Preparation of cationic liposome and liposome-DNA complex

The cationic liposome is composed of DOTAP: Chol. Liposome was preparated according to our previous methods [13]. Briefly, DOTAP and cholesterol were mixed at equimolar concentrations, then dissolved in chloroform. The mixture was evaporated to form a film, which was then hydrated in 5% glucose solution. The solution was first rotated at 50°C for 45 min in a water bath, then at 35°C for another 10 min. It was left overnight and sonicated at 50°C for 5 min. After sonication, the solution was put into a tube and heated at 50°C for 10 min. Then, the mixture was sequentially (five times at 0.2 μm and three times at 0.1 μm) extruded through polycarbonate membrane to decrease size using syringes. The size range is 100± 20 nm. The final cationic liposome was a small multilamellar liposome. Plasmids were packaged with liposome to form DNA–liposome complexes according to the previous report [2], then used for in vivo experiments.