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DevelopmentofUltrasound-SwitchableFluorescenceImagingContrastAgentsBasedonThermosensitive

PolymersandNanoparticles

BingbingCheng,Ming-YuanWei,YuanLiu,HarishPitta,ZhiweiXie,YiHong,KytaiT.Nguyen,andBaohongYuan

(InvitedPaper)

Abstract—Inthispaper,we rstintroducedarecentlydevel-opedhigh-resolution,deep-tissueimagingtechnique,ultrasound-switchable uorescence(USF).TheimagingprinciplesbasedontwotypesofUSFcontrastagentswerereviewed.Toim-proveUSFimagingtechniquesfurther,excellentUSFcon-trastagentsweredevelopedbasedonhigh-performancether-moresponsivepolymersandenvironment-sensitive uorophores.Herein,suchcontrastagentsweresynthesizedandcharacter-izedwith vekeyparameters:1)peakexcitationandemis-sionwavelengths(λexandλem);2)the uorescenceinten-sityratiobetweenon-andoff-states(IOn/IO );3)the uo-rescencelifetimeratiobetweenon-andoff-states(τOn/τO );4)thetemperaturethresholdtoswitchon uorophores(Tth);and5)thetemperaturetransitionbandwidth(TBW).Wemainlyinvestigated uorescenceintensityandlifetimechangesoffourenvironment-sensitivedyes[7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide(DBD-ED),St633,Sq660,andSt700]asafunctionoftemperature,whilethedyewasattachedtopoly(N-isopropylacrylamide)linearpolymersorencapsulatedinnanoparticles.Six uorescenceresonanceenergytransfersystemswereinventedinwhichboththedonor(DBD-EDorST425)andtheacceptor(Sq660)wereadopted.OurresultsindicatethatthreeF¨orsterresonanceenergytransfersystems,wherebothIOn/IO andτOn/τO arelargerthan2.5,arepromisingforapplicationinfuturesurfacetissuebioimagingbytheUSFtechnique.

IndexTerms—Bioimaging,nanomaterials,F¨orsterresonanceenergytransfer(FRET),environment-sensitive,thermosensitive.

I.INTRODUCTION

I

ManuscriptreceivedAugust1,2013;revisedSeptember1,2013;acceptedSeptember3,2013.DateofpublicationSeptember24,2013;dateofcurrentversionOctober25,2013.ThisworkwassupportedinpartbyfundingfromtheNIH/NIBIB7R15EB012312-02(B.Yuan),theCPRITRP120052(B.Yuan),andtheNSFCBET-1253199(B.Yuan).B.ChengandM.-Y.Weicontributeequallytothiswork.Correspondingauthor:B.Yuan,baohong@uta.edu.

B.Cheng,M.-Y.Wei,Y.Liu,andB.YuanarewiththeUltrasoundandOpticalImagingLaboratory,DepartmentofBioengineering,TheUniversityofTexasatArlington,Arlington,TX76010,USA,andalsowiththeJointBiomedicalEngineeringProgram,TheUniversityofTexasatArlingtonandTheUniversityofTexasSouthwesternMedicalCenteratDallas,TX75390,USA(e-mail:bingbing.cheng@mavs.uta.edu;mywei@uta.edu;yuan.liu@mavs.uta.edu;baohong@uta.edu).

H.Pitta,Y.Hong,andK.T.NguyenarewiththeDepartmentofBioengi-neering,TheUniversityofTexasatArlington,Arlington,TX76010,USA,andalsowiththeJointBiomedicalEngineeringProgram,TheUniversityofTexasatArlingtonandTheUniversityofTexasSouthwesternMedicalCenteratDallas,TX75390USA(e-mail:harish.pitta@mavs.uta.edu;yihong@uta.edu;knguyen@uta.edu).

Z.XiewaswiththeDepartmentofBioengineering,TheUniversityofTexasatArlington,Arlington,TX76010,USA,andwiththeJointBiomedicalEngineeringProgram,TheUniversityofTexasatArlingtonandTheUniversityofTexasSouthwesternMedicalCenteratDallas,TX75390USA.HeisnowwiththeDepartmentofBioengineering,ThePennsylvaniaStateUniversity,UniversityPark,PA16802USA(e-mail:zackxie@http://wendang.chazidian.com).

Colorversionsofoneormoreofthe guresinthispaperareavailableonlineathttp://wendang.chazidian.com.

