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Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Published: December 19th, 2020



1PHYMEDEXP, INSERM U1046 - CNRS UMR 9214 - University of Montpellier

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....

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1. Preparation of Large Unilamellar Vesicles (LUVs)

  1. Prepare LUVs for their use as cell membrane mimics for fluorescence leakage assay.
  2. Mix with a Hamilton glass syringe phosphatidylcholine (DOPC, 786.11 g/mol), sphingomyelin (SM, 760.22 g/mol) and Cholesterol (Chol, 386.65 g/mol) at the molar ratio 4:4:2. The lipid solution is obtained from a stock solution of each lipid solubilized in a methanol/chloroform (3/1; volume/volume) solvent at 25 mg/mL in a 25 mL glass round-bottom flask. Based on 4 .......

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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.......

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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.......

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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).


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Name Company Catalog Number Comments
25 mL glass round-bottom flask Pyrex
8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS) Invitrogen A350 Protect from light 
Chloroform  Sigma-Aldrich 288306
Cholesterol Sigma-Aldrich C8667
DOPC (dioleoylphosphatidylcholine) Avanti Polar 850375P Protect from air
Extruder Avanti Polar 610000
Fluorimeter PTI Serlabo
50 µL glass syringe Hamilton 705N
HEPES Sigma-Aldrich H3375
LabAssay Phospholipid  WAKO  296-63801
liquid chromatography column Sigma-Aldrich
Methanol Carlo Erba 414902
Nuclepore polycarbonate membrane (0.1 µm pore size, 25 mm diameter) Whatman 800309
polystyrene cuvette, 10 x 10 x 45 mm Grener Bio-One 614101
polystyrene semi-micro cuvette, DLS Fisher Scientific FB55924
p-xylene-bispyridinium bromide (DPX) Invitrogen X1525 Protect from light 
quartz fluorescence cuvette Hellma 109.004F-QS
rotavapor system  Heidolph Z334898
Sephadex G-50 resin Amersham 17-0042-01
Sodium azide (NaN3) Sigma-Aldrich S2002
Sodium chlorid (NaCl) Sigma-Aldrich S5886
Sonicator bath USC300T VWR 142-6001
Sphingomyelin Avanti Polar 860062P Protect from air
Triton X-100  Eromedex 2000-B
Zetaziser NanoZS  Malvern ZEN3500

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