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Extracellular acidification indicates a metabolic shift in cancer cells and is, along with tissue hypoxia, a hallmark of tumor malignancy. Thus, non-invasive mapping of extracellular pH (pHe) is essential for researchers to understand the tumor microenvironment and to monitor tumor response to metabolism targeting drugs. While electron paramagnetic resonance (EPR) has been successfully used to map pHe in mouse xenograft models, this method is not sensitive enough to map pHe with a moderate amount of exogenous pH-sensitive probes. Here, we show that a modified EPR system achieves twofold higher sensitivity by using the multiple harmonic detection (MHD) method and improves the robustness of pHe mapping in mouse xenograft models. Our results demonstrate that treatment of a mouse xenograft model of human-derived pancreatic ductal adenocarcinoma cells with the carbonic anhydrase IX (CAIX) inhibitor U-104 delays tumor growth with a concurrent tendency toward further extracellular acidification. We anticipate that EPR-based pHe mapping can be expanded to monitor the response of other metabolism-targeting drugs. Furthermore, pHe monitoring can also be used for the development of improved metabolism-targeting cancer treatments.

Air pollution accounts for more than 7 million premature deaths worldwide. Using ultrafine carbon black (CB) and ozone (O3) as a model for an environmental co-exposure scenario, the dose response relationships in acute pulmonary injury and inflammation were determined by generating, characterizing, and comparing stable concentrations of CB aerosols (2.5, 5.0, 10.0 mg/m3), O3 (0.5, 1.0, 2.0 ppm) with mixture CB + O3 (2.5 + 0.5, 5.0 + 1.0, 10.0 + 2.0). C57BL6 male mice were exposed for 3 h by whole body inhalation and acute toxicity determined after 24 h. CB itself did not cause any alteration, however, a dose response in pulmonary injury/inflammation was observed with O3 and CB + O3. This increase in response with mixtures was not dependent on the uptake but was due to enhanced reactivity of the particles. Benchmark dose modeling showed several-fold increase in potency with CB + O3 compared with CB or O3 alone. Principal component analysis provided insight into response relationships between various doses and treatments. There was a significant correlation in lung responses with charge-based size distribution, total/alveolar deposition, oxidant generation, and antioxidant depletion potential. Lung tissue gene/protein response demonstrated distinct patterns that are better predicted by either particle dose/aerosol responses (interleukin-1β, keratinocyte chemoattractant, transforming growth factor beta) or particle reactivity (thymic stromal lymphopoietin, interleukin-13, interleukin-6). Hierarchical clustering showed a distinct signature with high dose and a similarity in mRNA expression pattern of low and medium doses of CB + O3. In conclusion, we demonstrate that the biological outcomes from CB + O3 co-exposure are significantly greater than individual exposures over a range of aerosol concentrations and aerosol characteristics can predict biological outcome.

Background We investigated the role of A2B-adenosine receptor (A2BAR) in regulating immunosuppressive metabolic stress in the tumor microenvironment (TME). Novel A2BAR antagonist PBF-1129 was tested for anti-tumor activity in animals and evaluated for safety and immunological efficacy in a phase-I clinical trial in non-small-cell lung cancer (NSCLC) patients. Methods Anti-tumor efficacy of A2BAR antagonists and impact on metabolic and immune TME was evaluated in lung, melanoma, colon, breast and EGFR-inducible transgenic cancer models. Employing Electron Paramagnetic Resonance, we assessed changes in TME metabolic parameters including pO2, pH, and inorganic phosphate (Pi) during tumor growth and evaluated the immunological effects of PBF-1129, including its pharmacokinetics, safety, and toxicity in NSCLC patients. Results Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial Pi emerged as a correlative and cumulative measure of TME stress and immunosuppression. A2BAR inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ecto-nucleotidases, increased expression of adenosine deaminase (ADA), decreased tumor growth and metastasis, increased IFN-γ-production and enhanced the efficacy of anti-tumor therapies following combination regimens in animal models (anti-PD-1 vs. anti-PD-1 plus PBF-1129 treatment hazard ratio [HR] = 11.74, 95% CI = 3.35 to 41.13, n = 10, P Conclusions Data identify A2BAR as a valuable therapeutic target to modify metabolic and immune TME to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Low-field electron paramagnetic resonance spectroscopy paired with pTAM, a mono-phosphonated triarylmethyl radical, is an unmatched technique for concurrent and non-invasive measurement of oxygen concentration, pH, and inorganic phosphate concentration for in vivo investigations. However, the prior reported synthesis is limited by its low yield and poor scalability, making wide-spread application of pTAM unfeasible. Here, we report a new strategy for the synthesis of pTAM with significantly greater yields demonstrated on a large scale. We also present a standalone application with user-friendly interface for automatic spectrum fitting and extraction of pO2, pH, and [Pi] values. Finally, we confirm that pTAM remains in the extracellular space and has low cytotoxicity appropriate for local injection., We report a new strategy for the synthesis of a mono-phosphonated triarylmethyl radical spin probe and a standalone application with a user-friendly interface for automatic spectrum fitting and extraction of pO2, pH, and [Pi] values.

