Application of Synchrotron-Based Spectroscopic Techniques XAS and XFM Towards Probing the Cellular Metallome

Abstract

The use of synchrotron-based experimental techniques has gained increasing recognition, as they are powerful tools to explore the cellular metallome. Metallomics, which encompasses all aspects of biological systems, especially the cell, involved in metal speciation and metabolism; and probes the spatial metal ion distributions within differing cell types, organelles and regulating proteins. As such, the precise imaging of metal ion and intracellular organelle distribution within the wider context of cellular regulation pathways and biological function is essential. Biological systems are inherently complex, with the metal of interest being contained within an intricate biological matrix. Synchrotron-based techniques have benefits that laboratory-based fluorescence experiments do not; reduced required sample preparation, direct metal distribution imaging, and the ability to characterise the chemical nature of the intracellular metal pool. This thesis describes the use of two synchrotron-based techniques; X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM), to investigate the effects of biometal-based agents on the intracellular metallome. Firstly, the intracellular fate of a luminescent rhenium(I) tricarbonyl tetrazolato complex probe within 22Rv1 human prostate epithelial carcinoma cells was explored. It was demonstrated that the cellular distribution of the luminescent imaging agent could be determined by monitoring the luminescence from the compound using optical microscopy and then correlated with the cellular distributions of rhenium and iodine contained in the species within the same samples as measured using microprobe XFM. A combination of XAS and XFM was employed to investigate the neuroprotective action purported for diphenyl diselenide. This was achieved by treating SH-SY5Y human neurocarcinoma epithelial cells with more water soluble diphenyl diselenide analogues; bis(2-aminodiphenyl)diselenide and bis(2-nitrodiphenyl)diselenide. These two studies showcased that the advantageous, simultaneous collection of metallome and mode of action information can provide a pathway to exploring neurological diseases and disorders, the production of better therapeutic agents, and could be used diagnostically in medicine. Finally, the anticancer action of NKP-1339, a KP1019 analogue, within A549 human lung adenocarcinoma epithelial cells was investigated. Initially a co-localisation of NKP-1339, or its ruthenium-containing metabolites, with intracellular iron and copper was observed. Subsequent attempts to visualise a mitochondrial compartmentalisation of the NKP-1339 or its ruthenium-based metabolites utilising a combination of optical and synchrotron-based fluorescence microscopy were unsuccessful. The studies into the Re-I probe and diphenyl diselenide analogues showcased the power of synchrotron-based spectroscopic techniques yielding mode-of-action information as well as visualising the effects of the biometal-containing agents on the intracellular metallome. The study into NKP-1339, while largely unsuccessful, did provide similar information related to the perturbation of the intracellular metallome while not yielding mode-of-action information.