侧链含酸敏感基因的PEG嵌段共聚物的制备、评价及基因传递的研究

发布时间：2018-06-06 05:03

本文选题：嵌段共聚物 + 原酸酯　；参考：《江南大学》2014年硕士论文

【摘要】：本文采用可逆加成-断裂链转移自由基聚合法(RAFT)合成了一种新型酸敏感嵌段共聚物。该共聚物由亲水的聚乙二醇链段(PEG)和由原酸酯环连接叔胺与甲基丙烯酰胺形成的链段(PAOE)组成，侧链具有确定的长度(链段数分别为22，48，66)。在中性pH条件下，这些嵌段共聚物可以与DNA结合，并有效压缩DNA形成纳米颗粒。随着聚合物链段数的增加，阳离子聚合物/DNA复合物粒径逐渐减小，最终稳定在200nm左右，复合物抗肝素钠静电排斥的稳定性逐渐增强。Zeta电位表明，在N/P比大于2:1时，阳离子聚合物/DNA复合物表面带正电荷。PAOE侧链上的原酸酯环的降解机理是环外降解，且环境中的酸性越强，原酸酯环降解速率越快，最终导致复合物快速分解并且释放出质粒DNA。体外细胞毒性试验(MTT法)研究表明，三个不同链段数的共聚物对COS7细胞和HEK293细胞几乎无细胞毒性，，仅在聚合物浓度高达1mg/mL时，聚合物表现出一定的细胞毒性，但细胞整体存活率均达到60%以上。本文还采用流式细胞仪检测基因传递效率，为考察基因载体的结构和基因传递效率之间的关系提供依据，研究结果表明PAOE的链段数较长时，细胞的基因传递效率更高，且HEK293细胞的基因传递效率高于COS7细胞，PEG-b-PAOE66/DNA复合物的N/P比为8:1时，复合物在HEK293细胞和COS7细胞中均取得最高的基因传递效率。综上所述，嵌段共聚物PEG-b-PAOE具有优良的稳定性、低细胞毒性及结合DNA的能力，有望成为pH敏感的新型非病毒基因载体。
[Abstract]:A novel acid sensitive block copolymer was synthesized by reversible addition fracture chain transfer free radical polymerization (RAFT). The copolymer consists of a hydrophilic polyethylene glycol segment (PEG) and a chain segment (PAOE) formed by a tertiary amine and methyl acrylamide by a proactive ester ring. The side chain has a definite length (the number of chain segments is 22,48,66, respectively).
Under the condition of neutral pH, these block copolymers can be combined with DNA and effectively compress DNA to form nanoparticles. With the increase of the number of polymer chain segments, the particle size of the cationic polymer /DNA complex decreases gradually and eventually is around 200nm. The stability of the electrostatic repulsion of the compound anti heparin sodium gradually strengthens the.Zeta potential, indicating that the N/P ratio is greater than the N/P ratio. At 2:1, the degradation mechanism of the original ring of the cationic polymer /DNA complex on the surface of the positive charge.PAOE side chain is extracellular degradation, and the stronger the acid in the environment, the faster the degradation rate of the ester ring, the final result in the rapid decomposition of the complex and the release of the plasmid DNA. in vitro cytotoxicity test (MTT method), which showed that the number of three different segment numbers The copolymer has almost no cytotoxicity to COS7 cells and HEK293 cells. Only when the concentration of the polymer is up to 1mg/mL, the polymer shows a certain cytotoxicity, but the overall survival rate of the cells is above 60%.
The flow cytometry is also used to detect gene transfer efficiency, which provides a basis for investigating the relationship between the structure of gene vector and the relationship between gene transfer efficiency. The results show that the cell gene transfer efficiency is higher when the number of PAOE segments is longer, and the gene transfer efficiency of HEK293 cells is higher than that of COS7 cells, and the N/P ratio of PEG-b-PAOE66/DNA complex is higher. At the time of 8:1, the compound obtained the highest gene transfer efficiency in both HEK293 cells and COS7 cells. To sum up, the block copolymer PEG-b-PAOE has excellent stability, low cytotoxicity and the ability to combine with DNA. It is expected to be a new non viral gene vector sensitive to pH.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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