Analysis of the Thermotropic Phase Transitions of Liposomal Phosphatidylcholine Using Differential Scanning Fluorimetry

Liposomes are small vesicles composed of lipid bilayers, which have been widely studied and are used in drug delivery systems (DDSs). The lipid bilayers, as two-dimensional liquid crystalline structures, show different phase states, and temperature-dependent phase transitions occur as a result of the thermotropic alteration of the physicochemical properties of the lipid bilayers, resulting in drastic changes in the morphology and dynamics of the fluctuations of the lipid bilayers. Analysis of the thermotropic phase behavior of the liposomal lipid bilayer is crucial for the development and application of functional liposomes for DDSs. We constructed a differential scanning fluorimetry (DSF) method that enabled observation and analysis of the thermotropic phase transitions and temperatures of liposomal lipid bilayers using a real-time PCR device and solvatochromic dyes, which have fluorescence characteristics that reflect alterations in the polar environment. This DSF method using Nile Red and a tandem thermal sequence enabled analysis of the phase transition temperatures of three liposomal phosphatidylcholines, and not only the T_m and T_p, but also the T_sub values, except for the T_p value of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, were clearly determined. Other solvatochromic dyes could not be used to determine the T_sub values clearly. The measured phase transition temperatures of three liposomal phosphocholines correlated well with the reported values. Our DSF method has several practical advantages over the typical thermal analytical method, differential scanning calorimetry, including reduced sample volume and analytical time, which may contribute to expanding the opportunities for the physicochemical analysis of liposomal lipid bilayers.