3.4. Membrane Fluidity

The membrane fluidity of all samples was measured by fluorescence anisotropy measurements. TMA–DPH was used as the fluorescent probe. The measurement was carried out according to the method described by Maherani et al. [54]. In brief, the solution of TMA–DPH (1 mM in ethanol) was added to the liposome suspension to reach a final concentration of 4 µM and 0.2 mg/mL for the probe and the lipid, respectively. The mixture was lightly stirred for 1 h at ambient temperature in the dark. Then, 180 µL of the solution was distributed into each well of a 96-well black microplate. The fluorescent probe was vertically and horizontally oriented in the lipid bilayer. The fluorescent intensity of the samples was measured with a Tecan INFINITE® 200 PRO (Grödig, Austria) equipped with fluorescent polarizers. Samples were excited at 360 nm and emission was recorded at 430 nm under constant stirring at 25 °C. The Magellan 7 software was used for data analysis. The polarization value (P) of TMA–DPH was calculated using the following equation:

where I_II is the fluorescent intensity parallel to the excitation plane, I_⊥ the fluorescent intensity perpendicular to the excitation plane, and G is the factor that accounts for transmission efficiency. Membrane fluidity was defined as 1/P. The results were measured in triplicate.