高压乳匀法制备中药固体脂质纳米粒.doc

高压乳匀法制备中药固体脂质纳米粒作者厉英超1董蕾2贾皑1苌新明1薛挥1西安交通大学医学院1第一附属医院消化内科，陕西西安7100612第二附属医院消化内科，陕西西安710004摘要目的采用高压乳匀法将中药有效成分包载于固体脂质纳米粒SLN，并研究制备的纳米粒的主要性质。方法选择水飞蓟宾SIL和汉防己甲素TET为模型药物，采用高压乳匀法将其分别包载于SLN。在电镜下观察其形态，以粒度分析仪和Zeta电位分析仪测定其粒径和Zeta电位，用葡聚糖凝胶柱层析法和HPLC测定其包封率和载药量，还观察了SLN的稳定性。结果高压乳匀法制备的SILSLN呈球状，形态规则，平均粒径为157±8nm，Zeta电位为35.36±2.68mV，包封率为95.64，载药量为4.63TETSLN呈片状存在，不规则，粒径较小，平均粒径为47±3nm，Zeta电位为32.99±2.54mV，包封率为97.82，载药量为4.76。SILSLN和TETSLN有较高稳定性。结论高压乳匀法适于制备包载中药的SLN。关键词水飞蓟宾汉防己甲素固体脂质纳米粒高压乳匀法中药现代化中图分类号R944文献标识码A文章编号16734254200605054104PreParationofsolidlipidnanoParticlesloadedwithtraditionalChinesemedicinebyhighpressurehomogenizationLIYingchao1DONGLei2JIAAi1CHANGXinming1XUEHui11DePartmentofGastroenterology,FirstAffiliatedHospital,MedicalCollegeofXianJiaotongUniversity,Xian710061,China2DePartmentofGastroenterology,SecondAffiliatedHospital,MedicalCollegeofXianJiaotongUniversity,Xian710004,ChinaAbstractObjectiveToinvestigatethepreParationofsolidlipidnanoParticlesSLNloadedwithtraditionalChinesemedicinesbyhighpressurehomogenization,andstudythephysicochemicalcharacteristicsoftheParticlesproducedbythismethod.MethodsThemodeltraditionalChinesemedicines,silibininSILandtetrandrineTET,wereincorporatedintoSLNseParatelybyhighpressurehomogenization.TransmissionelectronmicroscopewasemployedtostudytheshapeoftheParticles.ParticlecharacterizationsystemandzetapotentialanalyzerwereusedtostudythediameterandzetapotentialofSLNinthesuspension.Theentrapmentefficiencyanddrugloadingweredeterminedwiththesephadexgelchromatographyandhighperformanceliquidchromatography.ThestabilityofSLNwasalsostudied.ResultsTheSILSLNspreParedbyhighpressurehomogenizationweresphericalandregular.ThemeandiameterandzetapotentialofSILSLNindistilledwaterwere157±8nmand35.36±2.68mV,respectively.Theentrapmentefficiencywas95.64,andthedrugloadingwas4.63.TheTETSLNwasplateletshaped,irregularandsmaller.ThemeandiameterandzetapotentialofTETSLNwere47±3nmand32.99±2.54mV,respectively,withdrugloadingof4.76,andupto97.82ofTETwasincorporated.SILSLNandTETSLNhadgoodstability.ConclusionHighpressurehomogenizationisfeasibleforpreParingSLNloadedwithtraditionalChinesemedicines.KeywordssilibinintetrandrinesolidlipidnanoParticleshighpressurehomogenizationtraditionalChinesemedicinesSupportedbyScienceandTechnologyProjectofXianCityGG04133.LIYingchao1974,PhD,attendingphysician,specializedinpharmaceuticalresearchofliverfibrosis,Tel02985324001,Emaill_163.comCorrespondingauthorDONGLei,medicalprofessor,Tel0298727693629368,Emaildonglei4488sohu.comSolidlipidnanoParticlesSLNsareParticlesmadefromsolidlipidswithameandiameterofapproximately50to1000nmtoserveasanalternativecolloidalcarriersystemforcontrolleddrugdelivery1.ComParedwithotherParticulatecarriersSLNhasseveraladvantagesfordrugdeliverysuchasitsgoodbiocomPatibility2,biodegradability3,highbioavailability4,andeffectstargetingtheliverandspleen.Inrecentyears,markedlyincreasingstudiesonSLNhavebeenreported,especiallywiththemethodofhighpressurehomogenization5.Nevertheless,onlyafewinvestigationshavebeenconductedinregardwiththeincorporationofeffectivecomponentsoftraditionalChinesemedicinesintoSLN.SilymarinisapurifiedextractfromthemilkthistleSilybummarianumL.Gaertn,whichiscomposedofamixtureof4isomericflavonolignans,namelysilibininorsilybin,SIL,isosilibinin,silidianinandsilychristin.SIL,whichconstitutes6070ofthesilymarinmixture,hasbeenidentifiedasthemajoractivecomponent6.TetrandrineTETisabisbenzylisoquinolinealkaloidextractedfromthetraditionalChinesemedicinalherbRadixstephaniatetrandrae.SILandTETpossesswidespectrumsofpharmacologicalactivities7891011.ThesetwoeffectivecomponentsofthetraditionalChinesemedicineshavehighlipophilicityandareexcellentcandidatesforSLNencapsulation.Byusingthisdrugdeliverysystem,ahighbioavailabilityandanintravenousadministrationarepossible.Inthepresentstudy,SILSLNandTETSLNwerepreParedbyhighpressurehomogenization,andthephysicochemicalcharacteristicsoftheParticlesproducedbythismethodwereanalyzed.MATERIALSANDMETHODSDrugsandreagentsSIL95waspurchasedfromPanjinGreenBiologicalDevelopmentCo.Ltd.,China.TET98waspurchasedfromShanchuanBiologicalCo.Ltd.,Xian.CholesterinobtainedfromZhengxiangChemicalResearchInstitute,ShanghaiandstearicacidTiandaChemicalIndustryLtd.,TianjinwereusedseParatelyasthelipidmaterialsofSLN.SoybeanlecithinwasobtainedfromAuboxingCo.Ltd.,Beijing.Sephadexgel50waspurchasedfromTianjinChemicalIndustryLtd.MethanolHPLCgradeandabsolutealcoholwassuppliedbyXianChemicalIndustryLtd.GlycerinAmoyGlycerinIndustryLtd.wasusedasacoemulsifierinwaterphase.PreParationofSILSLNSIL75mg,cholesterin1.5gandsoybeanlecithin1.0gwereweighedpreciselywithelectronicbalanceBP121S,sartoriusLtd.,Germanyanddissolvedin10mlabsolutealcoholinwaterbathat70℃.AnaqueousphasewaspreParedbydissolving45mlglycerinin75mldistilledwater.Theresultantorganicsolutionwasrapidlyinjectedintothestirredaqueousphase80℃.Theresultingsuspensionwasstirredcontinuouslyat80℃for2h.TheoriginalSILSLNsuspensionwasthenloadedintoahighpressurehomogenizer15M8BA,APV,UK,5cyclesat50MPaandthesampleswerekeptat4℃.PreParationofTETSLNTET75mg,stearicacid1.5gandsoybeanlecithin1.0gwereweighedpreciselyandpreParedintoTETSLNsuspensionaccordingtothemethoddescribedabove.TransmissionelectronmicroscopyThemorphologyofSILSLNandTETSLNwasexaminedwithtransmissionelectronmicroscopeH600,Hitachi,JaPan.Thesampleswerestainedwith2m/Vphosphotungsticacidfor30sandplacedoncoppergridswithfilmsforviewing.MeandiameterandzetapotentialParticlecharacterizationsystemMastersizer2000,MalvernInstruments,UK,20nm2000μmandzetapotentialanalyzerZetasizerNano,MalvernInstruments,UKwereusedtostudythediameterandzetapotentialofSLNindistilledwater.ThreesamplesofSILSLN/TETSLNwerepreParedaccordingtothepreviouslydescribedmethodandeachsamplewasmeasured3timestocalculatethemeandiameterandzetapotential.EntrapmentefficiencyEEanddrugloadingDLofSILSLN.ChromatographicconditionThechromatographiccolumnofPlanetsilC184.6mm15cmwasusedwithmobilephaseofmethanol/0.1mol/Lphosphatebuffer35/65,V/V,pH3.0,flowrateof1.0ml/min,columntemperatureof40℃,anddetectionwavelengthof288nm.Thecontrolsolutions0.050,0.161,1.605,14.19,28.38,56.75,113.50μg/mlwaspreParedbydissolvingpreciselyweighedSILinthemobilephase.TheamountofSILenteringthereceptorcomPartmentwasdeterminedwithhighperformanceliquidchromatogramHPLC,LC2010,Shimadzu,JaPan.TheintegralcalculusofthechromatographicpeakareaAwasrecordedastheYaxis,andtheconcentrationofSILCastheXaxis.DrugrecoverywascalculatedfromthefollowingequationDrugrecoverymeasureddrugweightinSLN100/theoreticaldrugweightloadedinthesystem.TheSILSLNsuspensionwasseParatedbySephadexgel50columnchromatography.TheconcentrationsofSILinthesuspensionn1andfreedrugn2wereassayedbyHPLCafterdilutionwithmethanol.EEandDLcouldbecalculatedaccordingtothefollowingequationsEEn1n2/n1100,DLWdrugloadedinsystem/Wlipidmatrix100.EEandDLofTETSLNChromatographicconditionThechromatographiccolumnofSpherisorbODSC18250mm4.6mm,5μmwasusedwithmobilephaseofmethanol/ether/ethylaminevolumeproportionof10010.05andflowrateof1.0ml/minatroomtemperatureanddetectionwavelengthof282nm.Theregressionequation,percentagerecoveriesofTET,EEandDLofTETSLNweredeterminedandcalculatedaccordingtothemethodsandequationsdescribedpreviously.EvaluationofstabilitySILSLNandTETSLNwerestoredat37℃andtheParticlesizesweredeterminedafter7,45and90days,respectively,toevaluatetheirstability.StatisticalanalysisTheresultswerepresentedasMean±SD.StatisticalanalysiswasperformedusingStudentsttestwithP0.05afterstorageat37℃for90days.DISCUSSIONSLNsareacolloidalcarriersystemforcontrolleddrugdelivery,anditisclaimedthatSLNcombinestheadvantagesandavoidsthedisadvantagesofothercolloidalcarriers.Itsadvantagesincludethepossibilityofcontrolleddrugreleaseanddrugtargeting,increaseddrugstability,absenceofcarrierbiotoxicity,andlargescaleproductionandsterilization12.HighpressurehomogenizationhasemergedasareliableandpowerfultechniqueforSLNpreParation12.Inthepresentstudy,thismethodprovedtobefeasibleforpreParingSILSLNandTETSLN,whicharesmall,steadyandhighlyincorporated.ThissuccessindicatesthepossibilityofincorporatingvariouslipophiliceffectivecomponentsextractedfromthetraditionalChinesemedicinesinSLNbythismethod,whichmakepossiblehighbioavailability,controlleddrugrelease,drugtargeting,decreaseddrugtoxicitiesandminimizedsideeffects,andrepresentsasuccessfulattemptofnovelapproachtothemodernizationoftraditionalChinesemedicines.VariousfactorsmayinfluencetheParticlesizeinhighpressurehomogenization,including,forinstance,homogenizationpressure,numberofcycles,lipidsandemulsifiers/coemulsifiersused,andoperatingtemperature.Inthepresentstudy,thetwokindsofSLNwerepreParedunderidenticalconditionswithalmostthesamematerialsexceptthedrugsincorporatedinSLNandthelipidmaterial.ThedifferenceofthelipidscholesterinandstearicacidandtheinteractionsbetweenthedrugsandthelipidsmightenormouslycontributetothedifferencesofParticlesizes.However,thedetailedmechanismsremaintobefurtherinvestigated.TheshapeofSLNmaysignificantlydifferfromasphere.Lipidstendtocrystallizeintheplateletform12,13.WhatfactorscausethedifferentshapesofSLNWhichshapeisinfavorofdrugprotectionandcontrolledreleaseThesequestionshaveattractedincreasingattentioninrecentyears12,14.SilymariniscomposedmainlyofSIL,andtheextractsofmilk