NRmail.126.com

Zhuetal.
JNanobiotechnol(2019)17:44https://doi.
org/10.
1186/s12951-019-0473-3RESEARCHSmartnanoplatformforsequentialdrugreleaseandenhancedchemothermaleffectofdualdrugloadedgoldnanorodvesiclesforcancertherapyFalianZhu1,GuozhuTan1,YingtaoZhong1,YaodongJiang2,LuluCai3,ZhiqiangYu4,ShuwenLiu4*andFeiRen1*AbstractBackground:Thecombinationofmultiplechemotherapeuticshasbeenusedintheclinicforenhancedcancerchemotherapy,however,frequentrelapse,chemo-resistanceandsideeffectsremainstherapeutichurdles.
Thus,thedevelopmentofco-deliverysystemwithenhancedtargetingandsynergisticdifferentmodaltreatmentshasbeenproposedaspromisingstrategiesforintensiveimprovementofthetherapeuticoutcomes.
Results:Wefabricatedananocarrierbasedongoldnanorods(AuNRs),cRGDpeptide-modifiedandmulti-stimuli-responsivepaclitaxel(PTX)andcurcumin(CUR)releaseforsynergisticanticancereffectandchemo-photothermaltherapy(PTX/CUR/AuNRs@cRGD).
ThespecificbandingofcRGDtoαvβ3integrinreceptoronthetumorcellsurfacesfacilitatedtheendocytosisofPTX/CUR/AuNRs@cRGD,andthenear-infraredray(NIR)furtherenhancedthedrugreleaseandchemotherapeuticalefficiency.
Comparedtosingledrug,singlemodeltreatmentorundecorated-PTX/CUR/AuNRs,thePTX/CUR/AuNRs@cRGDwithamildNIRshowedsignificantlyenhancedapoptosisandSphasearrestinthreecancercelllinesinvitro,andimproveddrugaccumulationintumorsitesaswellastumorgrowthinhi-bitioninvivo.
Conclusions:Thetumortargetedchemo-photothermaltherapywiththesynergisticeffectofdualdrugsprovidedaversatilestrategyforprecisecancertherapy.
Keywords:Stimuli-responsive,Sequentialdrugrelease,Synergisticeffect,Dualdrug,Combinationtherapy,NIRphotothermaleffectTheAuthor(s)2019.
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BackgroundStimuli-responsivenanocarriershavebeenwidelyinvestigatedaspotentialcontrollabledrug-releaseandenhancedcancer-targetingdrugdeliverysystem(DDSs)inrecentyears[1–3].
Thereareseveralspecificmaterials,chemicalbondsorphysicalinteractionson/inthenano-carrierswhichcanrespondtoinherentbiologicalfactors(e.
g.
,enzymesandpH)[4,5]aswellasexternalsignals(e.
g.
,temperatureandlight)[6,7].
Asoneoftheidealexternalstimuli,illuminationpossessesminimallyinva-siveandtunablecharactersthatcanbeeasilyachievedbyadjustingtheirradiationintensity.
Inthisregard,nanocarrierswithintensiveabsorptionsinthenear-infrared(NIR)region(650–900nm)areoftendesignedforinvivoNIR-responsivedrugdelivery[8–10].
Inaddi-tion,NIRlightcanfacilitateplasmonicphotothermaltherapy(PPTT)fromtheutilizationofphoto-absorbingagentssuchasgoldnanorods(AuNRs),hollowgoldOpenAccessJournalofNanobiotechnology*Correspondence:liusw@smu.
edu.
cn;paper_mail@126.
com1DepartmentofPharmacy,NanfangHospital,SchoolofPharmaceuticalSciences,GuangdongProvincialKeyLaboratoryofNewDrugScreening,SouthernMedicalUniversity,Guangzhou510515,China4SchoolofPharmaceuticalSciences,GuangdongProvincialKeyLaboratoryofNewDrugScreening,SouthernMedicalUniversity,Guangzhou510515,ChinaFulllistofauthorinformationisavailableattheendofthearticlePage2of15Zhuetal.
JNanobiotechnol(2019)17:44nanospheresandgoldnanocages[11–16],therebycaus-ingtumorablation.
Unfortunately,becauseoftheuna-voidabledepth-dependentdecayoflightscatteringandabsorption,themainchallengeofthePPTTisthelimitedtissuepenetration,whichresultsintheincompletetumoreradicationindeeptissues[17,18].
Therefore,itisneces-sarytodevelopthenovelsynergisticorcombinedthera-pieswithPPPTotherthanitalone.
ThecombinationofchemotherapyandPPTTsupplyfeasibilitytoenhancetheefficiencyofdrugdeliveryandpromoteintracellularuptakethankstoPPTTinducedchangesofcellmembranepermeability[19,20].
Besides,thedesignsystemofcombinedPPTTwithchemotherapythatacquiresstimuli-responsive,ultimatelyresultingintumorablationandprovidingmoreopportunitiesforon-demandtherapy[21].
Tothisend,avarietyofAuNRssystemshavebeenproposedforPPTTandcarryinganti-cancerdrugs,selectivelyreleasedtheirpayloadsatthecontrolledNIRarea,resultinginsynergisticchemoandphotothermo-therapeuticefficacy[22–24].
Multiplechemotherapeuticscombinationiscommonlyusedinclinictoimprovethetherapeuticactivityandmit-igatethesideeffects.
Themutationsofcancercellscouldbereducedandthemultidrugresistance(MDR)couldbedelayedviathecombinationofdualdrugswhichtargetthedifferentsignalingpathwaysormolecularsites[25,26].
Paclitaxel(PTX),oneofthemostvitalanticancercompounds,canstabilizemicrotubules,arrestthecan-cercellcycleattheG2/Mphaseandinduceapoptosis[27,28].
Additionally,curcumin(CUR)isknowntohavebroadpharmacologicalfunctions,includingantican-cerandanti-inflammatoryfunctions,withoutanymajorsideeffects[29,30].
Recentstudiessuggestthatasimul-taneoustreatmentofpaclitaxelandcurcumininducesdown-regulationofthePI3K/Aktandnuclearfactor-κB(NF-κB)signalingpathways,whichleadtoDNAconden-sationandfragmentation,aswellasintracellularROSincreasing,sothatresultsinenhancedtumorcellapopto-sis,reducedthetoxicityofdrugscomparedtousingCURorPTXalone[31,32].
DuetotheintenselylipophiliccharactersofPTXandCUR,manytypesofDDSshavebeenfabricatedandsequentiallyadministeredtoimprovetheirbioavailability.
Althoughthereareafewnano-DDSsreportedtoimprovethewatersolubilityofPTXandCURandovercomeundesiredsideeffects,theirapplicationinclinicalcancerareinadequateduetosinglemodaltreat-mentsandsimilarmolecularsites[33–35].
Thus,all-in-onesystemwithmultiplechemotherapeuticsandPPTTcharacterfordrugdeliveryinacontrolledandtriggeredmannerandcombinationofdifferentmodaltreatmentshavebeenofprominentsignificancetorealizesynergisticeradicationofcancer.
Toaddressthesechallenges,wedesignedatumor-tar-getedandmulti-stimuli-responsiveAuNRsforthecom-binationofPPTTandchemotherapy,whichwasmodifiedbycRGDpeptidesandsimultaneouslydeliveredboththepaclitaxelandcurcuminwithindependentreleaseman-ner(designatedasPTX/CUR/AuNRs@cRGD)(Fig.
