Nano-emulsions of fluorinated trityl radicals as sensors for EPR oximetry.

This article reports the development and evaluation of two nano-emulsions (F45T-03/HFB and F15T-03/PFOB) containing fluorinated trityl radicals dissolved in perfluorocarbons. Preparation with a high-pressure homogenizer conferred sub-micronic size to both nano-emulsions. In vitro and in vivo EPR spectroscopy showed that the nano-emulsions had much greater oxygen sensitivity than the hydrophilic trityl, CT-03. In vivo experiments in rodents confirmed the ability of the nano-emulsions to follow the changes in oxygen concentration after induced ischemia. Histological evaluation of the tissue injected with the nano-emulsions revealed some acute toxicity for the F45T-03/HFB nano-emulsion but none for the F15T-03/PFOB nano-emulsion. These new formulations should be considered for further EPR oximetry experiments in pathophysiological situations where subtle changes in tissue oxygenation are expected.

Retrievable micro-inserts containing oxygen sensors for monitoring tissue oxygenation using EPR oximetry.

Tissue oxygenation is a crucial parameter in various physiopathological situations and can influence the therapeutic response of tumours. EPR oximetry is a reliable method for assessing and monitoring oxygen levels in vivo over long periods of time. Among the different paramagnetic oxygen sensors available for EPR oximetry, lithium phthalocyanine (LiPc) is a serious candidate for in vivo applications because of its narrow linewidth and its high signal-to-noise ratio. To enhance the biocompatibility of the sensors, fluoropolymer Teflon AF2400 was used to make cylindrical micro-inserts containing LiPc crystals. This new micro-pellet design has several advantages for in vivo studies, including the possibility of being able to choose the implant size, a high sensor content, the facility of in vivo insertion and complete protection with preservation of the oxygen sensor's characteristics. The response to oxygen and the kinetics of this response were tested using in vivo EPR: no differences were observed between micro-inserts and uncoated LiPc crystals. Pellets implanted in vivo in muscles conserved their responsiveness over a long period of time (approximately two months), which is much longer than the few days of stability observed using LiPc crystals without protection by the implant. Finally, evaluation of the biocompatibility of the implants revealed no inflammatory reaction around the implantation area.

Characterization of self-assembling copolymers in aqueous solutions using Electron Paramagnetic Resonance and Fluorescence spectroscopy.

Electron Paramagnetic Resonance and fluorescence spectroscopy have been used to determine the micropolarity and microviscosity of self-assembling systems based on mmePEG-p(CL-co-TMC) having different PEG chain lengths and different CL/TMC ratios and PEG/MOG/SA (45/5/50) polymers with different PEG chain lengths. Four reporter probes have been used: two spin probes, 16-doxyl stearic acid and 5-doxylstearic acid, and two fluorescent probes, pyrene and 1,3-bis(1-pyrenyl) propane (P3P). We found that the micelles based on mmePEG-p(CL-co-TMC) polymers are of a biphasic nature. The micelles are made of a hydrophilic corona with low viscosity while the core of the micelle is more hydrophobic and more viscous. The outer shell is made up of PEG chains, the hydrophobic part of the chains making the core. The partial hydration of the shell seems to lead to a looser chain network than that associated with deeper domains in the micelles. By contrast, in micelles composed of PEG/MOG/SA, there is no clear domain separation. This is consistent with a spatial configuration of random polymeric chains forming a loose network. In these micelles, the microviscosity is low and the hydrophobicity is high.

Development and evaluation of biocompatible films of polytetrafluoroethylene polymers holding lithium phthalocyanine crystals for their use in EPR oximetry.

