Interaction of oxygen-sensitive luminescent probes Ru(phen)32+ and Ru(bipy)32+ with animal and plant cells in vitro: Mechanism of phototoxicity and conditions for non-invasive oxygen measurements

Understanding the role of oxygen in the physiology, pathophysiology and radio- and chemosensitivity of animal cells requires accurate and non-invasive measurements of oxygen concentrations in the range of 0–2×10−4 M, in cells in vitro or in vivo. High resolution 3D imaging techniques could be particularly useful in investigating tissue oxygenation in vivo and in model tissues (multicellular spheroids) in vitro. The goals of this work were to develop microscopy techniques and (i) to define conditions under which two oxygen-sensitive luminescent dyes, Ru(bipy)32+ (tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate) and Ru(phen)32+ (tris(1,10-phenanthroline)ruthenium(II) chloride hydrate) can be used to probe oxygen concentrations within viable cells in vitro, when no phototoxic effects are evident, and (ii) to investigate the mechanism of phototoxicity once cell damage occurs. This report demonstrates that Ru(bipy)32+ and Ru(phen)32+ do not pass through intact biological membranes, do not cause measurable photodamage to plasma membranes at a concentration of 0.2 mM and, when loaded into endosomes, yield a strong luminescent signal. However, at an extracellular concentration of 1 mM, in the presence of 457-nm light, detectable amounts of both complexes accumulate at the plasma membrane and cause a loss of membrane integrity via a mechanism which may involve the generation of singlet oxygen.