Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape
Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape
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DOI：
10.1042/bj20101021
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发表时间：
2011-04-15
影响因子：
4.1
通讯作者：
Barnett, Stanley F.
中科院分区：
文献类型：
--
作者：
Bartz, Rene;Fan, Haihong;Barnett, Stanley F.
将 siRNA（小干扰 RNA）有效递送到细胞中需要将 siRNA 易位到细胞质中。一种潜在的递送策略使用细胞递送肽来促进这一步骤。在本文中，我们描述了一种两亲性肽的表征，该肽介导非共价结合的 siRNA 被细胞摄取并随后释放到细胞质中。中性和溶酶体（酸性）pH 下肽和肽/siRNA 混合物的生物物理表征表明仅在内体和溶酶体中形成α-螺旋结构。令人惊讶的是，尽管该肽增强了细胞对 siRNA 的摄取，但通过等温滴定量热法在中性 pH 条件下没有观察到 siRNA 和肽之间的直接相互作用。重要的是，我们证明肽介导的 siRNA 摄取是通过内吞作用发生的，并且通过应用新型内体逃逸测定和细胞分级技术，我们证明了内体和溶酶体完整性的 pH 依赖性改变以及随后 siRNA 和其他货物释放到胞质溶胶中。这些结果表明肽介导的 siRNA 递送是通过与细胞膜而不是与货物的 pH 依赖性和构象特异性相互作用来实现的。
Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of a-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.