Au CdHg Te quantum dots for in vivo tumor-targeted

ORIGINALPAPER
Au:CdHgTequantumdotsforinvivotumor-targeted
multispectralfluorescenceimaging

SihaiHan&YingMu&QiangyuanZhu&YiboGao
&

ZuhongLi&QinhanJin&WeiJin

Received:31December2011/Revised:24February2012/Accepted:29February2012/Publishedonline:25March2012
#
Springer-Verlag2012

AbstractNear-infraredgold-dopedCdHgTequantumdots
(QDs)withimprovedphotoluminescenceandbiocompatibil-
ityweredevelopedusinganaqueoussolutionroutewith
L
-

glutathioneand
L
-cysteineasstabilizers.As-preparedAu:CdHgTeQDswerecovalentlylinkedtoarginine–glycine–asparticacid(RGD)peptide,anti-epidermalgrowthfactorreceptor(EGFR)monoclonalantibody(MAb),andanti-car-cinoembryonicantigen-relatedcelladhesionmolecule-1(CEACAM1)MAbseparately.ThreeAu:CdHgTeQDbio-conjugates(QD800-RGD,QD820-anti-CEACAM1,andQD840-anti-EGFR)weresuccessfullyusedasprobesforinvivotumor-targetedmultispectralfluorescenceimagingofxenografts.FluorescencesignalsfromtheQDbioconjugatesusedtodetectthreetumormarkerswerespectrallyunmixed,andtheirco-localizationwasanalyzed.TheresultsindicatethatmultipletumormarkerscouldbesimultaneouslydetectedbymultispectralfluorescenceimaginginvivousingQDbio-conjugatesasprobes.Thisapproachhasexcellentpotentialasanimagingmethodforthenoninvasiveexplorationandde-tectionofmultipletumormarkersinvivo,therebysubstan-tiallyaidingthediagnosisofcancer.KeywordsAu:CdHgTe.Quantumdots.Tumor.Multispectral.Fluorescence.ImagingIntroductionMultispectralfluorescenceimaging(MSFI),whichcapturesfluorescenceimagedataatspecificspectralfrequencies,is

attractingextensiveinterestinthefieldsofbiomedicalimag-
ingandnanotechnologyapplications[1].MSFIinvivoisalso
apromisingtechniqueforsensitivedetectionofcancer[2].In
traditionalfluorescenceimaginginvivo,animportantlimiting
factoristhattheemissionspectraoffluorescentlabelsof
biologicalinterestwouldoverlapsignificantly,andthesefluo-
rescentsignalsmayalsobeobscuredbyautofluorescenceof
theanimal.MSFIoffersauniquesolutiontothisproblemby
utilizingfullimagingoverawiderangeofopticalfrequencies
andspectralunmixingalgorithms,andcouldsimultaneously
detectmultipletypesoftumormarkersinvivo,potentially
leadingtoimprovedaccuracyofcancerdiagnosis.
Fluorescentprobeswithgoodopticalpropertiesplayan
importantroleinMSFI.Quantumdots(QDs)aresemicon-
ductornanocrystalscharacterizedbysize-andcomposition-
dependentopticalpropertiessuchashighlevelsofbright-
nessandphotostability,broadexcitationspectra,andnarrow
emissionbands[3].Moreover,multipleemissionspectraof
severaldifferentQDscanbesimultaneouslyexcitedwitha
singleexcitationwavelength[4].ThesefeaturesmakeQDs
theidealcandidateforMSFI.AmongtheQDs,near-infrared
(NIR)QDsareparticularlysuitableforinvivofluorescence
imaging,becausemostbiologicaltissuesabsorbcompara-
tivelyweaklyintheNIRspectralrange(650–900nm).So
far,manyNIRfluorescentQDshavebeendeveloped,such
asCdHgTe[5,6],CdTe/CdSe[7],CdTe
1−xSex
/CdS[8],

InAs/InP/ZnSe[9],InAs
xP1−x/InP/ZnSe[10],CdTexSe1−x
/CdS

[11],CuInSe[12],Cu:InP/ZnSe[13],Hg
xCd1−x
Te/CdTe/

Cd
xZn1−xS[14],andCd3As2
[15]QDs.Althoughsomefunc-

tionalizedNIRQDshavebeenusedforinvivoimaging

S.Han:Y.Mu(*):Q.Zhu:Y.Gao:Z.Li:Q.Jin:W.Jin(*)
ResearchCenterforAnalyticalInstrumentation,
InstituteofCyber-SystemsandControl,StateKeyLab.
ofIndustrialControlTechnology,ZhejiangUniversity,
Hangzhou310058,China
e-mail:muying@zju.edu.cn
e-mail:jinweimy@gmail.com

