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

Summary

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.

Abstract

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.

Introduction

After their discovery in the nineties, cell-penetrating peptides (CPPs) were developed to promote an efficient cellular delivery of cargoes through the plasma membrane^1,2. 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....

Protocol

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 µmol of DOPC, 4 µmol of SM, and 2 µmol of Chol, the lipid solution is obtained from stock solution by mixing 126 µL, 11....

Discussion

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.......

Materials

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