Noninvasive detection of the endogenous free radical melanin in human skin melanomas using electron paramagnetic resonance (EPR).

We explored the capability of low-frequency Electron Paramagnetic Resonance (EPR) to noninvasively detect melanin (a stable semiquinone free radical) in the human skin. As previous in vitro studies on biopsies suggested that the EPR signal from melanin was different when measured in skin melanomas or benign nevi, we conducted a prospective first-in-man clinical EPR study in patients with skin lesions suspicious of melanoma. EPR spectra were obtained using a spectrometer operating at 1 GHz, with a surface coil placed over the area of interest. Two clinical studies were carried out: 1) healthy volunteers (n = 45) presenting different skin phototypes; 2) patients (n = 88) with skin lesions suspicious of melanoma (n = 100) requiring surgical resection. EPR data obtained before surgery were compared with histopathology results. The method was not sensitive enough to measure differences in melanin content due to changes in skin pigmentation. In patients, 92% of the spectra were analyzable. The EPR signal of melanin was significantly higher (p < 0.0001) in melanoma lesions (n = 26) than that in benign atypical nevi (n = 62). A trend toward a higher signal intensity (though not significant) was observed in high Breslow depth melanomas (a marker of skin invasion) than in low Breslow lesions. To date, no naturally occurring free radicals have been detected by low-frequency EPR systems adapted for clinical studies. Here, we demonstrated for the first time the ability of this technology to detect an endogenous free radical, opening new avenues for evaluating clinical EPR as a potential aid in the diagnosis of pigmented skin lesions.

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study.

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1 GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.

Preparation and evaluation of trityl-loaded lipid nanocapsules as oxygen sensors for electron paramagnetic resonance oximetry.

Oxygen is essential in physiology and pathophysiology. Electron paramagnetic resonance (EPR) oximetry, using oxygen sensitive paramagnetic materials, could be attractive for measuring oxygen in tissues. The aim of the present study was to assess the properties of lipid nanocapsules (LNCs) loaded with the nitroxide tempo-benzoate (TB) or tetrathiatriarylmethyl (TAM) radicals. LNCs loaded with the EPR probes were successfully prepared by the phase inversion process leading to nanocapsules of about 60 nm. LNCs protected the TB radical against reduction in vitro. The calibration of the EPR line width (LW) as a function of the pO2 showed a two-fold increase in sensitivity with TAM-LNC compared to hydrophilic trityl radical. The TAM-LNCs were evaluated in vivo. Contrarily to unencapsulated TAM, for which a rapid decrease in EPR signal was observed, the half-life of TAM-LNCs administered in muscles or in tumours exceeded an hour. Carbogen-challenges in mice demonstrated that the TAM-LNCs responded well to changes in oxygen environment. However, the apparent pO2 values acquired were higher than the expected physiological values. These results warrant further investigation in the formulation of stable nano-objects encapsulating EPR oxygen sensitive probes.

Nanomedicines and gene therapy for the delivery of growth factors to improve perfusion and oxygenation in wound healing.

Oxygen plays a key role in wound healing, and hypoxia is a major cause of wound healing impairment; therefore, treatments to improve hemodynamics and increase wound oxygenation are of particular interest for the treatment of chronic wounds. This article describes the roles of oxygen and angiogenesis in wound healing as well as the tools used to evaluate tissue oxygenation and perfusion and then presents a review of nanomedicines and gene therapies designed to improve perfusion and oxygenation and accelerate wound healing.

EPR monitoring of wound oxygenation as a biomarker of response to gene therapy encoding hCAP-18/LL37 peptide.

PURPOSE: To investigate the value of electron paramagnetic resonance oximetry to follow oxygenation in wounds treated by a plasmid-encoding host defense peptide hCAP-18/LL37. METHODS: Flaps were created on diabetic mice (7- or 12-week-old db/db mice) presenting different levels of microangiopathy. The hCAP-18/LL37-encoding plasmids were administered in wounds by electroporation. Low-frequency electron paramagnetic resonance oximetry using lithium phthalocyanine as the oxygen sensor was used to monitor wound oxygenation in flaps during the healing process. Flaps were analyzed by immunohistochemistry to assess hypoxia and cell proliferation. Kinetics of closure was also assessed in excisional skin wounds. RESULTS: A reoxygenation of the flap was observed during the healing process in the 7-week-old db/db treated mice, but not in the untreated mice and the 12-week-old mice. Histological studies demonstrated less hypoxic regions and higher proportion of proliferating cells in hCAP-18/LL37-treated flaps in the 7-week-old db/db treated mice compared with untreated mice. Consistently, the kinetics of excisional wound closure was improved by hCAP-18/LL37 treatment in the 7-week-old db/db but not in the 12-week-old mice. CONCLUSIONS: Oxygenation measured by electron paramagnetic resonance oximetry is a promising biomarker of response to treatments designed to modulate wound oxygenation. Magn Reson Med 79:3267-3273, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Factors Affecting the Quality of Tooth Enamel for In Vivo EPR-Based Retrospective Biodosimetry.