Exchange processes which include conformational change, protonation/deprotonation, and binding equilibria are routinely studied by 2D exchange NMR techniques, where information about the exchange of nuclei between environments with different NMR shifts is obtained from the development of cross-peaks. Whereas 2D NMR enables the real time study of millisecond and slower exchange processes, 2D ESR in the form of 2D-ELDOR (two-dimensional electron–electron double resonance) has the potential for such studies over the nanosecond to microsecond real time scales. Cross-peak development due to chemical exchange has been seen previously for semiquinones in ESR, but this is not possible for most common ESR probes, such as nitroxides, studied at typical ESR frequencies because, unlike NMR, the exchanging states yield ESR signals that are not resolved from each other within their respective line widths. But at 95 GHz, it becomes possible to resolve them in many cases because of the increased g-factor resolution. The 95 GHz instrumental developments occurring at ACERT now enable such studies. We demonstrate these new capabilities in two studies: (A) the protonation/deprotonation process for a pH-sensitive imidazoline spin label in aqueous solution where the exchange rate and the population ratio of the exchanging states are controlled by the concentration and pH of the buffer solution, respectively, and (B) a nitroxide radical partitioning between polar (aqueous) and nonpolar (phospholipid) environments in multilamellar lipid vesicles, where the cross-peak development arises from the exchange of the nitroxide between the two phases. This work represents the first example of the observation and analysis of cross-peaks arising from chemical exchange processes involving nitroxide spin labels.

Environmental inhalation exposures are inherently mixed (gases and particles), yet regulations are still based on single toxicant exposures. While the impacts of individual components of environmental pollution have received substantial attention, the impact of inhalation co-exposures is poorly understood. Here, we mechanistically investigated pulmonary inflammation and lung function decline after inhalation co-exposure and individual exposures to ozone (O3) and ultrafine carbon black (CB). Environmentally/occupationally relevant lung deposition levels in mice were achieved after inhalation of stable aerosols with similar aerodynamic and mass median distributions. X-ray photoemission spectroscopy detected increased surface oxygen contents on particles in co-exposure aerosols. Compared with individual exposures, co-exposure aerosols produced greater acellular and cellular oxidants detected by electron paramagnetic resonance (EPR) spectroscopy, and in vivo immune-spin trapping (IST), as well as synergistically increased lavage neutrophils, lavage proteins and inflammation related gene/protein expression. Co-exposure induced a significantly greater respiratory function decline compared to individual exposure. A synthetic catalase-superoxide dismutase mimetic (EUK-134) significantly blunted lung inflammation and respiratory function decline confirming the role of oxidant imbalance. We identified a significant induction of epithelial alarmin (thymic stromal lymphopoietin-TSLP)-dependent interleukin-13 pathway after co-exposure, associated with increased mucin and interferon gene expression. We provided evidence of interactive outcomes after air pollution constituent co-exposure and identified a key mechanistic pathway that can potentially explain epidemiological observation of lung function decline after an acute peak of air pollution. Developing and studying the co-exposure scenario in a standardized and controlled fashion will enable a better mechanistic understanding of how environmental exposures result in adverse outcomes., Graphical abstract Image 1, Highlights • Interaction with O3 mediates free radical production on the surface of carbon black (CB) particles. • Oxidants mediate co-exposure (CB + O3)-induced lung function decline. • EUK-134 (a synthetic superoxide-catalase mimetic) abrogates CB + O3-induced lung inflammation. • CB + O3 co-exposure induces greater lung inflammation than individual exposures. • Epithelial alarmin (TSLP) contributes significantly to the CB + O3 toxicity.

