Preparationofcross-linkedchitosanmicrospheresbyspraydrying:Effectofcross-linkingagentonthepropertiesofspraydriedmicrospheres
K.G.H.DESAI1&H.J.PARK1,21SchoolofLifeSciencesandBiotechnology,KoreaUniversity,Sungbuk-ku,Seoul,SouthKorea,and2SchoolofLifeSciencesandBiotechnology,KoreaUniversityandDepartmentofPackagingScience,ClemsonUniversity,Clemson,SC,USA
(Received4September2004;accepted10December2004)
Abstract
Chitosanmicrospherescross-linkedwiththreedifferentcross-linkingagentsviz,tripolyphosphate(TPP),formaldehyde(FA)andgluteraldehyde(GA)havebeenpreparedbyspraydryingtechnique.Theinfluenceofthesecross-linkingagentsonthepropertiesofspraydriedchitosanmicrosphereswasextensivelyinvestigated.Theparticlesizeandencapsulationefficienciesofthuspreparedchitosanmicrospheresrangedmainlybetween4.1–4.7mmand95.12–99.17%,respectively.Surfacemorphol-ogy,%erosion,%wateruptakeanddrugreleasepropertiesofthespraydriedchitosanmicro-sphereswasremarkablyinfluencedbythetype(chemicalorionic)andextent(1or2%w/w)ofcross-linkingagents.Spraydriedchitosanmicrospherescross-linkedwithTPPexhibitedhigherswellingcapacity,%wateruptake,%erosionanddrugreleaserateatboththecross-linkingextent(1and2%w/w)whencomparedtothosecross-linkedwithFAandGA.Thesphericityandsurfacesmoothnessofthespraydriedchitosanmicrosphereswaslostwhenthecross-linkingextentwasincreasedfrom1to2%w/w.Releaserateofthedrugfromspraydriedchitosanmicrospheresdecreasedwhenthecross-linkingextentwasincreasedfrom1to2%w/w.Thephysicalstateofthedruginchitosan-TPP,chitosan-FAandchitosan-GAmatriceswasconfirmedbytheX-raydiffrac-tion(XRD)studyandfoundthatthedrugremainsinacrystallinestateevenafteritsencapsulation.Releaseofthedrugfromchitosan-TPP,chitosan-FAandchitosan-GAmatricesfollowedFick’slawofdiffusion.
Keywords:Chitosanmicrospheres,spraydrying,tripolyphosphate,formaldehyde,gluteraldehyde,swelling,erosion,sustainedrelease
Introduction
Therehasbeenconsiderableinterestinrecentyearsindevelopingcontrolledorsustaineddrugdeliverysystemsbyusingbiopolymers.Controlledorsustainedreleasedrugsprovidemanyadvantagesincomparisonwithconventionalforms:reducedsideeffects,drug
Correspondence:ProfessorH.J.Park,307,SchoolofLifeSciencesandBiotechnology,KoreaUniversity,1,5-Ka,Anam-Dong,Sungbuk-ku,Seoul-136-701,SouthKorea.Tel:82232903450.Fax:8229535892.E-mail:hjpark@korea.ac.krISSN0265-2048print/ISSN1464-5246online#2005Taylor&FrancisDOI:10.1080/02652040500100139
378K.G.H.Desai&H.J.Park
concentrationkeptateffectivelevelsinplasmaandimprovedutilizationofdruganddecreasethedosingtimes(Kimetal.2002).Ofthedifferentdrugdeliverysystems,nanoormicroparticlesbaseddrugdeliverysystemsgainedsignificantimportance(RaviKumar2000).Withtheattractivepropertiesandwiderapplicationrange,theyoccupyuniquepositionindrugdeliverytechnology(RaviKumar2000).Theuseofmicrospheres-basedtherapyallowsdrugreleasetobecarefullytailoredtothespecifictreatmentsitethroughthechoiceofappropriateformulationvariables.Usinginnovativemicroencapsulationtechnologiesandbymodifyingthepolymericmatrix,microspherescanbedevelopedintoanoptimaldrugdeliverysystemwhichwillprovidedesiredreleaseprofile(Benita1996;RaviKumar2000).
Chitosan,anaturalbiopolyaminosaccharide,isobtainedbyalkalinedeacetylationofchitinthatisfoundwidelyinnature.Chitosanhasattractedsignificantinterestinrecentyears.Thisislargelyduetotheproposednovelapplicationofthepolymerinphar-maceutical,foodandvariousindustrialandbiotechnologicalfields.Theseapplicationsarepossiblebecauseofthepolymerreactivegroupsandtheirbiodegradability,lowtoxicityandbiocompatibility(HejaziandAmiji2003).Duetotheeasyavailabilityoffreeaminogroupsinchitosan,itcarriesapositivechargeand,thus,inturnreactswithmanynegativelychargedsurfaces/polymers(Koetal.2002).Thisprinciplehasbeenusedfortheproduc-tionofchitosanmicrocapsulesandmicrospherestocontroldrugrelease(HejaziandAmiji2003).Chitosanmicrospheresaremostwidelystudieddrugdeliverysystemsforthecontrolledreleaseofdrugsviz.antibiotics,anti-hypertensiveagents,anti-canceragents,anti-inflammatoryagents,proteins,peptidedrugsandvaccines(Sinhaetal.2004).Chitosanmicrospherescanbesynthesizedbyanumberofdifferenttechniquessuchassolventevaporation,spraydrying,coacervation,emulsification/internalgelationandsuspen-sioncross-linking(Sinhaetal.2004).
