photo-channels

1-Scientific background and objectives : Control and understanding of the ubiquitous functions of lipid membranes forming domains is among the more fundamental and practical challenge in membrane biophysics. Assemblies and lateral heterogeneities provide remarkable functions, including the regulation of channels and permeability (e.g. pore formation by antimicrobial peptides pipes are developed in pharmaceutics). Recent investigations of amphiphilic copolymers binding onto lipid vesicles show similar control of membrane properties (domains&well-define pores) by much simpler macromolecules devoid of even a secondary structure. Of practical importance, similar macromolecules have been patented for drug delivery and release of liposomes content in cytosol after cellular uptake. Little is known to date on the physics of their anchoring and effect on membranes, but their hydrophobicity appears a critical parameter. We recently showed that such polymers impart bilayers with the ability to transiently form nano-channels with no disruption of membrane. We obtained similarly the permeabilisation of plasmamembranes of mammalian cells (HEK, COS). The milder impact of polymers as compared to detergents, absence of membrane solubilisation, and the remarkable responsiveness of amphipilic polymers open a route toward the development of new tools for handling membranes and cells, and would significantly help investigation on non-specific stabilisation of channels, provided that the properties of polymer can be controlled. We propose to develop macromolecules, whose hydrophobicity has been made light-sensitive. We seek at triggered permeabilisation of lipid membranes for applications to cell biology and liposome-carriers (e.g. ophthalmology). Reversible adjustment and rapid switch of the properties of membranes with exposure to light, would provide an unprecedented degree of control that bring a novel tool to characterise the dynamics and stability of the nano-channels formed by anchored macromolecules. 2-Description of the project, methodology : Amphiphilic polymers known to affect membranes and cells will be modified by grafting photochrome groups (e.g. hydrophobic azobenzene) to obtain light-responsive macromolecules. The modification procedure was developed by the coordinator and is published. The project will address in this context the following points: (i) Obtaining high magnitude of membrane response to light-exposure, both in liposomes and planar bilayers ; and characterizing the efficient structures. (ii) Permeabilising mammalian cells, with the aim of minimal perturbation of intra-cytosol functions. Using giant vesicles (GUV), thethered bilayers and black films, we would consider the physics of pore opening&closure (conductivity isolated pores, lipid translocation), and physical-chemistry of anchoring and permeability (dynamics and surface density of polymers by fluorescence correlation, kinetics of leakage of isolated GUV). Our approach gather together chemists (polymer synthesis and adjustment of structure), physicists (study of single pore dynamics), physical-chemists (colloids and liposomes), and biologists (permeabilisation of cells and preservation of cytosolic functions, proof of concept using the example of G-protein coupled receptors). Stimulation that is both specific and sharply focused would made possible totally new investigation on pore formation, including their dynamics, by the use of light beams to trigger the onset of phenomena, and eventually cycles. Polymer tools for mild and targeted (light-triggered) 'poration' of cells, and liposomes delivery of drugs could readily be made available at large scale for interested users. 3-Expected results : (i) Rapidly switch the release of internal compartment of lipid vesicles upon exposure to UV-visible light (possible development of liposomes for drug release in ophthalmology) (ii) Reversal of permeability to the impermeable state of the bilayer by exposure to another wavelength (reversibility of light-triggered hydrophobicity is achieved) (iii) Taking advantage of the degree of control so achieved to study pore opening/closure on black lipid membranes and GUVs. The investigation would benefit of the unprecedented possibility to swing (even cycles) specifically the properties of polymer, in absence of additives and with no other perturbation of lipids. (iv) Permeation of mammalian cells with substrates introduced in their cytosol at controlled time (and eventually targeting a few cells in a tissue), to follow the expression of functions in the internal leaflet (here G-protein coupled receptors). Proof of concept include toxicity assays and low perturbation of membrane proteins in presence of polymer, at condition of weak or no permeabilisation (UV exposure).