聚马来酸烷基酯—侧链烷基氨阳离子聚合物载体库的平行合成及在肿瘤基因治疗中的应用

发布时间：2018-03-19 02:33

本文选题：基因输送　切入点：阳离子聚合物　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：基因治疗是指将外源治疗基因输送到靶细胞、以纠正或补偿基因缺陷的一种遗传水平上的治疗方法。在过去的几十年中,基因治疗已被证实是一种有效的具有低副作用的癌症治疗新手段。非病毒基因输送载体包括阳离子聚合物、脂质体、树枝状大分子和多肽等,因其具有生物相容性良好、免疫原性低、易于化学修饰和制备方便等特点而成为各大科研机构的研究热点。然而与病毒载体相比,其临床应用受限于低效的基因转染效率。阳离子聚合物通过静电相互作用中和负电性的DNA将其压缩成纳米颗粒,保护DNA免受降解,同时促进细胞摄取,将DNA输送到靶细胞中进行转染。迄今为止,已有大量的工作致力于通过平行合成的方式筛选出具有高转染效率的阳离子聚合物。平行合成作为一种能够通过调整单体结构快速合成大量具有功能多样性聚合物的手段已经引起越来越多的关注。在高通量筛选后,可挑选出具有高转染效率的聚合物,并且可以清晰地阐明聚合物结构和转染效率之间的关系,这对于今后设计高效转染效率的非病毒基因输送载体具有重要意义。目前用于平行合成的方法往往局限于胺与丙烯酸酯或环氧反应,氨基硫醇与吖内酯加成后的开环聚合反应或passerini反应。因此我们迫切需要设计新型的载体合成路线来制备用于基因输送的一系列高转染效率的阳离子聚合物。在此,本论文报道一种后修饰的方法来简易合成一系列酯酶响应性的阳离子聚合物基因输送载体。我们通过巯烯点击反应将18种巯基胺侧链单体键合于7种聚马来酸烷基酯主链上,构建出一个包含126种可降解阳离子聚合物的基因输送载体库,从而克服现存载体库在功能和可扩展性方面的限制,并证明不同结构特征对基因输送的影响。首先,使用荧光素酶编码质粒作为报告基因,借助半高通量筛选的方法,评价阳离子聚合物的基因表达水平。在126种载体中,共有7种表现出高效的转染效率,同时具有良好的抗血清能力。其次,使用增强的绿色荧光蛋白编码质粒作为报告基因检测表达细胞的百分比来评估7种聚合物的转染效率。我们总结出聚合物结构对DNA包裹能力,转染效率,polyplex大小的影响。再次,构建HeLa腹腔瘤模型,评价转染效果最佳的载体G-1的抑瘤效果。G-1/pTRAIL纳米复合物能够成功将治疗基因TRAIL输送到肿瘤部位,表现出明显高于PEI25k/pTRAIL的抑瘤效率,同时没有毒副作用。
[Abstract]:Gene therapy is a genetic treatment that transports foreign therapeutic genes to target cells to correct or compensate for genetic defects. Gene therapy has been proved to be an effective new method for cancer treatment with low side effects. Non-viral gene delivery vectors include cationic polymers, liposomes, dendritic macromolecules and polypeptides because of their good biocompatibility. Because of its low immunogenicity, easy chemical modification and easy preparation, it has become a hot research topic in various scientific research institutions. However, compared with viral vectors, Its clinical application is limited by the low efficiency of gene transfection. Cationic polymers compress it into nanoparticles by electrostatic interaction and negatively charged DNA to protect DNA from degradation and promote cell uptake. Transfection of DNA into target cells. So far, A great deal of work has been done to screen cationic polymers with high transfection efficiency by parallel synthesis. As a kind of cationic polymer with high transfection efficiency, parallel synthesis can rapidly synthesize a large number of functional diversity polymerization by adjusting monomer structure. Material means have attracted more and more attention. After high-throughput screening, Polymers with high transfection efficiency can be selected and the relationship between polymer structure and transfection efficiency can be clearly clarified. This is of great significance for the future design of non-viral gene delivery vectors with high transfection efficiency. The current methods for parallel synthesis are often limited to the reaction of amine with acrylate or epoxide. The ring-opening polymerization or passerini reaction after addition of aminothiol with acridine. Therefore, we urgently need to design a novel carrier synthesis route to prepare a series of high transfection efficiency cationic polymers for gene transport. A series of esterase responsive cationic polymer gene delivery vectors were synthesized by a post-modification method. Eighteen mercapto amine side chain monomers were bonded to 7 polyalkyl maleate main chains by mercapto click reaction. A gene transfer vector library containing 126 biodegradable cationic polymers was constructed to overcome the functional and extensible limitations of the existing vector library and to prove the influence of different structural characteristics on gene delivery. Luciferase encoding plasmids were used as reporter genes to evaluate the gene expression level of cationic polymers by semi-high-throughput screening. Seven of 126 vectors showed high transfection efficiency. At the same time have good antiserum ability. Secondly, The transfection efficiency of seven kinds of polymers was evaluated by using enhanced green fluorescent protein coding plasmid as a percentage of reporter gene to detect expression cells. We summarized the effect of polymer structure on DNA encapsulation ability, transfection efficiency and the size of polyplex. HeLa peritoneal tumor model was constructed to evaluate the anti-tumor effect of the vector G-1. G-1 / pTRAIL nanocomplex could successfully transport the therapeutic gene TRAIL to the tumor site, which was significantly higher than that of PEI25k/pTRAIL, and had no toxic and side effects.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450

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