新型无机纳米载体的构建及其药物-基因的高效传递研究-曹霞

分类一号:一里车一一一UDC:-I密级:-一生浮一一编号刁耍卿李勒:缭叫一,,护利梦J}ANGSUUN}VERS}TY博士学位论文DISSER工绷几ON;八!!"新型无机纳米载体的构建及其卜匕夕-夕招华仁习:))药物/基因的高效传递研究学科专业:作者姓名:指导教师:答辩日期:l!备床检验诊断学曹霞徐希明教授2011年12月23日分类号断分UDe6,密级一一一么无一一编号业群血翅吵,新型无机纳米载体的构建及其药物/基因的高效传递研究FabricationofNovelInorganicNanoPartieulateCarriersandtheAPPlieationforEffieientDrug/GeneDelivery学生姓名:曹霞指导教师:徐希明专业名称:临床检验诊断学申请学位:博士论文答辩日期:20n.12.23学位授予单位:江苏大学ClassifiedIndex:UDC:FabrieationofNovelInorganicNanoParticulateCarriersandtheAPPlieationforEfficientDrug/GeneDeliveryByCaoXlaMajor:ClinicallaboratorydiagnosticsSuPervisor:Prof.XuximingProLYujiangnanJiangsuUniversityDee.,23.2011学位论文版权使用授权书本学位论文作者完全了解学校有关保留!使用学位论文的规定,同意学校保留并向国家有关部门或机构送交论文的复印件和电子版,允许论文被查阅和借阅"本人授权江苏大学可以将本学位论文的全部内容或部分内容编入有关数据库进行检索,可以采用影印!缩印或扫描等复制手段保存和汇编本学位论文"保密口,在年解密后适用本授权书"本学位论文属于不保密囚",如必心誉么太的学位论文作者签名:.飞.儿导师签名:乃万-厂甲0)签字日期:词11年陪月可日签字日期:习l/年肠月才日学位论文作者毕业后去向:工作单位:通讯地址:电话:邮编:独创性声明本人郑重声明:所呈交的学位论文,是本人在导师的指导下,独立进行研究工作所取得的成果"除文中已经注明引用的内容以外,本论文不包含任何其他个人或集体己经发表或撰写过的作品成果"对本文的研究做出重要贡献的个人和集体,均已在文中以明确方式标明"本人完全意识到本声明的法律结果由本人承担"学位论文作者签名:-青-氰日期:0!}年阵月〕丁户日江苏大学博士学位论文摘要纳米载体在药物/基因传递中具有特殊的价值和意义"纳米载体粒径大小在10一SOOIun,可将药物分子包裹其中或吸附在其表面,通过靶向分子与细胞表面特异性受体结合,在细胞摄取作用下进入细胞内,实现安全有效的靶向药物输送和基因治疗"无机纳米粒载体因具有生物安全!高效!价廉等优点,己成为近年来新型药物/基因传递系统研究的热点"本论文重点围绕新型无机纳米粒载体的构建及其在药物/基因传递中的应用开展研究,主要分为四个部分:第一部分纳米载体在药物/基因传递中的应用研究进展对纳米药物载体及基因载体的国内外研究现状进行了综述,主要内容包括:纳米载体的特点及制备新技术!纳米药物缓控释系统的优点!基因载体的分类与特点!非病毒纳米基因传递系统的优点!新型纳米载体在药物/基因传递系统中的应用进展!基于组织工程的三维支架以及软骨种子细胞的定向诱导分化研究进展等"文献综述为论文后续实验工作的开展奠定了基础"第二部分新型多孔二氧化硅纳米粒在药物传递中的应用研究以生物安全的硅胶为载体材料,运用纳米技术,自行研制出具有高效增溶!长效缓释作用的新型多孔二氧化硅纳米粒(poroussilicananopartieles,pSNs)"分别选择水溶性药物水飞蓟宾葡甲钱(silybinmeglumine,sLBM和难溶性药物水飞蓟宾(Silybin,SLB)为模型药物,研究PsNs新载体在水溶性药物和难溶性药物72h高效长效制剂开发中的应用可能,为3天给药1次的高效长效新制剂开发提供技术支撑"1.水溶性药物72h高效长效新制剂的研制:以水溶性药物水飞蓟宾葡甲钱)为模型药物(3d一SLBM)PSNs为载体,制备了水飞蓟宾葡甲钱72h"体外评价研究结果表明:3d一SLBM的主药含量为高效长效制剂29.46mg/50mg,载药量为58.91士0.39%,包封率为58.43士0.62%;体外释药特性研究结果显示,3d一SLBM中SLBM的释放适宜在低浓度的碳酸钠溶液中进行,72h累积释放大于80%;体内药动学研究结果显示:比格犬口服3d一SLBM,高效液相法测定犬体内血药浓度,3d一SLBM的Tmax24h与参比制剂Tmax0.5h相比大大增加,3d一SLBM的MRT38.89h与参比制剂的MRT7.31h相比显著延长,显示出长效新型无机纳米载体的构建及其药物噬因的高效传递研究缓释特征;3d一SLBM中游离药物的相对生物利用度达到803.99%;体外溶出结果与体内吸收具有良好的相关性,体外溶出结果可以间接反映其体内质量;药效实验结果表明:3d一SLBM组小鼠血浆中AST(291.83士76.03IU几)和ALT(774.92土223.85IU几)值明显低于CC从对照组(AST:901.00士174.32IU几,ALT:1997.58土335.08IU/L),具有统计学意义(p<0.01)"说明3d一sLBM的生物利用度高!保肝效果好"2.难溶性药物72h高效长效新制剂的研制:以难溶性药物水飞蓟宾为模型药物,综合运用固体分散速释技术!亲水凝胶骨架缓释技术和PSNS长效缓释技术制备了水飞蓟宾72h高效长效制剂(3d一SLB)"体外评价研究结果表明:3d一SLB中SLB的释放适宜在低浓度的碳酸钠溶液中进行,72h累积释放大于80%;体内药动学研究结果显示:比格犬口服3d一SLB,高效液相法测定犬体内血药浓度,3d一SLB的Tmax24h与参比制剂Tmaxlh相比大大增加,3d一SLB的MRrr32.15h与参比制剂的MRT6.nh相比显著延长,显示出长效缓释特征;3d一SLB中游离药物的相对生物利用度达到458.98%,体外溶出结果与体内吸收具有良好的相关性,体外溶出结果可以间接反映其体内质量;药效实验结果表明:3d一SLB小鼠血浆中AST(149.90士28.14IU/L)和ALT(843.33士169.18IU/L)值明显低于CCI;对照组(AST:901.00士174.32IU/L,ALT:1997.58士335.08IU/L),具有统计学意义(P<0.01)"说明3d一SLB生物利用度高!保肝性能优越"第三部分载基因纳米粒的制备及其在基因传递中的应用研究选择生物安全的磷酸钙纳米粒(ealeiumphosphatenanopartieles,CpNps)和PSNS,制备载基因DNA一CPNPS和DNA一PSNS;通过对载基因纳米粒进行修饰,成功制备了多糖修饰的DNA一CPNPS和钙离子化DNA一PSNs;着重研究了DNA一CPNPs!多糖修饰的DNA一CPNPS!钙离子化DNA一PSNs3种新型载基因纳米粒对干细胞的传递效果"1.DNA一磷酸钙纳米粒的制备及其干细胞的传递研究:用反相微乳法制备了DNA一CPNPS,对其进行表征,并将其应用于干细胞的转染"研究结果表明:DNA一CPNPS的粒径为20一50nm;琼脂糖电泳结果表明:磷酸钙:质粒质量比为2二1时,CPNPS携带质粒量最大,可有效阻滞DNA的迁移;活细胞工作站测定结果显示:游离DNA因没有纳米粒载体的作用,细胞未显红色,说明游离DNA江苏大学博士学位论文未进入细胞内,而DNA一CPNPsZh开始有少数细胞显红色,随着时间的推移,越来越多的外源基因进入细胞,说明纳米粒可有效携带基因进入细胞;激光共聚焦显微镜观察核转运结果显示:Zh未见细胞核内显红色,说明DNA一CPNPs此时尚未进入细胞核,4h进核亦不明显,sh有少量进核,18h可见细胞核内呈明显的红色,表明此时外源DNA在纳米粒作用下进核明显;活细胞工作站和共聚焦显微镜测定结果均显示纳米粒可被干细胞吞噬"MTT法测定细胞毒性,结果显示:DNA一CpNps的毒性明显低于市售转染试剂Lipo企etamineZ000(P<0.01);ELISA测定结果表明,DNA一CPNPS在干细胞中的转染效果与转染试剂Lipofeetamine200o相当(p>0.05)"实验研究结果表明:DNA一CpNps转染效率高!