湖南中医药大学第二届“三湘读书月”活动

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湖南中医药大学校权益部

2010-11-23

 

第二篇：纳米给药系统相关文献

Biomaterials33(2012)1573e1582

ContentslistsavailableatSciVerseScienceDirect

Biomaterials

journalhomepage:www.elsevi

er.com/locate/biomaterials

Gobletcell-targetingnanoparticlesfororalinsulindeliveryandthein?uenceofmucusoninsulintransport

YunJin1,YupinSong1,XiZhu,DanZhou,ChunhuiChen,ZhirongZhang,YuanHuang*

KeyLaboratoryofDrugTargetingandDrugDeliverySystem,MinistryofEducation,WestChinaSchoolofPharmacy,SichuanUniversity,No.17,Block3,SouthernRenminRoad,Chengdu610041,PRChina

articleinfo

Articlehistory:

Received27August2011Accepted27October2011

Availableonline16November2011Keywords:

CSKSSDYQCpeptideGobletcell-targetingMucus

Caco-2/HT29-MTXco-culturedcells

N-trimethylchitosanchloridenanoparticles

abstract

Thepresentstudywastodemonstratetheeffectsofgobletcell-targetingnanoparticlesontheoralabsorptionofinsulininvitro,exvivoandinvivo,andidentifythetargetingmechanismaswellasthein?uenceofmucus.Theinsulinloadednanoparticleswerepreparedusingtrimethylchitosanchloride(TMC)modi?edwithaCSKSSDYQC(CSK)targetingpeptide.Comparedwithunmodi?ednanoparticles,theCSKpeptidemodi?cationcouldfacilitatetheuptakeofnanoparticlesinvilli,enhancethepermeationofdrugsacrosstheepithelium,meanwhile,induceasigni?cantlyhigherinternalizationofdrugsviaclathrinandcaveolaemediatedendocytosisongobletcell-likeHT29-MTXcells.IntransportstudiesacrossCaco-2/HT29-MTXco-culturedcellmonolayer(simulatingintestinalepithelium),theCSKpeptidemodi?cationalsoshowedenhancedtransportability,evenifthetargetingrecognitionwaspartiallyaffectedbymucus.Moreover,itwasfoundtheexistenceofmucuswaspropitioustothetransportofinsulinfrombothmodi?edandunmodi?ednanoparticles.Inthepharmacologicalandpharmacokineticstudiesindiabeticrats,theorallyadministratedCSKpeptidemodi?ednanoparticlesproducedabetterhypoglycemiceffectwitha1.5-foldhigherrelativebioavailabilitycomparedwithunmodi?edones.Inconclusion,CSKpeptidemodi?edTMCnanoparticlesshowedsuf?cienteffectivenessasgobletcell-targetingnanocarriersfororaldeliveryofinsulin.

ó2011ElsevierLtd.Allrightsreserved.

1.Introduction

Oraladministrationofinsulinwouldbebene?cialtodiabeticpatients,asitnotonlyalleviatesthepaincausedbyinjections,but

Abbreviations:CSK,CSKSSDYQC;TJs,tightjunctions;NPs,nanoparticles;TMC,N-trimethylchitosanchloride;INS,insulin;FITC,?uoresceinisothiocyanate;Rho-UEA-I,rhodamine-conjugatedulexeuropaeusagglutininIlectin;MTT,3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide;AMCA-INS,7-amino-4-methy-(3-coumaringlacetic)acid-conjugatedinsulin;EDC$HCl,1-[3-(dimethyla-mino)propyl]-3-ethylaarbodiimidehydrochloride;NHS,N-hydroxysuccinimide;DMEM,dulbecco’smodi?edeaglemedium;CS,chitosan;CH3I,methyliodide;NaOH,sodiumhydroxide;NMP,N-methylpyrrolidone;DQ,degreeofquaterniza-tion;1HNMR,1Hnuclearmagneticresonance;FT-IR,Fouriertransforminfrared;TPP,tripolyphosphate;HCl,hydrochloricacid;HPLC,reverse-phasehighperformanceliquidchromatography;EE,entrapmentef?ciency;PS,physiologicalsaline;PBS,phosphatebuffersolution;PFA,paraformaldehyde;OCT-compound,optimalcuttingtemperature-compound;HEPES,4-(2-hydroxyethyl)-1-piperazineethansulfonicacid;CLSM,confocallaserscanningmicroscope;EDTA,ethylenediaminetetraaceticacid;HBSS,hank’sbalancedsaltsolution;PMSF,phenylmethanesulfonyl?uoride;BCA,bicinchoninicacid;TEER,transepithelialelectricresistance;MW,molecularweight;Papp,apparentpermeabilitycoef?cient.

*Correspondingauthor.Tel./fax:t862885501617.

E-mailaddress:huangyuan0@(Y.Huang).1

The?rsttwoauthorscontributedequallytothis

work.0142-9612/$eseefrontmatteró2011ElsevierLtd.Allrightsreserved.doi:10.1016/j.biomaterials.2011.10.075

canalsomimicthephysiologicalfateofinsulinandmayprovideabetterglucosehomeostasis[1].Nanocarrierisconsideredtobeapromisingvehiclefororaldeliveryofinsulin.Itcanofferdrugprotectionandfacilitatedrugabsorptionthroughtheintestinalmucosa.However,oneofthegreatestchallengesindevelopinganef?cientnanocarrierfororaldeliveryistoovercometheabsorptionbarrierofintestinalmucosa,consistingofintestinalepithelialcellsaswellasmucuslayer[2].

Nanocarriersmodi?edwithspeci?cligandstargetedtothereceptorsonthesurfaceofepithelialcellsareproposedtofacilitatetheoralabsorptionofpeptidesandproteins[2].However,onlylimitedkindsoftargetingagents,suchaslectins,vitaminB12andtransferrin,havebeenreportedtotargettoenterocytesorMcells[3e6].Meanwhile,thefact,beingoftenneglected,isthattherealeffectofepithelialcell-targetingmightbegreatlyaffectedbythemucuslayerpresentingonepithelium.Mucus,secretedbygobletcells,isacomplexandrobustbarrier,whichprotectsepithelialsurfacebyrapidlytrappingandremovingforeignparticles[7],thuscouldserveasaphysicalbarriertothetargetingrecognition.Lectin-conjugatedparticleswerereportedtoshowcross-reactionwithmucusandtheirtargetingef?ciencieswerediminishedbymucus[8].Furthermore,theeffectsofmucuslayeronthemostreported

1574Y.Jinetal./Biomaterials33(2012)1573e1582

targetingnanocarrierswerekeptunknownsincetheinvitroeval-uationsofmostepithelium-targetingsystemswereperformedontheCaco-2cellmodelwhichpossessesnomucus[9e11].Therefore,itisimportanttoexploitmoreeffectivetargetingligandswhichpossesshighspeci?cityforrecognition,soastoprovidesuf?cienttargetingeffectivenesswhileatthesametimeavoidbeingblockedbymucus.Meanwhile,evaluationonthetargetingef?ciencyandmechanismofthetargetingsysteminthepresenceofmucusisconsideredasanotherimportantissue.

Recently,aCSKSSDYQC(CSK)peptideidenti?edfromarandomphage-peptidelibrarythroughaninvivophagedisplaytechniquewasfoundtohaveaf?nitywiththegobletcellwhichisthesecondlargestpopulationofintestinalepithelialcells.ThepeptidecouldfacilitatethetransportofM13bacteriophageacrosstheintestinalepitheliumbyitsgobletcell-targetingproperty[12].ThisstudyimpliesCSKpeptidecouldbeusedasapotentialgobletcell-targetingligandfororaldeliveryofnanocarriers.However,nofurtherresearchwasreported.

