卓仁禧院士-用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件

用于基因和抗癌药物控制释放高分子材料研究卓仁禧院士生物医用高分子材料教育部重点实验室武汉大学化学与分子科学学院卓仁禧院士_用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件1一.高分子基因治疗载体研究病毒载体和非病毒载体的比较效率低安全容易制备，储存和质量控制传递的DNA大小从20个碱基对到几十万个碱基对非病毒载体安全隐患DNA大小受限制免疫原性高效病毒载体缺点优点载体一.高分子基因治疗载体研究效率低安全非病毒安全隐患高效病2

非病毒载体阳离子脂质体

高分子:阳离子高分子水溶性非离子高分子高分子基质非病毒载体阳离子脂质体3常用的聚阳离子基因载体Poly[-(4-amino-butyl)-L-glycolicacid]SWKim,UtahUnivPoly-L-lysinePolyethylenimines(PEI)BehrJP,FranceDendrimerSzokaUCSF&BakerJUnivMichPolyphosphoesterandPolyphosphoamideLeongKW,JohnsHopkingUnivLangerR,MIT常用的聚阳离子基因载体Poly[-(4-amino-but4从新的起始核合成聚酰胺胺树型高分子基因载体从新的起始核合成聚酰胺胺树型高分子基因载体5DendrimerGT5DendrimerGT56TransfectionofHEK293cellswithpRELuc/dendrimerGTcomplexes合成的树型高分子的转染效率与目前最好的阳离子载体PEI相当，但是比PEI的毒性低得多CytotoxicityofdendrimerGT3~GT5measuredbytheMTTassay.PEIandPLLwereusedascontrols

TransfectionofHEK293cells7肝细胞靶向基因载体

肝细胞靶向基因载体81.0E+041.0E+051.0E+061.0E+071.0E+081.0E+090246810N/PRLU/mgproteinGT5GT5-Gal5%TransfectionofHepG2cellswithpRELuc1.0E+041.0E+051.0E+061.0E+071.9TransfectionofNIH3T3cellswithpRELucTransfectionofNIH3T3cells10阳离子聚磷酸酯基因载体阳离子聚磷酸酯基因载体11卓仁禧院士_用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件12PPA-DPAGal-PPAPPA-DPAGal-PPA13Gal-PEG-PPAGal-PEG-PPA14liverspleenlungkidneyheart103105104102RLU/mgproteinPPAGal-PPAGal-PEG-PPA

LuciferaseExpressioninmouseafterintraportalveininjectionofcomplexescontaining50µgplasmidliverspleenlungkidneyheart103115二.药物控释生物可降解高分子材料研究聚酯(聚乳酸,聚乙醇酸及其共聚物;聚己内酯,聚羟基丁酸及其共聚物)聚酸酐聚碳酸酯及其共聚物聚磷酸酯及其共聚物纳米药物控制释放温度敏感快速响应PNIPA水凝胶Poly(lactide-co-glycolide)PLGAPolylactidePLAPolyglycolidePGA二.药物控释生物可降解高分子材料研究Poly(lacti16聚碳酸酯及其共聚物BTMCPTMC卓仁禧,王旭立,刘立建.５－苄氧基－三亚甲基碳酸酯及其制备方法。中国专利ZL01114284.7C07D319/04

卓仁禧，王旭立，刘立建。聚（５－取代基－三亚甲基碳酸酯）及其制备方法和用途。ZL01114285.5聚碳酸酯及其共聚物BTMCPTMC卓仁禧,王旭立,刘立建.17InvitrocytotoxicityofPBTMCandPHTMC

