Chems

All lipids required for liposome synthesis, i.e., DSPC (#850365), Chol (#700000), CHEMS (#850524), DSPE-PEG_2kDa (#880120), were purchased from Avanti Polar Lipids. Lipids were prepared as stock solutions at 5 mg/mL in CHCl₃: MeOH = 4 : 1 (v/v) and stored at −20°C before use. To make the liposomes, the lipid stock solutions were warmed to room temperature and transferred into a round bottom flask using glass syringes (Hamilton). Organic solvents were carefully removed using a rotary evaporator to allow the deposition of a uniform, thin lipid film in a round bottom flask while drying under compressed nitrogen. The film quality was visually inspected and then rehydrated in a 100 mM citric acid-sodium citrate buffer solution (pH 4) at 65°C in a water bath to keep the lipid concentration < 20 mM. The hydrated suspension was dispersed by vortexing to yield a milky liposomal suspension. Liposomes were extruded 10 times, using benchtop extruders (#61000, Avanti and #LF-50, Avestin, Inc.) to pass the liposomes through series of polycarbonate filters (Whatman^® Nuclepore filters) with a progressive decrease in pore sizes (1000 nm, 800 nm, 400 nm, 200 nm, and 100 nm). This ultimately brought the liposome size down to 100-120 nm before storage in sterilized containers.