Chitosanisacationicpolymer,whichisthesecondmostabundantpolymerinnatureaftercellulose.Itpossessesseveralpropertiessuchasbiocompatibility,biodegradability,permeationenhancingeffectviaopeningthetightjunctions(TJs)betweentheintestinalepithelialcells.Thesepropertiesmakeitasuitablecarriermaterialfororaldeliveryofpeptideandproteindrugs[13].Kisselhascomparedtheuptakeofchitosannanoparticles(NPs),poly-styreneNPsandPLA-PEGNPsbymucus-secretingMTX-E12cells[14].Althoughitwasalsofoundpartiallyboundtomucus,theinternalizedfractionofchitosanNPswasstillhigherthantheothertwotestedNPs,demonstratingthatchitosanNPscouldhavecomparativelyhighercapabilitytopenetratethroughthemucuslayer.Unfortunately,chitosanalwaysbecomesinsolublebyitsdeprotonationmechanismatneutralandbasicpHenvironment,resultinginthedecreaseofabsorptionenhancingability[15].N-trimethylchitosanchloride(TMC),apartiallyquaternizedderivativeofchitosan,ischaracterizedbygoodsolubilityandpermeationenhancingeffectinphysicalpH.Hence,modifyingTMCnanoparticleswithepithelium-targetingligandmightbeaneffec-tivemeansfortheoraldeliveryofpeptidesandproteins.

Therefore,theaimofthepresentstudywastoestablishgobletcell-targetingnanoparticlesusingCSKpeptidemodi?edTMCmaterial(TMC-CSK),evaluatetheireffectivenessasoralinsulin(INS)vehicles,andinvestigatethein?uenceofmucusonthetar-getingeffect.Twointestinalloopligatedmodels(invivoandexvivo)wereusedfortheabsorptionstudiesof?uorescentblankanddrugloadedNPs.Mucus-producingHT29-MTXcellsandtheco-incubatedCaco-2/HT29-MTXcellmodelwhichsimulatedtheepitheliumwereappliedfortheinvitroevaluationsoftargetingef?ciencyandthein?uenceofmucus.Finally,hypoglycemiceffectandrelativebioavailabilityofthetargetingNPsweretestedindiabeticrats.Inall,thegobletcell-targetingnanoparticlesandthein?uenceofmucusontheepithelium-targetingnanocarrierswerestudiedforthe?rsttime.

2.Materialsandmethods2.1.Materialsandanimals

Chitosan(deacetylationdegree>90%andmolecularweightof400kDa)wasprovidedbyAKBiotechCo.,Ltd.(Shandong,China).Porcineinsulin(30IU/mg)waspurchasedfromWanbangBio-ChemicalCo.,Ltd.(Jiangsu,China).CSKSSDYQCpeptidewaschemicallysynthesizedbyKaijieBio-pharmaceuticalsCo.,Ltd.(Sichuan,China).Fluoresceinisothiocyanate(FITC),rhodamine-conjugatedulexeuropaeusagglutininIlectin(Rho-UEA-I)and3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)wereallpurchasedfromSigmaeAldrich(St.Louis,MO,USA).N-Acetyl-L-CysteinewasobtainedfromAladdinChemistryCo.,Ltd.(Shanghai,China).7-Amino-4-methy-(3-coumaringlacetic)acid-conjugatedinsulin(AMCA-INS)wassynthesizedbyYoukebiotechCo.,Ltd.(Shanghai,China).

1-[3-(Dimethylamino)propyl]-3-ethylaarbodiimidehydrochloride(EDC$HCl)wasgainedfromMeapeoCo.,Ltd.(Shanghai,China).N-Hydroxysuccinimide(NHS),N-methylpyrrolidone,iodomethaneandacetonitrilewereallobtainedfromKelongchemicalCo.,Ltd(Chengdu,China).Otheragentswereallanalysisgrade.

Caco-2cellsweregainedfrominstituteofBiochemistryandCellBiology(Shanghai,China).HT29-MTXcelllinewasakindgiftfromDr.TheclaLesuf?eur(INSERM,Paris,France).TheywerebothculturedinDulbecco’sModi?edEagleMedium(DMEM)(Gibco,GrandIsland,NY,USA)containing10%fetalcalfserumand1%non-essentialamino(Hyclone,Logan,UT,USA).

MaleSpragueeDawleyratsweighing250?20gweresuppliedbyExperimentalAnimalCenterofSichuanUniversity(protocolnumberforanimalstudy:CSDGZ-10).Theratswerehousedataroomtemperatureof22?2??Candarelativehumidityof50?10%.

2.2.SynthesisofTMCandTMC-CSK

TMCandTMC-CSKweresynthesizedastheschematicroute(SupportinginformationFig.S1).Brie?y,TMCwasobtainedbymethylationofaminegroupsofchitosan(CS)withmethyliodide(CH3I)inastrongbasesolution(sodiumhydroxide,NaOH)usingN-methylpyrrolidone(NMP)assolvents.Thereactionproceededfor45minat60??C.Theproductwaspuri?edbydialysisandthenlyophilized(SNL216V,SavantInstrumentsInc.,NY,USA).Thedegreeofquaternization(DQ)wascalculatedfromtheintegrationof1Hnuclearmagneticresonance(1HNMR,UNITYINOVA-400,VarianInc.,CA,USA)[16].

TheobtainedTMCwassubsequentlyconjugatedwithCSKpeptideviaamidebondformedamongtheresidualprimaryaminogroupsonTMCandcarboxylgroupsonCSKpeptide.TMC(0.27mmolofprimaryaminogroups),EDC$HCl(0.45mmol)andNHS(0.45mmol)weredissolvedin12mlofwater(pH8.0)and?lledwithnitrogen.ThentheCSKpeptidewasaddedata?nalamountof0.045mmol.Thereactionwasconductedatambienttemperaturefor3daysindark.Subsequently,theproductwasdialyzed,lyophilizedandstoredat4??C.TheobtainedTMC-CSKwasidenti?edby1HNMRandFouriertransforminfrared(FT-IR)spectra.Thecontentoftheconjugatedpeptidewasdeterminedthroughtheaminoaciddetection(835-50,HitachiCo.,Tokyo,Japan).

Moreover,FITC-labeledTMCandTMC-CSK(FITC-TMCandFITC-TMC-CSK)werepreparedbasedonthereactionbetweentheisothiocyanategroupsofFITCandtheprimaryaminogroupsofTMCasreported[17].2.3.PreparationofNPs

2.3.1.PreparationofblankNPs

The?uorescentblankNPspreparedusingFITC-TMCandFITC-TMC-CSK(FITC-TMCNPsandFITC-TMC-CSKNPs)werepreparedusingionotropicgelationmethod[18].PluronicF68wasusedasastericstabilizer[19].Brie?y,1mg/mloftripoly-phosphate(TPP)solutioncontainingpluronicF68(0.4%,w/v)wasaddeddrop-wiselytoFITC-TMCorFITC-TMC-CSKsolution(1mg/ml)ataratioof1:5(v/v).Then,thesolutionwasstirredatroomtemperaturefor20min,yieldinganopal-escentsuspension.Thesuspensionwasultracentrifugedat10,000rpmfor30min(AllegraX-22R,BeckmanCoulterInc.,CA,USA).Thesupernatantwasdiscardedandthenanoparticlesformedwereresuspendedforfurtheruse.

