Liposomes of soybean phosphatidylcholine/cholesterol (3:2 molar ratio) were prepared by the thin film hydration method as described previously.23 (link) Briefly, the above lipids were dissolved in dichloromethane/ethanol (1:2 V/V) and dried to form a thin lipid film in a rotary evaporator (EYELA, Tokyo, Japan). Then the film was hydrated with 155 mM ammonium sulfate, and the liposome suspension was extruded through a polycarbonate membrane (200 nm) for ten times using an extruder (Avestin, Ottawa, ON, Canada).
Afterward, DOX was remotely loaded into the liposomes via the ammonium gradient method.11 (link),23 (link) Liposomes were eluted through a Sephadex G 75 column (GE Healthcare) preequilibrated with a HEPES buffer (20 mM HEPES and 144 mM NaCl, pH 7.4). DOX was added to the liposomes and incubated at 60°C for 20 minutes. Free DOX was removed by Sepharose CL-4B column.
Afterward, DOX was remotely loaded into the liposomes via the ammonium gradient method.11 (link),23 (link) Liposomes were eluted through a Sephadex G 75 column (GE Healthcare) preequilibrated with a HEPES buffer (20 mM HEPES and 144 mM NaCl, pH 7.4). DOX was added to the liposomes and incubated at 60°C for 20 minutes. Free DOX was removed by Sepharose CL-4B column.
