Published: December 19th, 2020
The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.
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.
After their discovery in the nineties, cell-penetrating peptides (CPPs) were developed to promote an efficient cellular delivery of cargoes through the plasma membrane1,2. CPPs are usually short peptides, generally 8 to 30 amino acids, having a wide variety of origins. They were first defined as "direct-translocating" carriers, meaning they were able to cross the plasma membrane and to transfer a cargo into cells independently of any endocytotic pathway neither energy requirement nor receptor involvement. However, further investigations revealed that these first observations mainly came from fluorescen....
1. Preparation of Large Unilamellar Vesicles (LUVs)
The principle of the fluorescence leakage assay is shown in the Figure 1. In detail, large unilamellar vesicles (LUVs) encapsulating a fluorescent dye and a quencher (no fluorescence signal) are treated with the biomolecule of interest. Due to the interaction of the peptide with lipid membranes, which could imply membrane permeability, reorganization or even rupture, the fluorescence dye and the quencher are released from the LUVs. Subsequent dilutions in the.......
The presented fluorescence leakage assay is a simple and fast method to address membrane destabilization by cell-penetrating peptide. Easy to do, it also enables an indirect comparison between different membrane-interacting peptides or other membrane-interacting molecules. Concerning critical steps of the protocol, as this assay provides relative values between the baseline (LUVs alone) and maximal fluorescence release (Triton condition), we usually evaluate the concentration of LUVs using the phospholipid quantification.......
The authors would like to thank Emilie Josse for the critical review of the manuscript. This work was supported by the foundation "La Ligue contre le Cancer", the "Fondation ARC pour la Recherche sur le Cancer", and the "Centre National de la Recherche Scientiﬁque" (CNRS).....
|25 mL glass round-bottom flask
|8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS)
|Protect from light
|Protect from air
|50 µL glass syringe
|liquid chromatography column
|Nuclepore polycarbonate membrane (0.1 µm pore size, 25 mm diameter)
|polystyrene cuvette, 10 x 10 x 45 mm
|polystyrene semi-micro cuvette, DLS
|p-xylene-bispyridinium bromide (DPX)
|Protect from light
|quartz fluorescence cuvette
|Sephadex G-50 resin
|Sodium azide (NaN3)
|Sodium chlorid (NaCl)
|Sonicator bath USC300T
|Protect from air
Copyright © 2024 MyJoVE Corporation. All rights reserved