2.3.2.PreparationofINS-loadedNPs

Thenon-?uorescentINS-loadedNPspreparedusingTMCandTMC-CSK(TMCINSNPsandTMC-CSKINSNPs)werepreparedasdescribedpreviously[20].Brie?y,INSwasdissolvedinhydrochloricacid(HCl)solution(0.01N,pH2.0)ataconcen-trationof1mg/ml,andthepHwasadjustedto8.0using1MNaOHsolution.Then,theINSsolutionwaspre-mixedwithTPPsolution(1mg/ml)containingpluronicF68(0.4%,w/v)ataratioof1:2.5(v/v).Thepre-mixedsolutionwasaddeddrop-wiselytoTMC-CSKaqueoussolution(1mg/ml)atanequalvolume.Themixturewasstirredatroomtemperaturefor20min,yieldinganopalescentsuspension.Theresultantsuspensionwasultracentrifugedat10,000rpmfor30minat4??C.Thesupernatantwasremovedforthedeterminationofentrapmentef?ciencyandthenanoparticlesformedwereresuspendedforfurtheruse.

Inaddition,fortheobservationandevaluationofINSinthefollowingtests,itwaslabeledwithFITC(FITC-INS)asdescribedinprevioussectionandtheFITC-INSloadedNPspreparedusingTMCandTMC-CSK(TMCFITC-INSNPsandTMC-CSKFITC-INSNPs)wereformedusingtheabove-mentionedmethod.2.4.CharacterizationofNPs

2.4.1.Sizeandzetapotential

TheblankNPsanddrugloadedNPswerecharacterizedfortheirsizeandzetapotentialwithaMalvernZetasizeNanoZS90(MalvernInstrumentsLtd.,Malvern,UK).Allmeasurementswereperformedintriplicate.

2.4.2.Entrapmentef?ciencyofdrugloadedNPs

TheamountoffreeINSinthesupernatantofnon-?uorescencelabeledNPswasmeasuredbyareverse-phasehighperformanceliquidchromatography(HPLC)method(Agilent1200series,CA,USA).SeparationwasachievedonaDiamosilC18

Y.Jinetal./Biomaterials33(2012)1573e1582

1575

column(150mm?4.6mm,5mm)withmobilephaseofacetonitrile-water(28:72,contained0.2MNa2SO4andthepHwasadjustedto2.3withphosphoricacid)andthedetectionwavelengthwassetat214nm[21].

Besides,theamountoffreeFITC-INSinthesupernatantof?uorescence-labeledNPswasmeasuredby?uorospectrophotometer(ShimadzuCorp.,Tokyo,Japan)andtheexcitationandemissionwavelengthsweresetat488and516nm,respectively.

Theentrapmentef?ciency(EE%)ofdrugloadedNPswascalculatedasfollowing[22]:EE%?

totalamountofinsulinàfreeinsulin

totalamountofinsulin

?100

2.4.3.StabilityofINSinNPs

TheactivityofINS-loadedTMCNPswasexaminedbyanHPLCmethodaccordingtotheU.S.Pharmacopeia[23]andthestudiesofHoyerGLandTiyaboonchaiW[24,25].Brie?y,200mg/mlofstandardinsulinwaspreparedin0.01NHClsolution.TMCINSNPscontaining200mgofINSweredissolvedin1.0mlof0.01NHClsolution.Thenthesamplewascentrifugedat10,000rpmfor20minbeforesubjectiontoHPLCdetermination.Thepotencyofinsulinwascalculatedasthefollowingequation:Ct?Cs?Rt=Rs

whereCtstandsforthepotencyoftestsampleandCsisthepotencyofstandardINS.RtispeakareaoftestsampleandRsispeakareaofstandardINS.

2.4.4.Mucinadsorption

ThetwostudiedNPs(TMCINSNPsandTMC-CSKINSNPs)werecentrifugedandresuspendedin5mlofmucinsolution(0.5mg/ml,pH5.5)andincubatedat37??Cfor5h.Thenthedispersionswerecentrifugedandthefreemucincontentinthesupernatantwasmeasuredbyacolorimetricmethodusingperiodicacid/schiffstaining[26].Testswereperformedintriplicateforeachsample.2.5.Absorptionstudiesintheligatedintestinalloops

2.5.1.Absorptionstudiesinvivo

TheinvivouptakeofblankanddrugloadedNPswerequalitativelyevaluatedusingtheligatedintestinalloopsmodelinvivo.AllexperimentswereapprovedbytheInstitutionalAnimalCareandUseCommitteeofSichuanUniversity.MaleSpragueeDawleyratsweighing250?20gwerefastedovernightbeforeexperi-ments,butallowedfreeaccesstowater.Theratwasanesthetizedwithpentobarbitalsodium(0.04mg/kg),andthen2-cmsectionsofileumfromsmallintestinalloopweremadeandwashedwithphysiologicalsaline(PS),thenligatedatbothends.NPssolution(resuspendedinphosphatebuffersolution(PBS),0.2ml)withequal?uo-rescentintensitywasinjectedintotheloop.Atdifferenttimepoints(0.5,1,2and3h),ratwassacri?cedbycervicaldislocationandthesectionofeachloopwasremoved,extensivelywashedusingPBS.ThewashedPBSofblankNPsgroupatthetimeintervalof3hwascollectedforquantitativelymeasurementby?uorospec-trophotometer.Subsequently,theremovedloopsofbothblankanddrugloadedNPsafterwashingateverytimepointwere?xedby4%paraformaldehyde(PFA)for2handimmersedin30%sucroseat4??Covernight.Sampleswerefrozenquicklyinliquidnitrogen-cooledOCT-compound(OptimalCuttingTemperature-compound,‘Tissue-Tek’,MilesLaboratoriesInc.,Indiana,USA).Thesmallintestinaltissue-sectionswerepreparedbyCryotome(HM505E,E-Microm,Zeiss,Walldorf,Germany)in5mmthicknessandputontothegelatin-coatedslideglasses.Thetissue-sectionswerewashedwithdistilledwaterforseveraltimes.MucusdropletsofgobletcellswerelabeledbyRho-UEA-Idilutedin4-(2-Hydroxyethyl)-1-piperazineethansulfonicacid(HEPES)buffer(1:100,v/v)at4??Covernightandwashedwithdistilledwater.Thetissue-sectionswerevisualizedusingconfocallaserscanningmicroscope(CLSM,Live5DUO,CarlZeiss,Jena,Germany)[12,27].2.5.2.AccumulativepermeationstudiesofINS-loadedNPsexvivo

Tofurtherinvestigatethe?nalabsorptionofdrugsfromCSKpeptidemodi?edNPs,FITC-INStransportedacrosstheepithelialmucosaofratileumexvivowasquantitativelymonitoredasdescribedbyYin[28].Brie?y,ratsweresacri?cedand2-cmsectionsofileumloopwerecutout.TMCFITC-INSNPsorTMC-CSKFITC-INSNPs(0.2ml)usedinsection2.5.1wassyringedintotheloop,whichwasincubatedinKreb’-Ringerbufferat37??Cwithsmoothshaking.Ateachtimeintervals(0.5,1,2and3h),theincubationbufferwastakenforthedeterminationofFITC-INSby?uo-rospectrophotometer.Thesamevolumeoffreshbufferwasadded.Alloftheexperimentswereperformedintriplicate.2.6.Cellstudies

2.6.1.Cellculture

Thehumancolonadenocarcinomacells,Caco-2andHT29-MTXcells,werecultivatedseparatelyin75cm2culture?asksusingDMEMsupplementedwith10%fetalbovineserum,1%non-essentialaminoacids,1%L-glutamine,penicillin(100IU/

ml)andstreptomycin(100mg/ml).Bothculturesweremaintainedat37??C,95%relativehumidityand5%CO2.Priortothetest,cellswereharvestedusing0.25%trypsincontaining0.05mMethylenediaminetetraaceticacid(EDTA)anddilutedatadensityof5?104cells/mlforMTTanduptakeassays.Then,thecellsuspensionswererespectivelyseededonto96-wellplates(Corning,NY,USA),andincubatedfor2daysbeforecytotoxicitytestsand7daysbeforeuptakestudies.Fortransportassays,theCaco-2òandHT29-MTXcellsweremixedataratioof1:1andseededontotheTranswellchambersconsistingofpolycarbonatemembrane(0.4mminporesize,0.33cm2ofòcellgrowtharea,Costar)atadensityof3?104cells/well.Then,theTranswellchamberswereplacedinto24-wellplates,intowhichtheDMEMwereadded.Thecellswereallowedtogrowanddifferentiatefor21daysbeforeuse[29].2.6.2.Cytotoxicity

