金纳米颗粒通过腙键负载DOX进行肿瘤干细胞治疗 - 图文

840T.-M.Sunetal./Biomaterials35(2014)836e845

Fig.3.DOX-Hyd@AuNPsovercomedrugresistanceofCSCs.(a)DOX-Hyd@AuNPsovercometheDOXef?uxabilityofP-gpofCSCs.Dataareshownasmeans?s.e.m.(n?3).*P<0.005.(b)ExpressionofABCB1inadherentcellsandmammospherecellsdeterminedbyquantitativereal-timePCR(mRNA,left)andwesternblot(protein,right).Dataareshownasmeans?s.e.m.(n?6).(c)ConfocallaserscanningmicroscopyimagesofthelocalizationofDOX[13]andactivatedP-gp(green)inMDA-MB-231,BT-474,andMCF-7CSCsafterincubatingwithPBS,mPEG@AuNPs,freeDOX,DOXtmPEG@AuNPs,orDOX-Hyd@AuNPsfor2h.TheactivatedP-gpwasmarkedwithP-gpantibody(UIC2)(green).CellnucleiwerestainedwithDAPI(blue).Scalebar?4mm.(Forinterpretationofthereferencestocolorinthis?gurelegend,thereaderisreferredtothewebversionofthisarticle.)

mRNAandthecorrespondingencodedP-gpproteinlevels(Fig.3b),whichisconsistentwiththeintrinsicdrugresistancenatureofCSCs.TheactivatedP-gp,whichisover-expressedonthemem-braneofmammospherecells,wasvisualizedusingUIC2antibody(green)labeling(Fig.3c).DOXwasfoundtobindtoP-gpwhentheCSCcellsweretreatedwithfreeDOXorDOXtmPEG@AuNPsfor2hat37??C.ThiswaseventuallyexocytosedfromtheCSCcells,meaningthatthe?uorescenceofDOXwasmerelyobservedinthecytoplasm.Incontrast,althoughpartialDOXboundtotheactivatedP-gponthecellmembraneofCSCs,aremarkablyincreasedamountofDOX?uorescencewasobservedintheircytoplasm(Fig.3c)whentheCSCsweretreatedwithDOX-Hyd@AuNPsunderidenticalconditions,indicatingthatDOX-Hyd@AuNPscanbypasstheef?uxofP-gp.3.2.InhibitionofmammosphereformationandcancerinitiationactivityWenextexaminedwhethertheenhanceddeliveryofDOXintoCSCsbyDOX-Hyd@AuNPswouldreducethestemnessinbreastmammospherecells.Wetreatedthethreemammospherecellswiththevariousformulationsfor2h,andthentestedtheirabilitytoregeneratemammosphereaswellasthecellnumberineachregeneratedmammosphereonthe20thdayaftertreatments.Therewasnosigni?cantdifferenceintheregenerationofmammosphereorthecellnumberperregeneratedmammospherebetweenPBS,mPEG@AuNPs,freeDOX,andDOXtmPEG@AuNPstreatments.Conversely,treatmentwithDOX-Hyd@AuNPsledtoareductionofregeneratedmammosphereby40?0%(Fig.4a).Inaddition,thecellnumberperregeneratedmammospherewasreducedtoapproximately35?0%comparedwiththecontrol(Fig.4b).TheseresultssuggestthatDOX-Hyd@AuNPstreatmentofCSCsreducedthequantityofcellscapableofmammosphereregenerationwhichre?ectstheself-renewalabilityofCSCsinvitro[33,37,44,45].Forcon?rmation,wedeterminedtheminimalfrequencyofCSCswithinthetreatedthreemammospherecellpopulationbyinvitroLDA[33].ThenumberofMDA-MB-231mammospherecellscapableofregeneratingonemammosphereperwellafter10daysculturewascalculatedtobe10forPBSand14forbothfreeDOXandDOXtmPEG@AuNPs,respectively.However,DOX-Hyd@AuNPstreatmentincreasedthenumberofmammospherecellsto28perwelltoregenerateonemammosphere(Fig.S4).SimilarresultswereobservedfromthetreatedBT-474andMCF-7mammospherecells(Fig.S4),indicatingthatDOX-Hyd@AuNPssigni?cantlydecreasesT.-M.Sunetal./Biomaterials35(2014)836e845841

Fig.4.DOX-Hyd@AuNPssigni?cantlyaffectthestemnessofCSCs.(a)Quanti?cationoftheabilitytoregeneratemammosphereand(b)therelativecellnumberperregeneratedmammosphereofMDA-MB-231,BT-474orMCF-7mammospherecellsonday20afterincubationwithPBS,freeDOX,DOXtmPEG@AuNPs,andDOX-Hyd@AuNPsfor2h.Dataareshownasmeans?s.e.m.(n?3).*P<0.005.(c)IncidenceoftumorsofMDA-MB-231mammospherecellsseriallytransplantedintoNOD/SCIDmice.Thecellswerepre-treatedwithPBS,freeDOX,DOXtmPEG@AuNPs,andDOX-Hyd@AuNPs.Tumorsweremonitoredevery3daysbyobservationandpalpationforupto60days.

