Neurotransmitter-loaded liposomes were prepared passively by thin lipid film hydration. A solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dioleoyl-sn-glycero-3-phosphethanolamine (DOPE) and cholesterol (60:20:20 mole ratio) in chloroform was dried in a round-bottom flask by rotary evaporation until a lipid film was obtained (~3 h). The dried lipid cake was re-hydrated to obtain a liposome suspension by gently mixing with 1.5 mL of the hydration buffer and then left to stand for 30 min under argon gas at room temperature. The liposome suspension was freeze-thawed in liquid nitrogen 3 to 5 times to form multilamellar vesicles, and then extruded 11 times through double polycarbonate membranes of 0.4μm pore size with an Avanti Mini-Extruder (Avanti Polar Lipids, Inc., USA). This procedure yielded liposomes with a mean diameter of 376 nm, as measured by dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern Inc., Malvern, UK). Removal of free DA or 5-HT outside liposomes was achieved by gel filtration on a Sephadex G-25 column which was pre-equilibrated in advance.