Electron paramagnetic resonance (EPR) oximetry is a powerful technology that allows the monitoring of oxygenation in tissues. The measurement of tissue oxygenation can be achieved using lithium phthalocyanine (LiPc) crystals as oxygen reporters. In order to have biocompatibility for the sensing system and to assure long-term stability in the responsiveness of the system, we developed films of Teflon AF 2400 with embedded LiPc crystals. These systems can be used as retrievable inserts or parts of an implantable resonator or catheter. Atomic force microscopy studies revealed that the surface of the films was regular and planar. The response to oxygen of the sensor (EPR linewidth as a function of pO(2)) remained unchanged after implantation in mice, and was not affected by sterilization or irradiation. The use of resonators, holding LiPc embedded in Teflon AF 2400, implanted in the gastrocnemius muscle of rabbits allowed the monitoring of oxygen during several weeks. Several assays also demonstrated the biocompatibility of the system: (1) no hemolytic effect was noted; (2) no toxicity was found using the systemic injection test of extracts; (3) histological analysis in rabbit muscle in which the films were implanted for 1 week or 3 months was similar to standard polyethylene biocompatible devices. These advanced oxygen sensors are promising tools for future pre-clinical and clinical developments of EPR oximetry. These developments can be applied for other applications of biosensors where there is a need for oxygen permeable membranes.

Non-invasive determination of the irradiation dose in fingers using low-frequency EPR.

Several reports in the literature have described the effects of radiation in workers who exposed their fingers to intense radioactive sources. The radiation injuries occurring after local exposure to a high dose (20 to 100 Gy) could lead to the need for amputation. Follow-up of victims needs to be more rational with a precise knowledge of the irradiated area that risks tissue degradation and necrosis. It has been described previously that X-band electron paramagnetic resonance (EPR) spectroscopy could be used to assess the dose in irradiated amputated fingers. Here, we propose the use of low-frequency EPR spectroscopy to evaluate non-invasively the absorbed dose. Low-frequency microwaves are indeed less absorbed by water and penetrate more deeply into living material (approximately 10 mm in tissues using 1 GHz spectrometers). This work presents preliminary results obtained with baboon and human fingers compared with human dry phalanxes placed inside a surface-coil resonator. The EPR signal increased linearly with the dose. The ratio of the slopes of the dry bone to whole finger linear regression lines was around 5. The detection limit achievable with the present spectrometer and resonator is around 60 Gy, which is well within the range of accidentally exposed fingers. It is likely that the detection limit could be improved in the future, thanks to further technical spectrometer and resonator developments as well as to appropriate spectrum deconvolution into native and dosimetric signals.

Removal of local field gradient artefacts in BOLD contrast imaging of head and neck tumours.

Monitoring of oxygenation in tumours is an important issue in predicting the success of anti-cancer treatments such as radiotherapy. Gradient echo (GE) imaging sequences can be used for monitoring changes in tumour blood flow and oxygenation. However, the application of this method in head and neck tumours is hampered by significant artefacts and losses of the MR signal near air-tissue interfaces. We investigated the usefulness of a gradient-echo slice excitation profile (GESEPI) sequence that should keep the oxygen contrast while recovering the signal loss caused by susceptibility artefacts. A tumour model was implanted in the neck and in the leg of mice. MR imaging was performed at 4.7 T. GE and GESEPI sequences were used for monitoring the blood oxygen level dependent (BOLD) contrast after carbogen breathing. The pO2 was also monitored in tumours using an OxyLite probe (Oxford Optronics). Using the tumours implanted in the leg, we found that the variations of signal intensity after carbogen breathing were similarin both sequences. In the tumour implanted in the neck, it was possible, using GESEPI sequences, to recover the signal loss caused by susceptibility artefacts and to monitor the effect of carbogen-induced changes in the tumour.

Development of biocompatible oxygen-permeable films holding paramagnetic carbon particles: evaluation of their performance and stability in EPR oximetry.

