一种新型喜树碱衍生物纳米乳的制备性质考察及其抗肿瘤活性的研究整理版.docx

一种新型喜树碱衍生物纳米乳的制备性质考察及其抗肿瘤活性的研究整理版.docx

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一种新型喜树碱衍生物纳米乳的制备性质考察及其抗肿瘤活性的研究整理版

Formulationdevelopmentandanti-tumoractivityofalipidnanoemulsionforanovelhydrophobicanticancerdrug

MinHan,1Cai-XiaHe,1Qiu-LiFang,1Xiao-ChunYang,2Qing-QingZhao,1Qiao-JunHe,2Yong-ZhouHu,3Wen-QuanLiang,1BoYang,2Jian-QingGao1,

1InstituteofPharmaceutics,CollegeofPharmaceuticalSciences,ZhejiangUniversity,Hangzhou,China.

2InstituteofPharmacology&ToxicologyandBiochemicalPharmacy,CollegeofPharmaceuticalSciences,ZhejiangUniversity,Hangzhou,China.

3InstituteofMaterialMedica,CollegeofPharmaceuticalSciences,ZhejiangUniversity,Hangzhou,China.

ABSTRACT:

OBJECTIVEAnoil/waternanoemulsionwasdevelopedinthepresentstudytoenhancethesolubility,stabilityandanti-tumoractivityofanovel10-methoxy-9-nitrocamptothecin（MONCPT）.METHODSMONCPTnanoemulsionwaspreparedusingLipoidE80andcremophorELasmainemulsifiersbymicrofluidization.Thedropletsizeofthenanoemulsionwasmeasuredbydynamiclightscattering.Invitrodrugreleasewasmonitoredbymembranedialysis.KineticsofMONCPTtransformedintocarboxylicsaltwasperformedinphosphatebufferatdifferentpH.HemolysisofMONCPTnanoemulsionwasconductedinrabbiterythrocytes.SolubilizationcharacterofMONCPTinnanoemulsionwasexperimentedusingnileredasasolvatochromicprobe.InvitrocytotoxicityofthenanoemulsionwasmeasuredinA549andS180cellsusingSulforhodamineBproteinstainmethod,andsuppressionrateoftumorgrowthwasinvestigatedinS180-bearingmice.ThecellcycleeffectsofMONCPTnanoemulsiononS180cellswereanalyzedby flow cytometry.DistributionofthenanoemulsioninA549cellswasalsoinvestigatedbyfluorescenceimage.RESULTSMONCPTisincorporatedinthenanoemulsioninformoflactonewithconcentrationof489µg/mL,morethan200foldshigherthanthatinwater.ExperimentsusingnileredasasolvatochromicprobeindicatedthatmoreMONCPTmightbelocatedintheinterfacialsurfactantlayerofthenanoemulsionthanthatindiscreteoildropletorcontinuousaqueousphase.NanoemulsioncouldreleaseMONCPTinasustainedway,anditwasfurthershowntonotablypostponethehydrolysisofMONCPTwithlongerhydrolysishalf-lifetime（11.38h）innanoemulsionatpH7.4thanthatofMONCPTsolution（4.03h）.NoobvioushemolysiswascausedbyMOCPTnanoemulsioninrabbiterythrocytes.MONCPTnanoemulsionshowedamarkedincreaseincytotoxicactivity,23.6foldsand28.6foldsinS180cellsandA549cellsrespectivelyviaarrestingthecellatG2phase,comparedtothatinducedbyMONCPTinjection.Itcorrelatedwelltotheinvivoanti-tumoractivityofMONCPTnanoemulsionwithsuppressionrateof93.6%,whilethatofMONCPTinjectionwasonly24.2%atthesamedosage.Moreover,nanoemulsionexhibitedenhancedcapabilityofdeliveringdrugintomalignantcell'snucleusinvitro.CONCLUSIONThenanoemulsionpreparedexhibitedanimprovedMONCPTsolubility,stabilityandanti-tumoractivity,providingapromisingcarrierforcancerchemotherapyusingMONCPT.

