Crombez Laurence, Divita Gilles
Department of Molecular Biophysics and Therapeutics, Centre de Recherches de Biochimie Macromoléculaire, Montpellier, France.
Methods Mol Biol. 2011;683:349-60. doi: 10.1007/978-1-60761-919-2_25.
The development of short-interfering RNA (siRNA) has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of siRNA together with the low permeability of the cell membrane to negatively charged molecules, remain major obstacles to clinical development. So far there is no universal method for siRNA delivery as they all present several limitations. Several non-viral strategies have been proposed to improve the delivery of synthetic siRNAs in both cultured cells and in vivo. Cell-penetrating peptides (CPPs) or protein transduction domains (PTD) constitute very promising tools for non-invasive cellular import of siRNA and non-covalent CPP/PTD-based strategies have been successfully applied for ex vivo and in vivo delivery of therapeutic siRNA molecules. We recently described a new peptide-based system, CADY, for efficient delivery of siRNA in both primary and suspension cell lines. CADY is a secondary amphiphatic peptide able to form stable non-covalent complexes with siRNA and to improve their cellular uptake independently of the endosomal pathway. This chapter describes easy to handle protocols for the use of the CADY-nanoparticle technology for the delivery of siRNA into both adherent and suspension cell lines. It will also highlight different critical points in the peptide/siRNA complex preparation and transfection protocols, in order to obtain siRNA-associated interfering response at low nanomolar concentration.
短干扰RNA（siRNA）的发展为针对导致病理疾病的特定基因进行治疗性靶向提供了巨大希望。然而，siRNA较差的细胞摄取以及细胞膜对带负电荷分子的低渗透性，仍然是临床开发的主要障碍。到目前为止，还没有通用的siRNA递送方法，因为它们都存在一些局限性。已经提出了几种非病毒策略来改善合成siRNA在培养细胞和体内的递送。细胞穿透肽（CPPs）或蛋白质转导结构域（PTD）是非侵入性细胞导入siRNA非常有前景的工具，基于非共价CPP/PTD的策略已成功应用于治疗性siRNA分子的离体和体内递送。我们最近描述了一种新的基于肽的系统CADY，用于在原代细胞系和悬浮细胞系中高效递送siRNA。CADY是一种二级两亲性肽，能够与siRNA形成稳定的非共价复合物，并独立于内体途径改善其细胞摄取。本章描述了使用CADY纳米颗粒技术将siRNA递送至贴壁细胞系和悬浮细胞系的易于操作的方案。它还将强调肽/siRNA复合物制备和转染方案中的不同关键点，以便在低纳摩尔浓度下获得与siRNA相关的干扰反应。
