Copper ion-mediated liposomal encapsulation of mitoxantrone: The role of anions in drug loading, retention and release

Besides pH gradient, other transmembrane gradients such as metal ion gradient could be also employed to load drugs into liposomes. In pH gradient method, anions have an important role since they could form specific aggregates with drugs, and then affect drug release kinetics from vesicles. To explore the role of anions in metal ion gradient method, copper ion-mediated mitoxantrone (MIT) loading was investigated systematically. When empty liposomes exhibiting a transmembrane copper ion gradient (300 mM) were mixed with MIT in a molar ratio of 0.2:1, after 5 min incubation at 60 °C, >95% MIT could be loaded into vesicles and the encapsulation was stable, regardless of the kinds of anions and initial intraliposomal pH values. The encapsulation ratio decreased with increased MIT/lipid molar ratio. But even when the molar ratio increased to 0.4, >90% encapsulation could still be achieved. In the presence of nigericin and ammonium, the drug loading profiles were affected to different degree with respect to both drug loading rate and encapsulation ratio. Relative to CuSO4-containing systems, CuCl2 mediated MIT loading was unstable. Both nigericin and ammonium could alter the absorption spectra of liposomal MITs loaded with CuSO4 gradient. In vitro release studies were performed in glucose/histidine buffer and in 50% human plasma using a dialysis method. In both of release media, CuCl2-containing vesicles displayed rapid release kinetics in comparison with CuSO4 systems; and during the experiment period, MIT was lost from the vesicles continuously. When the formulations were injected into BDF1 mice at a dose of 4 mg/kg, all the liposomal formulations exhibited enhanced blood circulation time, with half-life values of 6.8–7.2 h, significantly compared to the rapid clearance of free-MIT. In L1210 ascitic model, CuCl2 formulation was more therapeutically active than CuSO4 formulation. At a dose of 6 mg/kg, the treatment with CuCl2 formulation resulted in a median survival time of 21 days, considerably larger than that of CuSO4 groups (15 days). Based on these data, it was concluded that during the drug loading process, a dynamic transmembrane pH gradient is generated and intraliposomal pH might affect the complexation manner in which Cu2+ binds MIT. Owing to the presence of pH gradient, after the accumulation within vesicles, a part of MIT will be protonated and precipitated by sulfate. Accordingly, the aggregation status of MIT inside CuSO4 system was more complicated than that in CuCl2 vesicles. The difference in physical status of MIT aggregates affects not only the drug release rate, but also their therapeutic effects.