多功能共聚物抗癌藥偶聯(lián)物介導(dǎo)靶向藥物基因共傳輸抗腫瘤新生血管生成治療的研究

Amultifunctionalcopolymer-anticancerconjugatemediatedtargetedco-deliveryofdrugandgeneforsynergistictumoranti-angiogenesistherapyFangrongZhang,JianpingZhou*(StateKeyLaboratoryofNaturalMedicines,DepartmentofPharmaceutics,ChinaPharmaceutical24Tongjiaxiang,Nanjing210009,China)多功能共聚物-抗癌藥偶聯(lián)物介導(dǎo)靶向藥物/基因共傳輸抗腫瘤新生血管生成治療的研究王偉*，張芳榕，周建平*巷24號，南京210009，中國）Amultifunctionalcopolymer-anticancerconjugatechitosan-graft-polyethyleneimine-candesartan(CPC)containinglowmolecularweightchitosan(CS)backboneandpolyethyleneimine(PEI)armswithcandesartan(CD)conjugatedviaanamidebondwasfabricatedasatargetedco-deliverynanovectorofdrugandgeneforpotentialcancerHere,CDwasutilizedtospecificallybindtooverexpressedangiotensinIItype1receptoroftumorcells,strengthenendosomalbufferingcapacityofCPCandsuppresstumorangiogenesis.Theself-assembledCPC/pDNAcomplexesexhibiteddesirableandFoundationitems:theNationalNaturalScienceFoundationofChina(Nos.and81273469),theNaturalScienceFoundationofJiangsuProvince(No.BK2011624),theMinistryofEducationDoctoralProgramofHigherSpecializedResearchFundproject(Nos.*Correspondingauthors.+862583271102.E-mailaddresses:wangcpu@163zhoujp60@163(JianpingZhou).homogenousparticlesize,moderatepositivecharges,superiorandefficientreleaseofdrugandgeneinFlowcytometryandconfocallaserscanningmicroscopyanalysesconfirmedthatCD-targetedfunctionandCD-enhancedbufferingcapacityinducedhightransfection,specificcellularuptakeandefficientintracellulardeliveryofCPC/pDNAcomplexesinAT1R-overexpressedcells.Inaddition,CPC/wt-p53complexesco-deliveringCDandwildtypep53(wt-p53)geneachievedsynergisticangiogenesissuppression,ascomparedtomono-deliveryandmixed-deliverysystems.ThesefindingssuggestedthatCPCcouldbeanidealtumor-targetingnanovectorforsimultaneoustransferofdrugandgene.Keywords:Multifunctionalcopolymer-anticancerconjugate;Candesartan;typep53;AngiotensinIItype1receptortargeting;Co-delivery;anti-angiogenesis1.IntroductionAntiangiogenictherapyhasbeenrecognizedasaneffectiveantitumortreatmentstrategysinceJudahFolkman’slong-standingvisionofangiogenesisasatherapeutictarget[1].recentattemptsinrestoringp53functionusinggenedeliverysystemshavebeenreportedtosensitizecancercellstowardsanticancerdrugsandsuppressangiogenesis[2].Tumor-associatedangiogenesiswasinhibitedbythestableexpressionofexogenouswildtypep53(wt-p53)genewhichcoulddownregulatevascularendothelialgrowthfactor(VEGF)expressionbythedirectinteractionwithVEGFpromoterorindirectpathways(wt-p53-activatedsignalingcascades)[3].Ontheotherhand,co-deliverysystemssimultaneouslytransportinganticancerdrugandgeneintothesamecancercellsortissuesbymultifunctionalnanovectorsmayprovideanewparadigmincancertreatment,owingtotheirlowtoxicityandhightherapySotheco-deliveryofdoxorubicinandp53geneusingcationicβ-cyclodextrin-polyethyleniminevectors[4]ordouble-walledmicrospheres[5],andtheco-deliveryofpaclitaxelandp53genebysupramolecularself-assemblies[6]havebeensuccessfullydevelopedtoachievesynergistictherapeuticeffects.date,chitosan(CS),anaturallinearpolysaccharide,hasattractedmuchattentionasadrugandgenedeliveryvectorduetoitsspecialproperties,suchaseaseofmodification,biodegradabilityandlowimmunogenicity.However,thepotentialdrawbacksofCS,e.g.,poorratherlowtransfectionefficiencyandnon-specificityhavepreviouslylimiteditsextensiveapplication.overcomethesedilemmas,bothlowmolecularweightCSandaseriesofCSderivativeshavebeenproposed.Forinstance,CSwithamolecularweightof10kDa[7]wasreportedtoenhanceitswaterAlso,toimprovegenetransfectionCSgraftedwithimidazolemoieties[8]andcell-specificpolymersorgroups[9]werereported.Nevertheless,therearestillincreasingdemandsonCSderivativeswithallofdesirableproperties(watersolubility,hightransfectionefficiencyandfunctionality)forefficientandsafedrugand/orgeneInthiswork,wesuccessfullydevelopedanewmultifunctionalcopolymer-anticancerconjugatecalledchitosan-graft-polyethyleneimine-candesartan(CS-g-PEI-CD,CPC)toconstructaplatformforthetargetedco-deliveryofdrugandgenetotumorcells(Scheme1).Subsequently,thebufferingcapacityofCPCconjugatesandphysicochemicalcharacteristicsofCPC/pDNAcomplexeswereinvestigatedindetail.Furthertothis,thegenetransfectionmediatedbyCPCconjugatesunderdifferentconditionsandintracellulartraffickingofCPC/pDNAcomplexeswerestudiedinvitrotooptimizetheweight/weight(w/w)ratioofCPCconjugatestopDNAinCPC/pDNAcomplexesformulationandelucidatethemechanismsofeffectivetransfection.Meanwhile,theinhibitoryeffectsofCPC/wt-p53complexesonangiogenesiswerealsoexploredinvitro.Scheme1.Schematicillustrationofself-assemblyformation,accumulationattumortissue,intracellulartraffickingpathwayandsynergisticanti-angiogenesismechanismofCPC/wt-p53complexesasamultifunctionalco-deliverysystemfordrugandgene.2.Materialsandmethods2.1.MaterialsBranchedpolyethyleneimine(PEI,1.8Kand25K),amidaseandbafilomycinA1werepurchasedfromSigma-Aldrich(St.Louis,MO,USA).Chitosan(CS,10kDa,deacetylationdegree:92.53%)andcandesartan(CD)weresuppliedbyZhejiangBiochemicalCo.,Ltd.(Zhejiang,China)andAstraZeneca(Hamburg,Germany),respectively.DNaseI,LipofectamineTM2000,YOYO-1,Hoechst33258andTrackerRedwereofferedbyInvitrogenCo.(Carlsbad,CA,USA).RevertAidTMReverseTranscriptasekitandSYBRGreenKitwerepurchasedfromThermoFisherScientificInc.