B24-Cryo Soft X-ray Tomography
I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[22696, 21505, 25824]
Open Access
Abstract: The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species.
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Nov 2021
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Open Access
Abstract: Organo-osmium(II) 16-electron complexes [OsII(η6-arene)(R-PhDPEN)] (where η6-arene = para-cymene or biphenyl) can catalyze the reduction of prochiral ketones to optically pure alcohols in the presence of a hydride source. Such complexes can achieve the conversion of pyruvate to unnatural d-lactate in cancer cells. To improve the catalytic performance of these osmium complexes, we have introduced electron-donor and electron-acceptor substituents (R) into the para (R1) or meta (R2) positions of the chiral R-phenyl-sulfonyl-diphenylethylenediamine (R-PhDPEN) ligands and explored the reduction of quinones, potential biological substrates, which play a major role in cellular electron transfer chains. We show that the series of [OsII(η6-arene)(R-PhDPEN)] derivatives exhibit high turnover frequencies, enantioselectivities (>92%), and conversions (>93%) for the asymmetric transfer hydrogenation (ATH) of acetophenone-derived substrates and reduce duroquinone and menadione to their di-alcohol derivatives. Modeling of the catalysis using density functional theory (DFT) calculations suggests a mechanism involving formic acid deprotonation assisted by the catalyst amine groups, phenyl-duroquinone stacking, hydride transfer to OsII, possible CO2 coordination, and tilting of the η6-arene ring, followed by hydride transfer to the quinone. These findings not only reveal subtle differences between Ru(II) and Os(II) catalysts, but also introduce potential biological applications.
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Aug 2021
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I08-Scanning X-ray Microscopy beamline (SXM)
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James
Everett
,
Frederik
Lermyte
,
Jake
Brooks
,
Vindy
Tjendana-Tjhin
,
Germán
Plascencia-Villa
,
Ian
Hands-Portman
,
Jane M.
Donnelly
,
Kharmen
Billimoria
,
George
Perry
,
Xiongwei
Zhu
,
Peter J.
Sadler
,
Peter B.
O'Connor
,
Joanna F.
Collingwood
,
Neil D.
Telling
Diamond Proposal Number(s):
[15854]
Open Access
Abstract: The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero–oxidation state) metallic Cu0 accompanying ferromagnetic elemental Fe0 in the human brain. These nanoscale biometal deposits were identified within amyloid plaque cores isolated from Alzheimer’s disease subjects, using synchrotron x-ray spectromicroscopy. The surfaces of nanodeposits of metallic copper and iron are highly reactive, with distinctly different chemical and magnetic properties from their predominant oxide counterparts. The discovery of metals in their elemental form in the brain raises new questions regarding their generation and their role in neurochemistry, neurobiology, and the etiology of neurodegenerative disease.
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Jun 2021
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I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[19838, 20548]
Open Access
Abstract: Transition metal complexes are often prodrugs which undergo activation by ligand exchange and redox reactions before they interact with target sites. It is therefore important to understand the roles of both the metal and the ligands in their activation, especially in cells. Here we use a combination of synchrotron nanoprobe X-ray fluorescence (XRF) from Os L3M5 and Br KL3 emissions and inductively coupled plasma-mass spectrometry (ICP-MS) detection of 189Os, 79Br, and 127I, to investigate the time-dependent accumulation and localization of osmium as well as the monodentate ligand and the chelated phenylazopyridine in A2780 human ovarian cancer cells treated with the potent anticancer complexes [Os(η6-p-cymene)(4-R2-phenyl-azopyridine-5-R1)X]PF6, with R2 = NMe2 or OH, R1 = H or Br, and X = Cl or I. The data confirm that the relatively inert iodido complexes are activated rapidly in cancer cells by release of the iodido ligand, probably initiated by attack by the intracellular tripeptide glutathione (γ-L-Glu-l-Cys-Gly) on the azo double bond. The bond between osmium and the azopyridine appears to remain stable in cells for ca. 24 h, although some release of the chelated ligand is observed. Interestingly, the complexes seem to be degraded more rapidly in normal human cells, perhaps providing a possible mechanism for selective cytotoxicity towards cancer cells.
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Jun 2021
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I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[20552]
Open Access
Abstract: Most metallodrugs are prodrugs that can undergo ligand exchange and redox reactions in biological media. Here we have investigated the cellular stability of the anticancer complex [OsII[(η6‐p‐cymene)(RR/SS‐MePh‐DPEN)] [1] (MePh‐DPEN=tosyl‐diphenylethylenediamine) which catalyses the enantioselective reduction of pyruvate to lactate in cells. The introduction of a bromide tag at an unreactive site on a phenyl substituent of Ph‐DPEN allowed us to probe the fate of this ligand and Os in human cancer cells by a combination of X‐ray fluorescence (XRF) elemental mapping and inductively coupled plasma‐mass spectrometry (ICP‐MS). The BrPh‐DPEN ligand is readily displaced by reaction with endogenous thiols and translocated to the nucleus, whereas the Os fragment is exported from the cells. These data explain why the efficiency of catalysis is low, and suggests that it could be optimised by developing thiol resistant analogues. Moreover, this work also provides a new way for the delivery of ligands which are inactive when administered on their own.
