Department of Chemical Engineering, University of Waterloo, Waterloo, ON, N2L 3G1, Canada.
J Phys Chem B. 2012 Nov 8;116(44):13183-91. doi: 10.1021/jp3072553. Epub 2012 Oct 30.
RNA interference has emerged as a powerful tool in biological and pharmaceutical research; however, the enzymatic degradation and polyanionic nature of short interfering RNAs (siRNAs) lead to their poor cellular uptake and eventual biological effects. Among nonviral delivery systems, cell-penetrating peptides have been recently employed to improve the siRNA delivery efficiency. Here we introduce an 18-mer amphipathic, amino-acid-pairing peptide, C6, as an siRNA delivery carrier. Peptide C6 adopted a helical structure upon coassembling with siRNA. The C6-siRNA coassembly showed a size distribution between 50 and 250 nm, confirmed by dynamic light scattering and atomic force microscopy. The C6-siRNA interaction enthalpy and stoichiometry were 8.8 kJ·mol(-1) and 6.5, respectively, obtained by isothermal titration calorimetry. A minimum C6/siRNA molar ratio of 10:1 was required to form stable coassemblies/complexes, indicated by agarose gel shift assay and fluorescence spectroscopy. Peptide C6 showed lower toxicity and higher efficiency in cellular uptake of siRNA compared with Lipofectamine 2000. Fluorescence microscopy images also confirmed the localization of C6-siRNA complexes in the cytoplasm using Cy3-labeled siRNAs. These results indicate high capabilities of C6 in forming safe and stable complexes with siRNA and enhancing its cellular uptake.
RNA 干扰已成为生物和药物研究中的有力工具；然而，短干扰 RNA（siRNA）的酶降解和多阴离子性质导致其细胞摄取能力差，最终生物学效应也不佳。在非病毒递送系统中，细胞穿透肽最近被用于提高 siRNA 的递送效率。在这里，我们介绍一种 18 个氨基酸的两亲性氨基酸配对肽 C6，作为 siRNA 的递送载体。肽 C6 在与 siRNA 共组装时采用螺旋结构。通过动态光散射和原子力显微镜证实，C6-siRNA 共组装的粒径分布在 50 至 250nm 之间。通过等温滴定量热法获得 C6-siRNA 相互作用焓和化学计量比分别为 8.8kJ·mol(-1)和 6.5。琼脂糖凝胶电泳和荧光光谱表明，形成稳定共组装体/复合物所需的最小 C6/siRNA 摩尔比为 10:1。与 Lipofectamine 2000 相比，肽 C6 具有更低的毒性和更高的 siRNA 细胞摄取效率。使用 Cy3 标记的 siRNA 的荧光显微镜图像也证实了 C6-siRNA 复合物在细胞质中的定位。这些结果表明 C6 具有与 siRNA 形成安全稳定复合物并增强其细胞摄取能力的高能力。
