Preparation of Liposomes

Each 100 µL of the DDA liposome (250 µg DDA) and the DMT liposome (250 µg DDA, 25 µg MPLA, and 50 µg TDB) were prepared by lipid film hydration method as previously described (20, 22, 30). Briefly, a batch of 7,500 mg DDA (Sigma-Aldrich, MO, USA) alone, or with 750 mg MPLA (Avanti Polar Lipids Inc., AL, USA) and 1,500 mg TDB (Avanti Polar Lipids Inc., AL, USA), were firstly dissolved in 10 mL chloroform/methanol (9:1, by volume) and then evaporated by blowing N₂. Sample was maintained overnight under low-pressure condition to form a membrane. Then, liposome was rehydrated in 10 mM sterile Tris-buffer (pH 7.4) at 60°C for 60 min and vortexed every 10 min. pCMFO/DDA and pCMFO/DMT were prepared by emulsifying 100 µL of corresponding liposome with 100 µL of the plasmid pCMFO solution (50 μg/100 μL).