Characterization of phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers using laurdan fluorescence with log-normal multipeak analysis

Phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers combining polybutadiene-block-polyethylene oxide (PBdPEO), egg sphingomyelin (egg SM), and cholesterol were studied with fluorescence spectroscopy and microscopy for comparison to lipid bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), egg SM, and cholesterol. Laurdan emission spectra were decomposed into three lognormal curves. The temperature dependence of the ratios of the areas of the middle and lowest energy peaks revealed temperature break-point (T_break) values that were in better agreement, compared to generalized polarization inflection temperatures, with phase transition temperatures in giant unilamellar vesicles (GUVs). Agreement between GUV and spectroscopy results was further improved for hybrid vesicles by using the ratio of the area of the middle peak to the sum of the areas all three peaks to find the T_break values. For the hybrid vesicles, trends at T_break are hypothesized to be correlated with the mechanisms by which the phase transition takes place, supported by the compositional range as well as the morphologies of domains observed in GUVs. Low miscibility of PBdPEO and egg SM is suggested by the finding of relatively high T_break values at cholesterol contents greater than 30 mol%. Further, GUV phase behavior suggests stronger partitioning of cholesterol into PBdPEO than into POPC, and less miscibility of PBdPEO than POPC with egg SM. These results, summarized using a heat-map, contribute to the limited body of knowledge regarding the effect of cholesterol on hybrid membranes, with potential application toward the development of such materials for drug delivery or membrane protein reconstitution.