Fluorescein diacetate 5 maleimide

To prepare a fluorescein-LL-37 conjugate, 127 mg (31 μmol) of a modified LL-37 (CSG-LL37), as we have described previously [22, 26] , was dissolved in ~10 ml of water for injection and drop-wise added to a 5 ml of solution of 75 mg (147 μmol) of fluorescein diacetate 5-maleimide (Sigma-Aldrich, Sweden) in DMSO. The resulting reaction mixture was stirred at room temperature in the dark overnight and purified by dialysis (SnakeSkin™ Dialysis Tubing, 3.5 kDa MW cutoff, 22 mm, ThermoFisher Scientific, Sweden) against Millipore water (pH 7), for several days. The dialyzed solution was then freeze-dried to obtain 76 mg of a fluffy yellow solid, which was re-dissolved in definite volume of water for further use.
In order to label the liposomes cargo (single label, green) the fluorescein-LL-37 conjugate was co-encapsulated (10% w/w) with LL-37. Double labeled liposomes (green and red) were obtained by encapsulating fluorescein-LL-37 in liposomes composed of 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine Rhodamine B sulfonyl) ammonium salt (PE-Rhodamine, Avanti Polar Lipids, Inc., AL, USA) at a molar ratio of 0.05:20:1:10, to DSPC, DSPE-PEG and cholesterol, respectively. Single labeled empty liposomes were also prepared using PE-Rhodamine as a membrane label.