thestemnessofCSCsincomparisonwithfreeDOXandDOXtmPEG@AuNPs.WefurtherdeterminedthenumberofcellsrequiredtogeneratetumorsandCSCfrequencyinvivo.MDA-MB-231mammospherecellsweretreatedwithvariousformulationsfor2h.Equalcellnumbersfromeachofthetreatmentgroupswereinjectedinto10non-obesediabetic/severecombinedimmunode?cient(NOD/SCID)miceatfourdifferentcellnumbers,andtumorformationwasassessedafterby60days.TreatmentwithDOX-Hyd@AuNPssigni?cantlyreducedthetumorformationabilityofthecells,withtheCSCfrequency32efoldlowerthanthePBScontrolgroupandapproximately18efoldlowerthanfreeDOXandDOXtmPEG@AuNPs(Fig.4c).TounderstandwhethertheeffectofDOX-Hyd@AuNPsondecreasingthestemnessofCSCsisduetotheenhancedDNAdamageinducedbyDOXthatisreleasedintracellularly,weincu-batedMDA-MB-231mammospherecellsfor72hwithDOX-Hyd@AuNPsatanequivalentDOXdoseof1.3mg/ml.ThisisequivalenttotheIC50offreeDOXtreatmentinMDA-MB-231mammospherecells,andthistestwasusedtodeterminedtheDNAdouble-strandbreakage(DSB)markedbytheserine-139-phosphorylatedhistoneH2AX(g-H2AX)[46].ItwasobservedthatmuchmorepronouncedDNAdamage(higherMFIofg-H2AX)occurredwhenMDA-MB-231mammospherecellsweretreatedwithDOX-Hyd@AuNPsincomparisonwithothercontrols(Fig.S5),implyingthatDOX-Hyd@AuNPsmayinduceDNADSBinbreastCSCsfollowingintracellularDOXreleasefromthenanoparticles.TheeffectofDOXdeliverytobreastCSCsbynanoparticleswasfurtherdeterminedinvivo.MDA-MB-231,BT-474,andMCF-7breastmammospherecells(2?105)wereimplantedinthemammaryfatpadsof6NOD/SCIDmicepergroup.Themicewerethengivencontinuoustherapy(1injectionperdayatequivalentDOXdosesof0.3mg/kg)viatailveininjection,startingfromthe?rstdayaftermammospherecellsimplantation.Itisdemonstratedthatsus-tainedadministrationwithDOX-Hyd@AuNPsresultedinadelayintheonsetoftumorformationaswellasadecreaseintumorsize(Fig.5).AftertreatmentwithDOX-Hyd@AuNPs,tumorformationwasobservedin3outof6micereceivedMDA-MB-231mammo-spherecellsimplantation,whilenoneoftumorwasobservedwhenthemicereceivedBT-474orMCF-7mammospherecellsimplan-tation.Onthecontrary,therewasnosigni?cantdelayinthetumorformationbetweenPBS,mPEG@AuNPs,freeDOX,andDOXtmPEG@AuNPstreatments.TheseresultssuggestedthatDOX-Hyd@AuNPstreatmentcandirectlyaffectbreastCSCsinvivo,resultingintheprolongedtumorsuppression.3.3.TumorsuppressionandprolongedtumorsuppressionWenextinvestigatedwhetherDOX-Hyd@AuNPscouldenhancethedeliveryofDOXintotheengraftedbreasttumorsinvivo.WetreatedMDA-MB-231tumor-bearingmicewithdifferentformula-tionsviaintravenousinjectionatequivalentDOXdosesof1mg/kg,andanalyzedtheamountofDOXintumorsat24hpostinjection.842T.-M.Sunetal./Biomaterials35(2014)836e845

Fig.5.InhibitionoftumorformationofMDA-MB-231,BT-474orMCF-7mammospherecellsbyDOX-Hyd@AuNPsincomparisonwithotherformulations.Dataareshownasmeans?s.e.m.(n?6).

