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Silica−Polymer Dual .pdf

Silica−Polymer Dual .pdf

上传者: 抽象画-倩倩 2018-01-12 评分 0 0 0 0 0 0 暂无简介 简介 举报

简介:本文档为《Silica−Polymer Dual pdf》,可适用于自然科学领域,主题内容包含SilicaPolymerDualLayerEncapsulatedQuantumDotswithRemarkableStabilityXiaog符等。

SilicaPolymerDualLayerEncapsulatedQuantumDotswithRemarkableStabilityXiaogeHuandXiaohuGao*DepartmentofBioengineering,UniversityofWashington,WilliamHFoegeBuildingNM,Seattle,Washington,UnitedStatesSemiconductorQDshaveattractedgreatinterestinbiologyandmedicineinrecentyearsbecauseoftheirfascinatingopticalandelectronicpropertiesthatarenotavailablefromconventionalimagingagentsHowever,alongstandingissueforchemicalsensitivityandinstabilityunderdifferentenvironmentshampersQDapplicationinquantitativeimaging(ahallmarkofmodernbiology)andinvivodiagnosticsDegradationmightprecludeQDprobesfromaccuratequantitativeanalysisduetofluorescencefluctuationandresultinpotentialtoxicitywhenappliedinvivo(currentlyhighqualityQDsaremostlymadefromcarcinogenicchemicalssuchascadmium)Nanoparticleencapsulationtechnologiesbasedonsmallmoleculeligands,silica,andamphiphilicpolymersproducehighlywatersolubleandbrightQDs,butnoneofthemiscapableofprotectingQDsfromchemicalinduceddegradationorsurfacemodificationStabilityofQDfluorescenceisofparticularimportanceforquantitativeimagingandanalysis,whendataobtainedunderdifferentconditionsarecomparedHowever,oftenQDopticalpropertiesdependonthebuffersandsolventsusedandfluorescencemightdropdrasticallyupontreatmentwithlowpHsolutionsorbioconjugationreagentsIngeneral,QDfluorescenceisquenchedinacidicsolutionsandenhancedinbasicsolutionsUndercomplexinvivoconditions,theissueofchemicalinstabilitybecomesanevengreaterconcernasQDdegradation(indicatedbyfluorescencechanges)mightresultinreleaseofheavymetalionsandseveretoxicityForexample,DerfusetalhasshownthatCdSeQDscappedwithsmallmoleculemercaptocompoundsaredeterioratedunderultravioletilluminationandreleaseCdionsUnderinvivoconditions,releasedCdionstightlybindtolargeplasmaproteins,suchasmetallothionein,andcannotbeefficientlyclearedoutofthebodyTheexcretionofCdmetallothionein,primarilythroughurine,isextremelyslowwithabiologichalflifeinthekidneyofyearsQuickclearanceofintactQDsmightprovideonepossiblesolutiontothisproblemBreakthroughworkbyFrangioni,Bawendi,andcoworkersdemonstratedthatQDswithzwitterionicsurfaceandhydrodynamicdiametersmallerthannmcouldberapidlyandefficientlyeliminatedviaurinaryexcretionUnfortunately,rapidrenalclearanceisoftenundesirableforinvivoimagingandtherapeuticdeliveryFurthermore,fortargetedimaginganddelivery,functionalizationofQDswithtargetingligands(eg,antibodies,peptides,andaptamers)yieldssizesbeyondtherenalclearancethresholdandleadstoQDuptakebythereticuloendothelialsystems(RES)Thesetrappednanoparticles,dependingonsizeandsurfaceprop*AddresscorrespondencetoxgaouwashingtoneduReceivedforreviewJuly,andacceptedSeptember,PublishedonlineSeptember,nncopyAmericanChemicalSocietyABSTRACTSemiconductorquantumdots(QDs)areimportantfluorescentprobesduetotheirhighbrightness,multiplexingcapability,andphotostabilityHowever,applicationsinquantitativeandinvivoimagingarehamperedbytheirsensitivitytochemicalenvironmentsandpotentialtoxicityHerewereportasurprisingfindingthatthecombinationofsilicaandamphiphilicpolymercanstabilizeCdSeZnSQDsinabroadrangeofchemicalconditionsincludingstrongacidicsolutions,whichisunavailableforanyofthecurrentencapsulationtechnologies(eg,mercaptocompounds,silica,andamphiphilicpolymers)usedaloneWefurtherdemonstratetheuseoftheseultrastableQDsasinternalreferencesinpHsensingapplicationsWeexpectthisworkwillopenexcitingopportunitiesforinvivoandquantitativeapplications,andmayhelpsolvethetoxicityproblemofQDsKEYWORDS:quantumdotsmiddotimagingmiddotnanotoxicitymiddotsilicamiddotamphiphilicpolymermiddotsensingARTICLEVOLNOHUANDGAOwwwacsnanoorgerties,couldremaininanimalandhumanbodiesforlongperiodsbeforefinalclearanceIndeed,recentinvestigationbyFitzpatricketalshowedthatsystemicallyadministeredQDspersistedandretainedfluorescenceforuptotwoyearsinmiceItwasalsoobservedthatthetraditionalamphiphilicpolymerencapsulatedQDsexhibitedsignificantspectralblueshift,suggestingQDdegradationThisresultisperhapsnotsurprisingsinceQDsarenotstableinacids,buttheircellentryisprimarilythroughendocytosis,aprocesswhichinvolvesacidiccellularcompartmentssuchaslatestageendosomeandlysosomeNovelsurfacechemistrymightprovidewaysforacceleratedinvivoclearanceofnanomaterialsForexample,nearcompletesecretionhasbeenobservedforcarbonnanotubescoatedwithbranchedpolyethyleneglycol(PEG)withintwomonthsInthiscontext,akeychallengeistoengineerastablecoatingthatwillmaintainQDintegrityandopticalpropertiesundercomplexchemicalenvironments,inparticularacidicsolutionsThisgoalcannotbeachievedwithcurrentcappingmaterialsbasedonsmallmoleculemercaptoligands,silica,andamphiphilicpolymersHerewereportanewstrategyforpreparationofultrastableQDsbycombiningthesilicaandamphiphilicpolymerencapsulationtechniquesTooursurprise,althoughneithermaterialprotectsQDsfromharshchemicaltreatments,theircombinationcanprotectQDstosuchadegreethatQDfluorescenceremainsstableevenwhentreatedwithpHacidicsolutionsWefurtherdemonstratethepHsensingapplicationofthistechnologybycombiningtheultrastableQDwithapHsensitivedye(eg,fluoresceinderivative)Uptodate,ahandfulofpapershavereportedtheuseofQDsforpHsensingbasedoneithertheirpHdependentfluctuationofabsolutefluorescenceintensityortheFRETbasedratiometricmeasurementsbetweenconjugatedQDsanddyes,Despitetheserecentsuccesses,akeylimitationsharedbyvirtuallyallcurrentapproachesisthattheQDfluorescenceisnotonlysensitivetopHbutalsoaffectedbyothercompoundsinsolutionTherefore,forcomplexsamples,itwouldbeverydifficulttodistinguishthepHeffectfromotherfactorsTheultrastableQDsreportedherewillhelpsolvethisproblemRESULTSANDDISCUSSIONSynthesisandcharacterizationofultrastableQDsFigureillustratesthemajorstepsofthenewQDencapsulationtechnologyThekeytooursuccessisadoublephasetransferprocessHydrophobicQDsarefirstencapsulatedwithsilicashells(QDSiO)basedonawellestablishedreversemicroemulsionmethod,,IncontrasttopriorartswheresilicacoatedQDsaredirectlyusedforapplications,thehydrophilicQDSiOisthenconvertedbacktowaterinsolublebygraftingFigureSchematicillustrationofadoublephasetransferprocedureforQDSiOPEPEGsynthesisSinglehydrophobicQDsareencapsulatedwithinalayerofhydrophilicsilica,followedbysurfacemodificationwithahydrophobicsilane,OTMSThehydrophobicQDSiOCisthensolubilizedwithPEPEGFigureisnotdrawntoscaleFigureCharacterizationofQDSiOPEPEGTEMimagesof(a)CdSeZnSQDsdispersedinchloroform,(b)QDSiOinethanol,(c)QDSiOCinchloroform,and(d)QDSiOPEPEGinwaterScalebar,nm(e)DLSmeasurementofQDSiOandQDSiOPEPEGshowingahydrodynamicdiameterofandnm,respectively(f)Absorbance(blue)andfluorescence(red)spectraoftheQDSiOPEPEGnanoparticlesARTICLEwwwacsnanoorgVOLNOndashthesilicasurfacewithlongchainhydrocarbons(QDSiOC)Finally,thehydrophobicQDSiOCismadehydrophilicagainwithamphiphiliclipidPEGmolecules(QDSiOPEPEG,completechemicalnameoftheamphiphiliclipidPEGorPEPEGisprovidedintheMethodssection)FunctionalgroupsattheterminalofthePEGdomaincouldenablefurtherconjugationoftargetingmoleculesonthesurfaceofthenanoparticlesNotethatalthoughonlyPEPEGisillustratedhere,theselectionofamphiphilicmaterialsforoutersurfacecoatingisflexibleForexample,amphiphilicpolymersproducesimilarresults,,Figureadshowrepresentativetransmissionelectronmicroscopy(TEM)imagesofthedoubleprotectedQDSiOPEPEGnanoparticlescomparedwiththeoriginalorganicsolubleQDs,QDSiO,andQDSiOCAllthreesamplesofsilicacoatedQDsappearuniforminsizeandwelldispersed,withthemajorityofthesilicananoparticlescontainingasingleQDinthecoreandafewcontainingtwoQDsTheoverallparticlesizeisnmandtheshellthicknessisnmmeasuredfromtheTEMimagesNotethatthesilicashellthicknesscanbetunedbetweenandnmbyvaryingtheamountofTEOSprecursorandQDconcentration(Figure)nevertheless,thefollowingdiscussionisbasedononeshellthickness,nmAfterfurthersurfacemodificationwithCandPEPEG,theselayersarenotvisibleunderTEMbecausetheorganicmoleculesarenotelectrondensematerialsApparently,themultistepmodificationofQDsdoesnotcauseaggregation,whichisalsoconfirmedbydynamiclightscattering(DLS)measurementinanaqueousenvironmentTheDLSresultsshowedahydrodynamicdiameterofnmforQDSiOandnmforQDSiOPEPEG(Figuree),muchlargerthantheparticledrysizeThisisbecausethenanoparticlesarepegylatedandchargedinsolution,creatinganelectricaldoublelayersurroundingthenanoparticles,andconsequentlyincreasingthecolloidalhydrodynamicradiuscomparedtotheactualsizeSpectroscopicmeasurementsshowthatthedistinctiveabsorptionandemissionprofilesofQDsafterSiOandPEPEGcoatingarewellpreserved(Figuref)CharacterizationofChemicalStabilityNext,wesystematicallycomparedthechemicalstabilityofourQDSiOPEPEGwithQDscoatedwithtraditionalmaterialsForbiologicalapplications,QDsshouldbeatleaststablebetweenpHand,because(i)mostbioconjugationreactionsareperformedinthispHrangeand(ii)pHvaluesfoundinhumanbodyalsofallinthisrangeForexample,thepHofbloodisaroundneutralwithavalueof,whereasthatoflatestageendosmeandlysosomeisaboutThemostbasicenvironmentcanbefoundinthepancreas,whosesecretionsareofpHInrarecasesifQDsareevergoingtobeusedforgastrointestinal(GI)tractimaging,thegastricenvironmenthaspHvaluesaroundCertainly,theexposuretogastricacidislikelytobeshort,beforedotsexitthebodyorareuptakenbycellsGivenawiderangeofpotentialapplications,itishighlydesirabletomakeultrastablewatersolubleQDstominimizefluorescencefluctuationandtoxicitycausedbyCdreleaseIndeed,thesignificanceofmakingstableQDs,withaparticularfocusonstabilityinacids(majorreasonforQDetchingandCdrelease),isalsorecognizedbyotherresearchersRecentworksbyMattoussi,Nie,andcoworkerssuggestedthatQDscoatedwiththiolatedPEGandpolyethyleneimine(PEI),aremoreacidresistantthanwithsmallmoleculemercaptocompoundsoramphiphilicpolymersWehaveconfirmedthestabilityimprovementwiththesecoatingsHowever,detailedquantitativespectroscopicmeasurementsrevealedthatQDfluorescencequenchingatlowpHwasstillsignificant(atpH),similartothetraditionalmercaptoaceticacid(MAA),silica,PEPEG,andPMAT(poly(maleicanhydridealttetradecene))coatedQDs(Figure)Incontrast,thefluorescenceofourQDSiOPEPEGremainedconstantfrompHto,andchangedlessthanatpHandafterhincubation(normalizedbyfluorescenceatpH)AlthoughhigherpHvaluesarenotgenerallyencounteredinbiologicalexperiments,westillextendedthestabilitytesttoafullspectrumofpHFigureTEMimagesofQDSiOcoatedwithsilicashellsofvariousthickness(a),(b),(c),and(d)nmARTICLEVOLNOHUANDGAOwwwacsnanoorg()AdecreaseinfluorescenceintensitywasobservedatpHand,likelyduetoinstabilityofsilicainbasicsolutions,andgraduallyincreasedundermorebasicconditionsSuchincreaseinQDfluorescencemightbeassociatedwithpartialdegradationofthesilicashellinstrongbasesfollowedbyregainedQDsensitivitytoenvironmentAsafluorescentlabelingreagent,QDsareoftensubjecttobioconjugationinvolvingbiomoleculesandchemicalcrosslinkers,andthusitisimportantforQDstomaintaintheirfluorescenceinthepresenceofcommonbioconjugationreagentsComparedwiththetraditionalsurfacecappingmethods,thenewQDSiOPEPEGexhibitedremarkablestabilityFigureshowsthatonlythefluorescenceofQDSiOPEPEGremainsconstantunderdifferentchemicaltreatments,whereasotherQDspecimensexhibitdeteriorationinopticalpropertiesThischemicalstabilitycouldhaveimportantapplicationsinquantitativecellularandmolecularimagingThemechanismofthishighlevelprotectionbythesilicaamphiphilicpolymercoatingisnotentirelyunderstoodatthistimeWhensilicaorPEPEGisusedalone,neithermaterialprotectsQDsfromacidorchemicalinducedquenchingandetching,but,whencombined,thecoatinglayerbecomessignificantlylesspermeabletowatersolublechemicalsItisknownthatsilicashellspreparedwiththeStoberchemistryoritsderivativeshaveaporosityofwithporesontheorderofacoupleofnanometers,allowingionstodiffusethroughInthedoublephasetransferprocessreportedhere,thesilicashellsarefirstmodifiedwithhydrocarbons,whichlikelytoreactnotonlyontotheoutersurfaceofthesilicashellbutalsoontotheporewalls(inasimilarwaytopreparationofreversephasechromatographyresins),thusinhibitingdiffusionofwatersolublecompoundsthroughtheshellThisandotherpossiblemechanismsdeservefurthersystematicstudiesNevertheless,followingsolubilizationwiththeamphiphilicPEPEGmolecules,QDsbecamebothcolloidallyandchemicallystableThecombinationofthesilicaencapsulationandthehydrophobicdoublelayerprovidesarobust,inertshelllayeragainstnanoparticledegradationThepresenceofPEGchainsnotonlyimpartsthewatersolubilityofparticles,butalsoflexiblebioconjugationsinceCOOH,NH,andOHterminatedPEGarewidelyavailableCytotoxicityAsoneofthemajorpotentialapplicationsforultrastableQDsisinvivoimaginganddrugdelivery,wefurtherprobedthecytotoxicityofQDSiOPEPEGinLNCaPcellsPleasenoteFigurepHstabilitycomparisonofQDSiOPEPEGwithQDswithtraditionalsurfacecoatings,QDSHPEG,QDMAA,QDPEPEG,QDPMAT,QDPEI,andQDSiOThepanelsontherightshowthecorrespondingfluorescenceimagesofQDsdispersedinpHtosolutions(illuminatedwithanmhandheldUVlamp)FigureStabilitycomparisonofthevariousQDstreatedwithcommoncrosslinkingreagentsinbioconjugation,mMEDC,NHS,SIA,EMCH,mMsulfoSMCCandBS,mgmLbiotinandstreptavidinThecolorcodesofthesamplesareshowninthefirstpanelARTICLEwwwacsnanoorgVOLNOndashthattheLNCaPcellsmerelyserveasamodelAdditionalcelltypesderivedfrommultipletargettissueswillbeneededformorestringenttoxicitytestsBelowgmL(approximatelynMmeasuredwithUVabsorptionatfirstextinctionpeakoftheQDs),cellviabilityafterincubationwithQDSiOPEPEGforhisaboveThisisinthetypicalconcentrationrangeforcellularstainingwithQDbioconjugatesorQDsdilutedinbloodcirculationunderinvivoconditionsAtelevatedconcentrations(eg,gmL),cellviabilitydecreasestoapproximatelyafterhincubationNevertheless,comparedwithplainsilicananoparticles(noQDsinside)ofsimilarsizesandsurfacecoating(SiOPEPEG)(SupportingInformation,FigureS),theconcentrationdependenttoxicityofQDSiOPEPEGshowsvirtuallyanidenticaltrendtothatofSiOPEPEG(Figure),suggestingthattheobservedtoxicityisonlyduetothepresenceofcolloidalnanoparticlesinsolutionbutnotQDdegradationandCdrelease,whichalsoconfirmsthestabilityofQDSiOPEPEGinbiologicalsystemspHSensingBasedonthisnewseriesofultrastableQDs,weshowthattheycanbeusedforpHsensingapplicationsandserveasaninternalreferencePreviouslyanumberofgroupshavebuiltQDbasedpHsensorsbecauseQDsaresensitivetochemicalsinthesurroundingenvironmentsuchasacids,bases,ions,andproteinsForexample,pHvaluescanbedirectlycorrelatedwithQDfluorescenceintensitybecauseQDfluorescence,ingeneral,isquenchedinacidsandisenhancedinbasesHowever,thisdirectmeasurementislimitedtoconditionswhenthetotalQDquantityisfixedUnderdynamicconditionssuchasQDendocytosisandexocytosis,itwouldbeverydifficulttocorrelatepHvalueswithabsoluteQDfluorescenceAmoreelegantandrobustapproachistouseratiometricmeasurementssuchasbyusingQDdyeFRET(fluorescenceresonanceenergytransfer)pairs,becausefluorescenceintensityratiosareirrespectiveofchangesinprobequantity,excitationintensity,anddetectorsensitivity,However,asaforementioned,QDfluorescencefluctuationisnotspecifictopHvalues(orconcentrationsofHandOH)ThepresenceofotherionsormoleculescouldalsochangethefluorescenceintensityratiosandconsequentlyleadtomisinterpretationofexperimentalresultsHere,wedesignanovelratiometricpHsensorwhereultrastableQDsareonlyusedasinternalreferencesApHsensitivefluorophore,=aminomethylfluorescein,hydrochloride(AMF),isimmobilizedtothesurfaceofQDSiOPEPEGviaanamidebondInsuchaconfiguration,FRETbetweenthedyeandQDshouldhaveminimaleffectontheratiometricmeasurementbecauseofthelargeseparationbetweenthecoreQDsandsurfaceattacheddyemoleculesandtheefficientexcitationofQDs(duetotheQDsrsquobroadabsorptionprofile)regardlessofenergydonationfromdyemoleculesAsshowninFigurea,AMFfluorescencehasasharptransitionbetweenpHvalues,whichserveFigurepHsensingusingQDAMFdualcolorsensor:(a)pHdependentemissionofAMFfluorescencemeasuredatthepeak(inset,arepresentativeAMFspectrum)(b)fluorescenceemissionofQDAMFconjugatesatvariouspHvaluesThecontributionsfromQDandAMFareseparatedfromthecompositeemissionspectraforaccuratemeasurementofthepeakintensitiesWhileQDfluorescenceremainsconstant,fluorescencefromAMFfluctuateswithpH(c)WorkingcurveforpHmeasurementproducedwithAMFQDfluorescenceintensityratios(II)versuspHvalues(d)CorrelationofknownpHvaluesandthosemeasuredwiththeQDAMFpHsensorusingtheworkingcurveinpanelcFigureCytotoxicityevaluationofQDSiOPEPEGcomparedwithSiOPEPEG(noQDdoping)DosedependentviabilityevaluationofLNCaPcellstreatedwithQDSiOPEPEG(red)andSiOPEPEG(blue)ofthesamesizeThetoxicitiesofthetwosamplesexhibitnearlyidenticaltrendofdosedependentbehavior,indicatingthatthetoxicityisduetothecolloidalparticles,butnottheQDsdopedinsideARTICLEVOLNOHUANDGAOwwwacsnanoorgasthedynamicworkingrangeTheoverallfluorescenceofQDAMFconjugatescanbereadilyfittedusingalinearcombinationofAMFandQDbasedonpreviouslypublishedprocedures(Figureb)TheworkingcurveisplottedusingthefluorescenceintensityratiosofAMFQDandcorrespondingpHvalues(Figurec)WhenaseparatesetofsolutionsofdifferentpHaremeasuredusingthisworkingcurve,nearlyperfectcorrelation(Figured)isobservedforthemeasuredvaluesusingtheQDAMFsensorandtherealvalues(buffersofknownvaluesandconfirmedwithpHpapers)CONCLUSIONWehavedevelopedanewmethodforpreparationofultrastableQDsbycombiningtwocurrentencapsulationtechnologiesbasedonsilicashellsandamphiphilicpolymersSurprisingly,thissynergisticcombinationyieldsQDswithsignificantlyimprovedresistancetoharshchemicaltreatmentincludingstrongacids,whichhasneverbeenachievedpreviouslyusingeithercoatingmaterialaloneWefurtherdemonstratedapplicationsoftheultrastableQDsforpHsensingIncontrasttopreviousreports,theQDsusedhereareinsensitivetoenvironmentchangesandonlyserveasaninternalreferenceThisfeaturecouldopennewopportunitiesinsensingapplicationsincomplexbiologicalfluidswhenHandOHarenottheonlysolutesWealsonotethatthecurrentworkismainlyfocusedontechnologydevelopmentandcharacterizationofnanoparticlepropertiesApplicationsofthisnewclassofQDsinquantitativeimaging,theirinvivobehaviors,andlongtermtoxicityarecurrentlyunderinvestigationMATERIALSANDMETHODSChemicalsandInstrumentsUnlessspecified,chemicalswerepurchasedfromSigmaAldrich(StLouis,MO)andusedwithourtherpurificationEthyldimethylaminopropylcarbodiimidehydrochloride(EDC),NHydroxysuccinimide(NHS)bissulfosuccinimidylsuberate(BS),Nsuccinimidyliodoacetate(SIA),Nemaleimidocaproicacidhydrazide,trifluoroaceticacidsalt(EMCH),Nhydroxysulfosuccinimideestersofbiotin(sulfoNHSbiotin),and(SulfosuccinimidylNmaleimidomethylcyclohexanelcarboxylate)(SulfoSMCC)werepurchasedfromThermoScientific(Rockford,IL)Streptavidinand=(aminomethyl)fluorescein,hydrochloride)wasobtainedfromInvitrogen(Carlsbad,CA)(,DistearoylsnglycerophosphoethanolamineNcarboxy(polyethyleneglycol)(PEPEG)waspurchasedfromAvantiPolarLipidsTOPOcoatedCdSeZnScoreshellQDswereprovidedbyOceannanotech,LLCasagiftAllchemicalswereusedasreceivedAFluoromaxfluorometer(HoribaJobinYvon,Edison,NJ)wasusedtocharacterizetheemissionspectraofQDsTEMimageswereobtainedonaCMtransmissionelectronmicroscope(PhilipsEO,Netherlands)TruecolorfluorescenceimageswereobtainedwithaNikondigitalcameraSynthesisofSilicaCoatedQDsCdSeZnSQDswereincorporatedinsilicaspheresbyareversemicroemulsionmethoddescribedbyNannetalBriefly,mLofIGEPALCOwasaddedtomLofcyclohexane,followedbytheadditionofnmolofQDs(inLchloroform),LTEOS,andLammoniaaqueoussolution()Betweeneachchemicaladdition,thereactionmixturewasstirredforminThefinalmixture

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