【作者】 朱彩虹；

【导师】 朱秀林； 钟志远；

【作者基本信息】 苏州大学 ， 高分子化学与物理， 2010， 博士

【摘要】 基因疗法可用于治疗多种人类疾病如遗传性疾病、肿瘤、心血管疾病等。然而,由于缺乏高效无毒基因运送系统,基因疗法还没有在临床上得到广泛应用。近几年来,高分子非病毒基因载体因其众多的优点如低免疫反应、可运载任何尺寸的基因、外源基因整合几率低、制备简单方便等受到青睐。其中,聚甲基丙烯酸-N,N-二甲氨基乙酯(PDMAEMA)是一种常用的聚合物基因载体,高分子量的PDMAEMA(Mw>300kDa)可有效与DNA复合,在内涵体pH条件下具有强的缓冲能力,对多种细胞表现出较好的转染效率。但是,PDMAEMA不易降解,高分子量的PDMAEMA作为基因载体可能导致短期或长期的细胞毒性。此外,与其他阳离子聚合物相似,PDMAEMA/DNA复合物在血清条件下稳定性差,体内转染效率低。近年研究表明生物可降解PDMAEMA聚合物可望降低细胞毒性和／或提高转染效率。PDMAEMA与其他高分子基因载体如PEI和PAMAM相比,具有易于合成,结构和分子量可方便得到控制的优点。本论文采用可逆加成-断裂链转移(RAFT)活性自由基聚合的方法合成了一系列结构组成明确、分子量可控的新型PDMAEMA共聚物基因载体。主要包含以下四部分的工作：1.合成了可以同时运输siRNA和疏水性抗癌药物紫杉醇进入癌细胞的生物可降解阳离子聚合物PDMAEMA-PCL-PDMAEMA胶束。该阳离子胶束的细胞毒性比25kDa PEI低,能运载GFP siRNA进入MDA-MB-435-GFP细胞,基因沉默效率最高达70%以上；而25kDa bPEI在最佳N/P比条件下基因沉默效率约为40%,20kDaPDMAEMA均聚物在最高N/P比为36/1时,仍不能抑制细胞GFP的表达。另外,对人前列腺癌细胞PC-3的体外转染实验和共聚焦显微观察都表明该阳离子聚合物胶束可以同时运输紫杉醇和VEGF siRNA进入癌细胞,能更加有效地抑制细胞的VEGF表达(基因沉默效率85%),有效结合了药物治疗和基因治疗,是一种具有应用前景的载体。2.合成了新型还原敏感的三嵌段共聚物PDMAEMA-SS-PEG-SS-PDMAEMA作为基因载体。该三嵌段共聚物可以有效地压缩DNA,复合物粒径小于120nm；复合物的表面电位接近于中性,在0-+6mV之间,远小于PDMAEMA均聚物与DNA形成的复合物的表面电荷ca.+15mV；复合物在生理盐环境下稳定性好。动态激光光散射和凝胶电泳阻滞实验结果表明,在模拟细胞内的还原环境下,可以去PEG屏蔽,导致DNA解离。可还原降解PDMAEMA-SS-PEG-SS-PDMAEMA在血清存在下的转染效率最高时比不可还原降解PDMAEMA-PEG-PDMAEMA的转染效率高28倍,是有一种具有应用前景的基因载体。3.合成了分子量和结构组成可控的侧链含伯胺基团的PDMAEMA共聚物作为基因载体。共聚物在N/P比为3/1时能有效压缩DNA,复合物粒径小于PDMAEMA均聚物与DNA的复合物粒径。共聚物保持了PDMAEMA均聚物的低细胞毒性。在无血清条件下的转染效率顺序为P(DMAEMA/AHMA)>P(DMAEMA/AEMA)> PDMAEMA,共聚物P(DMAEMA/AHMA)的转染效率最高时是PDMAEMA均聚物在同等条件下的24倍；共聚物在有血清存在下的转染效率与25kDa PEI相当或者更高。4.合成了可还原降解的PDMAEMA-SS-PCL-SS-PDMAEMA阳离子聚合物胶束作为基因载体。嵌段共聚物是以含硫硫键的聚己内酯大分子RAFT试剂CPADN-SS-CL-SS-CPADN为链转移剂,通过RAFT聚合制备得到。阳离子胶束可以有效地压缩DNA,复合物表面携带正电荷,在生理盐环境条件下稳定性好。动态激光光散射和凝胶电泳阻滞实验结果表明,在模拟细胞内的还原环境下可以使DNA解离。可还原降解的阳离子聚合物胶束的细胞毒性比不可还原降解的阳离子胶束的细胞毒性要小得多,在浓度为12μg/mL时,细胞的存活率仍在80%以上,是有一种具有潜力的基因载体。更多还原

