PHYMEDEXP, INSERM U1046 - CNRS UMR 9214 - University of Montpellier.
PHYMEDEXP, INSERM U1046 - CNRS UMR 9214 - University of Montpellier;
J Vis Exp. 2020 Dec 19(166). doi: 10.3791/62028.
Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.
细胞穿透肽（CPPs）被定义为能够穿过质膜并将货物转移到细胞内的载体。这种活性的一个主要共同特征是 CPPs 与质膜（脂质）的相互作用，更具体地说是与膜本身的细胞外基质成分（硫酸乙酰肝素）的相互作用。事实上，无论直接转位还是依赖内吞作用的内化，脂质双层都参与内化过程，无论是在质膜水平还是在细胞内运输（内体小泡）水平。在本文中，我们提出了一个详细的方案，描述了大单层囊泡（LUVs）制剂、纯化、表征以及在荧光渗漏测定中的应用的不同步骤，以检测 CPP 与膜的可能不稳定/相互作用，并探讨它们在内化机制中的作用。生成具有反映质膜含量的脂质组成的 LUVs，以包裹荧光染料和淬灭剂。肽在囊外介质中的加入以及肽与 LUVs 的相互作用诱导可以以剂量依赖的方式显著增加荧光，从而揭示渗漏。这里提供了最近开发的色氨酸（W）和精氨酸（R）丰富的两亲肽（WRAPs）的示例，它们在各种细胞系中表现出快速有效的 siRNA 传递。最后，讨论了这些相互作用的性质和对脂质的亲和力，以理解和改善膜转位和/或内体逃逸。
