Preparation and Characterization of DMU-214-Loaded Liposomes

DMU-214-loaded liposomes were prepared by a thin lipid film hydration method followed by extrusion as described here (Skupin-Mrugalska et al., 2021 (link)) by using chloroform solutions of DMPC, DPPC, POPC (50 mg/ml), POPG (50 mg/ml), DMU-214 (10 mg/ml). DMU-214 was loaded passively into vesicles during hydration. Appropriate volumes of stock solutions were mixed, and then chloroform was evaporated under gradually reduced pressure at 40 °C in a round-bottomed flask. The resulting lipid film was then hydrated in PBS buffer pH 7.4. The resulting liposome suspension was passed 21 times through the polycarbonate membrane (Whatman, Kent, UK) with pore diameters of 100 nm using a syringe extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA). Unbound material was separated from liposomes by fast ultrafiltration using Amicon Ultra 2 ml centrifugal filters with molecular weight cutoff (MWCO) 50 kDa (Merck KGa, Darmstadt, Germany). The amount of DMU-214 incorporated into liposomes was determined by the chromatographic method (paragraph 2.5). Encapsulation efficiency EE (%) was calculated according to Eq. (1): EE (%) = (C_m/C_i) x 100 (1), where C_m is the concentration of DMU-214 loaded into liposomes, determined by HPLC, C_i is the initial (maximum) concentration of DMU-214 added in the liposomal formulation. The experiment was performed in triplicates.