卓仁禧院士-用于基因和抗癌药物操纵开释高分子资料研究(PPTminimizer)[精品]课件.ppt

1、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效率低安全安全容易制备，储存和质量控制容易制备，储存和质量控制传递的传递的DNADNA大小从大小从2020个碱基对个碱基对到几十万个碱基对到几十万个碱基对非病毒非病毒载体载体安全隐患安全隐患DNADNA大小受限制大小受限制免疫原性免疫原性高效高效病毒病毒载体载体缺点缺点优点优点载体载体 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 常用的聚阳离子基因载体OOH2NnH2NNHNNHNHNH

2、2NHNNNHHNNHPoly-(4-amino-butyl)-L-glycolic acidS W Kim, Utah UnivPoly-L-lysinePolyethylenimines (PEI)Behr J P, FranceNHOH2NnDendrimerSzoka UCSF & Baker J Univ MichNHHNOOHN(CH2)4NH2OHNNxyOPOOONH3ClnPPEOPOONnH2NNH2PPA-SPPolyphosphoester and PolyphosphoamideLeong K W, Johns Hopking UnivLanger R, MIT从新的

3、起始核合成聚酰胺胺树型高分子基因载体HNHNNHHNHOOHHOHOOHOHOHOHHOHOCOOMeCOOMeMeOOCNHNONNHHNOONNHHNOONNHHNOONNHHNOONH2NH2NNHHNOONH2NH2NNHHNOONH2NH2NNHNHOONH2NH2HNONNHHNOONNHHNOONNHHNOONNHHNOOH2NNH2NNHHNOOH2NNH2NNHHNOOH2NNH2NNHHNOONH2NH2NHOONNHONHNNHOONHNNHOONHNNHOONHNNHOONH2NH2NHNNHOONH2NH2NHNNHOONH2H2NNHNNHOONH2H2NHNONN

4、HNHOONNHNHOONNHNHOONNHHNOONH2NH2NNHHNOONH2NH2NNHNHOONH2NH2NHNNHOONH2NH2HNOOCNNHONHNNHOONHNNHOONHNNHOONHNNHOOH2NH2NNHNNHOOH2NH2NNHNNHOOH2NH2NNHNHNOOH2NH2NNHONHNNHOONHNNHOONHNNHOONHNNHOONH2H2NNHNNHOONH2H2NNHNNHOONH2H2NNHNNHOONH2H2NHNOONHNONNHHNOONNHHNOONNHHNOONNHHNOOH2NNH2NNHHNOOH2NNH2NNHHNOOH2NNH2NNH

5、HNOOH2NNH2NHONHNHNOONHNHNOONHNHNOONHNNHOOH2NH2NNHNNHOOH2NH2NNHNHNOOH2NH2NNNHHNOOH2NH2NNHOODendrimer GT51.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+091.00E+10Naked DNATransfastPEIPLLGT3GT4GT5RLU/mg proteinTransfection of HEK 293 cells with pRE Luc/dendrimer GT complexes合成的树型高分子的转染效率与目前最好的阳离

6、子载体PEI相当，但是比PEI的毒性低得多020406080100120050100150200Dendrimer concentration (ug/mL)Percent relative viabilityGT3GT4GT5PLLPEICytotoxicity of dendrimer GT3GT5 measured by the MTT assay. PEI and PLL were used as controls 肝细胞靶向基因载体肝细胞靶向基因载体 OOOHOOHOHOHOHOHHOHOPAMAM dendrimer+NaBH3(CN)Dendrimer-GalGT5-Gal 5%

7、GT5-Gal 10%GT5-Gal 20%1.0E+041.0E+051.0E+061.0E+071.0E+081.0E+090246810N/PRLU/mg proteinGT5GT5-Gal 5%Transfection of HepG2 cells with pRE Luc1.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+090246810N/PRLU/mg proteinGT5GT5-Gal 5%Transfection of NIH 3T3 cells with pRE Luc阳离子聚磷酸酯基因载体OPOOO(CH2)nNR

8、H2ClnPPE-HA: R=H, n=6PPE-MEA: R=Me, n=2PPA-DPAGal-PPAOCH2CHCH3OPONH2NNHOCH2CHCH3OPONH2NNH2xyOOHOHHOOHNHOCH2COPEGSOOGal-PEG-PPAliverspleenlungkidneyheart103105104102RLU/mg proteinPPAGal-PPAGal-PEG-PPA Luciferase Expression in mouse after intraportal vein injection of complexes containing 50 g plasmid

9、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)PLGAPolylactidePLAPolyglycolidePGAOOOOCH2Ph聚碳酸酯及其共聚物BTMCOC OCH2CH2CH2OnPTMCOC OCH2CHCH2OxOHPHTMC卓仁禧,王旭立,刘立建. 苄氧基三亚甲基碳酸酯及其制备方法。中国专利 ZL 01 1 14284.7 C07D319 /04 卓仁禧，王旭立，刘立建。聚（取代基

10、三亚甲基碳酸酯）及其制备方法和用途。ZL01114285.5 In vitro cytotoxicity of PBTMC and PHTMC COS7 cells. Incubation time: 24 h. Temp.: 37oCPLGA(75:25)20406080100120100502512.56.25 3.1250Polymer concentration (mg/ml)Cell viability (%)PBTMC20406080100120100502512.56.25 3.1250Polymer concentration (mg/ml)Cell viability (%)