AnalBioanalChem(2012)403:1343–1352
DOI10.1007/s00216-012-5921-y
studies[5,8,9,16–19],fewofthemwereappliedtotumor-
targetedMSFI.
Fluorescentmetal(e.g.,AuandAg)nanoclusters
(NCs),asanothertypeofnovelfluorescencenanostruc-ture,havealsoattractedagreatdealofinterestinrecentyears[20–27].MetalNCshaveultra-smallsize(below2nm),goodbiocompatibility,andsize-dependentphoto-luminescence(PL),makingthempotentialfluorescentprobesforbiolabeling[25,28,29]andimaging[20,22,23,26,30].However,metalNCsgenerallyhavelowerquantumyields(below2%)[22],limitingtheirapplicationsinimaginginvivo.Inthepresentstudy,goldwasfirstlydopedintoCdHgTeNIRQDsresultinginenhancedPLandlowercytotoxicity.TheAudopantconcentration(6.2%relativetotheCdmolarconcentration)wasverifiedbyinductivelycoupledplasmamassspectrometry(ICP-MS).Goldhasbeendemonstratedtobehighlystableandbiocompatible[31,32].AlthoughAuhasbeendopedintoCdHgTebulksemiconductorsinpreviousresearch[33–35],dopingofAuintoCdHgTeQDshasnotbeenreported.TheAu:CdHgTeQDsweresynthesizedbyanaqueoussolutionrouteusingL-glutathioneandL-cysteineasstabilizers,whichhavebeenusedtopreparehigh-qualityZn1–xCdxSe[36]andCdTeQDs[37–39].As-preparedAu:CdHgTeQDswithtunableNIRfluorescence(740–840nm)weremonodispersedandverycrystalline.Opticalcharacter-izationandcellviabilityanalysisindicatedthatAu:CdHgTeQDshaveenhancedPLandlowercytotoxicitythanCdHgTeQDs.Thequantumyield(QY)oftheAu:CdHgTeQDsreached48%,whichwas6percentagepointshigherthanthatofundopedCdHgTeQDs(42%),andabout24timesashighasthatofreportedAuNCs(below2%)[22].AlthoughMSFIwithQDshasbeenappliedtodetectmul-tipletargetsonafixedtissuesection[40]andsimultaneousimagingofdifferentlymphaticbasinsinvivo[4],noninvasivesimultaneousdetectionofmultipletumormarkersbyinvivoMSFIwithQDshasrarelybeenreported.Inthiswork,threeAu:CdHgTeNIRQDbioconjugateswithdifferentemissionspectraweresuccessfullyusedinMSFIofsubcutaneouslyxenograftedtumorsinmice,andthreetumormarkersweresimultaneouslydetectedinvivo.MaterialsandmethodsMaterialsAnalyticalgradepotassiumborohydride(KBH4),tellurium(Te)powder,sodiumhydroxide,cadmiumchloride(CdCl2·2.5H2O),mercuricchloride(HgCl2),chloroauricacidtetrahydrate(AuCl3·HCl·4H2O),L-glutathione,andL-cysteinewerepurchasedfromSinopharmChemicalReagentCo.,Ltd.(SCRC).Waterusedinsynthesiswashighpuritygradewithaconductivityof18.2MΩcm.N-Hydroxysuccinimide(NHS)and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC)werepurchasedfromSangonBiotech(Shanghai)
Co.,Ltd.TheRGDpeptidesweresynthesizedbySangon
Biotech(Shanghai)Co.,Ltd.Anti-epidermalgrowthfactor
receptor(EGFR)monoclonalantibody(MAb)andanti-
carcinoembryonicantigen-relatedcelladhesionmolecule1
(CEACAM1)MAbwerepurchasedfromEpitomics,Inc.
ThehumanlungepithelialcarcinomaA549cellswere
obtainedfromStemCellBank,ChineseAcademyofScien-
ces.Cellcultureproducts,unlessspecified,werepurchased
fromGibco.TheICRmice(about20geach)wereobtained
fromtheLaboratoryAnimalCenterofZhejiangProvince
(Hangzhou,China)andfedintheLaboratoryAnimalCenter
ofZhejiangUniversity.Thenudemice(about20geach)were
obtainedfromShanghaiLaboratoryAnimalCenterofChina
AcademyofScience(Shanghai,China)andfedintheLabo-
ratoryAnimalCenterofZhejiangUniversity.

Instrumentation
AUV–Visspectrophotometer(UV-2550)andaspectroflu-
orometer(RF-5301PC)werefromShimadzu.Theslit
widthsattheexcitation(ex)andemission(em)ofthe
spectrofluorimeterareall1.5nmtoobtainthefluorescence
spectra.Thetransmissionelectronmicroscope(TEM,JEM-
1230)wasfromJeol.TheX-raydiffractometer(X'Pert
PRO)wasfromPANalytical.AnAntonPaarMultiwave
3000systemwasusedtodigestQDssample.Theinduc-
tivelycoupledplasmamassspectrometer(ICP-MS,Agilent
G3271A)systemwasusedtodetermineelementconcentra-
tionsintheAu:CdHgTeQDsprepared.TheMaestro™in
vivoimagingsystemwasfromCRI,Inc.

Methods
SynthesisofAu:CdHgTeQDs
TheAu:CdHgTeQDsweresynthesizedbyanaqueous
solutionroute[5,6,36,41–54]with
L-glutathioneandL
-

cysteineasstabilizers[37].Inatypicalexperiment,the
procedureswereasfollows:(1)0.20mmolCdCl
2·2.5H2
O,

0.06mmol
L-glutathione,0.24mmolL
-cysteine,60μmol

HgCl
2,and15μmolAuCl3·HCl·4H2
Oweredissolvedin

200mLwater.ThepHofthemixedsolutionwasadjustedto
10with1MNaOHsolution.(2)Airwaspurgedbybub-
blingnitrogen(N
2
)for30min,afterwhich100μmolfreshly

preparedKHTewasquicklyinjectedintothestirredreaction
system.(3)Thereactionmixturewasrefluxedfor5–60min
at96°CunderaslowN
2
flowtoaffordthewater-soluble,L-

cysteine-and
L
-glutathione-cappedAu:CdHgTeQDs.

1344S.Hanetal.