Coexistence of lipid phases in multilayer phospholipid films probed by Raman mapping

Biomimetic phospholipid mixtures are actively used as models of biological membranes and materials for drug delivery in biomedical tasks. One of the essential properties of membranes formed from complex phospholipid mixtures is the equilibrium coexistence of domains of different phases. Studying the conformational state and chemical content of different phases is of great interest in membrane biophysics. We propose an approach for studying phase coexistence in stacked phospholipid bilayers using Raman mapping. For this purpose, phospholipid multilayer films were formed in which the domains of the same phase were self-aligned in stacks. Raman spectra with a high spectral resolution and signal-to-noise ratio obtained on these samples made it possible to estimate the chemical composition and conformational state of lipids in domains of different phases. For the ternary mixture 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC-d62)/cholesterol (Chol) used in our demonstration, the phase diagram was studied and the effect of hydration on lipid phase separation was revealed. For the hydrated films, the obtained phase diagram is in qualitative agreement with the previous data obtained using ²H NMR. In dry films, phase separation is observed for all investigated compositions, with a tendency to form a phase with a high fraction of DPPC-d62. The use of multilayer phospholipid films makes it possible to release the potential of Raman microspectroscopy to study the phase diagrams of phospholipid mixtures under various experimental conditions.