Influence of Polycation Molecular Weight on Poly(2-dimethylaminoethyl methacrylate)-Mediated DNA Delivery In Vitro
Influence of Polycation Molecular Weight on Poly(2-dimethylaminoethyl methacrylate)-Mediated DNA Delivery In Vitro
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DOI：
10.1021/bm9000124
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发表时间：
2009-05-01
影响因子：
6.2
通讯作者：
Long, Timothy E.
中科院分区：
文献类型：
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作者：
Layman, John M.;Ramirez, Sean M.;Long, Timothy E.
建立明确的结构-性质-转染关系是开发用于非病毒基因治疗的临床相关聚合物的关键步骤。在这项研究中，我们确定了聚（2-二甲基氨基乙基甲基丙烯酸酯）（PDMAEMA）的分子量对细胞毒性，DNA结合，并在体外质粒DNA递送效率在人脑微血管内皮细胞（HBMEC）的影响。采用自由基聚合法合成了重均分子量为43 000 ~ 915 000 g/mol的PDMAEMA。MTT和LDH试验表明，低分子量PDMAEMA（M-w = 43 000 g/mol）的毒性略低于高分子量（M-w > 112 000 g/mol），主要毒性模式是细胞膜不稳定。电泳凝胶位移试验表明，所有PDMAEMA分子量完全结合质粒DNA。然而，肝素竞争性结合实验显示，较高分子量的PDMAEMA（M-w = 915 000 g/mol）对质粒DNA的结合亲和力大于较低分子量的PDMAEMA（M-w = 43 000 g/mol）。发现PDMAEMA的分子量对转染效率具有显著影响，并且荧光素酶报告基因表达作为增加分子量的函数而增加。然而，细胞摄取的复合物被确定为是不敏感的PDMAEMA分子量。此外，我们的数据没有关联的多聚物的大小与转染效率。总的来说，我们的数据表明，涉及内体释放和DNA酶抗性的多聚物的细胞内命运对总体转染效率比进入屏障（例如多聚物大小）更重要。
Establishing clear structure-property-transfection relationships is a critical step in the development of clinically relevant polymers for nonviral gene therapy. In this study, we determined the influence of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) molecular weight on cytotoxicity, DNA binding, and in vitro plasmid DNA delivery efficiency in human brain microvascular endothelial cells (HBMEC). Conventional free radical polymerization was used to synthesize PDMAEMA with weight-average molecular weights ranging from 43 000 to 915 000 g/mol. MTT and LDH assays revealed that lower molecular weight PDMAEMA (M-w = 43 000 g/mol) was slightly less toxic than higher molecular weights (M-w > 112 000 g/mol) and that the primary mode of toxicity was cellular membrane destabilization. An electrophoretic gel shift assay revealed that all PDMAEMA molecular weights completely bound with plasmid DNA. However, heparin competitive binding experiments revealed that higher molecular weight PDMAEMA (M-w = 915 000 g/mol) had a greater binding affinity toward plasmid DNA than lower molecular weight PDMAEMA (M-w = 43 000 g/mol). The molecular weight of PDMAEMA was found to have a dramatic influence on transfection efficiency, and luciferase reporter gene expression increased as a function of increasing molecular weight. However, cellular uptake of polyplexes was determined to be insensitive to PDMAEMA molecular weight. In addition, our data did not correlate polyplex size with transfection efficiency. Collectively, our data suggested that the intracellular fate of the polyplexes, which involves endosomal release and DNase resistance, is more important to overall transfection efficiency than barriers to entry, such as polyplex size.