M Farrokhnia

Marine Derived Polysaccharides forBiomedical Applications

M. Farrokhnia

Introduction� Polysaccharide-based biomaterials are an emerging class

in several biomedical fields such as tissue regeneration,particularly for cartilage, drug delivery devices and gelentrapment systems for the immobilization of cells.

� The main functions played by polysaccharides in Natureare either storage or structural functions.

� Seaweeds are the most abundant source of polysaccharides,as alginates, agar and agarose as well as carrageenans.

� Agar (or agar-agar): unbranched polysaccharideobtained from the cell membranes of some species ofred algae, primarily from the genuses Gelidium andGracilaria.

� It is used in : gelatin and thickener in food industry,g y,and as a gel for electrophoresis in microbiology.

� Chemistry: galactose sugar molecules

� Carrageenans : polysaccharides of galactan withalternating 1,3- and 1,4-linked galactose residues,which fill spaces between the cellulosic plant structureof seaweeds.

� They are used : in the food processing industry fortheir gelling, thickening and stabilizing properties.

� Exopolysaccharides (EPSs) : high molecular weightcarbohydrate polymers.

� Origin: They make up a substantial component of theextracellular polymers surrounding most microbialextracellular polymers surrounding most microbialcells in the marine environment.

� They are used in : adhesives, textiles, pharmaceuticalsand medicine for anti-cancer, food additives.

� Chitin : is the second most abundant organiccompound in nature after cellulose.

� It is widely distributed in marine invertebrates, insects,fungi and yeastfungi, and yeast.

� Chitosan : poly-cationic biopolymer is generallyobtained by alkaline deacetylation of chitin, which isthe main component of the exoskeleton of crustaceans,such as shrimps

Modification Strategies of Marine Polysaccharid

� Needs for chemical modification: the improvement ofmechanical properties, biocompatibility, solubility, controlof biodegradability and manufacturing and shaping.g y g p g

� In particular, modification of alginate and chitin/chitosancan follow different approaches:

�Blending or chemical linkages with synthetic biopolymers.�Surface coating of micro- or nano-spheres with

biocompatible synthetic polymers.p y p y�Cross linking with different physical or chemical reagents.�Hydrophobization through alkylation reactions.�Modulation of guluronic/mannuronic ratio, or of

deacetylation degree, respectively.

Polysaccharides as biomaterialsAlginate� It enhances efficient treatment of esophageal reflux,

creates multi-quality calcium fibers for dermatology andwound healing.

Alginate : applications

�It is used for high- and low gel strength dental impressionmaterials.

�Alginate is an effective natural disintegrate, tablet binderand offers an attractive alternative for sustained-release

tsystems.�It forms hydrogels under relatively mild pH and

temperature.�It is regarded as non-toxic, biocompatible, biodegradable,

less expensive and abundantly available in nature.

Alginate is very suitable for controlled delivery of drugsand other biologically active compounds and for the

encapsulation of cells

� Calcium alginate is a natural haemostat, so alginate baseddressings are indicated for bleeding wounds . The gelforming property of alginate helps in removing thedressing without much trauma.

� The biopolymer alginate exhibits, like pectin and others,the effect of ionotropic gelation if multivalent cationsdiffuse directed from one side into the sol. During this sol-gel-transition channel like pores are created.

� The dimensions of these pores can be influenced by thechemical conditions, for example concentration of the sol orgelling agent, nature and conformation of the alginate and pHor temperature.

� For tissue engineering of bone, the phenomenon ofchannel-pore structure developed upon cross-linking wasintroduced as biomaterials by exchanging the toxic copperions as gelling agent for calcium ions and studying thecomposite hydrogels.

Chitin and chitosan� Chitin is almost solely used as a raw material for the

production of chitosan and other derivatives.

Chitin application:� Some wound-covering materials have been developed

from chitin non-woven fabrics and threads.

� Chitin is also used as an excipient and drug carrier in film,gel or powder form for applications involve inmucoadhesivity.

� Chitosan is widely employed in many biomedical fields.

� In association with hydroxyapatite composite bone-fillingmaterial, which forms a self-hardening paste for guidedtissue regeneration in treatment of periodontal bonydefects.

� Like alginate, chitosan has the characteristic of forminggels in addition to possessing viscosity-related properties,complete biodegradability, and even anti-tumor influence.

� Chitosan possesses bioadhesive properties which make it ofinterest in bioadhesive sustained release formulation required.

� Many chitosan derivatives are also biocompatible and non-toxic with living tissues.

� Chitosan-calcium phosphate (CP) composites appear to havea promising clinical application.

