Covalent LBL assembly of hyperbranched polymers on alginate microcapsules to impart stability

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MaterialsChemistry

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Citethis:J.Mater.Chem.B,2014,2,8208

Covalentlayer-by-layerassemblyofhyperbranchedpolymersonalginatemicrocapsulestoimpartstabilityandpermselectivity?

aabcabd

K.M.Gatta´s-Asfura,M.Valdes,E.CelikandC.L.Stabler*

Themicroencapsulationofcellshasshownpromiseasatherapeuticvehicleforthetreatmentofawidevarietyofdiseases.Whilealginatemicrocapsulesprovideanidealcellencapsulationmaterial,polycationscoatingsarecommonlyemployedtoenhancestabilityandimpartpermselectivity.Inthisstudy,functionalizedhyperbranchedalginateanddendrimerpolymerswereusedtogeneratediscreetnanoscalecoatingsontoalginatemicrobeadsviacovalentlayer-by-layerassembly.ThebioorthogonalStaudingerligationschemewasusedtochemoselectivelycrosslinkazidefunctionalizedhyperbranchedalginate(alginate-hN3)to1-methyl-2-(diphenylphosphino)terephthalate(MDT)linkedPAMAMdendrimer(PAMAM-MDT).Covalentlayer-by-layerdepositionofPAMAM-MDT/alginate-hN3coatingsontoalginatemicrobeadsresultedinhighlystablecoatings,evenaftertheinneralginategelwaslique?edtoformmicrocapsules.Thepermselectivityofthecoatedmicrocapsulescouldbemanipulatedthroughthe

Received28thJuly2014

Accepted20thSeptember2014DOI:10.1039/http://wendang.chazidian.com/MaterialsB

chargedensityofthePAMAM,thenumberoflayersdeposited,andthelengthofthefunctionalarms.ThecytocompatibilityoftheresultingPAMAM-MDT/alginate-hN3coatingwasevaluatedusingabetacellline,withnosigni?cantdetrimentalresponseobserved.Thebiocompatibilityofthecoatingsinvivowasalsofoundcomparabletouncoatedalginatebeads.Theremarkablestabilityandversatilenatureofthesecoatingsprovidesanappealingoptionforbioencapsulationandthereleaseoftherapeuticagents.

Introduction

Thetransplantationofcellstodelivertherapeuticagents,suchastrophicfactors,hormones,antigens,andproteins,isanextensivelyinvestigatedapproachwithbroadclinicalapplica-tions.1–9Whenusedasadeliveryagent,encapsulationofthesecellswithinsemipermeablebiomaterialsprovidesanattractiveapproachtoprotecttheforeigncellsfromthehostimmunesystem,wheretheencapsulationbiomaterialpermitsthedi?usionofthetherapeuticagentintothesurroundingmilieubutpreventsdirecthostcellinltration.OfparticularinteresttotheeldistheuseofencapsulatedinsulinsecretingcellsforthetreatmentofType1DiabetesMellitus(TIDM).Multiplestudieshavedemonstratedthereversalofdiabetesuponthetrans-plantationofencapsulatedpancreaticislets,exhibitingthe

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DiabetesResearchInstitute,UniversityofMiami,Miami,FL33136,USA

DepartmentofBiomedicalEngineering,UniversityofMiami,CoralGables,FL33146,USA.E-mail:cstabler@miami.edu

DepartmentofMechanicalandAerospaceEngineering,UniversityofMiami,CoralGables,FL33146,USA

cd

DepartmentofSurgeryandDepartmentofBiochemistryandMolecularBiology,MillerSchoolofMedicine,UniversityofMiami,Miami,FL33136,USA?Electronicsupplementary10.1039/c4tb01241k

information

(ESI)

available.