KEYWORDS:

emulsion;invitrorelease;stability;nilered;anti-tumor;cellcycle

一种新型喜树碱衍生物纳米乳的制备、性质考察及其抗肿瘤活性研究

韩旻1，何彩霞1，方秋黎1，杨小春2，赵青青1，何俏军2，胡永洲3，梁文权1，杨波2，高建青1，*（1.浙江大学药学院药物制剂研究所；2.浙江大学药学院药理毒理与生化药学研究所；3.浙江大学药学院药物研究所。

杭州310058）

摘要：

目的制备一种新型喜树碱衍生物（10-甲氧基-9-硝基喜树碱）的纳米乳剂，以提高药物的溶解度、稳定性和抗肿瘤活性。

方法以LipoidE80和cremophorEL为乳化剂，通过微射流制备载药纳米乳剂，考察乳剂的粒径、开环动力学、体外溶出和溶血情况。

以尼罗红荧光探针作为溶剂化变色染料探讨乳剂的增溶性质，研究药物乳剂的体内外抗肿瘤活性，并通过流式分析考察乳剂对S180细胞周期的影响，另外以尼罗红为荧光探针研究乳剂对其在A549细胞核内分布的影响。

结果乳剂中药物浓度为489µg/mL，显著高于其水中溶解度（2.61µg/mL），深入研究发现更多的药物被增溶在乳剂中的油水界面膜内。

乳剂可以延缓药物的释放，另外在pH7.4缓冲液中，乳剂中药物的开环半衰期（11.38h）较其水溶液（4.03h）显著延长。

乳剂对体外家兔血红细胞无明显溶血作用，并可通过作用于细胞的G2期（S180细胞）发挥其良好的体内外抗肿瘤活性。

与其注射液相比，其体外对S180和A549细胞的杀伤作用可分别提高23.6和28.6倍，乳剂的S180小鼠体内抑瘤率为93.6%，明显高于其注射剂的24.2%。

另外与注射剂相比，乳剂还显示出一定的细胞核内递送药物的能力。

结论所制备的乳剂可明显提高10-甲氧基-9-硝基喜树碱的溶解度、稳定性和抗肿瘤活性。

关键词：

乳剂；体外溶出；稳定性；尼罗红；抗肿瘤；细胞周期

1INTRODUCTION

Knownasimportantanticancerdrugs,camptothecinderivatives,suchasirinotecan,GG-211,dx-8951f,topotecan,9-nitrocamptothecinand10-hydroxycamptothecinhaveattractedextensiveattentionfortheirbindingandinhibitionoftopoisomeraseⅠ（TopoⅠ）whichdistributedextensivelyintumorcells[1,2].However,theresponserateandoverallsurvivalratehavenotbeenimprovedsubstantiallyformostofthemcomparedtothatofcamptothecin[3].Although9-nitrocamptothecinpossessesapotentactivitywithlowtoxicity,difficultinpreparinglimitsitsresearchandclinicalapplication.Theintroductionofanitroatposition9,suchas9-nitrocamptothecin,hasshownsatisfactoryactivitywithlowtoxicity.However,9-nitrocamptothecinwasdifficulttopreparebynitrationofcamptothecinbecause12-nitrocamptothecinwasthemainproduct[4].Incontrast,10-alkoxy-9-nitrocamptothecin（MONCPT）couldeasilybepreparedfrom10-hydroxycamptothecin[5,6],whichpossessesapotentanti-tumoractivityduetoitsregulationofcellcycleorpotentialpropertyforinhibitingangiogenesisasrevealedinourpreviousstudies[7,8].However,poorsolubilityandstabilityofMONCPTlikeothercamptothecinderivativeslimitsitsfurtherinvivoresearches.Althoughtransformationofalactoneringcontainedincamptothecinderivativesintoanopen-ringhydroxycarboxylicacidinalkalinesolventsmayenhanceitssolubilitydramatically,aninevitabledecreaseinactivityorincreaseintoxicitymakesitinappropriateforapplication[9-11].

Meanwhile,considerableemphasishasbeengiventodevelopemulsionsystemsasdrugcarriersystembecauseofitsimprovedsolubilizationforwater-insolubleand/oroilinsolubleactivecompounds,modifieddrugreleasecharacteristic,protectingdrugfromtransformationanddeliveringdrugsintosometissuesspecificallyafterintravenousadministration[12-16].Beingaclassofstableemulsionscomposedofdiscreteoilphaseandcontinuousaqueousphaseseparatedbyamonolayerofsurfactantswithparticlediameterusuallylessthan100nm[17],nanoemulsionhavebeenshowntoincreasebioavailabilityandefficacyofanumberofcompoundssuchasinsulinandpaclitaxel[18,19].

