation. Molecular weight of native chitin is usually larger than one million Daltons

while commercial chitosan products have the molecular weight range of 100,000 –

1,200,000 Daltons, depending on the process and grades of the product [26]. In general,

high temperature, dissolved oxygen, and shear stress can cause degradation of chitosan.

For instance at a temperature over 280ºC, thermal degradation of chitosan occurs and

polymer chains rapidly break down, thereby lowering molecular weight [27]. The


32

molecular weight of chitosan can be determined by methods such as chromatography,

light scattering, and viscometry [32, 81].

Although the primary structure of chitosan is a backbone of (1-4)-β-D-

glucosamine residues randomly acetylated to various extents, the name chitosan in fact a

collective term for deacetylated chitins differing in terms of crystallinity, optical

characteristics, degree of acetylation, impurity content and average molecular weights.

Production methods and origins are mainly responsible for the above differences, which

are encountered in chitosans. If quality control standards are sought for properly

characterized chitosans, especially for use in specialized applications, a reliable method

of molecular weight determinations is desirable. Methods currently used are based on

viscometric measurements. Chitosan molecular weight distributions have been obtained

using, High Performance Liquid Chromatography (HPLC). The weight average

molecular weight of chitin and chitosan has been determined by light scattering. As has

been well documented, viscometry is the simplest and the most effective method for

determining Mw of polymers [82]. For a linear chain polymer the relationship between

intrinsic viscosity, [η] and Mw, is shown by the Mark–Houwink equation [83].

[η] = kMw a

where, k and a are constants independent of Mw over a wide range. They are

affected by solvent conditions such as temperature, pH and ionic strength. For chitosan in

solvent, 0.1M acetic acid/0.2M NaCl, k and a values have been reported as 1.81 × 10−5

and 0.93 at 25 ºC, respectively.

1.4.6.2. Viscosity Just as with other food matrices, viscosity is an important factor in the

conventional determination of molecular weight of chitosan and in determining its

commercial applications in complex biological environments such as in the food system.

Higher molecular weight chitosans often render highly viscous solutions, which may not

be desirable for industrial handling. Some factors during processing such as the degree of


33

deacetylation, molecular weight, concentration of solution, ionic strength, pH, and

temperature affect the production of chitosan and its properties. For instance, chitosan

viscosity decreases with an increased time of demineralization [51]. Viscosity of chitosan

in acetic acid tends to increase with decreasing pH but decrease with decreasing pH in

HCl, giving rise to the definition of ‘Intrinsic Viscosity’ of chitosan which is a function

of the degree of ionization as well as ion strength. Bough et al. found that

deproteinization with 3% NaOH and elimination of the demineralization step in the chitin

preparation decrease the viscosity of the final chitosan products [41]. Moorjani et al also

stated that it is not desirable to bleach the material at any stage since bleaching

considerably reduces the viscosity of the final chitosan product [51].

Similarly, No et al demonstrated that chitosan viscosity is considerably affected

by physical (grinding, heating, autoclaving, ultrasonication) and chemical (ozone)

treatments, except for freezing, and decreases with an increase in treatment time and

temperature [85]. Chitosan solution stored at 4ºC is found to be relatively stable from a

viscosity point of view. The effect of particle size on the quality of chitosan products was

investigated by Bough et al [41], who reported that smaller particle size (1mm) results in

chitosan products of both higher viscosity and molecular weight than those of either 2 or

6.4 mm particle size. They further enumerated that a larger particle size requires longer

swelling time, resulting in a slower deacetylation rate. But, in contrast, Lusena and Rose

reported that the size of chitin particle within the 20-80 mesh (0.841-0.177 mm) range

had no effect on the viscosity of the chitosan solutions [84]. The viscosity parameters K

and ‘a’ for chitin in DMAc-5% LiCl solution are K=2.2 x 10 -4 dLg-1, a = 0.88 [85].

1.4.6.3. Solubility Chitin is a highly resistant biopolymer and is insoluble in water and most organic

solvents. It is soluble in hexafluoro isopropanol, hexafluoro acetone, chloro alcohols in

conjunction with aqueous solutions of mineral acids and dimethyl acetamide (DMAC)

containing 5% lithium chloride [86]. The solubility of chitin can be enhanced by

introducing bulky acyl residues into the polymer (as in butyrylchitin and valeroylchitin).


34

However, if modification is carried out with shorter-chain carboxylic acids (as in

acetylchitin), the solubility remains poor. By substituting the acetyl residues partially by

butyryl residues (mixed ester formation), exclusive use of the bulky carboxylic acids can

be avoided and yet good solubility is achieved [87]. Ravindra et al has reported a method

for the determination of the solubility parameters of chitin and chitosan by group

contribution method and they compared the values with those determined form intrinsic

viscosity, surface tension, the Flory-Huggins interaction parameter and dielectric constant

values [88]. Young and Kim described the synthesis of aminoethyl-chitin in order to

increase both the solubility and antimicrobial action of chitin [89]. The chitin became

soluble in dilute acetic acid at the DD of 28% or over and soluble in water at the DD of

49%. The solubility of the partially deacetylated chitins had a close relationship with their

crystal structure, crystallinity, and crystal imperfection as well as the glucosamine

content. The wide-angle X-ray diffractometry (WAXD) revealed that the chitin with 28%

DD retained the crystal structure of ά-chitin with significantly reduced crystallinity and

perfection of the crystallites. The water-soluble chitin of 49% DD had a new crystal

structure similar to that of β-chitin rather than either ά-chitin or chitosan, suggesting that

the homogeneous deacetylation transformed the crystal structure of chitin from the ά to

the β form. Some hydrogen bonds existing in raw ά-chitin were found to be missing at a

DD of 49% [90].

While chitin is insoluble in most organic solvents, chitosan is readily soluble in

dilute acidic solutions below pH 6.0. Organic acids such as acetic, formic, and lactic

acids are used for dissolving chitosan. Chitosan is soluble in dilute acids such as acetic

acid, formic acid etc. The dissociation of chitosan in N-methyl morpholine-N-Oxide

(NMMO)/H2O has been reported by Dutta et al [91]. Chitosan is soluble in dilute acids

on account of protonation of free amine groups.

The most commonly used is 1% acetic acid solution at about pH 4.0 as a

reference. Chitosan is also soluble in 1% hydrochloric acid but insoluble in sulfuric and

phosphoric acids. Solubility of chitosan in inorganic acids is quite limited. Concentrated

acetic acid solutions at high temperature can cause depolymerization of chitosan [92].


35

Above pH 7.0 chitosan does not exhibit a stable solubility. At higher pH, precipitation or

gelation tends to occur and the chitosan solution forms poly-ion complex with anionic

hydrocolloid resulting in the gel formation [93]. The concentration ratio between chitosan

and acid is of great importance to impart desired functionality [65]. At concentrations as

high as 50 percent organic solvent, chitosan still works as a viscosifier causing the

solution to remain smooth. Trimethylsilylation of chitosan with hexamethyldisilazane and

chlorotrimethylsilane was attained by Kurita et al and the resulting silylated derivative

expressed improved solubility and proved to be a convenient precursor for modification

reactions [94].

1.4.6.4. Crystalline structure Chitin is a linear polysaccharide consisting of β-1, 4-linked N-acetyl-D-

glucosamine. On the basis of its crystalline structures, chitin is classified into three forms,

α, β and γ chitins. The most abundant form is α-chitin, where the molecules are aligned in

an anti parallel fashion. This molecular arrangement is favourable for the formation of

strong intermolecular hydrogen bonding, and α-chitin is the most stable form of the three.

In β-chitin, the molecules are packed in a parallel way, resulting in a weaker inter-

molecular force. β -chitin is therefore less stable than α-chitin and can be transformed

into α-chitin. γ- chitin is considered to be a mixture of α and β forms and has both

parallel and anti parallel arrangements. Ogawa and Yui stated that the three structural

forms of chitosan (hydrated and anhydrous crystal, and non crystal) could be examined

easily by measuring the x-ray powder diffraction pattern of a chitosan sample [95]. The

hydrated crystal showed a strong reflection at an angle 2θ of 10.4° and the other peaks

more weakly at 20° and 22°. The anhydrous crystal exhibited a strong peak at 2θ of 15°

and a peak supplemented at 20º. An amorphous chitosan does show any reflective, but

they ascertained that it exhibits a broad halo at 2θ of around 20º. Piron et al reported that

the dissolution of chitosan involves the progressive disappearance of the peak at 2θ ~ 22°

and suggested that polymer swelling destroys the residual crystallinity increasing the

accessibility of solute to sorption sites [96].


36

1.4.6.5. Hydrophilicity Like other polysaccharides α-chitin is hygroscopic. β –chitin shows much higher

hygroscopicity because of the loose arrangement of its molecules and the cotton like fluffy

state compares with the more solid state of α-chitin. Chitosan derived from α-chitin shows

higher hydrophilicity than α-chitin, but the reverse is true of the chitosan derived from β –

chitin. The water soluble chitin is highly hygroscopic and almost like hyaluronic acid.

1.4.6.6. Biodegradability Both chitin and chitosan are degraded in nature by many varities of micro

organisms. Most of the chitinases in microorganisms hydrolyze N-acetyl- β-1, 4-

glucosaminide linkages randomly. Chitinases also are present in higher plants, even

though plants do not have chitin as a structural component. This characteristic may be

related to the self defence activity of plants against pathogenic microbes and insects that

have chitin. Chitinases have been studied extensively, but not much is known about

chitosanases. The degree of deacetylation of chitin affects its biodegradability, and the

sample with a degree of deacetylation of 0.7 is most susceptible to lysozyme.