See

DOI:

promiseofthisapproachforstabilizingbloodglucoselevelsintheabsenceofanti-rejectiontherapy.10–13

Alginateisoneofthemostwidelyusedbiomaterialsforcellularencapsulation.14–17Alginateisacollectivetermforafamilyofpolysaccharidesderivedfrombrownalgae.Alginatechainsarelinearbinaryco-polymersofb-D-mannuronic(M)anda-L-guluronic(G)acids,withavariationofsequentialarrange-mentandcompositiondependingontheirsource.18,19Thegelationofalginatemicrobeadsiscommonlyachievedbyexposuretodivalentcations(typicallyCa2+orBa2+);however,theresultingalginatehydrogellacksthelong-termstabilitytowithstandthemechanicalandchemicalstrainsassociatedwithimplantation,wheremicrobeaddegradationandrupturecommonlydevelopsduetotheslowexchangeofcationswithsodiumionsunderphysiologicalconditions.20,21

Stabilizationofalginatemicrobeadsiscommonlyachievedbytheassemblyofnanotomicroscalepolyelectrolytecoatingsontheouteralginatesurface.Theseareassembledthroughlayer-by-layerdepositionofpolymersofalternatingcharges,wheretherstlayerdepositedisapolycation,duetotheanionicnatureofalginate.Polycationstypicallyusedforalginatecoat-ingsarepoly-L-lysineandpoly-L-ornithine.22,23Followingdepo-sitionofthepolycationlayer,anallayerofalginateisthendeposited.Althoughassembledinalayer-by-layermanner,it

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hasbeendeterminedthatthesecoatingscomplextoformasinglepolycation/polyanionshell.16,24,25Assuch,polycationscontainedwithinthecoatingsareexposedontheoutersurfaceofthecapsule.Thisinvariablyleadstothewelldocumenteddecreaseinbiocompatibilityandincreasedcytotoxicityofalgi-natecapsulescoatedwiththesepolycations.26–30

Inthisstudy,wesoughttoexplorethepotentialofcovalentlayer-by-layerassemblyofcomplementarypolymersforstabili-zationofalginatecapsules.Covalentlayer-by-layerassemblyprovidesameantoenhancethestabilityofcoatingsthroughinterpolymercovalentbonding,aswellasintimatenanoscalecontroloverlayerthickness.31–34Further,thisapproachprovidesexibilityinpolymerselection,wheremoderatelychargedory on 07/05/2015 11:58:16.

evenneutralpolymerscanbeemployed.Herein,http://wendang.chazidian.comyer-by-layerassemblyofhyperbranchedand/ordendrimerpolymers,rstexploredbyDecherandco-workers,35,36arehighlydesirableforbiomedicalapplications,astheirfacilenatureprovidessubstantialvariationinfunctionandstructure,whiletheirhighdensityofterminalgroupsprovidesincreasedavailabilityforinteractionwithalternatinglayers.37Inourlaboratory,wehavedevelopedhyperbranchedalginateandpoly(amidoamine)(PAMAM)dendrimersfunc-tionalizedwiththecomplementaryStaudingerligationreactivegroups,azideand1-methyl-2-(diphenylphosphino)tere-phthalate(MDT),respectively(Fig.1A).ThebioorthogonalStaudingerligationschemeforcovalentbondformationisparticularlyappealingforbiologicalstudies,asthereactioncanproceedinanaqueousenvironment,underdenedenviron-mentalconditions(e.g.temperature25–37??C,pH7–7.5,osmolarity$300mOsM),andinvolvenon-nativechemicalhandlesandnontoxiccatalystsand/orreactionby-products.38,39Wehavedemonstratedthecapacityofthesehyperbranchedpolymerstoformstable,discreet,andnanoscalecoatingsthroughcovalentlayer-by-layerassembly,bothonidealizedsurfaceandcellclusters.40Herein,weappliedthesecoatingstoalginatemicrobeads,toevaluatetheirpotentialforprovidingastable,permselective,andbiocompatiblecoating.Physi-ochemicalpropertiesoftheresultingcoatingwereevaluatedviapermeability,swelling,andcytotoxicitystudies.Further,biocompatibilityofthecoatingswasevaluatedinarodentmodel.Thepotentialofthesenanoscale,covalent,layer-by-layerassembledcoatingstoprovideausefulplatformforbio-encapsulationisdiscussed.

