Accumulation of cell‐penetrating peptides in large unilamellar vesicles: A straightforward screening assay for investigating the internalization mechanism

Abstract

The internalization of cell-penetrating peptides (CPPs) into liposomes (large unilamellar vesicles, LUVs) was studied with a rapid and robust procedure based on the quenching of a small fluorescent probe, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Quenching can be achieved by reduction with dithionite or by pH jump. LUVs with different compositions of phospholipids (PLs) were used to screen the efficacy of different CPPs. In order to "validate" the composition of the membrane models, a control cationic peptide, which does not enter eukaryotic cells, was included in the study. It was found that pure DOPG or DOPG within ternary mixtures with cholesterol are the most appropriate models for studying CPP translocation. An anionic lipid, such as DOPG, is required for the adsorption of the basic peptides on the surface of LUVs. In addition, it acts as transfer agent through the lipid bilayer. A fluid phase and/or the presence of phase defects also appear mandatory for the internalization to occur. The neutralization of charges within an inverted micelle demonstrated in the case of DOPG and also proposed for a ternary mixture of PLs might not be the only mechanism for the CPP translocation. Finally, it is shown that oleic acid facilitates the entry inside LUVs in gel phase of a series of cationic peptides including CPPs and also the negative control peptide PKCi.

Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

“…Translocation, defined as the movement of a molecule across a bilayer, has been studied for many CPPs using a variety of techniques[8,51,65,71,72]. Yet, there is no consensus about which CPPs can translocate across synthetic bilayers and which, if any, do so without simultaneous membrane disruption.…”

“…At low or moderate peptide concentrations, or in bilayers with low anionic lipid content, CPPs such as Arg9, tat , and penetratin neither translocate nor disrupt membranes[8,52,56,72]. A novel fluorescence approach[71] to measure CPP translocation across synthetic bilayers was recently applied to Arg 9 tat , TP2 and other CPPs. All were found to rapidly cross anionic large unilamellar vesicles without permanent disruption of bilayer structure[52,56,71], although transient disruption was not assessed.…”

“…A novel fluorescence approach[71] to measure CPP translocation across synthetic bilayers was recently applied to Arg 9 tat , TP2 and other CPPs. All were found to rapidly cross anionic large unilamellar vesicles without permanent disruption of bilayer structure[52,56,71], although transient disruption was not assessed. For tat , Arg9 and other highly cationic CPPs, the extent of translocation increases sharply with increasing peptide concentration, suggesting that the translocating species is a peptide multimer[52].…”

Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes

“…Reduction of NBD could possibly be achieved according to aH 2 -mediatedp rocess, and/ or OH À that is known to switch off the NBD fluorescence through ap Hj ump. [45,46] However,t he pH increasep ossibly triggered through the productiono fh ydroxyl anionsw ould be compensated by the generation of protons at the anode through water oxidation since the auxiliary and working electrodes are not separated in the well. Additionally,w es howed that bubbling H 2 in aN BD-glycine-containings olution did not quencht he fluorescence.…”

Electrochemical quenching of the fluorescence produced by NBD-labelled cell penetrating peptides: A contribution to the study of their internalization in large unilamellar vesicles

“…In this context, fluorescence methods have been intensively used to monitor and quantify the amount of internalized peptides within liposomes during a given incubation period vis-à-vis those located outside [8][9][10][11]. NBD (7-nitrobenz-2-oxa-1,3-diazole) is a small fluorescent probe originally introduced to label proteins through binding to their hydrophobic regions [12].…”