We prepared a liposome solution (10 mg/ml) by first dissolving 5 mg of lipid of interest into a 5-ml round bottom flask in a dichloromethane/MeOH (7:3) solution. A thin lipid film was achieved by evaporating the solvent using a rotary evaporator (Buchi RE-111) and then by drying further with a high-vacuum pump (Welch 1402) for 4 hours. The thin lipid film was then hydrated at 10 mg/ml in either 10 mM Hepes buffer (150 mM KCl, pH 7.0), phosphate-buffered saline (PBS) (1×, pH 7.4), or a solution of PBS containing 100 mM CF by vortexing the solution for 30 s, followed by sonication in a water bath sonicator (Branson 2510) for 30 min. After sonication, the lipid mixture underwent five freeze-thaw cycles that consisted of 2 min at −78°C, followed by 2 min at 50°C. The liposomal suspension was then successively extruded (Avanti Mini Extruder) through 400- and 200-nm polycarbonate membranes (51 times for each membrane) to generate ~200-nm-diameter liposomes. Smaller liposomes (~80 nm diameter) were produced using additional extrusion with 100- and 50-nm polycarbonate membranes (51 times for each membrane). Liposome radii are shown in fig. S2. For CF-encapsulated liposomes, free CF was removed by gel filtration through a Sephadex G-100 column eluted with PBS (1×, pH 7.4), and the lipid solution was then stored at 4°C in a Protein LoBind Eppendorf tube.

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