1).
Topreparethenanocarrier,thiol-end-functionalizedcurcuminconjugatesweresynthesizedbycovalentlylinking11-mercaptoundecanoicacid(MUA)tocur-cumin(MUA–Cur)viaanesterbond.
MUA–CurandthiolendfunctionalizedPEGwasbothmodifiedontheAuNRsbyreplacingthecetyltrimethylammoniumbro-mide(CTAB)onthesurfaceofAuNRs.
PTXwasloadedinthehydrophobicalkanethiol-conjugatedpoly(ethyleneglycol)(PEG)monolayer.
ThecurcuminwassupposedtobereleasedastheesterbondsofMUA–Curdissociatedintheesterase-enrichedtumormicroenvironments.
ThePTXwascellulardeliveredthroughpartitioningofthedrugwithinthelipophilicplasmamembrane.
ThecRGDpeptidesspecificallybindtotheαvβ3integrinrecep-torsover-expressedontumorcells,thus,toenhancethetumorcelluptakeofthetherapeutics.
Moreover,theNIRlaserirradiationisappliedforphotothermaltherapyandfurthercontrolthereleaseofresidualPTXandCURon-demand.
Basedonthesehypotheses,thisuniquePTX/CUR/AuNRs@cRGDwasbelievedtoexhibitasuperiorstabilityandimprovedonsitedrugdeliverywithoutaprematuredosereductionunderphysiologicalcondi-tions,andintegratetheadvantagesoftheactive-target-ing,photo-chemotherapyandcontrolleddrugrelease.
Wesystematicallyinvestigatedtheinvitroevaluationincludingpresupposition,drugrelease,cellinternaliza-tion,tumorcellcytotoxicity;andinvivoantitumoreffi-cacyandsafetyofPTX/CUR/AuNRs@cRGD.
Theresultsexhibitedasufficientablationoftumorsviathesynergis-ticeffectanddemonstratedthatPTX/CUR/AuNRs@cRGDmightbeapromisingcandidateasananocarrierforchemophotothermalsynergisticcancertherapy.
MaterialsandmethodsMaterialsThechemicalsusedtopreparethegoldnanorodswereallpurchasedfromSigma-Aldrich(StLouis,MO,USA).
Dualfunctionalpoly(ethyleneglycol)(NH2–PEG–COOH,MW5000Da)andmPEG–NH2(MW5000Da)wereobtainedfromLaysanBio(Arab,AL,USA).
Pacli-taxelwasaproductofCivichemicaltechnologyCo.
,Ltd.
(Shanghai,China).
CurcuminwasobtainedfromTCI-Chemicalindustry(Tokyo,Japan).
c(RGDyK)waspurchasedfromtheChinaPeptides(Shanghai,China).
Calcein-AMandpropidiumiodide(PI)wereobtainedfromSigma-Aldrich(StLouis,MO,USA).
AllotherPage3of15Zhuetal.
JNanobiotechnol(2019)17:44solventsandreagentswereofanalyticalgradeanduseddirectly.
PreparationandcharacterizationofgoldnanorodsAuNRsweresynthesizedinaccordancewiththeseed-mediatedgrowthmethodusedinpreviousreports[36].
TheAuNRswerecharacterizedusingtheUV–Vis–NIRabsorptionprofilefromaspectrometer(Agilent8453,SantaClara,CA).
TheshapeoftheAuNRswasdeter-minedusingtransmissionelectronmicroscopy(TEM)(TecnaiG2Spirit,Hillsboro,OR).
ThesizeandzetapotentialoftheAuNRsweremeasuredusingdynamiclightscattering(DLS)(MalvernzetasizerNanoS90,Mal-vern,UK).
SynthesisandcharacterizationofMUA–CurInbrief,MUA(1.
091g,5mM),curcumin(3.
684g,10mM),and4-(dimethylamino)pyridine(DMAP)(0.
305g,2.
5mM)weredissolvedin120mLofanhy-drousdichloromethane(DCM)and15mLofdimethylformamide(DMF),followedbythedropwiseinjectionofDCC(1.
288g,6.
25mM)in20mLofanhydrousDCM.
Themixturewasreactedinanice-bathfor2handthenstirredfor5–6hatroomtemperature.
Thin-layerchro-matography(TLC)wasusedtomonitortheprogressofthereaction.
ToremovetheexcessDCM,thereactionmixturewasdriedusingarotaryevaporatoruntilreac-tioncompletion.
Thecrudeproductwaspurifiedthroughsilicagelcolumnchromatographywithagradientelu-tion.
Abrightyellowfinalproductwasobtainedbysol-ventevaporationandre-crystallization.
1HNMRanalysisoftheMUA–CurverifiedthattheMUAwassuccessfullyconjugatedwiththecurcumin.
SynthesisofMUA–PEGMUA–PEG–COOHwassynthesizedbycovalentbond-ingtheaminogroupofbifunctionalPEG(H2N–PEG–COOH)withthecarboxylicgroupsofMUA.
Briefly,MUA(1mM),H2N–PEG–COOH(10mg),andDMAP(0.
1mg)weredissolvedinanhydrousDCMinthepres-enceofDCC(0.
8mg)asacatalyst.
Thereactionmix-turewasstirredfor12hindarkness,andthesolventwasevaporated.
Toseparatetheexcessorganicprecipi-tant,theresiduewasdissolvedindeionizedwaterandFig.
1SchematicillustrationofPTX/CUR/AuNRs@cRGDasnovelvariousstimuli-responsivetheranosticnanoplatformsforcancertreatmentPage4of15Zhuetal.
JNanobiotechnol(2019)17:44filteredthrougha0.
2μmmembranefilter,followedbytheadditionofDTT(1mM)toreducethethiol.
Aftera30minreductioninthedark,theproductwascollectedbycentrifugation(8500rpm,20min)withacentrifugalfilter(MWCO3000Da,Millipore)andthenwashedwithdeionizedwaterthreetimes.
mPEG–MUAwaspreparedfromthemPEG–NH2andMUAusingidenticalproce-dures,andthetwolyophilizedsampleswerecharacter-izedby1HNMR.
cRGDpeptidetargetingmodificationTostabilizethecRGDpeptidemodificationsystem,theAuNRsweresuccessivelyconjugatedwithtwotypesofMUA–PEG(mPEG–MUAandMUA–PEG–COOH).
Firstly,0.
2mLof1mMmPEG–MUAwasaddedto10mLof0.
5nMAuNRs.
After10minofstirring,themixturewascentrifuged(14,000rpm)for10minandreconstitutedin10mLofdeionizedwater.
Subsequently,0.
2mLof1mMMUA–PEG–COOHwasintroducedandstirredfor1h,followedbycentrifugation(14,000rpm,10min)andre-dispersalinanidenticalvolumeofdeion-izedwater,whichformedamixedlayerofmPEG–MUAandMUA–PEG–COOHonthesurfaceofAuNRs.
ThependantcarboxylicgroupsofMUA–PEG–COOHallowedthemodificationoftheaminogroupsofthecRGDpeptide.
ThePEG-modifiedAuNRswereacti-vatedbyEDC(1mg/mL)andNHS(50μL)for5min;theactivatedAuNRswereaddedto100μLofcRGDyKpep-tidesolution(0.