COS7cells.Incubationtime:24h.Temp.:37oCInvitrocytotoxicityofPBTMC18BTMCTMCPoly(HTMC-co-TMC)Sn(Oct)2150oCDeprotectionBTMCTMCPoly(HTMC-co-TMC)Sn(Oct19DependenceofhydrophilicityoncopolymercontentDependenceofhydrophilicityo20Invitrodegradabilityofcopolymers,PBSpH7.4，37oC0246810121416182022242628020406080100P(HTMC-co-TMC)10/90PTMCP(HTMC-co-TMC)50/50WeightLoss(%)Time(day)PHTMC100%-OHwithout-OH50%-OH10%-OHInvitrodegradabilityofcopo215-Fureleaseprofilesofcopolymers,PBSpH7.4，37oC5-Fureleaseprofilesofcopol22ZhangY,ZhuoRX,Synthesisanddrugreleasebehaviorofpoly(trimethylenecarbonate)–poly(ethyleneglycol)–poly(trimethylenecarbonate)nanoparticles,Biomaterials(Inpress)PTETtriblockcopolymer纳米药物控制释放ZhangY,ZhuoRX,Synthesisa23SynthesisandpropertiesofPTETtriblockcopolymersSynthesisandpropertiesofPT24Particlesizes,drugloadingcontent,loadingefficiency,andyieldofthePTETpolymericnanoparticlesPolymericnanoparticleswerepreparedbythedialysistechnique.Drug:methotrexate(MTX)Particlesizes,drugloadingc25TEMimageofPTET-7nanoparticlesloadedmethotrexate(MTX)TEMimageofPTET-7nanopartic26EffectofmolecularweightofPEGinthecopolymersontheMTXreleaseratePBS,pH=7.4,37ºC.PEG6000PEG4000PEG2000Effectofmolecularweightof27EffectofcontentofPEGblockinthecopolymersontheMTXreleaseratePBS,pH=7.4,37ºC.PEG:6.17wt%PEG:24.12wt%PEG:28.94wt%PEG:54.26wt%EffectofcontentofPEGblock28温度敏感快速响应PNIPA水凝胶A混合溶剂法B相分离法C使用成孔剂:如PEGD形成互穿网络在温度敏感水凝胶领域发表SCI论文30多篇。温度敏感快速响应PNIPA水凝胶A混合溶剂法29卓仁禧院士_用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件30靶向性抗癌药物5-Fu抗癌药物PMliver-targetinggroup肝靶向高分子抗癌药物靶向性抗癌药物5-Fu抗癌药物PMliver-targe31肝靶向高分子抗癌药物5-Fu-(99mTc–DTPA)-PAEA-PM注射后不同时间在大鼠体内各器官的分布肝靶向高分子抗癌药物5-Fu-(99mTc–DTPA)-3262.5μg/mLBel-7204体外凋亡率:51%135μg/mLBel-7204体外凋亡率:62%Control肝靶向高分子抗癌药物5-Fu-PAEA-PM对人体肝癌细胞(Bel-7204)的体外凋亡实验的荧光照片62.5μg/mL135μg/mLControl肝靶向高分子33P(TMC-co-EEP)PorcinePancreasLipase(PPL)CandidaRugosaLipase(CL)Al(OiPr)3酶催化开环聚合反应P(TMC-co-EEP)PorcinePancreas34Ring-openingcopolymerizationofTMCandEEPcatalyzedbyenzymesMw/MnMnYield(%)Time(d)Molarratio(TMC:EEP)[E](mg/mmol)Lipase-<500-210:510--<500-210:110-1.69641042.1210:510CL1.83644062.0210:310CL2.23786075.6210:110CL2.18323019.8210:510PPL1.66802060.1210:310PPL1.711025082.8210:110PPL2.63573060.2410:210CL2.97601057.0310:210CL1.99683050.2110:210CL2.75343065.2410:210PPL1.91398072.3310:210PPL2.47597056.2110:210PPL1.81532860.2210:250CL1.87650057.6210:225CL2.27649058.3210:210CL2.43623036.5210:225PPL2.31933068.9210:210PPLRing-openingcopolymerization35PolymerizationcatalyzedbyPPLimmobilizedonsilicamicroparticles(PISM)

PolymerizationcatalyzedbyPP36EffectoflipaseconcentrationonMnandyield0.00.51.01.52.02.55000100001500020000250003000035000400004500050000Concentration(wt%)Molecularweight3035404550556065707580859095100Yield(%)MnYieldEffectoflipaseconcentration37TimedependenceofMnandyieldduringpolymerization1020304050601000012000140001600018000200002200024000Time(h)Mn404550556065707580859095100YieldMnYieldTimedependenceofMnandyiel38DependenceofMnandyieldontheimmobilizedlipasePPLrecyclingtimeRecyclingTimeLipaseConc.(wt%)MnMw/MnYield(%)I1.0147001.9270II1.0254001.7775III1.0308002.1181IV1.0452002.0178VI1.0617001.9483VII1.0630002.5381VIII1.0640002.2072VIII0.1874002.0669Reactiontemperature:100℃.Reactiontime:24hDependenceofMnandyieldon39PolymerizationofDTCcatalyzedbyPPLAimmobilizedonsilicamicroparticles

