SOX71, A Biocompatible Succinyl Derivative of the Triarylmethyl Radical OX071 for In Vivo Quantitative Oxygen Mapping Using Electron Paramagnetic Resonance.

PURPOSE: This study aimed to develop a biocompatible oximetric electron paramagnetic resonance (EPR) spin probe with reduced self-relaxation, and sensitivity to oxygen for a higher signal-to-noise ratio and longer relaxation times at high oxygen concentration, compared to the reference spin probe OX071. PROCEDURES: SOX71 was synthesized by succinylation of the twelve alcohol groups of OX071 spin probe and characterized by EPR at X-Band (9.5 GHz) and at low field (720 MHz). The biocompatibility of SOX71 was tested in vitro and in vivo in mice. A pharmacokinetic study was performed to determine the best time frame for EPR imaging. Finally, a proof-of-concept EPR oxygen imaging was performed on a mouse model of a fibrosarcoma tumor. RESULTS: SOX71 was synthesized in one step from OX071. SOX71 exhibits a narrow line EPR spectrum with a peak-to-peak linewidth of 66 mG, similar to OX071. SOX71 does not bind to albumin nor show cell toxicity for the concentrations tested up to 5 mM. No toxicity was observed after systemic delivery via intraperitoneal injection in mice at twice the dose required for EPR imaging. After the injection, the probe is readily absorbed into the bloodstream, with a peak blood concentration half an hour, post-injection. Then, the probe is quickly cleared by the kidney with a half-life of ~ 45 min. SOX71 shows long relaxation times under anoxic condition (T1e = 9.5 µs and T2e = 5.1 µs; [SOX71] = 1 mM in PBS at 37 °C, pO2 = 0 mmHg, 720 MHz). Both the relaxation rates R1e and R2e show a decreased sensitivity to pO2, leading to twice longer relaxation times under room air conditions (pO2 = 159 mmHg) compared to OX071. This is ideal for oxygen imaging in samples with a wide range of pO2. Both the relaxation rates R1e and R2e show a decreased sensitivity to self-relaxation compared to OX071, with a negligible effect of the probe concentration on R1e. SOX71 was successfully applied to image oxygen in a tumor. CONCLUSION: SOX71, a succinylated derivative of OX071 was synthesized, characterized, and applied for in vivo EPR tumor oxygen imaging. SOX71 is highly biocompatible, and shows decreased sensitivity to oxygen and self-relaxation. This first report suggests that SOX71 is superior to OX071 for absolute oxygen mapping under a broad range of pO2 values.

Toward a Nanoencapsulated EPR Imaging Agent for Clinical Use.

PURPOSE: Progress toward developing a novel radiocontrast agent for determining pO2 in tumors in a clinical setting is described. The imaging agent is designed for use with electron paramagnetic resonance imaging (EPRI), in which the collision of a paramagnetic probe molecule with molecular oxygen causes a spectroscopic change which can be calibrated to give the real oxygen concentration in the tumor tissue. PROCEDURES: The imaging agent is based on a nanoscaffold of aluminum hydroxide (boehmite) with sizes from 100 to 200 nm, paramagnetic probe molecule, and encapsulation with a gas permeable, thin (10-20 nm) polymer layer to separate the imaging agent and body environment while still allowing O2 to interact with the paramagnetic probe. A specially designed deuterated Finland trityl (dFT) is covalently attached on the surface of the nanoparticle through 1,3-dipolar addition of the alkyne on the dFT with an azide on the surface of the nanoscaffold. This click-chemistry reaction affords 100% efficiency of the trityl attachment as followed by the complete disappearance of the azide peak in the infrared spectrum. The fully encapsulated, dFT-functionalized nanoparticle is referred to as RADI-Sense. RESULTS: Side-by-side in vivo imaging comparisons made in a mouse model made between RADI-Sense and free paramagnetic probe (OX-071) showed oxygen sensitivity is retained and RADI-Sense can create 3D pO2 maps of solid tumors CONCLUSIONS: A novel encapsulated nanoparticle EPR imaging agent has been described which could be used in the future to bring EPR imaging for guidance of radiotherapy into clinical reality.

A new 13C trityl-based spin label enables the use of DEER for distance measurements.

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, 13C1-mOX063-d24, with a 13C isotope as the radical center. Due to the resolved hyperfine splitting, the spectrum is sufficiently broadened to permit DEER-based experiments at Q-band spectrometers. Additionally, this new label can be incorporated orthogonally with Cu(II)-based protein label. The orthogonal labeling scheme enables DEER distance measurement at X-band frequencies. Overall, the new trityl label allows for DEER-based distance measurements that complement existing TAM-label DQC and RIDME experiments.

Biocompatible Monophosphonated Trityl Spin Probe, HOPE71, for In Vivo Measurement of pO2, pH, and [Pi] by Electron Paramagnetic Resonance Spectroscopy.