5μMindeionizedwater).
Themixturewasgentlystirredfor2handcollectedbycentrifugation(14,000rpm,10min).
PaclitaxelandMUA–CurcoloadingForthedualdrugloading,12μLof5mMPTXinDMSOand100μLof10mMMUA–CurinDMSOweresimul-taneouslyaddedto2mLof0.
5nMcRGD-modifiedAuNRs;thesolutionwasstirredfor1hindarkness.
Thefinalproduct(PTX/CUR/AuNRs@cRGD)wascollectedbycentrifugation(14,000rpm,10min)andwaswashedoncewithdeionizedwater.
TheUV–Vis-NIRabsorptionprofile,sizeandzetapotentialwereobtainedusinganUV–Vis–NIRabsorptionspectrometerandaDLSMal-vernZetasizer.
TheamountofPTXorCURloadedinthePTX/CUR/AuNRs@cRGDwasevaluatedbyHPLC(Shi-madzu,LC-20ATJapan)throughareverse-phaseC18col-umn(4.
6mm*150mm,poresize5μm,Kromasil).
ThemobilephaseofPTXwasamixtureofacetonitrile:water(50:50,v/v)deliveredataflowrateof1.
0mL/min.
PTXwasdetectedat227nmwithanultraviolet–visibledetec-tor.
Amixtureofacetonitrile/watercontaining4%v/vofaceticacidwasusedasthemobilephaseofCUR.
The0.
8mL/minflowrateandawavelengthof430nmweresettotesttheCURloadingefficiency.
TodeterminethesurfacedensityofcRGDpeptidesboundonthePTX/CUR/AuNR@cRGD,wemeasuredtheamountsofcRGDthroughBCAassay.
Basedonthemanufacturer'spro-tocols,thereagentAwasmixedwithreagentBasBCAworkingsolution,then,1nMPTX/CUR/AuNR@cRGDwereaddedinto96-wellplates,followedbyculturedwith200μLBCAworkingsolutionat37°C.
Theassaywasperformedat570nmusingamicroplatereaderafter30minco-incubated.
DiR/PEG/AuNRs@cRGDorDiR/PEG/AuNRswerepreparedthroughtheidenticalproce-dures,DiRwereusedtoreplacePTXandwithoutaddi-tionMUA–Cur.
DiRisoneofNIRresponsivefluorescentprobetobeusedasmarkerforbio-distributionofDDSs.
DrugreleaseinvitroThereleaseofMUA–CurfromthePTX/CUR/AuNRs@cRGDwasmeasuredinthepresenceofdifferentconcen-trationsofesterase.
PTX/CUR/AuNRs@cRGDinwaterwasmixedwithesteraseinthetube,andthereleasemix-turewaskeptinthedarkat37°C.
Ateverygiventime,thetubeswerecentrifuged(2500rpm)for5min,andthesupernatantswerecollectedtoanalyzetheamountsofCURandPTXreleasedusingHPLC.
PTX/CUR/AuNRs@cRGDwasgentlyintroducedintoanidenticalvol-umeofDCM.
Afterthespecifiedtimeintervals,thetopDCMphasewascollected,andthePTXintheorganicsolutionwasseparatedbycentrifugation.
Toevaporatethesolvent,theresiduewasre-dispersedinmethanolandmeasuredbyHPLCtoestimatethecumulativepercent-agesofPTXandCURthathadbeenreleased.
CellcultureHepG2,KBandA549cellswereallpurchasedfromAmericanTypeCultureCollection(ATCC)(Manas-sas,USA).
TheHepG2andKBcellsweremaintainedinDMEMmedium,andtheA549cellswereculturedinRPMI1640medium.
Theabovemediumwassupple-mentedwith10%(V/V)FBS,100U/mLpenicillin,and100U/mLstreptomycin.
Thecellswereincubatedat37°Candahumidifiedatmospherecontaining5%CO2.
IntracellularuptakeFortheintracellularuptakestudies,theA549cellswereincubatedwithPEG/AuNRs,PTX/CUR/AuNRsorPTX/CUR/AuNRs@cRGDfor3,6,and12h.
Then,theexperimentalmediumwasremovedandthecellswerewashedthreetimeswithPBS.
ThetreatedcellsweredigestedwithtrypsinincludingEDTA,centrifuged,andharvestedaspellets.
Thesecellpelletswerefixedin2.
5%fixationglutaraldehyde,dehydrated,embeddedandcut,followedbyTEMobservationwithatransmissionelec-tronmicroscope(HitachiH-7500,Japan)at80kV.
Page5of15Zhuetal.
JNanobiotechnol(2019)17:44ConfocalmicroscopicstudieswerecarriedouttodetecttheinvitrocellularuptakeofPTX/CUR/AuNRs@cRGDandPTX/CUR/AuNRsinA549cells.
Briefly,A549cellswereculturedovernightonglassbottomdishesandtreatedwithPTX/CUR/AuNRs@cRGDorPTX/CUR/AuNRs(CURconcentrations:3.
125μM,650pMwithrespecttoAuNRsconcentration)for6hat37°C.
Then,thecellswerewashedwithPBS.
Thefluorescencewasvisualizedusingaconfocallaserscanningmicroscopy(OlympusFV1000,Tokyo,Japan).
TherapeuticefficacyinvitroTheA549,HepG2andKBcells(8*103cells/well)wereseededin96-wellplatesandallowedtoadhereat37°Cfor24h.
Then,theseedingmediumwasremovedandaddedtotheexperimentalmediumcontainingvariousconcentrationsofAuNRsCUR/AuNRs,PTX/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD(25–400pM,withrespecttoAuNRsconcentration).
Afterincubation,thecellviabilitywasevaluatedbycck-8assay.
Todeterminetheeffectsofcombiningchemo-therapyandPPTT,A549,HepG2orKBcellswereseededin96-wellplatesandculturedwith100μLofanexperi-mentalmediumthatcontainedequivalentconcentrationsofPTX/CUR/AuNRs,PEG/AuNRsorPTX/CUR/AuNRs@cRGD.
After24hofincubation,thePEG/AuNRsandPTX/CUR/AuNRs@cRGDgroupswereirradiatedwithaNIRlaser(λ=808nm;beamsize,5mm;powerintensity,0.
55–0.
7Wcm2)(Diodelaser,ChangchunNewIndustriesOptoelectronicsTech.
Co.
Ltd,China)for10min.
Thelasertreatmentcellswerefurtherincubatedfor1hwiththedrug-containingmedium,afterwhicheveryexperimentalgroupofcellswaswashedusingPBSandincubatedinfreshmediumfor48h.
Thecellviabilitywasalsodeterminedthroughthecck-8assay.
AnannexinV-FITCapoptosisdetectionkitwasusedtoassesstheapoptosisofthreetypesofcancercellsafterincubationwithnanocarrier.
Thecellcyclearrestwasperformedusingacellcycledetectionkit.
Accordingtothestandardprotocol,theA549,HepG2andKBcellswereincubatedwiththeCUR/AuNRs,PTX/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD.
Subsequently,PBSwasusedtowashthecellsandcooled70%ethanolwasaddedtofixthecellsfor4h.
Toremovetheethanolthroughcentrifugation,RNaseandpropiduiumiodidewereintro-ducedintothefixedcells.