DigitalObjectIdenti er10.1109/JSTQE.2013.2280997

TISalwaysintriguingtorevealinformationindeeptissuebynoninvasivelyimagingtechniques,whichiscriticalforstudyingtissuestructures,functions,anddysfunctions[1],[2].However,mostbiologicaltissuesareopticallyopaquetohumaneyes.Therefore,animagingtechniqueisindispensable[2].Op-ticalmicroscopy,suchasconventionalwide- eldmicroscopyandconfocalormultiphotonmicroscopy,isexcellentinspatialresolution(<1μm)andcanprovidesubcellularimages[3],[4].However,itsimagingdepthissigni cantlylimitedinopaquebiologicaltissues(<1mm)duetostronglightscattering[4].Besidescellularorsubcellularinformationatverysuper cialtissue,deeptissueinformationisalsoveryattractivebutisex-tremelydif culttodetectusingcurrentmicroscopicimagingtechniques[3].Therefore,deep-tissue( 1mm)high-resolutionimagingisdesirableforbothtissuebiologystudiesandpreclin-ical/clinicalapplications[1]–[3],[4].

Opticalandultrasonictechniquesarecommonlyusedfornoninvasivetissueimaging[5]–[7].Theysharemanyfeatures,suchascostef ciency,safety,and exibilityintheselectionofthewell-developedandinexpensiveimagingcontrastagents[8].Theyarealsocomplementary.Forexample,imagingdeeptissue(30–50mm)opticaltechniqueshaveverylowspatialresolution(3–5mm)duetostronglightscattering[8];however,ultra-soundismuchlessscatteredbytissueandhasrelativelyhigherspatialresolution(belowafewhundredmicrometers)[5],[6],[9].Microbubblesarecommonlyusedasultrasoundcontrastagentsandusuallyrestrictedinsideatissuevascularsystembe-causeoftheirmicrometersize[5].However,opticalcontrastagentscansimultaneouslyimagebothvascularandextravascu-larmoleculartargetsviaspectroscopictechniques,becauseoftheirrelativelysmallsize( 1micron)[1]–[3],[6],[8]–[10].Therefore,ultrasoundandopticalhybridimagingtechniques,suchasphotoacousticimagingandultrasound-medicatedopti-calimaging,havebeenintensivelydevelopedduringthepastyears[5],[6],[11],[12].Thesehybridtechniquestakeadvan-tageofbothtechniquesandachieveuniquefeaturesthatareunabletobeachievedbyindividualones[6],[12].

Toquantitativelycomparethesetechniques,depth-to-resolutionratio(DRR)isusuallyadoptedandhighDRRispreferred[6],[13].Fig.1schematicallysummarizedthemajoroptical-andultrasonic-relatedimagingtechniques.Notethat

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Fig.1.

Majorrelatedopticalandultrasonicimagingtechniques.

thevaluesofDRRlistedinFig.1areforageneralcompari-sonamongdifferenttechniquesandthespeci cvalueforeachtechniquemayvaryfordifferentapplications.Diffuseopticaltomography(DOT)andlaminaropticaltomography(LOT)aretwopureimagingtechniques(meaningonlyopticaltechniquesareused)andcanimageseveralmillimetertocentimeterdeeptissuewithlowresolution(submillimetertomillimeters)[7],[10],[14].TheyareroughlylocatedonthelineofDRR=10.ThisDRRisfundamentallylimitedbytissuelightscatter-ing[8].Low-frequencyultrasound,high-frequencyultrasound,photoacoutictomography,photoacousticmicroscopy,andopti-calcoherencetomography(OCT)haveimprovedtheDRRupto～100[6],[13].ThisDRRisfundamentallylimitedbyacous-ticdiffraction(exceptOCT)[6],[13].Opticalmicroscopy,suchasopticalcoherencemicroscopy,two-photonmicroscopy,andconfocallaserscanningmicroscope,maytheoreticallylocatearoundthelineofDRR=1000,butwithalimitedimagingdepth(<1mm,seethehorizontaldashedline)[4],[15],[16].ThisDRRisfundamentallylimitedbytheopticaldiffractionandscattering[4],[15].Tobreakthelightdiffractionlimit,super-resolutionopticalmicroscopyhasbeenintensivelydevelopedrecently[17]–[20],suchasstimulatedemissiondepletionmi-croscopy,photoactivationlocalizationmicroscopy,andstochas-ticopticalreconstructionmicroscopy[21],[22].Whilethesesuperresolutiontechniquescanprovidemuchhigherspatialres-olution(tensofnanometers)thanconventionalopticalmicro-scopes,theirimagingdepthsareusuallylimited(tensofmicrom-eters)[21],[22].Therefore,theDRRmayremain～1000[23](notshowninFig.1).

