论文部分内容阅读
背景:病毒类基因载体虽然具有较高的转染效率,其安全性一直受到人们的质疑。目的:以阳离子磷酸胆碱聚合物作为基因药物运输的载体,研究其对人类c-myc反义寡核苷酸(AS-ODN)的负载和运输能力,并对其安全性和有效性进行评价,方法:应用原子转移自由基聚合法合成具有高度生物相容性的双嵌段磷酸胆碱聚合物MPC30-DEA70,并对其溶液进行表征。用DNA凝胶电泳法对MPC30-DEA70与针对AS-ODN生成不同N/P比值的基因复合物进行表征。将MPC30-DEA70/AS-OD基因复合物转染至体外培养的HEK293细胞,检测其细胞相容性、转染效率和细胞内转运机制。结果与结论:MPC30-DEA70在pH=4.0-11.0范围内的0.01 mol/L PBS中显示出高度水溶性,其正电性随pH值降低而增强。MPC30-DEA70/AS-ODN基因复合物在DNA凝胶电泳中显示出不同程度的电泳迟滞,随电性增强而显著。MTT法检测显示MPC30-DEA70对HEK293细胞株的毒性呈剂量依赖性,高浓度下细胞毒性显著增强。流式细胞术检测发现复合物的转染随N/P比值增加而显著增强;共聚焦显微镜观察到AS-ODN分子在细胞核内的转运,提示其有效的核定位。证实新型阳离子磷酸胆碱聚合物可以有效负载和运输反义寡核苷酸,是一种高效安全的非病毒类转基因载体。
Background: Although the viral gene vector has high transfection efficiency, its safety has been questioned. OBJECTIVE: To evaluate the safety and efficacy of cationic phosphorylated choline polymer as a carrier for gene delivery of human c-myc antisense oligonucleotides (AS-ODNs) METHODS: A highly biocompatible diblock phosphocholine polymer MPC30-DEA70 was synthesized by atom transfer radical polymerization. The solution was characterized. Characterization of the gene complex of MPC30-DEA70 with different N / P ratios against AS-ODN was performed by DNA gel electrophoresis. The MPC30-DEA70 / AS-OD gene complex was transfected into HEK293 cells cultured in vitro and its cytocompatibility, transfection efficiency and intracellular transport mechanism were tested. RESULTS AND CONCLUSION: MPC30-DEA70 showed a high degree of water solubility in 0.01 mol / L PBS with pH = 4.0-11.0. The positive charge of MPC30-DEA70 increased with the decrease of pH. The MPC30-DEA70 / AS-ODN gene complex showed different degrees of electrophoretic hysteresis in DNA gel electrophoresis, with significant enhancement of electrical properties. MTT assay showed that the toxicity of MPC30-DEA70 on HEK293 cell line was dose-dependent, and cytotoxicity was significantly enhanced at high concentration. Flow cytometry showed that the transfection of the complex was significantly enhanced with the increase of the N / P ratio. The translocation of AS-ODN molecules in the nucleus was observed by confocal microscopy, indicating that it was effective nuclear localization. It is confirmed that the novel cationic phosphocholine polymer can effectively transport and transport antisense oligonucleotides and is a highly efficient and safe non-viral gene delivery carrier.