4.1. Chemicals and Liposome Preparation

Phosphatidylcholine (PC), lyso-phosphatidylcholine (lysoPC), cholesterol (chol) and phosphatidylethanolamine (PE) were purchased from Avanti Polar Lipids (Amsterdam, Netherlands) and were of analytical grade. Fluorescent probe, fluorescein-PE and carboxy-fluorescein (>99%) were purchased from Molecular Probes (Eugene, OR, USA). All experiments were carried out in the deionized water (conductivity less than 0.1 µS). In order to generate the osmotic pressure difference across the lipid bilayer the water solutions of KCl, NaCl or sucrose (POCH, Gliwice, Poland, 99.99%) were used. Liposomes were prepared by the extrusion method [90]. In short, the appropriate amounts of lipids dissolved in chloroform were mixed together (when needed, with the addition of 0.1 mol% of fluorescein-PE) and the organic solvent was than evaporated using the stream of nitrogen. To remove the remnants of the organic solvent, the lipid film was left overnight under vacuum. The resulting dry lipid film was hydrated and agitated for 2 h to obtain milky multilamellar vesicles suspension. Next, the suspension was extruded through the 100 nm polycarbonate membrane filters (Whatman, Maidsone, UK). In samples where the entrapment of carboxy-fluorescein was needed, the dry lipid film was hydrated with aqueous phase with the fluorescent dye (0.05 M) added. The non-encapsulated dye was removed by passing the vesicle suspension through the column (Sephadex 25G; Cytiva, Marlborough, MA, USA), as described in detail elsewhere [45]. The quality of the liposome suspension was tested prior and after each measurement using the dynamic light scattering technique (NanoSizer ZS, Malvern, UK). Typically, the average vesicle size was equal to 110 nm ± 10 nm with the polydispersity index always smaller than 0.12. Liposome size depended on the lipid type and composition to the small extent. Vesicles size distribution was also measured as a function of pressure difference and time after application of the osmotic pressure difference. The pH of buffer solutions for proton transport measurements were controlled with pH-meter, calibrated for pH 4, 7 and 11 prior each measurement.