Gene Delivery System Using Thermo-responsive Polymers

合成了一种温敏性共聚物聚（N-异丙基丙烯酰胺（IPAAm）-co-2-（二甲氨基）乙基甲基丙烯酸酯（DMAEMA）-co-甲基丙烯酸丁酯（BMA）），并研究了其在不同温育温度下的体外基因转染效率。当DMAEMA和BMA的摩尔比分别为8%和11%时，共聚物P（IP-8DA-11BM）的最低临界溶解温度（LCST）为21℃，在21℃以上不溶，在21℃以下可溶。该共聚物与质粒DNA复合。以编码β-半乳糖苷酶的pCMV-βgal质粒作为报告基因，在COS-1细胞中检测聚合物-质粒复合物的转染效率。PDMAEMA均聚物在37℃孵育48 h的转染效率高于20℃孵育3 h和37℃孵育45 h的转染效率。而P（IP-8DA-11BM）在20℃孵育3 h和37℃孵育45 h的转染效率明显高于37℃孵育48 h。通过优化复合物与细胞的孵育时间，这种基因表达增强增加到8.6倍。这种通过降低温度而提高的转染效率被认为是由于P（IP-8DA-11BM）-DNA复合物的适当形成/解离控制。

A thermo-responsive copolymer, poly(N-isopropylacrylamjde (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)) was synthesized, and its in vitro gene transfection efficiency at different incubation temperatures was evaluated. A copolymer containing 8 mol% of DMAEMA and 11 mol% of BMA (P(IP-8DA-11BM)) had a lower critical solution temperature (LCST) at 21℃, therefore, the copolymer was insoluble above 21℃ and soluble below 21℃. This copolymer was complexed with plasmid DNA. Transfection efficiency of polymer-plasmid complexes was evaluated in COS-1 cells using pCMV-βgal plasmid, encoding for β-galactosidase as a reporter gene. Transfection efficiency of PDMAEMA homopolymer incubated at 37℃ for 48h was higher than that incubated at 20℃ for 3h and 37℃ for 45h. In contrast, transfection efficiency of P(IP-8DA-11BM) incubated at 20℃ for 3h and 37℃ for 45h was much higher than that incubated at 37℃ for 48h. This gene expression enhancement was increased to 8.6-fold by optimizing incubation period of the complex with cells. Such increased transfection efficiency by lowering temperature is considered to be due to appropriate formation/dissociation control of P(IP-8DA-11BM)-DNA complexes.