Althoughnumeroustechniquesareavailableforthesynthesisofmicroparticles,spraydryingtechniqueiswidelyusedinthepharmaceuticalindustriesbecauseofitsnumerousadvantagesoverothermethods.Theadvantageofspraydryingtechniqueforapplica-tiontomicroencapsulationisthatitisreproducible,rapidandeasytoscaleup(Masters1991;Benita1996;Heetal.1999;Sinhaetal.2004).Spraydryingtechniquecanbeusedtoproducedrypowders,granulesoragglomeratesfromdrug-excipientsolutionsandsuspensions(WangandWang2002).Theparticlesizeofthemicroparticlespreparedbyspraydryingtechniquerangedfrommicronstoseveraltensofmicronsandhadarelativelynarrowdistribution(Masters1991).Recently,anumberofarticleshavebeenpublisheddescribingthepreparationofcontrolledreleasemicroparticlesbysuchaspraydryingtech-nique.Forexample,microparticlescomposedofthewatersolublepolymersusedasthecarrierforintra-articulardeliveryofdexamethasone(Pavenettoetal.1994)orsustainedreleasedosageform(MaaandPrestrelski2000)forthedeliveryofacetazolaminde(DiMartinoetal.2001),butorophanol(ChangandLi2000),dexamethasonehydrochlorideandtoremifenecitrate(Kortesuoetal.2000),erythromycinandclarithromycin(Zgoullietal.1999).Waterinsolublepolymerpolylacticacidorpoly(lactideco-glycolide)waspreparedforthedeliveryofrifampicin(Bainetal.1999a,b,O’HaraandHickey2000)andgentamycin(Prioretal.2000).
Ideally,adeliverysystemmightbedevelopedtoreleaseadrugatpreciselytherateitisrequiredfordifferentapplication.Chitosan-basedmicrosphereshavebeenpreparedbyspraydryingtechnique(Heetal.1999;Huangetal.2002;2003a,b;Filipovic-Grcicetal.2003).However,asanunfavourablefactor,spraydriedchitosanmicrospheresswellquicklyinwaterandreleasetheencapsulateddrugimmediately(Gentaetal.1995).Thedrugreleasekineticsfromspraydriedchitosanmicrospheresisaffectedbytheconcentration
Preparationofcross-linkedchitosanmicrospheres379
andmolecularweightofthechitosan,solubilityofthedrugs,especiallythechitosanmatrix(cross-linkedornon-cross-linked)(Heetal.1999).Therefore,non-cross-linkedspraydriedchitosanmicrospheresareunsuitableforsustaineddrugdelivery(Gentaetal.1995;Heetal.1999).Inordertostabilizethespraydriedchitosanmicrospheres,cross-linkingagentssuchasGAandFAhavebeenused(Heetal.1999).Inanearlierwork(DesaiandPark2005),TPPhasbeendemonstratedasanewstabilizingagentforthepreparationofchitosanmicrospheresbyspraydryingtechniquecoveringtheinfluenceofconcentrationandmolecularweightofthechitosananddrugloadingonthepropertiesofspraydriedchitosan-TPPmicrospheres.However,theeffectofdifferentcross-linkingagentsonthepropertiesofspraydriedchitosanmicrospheresisnotstudiedsofar.
Incontinuationoftheongoingprogrammeofresearchtodevelopthechitosanbasedmicrospheresforthereleaseofdrugs(Koetal.2002;Leeetal.2003),onenowreportstheinfluenceofthreedifferentcross-linkingagents(TPP,FAandGA)ontheproper-ties(%encapsulationefficiency,size,surfacemorphology,%erosion,%swellingandreleasebehaviour)ofspraydriedchitosanmicrospheres.Therefore,theobjectiveofthepre-sentstudywastopreparethechitosan-TPP,chitosan-FAandchitosan-GAmicrospheresbyspraydryingtechniquethroughanovelprocessaswellastoexaminetheinfluenceoftheabovementionedthreedifferentcross-linkingagentsonthepropertiesofthuspreparedmicrospheres.Acetaminophenwasusedasamodeldrugcandidate.Materialsandmethods
Materials
Acetaminophen(99.5%purity)waspurchasedfromKantoChemicalCo.,Inc.(Tokyo,Japan).Chitosan(mediummolecularweight)waspurchasedfromSigma-AldrichChemie(Steinheim,Germany).Theaveragemolecularweightofchitosanwasdeterminedbybatchmodemethodusingmulti-anglelaserlightscattering(MALLS)photometeraccordingtothemethodofChenandTsaih(1998).Theaveragemolecularweightofthechitosanwasfoundtobe1.336Â106.The%N-deacetylationofchitosanwasdeterminedbythe1NMRspectroscopymethod(Hiraietal.1991;Lavertuetal.2003).Thedegreeofdeacetylationofthechitosanwasfoundtobe82.10%.FA(35%)andGA(25%)werepurchasedfromShowaChemicals(Japan).Allotherchemicalswereofanalyticalgradeandusedasreceived.Ultrapurewater(Millipore,USA)waterwasusedthroughoutthestudy.