InFig.1,thehorizontalaxisrepresentsthespatialresolutioninmicrometers(μm)andtheverticalaxisindicatestheimagingdepthinmillimeter(mm).Thefour45 -tilteddashedlinesrepre-sentDRR=10,100,500,and1000,respectively.Theoretically,theyellowareahasbeencoveredbytheaforementionedimag-ingtechniques.Thelightgreenareahasnotbeencoveredduetovariousfundamentalphysicslimits.Thethirdarea,showninred,hasaDRRbetween100and1000.Currently,opticaltech-niquesbasedonthedetectionofscatteredlight(suchasDOTandLOT)havedif cultiesinreachingthisareaduetostrongtissuelightscattering[7],[8],[10],[12],[14].Ultrasoundandphotoacoustictechniquesaredif culttoreachbeyondtheareaofDRR>200(forimagingdepth>1mm)becauseofthefundamentalphysicslimitoftheacousticdiffraction[6],[13].Accordingly,afundamentalquestioniswhetheritisfeasible

to

Fig.2.SchematicdiagramsshowthebasicprinciplesofUSFimaging.Theleftpanelshowsthecasewhenultrasoundtransducer(UST)isOFFandthe uorophoresareOFF.TherightpanelrepresentsthattheUSTisONandsome uorophoresinthefocalvolumeareswitchedON.OC:opticalcondenser;AC:acousticcoupling.

developanewimagingmethodthatislocatedinsidetheredareawithaDRR>200(breakingtheacousticdiffractionlimitinultrasoundandPAtechniques)andanimagingdepth>>1mm(breakingtheimagingdepthlimitinopticalmicroscopy).Toaddressthisquestion,onemayneedtothinkoutsidetheboxtodevelopsometechniquesthatarefundamentallydifferentfromcurrentlyexistingimagingtechniques.Recently,weproposedanddevelopedafundamentallydifferenttechnique,ultrasound-switchable uorescence(USF)imaging[17]–[19].IthasbeendemonstratedthattheUSF-basedimagingtechniquehaspoten-tialtobreaktheacousticdiffractionlimitandtoreacharelativelyhigherspatialresolutioncomparedwithsimilarultrasoundandphotoacoustictechniques[17],[18].Inthispaper,ageneralin-troductiontotheUSFimagingprincipleisgiveninSectionII.InSectionIII,ourrecentprogressinthedevelopmentoftheUSFcontrastagents,oneoftheUSFimagingkeys,ispresented.TheconclusionsaregiveninSectionIV.

II.PRINCIPLEOFUSFIMAGING

A.BasicUSFImagingPrinciples

Thebasicmechanismshavebeendiscussedinourrecentpublicationsin[17],[19],[20],and[24].Brie y,USFimag-ingrequirestwobasicelements:1)imagingcontrastagentsand2)anacoustic-opticalimagingsystem.Theimagingprincipleistouseafocusedultrasoundbeamtoexternallyandlocallycontrol uorophoreemissionfromasmallvolume(closetoorevensmallerthantheultrasoundfocalvolume)[17],[19],[20],[24].TheimagingprincipleisschematicallyshowninFig.2.Ideally,withoutapplyingultrasonicenergy,thecontrastagentsshouldbedark(OFF,no uorescenceemission).Anydetected uorescencesignalshouldbeconsideredasnoiseandmaybegeneratedfromtissueauto uorescenceand/orimper-fectUSFcontrastagents,whichshouldbeextremelyweakinUSFimaging.WhentheultrasonicenergyisturnedONandfocusedinsidethesample,USFcontrastagentsinasmallvol-ume(usuallywithintheultrasoundfocalvolume)areswitchedONand uoresce.Byscanningtheultrasoundfocus,thedistri-butionoftheUSFcontrastagentscanbeimaged[17],[19].Currently,twotypesofUSFcontrastagentshavebeende-veloped:1) uorophore-quencher-labeledmicrobubbles(F–Qmicrobubbles)[19],[25]–[27]and2) uorophore-labeledther-mosensitivepolymersor uorophore-encapsulatednanoparti-cles(NPs)[17],[20].

