Search

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.

We report an isotopologue of the trityl radical OX071, labeled with 13C at the central carbon highly sensitive to molecular tumbling.

Triarylmethyl (TAM)-based labels, while still underutilized, are a powerful class of labels for pulsed-Electron Spin Resonance (ESR) distance measurements. They feature slow relaxation rates for long-lasting signals, high stability for cellular experiments, and narrow spectral features for efficient excitation of the spins. However, the typical narrow line shape limits the available distance measurements to only single-frequency experiments, such as Double Quantum Coherence (DQC) and Relaxation Induced Dipolar Modulation Enhancement (RIDME), which can be complicated to perform or hard to process. Therefore, widespread usage of TAM labels can be enhanced by the use of Double Electron-Electron Resonance (DEER) distance measurements. In this work, we developed a new spin label

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.

A stable triarylmethyl spin probe whose electron paramagnetic resonance (EPR) spectrum is highly sensitive to molecular tumbling is reported. The strong anisotropy of the hyperfine coupling tensor with the central carbon of a 13 C1 -labeled triarylmethyl radical enables the measurement of the probe rotational correlation time with applications to measure microviscosity and molecular dynamics.

Dynamic nuclear polarization (DNP) is an NMR sensitivity enhancement technique that mediates polarization transfer from unpaired electrons to NMR-active nuclei. Despite its success in elucidating important structural information on biological and inorganic materials, the detailed polarization-transfer pathway from the electrons to the nearby and then the bulk solvent nuclei, and finally to the molecules of interest-remains unclear. In particular, the nuclei in the paramagnetic polarizing agent play significant roles in relaying the enhanced NMR polarizations to more remote nuclei. Despite their importance, the direct NMR observation of these nuclei is challenging because of poor sensitivity. Here, we show that a combined DNP and electron decoupling approach can facilitate direct NMR detection of these nuclei. We achieved an ∼80 % improvement in NMR intensity via electron decoupling at 0.35 T and 80 K on trityl radicals. Moreover, we recorded a DNP enhancement factor of urn:x-wiley:09476539:media:chem202202556:chem202202556-math-0001 ∼90 and ∼11 % higher NMR intensity using electron decoupling on paramagnetic metal-organic framework, magnesium hexaoxytriphenylene (MgHOTP MOF).

A perchlorotriarylmethyl tricarboxylic acid radical 99% enriched in (13)C at the central carbon ((13)C(1)-PTMTC) was characterized in phosphate buffered saline solution (pH = 7.2) (PBS) at ambient temperature. Samples immobilized in 1:1 PBS:glycerol or in 9:1 trehalose:sucrose were studied as a function of temperature. Isotope enrichment at C(1) creates a trityl that can be used to accurately measure microscopic viscosity. Understanding of the impact of the (13)C hyperfine interaction on electron spin relaxation is important for application of this trityl in oximetry and distance measurements. The anisotropic (13)C(1) hyperfine couplings (A(x) = A(y) = 24 ± 2 MHz, A(z) = 200 ± 1 MHz) are larger than for the related (13)C(1)-perdeuterated Finland trityl ((13)C(1)-dFT) and the g anisotropy (g(x) = 2.0013, g(y) = 2.0016, g(z) = 2.0042) is slightly larger than for (13)C(1)-dFT. The tumbling correlation times (τ(R)) for (13)C(1)-PTMTC are 0.20 ± 0.02 ns in PBS and 0.40 ± 0.05 ns in 3:1 PBS:glycerol, which are shorter than for (13)C(1)-dFT in the same solutions. T(1) for (13)C(1)-PTMTC is 3.5 ± 0.5 μs in PBS and 5.3 ± 0.4 μs in 3:1 PBS:glycerol, which are shorter than for (13)C(1)-dFT due to faster tumbling, larger anisotropy of the (13)C(1) hyperfine, and about 30% larger contribution from the local mode. In immobilized samples T(1) for (13)C(1)-PTMTC is similar to that for (13)C(1)-dFT and other trityls without chlorine or (13)C(1) substituents, indicating that the (13)C(1) and Cl substituents on the phenyl rings have little impact on T(1). The temperature dependence of T(1) was modeled with contributions from the direct, Raman, and local mode processes. Broadening of CW linewidths of about 0.6 G in fluid solution and about 2 G in rigid lattice is attributed to unresolved (35,37)Cl hyperfine couplings.

