Synthesis,structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthesized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated. The compounds are potential candidates for targeted spin probes in EPR imaging.     

Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging

Tetrathiatriarylmethyl radicals are ideal spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of affordable large-scale syntheses. We report the large-scale synthesis of the Finland trityl radical using an improved addition protocol of the aryl lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivo applications.     

Electron paramagnetic resonance imaging of tumor pO₂

Electron paramagnetic resonance imaging (EPRI) can be used to noninvasively and quantitatively obtain three-dimensional maps of tumor pO?. The paramagnetic tracer triarylmethyl (TAM), a substituted trityl radical moiety, is not toxic to animals and provides narrow isotropic spectra, which is ideal for in vivo EPR imaging experiments. From the oxygen-induced spectral broadening of TAM, pO? maps can be derived using EPRI. The instrumentation consists of an EPRI spectrometer and 7T magnetic resonance imaging (MRI) system both operating at a common radiofrequency of 300 MHz. Anatomic images obtained by MRI can be overlaid with pO? maps obtained from EPRI. With imaging times of less than 3 min, it was possible to monitor the dynamics of oxygen changes in tumor and distinguish chronically hypoxic regions from acutely hypoxic regions. In this article, the principles of pO? imaging with EPR and some relevant examples of tumor imaging are reviewed.     

Application of a trityl-based radical probe for measuring superoxide

The use of triarylmethyl (trityl) free radical, TAM OX063, for detection of superoxide in aqueous solutions by electron paramagnetic resonance (EPR) spectroscopy was investigated. TAM is paramagnetic (EPR active), highly soluble in water and exhibits a single sharp EPR peak in aqueous media. It is also highly stable in presence of many oxidoreductants such as ascorbate and glutathione that are present in the biological systems. TAM reacts with superoxide with an apparent second order rate constant of 3.1 × 10³ M⁻¹ s⁻¹. The specific reactivity of TAM with superoxide, which leads to loss of EPR signal, was utilized to detect the generation of superoxide in various chemical (light/riboflavin/electron/donor), enzymatic (xanthine/xanthine oxidase), and cellular (stimulated neutrophils) model systems. The changes in the EPR line-width, induced by molecular oxygen, were utilized in the simultaneous determination of consumption of oxygen in the model systems. The effects of flux of superoxide and concentration of TAM on the efficiency of detection of superoxide were studied. The use of TAM for detection of superoxide offers unique advantages namely, (i) the utilization of very low concentration of the probe, (ii) its stability to bioreduction, and (iii) its use in the simultaneous determination of concentrations of superoxide and oxygen.     

In vivo imaging of free radicals: Applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spatial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

In vivo imaging of free radicals: applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spa-tial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

First combined in vivo X-ray tomography and high-resolution molecular electron paramagnetic resonance (EPR) imaging of the mouse knee joint taking into account the disappearance kinetics of the EPR probe

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 μm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.     

Injectable LiNc-BuO loaded microspheres as in vivo EPR oxygen sensors after co-implantation with tumor cells

Electron paramagnetic resonance (EPR) oximetry is a technique which allows accurate and repeatable oxygen measurements. We encapsulated a highly oxygen sensitive particulate EPR spin probe into microparticles to improve its dispersibility and, hence, facilitate the administration. These biocompatible, non-toxic microspheres contained 5–10 % (w/w) spin probe and had an oxygen sensitivity of 0.60±0.01 µT/mmHg. To evaluate the performance of the microparticles as oxygen sensors, they were co-implanted with syngeneic tumor cells in 2 different rat strains. Thus, tissue injury was avoided and the microparticles were distributed all over the tumor tissue. Dynamic changes of the intratumoral oxygen partial pressure during inhalation of 8 %, 21 %, or 100 % oxygen were monitored in vivo by EPR spectroscopy and quantified. Values were verified in vivo by invasive fluorometric measurements using Oxylite probes and ex vivo by pimonidazole adduct accumulation. There were no hints that the tumor physiology or tissue oxygenation had been altered by the microparticles. Hence, these microprobes offer great potential as oxygen sensors in preclinical research, not only for EPR spectroscopy but also for EPR imaging. For instance, the assessment of tissue oxygenation during therapeutic interventions might help understanding pathophysiological processes and lead to an individualized treatment planning or the use of formulations with hypoxia triggered release of active agents.     

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.     

Comparison of different methods for measuring the superoxide radical by EPR spectroscopy in buffer,cell lysates and cells

Abstract

As superoxide anion is of keen interest in biomedical research, it is highly desirable to have a technique allowing its detection sensitively and specifically in biological media. If electron paramagnetic resonance (EPR) techniques and probes have been individually described in the literature, there is actually no comparison of these techniques in the same conditions that may help guiding researchers for selecting the most appropriate approach. The aim of the present study was to compare different EPR strategies in terms of sensitivity and specificity to detect superoxide (vs. hydroxyl radical). Three main classes of EPR probes were used, including paramagnetic superoxide scavengers (such as nitroxides TEMPOL and mitoTEMPO as well as trityl CT-03), a spin trap (DIPPMPO), and diamagnetic superoxide scavengers (such as cyclic hydroxylamines CMH and mitoTEMPO-H). We analysed the reactivity of the different probes in the presence of a constant production of superoxide or hydroxyl radical in buffers and in cell lysates. We also assessed the performances of the different probes to detect superoxide produced by RAW264.7 macrophages stimulated by phorbol 12-myristate 13-acetate. In our conditions and models, we found that nitroxides were not specific for superoxide. CT-03 was specific, but the sensitivity of detection was low. Comparatively, we found that nitrone DIPPMPO and cyclic hydroxylamine CMH were good candidates to sensitively and specifically detect superoxide in complex biological media, CMH offering the best sensitivity.     

