Novel types of polysaccharidic assemblies

Mocromol.Chem.Phys.196,2873-2880(1995)

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Novel types of polysaccharidicassemblies Dedicatedto the memory of Prof. Mario Farina Vittorio Crescenzix, David Imbriacq Alberto Ci.ferri^)

Cristóbal Ldrez Veldsquez,Mariella Dentini,

ChemistryDepartment,University"La Sapienza",Rome,00185,Italy (Received: January23, 1995;revisedmanuscriptof May 9, 1995) SUMMARY New typesof polysaccharidic assemblies basedon chitosanand unchargedor charged derivatives of scleroglucan are presented. havinga sphericolskinMacroscopicassemblies like shapewerepreparedby controlledmixing of two polyelcctrolytc solutions.Hydrogels bascdcxclusively on the abovcnaturalderivatizedpolysaccharides crosslinkedvia simple chemicalprocesses, avoidingthe useofextraneousreticulatingagentsand organicsolvents, arealsodescribed.Thesebiocompatiblematerialsmay,eventually, be usedin drug release formulations or for the incapsulationof bioactive macromolecules.fhc intcresting possibilityexiststo study the micelleformationof amphiphileswithin the sphericalskins. Introduction Composite hydrogelsare a classof materialshaving grcat biological and practical relevance. In addition, the mechanismcontrolling the assemblyingof rigid polyelectrolyteswith more flexiblepolymcrs,or with polyelectrolytes bearingoppositecharges,is a subject of renewedtheoreticalsignificanccr). We prcscnt results illustrating ncw types of polysaccharidicasscmblicsbascd on cationic chitosanand unchargedor chargedderivativesof scleroglucan.The first type of assembly,involving interpenetratingnetworkscharacterizedby low chargedensity, i s d e s c r i b e di n s e c t i o nA . SectionB describespeculiarskin-likesphericalshapesofpolysaccharidepolyelectrolyte complexeswhich may include a polyelectrolyteof high charge density or an interpenetratingnetwork. The skin-like complex acts as a permeablemembrane. Results and discussion A) Interpenetrating polymer nelworks Typical synthetic assembliesbased on chain molecules belonging to the class of polymeric networks (e.g. materials behaving as chemical gels) are normally obtained by random interconnection of the chains of a single reactivepolymer with crosslinking agents or, more directly, by copolymerization of a given difunctional monomer with appropriateamounts of a comonomer with functionality >22).

u) Institute of Industrial Chemistrv.Universitv of Genoa. Genoa. Italv O 1995,Hüthig & Wepf Verlag,Zug

ccc 1022-1352/95 /$t0.00

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V. Crescenzi,D. Imbriaco, C. Lárez Velásquez,M. Dentini, A. Ciferri