Thepercentagesofcellsinthedifferentphaseswereexaminedusingaflowcytometer.
InvivoNIRFimagingToevaluatewhetherthenanocarriertargetedthetumor,invivoNIRFimagingwasperformed.
BALB/cnudemicebearingA549tumors(female,20–25g)wereinjectedwith200LofDiR/PEG/AuNRs@cRGDorDiR/PEG/AuNRs(2nM)throughthetailvein.
TheNIRFimagingofmicewasmonitoredusingasmall-animalimagingsystemwithanexcitationwavelengthof780nmat2,4,6and8hafterinjection.
Then,themiceweresacrificed,andthebio-distributionsofDiR/PEG/AuNRs@cRGDorDiR/PEG/AuNRswereinvestigatedandimagedusingtheremovedtumortissuesandmajororgans(heart,liver,spleen,lungsandkidneys).
InvivoantitumorefficacyAllanimalprocedureswereincompliancewiththeChinaCouncilonAnimalCareandUseprotocol.
Theexperimentalprocedureswereperformedinaccord-ancewiththeGuidelinesfortheInstitutionalAnimalCareandUseCommitteeofSouthernMedicalUniver-sity(Guangzhou,China)andapprovedbytheAnimalEthicsCommitteeofSouthernMedicalUniversity.
ToexaminetheantitumoreffectofPTX/CUR/AuNRs@cRGDinvivo,atumorxenograftanimalmodelwasestablished.
BALB/cnudemicebearinganA549tumorwererandomizedandassignedintofivegroups,withfivemiceineverygroup.
Whenthetumorvolumeincreasedto~120mm3,thefivegroupswereinjectedwith200LofPBS,the200LmixtureofMUA–CurandPTX(dissolvedinDMSOanddilutedwithPBS,0.
91mgkg1forMUA–Cur,1.
00mgkg1forPTX),PEG/AuNRs@cRGDorPTX/CUR/AuNRs@cRGD(200μL,thesamedosageofAuNRs2.
5mgkg1).
Thephotothermaltherapygroupswereirradiatedfor5minwithaNIRlaser(0.
95Wcm2)at24hafterincuba-tionwiththenanocarrier.
Thetumorsizewasobservedusingacaliperandthebodyweightsweremeasuredeverydayforthefollowing15days.
Thetumorvol-umeswerecalculatedastumorvolume=(tumorlength)*(tumorwidth)2/2.
Attheendoftreatment,themiceweresacrificed,andthetumorsandorgans(heart,liver,spleen,lungsandkidneys)wereremoved.
Toevaluatethebiocompatibilityofthenanocarrier,thecollectedtissues,includingthetumors,werewashedusingcoldPBS,fixedwiththeformalin,andembeddedinparaffin,H&Estainandobservationsusinganopticalmicroscope(OlympusIX71,Japan)wereperformed.
StatisticalanalysisAlldataareexpressedasthemean±standarddeviation.
Aone-wayANOVAfollowedbyDunnett'smultiplecom-parisontestandapairedt-testwereusedtoanalyzethestatisticalcomparisons.
Astatisticallysignificantdiffer-encewasdenotedwhenthePvaluewaslessthan0.
05.
Page6of15Zhuetal.
JNanobiotechnol(2019)17:44ResultsanddiscussionPreparationandcharacterizationofPTX/CUR/AuNRs@cRGDBasedontheseed-mediatedgrowthmethod,AuNRswereobtainedandcharacterizedusingtheUVabsorp-tionspectrum(Additionalfile1:FiguresS1A,S1B).
ThepreparedAuNRspossessedalocalizedsurfaceplasmonresonance(SPR)peakaround780nmandanaspectratioof3.
7.
TheAuNRshadanaveragesizeof55nm*15nmandapositivechargeof+38mVbecauseofthecationicsurfactantcoating(i.
e.
,CTAB).
ThesuccessfulformationoftheMUA–CurandMUA–PEG–COOHwasconfirmedby1HNMRanalysis.
The1HspectraoftheMUA–Curshowedthecharac-teristicpeakofcurcuminat9.
83ppm,doubletpeaksat3.
82ppmandthepeakofMUAat1.
29~1.
34ppm(Additionalfile1:FigureS1C).
Thetypical1HNMRpeaksofMUA–PEG–COOHareascribedtoacom-binationofthoseofMUAandH2N–PEG–COOH,showingthesuccessfulconjugationofMUAandH2N–PEG–COOH(Additionalfile1:FigureS1D).
Forbiomedicalapplications,AuNRswerefunctional-izedwithtwotypesofligandstoreplacetheCTAB,theMUA–CurandMUA–PEG–COOHweresuccessivelyanchoredontotheAuNRsbythoilatedlinkers,duetothehighaffinityofgoldforthiols.
TheMUA–CurandMUA–PEG–COOHcouldformahydrophobicregiononthesurfacesofAuNRs,whichfacilitatedthePTXloading.
Figure2adisplayedtheabsorptionprofilesofAuNRs,PTX,MUA–Cur,PEG/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD.
ComparedtoPEG/AuNRs,thePTX/CUR/AuNRshadadditionalabsorptionpeaksat232nmand416nmfromthePTXandMUA–Cur,respectively,thusconfirmingthesuc-cessofdualdrugloading.
ThependantcarboxylicgroupofMUA–PEG–COOHallowedthecovalentattachmentoftheaminogroupsofcRGDforsurfacemodification,therebyimprovingtheinvivobehaviorofPTX/CUR/AuNRs@cRGD.
Consequently,theabsorptionprofileof400600800100012000.
00.
20.
40.
60.
81.
0NormalizedAbsorbanceWavelength(nm)AuNRsPEG/AuNRsPTX/CUR/AuNRsPTXMUA-CurPTX/CUR/AuNRs@cRGDa020406080100Size(nm)CTAB/AuNRsPEG/AuNRsPTX/CUR/AuNRsPTX/CUR/AuNRs@cRGDb-10010203040CTAB/AuNRsPEG/AuNRsPTX/CUR/AuNRsPTX/CUR/AuNRs@cRGDZetapotential(mV)CdFig.
2CharacterizationofAuNRs.
aUV–Vis-NIRabsorptionprofilesofAuNRs,PTX,MUA–Cur,PEG/AuNRsbeforeandaftercRGDcoating.
bHydrodynamicdiametersofCTAB/AuNRs,PEG/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD.
cZetapotentialofCTAB/AuNRs,PEG/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD.
dTEManalysisofPTX/CUR/AuNRs@cRGDPage7of15Zhuetal.
JNanobiotechnol(2019)17:44PTX/CUR/AuNRs@cRGDindicateda12nmredshift(from772to784nm)oftheoriginalbasis.
TheredshiftoftheSPRbandofPTX/CUR/AuNRs@cRGDafterbeingfunctionedwithcRGDwasattributedtothevarietydielectricconstantofthesurroundingsoftheAuNRs.
Toverifysuccessfulsurfacemodifications,thesizeandzetapotentialsoftheAuNRsatthedifferentfunctionalstageswerealsomeasured.
TheDLShistogramsofPEG/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGDhadaverageparticlesizeof78.
08±1.
64,91.
74±4.
66and93.
76±3.
43nm,respectively(Fig.
2b).
