Photophysical Characterization and Microscopy Application of an Anthracene Analogous of Laurdan

Abstract

Design of new bio-nano hybrid systems calls for understanding and accounting for the influence of a nanostructured support and nanoconfinement on the structure and biophysical properties of lipid bilayer hybrid systems and membraneprotein interface. Here we report on spin-labeling EPR studies of pH-sensitive lipids and specifically labeled protein side chains to assess effects of solid inorganic interface, specifically, silica support in a form of monodispersed nanoparticles ranging from 20 to 300 nm in diameter on the surface electrostatic potential of lipid bilayers associated with the particles and effective pKa of the membrane-burred peptide ionisable sidechains. We have shown that bilayers formed from zwitterionic or mixed lipids on silica nanoparticle surfaces possess a higher negative electrostatic potential than the unsupported bilayers with the potential of mixed bilayers containing negatively charged lipids being significantly more sensitive to the silica support. Effect the silica nanoparticle size on the lipid bilayer surface electrostatic potential was also observed for particles smaller than 100 nm. pH-sensitive EPR probes were then employed to label model WALP peptide known to form an a-helix when integrated into a lipid bilayer. The silica support exerted pronounced effects on WALP dynamics and the effective pKa of the ionizable probe. It was demonstrated that the silica nanoparticles shift the effective pKa of the ionizable nitroxide probe in a membrane depth-dependent manner. Upon protonation of the membraneburred model ionisable sidechain the silica support caused significant changes in the membrane association of WALP peptide that are not observed when WALP is integrated into unilamellar phospholipid vesicles of similar curvature. Lipid metabolism is characterized by a complex set of sequential reactions which underpin many biological processes, including the maintenance of the cellular energy balance and the intercellular communication through the socalled lipid signalling. The lipid composition and distribution within cells change following chemical and physical interactions with the surrounding environment in response to specific needs or conditions, as is the case of the activation of lipolysis or storage pathways. The method here presented allows characterizing some of these processes through the evaluation of the microenvironment polarity within the cell membranes with a high spatial and temporal resolution. This is achieved by using an algorithm based on the phasor approach to the spectral imaging. As a result, a metabolic parameter is furnished to enable a quantitative assessment of the storage of fatty acids in triacylglycerols in the form of lipid droplets or the triacylglycerols hydrolysis in response to energetic demands, as well as changes in the global and local distribution of polar and non-polar lipids with a sub-micrometre resolution. The method has been applied both to tissues and cell cultures to study the reaction of the systems to lipid metabolism disorders or nutritional ...