Intracellular Dynamic Disentangling of Doxorubicin Release from Luminescent Nanogold Carriers by Fluorescence Lifetime Imaging Microscopy (FLIM) under Two-Photon Excitation

Abstract

There is still a lack of available techniques 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 kinetics of doxorubicin (DOX) and gold nanoparticle (AuNP) carriers 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 onto the AuNPs was studied by spectroscopic methods and steady-state fluorescence, and the nanosystem pH-responsive character was validated under simulated biological conditions at different pH values (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 of the discrimination of the different states of the drug in the internalization path, i.e., released versus loaded, the kinetics of AuNPs@gelatin–hyd–DOX cellular uptake and DOX release was compared to that of free DOX, resulting in 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 noninvasive, high-resolution imaging of cells with improved capabilities over current one-photon-excited FLIM.

Mechanistic Insights into the Release of Doxorubicin from Graphene Oxide in Cancer Cells

“…Additionally, our data do not contradict previous evidences showing that GO likely binds to integrins at the plasma membrane of cancer cells, activates the integrin-FAK-Rho-ROCK pathway and makes cancer cells more susceptible to chemotherapeutic agents [31]. From a methodological point of view, our results corroborate the idea that FLIM can represent a quantitative platform to analyze DOX cellular uptake and release from nanocarriers, in line with a growing body of evidences from the literature (see for instance References [29,30,32,33]).…”

Au nanostar nanoparticle as a bio-imaging agent and its detection and visualization in biosystems

“…For example, the analysis of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate redox couples [37] or the analysis of distribution of flavin adenine dinucleotide, elastic fibers, keratin, and others [38] is of particular use in the aforementioned studies. However, while previously using gold nanoparticles as a fluorescent marker, FLIM was demonstrated for cells [23,28,29], we have extended the studies to include tissue models with using Au nanostars.…”

“…In the present work, we focus on fluorescence lifetime imaging at two-photon excitation to visualize AuNSt in biological systems of different structures and thickness to investigate the possibilities of using AuNSt as an imaging agent in bio applications. Although Au nanoparticles have promising potential for bio-imaging and biosensing at two-photon excitation, only a few works concerning two-photon FLIM have been recently reported [28][29][30] -nanospheres [28][29][30], nanorods and nanotriangles [29], not Au nanostars. In this work, a cultured cancer cell line (A549 cell human lung cancer cell), tissue (murine skin) and a three-dimentional (3D) cellular model (multi cellular tumor spheroid, MCTS) were interacted with AuNSt.…”

Emulsion Techniques for the Production of Pharmacological Nanoparticles

“…Fluorescence lifetime is highly dependent on the local microenvironment of molecules but independent on dye concentration, photo‐bleaching, and excitation intensity. Therefore, FLIM can indicate changes of microenvironment around drugs, reflecting intercellular drug release and transport inside the cells . Zhou et al used FLIM to monitor the dynamic change of DOX fluorescence lifetime in intercellular environments .…”