In addition, various types of networks can be prepared by allowing one polymeric component to grow (e.g. via free radical polymerization of the relevantvinyl monomer) inside and throughout a preexisting polymeric phase (normally a synthetic polymer, but also certainbiopolymersand derivatives,e.g. gelatin,celluloseestersand carboxymethylcellulose)thereby forcing different macromoleculesto interdigitate in intimate and stable fashionr). Networks3) in which both components are individually crosslinked have been mixture in a obtained by the in situ polymerizationof an acrylamide/bis(acrylamidc) gelation medium followed by crosslinking of the gelatin chains with glutaraldehydea'sr.To our knowledge,however,in the few casesin which two diff'erentbiopolyhave been so far involved in the mers (e.g. gelatin and carboxymethylcelluloseb)) synthesisof networks, they were not interconnectedby covalentlinkages. In a researchon novel biocompatible hydrogels- with potential in biomcdical sectors- basedexclusivelyon polysaccharidcsand synthesizedwithout recurring to extraneouscrosslinkingagentsor organic solvents,we have found that nctworks with the characteristicsof hydrophilic, highly water swellable hydrogels based on the reaction between two different water soluble polysaccharidicspeciescan be easily obtained. In these networks the two polymer components are linked together and thereforewe rcfcr to them as co-networks.In particular,chitosanand the dialdehyde obtainable from scleroglucanby controlled periodate oxidation (scleroglucanclialdehydeT))appear as particularly useful starting polymers (Fig. l). Thc overall processbasically consistsin the mixing at room temperatureof an aqueoussolution of chitosanin equimolar or.-lacticacid (polymer concentration2rb (0.84m0rv/r ; w/v; degreeof acetylation,d. a., 0. I 5) and of solid scleroglucandialdehyde degreeoo f x i d a t i o n , d . o x . , 0 . 9 2 8 ' e t ¡ . A f t e r h o m o g e n i z a t i o n t h c p H i s e l e v a=t eI 0d t o thereby allowing rcaction between the free amino groups of chitosan and the free aldehyde groups of the scleroglucanderivative to take place. This is follorved b¡' reductionof the resultingSchiff's baseswith NaBH,CN and finally, extensivedialysis againstdistilled water. A tentativcpictureof the resultingco-networkis schematicallydrawn in Fig. 2 acting upon information on the structure of derivativesof simple amincs with sucrose dialdehydesr0).Samples with diff'erent degreesof crosslinking can be obtained by changingthe molar ratio of reactingpolymers(ratio, R, of amino to aldehydegroups). In addition, if R > 1, the hydrogelcan bear positivecharges(protonation of excess amino groups) whereasif R < l, easypost-reticulationoxidation of unreactedaldehyde groups (aqueous sodium chloriteT))with the formation of -COOH groups cont'ersa net negativechargeto the co-network.In all cases,the co-networksare stable and resist unaltered repeatedswelling-deswellingcyclesand/or exposureto pH from 2 to 9. To exemplify our findings concerning the characterization of a number of samples of the new networks, a f'ewresultsare shown in Figs. 3-5. Fig. 3 showsthat the hydogel swelling capacity is remarkable, as expected,considering the highly hydrophilic characterof both polysaccharidicpartners, and that swelling strongly dependson the pH of the bathing solution.

Novel types of polysaccharidic asscmblies

2875

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Data in Fig. 4 indicate that the releaseof the anionic drug disodium cromoglicate (DSCG) from a co-network bearing positive charges (NHr* groups) exhibits an inlercstingtime coursewhich, in addition, should be amenableto control by changing pH andlor ionic strengthof the externalaqueousphase.Application of the new conetworksin drug releaseformulation should be f'aciiitatedby the bicompatibility of the solvent (or-lactic acid) and of the two starting polsaccharideswhich may result only slightly deteriorated,if at all, by the crosslinkingprocedureoutlined above. Fig. 5 showsthe microscopicfeaturesof a co-network sample:theseare typical of very porous polymeric materials.

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Fig. 3. (a) Dynamicswellingin rvaterof a purified, freeze-dried co-networksample(see text) preparedby reactingchitosanand scleroglucandialdehyde at a stoichiometricratio (R) of amino to aldehydegroups equal to 6. tV is the sampleweight at time t; (b) pH dependentswellingof rhe samesample. Ilzois rhe equilibrium weight in water. All expenmentswereconductedat 25.C

B) Polysaccharide polyelectrolyte complexes In addition to the co-network, it is possible to obtain other interesting types of assemblieswhich involve chitosan and scleroglucan.For instanceif a chitosan aqueous solution is added in the form of finely divided drops ro a .,sclerox" (Fig. 1(c)) aqueous

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Novel types of polysaccharidic assemblies

DSCG

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Fig. 4. Fractionof disodium cromoglicate(DSCG) released(% w,/w) as a function of time from a conetwork obtained from c h i t o s a na n d s c l e r o g l u c a n dialdehyde.R - 7.3

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Fig.5. SEMpictureofaco-networksampleobtainedfromchitosanandscleroglucandialdehyde

solulion, the resultingpolyelectrolytecomplex assumesa macroscopic,sphericalskinlike form (Fig. 6). Addition of an anionic dye (fluoresceine)to the external aqueous solution in equilibrium with such spherical beads with a chitosan solution inside, resulted in a slorv,quantitative transfer of dye molecules inside the spheres. These sphericalassembliescontaining inside aqueouschitosan can be used, as in casesalready reported by others working with chitosan and alginatesr'),to encapsulate bioactivespeciessuch as drugs and enzymes. Alternatively, by reversingthe mixing protocol, droplets can be made encapsulating sclerox rather than chitosan solutions: in both casesthe assembliesmay be used for

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V. Crescenzi, D. Imbriaco, C. Lárez Velásquez, M. Dentini, A. Ciferri