Inpresentstudy,nanoemulsionincorporatingMONCPTwasdevelopedtoachieveapotentialparenteraldeliverysystemforMONCPT.Theinvestigationfocusesonthereleaseandstabilityofthedrugencapsulatedinnanoemulsion,aswellasitsanti-tumoractivitybothinvitroandinvivo.Furthermore,nileredwasusedasasolvatochromicprobetoinvestigatethemechanismofsolubilityenhancement.

Inpresentstudy,nanoemulsionincorporatingMONCPTwasdevelopedtoachieveapotentialparenteraldeliverysystemforMONCPT.Theinvestigationfocusesonthereleaseandstabilityofthedrugencapsulatedinnanoemulsion,aswellasitsanti-tumoractivitybothinvitroandinvivo.Furthermore,nileredwasusedasasolvatochromicprobetoinvestigatethemechanismofsolubilityenhancement,andthedistributionofnanoemulsionincellnucleus.

2MATERIALSANDMETHODS

2.1Materials

LipoidE80waspurchasedfromLipoidAG（Ludwigshafen,Germany）,whichconsistsof82.4%phosphatidylcholineand8.0%phosphatidylethandamineasmentionedinmanufacture’sspecification.CremophorELwasfurnishedbyBASF（Parsippany,NJ.USA）.SoybeanoilwaspurchasedfromTielingbeiyapharmaceuticalCo.（Tieling,China）.GlycerolformalwaskindlyprovidedbyElementis（London,England）.SulforhodamineB（SRB）waspurchasedfromSigma（StLouis,MO,USA）.Allotherchemicalsandreagentsusedwereofanalyticalorchromatographicgrade.

2.2AssayofMONCPT

Highperformanceliquidchromatography（HPLC）systemwascomposedofanAgilentG1310Apump,anAgilentG1314AVWDdetectorandaDiamonsil®C18reversephasecolumn（4.6×150mm,5um,DikmaTechnologies）.Themobilephaseconsistedofacetonitrile-1%triethylamineindistilledwater（32:

68）withpHadjustedto5.5byglacialaceticacid.Chromatographywasperformedat40℃andwavelengthwassetto383nm.

2.3PreparationofMONCPTnanoemulsion

0.27glipoidE80,0.36gcremophorEL,0.15gethanol,and15mgMONCPTweredissolvedinto1.8gsoybeanoilat70℃.6.0gPEG400,0.66gglyceroland6.0gglycerolformalweredissolvedin15gpurifiedwaterasaqueousphase.Aftermixingaqueousphasewithoilphaseat70℃,ThecoarsenanoemulsionwasobtainedbyhomogenizationusingaFJ-200highshearmixer（12000rpm,3min）（shanghaispecimenmodelLtd.,shanghai,china）,thenitwasfurtheremulsifiedinaM110Lmicrofluidizersystem（MicrofluidicsCo.,American）under10microfluidizationcyclesatapressureof12,000psiat4℃toobtainfinalnanoemulsionwithMONCPTconcentrationof0.489mg/ml.Ethanolwasusedasco-surfactant,andPEG400,glycerolformalwereappliedassolubilizerandstabilizerintheformulation.

2.4CharacteristicevaluationofMONCPTandnanoemulsion

SolubilityofMONCPTindifferentsolventswasmeasuredbyshaking3mlsolventsaddedwithexcessamountofMONCPTat37℃for48hawayfromlight.Thesuspensionwascentrifugedat8,000×gfor10minandfilteredthrougha0.22µmmembrane,thenMONCPTconcentrationinsampleswasdeterminedbyHPLC.N-octanol/waterpartitioncoefficient（P）ofMONCPTwasdeterminedbyshaking-flaskmethod.

Followingappropriatedilutionwithdistilledwater,MONCPTnanoemulsiondropletsizewasdeterminedbydynamiclightscattering（MALVERNNanoZS®,Malvern,UK）.ThenanoemulsionprofileswascheckedunderJEM1230electronmicroscope（JapanElectronOpticsLaboratory,Tokyo,Japan）andpHwasmeasuredusingapH-meteratroomtemperature.

InvitroreleaseofMONCPTwasmonitoredbyreversed-dialysisbagmethodat37℃undershakingat75rpmusingphosphate-bufferedsaline（PBS）asasinksolutionatpH7.0[20].DialysisbagwithaMWcut-offbetween12kand14kwasfilledwith10mLPBSand