Preparation and characterization of doxorubicin-containing liposomes. II. Loading capacity, long-term stability and doxorubicin-bilayer interaction mechanism

Doxorubicin loading capacity was determined for negative (phosphatidylcholine-cholesterol-phosphatidylserine, 10:4:1) and positive (phosphatidylcholine-cholesterol-stearylamine 10:4:3) liposomes prepared according to the “film” method with doxorubicin added to the phospholipids before film formation. Doxorubicin association depended on the initial ratio of doxorubicin/phospholipid, the charge and the size. The maximum loading capacity was 60–75 mmol doxorubicin/mol phospholipid for the negative and about 55 mmol doxorubicin/mol phospholipid for positive, non-filtered, multilamellar liposomes. Filtration or ultrasonication/ultracentrifugation reduced the doxorubicin-bilayer interaction. Loss on storage of doxorubicin from the filtered, multilamellar, or unilamellar vesicles decreased in the following order: filtered negative < unilamellar positive < unilamellar negative vesicles. Positive unilamellar vesicles increased in particle size on storage. Negative liposomes were stable. ζ-Potentials of positive or negative liposomes did not depend on the presence of associated doxorubicin. This makes a purely electrostatic mechanism of interaction between the drug and the bilayer unlikely. If doxorubicin was added to the phospholipid film in the hydration buffer a dramatic drop in doxorubicin-bilayer association was observed for positive liposomes. For negative liposomes the loading efficiency was unaffected