Introduction of mercapto groups also enhances susceptibility to lysozyme. These results

interpreted in terms of the destruction of crystalline structure of O-carboxy methyl chitin

which has the substituents mainly at C-6 is degraded by lysozyme more readily than the

original chitin but the degradation may be slower if the carboxy methyl group is at C-3.

Ratajska, et al studied the biodegradation of chitosan in an aqueous medium [97].

Ramesh et al characterised chitosan films after in vivo implantation in mice [98].

Ratajska and Boryniec studied the physical and chemical aspects of natural polymers and

found that the biodegradation of chitosan was dependent on both the origin and properties

of the samples, as well as on the degree of their deacetylation and conditions of the

biodegradation process. Research into the biodegradation of mixtures of synthetic and

natural polymers with the use of the method of light transmittance led to the general

conclusion that the biodegradation process was largely dependent on the type of the


37

synthetic polymer as well as on conditions of the process [99]. Tomihata and Ikada

reported that the in vitro and in vivo degradation of chitosan films prepared by solution

casting method occurred less rapidly as the degree of deacetylation increased, and films

that were greater than 73% deacetylated, showed slower biodegradation [100]. Wang etal

compared the chitosan and collagen sponges as haemostatic dressings. After implantation

in rabbit muscles they found that the chitosan sponge was degraded much slower than the

collagensponge, while tissue responses for the chitosan sponges were significantly

greater than for the collagen sponges [101].

1.4.6.7. Water Binding Capacity (WBC) and Fat Binding Capacity (FBC) Water uptake of chitosan was significantly greater than that of cellulose and even

chitin [22]. Basically, WBC for chitosan ranges between 580 to 1150% with an average

of about 70%, according to Rout [27]. He also noted that reversing the sequence of steps

such as demineralization (DM) and deproteinization (DP) had a pronounced effect on

WBC and FBC. DP of demineralized shell also gives higher WBC compared to the

process when DM of the deproteinized shell is conducted. Besides, the process of

decolouration (DC) also causes a decrease in WBC of chitosan than those of unbleached

crawfish chitosan. The fat uptake of chitin and chitosan ranges from 315 to 170% with

chitosan having the lowest and chitin the highest fat uptake [102].

1.4.6.8. Antimicrobial Properties Recent studies in antibacterial activity of chitosan have revealed that chitosan is

effective in inhibiting growth of bacteria. The antimicrobial properties of chitosan depend

on its molecular weight and the type of bacterium. For gram-positive bacteria, chitosan

with 470 KDa was the most effective, except for Lactbacillus sp., whereas for gram-

negative bacteria, chitosan with 1,106 KDa was effective. Chitosan generally showed

stronger bactericidal effects for gram-positive bacteria (Listeria monocytogenes, Bacillus

megaterium, B. cereus, Staphylococcus aureus, Lactobacillus plantarum, L. brevis, and

L. bulgaris) than for gram-negative bacteria (E.coli, Pseudomonas fluorescens,


38

Salmonella typhymurium, and Vibrio parahaemolyticus) in the presence of 0.1% chitosan

[103]. Koide reported that chitin and chitosan in vitro show antibacterial and anti-yeast

activities [104]. One of chitosan derivatives, i.e., N-carboxybutyl chitosan, was tested

against 298 cultures of different pathogenic microorganisms that showed bacteriostatic

and bactericidal activities, and there were marked morphological alterations in treated

microorganisms when examined by electron microscopy [105]. Conversely, growth

inhibition and inactivation of mould and yeasts seem to depend on chitosan

concentration, pH, and temperature [27]. According to Cuero, the antimicrobial action of

chitosan is influenced by intrinsic and extrinsic factors such as the type of chitosan (e.g.,

plain or derivative), degree of chitosan polymerization, host nutrient constituency,

substrate chemical and/ or nutrient composition, and environmental conditions such as

substrate water activity [106]. In an extensive research by Tsai and Su on the

antimicrobial activity of chitosan prepared from shrimp against E.coli, they found that

higher temperature and acidic pH of foods increased the bactericidal effect of chitosan

[107]. They also explained the mechanism of chitosan antibacterial action involving a

cross-linkage between polycations of chitosan and the anions on the bacterial surface that

changes membrane permeability. Chitosan has been approved as a food additive in Korea

and Japan since 1995 and 1983, respectively [108, 109, 103]. Higher antibacterial activity

of chitosan at lower pH suggests that addition of chitosan to acidic foods will enhance its

effectiveness as a natural preservative [103].

1.4.6.9. Film Forming Property Chitosan has an ability to form film which makes it suitable for use as food

preservation for control of psychotropic pathogen in fresh/ processed meat and fish

products packaged under modified atmosphere [110]. According to Charles et al, the

most potential application of chitosan is as a coating agent in the area of fruit

preservation [111]. The biodegradability of chitosan is one of the most advantageous

features for concern of the environmental damage occurring by improper disposal of

petrochemical based plastics [112]. N, O-carboxymethyl chitosan can form a strong film

that is selectively permeable to such gases as oxygen and carbon dioxide. Apples coated


39

with this material remain fresh for up to six months. The chitosan coating has been shown

to delay ripening of banana for up to 30 days whereas chitosan film manifests a slightly

yellow appearance, with the color darkening as thickness increased [113].

1.4.7. Chitin Oligomers

Scheme 5. Pathway for the conversion of chitin and chitosan into their oligomers by enzymatic means.

Chitin and chitosan backbones are hydrolysed with hot hydrochloric acid to afford

D-glucosamine. Degradation under milder conditions affords mixtures of oligomers

consisting of N-acetyl-D-glucosamine and D-glucosamine respectively. The resulting

oligomers are separated by column chromatography/SEC. Enzymatic degradation of

chitin is another way to prepare oligomers. The addition of some inorganic salts was

shown to effectively accelerate the degradation of chitosan under microwave irradiation.

The molecular weight of the degraded chitosan obtained by microwave irradiation

was considerably lower than that obtained by traditional heating [114].


40

1.4.8. Water Soluble Chitin When chitin is steeped in concentrated aqueous sodium hydroxide and treated

with crushed ice, an alkaline chitin solution results. Under these conditions, deacetylation

proceeds efficiently. An alkali chitin solution in 10% sodium hydroxide left for 70 h at

room temperature gives a product with about 50% deacetylation that is soluble in neutral

water. Higher or lower deacetylations fail to lead to complete solubilisation, and the

degree of deacetylation should be 0.45-0.55 for water solubility. Alternatively, random

N-acetylation of chitosan to a degree of acetylation of about 0.5 gives rise to a water

soluble product [115].

O

NH2

OH

OH

O

Ac2O

Pyr

O

NHAcOH

OH

l

O

NH2

OH

OH

m

Scheme 6. Preparation of water soluble chitin (l~m), from chitosan

1.4.9. Chemical Modifications Because chitin and chitosan have strong intermolecular forces and highly

crystalline structures they are insoluble in most organic solvents and much less accessible

to potential reactants than cellulose. Therefore, modifications of chitin and chitosan

generally have been performed under heterogeneous conditions with a few exceptions.

Chitin and chitosan have been modified via a variety of chemical methods. Some authors

have reviewed the methods [116, 117] and Roberts have explained the modification

reactions in his source-book Chitin Chemistry [118]. Of the various possible

modifications (e.g., nitration, phosphorylation, sulphation, xanthation, acylation,

hydroxyalkylation, Schiff’s base formation and alkylation) graft copolymerization is

expected to be one of the most promising approaches to a wide variety of molecular

designs leading to novel types of hybrid materials, which are composed of bio- and


41

synthetic-polymers [117-122]. This modification technique, as foreseen by Kurita, will likely

find new applications in some fields including water treatment, metal cation adsorption,

toiletries, medicine, agriculture, food processing and separation [117].

1.4.9.1. Deacetylaton

O

NHAcOH

OHOH- O

NHAcOH

OHO

NH2

OH

OH

nl m

Scheme 7. Deacetylation of chitin

Deacetylation of chitin is effected with strong aqueous bases such as sodium and

Potassium hydroxides. When deacetylation is conducted with dilute alkali (20-30%

NaOH) at gentle reflux, the degree of deacetylation levels off early. This result suggests

that deacetylation under these conditions proceeds preferentially in amorphous regions of

chitin and the products are supposed to be block type copolymers composed of N-acetyl-

D-glucosamine and D-glucosamine. Deacetylation with 40% sodium hydroxide at 130°C

is quite satisfactory and the deacetylation degree increases with time without leveling off.

During the deacetylation process, however, degradation of the main chain occurs as

evident from considerable decrease in the molecular weight.

β –chitin shows much higher reactivity than α-chitin in deacetylation because of

relatively weak intermolecular hydrogen bonding, but β –chitin tends to discolour during

the reaction. Deacetylation proceeds more efficiently in homogeneous alkali chitin

solution. Under these homogeneous conditions, deacetylation was considered to take

place randomly along the main chain, and the samples with about 50% deacetylation

were water-soluble. Partially deacetylated chitin with degrees of deacetylation <0.45 or

>0.55 showed lower solubility. The water solubility of the samples with a degree of


42

deacetylation about 0.5 is ascribable to the greatly enhanced hydrophilicity due to the

random distribution of acetyl groups at half of the amino groups. Interestingly, samples

with the same degree of deacetylation prepared by conventional heterogeneous hydrolysis

are not soluble owing to the block-wise distribution of the acetyl groups.

