卓仁禧院士-用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件

用于基因和抗癌藥物控制釋放高分子材料研究卓仁禧院士生物醫(yī)用高分子材料教育部重點(diǎn)實(shí)驗(yàn)室武漢大學(xué)化學(xué)與分子科學(xué)學(xué)院卓仁禧院士_用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件1一.高分子基因治療載體研究病毒載體和非病毒載體的比較效率低安全容易制備，儲(chǔ)存和質(zhì)量控制傳遞的DNA大小從20個(gè)堿基對(duì)到幾十萬個(gè)堿基對(duì)非病毒載體安全隱患DNA大小受限制免疫原性高效病毒載體缺點(diǎn)優(yōu)點(diǎn)載體一.高分子基因治療載體研究效率低安全非病毒安全隱患高效病2

非病毒載體陽離子脂質(zhì)體

高分子:陽離子高分子水溶性非離子高分子高分子基質(zhì)非病毒載體陽離子脂質(zhì)體3常用的聚陽離子基因載體Poly[-(4-amino-butyl)-L-glycolicacid]SWKim,UtahUnivPoly-L-lysinePolyethylenimines(PEI)BehrJP,FranceDendrimerSzokaUCSF&BakerJUnivMichPolyphosphoesterandPolyphosphoamideLeongKW,JohnsHopkingUnivLangerR,MIT常用的聚陽離子基因載體Poly[-(4-amino-but4從新的起始核合成聚酰胺胺樹型高分子基因載體從新的起始核合成聚酰胺胺樹型高分子基因載體5DendrimerGT5DendrimerGT56TransfectionofHEK293cellswithpRELuc/dendrimerGTcomplexes合成的樹型高分子的轉(zhuǎn)染效率與目前最好的陽離子載體PEI相當(dāng)，但是比PEI的毒性低得多CytotoxicityofdendrimerGT3~GT5measuredbytheMTTassay.PEIandPLLwereusedascontrols

TransfectionofHEK293cells7肝細(xì)胞靶向基因載體

肝細(xì)胞靶向基因載體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陽離子聚磷酸酯基因載體陽離子聚磷酸酯基因載體11卓仁禧院士_用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件12PPA-DPAGal-PPAPPA-DPAGal-PPA13Gal-PEG-PPAGal-PEG-PPA14liverspleenlungkidneyheart103105104102RLU/mgproteinPPAGal-PPAGal-PEG-PPA

LuciferaseExpressioninmouseafterintraportalveininjectionofcomplexescontaining50μgplasmidliverspleenlungkidneyheart103115二.藥物控釋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;聚己內(nèi)酯,聚羥基丁酸及其共聚物)聚酸酐聚碳酸酯及其共聚物聚磷酸酯及其共聚物納米藥物控制釋放溫度敏感快速響應(yīng)PNIPA水凝膠Poly(lactide-co-glycolide)PLGAPolylactidePLAPolyglycolidePGA二.藥物控釋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,劉立建.５－芐氧基－三亞甲基碳酸酯及其制備方法。中國專利ZL01114284.7C07D319/04

卓仁禧，王旭立，劉立建。聚（５－取代基－三亞甲基碳酸酯）及其制備方法和用途。ZL01114285.5聚碳酸酯及其共聚物BTMCPTMC卓仁禧,王旭立,劉立建.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納米藥物控制釋放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溫度敏感快速響應(yīng)PNIPA水凝膠A混合溶劑法B相分離法C使用成孔劑:如PEGD形成互穿網(wǎng)絡(luò)在溫度敏感水凝膠領(lǐng)域發(fā)表SCI論文30多篇。溫度敏感快速響應(yīng)PNIPA水凝膠A混合溶劑法29卓仁禧院士_用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件30靶向性抗癌藥物5-Fu抗癌藥物PMliver-targetinggroup肝靶向高分子抗癌藥物靶向性抗癌藥物5-Fu抗癌藥物PMliver-targe31肝靶向高分子抗癌藥物5-Fu-(99mTc–DTPA)-PAEA-PM注射后不同時(shí)間在大鼠體內(nèi)各器官的分布肝靶向高分子抗癌藥物5-Fu-(99mTc–DTPA)-3262.5μg/mLBel-7204體外凋亡率:51%135μg/mLBel-7204體外凋亡率:62%Control肝靶向高分子抗癌藥物5-Fu-PAEA-PM對(duì)人體肝癌細(xì)胞(Bel-7204)的體外凋亡實(shí)驗(yàn)的熒光照片62.5μg/mL135μg/mLControl肝靶向高分子33P(TMC-co-EEP)PorcinePancreasLipase(PPL)CandidaRugosaLipase(CL)Al(OiPr)3酶催化開環(huán)聚合反應(yīng)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致謝武漢大學(xué)生物醫(yī)用高分子材料教育部重點(diǎn)實(shí)驗(yàn)室的教師、博士研究生、碩士研究生國家科技部973項(xiàng)目國家自然科學(xué)基金重點(diǎn)項(xiàng)目、面上項(xiàng)目資助致謝43用于基因和抗癌藥物控制釋放高分子材料研究卓仁禧院士生物醫(yī)用高分子材料教育部重點(diǎn)實(shí)驗(yàn)室武漢大學(xué)化學(xué)與分子科學(xué)學(xué)院卓仁禧院士_用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件44一.高分子基因治療載體研究病毒載體和非病毒載體的比較效率低安全容易制備，儲(chǔ)存和質(zhì)量控制傳遞的DNA大小從20個(gè)堿基對(duì)到幾十萬個(gè)堿基對(duì)非病毒載體安全隱患DNA大小受限制免疫原性高效病毒載體缺點(diǎn)優(yōu)點(diǎn)載體一.高分子基因治療載體研究效率低安全非病毒安全隱患高效病45

