In vitro and in vivo studies of new photoluminescent oxygen sensors for non-invasive intravascular pO 2 measurements

The concentration of oxygen and its rate of consumption are important factors playing a role in PDT and radiotherapy. One of the methods for measuring the tissular oxygen partial pressure (pO 2) is based on the use of luminophores presenting an oxygen-dependent quenching of their phosphorescence. The time-resolved luminescence spectroscopy of palladium (PdTCPP) or ruthenium (RuDPP) porphyrin complexes is used for this purpose. Unfortunately, these porphyrin derivatives are phototoxic and leak rapidly out of the blood vessels, making them unsuitable for measuring tissular and or intravascular pO 2. Therefore, this research aimed at developing and testing new biocompatible, non-phototoxic oxygen sensors based on palladium complexes incorporated into oxygen permeable, polysaccharide-based nanoparticles appropriate for noninvasive in situ and in vivo measurements of the pO 2. In vitro studies, performed with an optical fiber-based time-resolved spectrophotometer, showed that the incorporation of such pO 2 probes in nanovectors reduces their sensitivity to oxygen as well as their photobleaching by less than one order of magnitude. However, in vivo biocompatibility studies performed on the chick's embryo chorioallantoic membrane (CAM) model demonstrated that the luminescence of those oxygen probes tends to be heterogeneously distributed within the vasculature. In addition, these probes induce a 'clumping tendency', resulting in a more or less decreased viability of the embryos. © 2009 Copyright SPIE - The International Society for Optical Engineering.

Effect of PpIX photoproducts formation on pO2 measurement by time-resolved delayed fluorescence spectroscopy of PpIX in solution and in vivo

Emmanuel Louis Arthur Gerelli, Veronika Huntosova, Georges Wagnières, Matthieu Zellweger

The measurement of Protoporphyrin IX delayed fluorescence lifetime is a minimally invasive method for monitoring the levels of oxygen in cells and tissues. The excitation of Protoporphyrin IX during this measurement can lead to the formation of photoproducts in vitro and in vivo. The influence of their luminescence on the measured Protoporphyrin IX delayed fluorescence lifetimes was studied in solution and in vivo on the Chick's chorioallantoic membrane (CAM) model under various oxygen enriched air conditions (0mmHg, 37mmHg and 155mmHg). The presence of photoproducts disturbs such measurements since the delayed fluorescence emission of some of them spectrally overlaps with that of Protoporphyrin IX. One possible way to avoid this obstacle is to detect Protoporphyrin IX's delayed fluorescence lifetime in a very specific spectral range (620-640nm). Another possibility is to excite Protoporphyrin IX with light doses much lower than 10J/cm(2), quite possibly as low as a fraction 1J/cm(2) at 405nm. This leads to an increased accuracy of pO2 detection. Furthermore, this method allows combination of diagnosis and therapy in one step. This helps to improve detection systems and real-time identification of tissue respiration, which is tuned for the detection of PpIX luminescence and not its photoproducts.

Elsevier Science Sa2016

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

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

Veronika Huntosova, Georges Wagnières

Detection of tissue and cell oxygenation is of high importance in fundamental biological and in many medical applications, particularly for monitoring dysfunction in the early stages of cancer. Measurements of the luminescence lifetimes of molecular probes offer a very promising and non-invasive approach to estimate tissue and cell oxygenation in vivo and in vitro. We optimized the evaluation of oxygen detection in vivo by [Ru(Phen)3]2+ in the chicken embryo chorioallantoic membrane model. Its luminescence lifetimes measured in the CAM were analyzed through hierarchical clustering. The detection of the tissue oxygenation at the oxidative stress conditions is still challenging. We applied simultaneous time-resolved recording of the mitochondrial probe MitoTrackerTM OrangeCMTMRos fluorescence and [Ru(Phen)3]2+ phosphorescence imaging in the intact cell without affecting the sensitivities of these molecular probes. [Ru(Phen)3]2+ was demonstrated to be suitable for in vitro detection of oxygen under various stress factors that mimic oxidative stress: other molecular sensors, H2O2, and curcumin-mediated photodynamic therapy in glioma cancer cells. Low phototoxicities of the molecular probes were finally observed. Our study offers a high potential for the application and generalization of tissue oxygenation as an innovative approach based on the similarities between interdependent biological influences. It is particularly suitable for therapeutic approaches targeting metabolic alterations as well as oxygen, glucose, or lipid deprivation.

2021