Liposomes preparation

Liposomes were prepared by initially drying a lipid mixture in chloroform (Lipid Composition 1 [LC 1]: cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE], 1,2-dimyristoyl-sn-glycero-3-phosphate [sodium salt] [DMPA], molar ratio [2-4-2-2]; LC 1′: LC 1 + 1 mg/ml monophosphoryl lipid A [MPLA]; LC 2: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine [POPE], 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) [sodium salt] [POPG], E. coli cardiolipin [CL], molar ratio [6-2-2]; LC 2′: LC 2 + 1 mg/ml MPLA; LC 3: POPE, POPG, E. coli CL, DMPA, molar ratio [6-2-1-1]; LC 3′: LC 3 + 1 mg/ml MPLA) (Avanti Polar Lipids) by evaporation under a nitrogen stream. Residual traces of chloroform were removed using a vacuum pump. The lipidic film was hydrated in 500 μl of Tris buffer (50 mM, pH 7.5) by pipetting and vortexing followed by four freeze/thaw cycles in liquid nitrogen. The lipidic mixture was extruded using an extruder (Avanti Polar Lipids) to produce liposomes with an average diameter of 200 nm. Liposomes were stored at 4°C.