Carbon DotsImaging in Vivo

CarbonDotsforOpticalImaginginVioShngTaoYang,†,‡LiCao,†PngjuG.Luo,†FushnLu,†XinWang,†HaifangWang,,‡,MohammdJ.Mziani,†YuanfangLiu,‡,GangQi,†andYaPingSun,†DpartmntofChmistryandLaboratoryforEmrgingMatrialsandTchnology,ClmsonUniVrsity,Clmson,SouthCarolina296340973,BijingNationalLaboratoryforMolcularScincs,DpartmntofChmicalBiology,CollgofChmistryandMolcularEnginring,PkingUniVrsity,Bijing100871,China,andInstitutofNanochmistryandNanobiology,ShanghaiUniVrsity,Shanghai200444,ChinaRcidJun13,2009;Email:haifangw@pku.du.cn;syaping@clmson.duThrhasbnsignificantrcntintrstinthdlopmntofhighlyfluorscntnanomatrialsascontrastagntsforopticalimaginginio.1Thimagingagntsshouldidallybbright,nontoxic,biocompatibl,andstablagainstphotoblaching.Amongthosxtnsilystudidaronsbasdonsmiconductorquantumdots(QDs)suchasCdSZnS.2ThrationalforthusofQDsorconntionalorganicdysisnowgnrallyaccptdinthlitratur.3ThraralradysuccssfulinioimagingdmonstrationsofQDsontumorasculatur,tumorspcificmmbranantigns,sntinllymphnods,andsoon.2,4SmiconductorQDscontainingcadmiumorothrhaymtalsarunfortunatlyknownforthirsignificanttoxicitynatrlatilylowconcntrations,5,6whichmayproprohibititoanypatintstudis.Thrfor,thsarchforbnignaltrnatishascontinud.Ofparticularintrstandsignificancwasthrcntfindingthatsmallcarbonnanoparticlscouldbsurfacpassiatdbyorganicorbiomolculstobcomstronglyfluorscnt.7Thsfluorscntcarbonnanoparticls,7,8dubbd“carbondots”(CDots,Schm1),wrfoundtobphysicochmicallyandphotochmicallystablandnonblinking.ThcarbonparticlcorcouldalsobdopdwithaninorganicsaltsuchasZnSbforthsurfacfunctionalizationtosignificantlynhancthfluorscncbrightnss(CZnSDots,Schm1).9Thscarbondotshasuccssfullybnusdforinitrocllimagingwithbothonandtwophotonxcitations.7,9,10Carbonishardlyconsidrdasanintrinsicallytoxiclmnt.AailablrsultsfromthongoingtoxicityaluationofoligomricPEGfunctionalizdCDots7inmichasuggstdnomaningfultoxicffcts,11raisingthprospctforiniobiocompatibilityandussofcarbondots.Hrwrportthfirststudyofcarbondotsforopticalimaginginio.Thrsultssuggstthatthcarbondotsarnotonlybrightlyfluorscntinsolution,asrportdpriously,7,9butalsowllbhadascontrastagntsinlimic.CDotsandCZnSDotswithPEGdiamin,H2NCH2(CH2CH2O)nCH2CH2CH2NH2(n35,PEG1500N),asthsurfacpassiationagntwrprpardandcharactrizdaspriouslyrportd.7,9,10ShowninFigur1arrprsntatiatomicforcmicroscopy(AFM)andhighrsolutiontransmissionlctronmicroscopy(HRTEM)imagsofthcarbondots.Bothsamplswrradilysolublinwatrtoformstablaquoussolutionssuitablforthariousinjctionsdscribdblow.Forsubcutanousinjction,fmalDBA1mic(∼25g)wrshadinthbackarasurroundingthinjctionpoint.UponinjctionofaCDotssolution(30µgcarboncorquialntin30µL)oraCZnSDotssolution(65µgin30µL),thmicwrimagdinaLumazonFAinioimagingsystm(MAGBiosystms)with470nm(∼40nmfwhm)xcitationand525nm(∼47nmfwhm)missionfiltrs.AsshowninFigur2,thfluorscncimagsofthsubcutanouslyinjctdmicxhibitdbrightmissionsfromCDotsandCZnSDots.Thrlatilystrongrfluorscncfromthlattrisconsistntwiththpriouslyrportdsolutionphasrsults.9Thinjctdcarbondotsinmicdiffusdrlatilyslowly,andthfluorscncfaddat∼24hpostinjction.Thcarbondotscouldbxcitdatlongrwalngthsforrdfluorscncmission.Forthsamsubcutanousinjctionintomic,thimagingrsultswith545nm(∼29nmfwhm)xcitationand620nm(∼59nm)missionfiltrsalsoxhibitdsignificantfluorscncfrombothCDotsandCZnSDots(Figur2).ThbrightrgrnfluorscncofCZnSDotswasusdinthimagingtotrackthmigrationthroughlymphssls.Uponintrdrmalinjctionintothfrontxtrmity(10µgin10µL),thcarbondotsmigratdalongtharm(Figur3).Unliksmiconductorquantum†ClmsonUnirsity.‡PkingUnirsity.ShanghaiUnirsity.Schm1Figur1.AFMtopographyimagsof(lft)CDotsand(right)CZnSDotsonmica;thinstsshowcorrspondingHRTEMimagsofindiidualdots.PublishdonWb0724200910.1021ja904843xCCC:$40.752009AmricanChmicalSocity113089J.AM.CHEM.SOC.2009,131,11308–11309dotssuchasCdSZnS,whichcanmigrattoaxillarylymphnodsinminuts,4cthobsrdmigrationofthcarbondotswasslowr.Thiscouldbdutothsmallsizsofcarbondots(onthordrof45nm)andorthsurfacfunctionalizationbythPEGs,whosprotinrsistanccharactristicsmightrducintractionsofthcarbondotswithlymphclls.Thaxillarylymphnodswrharstdanddissctdat24hpostinjctionandxhibitdradilydtctdfluorscncfromthcarbondots(Figur3).ACDotssolution(440µgin200µL)wasintranouslyinjctdintomicforwholbodycirculation.Thabdomnwasshadforfluorscncdtctionofthdotstrappdinorgansduringthcirculation,butonlymissionsfromthbladdrarawrobsrd(Figur4).At∼3hpostinjction,brightfluorscncinthurinbcamisiblinthimagingfacility(Figur4).Thrsultssuggstthatthintranouslyinjctdcarbondotsarprimarilyxcrtdiaurin,anxcrtionpathwaythathasbnwidlyrportdinthlitraturforPEGylatdnanoparticls,spciallyforrysmallparticlslikthonsusdhr.12Thorganswrharstdat4hpostintranousinjctionforimaginganalyssxio.Onlythdissctdkidnysandlirxhibitdmaningfulfluorscncfromthcarbondots,whichwasbrightrinthformr(Figur4),consistntwiththurinxcrtionpathway.Thrlatilywakfluorscncinthdissctdlirwasanindicationofalowaccumulationllofthcarbondots.Whilgnrallysignificanthpaticuptakofnanoparticlsandnanotubshasbnwidlyobsrdanddiscussdinmanystudis,13thlowaccumulationhrmightagainbattributdtothffctisurfacPEGylationthatprobablyrducdthprotinaffinityandmadthcarbondotsstalthywithrspcttohpaticuptak.AllofthrportdanimalxprimntswrprformdatClmsonUnirsitybystrictlyfollowingthIACUCapprodprotocols.Duringthxprimnts,noanimalxhibitdanysignofacuttoxicologicalrsponss.Insummary,thrsultsrportdhrdmonstratthatcarbondotsinjctdinariouswaysintomicrmainstronglyfluorscntinio,which,coupldwiththirbiocompatibilityandnontoxiccharactristics,mightoffrgratpotntialforopticalimagingandrlatdbiomdicalapplications.Acknowldgmnt.WthankHilaryHicksofMAGBiosystmsforxprimntalassistanc.ThisworkwassupportdprimarilybyaSusanG.KomnforthCurPostdoctoralFllowshipAward(L.C.andY.P.S.)andthNIH(Y.P.S.).H.W.andY.L.alsoacknowldgfinancialsupportfromthNSFCandChinaMinistryofScincandTchnology.SupportingInformationAailabl:Compltrf7andadditionalxprimntaldtails.ThismatrialisaailablfrofchargiathIntrntathttp:pubs.acs.org.Rfrncs(1)D,M.;Ghosh,P.S.;Rotllo,V.M.AdV.Matr.2008,20,4225–4241.