Continuous-flow electron spin resonance microfluidics device with sub-nanoliter sample volume.

This paper presents a novel continuous-flow electron spin resonance (ESR) microfluidic device designed for both continuous-wave (CW) and pulsed ESR measurements on sub-nanoliter liquid samples. The system integrates a planar surface microresonator (ParPar type) operating at ~9.4 GHz with a precision-fabricated quartz microfluidic chip, enabling spatial confinement of the sample within the resonator's microwave magnetic field hotspot while minimizing dielectric losses.

Development of uMUC-1 Targeted NEMO Particles with pH-Activatable MRI Signals for Enhanced Detection of Malignant Breast Cancer Cells.

Magnetic resonance imaging (MRI) detects more breast cancers than mammography due to its superior soft tissue contrast; however, it still misdiagnoses 40% of benign tumors as malignant due to clinically used nonspecific contrast agents (e.g., gadolinium chelates).

Reaction of polarizing agent Ox063 with pyruvic acid under standard sample preparation protocol for dissolution dynamic nuclear polarization.

Dynamic nuclear polarization is a technique that significantly enhances signal intensity in nuclear magnetic resonance spectroscopy and imaging. In a DNP experiment, a sample of interest is doped with a radical, and microwaves are applied in a strong magnetic field, leading to an increase in nuclear spin polarization. Notably, the potential reactions between the sample and the polarization agent are rarely considered.

Operando detection of dissolved oxygen in fluid solution using a submersible rapid scan EPR on a chip dipstick sensor.

Electron paramagnetic resonance (EPR) is an accurate and efficient technique to probe unpaired electrons in many applications across materials science, chemistry, and biology. Dynamic processes are investigated using EPR; however, these applications are limited by the use of resonator-based spectrometers such that the entire process must be confined to the resonator. The EPR-on-a-chip (EPRoC) device circumvents this limitation by integrating the entire EPR spectrometer into a single microchip.

Line-Shifting Triarylmethyl Radicals for Imaging of Enzyme Activity Using Overhauser-Enhanced Magnetic Resonance Imaging: Application to Alkaline Phosphatase.

Enzyme catalytic activities are critical biomarkers of tissue states under physiological and pathophysiological conditions. However, the direct measurement and imaging of enzyme activity remains extremely challenging. We report the synthesis and characterization of the first stable triarylmethyl (TAM) radical substrate of alkaline phosphatase (TAM-ALPs).

Evaluation of Mesoporous Silica Nanoparticles as Carriers of Triarylmethyl Radical Spin Probes for EPR Oximetry.

measurement and mapping of oxygen levels within the tissues are crucial in understanding the physiopathological processes of numerous diseases, such as cancer, diabetes, or peripheral vascular diseases. Electron paramagnetic resonance (EPR) associated with biocompatible exogenous spin probes, such as Ox071 triarylmethyl (TAM) radical, is becoming the new gold standard for oxygen mapping in preclinical settings. However, these probes do not show tissue selectivity when injected systemically, and they are not cell permeable, reporting oxygen from the extracellular compartment only.

EPR Viscometric Measurements Using a C-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.

Sensitive simultaneous measurements of oxygenation and extracellular pH by EPR using a stable monophosphonated trityl radical and lithium phthalocyanine.

The monitoring of acidosis and hypoxia is crucial because both factors promote cancer progression and impact the efficacy of anti-cancer treatments. A phosphonated tetrathiatriarylmethyl (pTAM) has been previously described to monitor both parameters simultaneously, but the sensitivity to tackle subtle changes in oxygenation was limited. Here, we describe an innovative approach combining the pTAM radical and lithium phthalocyanine (LiPc) crystals to provide sensitive simultaneous measurements of extracellular pH (pH) and pO.

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).

Toward a Nanoencapsulated EPR Imaging Agent for Clinical Use.

Purpose: Progress toward developing a novel radiocontrast agent for determining pO 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 O to interact with the paramagnetic probe.

Dose-Specific Intratumoral GM-CSF Modulates Breast Tumor Oxygenation and Antitumor Immunity.

GM-CSF has been employed as an adjuvant to cancer immunotherapy with mixed results based on dosage. We previously showed that GM-CSF regulated tumor angiogenesis by stimulating soluble vascular endothelial growth factor (VEGF) receptor-1 from monocytes/macrophages in a dose-dependent manner that neutralized free VEGF, and intratumoral injections of high-dose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic polyoma middle T Ag (PyMT) triple-negative breast cancer (TNBC). In this study, we assessed both immunoregulatory and oxygen-regulatory components of low-dose versus high-dose GM-CSF to compare effects on tumor oxygen, vasculature, and antitumor immunity.

In Vivo Electron Paramagnetic Resonance Molecular Profiling of Tumor Microenvironment upon Tumor Progression to Malignancy in an Animal Model of Breast Cancer.

Purpose: Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy.

Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

Background: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer.

Methods: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models.

A new C 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.

Biocompatible Monophosphonated Trityl Spin Probe, HOPE71, for In Vivo Measurement of pO, pH, and [P] 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.

Synthesis and characterization of a biocompatible C isotopologue of trityl radical OX071 for EPR viscometry.

We describe the synthesis, characterization, and application of an isotopologue of the trityl radical OX071, labeled with C at the central carbon (C). This spin probe features large anisotropy of the hyperfine coupling with the C ( = 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 measurement of tissue microviscosity by EPR.

Observing Nearby Nuclei on Paramagnetic Trityls and MOFs via DNP and Electron Decoupling.

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.

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

Low-field electron paramagnetic resonance spectroscopy paired with , a mono-phosphonated triarylmethyl radical, is an unmatched technique for concurrent and non-invasive measurement of oxygen concentration, pH, and inorganic phosphate concentration for investigations. However, the prior reported synthesis is limited by its low yield and poor scalability, making wide-spread application of unfeasible. Here, we report a new strategy for the synthesis of with significantly greater yields demonstrated on a large scale.

Formulation and In Vitro Characterization of PLGA/PLGA-PEG Nanoparticles Loaded with Murine Granulocyte-Macrophage Colony-Stimulating Factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated notable clinical activity in cancer immunotherapy, but it 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.

Biological Applications of Electron Paramagnetic Resonance Viscometry Using a C-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 C-labeled trityl spin probe (C-dFT) is highly sensitive to the local viscosity of its microenvironment. In the present study, we demonstrate that X-band (9.

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

Sensitive 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 (). This property leads to a sufficiently intense ESR signal for reliable distance measurements.

C 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 C at the central carbon (C-dFT) were measured in phosphate buffered saline (pH = 7.2) (PBS) solution at X-band. The anisotropic C hyperfine (A = A = 18 ± 2, A = 162 ± 1 MHz) and g values (2.

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.