An electron paramagnetic resonance (EPR) imaging system has been custom built for use in pre-clinical and, potentially, clinical studies. Commercial standalone modules have been used in the design that are MATLAB-controlled. The imaging system combines digital and analog technologies. It was designed to achieve maximum flexibility and versatility and to perform standard and novel user-defined experiments. This design goal is achieved by frequency mixing of an arbitrary waveform generator (AWG) output at the intermediate frequency (IF) with a constant source frequency (SF). Low noise SF at 250, 750, and 1000 MHz are available in the system. A wide range of frequencies from near-baseband to L-band can be generated as a result. Two-stage downconversion at the signal detection side is implemented that enables multi-frequency EPR capability. In the first stage, the signal frequency is converted to IF. A novel AWG-enabled digital auto-frequency control method that operates at IF is described that is used for automatic resonator tuning. Quadrature baseband EPR signal is generated in the second downconversion step. The semi-digital approach of mixing low-noise frequency sources with an AWG permits generation of arbitrary excitation patterns that include but are not limited to frequency sweeps for resonator tuning and matching, continuous-wave, and pulse sequences. Presented in this paper is the demonstration of rapid scan (RS) EPR imaging implemented at 800 MHz. Generation of stable magnetic scan waveforms is critical for the RS method. A digital automatic scan control (DASC) system was developed for sinusoidal magnetic field scans. DASC permits tight control of both amplitude and phase of the scans. A surface loop resonator was developed using 3D printing technology. RS EPR imaging system was validated using sample phantoms. In vivo imaging of a breast cancer mouse model is demonstrated.

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.

Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CBox) as a model particle. Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CBox particles (20 μg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure. XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CBox particles. Treatment with CBox demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CBox-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner. In summary, this study mechanistically demonstrated an increased biological potency of CBox particles and established the role of macrophage-released chemical mediators in endothelial damage., Graphical abstract Image 1, Highlights • Exposure to ozone oxidizes carbon black nanoparticles. • Oxidized carbon black particles demonstrate enhanced oxidant production. • Oxidized carbon black particles exposed macrophages secreted CXCL9/10/11 activate CXCR3 on endothelial cells. • CXCR3 activation mediate increased endothelial monolayer permeability and migration impairment.

Nitroxyl radicals, NRs, represent the most diverse class of stable organic radicals varying in stability, spectral properties and functionality which have been successfully used as spin labels and probes in numerous electron paramagnetic resonance (EPR)-based spectroscopic and imaging applications. The stability of N–O group of the NRs allows chemical reactions which do not destroy the radical center and, therefore, opens the opportunity to design NR probes for sensing specific chemical reagents such as oxygen, protons (pH), glutathione, nitric oxide and specific enzymatic activities. In this chapter we overview the principles of NR applications as probes to assess the local chemical microenvironment with a focus on their in vivo functional imaging.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1αfl/fl/LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2αfl/fl/LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α–deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α–deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α–deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical 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 towards 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 ×10(8) M(−1)s(−1) leading to a tri-sulfonated 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-, P- nucleophiles opening access to various mono-functionalized sulfonated trityl radicals.

The partial pressure of oxygen (pO(2)) and the extracellular pH in the tumour microenvironment are essential parameters for understanding the physiological state of a solid tumour. Also, phosphate-containing metabolites are involved in energy metabolism, and interstitial inorganic phosphate (Pi) is an informative marker for tumour growth. This article describes the simultaneous mapping of pO(2), pH and Pi using 750 MHz continuous-wave (CW) electron paramagnetic resonance (EPR) and a multifunctional probe, monophosphonated trityl radical p(1)TAM-D. The concept was demonstrated by acquiring three-dimensional (3D) maps of pO(2), pH and Pi for multiple solution samples. This was made possible by combining a multifunctional radical probe, fast CW-EPR spectral acquisition, four-dimensional (4D) spectral-spatial image reconstruction, and spectral fitting. The experimental results of mapping pO(2), pH and Pi suggest that the concept of simultaneous mapping using EPR is potentially applicable for the multifunctional measurements of a mouse tumour model.