1.4.9.2. Acylation

O

NHAcOH

OH

l

O

NH2

OH

OH

m

AC2O

MeSO3H

O

NHAcOH

OH

n

O

NHAcOH

OH

l

O

NH2

OH

OH

m

AC2O O

NHAcOH

OH

n

MeOH

Scheme 8. Peracetylation and N-acetylation of partially deacetylated chitin

Because α-chitin is not soluble in most solvents suitable for acylation, extensive

acetylation is attained only under rather severe conditions (eg: with acetic anhydride and

hydrogen chloride) [123].

Full acetylation is possible in methane sulfonic acid with acetic anhydride. The reaction

mixture is heterogeneous initially but the acetylated derivative goes into solution as the degree

of acetylation increases. A mixture of trichloroacetic acid and 1,2-dichloroethane dissolves

chitin, and so acetylation proceeds in this solution [124]. These solvent systems however are

strongly acidic and cause degradation of the backbone. DMAC containing lithium chloride also

allows reactions in solution, and acylated or carbamoylated chitin were prepared as models for

controlled release herbicides [116].

Hirano et al studied the effect of N-acylation on the property of chitosan

filaments. Their filament tenacity and elongation values were little influenced by the N-


43

acylation [125]. N-acylation of chitosan with various fatty acid (C6-C16) chlorides

increased its hydrophobic character and made important changes in its structural features.

Unmodified chitosan exhibited a low degree of order (DO) and a weak tablet crushing

strength. Chitosan acylated with a short chain length (C6) possessed similar properties,

but exhibited significant swelling. Acylation with longer side chains (C8-C6) resulted in

a higher DO and crushing strength but lower swelling. The best mechanical

characteristics and drug release properties were found for palmitoyl chitosan (substitution

degree 40-50%) tablets with 20% acetaminophen as a tracer [126]. Complete N-acylation

of chitosan has been achieved by treating with cyclic acid anhydrides in aqueous

homogeneous media at pH 4 to 8 [127].

Novel N-saturated fatty acyl derivatives of chitosan soluble in water and in

aqueous alkaline and acid solutions were prepared in 45-72% yields by N-deacylation of

sodium N-acylchitosan salts, and in 75-85% yields by N-acylation of chitosan by Hirano

et al [125].

Although β –chitin is also not soluble in common solvents, it swells considerably

in many solvents including methanol. Free amino groups present in β –chitin can thus be

fully acetylated directly in methanol with acetic anhydride to give structurally uniform

chitin, poly (N-acetyl-D-glucosamine).

α-chitin does not undergo acetylation under similar conditions. When β –chitin is

treated with acetic anhydride in pyridine in the presence of 4-dimethyl aminopyridine,

fully acetylated chitin is prepared quite readily, and this result indicates much higher

potential of β –chitin as a versatile starting material for chemical modifications.

Chitosan is soluble in aqueous acetic acid and does not precipitate in dilution with

an equal amount of methanol; therefore, N-acylation of chitosan is facile. With

carboxylic acid anhydrides or chlorides, acylation proceeds smoothly at the free amino

groups and then more slowly at hydroxyl groups, but gelation is usually observed during

reaction as a result of reduced solubility. Chitosan is also acylated with long chain


44

aliphatic acid chlorides in pyridine-chloroform and a soluble derivative with a degree of

acylation 4 was obtained.

The water soluble chitin can be selectively N-acetylated in homogeneous solution

with acetic acid and dicyclohexyl carbodiimide to give fully N-acetylated chitin. With

highly swollen water soluble chitin gel in pyridine or DMAc, many modification

reactions can be conducted in almost homogeneous solutions under mild conditions. An

example is acylation with aromatic anhydrides such as phthalic, trimellitic and

pyromellitic anhydrides. In these cases, acylation followed by dehydration gives rise to

the formation of the imide derivatives. The phthaloyl derivative was soluble in dimethyl

sulfoxide (DMSO). The trimellitic and pyromellitic derivatives were reactive toward

nucleophiles due to the presence of carboxyl or acid anhydride groups [128].

O

NHAcOH

OH

l

O

NH2

OH

OH

m

Ar O

O

O

Pyr

O

NHAcOH

OH

l

O

NHOH

OH

mO=C

ArCOOH

-H2O

O

NHAcOH

OH

l

O

NOH

OH

m

Ar

OO

Ar =

COOH

C

O

C

O

O

Scheme 9. Acylation of the water soluble chitin with cyclic acid anhydrides

To further improve solubility in organic solvents, N-phthaloylation of 100%

deacetylated chitosan has been examined in detail. The reaction is accomplished by


45

treating a chitosan suspension in dimethylformamide (DMF) with excess phthalic

anhydride at 130°C. The resulting fully N-phthaloylated chitosan proved to be readily

soluble in polar solvents such as pyridine, DMF, DMAc and DMSO [129]. Also,

N-Phthaloylation proceeds more readily with chitosan derived from β–chitin than that

from α-chitin, a result indicating that the difference in crystalline structures of chitins

affects their reactivity even after deacetylation [130]. With N-phthaloyl chitosan, various

modification reactions proceed quantitatively and regioselectively in homogeneous

solutions in organic solvents. Tritylation for example, quantitatively takes place at C-6

hydroxyl groups in pyridine.

O

NH2

OH

OH

O

C

O

C

O

O

DMF

O

NphthOH

OH

O

TrCl

Pyr

O

NphthOH

OTr

O

N2H2

H2O

O

NH2

OH

OTr

O

O

NH2

OH

OTr

O

1) SO3-Pyridine

2) CHCl2COOH

O

NH

NaO3SO

OTr

O

0SO3Na

Trityl-chitosan 3-O, 2-N-Chitosan sulphate

Scheme 10. Preparation of N-phthaloyl-chitosan and its etherifications

The phthaloyl group can be removed with hydrazine to generate the free amino

group. Dephthaloylation of N-phthaloyl-6-trityl-chitosan, for example gives 6-trityl-

chitosan. 6-Trityl-chitosan can be transformed into amphiphilic derivatives by

regioselective introduction of palmitoyl groups at the C-2 amino and C-3 hydroxyl

groups, followed by detritylation and then sulfation at the C6 hydroxyl group. The


46

resulting derivative shows characteristic amphiphilic properties and forms Langmuir

monolayers with high collapse pressures [131].

1.4.9.3. Tosylation Tosylation of chitin was accomplished by interfacial condensation to give

tosylchitins expected to be useful as soluble and reactive precursors for chemical

manipulations under mild conditions [132]. Incorporation of p-toluenesulfonyl (tosyl)

groups into chitin is useful because the tosyl group enhances solubilization and tosylates

are highly reactive. Although tosylation of chitin is quite sluggish in pyridine even at

100°C, it proceeds smoothly in a two-phase mixture of an aqueous alkali chitin solution

and tosyl chloride in chloroform [115].

O

NHAcOH

OH

n

TsCl

OH-

O

NHAcOH

OTs

n

O

NHAcOH

I

n

O

NHAcOH

Me

n

O

NHAcOH

SH

n

NaINaBH4

1)CH3COSK2) NaOMe

Scheme11. Preparation of tosyl-chitin and its modifications

Tosyl groups had been introduced at the C-6 positions. Tosyl-chitins with

substitution degrees >0.5 are soluble in polar organic solvents such as DMSO.

1.4.9.4. Alkylation Hydroxy ethylation (or glycolation) of chitin is effected by treating alkali-chitin

with ethylene oxide to give hydroxyl-ethyl chitins or glycol-chitins. However, the

reaction is carried out under strongly alkaline conditions; N-deacetylation also takes

place at the same time.


47

O

NHAcOH

OH

n

O

NaOH

O

NHROH

n

O(CH2CH2OH)n

R = AC or H

Scheme12. Preparation of hydroxyethyl-chitin

Moreover, ethylene oxide may polymerise and result in the introduction of

oligoethylene glycol side chains as observed in the hydroxyl ethylation of cellulose under

similar conditions. The derivatives are readily soluble in water and are used as substrates

for assaying chitinolytic enzymes. In place of ethylene oxide, 2-chloro ethanol can be

used. In a similar manner, propylene oxide gives hydroxyl-propyl chitin. Monoaldehydes

have been synthesized from tri- and tetra (ethylene glycol) monosubstituted derivatives

and introduced into chitosan by the reductive alkylation technique to give comb-shaped

polysaccharide hybrids [132]. The reaction of chitosan with the aldehydes in the presence

of sodium cyanoborohydride proceeded efficiently to give chitosan derivatives having

oligo (ethylene glycol) side chains at the amino groups. The products were characterized

by high affinity for organic solvents as well as water in sharp contrast to the original

chitin and chitosan. They showed significant adsorption capacity toward metal cations.

Hydroxy propylation of chitosan preferentially takes place at hydroxyl groups under

alkaline conditions, but N-alkylation occurs almost exclusively under acidic conditions

[133]. Hydroxy alkylation is also possible with glycidol or 3-chloropropane-1, 2-diol

(glycerol ά-monochlorohydrin). The resulting chitosan derivatives are soluble in water

and attractive as components of toiletries.