TheaveragesizeoftheAuNRs(46.
68±0.
42nm)increased30nmwhenconjugatedwiththeMUA–PEG–COOH,andwasfurtheraugmentedby15nmaftercRGDcoating.
ThehydrodynamicsizedistributioncurveandPDIvalueofeachnanoparticle(CTAB/AuNRs,PEG/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD)wereshowninAdditionalfile1:FigureS2.
WhenthePEGreplacedCATBtostabilizetheAuNRs,theparticledis-persitydecreasedalittle.
AfterthePTX/CUR/AuNRsmodificationwithcRGD,thePTX/CUR/AuNRs@cRGDwasabout0.
15smallerthantheirPTX/CUR/AuNRsintheparticledispersity.
TheseresultsindicatethatthehydrophilicPEGexteriorandcRGDmodificationmakethesystemphysiologicallystable.
ThezetapotentialofthePEG/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGDweredeterminedtobe11.
70,1.
75,and8.
42mV,respectively(Fig.
2c).
Thesystemfirstlyunder-wentthereversionofthechargeaftertheMUA–PEG–COOHmodification,whilethezetapotentialreturnedtopositiveafterconjugatingwithcRGD.
Together,theabsorptionspectrum,DLSandzetapotentialresultswerecompellingproofofthesuccessfulpreparationofthenanocarrier.
ComparetoAuNRs,TEMofPTX/CUR/AuNRs@cRGDpresentednoapparentchangesaftermodi-fication(Fig.
2d).
Additionally,ithasbeenreportedthatthethiol/disulfideredoxcouplecancoexistonthegoldnanoparticlessurfaceandisfavoredbythedisulfidefor-mationforthehigh-densitygoldsurfacecoating[37].
TheloadingefficiencyofPTXandMUA–Curwere7.
6%(wt%)and16.
43%(wt%),respectively,examinedbyHPLC(Additionalfile1:FigureS3).
Itwasfoundthat4.
56*103PTXand9.
85*104CURwassimultaneouslyloadedperAuNRinthesystemofPTX/CUR/AuNRs@cRGD(ThedetailedcalculationofthenumberofdrugperAushowedinAdditionalfile1).
WemeasuredthesurfacedensityofcRGDpeptidesandtheresultsindicatedthatcRGDpeptidesefficiencywas37.
3%.
ControlleddrugreleaseandcellularuptakeinvitroBasedonourdesignconception,thereleasebehav-iorsofPTXandCURfromPTX/CUR/AuNRs@cRGDshouldbeindependentandhavenoinfluenceoneachother.
Tovalidatethishypothesis,weevalu-atedthePTXorCURreleaseunderdifferentcondi-tions.
TheCURreleasewasexaminedinthepresenceofesteraseduetothecovalentbondingofMUA–Cur,andPTXwasexpectedtoexhibitnosignificantreleaseinthesamesituation.
Fordifferentconcentrationsofesterase,thereleasecurvesofCURpresenteddiffer-entreleaseefficacies;ahighconcentrationesteraseinthetumormicroenvironmentresultedinanincreaseddrugrelease[38,39].
TheCURreleaseachievedapla-teauandkeptasteadyandslowreleaseaftertherapidreleaseforthefirst4days(showninFig.
3a).
Incon-trast,noobviousPTXwasdeliveredundertheCURreleasecondition.
Asahydrophobicdrug,thePTXinoursystemprovidesanopportunityforreleaseintothe020406080100withoutesterase0.
5mg/ml0.
75mg/ml1.
0mg/mlPTXReleasedAccumulateddrugReleased(%)Time(days)aCURRelease02468100123456020406080Time(h)PBS-PTXMedium-PTXSerum-PTXDCM-PTXDCM-CURbAccumulateddrugreleased(%)Fig.
3Thecumulativedrugreleaseprofilesunderdifferentreleasemanner.
aThecumulativereleaseofcurcuminandpaclitaxelfromPTX/CUR/AuNRs@cRGDinthepresenceofdifferentconcentrationsofesterase.
bThecumulativereleaseprofileofPTXandCURfromPTX/CUR/AuNRs@cRGDPage8of15Zhuetal.
JNanobiotechnol(2019)17:44cellviathedetachmentofthedrugwithinthelipophilicplasmamembrane[40].
Toimitatethelipidmembrane,thePTXreleasewasestimatedusingawater/DCMtwo-phasesystem;thePTXwasprogressivelyreleasedfromthePTX/CUR/AuNRs@cRGDandassumedaclearbi-phaseprofile(Fig.
3b).
After6hofexposuretoDCM,morethan70%oftheloadedPTXwasreleased,andatraceamountofAuNRsappearedintheDCM,indicatinganefficientrelease.
WealsoobservedthatalmostnoCUR(<5%)wasreleasedfromthePTX/CUR/AuNRs@cRGDwithouttheesteraseovertime,demonstratingthattheloadeddualdrugsfolloweddifferentreleasepatterns,withtheCURrelyingonenzymehydrolysisandthePTXonhydrophobicinter-actions(Additionalfile1:FigureS4).
ToaccesswhetherthetemperatureincreaseinducedbylightexposureonAuNRsacceleratethedrugrelease,weadditionallyexaminedthedrugreleasecurveunderlightirradiation(Additionalfile1:FigureS5).
Itwasfoundthatprogres-sivereleaseofCURfromthePTX/CUR/AuNRs@cRGDclosedto85%atthefifthdaysunderlaserexposure,thelaserirradiationfacilitatedthePTXrelease,theaccumulateddrugreleasewas94%at6h.
TheresultssuggestedtheNIRfurtheracceleratedthedrugreleaseandenhancedchemotherapeuticalefficiency.
Asweknow,intracellularuptakeisanessentialproce-dureforthesubsequentstimuli-responsivedrugrelease.
ThelocationofPTX/CUR/AuNRs@cRGDwithinthecellswasobservedbyTEMimaging,A549cellswerecho-senduetotheirover-expressingofαvβ3integrinrecep-tor.
NoPEG/AuNRsparticleswereidentifiedwithintheobtainedimages(datanotshown),whichmightduetotheirnegativesurfacecharge.
AsmallamountofPTX/CUR/AuNRswerefoundinthecytoplasmafter6hco-incubation.
TheseobservationsagreedwiththeincreaseduptakeofPTX/CUR/AuNRsbyA549lungepithelialcellsuponintravascularinjection(Fig.
4a)[41].
Inter-estingly,theclustersofPTX/CUR/AuNRs@cRGDbothclosetothecellmembrane(Fig.
4b)andwithintheendo-somalcompartments(Fig.
4c)wereobservedafter6hco-incubation,suggestingthatthePTX/CUR/AuNRs@cRGDprovidedarelativelygoodcolloidalstability,andsubsequentlyenhancedthecellularuptake[42].
Moreo-ver,intracellularPTX/CUR/AuNRs@cRGDcouldbeFig.
4TEMimagesshowthelocalizationofPTX/CUR/AuNRsorPTX/CUR/AuNRs@cRGDinsideanA549cell.
aCellularinternalizationofPTX/CUR/AuNRsinsidetheA549cytoplasm.
bImageoftheclustersofPTX/CUR/AuNRs@cRGDlocatedonthecellmembrane.
cImageoftheclustersofPTX/CUR/AuNRs@cRGDlocatedattheendosomes.
dCellularinternalizationofPTX/CUR/AuNRs@cRGDinsidetheA549cytoplasm.
