Liposome Cell Uptake Assay

Liposomes were stained with 0.25% nile red (Sigma-Aldrich) for 24 h at room temperature. Free nile red was removed from liposomes by centrifugation at 3000 xg for 30 min in filter membranes (Amicon, Sigma-Aldrich, molecular cutoff: 10,000 MWCO). 10⁶ HMC3 or SVGA cells were seeded on glass cover slips and grown for 24 h. Stained liposomes (or the nile red staining solution for control) were applied to the cells for 24 h in concentrations corresponding to 0.01 µM free curcumin. Cells were fixed with 4% paraformaldehyde (PFA, in phosphate-buffered saline, PBS), rinsed with PBS (3x), incubated with Alexa Fluor 647 labelled wheat germ agglutinin (Thermo Fisher scientific, 1:200) for 1 h, rinsed with PBS (3x). Then, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, 30 min) and cover slips were embedded after rinsing with PBS and distilled water using Shandon Immumount (Thermo Fisher scientific). Cover slips were inspected and documented using an Axiovert 200M microscope with Apotome (Zeiss, Oberkochem, Germany).