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Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.

Measurement of thiol-disulfide redox status is crucial for characterization of tumor physiology. The electron paramagnetic resonance (EPR) spectra of disulfide-linked dinitroxides are readily distinguished from those of the corresponding monoradicals that are formed by cleavage of the disulfide linkage by free thiols. EPR spectra can thus be used to monitor the rate of cleavage and the thiol redox status. EPR spectra of (1)H,(14)N- and (2)H,(15)N-disulfide dinitroxides and the corresponding monoradicals resulting from cleavage by glutathione have been characterized at 250 MHz, 1.04 GHz, and 9 GHz and imaged by rapid-scan EPR at 250 MHz., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Projections for 2D spectral-spatial images were obtained by continuous wave and rapid-scan electron paramagnetic resonance using a bimodal cross-loop resonator at 251MHz. The phantom consisted of three 4mm tubes containing different (15)N,(2)H-substituted nitroxides. Rapid-scan and continuous wave images were obtained with 5min total acquisition times. For comparison, images also were obtained with 29s acquisition time for rapid scan and 15min for continuous wave. Relative to continuous wave projections obtained for the same data acquisition time, rapid-scan projections had significantly less low-frequency noise and substantially higher signal-to-noise at higher gradients. Because of the improved image quality for the same data acquisition time, linewidths could be determined more accurately from the rapid-scan images than from the continuous wave images., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Electron spin relaxation times at 295 K were measured at frequencies between 250 MHz and 34 GHz for perdeuterated 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (PDT) in five solvents with viscosities that result in tumbling correlation times, τR, between 4 and 50 ps and for three (14)N/(15)N pairs of nitroxides in water with τR between 9 and 19 ps. To test the impact of structure on relaxation three additional nitroxides with τR between 10 and 26 ps were studied. In this fast tumbling regime T2(-1)~T1(-1) at frequencies up to about 9 GHz. At 34 GHz T2(-1)>T1(-1) due to increased contributions to T2(-1) from incomplete motional averaging of g-anisotropy, and T2(-1)-T1(-1) is proportional to τR. The contribution to T1(-1) from spin rotation is independent of frequency and decreases as τR increases. Spin rotation dominates T1(-1) at 34 GHz for all τR studied, and at all frequencies studied for τR=4 ps. The contribution to T1(-1) from modulation of nitrogen hyperfine anisotropy increases as frequency decreases and as τR increases; it dominates at low frequencies for τR>~15 ps. The contribution from modulation of g anisotropy is significant only at 34 GHz. Inclusion of a thermally-activated process was required to account for the observation that for most of the radicals, T1(-1) was smaller at 250 MHz than at 1-2 GHz. The significant (15)N/(14)N isotope effect, the small H/D isotope effect, and the viscosity dependence of the magnitude of the contribution from the thermally-activated process suggest that it arises from intramolecular motions of the nitroxide ring that modulate the isotropic A values., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Electron spin relaxation times of perdeuterated tempone (PDT) 1 and of a nitronyl nitroxide (2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) 2 in aqueous solution at room temperature were measured by 2-pulse electron spin echo (T(2)) or 3-pulse inversion recovery (T(1)) in the frequency range of 250 MHz to 34 GHz. At 9 GHz values of T(1) measured by long-pulse saturation recovery were in good agreement with values determined by inversion recovery. Below 9 GHz for 1 and below 1.5 GHz for 2,T(1)~T(2), as expected in the fast tumbling regime. At higher frequencies T(2) was shorter than T(1) due to incomplete motional averaging of g and A anisotropy. The frequency dependence of 1/T(1) is modeled as the sum of spin rotation, modulation of g and A-anisotropy, and a thermally-activated process that has maximum contribution at about 1.5 GHz. The spin lattice relaxation times for the nitronyl nitroxide were longer than for PDT by a factor of about 2 at 34 GHz, decreasing to about a factor of 1.5 at 250 MHz. The rotational correlation times, τ(R) are calculated to be 9 ps for 1 and about 25 ps for 2. The longer spin lattice relaxation times for 2 than for 1 at 9 and 34 GHz are due predominantly to smaller contributions from spin rotation that arise from slower tumbling. The smaller nitrogen hyperfine couplings for the nitronyl 2 than for 1 decrease the contribution to relaxation due to modulation of A anisotropy. However, at lower frequencies the slower tumbling of 2 results in a larger value of ωτ(R) (ω is the resonance frequency) and larger values of the spectral density function, which enhances the contribution from modulation of anisotropic interactions for 2 to a greater extent than for 1., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Optimization of nitroxides as probes for EPR imaging requires detailed understanding of spectral properties. Spin lattice relaxation times, spin packet line widths, nuclear hyperfine splitting, and overall lineshapes were characterized for six low molecular weight nitroxides in dilute deoxygenated aqueous solution at X-band. The nitroxides included 6-member, unsaturated 5-member, or saturated 5-member rings, most of which were isotopically labeled. The spectra are near the fast tumbling limit with T(1)∼T(2) in the range of 0.50-1.1 μs at ambient temperature. Both spin-lattice relaxation T(1) and spin-spin relaxation T(2) are longer for (15)N- than for (14)N-nitroxides. The dominant contributions to T(1) are modulation of nitrogen hyperfine anisotropy and spin rotation. Dependence of T(1) on nitrogen nuclear spin state m(I) was observed for both (14)N and (15)N. Unresolved hydrogen/deuterium hyperfine couplings dominate overall line widths. Lineshapes were simulated by including all nuclear hyperfine couplings and spin packet line widths that agreed with values obtained by electron spin echo. Line widths and relaxation times are predicted to be about the same at 250 MHz as at X-band., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 μM nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals.

