Supramolecular organization of S12363-liposomes prepared with two different remote loading processes

The S12363 anticancer drug was encapsulated into liposomes in an attempt to increase its therapeutic index. Loading of S12363 was achieved using two different processes based on the formation of either a pH gradient or an ammonium gradient between the acidic inner liposomal compartment and the basic outer phase. High encapsulation yields (>90%) were obtained using both processes for sphingomyelin/cholesterol/cholesterol-PEG vesicles. Spectrofluorimetry measurements have shown that liposomes were characterized by an internal pH around 4 for both loading processes. This internal pH was stable over a period of at least 20 days. Differential scanning calorimetry coupled with time-resolved synchrotron X-ray diffraction was used to study the drug/carrier supramolecular organization. In ammonium sulfate, S12363 was inserted into the bilayer in the vicinity of the polar headgroup. In citrate buffer, S12363 was mainly adsorbed at the water-lipid interface. The drug partitioning into the membrane was inhomogeneous and led to the formation of drug-rich and drug-poor domains. This effect was enhanced in the presence of cholesterol, especially in ammonium sulfate. To conclude, for both processes, the encapsulated drug was found inside the liposome aqueous core but strongly interacting with the membrane.