Preparationofcross-linkedchitosanmicrospheresbyspraydrying
Modeldrug,acetaminophen(0.5%w/v)wasdissolvedin300mlof1%v/vaceticacidsolution.Thenthechitosanwasdissolvedinthedrugsolutionbystirringitovernight.About10mlofdifferentcross-linkingagents(seeTableI)(TPPorGAorFA)wasaddeddropwiseintotheaqueouschitosan-drugsolutionwithconstantstirringat8000rpmfor30minusingaYoungJiHMZ20DNstirrer(HanaInstruments).Thuspreparedchitosan-TPP-drugorchitosan-GA-drugorchitosan-FA-drugsolutionwasthenspraydriedtoobtainthecross-linkedchitosanmicrospheresloadedwiththedrug.SpraydryingwasperformedusingaSD-05spraydrier(LabPlant,UK),withastandard0.5mmnozzle.Spraydryingconditionssuchasinlettemperature,liquidflowanddryingairflowweresetat170C,2mlminÀ1,1.2m3minÀ1,respectively.Theatomizingairpressurewas60kPa.Whentheliquidwasfedtothenozzlewithperistalticpump,atomizationoccurredbytheforceofthecompressedair,disruptingtheliquidintosmalldroplets.Thedroplets,togetherwith
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TableI.Preparationofcrosslinkedchitosanmicrospheresbyspraydryingmethod.
FormulationChitosanChitosancodeconcentration(%w/v)molecularweightF1F2F3F4F5F6
1.01.01.01.01.01.0
1.336Â1061.336Â1061.336Â1061.336Â1061.336Â1061.336Â106Cross-linking
agentTripolyphosphateTripolyphosphateFormaldehyde(35%)Formaldehyde
Gluteraldehyde(25%)Gluteraldehyde
Cross-linkingDrug
extent(%w/w)loading(%w/v)
121212
0.50.50.50.50.50.5
Model drug solution (0.5% w/v) (Acetaminophen) Chitosan (1.0% w/v) Stirring (8 h) Chitosan-drug solution TPP or GA or FA solution (10 ml)Dropwise Homogenization 8000 rpm, 30 min Chitosan-TPP-drug or chitosan-GA-drug orchitosan-FA-drug solution Spray dryingIn let temperature 170°C Liquid flow rate 2 ml/min Drying air flow 1.2 m3/min Spray dried chitosan-TPP or chitosan-FA or chitosan-GA
microspheres loaded with acetaminophen
Figure1.Preparationprocessofcross-linkedchitosanmicrospheresbyspraydrying.
hotair,wereblownintoachamberwherethesolventinthedropletswasevaporatedanddischargedoutthroughanexhausttube.Thedryproductwasthencollectedinacollectionbottle.Thepreparationprocessofcross-linkedchitosanmicrospheresthroughanovelprocessbyspraydryingmethodisshowninFigure1.
Loadingefficiency
About10mgofdrug-loadedchitosan-TPPorchitosan-GAorchitosan-FAmicro-spheresweredissolvedin50mlof0.1NHCl.Thesolutionwaspassedthrough
Preparationofcross-linkedchitosanmicrospheres381
a0.22mmmembranefilter(Millipore,USA)andthenthedrugcontentwasassayedbymeasuringtheabsorbanceat201nm(maxofacetaminophenin0.1NHCl)aftersuitabledilutionusingUVspectrophotometer(Shimadzu1601PC,Japan).Experimentswereperformedintriplicate(n¼3)andloadingefficiencieswerecalculatedusingequation(1).
Loadingefficiencyð%Þ¼
Calculateddrugconcentration
Â100
Theoreticaldrugconcentration
ð1Þ
Measurementofparticlessize
Spraydriedchitosanmicrospheresexhibitedquickswellinginliquidmediumand,hence,sizescouldnotbedeterminedusingalaserdiffractiontechniqueinaparticlesizeanalyser.Therefore,theparticlesizewasdeterminedbymicroscopy.Briefly,$5mgofcross-linkedchitosanmicrospheresweretakenonaglassslideandsizesof$200sphericalparticlesweremeasuredeachtime(n¼3)byusingabiologicalmicroscope(Olympus,Japan).
Erosionstudy
Spraydriedplacebochitosan-TPP,chitosan-FAandchitosan-GAmicrospheres(200mg)wereimmersedinphosphatebuffersolution(pH7.4)andstirredat100rpmfor6h.After6h,microsphereswereseparatedbycentrifuge(3000rpm,10min)anddriedinatemperaturecontrolledoven(JEIOTECH,FO600M,SouthKorea)at40Cfor24htodrythemicrospherescompletely;thesewereweighedtocalculatethemassloss.
Surfacemorphology
Thesurfacemorphologyofthespraydriedchitosanmicrospherescross-linkedwithTPP,FAandGAwasexaminedbymeansofHitachi(Japan)scanningelectronmicroscope.Thepowderswerepreviouslyfixedonabrassstubusingdouble-sidedadhesivetapeandthenweremadeelectricallyconductivebycoating,inavacuum,withathinlayerofplatinum($3–5nm),for100sandat30W.Thepicturesweretakenatanexcitationvoltageof15kvandamagnificationof1.8,2,3.5,4,4.5or800k.
