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FULLPAPER2016WILEY-VCHVerlagGmbH&Co.
KGaA,Weinheim1040wileyonlinelibrary.
comCancerCellInternalizationofGoldNanostarsImpactsTheirPhotothermalEfciencyInVitroandInVivo:TowardaPlasmonicThermalFingerprintinTumoralEnvironmentAnaEspinosa,AmandaK.
A.
Silva,AnaSánchez-Iglesias,MarekGrzelczak,ChristinePéchoux,KarineDesboeufs,LuisM.
Liz-Marzán,andClaireWilhelm*DOI:10.
1002/adhm.
201501035Goldnanoparticlesareprimecandidatesforcancerthermotherapy.
How-ever,whiletheultimatetargetfornanoparticle-mediatedphotothermaltherapyisthecancercell,heatingperformancehasnotpreviouslybeenevaluatedinthetumoralenvironment.
Asystematicinvestigationofgoldnanostarheat-generatingefciencyinsituispresented:notonlyincancercellsinvitrobutalsoafterintratumoralinjectioninvivo.
Itisdemonstratedthat(i)inaqueousdispersion,heatgenerationisgovernedbyparticlesizeandexcitinglaserwavelength;(ii)incancercellsinvitro,heatgenerationisstillveryefcient,butirrespectiveofbothparticlesizeandlaserwavelength;and(iii)heatgenerationbynanostarsinjectedintotumorsinvivoevolveswithtime,asthenanostarsaretrafckedfromtheextracellularmatrixintoendosomes.
Theplasmonicheatingresponsethusservesasasignatureofnanoparticleinternalizationincells,bringingtheultimategoalofnanopar-ticle-mediatedphotothermaltherapyastepcloser.
1.
IntroductionWhenmalignanttumorsaredeeplyembeddedwithinthebodyorentangledaroundvitalorgans,makingtheminop-erable,non-invasivethermaltherapypromisestoyieldbetterpatientoutcomesandfeweradverseeffectsthanconven-tionaltreatments.
[1]Thermaltherapycaninictdirect,irreversiblecancercelldamagebydisruptingtheirmembranes[2]orbydenaturingproteinsandDNA.
[3]Thermaltherapycanalsobeusedtoenhancecancercellsensitivitytoradia-tionandchemotherapy.
[4]Avarietyofheatsourceshavebeeninvestigated,includinglasers,[5]microwaves,[6]andfocusedultrasound,[7]buttheseapproachesalsodamagehealthytissuessituatedbetweenthesourceandthetarget.
[8]Incontrast,energy-absorbingnanoparticlescanbeusedtoinduceheatingrestrictedtothetargettissue.
[9]Nanoparticle-basedthermaltherapyisdividedintotwomaincategories:magnetothermalandphotothermaltherapy.
Magnetothermaltherapy,alsocalledmagnetichyperthermia,isbasedontheuseofahigh-frequencyalternatingmagneticeldtoexcitemagneticnanoparticlesandtherebygeneratelocalheating.
[10]Decadesofresearchhavemovedtheconceptofmagnetothermaltherapyforwardfrominvitromechanisticstudies[11,12]topreclinicalinvestigations.
[13,14]Photothermaltherapyisamorerecentconceptinwhichlightisconvertedtoheatbyplasmonicnanoparticles,[15]semicon-ductornanocrystals,[16]orcarbon-basednanosystems.
[17]Inrecentworks,magnetichyperthermiacouldbecombinedwithphotothermiatoamplifyheatgeneration.
[18,19]Theuseoflasersourcesat650–950nm(rstnearinfrared(NIR)window)and1000–1350nm(secondNIRwindow)ensuresminimallightabsorptionbysurroundingtissues.
[20]Effortstoimprovelight-to-heatconversionefciencyhavefocusedonthesize,shape,orsurfacecoatingofplasmonicnanoparticles,aswellassurfaceplasmonresonancesintheNIR.
[21]Goldnanostarsareprom-isingcandidates,astheirplasmonresonancespectraandlightabsorptionefciencycanbepreciselytunedbyadjustingtheirwidth,length,ornumberofspikes.
[22,23]Despiteadvancesinnanoparticledesign,thefactorsdeter-mininglight-to-heatconversionefciencyinthetumorenvi-ronment,whichistheultimatedestinationofnanoparticlesforDr.
A.
Espinosa,Dr.
A.
K.
A.
Silva,Dr.
C.
WilhelmLaboratoireMatièreetSystèmesComplexes(MSC)UMR7057CNRSandUniversitéParisDiderot75205Pariscedex13,FranceE-mail:claire.
wilhelm@univ-paris-diderot.
frDr.
A.
Sánchez-Iglesias,Dr.
M.
Grzelczak,Prof.
L.
M.
Liz-MarzánBioNanoPlasmonicsLaboratoryCICbiomaGUNEPaseodeMiramón182,20009Donostia,SanSebastián,SpainDr.
M.
Grzelczak,Prof.
L.
M.
Liz-MarzánIkerbasque,BasqueFoundationforScience48013Bilbao,SpainDr.
C.
PéchouxGABI,INRA–MIMA2-METAgroParisTech,UniversitéParis-Saclay78350Jouy-en-Josas,FranceDr.
K.
DesboeufsLISA,CNRSUMR7583UniversitéParis-DiderotetUniversitéParis-EstCréteil,61avduGénéraldeGaulles94010Créteil,FranceProf.