ThecytotoxicityofthesynthesizedpolymerwasevaluatedusingMTTassaywithHT29-MTXcellsandCaco-2cells,respectively.Priortothetest,themediumin96-wellplateswasremoved.Subsequently,thecellswerewashedwithPBSandincu-batedwith100mlofTMCorTMC-CSKsolutionsinHank’sbalancedsaltsolution(HBSS)for3h(concentrationsofTMCandTMC-CSKwere0.125,0.25,0.5,1and2mg/ml,thepHwassetat7.0)andthensubmittedtoMTTassay.HBSSwasusedasreferencefor100%cellviability[28].Testswereperformedintriplicateforeachsample.

2.6.3.UptakestudywithHT29-MTXcells

InordertoestimatethetargetingeffectofCSKpeptidemodi?cationontheinternalizationofTMCFITC-INSNPsinvitroandtherelevantmechanism,uptakestudieswereconductedonthegobletcell-likeHT29-MTXcellsasdescribedprevi-ously[14].

ToremovethemucussecretedontheapicalsideofHT29-MTXcells,thecellswerepre-incubatedwith10mMacetylcysteinedissolvedinHBSSandkeptunderagitationfor60minat37??Cbeforestudy.Subsequently,thecellswererinsedwithHBSSandallowedtoequilibrateat37??Cfor30min,andthenincubatedwith100mlTMCFITC-INSNPsorTMC-CSKFITC-INSNPssuspension(atadoseof220mg/mlofFITC-INS)intransportbuffer(HBSS)at37??C.Toinvestigatethein?uenceofincu-bationtime,thesupernatantswereremovedatpredeterminedtime(0.5,1,2and3h),thenthecellswerewashedtwicewithice-coldPBS.Celllysiswasachievedwithlysisbuffercontaining1%TiritonX-100,50mMHEPES(pH7.5),150mMNaCl,2mMNa3VO4,100mMNaF,100units/mlaprotinin,20mMleupeptinand0.2mg/mlphe-nylmethanesulfonyl?uoride(PMSF)[30].Thenthecellassociated?uorescencesandproteinsweredeterminedbyVarioskanFlash(ThermoFisherScienti?c,MA,USA)andbicinchoninicacid(BCA)assaykit(KeyGenBiotechCo.,Ltd.,Nanjing,China),respectively.TheamountsofuptakewereexpressedasthequantityofFITC-INSassociatedwith1mgofcellularprotein.

WiththepurposeofidentifyingthepossibleinternalizationmechanismofNPsbyHT29-MTXcells,thecellswereincubatedindifferentconditionsortreatedwithspeci?cagents.(I)Tostudytheeffectoftemperatureoncellularuptake,cellswereincubatedwithtestsamplesat4??Cfor3h.(II)Totesttheeffectofsodiumazide,anactivetransportinhibitor,100mMsodiumazidewasdissolvedintheNPssuspensionandincubatedwithcellsfor3hat37??C.(III)Tostudytheeffectofprotaminetreatment,anadsorptive-mediatedendocytosisinhibitor,1mMprotaminesulfatewasdissolvedintheNPssuspensionandincubatedwithcellsfor3hat37??C.(IV)Toinvestigatetheeffectofdesulfurization,cellswerepre-incubatedwith35mMsodiumchlorateincellculturemediumfor48h,rinsedtwicewithtransportbufferandincubatedwiththeNPssuspensionfor3hat37??C[31].(V)TostudythecompetitiveinhibitioneffectoffreeCSKpeptide,cellswerepre-incubatedwithCSKpeptide(0.06mM)inthecellculturemediumfor1h,rinsedtwicewithtransportbufferandincubatedwiththeNPssuspensionfor3hat37??C.(VI,VII)Toexaminetheeffectsofchlorpromazineand?lipin,whichweretheclathrinandcaveolaeendocytosisinhibitors,cellswereincubatedwith10mg/mlchlorpromazineor1mg/ml?lipindissolvedinNPssuspensionandtreatedfor3hat37??C[32].Inaddition,thecontrolsampleswereconductedat37??Cfor3hwithoutanytreatment,andtheresultsoftheinhibitiontestswerepresentedasthepercentageofthatinternalizedincontrol.

2.6.4.Transportthroughtheco-culturedCaco-2/HT29-MTXcellmonolayer

TofurtherinvestigatethetransportofCSKpeptidemodi?edNPsacrosstheepithelialcellsandthein?uenceofmucusonthetargetingrecognition,theco-culturedcellòmonolayerwasincubatedfor21daysafterbeingseededontheTranswellinserts,mimickingtheintestinalepitheliumwithbothepithelialcellsandmucuslayer.Theintegrityofthemonolayerwascheckedusingpropranololwhichismainlyabsorbedbythetranscellularrouteandatenololwhichismainlytransportedthroughtheparacellularpathway[33].

Thein?uenceofincubationtimeontransportwasinvestigated.Priortothestudies,themediumsintheapicalandbasolateralchamberswerereplacedwithpre-warmedHBSS.ThecellswererinsedwithHBSSandallowedtoequilibrateat37??Cfor30min,andthenincubatedwith100mlofTMCFITC-INSNPsorTMC-CSKFITC-INSNPssuspension(220mg/mlofFITC-INS)intransportbufferat37??C.Atthedeterminedtimepoints(0.5,1,2and3h),0.2mlofsamplesweretakenfromthebasolateralchamberandequalvolumesofHBSSweresupplemented.TheamountoftransportedFITC-INSwasdeterminedusingVarioskanFlash,andtheaccumulative

1576Y.Jinetal./Biomaterials33(2012)1573e1582

transportofFITC-INSwascalculated.Simultaneously,thetransepithelialelectricresistance(TEER)valuesweremeasuredwithaMillicell-ERS(Millipore,MA,USA).

Toinvestigatethein?uenceofmucuslayer,thetransportofthetwostudiedNPswerealsoconductedontheco-culturedmodelintheabsenceofmucus(removedusingthemethoddescribedabove).Andforfurtherstudies,transportprocedurewascarriedoutat4??CorwiththeexistenceoffreeCSKpeptideastheinhibitionagent(0.06mM)whichadministered30minbeforetest.

2.7.Pharmacologicalandpharmacokineticalstudies

2.7.1.Diabetesinducing

Diabeteswasinducedinratsbyaninjectionofstreptozotocin(65mg/kg)dis-solvedina10mMcitratebuffer(pH4.5)aspreviouslydescribed[21].Thebloodglucoselevelwasdeterminedusingaglucosemeter(JPS-6,YichengBiotech.Co.Ltd.,Beijing,China).Ratswereconsideredtobediabeticwhentheirfastingglycemiawashigherthan16.0mMoneweekafterstreptozotocintreatment.