BimmobilizedonsilicananoparticlesPolymerizationofDTCcatalyze40RecyclingtimeofIMPPLvsMnofPoly(DTC)obtainedat130°Cfor4day-polymerization

RecyclingtimeofIMPPLvsMn41Poly(DTC)TheoriginalIMPPLThefirstrecyclingIMPPLThesecondrecyclingIMPPLThethirdrecyclingIMPPL

1752cm-1Poly(DTC)1752cm-142致谢武汉大学生物医用高分子材料教育部重点实验室的教师、博士研究生、硕士研究生国家科技部973项目国家自然科学基金重点项目、面上项目资助致谢43用于基因和抗癌药物控制释放高分子材料研究卓仁禧院士生物医用高分子材料教育部重点实验室武汉大学化学与分子科学学院卓仁禧院士_用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件44一.高分子基因治疗载体研究病毒载体和非病毒载体的比较效率低安全容易制备，储存和质量控制传递的DNA大小从20个碱基对到几十万个碱基对非病毒载体安全隐患DNA大小受限制免疫原性高效病毒载体缺点优点载体一.高分子基因治疗载体研究效率低安全非病毒安全隐患高效病45

非病毒载体阳离子脂质体

高分子:阳离子高分子水溶性非离子高分子高分子基质非病毒载体阳离子脂质体46常用的聚阳离子基因载体Poly[-(4-amino-butyl)-L-glycolicacid]SWKim,UtahUnivPoly-L-lysinePolyethylenimines(PEI)BehrJP,FranceDendrimerSzokaUCSF&BakerJUnivMichPolyphosphoesterandPolyphosphoamideLeongKW,JohnsHopkingUnivLangerR,MIT常用的聚阳离子基因载体Poly[-(4-amino-but47从新的起始核合成聚酰胺胺树型高分子基因载体从新的起始核合成聚酰胺胺树型高分子基因载体48DendrimerGT5DendrimerGT549TransfectionofHEK293cellswithpRELuc/dendrimerGTcomplexes合成的树型高分子的转染效率与目前最好的阳离子载体PEI相当，但是比PEI的毒性低得多CytotoxicityofdendrimerGT3~GT5measuredbytheMTTassay.PEIandPLLwereusedascontrols

TransfectionofHEK293cells50肝细胞靶向基因载体

肝细胞靶向基因载体511.0E+041.0E+051.0E+061.0E+071.0E+081.0E+090246810N/PRLU/mgproteinGT5GT5-Gal5%TransfectionofHepG2cellswithpRELuc1.0E+041.0E+051.0E+061.0E+071.52TransfectionofNIH3T3cellswithpRELucTransfectionofNIH3T3cells53阳离子聚磷酸酯基因载体阳离子聚磷酸酯基因载体54卓仁禧院士_用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件55PPA-DPAGal-PPAPPA-DPAGal-PPA56Gal-PEG-PPAGal-PEG-PPA57liverspleenlungkidneyheart103105104102RLU/mgproteinPPAGal-PPAGal-PEG-PPA

LuciferaseExpressioninmouseafterintraportalveininjectionofcomplexescontaining50µgplasmidliverspleenlungkidneyheart103158二.药物控释生物可降解高分子材料研究聚酯(聚乳酸,聚乙醇酸及其共聚物;聚己内酯,聚羟基丁酸及其共聚物)聚酸酐聚碳酸酯及其共聚物聚磷酸酯及其共聚物纳米药物控制释放温度敏感快速响应PNIPA水凝胶Poly(lactide-co-glycolide)PLGAPolylactidePLAPolyglycolidePGA二.药物控释生物可降解高分子材料研究Poly(lacti59聚碳酸酯及其共聚物BTMCPTMC卓仁禧,王旭立,刘立建.５－苄氧基－三亚甲基碳酸酯及其制备方法。中国专利ZL01114284.7C07D319/04

卓仁禧，王旭立，刘立建。聚（５－取代基－三亚甲基碳酸酯）及其制备方法和用途。ZL01114285.5聚碳酸酯及其共聚物BTMCPTMC卓仁禧,王旭立,刘立建.60InvitrocytotoxicityofPBTMCandPHTMC