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Feb 2021
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I08-Scanning X-ray Microscopy beamline (SXM)
I10-Beamline for Advanced Dichroism
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Open Access
Abstract: Atypical low-oxidation-state iron phases in Alzheimer’s disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide β-amyloid (Aβ) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aβ and ferritin resulted in the conversion of ferritin’s inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aβ in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.
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Jun 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[15230]
Open Access
Abstract: Background: Neuromelanin-pigmented neurons, which are highly susceptible to neurodegeneration in the Parkinson’s disease substantia nigra, harbour elevated iron levels in the diseased state. Whilst it is widely believed that neuronal iron is stored in an inert, ferric form, perturbations to normal metal homeostasis could potentially generate more reactive forms of iron capable of stimulating toxicity and cell death. However, non-disruptive analysis of brain metals is inherently challenging, since use of stains or chemical fixatives, for example, can significantly influence metal ion distributions and/or concentrations in tissues. Aims: The aim of this study was to apply synchrotron soft x-ray spectromicroscopy to the characterisation of iron deposits and their local environment within neuromelanin-containing neurons of Parkinson’s disease substantia nigra. Methods: Soft x-ray spectromicroscopy was applied in the form of Scanning Transmission X-ray Microscopy (STXM) to analyse resin-embedded tissue, without requirement for chemically disruptive processing or staining. Measurements were performed at the oxygen and iron K-edges in order to characterise both organic and inorganic components of anatomical tissue using a single label-free method. Results: STXM revealed evidence for mixed oxidation states of neuronal iron deposits associated with neuromelanin clusters in Parkinson’s disease substantia nigra. The excellent sensitivity, specificity and spatial resolution of these STXM measurements showed that the iron oxidation state varies across sub-micron length scales. Conclusions: The label-free STXM approach is highly suited to characterising the distributions of both inorganic and organic components of anatomical tissue, and provides a proof-of-concept for investigating trace metal speciation within Parkinson’s disease neuromelanin-containing neurons.
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May 2020
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[18339, 19615]
Open Access
Abstract: The organoiridium complex Ir[(C,N)2(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (1O2) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of 1O2 generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.
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Mar 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[15230, 15854, 20809, 24526, 24531]
Open Access
Abstract: A hallmark of Parkinson’s disease is the death of neuromelanin‐pigmented neurons, but the role of neuromelanin is unclear. Lack of a neuromelanin‐specific marker was highlighted over 30 years ago, yet in‐situ characterization of neuromelanin remains dependent on detectable pigmentation, rather than direct quantification of neuromelanin. We show that direct, label‐free nanoscale visualization of neuromelanin and associated metal ions in human brain tissue can be achieved using synchrotron Scanning Transmission X‐ray Microscopy (STXM), via a characteristic feature in the neuromelanin x‐ray absorption spectrum at 287.4 eV that is also present in iron‐free and iron‐laden synthetic neuromelanin. This is confirmed in consecutive brain sections by correlating STXM neuromelanin imaging with silver nitrate‐stained neuromelanin. Analysis suggests that the 1s ‐ σ* (C‐S) transition in benzothiazine groups accounts for this feature. This advance in visualizing neuromelanin illustrates the wider potential of STXM as a label‐free spectromicroscopy technique applicable to both organic and inorganic materials.
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Mar 2020
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I18-Microfocus Spectroscopy
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Open Access
Abstract: Iron is an essential element, and cornflake-style cereals are typically fortified with iron to a level up to 14 mg iron per 100 g. Even single cornflakes exhibit magnetic behaviour. We extracted iron microparticles from samples of two own-brand supermarket cornflakes using a strong permanent magnet. Synchrotron iron K-edge X-ray absorption near-edge spectroscopic data were consistent with identification as metallic iron, and X-ray diffraction studies provided unequivocal identification of the extracted iron as body-centred cubic (BCC) α-iron. Magnetometry measurements were also consistent with ca. 14 mg per 100 g BCC iron. These findings emphasise that attention must be paid to the speciation of trace elements, in relation to their bioavailability. To mimic conditions in the stomach, we suspended the iron extract in dilute HCl (pH 1.0–2.0) at 310 K (body temperature) and found by ICP-MS that over a period of 5 hours, up to 13% of the iron dissolved. This implies that despite its metallic form in the cornflakes, the iron is potentially bioavailable for oxidation and absorption into the body.
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Mar 2020
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