L.
M.
Liz-MarzánBiomedicalResearchNetworkingCenterinBioengineeringBiomaterialsandNanomedicine(CIBER-BBN)50018Aragon,SpainAdv.
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KGaA,Weinheimaccomplishingtherapy,remaintobefullyelucidated.
Indeed,mostheatingstudieshaveusedaqueousnanoparticledisper-sion,whichfailtotakeintoaccounttheeffectofthebiologicalenvironment.
Forexample,magneticnanoparticlesareknowntobehavedifferentlyfollowingproteincoronaformationorcon-nementintocellularcompartmentssuchasendosomesandlysosomes.
[11,24,25]Indeed,magneticnanoparticlesundergotheformationofaggregatesinthebiologicalenvironment,whichleadstoadivergenceinheatingefciencywhencomparingnanoparticlecharacterizationinaqueousdispersionandrealperformanceincells.
Decadesofintensestudieshaveshownthattheefciencyoflightenergyconversionintoheatusingplasmonicparticlesincreaseswithdecreasinginterparticledistances,i.
e.
,whentheparticlesareinanaggregatedstate.
Toenhancesuchpropertiesinabiologicalsystem,nanoparticlescanbepre-aggregated,thuscontrollingbeforehandtheopticalpropertiesandaddressingthemintothedesiredsystem.
Asanexample,Niuandco-workers[26]developedplasmonicvesicleswithstrongplasmoniccouplingthatservednotonlyforinvivoimagingbutalsoforremotedrugdelivery.
Nevertheless,suchanapproachof"pre-aggregation"issyntheticallychallengingandrequirescomplexsurfacechemistry.
Weproposeadifferentstrategy,inwhichlivingcells(invivo)takeuptheinitiallystablenanoparticlesandaggregatetheminsitu,inducingplasmoncouplingandthere-forechangingtheirefciencytowardhyperthermiatreatment.
Wethusinvestigatedthelight-to-heatconversionefciencyofgoldnanostarsinenvironmentsofincreasingcomplexity,fromaqueousdispersiontocancercellsinvitroandthentosolidtumorsinvivo.
Thisseriesofheatingmeasurementsrevealedathermalngerprintofplasmonicnanostarinternalizationbycancercells.
2.
HeatingEfciencyofGoldNanostarsinAqueousDispersionisSizeandLaser-DependentWesynthesizedapanelofvedifferentAunanostarswithsizesrangingfrom25to150nm(Figure1A).
Transmissionelectronmicroscopy(TEM)showsthatthenanoparticlescom-priseacentralcorefromwhichmultiplesharptipsprotrude,withnarrowsizedistributions:27.
1±2.
2nm(25-nmsample),52.
6±3.
4nm(55-nmsample),85.
9±4.
5nm(85-nmsample),121.
6±2.
9nm(120-nmsample),and150.
9±2.
4nm(150-nmsample).
SizedistributionsaredisplayedinFigureS1,Sup-portingInformation.
Z-potentialanddynamiclightscattering(DLS)ofthedifferentAunanostarsareshowninTableS1,SupportingInformation.
Asexpected,thevesamplesdisplaydistinctUV–vis–NIRopticalspectra,allofthemfeaturingabroadplasmonband(Figure1B).
The25-nmsampleyieldedtwoabsorbancemaximaataround550nmand700nm,whichwereattributedtoplasmonmodeslocalizedatthecoreandthetips,respectively.
[27,28]Asthenanostardiameterincreased,togetherwiththenumberofspikes,theinuenceofthecoreontheextinctionspectrumgraduallydeclines,[28]eventuallyresultinginasingleband.
Regarding55-nm,85-nm,120-nm,and150-nmnanostars,thesingleabsorbancebandgraduallyshiftstowardtheNIRregion,at790,800,900,and950nm,respectively,conrmingthatnanostarplasmonresonancesarestronglydependentonsizeandmorphology,especiallyspikelengthandnumberofspikes.
[23,29,30]Thephotothermalheatingefciencyofthenanostarsam-ples,dispersedinwateratthesameAuconcentrationof0.
75*103M,wasmonitoredwithaninfrared(IR)thermalcamera(imagesareshowninFigure1CandFigureS2,Sup-portingInformation)duringlaserirradiationat680,808,and1064nm,atthesamepowersettingof1Wcm2.
Thetempera-turewasobservedtoriseasafunctionoftimeatallwavelengthsandwithallvesamples,reachingsaturationafter5–10min(seetypicaltemperaturecurvesinFigure1DandFigureS3,SupportingInformation).
Theaveragevaluesoftemperatureincrementrecordedafter1min,ΔT1min,aswellasthetem-peratureincreasereachedatsaturationΔTplateauareplottedinFigure1E,F,respectively.
Aremarkablecorrelationbetweenthelight-to-heatconversionprolesandtheUV–vis–NIRabsorp-tionspectrawasobserved(seeFigureS4,SupportingInfor-mation,forquantitativecomparison).
Forinstance,the25-nmnanostarsdisplayedthebestheatingefciency(ΔT1min=19°C;ΔTplateau=44°C)whenirradiatedat680nm,closetothemax-imumofthelocalizedsurfaceplasmonresonance(LSPR)band.