Reductive alkylation is another interesting procedure for N-alkylation. Chitosan is first

treated with an aldehyde to give an imine (Schiff base), which is converted into N-alkyl

derivative by reduction with sodium cyanoborohydride. With reducing sugars including

glucose, galactose, lactose and cellobiose in the presence of sodium cyanoborohydride, sugar

groups are introduced into chitosan through reductive alkylation [134, 135].


48

O

OH

OH

n

NH2

O

O

OH

OH

OHO

O

OHHO

OH

OH

O

OH

OH

n

NH

O

OHHOO

OH

OH

OH

O

OHOH

NaCNBH3

OH

OH

Scheme13. Reductive alkylation of chitosan with sugars

The degrees of substitution are generally high and the derivatives are soluble in water

or dilute acids. The derivatives are interesting as water soluble polysachharides with peculiar

viscosity behaviour. An aqueous solution of chitosan-lactose showed unusual non-Newtonian

features such as low-shear Newtonian behavoiour, a medium shear viscisoty increase

(dialatancy), and a high-shear viscosity drop (pseudo plasticity) [136].

When chitosan is treated with aliphatic aldehydes and then with a reducing agent,

N-alkylation takes place. In this manner alkyl groups such as methyl, ethyl and propyl are

introduced. The resulting derivatives are soluble in dilute acetic acid and have film

forming properties. They absorb various metal cations [137].

1.4.9.5. Carboxylation Carboxymethylation of chitin proceeds with alkali chitin and monochloroacetic

acid. Because of the strongly alkaline conditions; N-deacetylation also takes place and

leads to the formation of amphoteric polymers having both carboxyl and amino groups.

The degree of deacetylation may reach >0.5. Carboxymethylation is supposed to proceed

preferentially at C-6 as implied from the results of backbone hydrolysis.

Carboxymethylated chitin is soluble in water. When chitosan is treated with

monochloroacetic acid, N-, O-dicarboxymethylated chitosan is produced.


49

O

NHAcOH

OH

n

O

ClCH2CO2H

NaOH

O

NHROH

OCH2CO2H

n

O

R= Ac or H

Sceheme14. Carboxymethylation of chitin

1.4.9.6. Sulphation Sulphation of chitin and chitosan was studied extensively, primarily for preparing

anticoagulants, because of the structural similarities to heparin. The use of various

sulphating agents including concentrated or fuming sulfuric acid and sulphur trioxide

with pyridine, trimethyl amine, DMF or sulphur trioxide was reported, but

chlorosulphonic acid is most widely employed. In chitin, hydroxyl groups are sulphated,

whereas with chitosan, sulphation occurs at both hydroxyl and amino groups.

O

NH2

OH

OTr

O

1) SO3-Pyridine

2) CHCl2COOH

O

NH

NaO3SO

OTr

O

0SO3Na

Trityl-chitosan 3-O, 2-N-Chitosan sulphate

Sceheme15. Sulphation of chitosan

1.4.9.7. Schiff Base Formation Free amino groups of chitosan react with an aldehyde to give the corresponding

Schiff base. The resulting imine linkage is fairly stable in neutral and alkaline solutions,

but under acidic conditions, it is readily hydrolyzed. Therefore amino groups can be

protected with aldehydes to allow hydroxyl groups to be modified. Reactions of chitosan


50

with aldehydes are carried out conveniently in a mixture of aqueous acetic acid and

methanol to give derivatives with a degree of substitution < 1.0. During the reaction, the

originally homogeneous solution gels due to poor solubility of the Schiff base [116].

1.4.9.8. Quaternary Salt Formation Because of the presence of the free amino groups, chitosan forms salts with

organic and inorganic acids. This characteristics account for the solubility of chitosan in

aqueous acid solutions. It is readily soluble in hydrochloric or nitric acid (< 1%), less

soluble in phosphoric acid, and not at all soluble in sulphuric acid. Many aqueous organic

acids dissolve chitosan; and formic, acetic, lactic, and pyruvic acids are frequently used

to make solutions.

Chitosan and some derivatives form polyion complexes with polyanions. On

mixing a chitosan solution with polyanion solution, a polyion complex precipitates. The

compositions and properties vary widely depending on concentration, molar ratio, pH,

and order of mixing. Various polyanions, including dextran sulphate, carboxymethyl-

dextran, carboxymethyl cellulose, carboxymethyl chitosan, alginic acid, poly (aspartic

acid), keratin derivatives, and poly(acrylic acid), have been used for this purpose. The

resulting complexes are useful as anticoagulant biomedical materials, microcapsule walls,

membranes, and carriers for drug-delivery systems.

Synthesis of novel quaternary chitosan derivatives via N-chloroacyl-6-o-

triphenylmethylchitosans has been reported by Jukka Holappa et al [138]. Full

substitutions were obtained from the quaternization reactions and the obtained quaternary

chitosan derivatives were water-soluble. Chitosan derivatives have been evaluated to

overcome chitosan’s limited solubility and effectiveness as absorption enhancer at neutral

pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC)

has been synthesized at different degrees of quaternization [139]. This quaternized

polymer forms complexes with anionic macromolecules and gels or solutions with

cationic or neutral compounds in aqueous environments and neutral pH values. TMC has


51

been shown to considerably increase the permeation and/or absorption of neutral and

cationic peptide analogs across intestinal epithelia. The mechanism by which TMC

enhances intestinal permeability is similar to that of protonated chitosan.

1.4.9.9. Graft Copolymers of Chitosan A graft copolymer is a macromolecular chain with one or more species of block

connected to the main chain as side chain(s). Thus, it can be described as having the

general structure shown in Scheme 16, where the main polymer backbone poly (A),

commonly referred to as the trunk polymer, has branches of polymer chain poly (B)

emanating from different points along its length.

Scheme 16. Graft copolymer formation

The common nomenclature used to describe this structure, where poly(A) is

grafted with poly(B), is poly(A)-graft-poly(B), which can be further abbreviated as

poly(A)-g-poly(B). Grafting of synthetic polymer is a convenient method to add new

properties to a natural polymer with minimum loss of the initial properties of the


52

substrate. Chitin and chitosan possess aforesaid useful properties that render them

interesting starting materials for the synthesis of graft copolymers. Most of the

copolymers are prepared through graft polymerization of vinyl monomers onto the

biopolymer backbone [140].

1.4.9.10. Vinyl Graft Copolymerization Graft copolymers are prepared by first generating free radicals on the biopolymer

backbone and then allowing these radicals to serve as macro initiators for the vinyl (or

acrylic) monomer (Scheme 16). Mino and Kaizerman firstly reported this approach in

1958 for graft copolymer preparation using a ceric ion redox initiating system [141].

Then, the chemistry and technology of the radical graft copolymerization technique [122,

142] was developed especially in the case of cellulose and starch [143-145]. Generally,

free radical initiated graft copolymers have medium to high molecular weight branches

that are infrequently spaced along the polysaccharide backbone [143]. The

copolymerizations can also be initiated anionically by allowing monomer to react with an

alkali- metal alkoxide derivative of polysaccharide. However, this method has not

progressed due to difficulty of the process and the low molecular weight of the grafted

branches [145]. The properties of the resulting graft copolymers may be controlled

widely by the characteristics of the side chains including molecular structures, length,

number, and frequency.

One of the most important features of graft polymerization is unwanted

concomitant formation of homopolymer, homopoly (B) that is not chemically bonded to

the substrate poly (A). Homopolymer can result if the initiator used is one that produces

free radicals in solution (in the presence of vinyl monomer B initiating

homopolymerization) before creating the macro radicals. Once a grafted chain has been

initiated and begins to propagate, chain transfer from the growing grafted chain end can

occur with some species in the medium to yield free radicals that could initiate the

growth of homopoly (B) chains [122].


53

To evaluate the efficiency of the graft copolymerization, the homopolymer is

extracted with an appropriate solvent. Then, the homopolymer percentage (Hp%) and

other various grafting compositional parameters are calculated. Although there are no

unified definitions for calculating the parameters, the most frequently reported

expressions for all kinds of graft copolymerizations are as follows [122, 144,146].

Graft yield (G%) = 100 (W3-W0) / W0 ------------- (1) Add-on (Ad%) = 100 (W3-W0) / W3 --------------- (2) Hp% = 100 (W2-W3) / W2 or, Hp% = 100 W4/ W1 --------------- (3)

where W0, W1, W2, W3 and W4 designate the weight of the original substrate,

monomer charged, total product (i.e., copolymer and homopolymer), pure graft

copolymer, and homopolymer, respectively.

The various initiating systems employed to graft copolymerize different vinyl

monomers onto chitin or chitosan can be categorized to two main classes, i.e. chemical

initiation and radiation initiation.

1.4.9.10.1. Chemically-Initiated Vinyl Polymerization Among the variety of chemical reagents reported for initiating the vinyl monomer

graft copolymerization onto chitin/chitosan, ceric ion initiation and Fenton’s initiation are

the most important systems. Cerium in its tetravalent state is a versatile oxidizing agent

used most frequently in the graft copolymerization of vinyl monomers onto cellulose and

starch. It forms a redox pair with the anhydroglucose units of the polysaccharide to yield

the macro radicals under slightly acidic conditions [141, 142, 144- 147]. As with

cellulose and starch, the ceric ion has been a useful initiation method for graft

copolymerizing chitin and chitosan with typical vinyl monomers due to the similarities in

the chemical structures of these polysaccharides [148-153].


