Liposome preparation

The extrusion method for liposome preparation has been well documented by others [19]. Briefly, DSPC and Chol were removed from the freezer and placed in a desiccator for 2 h before being weighed and dissolved in chloroform at a 55:45 mole ratio. A non-exchangeable and non-metabolizable lipid marker ³H-CHE was incorporated into the chloroform mixture to achieve a specific activity of approximately 0.025-μCu/mmol total lipid. The solution was dried from chloroform using nitrogen gas and a thin film was generated with further drying under high vacuum for at least 3 h. The lipid film was then rehydrated at 65 °C with unbuffered 300-mM CuSO₄ (pH 3.5). The resulting multilamellar vesicles underwent 5 freeze (in liquid nitrogen) and thaw (65-°C water bath) cycles [20]. The vesicles were then placed in an extruder (Evonik Transferra Nanosciences, Vancouver) and extruded at 65 °C through stacked 0.1-μm polycarbonate filters at least ten times. The size of the resulting liposomes was determined using quasi-electric light scattering (ZetaPals, Brookhaven). The unencapsulated copper was removed by exchanging the sample into a sucrose (300 mM), HEPES (20 mM), and EDTA (15 mM) buffer (SHE buffer, pH 7.4) by passing the sample through a Sephadex G-50 column equilibrated with the buffer. The resulting solution was then dialyzed against a sucrose (300 mM) and HEPES (20 mM) buffer (SH buffer, pH 7.4) and concentrated using tangential flow to the desired liposomal lipid concentration required for experimental studies. Liposomal lipid concentration was determined by vortexing an aliquot of the liposome solution with scintillation cocktail and measuring ³H-CHE by liquid scintillation counting (Packard 1900TR Liquid Scintillation Analyzer).