4.3. Preparation of Liposomes by Thin-Film Hydration

For preparing the liposomes, we used the thin-film hydration method described by Bangham et al. [43] with slight modifications. BBR, HSPC, DSPG, SDC and α-tocopherol were dissolved in the mixture of methanol and chloroform at a volume ratio of 2.5:1. This solution was evaporated at 50–55 °C under vacuum (300–350 mPa) to form a thin-film layer on the wall of a flask in a rotary evaporator. The rotation rate of an evaporation flask was 150 rpm. The rotary evaporation was continued until all trace of organic solvents in the wall of a flask was removed to ensure the absence of the solvents. The dried thin-film layer was then hydrated completely by distilled water to form the suspension of liposomes. Finally, the size of the liposomes was reduced by extruding through a polycarbonate membrane with a pore size of 0.4 μm using a mini-extruder (Avanti Polar Lipids, Inc., Alabaster, AL, USA) equipped with the Hamilton syringe (The Hamilton Company, Boston, MA, USA). The suspensions were stored at 2–8 °C prior to characterization.