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Fig.3.SchematicdiagramshowstheconceptofUSFbasedon uorophore-quencher-labeledmicrobubbles;F, uorophores;andQ,quenchers.Anultra-soundpressurepulseswitches“ON”the uorophores.ThedottedcylinderrepresentsultrasoundfocalzoneinwhichtheultrasoundinteractswithF–Qmicrobubbles.Thegreen-dottedarrowsindicatetheexcitationlight.Thedottedorangecirclesandarrowsrepresentthe uorescenceemissionfromtheexpanded(switchedON)microbubbles.

Inthe rsttype, uorophoresandquenchersareattachedonthemicrobubblesurfaceviavarioustypesoflabelingtech-niques.Initially,the uorophoresaresigni cantlyquenchedbyquenchers(orviaself-quenching)sothatnoorveryweak uo-rescencecanbedetected.ToswitchONthe uorescencesignal,ashortandfocusedultrasound(mechanical)pressurepulseisusedtosigni cantlyexpandmicrobubbles.Therefore,theaver-agemoleculardistancebetweenthe uorophoresandquenchersonthemicrobubblesurfacecanbesigni cantlyincreaseddur-ingtheexpansioncycles(orthesurfaceconcentrationofthe uorophoresonthebubblesurfacecanbesigni cantlyreducedifonlyonetypeof uorophoresislabeled).Thus,thequenchingef ciencyisdramaticallyreduced,whichcanswitchONthe u-orescencefromthe uorophores[19],[25]–[27].Fig.3displaysaschematicdiagramtoshowthisconcept.ThelargeF–Qmi-crobubblesrepresentthatanegativeultrasoundpressurecyclesigni cantlyincreasesthebubblesizeandreducesthequench-ingef ciencysothatthe uorophorescanemit uorescencesignal.ThesmallF–Qmicrobubblesarelocatedoutsideoftheultrasoundfocalvolumesothe uorophoresremainquenched(OFF)[19],[24].

Inthesecondtype,polarity-sensitive uorophores(high-quantumyieldsinlowpolarityenvironment)areeithercon-jugatedonthechainofthermosensitivepolymers[seeFig.4(a)][17]orencapsulatedintoNPsthataremadeofthermosensitivepolymers[seeFig.4(b)][20].Tocontrolthe uorescencesignal,arelativelylongandfocusedultrasoundpulse(rangingfromafewtohundredsofmilliseconds)withhighintensityisadoptedtoheatthesampleuptoafewdegreesCelsiusinthefocalvolume[17].WhenthetemperatureToftheUSFcontrastagentsisheatedupabovethelowercriticalsolutiontemper-ature(LCST)ofthethermosensitivepolymersorNPs,thesepolymersorNPsexperienceareversiblephasetransition.Thisphasetransition(betweenthetwostatesofT<LCSTandT>LCST)leadstoasigni cantchangeinthepolaritymicroen-vironmentofthepolymerorNP.Thus,the

polarity-sensitive

Fig.4.SchematicdiagramsshowingtheUSFprinciplebasedon(a)apolymerchainstructureand(b)anNPstructure.Adoptedfrom[20].