Experimental

Materialssourcing,polymerfabrication,andsolutionsForformationofalginatebasedhydrogels,alginateoralginatefunctionalizedwithazide(butnothyperbranched),sourcedfromUPMVG(Pronova,NovaMatrix,NJ),wasused.ThecontentanddistributionofMandGunitsweredeterminedby1HNMRspectroscopyandprovidedbythemanufacturer.ForUPMVG,thefractionofGunits(FG)was0.6889.ThefractionofdiadsFGG,FMG/GM,andFMMwere0.58,0.11,and0.20,respectively.The

Thisjournalis©TheRoyalSocietyofChemistry2014Fig.1

(A)SchematicofStaudingerligationreactionunderaqueous

conditions.(B)Schematicofultrathincoatingassemblyontoalginatemicrobeads.Alginatemicrobeadswere?rstincubatedinMDTfunc-tionalizedPAMAM(1).Subsequently,hyperbranchedalginateazidewascovalentlylinkedtotheexposedMDTfunctionalizedPAMAMcoating(2).InterlayercovalentbondswereformedbetweencomplementaryazideandMDTgroupsviaStaudingerligation.Additionallayerswerebuiltviastep-wiseincubationofMDTfunctionalizedPAMAM(1)andhyperbranchedalginateazide(2),untildesirednumberoflayerswereachieved.(C)Visualizationofcoatinggeneratedonalginatemicrobeadsviastep-wiselayerformationusinghyperbranchedalgi-nateazide(labeledwithFITCforconfocalimaging)andPAMAM15/0,PAMAM15/20,orPAMAM15/40.Imageswerecollectedafter2,4,and6layerswereapplied.Scale¼200mm.

fractionoftriadsFGGG,FMGG/GGM,FMGMwere0.54,0.039,and0.070,respectively,withaNG>1of15.82.Synthesisofalginate-N3(nothyperbranched)usedtofabricatemicrobeadswaspreparedaspreviouslydescribed.41,42Inbrief,50mgofUPMVGsodiumalginate,14mgNHSand62mgMESweredissolvedin5mLde-ionizedwater.A200mLsolutionof376mMNÀ13-PEG-NH2(Mw372gmol)wasadded,followedby116mgEDC(116mgin200mLwater,0.60mmol,preparedfresh),25minstirring,andaddedslowly(within20min)270mLof1MNaOH.Puri-cationwasachievedvia4daysdialysis(10000MWCOmembrane)against600mLwater,whichwaschanged

three

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timesaday.Duringtherst3days,NaOH(5mLof5M)andNaCl(250mLof4M)http://wendang.chazidian.comrgerbatchsizes(10Â)werealsofabri-catedusingthismethod.Theremainingsolutionwaslteredthrougha0.2mmmembrane(PallCorp)andfreeze-dried.Purityandfunctionalizationhasbeenreportedpreviously,9wherepuritywasaminimumof97%,withanaverageazidemodi-cationof11%(percentmodicationofalginatecaboxylategroups)or149N3peralginatemolratio.

