From synthetic identity to biological function of liposomal Doxorubicin: a biophysical study

The successful trajectory of liposome-encapsulated doxorubicin (e.g. FDA-approved Doxil®) as an anticancer nanodrug in clinical applications is countered by in vitro cell-viability data suggesting its reduced efficacy, compared to the non-encapsulated drug, in promoting cell death. No report thus far provided a mechanistic explanation for this apparently discordant evidence. By exploiting the intrinsic fluorescence of Doxorubicin and time-resolved optical microscopy, here we dissect the uptake and intracellular processing of the drug, both liposome-encapsulated (hereafter ‘L-DOX’) and non-encapsulated (or ‘free’, hereafter ‘F-DOX’), in different in vitro cellular models. Cell-entry of L-DOX is found to lead to rapid, energy- and temperature-independent release into the cytoplasm of crystallized Doxorubicin nanorods, which then rapidly disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane, simultaneously releasing active drug monomers. A steady state is observed in which the continuous supply of crystal nanorods from incoming liposomes is counterbalanced by the concentration-driven efflux to the extracellular medium of all drug species, including active drug monomers. Present findings underscore how encapsulation impairs the efficient delivery of Doxorubicin to its intended target, the cell nucleus.