Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery
Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery
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DOI：
10.1021/bc0602026
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发表时间：
2006-09-20
影响因子：
4.7
通讯作者：
Feijen, Jan
中科院分区：
文献类型：
--
作者：
Christensen, Lane V.;Chang, Chien-Wen;Feijen, Jan
聚（酰胺基乙烯亚胺）聚合物是一种新型拟肽聚合物，含有多个二硫键（SS-PAEI），设计用于在将质粒 DNA（pDNA）递送到细胞后降解，并研究其作为触发细胞内基因递送的新载体。更具体地说，通过胱胺双丙烯酰胺（CBA）和三种不同的乙烯胺单体（即乙二胺（EDA）、二乙烯三胺（DETA）或三乙烯四胺（TETA）之间的迈克尔加成反应合成了三种SS-PAEI。通过H-1 NMR证实加成反应完成。分别通过凝胶渗透色谱（GPC）、酸碱滴定和液相色谱-质谱（LC-MS）测定每种SS-PAEI的分子量、缓冲容量和相对支化度。通过凝胶电泳、粒度和 zeta 电位测量来分析聚合物/ pDNA 复合物（聚合复合物）的物理化学特性。所有三种 SS-PAEI 都能有效地复合 pDNA，形成直径小于 200 nm 且表面正电荷类似于 32 mV 的纳米颗粒。使用小鼠胚胎成纤维细胞 (NIH3T3)、原代牛主动脉内皮细胞 (BAEC) 和大鼠主动脉平滑肌细胞 (A7R5) 系评估 SS-PAEI 的体外基因转移特性。有趣的是，基于所有三种 SS-PAEI 的聚合复合物均表现出非常高水平的报告基因表达，转染效率比聚乙烯亚胺 25k 高出近 20 倍。在转染培养基中存在10%血清的情况下仍能保持高转染效率。此外，使用标记 pDNA 的共聚焦显微镜实验表明，与 PEI 相比，SS-PAEI 的聚合复合物显示出更大的 pDNA 细胞内分布，这很可能是由于环境触发的释放。因此，SS-PAEI 是一类新型转染剂，可促进基因高表达，同时保持低水平的毒性。
Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More specifically, three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers, i.e., ethylenediamine (EDA), diethylenetriamine (DETA), or triethylenetetramine (TETA). Complete addition reactions were confirmed by H-1 NMR. The molecular weight, buffer capacity, and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC), acid-base titration, and liquid chromatography-mass spectroscopy (LC-MS), respectively. Physicochemical characteristics of polymer/ pDNA complexes (polyplexes) were analyzed by gel electrophoresis, particle size, and zeta-potential measurements. All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of similar to 32 mV. The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3), primary bovine aortic endothelial cell (BAEC), and rat aortic smooth muscle cell (A7R5) lines. Interestingly, polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k. The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium. Furthermore, confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI, most likely due to environmentally triggered release. Therefore, SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.