As described by Ralston et al.,
19 20 5,6-carboxyfluorescein (Molecular Probes, Junction City, OR), with an excitation peak of 490 nm, was purified and diluted to approximately 100 mM. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylglycerophosphocholine (DSPC) (all from Genzyme, Liestal, Switzerland), and myristoylpalmitoylphosphatidylcholine (MPPC; Avanti, Alabaster, AL) were used without further purification. Liposomes were prepared by using a method previously described.
15 21 Briefly, lipids (dissolved in chloroform and methanol) were dried to a thin film by rotary evaporation under vacuum. A 100-mM solution of CF was mixed with the dried lipid film, and the mixture was subjected to five freeze–thaw cycles. This process was followed by extrusion sizing in a thermobarrel extruder (Lipex Biomembranes, Vancouver, British Columbia, Canada) through a stack of two 25-mm, 0.2-μm polycarbonate membranes (Millipore, Bedford, MA) 10 times to yield large unilamellar vesicles. Free CF was removed through a resin column (Sephadex G-50; Pharmacia Biotech, Uppsala, Sweden).
Five types of liposomes were prepared as follows: (1) 40°C liposome (Tc = 40°C), DSPC, DPPC, DPPG, and MPPC at 0:16:3:1 (M/M); (2) 46°C liposome (Tc = 46°C), DSPC, DPPC, DPPG, MPPC at 47:43:10:20 (M/M); (3) 47°C liposome (Tc = 47°C), DSPC, DPPC, DPPG, MPPC at 47:43:10:10 (M/M); (4) 48°C liposome (Tc = 48°C), DSPC, DPPC, DPPG, MPPC at 47:43:10:3 (M/M); and (5) 52°C liposome (Tc = 52°C), DSPC, DPPC, DPPG, MPPC at 16:2:2:1 (M/M).