Preparation of liposomes

For preparation of DOTAP:modified PAA liposomes (10 mg/ml based on DOTAP), 10:1 mole ratio of DOTAP and lipopolymer were dissolved in methanol-chloroform (1:1 v/v) solvent. After complete dissolution, the organic solvent was evaporated by rotary evaporator (Heidolph, Germany) in order to form a thin lipid film. The thin film was then hydrated by deionized water at 50 °C and the container was placed in bath sonicator (40 °C) (Branson, USA) to form the liposomal vesicles. To prepare DOTAP: cholesterol liposome (10 mg/ml based on DOTAP), 1:1 molar ratio of DOTAP and cholesterol were dissolved in the same organic solvent and liposomes were prepared in the same procedure described above.

In order to reduce the size, liposomal formulations were extruded through 800, 400 and 100 nm polycarbonate membranes repeatedly at 50 °C using Thermobarrel extruder (Northern Lipid, Canada).