(Shanghai,China).Allotherreagentswereofanalyticalgradeandusedwithoutfurtherpurification.2.2.CellcultureHumanpancreaticadenocarcinomacelllinehumanbreastadenocarcinomacelllineMCF-7,humanembryonickidneyepithelialcelllineHEK293andhumanumbilicalveinendothelialcellsHUVECswerepurchasedfromtheCellBankofShanghaiInstituteofBiochemistryandCellChineseAcademyofSciences(Shanghai,China)andculturedinDMEMmedium(Gibco,USA)supplementedwith10%FBS(HyClone,USA),100U/mlpenicillinand100μg/mlstreptomycininahumidifiedatmosphereof95%air/5%CO2incubatorat37Allexperimentswereperformedoncellsinthelogarithmicphaseofgrowth.2.3.SynthesisofCS-g-PEI-CD(CPC)conjugatesandCS-g-PEI(CP)copolymers2.3.1.SynthesisofPEI-CDCDmg,mmol)wasdissolvedin3mlofThesolutionwasstirredonicefor5minfollowedbydrop-wiseadditionofasolutionofEDCmg,mmol)andNHSmg,mmol)in6mlofDMFwithagitation.Thereactionwasallowedtoproceedfor2hatroomtemperaturetoactivatecarboxylgroupsofCD.theabovesolutionwasaddedtoPEI(170.28mg,3.96mmol)dissolvedinmixedsolventofDMF/H2O(2:1,v/v),andthereactionwaskeptfor24hatwithstirring.Subsequently,thereactionmixturewasprecipitatedwithanexcessofcoldacetonetoremoveresidualCDandtheprecipitationwasfurtherpurifiedbydialysisagainstdeionizedwater(DW)(MWCO1000,3×2L).theresultingsolutionwaslyophilizedandstoredat-20°Cuntilfurtheruse.2.3.2OxidizationofCSpotassiumperiodatemg,mmol)dissolvedinacetatebuffer(pH4.5)wasdropwiseaddedintoCS(150mg,mmol)dissolvedinthesamesolventaftereachsolutionwasdegassedwithN2andadjustedto4°C.Aftermixing,thereactionwasperformedfor12hat4°Cindark,terminatedwithethyleneglycol(10%,v/v).Thesolutionwasdialyzed(MWCO3500)exhaustivelyagainstMNaClandthenDWfor24hrespectivelytoensurecompleteremovalofsmallmolecularimpurities,followedbylyophilizationandstorageat-20°C.2.3.3SynthesisofCPCconjugatesandCPcopolymersPEI-CD(112.5mg,0.056mmol)orPEI(27mg,0.63mmol)dissolvedindifferentvolumesofDWwasslowlyaddedto9mlofaqueoussolutioncontainingO-CS(75mg,mmolofaldehydegroups).Thereactionmixturewasgentlystirredfor48hat4°C.Afterthat,thesolutionofsodiumborohydride(68.09mg,mmol)wasdrop-wiseaddedtoabovesolutionstoreduceiminebond.Thereactioncontinuouslyproceededfor12hatwithgentleagitation.TheresultingcrudeproductwaspurifiedbydialysisinthesamemannerasdescribedinSectiontoremoveunreactedPEI-CDorPEI.Followingdialysis,thepolymersolutionswerelyophilizedandthefinalproductwasstoredat-20°C.2.4.CharacterizationofCPCconjugatesandCPcopolymersThechemicalstructuresofPEI-CD,O-CS,CPandCPCdissolvedinD2Orespectivelywereconfirmedby1HNMRat300MHz600,Bruker,Germany)withCDdissolvedinDMSO-d6andCSascontrols.Furthermore,nitrogencontentinPEI-CD,CPandCPCwasdeterminedusinganautomaticelementalanalyzerELIII,Elementar,Germany).ThedegreeofoxidizationofCS(dialdehydesrelativetotheglucosamineresiduesofCS,mol%)andthedegreeofconjugationofCDtoCSbackbone(CD/glucosamineresiduesofCS,mol%)wereevaluatedbasedontherelativepeakareaofthecorrelatedgroupsby1HNMR.ThedegreeofgraftingofPEItoCSbackbone(percentageofaveragechitosanglucosamineresiduesgraftedwithPEI,mol%)wasdeterminedbyapreviouslyreportedmethod[10].2.5.Determinationofbufferingcapacity10mlof0.15MaqueousNaClsolutioncontaining10mgpolymerswasadjustedtoaninitialpHofbyMNaOHandthentitratedusingMHCluntilthepHdeclinedto3.ThepHofallsolutionswasmeasuredbyapHmeter(pHS-25B,Dapu,China)aftereachadditionofMHCl(100μl).MNaClsolutiontitratedinthesamewaywastakenasablankcontrolandthebufferingcapacityofCSoverthepHrange(7.4~3.0)wasmeasuredowingtoitspoorsolubilityinthebasicsolution.2.6.Preparationandcharacteristicsofpolymer/pDNAcomplexes2.6.1.PreparationofCPC/pDNAcomplexesCPC/pDNAcomplexeswerepreparedatvariousweight/weight(w/w)ratiosofCPCconjugatestopDNA.CPCconjugatesandpDNAweredissolvedin20mMHEPESbuffer(pH7.4),respectively.obtainspecifiedw/wratiosofCPCconjugatestopDNA,theconjugatesstocksolutions(0.2mg/ml)wasaddedrapidlyto2μlofpDNAsolutionμg/μl),followedbyvortextoinduceself-assemblyofcomplexes.Theresultingsampleswereincubatedfor30minatpriortouse.CP/pDNAcomplexeswerepreparedasacontrol.2.6.2.GelretardationassayThecomplexescontaining0.6μgpDNAwereformulatedin20mMHEPESbuffer(pH7.4)and10mMsodiumacetatebuffer(pH5.5)atvariousw/wratios(0.25~2).Then,thecomplexessolutionswereelectrophoresedona0.8%agarosegelcontaining5μl/100mlGoldview(Amresco,USA)in0.5×(TBE)bufferat90Vfor60min.ThepDNAretardationwasvisualizedusingGel-Proanalyzer(Genegenius,Syngene,UK).2.6.3.Measurementofparticlesize,zetapotentialTheparticlesizeandzetapotentialoftheCP/pDNA(3μg)andCPC/pDNA(3μg)complexespreparedatdifferentw/wratiosorpHweremeasuredintriplicatebydynamiclightscattering(DLS)usingaMalvernZetasizer(NanoZS-90,Malverninstruments,UK)at25°Cwith90°scatteringangle.ThepHwasadjustedbyMHCl.2.6.4Saltstabilityofcomplexes50μlofpDNAsolution(0.1μg/μl)wasmixedwithequalvolumeofCPCorCPsolutiontoformulatecomplexesatthespecifiedw/wratio(w/w:5forCPC/pDNAcomplexesand4forCP/pDNAcomplexes).Afterincubationatfor30min,thesolutionsofcomplexesweredilutedwith400μlof500μlof2×PBSwasaddedintoabove-mentionedsolutionstogenerate1×PBSbuffersolutions(150mMPBS).Theparticlesizeofcomplexeswasmeasuredatprearrangedtimeintervals(0,1,2,4,8,12and24h).2.6.5.InvitroreleaseofCDfromcomplexestriggeredbyamidase10mlofCPC/pDNAcomplexessolutionswithdifferentconcentrationsofCD(10,50,100μM)wasaddedtotheequalvolumeofamidase(10μM)solution.Theresultingsolutionwasincubatedat37°Cwithgentleagitation.Atpredeterminedtimepoints,aliquots(1ml)withdrawnweremixedwith1mlofacetone/methanol(1/1,-20°C)toextractreleasedCD,andprecipitateamidaseandcomplexes.Then,thesupernatantharvestedbycentrifugationwasloadedontoHPLCsystemShimadzu,Japan)withEuropspher-100C18column(250mm×4.6mm,5μm)andelutedwithamobilephaseofmethanol/potassiumdihydrogenphosphatebuffer(66/34,pH2.5)at25°Candml/minofflowrate.Anexcitationandemissionwavelengthof265nmand395nmwereappliedforthemeasurementofCDconcentrations.2.7.InvitrocytotoxicityCells(1×104cells/well)wereseededinto96-wellplatesandincubatedovernight.CPCandPEI25Kpolymersatpredeterminedconcentration(5~100μg/ml)wereaddedtoeachwellinreplicatesofsixandincubatedfor48h,respectively