Malvern zeta sizer 4 spectrometer

The appropriate lipids were mixed in organic solution and the solvent was evaporated to dryness under a N₂ stream. Then the sample was kept under vacuum for 1 h to remove solvent traces. The lipids were swollen in Assay Buffer in order to obtain multilamellar vesicles (MLVs). Large unilamellar vesicles (LUV) were produced from MLV according to the extrusion method described by Mayer et al. (1986) [60 ]. They were subjected to 10 freeze/thaw cycles, and then extruded using a LIPEX Liposome Extrusion System (Evonik Health Care, Essen, Germany) with a 0.1-μm pore size Nuclepore filters (Whatman, 110,605). Small unilamellar vesicles (SUV) were obtained by sonicating MLV with a probe tip sonicator (MSE Soniprep 150, MSE, UK) for 20 min (10 sec on, 10 sec off) on ice. Vesicle size was checked by quasi-elastic light scattering using a Malvern Zeta-Sizer 4 spectrometer (Malvern Instruments, Malvern, UK). LUV had an average diameter of ≈100 nm and SUV average diameter was ≈50 nm. Phospholipid concentration was determined by phosphate analysis [61 ].