Coumarin

Fluorescent liposomes containing the lipophilic markers L-α-phosphatidylethanolamine-N (lysamine rhodamine B sulfonyl (rhodamine) (Avanti Lipids, Alabama, USA) or coumarin (Sigma Aldrich, St. Louis, USA) were prepared as described previously with 0.02 rhodamine or 0.03 coumarin (mol% of total structural lipids). To evaluate the influence of liposome composition on cell internalization, 1.5 × 10⁴ HaCaT and Balb/c 3T3-A31 cells were plated in individual 35 mm plates and incubated at 37 °C with 5% CO₂ for 12 h. After removal of the culture medium, cells were incubated with labeled liposome formulations at 100 µM AA diluted with DMEM with 2% FBS for 3 h. The liposome dispersing medium was used as a control. HaCaT and NIH/3T3 cells were then washed, fixed with 4% paraformaldehyde in 10 mM Tris-HCl, and labeled with 0.2 μg/ml 4, 6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, USA). Fluorescence microscopy was performed using excitation/emission of 350–470 nm for DAPI, 578–603 for rhodamine-labeled liposomes and 490–525 nm for coumarin-labeled liposomes. Random images of three fields were recorded by replica with a 20x objective of a Leica DMI4000B microscope (Wetzlar, Germany). The observed fluorescence inside keratinocytes and fibroblasts was considered as a result of the cellular uptake of labeled liposomes.