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.

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.

Sensitive in-cell distance measurements in proteins using pulsed-Electron Spin Resonance (ESR) require reduction-resistant and cleavage-resistant spin-labels. Among the reduction-resistant moieties, the hydrophilic trityl core known as OX063 is promising due to its long phase-memory relaxation time (T(m)). This property leads to a sufficiently intense ESR signal for reliable long-distance measurements. Furthermore, the T(m) of OX063 remains sufficiently long at higher temperatures, opening the possibility for measurements at temperatures above 50 K. In this work, we synthesized deuterated OX063 with a maleimide linker (mOX063-d(24)). We show that the combination of the hydrophilicity of the label and the maleimide linker enables high protein labeling that is cleavage-resistant in-cells. Distance measurements performed at 150 K using this label are more sensitive than the measurements at 80 K. The sensitivity gain is due to the significantly short longitudinal relaxation time (T(1)) at higher temperatures, which enables more data collection per unit of time. In addition to in vitro experiments, we perform distance measurements in Xenopus laevis oocytes. Interestingly, the T(m) of mOX063-d(24) is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d(24) provides highly sensitive distance measurements both in vitro and in-cells.

Soluble stable radicals are used as spin probes and spin labels for in vitro and in vivo Electron Paramagnetic Resonance (EPR) spectroscopy and imaging applications. We report the synthesis and characterization of a perchlorinated triarylmethyl radical enriched 99% at the central carbon, 13C1-PTMTC. The anisotropy of the hyperfine splitting with the 13C1 (Ax=26, Ay=25, Az=199.5 MHz) and the g (gx=2.0015, gy=2.0015, gz=2.0040) are responsible for a strong effect of the radical tumbling rate on the EPR spectrum. The rotational correlation time can be determine by spectral simulation or via the linewidth after calibration. As spin probe 13C1-PTMTC can be used to measure media microviscosity with high sensitivity. Bound to a macromolecule as spin label, 13C1-PTMTC could be used to study local mobility and molecular interactions.

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) has demonstrated notable clinical activity in cancer immunotherapy, but is limited by systemic toxicities, poor bioavailability, rapid clearance, and instability in vivo. Nanoparticles (NPs) may overcome these limitations and provide a mechanism for passive targeting of tumors. This study aimed to develop GM-CSF-loaded PLGA/PLGA-PEG NPs and evaluate them in vitro as a potential candidate for in vivo administration. NPs were created by a phase-separation technique that did not require toxic/protein-denaturing solvents or harsh agitation techniques and encapsulated GM-CSF in a more stable precipitated form. NP sizes were within 200 nm for enhanced permeability and retention (EPR) effect with negative zeta potentials, spherical morphology, and high entrapment efficiencies. The optimal formulation was identified by sustained release of approximately 70% of loaded GM-CSF over 24 hours, alongside an average size of 143 ± 35 nm and entrapment efficiency of 84 ± 5%. These NPs were successfully freeze-dried in 5% (w/v) hydroxypropyl-β-cyclodextrin for long-term storage and further characterized. Bioactivity of released GM-CSF was determined by observing GM-CSF receptor activation on murine monocytes and remained fully in-tact. NPs were not cytotoxic to murine bone marrow-derived macrophages (BMDMs) at concentrations up to 1 mg/mL as determined by MTT and Trypan Blue exclusion assays. Lastly, NP components generated no significant transcription of inflammation-regulating genes from BMDMs compared to IFNγ+LPS “M1” controls. This report lays the preliminary groundwork to validate in vivo studies with GM-CSF-loaded PLGA/PEG-PLGA NPs for tumor immunomodulation. Overall, these data suggest that in vivo delivery will be well tolerated.

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.

A stable triarylmethyl spin probe whose Electron Paramagnetic Resonance (EPR) spectrum is highly sensitive to molecular tumbling is reported. The strong anisotropy of the hyperfine coupling tensor with the central carbon of a (13)C(1)-labeled triarylmethyl radical enables the measurement of the probe rotational correlation time with applications to measure microviscosity and molecular dynamics.

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.

