Structure-function relationships of gene delivery vectors in a limited polycation library
Structure-function relationships of gene delivery vectors in a limited polycation library
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DOI：
10.1016/j.jconrel.2004.11.028
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发表时间：
2005-03-02
影响因子：
10.8
通讯作者：
Putnam, D
中科院分区：
文献类型：
--
作者：
Chen, DJ;Majors, BS;Putnam, D
合成了13个聚赖氨酸-接枝-咪唑乙酸缀合物的文库，以检查聚合物分子量、侧链取代和DNA：聚合物比率对细胞毒性、转染效率和聚阳离子-DNA相互作用的集体影响。一般来说，这些聚阳离子的理化特性和基因转移能力之间的关系出现非线性。这些聚合物的体外细胞毒性降低，而总蛋白表达增加，分子量降低，咪唑含量增加。然而，流式细胞术实验表明，标记基因表达的增加并不总是与转染的细胞总数相关，即使使用类似的聚合物结构进行转染。荧光素酶基因表达的最大水平介导的转染与低分子量，高咪唑含量（9400 M，95摩尔%咪唑）聚合物。溴化乙锭荧光猝灭法测定的DNA缩合程度也随聚合物分子量的降低和咪唑含量的增加而降低。DNA和聚阳离子之间的相对结合亲和力，通过竞争性结合在合成的聚阴离子的存在下测量，随着聚合物分子量的降低而降低;然而，相对亲和力也似乎随着咪唑的增加而增加，这表明静电的贡献不是唯一负责DNA-聚阳离子结合相互作用。这种有限的文库和相应的结构/功能分析形成了基础，在此基础上可以设计和评估更大、更全面的聚阳离子文库，以进一步了解聚阳离子转染试剂的功能。(c)2004 Elsevier B. V.保留所有权利。
A library of 13 polylysine-graft-imidazoleacetic acid conjugates was synthesized to examine the collective effects of polymer molecular weight, side chain substitution, and DNA:polymer ratio on cytotoxicity, transfection efficiency, and polycation-DNA interaction. In general, the relationships between the physicochemical characteristics and the gene transfer capabilities of these polycations appear nonlinear. The in vitro cytotoxicity of these polymers decreased, while total protein expression increased, with decreasing molecular weight and increasing imidazole content. Flow cytometry experiments indicated, however, that an increase in marker gene expression does not always correlate with the total number of cells transfected, even when similar polymer structures are used for transfection. The maximum level of luciferase gene expression was mediated by transfection with a low molecular weight, high imidazole content (9400 M, 95 mol% imidazole) polymer. The extent of DNA condensation, as determined by ethidium bromide fluorescence quenching, also decreased with decreasing polymer molecular weight and increasing imidazole content. Relative binding affinity between DNA and the polycations, measured via competitive binding in the presence of a synthetic polyanion, decreased with decreasing polymer molecular weight; however, the relative affinity also appeared to increase with increasing imidazole, suggesting that electrostatic contributions are not solely responsible for DNA-polycation binding interactions. This limited library and corresponding structure/function analysis forms the foundation upon which larger, more comprehensive polycationic libraries can be designed and evaluated to further understand how polycation transfection reagents function. (c) 2004 Elsevier B.V. All rights reserved.