Preparation of peptide-liposomal drugs

A lipid film hydration method was used to prepare PEGylated liposomes composed of distearoylphosphatidylcholine, cholesterol, and mPEG2000-DSPE, which were then used to encapsulate doxorubicin (3:2:0.3 molar ratio) or vinorelbine (3:2:0.15 molar ratio). The lipid films were hydrated at 60°C in 250 mM ammonium sulfate or 300 mM ammonium salts of 5-sulfosali-cylic acid solution, and were extruded through polycarbonate membrane filters with a pore size of 0.1 µm using high-pressure extrusion equipment (Lipex Biomembranes, Vancouver, BC, Canada) at 55°C. Doxorubicin or vinorelbine were encapsulated by a remote loading method, at concentrations of 1 mg or 3.5 mg per 10 µmol of phospholipid, respectively. The final concentration of liposome was estimated by phosphate assay. The peptide-PEG-DSPE was subsequently incorporated into pre-formed liposomes by co-incubation at 60°C, the transition temperature of the lipid bilayer, for 0.5 h with gentle shaking. Sepharose 4B (GE Healthcare) gel filtration chromatography was used to remove released free drug, unconjugated peptides, and unincorporated conjugates. Doxorubicin concentrations in the fractions of eluent were determined by measuring fluorescence at λEx/Em = 485/590 nm using a spectrofluorometer (Spectra Max M5, Molecular Devices). Vinorelbine concentrations were determined using the HPLC method.