uorophoresare“OFF”whenT<LCSTand“ON”whenT>LCST,whichshowsaswitch-like uorescenceemissionprop-erty[17],[20],[28].Speci cally,whenT<LCST,thepolymerchainiselongated,whichiscalledacoilstate.WhenT>LCST,thepolymerchainshrivelsintoaninsolubleglob,whichiscalledaglobulestate.Becausethepolarityofthemicroenvironmentissigni cantlychangedbetweenthetwostates,thepolarity-sensitive uorophoresshowaswitch-like uorescenceemissionproperty[28].AsimilarmechanismappliestotheNPs.WhenT<LCST,NPsarein atedwithpolarizedsolventmoleculesthatquenchthe uorophores.WhenT>LCST,theNPsaredra-maticallyshrunkandthepolarizedsolventmolecules(usuallywithmuchlowermolecularweightthanthe uorophores)aresqueezedout[29],[30].Thus,the uorophores uorescesignif-icantly.ThisconceptisschematicallydisplayedinFig.4[20].Ahigh-intensityfocusedultrasoundtransducercanbeusedtoexternallyandrapidlyincreasethetemperatureofthetissueabovethethresholdtemperaturetoswitchONthe uorophores(duetotissueabsorptionofacousticenergy)[17],[18].Afterultrasoundexposure,thethermalenergyisdiffusedquicklyandthetemperaturerecoverstobackgroundtemperature.Thus, u-orophoresareswitchedOFF.The uorophoresoutsidethefocalzonealwaysremainOFFduetoT<LCST[17].B.MechanismsofBreakingAcousticDiffractionLimitWhenadopting uorophore-labeledthermosensitivepoly-mersorNPs,thespatialresolutioncanbefurtherimprovedbasedontwouniquemechanisms,ashasbeendemonstratedrecently[17],[18].1)Whenanonlinearacousticeffectoccurs,bothlateralandaxialacousticandthermalfocalsizesaredra-maticallyreducedbelowthediffraction-limitedsize.ThismeansthespatialresolutionoftheUSFtechniquecanbehigherthantheultrasoundandPAtechniqueswhenusingthesameultra-soundfrequency[18].2)UnlikeultrasoundandPAtechniques,thespatialresolutionoftheUSFtechniquedependsonthesizeoftheregionwherethe uorophorescanbeswitchedON[17].Becauseoftheexistenceofathresholdofultrasound-induced

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temperaturetoswitchON uorophores,USF uorophorescanbeswitchedONonlyinavolumewhereultrasoundenergyisabovethethreshold.Thus,thesizeoftheregionwhere uo-rophorescanbeswitchedONisusuallysmallerthantheactualfocalsizeoftheultrasound[17].Withappropriateselectionofthethresholdandultrasoundpower,thespatialresolutionofUSFtechniquecanbefurtherimprovedincomparisonwiththespatialresolutiondeterminedbythenonlinear-effect-producedfocalsize.

III.THERMOSENSITIVEPOLYMER-ORNP-BASED

USFCONTRASTAGENTS

A.WhatisanIdealUSFContrastAgent?

TocomparedifferentUSFcontrastagentsquantitatively, veparametershavebeende nedinourpreviouswork[20]:1)peakexcitationandemissionwavelengths(λexandλem);2)the u-orescenceintensityratiobetweenon-andoff-states(IOn/IO );3)the uorescencelifetimeratiobetweenon-andoff-states(τOn/τO );4)thetemperaturethresholdtoswitchON uo-rophore(Tth);and5)thetemperaturetransitionbandwidth(TBW).Toachievethebestsignal-to-noiseratio(SNR),anidealUSFλcontrastagentshouldhavethefollowingproperties:1)bothexandλemarelocatedatredornear-infrared(NIR)regionstoavoidsigni canttissueabsorption(thereforelargepenetra-tiondepth)andauto uorescence(thereforesmallbackground uorescencenoise);2)anIOn/IO ,aslargeaspossible,andτOn/τO reducebackground uorescencegeneratedfrom u-orophoresattheoff-stateandincreasetheon-to-offratio(orSNR);3)fordifferentfutureapplications,Tthshouldbead-justableroughlyinarangeof25–42 Cforbothphantom(atroomtemperature)andinvivostudies(>physiologicalbodytemperature,～37 C);4)TBWshouldbeasnarrowaspossible(typicallyafewdegreeCelsius)toavoidtissuethermaldam-age,and5)ifpossible,the uorescenceintensityaton-stateitself(IOn)andthe uorescencelifetimeaton-stateitself(τOn)shouldbeaslargeaspossibletoincreasesignalstrengthand uorescenceemissiondecaytime,whichcanhelptoimproveSNR.Inpractice,ifsimultaneouslyachievingallthebestval-uesoftheaboveparametersisdif cult,parameteroptimizationbasedonspeci capplicationsshouldbeconsidered.

ThepromisingresultsoftheUSFimagingtechniqueheav-ilyrelyonexcellentanduniqueUSFcontrastagents.There-fore,synthesisofnewUSFcontrastagentsiscriticalforthefurtherdevelopmentofthisnewimagingtechnique.Inthefol-lowingsectionsofthispaper,newlysynthesized uorophore-labeledthermosensitivepolymersandNPsforUSFimagingarepresented.B.Materials

N-isopropylacrylamide(NIPAM),N-tert-butylacrylamide(TBAm),acrylamide(AAm),acrylicacid(AAc),allylamine(AH),N,N,N’,N’-tetramethylethylenediamine(TEMED),ammoniumpersulfate(APS),N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimidehydrochloride(EDC),sodiumdodecylsulfate(SDS),N,N’-methylenebisacrylamide(BIS),and7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide(DBD-ED)werepurchasedfromSigma-AldrichCorporate(St.

