侧链含咪唑取代基聚(L-天冬酰胺)高效基因载体研究

POLYASPARTAMIDES GRAFTED WITH 1-(3-AMINOPROPYL)IMIDAZOLE AS LESS TOXIC AND HIGHLY EFFICIENT GENE CARRIERS

通过氨基引发聚丁二酰亚胺(PSI)开环反应,制备了系列侧链含氨乙基和咪唑丙基的聚(L-天冬酰胺)共聚物(P1～P5).该系列聚合物不仅具有极低的细胞毒性,而且随侧链中咪唑取代基含量的增加,聚合物在pH 5～8范围内缓冲能力显著提高.通过凝胶电泳、粒径和电位分析等研究了聚合物与质粒DNA的相互作用.结果表明,所有聚合物均可以在较低N/P比有效结合DNA.在N/P比高于5时,形成粒径在100 nm以下、表面电荷为+20 mV左右的稳定复合物纳米粒子.聚合物介导荧光素酶基因(pGL-3)和增强型绿色荧光蛋白基因(pEGFP-C1)体外转染293T细胞和COS-7细胞的结果表明,侧链中引入咪唑取代基显著提高了聚合物的转染能力.通过优化侧链氨基/咪唑取代基比例,获得转染效率最高的聚合物P4.P4介导pGL-3和pEGFP-C1在细胞中的表达与聚乙烯亚胺(PEI)相当、甚至是PEI的2～3倍.

Polyaspartamides containing pendant imidazolyl propyl groups and amino ethyl groups were synthesized via ring-opening of polysuccinimide（PSI） reacting with N-Boc protected ethylenediamine and 1-（3-aminopropyl）imidazole.Polyaspartamides（P1~P5） with different grafting ratios were characterized by FTIR and 1H-NMR spectra,which were well in accordance with the expected chemical structures.The cytotoxicity of polymers was evaluated by MTT assay.Compared to the commercial branched polyethyleneimine（PEI）（25 kDa） with an IC50 of 15 μg/mL,P1~P5 showed no significant cytotoxicity against both 293T cells and COS-7 cells.More than 90% cells still retained their metabolic ability even at the polymer concentration up to 0.8 mg/mL.The introduction of imidazolyl group into polymer side chains make them possess high buffer capacity between pH 5 and pH 7,which was 2~5 times higher than that of PEI.Gel electrophoresis assay and dynamic light scattering measurements were performed to characterize the polycation/DNA complexes.At an N/P ratio of above 5,all of the five polyaspartamide-based polycations can condense plasmid DNA to form nanosized（100 nm） polyelectrolyte complexes with positive surface charge（＋20 mV）.The transfection assay demonstrated that the transfection efficiencies of foreign DNA in 293T cells and COS-7 cells increased with increasing the contents of imidazolyl graft of polyaspartamides.P4 with a grafting ratio of 1/3.7（amino ethylene group to imidazolyl propyl group） showed the highest ability to deliver pGL-3 and pEGFP-C1 into both 293T cells and COS-7 cells.At the optimal N/P ratios,the gene expression efficiencies mediated by P4 were comparable to or even 2~3 times higher than that of PEI.The results suggest that imidazole-containing polyaspartamides are a very promising class of novel polycations for highly efficient and less toxic gene delivery.The structure-gene delivery ability relationship of imidazole-grafted polyaspartamides will provide valuable insight into the development of novel nonviral gene carrier.