Liposomes preparation

Fluorescent- and gadolinium-labeled liposomes were prepared by the thin film hydration method. A rhodamine-labeled lipid (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), DPPE-Rhodamine) and a gadolinium labeled lipid ((DTPA-bis(stearylamide) (gadolinium salt), Gd-DTPA-BSA) was used to incorporate rhodamine and gadolinium on the head groups of the lipid bilayer. The lipid composition was a molar ratio of 58.4-59.5 mol percent DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), 35 mol percent Gd-DTPA-BSA, 5 mol percent DSPE-PEG2k (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (ammonium salt)), and 0.5-1.6 mol percent DPPE-Rhodamine. Lipids were dissolved in a 2:1 volume ratio of chloroform:methanol which was evaporated over a stream of argon gas and left in a vacuum desiccator overnight to remove residual solvent. The lipid thin film was rehydrated with PBS for 2 hours at 66°C with occasional stirring and pipetting. The lipid solution was sonicated at 20% power for 2 min with a probe sonicator, and extruded sequentially through membrane filters with 1.0 um pores (4×), 0.4 um pores (6×), and 0.2 um pores (8×) to prepare liposomes with a mean diameter of 130-150 nm (Group B). To prepare liposomes with a mean diameter of 70-85 nm (Group A), the lipid solution was extruded in the same manner with additional extrusion through membrane filters with 0.1 um pores (8×), and 0.05 urn pores (12×). The size of the liposomes was measured by dynamic light scatterin. The administered dose was similar for group A (0.56±0.03 mmol total lipid/kg body weight) and group B (0.51±0.08 mmol total lipid/kg body weight). The calculated administered dose assumes no loss of lipid due to extrusion.