Blanc SIMI 8 - Blanc - SIMI 8 - Chimie du solide, colloïdes, physicochimie

Electrochemistry and LIpid architectures for Peptide Translocation Investigation and Control – ELIPTIC

Molecular characterization of the processes involved in the translocation of therapeutic molecules across membranes

Transport of active molecules across biological membranes is a central issue for the success of many pharmaceutical strategies such as epigenetic therapies which opens new perspectives in cancer treatments.

Development of original analytical tools to investigate the transport of cationic peptides across membranes.

The Cell Penetrating Peptides (CPPs) have become one of the most popular and efficient shuttle to access the intracellular medium and their discovery represents a major breakthrough for the transport of active molecules (drugs, proteins, nanoparticles,…). In this context, providing information on the internalization mechanisms as well as on the CPPs transport kinetics is a crucial issue for the development of new pharmaceutical strategies. Our objective is devoted to the development of original analytical tools based on electrochemical and/or fluorescence techniques to analyze the consequences of peptides/lipids interactions on the supramolecular scale. This project is primarily aimed at developing new advances in analytical techniques via the coupling of molecular electrochemistry with the “patch-clamp” technique, the electrochemical impedance spectroscopy, and fluorescence techniques such as confocal microscopy. Beyond the cell penetrating peptides, this project will allow, in the future, the investigation of mechanisms that are involved in the transport of any type of drug/biomolecule across biological membranes.

This project focuses on the following items : (i) synthesis of a family of cationic peptides bearing a fluorescent or a redox probe, and investigations of their bio-physico-chemical properties, (ii) electrochemical studies of the interactions between these peptides and model membranes such as phospholipid vesicles of defined composition, (iii) characterization of specific peptides / lipids assemblies by electrochemical impedance spectroscopy (EIS) and electrochemical microscopy (SECM), (iv) electrochemical studies of nano-flow cationic peptides either with antimicrobial or cell penetrating properties through a lipid bilayer suspended above a micro-hole or obtained by «patch« of giant vesicles (v) development of analytical tools combining electrochemistry and fluorescence (TIRF and confocal microscopies) to obtain kinetic information and spatial resolution on the translocation process in both ranges of short and long time.eptides / lipids assemblies by electrochemical impedance spectroscopy (EIS) and electrochemical microscopy (SECM), (iv) electrochemical studies of nano-flow cationic peptides either with antimicrobial or cell penetrating properties through a lipid bilayer suspended above a micro-hole or obtained by «patch« of giant vesicles (v) development of analytical tools combining electrochemistry and fluorescence (TIRF and confocal microscopy) to obtain kinetic information and spatial resolution on the translocation process in both ranges of short and long time.

Novel integration of patch-clamp and amperometry provided a powerful mean for quantifying, in real time, molecular species fluxes across real cell and artificial membranes. The patch-clamp technique allows stretching a piece of real or artificial membrane (by excision of a real or artificial membrane such as a vesicle) at the tip of a glass micropipette; on the other hand, an ultra-microelectrode positioned in front of the suspended membrane allows an efficient detection of a molecule crossing the suspended membrane. This method which has been experimentally validated in the presence of “classical” redox probes (ferrocene methanol, hydroquinone, …) and of lipid bilayer involving various phospholipids (DPhPC, DOPC, DOPG) can be now used to monitor and quantify the transport of high added value biomolecules such as cell penetrating peptides.
Fluorescence spectroscopy also allowed the quantification of various cell penetrating peptides crossing large unilamellar vesicles. It was notably shown that the direct translocation is a rapid process which leads within a few minutes to intravesicular accumulation up to 0.5 mM. Interestingly, the role of anionic phospholipid flip-flopping in the translocation process was also ascertained. Indeed, the peptide intravesicular concentration is correlated with the amount of phospholipids that had been transferred from the external to the internal leaflet of the lipid bilayer demonstrating thus that CPPs are transported by phospholipids.

A certain number of important scientific breakthroughs can be anticipated as outcomes of this project such as advances in analytical techniques (molecular electrochemistry, electrochemical impedance spectroscopy, confocal microscopy, …) as well as investigations of peptide/membrane interactions via the synthesis and electrochemical and/or fluorescence characterization of original cationic peptides bearing an appropriate probe. In the future, this work will allow the investigation of mechanisms that are involved in the transport of any drug/biomolecule and will bring crucial information for the development of new pharmaceutical strategies.

Novel integration of patch-clamp and amperometry provided a powerful mean for quantifying, in real time, molecular species fluxes across real cell and artificial membranes: Angew. Chem. Int. Ed. 53 (2014) 3192.
The direct translocation of cell-penet

This project is aimed at characterizing at the molecular and nanomolecular levels the processes involved in the translocation of cationic peptides, such as the cell penetrating peptides (CPPs), and in the permeabilization of membranes such as the antimicrobial peptides (AMPs). These studies will be conducted on different types of model membranes with various controlled phospholipidic compositions. In recent years, the interest of the scientific community for CPPs has grown because they are able to efficiently cross the cell membrane and thus to shuffle active molecules into cells. The penetration of cationic peptides through cell membranes is a complex process, associated with the high molecular diversity of the peptide sequences. Despite the development of numerous both in vivo and in vitro studies, mainly based on fluorescence techniques, the mechanism(s) involved in the trans-membrane transfer of cationic peptides is still under debate.
In this context, we propose to develop a new system based on the combination of electrochemical and fluorescence techniques to analyze the consequences of peptides / lipids interactions on the supramolecular scale by following simultaneously the translocation of cationic peptides through membranes (CPPs) and the alteration of membrane permeability via the formation of pores (AMPs). We propose to develop a novel method for a fast screening of peptides families to differentiate these two extreme properties.
This project is primarily aimed at developing new advances in analytical techniques via the coupling of molecular electrochemistry with electrochemical impedance spectroscopy (EIS) and fluorescence techniques such as confocal microscopy or TIRF microscopy (Total Internal Reflection Fluorescence).
This project will focus on the following items: (i) synthesis of a family of cationic peptides with a fluorescent probe or redox properties and studies of bio-physico-chemical properties, (ii) electrochemical studies of the interactions between these peptides and model membranes such as phospholipid vesicles of defined composition, (iii) characterization of specific peptides / lipids assemblies by electrochemical impedance spectroscopy (EIS) and electrochemical microscopy (SECM), (iv) electrochemical studies of nano-flow cationic peptides either with antimicrobial or cell penetrating properties through a lipid bilayer suspended above a micro-hole or obtained by "patch" of giant vesicles (v) development of analytical tools combining electrochemistry and fluorescence (TIRF and confocal microscopy) to obtain kinetic information and spatial resolution on the translocation process in both ranges of short and long time.

Project coordinator

Groupe d'électrochimie, UMR 8640 "Pasteur" (Laboratoire public)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Groupe d'électrochimie, UMR 8640 "Pasteur"
Laboratoire des Biomolécules - UMR 7203
Laboratoire Interface et Systèmes Electrochimiques - UPR 15

Help of the ANR 500,000 euros
Beginning and duration of the scientific project: October 2012 - 36 Months

Useful links