Dynamic nuclear polarization (DNP) has evolved as the method of choice to enhance NMR signal intensities and to address a variety of otherwise inaccessible chemical, biological and physical information. Despite its success, there is no detailed understanding of how the large electron polarization is transferred to the surrounding nuclei or where these nuclei are located relative to the polarizing agent. We have previously shown using three-spin solid effect that the size of the spin diffusion barrier surrounding the trityl radical in a glassy glycerol-water matrix is < 6 Å. In this contribution, we demonstrate that a combined DNP and electron decoupling approach enables direct NMR detection of intramolecular 1H’s on the trityl radicals. Although the 1H’s reside outside of the spin diffusion barrier, they are still broadened by the electron-nuclei hyperfine interaction, which can be quenched using electron decoupling. We achieved an ~80% improvement in the NMR peak intensity using a ~2 MHz microwave Rabi frequency at 0.35 T and 80 K. The direct detection of 1H’s closest to the unpaired electrons allows us to extract the relaxation parameters of the 1H’s and shed light on the spin diffusion pathways from the unpaired electron to the bulk nuclei. This information could provide insights important in designing more robust DNP polarizing agents. Finally, we showed that the electron decoupling method can be applied to study magnesium hexahydroxytriphenylene metal-organic framework (MgHHTP MOF).

Dynamic nuclear polarization (DNP) has evolved as the method of choice to enhance NMR signal intensities and to address a variety of otherwise inaccessible chemical, biological and physical information. Despite its success, there is no detailed understanding of how the large electron polarization is transferred to the surrounding nuclei or where these nuclei are located relative to the polarizing agent. We have previously shown using three-spin solid effect that the size of the spin diffusion barrier surrounding the trityl radical in a glassy glycerol-water matrix is < 6 Å. In this contribution, we demonstrate that a combined DNP and electron decoupling approach enables direct NMR detection of intramolecular 1H’s on the trityl radicals. Although the 1H’s reside outside of the spin diffusion barrier, they are still broadened by the electron-nuclei hyperfine interaction, which can be quenched using electron decoupling. We achieved an ~80% improvement in the NMR peak intensity using a ~2 MHz microwave Rabi frequency at 0.35 T and 80 K. The direct detection of 1H’s closest to the unpaired electrons allows us to extract the relaxation parameters of the 1H’s and shed light on the spin diffusion pathways from the unpaired electron to the bulk nuclei. This information could provide insights important in designing more robust DNP polarizing agents. Finally, we showed that the electron decoupling method can be applied to study magnesium hexahydroxytriphenylene metal-organic framework (MgHHTP MOF).

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.

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.

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

Purpose The ability to assess the extracellular pH (pHe) is an important issue in oncology, because extracellular acidification is associated with tumor aggressiveness and resistance to cytotoxic therapies. In this study, a stable triphosphonated triarylmethyl (TPTAM) radical was qualified as a pHe electron paramagnetic resonance (EPR) molecular reporter. Methods Calibration of hyperfine splitting as a function of pH was performed using a 1.2-GHz EPR spectrometer. Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was used as an extracellular paramagnetic broadening agent to assess the localization of TPTAM when incubated with cells. In vivo EPR pH-metry was performed in MDA, SiHa, and TLT tumor models and in muscle. Bicarbonate therapy was used to modulate the tumor pHe. EPR measurements were compared with microelectrode readouts. Results The hyperfine splitting of TPTAM was strongly pH-dependent around the pKa of the probe (pKa = 6.99). Experiments with Gd-DTPA demonstrated that TPTAM remained in the extracellular compartment. pHe was found to be more acidic in the MDA, SiHa, and TLT tumor models compared with muscle. Treatment of animals by bicarbonate induced an increase in pHe in tumors: similar variations in pHe were found when using in vivo EPR or invasive microelectrodes measurements. Conclusion This study demonstrates the potential usefulness of TPTAM for monitoring pHe in tumors. Magn Reson Med 77:2438-2443, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

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.

