Hydrogenated phospholipids

Liposomes were prepared by the film-hydration method, using hydrogenated phospholipids (LIPOID Gmbh, Germany) [36 (link)]. For the preparation of the negatively charged liposomes (PS-liposomes), saturated egg phosphatidylcholine, saturated egg phosphatidylserine, and cholesterol (Sigma-Aldrich) (7 : 2 : 1 molar ratio) or neutral liposomes (PC-liposomes-control group), phosphatidylcholine, and cholesterol (9 : 1) were dissolved in chloroform : methanol (1 : 1 ratio). The solution was further sonicated for 10 minutes. The mixture was evaporated in a rotary evaporator at 55°C at 60 rpm for 40 minutes in a vacuum and protected from light. A preheated (55°C) solution of 2.25% glycerol (9 mL) was added to the lipid film using glass beads. The swelling process of the preformed liposomes was performed in a rotary evaporator at 55°C at 80 rpm for 60 minutes without a vacuum. The surface charge of neutral and negatively charged liposomes was previously confirmed using the zeta potential analysis, in a Zeta Plus Analyzer (BrookHaven Instr. Corp.). The samples were diluted in 1 mL of 3 mM KCl as recommended by the manufacturer. The diluted samples were then analyzed for ten cycles with a voltage of 4 mV. The phospholipid content of the liposomal formulations was determined by the Stewart assay [37 (link)].

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (thiazol blue; MTT), sodium dodecyl sulfate (SDS), M-199 medium, RPMI-PR-1640 medium (w/o phenol red), and cholesterol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Hydrogenated phospholipids were kindly donated by Lipoid GmbH (Ludwigshafen, Germany). Sertraline and other analytical reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Molecular biology reagents are purchased from Life Technologies, and CBA (cytometric beads array) was purchased from BD (San Jose, CA, USA).