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 (DOXc >98%), and two minor fractions of free DOX (DOXf∼1%), and DOX associated with the liposomal membrane (DOXb <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.

The supramolecular processing of liposomal doxorubicin hinders its therapeutic efficacy in cells

“…At this point, it remains to be demonstrated whether the released material (i.e., nanorod crystals) eventually evolves over time into some other drug form. In this regard, we recently demonstrated that L-DOX synthetic identity evolves spontaneously in cuvette at 37°C 23 (top-down approach, schematics in Figure 3 A) generating drug products with an FLIM signature in cuvette ( Figure S13 ) compatible with that measured after sonication ( Figure S12 ). Upon administration to cells, the products of L-DOX spontaneous evolution, similar to those obtained by sonication, show the ability to cross the cell membrane and localize exclusively in the cytoplasm (see an exemplary image in Figure 3 B) with a phasor FLIM signature coincident to that of L-DOX in cells at early times ( Figure 3 C).…”

Phasor identifier: A cloud-based analysis of phasor-FLIM data on Python notebooks

“…5 , we present the potential of this approach through a case study involving the storage of Doxoves at different temperatures (4°C and 37°C) for a duration of 120 days. This study provides valuable insights into the behavior of the three molecular species under varying storage conditions ( 61 ). In Fig.…”

Phasor-FLIM-guided unraveling of ATRA supramolecular organization in liposomal nanoformulations

“…38 Moreover, there's no guarantee that the manufacturer's formulation protocol facilitates drug interaction within the nanoparticles’ interior. A clear example of this, as recently revealed by FLIM analysis, 13,39 is liposomal doxorubicin: because of the active-loading protocol used by the manufacturer, the drug is mostly in a precipitated semi-crystalline form and interacts with the membrane much less than it would do based on its amphipathic properties. It should be noted that previous FLIM-based case reports on encapsulated drugs (doxorubicin 13 and irinotecan 24 ) lacked a convincing experimental validation of FLIM sensitivity to drug–membrane interaction, which is instead provided here by independent SAXS- and nanoDSC-based analyses.…”