Conversely,theweakestheatingeffect(ΔT1min=3°C;ΔTplateau=6°C)wasobservedatthewavelengthatwhichspectralabsorb-ancewaslowest(1064nm).
Similarresultswereobtainedwiththe85-nmnanostarsample,forwhichthemaximumoftheLSPRbandwasnear808nm.
Thissampleshowedaveragetemperatureincrementsof(ΔT1min=13°C;ΔTplateau=33°C),(ΔT1min=18°C;ΔTplateau=42°C),and(ΔT1min=5°C;ΔTplateau=10°C)atlaserwavelengthsof680,808,and1064nm,respec-tively.
Withthe150-nmnanostarsample,becausethelaserwavelengthsettingscorrespondedtoneitherthemaximumnortheminimumabsorption,ΔTvaluesweresimilaratallthreewavelengths((ΔT1min=10°C;ΔTplateau=28°C),(ΔT1min=13°C;ΔTplateau=30°C),(ΔT1min=16°C;ΔTplateau=29°C)for680,808,and1064nm,respectively).
AsshowninFigure1E,F,foragivenlaserwavelength(dottedlinesonthegraphs),theheatingefciencieswerered-shiftedasthenanostarsizeincreased.
At680nm,the25-nmsamplewasmostefcient,whereasthe150-nmsampledominatedat1064nm.
Theintermediatesizes(55,85,and120nm)weresimi-larlyefcientat808nm,andallthreeweremoreefcientthanthe25and150nmsamples.
Thus,precisenanostardesignfeaturesresultindistinctandwell-denedphotothermalconversionpro-les,whichisconsistentwiththeirabsorptionspectralfeatures.
Importantly,excellentabsoluteheatingefciencywasobtainedattheappropriatelasersettings.
Toeliminatetheinuenceoftheexperimentalconditions(sampleconcentra-tion,geometrydeterminingthesaturationtemperature),thisheatingefciencyisexpressedwithaparametercalledthespe-cicabsorptionrate(SAR,expressedinWg1ofAu,seetheSupportingInformation),whichiscalculatedfromtheinitialslopeofthetemperaturecurvesasafunctionoftime(valuesarepresentedinFigure1E).
Underoptimalconditions,allvesamplesreachedaSARofnearly10kWg1atalaserpowerof1Wcm2.
ThisisoneofthehighestSARvaluesreportedatthislaserpower.
Highervalues,suchas430±40and190±20kWg1(at800nm),havebeenreportedwithgoldnanorodsandnanostars,respectively,butatmuchstrongerlaserpowers(13Wcm2).
[2,31]Adv.
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KGaA,WeinheimAscancercellsarethemaintargetofnanostar-basedphoto-thermaltherapy,wenextinvestigatedAunanostarheatingef-ciencyinvitro(nanostarsinternalizedbycancercells),andinvivo(afterintratumoralinjection).
3.
InteractionofAuNanostarswithCancerCells:InternalizationandHeatingEfciencyThephotothermalconversionefciencyofthethreemostrep-resentativenanostarsamplescoveringthewholesizerangewereselected(25,85,and150nm)andrstmonitoredinvitro,usingthehumanprostatecancercelllinePC3.
NanostaruptakewasinvestigatedbyTEMusingxedsamplesandAuelementalanalysis(seetheSupportingInformation).
Figure2A(andFigureS5,SupportingInformation)showscellsincubatedwithnanostarsincompleteculturemediumDulbecco'smodiedEagle'smedium(DMEMsupplementedwith10%fetalbovineserum(FBS))overnight(16h)at[Au]=0.
02*103M.
Thenanostars,visualizedaselectron-densespots,werealllocatedwithinendosomes,closetooneanother.
Nonanostarswerefoundintheextracellularmediumorattachedtothecelloutermembrane.
TheseobservationsareinagreementwiththoseofpreviousstudieswhereaccumulationofAunanostarswasobservedbyTEMwithinendosomes.
[32,33]Elementalanalysis(inductivelycoupledplasmaatomicemissionspectroscopy(ICP-AES))conrmedsuchanef-cientcellularuptake,whichincreasedwithnanostarsizefrom1.
4±0.
1pgAu/cellforthe25-nmsampleto2.
6±0.
3and3.
0±0.
3pgAu/cellforthe85and150nmsamples,respectivelyAdv.
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Goldnanostarsinaqueousdispersion:inuenceofnanostarsizeandlaserexcitationwavelengthonplasmonicresonancesandheatingefciency.
A)RepresentativeTEMimagesofthevesamplesofAunanostars,atlow(top,scalebar=200nm)andhigh(bottom,scalebar=50nm)magnication.
B)UV–vis–NIRabsorptionspectraofthesamesamples:theLSPRbandshiftsfrom600to1000nmasthenanostarsizeincreases.
C)Infraredthermalimagesofaqueousdispersions(150L)ofAunanostars(25,85,and150nmsamples)at[Au]=0.
75*103M,after1minoflaserirradiationat680,808,and1064nmand1Wcm2.
D)Temperatureincreasecurvesforthe25,85,and150nmsamplesuponirradiationat808nm(1Wcm2).
E,F)Temperatureincrementsachievedwithallvesamples(25,55,85,120,and150nm)asafunctionoflaserwavelength(680,808,and1064nm),afterE)1min(ΔT1min)orF)10min(plateau,ΔTplateau)ofexposureat1Wcm2.