� Chemical modificated HA (hyaluronic acid)-chitin andchitosan-HA material were reported to be osteoinductive anchitosan HA material were reported to be osteoinductive anexhibited rapid degradation and neovascularization in vivo.

Chemical modification� Aqueous carbodiimide chemistry, using 1-ethyl

(dimethylaminopropyl) carbodiimide (EDC) as watersoluble carbodiimide, is widely used to couple carboxylicgroups on alginate with molecules containing primary orsecondary amines.(essential for the gelation process)

The reaction is often used for hydrophobization of alginate by reactionwith medium-long chain (C8- C16) alkylic amines.Hydrophobization of alginate by insertion of alkylic chains iscommonly reported to promote protein absorption and, consequently,cell anchorage. For several applications, as additive in food andcosmetics, the gelling ability of alginate may be inhibited by chemicalmodification with propylene oxide.

PGA (propylene glycol alginate) is the only, commerciallyavailable, chemically modified alginate (coded as E405).

Alginate-based materials for drug-deliveryapplications

� In particular proteins can be loaded and released byalginate matrices without loss of their biologicalactivity because of the relatively mild gelation processof gelation.

� Alginate with polyethyleneglycol (PEG). Alginate gelscan act as core materials in this application, whilePEG, which exhibits certain useful properties such asprotein resistance, low toxicity and immunogenicity,together with the ability to preserve the biologicalproperties of proteins.

� A chitosan/PEG-alginate microencapsulation processapplied to biological macro-molecules such as albumin,was reported to be a good candidate for oral delivery ofbioactive peptides.

� In general, drugs with non-favourable solid state properties,such as low solubility, benefit from encapsulation in anamorphous gel matrix. Recently, the synthesis of alginatebearing cyclodextrin (CD) molecules covalently linked onpolymer chains for a sustained release of hydrophobic drugshas been reported.

� The main shortcomings of alginate devices are their rapiderosion at neutral pH and low adhesion to mucosal tissues, whichis further reduced upon crosslinking.

� Modification of polysaccharides by introducing acrylic polymerchains is used to obtain a finer control over drug release rate andchains is used to obtain a finer control over drug release rate andto improve adhesion to biological substrates.

� Hydrogels based on crosslinked poly(acrylic acid) havebeen reported to adhere to mucus providing a barrieragainst irritations and inflammations of membranes of thegastrointestinal system. Acrylic polymers containingamine functionality, a poly(dimethylaminoethylacrylate,DMAEA), in combination with glycolic residues havebeen demonstrated to show good bioadhesion andmucoadhesion.

� The results show that modification with basic moietiesdecreases polymer hydrophilicity, particularly at pH1.2. This may be due to a partial shielding ofcarboxylic functions that interact with protonatedamines.

� At pH 6.8 the decrease in the amount of positivecharges in the network disturbs the intermolecularbonding between alginate chains and results.

� Drug release studies were performed using a freelysoluble, unionized drug molecule, namely 7-(β-hydroxyethyl)theophylline (ETO), as release fromalginate matrices is strongly affected by the formation ofintermolecular bonds between positively-charged drugmolecules and AA networkmolecules and AA network.

� The introduction of pAcrAc or pDMAEA chains mayimprove chain mobility and, in turn, increase the numberof interconnections between the tablet and the substrate,leading to better adhesion. This behavior is welcome forany application in which the systems are used as drugdelivery in contact with mucosaedelivery in contact with mucosae.

Alginate for cell immobilization:

� The main drawback of alginate matrix gels : high density ofnetwork, which limits the cell growth.

� Cell anchorage, a strict requirement for survival, is limitedon alginate gels, because of its hydrophilic nature. PEGcopolymers are used to improve the biocompatibility ofpolysaccharides. Several PEG-alginate systems for cellentrapment have been reported .

� The presence of grafted PEG molecules inside alginate gels isthought to increase the pores dimension and induce improvedcell anchorage. Gelation ability of alginate is not significantlyaffected by the presence of grafted PEG molecules.

Chitin and chitosan: structure andchemical modification� Studies of Asford and co-workers demonstrated that

chitin represents 14-27% and 13-15% of the dry weightof shrimp and crab processing wastes, respectively.

