Preparation and Characterization of Lipid Vesicles

1-palmitoyl,2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), chicken egg yolk sphingomyelin (SM), and cholesterol (Chol) were purchased from Sigma-Aldrich (Darmstadt, Germany). The lipids were mixed in chloroform with desired lipid ratios (Table 1). The lipid films were formed by slowly removing the solvent under a gentle nitrogen stream for 1 h. PBS solution, pH 7.4 (Sigma-Aldrich, Darmstadt, Germany) was added to the lipid films. In order to form lipid vesicles, the lipid films were incubated with PBS at above the melting transition temperature (T_m) for 2 h and intermittently vortexed during incubation. Large unilamellar vesicles (LUVs) of POPC and POPC/Chol vesicles were produced by repeatedly pressing through a polycarbonate membrane 21 times at room temperature with a mini-extruder (Avanti, Birmingham, AL, USA). The sizes of the POPC and POPC/Chol vesicles were approximately 120–130 nm with a polydispersity index (PDI) of about 0.15. The LUVs of the POPC/DPPC, POPC/SM and DOPC/SM vesicles were obtained via tip sonication with a 50% duty cycle for 10 min (Branson sonifier, St. Louis, MO, USA). The sizes of the POPC/DPPC, POPC/SM and DOPC/SM vesicles were approximately 95–105 nm with a PDI of about 0.39. The vesicle sizes were determined via dynamic light scattering with a Zetasizer Nano ZS (Malvern Instrument, Malvern, Worcestershire, UK). The lipid vesicles were kept at temperatures higher than Tm and used within a week.