Nanoprobe XRF analysis reveals the intracellular distribution of organometallic rhenium complexes with different directing ligand

The intracellular localization of metal-based anticancer agents can provide important indications for their biological mode of action and intracellular targets. Here, we investigated the uptake, the intracellular distribution and cellular responses to three cytotoxic rhenium 3 complexes with different ligand environments in HeLa cells using cryogenic X-ray fluorescence (XRF) nanoanalysis combined with fluorescence microscopy. Despite their distinct ligand functionalities and expected targeting properties, all three Re complexes showed a similar intracellular distribution. The close agreement between fluorescence microscopy and XRF imaging of Re and Br supports the preservation of the spatial association of the complexes and their ligands following cellular uptake. Quantitative pixel-by-pixel XRF analysis at the single-cell level revealed elevated intracellular potassium levels after treatment, consistent with a global cellular stress response, but no local correlation between Re and K. In contrast, Zn showed a spatial correlation with Re, pointing to Zn-rich cellular structures as potential sites of interaction. The presented work characterizes the uptake of medicinally relevant metal-based drug candidates containing unnatural metals with different ligand spheres and correlates the data with the resulting endogeneous biometal levels and their localization. The observed Re–Zn association provides a basis for investigating potential Zn-associated targets of emerging metallodrugs.