Layman John M, Ramirez Sean M, Green Matthew D, Long Timothy E
Department of Chemistry, Macromolecules and Interfaces Institute, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Biomacromolecules. 2009 May 11;10(5):1244-52. doi: 10.1021/bm9000124.
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.
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。在本研究中，我们测定了聚（甲基丙烯酸2-二甲基氨基乙酯）（PDMAEMA）分子量对人脑微血管内皮细胞（HBMEC）的细胞毒性、DNA结合以及体外质粒DNA递送效率的影响。采用传统自由基聚合法合成了重均分子量范围为43,000至915,000 g/mol的PDMAEMA。MTT和LDH测定表明，较低分子量的PDMAEMA（M(w)=43,000 g/mol）的毒性略低于较高分子量的（M(w)>112,000 g/mol），且主要毒性模式是细胞膜去稳定化。电泳凝胶迁移试验表明，所有PDMAEMA分子量均能与质粒DNA完全结合。然而，肝素竞争性结合实验表明，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有显著影响，并且荧光素酶报告基因表达随分子量增加而增加。然而，确定多聚体的细胞摄取对PDMAEMA分子量不敏感。此外，我们的数据未显示多聚体大小与转染效率之间存在相关性。总体而言，我们的数据表明，多聚体的细胞内命运，包括内体释放和对DNA酶的抗性，对整体转染效率比进入障碍（如多聚体大小）更重要。
