85HIGHLIGHTS 2020

Mapping the 3D orientation of nanocrystals and nanostructures in human bone: indications of novel structural features, T. A. Grünewald (a), M. Liebi (b), N.K. Wittig (c), A. Johannes (a), T. Sikjaer (d), L. Rejnmark (d), M. Rosenthal (a),

M. Guizar-Sicairos (e), H. Birkedal (c) and M. Burghammer (a), Sci. Adv. 6, eaba4171 (2020); https://doi.org/10.1126/sciadv. aba4171. (a) ESRF (b) Chalmers University of Technology,

Gothenburg (Sweden) (c) Department of Chemistry and iNANO, Aarhus University (Denmark) (d) Aarhus University Hospital (Denmark) (e) Paul Scherrer Institut, Villigen (Switzerland)

[1] N.K. Wittig et al., ACS Nano 13, 12949-12956 (2019). [2] M. Liebi et al., Nature 527, 349-352 (2015).

CORRELATIVE 3D CRYO X-RAY IMAGING FOR SUBCELLULAR LOCATION OF A HIGHLY POTENT ORGANO-IRIDIUM ANTICANCER COMPOUND

Iridium complexes are emerging as potent cytotoxic drugs for cancer treatment. A potent iridium compound has been designed, whose intracellular fate and concentration within whole hydrated cells was determined by a new nanometric 3D cryo X-ray correlative approximation, using cryo soft X-ray tomography and cryo hard X-ray fluorescence tomography.

PRINCIPAL PUBLICATION AND AUTHORS

REFERENCES

Iridium compounds have emerged as some of the most potent metal-based drug candidates, acting by a mechanism likely to involve disruption of the intracellular redox balance. In the search to design more potent and selective metallodrugs, a new family of highly cytotoxic Ir(III)-C5Me5 derivatives [1] has been created. The extraordinary structural feature of these compounds resides in bearing a metal centre sheltered by two chelate rings, as shown in compound ACC25 in Figure 69.

Cell viability assays showed that ACC25 exhibits high potency towards all human cancer cell lines tested (ovarian, colon and breast) in the submicromolar range (0.11 to 0.82 µM). Cytotoxicity is as high as 247 times that of cisplatin. The compound was highly internalised, especially in MCF7 breast cancer cells, which relates to its high cytotoxicity.

Intracellular drug localisation and quantification is a mandatory step to improve drug design. A correlative approach that provides information about cell ultrastructure and metallodrug localisation and quantification is ideal to unambiguously determine the drug s intracellular fate. A new three-dimensional (3D) correlative approximation has been developed, combining cryo soft X-ray tomography (cryo-SXT) and cryo hard X-ray fluorescence (cryo-XRF). Cryo-SXT at ALBA synchrotron provided the 3D cell ultrastructure in its close-to-native state, avoiding the use of chemical fixation artefacts, and was capable of providing volumes at spatial resolutions of 30 nm in whole cells, allowing for the identification of subcellular compartments [2].

Cryo-XRF nanoimaging performed at ESRF beamline ID16A quantitatively traced the intracellular distribution of multiple elements at detection limits in the range of ppm in a label- free fashion and in the native state.

Using this correlative approach, the intracellular accumulation of the iridium compound was investigated. Cells were vitrified and imaged by cryo-epifluorescence microscopy and then transferred to the cryo-SXT microscope (Figure 70). Several cytomorphological regulated cell-death markers were found in treated cells, notably, swelling mitochondria with highly degenerated cristae. Samples were transferred to the cryo-XRF beamline to perform experiments at 17 keV energy. Analysis of cryo- XRF data on different elements showed that the ratios between mitochondrial and cytoplasmic Ca and K were higher in iridium-treated cells than in non-treated cells. The overlaying of the 3D distribution of the iridium signal with the

Fig. 69: a) X-ray crystal structure of iridium(III) cyclopentadienyl complex ACC25, of formula [Ir(h5:κ1-C5Me4CH2C5H4N)(phpy)]PF6 (phpy = 2-phenylpyridine;

Ir, orange; N, blue; C, grey). Thermal ellipsoids show 50% probability. Hydrogen atoms and counter ions (PF6-) have been omitted for clarity. b) Schematic

representation of ACC25 highlighting the two five-membered metal chelates.