2.7.2.Hypoglycemiceffectandrelativebioavailabilitystudiesafteroraladministration

PriortooraldeliveryofdrugloadedNPs,0.5mlofNaHCO3solutionwasintra-gastricallyadministrated.Thendiabeticratswereadministratedwiththefollowingformulations:oraladministrationwithTMC-CSKINSNPs(50.0IU/kgofINS),TMCINSNPs(50.0IU/kgofINS),INSsolution(50.0IU/kg),physiologicalsalineandsubcutaneouslyinjectionwithINSsolution(5.0IU/kg).Bloodsampleswerecollectedfromthetailveinsofratspriortodrugadministrationandatdifferenttimeintervals(1,2,3,4,6,8and10h)afterdosing.Thebloodglucoselevelswerethendetermined.Theareaundertheserumglucoseconcentration-timecurveover10hwascalculatedaccordingtothelineartrapezoidalrule.Thetotaldecreases(D%)inserumwerecalculatedusingamodi?cationmethodasfollows[34]:D%?

AUCephysiologicalsalineTàAUCetestT

AUCephysiologicalsalineT

?100

whereAUCisthetotalareaunderthecurveofplasmaglucoseconcentrationvs.time.

FortheanalysisofplasmaINSlevels,bloodsampleswerecentrifuged(3000rpm,5min)andsubsequentlyquanti?edusinganappropriateinsulinELISAkit(R&DSystem,Inc.,MN,USA).Therelativebioavailability(F%)ofthetestedNPsafteroraladministrationwascalculatedusingthefollowingformula[35].F%?

AUCeoralT?DoseescT

AUCescT?DoseeoralT

?100

whereAUCisthetotalareaunderthecurveofplasmainsulinconcentrationvs.time.

3.Resultsanddiscussion

3.1.Characterizationofsynthesizedpolymers

ChitosanwasconvertedintoTMCtoimproveitswatersolubilityatneutralandbasicpHenvironment.ItwasreportedtheincreasedsolubilityofTMCcanevenbeobservedataDQaslowas10%[36].Consequently,inordertoretainasmuchastheamountoffreeaminogroupstofacilitatethereactionbetweenTMCandCSKpeptide,TMCwithaDQof12.7%waschosen.

TMC-CSKwassynthesizedbycouplingtheaminogroupsofTMCwiththecarboxylgroupsintheC-terminalofCSKpeptide.Andtheself-couplingofCSKpeptidewaspreventedviacontrollingthefeedingmolarratioofTMCandCSKpeptideat6:1.1HNMRandFT-IRspectrawereshowninsupportinginformationFig.S2andS3.Resultsof1HNMRdeterminationhadcon?rmedtheconjugationofTMCwithCSKpeptidebythecharacteristicpeaksat6.752and7.023ppmfortwoprotonsofbenzeneringoftyrosineinCSKpeptidesequence,respectively.TheotherprotonpeaksofCSKpeptiderangedfrom0.9to4.7ppmwereundistinguishedduetotheoverlapofglucoseunitpeaksofTMC.Besides,comparedtoFT-IRspectrumofTMC,thecharacteristicpeakofbenzeneringat798.15cm-1forTMC-CSKwasobserved,againindicatingtheconjugationofCSKpeptidewithTMC.Then,accordingtotheresultofaminoaciddetection,thecontentofCSKpeptideinTMC-CSKpolymerwascalculatedtobe0.09mmol/g.Meanwhile,theFITC-labeledpolymerswerealsocharacterized(datanotshown).

3.2.CharacterizationofNPs

Sinceproteinsareliabletobedegradedandeasilyinactivated,themildionotropicgelationmethodissuitableforthepreparationofproteinloadedNPs[17].

ThecharacteristicsofblankanddrugloadedNPs(includingFITC-labeledNPsandnonFITC-labeledNPs)weresummarizedinTable1.Whencomestothe?uorescence-labeledbatches,thedrugloadedNPspossessedbothlargerparticlesizeandlowerzetapotentialcomparedwithblankbatches,duetotheentrapmentofnegativelychargedFITC-INS.Otherwise,itwasclearthatalltheblankNPsanddrugloadedNPspreparedbyTMC-CSKexhibitedlargersizeandlowerzetapotentialincomparisonwithNPspreparedbyTMC.ThismightbeduetotheintroductionofCSK,apeptidewithmolecularweight(MW)of1018Daandnegativecharge.Moreover,thenon-?uorescence-labeledTMCINSNPsandTMC-CSKINSNPsshowedsizeof318.1?11.5and342.0?5.8nm,respectively,aswellasasimilarEE%.Besides,thehigherEE%ofFITC-INSincorporatedNPs(around87%)comparedwithINS-loadedNPs(around55%)wasprobablyascribedtotheexposednegativechargesendowedbythefreecarboxyandphenolichydroxylgroupsofFITCwhichcouldalsointeractwithTMC.

ThepotencyofstandardINSwas30.0IU/mg.AccordingtotheresultsofHPLCspectra,thepotencyofINSextractedfromtheNPswascalculatedtobe29.30?0.18IU/mg,andtheINSrecoverywas97.68?0.59%.Therefore,itcouldbeconcludedthattheactivityofINSinNPswasmainlyremained,againsuggestingtheionicgela-tionmethodwassuitablefortheincorporationofpeptideandproteindrugs.

Themucinadsorptionwasmeasuredtoevaluatethein?uenceofCSKpeptidemodi?cationonthemucoadhesivepropertiesofTMCINSNPs.TheresultsshowedtheamountofmucinabsorbedbyTMCINSNPswas0.48?0.01mg/ml,while0.35?0.04mg/mlforTMC-CSKINSNPs(P<0.05).SincetheinteractionbetweenmucinandNPswasmainlydeterminedbyparticlesizeandchargeinteraction,theCSKpeptidemodi?edNPswithlargersizeandlowerzetapotentialmayleadtolessabsorptionwithmucin.3.3.Absorptionstudiesinligatedileumloop

3.3.1.TheabsorptionofblankanddrugloadedNPsinvivo

TheabsorptionofNPswasqualitativelyobservedinthevilliofileumloopbyCLSM.Figs.1and2showedtheabsorptionofmaterialsfromblankNPs(FITC-TMC-CSKNPsandFITC-TMCNPs)andFITC-INSfromdrugloadedNPs(TMC-CSKFITC-INSNPsandTMCFITC-INSNPs)atdifferenttimepoints(0.5,1,2and3h),respectively.

Itwasobservedtheabsorptionofallsamplesexhibitedtime-dependentcharacteristicsandthemaximalsignalswerepre-sentedat3h.Modi?cationofbothblankanddrugloadedNPswith

Table1

Characterizationofnanoparticles.NPs

Size(nm)PDI

ZetapotentialEE%(mV)FITC-TMCNPs183.3?6.50.186?0.02210.7?0.3eFITC-TMC-CSK223.1?11.00.179?0.0754.1?0.1e

NPs

TMCFITC-INS298.4?10.40.204?0.0077.0?0.287.5?1.4%NPsTMC-CSK

324.4?10.70.225?0.0213.5?0.388.7?3.1%FITC-INSNPsTMCINSNPs318.1?11.50.215?0.0144.3?0.456.9?2.6%TMC-CSKINS342.0?5.8

0.217?0.022

3.0?0.1

55.4?2.7%

NPs

Y.Jinetal./Biomaterials33(2012)1573e15821577

Fig.1.LocalizationofblankNPspreparedusingFITC-TMC-CSK(a)andFITC-TMC(b)invilliofileumat0.5,1,2and3h.Red?uorescencereferstothemucusdropletsofgobletcells,andgreen?uorescencereferstoFITC.(Forinterpretationofthereferencestocolourinthis?gurelegend,thereaderisreferredtothewebversionofthis

article.)