Electron spin relaxation times for perdeuterated Finland trityl 99% enriched in (13)C at the central carbon ((13)C(1)-dFT) were measured in phosphate buffered saline (pH = 7.2) (PBS) solution at X-band. The anisotropic (13)C(1) hyperfine (A(x) = A(y) = 18±2, A(z) = 162 ±1 MHz) and g values (2.0033, 2.0032, 2.00275) in a 9:1 trehalose:sucrose glass at 293 K and in 1:1 PBS:glycerol at 160 K were determined by simulation of spectra at X-band and Q-band. In PBS at room temperature the tumbling correlation time, τ(R), is 0.29 ± 0.02 ns. The linewidths are broadened by incomplete motional averaging of the hyperfine anisotropy and T(2) is 0.13 ± 0.02 μs, which is shorter than the T(2) ~ 3.8 μs for natural abundance dFT at low concentration in PBS. T(1) for (13)C(1)-dFT in deoxygenated PBS is 5.9 ± 0.5 μs, which is shorter than for natural abundance dFT in PBS (16 μs) but much longer than in air-saturated solution (0.48 ± 0.04 μs). The tumbling dependence of T(1) in PBS, 3:1 PBS:glycerol (τ(R) = 0.80 ± 0.05 ns, T(1) = 9.7 ± 0.7 μs) and 1:1 PBS:glycerol (τ(R) = 3.4 ± 0.3 ns, T(1) = 12.0 ± 1.0 μs) was modeled with contributions to the relaxation predominantly from modulation of hyperfine anisotropy and a local mode. The 1/T(1) rate for the 1% (12)C(1)1-dFT in the predominantly (13)C labeled sample is about a factor of 6 more strongly concentration dependent than for natural abundance (12)C(1)-trityl, which reflects the importance of Heisenberg exchange with molecules with different resonance frequencies and faster relaxation rates. In glassy matrices at 160 K, T(1) and T(m) for (13)C(1)-dFT are in good agreement with previously reported values for (12)C(1)-dFT consistent with the expectation that modulation of nuclear hyperfine does not contribute to electron spin relaxation in a rigid lattice.

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.

Microfluidics is a well-established technique to synthesize, process, and analyze small amounts of materials for chemical, biological, medical, and environmental applications. Typically, it involves the use of reagents with a volume smaller than ~ 1 micro-l—ideally even nano- or picoliters. When the sample of interest contains paramagnetic species, it can in principle be quantified and analyzed by electron spin resonance (ESR) spectroscopy. However, conventional ESR is typically carried out with a sample volume of ~ 1 ml, thereby making it incompatible with most microfluidics applications. Here we show that by using a new class of miniature surface resonators combined with photolithography to prepare microfluidic patterns, ESR can be applied to measure small liquid samples, down to picoliter volumes, without considerably sacrificing concentration sensitivity. Our experiments, carried out with resonators whose mode volumes range from ~1 to 3.6 nL, showed that with a sample volume of ~0.25 nL good signals could be obtained from solutions with spin concentrations of less than 0.1 μM. The advantage of using microfluidics ESR is evident in our work, not only because it facilitates the use of a very small sample volume, but also because it makes it possible to apply huge ( ~ 1000 T/m) and fast ( ~ 1 μs) pulsed magnetic field gradients to the sample. This is a key capability to measuring unique properties such as nanoscale real-space diffusion and quantum spin diffusion. All our experiments are performed at room temperature, making our technique compatible with future microfluidics applications that might employ a complete system of compact resonators, microfluidic chips, miniature magnets, and a compact ESR-on-a-chip spectrometer. This could result in a completely new approach to processing and measuring paramagnetic liquid samples for use in a variety of chemical, biological, medical, and environmental applications.

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 (versus 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 analyzed 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 (PMA). 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.

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.

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.

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 ability to assess the extracellular pH (pHe) is an important issue in oncology, because extracellular acidification is associated with tumor aggressiveness and resistance to cytotoxic therapies. In this study, a stable triphosphonated triarylmethyl (TPTAM) radical was qualified as a pHe electron paramagnetic resonance (EPR) molecular reporter. Calibration of hyperfine splitting as a function of pH was performed using a 1.2-GHz EPR spectrometer. Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was used as an extracellular paramagnetic broadening agent to assess the localization of TPTAM when incubated with cells. In vivo EPR pH-metry was performed in MDA, SiHa, and TLT tumor models and in muscle. Bicarbonate therapy was used to modulate the tumor pHe. EPR measurements were compared with microelectrode readouts. The hyperfine splitting of TPTAM was strongly pH-dependent around the pKa of the probe (pKa = 6.99). Experiments with Gd-DTPA demonstrated that TPTAM remained in the extracellular compartment. pHe was found to be more acidic in the MDA, SiHa, and TLT tumor models compared with muscle. Treatment of animals by bicarbonate induced an increase in pHe in tumors: similar variations in pHe were found when using in vivo EPR or invasive microelectrodes measurements. This study demonstrates the potential usefulness of TPTAM for monitoring pHe in tumors. Magn Reson Med 77:2438-2443, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Tetrathiatriarylmethyl (TAM) radicals are favourite spin labels for electron paramagnetic resonance imaging (EPRI). In this work, we report a straightforward synthesis of new TAM radicals bound to an Arg-Gly-Asp (RGD)-peptido-mimetic known to be a cell-targeting agent. These new radicals are stable in solution, and sensitive to oxygen, and their experimental EPR data is consistent with density functional theory (DFT) calculations.

