2.1. Liposome Preparation and Characterization

Liposomes were prepared by the thin film hydration method ^20–22. A lipid mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids, Alabaster, AL, USA) and cholesterol (Sigma, St. Louis, MO, USA) 2:1 molar ratio was dissolved in a 9:1 chloroform : methanol solution. The lipid mixture underwent vacuum evaporation to form a dry lipid film. T-butanol was used to dissolve the lipid thin film and freeze-dried to form a lipid cake. The lipid cake was hydrated with phosphate buffered saline (PBS), DMED solution (1 mg/mL PBS; Sigma, St. Louis, MO, USA), DexP solution (50 mg/mL PBS; Sigma, St. Louis, MO, USA), or 250 mM (NH₄)₂SO₄. The liposomes then underwent ten freeze-thaw cycles followed by dialysis against PBS for 48 hours. Bup (Sigma, St. Louis, MO, USA) was loaded into liposomes using a previously reported remote loading technique ²³. Briefly, after dialysis in PBS, the liposomes that were hydrated with 250 mM (NH₄)₂SO₄ were loaded with Bup upon incubation with a 50 mg/mL Bup hydrochloride solution at 50°C for 1 h. The liposomes were then dialyzed against PBS for 48 hours.

Liposome size was determined by dynamic light scattering (Delsa Nano, Beckman Coulter, Brea, CA, USA). Drug loading was determined by HPLC after disrupting the liposomes with 100 mM octyl β-D-glucopyranoside (Sigma, St. Louis, MO, USA).