Louis,MO,USA).SeTau425mono-N-hydroxysuccinimide(NHS),Square660mono-NHS,Seta700mono-NHS,Seta633mono-NHS,andSquare660mono-NH2werepurchasedfromSETABioMedicals(Urbana,IL,USA),anddenotedasST425,Sq660,St700,Sq633,andSq660arespectively(notethatSq660andSq660ahavethesameabsorbanceand uorescencespectra/lifetime).Allchemicalswereuseddirectlywithoutfurtherpuri cation.

C.Methods

Inthisstudy,poly(N-isopropylacrylamide)(PNIPAM)http://wendang.chazidian.comparedwithotherthermoresponsivepolymers,(i.e.,Pluronic[31]andpoly-N-vinylcaprolactam,[32]),thisisbecause1)ithasbetterperfor-manceofstructurechangefromacoilstatetoaglobulestate,2)ithasrelativenarrowertemperaturetransitionbandwidth(TBW),3)themethodsofadjustLCSTofaPNIPAMpolymerhavebeenwelldeveloped[29],[30];andtheLCSTcanbeadjustedfrom20to49 C[29],[30],[33],whichisbene cialforbothinvitroandinvivostudies,4)itcanbecopolymerizedwithothermaterials,includingamine-containingorcarboxyl-containingmonomers,whichenablesconjugationbetweenthermosensitivepolymersandenvironment-sensitive uorophoreswithfunctionalgroups.Fourpolarity-sensitive uorophores(DBD-ED,St633,Sq660,andSt700)areeitherattachedtoPNIPAMlinearpoly-merorencapsulatedinPNIPAMNPsforinvestigatingtheir uorescenceintensityandlifetimeasafunctionoftempera-ture.Inaddition,sixF¨orsterresonanceenergytransfer(FRET)systems(includingbothpolymerchainandNPstructures)aredesigned,synthesized,andcharacterizedinwhichDBD-EDorST425isusedasthedonorandtheSq660(a)astheacceptor.Allthesedyeswiththedesiredfunctiongroupsarecommerciallyavailableandarefoundpolaritysensitiveinboth uorescenceintensityandlifetime.

1)USFContrastAgentsBasedonLinearThermosensitivePolymersasFluorophoreCarriers:Fig.5showsthestructuresofthethreetypesof uorophore-labeledlinearpolymerstruc-tures,whichincludedonoronly,acceptoronly,andFRETsys-tems.Ingeneral,thethermosensitivelinearpolymeris rstsyn-thesized,andthen, uorophoresaregraftedintothepolymerbycovalentbinding(conjugation).Thedonorhasshortexcita-tion/emissionwavelengthsinvisiblelight,whiletheacceptorhasared/NIRemission(longwavelength).Ashortwavelengthexcitationlight(fordonor)isusedtoexcitethesystem,sothatthereisasmallamountofacceptor uoresced.Whenthepoly-mer( uorophorescarrier)shrivelsintotheglobulestate,donorsandacceptorsgetclosertoeachother,leadingtoFRETfromthedonortotheacceptor.Therefore,theemissionoftheacceptor(inlongwavelength)canbeobserved.

a)SynthesisofThermosensitiveLinearPolymers:AsshowninFig.6,threecomponentsofthepolymerareneces-sarilyincluded:1)mainthermosensitiveunit,i.e.,NIPAM;2)LCST-controllingunit,i.e.,TBAmorAAm;and3)functionalunit,i.e.,AAcorAH.AAmmonomerhasaminegroup,theactivityofwhich,however,isquiteinertintheamideform.Wewilldiscussthefunctionsofthesethreecomponentsinthefollowingsections.

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Fig.5.Schematicdiagramsofthe uorophore-labeledlinearpolymersystems.Fromtoptobottom:donoronly,acceptoronly,andFRET

system.

Fig.6.Schematicdiagramofthecompositionofpolymersinthisstudy.NI-PAM,TBAm,AAm,AAc,H.Dyesareattachedtothepolymerviapostlabelingconjugation.