毒性低,可以作为一种生物安全的非病毒基因载体"2.多糖修饰的DNA一磷酸钙纳米粒的制备及其干细胞的传递研究:选择DNA一CPNPS,通过对载基因纳米粒进行修饰,成功制备了多糖修饰的DNA一CPNPS,初步研究了多糖修饰的DNA一CPNPS在千细胞中的转运情况"透射电镜测定结果表明:多糖修饰的DNA一CPNPs呈球型!粒径分布稳定!分散均一,粒径在50nm左右;ELISA测定结果表明:与市售脂质体lipofectamine2000相比,多糖修饰的DNA一CPNPS在干细胞中具有更高的转染效率"3.钙离子化DNA一多孔二氧化硅纳米粒的制备及其性能评价:选择PSNS为载体,通过对其进行修饰,成功制备了钙离子化DNA一PSNS,初步研究了钙离子化DNA一PSNS在干细胞中的转运"琼脂糖电泳结果表明:钙离子化的PSNS能够与质粒DNA较好的结合,阻滞其电泳迁移,而未修饰的PSNS与DNA结合能力弱,不能有效阻滞DNA迁移;EDS能谱分析结果表明:钙离子有效结合到PSNs上;倒置荧光显微结果表明:与DNA一PSNs相比,钙离子化DNA一PSNs能够转染更多的细胞,说明钙离子化DNA一PSNS可以作为一种有效的非病毒基因载体"第四部分三维纳米基因传递系统的构建及其在干细胞诱导分化中的应用研究将细胞外基质修饰纳米粒技术与基于组织工程的三维支架技术相结合,构建非病毒三维纳米基因传递系统"以细胞外基质(ECM)成分为支架材料,制备嵌合有DNA一CPNPS的三维支架,成功构建了兼具高效!缓释特征的非病毒三维纳米基因传递系统(3dimensiona一nanoparticlesgenedeliverysystem,3D一NGDs)"1.三维纳米基因传递系统的构建及其性能评价:以细胞外基质劲附实验为新型无机纳米载体的构建及其药物恩因的高效传递研究基础,选择对干细胞豁附及增殖效果好的纤维粘结蛋白!胶原为支架材料,制备胶原/FN/壳聚糖三维支架;在此基础上,采用冷冻干燥法制备嵌合DNA一CPNPs的三维纳米基因传递系统(3D一GDS),并观测其孔径!孔隙率!吸水率!保水率,结果表明:3D一GDs中存在较大孔径(扒00林m)的孔道,吸水率和保水率与支架材料的配比有关"研究结果表明:3D一NGDS多孔!生物安全,适宜干细胞生长"2.三维纳米基因传递系统的基因转运研究:以碘化丙咤标记基因,通过活细胞工作站观测,结合免疫组化的方法,对3D一GDS中基因的细胞内转运进行了研究;运用PicogreenDye法测定不同支架不同时间培养液中DNA的含量,绘制DNA累积释放曲线,结果显示3D一GDS中DNA累积释放率3d达到33.4%,Zld达到69.7%;而游离DNA一三维支架3dDNA累积释放率达到87.7%,3D一GDS对DNA具有良好的缓释作用"ELISA法测定二维体系及3D一NGDS3一巧天细胞表达的TGF一田浓度,结果表明:3D一GDS在3一巧天内,蛋白表达水平维持在10ng/ml左右,其中第6天表达浓度达到12石n留ml,显著高于二维转染体系(P<0.01),达到了外加TGF一印因子有效浓度,显示出明显的高效特征"通过观测干细胞在二维及3D一NGDS中转染巧天的甲苯胺蓝(GAG)染色图及n型胶原免疫组化染色图,结果表明:二维单层培养细胞的GAG染色和n型胶原免疫组化染色均为阴性,干细胞在3D一NGDS中培养巧天后,GAG染色和n型胶原免疫组化染色均为阳性,说明种子细胞已成功分化成软骨细胞,分泌软骨细胞特征性的细胞外基质:H型胶原和GAG"三维纳米基因传递系统作为一种新型的非病毒基因载体,可以实现外源基因在种子细胞中的高效持续表达,为后续组织工程和再生医学研究提供新技术!新思路"关键词:无机纳米载体,多孔二氧化硅纳米粒,磷酸钙纳米粒,药物传递系统,基因传递系统,72h高效长效药物新制剂,非病毒三维纳米基因传递系统,干细胞,组织工程三维支架"江苏大学博士学位论文ABSTRACTNano一sealedearriershavespeeialvalueand51助ifieanceindrug/genedeliv卿.TheParticlesizeofnano一earriersrangesfrom10to500nm.Drugmoleeuleseouldbeencapsulatedintheearrierorabsorbedonthesurfaee.SafeandeffeetivetargeteddrugdeliveryandgenetherapyeouldbeachievedbythecombinationoftargetingmoleculeswiththesPeeificreeePtorsoneellsurfaeefollowedbyenieringintoeellsviaeellularuPtake.InorganienanoPartielesenjoyingsuchadvantagesasbiosafety,high一efficacyandlowPrieehavebecomethehotsPotofnewdrug/genedeliverysystemstudyinrecentyears.ThisPaperfoeusesontheeonstrUetionofnovelinorganienanoParticlesandtheinvestigationoftheirapPlieationindrug/genedelivery.TherearemainlyfourPartsinthisthesis.PartOneResentAdvaneesontheAPPlieationofNano一SealedCarriersforDrug/GeneDeliveryThisParthassummarizedtheresearchstatusofnano一sealeddrugandgeneveetorsovertheworld,ineludingeharaeteristicsandnewPreParationteehnologyofnano一sealedvectors,advantagesofnanodrug一sustained一releasesystem,elassificationandeharaeteristiesofgeneveetors,meritsofnonviralnanogenedeliverysystem,theapPlieationofnewnanoveetorsindrug/genedeliverysystem,andProgressofstudyonthree一dimensionalseaffoldbasedontissueengineeringandinducingoriented一differentiationofseededchondroeyte.TheliteraturereviewhaslaidafoundationforthedeveloPmentofsubsequentworkinthisstudy.Partl!voNovelPorousSilieaNanoParticlesandtheAPPlieationinSustainedDrugDeliveryAnewPoroussilieananoPartielewithhighsolubilizationeffieientandlong一termsustained一releaseeffeethasbeenPreParedinthisstudybasedonnanoteclmologyusingsilieaastheearriermaterialswhieh15biologicallysafe.Thewater一solubledrugV2新型无机纳米载住今鱼步匆壑些兰壑些些量蟹竺些1一一一一一silybinmeglumineandPoorlywater-solubledrugsilybinareemPloyedasmodeldrugsresPeetivelytoinvestigatethePossibilityofthenovelPoroussilieananoP叭ielestodeveloplong一tennsustainedreleasesystemfor72hofbothwater-solubleandPoorlywater-solubledrugs,which初11ProvidePromisingfutureforthedeveloPmentofhigh一effieacyandlong一actingPreParationsafteroraladministrationoneeevery3days.1.DeveloPmentof72hlong一lastingandhigh一effieacyPreParationforwater-solubledrugs.