In vivo electron paramagnetic resonance biodosimetry on tooth enamel is likely to be an important technology for triage of overexposed individuals after a major radiological incident. The accuracy and robustness of the technique relies on various properties of the enamel such as the geometry of the tooth, the presence of restorations, whitening treatments or exposition to sunlight. Those factors are reviewed, and their influence on dosimetry specifically for triage purposes is discussed.

Application of Electron Paramagnetic Resonance (EPR) Oximetry to Monitor Oxygen in Wounds in Diabetic Models.

A lack of oxygen is classically described as a major cause of impaired wound healing in diabetic patients. Even if the role of oxygen in the wound healing process is well recognized, measurement of oxygen levels in a wound remains challenging. The purpose of the present study was to assess the value of electron paramagnetic resonance (EPR) oximetry to monitor pO2 in wounds during the healing process in diabetic mouse models. Kinetics of wound closure were carried out in streptozotocin (STZ)-treated and db/db mice. The pO2 was followed repeatedly during the healing process by 1 GHz EPR spectroscopy with lithium phthalocyanine (LiPc) crystals used as oxygen sensor in two different wound models: a full-thickness excisional skin wound and a pedicled skin flap. Wound closure kinetics were dramatically slower in 12-week-old db/db compared to control (db/+) mice, whereas kinetics were not statistically different in STZ-treated compared to control mice. At the center of excisional wounds, measurements were highly influenced by atmospheric oxygen early in the healing process. In pedicled flaps, hypoxia was observed early after wounding. While reoxygenation occurred over time in db/+ mice, hypoxia was prolonged in the diabetic db/db model. This observation was consistent with impaired healing and microangiopathies observed using intravital microscopy. In conclusion, EPR oximetry using LiPc crystals as the oxygen sensor is an appropriate technique to follow wound oxygenation in acute and chronic wounds, in normal and diabetic animals. Nevertheless, the technique is limited for measurements in pedicled skin flaps and cannot be applied to excisional wounds in which diffusion of atmospheric oxygen significantly affects the measurements.

Tooth Retrospective Dosimetry Using Electron Paramagnetic Resonance: Influence of Irradiated Dental Composites.

In the aftermath of a major radiological accident, the medical management of overexposed individuals will rely on the determination of the dose of ionizing radiations absorbed by the victims. Because people in the general population do not possess conventional dosimeters, after the fact dose reconstruction methods are needed. Free radicals are induced by radiations in the tooth enamel of victims, in direct proportion to dose, and can be quantified using Electron Paramagnetic Resonance (EPR) spectrometry, a technique that was demonstrated to be very appropriate for mass triage. The presence of dimethacrylate based restorations on teeth can interfere with the dosimetric signal from the enamel, as free radicals could also be induced in the various composites used. The aim of the present study was to screen irradiated composites for a possible radiation-induced EPR signal, to characterize it, and evaluate a possible interference with the dosimetric signal of the enamel. We investigated the most common commercial composites, and experimental compositions, for a possible class effect. The effect of the dose was studied between 10 Gy and 100 Gy using high sensitivity X-band spectrometer. The influence of this radiation-induced signal from the composite on the dosimetric signal of the enamel was also investigated using a clinical L-Band EPR spectrometer, specifically developed in the EPR center at Dartmouth College. In X-band, a radiation-induced signal was observed for high doses (25-100 Gy); it was rapidly decaying, and not detected after only 24 h post irradiation. At 10 Gy, the signal was in most cases not measurable in the commercial composites tested, with the exception of 3 composites showing a significant intensity. In L-band study, only one irradiated commercial composite influenced significantly the dosimetric signal of the tooth, with an overestimation about 30%. In conclusion, the presence of the radiation-induced signal from dental composites should not significantly influence the dosimetry for early dose assessment.