.20μlofMTT(5mg/ml)inPBSwasaddedandincubatedforextra4h.eachwellwasreplacedwith100μlofDMSOandmeasuredat570nmusingaUniversalMicroplateReader(EL800,BIO-TEKInstrumentsInc.,USA).2.8.InvitrogenetransfectionstudiesCells(1×105cells/well)wereseededin6-wellplatesfor24htoreach70-80%confluenceatthetimeoftransfection.Priortotransfection,themediawerediscardedandcellswerewashedwithPBS.Then,thecellswereincubatedin2mloffreshserum-freemediacontaining400μlofCPC/pDNA(4μg)complexesatvariedw/wratiosfor6hforeffectiveuptakeofcomplexes.thetransfectionmediawereexchangedforfreshcompletegrowthmediaandthecellswereincubatedforadditional48h.TransfectionwithnakedpDNA,CP/pDNA,PEI25K/pDNAandLipofectamineTM2000/pDNAcomplexespreparedaccordingtothestandardprotocolwereusedascontrols.TheexpressionofGFPincellswasvisualizedandphotographedbyaconfocallaserscanningmicroscope(CLSM,LeicaTCSSP5,Germany).ThetransfectionefficiencydefinedasthepercentageofGFPpositivecellswasalsoquantifiedbyflowcytometer(BDUSA).Furthermore,thecompetitivebindingassayswerecarriedouttoevaluatetheactivetargetingofCPC-basedpDNAcomplexesagainstcells.CellswerepretreatedwithfreeCD(5,10μM)for1htosaturateoncellsurfacebeforethecomplexeswereadded.Ontheotherhand,tovalidatetheCD-enhancedendosomebufferingcapacityofCPCconjugatesatcelllevel,thetransfectionactivityofCPC/pDNAcomplexesinthepresenceofbafilomycinA1wastestedincells.Indetail,bafilomycinA1(200nM)inDMSOwasaddedafterdilutionofthecomplexeswithmedia,justbeforeadditionofthemediatothecellsintransfectionexperiment.2.9.IntracellulartraffickingThepDNAwaslabeledwithfluorescentdyeYOYO-1asdescribedpreviously[11].cells(1×105cells/dish)wereseededinto35-mmglass-bottomculturedishes(Greiner,Germany)andincubatedfor24h.Then,cellswithorwithoutpretreatmentoffreeCD(10μM)for1hwereincubatedwithYOYO-1-labeledCPC/pDNA(5μg)complexesat37°C.Atprearrangedtimeintervals,cellswererinsedtwicewithcoldPBStoremovenon-ingestedcomplexes,stainedwithTrackerRed(50nM)for30minat37°C,washedwithcoldPBSforthreetimesandfixedwith4%paraformaldehydefor10minat37°C.thefixedcellswerecounterstainedwithHoechst33258(10μg/ml)for15minandobservedbyCLSM.2.10.InvitroangiogenesisassayAngiogenesisassayinvitrowasperformedwithagelofbasementmembranetoevaluatetheinfluenceofCPC/wt-p53complexesontubularstructureformationduringangiogenesis.LiquefiedECMatrixTMwasplacedinpre-cooled96-wellplates(50μl/well),followedbysolidificationat37°Cfor30min.HUVECs(3×104cells/well)wereaddedtothepre-coated96-wellplatesandculturedinthemediacontainingCP/wt-p53complexes,CPC/pDNAcomplexes,CP/wt-p53complexesplusfreeCDorCPC/wt-p53complexesfor24h.Concentrations/amountsofdrugdeliverysystemscomposedofCDand/orwt-p53ineachwellwere2μμgforwt-p53gene.Thecellswerephotographedusinganinvertedbrightfieldmicroscope(NikonECLIPSETE-2000U,Japan)andtheimages(100×)weresoftware.StatisticalanalysisQuantitativedatawerepresentedasmean±standarddeviation(S.D.)fromtriplicateexperimentsperformedinaparallelmannerunlessotherwisenoted.Statisticalsignificancewastestedbytwo-tailedt-testorone-way*P<0.05,**P<or***P<wasconsideredstatisticallysignificant.3.Resultsanddiscussion3.1.SynthesisandcharacterizationofCPCconjugatesandCPcopolymersBranchedPEIasaconnectingbridgewasusedtoconjugatelowmolecularweightCSbackboneandCDligandthroughitsprimaryamines.Usingthisapproach,partoftheprimaryaminegroupsofPEIwasmodifiedwithCDbyEDCreaction,resultinginformationofamidebond-bearingPEI-CD.Then,PEI-CDwasgraftedontoO-CSpreparedbypotassiumperiodateoxidationreactiontosynthesizeCPCconjugatesbyreductiveaminationreaction.Asanegativecontrol,CPcopolymerswerealsosynthesizedbyreductiveaminationreactionbetweendialdehydegroupsofO-CSandfreeaminesofPEI.ThechemicalstructureofPEI-CDwasverifiedby1HNMR.Ascomparedtothe1HNMRspectrumofCD(Fig.1A),thecharacteristicpeaksofCDatppm(t,-CH3),5.08ppm(s,-OCH2-)and6.67-7.69ppm(m,Ar-H),andrepeatunitsat2.52-3.47ppm(m,-NHCH2CH2-)appearedinthe1HNMRspectrumofPEI-CD(Fig.1B).Thecompositionofsynthesizedpolymersbyperiodateoxidationreactionwasanalyzedby1HNMR.Thepeakatδ3.18ppm(s,H2)(Fig.1D)wassignificantlyattenuatedcomparedtothatinFig.1C,suggestingtheformationofO-CSwithdialdehyderesidues.ThedegreeofoxidizationofCScalculatedbyintegralvaluesfromtheprotonpeakofH2from1HNMRwas28.92%.TheappearanceofcharacteristicprotonpeaksofPEIatδppm(m,-NHCH2CH2-)confirmedthatPEIwasintroducedtoCS(Fig.1E),implyingthesynthesisofCPcopolymers.Meanwhile,asshowninFig.thepeaksatδppm(s,-COCH3,CS),3.63-3.89ppm(m,H3-6,CS),1.27-1.39ppm(t,-CH3,CD),ppm(m,Ar-H,CD)andppm(m,-NHCH2CH2-,PEI)revealedthattheCPCconjugateswereproducedundermildconditions.Fig.1.1H-NMRspectraofCD(A),PEI-CD(B),CS(C),O-CS(D),CP(E)andCPC(F).3.2.BufferingcapacityofCPCconjugatesAspresentedinFig.2A,thebufferingcapacityofCSwashighlyweakfromneutral(pH7.4)toacidic(pH5.1)condition.CPCexhibitedstrongerbufferingcapacityincomparisonwithCS,PEI1.8K,CPandPEI25K.3.3.FormationandcharacterizationofCPC/pDNAcomplexesAsshowninFig.2B-aandb,themigrationofpDNAwascompletelyretardedwhencomplexeswereformedatthesamew/wratioofforCPandCPCunderphysiologicalpHconditions(pH7.4).Interestingly,CP/pDNAandCPC/pDNAcomplexespreparedundermildlyacidicconditions(pH5.5)showednomigrationofpDNA,andonlyfaintbandswerefoundattheloadingslotataw/wratioofforCPandforCPC,aspointedbyarrowsinFig.2B-candd.DLSmeasurementrevealedthatparticlesizeofCPC/pDNAcomplexesatpHdecreasedwiththeincreaseinw/wratiooveraspecificrangeandwasminimum(~150nm)atthew/wratioof5(Fig.2C-a).AsimilartrendwasobservedinCP/pDNAcomplexesandtheparticlesizeofCP/pDNAcomplexesreachedminimum(~130nm)atthew/wof4.Ontheotherhand,thezetapotentialofcomplexesincreasedwithincreasingw/wratios,butthezetapotentialofCPC/pDNAcomplexes(<+20mV)atalldetectedw/wratioswaslowerthanthatofCP/pDNAcomplexes(Fig.2C-a).Asexpected,withdecreaseofpHfromto5thezetapotentialofthecomplexesincreasedfrom+18mVto+30mVforCP/pDNAcomplexes,andfrom+17mVto+39mVforCPC/pDNAcomplexes,respectively(Fig.2C-b).Thechangeinparticlesizeofthecomplexesisgenerallymonitoredunderphysiologicalsaltcondition(150mMPBS)asasimplebutessentialindextoestimatethestabilityofcomplexesinItwasfoundthatparticlesizeofCPC/pDNAcomplexesatthew/wratioof5remainedrelativelystableinPBSwithin24handwaschangedto~154nmwithapproximatelyafluctuationof2.67%,whilethatofCP/pDNAcomplexeswaschangedto~140nmwithanincreaseof7.69%(Fig.2C-c).Fig.2.(A)Acid-basetitrationprofilesofCS,PEI1.8K,CPCandPEI25K.(B)EffectsofdifferentpHvaluesonformationofCPC11/pDNAcomplexesatthew/wratioof0.25,0.5,0.75,1,and2,respectively.(C)intheparticlesize,zetapotentialofCP/CPC.3.4.InvitroreleaseAsshowninFig.3,theCDreleasedfromcomplexesintheabsenceofamidasewasnegligible(only1.56%)within24h,revealingthattheamidebondinCPCconjugateswashighlystable.Bycontrast,with10μMamidase,thereleaseofCDfromcomplexesattheCDconcentrationrangefrom100μMto10μMwasdramaticallyaccelerated,indicatingthehypersensitivecleavagereactionofamidebondinresponsetoamidase.Inparticular,thepresenceofamidaseresultedinthefastreleaseofCD(90.41%)fromcomplexeswithCDconcentrationof10μMfor12hincubation.Fig.3.Inamidase-triggeredreleaseofCDfromCPC/pDNAcomplexeswithdifferentconcentrationsofCD(10,50,100μM).3.5.InvitrocytotoxicitystudiesofCPCconjugatesAsshowninFig.4,significantinhibitoryeffectsofPEI25KonMCF-7andHEK293cellswereobserved,risingfromitsconsiderablesurfacepositivechargesandsubstantialprimaryaminesdisruptingPKCfunctionthroughdisturbanceofproteinkinaseactivity[12].contrast,thecellviabilityinalltestedcelllinesexposedtoCPorCPCpolymerswasapproximately90%ataconcentrationof20μg/ml,whichwasstillhigherthanthatadoptedinthefollowingstudies.Fig.4.ThecytotoxicityofCPC5,CPC11,CPC16andPEI25Kagainst(A),MCF-7(B)andHEK293(C)cellsafter48htreatmentwasevaluatedbyMTT3.6.InvitrotransfectionstudiesAsshowninFig.5A,GFPexpressionmediatedbyCPC/pDNAcomplexeswashigherthanthatbynakedpDNA,CP/pDNAandPEI25K/pDNAcomplexesat48hpost-transfection.Furthermore,itwasfoundthatthetransfectionefficiencyofCPC/pDNAcomplexesinMCF-7andHEK293cellsgraduallylowered,andsimilarresultswereobservedinFig.5B.TheCPC-mediatedGFPexpressionefficiencyincells(~35.82%)wasmuchhigherthanthatinHEK293cells(3.31-fold,P<0.01)andMCF-7cells(1.59-fold,P<0.05).Inaddition,CPC/pDNAcomplexesshowed16.47-fold,5.75-foldand2.18-foldincreasesintransfectionefficiencycomparedwithCP/pDNAcomplexesinMCF-7andHEK293cells,respectively.Meanwhile,Fig.5CdisplayedflowcytometrichistogramprofilesoffluorescenceintensityofGFPpositivecells.cellstreatedwithCPC/pDNAcomplexes±12.1%)displayed27.65-foldhighermeanfluorescenceintensitythanthatwithCP/pDNAcomplexestreatment±2.2%).furtherdemonstratetheCD-mediatedtargeteddeliveryofCPC/pDNAcomplexesviaweblockedincellswithvariousconcentrationsoffreeCD,followedbytransfectionwithCP/pDNAorCPC/pDNAcomplexes.AsshowninFig.6A,thetransfectionefficiencymediatedbyCD-containedCPCconjugatesremarkablydecreasedinaCDconcentration-dependentmannerwhencellswerepre-treatedwithfreeCD.Incontrast,freeCDshowednoinhibitoryeffectonthetransfectionefficiencyofCP/pDNAcomplexes.verifytheCD-enhancedbufferingcapacityofCPCconjugateswasresponsibleforelevatedtransfectionthetransfectionexperimentincellswasconductedintheabsenceandpresenceofbafilomycinA1whichwasaspecificinhibitorofvacuolar-typeandhasbeenreportedtoreducePEI-mediatedtransfectionefficiencybyinhibitingacidificationintheendosomesandlysosomes[13].AsshowninFig.6B,bafilomycinA1considerablyinhibitedtransfectionofCPC/pDNAandPEI25K/pDNAcomplexesbutnotCP/pDNAcomplexes.Fig.5.InGFPexpressionmediatedbyCPC/pDNA(4μgpEGFP-C3)complexes(theoptimalw/wratio:5,5and4inMCF-7andHEK293cells,respectively).Fig.6.Competitivebindingandprotonpumpinhibitionassaysbyflow3.7.IntracellulartraffickingofCPC/pDNAcomplexesAsshowninFig.7,theintracellulargreenfluorescencedotsinducedfromYOYO-1-labeledCPC/pDNAcomplexesandmergedfluorescencedots(yellowandcyanfluorescencedots)weresignificantlydecreasedateachtimepointwhencellswerepretreatedwithfreeCD,indicatingthatanendocytosismechanismwasinvolvedinthecellularuptakeofcomplexes.astimeextendedfrom2hto6h,moregreenpunctatescouldbeobservedincells,meanwhile,theredandgreenpunctatesweredistinctlyseparatedandmoreparticles(greendotsinmergedimages)werereleasedintocytosol.Inparticular,at6h,aportionofgreendotswerelocalizedinthenucleusandcyanfluorescencewasfound.Fig.7.IntracellulartraffickingoftheYOYO-1-labledCPC/pDNAcomplexesincellsatvarioustimeobservedbyCLSM.3.8.AngiogenesisassayinvitroFig.8Ashowedthatuntreatedcellsformedinterconnectingnetworksofcapillarytubes.Bycomparison,allofformulationsexerted,tosomeextent,suppressiveeffectontubularstructureformation.Thetubeformation-inhibitoryeffectofdifferentformulationsfollowedanorder:CPC/wt-p53complexes(co-deliverysystem)>CP/wt-p53complexesplusfreeCD(mixed-deliverysystem)>CPC/pDNAcomplexes(mono-deliverysystem)>CP/wt-p53complexes(mono-deliverysystem).Indetail,CPC/wt-p53complexesshowedaninhibitionrateof85.73%fortubeformationinHUVECs(Fig.8B).Fig.8.Inangiogenesis4.ConclusionsInthisamultifunctionalcopolymer-anticancerconjugate(CPC)basedontargetingenhancedendosomalbufferingcapacityandcombinedtumortherapystrategywassuccessfullyconstructedforsimultaneouslytumor-targeteddeliveryofCDandwt-p53gene.Thewell-formedCPC/pDNAcomplexesexhibitedsmallparticlesize,reasonablepositivecharges,excellentsaltandeffectivereleaseofdrugandgeneinCD-functionalizedCPCconjugatespotentiatedspecificcellularuptakeandeffectivelyintracellulardel