11、PHTMC20406080100120100502512.56.253.1250Polymer concentration (mg/ml)Cell viability (%)OOCOCH2PhOOOCOCOCH2CHCH2OCH2PhOCO(CH2)3OOOyx+COCH2CHCH2OHOCO(CH2)3OOOyxBTMCTMCPoly(HTMC-co-TMC)Sn(Oct)2150oCDeprotection0501001502000102030405060708090100Mole percentage of -OH groupsWater absorption(Wt%)Dependenc

12、e of hydrophilicity on copolymer contentIn vitro degradability of copolymers, PBS pH7.4，37oC0246810121416182022242628020406080100P(HTMC-co-TMC) 10/90PTMCP(HTMC-co-TMC) 50/50Weight Loss (%)Time (day)PHTMC100% -OHwithout -OH50% -OH10% -OH024681012141601020304050Poly(HTMC-co-TMC)(50:50)5-Fu release per

13、cent (%)Time (day)Poly(BTMC-co-TMC)(50:50)5-Fu release profiles of copolymers, PBS pH7.4，37oCOOO+HOOHnSn(Oct)2TMCPEGHOOOOOOOOOHmnm150 CPTETZhang Y, Zhuo R X, Synthesis and drug release behavior of poly (trimethylene carbonate)poly (ethylene glycol)poly (trimethylene carbonate) nanoparticles, Biomate

14、rials (In press) PTET triblock copolymer纳米药物控制释放Synthesis and properties of PTET triblock copolymers Particle sizes, drug loading content, loading efficiency, and yield of the PTET polymeric nanoparticlesPolymeric nanoparticles were prepared by the dialysis technique.Drug: methotrexate (MTX)TEM imag

15、e of PTET-7 nanoparticles loaded methotrexate (MTX)Effect of molecular weight of PEG in the copolymers on the MTX release ratePBS, pH =7.4, 37 C.PEG 6000PEG 4000PEG 2000Effect of content of PEG block in the copolymers on the MTX release ratePBS, pH =7.4, 37 C.PEG: 6.17 wt%PEG: 24.12 wt%PEG: 28.94 wt

16、%PEG: 54.26 wt%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。靶向性抗癌药物 5-Fu抗癌药物PM liver-targeting group肝靶向高分子抗癌药物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0 05 510101515202025253030 B l o o d H e a r t L i v e r S p l e e n L u n g K i d n e y I n t e s t i n e M u

17、 s c l e B o n eB l o o d H e a r t L i v e r S p l e e n L u n g K i d n e y I n t e s t i n e M u s c l e B o n e%ID/g%ID/g10 min20 min30 min60 min90 min120min180min240min 62.5g/mLBel-7204体外凋亡率:51%135g/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P(TMC-co-EEP)OOCOO

18、OPOCH2CH3O+CatalystBulkPO (CH2)2OC O CH2CH2CH2OOCH2CH3OmnOPorcine Pancreas Lipase (PPL) Candida Rugosa Lipase (CL)Al(OiPr)3酶催化开环聚合反应Ring-opening copolymerization of TMC and EEP catalyzed by enzymesMw/MnMnYield (%)Time(d)Molar ratio(TMC:EEP)E (mg/mmol)Lipase-500-210:510-500-210:110-1.69641042.1210:51

19、0CL1.83644062.0210:310CL2.23786075.6210:110CL2.18323019.8210:510PPL1.66802060.1210:310PPL1.711025082.8210:110PPL2.63573060.2410:210CL2.97601057.0310:210CL1.99683050.2110:210CL2.75343065.2410:210PPL1.91398072.3310:210PPL2.47597056.2110:210PPL1.81532860.2210:250CL1.87650057.6210:225CL2.27649058.3210:2

20、10CL2.43623036.5210:225PPL2.31933068.9210:210PPLOOCOOCH2CH2CH2OCnPISMOPolymerization catalyzed by PPL immobilized on silica microparticles (PISM) Effect of lipase concentration on Mn and yield0.00.51.01.52.02.55000100001500020000250003000035000400004500050000Concentration(wt%)Molecular weight3035404

21、550556065707580859095100Yield(% )MnYieldTime dependence of Mn and yield during polymerization1020304050601000012000140001600018000200002200024000Time(h)Mn404550556065707580859095100YieldMnYieldDependence of Mn and yield on the immobilized lipase PPL recycling timeRecycling TimeLipase Conc. (wt%)MnMw

22、/MnYield (%)I1.0147001.9270II1.0254001.7775III1.0308002.1181IV1.0452002.0178VI1.0617001.9483VII1.0630002.5381VIII1.0640002.2072VIII0.1874002.0669Reaction temperature: 100. Reaction time: 24hOOOIMPPLOOOnPolymerization of DTC catalyzed by PPLA immobilized on silica microparticles B immobilized on sili

23、ca nanoparticles 012350001000015000200002500030000350004000045000Mn (g/mol)Recycling times 2.0wt.-% 1.0wt.-% 0.5wt.-%Recycling time of IMPPL vs Mn of Poly(DTC) obtained at 130C for 4 day-polymerization 4000350030002500200015001000EDCBWave number cm-1APoly (DTC)The original IMPPL The first recycling IMPPLThe second recycling IMPPL The third recycling IMPPL 1752cm-1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