摘要/Abstract

引入快速、主动释放基因的机制是提升非病毒基因递送效率的关键. 本研究以2-甲基丙烯酰氧乙基磷酰胆碱(MPC)与(2-丙烯酰基)乙基(硼酸苄基)二乙基溴化铵(BD)为单体, 基于可逆加成-断裂链转移聚合(RAFT)反应制备具有活性氧响应性质的聚阳离子嵌段共聚物pM-pBD. 通过静电作用, 阳离子共聚物pM-pBD能够与带负电荷的DNA以分子自组装的方式形成纳米复合物. 其中, 阳离子pBD片段具有活性氧触发电荷反转的特性, 因此, 有助于获得活性氧触发的静电组装复合体结构解离, 从而实现可控基因释放的功能. 理化表征结果表明, pM-pBD与质粒DNA静电复合后能够形成粒径约为99.1 nm, ζ电位约为+13.8 mV, 显微形貌近似球形的纳米复合物. 在加入促过氧化氢产生的抗坏血酸后, 上述pM-pBD基因递送系统的转染效率得到了显著的提升. 因此, 本研究创制的pM-pBD为基因递送系统的可控释放提供了新的解决方案.
关键词: 聚阳离子, 电荷反转, 基因递送, 活性氧响应, 抗坏血酸
The development of rapid and active gene release function is the key to improve the efficiency of non-viral gene delivery system. Herein, a novel reactive oxygen-responsive cationic block copolymer non-viral gene delivery vector (termed as pM-pBD) consisting of biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC) segment and charge reversible poly[(2-acryloyl)-ethyl-(boronic acid benzyl)-diethylammonium bromide] segment (pBD) was synthesized via reversible addition-fragmentation chain transfer polymerization (RAFT). Dynamic light scattering (DLS) assay and ζ potential measurements, transmission electron microscope (TEM) and gel electrophoresis were performed to characterize pM-pBD/(plasmid DNA, pDNA) complexes. Gel electrophoresis retardation assay displayed that pM-pBD can firmly bind pDNA through electrostatic interaction even in the presence of high concentration of heparin, but capable of releasing pDNA in response to reactive oxygen (such as H₂O₂). The pM-pBD/pDNA complexes show low cytotoxicity against HeLa cells, even at high weight ratio of pM-pBD and pDNA (N/P) (up to 16) demonstrated by MTT (methylthiazolyldiphenyl- tetrazolium bromide) assay. The pM-pBD/pDNA complexes demonstrated appreciable colloidal stability in the presence of 10% fetal bovine serum. The pM-pBD/pDNA complexes at N/P ratio of 3 displayed spherical morphologies with average diameter of approximate 99.1 nm and ζpotential of approximate +13.8 mV. However, once upon incubation in presence of 1 mmol/L H₂O₂, the diameter of pM-pBD/pDNA complexes at N/P ratio of 3 was enlarged to 330 nm and ζ potential was reversed to –3.91 mV due to charge reversal of BD responsive to reactive oxygen. Flow cytometry revealed uptake efficiency (88.9%) and the highest transfection efficiency (31.2%) for pM-pBD at N/P ratio of 3 and 16, respectively. The transfection efficiency of pM-pBD/pDNA against HeLa cells was observed to be significantly augmented (1.5-fold) after the addition of ascorbic acid, which could stimulate the production of hydrogen peroxide. Therefore, pM-pBD represented intriguing utilities in fabrication of non-viral gene delivery systems, which enable spatiotemporal control of gene release and thereby facilitated the subsequent transcription machinery.
Key words: polycation, charge reversal, gene delivery, ROS-responsive, ascorbic acid


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