Ammonium sulfate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandoliposomes

The amphipathic anthracycline base doxorubicin (DXR) was accumulated in the aqueous phase of the liposomes where it reached a level as high as 100-fold its concentration in the remote loading medium. Most of the intraliposomal DXR was present in an aggregated state. Efficient (>90% and stable loading into the liposomes' and ligandoliposomes' aqueous phase was obtained by using gradients of ammonium sulfate in which the ammonium sulfate concentration in the liposomes was higher than its concentration in the extraliposomal medium [(NH₄)₂SO₄)lip. ≫ [(NH₄)₂SO₄)med.]. The "remote" loading is a result of the DXR exchange with ammonia from (NH₄)₂SO₄. Both the ammonium and sulfate contribute to high level and stability of the loading. The ammonium sulfate gradient method differs from most other chemical approaches used for remote loading of liposomes since it neither requires to prepare the liposomes in acidic pH, nor to alkalinize the extraliposomal aqueous phase. Although most of the intraliposomal DXR is present in an aggregated gel-like state, the drug is bioavailable. This approach permits the preparation of DXR-loaded liposomes of a broad spectrum of types, sizes, and composition, including sterically-stabilized liposomes, immunoliposomes, and sterically-stabilized immunoliposomes. Due to the long shelf stability (>6 mo), no "bedside" remote loading is required immediately before patient treatment, and the formulation is ready for injection. The stable encapsulation of the doxorubicin in an aggregated form also permits freezing and lyophilization of the liposomes with only minimal drug release. The loading by ammonium sulfate gradient approach meets all pharmaceutical requirements; it has brought the clinical use of DXR-loaded sterically-stabilized liposomes to reality.