Esterified trityl radicals as intracellular oxygen probes

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O₂ concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O₂ concentrations and the effects of menadione and KCN on the rates of O₂ consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O₂ concentration and consumption.     

Triarylmethyl-based biradical as a superoxide probe

Abstract

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM–TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7?±?0.2)?×?10³ M^?1 s^?1) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.     

A highly sensitive biocompatible spin probe for imaging of oxygen concentration in tissues

The development of an injectable probe formulation, consisting of perchlorotriphenylmethyl triester radical dissolved in hexafluorobenzene, for in vivo oximetry and imaging of oxygen concentration in tissues using electron paramagnetic resonance (EPR) imaging is reported. The probe was evaluated for its oxygen sensitivity, biostability, and distribution in a radiation-induced fibrosarcoma tumor transplanted into C3H mice. Some of the favorable features of the probe are: a single narrow EPR peak (anoxic linewidth, 41 microT), high solubility in hexafluorobenzene (>12 mM), large linewidth sensitivity to molecular oxygen ( approximately 1.8 microT/mmHg), good stability in tumor tissue (half-life: 3.3 h), absence of spin-spin broadening (up to 12 mM), and lack of power saturation effects (up to 200 mW). Three-dimensional spatial and spectral-spatial (spectroscopic) EPR imaging measurements were used to visualize the distribution of the probe, as well as to obtain spatially resolved pO(2) information in the mice tumor subjected to normoxic and hyperoxic treatments. The new probe should enable unique opportunities for measurement of the oxygen concentration in tumors using EPR methods.     

EPR Oximetry Sensor—Developing a TAM Derivative for In Vivo Studies

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1?mM PBS, pH?=?7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH?=?7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250?mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.     

Synthesis and characterization of a perchlorotriphenylmethyl (trityl) triester radical: a potential sensor for superoxide and oxygen in biological systems

Synthesis and characterization of an inert perchlorotriphenylmethyl triester radical, PTM-TE, are reported. PTM-TE was prepared by a facile 3-step synthesis using Friedel-Crafts reaction of tetrachlorobenzene with chloroform followed by ethoxycarbonylation and subsequent oxidation. PTM-TE is paramagnetic and exhibits a single sharp EPR spectrum. In solution, the EPR linewidth of PTM-TE is highly sensitive to the dissolved oxygen content, thus enabling accurate measurement of oxygen concentration (oximetry). In addition, the radical also shows high reactivity towards superoxide. The ester radical has the potential for use as a high-sensitive probe for determination of oxygen concentration and superoxide in biological systems.     

CW EPR and 9 GHz EPR imaging investigation of stable paramagnetic species and their antioxidant activities in dry shiitake mushroom (Lentinus edodes)

We investigated the antioxidant activities and locations of stable paramagnetic species in dry (or drying) shiitake mushroom (Lentinus edodes) using continuous wave (CW) electron paramagnetic resonance (EPR) and 9?GHz EPR imaging. CW 9?GHz EPR detected paramagnetic species (peak-to-peak linewidth (ΔH_pp)?=?0.57?mT) in the mushroom. Two-dimensional imaging of the sharp line using a 9?GHz EPR imager showed that the species were located in the cap and shortened stem portions of the mushroom. No other location of the species was found in the mushroom. However, radical locations and concentrations varied along the cap of the mushroom. The 9?GHz EPR imaging determined the exact location of stable paramagnetic species in the shiitake mushroom. Distilled water extracts of the pigmented cap surface and the inner cap of the mushroom showed similar antioxidant activities that reduced an aqueous solution of 0.1?mM 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. The present results suggest that the antioxidant activities of the edible mushroom extracts are much weaker than those of ascorbic acid. Thus, CW EPR and EPR imaging revealed the location and distribution of stable paramagnetic species and the antioxidant activities in the shiitake mushroom for the first time.     

EPR imaging and HPLC characterization of the pigment-based organic free radical in black soybean seeds

We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9?GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn^2+?species based on the g values and hyperfine structures. The signal from the stable radical was noted at g?≈?2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product.     

Cellular uptake of electron paramagnetic resonance imaging probes through endocytosis of liposomes

Electron paramagnetic resonance imaging (EPRI) allows detection and localization of paramagnetic spin probes in vivo and in real time. We have shown that nitroxide spin probes entrapped in the intracellular milieu can be imaged by EPRI. Therefore, with the development of a tumor-targetable vehicle that can efficiently deliver nitroxides into cells, it should be possible to use nitroxide spin probes to label and image cells in a tumor. In this study, we assess the potential of liposomes as a delivery vehicle for imaging probes. We demonstrate that liposomes can stably encapsulate nitroxides at very high concentrations (> 100 mM), at which nitroxides exhibit concentration-dependent quenching of their EPR signal—a process analogous to the quenching of fluorescent molecules. The encapsulating liposomes thus appear spectroscopically “dark”. When the liposomes are endocytosed and degraded by cells, the encapsulated nitroxides are liberated and diluted into the much larger intracellular volume. The consequent relief of quenching generates a robust intracellular nitroxide signal that can be imaged. We show that through endocytosis of nitroxide-loaded liposomes, CV1 cells can achieve intracellular nitroxide concentrations of ∼ 1 mM. By using tissue phantom models, we verify that this concentration is more than sufficient for in vivo EPR imaging.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

Abstract

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG_2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.