CSKpeptidecouldpromotetheirabsorptionbyvilliateachtime

point,comparedwithunmodi?edNPs,suggestingthelatent

absorptionenhancingabilityofCSKpeptidemodi?cation.Inter-

estingly,althoughenterocytesarequiteoutnumberedgobletcells

inepithelium,mostoftheintensiveFITC-TMC-CSKsignals(green)

wereassociatedwiththelocationofgobletcells(red)andtheoverlaydisplayedyellowcolor,whereasFITC-TMCsignalsweredispersedlydistributed(magni?edpicturesinFig.1).Thisman-ifestedthepossiblegobletcell-targetingeffectofCSKpeptidemodi?edNPswhichfacilitatedtheabsorption.Ontheotherhand,Fig.2exhibitedthattheFITC-INSpermeateddeeplyintovilli,indicatingtheirwellabsorptioninvivo

.

Fig.2.LocalizationofFITC-INSloadedNPspreparedusingTMC-CSK(a)andTMC(b)invilliofileumat0.5,1,2and3h.Red?uorescencereferstothemucusdropletsofgobletcells,andgreen?uorescencereferstoFITC.(Forinterpretationofthereferencestocolourinthis?gurelegend,thereaderisreferredtothewebversionofthisarticle.)

1578Y.Jinetal./Biomaterials33(2012)1573e1582

TogetavisualizationofthedistributionofTMC-CSK(material)andINS(drug)invilli,double?uorescentTMC-CSKINSNPspreparedwithAMCA-labeledINS(blue)andFITC-labeledTMC-CSK(green)wereusedininvivouptakestudy.AsshowninFig.3,thebluesignalsofINSpermeateddeeplyintovilli,suggestedthedrugswerewellabsorbed,whilethegreensignalsofTMC-CSKwereonlypresentedinthedistalendandinsideedgeofvilli.ItcouldbeinterpretedthataftertheTJsoftheepitheliumwereopened,INSwithMWofonly5800Dawasmorepriortopassthroughtheintestinalepithelialcellsbyparacellularandtranscellularpath-ways,hencepermeateddeeplyintovilli,whereasTMC-CSKmate-rialswithpositivechargeswere?xedabundantlyonthenegativelychargedinteriorsitesofTJsofepithelialcells[2].

Afterinjectedintoloop,nanoparticlesmayundergo:(1)remainintheintestinallumen;(2)adheretomucin?berandtrappedinmucus;(3)penetratethroughthemucuslayerforpossibleentrytotheunderlyingepithelial[37,38].Tofurtherverifytheaboveexperiments,theamountsofNPsremainedinlumenandtrappedinmucusweremeasuredforinvestigation.TheresultsshowedthattheamountofFITC-TMCNPsremainedinlumenandtrappedinmucuswas2.2-folderhigherthanFITC-TMC-CSKNPs.Inotherword,theamountofFITC-TMC-CSKNPspenetratedthroughthemucuslayerwashigherthantheunmodi?edones,againimplyingthebetterabsorptionpropertyoftargetingNPsacrossthemucuslayerendowedbyCSKpeptidemodi?cation.

3.3.2.Theaccumulativetransportofdrugsexvivo

ThetransportofNPsacrossintestinalepitheliumintothebloodistheultimatedestination.Hence,theaccumulatedINSpermeationexvivoinligatedileumloopwasconductedtofurtherinvestigatethepermeationabilityofTMC-CSKFITC-INSNPsacrosstheepithelium.ComparedtoTMCFITC-INSNPs,TMC-CSKFITC-INSNPsenhancedtheaccumulativeamountofFITC-INSpermeatedthroughratileum(SupportinginformationFig.S4).ThepermeatedFITC-INSfromCSKpeptidemodi?edNPsat3hwas74.87?10.45ng,whichwas1.7-foldhigherthanthatofunmodi?edNPs(44.91?0.70ng,P<0.05).Theresultsrevealedtheabsorptionenhancingabilityof

Fig.3.DistributionofAMCA-INSandFITC-TMC-CSKofdrugloadedNPsinvilliofileum.Red?uorescencereferstothemucusdropletsofgobletcells,green?uorescencereferstoFITC-TMC-CSK,andblue?uorescencereferstoAMCA-INS.(Forinterpretationofthereferencestocolourinthis?gurelegend,thereaderisreferredtothewebversionofthis

article.)

CSKpeptidemodi?edNPsacrosstheepitheliumassignedbytheirtargetingeffect.

3.4.Cytotoxicityevaluationsofpolymers

Toevaluatethesafetyofthesesynthesizedpolymers,cellularviabilityofHT29-MTXandCaco-2cellsweretestedseparatelyusingMTTassay.BothTMCandTMC-CSKexhibiteddose-dependentcytotoxicityfrom0.125to2mg/mlforHT29-MTXandCaco-2cellsinMTTassay(SupportinginformationFig.S5).Besides,theresultssuggestedthatthemodi?cationofTMCwithCSKpeptideledtoadecreaseincytotoxicity,whichmightbeascribedtotheshieldingofpositivechargesonTMCbythenegativechargesofCSKpeptide.

3.5.UptakestudyanditsmechanisminHT29-MTXcells

TheresultsofinvivoandexvivoligatedintestinalmodeltestsshowedthepotentialtargetofCSKpeptidetogobletcellswhereasitsspeci?ctargetingroutewasstillunknownaccordingtoKang[12].Therefore,thecellularuptakeandthepossiblemechanismsforCSKpeptidemodi?edandunmodi?edTMCFITC-INSNPswereinvestigatedwithHT29-MTXcellswhichcompriseapproximately80%maturegobletcellswithTJsinmonolayer.

TheamountsofFITC-INSinternalizedbyHT29-MTXcellsatdifferentincubationtimeswereshowninFig.4.TheuptakeofFITC-INSexhibitedtime-dependentpropertiesforallthetestsamples.TMC-CSKFITC-INSNPssigni?cantlyimprovedtheinternalizationofdrugsto15.95?3.15and19.18?0.99mg/mgproteinat2and3h,respectively,whichweresigni?cantlyhigherthanthoseofTMCFITC-INSNPs(9.51?1.21and12.11?3.02mg/mgproteinat2hand3h,P<0.05).TheresultsindicatedtheCSKpeptidemodi?cationcouldgreatlyenhanceHT29-MTXcellsinternalizationofTMCNPs.Themechanismsweresubsequentlyinvestigatedunderdifferentconditions.AsshowninFig.5,theuptakeofFITC-INSinHT29-MTXcellswassigni?cantlyreducedat4??CorwiththeadditionofsodiumazideforTMCFITC-INSNPs(P<0.001)andTMC-CSKFITC-INSNPs(P<0.05),bothofwhichcouldblocktheactivetransportprocesses[14].Besides,theadsorptive-mediatedendocytosisinhibitor,protamine,ledtothestrongestdecreasedinternalizationto8.84?3.80%and17.41?6.93%forunmodi?edandmodi?edNPs,respectively(P<0.001),whichmightbeduetothecompetitionofadsorptiveendocytosis[39].Theseresultsdemonstratedthattheactivetransportprocesses,involvingadsorptiveendocytosis,mightplayanimportantroleontheuptakeofbothNPs.Meanwhile,theinternalizationofthetwo

NPs

Fig.4.UptakeofTMCFITC-INSNPs,TMC-CSKFITC-INSNPsandFITC-INSsolutionbyHT29-MTXcellsatdifferentincubationtime(Mean?SD,n?3e5).*:P<0.05,#:P<0.001.

Y.Jinetal./Biomaterials33(2012)1573e15821579

Fig.5.UptakeofTMCFITC-INSNPsandTMC-CSKFITC-INSNPsbyHT29-MTXcellsatdifferentconditions(Mean?SD,n?3e5).Signi?cantdifferencefromcontrol.*:P<0.05,#:P<

0.001.

decreasedmarkedlywiththepretreatmentofsodiumchlorate,aninhibitorofglycosaminoglycansulfation,whichcouldaffecttheelectrostaticinteractionsbetweenpositivechargedTMCandthenegativechargedglycocalyxontheapicalmembrane,thusin?u-encetheinternalizationofcells[14].

