Degree
Doctor of Philosophy (PhD)
Department
Department of Chemistry
Document Type
Dissertation
Abstract
To satisfy the requirements of clinicians charged with locating and assessing diseased tissue in the human body via new qualitative and quantitative visualization and imaging modalities that hinge on the successful creation of novel imaging agents, the cellular and tissue transport properties of those entities must permit access to diseased tissue. For imaging approaches employing target-activated fluorescence probe-based readout across a range of emission energies, ready access of probes to diseased tissue depends on their fundamental physicochemical properties in conjunction with those of the biological milieu, which for 3-dimensional tissue mimics can be complex. Here, a set of computational and empirical approaches are described in an effort to gain fundamental knowledge about the transportation of cytosolically activated fluorescence-based molecular probes into cells in 2-dimensional and 3-dimensional mammalian cell cultures, as well as their ability to traverse between cultured cells in 3-dimensions and gain access to the interior of cells, present deep within tissue mimics. While such knowledge is critical for identifying a variety of diseased tissues, the fields of fluorescence-guided surgery (FGS) for cancer imaging and monitoring of disease-linked enzyme activities during disease treatment will be transformed by an understanding of how activatable probes can be designed and synthesized to reach diseased cells present deep within tissue. In this study, I build on successes of the McCarley research lab with small-molecule imaging probes whose energy and intensity of emission are selectively altered by an intracellularly present cancer-marker enzyme activation, namely, 2-electron reduction via human NAD(P)H: quinone oxidoreductase isoenzyme-1 (hNQO1) to yield a reporter variant. The computed physicochemical properties of six probes and reporter variants—with emission spanning the visible and near-infrared—are examined in the context of their transport properties and ultimate organelle fate using two-dimensional cell cultures and three-dimensional, lab-grown multicellular tumor spheroids (MCTSs) of human colorectal adenocarcinoma cells (HT-29). Examination of transport routes in two-dimensional cultured HT-29 cells is achieved with appropriate immunostaining after incubating in appropriate probe/reporter solutions and additional organelle-tracker molecules, followed by inspection with confocal laser scanning and spinning disk confocal microscopy, equipped with super-resolution by optical re-assignment (SoRa) functionality. Information about probe/reporter location in HT-29 MCTSs is obtained performing identical staining experiments in concert with light sheet fluorescence and advanced X resonant or AXR point scanning confocal microscopy.
Date
3-26-2025
Recommended Citation
Mukherjee, Shibalik, "Toward an Understanding of the Transport Routes of Enzyme-responsive, Fluorescent Molecular Probes in 3-Dimensional Tumor Mimics" (2025). LSU Doctoral Dissertations. 6710.
https://repository.lsu.edu/gradschool_dissertations/6710
Committee Chair
McCarley, Robin L.
Included in
Analytical Chemistry Commons, Medicinal-Pharmaceutical Chemistry Commons, Organic Chemistry Commons