Swellingstudy
Thedynamicswellingpropertiesofthespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresinthedissolutionmedium(phosphatebuffersolution,pH7.4)weredetermined.Spraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresofknownweight(200mg)withoutcontainingthedrugwereplacedinphosphatebuffersolution(pH7.4)foraperiodof6h.Theswollenchitosan-TPP,chitosan-FAandchitosan-GAmicrosphereswerecollectedbyacentrifugeandthewetweightoftheswollenmicrosphereswasdeterminedbyfirstblottingtheparticleswithfilterpapertoremoveabsorbedwateronsurfaceandthenweighingimmediatelyonanelectronicbalance.Theweightoftheswollenmicrosphereswasdeterminedatapre-determinedtimeperiod(0.5,1,2,4and6h)toaccuracyof0.01mgusinganelectronicbalance.Thepercentage
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ofswellingofthecross-linkedspraydriedchitosanmicrospheresinthedissolutionmediawasthencalculatedusingequation(2).
WtÀWo
Â100ð2ÞSSW¼
WowhereSSWisthepercentageofswellingofspraydriedchitosan-TPPorchitosan-FAorchitosan-GAmicrospheres,WtdenotestheweightofthespraydriedmicrospheresattimetandWoistheinitialweightofthemicrospheres.Equilibriumwateruptake(%wateruptake)ofthecross-linkedchitosanmicrospheres(placebo)werealsodeterminedbymeasuringtheextentofswellingofthematricesinphosphatebuffersolution(pH7.4).Toensurecompleteequilibrium,thesampleswereallowedtoswellfor24h.
X-raydiffraction
Thephysicalstateofthemodeldrug(acetaminophen)inthespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmatriceswasassessedbyXRDstudies.X-raypowderdiffractionpatternsofneatacetaminophen,spraydriedplacebochitosanmicrospheresandchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresloadedwithdrugwereobtainedatroomtemperatureusingaPhilipsX’PertMPDdiffractometer(Philips,TheNetherlands)withCoasanodematerialandgraphitemonochromatoroperatedatavoltageof40kV.Thesampleswereanalysedinthe2anglerangeof2–60andtheprocessparametersweresetasscanstepsizeof0.025(2),scansteptimeof1.25sandtimeofacquisitionof1h.
Invitrodrugrelease
Theinvitroreleaseofthemodeldrug(acetaminophen)fromspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrosphereswasdeterminedusingadissolutionapparatus(TW-SM,WoojuScientific,Co.,Korea).Inordertosuspendthespraydriedchitosanmicrospheresinthedissolutionmedium,25mgofmicrospheresweretakenintothepreviouslysoakedcellulosedialysisbag(molecularweightcut-off8000)containing3mlofdissolutionmediaandtiedtothepaddle.Theinvitroreleasestudiesofacetaminophenwerecarriedoutatpaddlerotationof100rpmin900mlofphosphatebuffersolution(pH7.4and37C).Analiquotofthereleasemedium(5ml)waswithdrawnthroughasamplingsyringeattachedwith0.22mmmembranefilter(Millipore,USA)atpre-determinedtimeintervals(0.5,1,2,3,4,5and6h)andanequivalentamountoffreshdissolutionmediumwhichwaspre-warmedat37Cwasreplaced.Collectedsampleswerethenanalysedforacetaminophencontentbymeasuringtheabsorbanceat202nm(maxofacetaminopheninphosphatebuffersolution(pH7.4))aftersuitabledilutionusingUVspectrophotometer(Shimadzu1601PC,Japan).Invitroreleasestudieswereperformedintriplicate(n¼3)foreachmicrosphereformulationinanidenticalmanner.Resultsanddiscussion
Preparationofcross-linkedchitosanmicrospheresbyspraydrying
Chitosaniscurrentlyreceivingagreatdealofinterestinpharmaceuticalapplications.Themainreasonsforthisincreasingattentionarecertainlyitsinterestingintrinsicproperties.Spraydryingisawell-knownprocess,whichisusedtoproducedrypowders,granulesoragglomeratesfromdrugexcipientsolutionsandsuspensions(Heetal.1999).
Preparationofcross-linkedchitosanmicrospheres383
Recently,anumberofarticleshavebeenpublisheddescribingthepreparationofchitosanmicrospheresbysuchaspraydryingmethod(Gentaetal.1995;Heetal.1999;Huangetal.2002;2003a,b;Filipovic-Grcicetal.2003).Thedrugreleasekineticsfromchitosanmicrospheresisaffectedbytheconcentrationandmolecularweightofthechitosan,solubilityofthedrugandencapsulatingprocess,especiallythecross-linkedchitosanmatrixdensity(Sinhaetal.2004).Non-cross-linkedspraydriedchitosanmicrospherescannotbekeptsuspendedinwaterbecauseofswellinganddissolution.Asaresult,thereleaserateofthedrugfromnon-cross-linkedspraydriedchitosanmicrospheresisrapid(morethan90%ofthedrugreleasewithin0.5h)(Gentaetal.1995).Therefore,non-cross-linkedspraydriedchitosanmicrospheresareunsuitableforthesustainedreleaseapplication.Todate,themostcommoncross-linkersusedwithchitosanarechemicalcross-linkingagents(FAandGA)andioniccross-linkingagents(genipin,TPP).Theirreactionwithchitosaniswell-documented(Bergeretal.2004).Morerecently,TPPhasbeendemonstratedasanewstabilizingagentforthepreparationofchitosanmicrospheresbyspraydryingmethod(DesaiandPark2005).However,comparativestudy,i.e.theinfluenceofdifferentcross-linkingagents(TPP,FAandGA)onthepropertiesofspraydriedchitosanmicrospheresisnotstudiedsofar.Therefore,thispaperrevealstheireffectsonthesize,%encapsulationefficiency,%swelling,%erosion,surfacemorphologyandreleasebehaviourofthespraydriedchitosanmicrospheres.Thus,cross-linkedchitosanmicrospheresbyspraydryingmethodwerepreparedasanefforttoexplorethepossibilityofthembeingusedassustainedreleasecarriersfordrugs.
Propertiesofspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheres
Thepropertiesofspraydriedchitosanmicrospheres(yield,meansizeandencapsulationefficiency)loadedwiththedrugarepresentedinTableII.Theyield(%)ofthespraydriedchitosanmicrospherescross-linkedwithdifferentcross-linkingagentsrangedmainlybetween39.3–45.8%.Generally,yieldofthemicrospherespreparedbythespraydryingmethoddependsuponthespraydryingconditions(inlettemperature,flowrateandcompressedairflow).Underthepresentspraydryingconditions,yieldofdrugloadedchitosanmicrospheresdidnotvarymuchwiththevaryingamountofdifferentcross-linkingagents.Theparticlesizeofthespraydriedchitosanmicrospherescross-linkedwithTPP,FAandGArangedbetween4.1–4.7mm.However,theparticlesizedecreasedslightly(seeTableII)whenthecross-linkingextentofTPP,FAandGAincreasedfrom1to2%w/w.Generally,thespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospherescouldbeproducedwithhigher(95.12–99.17%)encapsulationefficiencies.However,asthe
TableII.Resultsofmeanparticlesize,%encapsulationefficiency,%yield,%erosionand%wateruptakeofspraydriedchitosan-TPP(F1andF2),chitosan-FA(F3andF4)andchitosan-GA(F5andF6)microspheres(ValuesareexpressedasmeanÆstandarddeviation).FormulationcodeF1F2F3F4F5F6
Mean
particlesize(mm)
4.5Æ0.84.1Æ0.54.7Æ0.74.3Æ0.34.6Æ0.84.2Æ0.4
Encapsulationefficiency(%)99.17Æ1.096.42Æ1.499.03Æ1.395.12Æ0.698.81Æ1.195.61Æ1.2
Yield(%)41.243.540.439.345.842.6
%Erosion10.1Æ1.48.8Æ1.16.8Æ1.35.1Æ0.73.7Æ0.82.9Æ0.6
%Wateruptake365.2Æ11.3340.3Æ5.3312.6Æ4.8295.8Æ7.6266.2Æ4.6245.2Æ8.1
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cross-linkingextentofTPP,FAandGAincreasedfrom1to2%w/w,theencapsulationefficienciesofspraydriedchitosanmicrospheresdecreasedslightly(seeTableII).
Surfacemorphology
Thesurfacemorphologiesofspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresareshowninFigures2and3.Thenewprocessofpreparationofchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresbyspraydryingmethodproducedthemicrosphereswithsphericalshapeandsmoothsurface.However,well-definedchangeinthesurfacemorphologyofspraydriedchitosanmicrosphereswasobservedwhenthecross-linkingextentofTPP,FAandGAwasincreasedfrom1to2%w/w.Forinstance,microspherescross-linkedwith1%w/wofTPPorFAorGAwerecompletelysphericalandhadasmoothsurface(seeFigure2(a,bandc)).Whereas,inthecaseofmicrospherescross-linkedwith2%w/wofTPPorFAorGA,althoughthemicrosphereswerespherical,buthadadepressed(wrinkles)surface.However,spraydriedchitosanmicrospherespreparedwithFAorGAhadmoredepressedsurfaces(seeFigure3(bandc))thanthosepreparedwithTPP(Figure3(a)).ThepicturesoftheSEMstudyrevealedthatchemicalcross-linking(GAorFA)hadaremarkableeffectonthesurfacemorphologyofspraydriedchitosanmicrospheresthantheioniccross-linking(TPP).Ontheotherhand,drug(acetaminophen)crystalswereadheredtothesurfaceofthespraydriedchitosanmicrospheresduetoitsextremecrystallinenature.
Erosionofmatrices
Thechitosanmicrospherespreparedbycross-linkingwithdifferentcross-linkingagentsarestirredindissolutionmedia(phosphatebuffer,pH7.4)for6h.Theseresults,giveninTableII,indicatethaterosiondecreaseswithanincreaseincross-linkingofthematrices.Amongthethreecross-linkingagentsused,GA-cross-linkedmicrospheresshowtheleasterosion,i.e.2.9–3.7%,buttheFA-andTPP-cross-linkedmicrospheresshowerosionsof5.1–6.8%and8.8–10.1%,respectively.ThepercentageerosionofTPP-cross-linkedmicrospheresishigherthantheFA-andGA-cross-linkedmatricesbecausethelattercasesinvolvesactualchemicalreactionsbetweenFAorGAandchitosanand,therefore,FA-chitosanorGA-chitosanisastrongerandmorerigidmatrixthantheionically(TPP)cross-linkedmatrix.Thesurfacemorphologyofspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresaftertheirerosionstudywasalsoexaminedbySEMandthesamearepresentedinFigure4.Itcanbeseenthatthechitosan-TPPmicrospheres(Figure4(a))didnotmaintaintheformofsphereindicatingthepoorrigidityofthespraydriedchitosan-TPPmatrix.Spraydriedchitosan-FA(Figure4(b))andchitosan-GA(Figure4(c))microspheresmaintainedtheirsphericalshapeevenafter6h,indicatingthemorerigidityofthechitosan-FAandchitosan-GAmatrices.