(2)Michalt,X.;Pinaud,F.F.;Bntolila,L.A.;Tsay,J.M.;Doos,S.;Li,J.J.;Sundarsan,G.;Wu,A.M.;Gambhir,S.S.;Wiss,S.Scinc2005,307,538–544.(3)(a)Aliisatos,A.P.Scinc1996,271,933–937.(b)Chan,W.C.W.;Ni,S.Scinc1998,281,2016–2018.(4)(a)Smith,B.R.;Chng,Z.;D,A.;Koh,A.L.;Sinclair,R.;Gambhir,S.S.NanoLtt.2008,8,2599–2606.(b)Gao,X.;Cui,Y.;Lnson,R.M.;Chung,L.W.K.;Ni,S.Nat.Biotchnol.2004,22,969–976.(c)Kim,S.;Lim,Y.T.;Soltsz,E.G.;Grand,A.M.D.;L,J.;Nakayama,A.;Parkr,J.A.;Mihaljic,T.;Laurnc,R.G.;Dor,D.M.;Cohn,L.H.;Bawndi,M.G.;Frangioni,J.V.Nat.Biotchnol.2004,22,93–97.(5)Hardman,R.EnViron.HalthPrspct.2006,114,165–172.(6)Lin,P.;Chn,J.W.;Chang,L.W.;Wu,J.P.;Rdding,L.;Chang,H.;Yh,T.K.;Yang,C.S.;Tsai,M.H.;Wang,H.J.;Kuo,Y.C.;Yang,R.S.H.EnViron.Sci.Tchnol.2008,42,6264–6270.(b)Gys,J.;Nmmar,A.;Vrbkn,E.;Smoldrs,E.;Ratoi,M.;Hoylarts,M.F.;Nmry,B.;Hot,P.H.M.EnViron.HalthPrspct.2008,116,1607–1613.(7)Sun,Y.P.;tal.J.Am.Chm.Soc2006,128,7756–7757.(8)(a)Liu,H.;Y,T.;Mao,C.Angw.Chm.,Int.Ed.2007,46,6473–6475.(b)Zhou,J.;Bookr,C.;Li,R.;Zhou,X.;Sham,T.K.;Sun,X.;Ding,Z.J.Am.Chm.Soc.2007,129,744–745.(c)Zhao,Q.L.;Zhang,Z.L.;Huang,B.H.;Png,J.;Zhang,M.;Pang,D.W.Chm.Commun.2008,5116–5118.(d)Bourlinos,A.B.;Stassinopoulos,A.;Anglos,D.;Zboril,R.;Karakassids,M.;Giannlis,E.P.Small2008,4,455–458.()Hu,S.L.;Niu,K.Y.;Sun,J.;Yang,J.;Zhao,N.Q.;Du,X.W.J.Matr.Chm.2009,19,484–488.(9)Sun,Y.P.;Wang,X.;Lu,F.;Cao,L.;Mziani,M.J.;Luo,P.G.;Gu,L.;Vca,L.M.J.Phys.Chm.C2008,112,18295–18298.(10)Cao,L.;Wang,X.;Mziani,M.J.;Lu,F.;Wang,H.;Luo,P.G.;Lin,Y.;Harruff,B.A.;Vca,L.M.;Murray,D.;Xi,S.Y.;Sun,Y.P.J.Am.Chm.Soc.2007,129,11318–11319.(11)Yang,S.T.;Wang,X.;Wang,H.;Lu,F.;Luo,P.G.;Cao,L.;Liu,J.H.;Liu,Y.;Chn,M.;Huang,Y.;Sun,Y.P.Unpublishdrsults.(12)Choi,H.S.;Liu,W.;Misra,P.;Tanaka,E.;Zimmr,J.P.;Ip,B.I.;Bawndi,M.G.;Frangioni,J.V.Nat.Biotchnol.2007,25,1165–1170.(13)Li,S.D.;Huang,L.Mol.Pharm.2008,5,496–504.JA904843XFigur2.Subcutanousinjctionof(top)CDotsand(bottom)CZnSDots:(a)brightfild,(b,d)asdtctdfluorscnc(xcitationmissionwalngthsindicatd),and(c,)colorcoddimags(ImagJfromNIH).Figur3.IntrdrmalinjctionofCZnSDots:(a)brightfild,(b)asdtctdfluorscnc,and(c)colorcoddimags.Insts:dissctd(inthcircldara)axillarylymphnod(LN).Figur4.IntranousinjctionofCDots:(a)brightfild,(b)asdtctdfluorscnc(Bl,bladdr;Ur,urin),and(c)colorcoddimags.Thsamordrisusdforthimagsofthdissctdkidnys(a′c′)andlir(a′′c′′).J.AM.CHEM.SOC.9VOL.131,NO.32,200911309COMMUNICATIONS