54

The mechanism of initiation for chitosan is believed to begin with a complex

formation of Ce4+ with the primary amine and the hydroxyl groups at the C-2 and C-3

positions, respectively. The radicals responsible for the initiation of grafted copolymer chains

using vinyl monomer are produced from the complex dissociation. A general mechanism for

the reaction proposed recently by the Pourjavadi et al [151] is shown in Scheme 17.

Chitosan

Scheme17. General mechanism for ceric-initiated graft copolymerization

of a typical vinyl monomer, acrylonitrile (AN), onto chitosan

Kim et al reported the ceric-induced graft copolymerization of N-

isopropylacrylamide (NIPAM) onto chitosan at 25ºC to prevent a high level of

homopolymer formation [154]. Vinyl acetate (VAc), a less reactive monomer than

acrylates, was also recently graft copolymerized onto chitosan by CAN in dispersion

medium at 60ºC [155, 156]. The monomer 4-vinylpyridine (4VP) is another non-acrylic

monomer graft polymerized onto chitosan [157] under homogeneous conditions. Acrylic

and methacrylic acids were graft polymerized onto chitosan by Shantha et al [158] to


55

tailor drug carriers. A sulphobetain methacrylic monomer, N,N1-dimethyl-N-

methacryloxyethyl-N-(3-sulphopropyl) ammonium, was recently reported to be graft

polymerized onto chitosan by ceric ion initiation [159]. Chemically modified chitosan

microspheres were synthesized by graft copolymerization of a bifunctional

macromolecular monomer, poly(ethylene glycol) diacrylate onto chitosan backbone using

CAN [160]. Acrylic acid was graft copolymerized onto chitin using CAN by other

workers as well [161].

As an alternative method for grafting, a variation of Fenton’s reagent has been

investigated. Thus, potassium persulphate (KPS) and ferrous ammonium sulphate are

combined in a redox reaction that ultimately produces hydroxyl radicals being able to

form chitin macro radicals. Chitosan has been subjected to graft copolymerization,

comparatively with MA and MMA monomers using KPS alone and KPS coupled with

various reducing agents [162]. Hsu et al found that chitosan was degraded by KPS in

aqueous media via free radical mechanism [163]. This is an important point that should

be taken into account in all the persulphate containing initiating systems. Most recently,

KPS-initiated graft copolymerization of acrylonitrile (AN) and MMA onto chitosan was

reported [164]. A novel redox system, Cu (III)-chitosan, was employed to initiate the

graft copolymerization of MA onto chitosan in alkali aqueous media [165].

Azobisisobutyronitrile (AIBN), ammonium persulphate (APS), and hydrogen

peroxide (H2O2) are commonly employed to graft copolymerize vinyl monomers onto

chitin and chitosan [140, 72]. Under heating (or irradiation), the first radicals produced by

these systems occur from homolytic bond scissions of the initiator, whereby these

radicals subsequently react with the monomer to initiate the polymerization. For typical

graft copolymerization, these radicals provided by the initiator, in addition to reacting

directly with vinyl monomer, abstract hydrogens from chitin or chitosan creating macro

radicals that are capable of initiating a grafted chain with vinyl monomers. Vinyl acetate,

AN, MA, and MMA are graft polymerized onto chitosan using this kind of initiation

system. Tributylborane (TBB) was also utilized for initiating the grafting onto chitosan

[166]. But this technique produces good proportion of homopolymers.


56

1.4.9.10.2. Radiation-Initiated Vinyl Polymerization Both high- and low-energy radiation may be used for graft copolymerization of

vinyl monomers onto polysaccharides. Employing high-energy radiation (e.g., β, γ , X-

ray) is an efficient basic method for initiating radical graft polymerization onto

polysaccharides. Although the radiation-based grafting is cleaner and more efficient in

this regard than chemical initiation methods, they are harder to handle under technical

conditions [167]. Pengfei et al recently reported the γ –radiation induced graft

copolymerization of styrene onto chitin and chitosan powder [168]. Singh and Ray graft

copolymerized 2-hydroxyethylmethacrylate (HEMA) onto chitosan films using 60Co

gamma radiation to improve their blood compatibility [169]. Chitosan films have also

been subjected to gamma radiation-induced graft copolymerization of the vinyl monomer

N, N-dimethylaminoethyl methacrylate [170]. Low energy photons may also initiate the

polymerization of a vinyl or acrylic monomer if the irradiation is carried out in the

presence of an activator (photosensitizer). Such a sensitizer must become active on

exposure to the particular wavelength range of the incident radiation [171]. Irradiation

with low energy radiation, i.e. visible or ultraviolet (UV) lights, usually in the presence of

a photosensitizer such as benzophenone or azo compounds, is a rarely used method for

grafting onto chitin/chitosan. UV-initiated graft copolymerization of MMA onto chitosan

has been reported [172].

1.4.9.11. Non-vinyl Graft Copolymerization

1.4.9.11.1. Graft Copolymerization via Polycondensation Condensation polymerization has not been widely used for preparing graft

copolymers of polysaccharides usually due to susceptibility of the saccharide backbone to

high temperature and harsh conditions of the typical polycondensation reactions.

However, lactic acid (LA) was successfully graft copolymerized onto chitosan through

condensation polymerization of D, L-lactic acid in the absence of a catalyst [173]. So, the

bio-active and compatible polymer polylactic acid (PLA) is coupled to the biopolymer

chitosan through an amide linkage.


57

1.4.9.11.2. Graft Copolymerization via Oxidative Coupling With a view to prepare conductive polymers, polyaniline was grafted onto

chitosan [174]. On the treatment of chitosan in aqueous acetic acid solution with aniline

in the presence of APS, polyaniline side chains were introduced at the amino groups.

Chitosan-g-polyaniline was fabricated into films and fibres, but the properties varied

according to the ratio of amino group to aniline in the grafting reaction.

The reactions of chitin and chitosan discussed so far stimulate further research on

them to use these precious renewable biomaterials in the fast growing biomedical and

industrial technology.

1.5. Applications of Chitin and Chitosan The solubility is the major limiting factor for processing chitin in various

applications [175]. Chitosan is considered as a potential polysaccharide because of its

free amino groups that contribute to polycationic, chelating, and dispersion forming

properties along with ready solubility in dilute acetic acid. Chitosan possesses

exceptional chemical and biological qualities that can be used in a wide variety of

applications, ranging from biomedical, pharmaceutical and cosmetic products to water

treatment and plant protection. In different applications, different properties of chitosan

are required. These properties change with e.g., degree of deacetylation and molecular

weight.

1.5.1. Medical Applications

1.5.1.1. Wound Management Rapid wound healing is desirable for patients, especially those suffering from

diabetes as they show an extremely slow rate of healing [176]. Wound dressings are used

to protect the site of injury from further insult, contamination and infection that may

impede healing. The ideal wound dressing would also facilitate and accelerate wound

healing [177]. Chitin and chitosan are widely used as wound dressings and as a


58

haemostatic agent. The N-acetyl glucosamine (NAG) present in chitin is a major

component of dermal tissue and its presence is essential for repair of scar tissue. It is

possible that chitin derivatives, being easily degraded by lysozyme naturally present in

wound fluid, could promote wound healing by functioning as a controlled delivery source

for NAG to the healing wound [176]. Development and in vitro evaluation of chitosan-

eudragit RS 30D composite was reported by Wittaya-areekul et al [178]. A novel

asymmetric chitosan membrane consisting of skin surface on top layer supported by a

macroporous sponge-like sublayer has been designed. The chitosan membrane showed

controlled evaporative water loss, excellent oxygen permeability and promoted fluid

drainage ability, at the same time effectively inhibiting invasion of exogeneous

microorganisms [179]. Another study reported by the same group involves a novel

bilayer chitosan membrane prepared by a combined wet/dry phase inversion method and

evaluated as a wound dressing [180]. The wound healing rate was found to be fast for

water soluble chitin solution. Fluid absorbing chitin beads has also been proposed as

wound dressing material [181]. N-Carboxybutyl chitosan is water soluble and its gel

forming ability permits the absorption of wound exudates [182]. The inclusion of

antimicrobial agents into wound dressings is another strategy that has been investigated.

Novel chitosan wound dressings loaded with minocycline for the treatment of severe burn

wounds was reported by Aoyagi et al [183]. Silver sulfadiazine was added to a β-chitin-

polyethylene glycol gel and freeze-dried to form the dressing. Results from animal

studies indicated infection controlled wound healing. In another study, a chlorhexidine

containing chitosan-based wound dressing was shown to have antibacterial efficacy

towards the primary wound bed bacteria, Pseudomonas aeruginosa and Staphyloccocus

aureus [184].

Lots of studies are reported in the literature about chitosan wound dressings [185-

189]. A dressing made of chitosan-collagen sponge and its clinical curative effects was

studied by Ye et al [190]. Drug-impregnated polyelectrolyte complex (PEC) sponge

composed of chitosan and sodium alginate was also prepared for wound dressing

application [191]. Research suggests that chitin and chitosan can make lenses more


59

permeable to oxygen than other lens materials. This could be particularly useful for

injured eyes, because chitin and chitosan also help to heal the wounds. In addition, chitin

does not adhere to eye wounds; that makes removing the lenses from the eyes safer. Tests

with rabbits have shown that chitosan lenses accelerate the healing of the cornea. When

chitosan is used, more collagen is laid down and more fibroblasts enter the area of the

wound. This technology has particular promise in soft contact lenses.