Scalebar:200nmPage9of15Zhuetal.
JNanobiotechnol(2019)17:44easilyfoundwithintheclearendosomalmembrane,thussupportingendocytosisasapossiblemechanismforthecellularuptakeandinlinewiththeliteraturedata[43].
Additionally,asmallfractionofPTX/CUR/AuNRs@cRGDappearedtobedispersedwithinthecytoplasmafter12hco-incubation,whichwasanindicativeofsup-plementarymechanismsofinternalization,suchasdif-fusionandendosomalrelease(Fig.
4d).
WhentheA549cellswerepretreatedwithanexcessoffreecRGDpep-tidestoretardαvβ3integrin,theuptakequantityofPTX/CUR/AuNRs@cRGDwassignificantlydecreased,thusverifyingthattheintracellularuptakeofPTX/CUR/AuNRs@cRGDwasprimarilyreliantonthespecialtargetoftheαvβ3integrin.
ThewholecellimagewasshowninAdditionalfile1:FigureS6,inwhichthecellmem-branebeapparentlyobserved.
TohaveastraightforwardinsightintothecellularuptakebehaviorofPTX/CUR/AuNRs@cRGDbymultiplecells,weprovidetheconfo-calimagesinA549cells.
AsshowninAdditionalfile1:FigureS7,strongerbluefluorescencesignalsfromCURwasdetectedforPTX/CUR/AuNRs@cRGDincompari-sonwiththePTX/CUR/AuNRs.
ItcanbeobservedthatPTX/CUR/AuNRs@cRGDorPTX/CUR/AuNRsdis-tributeinthenucleoplasm.
ThemodificationwithcRGDfacilitateinternalizedbyA549cellswhichcontainαvβ3integrinreceptor,whilePTX/CUR/AuNRspresentacer-tainamountofuptake.
Recentreportsshowedthatrod-shapedparticlesatthenanoscalehavebeenexhibitedtoshowhighercellularuptakeandexcellentabilityoftar-getingintumorxenografts[41,44].
Inthepreviousstudy,goldnanorodscouldloweredclearanceofmacrophageandfurtherresultinhighertumoraccumulation[45,46].
Therefore,ourresultscoherentlyindicatedthattherod-shapedparticles(AuNRs)associatedwithgreaterinternalizationrates.
ThecellularuptakeofPTX/CUR/AuNRs@cRGDwasinhibitedbycRGDpre-treatedA549cells.
ChemotherapyandphotothermaleffectsinvitroThechemotherapyeffectofAuNRsCUR/AuNRs,PTX/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGDwereexaminedonthreecancercelllines(A549,KBandHepG2)usingacck-8assay.
AsshowninFig.
5andAdditionalfile1:FigureS8,cellviabilityexhibitedareductioninbothtime-anddose-depend-entmanner,andremarkablecytotoxicitywasobservedforincubationwithPTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD.
Asexpected,CUR/AuNRsshowedmildcytotoxicitycomparewiththePTX/AuNRs,duetotheformer'santicancercapacityandsustainedreleasefromthesystem.
Notably,thedualdrug-loadedsystem(PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGD)exhibitedahighercytotoxicitythanthesingledrug-loadedsystem(CUR/AuNRsorPTX/AuNRs),despitethecellorigins.
Toestimatewhetherthedual-drug-loadedsystemhadthesynergisticeffects,wecal-culatedthecombinationindex(CI).
TheCIvaluewasquantifiedusingthefollowingequation:TheequationwhereaandbaretheIC50ofthePTXandCURinthedualdrug-loadedsystemandAandBaretheIC50ofthePTXandCURinthesingledrug-loadedsys-tem.
CIvalueslessthan1meantsynergism(Additionalfile1:TableS1).
TheCIvaluesforPTX/CUR/AuNRs@cRGDinthethreecancercelllineswerealllowerthan1,thusdemonstratingthattheenhancedcellmortalitywasascribedtothesynergisticeffectoftheCURandPTX[31].
Therefore,PTX/CUR/AuNRs@cRGDwascapableofachievingahighdualdrugloadingefficiencyandsyn-ergisticeffectsofthecombineddrugs.
Inlightofthechemotherapyresults,weinvestigatedtheeffectofPTX/CUR/AuNRs@cRGDonthePPTTandcombinationtherapy.
Asbeingknown,thephoto-thermalconversionefficiencyofAuNRs(Additionalfile1:FigureS9)shouldbeassessedbeforePPTTther-apy.
TheAuNRs,PBSandDMEMwereirradiatedwithan808nmNIRlaseratpowerdensitiesof0.
5,0.
7and0.
9Wcm2.
NoobvioustemperatureelevationoccurredforPBSandDMEM;whereasthetemperaturesoftheAuNRsincreasedrapidlyandevidently.
Aftera5minexposurewithlaserirradiationat0.
7Wcm2,thetem-peratureoftheAuNRsincreasedto45°Candreachedaplateau,indicatingthattheas-preparedAuNRspre-sentedanoutstandingNIRlight-responsivecapacity,whichencouragedustoperformthePPTTinvitro.
ForthePPTTtherapy,threecancercellswereincubatedwithPEG/AuNRsorPTX/CUR/AuNRs@cRGDfor24h,andexposedtotheNIRlaserfor5min,andthecellviabilitywasdetected.
Inaccordancewithexpectation,thehighconcentrationofAuNRsandtheincreasedilluminationintensityexertedaseverecytotoxiceffect.
Attractively,thetreatmentwithPTX/CUR/AuNRs@cRGDpluslaserirradiationresultedinthestrongestcytotoxicityamongallthetreatmentgroups(P<0.
05).
ThecellviabilityofPTX/CUR/AuNRs@cRGDwasapproximately10%,implyingexcellenttumorablationfromthecombinedeffectofthePPTTandchemotherapy(Fig.
6).
Wealsoevaluatedthesynergisteffectindex(Additionalfile1:TableS2);andtheresultsshowedthatasynergisticeffectwasfoundbetweenthechemotherapyandPPTTregard-lessofthecelltypesortheconcentrationofAuNRs.
Infact,inadditiontothecelleradicationduetothePPTT,anenhancedandcontrollabledrugreleaseaswellasthechemo-sensitizerderivedfromhyperthermiaalsocausedthecancercelldeaths[6].
(1)CI=aA+bBPage10of15Zhuetal.
JNanobiotechnol(2019)17:44Todeterminethepossiblemechanismofthecombin-ingtherapy,weassessedthecellapoptosisandnecrosisviaflowcytometryanalyses.
AnnexinV-FITC/PIdouble-stainedcellswereidentifiedtoanddistinguishtheapop-toticandnecroticcells.
Comparedwitheachsingledrug,chemotherapywithco-delivereddrugsrevealedenhancedcellapoptosis(Q2+Q3)(A549:71.
95%;HepG2:61.
63%;KB:64.
34%),whereasthetotalapoptoticratioofthecombinationtreatmentwithPPTTandchemotherapyfurtherincreasedto82.
88%(A549),69.
21%(HepG2),and85.
51%(KB)(Additionalfile1:FigureS10).
Theaboveresultsclearlydemonstratedthatdual-therapytreat-mentcouldimprovethetherapeuticefficiencyofPTX/CUR/AuNRs@cRGDbyinducingmorecellapoptosis.