Ultrasmall particles of iron oxide (USPIOs) coated with 3,3'-bis(phosphonate)propionic acid were covalently coupled to a home-made Arg-Gly-Asp (RGD) peptidomimetic molecule via a short oligoethylene-glycol (OEG) spacer. The conjugation rate was measured by X-ray photoelectron spectroscopy (XPS). The particle size and magnetic characteristics were kept. Our novel conjugate targeted efficiently Jurkat cells (increase of 229% vs the control).

This article reports the development and evaluation of two nano-emulsions (F45T-03/HFB and F15T-03/PFOB) containing fluorinated trityl radicals dissolved in perfluorocarbons. Preparation with a high-pressure homogenizer conferred sub-micronic size to both nano-emulsions. In vitro and in vivo EPR spectroscopy showed that the nano-emulsions had much greater oxygen sensitivity than the hydrophilic trityl, CT-03. In vivo experiments in rodents confirmed the ability of the nano-emulsions to follow the changes in oxygen concentration after induced ischemia. Histological evaluation of the tissue injected with the nano-emulsions revealed some acute toxicity for the F45T-03/HFB nano-emulsion but none for the F15T-03/PFOB nano-emulsion. These new formulations should be considered for further EPR oximetry experiments in pathophysiological situations where subtle changes in tissue oxygenation are expected.

A stable RGD-conjugated triarylmethyl (TAM) radical 3 has been synthesized in a straightforward three-step sequence. CT-03 ‘the Finland trityl’ which is a very popular contrast agent for electron paramagnetic resonance imaging has been covalently bound to the NH2-ending of a pentapeptide commonly used to target integrins and confers tissue selectivity. Moreover, this new TAM radical is stable and sensitive to molecular oxygen.

Synthetically useful unsymmetrical bis-silyl or silylstannyl isobutene derivatives are conveniently prepared from selenoisobutene 5, a versatile equivalent of the isobutene dianion. Sequential treatment of this selenated compound with n-butyl-lithium and the required chlorosilanes or chlorostannanes leads efficiently to the corresponding functionalized isobutenes.

Tris(tetrathioaryl)methanols and tris(tetrathioaryl)methanes are direct precursors and metabolites, respectively, of tris(tetrathioaryl)methyl radicals, which are very attractive paramagnetic spin probes for many magnetic resonance applications. Due to steric hindrance, these propeller-shaped molecules display slow interconversion between their two enantiomeric conformations (right-handed and left-handed propeller conformations). Syntheses and HPLC resolutions carried out on a chiral stationary phase have been achieved. The influence of the hybridization of the central carbon atom as well as the para substitutient on the conformational stability were experimentally and theoretically studied. Depending on their particular structure, the free activation energy for the isomerization process determined by kinetics of racemization varies from 24.4 to 26.4 kcal mol -1 and corresponds to racemization half-lives of 12 and 343 h, respectively. Computational and NOESY/exchange spectroscopy (EXSY) studies are consistent with a two-ring flip mechanism for the isomerization process of tris(tetrathioaryl)methanols, radicals and tris(tetrathioaryl) methanes. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

We report that tetrathiatriarylmethyl (trityl) EPR probes are chiral molecules at room temperature, the two stereoisomers that differ in their helicity being configurationally stable enough to be separated and stored independently.

Tetrathiatriarylmethyl radicals are attractive spin probes extensively used in biomedical magnetic resonance applications. We report a straightforward synthesis of two original tetrathiatriarylmethyl radicals incorporating, respectively, 15 and 45 fluorine atoms, and thus possessing a high affinity to fluorous media. F15T-03 and F45T-03 exhibit a single sharp EPR spectrum and their EPR line broadening is highly sensitive to molecular oxygen. These spin probes are specially designed for assessment of tumor oxygenation using perfluorocarbon formulations.

A new water soluble phosphonated tetrathiatriarylmethyl radical has been synthesized and its application for pH measurement in a physiological range by EPR is reported.