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In-cell synchrotron investigations of precious-metal anticancer complexes
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Bolitho, Elizabeth May (2020) In-cell synchrotron investigations of precious-metal anticancer complexes. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3518274~S15
Abstract
Platinum (II) anticancer drugs are used in over 50% of all chemotherapeutic treatments worldwide, however, platinum-resistance is a major clinical concern. Platinum-group complexes are of significant interest in the development of anticancer drugs which overcome such resistance, with improved selectivity for cancer vs. healthy cells, ultimately enhancing the drug efficacy and reducing patient side effects. In this thesis, three families of metal complexes have been investigated: (i) OsII half-sandwich complexes for catalytic therapy, (ii) cyclometallated IrIII complexes for photodynamic therapy (PDT) and (iii) diazido-PtIV prodrugs for photoactivatable chemotherapy (PACT). Determination of the chemical speciation of such complexes in cancer cells is essential for understanding the antiproliferative mechanisms of action. Herein, the speciation, distribution and stability of such complexes in cancer cells have been probed using Inductively-Coupled Plasma Mass Spectrometry (ICP-MS), synchrotron X-ray Fluorescence (XRF), Differential Phase Contrast (DPC) imaging and synchrotron X-ray Absorption Near Edge Structure (XANES) spectroscopy. Complementary to this, cryogenic fluorescence microscopy, Structured Illumination Microscopy (SIM) and synchrotron soft X-ray Tomography (XRT) have been used to investigate the 3D drug distribution and morphological changes in cancer cells down to a subcellular resolution (40 nm).
OsII half-sandwich complexes of the general formula OsII[(η6-pcymene)(BsDPEN)] (where BsDPEN=benzyldiphenylethylene diamine) show promise as catalysts for transfer hydrogenation in cancer cells. In this thesis a total of six (1S, 2S) or (1R, 2R)-(R)BsDPEN ligands and their corresponding OsII[(η6-pcymene)((R)BsDPEN)] complexes have been synthesized and characterized (where R=Br, I, OCH3). These complexes exhibited moderate antiproliferative potencies (IC50~20-30 μM) against four human cancer cell lines. A 16-electron Br-labelled complex (OsII[(η6-p-cymene)(p-Br)BsDPEN)]) showed similar chemical properties (x-ray crystal structure, DFT, LogP, solution stability and transfer hydrogenation catalytic activity) to that of parent compound OsII[(η6-p-cymene)((CH3)BsDPEN)]. The Br-probe did not significantly alter the in vitro activity, or in-cell catalytic activity, thus, was a suitable analogue to probe the intracellular stability and distribution these complexes by ICP-MS (189Os; 79Br) and synchrotron-XRF (Os L3M5=8.91 keV; Br KL3=11.92 keV). The Os and the Br-labelled sulfonamide ligand strongly co-localised in the cytoplasm. ICP-MS and synchrotron-XRF revealed significantly higher levels of the Br-labelled sulfonamide ligand compared to the Os, implying intracellular degradation. Surprisingly, the Br-labelled sulfonamide ligand was distributed in cell nuclei, whereas Os was not, but it is not yet known whether this contributes to the anticancer activity. Synchrotron-XRF also revealed the co-localisation of Os with the Br-labelled ligand in lysosomes.
Cyclometallated luminescent IrIII complexes show promise as photosensitizers for PDT. Potent complex [IrIII(C,N)2(O,O)] (where C, N=2-phenylpyridine and O,O=diketonate) can generate toxic singlet oxygen (1O2) and was investigated by cryo-XRT, revealing significant 3D mitochondrial damage in cancer cells treated with this photosensitiser, likely caused by the selective generation of 1O2 in close proximity to mitochondria. Additionally, the stability and localisation of a potent photodynamic-photochemotherapeutic IrIII-PtIV conjugate under dark conditions was investigated in cancer cells using synchrotron-XRF. This revealed significantly higher levels of Pt vs. Ir, suggestive of in-cell complex cleavage. Pt was distributed homogenously in cells, whereas Ir localised in organelle-like cytoplasmic regions, providing evidence for a multi-targeting mechanism of action.
The antiproliferative mechanism of action of a potent coumarin-labelled diazido-PtIV prodrug trans, trans, trans-[PtIV(OH)(coumarin)(N2)2(pyridine)2] was investigated using cryo-SIM and cryo-XRT. Cryo-XRT revealed the presence of endosomes in treated cells exposed to dark conditions, suggesting that active processes may be involved in the efflux of PtIV prodrugs prior to light activation. Upon irradiation, cells treated with this diazido-PtIV prodrug caused severe cellular damage, including vacuolisation, membrane-blebbing, vesicle-shedding and increased cell granularity. Synchrotron-XRF demonstrated the targeting of cell nuclei by diazido-PtIV prodrugs, in addition to the increased Pt accumulation upon blue light irradiation. XANES studies suggest the presence of predominantly PtIV vs. PtII in treated cancer cells exposed to blue light.
This thesis has utilized a variety of advanced synchrotron imaging techniques to probe the intracellular distribution, speciation and cell damage induced by Os, Ir and Pt complexes in cancer cells in their near-native state, demonstrating how new biological imaging techniques can be exploited in metals in medicine.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QC Physics Q Science > QD Chemistry R Medicine > RC Internal medicine |
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Library of Congress Subject Headings (LCSH): | Cancer -- Chemotherapy, Platinum -- Therapeutic use, Drug resistance in cancer cells, Synchrotrons | ||||
Official Date: | June 2020 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Sadler, P. J. ; Quinn, Paul | ||||
Sponsors: | Diamond Light Source (Firm) ; University of Warwick ; Engineering and Physical Sciences Research Council ; Cancer Research UK | ||||
Extent: | viii, 329 leaves : illustrations | ||||
Language: | eng |
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