Forformationofcovalentlayer-by-layercoatingsontothealginatehydrogelmicrobead,hyperbranchedalginate-azide(termed“alginate-hN3”inthisstudy)andfunctionalizedPAMAMs(labeledaccordingtothedegreeoffunctionalizationofterminalgroupswithMDT/glutaricanhydride,seeTable1)werepreparedaspreviouslydescribed.40Foralginate-hN3,UPVLVGsodiumalginate(PRONOVA,NovaMatrix)wasused.ForUPVLVG,thefractionofGunits(FG)was0.6661.ThefractionofdiadsFGG,FMG/GM,andFMMwere0.56,0.11,and0.22,respec-tively.ThefractionoftriadsFGGG,FMGG/GGM,FMGMwere0.51,0.043,and0.067,respectively,withaNG>1of13.88.Briey,800mgof1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimidehydro-chloride(EDC)followedby200mgof3,5-dicarboxy-phenylglycineamide(in3.8mLof0.42MNaOH,267mLminÀ1)wereaddedto200mgUPVLVGalginate,16mgofN-hydroxy-succinimide(NHS),and240mgof2-(N-morpholino)ethane-sulfonicacid(MES)dissolvedin20mLofwater.Aer25min,240mLof5MNaOHwasaddedat2.67mLminÀ1.Hyper-branchedalginatewasprecipitatedwith40mLacetone,dis-solvedin4mLof50mMNaCl,precipitatedwith8mLacetone,anddriedunderreducedpressure.Next,50mgofhyper-branchedalginateand50mgofH2N-PEG-N3(Mw372gmolÀ1)werereactedwith125mgEDCin5mLpuriedwatercon-taining5mgNHS,30mgMES,andeither0or2mgofuo-rescein-5-thiosemicarbazide.Aer20min,30mLof5MNaOHwasaddedat1mLminÀ1.Hyperbranchedalginate-azidewasprecipitatedwith20mLacetone,dissolved/precipitatedtwicein3mLof50mMNaCl/15mLacetone,driedunderreducedpressure,puriedbydialysis(10kDaMWCO,against500mLwaterreplacedevery20minfor2h,10mLNaOHand200mL1MNaClwereaddedtothealginatesolutionduringthersthourevery20min),lter-sterilized,andfreeze-dried.

Forthefunctionalizationofpoly(amidoamine)(PAMAM)dendrimers,generation5PAMAM(1mLof5%inCH3OH,39.85mg)wasreactedwith14mgof4-pentauorophenylesterof1-methyl-2-(diphenylphosphino)terephthalicacidin0.5mLanhydrousdichloromethane(DCM,injecteddrop-wise)for30minunderargonatmosphere(PAMAM15/0).ForPAMAM

Table1PropertiesofselectedPAMAMderivativeswithvaryingMDTandGAfunctionalization.Chargeandmolecularweightwerecalcu-latedasdescribedinpreviouspublication40

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15/20or15/40,25mLoftriethylaminein0.5mLDCMwasinjectedfollowedby4or8mg(respectively)ofglutaricanhy-dridein0.5mLDCM(injecteddrop-wise).Aer30min,20mLglacialaceticacidwasadded,theproductprecipitatedwith10mLdiethylether,dissolved/suspendedin1mLof50%v/vDCMinmethanol,precipitatedwith10mLdiethylether,driedunderreducedpressure,dissolvedin2mLwaterwith20mLglacialaceticacid,puriedbydialysis(10kDaMWCO,against500mLwaterreplacedevery20minfor2h).100mLof1MNaClwasaddedtothealginatesolutionevery20minforthersthour,lter-sterilized,andfreeze-dried.ATR-FT-IRspectracharacter-izedMDTgroup(1719,746,and697cmÀ1)andamidebonding(1637and1539cmÀ1),whilethedegreeoffunctionalizationwasdeterminedviaNMRpeakintegrationratiobetweenaromatic(d6.5–8.2ppm)orGA(d1.83ppm)againstPAMAM(d2.07–3.66ppm)protons.CharacterizationdataofselectedMDTandGAfunctionalizedPAMAMdendrimersaresummarizedinTable1.Ofnote,thesurfacenetchargelistedforthespecicdendrimerassumeseveryprimaryaminoorcarboxylategroupischarged,thusthetruenetchargedependsonpHofthesolution.Fabricationofalginateoralginate-azidemicrobeadswithoutandwithbetacells