In vivo quantitation of O(2) in brain has been hindered by a lack of suitable imaging modalities. Development of low-frequency electron paramagnetic resonance (EPR) spectrometers that can detect free radicals in animals in real time makes it feasible to image paramagnetic oximetry probes such as nitroxides in brain tissue. We have shown that masking the carboxyl group of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 1) as an esterase-labile acetoxymethyl ester yields 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 2). Nitroxide 2 can cross the blood-brain barrier and is then hydrolyzed in situ by esterases to regenerate nitroxide 1, which becomes entrapped in brain tissue. Seeking to improve the loading of nitroxides into brain, we synthesized the more lipophilic pentanoyloxymethyl ester, 3-pentanoyloxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 3). We report that the higher lipophilicity of nitroxide 3 does not significantly increase its ability to generate EPR signals in the mouse brain. Therefore, irrespective of whether nitroxide 2 or 3 was injected, similar levels of nitroxide were entrapped in brain tissue. These findings suggest that nitroxides 2 and 3 perform comparably well as proimaging agents for measuring O(2) distribution in brain., ((c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association)

Nitroxides with narrow linewidths are essential for low-frequency EPR spectroscopy and in vivo EPR imaging. In developing a framework for designing narrow-line nitroxides, we sought to understand the unexpectedly narrow line width of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxyl (5). Computational modeling revealed that the carbonyl double bond in the 4-position allows conformational diversity that results in the observed narrowing of the EPR spectral line. In view of this finding, we synthesized two new nitroxides bearing an exocyclic double bond: 4-methoxycarbonylmethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl (7) and 4-acetoxymethoxycarbonylmethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl (9). These nitroxides, like nitroxide 5, exhibited narrow linewidths-consistent with the results of modeling. Nitroxide 8 (4-carboxymethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl), as a prototype, allows for a variety of structural diversity, such as nitroxide 9,that can, for instance, target tissue compartments for in vivo EPR imaging.

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.

Measurement of O(2) concentration and distribution in brain is essential to understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that can potentially map O(2) concentration in the brain. In a previous study, we demonstrated that, after intraperitoneal administration of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (1) to mice, this nitroxide crossed the blood-brain barrier into brain tissue where, after hydrolysis, 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (2) was liberated and entrapped. This pilot study suggested that nitroxide 1 is a proimaging agent that can deliver nitroxide 2 to brain tissue, where O(2) levels can be estimated. In the present study, we conducted a series of pharmacokinetic and pharmacodynamic experiments designed to assess the uptake of structurally disparate nitroxides into brain tissue and retention, after hydrolysis, of the anions of the corresponding nitroxide acids. From these findings, nitroxide 1 and trans-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (5) meet the requirement as EPR proimaging agents for mapping O(2) distribution in the brain following stroke.

Measurement of oxygen concentration and distribution in the brain is essential for understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that potentially can be used to map O(2) concentration in the brain. We examined two nitroxides, 3-methoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [2] and 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [3], as pro-imaging agents to deliver 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] across the blood-brain barrier (BBB). In primary cultured neurons, nitroxide [3] but not [2] was hydrolyzed by intracellular esterases to [1], which, being anionic at physiologic pH, was well retained intracellularly. In contrast, [2] was not well retained by neurons. In vivo pharmacokinetic and pharmacodynamic studies in mice suggested that esterase-labile nitroxide [3] crossed the BBB, and was converted to [1] and retained. Retention occurred in brain tissue and not in the extensive vasculature, as evidenced by the fact that removal of blood by whole-body saline perfusion did not eliminate the nitroxide EPR signal from the brain. The EPR linewidths of [1] and [3] were more O(2)-sensitive than that of the commonly-used oximetry probe 4-oxo-2,2,6,6-tetramethylpiperidine-d(16)-1-(15)N-oxyl [4]. Moreover, we used [3] in vivo to estimate O(2) concentration in mouse brains. These results indicate that nitroxide [3] could be useful for mapping O(2) distribution in the brain following stroke., (Copyright 2006 Wiley-Liss, Inc.)

Given that spin trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for spin trapping O(2)(*-) compared to the well-studied spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for spin trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known spin traps were also undertaken.

