Here, we examine the photophysical properties of five ruthenium(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and functionalized bipyridine (R₁bpy-R₂, where R₁ = H or CH₃, R₂ = H, CH₃, COO⁻,4-[3-(2-nitro-1H-imidazol-1-yl)propyl] or 1,3-dicyclohexyl-1-carbonyl-urea) towards development of luminescence probes for cellular imaging. These complexes have been shown to interact with albumin and the formed adducts exhibited up to eightfold increase in the luminescence quantum yield as well as the average lifetime of emission. It was demonstrated that they cannot bind to DNA through the intercalation mode and its luminescence in the presence of DNA is quenching. Cell viability experiments indicated that all complexes possess significant dose-dependent cytotoxicity (with IC₅₀ 5–19 μM) on 4T1 breast cancer cell line and their anti-proliferative activity correlates very well with their lipophilicity. Cellular uptake was studied by measuring the ruthenium content in cells using ICP-MS technique. As expected, the better uptake is directly related to higher lipophilicity of doubly charged ruthenium complexes while uptake of monocationic one is much lower in spite of the highest lipophilicity. Additionally staining properties were assessed using flow cytometry and fluorescence microscopy. These experiments showed that complex with 1,3-dicyclohexyl-1-carbonyl-urea substituent exhibits the best staining properties in spite of the lowest luminescence quantum yield in buffered solution (pH 7.4). Our results point out that both the imaging and cytotoxic properties of the studied ruthenium complexes are strongly influence by the level of internalization and protein interaction.

Hypoxia is one of the hallmarks of the tumor microenvironment and can be used in the design of targeted therapies. Cellular adaptation to hypoxic stress is regulated by hypoxia-inducible factor 1 (HIF-1). Hypoxia is responsible for the modification of cellular metabolism that can result in the development of more aggressive tumor phenotypes. Reduced oxygen concentration in hypoxic tumor cells leads to an increase in oxidoreductase activity that, in turn, leads to the activation of hypoxia-activated prodrugs (HAPs). The same conditions can convert a non-fluorescent compound into a fluorescent one (fluorescent turn off–on probes), and such probes can be designed to specifically image hypoxic cancer cells. This review focuses on the current knowledge about the expression and activity of oxidoreductases, which are relevant in the activation of HAPs and fluorescent imaging probes. The current clinical status of HAPs, their limitations, and ways to improve their efficacy are briefly discussed. The fluorescence probes triggered by reduction with specific oxidoreductase are briefly presented, with particular emphasis placed on those for which the correlation between the signal and enzyme expression determined with biochemical methods is achievable.