几klngthewater-solubledrugsilybinmeglumine(SLBM)asthemodeldrugandPoroussilieananoPartielesasearriers,wePreParedSLBM一loadednanoP叭ieles(3d一SLBM).Theresultsofinvitroevaluationrevealedthattheamountofdruginthed扭g一loadednanoPartieleswas29.46mg/50mg,thedrugloadingratewas58.91土0.39%,andeneapsulatingratewas58.43士0.62%.Theinvestigationofinvitrodissolutionsho丫vedthatloweoneentrationsodiumcarbonatesolution认,asmostsuitableforthereleaseofSLBMfrom3d一SLBM,withthe72haccumulativereleaserateover80%.FortheinvivoPharnnacokinetiesstudy,drugwasadministratedorallytobeagledogsandhighPerformaneeliquidcliromatography(HPLC)wasusedtodeterminethePlasmaeoneenirationinbeagledogs.TheresultsofinvivostudydemonstratedthattheTmaxof3d一SLBMwas24h,significantlyincreasedcomParedwiththatofthereferencePreParation(Tmax0.5h).Furthermore,eomParedwiththeMRT7.3lhofthereferencePreParation,theMR月,of3d一SLBMwasnotieeablyProlongedto38,89h,revealinglong一lastingsustainedreleaseeharaeteristie.Additionally,therelativebioavailabilityofthefreedrugfrom3d一SLBMreaehed803.99%.Interestingly,theinvitrodissolutionandinvivoabsorptionshowedagoodeorrelation,that15,theinvivoabsorptionProfileeanberefleetedindireetlyfromtheinvitrodissolutionProfile.TheresultsofhePatieProteetionshowedthatAsT(291.83士76.03Iu/L)andALT(774.92士223.85IU/L)levelintheplasmaofmicewhichweregiven3d一SLBMweresignifieantlylowerthanthatofmieeinCC14treatedgrouP(AST:901.00士174.32IU/L,ALT:1997.58士335.08IU/L),withstatistiealsignifieance(P<0.01).Alltheseresultsindicatedthat3d一SLBMPossessedhighVI江苏大学博士学位论文bioavailabilityandexeellenthePatieProteetioneffeet.2.DeveloPmentof72hlong一lastingandhigh一effieaeyPreParationforPoorlywater-solubledrugs.A3一dayreleaseformulationofsilybin(3d一SLB)withhigh一effieacy,long一acting,andslow一releaseProPertieswasPreParedinthisstudybyeombineduseofsilybinsoliddisPersion,silybinloadedsilieananoPartielesandslow一releasematrixmaterial.SilybinwasemPloyedasthemodeldrug.TheresultofinvitroevaluationdemonstratedthatSLBwasapttoreleasefrom3d一SLBinloweoneentrationsodiumearbonatesolution,Withtheaeeumulatedreleaserateover80%.TheresultsofinvivoPharnnaeokinetiesresearehshowedthatTmaxof3d一SLBwas24h,whlehwasmuehlaterthanthatofthereferencePreParation(Tmaxlh).TheMRTof3d一SLBwas32.15h,greatlyProlongedthantheMRT6.llhoftherefereneePreParation,indicatingthelong一lastingsustainedreleaseProfile.Moreover,thebioavailabilityofthefreedrugreleasedfrom3d一SLBreaehed458.98%;there15agoodcorrelationbetweentheinvitrodissolutionandinvivoabsorption,that15,theinvivoabsorptionProfileeanberefleetedindirectlyfromtheinvitrodissolutionProfile.TheresultsofdrugeffeetshowedthatAST(149.90士28.14IU/L)andALT(843.33士169.18IU/L)levelsinthePlasmaofmicewhiehwereadministered3d一SLBMweresignifieantlylowerthanthatofmieeinCC14treatedgrouP(AST:901.00士174.32IU/L,ALT:1997.58士335.08IU/L),withstatistiealsignifieanee(P<0.01).Alltheseresultsindieatedthat3d一SLBPossessedhighbioavailabilityandexeellenthePaticProteetioneffeet.PartThreePreParationofGeneLoadedNanoPartielesandtheAPPlieationinGeneDeliveryBiologieallysafeealciumPhosPhatenanoPartielesandPoroussilieananoParticleswereusedtoencapsulategenetoPrePareDNA一caleiumPhosPhatenanoPartielesandDNA一PoroussilicananoPartieles.PolysaccharidemodifiedDNA一ealeiumPhosPhatenanoPartielesandealcium一ionizedDNA一PoroussilieananoPartieleswerealsosuceessfullyPreParedbymodifieationofgene一loadednanoPartieles.ThisPartfoeusesonthetransfeetioneffeetofthleekindsofnewgene一loadednanoPartielesonV11了新型无机纳米载体的构建及其药物噬因的高效传递研究mesenehymalstemeells,ineludingDNA一caleiuxnPhosPhatenanoPartieles,PolysaecharidemodifiedDNA一ealeiumPhosPhatenanoPartielesandcaleium一ionizedDNA一PoroussilieananoParticles.1.PreParationofDNA一ealeiumPhosPhatenanoPartielesandtheaPPlicationingenedeliverytomesenchymalstemeells.DNA一caleiumphosphatenanoPartieleswerepreparedbyreversemieroemulsionmethod,eharacterized,andapPliedintransfeetionofmesenehymalstemeells.TheresultshowedthatthePartielesizeofDNA一ealciumPhosPhatenanoParticlesrangedfrom20to50nm.AgarosegeleleetroPhoresisfoundthatcaleiumPhosPhatenanoPartieleseouldloadthemaximumamountofDNAwhentheweightratioofealeiumPhosPhatetoPlasmidnanoparticleswiththisweightDNA152:1.Additionally,DNA一ealeiumratioeouldeffeetivelyretardPlasmidPlasmidDNAPhosPhatemigration.Thedeterminationofliveeellimagingrevealedthatfreehadalmostnotransfeetioneffectwithnostaininginthecells,indieatingthatfreeDNAeouldnotenterintocells.FortheDNA一ealeiumPhosPhatenanoPartielestransfeetedcells,afeweellswerestainedatZhPosttransfeetion.Withtimewentby,moreandmoreexogenousgeneenteredintocells,demonstratingthatDNA一ealeiumPhosPhatenanoPartieleseouldeffeetivelycarrygeneintoeells.LasereonfoealfluoreseeneemieroseoPydisPlayedthatnoredfloresceneewasobservedinthenucleiatZhPosttransfeetion,indicatingthatDNA一ealciumPhosPhatenanoPartieleswerenotinnucleiatthistime.At4hPosttransfeetion,therewasstillnoobviousredstaininginthenuelei.AtshPosttransfeetion,afewDNA一ealeiumPhosPhatenanoPartielesshowedinthenuelei.At1shPosttransfeetion,obviousredflorescencewasobservedinnuclei,indieatingthatexogenousgenehadenteredintoeellswiththehelPofealeiumPhosPhatenanoPartieles.TheresultsofbothliveeellimagingandlasereonfoealfluoreseencemicroseoPyindieatedthatDNA一ealeiumPhosPhatenanoPartieleseouldbePhagoeytosedbymesenehymalstemeells.CytotoxicitydeterminedbyMTTassayshowedthatDNA一caleiumPhosPhatenanoPartieleshadlowercytotoxieitythancolnmereiallyavailabletransfeetionagentLIPofeetamineZ000(P<0.01).TheresultofELISAtestrevealedthatDNA一ealciumPhosPhatenanoPartieleshadeomParableVlll江苏大学博士学位论文transfectioneffeetwithtransfeetionagentLIPofeetamine2000(P>0.05),whiehwasobviouslyhigherthanstandardealeiumPhosPhatetransfeetionageni(P<0.01).Altogether,DNA一ealciumPhosPhatenanoPartieleswithhightransfeetioneffieieneyandloweytotoxieityeouldbedeveloPedinioakindofbiologieallysafenonviralgeneVeCtof.2.PreParationofPolysaccharidemodifiedDNA一ealeiumPhosPhatenanoPartielesandtheaPPlieationingenedeliverytomesenehymalstemeells.ModifiedbyPolysaeeharide,DNA一ealeiumPhosPhatenanoPartieleswereemP10yedsuccessfullytoPreParePolysaeeharidemodifledDNA一ealciumPhosPhatenanoPartieles.PrimaryresearehhasbeenearriedoutaboutthetransPortationofPolysaeeharidemodifledDNA一ealeiumPhosPhatenanoPartielesinthenuelei.TransmissioneleetronmieroseoPydisPlayedthatthePolysaceharidemodifiedDNA-ealeiumPhosPhatenanoParticleswereofsPhericalshape,andthePartielesizewasabout50nmwhiehwereuniformlydistributedwithinanarrowrange.TheresultofELISAtestshowedthatPolysaeeharidemodifiedDNA一calciumPhosPhatenanoPartielesPossessedhighertransfeetioneffieieneyinmesene场malstemeellsthantheeonllllereiallyusedLIPofeetamine2000.3.PreParationandevaluationofealeium一ionizedDNA一PoroussilieananoPartieles.Calcium一ionizedDNA一PoroussilieananoPartielesweresueeessfullyPreParedbasedonthemodifieationofDNA一PoroussilieananoParticles.PrimaryinvestigationhasbeeneonduetedtostudythetransPortationofealeium一ionizedDNA一PoroussilicananoParticleseleetroPhoresisinsidemesenehymalstemeells.Theresultofagarosegeldemonstratedthatealeium一ionizedDNA一PoroussilieaeouldbewellcombinedwithPlasmidDNAandretardDNAmigration.nanoPartielesHowever,theunmodifiedDNA一PoroussilieananoPartieleshadaweakabilitytocombinePlasmidDNAandeouldnoteffeetivelyretardDNAmigration.TheresultofEDSrevealedthatealciumioneouldeffectivelyeombinewithPoroussilieananoPartieles.FluorescentmieroseoPyshowedthatealeium一ionizedDNA一PoroussilieananoParticleseouldtransfeetmoreeellsthanDNA一PoroussilicananoParticles,indieatingthatIX夕新型无机纳米载体的构建及其药物基因的高效传递研究ealeium一ionizedDNA一PoroussilieananoParticlescouldbeakindofeffeetivenonviralgenevector.PartFourConstruetionofThree一DimensionalNanoGeneDeliverySystemandItsAPPlieationintheDifferentiationofMesenehymalStemCellsCombiningtheteehnologyofPreParingextraeellularmatrix(ECM)modifiednanoPartielesandthxee一dimensionalscaffeldbasedontissueengineering:Weeonstruetedanonviral3dimensionalnanogenedeliverysystem(3D一NGDS),FollowingECMingredientsasscaflbldmaterial,DNA一ealciumPhosPhatenanoPartieleswereencapsulatedinthlee一dimensionalseaffoldtofabrieate3D一NGDS,andthecharacteristieswerethenevaluatedsuehasDNAreleasekineties,eelluPtake,genetransfectioneffieieney,MSCsdifferentiation.1.Construetionandevaluationofthree一dimensionalnanogenedeliverysystem.ChoosingmaterialsBasedontheresultsofECMadhesionexPeriment,FNandcollagen(C),whiehPossessgoodadhesionandProliferationabilityonmesenehymalstemeells,athree一dimensionalsea且bldwasPIeParedwitheollagen,FN,andehitosan.3D一NGDSwasProducedbyeneapsulatingDNA一calciumPhosPhatenanoPartielesinscaffoldusingfreezedryingtechnique.TheresultsofitsPoresize,Porosityrate,waterabsorptionrate,andwaterretainingrateshowedthatmostofthescaffoldPoresizewasabout100林minadditiontosomePoreswithlargerPoresize(>100林m):thewaterabsorptionandwaterretainingabilitieswererelatedtotheProPortioningofseaffoldmaterials.Inaword,thethree一dimensionalnanoseaffoldwasPorous,biosafeandsuitableforthegrowthofmesenchymalstemeells.2.Genedeliveryviathethree一dimensionalnanogenedeliverysystem.ByusingProPidiumiodidetomarkgene,eellularuPtakeofexogenousgeneinthethlee一dimensionalsystemwasinvestigatedviaeombinedemPloymentofliveeellimagingandimmunohistoehemistry.TheDNAeontentsindifferentthree一dimensionalScaffoldsWeredeterminedbyPicogreenDyemethodatdifferenttimePoints.DNAaeeumulativereleaseeurvesindlft七refltsea伍〕ldsshowedthattheDNAaeeumulativereleaserateofDNAeneapsulatedthxee一dimensionalseaffoldwas33.4%at3d,andX江苏大学博士学位论文69.7%at21d;forfreeDNA一three一dimen污ionalseaflbld,theDNAaeeumulativereleaseratewas87.7%at3d.Theresultindieatedthat3D一NGDShadgoodsustained一releaseeffeetonDNAdelivery.TGF一pleoncentrationexPressedbyeellsin3D一GDSfromday3today15wasdeterminedbyELISA,andtheresultrevealedthatduringthePeriodofday3today15,theTGF一plProteinexPressionlevelcouldmainiainatabout1ong/m1.Partieularly,theexPressionconeentrationatday6reaehed12.6ng加1,whiehwassignifieantlyhigherthanthatofthetwo一dimensionaltransfectionsystem(P<0.01).ThisProteinexPressionlevelreachedtheeffeetiveeoneentrationofTGF一plProteinnormallyaddedinmedium,showingobviouslyhigheffieiencyof3D一GDS.Mesenchymalstemeellsintwo一andthree一dimensionalnanoseaffoldsat15daysPosttransfectionwereobservedbyGAGstainingandtyPeIIcollageninununohistoehemistrystaining,andtheresultsfoundthattheGAGstainingandtyPe11collagenimmunohistoehemistrystainingoftheeellseulturedintwo一dimensionalmonolayerconditionwereallnegative,whileforthemesenehymalstemeellsculturedin3D一NGDSforIln们nunohistoehemistrystainingwere15days,theGAGstainingandtyPe11eollagenPositive.Theseresultsindieatethattheseededcellshavesuccessfullydifferentiatedintoehondroey-tes,whicheouldseereteECMwithehondrocyteeharaeteristics:tyPe11eollagenandGAG.Asakindofnewnonviralgenedeliveryearrier,3D一NGDSeouldachievehigheffieientandlong一lastingexPressionofexogenousgeneintheseededcells,wlliehProvidedanewideaandteelmologyforthesubsequentstudyontissueengineeringandregenerativemedicine.Keywords:inorganienanoPartieleearrier,PoroussilieananoPartieles,caleiumPhosPhatenanoParticles,drugdeliverysystem,genedeliverysystem,72hlong一aetingandhigh一effieaeyoraldrugdeliverysystems,non一viralthree一dimensionalnanogenedeliverysystem,mesenehymalstemeells,tissueengineeringXIlo新型无机纳米载体的构建及其药物冻因的高效传递研究目录第一章纳米载体在药物/基因传递中的应用研究进展......................................,,11纳米缓控释药物传递体系研究..........,,,.................................................,,11.1纳米药物载体的概况..,,,...............................................................,,11.2制备纳米粒的方法................................................................############,,11.3纳米药物载体的优势........................,,,,........................................,,21.4载药纳米粒在缓控释给药系统中的应用........................................,,21.5展望....................................................................................................,,32纳米制剂用于基因治疗.,,,,,,,,.,.,..,,..............,,,................................,,32.1基因治疗的概述.....................................................#.#########################,,32.2理想基因转运载体的特征..............................,,,...........................,,42.3基因载体的研究概况.............................,,,..######!#######################,###,,42.3.1病毒载体系统...........................................................#.#############,,42.3,2非病毒载体系统...............................................#######################,,42.4纳米基因载体系统......................................######################################,,62.5纳米材料的选择,.,.,.,二,.,.........................,,,.,######,##########################,,62.6展望....................................................................................................,,73组织工程软骨种子细胞的定向诱导分化..................................................,,83.1组织工程概述...................................................................................,,83.2种子细胞定向诱导分化成软骨方法................................................,,83.3展望.............................................................................,,,..............,,10第二章新型多孔二氧化硅纳米粒在药物传递中的应用研究.,,,................,,n1水溶性药物72h高效长效新制剂的研制...............................................,,H1.13d一SLBM的制备及体外质量评价...................,,,........................,,111.1.1实验材料................................................