Hyperthermia, as an adjuvant with radiation and chemotherapy, has shown promise in the treatment of cancer. The relevant biological effects of a hyperthermia treatment are both time and temperature-dependent, creating a need for accurate thermometry. We present a novel noninvasive thermometry modality that combines a temperature responsive biopolymer, the elastin-like polypeptide (ELP), and nitroxide to produce an ELP-nitroxide conjugate. When examined with electron paramagnetic resonance (EPR) spectroscopy, the ELP-nitroxide conjugate has temperature-dependent spectral line widths whose predictive accuracy is approximately 0.3 degrees C (80 microM). We believe that the temperature-dependent changes observed in the EPR spectrum are due to the combined effect of temperature, viscosity and effective radius on the rotational correlation time of the ELP-nitroxide conjugate.

The relaxivity and bioreduction rates of eight dendrimer-linked nitroxides varying in the number of nitroxides per molecule were measured and the potential use of these compounds as MR contrast agents was demonstrated. The T(1) and T(2) relaxivities, measured at room temperature and 1.5 T, varied linearly with the number of nitroxides per molecule for compounds with up to 16 nitroxides per molecule. Fourth-generation polypropylenimide- (DAB) and third-generation polyamidoamine- (PAMAM) dendrimer-linked nitroxides were found to have greater relaxivity than gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). The greater number of nitroxides per dendrimer increased relaxivity over that of a single nitroxide, allowing a decreased dose to achieve differential contrast with MR evaluations. Rates of nitroxide bioreduction were below detection threshold using EPR spectroscopy for generation 2 dendrimers and higher. A pilot assessment of in vivo cartilage uptake that compared intraarticular injection of three structurally different dendrimer-linked nitroxides with Gd-DTPA and with saline demonstrated high affinity of the DAB-dendrimer-linked nitroxides for normal rabbit articular cartilage. From these results, it is evident that target-specific dendrimer-linked nitroxides can be designed., (Copyright 2002 Wiley-Liss, Inc.)

Early degeneration of cartilage is accompanied by a loss of proteoglycans and consequent changes in the content of water. Conventional magnetic resonance imaging (MRI) cannot reliably detect this change, since the relaxation properties of the cartilage are dominated by its collagen content. The applicability of a positively charged nitroxide as an MRI contrast agent in detection of the content of the negatively charged proteoglycans within the cartilage was investigated. The results from both MRI and electron paramagnetic resonance (EPR) spectroscopy indicate that the accumulation of the contrast agent reflects the amount of proteoglycans within the cartilage, presumably due to the electrostatic interactions between the negatively charged proteoglycans and the positively charged nitroxide. Such a contrast agent could be useful in the detection and study of early stages of the degeneration of joints.

Central to the study of free radical processes is the ability to identify and localize their cellular site of formation. Under the best of experimental conditions, spin trapping/ESR spectroscopy can only characterize intracellular production of specific free radicals and confocal microscopy can only localize the site of their formation. In this article, we report on the development of a fluorophore-containing nitrone, alpha-[4-[5-((2-carboxy)phenyl)-5-hydroxy-4-oxo-3-phenyl)-2-pyrrolin+ -1-yl]phenyl]-N-(tert-butyl)nitrone sodium salt (4). This nitrone (4) reacts with alpha-hydroxyethyl radical with a second order rate constant of 1.7 x 10(5) M-1 s-1 to give a characteristic ESR spectrum. However, we were unable to decrease the fluorescence emission, due in part to the small concentration of nitroxide generated from the reaction of alpha-hydroxyethyl radical with nitrone (4). Using the fluorophore-containing nitroxide (7) as a model, we found that only 12% of the nitroxide needs to be reduced to give an almost 400% increase in the fluorescent emission of (7). Our findings suggest new approaches to the development of various fluorophore-containing nitrones that can both characterize specific free radicals and localize their site of intracellular formation.

Free radicals react with nitrones to form stable nitroxides which can be identified by ESR spectroscopy. Unfortunately, little is known regarding the pharmacological properties of these compounds. In this study, three commonly used nitrones, 5,5-dimethylpyrroline-N-oxide (DMPO), alpha-phenyl-tert-butylnitrone (PBN), and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), were found to induce relaxation of preconstricted isolated rat pulmonary artery rings. Additional experiments with PBN indicated that vasorelaxation could not be attributed to production of endothelial derived factors, prostaglandins, or free radicals. Patch-clamp techniques revealed reversible calcium channel blockade with PBN at a concentration below that needed to detect free radicals. Calcium channel blockade probably accounts for the vasorelaxation observed in the isolated ring preparations described here, and should be considered when using nitrone spin-traps both in in vivo and clinical studies.

The pharmacokinetics of two nitroxides were investigated in isolated rat hearts situated in a low-frequency electron spin resonance spectrometer. The spin labels 2,2,3,3,5,5-hexamethyl-1-pyrrolidinyloxy and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy were chosen for their physiochemical analogy to the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and its corresponding spin-trapped adduct, 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH). The bioreductive rates of the two nitroxides were measured during constant perfusion as well as during ischemia and are discussed in terms of a two-compartment pharmacokinetic model. These data provide information necessary to the design and application of spin traps to detect oxy radicals during reperfusion of ischemic tissue and suggest the feasibility of monitoring free-radical processes in intact, functioning mammalian tissues by using a low-frequency electron spin resonance spectrometer.