Overhauser-enhanced Magnetic Resonance Imaging (OMRI) is a double resonance technique applied for oxygen imaging in aqueous samples and biological tissues. In this report, we present an improved OMRI approach of oxygen measurement using the single line “Finland” trityl spin probe. Compared to a traditional approach, we introduced an additional mechanism of leakage of spin polarization due to an interaction of a spin system with oxygen. The experimental comparison of the new approach with an oxygen-dependent leakage factor to a traditional approach performed in phantom samples in vitro, and mouse tumor model in vivo, shows improved accuracy of determination of oxygen and contrast agent concentrations.

Enzyme activities are well established biomarkers of many pathologies. Imaging enzyme activity directly in vivo may help to gain insight into the pathogenesis of various diseases but remains extremely challenging. In this communication, we report the use of EPR imaging (EPRI) in combination with a specially designed paramagnetic enzymatic substrate to map alkaline phosphatase activity with a high selectivity, thereby demonstrating the potential of EPRI to map enzyme activity.

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.

The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T 2 * mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-(1-15N)-1-oxyl (2H,15N-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-1-oxyl (2H-DCP), were used in the study. A clonogenic cell survival assay was performed with the 2H-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected 2H,15N-DCP and 2H-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T 2 * mapping. 2H-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of 2H,15N-DCP in tumor tissues was 24.7 ± 2.9 min (mean ± standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the 2H,15N-DCP radical showed a plateau of 10.2 ± 1.2 min (mean ± SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T 2 * maps with 2H,15N-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. EPR signals of 2H,15N-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T 2 * mapping with CW-EPR-based SPI.

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.

We report the synthesis of hydroxyethyl tetrathiatriarylmethyl radicals OX063 and its deuterated analogue OX071 for biomedical EPR applications.

Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7 × 105 M-1 s-1 which is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.

Tissue oxygenation, extracellular acidity and tissue reducing capacity are among crucial parameters of tumor microenvironment (TME) of significant importance for tumor pathophysiology. In this paper we demonstrate the complementary application of particulate lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) and soluble nitroxide (NR) paramagnetic probes for monitoring of these TME parameters using electron paramagnetic resonance (EPR) technique. Two different types of therapeutic interventions were studied: hypothermia and systemic administration of metabolically active drug. In summary, the results demonstrate utility of EPR technique for noninvasive concurrent longitudinal monitoring of physiologically relevant chemical parameters of TME in a mouse xenograft tumor models including that under therapeutic intervention.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

The rechargeable aqueous zinc-ion batteries (ZIBs) based on the Zn/MnO2 couple and mildly acidic electrolyte have emerged as promising large-scale energy storage systems. This work reports an in situ electrochemical activation approach to oxidizing MnS into an electrochemically derived oxide (MnS-EDO), which unlocks its potential as high-performance cathodes for ZIBs. MnS-EDO contains fragmented layers with abundant defects and thus demonstrates large electrochemically active surface areas, high electrochemical reactivity, fast ion diffusion kinetics, accelerated charge transfer and exceptional structural robustness during cycling compared to α-MnO2. MnS-EDO exhibits a specific capacity of 335.7 mAh g−1 with ~100% capacity retention after 100 cycles at 0.3 A g−1, outstanding rate capability and long-term stability retaining 104 mAh g−1 after 4000 cycles at 3 A g−1. This work elucidates the underlying electrochemical insights and a hybrid discharge mechanism involving homogeneous Zn2+ intercalation at ~1.4 V and subsequent heterogeneous reactions of insertion of both H+ and Zn2+ at ~1.25 V. The ambiguities among Zn buserite, birnessite and zinc hydroxide sulfate are clarified. This work provides a simple and low-cost approach to unlocking the potential of MnS-EDO cathode for promising aqueous rechargeable ZIBs and sheds light on a mechanistic understanding of manganese oxide-based cathodes.

