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.

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.

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.

Cleavage-Resistant Protein Labeling With Hydrophilic Trityl Enables Distance Measurements In-Cell.

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 (Tm). This property leads to a sufficiently intense ESR signal for reliable distance measurements. Furthermore, the Tm 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-d24). 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 (T1) 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 Tm of mOX063-d24 is sufficiently long even in the crowded environment of the cell, leading to signals of appreciable intensity. Overall, mOX063-d24 provides highly sensitive distance measurements both in vitro and in-cells.

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.