Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors and normal tissues in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of 2H,15N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with L-buthionine sulfoximine (BSO) markedly altered the kinetic images.
This Feature overviews the basic principles of using stable organic radicals involved in reversible exchange processes as functional paramagnetic probes. We demonstrate that these probes in combination with electron paramagnetic resonance (EPR)-based spectroscopy and imaging techniques provide analytical tools for quantitative mapping of critical parameters of local chemical microenvironment. The Feature is written to be understandable to people who are laymen to the EPR field in anticipation of future progress and broad application of these tools in biological systems, especially in vivo, over the next years.
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 1H,14N- and 2H,15N-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.
X-band rapid-scan EPR was implemented on a commercially available Bruker ELEXSYS E580 spectrometer. Room temperature rapid-scan and continuous-wave EPR spectra were recorded for hydrogenated amorphous silicon powder samples. By comparing the resulting signal intensities the feasibility of performing quantitative rapid-scan EPR is demonstrated. For different hydrogenated amorphous silicon samples, rapid-scan EPR results in signal-to-noise improvements by factors between 10 and 50. Rapid-scan EPR is thus capable of improving the detection limit of quantitative EPR by at least one order of magnitude. In addition, we provide a recipe for setting up and calibrating a conventional pulsed and continuous-wave EPR spectrometer for rapid-scan EPR.
Stable tetrathiatriarylmethyl
radicals have significantly contributed to the recent progress in
biomedical electron paramagnetic resonance (EPR) due to their unmatched
stability in biological media and long relaxation times. However,
the lipophilic core of the most commonly used structure (Finland trityl)
is responsible for its interaction with plasma biomacromolecules,
such as albumin, and self-aggregation at high concentrations and/or
low pH. While Finland trityl is generally considered inert toward
many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl
with a high rate constant of 3.53 × 108 M–1 s–1, leading to a trisulfonated Finland trityl
radical. This newly synthesized highly hydrophilic trityl radical
shows an ultranarrow linewidth (ΔB
pp = 24 mG), a lower affinity for albumin than Finland trityl, and
a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl
radical can be considered as a superior spin probe in comparison to
the widely used Finland trityl. One of its potential applications
was demonstrated by in vivo mapping oxygen in a mouse
model of breast cancer. Moreover, we showed that one of the three
sulfo groups can be easily substituted with S-, N-, and P-nucleophiles,
opening access to various monofunctionalized sulfonated trityl radicals.
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