Hypoxia, acidosis, and elevated inorganic phosphate concentration are characteristics of the tumor microenvironment in solid tumors. There are a number of methods for measuring each parameter individually in vivo, but the only method to date for noninvasive measurement of all three variables simultaneously in vivo is electron paramagnetic spectroscopy paired with a monophosphonated trityl radical, pTAM/HOPE. While HOPE has been successfully used for in vivo studies upon intratissue injection, it cannot be delivered intravenously due to systemic toxicity and albumin binding, which causes significant signal loss. Therefore, we present HOPE71, a monophosphonated trityl radical derived from the very biocompatible trityl probe, Ox071. Here, we describe a straightforward synthesis of HOPE71 starting with Ox071 and report its EPR sensitivities to pO2, pH, and [Pi] with X-band and L-band EPR spectroscopy. We also confirm that HOPE71 lacks albumin binding, shows low cytotoxicity, and has systemic tolerance. Finally, we demonstrate its ability to profile the tumor microenvironment in vivo in a mouse model of breast cancer.

EPR Viscometric Measurements Using a 13C-Labeled Triarylmethyl Radical in Protein-based Biotherapeutics and Human Synovial Fluids.

The viscosity measurements are of clinical significance for evaluation of the potential pathological conditions of biological lubricants such as synovial fluids of joints, and for formulation and characterization of peptide- and protein-based biotherapeutics. Due to inherent potential therapeutic activity, protein drugs have proven to be one of the most efficient therapeutic agents in treatment of several life-threatening disorders, such as diabetes and autoimmune diseases. However, home-use applications for treating chronic inflammatory diseases, such as diabetes and rheumatoid arthritis, necessitate the development of high-concentration insulin and monoclonal antibodies formulations for patient self-administration. High protein concentrations can affect viscosity of the corresponding drug solutions complicating their manufacture and administration. The measurements of the viscosity of new insulin analogs and monoclonal antibodies solutions under development is of practical importance to avoid unwanted highly viscous, and therefore, painful for injection drug formulations. Recently, we have demonstrated capability of the electron paramagnetic resonance (EPR) viscometry using viscosity-sensitive 13C-labeled trityl spin probe (13C1-dFT) to report the viscosity of human blood, and interstitial fluids measured in various organs in mice ex-vivo and in anesthetized mice, in vivo. In the present work, we demonstrate utility of the EPR viscometry using 13C1-dFT to measure microviscosity of commercial insulin samples, antibodies solution, and human synovial fluids using small microliter volume samples (5-50 µL). This viscometry analysis approach provides useful tool to control formulations and administration of new biopharmaceuticals, and for evaluation of the state of synovial fluids of importance for clinical applications.

Synthesis and characterization of a biocompatible 13C1 isotopologue of trityl radical OX071 for in vivo EPR viscometry.

We describe the synthesis, characterization, and application of an isotopologue of the trityl radical OX071, labeled with 13C at the central carbon (13C1). This spin probe features large anisotropy of the hyperfine coupling with the 13C1 (I = 1/2), leading to an EPR spectrum highly sensitive to molecular tumbling. The high biocompatibility and lack of interaction with blood albumin allow for systemic delivery and in vivo measurement of tissue microviscosity by EPR.

Large-scale synthesis of a monophosphonated tetrathiatriarylmethyl spin probe for concurrent in vivo measurement of pO2, pH and inorganic phosphate by EPR.

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.

Perchlorinated Triarylmethyl Radical 99% Enriched 13C at the Central Carbon as EPR Spin Probe Highly Sensitive to Molecular Tumbling.

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 determined by spectral simulation or via the line width or the apparent Az after calibration, so the spin probe 13C1-PTMTC can be used to measure media microviscosity with high sensitivity.

Biological Applications of Electron Paramagnetic Resonance Viscometry Using a 13C-Labeled Trityl Spin Probe.

Alterations in viscosity of biological fluids and tissues play an important role in health and diseases. It has been demonstrated that the electron paramagnetic resonance (EPR) spectrum of a 13C-labeled trityl spin probe (13C-dFT) is highly sensitive to the local viscosity of its microenvironment. In the present study, we demonstrate that X-band (9.5 GHz) EPR viscometry using 13C-dFT provides a simple tool to accurately measure the microviscosity of human blood in microliter volumes obtained from healthy volunteers. An application of low-field L-band (1.2 GHz) EPR with a penetration depth of 1-2 cm allowed for microviscosity measurements using 13C-dFT in the living tissues from isolated organs and in vivo in anesthetized mice. In summary, this study demonstrates that EPR viscometry using a 13C-dFT probe can be used to noninvasively and rapidly measure the microviscosity of blood and interstitial fluids in living tissues and potentially to evaluate this biophysical marker of microenvironment under various physiological and pathological conditions in preclinical and clinical settings.

13C isotope enrichment of the central trityl carbon decreases fluid solution electron spin relaxation times.

Electron spin relaxation times for perdeuterated Finland trityl 99% enriched in 13C at the central carbon (13C1-dFT) were measured in phosphate buffered saline (pH = 7.2) (PBS) solution at X-band. The anisotropic 13C1 hyperfine (Ax = Ay = 18 ± 2, Az = 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, τR, is 0.29 ± 0.02 ns. The linewidths are broadened by incomplete motional averaging of the hyperfine anisotropy and T2 is 0.13 ± 0.02 µs, which is shorter than the T2 ~ 3.8 µs for natural abundance dFT at low concentration in PBS. T1 for 13C1-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 T1 in PBS, 3:1 PBS:glycerol (τR = 0.80 ± 0.05 ns, T1 = 9.7 ± 0.7 µs) and 1:1 PBS:glycerol (τR = 3.4 ± 0.3 ns, T1 = 12.0 ± 1.0 µs) was modeled with contributions to the relaxation predominantly from modulation of hyperfine anisotropy and a local mode. The 1/T1 rate for the 1% 12C1-dFT in the predominantly 13C labeled sample is about a factor of 6 more strongly concentration dependent than for natural abundance 12C1-trityl, which reflects the importance of Heisenberg exchange with molecules with different resonance frequencies and faster relaxation rates. In glassy matrices at 160 K, T1 and Tm for 13C1-dFT are in good agreement with previously reported values for 12C1-dFT consistent with the expectation that modulation of nuclear hyperfine does not contribute to electron spin relaxation in a rigid lattice.

Synthesis, Characterization, and Application of a Highly Hydrophilic Triarylmethyl Radical for Biomedical EPR.

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M-1 s-1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔBpp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.

A 13 C-Labeled Triarylmethyl Radical as an EPR Spin Probe Highly Sensitive to Molecular Tumbling.

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.

Development of multifunctional Overhauser-enhanced magnetic resonance imaging for concurrent in vivo mapping of tumor interstitial oxygenation, acidosis and inorganic phosphate concentration.

Tumor oxygenation (pO2), acidosis (pH) and interstitial inorganic phosphate concentration (Pi) are important parameters of the malignant behavior of cancer. A noninvasive procedure that enables visualization of these parameters may provide unique information about mechanisms of tumor pathophysiology and provide clues to new treatment targets. In this research, we present a multiparametric imaging method allowing for concurrent mapping of pH, spin probe concentration, pO2, and Pi using a single contrast agent and Overhauser-enhanced magnetic resonance imaging technique. The developed approach was applied to concurrent multifunctional imaging in phantom samples and in vivo in a mouse model of breast cancer. Tumor tissues showed higher heterogeneity of the distributions of the parameters compared with normal mammary gland and demonstrated the areas of significant acidosis, hypoxia, and elevated Pi content.

Dextran-conjugated tetrathiatriarylmethyl radicals as biocompatible spin probes for EPR spectroscopy and imaging.

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.

Synthesis of hydroxyethyl tetrathiatriarylmethyl radicals OX063 and OX071.

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

Oxygen-induced leakage of spin polarization in Overhauser-enhanced magnetic resonance imaging: Application for oximetry in tumors.

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.

Triarylmethyl-based biradical as a superoxide probe.

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.

Separation of superimposed images with subpixel shift.

The problem of the separation of superimposed images is considered in the particular case of a steady background and a foreground that is composed of different patterns separated in space, each with a compact support. Each pattern of the foreground may move in time independently. A single pair of these superimposed images is assumed to be available, and the displacement amplitude is typically smaller than the pixel size. Further, assuming that the background is smoothly varying in space, an original algorithm is proposed. To illustrate the performance of the method, a real test case of X-ray tomographic radiographs with moving patterns due to dust particles or surface scratches of optical elements along the beam is considered. Finally an automatic and simple treatment is proposed to erase the effects of such features.

On the use of flat-fields for tomographic reconstruction.

Seeking for quantitative tomographic images, it is of utmost importance to limit reconstruction artifacts. Detector imperfections, inhomogeneity of the incident beam, as classically observed in synchrotron beamlines, and their variations in time are a major cause of reconstruction bias such as `ring artifacts'. The present study aims at proposing a faithful estimate of the incident beam local intensity for each acquired projection during a scan, without revisiting the process of data acquisition itself. Actual flat-fields (acquired without specimen in the beam) and sinogram borders (when the specimen is present), which are not masked during the scan, are exploited to construct a suited instantaneous detector-wide flat-field. The proposed treatment is fast and simple. Its performance is assessed on a real scan acquired at ESRF ID19 beamline. Different criteria are used including residuals, i.e. difference between projections of reconstruction and actual projections. All confirm the benefit of the proposed procedure.