1.5.1.2. Chitin-Based Bone Substitutes Chitin has been applied both neat as well as in combination with calcium

compounds in orthopedic applications. Maeda et al were one of the first to use chitin in

the form of braided filaments, rods and powders. These substitutes were found to be

potentially suitable for sutures and temporary artificial ligaments for the knee joint [192].

In one study, N-carboxybutyl-chitosan was injected as a 2% solution into the meniscus

region of the rabbit knee. After 45 days, the meniscal tissue site was found to exhibit

structural repair processes [193]. Muzzarelli et al have also looked at the osteoinducting

properties of hydroxyapatite nails surface coated with chitosan. Chitosan-hydroxyapatite

nanocomposites have been prepared and were found to be mechanically flexible and

promoted bone formation [194]. A collagen-chitosan-hydroxyapatite system for bone

substitute was developed by Wang et al [195]. Bone regeneration by using growth factor

releasing chitosan based bone substitutes was studied by Lee et al [196]. The crystal

growth of calcium phosphate phases on gel chitosan membranes was studied by Ehrlich

et al [197]. A study by Ge et al demonstrates the potential of HA-chitin matrices as a

good substrate candidate for tissue engineered bone substitutes [198].

1.5.1.3. Tissue Engineering Tissue engineering is defined as ‘an interdisciplinary field that applies the

principles of engineering and the life sciences toward the development of biological

substitutes that restore, maintain and improve the functions of damaged tissues and

organs’. One of the strategies in tissue engineering is the use of biodegradable polymers


60

to form a porous matrix or scaffold onto which cells are seeded. In time the cells

proliferate into the scaffold to form a ‘tissue system’. In the ideal situation, the tissue

system after implantation into the body becomes integrated with the host tissue as the

scaffold gradually biodegrades. Chitosan is among one of the many candidates suitable as

a biodegradable polymer to form scaffolds in tissue engineering. The literature scan gives

several reports where chitosan scaffolds are used for various tissue engineering

applications [199-201]. Biodegradable nanotubes were fabricated through the layer-by-

layer (LbL) assembly technique of alternate adsorption of alginate and chitosan onto the

inner pores of polycarbonate template with the subsequent removal of the template. The

nanotubes were found to be cytocompatible and biodegradable [202]. It is readily

fabricated into various shapes and sizes, processed into fiber, knitted and weaved. This

provides the capability of pre-fabricating the scaffold in the shape of desired tissues or

organs that can include 3-D scaffold structures. Chitosan is also insoluble at the

physiological pH of 7 and therefore maintains its structure once formed. Furthermore, its

monomeric constituent is similar to the extracellular matrix environment of humans, and

when biodegraded, should generate non-toxic, non-harmful residues. The prospect to

chemically modify chitosan at its C-6 and N-2 positions to impart desired features offers

great flexibility to this biopolymer [3]. Controlled freezing followed by lyophilisation of

chitosan solutions and gels is the general method to fabricate porous chitosan scaffolds.

Chitin scaffolds has also been achieved using similar strategies of freezing and

lyophilisation [203]. Particularly promising has been the use of the cationic property of

chitosan as the basis to forming insoluble complexes with chondroitin-4-sulphate-A,

(CSA) to fabricate membranes for the growth of bovine articular chondrocytes [204]. The

chitosan-CSA hydrogel supported the maintenance of the articular chondrocyte

phenotype expression in morphology and mitosis. A bilayer chitosan film-sponge has

been produced that supports the growth and proliferation of human neofetal dermal

fibroblast [205]. Bagnaninchi et al designed a new porous chitosan scaffold with

microchannels (diameter: 250 µm), which allows primary porcine tenocytes to proliferate

in a bundle-like structure. The cell proliferation and extracellular matrix (ECM)

production within the microchannels were successfully assessed under sterile conditions


61

using optical coherence tomography (OCT) [206]. Interfacial polyelectrolyte complexation

(PEC) fiber has been proposed as a biostructural unit and biological construct for tissue

engineering applications, with its ability to incorporate proteins, drug molecules, DNA

nanoparticles, and cells. The results of the study by Evelyn et al showed that the chitosan-

alginate-heparin fibrous scaffold could be used in various tissue engineering applications

for its good biocompatible and blood compatible properties [207].

1.5.1.4. Controlled Drug Release Chitosan is a versatile carrier for biologically active species such as drugs due to

the presence of free amino groups as well as its low toxicity and degradability. The

degraded products of chitosan do not introduce any disturbance in the body. Hence, it can

be a suitable matrix, available in different forms, for sustained release of various drug

formulations. Various drugs may well be incorporated into chitosan matrix in a variety of

forms (viz, beads, films, microcapsules, coated tablets etc) for controlled release

therapies. Chitosan films were fabricated in order to deliver paclitaxel at the tumor site in

therapeutically relevant concentrations [208]. Chitosan-gelatin sponges are another

system that has been shown to give controlled release prednisolone [209]. A pH-sensitive

system utilizing an inorganic material tetra-ethyl-orthosilicate (TEOS) with chitosan has

been reported. TEOS formed the network structure into which chitosan is impregnated to

form the transparent IPN membrane. The membrane swells at low pH and shrinks at

physiological pH and is useful for bioseparation. When loaded with a drug, it can be used

as a delivery system [210]. There are several reports on the drug delivery applications of

chitosan and its derivatives [211, 212]. Kim et al focused on the galactose or mannose

ligand modification of chitosan for enhancement of cell specificity and transfection

efficiency via receptor-mediated endocytosis in vitro and in vivo [213].

1.5.1.5. Microspheres Microspheres and nanospheres are a popular method of effecting drug delivery

systems useful in parenteral applications. Reduction of harmful side effects on the gastric


62

mucosa has been the purpose for the encapsulation of the drug diclofenac sodium (DS) as

chitosan microspheres [214]. A slow release was obtained over 6 h in vitro. Other drugs

that have been incorporated using similar techniques include 5-fluorouracil, where the

drug release characteristics were again modified with other substances such as alginic

acid, chitin and agar [215]. Microparticles of chitosan have also been investigated for use

in oral vaccination [216]. The delivery of genetic materials for the treatment of hereditary

diseases is an application where the use of polycationic systems bound to DNA via ionic

interactions have been proposed. In a study on DNA-polycation nanospheres, Leong et al

reported the possible benefit of using chitosan as a delivery system [217]. Microspheres

of poly(ethylene oxide)-modified chitosan have also been developed for blood contact

applications [218]. Dass et al reported that when a plasmid expressing PEDF (Pigment

epithelium-derived factor) was encapsulated within two types of chitosan microparticles,

anti-invasion and increased adhesion of the osteosarcoma cell line SaOS-2 was noted.

The microparticle resulted in a decrease in primary tumour growth, reduced bone lysis

and reduced establishment of lung metastases in a clinically relevant orthotopic model of

osteosarcoma [219].

1.5.1.6. Artificial Kidney Membranes The active part of the artificial kidney is the semipermeable membrane. Chitosan

membranes have been proposed as artificial kidney membranes possessing high

mechanical strength in addition to permeability to urea and creatinine. Singh and Ray

have extensively reviewed the application of chitosan and modified chitosans as artificial

kidney membrane [176]. The permeability of modified chitosan membranes have been

reported by many authors [220-222]. Hirano and Noshiki have reported that chitosan is

thrombogenic in vivo [223]. In a two hour implantation study in veins, they report that

the chitosan membrane is thrombogenic but the N-acyl chitosans are nonthrombogenic.

Various modifications are suggested to dramatically improve the blood compatibility of

chitosan membranes without altering its superior permeability [224].


63

1.5.1.7. Absorbable Sutures With regard to absorbable sutures, there are commercially available suture

materials such as catgut, chromic catgut, polyglycolic acid and polylactic acid. All of

these materials are used for various types of surgical operations, but are not ideal as their

degradation products are harmful. It is reported that chitin is a suitable material for

absorbable, flexible sutures for use in contact with bile, urine and pancreatic juices, which

are problem areas with other absorbable sutures [176]. Fibers made of chitin and chitosan

have been useful as absorbable sutures and wound–dressing materials [225, 226, 24]

1.5.1.8. Antibacterial Properties Chitin from various sources is highly effective as an antigen when administered to

animals attacked by bacteria and fungi. The serum of warm-blooded animals containing

antibodies developed by chitin is useful for immunization of other animals against

parasitic attack and the associated diseases [34]. A number of reports show the

antibacterial property of chitosan against many microorganisms [227, 228], both gram

positive and gram negative bacteria and fungi [229]. Such an application stems from the

cationic charge of chitosan molecule to give rise to aggressive binding onto the microbial

cell surface, leading to gradual shrinkage of cell membrane and finally death of the cell.

This property of chitosan is useful in food preservation and food protection. It has been

reported that derivatives of chitosan have been found more effective than chitosan.

N-Carboxymethyl and N-carboxybutyl chitosans are more effective bacteriostatic agents

than chitosan. To enhance the antibacterial potency of chitosan, thiourea chitosan was

prepared by reacting chitosan with ammonium thiocyanate followed by its complexing

with Ag + [230].