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KGaA,Weinheim(graphshowninFigureS6A,SupportingInformation).
Uptakewaswithinthesamerange,whateverthenanostarsize.
Besides,asshownalsoinFigureS6B,SupportingInformation,nanostarinternalizationdidnotaffectcellmetabolicactivity,regardlessoftheparticlesize,inagreementwithpreviousreports.
[30,33,34]Itthenclearlyappearedthatnanostarheatingefciencywasmodiedfollowingcellularinternalization.
Nanostar-loadedcellswereharvestedinapellet.
ThenumberofcellspermLwasthendetermined(throughcellcountinginaMalassezchamber)tonallyobtaintheAumasspermL(Auconcen-trationofthepellet).
Theywereresuspendedinavolumeof150LtomatchtheAuconcentrationof0.
75*103M,thesameasusedfortheaqueousnanostardis-persion.
TheheatingwasrecordedwithIRcameraandthesameset-upastheoneusedfortheaqueousdispersions(Figure2B,CshowstypicalIRimagesandaveragetemper-atureincrease,respectively,after1minlaserexposure).
Atthehighestlaserwavelength(1064nm),the1mintemperatureincre-mentΔT1minrosefrom3upto13°Cwiththe25-nmnanostarsandfrom5upto13°Cwiththe85-nmnanostars,whileitdroppedfrom16downto11°Cwiththe150-nmnanostars.
Withthe25-nmnanostars,ΔT1minwasstillhigherat680nmthanat1064nm(15vs.
13°C),butitshighestvaluewasfoundat808nm(17°C).
Atawavelengthof680nm,thedeclineinheatingefciencywithsizeobservedinaqueousdispersion(nanostarsizeisrepresentedbydottedlinesinFigure2C)wasmuchlessmarkedwhenthenanostarswereconnedwithincells(datainaqueousdispersionareshownasemptybarsinFigure2Cforadirectcomparison).
Similarly,themaximumheatingobservedat808nmwiththeintermediate-sizenanostarsinaqueousdispersionwasnotobserved,andneitherwasthesize-dependentincreaseinheatingefciencyat1064nm.
Wethusconcludethatthewavelengthdependenceofnanostarheatingefciencywasreduceduponconnementinendosomalcompartments.
Indeed,whilethelaserwave-lengthmarkedlyinuencedheatingefciencyinaqueousdispersion(respective95%and80%decreasesinheatingefciencywiththe25-nmand85-nmnanostarsirradiatedwiththe"correct"and"incorrect"wavelengths),thiswasfarlessthecaseinthecellularenvi-ronment(20%and17%,respectively).
Finally,theheatingpower(SAR)(alsoshowninFigure2C)ofthedifferentnanostar-loadedcellsamplesfollowedthesametrend:theSARinsidecancercellswasmuchlessdependentonnanostarsizebutstillreachedhighvalues,intherangeof10kWg1.
Themostlikelyexplanationforthisattenuationofwavelengthdependenceuponcellularuptakeisnanostarconnementwithinendosomes.
Indeed,AunanoparticleaggregationinendosomeswasrecentlymeasuredandledtoLSPRred-shiftandbroadening.
[35]Similarly,usingplasmonicallyenhancedRayleighimagingoflivingcells,thesubcellularaggregationofAunanoparticlesinendosomeswasdemonstratedthroughthered-shiftandbroad-eningoftheplasmonband.
[36]ThemacroscopicUV–vis–NIRspectraofAunanostarsincellsareshowninFigureS7,Sup-portingInformation.
Whileacquisitionwashinderedbycel-lularbackgroundstructures,thesespectrarevealaatteningoftheextinctionbands,adeclineinthepeak-to-valleydistance,andared-shift.
Adv.
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InteractionofAunanostarswithcancercells,andintracellularheatingefciency.
A)TEMimagesofPC3cancercellsafterincubationwithAunanostars,atdifferentmagni-cations(scalebars2mand100nm).
B)Typicalinfraredthermalimagesofthecellsam-ples(about15millioncellscontainingAunanostarsdispersedin150LofPBS,adjustedtoaconcentrationof[Au]=0.
75*103M).
Fromtoptobottom,cellsincubatedwith25,85,and150nmnanostarsafter1minoflaserirradiationat680,808,and1064nmand1Wcm2.
C)Averagetemperatureincreaseforallcellsamplesasafunctionoflaserwave-length(680,808,and1064nm)after1minat1Wcm2.
Onthesamegraphthecalcu-latedvaluesfornanostarsinaqueousdispersionareshown(emptybars).
SAR(inkWg1,secondaryrightaxis)wasalsocalculated(seeExperimentalDetailsintheSupportingInfor-mation)forallcellularsamples.
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KGaA,Weinheim4.
HeatingEfciencyofAuNanostarsInVivo:EvolutionPost-injectionHeatingefciencyinvivowasevaluatedbyinjectingnanostars(atthesame[Au]=0.
75*103M)intoPC3tumorsinducedinmice.
Theuptakeandheatingefciencyof25,85,and150nmnanostarswereinvestigatedondays0and3post-injection.
IntratumoralnanostarlocalizationwasassessedbyTEM,andlocaltumoraltemperaturechangesweremonitoredinlivingmicewiththeinfraredthermalcameraduringtheentireperiodofirradiation.