The purpose of this study is to investigate localized delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential therapeutic modality in breast cancer to alleviate tumor hypoxia and immunosuppression. A therapeutic that has the potential to target and reverse these factors of the tumor microenvironment (TME) would aid in reducing tumor aggressiveness, immune escape, metastasis, and importantly, could potentially be administered as a pre-treatment to reduce resistance to standard therapies such as chemotherapy and radiation. We have previously reported that 100 ng of intratumorally delivered recombinant GM-CSF 1) altered the macrophage profile from immunosuppressive M2-like to immunosupportive M1-like and 2) inhibited angiogenesis and induced near anoxic levels in the tumor due to the dose-dependent production of soluble VEGFR-1 from tumor macrophages that sequestered VEGF. Our current focus is to determine if a dosage window of GM-CSF exists that may normalize leaky and dysfunctional tumor vasculature and alleviate hypoxia in tumors by lesser sVEGFR-1 production, and simultaneously convert the immune cell profile from immunosuppressive to immunosupportive to promote cytotoxic T cell killing. After intratumoral injections with either GM-CSF or saline 3x per week for 2.5 weeks in an orthotopic PyMT murine breast cancer model, we utilized a soluble multifunctional trityl probe and Electron Paramagnetic Resonance Spectrometry to measure intratumoral oxygen and pH in vivo in real time. Our findings demonstrate that intratumoral injections of 15 ng GM-CSF rescued tumor oxygen by ~16% and raised pH ~0.2 units, while 5 ng enhanced oxygen even further by ~40% and raised pH ~0.4 units. Additionally, tumor-associated macrophages (TAMs) that were isolated from Stage IV MMTV-PyMT tumors and treated with 5 ng/ml GM-CSF 3x per week for 2 weeks demonstrated a reduction in IL-10 expression as determined by intracellular staining and flow cytometry. Further, as a result of these data, we have also formulated and characterized GM-CSF-containing PLGA/PEG-PLGA nanoparticles in vitro with average sizes under 200 nm for the purpose of systemic but localized delivery of dose-optimized recombinant GM-CSF to tumors. Nanoparticles yielded an entrapment efficiency of 87.9% +/- 4.2 S.D. and release of functional GM-CSF in vitro. Overall, we have demonstrated the potential of locally delivered recombinant GM-CSF to alleviate tumor hypoxia and acidity in vivo and reverse immunosuppressive TAM phenotype ex vivo, and we have formulated nanoparticles encapsulating recombinant murine GM-CSF as another modality which will be utilized for localized delivery of GM-CSF to breast tumors to achieve similar effects. Citation Format: Nicole Mihalik, Andrey Bobko, Valery Khramtsov, Benoit Driesschaert, Tim D. Eubank. Localized delivery of granulocyte-macrophage colony-stimulating factor to alleviate tumor hypoxia and immunosuppression in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2672.

An electron paramagnetic resonance (EPR)-based method for noninvasive three-dimensional extracellular pH mapping was developed using a pH-sensitive nitroxyl radical as an exogenous paramagnetic probe. Fast projection scanning with a constant magnetic field sweep enabled the acquisition of four-dimensional (3D spatial +1D spectral) EPR images within 7.5 min. Three-dimensional maps of pH were reconstructed by processing the pH-dependent spectral information on the images. To demonstrate the proposed method of pH mapping, the progress of extracellular acidosis in tumor-bearing mouse legs was studied. Furthermore, extracellular pH mapping was used to visualize the spatial distribution of acidification in different tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells. The proposed EPR-based pH mapping method enabled quantitative visualization of regional changes in extracellular pH associated with altered tumor metabolism.

