PEGylated liposome preparation

Distearoyl phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG₂₀₀₀) and cholesterol (Avanti Polar Lipids, Inc.) were dissolved in chloroform at a 65:5:30 molar ratio, respectively. A dried lipid film was formed at 65 °C by rotary evaporation (Buchi, Rotavapor RII) and rehydrated in Tris-buffered saline (TBS, 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) at 65 °C to a final lipid concentration of 20 mg ml⁻¹. The liposomal suspension was submitted to 10 freeze/thaw cycles in liquid nitrogen and a heated water bath at 80 °C, followed by 21 extrusions at 75 °C through 400, 200 and 100 nm polycarbonate membranes each using a mini-extruder (Avanti Polar Lipids, Inc.). The final lipid concentration was measured by Bartlett's assay⁶⁸ and adjusted to 13.9 μmol ml⁻¹ with TBS before use.