� Chitosan is a fiber-like substance derived from chitin.Chitin and chitosan have similar chemical structures.Chitin is made up of a linear chain of acetyl glucosaminegroups, while chitosan is obtained by removing enoughacetyl groups for the molecule to be soluble in most diluteacids (deacetylation)acids.(deacetylation)

Definition and Composition of Chitosan: Degree ofDeacetylation (DD)

h i h i l ti� physicochemical properties� biodegradability� immunological activity

� In any case, the degree of deacetylation can be employedto differentiate between chitin and chitosan because itdetermines the content of free amino groups in thepolysaccharides. (Chitin with a degree of deacetylationof 75% or above is generally known as chitosan )

Advantages of chitosan :

� 1-In order to dissolve chitin, highly toxic solvents such aslithium chloride and dimethylacetamide are used whereaschitosan is readily dissolved in diluted acetic acid.

� 2-Chitosan possesses free amine groups which are anactive site in many chemical reactions.

� Problem: Both chitin and chitosan exhibit limitationsin their reactivity and process ability. The scarce watersolubility is the major limiting factor in theirapplication.

� Solution : Chemical modification to introduce aSolution : Chemical modification to introduce avariety of functional groups will be a key pointbecause such procedure would not change thefundamental skeleton of polymers and would keep theoriginal physicochemical and biochemical properties,depending on the nature of introduced group andfinally would bring new or improved properties.

Chitin chemical modification

� Carboxymethyl chitin by adding monochloroacetic acidto chitin previously treated with sodium hydroxide atdifferent concentrations, until a neutral viscous milkysolution was obtained. The water soluble product wascarboxymethyl chitin.

� This product activates peritoneal macrophages in vivo,suppresses the growth of tumor cells in mice, andstimulates nonspecific host resistance against EscherichiaColi infections.

� Chitin can be used in blends with natural or syntheticpolymers; it can be cross linked by epichlorhydrin andglutaraldehyde.

Chitosan chemical modification

� Esterification and etherification

� O-/N-carboxymethyl chitosan: wound dressings, artificialbone and skin, bacteriostatic agents, and bloodanticoagulants, due to its unique chemical, physical, andbiological properties especially its excellentbiological properties, especially its excellentbiocompatibility (controlled or sustained drug-deliveringsystems )

� N-carboxyalkylated chitosans were prepared via Schiffbase formation from carboxylic acids having aldehyde orketo groups. The resulting carboxyalkylated derivativesfind applications as biomedical materials and fungistaticfind applications as biomedical materials and fungistaticagents.

� O-carboxymethyl chitosan: water-soluble matrixpolymer for controlled drug release. OCM-chitosanmicrospheres containing antibiotic drug pazufloxacinmesilate were prepared by the emulsion method andsuccessively crosslinked with glutaraldehyde.y g y

Sulfonation :

� Sulfonation reactions of polysaccharides can give riseto a structural heterogeneity in polymer chain, but onthe other hand some structures that emerge fromthe other hand some structures that emerge fromrandom distribution can reveal good features forbiological functions.

� Sulphated chitosans, that represent the nearest structuralanalogues of the natural blood anticoagulant heparin, showanticoagulant, antisclerotic, antitumor and antiviralactivities.

� N-Alkyl-O-sulphate chitosan has an amphiphilic character� N Alkyl O sulphate chitosan has an amphiphilic characterdue to the presence of hydrophobic moieties, alkyl chains,and hydrophilic moieties, sulphate groups. Because of this,it has the capacity to form micelles in water and can be usedas a potential drug carrier.

Acylation :� acylation reactions by different acylating agents :

aliphatic carboxylic acid chlorides (hexanoyl,dodecanoyl and tetradecanoyl chlorides), cyclicanhydrides cyclic estersanhydrides, cyclic esters.

Why?� It induces a hydrophobic nature to the hydrophilic

chitosan backbone and to prevent particle aggregation.

� Sugar-modified chitosanThis type of modification has generally been used to introduce

cell-specific sugars onto chitosan.(Example: liver-specific drugcarrier in mice through a sialoglycoprotein receptor)

� Graft copolymerization reactions introduce side chainsand lead to the formation of novel types of tailored hybridmaterials composed of natural and synthetic polymers.

� It is useful in formation of inclusion complexes ,bacteriostatic effect to enhance adsorption properties.Although the grafting of chitosan modifies its propertiesmucoadhesivity, biocompatibility, and biodegradability.

� Chitosan crosslinkingIn the case of chitosan the amino groups of the polymermay allow the establishment of different types ofinteractions with both non-ionic and ionic drugs and alsoprovide pH-sensitive systems, which swell in gastric

di i ll i i ifi lconditions allowing a site-specific release .

Conclusion� The possibility of producing a variety of chemically

modified derivatives makes these polysaccharidesversatile biomaterials in almost all fields of biomedicalinterest.