Proton-electron double-resonance imaging (PEDRI) employs electron paramagnetic resonance irradiation with low-field magnetic resonance imaging so that the electron spin polarization is transferred to nearby protons, resulting in higher signals. PEDRI provides information about free radical distribution and, indirectly, about the local microenvironment such as partial pressure of oxygen (pOHigh-power radio frequency irradiation is needed for optimum signal enhancement, which may be harmful to living tissue by unwanted heat deposition. Free radical probes differ depending on the purpose of PEDRI. Some probes are less effective for enhancing signal than others, which can reduce image quality. It is so far not possible to image endogenous radicals by PEDRI because low concentrations and broad line widths of the radicals lead to negligible signal enhancement.PEDRI has similarities with electron paramagnetic resonance imaging (EPRI) because both techniques observe the EPR signal, directly in the case of EPRI and indirectly with PEDRI. PEDRI provides information that is vital to research on homeostasis, development of diseases, or treatment responses in vivo. It is expected that the development of new EPR techniques will give insights into novel PEDRI applications and vice versa. Antioxid. Redox Signal. 28, 1345-1364.

The advent of hybrid scanners, combining complementary modalities, has revolutionized the application of advanced imaging technology to clinical practice and biomedical research. In this project, we investigated the melding of two complementary, functional imaging methods: positron emission tomography (PET) and electron paramagnetic resonance imaging (EPRI). PET radiotracers can provide important information about cellular parameters, such as glucose metabolism. While EPR probes can provide assessment of tissue microenvironment, measuring oxygenation and pH, for example. Therefore, a combined PET/EPRI scanner promises to provide new insights not attainable with current imagers by simultaneous acquisition of multiple components of tissue microenvironments. To explore the simultaneous acquisition of PET and EPR images, a prototype system was created by combining two existing scanners. Specifically, a silicon photomultiplier (SiPM)-based PET scanner ring designed as a portable scanner was combined with an EPRI scanner designed for the imaging of small animals. The ability of the system to obtain simultaneous images was assessed with a small phantom consisting of four cylinders containing both a PET tracer and EPR spin probe. The resulting images demonstrated the ability to obtain contemporaneous PET and EPR images without cross-modality interference. Given the promising results from this initial investigation, the next step in this project is the construction of the next generation pre-clinical PET/EPRI scanner for multi-parametric assessment of physiologically-important parameters of tissue microenvironments.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO(2), pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO(2) and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.

Purpose Electron paramagnetic resonance (EPR) imaging in the spectral-spatial domain with application of soluble paramagnetic probes provides an opportunity for spatially resolved functional measurements of living objects. The purpose of this study was to develop EPR methods for visualization of oxygenation and acidosis of ischemic myocardium. Methods EPR oxygen measurements were performed using isotopically substituted 2H,15N-dicarboxyproxyl. The radical has an EPR line width of 320 mG and oxygen-induced line broadening of 0.53 mG/mm Hg, providing oxygen sensitivity down to 5 μM. pH measurements were performed using previously developed pH-sensitive imidazoline nitroxide. The radical has an EPR spectrum with pH-dependable hyperfine splitting, pK = 6.6, providing pH sensitivity of approximately 0.05 U in the physiological range. Results EPR imaging of isolated and perfused rat hearts was performed in the two-dimensional + spectral domain. The spatial resolution of the measurements was about 1.4 mm. Marked tissue hypoxia was observed in the ischemic area of the heart after occlusion of the left anterior descending coronary artery. Tissue oxygenation was partly restored upon reperfusion. EPR mapping of myocardial pH indicated acidosis of the ischemic area down to pH 6.7–6.8. Conclusion This study demonstrates the capability of low-field EPR and the nitroxide spin probes for mapping of myocardial oxygenation and pH. The developed approaches might be used for noninvasive investigation of microenvironment on living objects. Magn Reson Med, 2015. © 2015 Wiley Periodicals, Inc.

