Correlations between the rate of intracellular release of endocytosed liposomal Doxorubicin and cytotoxicity as determined by a new assay.

Previously, we showed that liposomes with surface-attached anti-CD19 were internalized into human B lymphoma cells through receptor-mediated endocytosis, resulting in improved anti-tumor efficacy 1-2 . In order to further increase the efficacy of antineoplastic drug-containing liposomes, we have taken advantage of this internalization process by producing triggered release liposomes that rapidly release drug from the enzyme-rich, acidic environment of lysosomes. To analyze the effectiveness of these triggered-release formulations, we developed a nuclear accumulation assay for doxorubicin (DXR) that allows us to determine the rate of cytoplasmic drug delivery subsequent to drug release from the endosomal/lysosomal compartments by examining the rate of accumulation of drug in cellular nuclei. We demonstrate the usefulness of this assay by comparing the kinetics of cytoplasmic drug delivery for DXR-containing, pH-sensitive, triggered release liposomes versus DXR-containing, non-sensitive, liposomal formulations. We see a significant correlation between the rate of nuclear accumulation of DXR and its in vitrocytotoxicity. This indicates that pH-sensitive formulations traffic drug to the cytoplasm and the nucleus significantly more rapidly than do non-sensitive formulations. We conclude that the development of triggered release liposomes is a promising strategy for further improving the therapeutic efficacy of liposomal antineoplastic drugs targeted selectively to cancer cells by surface-attached ligands that bind to internalizing epitopes.