Swellingbehaviour
Itisnotknownwhetherthemacro-molecularchainsofpolymerarefixedbyioniccross-linkingorchemicalcross-linking,theswellingabilityofthepolymerdecreases.However,theswellingabilityofspraydriedchitosanishigherthanthepurechitosan(Mietal.1999).Theswellingexperimentsofspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrosphereswereconductedindissolutionmedia(pH7.4).Theswellingcapacity(%)ofspraydriedchitosanmicrospherescross-linkedwithdifferentcross-linking
Preparationofcross-linkedchitosanmicrospheres385
Figure2.Scanningelectronmicroscopicpicturesofspraydriedchitosanmicrospherescross-linkedwith1%w/wTPP(a),FA(b)andGA(c).
agents(TPP,FAandGA)increasedwithtime.Theinfluenceofdifferentcross-linkingagents(TPP,FAandGA)atacross-linkingextentof1and2%w/wontheswellingbehaviourofspraydriedchitosanmicrospheresisdepictedinFigures5and6,respectively.FromFigures5and6,itisveryclearthattheswellingcapacityofthecross-linkedspray
386K.G.H.Desai&H.J.Park
Figure3.Scanningelectronmicroscopicpicturesofspraydriedchitosanmicrospherescross-linkedwith2%w/wTPP(a),FA(b)andGA(c).
driedchitosanmicrosphereswasinfluencedbythenatureofthecross-linking,i.e.chemicalorioniccross-linking.Forinstance,spraydriedchitosanmicrospherescross-linkedwithFAorGA(chemicalcross-linking)exhibitedlowerswellingcapacitywhencomparedtothosecross-linkedwithTPP(ioniccross-linking)(seeFigures5and6).Dependingonthenature
Preparationofcross-linkedchitosanmicrospheres387
Figure4.Scanningelectronmicroscopicpicturesofspraydriedchitosan-TPP(a),chitosan-FA(b)andchitosan-GAmicrospheresaftertheirerosionstudy(cross-linkingextent1%w/w).
ofthecross-linkers(chemicalorioniccross-linkingagents),themaininteractionsformingthenetworkarecovalentorionicbonds(Bergeretal.2004).Inthecaseofchemicalcross-linking,thealdehydegroupsformcovalentiminebondswiththeaminogroupsofchitosan,duetoresonanceestablishedwithadjacentdoubleethylenicbondsviaaSchiffreaction
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400
TPP
300
FAGA
Swelling (%)200
100
0
0.5
1
2
Time (h)
Figure5.Theinfluenceof1%w/wTPP,FAandGAontheswellingbehaviourofspraydriedchitosanmicrospheres.
46
(Bergeretal.2004).Thesealdehydesallowdirectreactioninaqueousmedia,undermildconditionswithorwithouttheadditionofauxiliarymoleculessuchasreducers(MonteiroandAiroldi1999;Bergeretal.2004).Ionicinteractionsbetweenthenegativechargesofthecross-linker(TPP)andpositivelychargedgroupsofchitosanarethemaininteractionsinsidethenetwork.Therefore,chemicallycross-linked(covalent)chitosanmatricesaremorerigidand,hence,these(chitosan-FAandchitosan-GA)matricesexhibitedlowerswellingcapacity(lowerwateruptake(seeTableII))thantheionicallycross-linked(chitosan-TPP)matrix.Ontheotherhand,astheextentofcross-linkingincreasedfrom1to2%w/w,theswellingcapacityofthespraydriedchitosanmicrospheresdecreasedconsiderably.Theseresultssup-portthatthemoretightlycross-linkedchitosan-TPPorchitosan-FAorchitosan-GAmatrixdoesnotswell(lowerwateruptake(seeTableII))asmuchasthelooselycross-linkedchitosan-TPPorchitosan-FAorchitosan-GAmatrix.AtlowerlevelsofTPPorFAorGA(i.e.lowercross-linkdensity),thenetworkislooseandhasahighhydrodynamicfreevolumetoaccommodatemoreofthesolventmolecules,therebyinducingchitosan-TPPorchitosan-FAorchitosan-GAmatrixswelling.Thewateruptakeinhydrogelsdependsupontheextentofhydrodynamicfreevolumeandavailabilityofhydrophilicfunctionalgroupsforthewatertoestablishhydrogenbonds.Higherwateruptakevalueswereobservedatlowerlevelsofcrosslinking(seeTableII)andviceversaobservedinthepresentstudyconfirmtheformationofrigidchitosan-TPPorchitosan-FAorchitosan-GAnetworksthroughanewprocessduetocross-linking.