Nitronyl nitroxides (NNs) are the paramagnetic probes that are capable of scavenging physiologically relevant reactive oxygen (ROS) and nitrogen (RNS) species, namely superoxide, nitric oxide (NO), and nitroxyl (HNO). NNs are increasingly considered as potent antioxidants and potential therapeutic agents. Understanding redox chemistry of the NNs is important for their use as antioxidants and as paramagnetic probes for discriminative detection of NO and HNO by electron paramagnetic resonance (EPR) spectroscopy. Here we investigated the redox properties of the two most commonly used NNs, including determination of the equilibrium and rate constants of their reduction by HNO and ferrocyanide, and reduction potential of the couple NN/hydroxylamine of nitronyl nitroxide (hNN). The rate constants of the reaction of the NNs with HNO were found to be equal to (1-2) × 10(4) M(-1)s(- 1) being close to the rate constants of scavenging superoxide and NO by NNs. The reduction potential of the NNs and iminonitroxides (INs, product of NNs reaction with NO) were calculated based on their reaction constants with ferrocyanide. The obtained values of the reduction potential for NN/hNN (E'0 ≈ 285 mV) and IN/hIN (E' ≈ 495 mV) are close to the corresponding values for vitamin C and vitamin E, correspondingly. The "balanced" scavenging rates of the NNs towards superoxide, NO, and HNO, and their low reduction potential being thermodynamically close to the bottom of the pecking order of oxidizing radicals, might be important factors contributing into their antioxidant activity.

Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

Electron paramagnetic resonance (EPR)-based spectroscopic and imaging techniques allow for the study of free radicals—molecules with one or more unpaired electrons. Biological EPR applications include detection of endogenous biologically relevant free radicals as well as use of specially designed exogenous radicals to probe local microenvironments. This Forum focuses on recent advances in the field of in vivo EPR applications discussed at the International Conference on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems (EPR-2017). Although direct EPR detection of endogenous free radicals such as reactive oxygen species (ROS) in vivo remains unlikely in most cases, alternative approaches based on applications of advanced spin traps and probes for detection of paramagnetic products of ROS reactions often allow for specific assessment of free radical production in living subjects. In recent decades, significant progress has been achieved in the development and in vivo application of specially designed paramagnetic probes as “molecular spies” to assess and map physiologically relevant functional information such as tissue oxygenation, redox status, pH, and concentrations of interstitial inorganic phosphate and intracellular glutathione. Recent progress in clinical EPR instrumentation and development of biocompatible paramagnetic probes for in vivo multifunctional tissue profiling will eventually make translation of the EPR techniques into clinical settings possible. Antioxid. Redox Signal. 28, 1341–1344.

Carboxy-substituted trityl (triarylmethyl) radicals are valuable in vivo probes because of their stability, narrow lines, and sensitivity of their spectroscopic properties to oxygen. Amino-substituted trityl radicals have the potential to monitor pH in vivo, and the suitability for this application depends on spectral properties. Electron spin relaxation times T1 and T2 were measured at X-band for the protonated and deprotonated forms of two amino-substituted triarylmethyl radicals. Comparison with relaxation times for carboxy-substituted triarylmethyl radicals shows that T1 exhibits little dependence on protonation or the nature of the substituent, which makes it useful for measuring O2 concentration, independent of pH. Insensitivity of T1 to changes in substituents is consistent with the assignment of the dominant contribution to spin lattice relaxation as a local mode that involves primarily atoms in the carbon and sulfur core. Values of T2 vary substantially with pH and the nature of the aryl group substituent, reflecting a range of dynamic processes. The narrow spectral widths for the amino-substituted triarylmethyl radicals facilitate spectral-spatial rapid scan electron paramagnetic resonance imaging, which was demonstrated with a phantom. The dependence of hyperfine splittings patterns on pH is revealed in spectral slices through the image.

An algorithm is derived and demonstrated that reconstructs an EPR spectral-spatial image from projections with arbitrarily selected gradients. This approach permits imaging wide spectra without the use of the very large sweep widths and gradients that would be required for spectral-spatial imaging with filtered back projection reconstruction. Each projection is defined as the sum of contributions at the set of locations in the object. At each location gradients shift the spectra in the magnetic field domain, which is equivalent to a phase change in the Fourier-conjugate frequency domain. This permits solution of the problem in the frequency domain. The method was demonstrated for 2D images of phantoms consisting of (i) two tubes containing (14)N and (15)N nitroxide and (ii) two tubes containing a pH sensitive trityl radical at pH 7.0 and 7.2. In each case spectral slices through the image agree well with the full spectra obtained in the absence of gradient.