Onday0,TEMimages(Figure3A,top)showedindividualnanostarsdispersedclosetovessels,throughouttheextra-cellularmedium,orwithinbundlesofcollagenbres(seealsoFiguresS8–S10,SupportingInformation).
Ofnote,nonanostarswereobservedinthecytoplasm.
Onday3,thenanostarswerelocatedsolelyinendosomes(Figure3A,bottomandFiguresS11–S14,SupportingInformation),insidetumorAdv.
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HeatingefciencyofAunanostarsintumorsinvivo:evolutionovertime.
A)TEMimagesofnanostarsonday0andday3afterintratumoralinjection.
B)Invivoinfraredthermalimagesoftumorsinjectedwith25,85,and150nmnanostarsandexposedfor1minto680,808,and1064nmlaserirradiationat1Wcm2,onday0(1hafterinjection,upperpanel)andonday3(lowerpanel).
C)Averagetumoraltemperatureincreaseasafunctionofthelaserwavelength(680,808,and1064nm)andnanostarsize,after1minofexposureat1Wcm2,onday0(left)andday3(right).
Dottedlinesservetovisualizetheevolutionofheatgenerationaccordingtonanostarsize,foragivenlaserwavelength(lightgrayfor680nm,grayfor808nm,anddarkgrayfor1064nm).
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KGaA,Weinheimcells,formingsmallrandomclusters,butretainingtheoriginalbranchedmorphology,i.
e.
,noreshapinghastakenplacewithinthetumor.
ThisisanimportantpointconsideringthatnanostarreshapingmayfurtherinuencenanoparticleLSPR.
[37]Thus,aspreviouslyobservedincancercellsinvitro,allAunanostarswereprocessedintosubcellularcompartmentsinvivowheretheyaccumulate.
Figure3Bshowsthermalimagesafter1minoftumorirradiationonday0(1hafterinjection)andonday3,whileFigure3CshowstheaveragetemperatureincrementΔT1min.
Onday0,thephotothermalconversionprolewassimilartothatofdispersednanostars(Figure3C,left).
Notethatthenanostardispersion(50–100L)at[Au]=0.
75*103Mwasinjectedintotumors.
Thevolumeinjecteddependedonthetumorvolume;Foreachtumor,thenanostarsinjectedvolumewasadjustedinordertokeepthedilutionfactorconstant.
Itclearlyappearsfromthethermalimagesthatheatingremainslocalizedclosetothesiteofinjection.
Inkeepingwiththisobservation,thevaluesoftemperatureincreasewerefoundtobeinthesamerangeasthoseobtainedindispersionorincancercells(at[Au]=0.
75*103M).
Forinstance,with25-nmnanostars,thetumortemperaturerose(atday0,rightafterinjection)byrespectively12,8,and5°Cafter1minofirradia-tion(1Wcm2)at680,808,and1064nm.
Inthesamecondi-tions,therespectivetemperatureincrementswereabout10,14,and5°Cwith85-nmnanostarsandabout6,10,and9°Cwith150-nmnanostars.
Untreatedtumorsexposedtolaserirradia-tionshowedanegligibletemperatureriseof3–4°C(FigureS15,SupportingInformation).
Theinvivolight-to-heatconversionproleevolvedwithtime,markedlydifferingbetweenday0andday3withboth25-nmand85-nmnanostars(Figure3C,right).
Remarkably,thephotothermalefciencyonday3showedthesamepatternasthatofnanostarsinternalizedbycancercellsinvitro.
ThisisinperfectagreementwithTEMobservations,whichshowedthatallnanostarswereconnedinendosomes.
Onday3therecordedtemperatureincrementswerethusinde-pendentofnanostarsizeandlaserwavelength.
5.
InVivoThermalSignatureofIntratumorLocalizationofPlasmonicNanoparticlesHere,wedemonstratethattheheatingefciencyatdifferentlaserwavelengthsremarkablymatchesthedegreeofplas-monicgoldnanostarsinternalizationbycancercellsinvivo.
Wethereforeprovideinformationonthenanoscalecellularfateofplasmonicnanoparticleswithinatumor,throughsimplemeasurementofheatgenerationuponirradiationatdifferentwavelengths,bycontrastwiththeinitialwavelength-dependentheatingproleinaqueousdispersion.
Therelationshipbetweenmacroscopicphysicalmeasure-ments(magnetic,optical,thermal,etc.
)andthenanoscalelocalizationofnanomaterialsinlivingenvironmentshasrarelybeenstudied.
Recently,DiCoratoetal.
[11]andSoukupetal.
[24]showedthatmagneticheatgeneration(magnetichyperthermia)canrevealthelocalorganizationofmagneticnanoparticles,providingamagneticheatingsignaturerelateddirectlytothenanoparticles'localanisotropyandinteractions.
Thesemeas-urementswereperformedinsituincellularsamples(invitro),withmagneticnanoparticlesconnedwithinendosomesandinveryclosemutualcontact.
Thestrongconnementofthepar-ticlesandtheirinteractionsprovokedstericfrustrationwhichinturnmodiedthemagneticdynamicsresponsibleforheatgeneration,resultinginadropinheatingefciencyaftercancercelluptake.
Here,wedemonstratethatintracellularconnementofplas-monicnanoparticlesinendosomesalsoimpactstheirphoto-thermalefciency.
Whereasmagnetichyperthermiameasure-mentshadpreviouslyonlybeenmadeinvitro,weshowthatthisthermalsignaturealsoexistsinvivo,aftercellularinter-nalizationwithinatumor.
Tothebestofourknowledge,thisistherstreportonphotothermalmeasurementsasawayforreal-timeprobingtheeffectofthebiologicalmicroenviron-mentontheplasmonicpropertiesofnanoparticlesinsitu.
Byprovidinginsightsintonanoparticles/microenvironmentinter-actions,thisapproachofferstheopportunitytonon-invasivelyassesscellularuptakeofnanoparticles.
Consideringinvivoapplications,depictingtheinterplayofnanoparticleswithbio-logicalmediaateachstepoftheirlifetimecycle(fromaqueousdispersiontocellinternalization)isacriticalpointforultimateimplementationofnanostar-mediatedphotothermaltherapysettings.
Besides,theseresultsareinagreementwiththeobservationthattheplasmoncharacteristicsofindividualgoldnanoparticlesevolvewhentheparticlesformclusters,[35]sometimesevenpro-ducingasynergisticeffect.
Thisiswhatisobservedhere,atagivenlaserwavelength,with25-nmgoldnanostars:whenirra-diatedat808nm,atthesameconcentration,thesenanostarsdeliveronlya6°Ctemperatureincreasewhenindividuallydis-persedinsolution,whilethetemperatureincrementleapstoalmost20°Cuponaggregationinsidecells.
6.
StrategySelectionforPhotothermalTherapyThisparticularcaseof25-nmnanostarsraisesquestionsastothebeststrategyfortranslatingplasmonicphotothermiafromthelaboratorytotheclinic.
Measurementsinaqueousdisper-sionssuggestedthat25-nmnanostarswouldnotbesuitableforinvivoapplications,giventheirlowNIRabsorption.
Indeed,at808nm,largernanostars(>50nm)yieldeda200%increaseinheatgeneration.
Yet,invivo,afterprocessingbycancercells(day3),25-nmnanostarswereasefcientas85-nmandeven150-nmnanostars.
Advancesincolloidchemistryhaveenabledthesynthesisofnanoparticleswithtightlycontrolledsizesandshapes,[38]allowingne-tuningoftheirphysicalproperties.
Herewedem-onstratethatgoldnanostarheatingefciencyindispersioncanbetunedbyadjustingtheirsizeorthelaserexcitationwave-length.
Althoughspectroscopyandthermalmeasurementshavebeenwidelyusedtocharacterizeandoptimizethedesignofplasmonicnanoparticlesinaqueousdispersion,weshowherethattherecordedphotothermalconversionefcienciesarenotpredictiveofthevaluesachievedineitherisolatedcellsorlivingtissues.
Moreover,weshowthat,regardlessoftheirsize,nanostarsconnedwithintumorcellendosomesexhibitsimilarheatingefcienciesuponnear-infraredlaserexcitation.
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KGaA,Weinheiminvivo,weproposethat25-nmnanostarsarethecandidatesofchoiceforphotothermaltumortherapy.
Fromapharmacokineticstandpoint,nanoparticlesizeisakeypropertyasitdeterminesinteractionswithcells,thevascula-tureandinterstitialtransport.
Indeed,thesizeofnanoparticlesdirectlyinuencestheinteractionwiththelocalfenestratedvasculature.
Nanoparticlesizeshouldbeinferiortovasculaturefenestrationcut-offinordertoallowtumoraccumulationbytheenhancedpermeabilityandretentioneffect.
[39]Consideringgoldnanoparticles,ithasbeenreportedthattheyshouldfeaturesub-100nmsizerangetobeabletocrosstumorvasculatureandmovethroughoutthetumorinterstitium.
[40]Inthisregardandtakingintoaccountthephotothermalconversionprolereportedhereinafterprocessingbycancercells,thesmallestgoldnanostarsinthe25–50nmsizerangeshoulddenitelybeconsideredbestsuitedforphotothermalcancertherapy.
Concerningtheinvivomodel,intratumoralinjectioninsubcutaneousmicetumorisaconvenientcancermodelforassessingnanoscalecellularheatingconversionofplasmonicnanoparticles.
Thismodelissuitabletotrackthenanophysicalpropertiesofnanomaterialsmeasuringthethermalefciencyfeatureinasupercial(accessibletolaserirradiation)andlocal-izedtumor,ensuringhighaccumulationofnanomaterials.
Inthecaseofsystemicadministration,theinjectednanoparticleswouldprobablybeuptakenbyseveralorganssuchaslungs,liver,andspleenandonlyafractionofitwouldreachthetumorsite.
Asaresult,wecouldexpecttoobservesimilarheatingconver-siontotheappliedlaserwavelengthsbutatareducedintensitydegree,asafunctionoftheamountofnanomaterialthatwouldreachthetumorsite.
However,itisnoteworthytomentionthatevenifnanoparticleswouldbedistributedthroughouttheorganism,theheatingeffectwouldremainconnedtotumorsiteasitistriggeredbylocallaserexposureatthetumorregion.
7.
ConclusionInsummary,bytestingavarietyofgoldnanostarswithaveragediametersrangingfrom25to150nm,andplasmonicreso-nancepeaksbetween500and1000nm,wefoundthatheatingefciencyinaqueousdispersiondependsonbothparticlesizeandexcitationlaserwavelength.
