Preparation and Characterization of Lipid Vesicles

Vesicles were prepared by the thin-film hydration method as described before^{34 (link)} to obtain a suspension of multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) of 100 nm size were obtained by extruding MLVs at least 21 times through two stacked polycarbonate filters and a permeable membrane with 100 nm pores (Nuclepore, Pleasanton, CA, USA). The whole procedure was carried out using an extruder (Avanti Polar Lipids Inc. Alabaster, AL, USA) filled with two 1.0 mL Hamilton syringes (Hamilton, Reno, NV, USA). Vesicles size was confirmed by means of dynamic light scattering (DLS) measurements, which showed a mean hydrodynamic radius consistent with the formation of LUVs. Liposomes with different lipid composition were prepared: POPC/POPG (8/2 mol/mol) and DPPC/POPG (8/2 mol/mol). Liposome samples in the presence of peptides were prepared by mixing appropriate amounts of peptide solution and liposomes suspensions to obtain the required lipid-to-peptide (L/P) ratio. The vesicles containing the fluorescent probe Laurdan were obtained by adding a solution of Laurdan in DMF to the lipid organic mixture at a lipid/Laurdan mole ratio of 30, while vesicles containing the fluorescent probe DPH were obtained by adding a solution of DPH in chloroform to the lipid organic mixture at a lipid/DPH mole ratio of 150.