Monitoring drug stability by label-free fluorescence lifetime imaging: a case study on liposomal doxorubicin

Abstract In a previous report, we demonstrated that Doxorubicin (DOX) intrinsic fluorescence can be exploited in combination with the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and quantitative absorption/fluorescence spectroscopy to resolve the supramolecular organization of the drug within its FDA-approved nanoformulation, Doxil®. The resulting ‘synthetic identity’ comprises three co-existing physical states of the drug within Doxil®: a dominating fraction of crystallized DOX (DOX c >98%), and two minor fractions of free DOX (DOX f ∼1%), and DOX associated with the liposomal membrane (DOX b <1%). This result serves as a benchmark here to address the time evolution of Doxil® synthetic identity. We probe the effect of temperature for a total duration of 6 months in a non-invasive way by FLIM. We confirm Doxil® stability if stored at 4°C, while we detect marked changes in its synthetic identity at 37°C: crystallized DOX gets progressively disassembled in time, in favor of the other two physical states, free and membrane-associated DOX. Our phasor-FLIM-based approach paves the way to time-resolved biochemical assays on the supramolecular organization of encapsulated fluorescent drugs potentially all the way from the production phase to their state within living matter.