Wealsostudiedtheunderlyingmechanismthroughthecellcycledistributionassayusingflowcytometry.
Com-paredtothesingledrugdeliverysystem,theco-deliverysystemarrestedmorecellsintheSphase.
ThenumberofSphasecellsinthePPTTandchemotherapytreatmentwascalculatedtobe56.
02%(A549),74.
90%(HepG2)and92.
99%(KB),respectively,whichwere31.
23%(A549),10.
1%(HepG2)and44.
02%(KB)higherthanthoseofthedual-drugchemotherapytreatments(Additionalfile1:FigureS11).
TheseresultsindicatedthatthePTX/CUR/AuNRs@cRGDtogetherwiththePPTTexhibitedanextensivetumorcell-killingefficiencybySphasearrestinthethreetestcancercelllines[47,48].
TumortargetingandantitumorefficacyinvivoToevaluatetheinvivobehaviorofPTX/CUR/AuNRs@cRGD,weestablishedsubcutaneousA549tumor-bearingmodels.
Whenthetumorvolumereached~120mm3,200μLofDiR/PEG/AuNRsorDiR/PEG/AuNRs@cRGDinPBSwasinjectedintravenouslyintothemice.
Wecollectedthetime-dependentbio-distributionofthenanoparticlesviaasmallanimalinvivoimagingsystem.
Figure7apresentstheimagesatdifferenttimepointsaftertheintravenousinjectionofthesample.
Obviously,DiR/PEG/AuNRs@cRGDdisplayedanotableaccumu-lationofnear-infraredfluorescence(NIRF)signalsat020406080100Concentration(pM)CellViability(%control)CUR/AuNRsPTX/AuNRsPTX/CUR/AuNRsPTX/CUR/AuNRs@cRGDAuNRsa020406080100Concentration(pM)CellViability(%control)CUR/AuNRsPTX/AuNRsPTX/CUR/AuNRsPTX/CUR/AuNRs@cRGDAuNRsb010020030040001002003004000100200300400020406080100Concentration(pM)CellViability(%control)CUR/AuNRsPTX/AuNRsPTX/CUR/AuNRsPTX/CUR/AuNRs@cRGDAuNRsCFig.
5ChemotherapeuticeffectsofA549,HepG2andKBtumorcells.
CellviabilityofaA549cells,bHepG2cells,cKBcellsincubatedwithACUR/AuNRs,PTX/AuNRs,PTX/CUR/AuNRsandPTX/CUR/AuNRs@cRGDfor48hPage11of15Zhuetal.
JNanobiotechnol(2019)17:44thetumors,andthesignalsgenerallyweakenedastimepassed,whereasDiR/PEG/AuNRsexhibitednoNIRFsignalsatthetumors.
Interestingly,strongerfluores-cencesignalsofDiR/PEG/AuNRs@cRGDwereobservedattumorsites2hafterinjectioncomparedwithuntar-getedofDiR/PEG/AuNRs;andthefluorescencesignalssequentiallystrengthenedandachievedthehighestlevelafter6hinjection.
Theincreasedtumoraccumulationaswellasretentionfavoredthegenerationofelevatedhyperthermiaandchemotherapyefficacy.
ToestimatetherelativetumorandorgandistributionofDiR/PEG/AuNRs@cRGD,themajororgansofthemicewerehar-vested2daysafterinjection,andthefluorescencesignalsoftheorganswereacquired(Fig.
7b).
WefoundthattheDiR/PEG/AuNRs@cRGDaccumulatedsignificantlyinthetumor,liverandspleen,butdidnotdistributetotheotherorgans.
TheseresultsimpliedthatDiR/PEG/AuNRs@cRGDcouldbestableinthebloodstreamandrapidlytargettumorswithoutobviousdamagetonormalorgans.
ConsideringthepossibilityofthedetachmentofDiRfromAuNRs,thequantitativebio-distributionofPTX/CUR/AuNRs@cRGDinvivowasestimatedat6hpost-injectionthroughanICP-MSanalysisofgoldcon-tentaccumulatedinthemajortissuesandtumor(Addi-tionalfile1:FigureS12).
TheresultsofPTX/CUR/AuNRs@cRGDexhibitedthattheaccumulationofAuinthe50pM100pM200pM400pM020406080100120++**###*#*A549celllaser0.
55W/cm2laser0.
7W/cm2concentrationcellviability(%control)ControlPTX/CUR/AuNRsPEG/AuNRs+laserPTX/CUR/AuNRs@cRGD+laserPTX/CUR/AuNRs@cRGD50pM100pM200pM400pM020406080100###***#*+laser0.
55W/cm2HepG2cellControlPTX/CUR/AuNRsPEG/AuNRs+laserPTX/CUR/AuNRs@cRGD+laserPTX/CUR/AuNRs@cRGD+laser0.
7W/cm2concentrationcellviability(%control)50pM100pM200pM400pM020406080100120####****+0.
55W/cm2laser+0.
7W/cm2laserKBcellControlPTX/CUR/AuNRsPEG/AuNRs+laserPTX/CUR/AuNRs@cRGD+laserPTX/CUR/AuNRs@cRGDconcentrationcellviability(%control)Fig.
6CombinationtherapyeffectsofthesmartnanocarrierstotheA549,HepG2andKBtumorcelllines.
PBSwasusedasacontrol.
Dataarepresentedasthemean±SD(n=5,*P<0.
05vsPTX/CUR/AuNRsand#P<0.
05vsPEG/AuNRs+laser)Page12of15Zhuetal.
JNanobiotechnol(2019)17:44liver,spleenandtumorswasmuchhigherthanthoseintheheart,lungsandkidneys,whereasAulevelsofPTX/CUR/AuNRsweremostlypresentintheliverandspleenofmice.
ItwasnoteworthythattheuptakeofPTX/CUR/AuNRs@cRGDinthetumorincreasedsignificantlycomparewiththePTX/CUR/AuNRs,likelyduetothecRGDmodification.
WefurtherexaminedthesynergisticefficacyofPTX/CUR/AuNRs@cRGDinvivo.
Themicebear-ingA549tumorswereinjectedwiththemixtureofMUA–CurandPTX,PEG/AuNRs@cRGDorPTX/CUR/AuNRs@cRGDandsalineasacontrol(Addi-tionalfile1:FigureS13).
TocombinethePPTTwiththetumorchemotherapy,weirradiatedthetumorusingan808nmNIRlaserfor5minatpowerdensityof0.
95Wcm2after24hinjection.
Thetumorvol-umesofeverygroupwererecordedduringthesubse-quent15daystoexaminetheantitumoreffect.
AsseeninFig.
8a,b,thePBScontrolgroupshoweda10-foldincreaseintumorvolume,indicatingnosuppressionoftumorgrowth,andthesimplemixtureofMUA–CurandPTXappearedpooranticancerpotency.
Theinhibi-tionoftumorgrowthinthephotothermaltreatedmice(PEG/AuNRs@cRGD+laser,inhibitionrate:53.
22%)wassimilartothetherapeuticeffectofdualdrugnano-chemotherapy(PTX/CUR/AuNRs@cRGD,inhibitionaLiverHeartb2h4h6h8hFig.