COS7cells.Incubationtime:24h.Temp.:37oCInvitrocytotoxicityofPBTMC61BTMCTMCPoly(HTMC-co-TMC)Sn(Oct)2150oCDeprotectionBTMCTMCPoly(HTMC-co-TMC)Sn(Oct62DependenceofhydrophilicityoncopolymercontentDependenceofhydrophilicityo63Invitrodegradabilityofcopolymers,PBSpH7.4，37oC0246810121416182022242628020406080100P(HTMC-co-TMC)10/90PTMCP(HTMC-co-TMC)50/50WeightLoss(%)Time(day)PHTMC100%-OHwithout-OH50%-OH10%-OHInvitrodegradabilityofcopo645-Fureleaseprofilesofcopolymers,PBSpH7.4，37oC5-Fureleaseprofilesofcopol65ZhangY,ZhuoRX,Synthesisanddrugreleasebehaviorofpoly(trimethylenecarbonate)–poly(ethyleneglycol)–poly(trimethylenecarbonate)nanoparticles,Biomaterials(Inpress)PTETtriblockcopolymer纳米药物控制释放ZhangY,ZhuoRX,Synthesisa66SynthesisandpropertiesofPTETtriblockcopolymersSynthesisandpropertiesofPT67Particlesizes,drugloadingcontent,loadingefficiency,andyieldofthePTETpolymericnanoparticlesPolymericnanoparticleswerepreparedbythedialysistechnique.Drug:methotrexate(MTX)Particlesizes,drugloadingc68TEMimageofPTET-7nanoparticlesloadedmethotrexate(MTX)TEMimageofPTET-7nanopartic69EffectofmolecularweightofPEGinthecopolymersontheMTXreleaseratePBS,pH=7.4,37ºC.PEG6000PEG4000PEG2000Effectofmolecularweightof70EffectofcontentofPEGblockinthecopolymersontheMTXreleaseratePBS,pH=7.4,37ºC.PEG:6.17wt%PEG:24.12wt%PEG:28.94wt%PEG:54.26wt%EffectofcontentofPEGblock71温度敏感快速响应PNIPA水凝胶A混合溶剂法B相分离法C使用成孔剂:如PEGD形成互穿网络在温度敏感水凝胶领域发表SCI论文30多篇。温度敏感快速响应PNIPA水凝胶A混合溶剂法72卓仁禧院士_用于基因和抗癌药物控制释放高分子材料研究-(minimizer)课件73靶向性抗癌药物5-Fu抗癌药物PMliver-targetinggroup肝靶向高分子抗癌药物靶向性抗癌药物5-Fu抗癌药物PMliver-targe74肝靶向高分子抗癌药物5-Fu-(99mTc–DTPA)-PAEA-PM注射后不同时间在大鼠体内各器官的分布肝靶向高分子抗癌药物5-Fu-(99mTc–DTPA)-7562.5μg/mLBel-7204体外凋亡率:51%135μg/mLBel-7204体外凋亡率:62%Control肝靶向高分子抗癌药物5-Fu-PAEA-PM对人体肝癌细胞(Bel-7204)的体外凋亡实验的荧光照片62.5μg/mL135μg/mLControl肝靶向高分子76P(TMC-co-EEP)PorcinePancreasLipase(PPL)CandidaRugosaLipase(CL)Al(OiPr)3酶催化开环聚合反应P(TMC-co-EEP)PorcinePancreas77Ring-openingcopolymerizationofTMCandEEPcatalyzedbyenzymesMw/MnMnYield(%)Time(d)Molarratio(TMC:EEP)[E](mg/mmol)Lipase-<500-210:510--<500-210:110-1.69641042.1210:510CL1.83644062.0210:310CL2.23786075.6210:110CL2.18323019.8210:510PPL1.66802060.1210:310PPL1.711025082.8210:110PPL2.63573060.2410:210CL2.97601057.0310:210CL1.99683050.2110:210CL2.75343065.2410:210PPL1.91398072.3310:210PPL2.47597056.2110:210PPL1.81532860.2210:250CL1.87650057.6210:225CL2.27649058.3210:210CL2.43623036.5210:225PPL2.31933068.9210:210PPLRing-openingcopolymerization78PolymerizationcatalyzedbyPPLimmobilizedonsilicamicroparticles(PISM)

PolymerizationcatalyzedbyPP79EffectoflipaseconcentrationonMnandyield0.00.51.01.52.02.550