EPR oximetry using paramagnetic particles relies on the measurement of the EPR linewidth, which is directly related to the pO2. It was previously found that some of the paramagnetic materials with optimal EPR spectroscopic properties in vitro may lose their responsiveness to oxygen in tissues (change of the calibration curve of the EPR linewidth as a function of the pO2). We hypothesized that coating paramagnetic particle materials could improve the stability of response, as well as the biocompatibility. In this study, very thin films holding paramagnetic materials were prepared with different biopolymers (cellulose acetate, cellulose triacetate, cellulose nitrate, silicone, and polyurethane) that already are accepted for clinical applications. Their performance was evaluated in EPR oximetry by measuring the stability of the calibration curves (EPR linewidth as a function of pO2) after a prolonged period in an aqueous environment (1 week in saline) or in vivo (implantation for 3 weeks under the skin of mice). We found that one type of silicone film was able to stabilize the responsiveness of an intrinsically unstable carbon material (a wood char).

Non invasive quantification of manganese deposits in the rat brain by local measurement of NMR proton T1 relaxation times.

Up to now, there is no reliable non invasive biomarker for the concentration of manganese (Mn) in the brain after intoxication to this metal. The aim of the present experimental study was to determine the predictive value of the localized measurement of the proton NMR relaxation time T1 as a quantitative estimation of the concentration of Mn in brain. The relationship of the proton relaxation rates (1/T1) was established in rat brain homogenates as a function of the Mn, iron, and copper concentration. Subsequently, an experimental model of Mn neurotoxicity was used: rats were stereotactically injected with increasing amounts of Mn2+ (as MnCl2) in the ventricles. After 3 weeks, local measurements of T1 were carried out in live rats. They were then sacrificed in order to sample the striatum, the cortex, and the cerebellum from the brain and to perform a quantitative determination of the concentration of Mn in these tissues by atomic absorption spectrometry (AAS). The results indicate excellent correlation coefficients between relaxation rates and tissue Mn concentrations (r= 0.84, 0.77 and 0.92 for the striatum, the cortex and the cerebellum, respectively). This methodology offers a unique toolfor monitoring the degree of Mn concentration in different areas of the brain in animal models of Mn intoxication. It will be useful for evaluating the efficacy of treatments aimed at decreasing the metal in the brain. The method could be potentially useful for being transposed in the clinical situation for monitoring Mn-exposed workers.

Changes in tumor oxygenation/perfusion induced by the no donor, isosorbide dinitrate, in comparison with carbogen: monitoring by EPR and MRI.

PURPOSE: In an effort to improve radiotherapy treatments, methods aimed at increasing the quantity of oxygen delivered to tumors were investigated. The aim of this study was to evaluate the effect of one nitric oxide (NO) donor (isosorbide dinitrate) on pO(2) and blood flow in a murine tumor model. The effect was compared to carbogen, used as a reference treatment. METHODS AND MATERIALS: Thirty-six liver tumors implanted in mouse thighs were imaged using magnetic resonance imaging (MRI) at 4.7 Tesla with dynamic Gd-DTPA and blood oxygen level-dependent (BOLD) contrast-enhanced imaging after administration of isosorbide dinitrate or carbogen. The effect on the pO(2) was also tested by EPR oximetry (1.1 GHz) on 52 mice. RESULTS: A significant increase in MRI intensity was observed for both treatments in comparison with the control group. EPR oximetry showed a dose-dependant increase in tumor pO(2) for isosorbide dinitrate (by 5.9 mmHg at 0.2 mg/kg) and a substantially greater change for carbogen breathing (by 23 mmHg). CONCLUSION: Both tumor blood flow and pO(2) were increased by isosorbide dinitrate and carbogen. Carbogen is more efficient than isosorbide dinitrate in increasing the BOLD image intensity, as well as the tumor pO(2), but as efficient as isosorbide dinitrate in the Gd-DTPA contrast-enhanced imaging. We conclude that the effects of carbogen on improving tumor pO(2) involve both improved blood flow and improved hemoglobin oxygenation, whereas the effects of isosorbide dinitrate are predominantly mediated by improved blood flow alone.