It has been reported that quaternary ammonium salt of chitosan exhibits good

antibacterial activities, for example, diethylmethylchitosan chloride showed higher

antibacterial activity than chitosan. Novel N, O-acyl chitosan derivatives were more

active against the gray mould fungus Botrytis cinerea and the rice leaf blast fungus


64

Pyricularia oryzae; hydroxypropyl chitosan grafted with maleic acid sodium killed over

99% of Staphylococcus aureus and E. coli within 30 min of contact at a concentration of

100 ng/ml; hydroxypropyl chitosan was a potent inhibitor of Azatobacter mali,

Clostridium diplodiella, Fusarium oxysporum and Pyricularia piricola. The degree of

substitution of hydroxypropyl group also influenced their antifungal activity. With regard

to their antifungal mechanisms, it was reported that these chitosan derivatives directly

interfered with fungal growth and activated several defense processes, such as

accumulation of chitinases, synthesis of proteinase inhibitors and induction of callous

synthesis. It was also noted that the antibacterial activity of chitosan derivatives increased

with increasing chain length of the alkyl substituent, and this was attributed to the

increased hydrophobicity [230].

1.5.1.9. Nanotechnology Micro- and nanoscale structures of chitosan were fabricated by nano imprinting

lithography and biochemically functionalized for bionanodevice applications. Chitosan

solutions were prepared and a nanoimprinting process was developed for it, where

chitosan solution is used as a functional resist for nanoimprinting lithography. A low

temperature (90 °C) and low pressure (5–25 psi) nanoimprinting with

polydimethylsiloxane mold could achieve not only microscale structures but also

nanoscale features such as nanowire and nanodots down to 150 nm dimensions. The

nanoimprinted structures were chemically modified and used for the immobilization of

protein molecules [231]. Nanoparticles made of chitosan, have shown promise as carriers

of anticancer drugs, antitumor genes, and other novel therapeutic agents. Qi et al [232]

has conducted a detailed study evaluating the effect of chitosan nanoparticles on human

liver cancer cells.

1.5.2. In Wastewater Treatment Among other polysaccharides chitosan is a positively-charged molecule, so it can

be used as a flocculating agent and can also act as a chelating agent and heavy metals


65

trapper. That is why it finds potential applications in water treatment [175]. In terms of

utilization, crawfish chitosan as a coagulant for recovery of organic compounds in

wastewater was demonstrated to be equivalent or superior to, the commercial chitosans

from shrimp and crab waste shell and synthetic polyelectrolytes in turbidity reduction

[28]. The wastewater released from food processing plants typically seafood, dairy or

meat processing industries contains appreciable amount of protein which can be

recovered with the use of chitosan; this protein, after drying and sterilization, makes a

great source of feed additives for farm animals [233].

Chitosan, due to its natural origin and being biodegradable, has proven to be a

most interesting alternative to synthetic chemical products in several points of view.

Treating wastewater using "greener" methods has become an ecological necessity.

Integrating a natural polymer made of crustacean residue into an existing system achieves

a two-fold purpose: it improves the effectiveness of water treatment while reducing or

even eliminating synthetic chemical products such as aluminum sulphate and synthetic

polymers. It reduces the use of alum by up to 60% and eliminates 100% of the polymers

from the treated water, improves system performance (suspended solids and chemical

oxygen demand), significantly reduces odour.

1.5.3. In Food Industry Chitosan is already used as a food ingredient in Japan, in Europe and in the

United States as a lipid trap, an important dietetic breakthrough. Since chitosan is not

digested by the human body, it acts as a fiber, a crucial diet component. It has the unique

property of being able to bind lipids arriving in the intestine, thereby reducing by 20 to

30% the amount of cholesterol absorbed by the human body. In solutions, chitosan has

thickening and stabilizing properties, both essential to the preparation of sauces and other

culinary dishes that hold their consistency well. Finally, as a flocculating agent, it is used

to clarify beverages. Because of its phytosanitary properties, it can be sprayed in dilute

form on foods such as fruits and vegetables, creating a protective, antibacterial, fungi


66

static film. Principal commercial applications of chitosan include: preservatives, food

stabilizers, animal feed additives, anti-cholesterol additives [234]

1.5.4. In Cosmetics Chitosan has been used extensively in hair care, especially commercial shampoos

and conditioners with chitosan as the main ingredient because of several advantages.

Among these advantages, chitosan is physiologically safe as it contains no harmful

monomers from any polymerization step. The other one is the ability to form films with

proteins which is more stable at high humidity, less statically charged during brushing

and combing than other traditional hair treated fixatives [233]. It is used in the

preparation of nail polish, fixtures, bath lotion, face, hand and body creams, toothpaste

and as a form enhancer. Today, chitosan is an essential component in skin-care creams,

shampoos, and hairsprays due to its antibacterial properties. Many patents have been

registered and new applications are just beginning to appear including the most highly

prized moisturizing and antibacterial properties. Applications include maintain skin

moisture, treat acne, tone skin, protect the epidermis, reduce static electricity in hair, fight

dandruff, improve suppleness of hair, make hair softer [234].

1.5.5. In Agriculture Chitosan offers a natural alternative to the use of chemical products that are

sometimes harmful to humans and their environment. Chitosan triggers the defensive

mechanisms in plants (acting much like a vaccine in humans), stimulates growth and

induces certain enzymes (synthesis of phytoalexins, chitinases, pectinases, glucanases,

and lignin). This new organic control approach offers promise as a biocontrol tool. In

addition to the growth-stimulation properties and fungi, chitosans are used for: seed-

coating, frost protection, bloom and fruit-setting stimulation, timed release of product

into the soil (fertilizers, organic control agents, nutrients), protective coating for fruits

and vegetables [234].


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1.5.6. Looking Forward…

Applications of chitosan are growing rapidly. Not only due to its multitude of

applications but due to increasing environmental awareness of the population,

biodegradable, and non-toxic products from ‘natural’ sources such as chitin and chitosan

are going to be more and more appealing for the replacement of synthetic compounds.

Moreover, in cosmetic and in biopharmaceutical industries, chitosan has exclusive

properties which are not found in other synthetic products [234].

1.6. Developments in Chemistry and Applications of Chitosan and

Chitosan Derivatives

The science and technology of chitosan a low cost, easily available biopolymer is

advancing quite rapidly as a result of expanding interest in this biopolymer, which has

unique characteristics. Chitosan seems to fulfil a number of demands in the highly

technological world. Khor has stated that the 21st century can be the century of chitin

taking place as an extraordinary material, because chitin and its derivatives have

exhibited high potential in a wide variety of fields including medical, pharmaceutical,

cosmetics, bio-related science and technology, food industry, agriculture and

environmental protection [31, 235]. It is non-toxic and biodegradable which has

increased its applicability in the pharmaceutical and biomedical fields. Ravikumar has

emphasized on the pharmaceutical applications of chitosan in his recent reviews [236-

238]. In a fascinating field like gene therapy, chitosan and its appropriate derivatives are

recently found to be excellent candidates for controlled gene delivery [239, 240].

Following are some of the recent works published on the modifications of chitosan and

some of the specific areas where chitosan and its derivatives can be applied.

A recent review by Zohuriaan-Mehr deals with the chemistry of modification of

chitin and chitosan via graft copolymerization with an emphasis on synthetic approaches.

The article includes the majority of published papers in the field [241]. Huang and Fang

have recently reported about the graft copolymer chitosan-g-poly(vinyl alcohol) which,

the authors claim to have nontoxicity, biodegradability, biocompatibility and a porous


68

structure as proved by scanning electron microscopy [242]. Wang et al explored the

usefulness of chitosan-poly(vinyl alcohol) films for the controlled release of bovine

serum albumin [243]. In another study, Don et al reported the preparation and

characterization of chitosan-g-poly(vinyl alcohol)/poly(vinyl alcohol) blends and showed

that the cellular compatibility of poly(vinyl alcohol) was improved due to the

incorporation of chitosan [244]. Modification of chitosan through blending or grafting

with PEG has been studied by many authors. Nasir et al characterized chitosan-

poly(ethylene oxide) blends for haemodialysis membranes [245]. Kolhe and Kannan

observed an improvement in ductility of chitosan on modification by blending and

grafting with PEG [246]. Composite membranes composed of poly(lactide-co-glycolide)

and PEG-g-chitosan were prepared by electrospinning and were found useful for the

sustained release of ibuprofen [247]. A new water-soluble derivative of chitosan, chitosan

oligoethylene oxide sulfonate was synthesized by Engibaryan et al. The formation of it

was confirmed by potentiometric titration and IR spectroscopy [248]. Synthesis of graft

copolymers of chitosan and PEG-CO2H incorporating biologically active molecules and

tags (mannose, cholesterol, a coumarin dye, and biotin) at the distal end of PEG have

been reported by Fernandez, et al [249]. Biodegradable chitosan-g-poly(L-lactide) hybrid

amphiphiles were prepared through direct grafting of a PLLA precursor to the backbone

of chitosan. Self-assembled nanoparticles could be generated by direct injection of these

graft copolymer solutions into distilled water, and their critical aggregation concentration

decreased with increasing number of PLLA grafts per chitosan. Significantly, this will

provide a convenient method not only to combine the bioactive functions of chitosan with

the good mechanical properties of biodegradable polymers, but also to generate

nanoparticles with adjustable sizes for targeted drug release [250]. Chitosan-based graft

copolymers (CECTS-g-PDMA) were synthesized through homogeneous graft

copolymerization of (N,N-dimethylamino)ethyl methacrylate (DMA) onto N-

carboxyethylchitosan (CECTS) in aqueous solution by using ammonium persulfate

(APS) as the initiator by Kang et al [251]. A thermo-responsive comb-like polymer with

chitosan as the backbone and pendant poly(N-isopropylacrylamide) (PNIPAM) groups

has been synthesized by grafting PNIPAM-COOH with a single carboxy end group onto


69

chitosan through amide bond linkages. The copolymer exhibits reversible temperature-

responsive soluble-insoluble characteristics with the lower critical solution temperature

(LCST) being at around 30ºC. A preliminary in vitro cell culture study has demonstrated

the usefulness of this hydrogel as an injectable cell-carrier material for entrapping

chondrocytes and meniscus cells. The hydrogel not only preserves the viability and

phenotypic morphology of the entrapped cells but also stimulates the initial cell-cell

interactions [252]. The graft copolymer, chitosan-g-polyethylene glycol (PEG), was

prepared through graft polymerization of PEG chains to chitosan due to the esterification

reaction between PEG and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA). The authors

claim that it is a potential method to combine chitosan with the hydrophilic synthetic

polymers [253]. A novel carboxymethylchitosan-g-poly(acrylic acid) (CMCTS-g-PAA)

super absorbent polymer was prepared through graft polymerization of acrylic acid onto

the chain of carboxymethylchitosan and subsequent crosslinking by Chen, and Tan. The

rate of water absorption of the polymer was high, and the swelling of the polymer fitted

the process of first dynamics. The swelling ratio of the polymer was pH-dependent [254].