................................,,n1.1.2实验方法................................................................................,,121.1.3实验结果................................................................................,,141.23d一SLBM的犬体内药动学评价...............................................,,,..,171.2.1实验材料...............................................................................,,171.2.2实验方法................................................................................,,171.2.3实验结果................................................................................,,181.33d一SLBM的药效学评价.................,,,..,,.,...............................,,211.3.1实验材料.................................................................................,,211.3.2实验方法....................,,,..,,.,..,,.,.,......,,,.,...,,,二,,,221.3.3实验结果..............,,,..,,....................................................,,23江苏大学博士学位论文2难溶性药物72h高效长效新制剂的研制...............................................,,242.13d一SLB的制备及药动学研究..........................................................,,242.1.1实验材料................................................................................,,242.1.2实验方法................................................................................,,252.1.3实验结果................................................................................,,262.23d一SLB的药效评价...............................................................,,,....,,282.2.1实验材料................................................................................,,282.2.2实验方法................................................................................,,282.2.3实验结果..........................,,,.........................................######,,293小结............................................................................................................,,30第三章载基因纳米粒的制备及其基因传递的应用研究.,,............................,,321DNA一磷酸钙纳米粒的制备及其干细胞中的传递研究...........................,,321.1实验材料..........................................................................................,,犯1.1.1主要试剂................................................................................,,321.1.2主要仪器................................................................................,,331.1.3主要试剂的配制.................,,,............................................,,331.2实验方法..........................................................................................,,341.2.1质粒DNA制备与纯化.........................,,,.,........................,,341.2.2酶切鉴定................................................................................,,341.2.3DNA一磷酸钙纳米粒的制备...................................................,,341.2.4细胞培养................................................................................,,341.2.5纳米粒与质粒的结合............................................................,,361.2.6二维纳米基因传递系统的研究............................................,,361.2.7毒性评价................................................................................,,361.2.8干细胞细胞转染....................................................................,,371.3实验结果..........................................................................................,,371.3.1质粒提取结果........................................................................,,371.3.2酶切实验结果........................................................................,,371.3.3DNA一磷酸钙纳米粒的分离纯化...........................................,,381.3.4DNA一磷酸钙纳米粒的表征...................................................,,381.3.5干细胞培养时首次换液时间的确定....................................,,40新型无机纳叁越鲤业些三塾竺翌鱼竺燮丝一一一一一1.3.6二维纳米基因传递系统的研究........................,,!............,,,401.3.7MTT结果,.............,,,.......................................,,,.........