Standardalginateoralginate-N3microbeadswerefabricatedusingamodicationoftheprotocoloriginallydevelopedbySun.43Topreparethealginatesolutions,1.0%w/valginateoralginate-N3wasdissolvedinphosphate-bu?eredsolution(PBS,Mediatech),thepHofthesolutionwasadjustedto11with5MNaOH,stirreduntilclear,andslowlyrecoveredtopH7.4viaequimolaramountofHCl.Aparallelairowbeadgenerator(Biorep,Inc.Miami,FL)wasusedtofabricatethemicrobeadsinBa2+containingMOPSbu?er.Thesizeofthedropletswascontrolledbyaparallelairowgenerator(10kPapressureofair;1inchdistancefromtheneedletogelationsolution)andmanualforceappliedtothesyringe.Thebeadswereexposedtothegelationsolutionfor10mintoformastablehydrogel.ThebariumsolutionwasaspiratedandthebeadswerethenrinsedwithPBSfourtimestoremoveexcessbarium.Forcoatingandpermselectivitystudies,resultingmicrobeaddiametersaver-aged757Æ35mm(n¼19).MicrobeadswerestoredinPBSpriortobeadswellingand/orchelationassays.

Formicrobeadscontainingcells,MIN6cellswereused.MIN6cells(subcloneC3,courtesyofDrValerieLillaandAle-jandraTomas)44wereculturedasmonolayersinT-asksandfedevery2–3dayswithfreshmediumcomprisedofDulbecco'smodiedEagle'smedium(DMEM)supplementedwith10%fetalbovineserum(FBS;Sigma),1%penicillin–streptomycin(P/S),1%L-glutamineand0.001%(v/v)b-mercaptoethanol.MIN6cellswereharvestedfrommonolayerculturesusingtrypsin–EDTA(Sigma,StLouis,MO)andsuspendedineitherpolymersolutionatadensityof2.5Â106cellspermLof1%w/valginatepolymersolution.Viablecellcountsformeasurementofloadingcelldensitywereperformedviatrypanblue(Sigma,StLouis,MO)exclusionmethod.Followingthehomogeneousdistributionofthecellswithineitheralginatesolution,thecell/polymersuspensionwasimmediatelyextrudedthroughtheair

PAMAM(ID)15/015/2015/40

MDT(%)141414

GA(%)02344

Netcharge+110+51À3

Mw(gmolÀ1)350263841241503

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owbeadgeneratortoformmicrobeads,asdescribedabove.Followingan8minexposuretothebariumgelationsolutionandPBSrinse(4times),thecell-containingmicrobeadswerewashedinculturemediadescribedabove.Beadsweresubse-quentlycoatedinPAMAM/alginatemicrobeadspriortocultureinnon-cellculturetreatedplatesinahumidied37??C,5%CO2/95%airincubatorfor48hpriortoassessment.Resultingmicrobeaddiametersforaveraged815Æ34mm(n¼24).Coatingofalginateoralginate-azidemicrobeadswithPAMAM/alginateorPLL/alginate

