Jazayeri Jalal A, Prankerd Richard J, Pouton Colin W
Department of Pharmaceutical Biology, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.
Bioconjug Chem. 2008 Apr;19(4):940-50. doi: 10.1021/bc700463q. Epub 2008 Mar 12.
Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.
由于非病毒表达载体具有安全性、稳定性以及适合作为大容量药物生产等特点，通过其进行基因治疗是非常理想的。然而，低转染效率仍然是其在非病毒基因递送应用中的一个限制因素。尽管该领域最近取得了进展，但仍有主要障碍需要克服。为了构建更高效的非病毒基因递送载体，我们设计了一系列新型脂肽转染剂，它们由一条烷基链、一个半胱氨酸、1至4个组氨酸和1至3个赖氨酸残基组成。设计这些脂肽是为了促进二聚化（通过半胱氨酸残基）、在中性pH下与DNA结合（利用带电荷的赖氨酸残基）以及实现内体逃逸（通过弱碱性的组氨酸残基）。对DNA/脂肽复合物的生物物理性质和转染效率进行了评估。脂肽构建体中所含氨基酸的数量和种类影响它们形成DNA/脂肽复合物的能力。随着脂肽中赖氨酸残基数量的增加，形成的DNA复合物变得更稳定，具有更高的zeta电位（颗粒表面电荷），并且产生的平均粒径更小（在电荷比为5.0时通常为110 nm，在电荷比为1.0时为240 nm）。脂肽部分中包含组氨酸对复合物形成的影响与赖氨酸相反，但对于高转染效率是必需的。在COS-7细胞中进行的体外转染研究表明，所有脂肽介导的编码荧光素酶的质粒pCMV-Luc的基因递送效率远高于没有内体逃逸系统的聚（L-赖氨酸）（PLL），在两种情况下略高于支链聚乙烯亚胺（PEI）。具有至少两个赖氨酸残基和至少一个组氨酸残基的脂肽与质粒DNA形成自发转染复合物，表明通过掺入组氨酸残基实现了内体逃逸。这些低分子量肽易于合成和纯化，并为转染复合物的作用机制提供了新的见解。