Fig.6ashowsthattheDOXlevelinthetumortissuewas955.1?64.8ng/gproteinformicereceivedasingleinjectionofDOX-Hyd@AuNPs,whilstthecorrespondinglevelsformicetreatedwithDOXaloneandDOXtmPEG@AuNPswereonly89.7?9.8ng/gand69.6?6.7ng/g,respectively.ThisdemonstratesthatDOX-Hyd@AuNPscanenhancetheaccumulationofDOXinbreasttu-mors,likelyduetotheenhancedpermeabilityandretentioneffectofnanoparticles.WefurtherdeterminedwhetherDOX-Hyd@AuNPsdeliveredmoreDOXintotheCSCsintumors.TheintracellularDOX?uores-cenceofCSCsde?nedbyCD44tCD24àALDHhiwasdeterminedby?owcytometricanalysis.Fig.6bshowsthattherelativemean?uorescenceintensityofDOXinCSCsaftertreatmentwithasingleinjectionofDOX-Hyd@AuNPswas2.01?0.38whencomparedwithPBStreatment(setas1.00).However,negligiblelevelsofDOXweredetectedintheCSCsfollowingsystemicadministrationofDOX(1.04?0.02)orDOXtmPEG@AuNPs(1.08?0.05).ToassesswhetherDOX-Hyd@AuNPscaninhibitbreasttumorgrowthfollowingsystemicadministration,wetreatedMDA-MB-231orthotopicxenografts-bearingmicewithDOX-Hyd@AuNPsorvariousotherformulationsthroughi.v.injectioneverydayfromthe10thdayafterxenograftimplantation.AsindicatedinFig.7a,DOX-Hyd@AuNPsatanequivalentDOXdoseof0.3mg/kgperinjectionsigni?cantlyreducedthetumorgrowth,whereastreatmentsoffreeDOXorDOXtmPEG@AuNPsatthesamedosesandfrequencyonlymoderatelyinhibitedtumorgrowth.Similarpatternswereobservedintheothertwobreastcancerorthotopicmodels(Fig.7bandc).Subsequently,weanalyzedthepercentageofCSCsmarkedwithCD44tCD24àALDHhiintheMDA-MB-231andBT-474tumormodelsandCSCsmarkedwithCD44tCD24àintheMCF-7tumormodelattheendoftherapy.IntheMDA-MB-231orthotopicxenograftmodel,DOX-Hyd@AuNPstreatmentresultedinadecreaseofpercentageofCSCpopulationintheresidualtumorcells,whichwas27.9?27.1%ofthatwithPBStreatment(setas100%)(Fig.7d).Incontrast,freeDOXorDOXtmPEG@AuNPstreatmentincreasedthispopulationbymorethanseventeen-fold(1804.1?35.5%and1706.3?49.4%,respectively,P<0.001).SimilartrendswerealsoobservedintheBT-474andMCF-7tumormodelsaftertreatment.ConsideringthattheenrichmentofCSCsinthetumoraftertreatmentmightleadtorapidtumorgrowthaftertreatment[28],theprolongedtumorsuppressionduringtheoff-therapystage(de?nedasthetimeafterthedrugtreatmentwasceased)wasexaminedbymeasuringtheslopeoftumorgrowthcurvesineachgroupfromday32today46(Fig.8).Asindicated,thereductionofCSCsinMDA-MB-231tumorscausedbyDOX-Hyd@AuNPstreat-mentsledtosigni?canttumorgrowthinhibitionafterdrugcessa-tion,showingarelativegrowthslopeof0.45tothatofPBStreatmentfromthetumorgrowthcurve.ThiswasmuchsmallerthanthatfollowingtreatmentswithfreeDOXorDOXtmPEG@AuNPstreatments(therelativegrowthslopeswereT.-M.Sunetal./Biomaterials35(2014)836e845843

Fig.8.Off-therapystudyofMDA-MB-231xenografttumorgroupsafterdifferenttreatments.Dataareshownasmeans?s.e.m.(n?6).

1.58and1.65tothatofPBStreatment,respectively).TheseresultsindicatethatDOX-Hyd@AuNPscanef?cientlyattenuatetumorgrowthafterdrugcessation,suggestingtheirtherapeuticpotentialintermsofkillingbreastCSCs.Fig.6.AccumulationofDOXinthe(a)tumortissueand(b)CSCsinthetumorsofMDA-MB-231xenograft-bearingmiceat24hposti.v.injectionofthevariousfor-mulations.Dataareshownasmeans?s.e.m.(n?3).*P<0.005.

4.DiscussionAlthoughdebatingontheCSCtheoryoverthepastfewyears,recentinvestigationshavetracedthecontributionofindividualFig.7.(aec)InhibitionofMDA-MB-231(a),BT-474(b),andMCF-7(c)xenografttumorgrowthbyDOX-Hyd@AuNPsincomparisonwithotherformulations.Dataareshownasmeans?s.e.m.(n?6).*P<0.0001.(d)AnalysesoftheCSCratioswithinMDA-MB-231,BT-474,andMCF-7tumorsafterthetumorsuppressionstudy.Dataareshownasmeans?s.e.m.(n?3).*P<0.005.