Forlayer-by-layerassemblyofPAMAMandalginate-hN3ontouorescenceconfocalmicroscopy.Confocalimageswerecross-sectionsat100mmdepthfrombottomofthemicrocapsules.ThedegreeofFITC-dextranpermeabilitythroughthedi?erentcapsuleswasquantiedbydividingthegreenluminosityinsidethebeadsoverthatontheoutsideutilizingthehistogramtoolofAdobePhotoshop,henceaccountingforvariationsinuores-centlabelconcentrationwiththechangeindextranmolecularweight.Valuesareexpressedastheaverageofmeasurementsfromatleastthreedi?erentbeadsperexperimentalcondition.Measurementswerecollectedagainaer24hofincubationtoverifythatequilibriumwasachievedwithinthat2hincubation.Variationbetween2and24hmeasurementswerelessthan8%.Thepermselectivityormolecularweightcut-o?ofthethealginatemicrobeads,microbeadswererstincubatedinthePAMAMpolymersolution(3mgmLÀ1inPBSbu?er)for10minat37??C,followingbyrinsing(3Â)withPBS.Subsequently,alayerofalginate-hN3wasaddedbyincubationofPAMAMcoatedbeadwithalginate-hN3polymersolution(3mgmLÀ1inPBSbu?er)for10minat37??C,followingbyrinsing(3Â)withPBS.Thisprocesswasrepeateduntilthedesirednumberoflayerswasachieved.AplasticdisposablePasteurpipetwasfrequentlyutilizedtostirandseparatethemicrobeadstopreventaggre-gation.Thepolymersolutionwassupplementedwithglucose(3mgdLÀ1)whencoatingmicrobeadscontainingMIN6cells.Microbeadscoatedwithpoly-L-lysineandstandardalginate(PLL/Alg)servedasthecontrolgroup.TogeneratethePLLcoating,microbeadswereincubatedin0.05%(wt/v)ofpoly-L-lysine(Mw30000–70000,SigmaCat#P2636)inPBSbu?erfor6minatroomtemperature.ExcessPLLwasrinsedawaywithmultiplewasheswithPBSbu?er.Microbeadsweresubse-quentlyincubatedin0.15%ofstandardalginate(UPMVG)for4minatroomtemperature,followedbyextensiverinsinginPBS.Cell-freecoatedmicrobeadswerestoredat37??CinPBS.Forcellloaded,microbeadswereculturedasdescribedabove.Forformationofmicrocapsules,coatedmicrobeadsweretreatedwith50mMEDTAsolutioninMOPSbu?er(1Â,pH7.4)for15mininordertochelatebariumionsfromthealginatecore.TheresultingcapsuleswererinsedthreetimeswithPBS(1Â,pH7.4)for5min.Followingrinsing,microbeadswereassessedforswelling.

Swellingandpermeabilityofcapsules

Forswellingtesting,thediameterofmultiple(atleast4)coatedbeadsinPBSwasmeasuredbeforeand1haerEDTAtreat-mentutilizingtheSZX7stereomicroscope(Olympus).Degreeofswellingwascalculatedfromtheratioofcapsulediameteroverbeaddiameter.

PermeabilitytestingwasconductedoncoatingsfollowingEDTAtreatment,tominimizethecontributionofthealginatehydrogelonpermselectivity.Forpermeabilitytesting,coatedalginateoralginate-N3microcapsules(followingEDTAtreat-ment)wereplacedonglassbottomPetridishesandincubatedfor2hin0.1mgmLÀ1ofFITC-dextranofdi?erentmolecularweights(4,10,40,70,150,250,500,or2000Â103gmolÀ1)inPBSbu?er.Ethidiumbromide(0.2mLofInvitrogenreddeadstainforcells)wasutilizedtouorescentlylabelthecapsules.Aer2h,microcapsulesweresubsequentlyimagedusing

Thisjournalis©TheRoyalSocietyofChemistry2014capsuleswasexperimentallyidentiedwhenthe%perme-abilityforaparticulardextranmolecularweightwas<10%.Thecorrespondinghydrodynamicradiusofthedextranwastheo-reticallydeterminedusingthefollowingpublishedformula:45

Rh¼0.026(Mw)0.495

(1)

whereRhisthehydrodynamicradiusofthedextraninnmandMwisthemolecularweightingmolÀ1.Atomicforcemicroscopy

Acommercialatomicforcemicroscope(MFP-3D-BIO,AsylumResearch)wasusedtoobtainhigh-magnicationimagesofthepolymercoatings.InordertoholdthesamplesteadyduringAFMimaging,themicrobeadsweretrappedonglassslidesbyagaroselms.Briey,acoatedmicrobeadwasplacedonaglassslideandawarmsolutionof1.5%agaroseinwaterwasappliedaroundthemicrobeadusingastereomicroscope.Themicrobeadwasheldstableinplaceuponagarosegelationbycooling.Carewastakennottocontaminatethemicrobeadsurfaceduringthisprocess.Theagaroseheightwas$75–80%ofthebead'sheight,whichcorrespondstoanexposureareaforAFMimagingof$640mm(seeESI?materialsforimagesofbeadsembeddedinagarose).TheAFMtipwaspositionedabovethetrappedmicrobeadimmersedinPBSandscannedinthecontactmodetoacquiresurfacetopography.Apyramidal,siliconnitrideAFMcantilevertip(nominalspringconstant0.01NmÀ1,MLCTseries,BrukerAFMProbes)wasusedtoimageallsamples.Allimageswereacquiredwithascansizeof10mmandaresolutionof256Â256points.RMSroughness(Rq)ofthesurfaceswasmeasuredontheAFMheightvimagesatuu

