Cholesterol → Sulfate Ion

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Liposomes with entrapped ammonium sulfate or ammonium polyacrylate were prepared from the lipid mixture of hydrogenated soybean phosphatidylcholine (Avanti PolarLipids, Ala., U.S.A.), cholesterol (Calbiochem, USA), and poly(ethylene glycol) (Mol. weight 2,000) derivative of distearoyl phosphatidyl ethanolamine (PEG-DSPE) (Sygena, Switzerland), at the molar ratio 60:40:6, by lipid film hydration, repetitive freezing-thawing at 60° C. (6 times) and extrusion through two stacked polycarbonate track-etched membranes with the pore size 100 nm at 60° C. (12 times). The bulk aqueous phase of the liposomes was exchanged by gel-filtration for the outer buffer, 0.2 M ammonium chloride, adjusted (when necessary) to pH 7.3 and buffered with 10 mM sodium hydroxyethylpiperazino-ethane sulfonate (HEPES). The inner (entrapped) solution had ammonium ion concentration of 200 milli-eqivalent/L, i.e. the same as the outer buffer (no ammonium ion gradient), and the anion composition and pH as indicated below. Polyacrylic acid with mol. weight of 2,000 (Aldrich Chemical Co.) was used. Doxorubicin was added to the liposomes at 2 mg for each 6-8 micro-mol of phospholipid, and incubated with shaking at 60° C., i.e. above the transition temperature of the lipid bilayer, for various times as specified below. At this temperature, doxorubicin did not form a detectable precipitate in the presence of sulfate anion, but was visibly precipitated by a polyacrylate anion. Doxorubicin-loaded liposomes were separated from the free drug by gel-chromatography on Sephadex G-75, eluted with the outer buffer. Liposomal doxorubicin was assayed by spectrophotometry at 485 nm, phospholipid was quantitated by molybdate method after acid digestion of the liposomes. The following results were obtained:

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