Nitric oxide, (·)NO, and product of its one-electron reduction, nitroxyl NO(-), are important molecules in the biochemistry of living organisms. At physiological conditions nitroxyl exists in its protonated form, HNO. Angeli's salt, AS, and diazeniumdiolates, NONOates, are widely used donors of HNO and (·)NO, correspondingly. In this work we observed oxidative decomposition of AS and PAPA NONOate in the presence of mild oxidizing agents, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, cPTIO, and 2,2'-azinobis(3-ethylbenzolthiazoline-6-sulfonate) radical, ABTS(·-). The observed unexpected fast oxidative decomposition of AS with release of NO instead of HNO suggests the need for a reevaluation of some of the biological effects of AS assigned to action of HNO. While oxidative decomposition of NONOate did not result in release of alternative NOx specimen but only (·)NO, it significantly affects the rates and stoichiometry of (·)NO release. In summary, possible contribution of oxidative decomposition of AS and NONOates should be taken into account upon interpretation of their actions in chemical and biological systems.

Pulse techniques in electron paramagnetic resonance (EPR) allow for a reduction in measurement times and increase in sensitivity but require the synthesis of paramagnetic probes with long relaxation times. Here it is shown that the recently synthesized phosphonated trityl radical possesses long relaxation times that are sensitive to probe the microenvironment, such as oxygenation and acidity of an aqueous solution. In principle, application of Fourier transform EPR (FT-EPR) spectroscopy makes it possible to acquire the entire EPR spectrum of the trityl probe and assess these microenvironmental parameters within a few microseconds. The performed analysis of the FT-EPR spectra takes into consideration oxygen-, proton-, buffer-, and concentration-induced contributions to the spectral shape, therefore enabling quantitative and discriminative assessment of pH, pO2, and concentrations of the probe and inorganic phosphate.

The in vivo quantification of extracellular pH (pHe) in tumours may provide a useful biomarker for tumour cell metabolism. In this study, we assessed the viability of continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy with a pH-sensitive nitroxide for the measurement of extracellular tumour pH in a mouse model. CW-EPR spectroscopy (750 MHz) of C3H HeJ mice hind leg squamous cell tumour was performed after intravenous tail vein injection of pH-sensitive nitroxide (R-SG, 2-(4-((2-(4-amino-4-carboxybutanamido)-3-(carboxymethylamino)-3-oxoproylthio)methyl)phenyl)-4-pyrrolidino-2,5,5-triethyl-2,5-dihydro-1Н-imidazol-1-oxyl) during stages of normal tumour growth and in response to a single 10-Gy dose of X-ray irradiation. An inverse relationship was observed between tumour volume and pHe value, whereby, during normal tumour growth, a constant reduction in pHe was observed. This relationship was disrupted by X-ray irradiation and, from 2–3 days post-exposure, a transitory increase in pHe was observed. In this study, we demonstrated the viability of CW-EPR spectroscopy using R-SG nitroxide to obtain high-sensitivity pH measurements in a mouse tumour model with an accuracy of

A disulfide-linked imidazoline nitroxide side chain (V1) has a similar and highly constrained internal motion at diverse topological sites in a protein, unlike that for the disulfide-linked pyrroline nitroxide side chain (R1) widely used in site directed spin labeling EPR. Crystal structures of V1 at two positions in a helix of T4 Lysozyme and quantum mechanical calculations suggest the source of the constraints as intra-side chain interactions of the disulfide sulfur atoms with both the protein backbone and the 3-nitrogen in the imidazoline ring. These interactions apparently limit the conformation of the side chain to one of only three possible rotamers, two of which are observed in the crystal structure. An inter-spin distance measurement in frozen solution using double electron–electron resonance (DEER) gives a value essentially identical to that determined from the crystal structure of the protein containing two copies of V1, indicating that lattice forces do not dictate the rotamers observed. Collectively, the results suggest the possibility of predetermining a unique rotamer of V1 in helical structures. In general, the reduced rotameric space of V1 compared to R1 should simplify interpretation of inter-spin distance information in terms of protein structure, while the highly constrained internal motion is expected to extend the dynamic range for characterizing large amplitude nanosecond backbone fluctuations.

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO2), extracellular acidosis (pHe), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO2, pHe and tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the “growth rate hypothesis” may underline its potential role in tumorigenesis and tumor progression.

The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time TIsotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(EPR signals of