Preparation and characterization of liposomes

DC-Cholesterol/DOPE, DOTAP and MVL5 (1:1:0.2) with or without membrane stain, DiI (ThermoFisher), were used to form a lipid film, hydrated with 1X phosphate buffered saline (PBS), sonicated and passed through a 0.1μm polycarbonate membrane followed by 0.05μm polycarbonate membrane, five times each, at 37° C using a nitrogen pressure operated Lipex extruder (Northern Lipids, Inc.)[24]. To prepare liposomes labeled with DiI, 1mg/ml DiI solution in chloroform was added to the lipid mixture and processed as described previously. The liposomes were then concentrated by passing first through a Sephadex G-25 medium (GE Life Sciences) column followed by concentration at 4°C in a centriprep30k concentrator (ThermoFisher). Liposomes were stored at 4°C for upto 4 weeks. Particle size, polydispersity index (PDI) and zeta potential analysis were performed using the PALS Zeta Potential Analyzer (Ver. 3.16; Brookhaven Instruments Corp.).