####,,421.3.SDNA一磷酸钙纳米粒纳米粒转染干细胞结果.......................,,432多糖修饰DNA一磷酸钙纳米粒的的制备及其千细胞中的传递研究.....,,432.1实验材料.............,,,..,,,.,,...........................................................,,432.1.1主要试剂.................................................................................,,432.1.2仪器..................................,,,...............................................,,442,2实验方法..........................................................................................,,442.2.1多糖修饰的DNA一磷酸钙纳米粒的制备.............................,,442.2.2多糖修饰的DNA一磷酸钙纳米粒的表征.............................,,442.2.3细胞转染..,,....................,,,................................................,,442.2.4统计学处理............................................................................,,442.3实验结果..........................................................................................,,452.3.1制备流程.............................................................,,"...........,,452.3.2多糖修饰的DNA一磷酸钙纳米粒形态观察.........................,,452.3.3ELISA测定各组TGF一印表达水平......................................,,453钙离子化多孔二氧化硅纳米粒的的制备及其性能评价........................,,463.1实验材料..........................................................................................,,463.1.1主要试剂................................................................................,,463.1.2仪器........................................................................................,,463.2实验方法..........................................................................................,,463,2.1钙离子化DNA一PSNs的制备...............................................,,463.2.2钙离子化DNA一PSNs的表征...............................................,,463.2.3质粒结合实验........................................................................,,463.2.4干细胞转染,,.....................................................................,,,二473.3结果与讨论......................................................................................,,474小结............................................................................................................,,48第四章三维纳米基因传递系统的构建及其在干细胞诱导分化中的应用研究,511三维纳米基因传递系统的构建及其性能评价.......................................,,511.1实验材料..........................................................................................,,511.1.1主要试剂...........................,,!..............................................,,511.1.2主要仪器!...................................................,,,.....................,,51江苏大学博士学位论文1.2实验方法..........................................................................................,,511.2.13D一GDS的构建.........................................,,,...................,,511.2.23D一GDS的质量评价...........................................................,,531.3实验结果.......................................................................................,,,二531.3.1三种不同方法制备的支架比较.........................................,,,二531.3.23D一NGDS的表征...,,,.........................................................,,5423D一GDS的基因转运研究.............................................,,,...................,,562.1实验材料.........................