However,whenthenanostarswereinternalizedbycancercellsandconnedinendosomes,sizeandwavelengthdependencearestronglyattenuatedorevensuppressed.
Attenuationwasalsoobservedinvivowhenthenanostarswereinjectedintosolidtumors,butonlyaftercellularuptake,whichisreectedinthetemperatureprolesobtainedatdifferentlaserwavelengths.
Inviewofthismeasuredinvivobehavior,weinferthatthemostimportantdesignfeatureofgoldnanostarsforthermotherapyisnottheiradjustedplas-monicpeakmeasuredinaqueousdispersion,butrathertheirsizeandcoatingtoensureoptimalbiodistributioninvivo.
8.
ExperimentalSectionGoldNanostarSynthesis—Chemicals:Gold(III)chloridetrihydrate(HAuCl4),sodiumcitratetribasicdihydrate,polyvinylpyrrolidone(PVP,averageMW=10000),N,N-dimethylformamide(DMF)werepurchasedfromSigma–Aldrich.
EthanolwaspurchasedfromSharlau.
Allreactantswereusedwithoutfurtherpurication.
GoldNanostarSynthesis—SynthesisofGoldSeeds(14and40nm):Twobatchesofcitrate-stabilizedgoldseedswereprepared.
Goldseedswithdiameterof14.
3±0.
2nmwerepreparedaccordingtothemethodbyEnustunandTurkevich.
[41]Theseedswithdiameterof40.
2±1.
2nmwerepreparedaccordingtomethodbyPuntesandco-workers.
[42]GoldNanostarSynthesis—FunctionalizationwithPVP:Citrate-stabilizedgoldseeds(14and40nm)werefunctionalizedwithPVPusingGrafmethod.
[43]TheamountofPVPwascalculatedtoprovide≈60moleculespernm2ofparticlessurface.
ThePVPwasdissolvedinwaterinultrasonicbathfor15min.
Subsequently,thesolutioncontainingPVPwasaddeddropwisetothesolutioncontainingcitrate-stabilizedgoldseedsunderstirring.
Toensuretheadsorptionofthepolymeronparticlessurface,thereactionmixturewasstirredovernightatroomtemperature.
Finally,thePVP-stabilizedgoldseedswerecentrifuged(14nm–4000rpm;40nm–1200rpm)andredispersedinethanol.
Thenalconcentrationofgoldwasadjustedto3*103Mforbothseedssolution.
GoldNanostarSynthesis—SynthesisGoldNanostars(25nm,55nm):GoldnanostarswerepreparedbyfollowingtheprotocolbyKumaretal.
[44]Briey,anaqueoussolutionofHAuCl4(0.
041mL,100*103M)wasmixedwithasolutionofPVP(15mL,10*103M)inDMF.
ThemixturewasstirreduntilcompletedisappearanceoftheAu+3CTTSbandat325nm,followedbyrapidadditionofgoldseeds(14nm)inethanolundervigorousstirring.
Toobtainnanostarswith25and55nmofdiameter,thevolumeofseedswasfoundtobe0.
292and0.
023mL,respectively.
Thecolorofthesolutionchangesfromcolorlesstobluewithin40min,indicatingtheformationofgoldnanostars.
Thesampleswerecentrifugedthreetimesandredispersedinwater.
GoldNanostarSynthesis—SynthesisGoldNanostars(85nm,120nm,150nm):AnaqueoussolutionofHAuCl4(0.
041mL,100*103M)wasmixedwithasolutionofPVP(15mL,10*103M)inDMF.
ThemixturewasstirreduntilcompletedisappearanceoftheAu+3CTTSbandat325nm,followedbyrapidadditionofgoldseeds(40nm)inethanolundervigorousstirring.
Toobtainnanostarswith85,125,and150nmofdiameter,thevolumeofseedswasfoundtobe0.
159,0.
053,and0.
026mL,respectively.
Thecolorofthesolutionchangesfromcolorlesstobluewithin40min,indicatingtheformationofgoldnanostars.
Thesampleswerecentrifugedthreetimesandredispersedinwater.
DynamicLightScatteringAnalysis:DLScurvesofthesizedistributionwereobtainedusingNanoSizer(Zeta-Sizer,MalvernInstrument,UK).
CellCulture,LabellingandUptakeAssays:Humanprostatecancercells(PC3,ATCCCRL-1435)wereculturedinDMEMsupplementedwith10%FBSand1%penicillin,andmaintainedat37°Cwith5%CO2untilconuence.
Cellswereincubatedwithnanostarsofdifferentsizes(from25to150nm)at[Au]=0.
02*103Mfor16hinRoswellParkMemorialInstitutemedium(RPMI)at37°Cintwo150-cm2asks(≈30millioncells).
Themediumwasthenremovedandthecellswerewashedwithculturemedium.
Labelledcellsweredetached,centrifugedandresuspendedinPBSforfurtherexperiments.
ElementalAnalysis:TheconcentrationofgoldinnanostaraqueousdispersionandcellswasanalyzedbyICP-AES.
ThesamplesweredigestedinconcentratedHNO3for1hat90–100°C,thenrecoveredanddilutedin1%HCl.
UV–vis–NIRSpectroscopy:OpticalabsorptionmeasurementsofAunanostarsinaqueousdispersionandincancercellsinvitrowerecarriedoutincommercialspectrophotometers(Agilent8453and50scanCary,Varian)inthe300–1100nmspectralrange.
Samplepreparation(preparationofacelllysateexcludingthenuclei)foracquiringthecellularspectraisdescribedinthecaptionofFigureS7,SupportingInformation.
CytotoxicityAssay:CellviabilityafterincubationwiththedifferentAunanostarswasevaluatedintheAlamarBlueassay(LifeTechnologies).
Labelledcellswereincubatedwith10%AlamarBlueinDMEMwithoutredphenolfor2handthentransferredtoa96-wellplateforanalysiswithamicroplatereader(BMGFluoStarGalaxy)atanexcitationwavelengthof550nmwithuorescencedetectionat590nm.
ViabilityAdv.
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KGaA,Weinheimwasdeterminedbycomparisonwithcontrolcells(100%).
Allreportedexperimentswereperformedintriplicate.
PhotothermalMeasurementsinAqueousDispersionandinCellsInVitro:PhotothermalmeasurementsweremadewithvisibleandNIRlasers(680,808,and1064nm;LaserComponentsS.
A.
S(France))withexternaladjustablepowersettings(0–5W).
Thesample(aqueousnanoparticledispersionorsuspensionofAunanostar-loadedcells)containedina0.
5-mLtubewasilluminatedat2.
5–3cmdistancewithalaserspotof1cm2.
Thelaserpowerwasxedat1Wcm2.
Thetemperatureelevationwasrecordedwithaninfraredthermalimagingcamera(FLIRSC7000)inrealtime,everysecond,inthetemperaturerangeof25to70°C.
Thetemperatureelevationwasmeasuredasafunctionoftime(dT/dt)attheinitiallinearslope(t≈30s)inordertoevaluatetheheatingeffectintermsofSAR,powerdissipationperunitmassofelement(Wg1).
SARwascalculatedusingthefollowingformula:SAR=CVmdTdtswheremisthetotalmassofgoldinthesample,Cisthespecicheatcapacityofthesample(Cwater=4185JL1K1,Ccell=4125JL1K1),andVsisthesamplevolume.
PhotothermalMeasurementsInVivo:Six-week-oldNavalMedicalResearchInstitute(NMRI)malenudemiceweighing20±1g,providedbyJanvierLaboratoriesFrance,werehostedinthefacilitiesofAnimalerieBuffon,InstituteJacquesMonod,Paris7University.
Theywereacclimatizedfor1weekbeforeuse,inkeepingwithEuropeanstandardsofanimalcareandwell-being.
Solidtumorswereinducedbysubcutaneousinjectionof2*106PC3humanprostatecarcinomacellsin100Lofphysiologicalsaline(PBS)intheleftandrightanks.
Whenthetumorsreachedavolumeofabout125mm3,theywereinjectedwith100LofAunanostars(25,85,or150nm)insalinedispersionat[Au]=0.
75*103M.
Twenty-onetumorsin12animalsweredividedintofourgroups:6tumorswereinjectedwith25-nmAunanostars,6with85-nmAunanostars,and6with150-nmAunanostars;3noninjectedtumorsservedascontrols.
Thetumorswereilluminatedwiththreelasers(680,808,and1064nm)at1Wcmatadistanceof3cmfor5minondays0,1,and3postinjection.
Thetumorsurfacetemperaturewasmonitoredwithaninfraredthermalcamera(FLIRSC7000,FLIRSystems,Inc.
),eachmeasurementbeingmadeintriplicate.
Duringthemeasurementstheanimalswereanesthetizedwithketamine/xylazine.
Theanimalsweresacricedwhencollateraltumorsreached1cm3.
TEM:TEMimagesofaqueousdispersionwereobtainedwithaJEOLJEM-1400PLUStransmissionelectronmicroscopeoperatingatanaccelerationof120kV(CICbiomaGUNE,Spain).
TEMmicrographsofAunanostarsincellswereacquiredusingaHitachiHT7700operatingat80kV(MIMA2platform,INRA,Jouy-en-Josas,France).
TumorcellswereincubatedwithAunanostarsandxedwith5%glutaraldehydein0.
1molL1sodiumcacodylatebuffer,thengraduallydehydratedinethanolandstainedwith1%osmiumtetroxideand1.
5%potassiumcyanoferrate.
ThesampleswereembeddedinEponandsectionedforanalysis.
Mousetumorswerecutinto1mm3pieces,xedwith2%glutaraldehydein0.
1Msodiumcacodylatebufferandkeptin0.
1Msodiumcacodylateand0.
2Msucrosebuffer,thenpostxedwiththesameprotocolasforisolatedtumorcellsbeforebeingcutintothinsections(70nm)forobservation.
SupportingInformationSupportingInformationisavailablefromtheWileyOnlineLibraryorfromtheauthor.
AcknowledgementsThisworkwassupportedbytheMarieCurieIntra-EuropeanProjectFP7-PEOPLE-2013-740IEF-62647.
TheauthorsaregratefultoA.
DjematfromAnimalerieBuffonforanimalcare.
L.
M.
L.
-M.
acknowledgesfundingfromtheEuropeanResearchCouncil(ERCAdvancedGrant#267867,Plasmaquo)andtheSpanishMinisteriodeEconomíayCompetitividad(MAT2013-46101-R).
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