非病毒載體陽離子脂質(zhì)體

高分子:陽離子高分子水溶性非離子高分子高分子基質(zhì)非病毒載體陽離子脂質(zhì)體46常用的聚陽離子基因載體Poly[-(4-amino-butyl)-L-glycolicacid]SWKim,UtahUnivPoly-L-lysinePolyethylenimines(PEI)BehrJP,FranceDendrimerSzokaUCSF&BakerJUnivMichPolyphosphoesterandPolyphosphoamideLeongKW,JohnsHopkingUnivLangerR,MIT常用的聚陽離子基因載體Poly[-(4-amino-but47從新的起始核合成聚酰胺胺樹型高分子基因載體從新的起始核合成聚酰胺胺樹型高分子基因載體48DendrimerGT5DendrimerGT549TransfectionofHEK293cellswithpRELuc/dendrimerGTcomplexes合成的樹型高分子的轉(zhuǎn)染效率與目前最好的陽離子載體PEI相當(dāng)，但是比PEI的毒性低得多CytotoxicityofdendrimerGT3~GT5measuredbytheMTTassay.PEIandPLLwereusedascontrols

TransfectionofHEK293cells50肝細(xì)胞靶向基因載體

肝細(xì)胞靶向基因載體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陽離子聚磷酸酯基因載體陽離子聚磷酸酯基因載體54卓仁禧院士_用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件55PPA-DPAGal-PPAPPA-DPAGal-PPA56Gal-PEG-PPAGal-PEG-PPA57liverspleenlungkidneyheart103105104102RLU/mgproteinPPAGal-PPAGal-PEG-PPA

LuciferaseExpressioninmouseafterintraportalveininjectionofcomplexescontaining50μgplasmidliverspleenlungkidneyheart103158二.藥物控釋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;聚己內(nèi)酯,聚羥基丁酸及其共聚物)聚酸酐聚碳酸酯及其共聚物聚磷酸酯及其共聚物納米藥物控制釋放溫度敏感快速響應(yīng)PNIPA水凝膠Poly(lactide-co-glycolide)PLGAPolylactidePLAPolyglycolidePGA二.藥物控釋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,劉立建.５－芐氧基－三亞甲基碳酸酯及其制備方法。中國專利ZL01114284.7C07D319/04

卓仁禧，王旭立，劉立建。聚（５－取代基－三亞甲基碳酸酯）及其制備方法和用途。ZL01114285.5聚碳酸酯及其共聚物BTMCPTMC卓仁禧,王旭立,劉立建.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納米藥物控制釋放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溫度敏感快速響應(yīng)PNIPA水凝膠A混合溶劑法B相分離法C使用成孔劑:如PEGD形成互穿網(wǎng)絡(luò)在溫度敏感水凝膠領(lǐng)域發(fā)表SCI論文30多篇。溫度敏感快速響應(yīng)PNIPA水凝膠A混合溶劑法72卓仁禧院士_用于基因和抗癌藥物控制釋放高分子材料研究-(minimizer)課件73靶向性抗癌藥物5-Fu抗癌藥物PMliver-targetinggroup肝靶向高分子抗癌藥物靶向性抗癌藥物5-Fu抗癌藥物PMliver-targe74肝靶向高分子抗癌藥物5-Fu-(99mTc–DTPA)-PAEA-PM注射后不同時(shí)間在大鼠體內(nèi)各器官的分布肝靶向高分子抗癌藥物5-Fu-(99mTc–DTPA)-7562.5μg/mLBel-7204體外凋亡率:51%135μg/mLBel-7204體外凋亡率:62%Control肝靶向高分子抗癌藥物5-Fu-PAEA-PM對(duì)人體肝癌細(xì)胞(Bel-7204)的體外凋亡實(shí)驗(yàn)的熒光照片62.5μg/mL135μg/mLControl肝靶向高分子76P(TMC-co-EEP)PorcinePancreasLipase(PPL)CandidaRugosaLipase(CL)Al(OiPr)3酶催化開環(huán)聚合反應(yīng)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