超支化聚己内酯/聚缩水甘油醚共聚物的合成及在基因传递载体方面的应用

【作者】 李晓慧；

【导师】 宋存先； 王亮； 杨菁；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2009， 博士

【摘要】 聚己内酯类材料由于其良好的生物相容性及长效降解性,在药物控释载体、基因转染等领域具有广泛的应用前景。聚缩水甘油醚是一类具有超支状结构,带有大量可修饰末端基团的高分子聚合材料,具有很好的溶解性和生物相容性。本研究合成了一种新型的超支化聚己内酯/聚缩水甘油醚共聚物（poly（epsilon-caprolactone）-b-hyperbranchedpolyglycidol,PCL-b-HPG）。PCL-b-HPG具有两亲性且带有大量羟基末端基团,能改善聚己内酯的亲水性和可修饰性。将PCL-b-HPG制备成纳米胶束,考察了其细胞毒性和基因转染效果,末端偶联RGD多肽,以期为开发新型靶向药物传递系统奠定基础。首先以辛酸亚锡（Sn（Oct）₂）为催化剂、月桂醇（1-Dodecanol）为引发剂,将己内酯单体聚合成一定分子量羟端基聚己内酯。随后采用阴离子聚合法,以二氧六环为溶剂,在萘钾为催化剂作用下,使羟端基聚己内酯引发缩水甘油醚聚合,合成了PCL-b-HPG。所得产物结构通过核磁共振氢谱（H¹NMR）、红外光谱（IR）和葡聚糖凝胶色谱（GPC）进行了表征。进一步对PCL-b-HPG嵌段共聚物反应条件进行考察,实验证实反应时间为5h,滴加速度小于1 mL/h时,反应条件最优。此外采用活性聚合法,以负离子络合型催化剂双金属μ—氧桥烷氧化物（[（n-BuO）₂AlO]₂Zn）催化ε—己内酯聚合,得到活性PCL预聚物,进一步引发缩水甘油醚聚合,成功的合成了带有大量端羟基的AB型嵌段共聚物PCL-b-HPG。与阴离子聚合法相比,通过“活”性聚合得到的嵌段共聚物的多分散度Mw/Mn为1.3左右,分散性较低。GPC、¹HNMR、DSC测定结果表明所得共聚物的结构基本符合预定结构。当己内酯和缩水甘油醚单体投料摩尔比为1:2时,反应时间10h,缩水甘油醚单体滴液速度为2 mL/h,达到最适反应条件。采用界面沉淀法成功制备了PCL-b-HPG纳米胶束。通过TEM、SEM和粒径分析仪观察,所得纳米胶束为球形,形状均匀,粒径为（170±10.0）nm。采用MTT法,检测了PCL-b-HPG纳米胶束的细胞毒性,并制备了载pEGFP-C1基因纳米胶束,所制备的纳米胶束的平均粒径约为225nm,pEGFP-C1质粒的含量约占2%,基因包埋效率在85%以上。凝胶阻滞电泳实验,显示以纳米胶束的形式装载基因,泳道中出现了明显的拖带,表明PCL-b-HPG纳米胶束能有效的包裹质粒DNA分子,体外EA.hy926细胞转染实验证实PCL-b-HPG载基因纳米胶束能够转染质粒DNA,转染效率约在15%。以N-甘氨酸马来酰亚胺直接偶联法和PCL-b-HPG羧化改性/酰胺键偶联法将RGD4C连接于PCL-b-HPG纳米胶束表面。通过IR、XPS等检测技术证实RGD成功连接于纳米粒表面。表面RGD化的纳米胶束可以识别肿瘤血管内皮上的整合素受体,使之有机会成为新型靶向药物传递系统。更多还原

【Abstract】 Biodegradable materials based on poly(ε-caprolactone) were widely used for drug delivery and gene transfection due to their good biocompatibility and long-term degradability. Polyglycidol is a soluble and biocompatible polymer which has hyperbranched architecture with numerous hydroxyl end groups.In present study,a novel di-block copolymer of poly(ε-caprolactone)-b-hyperbranched polyglycidol(PCL-b-HPG) was synthesized.Compared to PCL homopolymer,the PCL-b-HPG has improved solubility and modifiability owing to the introduced hydroxyl end groups.The PCL-b-HPG copolymer was used to prepare nanomicelles with reporter GFP gene and cytotoxicity and gene transfection efficiency of the gene nanomicelle were evaluated.The PCL-b-HPG-based nanomicelles were further chemically coupled with RGD4C peptide,which we thought to lay a preliminary foundation for developing new targeted drug delivery system.First,a hydroxyl-terminated PCL was prepared by ring-opening polymerization using 1-dodecanol as initiator and stannous octanoate as catalyst.The obtained PCL was treated with naphthalene potassium to get a PCL-based macro-initiator,which was used to initiate glycidol polymerization to form the amphiphilic copolymer of PCL-b-HPG.Chemical structure of the PCL-b-HPG was characterized by ~1H NMR,GPC and IR.Optimal parameters for the copolymerization procedure were established by series of experiments,i.e.1mL/hour of glycidol adding rate and 5 hours of total reaction time.A novel living copolymerization was designed for better control of the copolymer configuration.The living poly(ε-carprolatone) was synthesized by an ionic-coordination catalyst,named bimetallicμ-oxoalkoxides.The living system of PCL further initiated the polymerization of glycidol to form the block copolymer of PCL-b-HPG.In comparison with anion polymerization,PCL-b-HPG obtained from living copolymerization has lower polydispersity with Mw/Mn of 1.3.Within this system,optimal production conditions were set as follows:reaction time for 10 hours,glycidol adding rate at 2mL/hour and CL/glycidol ratio of 1:2.Chemical Structure of the resultant PCL-b-HPG was characterized by ~1H NMR, GPC,DSC and IR.Nanomicelles of the amphiphilic poly(epsilon-caprolactone)-b-hyperbranched polyglycidol(PCL-b-HPG) were prepared for further evaluation as targeted drug delivery vehicles.Size and morphology of the nanomicelles were characterized by TEM、SEM and DLS,respectively.The nanomicelles were spherical in uniform shape with diameter ranging from 170±10.0nm.Cytotoxicity of the PCL-b-HPG nanomicelles was evaluated by means of MTT assay.Subsequently,a reportr gene of pEGFP-C1 was loaded into the PCL-b-HPG nanomicelles of 225 nm in size.The loading efficiency is measured over 85%and the ratio of pEGFP-C1 to nanomicelles is about 1:50.In gel electrophoresis,obvious smears appeared when plasmid DNA was packaged into nanomicelles,indicating PCL-b-HPG-based nanomicelles have potential to serve as an efficient gene carrier.The yields of transfection efficiency were found to be 15%when the polyplexes were transfected to EA.hy926.These results demonstrated that the PCL-b-HPG/DNA nanomicelles are effective gene vector in vitro.RGD4C peptide was conjugated on the surface of PCL-b-HPG nanomicelle by a series of chemical reactions,such as direct coupling using N-glycine maleic anhydride as crosslinker, or modifying the hydroxyl end groups of the PCL-b-HPG molecules with carboxyl groups followed by coupling using acylamide chemical bond.The conjugation of RGD4C was confirmed by IR or XPS spectrum.It was hypothesized that the RGD4C-conjugated PCL-b-HPG nanomicelle be capable of specifically recognizing tumor neovascular endothelium,therefore may offer great opportunities for developing novel tumor-targeting drug delivery system.更多还原