In vivo measurement of tissue oxygenation by time-resolved luminescence spectroscopy: advantageous properties of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate

Measuring tissue oxygenation in vivo is of interest in fundamental biological as well as medical applications. One minimally invasive approach to assess the oxygen partial pressure in tissue (pO(2)) is to measure the oxygen-dependent luminescence lifetime of molecular probes. The relation between tissue pO(2) and the probes' luminescence lifetime is governed by the Stern-Volmer equation. Unfortunately, virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity. For that reason, we studied the oxygen sensitivity and phototoxicity of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate [Ru(Phen)] using a dedicated optical fiber-based, time-resolved spectrometer in the chicken embryo chorioallantoic membrane. We demonstrated that, after intravenous injection, Ru(Phen)'s luminescence lifetime presents an easily detectable pO(2) dependence at a low drug dose (1 mg/kg) and low fluence (120 mJ/cm(2) at 470 nm). The phototoxic threshold was found to be at 10 J/cm(2) with the same wavelength and drug dose, i.e., about two orders of magnitude larger than the fluence necessary to perform a pO(2) measurement. Finally, an illustrative application of this pO(2) measurement approach in a hypoxic tumor environment is presented. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)

Measurement of pO2 by luminescence lifetime spectroscopy: A comparative study of the phototoxicity and sensitivity of [Ru(Phen)3]2+ and PdTCPP in vivo

Emmanuel Louis Arthur Gerelli, Veronika Huntosova, Georges Wagnières

Dysfunctions in tissue metabolism can be detected at early stages by oxygen partial pressure (pO(2)) measurement. The measurement of emission lifetimes offers very promising and non-invasive approach to estimate pO(2) in vivo. This study compares two extensively used oxygen sensors and assesses their in vivo oxygen sensitivity and phototoxic effect. Luminescence lifetime of Ru-polypyridyl complex and of Pd-porphyrin is measured in the Chick's Chorioallantoic Membrane (CAM) model with a dedicated optical fiber-based, time-resolved spectrometer. The Pd-porphyrin luminescence lifetimes measured in the CAM model exposed to different pO(2) levels are longer and have a broader dynamic range (10-100 mu s) than those of Ru-polypyridyl complex (0.6-1 mu s). The combined statistical analysis based on an estimate of the kurtosis and skewness, bootstrapping method and routine normality tests is performed. The indicators of the averages and signal to noise ratio stability are also calculated. The combi-nation of several data processing allows selection of the better sensor for a given application. In particular, it is found that the advantage of Ru-polypyridyl complex over Pd-porphyrin is two-fold: i) Ru-polypyridyl complex datasets have consistently better statistical characteristics, ii) Ru-polypyridyl exhibits lower cytotoxicity.

Wiley-V C H Verlag Gmbh2016

In vivo measurement of the tissue oxygenation by time-resolved luminescence spectroscopy of protoporphyrin IX: Strategies to minimize artefacts associated with photoproducts

Emmanuel Louis Arthur Gerelli, Veronika Huntosova, Georges Wagnières

The determination of the oxygen partial pressure (pO(2)) in real time in living biological tissues is of high interest for numerous therapeutics, including photodynamic therapy (PDT) and radiotherapy. The minimally invasive and real-time measurement of the pO(2) also enables to obtain interesting fundamental information regarding the metabolic activities in cells and tissues. The development of time-resolved luminescence measurement (TRLM) methods combined with the availability of new oxygen-sensitive molecular probes is at the origin of the significant progress that have been achieved during these past decades to measure the pO(2) in living organisms. These probes include porphyrins, such as aminolevulinic acid-induced protoporphyrin IX (PPIX), which is an approved photosensitizer. Using the photosensitizer to probe the pO(2) is of high interest in PDT since the level of oxygen is measured at the precise location where the phototoxic mechanisms take place. However, PPIX has drawbacks to measure the pO(2) by TRLM, including its significant photobleaching. Since the PPIX excitation during pO(2) measurements leads to the generation of its photoproducts, we studied the impact of their luminescence on the measurement of the PPIX triplet state lifetime in solution and in vivo on the Chick's Chorioallantoic Membrane (CAM) model. We performed this study under various oxygen conditions. Our results indicate that perturbations induced by these photoproducts can be avoided if the PPIX luminescence is detected between 620 and 640 nm, or if PPIX is excited at 405 nm with light doses < 1 J/cm(2).

Spie-Int Soc Optical Engineering2017

Endosomes: Guardians Against [Ru(Phen)3]2+ Photo-action In Endothelial Cells During In Vivo pO2 Detection?

Veronika Huntosova, Georges Wagnières

Phototoxicity is a side-effect of in vitro and in vivo oxygen partial pressure (pO(2)) detection by luminescence lifetime measurement methods. Dichlorotris(1,10-phenanthroline)-ruthenium(II) hydrate (Ru(Phen)(3)) is a water soluble pO(2) probe associated with low phototoxicity, which we investigated in vivo in the chick's chorioallantoic membrane (CAM) after intravenous or topical administration and in vitro in normal human coronary artery endothelial cells (HCAEC). In vivo, the level of intravenously injected Ru(Phen)(3) decreases within several minutes, whereas the maximum of its biodistribution is observed during the first 2 h after topical application. Both routes are followed by convergence to almost identical "intra/extra-vascular" levels of Ru(Phen)(3). In vitro, we observed that Ru(Phen)(3) enters cells via endocytosis and is then redistributed. None of the studied conditions induced modification of lysosomal or mitochondrial membranes without illumination. No nuclear accumulation was observed. Without illumination Ru(Phen)(3) induces changes in endoplasmic reticulum (ER)-to-Golgi transport. The phototoxic effect of Ru(Phen)(3) leads to more marked ultrastructural changes than administration of Ru(Phen)(3) only (in the dark). These could lead to disruption of Ca2+ homeostasis accompanied by mitochondrial changes or to changes in secretory pathways. In conclusion, we have demonstrated that the intravenous injection of Ru(Phen)(3) into the CAM model mostly leads to extracellular localization of Ru(Phen)(3), while its topical application induces intracellular localization. We have shown in vivo that Ru(Phen)(3) induces minimal photo-damage after illumination with light doses larger by two orders of magnitude than those used for pO(2) measurements. This low phototoxicity is due to the fact that Ru(Phen)(3) enters endothelial cells via endocytosis and is then redistributed towards peroxisomes and other endosomal and secretory vesicles before it is eliminated via exocytosis. Cellular response to Ru(Phen)(3), survival or death, depends on its intracellular concentration and oxidation-reduction properties.

Royal Soc Chemistry2014

Endosomes: Guardians Against [Ru(Phen)3]2+ Photo-action In Endothelial Cells During In Vivo pO2 Detection?

Veronika Huntosova, Georges Wagnières

Royal Soc Chemistry2014