However,theadditionsofchlorpromazineand?lipinwerefoundtoinhibittheuptakeofTMC-CSKFITC-INSNPsto69.5?4.94%and65.16?6.01%inHT29-MTXcells(P<0.05),whiletheunmodi?edNPswerealmostunaffected.Chlorpromazineataconcentrationof6e10mg/mlcoulddisrupttheassemblyanddisassemblyofclathrinand?lipinataconcentrationof1mg/mlisknowntodisruptcaveolaestructurebybindingtocholesterolanddisorganizingthecaveolin[40].Hence,theseresultsimpliedthattheincreasedinternalizationofdrugsfromCSKpeptidemodi?edNPs,comparedwithunmodi?edones,mightbemediatedbycla-thrinandcaveolaeendocytosis.

Furthermore,theadditionoffreeCSKpeptidealsoinhibitedtheinternalizationofTMC-CSKFITC-INSNPs,whereasTMCFITC-INSNPswaskeptunin?uenced,furtherprovingtheexistenceofreceptorsonHT29-MTXcells.

Fromalltheresults,itcouldbeconcludedthattheCSKpeptidereceptormayexistonHT29-MTXcells.Themechanisminvestiga-tionsrevealedthatbesideoftheactivetransportprocessesandelectrostaticinteractionmediateduptakeofbothNPs,theenhancedinternalizationofCSKpeptidemodi?edNPsthanunmodi?edNPsmaybeinvolvedinbothclathrinandcaveolaemediatedendocytosis.

3.6.Transportstudyanditsmechanism

TheabsorptionofINSintocirculatorysystemmeansthatINStransportsacrossintestinalepithelialcellsintothebloodstream.Inotherwords,thepermeatedINSfromtheapicalside,eitherthroughthetranscellularwayortheparacellularway,shouldbereleasedanddeliveredtothebasolateralsideofmonolayers.

Therefore,inourstudy,theco-culturedcellmodelconsistingofbothabsorptiveenterocyte-likeCaco-2cellsandthemucus-producingHT29-MTXcellswereappliedforthe?rsttimetoeval-uatethetransportofTMCnanoparticles.Thisco-culturedmodelcouldbettersimulatetheintestinalepitheliumbyvirtueofnotonlythepresenceofmucuslayer,butalsoaTEERvalueclosetointestinalepithelium[29].Itseemstobeasuitableinvitromodeltoevaluatethepermeabilityoforaldeliveredvehiclesaswellasthein?uenceofmucus.Theapparentpermeabilitycoef?cient(Papp)ofatenololandpropranololfortheco-culturedcellmodelwere2.46?10à6and1.10?10à5cmsà1,respectively,whichimpliedtheintegrityofthecellmonolayer[29,41].Meanwhile,onlythecellmonolayerswithTEERvalueswithintherangeof300e450Ucm2wereused.

ThecumulativetransportedFITC-INSindifferentconditionswaspresentedinFig.6.Thetransportexhibitedtime-dependentandtemperature-dependentproperties.TheamountsofFITC-INSpermeatedthroughCaco-2/HT29-MTXcellmonolayerweregreatlyincreasedwiththemodi?cationofCSKpeptidewithorwithoutthepresenceofmucusateverytimepoint(P<0.05).Besides,thepermeationofFITC-INSfromCSKpeptidemodi?ed

NPs

Fig.6.TransportstudiesofTMCFITC-INSNPsandTMC-CSKFITC-INSNPsacrosstheco-incubatedCaco-2/HT29-MTXmonolayeratdifferentconditions(Mean?SD,n?3e5).*:P<0.05,#:P<0.001.

1580Y.Jinetal./Biomaterials33(2012)1573e1582

Fig.7.BloodglucoselevelsindiabeticratsfollowingoraladministrationofTMCINSNPsandTMC-CSKINSNPsataninsulindoseof50IU/kg(Mean?SD,n?4).Signi?cantdifferencefromINSsolution(Sol)(*)andfromTMCINSNPs(#):P<0.05.

inthepresenceofmucuswereinhibitedwiththeadditionoffreeCSKpeptideateachtimeinterval(P<0.05),whileTMCFITC-INSNPsremainedunaffected.

Inaddition,thetwostudiedNPsexhibitedasimilareffectonthereductionofTEERvalue(about50%reduction)oftheCaco-2/HT29-MTXcellmonolayerinthepresenceofmucus.However,itisknownthattheabilityofTMCNPstoreduceTEERvaluedependsonparticlezetapotential[42]anditwasworthnoticingthatCSKpeptidemodi?edNPsexhibitedaninferiorzetapotentialthanunmodi?edNPs(asshowninTable1).ThepossibleexplanationforthesimilareffectonthereductionofTEERvaluemightowntothehigheramountofCSKpeptidemodi?edNPs?ltratedthroughthemucusandaccumulatedontheapicalsideofcells,asmentionedinsection3.3.1,whichcouldcounteracttheinferiorzetapotentialofsinglenanoparticleandsubsequentlyfacilitatethepermeationofFITC-INS.Thisresultwasalsocon?rmedbythefactthatthetransportofCSKpeptidemodi?edNPswasremarkablysuppressedbytheadditionoffreeCSKpeptidewhichmightinducecompetitiveinhibition.

ThemechanismofabsorptionenhancingabilityofTMCNPswasreportedtobeacombinationofmucoadhesionandtransientopeningofTJsonthemucosalcellmembrane[43].TheionicattractionbetweenTMCandmucuscouldincreasethedrugconcentrationintheextracellularenvironmentwhichisfavorabletothedrugpermeation.Nevertheless,themucusmayalsorestricttheTMCNPs’abilityofopeningTJsbypreventingthemfromreachingcellsurfaces[44].SincetherewereindeedsomedivergentopinionsabouttheeffectofmucusonTMCNPsinpreviouslyliteratures[14,45,46],andthein?uenceofmucusonthetargetingeffectandrecognitionwasrarelyinvestigatedforthereportedepithelium-targetingligands,itwouldbehighlysigni?canttoclarifythein?uenceofmucusonthebehaviorofCSKpeptidemodi?edTMCNPs.

Inourstudies,itwasveryinterestingthatthetransportedamountofFITC-INSwasmuchhigherwiththepresenceofmucusforbothmodi?edandunmodi?edNPsateverytimeinterval(Fig.6),whichwasconsistentwiththereportofKissel[14].Theresultwasquitedifferentfromtheobservationsinpreviousstudies,inwhichthemucuslayeractedasadiffusionbarrierforNPs[46].However,itshouldbenotedthatinthesereports,Caco-2cellsandHT29cellswereseparatelyusedasmucus-freeandmucus-secretingcellmodels.Infact,HT29cellsnotonlyprovidedamucuslayerbutalsocouldresultinadistinctpermeationbehaviorbecauseitisadifferentcelltypefromCaco-2cells.

Therefore,inourtests,thecoherentcellmodelswereusedforthe?rsttimetoinvestigatethein?uenceofthemucusinthetrans-portstudiesbypreservingorremovingthemucusfromthecellmodel.

