An in vivo gene expressing system induced by external thermal stimuli

以n -异丙基丙烯酰胺、2-（二甲氨基）甲基丙烯酸乙酯和甲基丙烯酸丁酯为原料合成了热敏聚合物。该共聚物的相变温度为25℃。分析了其与质粒DNA的络合行为。当该聚合物与DNA以N/P电荷比为4时，形成的络合物的ζ电位为+14.1mV。该聚合物与DNA的结合亲和力在温度刺激下发生变化；温度越高亲和力越低。这种诱导的亲和力变化可能有助于DNA在温度下降时从复合物中释放出来。这种聚合物的亲和力变化比先前研究的聚合物更大，这些聚合物是用较低含量的2-（二甲氨基）甲基丙烯酸乙酯和甲基丙烯酸丁酯合成的，而不是本研究的聚合物。体外基因表达实验表明，该热响应性聚合物具有热响应性基因表达；20℃冷却3h后基因表达为两倍。该聚合物比先前研究的聚合物表现出更高的基因表达水平。此外，与未经处理的对照相比，该聚合物复合物的细胞毒性相当低，如80%的生物活性。这些结果表明，利用热响应性共聚物作为合成基因载体，良好地控制DNA结合变化有助于在体外获得高温控的低细胞毒性基因表达。

A thermo-responsive polymer was synthesized from N-isopropyl acrylamide, 2-(dimethylamino)ethyl methacrylate, and butylmethacrylate. This copolymer possessed a phase transition temperature at 25℃. Behaviors of its complexation with plasmid DNA were analyzed. When this polymer was complxed with DNA at a N/P charge ratio of 4, zeta-potencial of the formed complex was +14.1mV. Binding affinity of this polymer to DNA was revealed to change on temperature stimuli ; lower affinity at higher temperature. This induced change of affinity could contribute to DNA release from the complex on temperature drop. This affinity change for this polymer was larger than that obtained in previously studied polymers that synthesized with lower contents of 2-(dimethylamino)ethyl methacrylate and butylmethacrylate than the polymer of the present study.In vitro gene expression assay revealed that this thermo-responsive polymer exhibited thermo-responsive gene expression ; two-fold gene expression upon cooling at 20℃ for 3h. This polymer showed much higher level of gene expression than the previously studied polymer. Furthermore, cytotoxicity of this polymer complex was considerably low such as 80% bioactivity compared to the untreated control. These all results indicated that well controlled DNA binding change using the thermo-responsive copolymer as a synthetic gene vector contributed to obtain high and temperature-controlled in vitro gene expression with low cytotoxicity.