7RepresentativeNIRFimagesinvivo.
aNIRFimagesofA549tumor-bearingmiceinjectedwithDiR/PEG/AuNRsorDiR/PEG/AuNRs@cRGDat2,4,6and8hafterinjection(Thesemicircularregionindicatestumorsite).
bThemajororgandistributionofDiR/PEG/AuNRsorDiR/PEG/AuNRs@cRGDinthesubcutaneousA549tumormodelPage13of15Zhuetal.
JNanobiotechnol(2019)17:44rate:50.
50%),indicatingthatthesinglemodaltreat-menthadacomparableanticancerefficacy.
ItwasnotedthatthePTX/CUR/AuNRs@cRGDgroupwithNIRlightirradiationsignificantlyamplifiedthetumorabla-tioneffects(inhibitionrate:76.
67%)duetothecombi-nationofdualdrugchemotherapyandphotothermaltherapy(Fig.
8c).
Undermildlaserexposure,PTX/CUR/AuNRs@cRGDpossessedthehighestanticancerefficacyamongalltherapygroups,indicatingthattheNIRandtumormicroenvironmentresponsiveofPTX/CUR/AuNRs@cRGDpromotedtheon-demandreleaseandsynergisticeffectsofPTXandCURwithNIRirradiation.
Mostimportantly,PTX/CUR/AuNRs@cRGDpresentedanidealtumorablationefficacy,sug-gestingthatNIR-lightinducedanenhancementofthedualdrugdeliveryefficiencyandresultedinaremark-abletoxiceffecttowardcancercells[49–53].
Noobvi-ousbodyweightlosswasdetected15daysafterthetreatment,showingnosignificantorgandamageorabnormalitiesinthemicetreatedbythePTX/CUR/AuNRs@cRGD(Fig.
8d).
Toevaluatethepotentialsys-temictoxicityofPTX/CUR/AuNRs@cRGD,wecarriedouthistologicalexaminationsofthemajororgansviaH&Estaining(Additionalfile1:FigureS14).
ThetumortissueofthemicetreatedwithcombinationtherapyofPTX/CUR/AuNRs@cRGDandPPTTmanifestedaseverecancerapoptosis,cellshrinkageandnucleardamage,whichledtothelooseandfragiletumors.
Incontrast,inthemicetreatedwithsalineorthemixtureofMUA–CurandPTX,thetumorcellsemergedwithtypicalmorphologies,suchaslargecellnucleus,whichindicatedthecontinuousproliferationofthetumors.
Nonoticeableinflammatorylesionsororgandamagewereobservedinthecollectedmajororgans,demon-stratingtheexcellentbiocompatibilityandnegligiblesideeffectsofthePTX/CUR/AuNRs@cRGD.
0100200300400500600700*Tumorweight(mg)PBSMUA-Cur+PTXPTX/CUR/AuNRs@cRGDPEG/AuNRs@cRGD+laserPTX/CUR/AuNRs@cRGD+laser#a02468101214160400800120016002000TumorVolume(mm3)Time(days)PBSMUA-Cur+PTXPTX/CUR/AuNRs@cRGDPEG/AuNRs@cRGD+laserPTX/CUR/AuNRs@cRGD+laser*#b0246810121416181618202224262830Bodyweight(g)Time(days)PBSMUA-Cur+PTXPEG/AuNRs@cRGDPTX/CUR/AuNRs@RGDPTX/CUR/AuNRs@RGD+laserdcFig.
8Invivochemo-photothermalsynergistictherapyinmicebearingahumanlungcancerA549cellxenograft.
aTumorweightchangesofmiceinthedifferentgroups.
bChangesintumorvolume.
Dataarepresentedasthemean±SD(n=5,*P<0.
05vsPTX/CUR/AuNRs@cRGDand#P<0.
05vsPEG/AuNRs@cRGD+laser).
cPhotographoftheexcisedtumors.
dBodyweightofA549-xenograftedmicefollowingavarietyoftreatmentsPage14of15Zhuetal.
JNanobiotechnol(2019)17:44ConclusionsInconclusion,forcombinationofchemotherapyandPPTT,wefabricatedananocarrierbasedongoldnanorods,whichwasmultiplestimuli-responsiveforsequentialdualdrugreleaseandspecificallytumortar-getedbycRGDpeptidecoating.
Profitingfromthetumormicroenvironment-responsivecharacterofthepreparednanocarrierandtheintrinsicphotothermaleffectsofAuNRs,thesystempossessesadual-modeandindependentdrugreleasemanner:CURdependsonenzymehydroly-sis,andPTXreliesonhydrophobicinteractions.
ANIRlightlaserwellcontrolledthereleaseoftheremainderofthedrug.
ThecoatingofcRGDendowedtheAuNRswithbothenhancedtumorcellularinternalizationandnegligiblesideeffectstoothernormalorgans.
AlongwiththePPTT,synergeticanticancereffectinvitroandtumorablationwereobservedinmousemodels.
Thisstrategyintegratesthechemo-photothermaleffects,activetumortargeting,andmultiplestimuli-responsivedualdrugreleasetoprovideaversatileapproachtowardprecisecancernanotherapy.
AdditionalfileAdditionalfile1.
Additionalfiguresandtables.
Authors'contributionsLSandFRconceivedofthisworkanddraftedthismanuscript.
Inaddition,FZ,TGandZYperformedthepreparationandcharacteristicsofPTX/CUR/AuNRs@cRGD.
YJ,LCandZYhelpedwiththebiologicalstudyandperformedthestatisticalanalysis.
Allauthorsreadandapprovedthefinalmanuscript.
Authordetails1DepartmentofPharmacy,NanfangHospital,SchoolofPharmaceuticalSci-ences,GuangdongProvincialKeyLaboratoryofNewDrugScreening,South-ernMedicalUniversity,Guangzhou510515,China.
2DepartmentofUrology,NanfangHospital,SouthernMedicalUniversity,Guangzhou510515,China.
3PersonalizedDrugTherapyKeyLaboratoryofSichuanProvince,SichuanAcademyofMedicalSciencesandSichuanProvincialPeople'sHospital,SchoolofMedicine,UniversityofElectronicScienceandTechnologyofChina,Chengdu610072,Sichuan,China.
4SchoolofPharmaceuticalSciences,GuangdongProvincialKeyLaboratoryofNewDrugScreening,SouthernMedicalUniversity,Guangzhou510515,China.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
AvailabilityofdataandmaterialsAlldatageneratedoranalyzedduringthisstudyareincludedinthispublishedarticle.
ConsentforpublicationAllauthorsagreetobepublished.
EthicsapprovalandconsenttoparticipateAllanimalprocedureswereincompliancewiththeChinaCouncilonAnimalCareandUseprotocol.
TheexperimentalprocedureswereperformedinaccordancewiththeGuidelinesfortheInstitutionalAnimalCareandUseCommitteeofSouthernMedicalUniversity(Guangzhou,China)andapprovedbytheAnimalEthicsCommitteeofSouthernMedicalUniversity.
FundingThisresearchwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(GrantNo.
81402854),theNationalKeySpecialtyConstructionProjectofClinicalPharmacy(30305030698).
ThisworkwasalsosupportedbyagrantfromthePresidentFoundationofNanfangHospital,SouthernMedicalUniversity(2014033).
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpub-lishedmapsandinstitutionalaffiliations.
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