2.3. Liposome Suspension Characterization

Liposomes size distributions and polydispersity indexes (PDI) were measured with the dynamic light scattering method (DLS) (Zetasizer Nano ZS, Malvern, UK) after samples were diluted 50 times with the deionized water or buffer (Chempur, Łódź, Poland). All solutions before use were filtered through the cellulose membrane with 0.2 μm pores (VWR, Gdańsk, Poland).

The lipid concentration in the liposome suspension, before and after the extrusion process, was determined by the Stewart method [78]. Specifically, 40 µL of liposome suspension was added to 2 mL of chloroform, 2 mL of 0.4 M of ammonium ferrothiocyanate, and 0.1 M of ferric chloride hexahydrate solution. Then, the samples were vortexed and set aside for 15 min. The lipid concentration was calculated based on the determined value of absorbance (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany) and the calibration curve. The liposomes fluorescence was measured using a Fluoromax-4 Spectrofluorometer (Horiba, Tokyo, Japan). In the first step, the excitation and emission spectra of fluorophores, when in the lipid bilayer, were collected, and the wavelengths of maximum fluorescence intensities were determined. For NBD and Rh-B, the maximum fluorescence intensity was measured when the excitation/emission wavelength was equal to 467/531 and 569/589 nm, respectively. In experiments where the fluorescence resonance energy transfer between the two probes was evaluated, the excitation wavelength was set at 467 nm and emission spectra were acquired in the wavelength range from 520 to 630 nm.