【Abstract】 Gene therapy is one of the most promising treatments for various diseases such as cancers, cardiovascular diseases, and genetic disorders. The clinical applications of gene therapy, however, are restricted by shortage of safe, efficient gene delivery technology. In the past decade, polymer-based gene delivery systems have attracted great attention due to many advantages including improved safety, low immune responses, enabling repeated uses, and ease of production. Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) has been often studied for gene transfection.High molecular weight (Mw>300 kDa) PDMAEMA has show to display high buffer capacity at endosomal pH and mediates efficient transfection in various types of cells. However, PDMAEMA is not readily biodegradable, which may render long-term and/or acute toxicity. In addition, polyplexes of PDMAEMA expose insufficient colloidal and serum stability, which restricts their applications in vivo. In the past years, bioreducible and biodegradable PDMAEMA copolymers have been developed to achieve reduced toxicity and/or enhanced transfection activity. Furthermore, unlike other cationic polymers including PEI and PAMAM, PDMAEMA copolymers can be conveniently prepared with controlled macromolecular structures and compositions by living radical polymerization. In this paper, we adopted reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare several new types of PDMAEMA copolymers for gene delivery.First, biodegradable cationic micelles based on PDMAEMA-PCL-PDMAEMA triblock copolymers were designed for the combinatorial delivery of siRNA and paclitaxel into cancer cells. The cationic micelles revealed lower cytotoxicity than 25 kDa branched PEI. Notably, GFP siRNA complexed with micelle achieved over 70% silencing efficiency at an N/P ratio of 36/1 which is higher than 25kDa bPEI (40% silencing efficiency). In contrast,20 kDa PDMAEMA revealed no silencing effect at an N/P ratio of 36/1.Moreover, the combinatorial delivery of VEGF siRNA and paclitaxel resulted in significantly lower VEGF expression as compared to delivery of VEGF siRNA or paclitaxel alone, reaching a high silencing efficiency of ca.85%. Confocal laser scanning microscope (CLSM) studies revealed that nile red was delivered into MDA-MB-435-GFP cells and that GFP expression was significantly inhibited. These results demonstrated that cationic biodegradable micelles are highly promising for the combinatorial delivery of siRNA and lipophilic anticancer drugs.Second, bioreducible PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers were designed, prepared and investigated for gene transfection. Like the non-reducible analogues, PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers could effectively condense DNA into small particles with average diameters less than 120 nm and zeta potentials close to neutral (0-+6 mV) at and above an N/P ratio of 3/1. The resulting polyplexes showed excellent colloidal stability against 150 mM NaCl, which contrasts with polyplexes of 20 kDa PDMAEMA homopolymer. In the presence of 10 mM DTT, however, polyplexes of PDMAEMA-SS-PEG-SS-PDMAEMA were rapidly de-shielded and unpacked. Release of DNA in response to 10 mM DTT was further confirmed by gel retardation assays. Notably, in vitro transfection studies showed that reversibly shielded polyplexes afforded up to 28 times higher transfection efficacy as compared to stably shielded control under the same conditions. These results demonstrated that reversibly shielded DNA polyplexes have a great potential in achieving safe and efficient gene transfection.Third, a versatile family of PDMAEMA copolymers containing varying amounts of primary amino side groups were synthesized and investigated for in vitro gene transfection. The copolymer compositions were well controlled by feed ratios. These PDMAEMA copolymers could effectively condense DNA at an N/P ratio of 3/1 to give small sized polyplexes, which were smaller than for PDMAMEA. The polyplexes of these copolymers revealed similar level of cytotoxicity as those of PDMAEMA homopolymer. Interestingly, the in vitro transfection in COS-7 cells in serum free medium demonstrated significantly enhanced (up to 24-fold) transfection efficiencies for PDMAEMA copolymer polyplexes as compared to PDMAEMA control, with following the order of P(DMAEMA/AHMA)> P(DMAEMA/AEMA)> PDMAMEA. It is remarkable to note that in the presence of 10% serum, P(DMAEMA/AEMA) and P(DMAEMA/AHMA) displayed comparable or better transfection activity with respect to 25kDa PEI at its optimal formulation. These results suggest that cationic methacrylate copolymers are highly promising for development of safe and efficient non-viral gene transfer agents.Last, reduction-responsive cationic micelles based on PDMAEMA-SS-PCL-SS-PDMAEMA triblock copolymers were investigated as non-viral gene vectors. PDMAEMA-SS-PCL-PDMAEMA triblock copolymers were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization of dimethylaminoethyl methacrylate using CPADN-SS-PCL-SS-CPADN as RAFT agent. Both DNA complexation assay and gel retardation assay showed that the cationic micelles could effectively complex DNA at and above N/P ratios of 3/1. These resulting polyplexes showed good colloidal stability against 150 mM NaCl, which contrasts with polyplexes of 12 kDa PDMAEMA homopolymer. Notably, the polyplexes were prone to fast aggregation in the presence of 10 mM DTT, as further confirmed by gel retardation assay. Moreover, the reducible micelles from PDMAEMA-SS-PCL-SS-PDMAEMA revealed a lower cytotoxicity than corresponding non-reducible triblock copolymers.更多还原