Mayer s hematoxylin

Donepezil hydrochloride (DNP, ≥98%, HPLC), cholesterol (Chol, ≥99%), α-tocopherol (TOC), and thioflavin S were procured from Sigma-Aldrich (St. Louis, MO, USA). Soybean L-α-phosphatidylcholine (PC, 95%) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) was used as the main component of liposomes. Tetradecyltriphenylphosphonium bromide (TPPB-14) was synthesized according to the published method [74 (link)]. Chloroform and ethanol (HPLC) were purchased from JSC “№1 BASE Chemical reagents” (Staraya Kupavna, Russia). Rhodamine B (RhB) (Acros Organics, Morris Plains, NJ, USA) and fluorescent lipid DOPE-RhB (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) were used to visualize liposomes inside cells. Liposomal dispersions were prepared using ultrapure Milli-Q water purified by the Simplicity^® UV system (Millipore SAS, Molsheim, France). For visualization of the immunoexpression of synaptophysin, primary rabbit monoclonal antibody to synaptophysin (ab 32127) and secondary donkey anti-rabbit (Alexa Fluor^® 488) antibody (ab 150073) were purchased from Abcam (Cambridge, UK). Mayer’s hematoxylin was purchased from Biovitrum (Saint Petersburg, Russia).

Human skin fibroblasts (cell line FD2) and ECV cells (human umbilical vein endothelial cells) (Institute of Cytology of the Russian Academy of Sciences, Saint-Petersburg, Russia) were used as an in vitro model for toxicity analysis. Cells were cultivated in DMEM F12 with glutamine (Biolot, St. Petersburg, Russia), bovine serum 10% (Biolot, St. Petersburg, Russia), and fibroblast growth factor (for line FD2) at a concentration of 20 ng/mL (cat#PSG060-10. Lot#16F0519F2, Sci-Store, Skolkovo, Russia). Cells were incubated at 37 °C in 5% CO₂. To analyze the morphology, ECV cells grown on coverslips were fixed with 96% ethanol, stained for 30 s with Mayer’s hematoxylin (BioVitrum, St. Petersburg, Russia), and analyzed using a Leica DM 2500 (Leica microsystems, Wetzlar, Germany) light microscope. The MTT assay and apoptosis assay were used to determine toxicity.