Linearpolymersweresynthesizedthroughfreeradicalpoly-merization.Allreactionswerecarriedoutina250-mLSchlenktube.Thethreemainstepsare:1)Purgingprocedure:theso-lutionwas rstpurgedwithnitrogenfor10min.Whenaddinginitiator(APS)/accelerator(TEMED)intothesolution,oxygenwaspurgedoutbyvacuuming(1min)and llingwithnitrogen

(5s),whichwasrepeatedthreetimes;2)reactionconditions:4hwithstirringatroomtemperature;3)puri cationprocedure:thesamplewasdialyzedwithappropriatemolecularweightcut-off(MWCO)membraneforthreedaystoremovetheunreactedmonomers,initiator,andothersmallmolecules.ThesethreestepsarealsousedinthesynthesisofpolymerNPsinthefol-lowingsections.

UsingP(NIPAM-AAc200:1)asoneexample,ageneralprocedureisdescribedhere.Samplesof1.3644-gNIPAM(monomer)and4-μLAAc(monomer)atamolarratioof200:1weredissolvedwith50-mLdeionized(DI)waterinthetube.Alongwiththepurgingprocedure,0.067-gAPS(initiator)and51-μLTEMED(accelerator)wereaddedintothetube.Afterthereaction,thesamplewasdialyzedwitha3.5KMWCOmembrane.Theresultingsolutionwascollectedandfreeze-dried,whichthenwasreadyforfurtherconjugationwithamine- uorophores.FortheconjugationwithNHS- uorophores,theamine-functionalizedpolymerofP(NIPAM-AH)wassynthe-sizedbyfollowingthesameprotocolexceptusingAHinsteadofAAc.

Ourhypothesisisthatallofthepolarity-sensitive uo-rophoresgraftedintothepolymershouldbeembeddedwhenthepolymershrinks(formingahydrophobiccore,low-polarityenvironment),bywhichtheir uorescenceintensityandlife-timewouldbeincreasedtothemaximum.Ifany uorophoresareoutsideoftheglobule,i.e.,inahigh-polarityenvironment(exposedtothesolvent),nosigni cantincreasein uorescenceintensityorlifetimewouldbeobserved.Anextraamountofdyeisnotnecessaryalongthepolymerchain,andasare-sult,theratioofdye/polymerneedstobeoptimized.AsshowninFig.6,thepercentageofthefunctionalunitinthepoly-mercompositiondeterminedtheratioofdye/polymerintheconjugates.ToinvestigatetheeffectoftheamountofAAc,forinstance,anothertwopolymerswithdifferentratiosofNI-PAMtoAAcweresynthesizedbyfollowingthesameprotocol:P(NIPAM-AAc100:1)andP(NIPAM-AAc600:1).Sincetheratioofthemonomer(s)totheinitiatorremainedthesameinallthethreebatchesofP(NIPAM-AAc)polymers,thelengthofthethreepolymerswaslikelyincloserange.Therefore,thelowermolarratioofNIPAMtoAAcindicatedtheincreasedconjugatingsites(carboxylfromAAc)availableforamine-functionalized uorophores.The uorescenceintensityandlife-timeasafunctionoftemperatureweremeasuredforallthepolymers,andotherUSFparametersmentionedabovewerealsomeasured.

Tocontrolthetemperaturethreshold(LCST)ofthepolymers,theLCST-controllingunitAAmorTBAmwascopolymerizedwithNIPAM.Itwasfoundthataddinghydrophilicmonomers(suchasAAm)couldincreasetheLCST[29],[30],[33]andaddinghydrophobicmonomers(suchasTBAm)coulddecreasetheLCST.Moreimportant,theintroductionofTBAmmightfurtherimprovethehydrophobicityinsidetheglobulewhenthetemperature>LCST,whichcouldpotentiallyincreasethevaluesofIOn/IO andτOn/τO .Therefore,thefollowingpolymersweresynthesized:P(NIPAM-TBAm-AAc85:15:1),P(NIPAM-TBAm-AAc185:15:1),P(NIPAM-TBAm-AAc585:15:1),andP(NIPAM-AAm-AAc200:32:1).TheTBAmremainedat15%moleinthesecopolymersbecausewefoundthatTBAmcouldbewelldissolvedinDIwateratthisratio.