2.2.2. Preparation of Spherical Bilayers

The stock solutions for bilayers formation were composed of 20 mg cm−<sup>3</sup> of DOPC or DOPC-PS mixture in concentrations of 9:1 and 8:2 mol%, respectively. Chloroform phospholipids alone and phenolic acids solutions were transferred into a glass tube in amounts suitable to obtain the same concentration of CinA, *p*-CoA or FA as in liposome forming solutions. After mixing the compounds, the solvent was removed under a nitrogen flow, and the resulting dry residues were dissolved in a n-hexadecane-n-butanol mixture (10:1 by volume). All the bilayer-forming solutions were stored in darkness at a temperature of <4 ◦C in a refrigerator for at least 3 days before examination. To minimize the oxidation of lipids, the vessels containing final samples were filled with nitrogen.

Bilayers were prepared by the method of squeezing the solution, which enables creating spherical bilayers dividing two aqueous solutions. They were formed at the Teflon cap constituting a part of the measuring vessel. During membranes creation, the solvent mixture was removed from the lipid phase, resulting in membranes with the same composition as in stock solutions. Bilayers were monitored both electrically and optically during the entire process of formation. Measurements were initiated 10–15 min after the membranes turned completely black. Membrane images were taken by color CCD camera using the WinFast PVR program. The bilayer areas were calculated from the photographs, taking into consideration the spherical nature of the surface and employing the Makroaufmassprogram program [42]. The area of the spherical membranes was about 6 × 10−<sup>2</sup> cm<sup>2</sup> . More details regarding the procedure for the membrane formation are given in our previous works [31,36,37].