AnotherissueshouldbestressedthatthepermeationenhancingratioofCSKpeptidemodi?cation(theamountofpermeatedFITC-INSinCSKpeptidemodi?edNPsgroupversusthatoftheunmod-i?edNPsgroup)increasedfrom1.15to1.75during0.5e3hintheabsenceofmucus,whiledecreasedfrom2.98to1.30inthepres-enceofmucus.Itcouldbeinterpretedthat,intheabsenceofmucus,theincreasedpermeationenhancingratiomightbemediatedbythepredominantactiveroleofCSKpeptidemodi?cationduringthewholetransportprocess.Bycontrast,whenitcametothesituationwiththepresenceofmucus,thehighlypermeationenhancingratioat0.5h(2.98)mightbeduetothetargetingmodi?cation.However,withtheincreaseofincubationtime,theover-trappingeffectofmucuswasgraduallydominated,whichledtothedecreasedpermeationenhancingratio.Eventhough,signi?cantlyincreasedtransportofTMC-CSKFITC-INSNPswasstillobservedcomparedwithTMCFITC-INSNPsinthepresenceofmucusateverytimepoint(P<0.05),implyingthefeasibilityandeffectivenessofCSKpeptidemodi?cationonTMCNPs.

Ingeneral,CSKpeptidemodi?cationofnanoparticlescanenhancethedrugpermeationbytheirtargetingproperty,althoughitcouldbepartiallyin?uencedbymucuslayer.

3.7.HypoglycemiceffectandrelativebioavailabilityofdrugloadedNPs

ThepharmacologicaleffectsofTMC-CSKINSNPsandTMCINSNPswereevaluatedondiabeticratsafteroraladministration.AsshowninFig.7,bothTMC-CSKINSNPsandTMCINSNPsexhibitedrelativelystronghypoglycemiceffects(P<0.05)ascomparedwithinsulinsolutionat2hand3hpost-administration.Moreover,withthemodi?cationofCSKpeptide,TMC-CSKINSNPsshowedabetterhypoglycemiceffectwiththemaximalbloodglucosedepressionof28%at3h,comparedtoTMCINSNPs(20%,p<0.05).Furthermore,tointuitivelyelucidatethehypoglycemiceffectofCSKpeptidemodi?edNPs,theD%ofeachformulationwascalculated.ItwasobtainedthattheD%ofCSKpeptidemodi?edNPswere1.52-foldand33.90-foldhigherthannon-modi?edNPsandINSsolutions,suggestingimprovedpharmacologicaleffectbyCSKpeptidemodi?cation.Duringthisexperiment,thefastingbloodglucosedidnotreturntotheinitiallevelafter10h,sameasother

previous

Y.Jinetal./Biomaterials33(2012)1573e15821581

Fig.8.PlasmainsulinlevelindiabeticratsfollowingoraladministrationofTMCINSNPs,TMC-CSKINSNPs,INSsolution(Sol)(50IU/kg)andsubcutaneousinjectionofINSSol.(5.0IU/kg)aspositivecontrol(Mean?SD,n?4).Signi?cantdifferencefromINSSol:(*),P<0.001;(#),P<0.01.Signi?cantdifferencefromTMCINSNPs:(t),P<0.001;(q),P<0.01and(※),P<0.05.

Table2

Pharmacokineticparametersofinsulinindiabeticratsafterintragastricadministration(i.g.)ofTMC-CSKINSNPs,TMCINSNPs,INSsolutionandsubcutaneous(s.c.)administrationofINSsolution(n?4).

INSsolution(s.c.)

Dose(IU/kg)AUC(mg/L*h)Cmax(mIU/mL)Tmax(h)FR%

5.0

407.96?63.64110.91?14.721.0

100.00%

TMC-CSKINSNPs(i.g.)50.0

230.87?41.2362.87?3.362.05.66%

TMCINSNPs(i.g.)50.0

150.69?27.5639.57?2.642.03.69%

INSsolution(i.g.)50.0

16.10?32.794.01?5.065.30.39%

Cmax:maximumplasmaconcentration;Tmax:timeatwhichCmaxisattained;FR:relative

bioavailability.

reports,whichmightbetheconsequenceofthedualeffectsofhungerandhypoglycemicagents.

Furthermore,apharmacokineticanalysiswascarriedouttodeterminetherelativebioavailabilityofinsulinfromNPs.Fig.8showedtheplasmainsulinlevelsfollowingintragastricadminis-tration(i.g.)ofTMC-CSKINSNPs,TMCINSNPsandinsulinsolution,aswellassubcutaneousinjection(s.c.)ofinsulinsolution.ThepharmacokineticparameterswerelistedinTable2.BothTMC-CSKINSNPsandTMCINSNPsexhibitedsigni?canthigherplasmainsulinconcentrationcomparedwithinsulinsolutionat1e4hpost-administration(P<0.01).Furthermore,thehighestplasmainsulinconcentrationafteroraladministrationofTMC-CSKINSNPs(62.87?3.36mIU/mL)waspresentedat2h,whichwassigni?cantlyhigherthanthatofTMCINSNPs(39.57?2.64mIU/mL)(P<0.001).Inaddition,theAUCvalueoftheCSKpeptidemodi?edNPswas230.87?41.23mg/L*h,whichwas1.53-foldhigherthanunmodi-?edones(150.69?27.56mg/L*h).Finally,therelativebioavail-abilityofTMC-CSKINSNPs(5.66%)was14.5-foldand1.5-foldhigherthanthatofinsulinsolution(0.39%)andTMCINSNPs(3.69%),respectively.

Overall,theinvivopharmacologicalandpharmacokineticef?-caciesshowedgoodcorrelationswiththeimprovedabsorptionandtransportofINSintheligatedintestinalloopandcellstudiesinvitro.TheresultsofrelativebioavailabilityclearlyrevealedthatTMCNPsandTMC-CSKNPscouldpartiallyprotectINSfromenzymaticdegradationthroughthegastricintestinal(GI)tracttransitandfacilitateitsabsorptionbyepithelium.Moreover,themodi?cationwithCSKpeptidewouldfurtherimprovetheabsorptionofTMCNPsasanoraldeliveredvehicleforINS,whichwasveri?edtoberesultedbygobletcell-targetingef?ciency.Otherwise,tofurtherimprovethebioavailabilityoforaldeliveredINS,somestrategiessuchas

increasingthestabilityofTMCNPsorovercomingtheexcessivetrapofparticlesbymucuslayerareunderinvestigation.4.Conclusions

Inthepresentwork,gobletcell-targetingTMCnanoparticleswerepreparedandcharacterized.TheCSKpeptidemodi?cationcouldeitherpromotetheuptakeofnanoparticlesinvilliorenhancethepermeationofdrugsacrosstheepitheliumthanunmodi?edNPs.Meanwhile,comparedwithTMCFITC-INSNPs,theinternali-zationofdrugsfromTMC-CSKFITC-INSNPswassigni?cantlyhigherinHT29-MTXcells,whichwascon?rmedtoberesultedbyclathrinandcaveolaemediatedendocytosis.Inthetransportstudiesacrosstheco-culturedCaco-2/HT29-MTXcellmodel,CSKpeptidealsoshowedexcellenttransportenhancingabilityasatargetingagent,althoughthetargetrecognitionofCSKpeptidemodi?edNPscouldbepartiallyin?uencedbymucus.Moreover,theexistenceofmucuswaspropitioustothetransportofinsulinfrombothmodi?edandunmodi?ednanoparticles.Atlast,thehypoglycemiceffectandtherelativebioactivityofTMC-CSKINSNPsweresigni?cantlyimprovedbymodi?cationofCSKpeptide,suggestingthatCSKpeptidewasapotentgobletcell-targetingagentandcouldbeusedasaprom-isingligandfororaldeliveryofpeptidesandproteins.Acknowledgments

Wegratefullyacknowledge?nancialsupportfromtheNationalNaturalScienceFoundationofChina(81173010),agrantfromtheNationalS&TMajorProjectofChina(2009ZX09310-002)andYouthacademicleaderFoundationofSichuanprovince(09ZQ026-051).

1582Y.Jinetal./Biomaterials33(2012)1573e1582

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