Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota-Twin Cities, 308 Harvard St., SE, Minneapolis, Minnesota 55455, USA.
Pharm Res. 2010 Apr;27(4):683-98. doi: 10.1007/s11095-010-0058-1. Epub 2010 Feb 17.
Improved polycation-based non-viral DNA vectors were sought by preparing dendrimers with polyethylenimine cores surrounded by various shells incorporating structural features intended to facilitate steps in transfection mechanisms. Dendrimeric vectors were designed with (a) an outer oligocation shell, intended to facilitate DNA release inside cells, (b) a hydrophobic C-16 alkyl shell, and (c) a polycationic core, the latter two intended to combine lipid-depletion and osmotic burst endosome escape mechanisms, respectively, and were (d) attached through an a acid-cleavable linker reported to hydrolyze at endosomal pH values.
Vectors and DNA complexes were characterized by dynamic and static light scattering. Flow cytometry was used to quantitate transfection activity and cytotoxicity in CHO-K1 cells.
About 5-fold increased transfection activity was obtained for a vector constructed with an outer shell of oligocations attached through acid-cleavable linkers, relative to a control dendrimer with an acid-stable linker. The most effective oligocation component of outer shells tested was spermine. Neither modification was associated with increased cytotoxicity. This vector design did not permit an evaluation of the benefit of combining endosome release mechanisms.
Using acid-cleavable linkers to attach an outer shell of short, highly-charged oligocations to a PEI-based dendrimeric vector substantially increased transfection efficiency without increasing cytotoxicity.
通过制备具有聚亚乙基亚胺核心的树枝状大分子，并用各种外壳包围，这些外壳包含旨在促进转染机制步骤的结构特征，来寻找改良的基于聚阳离子的非病毒 DNA 载体。设计了具有（a）外层低聚阳离子壳的树枝状载体，旨在促进细胞内 DNA 的释放，（b）疏水性 C-16 烷基壳，和（c）聚阳离子核，后两者旨在分别结合脂质耗竭和渗透破裂内涵体逃逸机制，并通过报道在内涵体 pH 值下水解的酸可裂解接头（d）连接。
通过动态和静态光散射来表征载体和 DNA 复合物。流式细胞术用于定量测定 CHO-K1 细胞中的转染活性和细胞毒性。
与具有酸稳定接头的对照树枝状大分子相比，通过酸可裂解接头连接的带有外壳的短链、高电荷低聚阳离子的载体的转染活性增加了约 5 倍。测试的最有效的外壳低聚阳离子成分是 spermine。这两种修饰都与增加细胞毒性无关。这种载体设计不允许评估结合内涵体释放机制的益处。
使用酸可裂解接头将短的、高电荷的低聚阳离子外壳连接到基于聚亚乙基亚胺的树枝状大分子载体上，可显著提高转染效率而不增加细胞毒性。