Preparationofcross-linkedchitosanmicrospheres389
400
TPP
300
FAGA
Swelling (%)200
100
0
0.5
1
2Time (h)
Figure6.Theinfluenceof2%w/wTPP,FAandGAontheswellingbehaviourofspraydriedchitosanmicrospheres.
46
Physicalstateofthedrug
Thephysicalstateofadrugisalsoanimportantparameterwhileinvestigatingthedrugreleasekineticsfromnewlypreparedmicrospheres.Thephysicalstateofthedrugmayvaryfrommoleculardispersion(amorphous)towell-definedcrystallinestructures.Therefore,XRDanalysesofthespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrosphereswereperformedinordertocharacterizethephysicalstateoftheloadeddruginthespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmatrices.ThecharacteristicXRDpatternsofneatacetaminophen,spraydriedplacebochitosanmicrospheres,drugloadedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresarepresentedinFigure7.Characteristiccrystallinepeaksofacetaminophenwereobservedat2of12.14,13.13,15.56,16.76,18.23,20.38,23.0,24.37,26.6,27.232.48,32.64and36.89(Figure7(a))indicatingthepresenceofcrystallineacetaminophen.Underthepresentexperimentalconditions,spraydriedplacebochitosanmicrospheresdidnotshowanypeaks(Figure7(b)).Peaksofacetaminophencrystalsarealwayspresentindrugloadedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheres,evenifveryreducedinintensity.Typicaldiffrac-tionpatternoftheacetaminophen-loadedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresareshowninFigure7(c–e),respectively.Theseresultsindicatedthattheacetaminophenispresentinthecrystallineformevenafteritsencapsulationinthespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmatrices.
390K.G.H.Desai&H.J.Park
(e)(d)Intensity (c)(b)(a)2θ
Figure7.X-raydiffractogramsofneatacetaminophen(a),spraydriedplacebochitosanmicrospheres(b)andacetaminophenloadedspraydriedchitosan-TPP(c),chitosan-FA(d)andchitosan-GA(e)microspheres.
Invitrodrugrelease
Manyparametersdeterminethedrugreleasebehaviourfromchitosanmicrospheres.Theseincludeconcentrationandmolecularweightofchitosan,typeandconcentrationofcross-linkingagent,variablelikedrug-chitosanratio,etc.Thedrugreleasepatternfromchitosanmicrospherescanbetunedintoadesiredreleasebehaviourbyusingdifferentcross-linkingagents(chemicalorioniccross-linkingagents)atdifferentcross-linkingextent(Sinhaetal.2004).Whetheritisachemicalorioniccross-linking,thechitosannetworkexhibitavariedswellingbehaviour.Indeed,asaresultoftheincreaseordecreaseoftheswellingratio,onecanexpectthemeshsizeofthenetworktoalsoincreaseordecreaseconsiderably.Theswellingratiochangeofchitosantranslatesintoachangeinmeshsizeofthegel,whichmodulatesdrugrelease(Bergeretal.2004).Thedrugreleasepropertiesofthechitosanmicrospherescross-linkedwithdifferentcross-linkingagentsandpreparedbydifferenttechniques,especiallyemulsionorcoacervationorsolventevaporationorionotropicgelationmethod,isthoroughlystudied(Sinhaetal.2004).Whereastheinfluenceofwidelyusedcross-linkingagents(TPP,FAandGA)onthedrugreleasepropertiesofspraydriedchitosanmicrospheresisnotstudiedsofar.Hence,thispaperreportstheinfluenceofthesecross-linkingagents(TPP,FAandGA)atdifferentcross-linkinglevel(1and2%w/w)onthedrugreleasepropertiesofspraydriedchitosanmicrospheres.
TheinfluenceofTPP,FAandGAatacross-linkingextentof1and2%w/wonthedrugreleaserateofspraydriedchitosanmicrospheresispresentedinFigures8and9,respec-tively.FromFigures8and9,itcanbeemphasizedthatdrugreleaseratefromspraydriedchitosanmicrosphereswasinfluencedbythetypeandextentofcross-linkers.Forinstance,chitosan-TPPmicrospheresexhibitedhighercumulativeamountofdrugreleaseatboththecross-linkingextent(1and2%w/w)whencomparedtochitosan-FAandchitosan-GAmicrospheres.Releaseofthedrugfrommicrospheresdependsuponthetypeofmatrixanditsrigidity.Thereleaseofdrugfrommatrixinvolvesinitialswellingfollowed
Preparationofcross-linkedchitosanmicrospheres
100391
80Cumulative drug release (%)60GA40FATPP2000123Time (h)4567Figure8.Theinfluenceof1%w/wTPP,FAandGAontheacetaminophenreleasebehaviourfromspraydriedchitosanmicrospheres.
100
80
Cumulative drug release (%)60
40
GATPPFA20
00
1
2
3Time (h)
4
5
6
7
Figure9.Theinfluenceof2%w/wTPP,FAandGAontheacetaminophenreleasebehaviourfromspraydriedchitosanmicrospheres.
bydiffusionofdrug.Theanalysisofswellingbehaviourofspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresallowedonetogaininsightintothedrugreleasebehaviourfromthesematrices.Indeed,thehighertheswellingcapacityofthematrix,thehighertheamountofdrugdiffusedoutfromthematrix.Sincespraydriedchitosan-TPP
392K.G.H.Desai&H.J.Park
microspheresexhibitedhigherswellingcapacity(%),%wateruptakeand%erosion(seeTableII)whencomparedtochitosan-FAandchitosan-GAmicrospheres,thereleaserateofthedrugwascomparativelyhigher(seeFigures8and9)fromchitosan-TPPmicro-spheresthanthechitosan-FAandchitosan-GAmicrospheres.
