Intracellular dynamic disentangling of Doxorubicin release from luminescent nanogold carriers by Fluorescence Lifetime Imaging Microscopy (FLIM) under two-photon excitation.

There is still a lack of available technique to follow noninvasively the intracellular processes as well to track or disentangle various signals from the therapeutic agents at the site of action in the target cells. We present here the assessment of the intracellular kinetic of Doxorubicin (DOX) and gold nanoparticles carriers (AuNPs) by mapping simultaneously fluorescence and photoluminescence signals by Fluorescence Lifetime Imaging Microscopy under two-photon excitation (TPE-FLIM). The new nano-chemotherapeutic system AuNPs@gelatin-hyd-DOX has been fabricated by DOX loading onto the surface of gelatin biosynthesized AuNPs (AuNPs@gelatin) through a pH-sensitive hydrazone bond. The successful loading of DOX to the AuNPs was studied by spectroscopic methods and steady-state fluorescence and the nano-system pH-responsive character was validated in simulated biological conditions at different pH buffers (i.e. pH 4.6, 5.3 and 7.4). Considering that the fluorescence lifetime of DOX molecules at a specific point in the cell is a reliable indicator for the discrimination of the different states of the drug in the internalization path i.e. released vs loaded, the kinetics of AuNPs@gelatin-hyd-DOX cellular uptake and DOX release was compared to free DOX, resulting two different drug internalization pathways. Finally, cell viability tests were conducted against NIH:OVCAR-3 cell line to prove the efficiency of our chemotherapeutic nanosystem. TPE-FLIM technique could be considered promising for non-invasive, high-resolution imaging of cells with improved capabilities over current one-photon excited FLIM.