2.2.1. Preparation of Liposomes

Liposomes were prepared by the thin film hydration method to obtain small unilamellar vesicles (SUVs) [39]. Briefly, DOPC, PS, CinA, *p*-CoA or FA were dissolved in chloroform (anhydrous, ≥99%). Lipid alone and phenolic acids solutions were transferred into a glass tube in amounts suitable to obtain the appropriate concentrations of phenolic acids. The concentrations were chosen on the basis of the results of MTT assay performed on human glioblastoma cell lines subjected to CinA, *p*-CoA or FA treatment for 24 and 48 h. Regarding the IC<sup>50</sup> values and in order to choose the sublethal doses of phenolic acids, the concentrations of 1.0 and 5.0 mmol/dm<sup>3</sup> have been selected [10,40,41]. A thin lipid film was obtained after the organic solvent evaporation under an argon gas stream to remove any organic solvent residue. The dried lipid film was hydrated with an electrolyte solution (155 mM/L NaCl). Liposomes were formed by sonicating the suspension using the ultrasound generator (UD 20, Techpan, Warsaw, Poland). Sonication was repeated five times, 90 s each. Since heat is released during the process, cooling the suspension is mandatory. It was performed with an ice bath (a container with a mixture of ice and dry sodium chloride). The samples consisted of plain liposomes (10 mg of: DOPC, PS, 9:1 mol% DOPC:PS or 8:2 mol% DOPC:PS), as well as liposomes containing chosen phenolic acid. The liposomes were directly examined in the ELS apparatus.