??????????????????????????????5

erentlocationsusingtheequation;RtX?ðZiÀZaveÞ2

di?q¼

N

wherezaveistheaverageoftheelevation(zvalue)withinthegivenarea,ziistheelevationforagivenpoint,andNisthenumberofdatapointswithinthegivenarea.

Cytocompatibilityandinsulinreleaseofencapsulatedbetacells

Atwo-coloruorescentviabilityandcytotoxicitykit(MolecularProbes)wasusedtoevaluatecellviability.Viable(activeintra-cellularesterase)cellsstainuorescentgreenwithcalcein-AM

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anddead(lostplasmamembraneintegrity)cellsstainuores-centredwithethidiumhomodimer-1dye.Encapsulatedcellsanddyeswereco-incubatedinDMEMmediafor1hfollowedbyimagingwithconfocalmicroscope.Ofnote,interactionsbetweenthePAMAM/alginatecoatingandtheethidiumhomodimer-1dyehavebeendocumented.Hoechstdyewasaddedtolabelcellnucleiinane?orttodelineatecoatingstainingfromdeadcellstaining.

Glucosestimulatedinsulinrelease(GSIR)consistedofali-quoting130microbeadspergroup(127Æ37microbeads/group)in1mLKrebsbu?erwithlowglucose(60mgdLÀ1)for30minina12-wellplateandincubator.ThesamemicrobeadswerethenincubatedinKrebsbu?http://wendang.chazidian.comyercoatingsontothealginatemicrobeadswasexploredusingtwoalginatehydrogelplatforms:standardalginateoralginate-N3.Thealginate-N3employedformicrobeadgenerationwasnothyperbranched;aportionofcarboxylategroupsofalginateweresimplyfunctionalizedwithlinearH2N-PEG-N3.Bothmicrobe-adsweregeneratedbyionicgelation.ResultingmicrobeadswerecoatedwithlayersofPAMAMandalginate,functionalizedwithmethyl-2-diphenylphosphino-terephthalate(MDT)andazide,respectively,throughstep-wiseincubation(Fig.1B).Forallcoatings,PAMAMwasusedasthebaselayer(Fig.1).Inthisstudy,PAMAMdendrimer(generation5)wasused,whereby15%ofprimaryaminoendgroupsonPAMAMwasfunctional-izedwithMDT.40ToexplorethecontributionofnetchargeonThehighglucose(300mgdLÀ1)Krebsbu?erwascollectedandtheamountofinsulinwasdeterminedutilizingthemouseinsulinElisaquanticationkit.Biocompatibilityassay

AllanimalstudieswerereviewedandapprovedbytheUniversityofMiamiInstitutionalAnimalCareandUseCommittee.AllprocedureswereconductedaccordingtotheguidelinesoftheCommitteeonCareandUseofLaboratoryAnimals,InstituteofLaboratoryAnimalResources(NationalResearchCouncil,WashingtonDC).AnimalswerehousedwithinmicroisolatedcagesinVirusAntibodyFreeroomswithfreeaccesstoauto-clavedfoodandwaterattheDepartmentofVeterinaryResourcesoftheUniversityofMiami.MaleC57BL/6Jmice,between7–9weeksofage(JacksonLaboratory),wereusedasrecipients.Thisstrainhasademonstratedrobusthostresponsetomaterialsandisacommonchoiceusedforbiocompatibilitystudies,particularlythoseinvolvingalginatecapsules.46–48Groupsconsistedofuncoatedmicrobeads,6-layercoating,and7-layercoatingusingalternatePAMAM15/0andhyperbranchedalginate-azidelayers.Undergeneralanesthesia(isouraneUSP,Deereld,IL),alowermid-lineincisionwasmadeandmicrobeads(1–1.5mL)wereintroducedintotheperitonealcavity.Theincisionwasthenstapledclosed.Microbeadswereretrievedaer21dayspostimplantationvialavage,rinsedwithPBS,xed,sectioned,andstainedwithhematoxylinandeosinformicroscopicimaging.Statistics

