基因和药物共传递体系的制备与研究

发布时间：2018-06-11 15:37

本文选题：共聚物 + 电荷翻转　；参考：《长春理工大学》2014年硕士论文

【摘要】：本文以超支化聚乙烯亚胺(PEI)引发赖氨酸-N-内羧酸酐(Lys-NCA)和谷氨酸-N-内羧酸酐(Glu-NCA)单体开环聚合,得到具有pH响应的电荷翻转功能的聚乙烯亚胺-聚赖氨酸-聚谷氨酸无规共聚物(PELG),该共聚物可以作为纳米载体的遮蔽体系。利用乌头酸酐(CA)对抗肿瘤药物阿霉素(DOX)进行修饰,得到酸敏感的小分子药物乌头酸酐-阿霉素(CAD)。通过核磁、凝胶渗透色谱、红外光谱、质谱对目标产物进行了结构和分子量等一系列表征,证实了目标产物的成功合成。通过静电吸附制备了PELG/PEI/CAD载药复合物体系。Zeta电位测试证实了体系具有pH响应的电荷翻转功能。药物释放实验证实体系具有酸敏感的药物释放性能。细胞毒性实验证实载体材料的生物安全性良好,且PELG/PEI/CAD对HeLa细胞具有非常高的杀伤力。细胞内吞实验和共聚焦照片结果显示,体系在pH6.8(类似肿瘤微酸环境)时能够被肿瘤细胞高效内吞,大量的药物可被载体携带进入细胞并分布于细胞质及细胞核中。由于早期的工作已经验证了体系的载基因性能,所以本文中进一步主要研究基因和药物共传递体系PELG/PEI/(DNA+CAD)。Zeta电位测试证实了共传递体系具有pH响应的电荷翻转功能。DNA凝胶阻滞实验证明体系具有良好的DNA结合能力。药物释放实验证实该共传递体系具有酸敏感的药物释放功能。利用PELG/PEI/DNA对HepG2细胞进行DNA转染,体系展现出良好的细胞转染效率,并确定了最佳转染质量比：PELG/PEI/(DNA+CAD)共传递体系中,载入不同浓度的CAD对DNA的转染影响并不大,并确定了体系担载DNA和CAD的质量比。进一步利用p53基因作为治疗基因,制备PELG/PEI/(p53+CAD)基因和药物共传递体系。细胞毒性实验显示共传递体系的半数抑制浓度(IC50)均低于单独的载基因或载药体系,即共传递体系对肿瘤细胞的杀伤力最大。激光共聚焦照片证实基因和药物能被载体共同携带到肿瘤细胞中,且在pH6.8条件下,肿瘤细胞对体系复合物的内吞效率更高,细胞内定位的基因和药量更多。RT-PCR实验证实经过共载体系作用后的肿瘤细胞中抑癌基因p53的表达量显著增大。凋亡实验显示共载体系可使肿瘤细胞发生严重的凋亡,24h时可使80%的细胞凋亡,48h时癌细胞凋亡达到96%。该共传递体系在抗肿瘤治疗中将具有很高的潜力。
[Abstract]:The ring-opening polymerization of Lys-NCAand Glu-NCA-Glu-NCA) was initiated by hyperbranched polyimide (PEI). The polyethyleneimine-poly-lysine-poly (glutamic acid) random copolymer (PELGN) with charge flipping function in response to pH was obtained. The copolymer can be used as a shelter system for nanometer carriers. Aconitine anhydride (CAA) was used to modify adriamycin (DOX) to obtain aconitine-adriamycin (CAD), a small aconitine sensitive drug. The structure and molecular weight of the target products were characterized by NMR, gel permeation chromatography, infrared spectroscopy and mass spectrometry. The PELG- / PEI- / CAD drug-carrying complex system 路Zeta potential was prepared by electrostatic adsorption. The results showed that the system had the function of charge flipping in pH response. Drug release experiments confirmed that the system has acid-sensitive drug release performance. Cytotoxicity test showed that the carrier material had good biological safety, and PELG- / PEI / CAD had a very high bactericidal effect on HeLa cells. The results of endocytosis test and confocal photography showed that the system could be efficiently ingested by tumor cells at pH 6.8 (similar to the acid-like environment of tumor). A large number of drugs can be carried into cells by vectors and distributed in the cytoplasm and nucleus. Therefore, the further study of gene and drug codelivery system PELGR / PEI / P CADN. Zeta potential test confirmed that the co-transfer system has the charge flipping function of pH response. DNA gel block test proved that the system has good DNA binding ability. The drug release assay confirmed that the co-delivery system had acid-sensitive drug release function. Using PELG- PEI / DNA to transfect HepG2 cells, the system showed good transfection efficiency, and the best transfection quality was determined to be better than that in the cotransfer system of% PELG- PEI / DNA CAD, and the effect of different concentrations of CAD on DNA transfection was not significant. The mass ratio of DNA to CAD was determined. Furthermore, using p53 gene as therapeutic gene, PELGR / PEI / p53CAD gene and drug codelivery system were prepared. The cytotoxicity test showed that the IC50 of the co-delivery system was lower than that of the single gene carrier or drug carrier system, that is, the co-delivery system had the greatest cytotoxicity to tumor cells. Laser confocal imaging showed that genes and drugs could be carried into tumor cells by vector, and at pH 6.8, tumor cells had higher endocytosis efficiency to systemic complexes. RT-PCR results showed that the expression of tumor suppressor gene p53 in tumor cells was significantly increased after co-loading. Apoptosis assay showed that cocarrier system could induce severe apoptosis in tumor cells at 24 h, and the apoptosis rate of cancer cells reached 96% at 48 h after 80% apoptosis. The co-delivery system will have high potential in anti-tumor therapy.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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