Preparation and Characterization of Fluorescent Liposomes

Non-fluorescent liposomes were prepared as described above. 5(6)-Carboxyfluorescein (CF) (Merck; 100 mM CF stock, dissolved in 300 mM NaOH) was mixed in a 1:1 ratio with a buffer, containing 300 mM NaCl, and 100 mM Na₂HPO₄-NaH₂PO₄, pH 6. The dried lipid film was rehydrated with the 50 mM CF-stock solution, followed by sonication and extrusion. Finally, to remove the excess CF, the liposomes were applied onto a gravity flow desalting column (PD MiniTrap G25, Cytiva), pre-equilibrated with an elution buffer containing 150 mM NaCl, and 50 mM Na₂HPO₄-NaH₂PO₄, pH 6. Liposome content mixing fluorometry measurements were performed using a Varioskan LUX Fluorometer plate reader (ThermoFisher Scientific), similar to the R18-based assay in a 96-well format (200 µL reaction volume). Briefly, 490/515 nm excitation/emission wavelengths and 5 nm slits were used. The liposome mixture consisted of a 1:2 ratio between CF-encapsulating and empty LUVs (250 µM total lipid concentration), respectively, and measurements were performed in the elution buffer.