Multilamellar and/or unilamellar liposomes consisting of POPC (1 mM) alone or POPC (0.95 mM) and POPS (0.05 mM) were prepared according to previously described methods [20 –22 (link)]. Briefly, a mixture of the selected lipids at desired concentration was dissolved in chloroform saturated with argon to prevent oxidation. Chloroform was then evaporated with a stream of argon/nitrogen gas, and the lipid film that formed on the bottom of the test tube was thoroughly dried under reduced pressure for 12 h. A buffer solution was added to the dried lipids at a temperature higher than the main phase-transition temperature of the lipids used (room temperature was appropriate for the lipids used in this work), and the mixture was vortexed vigorously. To obtain unilamellar liposomes, the suspension was extruded using an extruder with needles (cat. no. 610000, Avanti Polar Lipids, Inc.) and filtered using filters with pore diameters of 0.1 μm and 0.2 μm (cat. nos. 610005 and 61006, Whatman, GE Healthcare Ltd., Little Chalfont, UK) above the phase-transition temperature of the lipids. The size distribution and stability of the liposomes were analyzed in 10× dilution using a Zetasizer Nano S particle size analyzer (Malvern Instruments, Malvern, UK).
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