AlldataanalysisusedExcel(Microso,USA),withstatisticalanalysisandgraphingviaGraphPadPrism(GraphPadSoware,Inc.,USA).DataarepresentedasmeanÆSEM.Resultswereanalyzedviaoneortwo-wayANOVA,withBonferroniorTukeypost-hostanalysis.Observationswereconsideredtobestatisti-callysignicantwithp<0.05.

Resultsanddiscussion

Formationofcoatingsontoalginatemicrobeads

Initialstudiessoughttoexplorethecapacitytocoathydrogelmicrobeads,inalayer-by-layermanner,usingcomplementaryfunctionalizedpolymersthatarecovalentlylinkedviaStau-dingerligation(Fig.1A).Theformationofcovalentlayer-by-

8212|J.Mater.Chem.B,2014,2,8208–8219layerformation,asubsetoftheremainingprimaryaminogroupsonthePAMAM-MDTwassubsequentlymodiedwithglutaricanhydride(GA).GAfunctionalizationwasvariedfrom0to20to40%,designatedasPAMAM15/0,PAMAM15/20,andPAMAM15/40,respectively,resultinginanetdendrimersurfacechargeof+110to+51toÀ3,aspreviouslypublished(seeTable1forsummary).40Forinterimlayers,hyperbranchedalginateazide,termedalginate-hN3,wasemployed(Fig.1B).Asprevi-ouslydescribed,acarbodiimide-basedcouplingprotocolwasutilizedtohypergrabranchedpolymericchainsof3,5-dicar-boxyphenylglycineamideontothealginatebackbone,whichweresubsequentlyfunctionalizedwithazidebytreatmentwithalinear,heterobifunctionalpoly(ethyleneglycol),expressingaprimaryaminoandanazidoendgroup.40ThemolecularweightofthePEG-N3linker,andhencethelengthofthehyper-branchedazidefunctionalizedarm,couldbemodiedtoexploreitsimpactontheresultingcoating.Molecularweightsof300and600gmolÀ1wereinvestigatedinthisstudy.Theuseofhyperbranchedanddendrimerpolymers,withincreasedpresentationofreactivegroups,wasfoundtoenhancethee?-ciencyandcompetencyofcovalentlayer-by-layerformationonidealizedplanarsurfaces.40

ResultingcoatingswerevisualizedwithconfocalmicroscopybyFITClabelingofthehyperbranchedalginateazide.Asillus-tratedinFig.1C,nanoscalecoatingsweregeneratedonalginatemicrobeads,withcoatinguniformityandintensityincreasingwithsubsequentlayers.IncreasednetpositivechargeofthePAMAMcorrelatedwithincreaseduniformityat2and4layers;however,by6layers,coatingsgeneratedbyanyofthePAMAMformswerecomparable,withuniformandcompletecoatingsobserved.Surprisingly,thepresenceofazidegroupsonthebasealginatemicrobeaddidnotsignicantlyimpactPAMAMdepo-sition.Resultingcoatingswerehighlystable,evenaerseveralweeksofstorageatroomtemperature.

StabilityofcoatingsfollowingliquicationofinneralginatecoreOfinteresttothegenerationofaversatilecoatingplatformistheimpactofthecoatingsonbeadstability.Toexplorethecontributionofthecoatingstoenhancealginatestability,algi-nateoralginate-N3microbeadswerechelatedusingEDTAfollowinglayer-by-layercoatingtoremovebariumionsandliquefytheinneralginatehydrogel,resultinginalginatemicrocapsules.Thestabilityofmicrocapsuleswasassessedby

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