Diffusionofthedrugfromthechitosanmatrixdependsmainlyonthecross-linkingdensity,whichisdeterminedbytheextentofcross-linking;,therefore,canbeusedtomoni-tordrugrelease(Bergeretal.2004).Inthepresentstudy,spraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrosphereswerepreparedattwocross-linkinglevels(1and2%w/w)tomodulatethedrugreleasebehaviour.Releaserateofthedrugfromspraydriedchitosanmicrospheresdecreasedastheextentofcross-linkingincreasedfrom1to2%w/w(seeFigures8and9).Asthecross-linkingextentincreasedfrom1to2%w/w,wateruptake(%)andswellingcapacity(%)ofthespraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresdecreased(seeTableII)and,hence,drugreleaseratedecreasedwithanincreasingextentofcross-linkingagents.Moreover,thehigherthecross-linkingdensity,thelowertheswellingabilityofthechitosanhydrogelsduetotheslowerrelaxationtimeofthepolymericchains,whichresultsinadecreaseddrug-releaserate(Sinhaetal.2004).
Furthermore,thein-vitroreleasedatahavebeenanalysedusingtheHiguchiequation(Mietal.1999)(Equation3)torevealthemechanismofdrugreleasefromspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheres.
Q¼kHt1=2
ð3Þ
whereQisthecumulativeamountofacetaminophenrelease(%)attimetandkHtheHiguchirateconstant.Thereleasedatawereplottedasthepercentageofacetaminophenreleaseagainstthesquarerootoftime.Linearitywasobservedwiththeplotssincethecorrelationcoefficient(R2)rangedfrom0.9903–0.9989(seeTableIII).Thisindicatesthatreleaseoftheacetaminophenfromspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmatricesfollowedFick’slawofdiffusion.Acikgozetal.(1996)alsoreportedsimilarfindingwherethebestforthereleaseofdiclofenacsodiumfromchitosanmicrosphereswasobtainedbytheHiguchiequation.
Inthepresentstudy,theoverallreleaseprocessofacetaminophenfromspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrospheresissomewhatbiphasicwithaninitialbursteffect,followedbyasubsequentslowerrelease.Theresultsoftheinvitrodrugreleasestudyindicatedthatthereleaseofacetaminophenfromspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmatricestakesplacethroughtwostepsas
TableIII.Kinetic(Higuchiplot)parametersofacetaminophenreleasefromspraydriedchitosan-TPP(F1andF2),chitosan-FA(F3andF4)andchitosan-GA(F5andF6)microspheres.
Higuchiplotparameters*
FormulationcodeF1F2F3F4F5F6
kH(%h1/2)57.6771.3853.9766.5651.4761.81
R20.99380.99230.99030.99090.99890.9903
*Dataareobtainedbyplottingmean(n¼3)ofcumulativedrugrelease(%)vssquarerootoftime.
Preparationofcross-linkedchitosanmicrospheres393
Step 1
Acetaminophen loaded spray dried chitosan-TPP or chitosan-FA or chitosan-GA
microspheres
Release of acetaminophen
from surface
Step 2
Diffusion of acetaminophen from swollen microsphere
Figure10.Releaseprocessofthedrug(acetaminophen)fromspraydriedchitosan-TPP,chitosan-FAandchitosan-GAmicrosphere.
depictedinFigure10.Thesurfaceadheredacetaminophenisreleasedquickly(43–66%ofdrugreleasewithin30min(seeFigures7and8))fromallthespraydriedchitosanmicrospheres(step1).Then,thereleaserateoftheacetaminophenfromswollenchitosan-TPP,chitosan-FAandchitosan-GAmicrospherewassustainedwhichismainlygovernedbythediffusionability(followedFick’slawofdiffusion,seeTableIIIforkineticparameters)oftheacetaminophenfromswollenchitosan-TPP,chitosan-FAandchitosan-GAmatricesintothedissolutionmedia(step2).Conclusions
Thepresentstudyinvestigatedtheeffectofdifferentcross-linkingagents(TPP,FAandGA)ontheproperties(meansize,encapsulationefficiency,surfacemorphology,erosion,swellingandinvitrodrugrelease)ofspraydriedchitosanmicrospheres.Thesecross-linkingagentshadaremarkableinfluenceonthesurfacemorphology,erosion,swellinganddrugreleasebehaviourofthemicrospheres.Itwasfoundthat%encapsulationefficiency,%erosion,%swelling,surfacemorphologyanddrugreleaserateofthespraydriedchitosanmicrospherescouldbeproperlycontrolledbyaddingdifferentcross-linkingagentsintothechitosansolutiontobespraydried.
Acknowledgements
ThisstudywassupportedbyagrantoftheKoreaHealth21R&DProject,MinistryofHealth&Welfare,RepublicofKorea(A050376).WealsothanktheKoreaScienceandEngineeringFoundation(KOSEF)forthefinancialsupport.
394K.G.H.Desai&H.J.Park
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