Fig. 6. Threebeadsin contact(magnification x 4 0 ) .T h e s k i n sc o n s i s t i nign r h ec h i t o s a n _ "sclerox" polyelectrolytecomplex are clearlv visible. Each sphericalbeaclcontainsa chitosansolution(scetext)

absorbing and releasing chargcd species such as heavy metal ions or bioactive compounds. wc are currently investigating the absorption of charged amphiphilic compounds and their micellizationwithin thc sphericalskin. More interestingly,if thc startingchitosansolution is addeclto a dilute scleroglucandialdehydesolution - so that the two polymers can mutualry crosslink - and the resulting mixture is afterwards drop-wise added to a sclerox s.lution, cach polyelectrolyte-complex sphericalskin now includesa tiny co-network phase. This is an easy way to impart a stablesphericar shape to a given co_network:thc diameter ol'the spheresand the thicknessof their skins can be controlled by the size of the drops and by polymer concentration, respectively.Likewise,metal ions (e.g. cu(lI)) and chargeddrugs could be absorbed within the complex assembry. Experimental part Mater¡als chitosan from chionocetesjaponicus crab(Katakura chikkarin co., Ltd, Tokyo,Japan) with a residualdegreeof acetylationof 0. r 5 was userlas received. The scleroglucansample (Actigum cs-r1), kindry provided by SANofi BIoINDUSTRIES, France),was purified from resiáualproreins, extensiverydialysedagainst distilledwaterand finalry feeze-dried.The sclerogrucandialdehyde and the ,,sclerox,,deriva_ trveswere preparedas describedelsewhereT). commercial NaBH,cN and ¡r_lactic acid wereboth from Fluka chemika. Disociiumcromoglicate was a Sigma product.

Novel types of polysaccharidic assemblies

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synthesis of fhe co-networks based on chitosan and screrogrucandiardehyde To a chitosanaqueoussolution (polymer2vo w/v; or-ractic acid r.lvo w/v) powdered scleroglucandialdehyde (degreeof oxidation 92v08 e\ was added at zi.c under stirring. In different experiments,the equivalentratio, R, of amino to aldehydegroups was always keptin the range6-8. ThepH of themixturewasslowlyadjusted to l0- ll (0.0grr.r NaoH): this leadsto graduarformation (about r5 h) of a snow-white network.To the latter,after rinsingwith warer,wasaddedan excess of NaBH.,cN. Then it wasthoroughlyrvashed and allowedto reachswellingequilibriumin water. Swelling measuremenfs To a freeze-driedco-nerwork sampre(R : 6; 0. I g) was added 20 mL of water ancl its weight(,/) wasdetermined,after wiping off excess surfacewater,at regulartime rntervals. Affer about2 hat25 'c the samplereachecr its equiribriumswelling(co'nstant weighl, r,2,,). Afterwards, the externalsolution was macleo.os u in Nacl (with negrigiblechangein swellingof the network)and the pH gradua[y decreasecr 'fhe to 2, approximately. weight of the samplewasdeterminedat eachintermediatepH value as outlineclabove. Time course of the releaseof DSCG A fiecze-dried co-nuwork sampre(R - 7.3)wasswollenin waterand equilibrated with an aqueousdisodiumcromoglicate (DSCC) solution.The samplewasthen immersedin a known amount of waterand the releaseof DSCCicletermined spectrophotometrically at regulartime intcrvals(absorbance readingsat ,l _ 239 nm). Potysacch or i de po lyelect ro !yf e com lexes ¡t A solutionof chitosan(2vo w/v, in equimolarHCI) was acldedcrrop-wise to a solution of "sclerox" (sodiumsalt;0.5q0w/v). Sphericarbeadswirh skins built up by the poly_ electrolytecomplexand encapsulating chitosansolutionwerefbrmed immecliately: thesc, after equilibriumsweiling,werecontactecl with ciiluteaqueoussolutionsof cucl, (0.02u) and of fluoresceine(0.01% w,zv),respectively. The accumulationof cu(rt) rons (blue coloured beads) leads to chitosanprecipitationinsiclethe beacrs.The upiate of aye molecules(yellowcolouredbeads)was rikewiseextensive but with no sign of precipitate formation. In a diff'erentexperiment, the startingchitosansolutionwasmixeclwith scleroglucan