Chitin, chitosan and their derivatives have been extensively used in various fields like,

biomedical and pharmaceutical, chemical and industrial, cosmetics, food industry, water

purification, agriculture etc. The inclusion of antimicrobial agents into wound dressings is

another strategy that has been investigated. Recently several studies on the use of

chitosan as a tissue engineering tool especially for artificial cartilage etc have been

published [255-259]. Differently proportioned porous chitosan/collagen scaffolds were

prepared by controlled freezing and lyophylization of corresponding composite solutions

for periodontal tissue engineering. Compared to a single component scaffold, the

addition of collagen to chitosan decreased the mean aperture, increased the swelling

ability and the addition of chitosan to collagen decreased the contraction. The adherence

and growth of periodontal ligament cells (PDLCs) cultured within the chitosan/collagen

scaffolds were better than those grown on single chitosan or collagen scaffolds [260].

Yang et al had shown that chitosan sponges can be used as effective scaffolding materials

for tissue engineered bone formation in vitro [261].


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Aparna et al evaluated the anti-bacterial properties and changes in physicochemical

properties of chitosan upon blending with synthetic polyester poly (-caprolactone) (PCL)

[262]. Chitosan could act effectively against typhoid-producing microorganisms [263].

Amphiphilic N-derivatives of chitosan containing C12 alkyl and carboxyl groups were

prepared by Stepnova et al. It was shown that the compounds obtained have fungicidal

activity and form intermolecular associates in solutions [264]. Yang Tsui-Chu et al. found

that the antibacterial activity of chitosan derivatives was affected by the degree of substitution

(DS) with disaccharide and the kind of disaccharide present in the molecule [265].

Numerous control or sustained delivery systems with chitosan have been

described in the literature [266-272]. Jochen Haas reported the preparation and

characterization of chitosan and trimethyl-chitosan-modified poly-(ε-caprolactone)

nanoparticles as DNA carriers [273]. A novel pH-sensitive nanoparticle system that is

suitable for entrapment of hydrophilic insulin but without affecting its conformation was

developed by Li et al. [274]. Jiang et al prepared chitosan-graft-polyethylenimine (CHI-

g-PEI) copolymer by an imine reaction between periodate-oxidized chitosan and

polyethylenimine (PEI) to improve the transfection efficiency. The results show that the

CHI-g-PEI copolymer has potential as a gene carrier in vitro [275]. Several novel

functionalized graft copolymer nanoparticles consisting of chitosan (CS) and the

monomer methyl methacrylate (MMA), N-dimethylaminoethyl methacrylate

hydrochloride (DMAEMC), and N-trimethylaminoethyl methacrylate chloride

(TMAEMC), which show a higher solubility than chitosan in a broader pH range, have

been prepared by free radical polymerization by Qian et al. [276]. The Protein-loaded

nanoparticles showed maximum encapsulation efficiency up to 100%. In vitro release

showed that these nanoparticles provided an initial burst release followed by a slowly

sustained release for more than 24 h. These graft copolymer nanoparticles enhanced the

absorption and improved the bioavailability of insulin via the gastrointestinal (GI) tract of

normal male Sprague-Dawley (SD) strain rats to a greater extent than that of the

phosphate buffer solution (PBS) of insulin. Methoxy poly(ethylene glycol) (mPEG)-

grafted chitosan (CP) was synthesized in order to make polymeric micelles encapsulating


71

all-trans retinoic acid (ATRA) based on polyion complex formation [277]. The loading

efficiency of micelle was higher than 80% (w/w) for all formulations. A migration test

was performed to investigate the inhibition of tumor cell invasion in vitro. The results

suggested that the polyion complex micelles were more effective at inhibiting tumor cell

migration than free ATRA. Agnihotri, and Aminabhavi describe the synthesis of

capecitabine-loaded semi-interpenetrating network hydrogel microspheres of chitosan-

poly(ethylene oxide-g-acrylamide) by emulsion crosslinking using glutaraldehyde [278].

Capecitabine was encapsulated into semi-IPN microspheres and percentage of

encapsulation efficiency varied from 79 to 87. In vitro release was studied in simulated

gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4). The release of capecitabine

was continued up to 10 h.

Chitosan and its derivatives are widely used in the area of nanotechnology in the

form of delivery systems for drugs, proteins, antigens, or genes [279, 280]. The research

in these area finds that chitosan nanoparticles could be a kind of promising agent for

further evaluations in the treatment of hepatocellular carcinoma [232].

1.7. Aim and Scope of the Present Study While many polysaccharides possess potentially useful biological activities,

relatively few also exhibit material properties that would allow their use as materials for

biomedical applications. Chitosan is one glycopolymer that exhibits both structural

potential and useful biological activity. Simple architectural modifications can be used to

tune the physical or biological properties of the polymer by creating new intra- or

intermolecular interactions [281]. The extended use of chitosan for certain applications in

the biomedical field has been limited by its thrombogenicity and uncontrollable

biodegradability. The positively charged chitosan molecules bond with red blood cells

forming artificial clots. Though numerous works have been reported on the synthesis and

characterization of organically modified chitosan, little work has been reported on the

synthesis, and systematic characterization, and studies to establish the structure-property

correlations of such modified systems.


72

The major objective of the present research work is to modify chitosan with various

polymers like, PMMA poly(HEMA), PEGm, PVAc, and PVOH by way of graft

copolymerization for the development of synthetic-natural hybrid polymer materials with

tailor-made properties. This warrants an elaborate and systematic characterization of the

copolymers using spectro-analytical, thermal and mechanical studies. To assess the utility of

the synthesized materials for possible biomedical application, biocompatibility of the

copolymers, like blood compatibility, cytotoxicity and biodegradability have been evaluated

using standard protocols. The copolymers were also tested for biomedical applications like

haemodialysis membranes, by studying their permeability to low molecular weight solutes

like urea, creatinine and glucose. The permeability towards high molecular weight solute

albumin is studied for assessing immunoisolatory properties. The suitability of the

copolymers for the preparation of microspheres was checked and the drug loading capacity of

these microspheres and their ability to release the encapsulated drug in a controlled manner

were studied in detail using a model antibiotic drug, ampicillin.

In this perspective, this investigation encompasses the synthesis of chitosan-g-

vinyl polymers, their characterizations and property evaluations. The results are detailed

in eight chapters. The first chapter compiles an exhaustive literature survey on chitosan

and its derivatives. A brief introduction to the general concepts of polymers forms a

prelude to this. In the backdrop of chitosan being the core subject of interest to

biomedical scientists, the scope and objectives of the present work is highlighted.

The chapter 2 is devoted to the general methodology adopted using various

experimental techniques for the total characterization of the newly synthesized

copolymers as potential biomaterials.

The chapter 3 describes the synthesis and characterization of chitosan-g-PMMA.

The details regarding the characterization of this graft copolymer is also discussed in this

chapter. The discussion also includes the drug delivery application.


73

The main focus of chapter 4 is the synthesis of HEMA grafted chitosan and its

characterization. HEMA is a hydrophilic molecule and it was of interest to study the

impact of its grafting onto chitosan and the basic characterization of the resultant

polymers and their potential as a haemodialysis membrane.

Chapter 5 accounts the synthesis and properties of the chitosan-g-polyethylene

glycolmonomethacrylate (PEGm). PEGm is a polymer of medium hydrophilicity. The

graft (PEGm) segments have a propensity for crystallization. The impact of these two

characteristics on the properties of the chitosan grafted with this polymer was a subject of

interest for detailed investigations.

The next attempt was the modification of chitosan using a highly hydrophilic

monomer vinyl alcohol. Since vinyl alcohol can not be directly grafted onto chitosan, a

route via the hydrolysis of chitosan-g-poly(vinyl acetate) was chosen. The

characterization and permeation properties of these two vinyl polymer grafted chitosan

are compared in chapter 6.

Chapter 7 illustrates a comparative study of the five different graft copolymers in

terms of their physico-chemical properties, biocompatibilities and permeability

properties. This chapter gives a systematic overview of the performance of these graft

copolymers as a biomaterial.

Chapter 8 gives the overall summary and conclusions of this investigation. The

prospects and scope for future research in this area are discussed in this chapter.

The results of the works have been published in international journals and

presented in national and international conferences, a list of which is given at the end of

this chapter.


74

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