Photosensitized Damage Inflicted on Plasma Membranes of Live Cells by An Extracellular Generator of Singlet Oxygen—A Linear Dependence of A Lethal Dose on Light Intensity

Abstract

We describe a study of the influence of a dose rate, i.e. light intensity or photon flux, on the efficiency of induction of a loss of integrity of plasma membranes of live cells in culture. The influence of a photon flux on the size of the light dose, which was capable of causing lethal effects, was measured in an experimental system where singlet oxygen was generated exclusively outside of live cells by ruthenium(II) phenantroline complex. Instantaneous, sensitive detection of a loss of integrity of a plasma membrane was achieved by fluorescence confocal imaging of the entry of this complex into a cell interior. We demonstrate that the size of the lethal dose of light is directly proportional to the intensity of the exciting light. Thus, the probability of a photon of the exciting light inflicting photosensitized damage on plasma membranes diminishes with increasing density of the incident photons.

Temperature and oxygen-concentration dependence of singlet oxygen production by RuPhen as induced by quasi-continuous excitation

“…This value is not negligible, and thus singlet oxygen production has to be considered when thinking of the possible mechanisms of RuPhen phototoxicity. 30 On the other hand, our preliminary results obtained with the CAM, 29 and the results of Dobrucki 28 and Asiedu et al 27 indicate that under certain conditions (RuPhen concentrations below 1 mg kg −1 of body weight and light doses of less than 10 J cm −2 at 470 nm) the photo-toxicity of RuPhen is very low. Logically, the damage induced by photo-excited RuPhen will depend on the concentration used.…”

Endosomes: guardians against [Ru(Phen)3]2+photo-action in endothelial cells during in vivo pO2detection?

“…We chose to avoid illumination on the spot of topical [Ru(Phen) 3 ] 2+ application because high [Ru(Phen) 3 ] 2+ concentrations applied on the CAM would induce extracellular membrane photo-degradation. 29,36 Furthermore, we expected more important photo-damage 2 h post intravenous [Ru(Phen) 3 ] 2+ application, since higher pO 2 is needed to effectively complete PDT. This could be the reason for explaining the reduced effect observed after topical [Ru(Phen) 3 ] 2+ application, as reported in Fig.…”

“…For this purpose we have chosen dichlorotris(1,10-phenanthroline)-ruthenium(II) hydrate ([Ru(Phen) 3 ] 2+ ). This compound has been used for pO 2 detection in vitro and in vivo 28,29,[36][37][38][39][40] for many years and it was shown to be much less cytotoxic (without illumination) than other RuPCs in several cell lines. 34,41 Our study clarifies controversies regarding the phototoxicity of [Ru(Phen) 3 ] 2+ , which has been observed upon extracellular photo-action in vitro 29,36 and in vivo.…”

“…This compound has been used for pO 2 detection in vitro and in vivo 28,29,[36][37][38][39][40] for many years and it was shown to be much less cytotoxic (without illumination) than other RuPCs in several cell lines. 34,41 Our study clarifies controversies regarding the phototoxicity of [Ru(Phen) 3 ] 2+ , which has been observed upon extracellular photo-action in vitro 29,36 and in vivo. 40 Our previous results, which are in agreement with reports published by other groups, showed that the photoactivation of [Ru(Phen) 3 ] 2+ may result in singlet oxygen production high enough to induce phototoxicity (with limited temperature-dependence), depending on the properties of its surrounding.…”

Influence of Oxidative Stress on Time-Resolved Oxygen Detection by [Ru(Phen)3]2+ In Vivo and In Vitro

“…It takes a long time (24 h) for [Ru(Phen) 3 ] 2+ to accumulate in cells [7]. However, straight nuclear localization of [Ru(Phen) 3 ] 2+ was observed in damaged cells due to the increased permeability of their membrane [7,9]. Interaction with DNA increases the [Ru(Phen) 3 ] 2+ luminescence intensity as well as its luminescence lifetimes [10,11].…”