3.2. Preparation of Liposomes (Lip)

The liposomes were prepared by the thin film hydration method in phosphate buffered saline (PBS), as described in our previous work [10]. In brief, DSPC, cholesterol, DDAB and DSPE-PEG-NH₂ with 6:4:1:0.5 molar ratios were dissolved in a 100 mL round-bottom flask containing 3 mL of chloroform/methanol (2:1(v/v)) mixture to prepare a 10 mM lipid solution. The lipid solution was converted into a lipid film using a roto-evaporator (EYELA N- 1200AVF, Tokyo, Japan) working at 100 psi and 55 °C. After 20 min evaporation, the film formed on the wall of the round-bottom flask was further dried overnight in a vacuum oven, for complete removal of trace organic solvents. Subsequently, the lipid film was hydrated for 20 min at 55 °C with 10 mL PBS in the round-bottom flask and the resultant liposome suspension was sonicated for 20 min using a Q700 ultrasonicator (Qsonica, Newtown, CT, USA) maintained at 30% amplitude and 5 s pulse on/off cycles. By extrusion of liposomes through a polycarbonate filter of 220 nm pore size in an Avanti^® Mini Extruder (Avanti Polar Lipids, Inc., Alabaster, AL, USA), we produced small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) from multilamellar vesicles (MLVs). Compared to the MLVs, the SUVs and LUVs are more stable and could achieve higher drug encapsulation efficiency.