Contrast Agents Based on Human Serum Albumin and Nitroxides for 1H-MRI and Overhauser-Enhanced MRI.

In cancer diagnostics, magnetic resonance imaging (MRI) uses contrast agents to enhance the distinction between the target tissue and background. Several promising approaches have been developed to increase MRI sensitivity, one of which is Overhauser dynamic nuclear polarization (ODNP)-enhanced MRI (OMRI). In this study, a macromolecular construct based on human serum albumin and nitroxyl radicals (HSA-NIT) was developed using a new synthesis method that significantly increased the modification to 21 nitroxide residues per protein. This was confirmed by electron paramagnetic resonance (EPR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry. Gel electrophoresis and circular dichroism showed no significant changes in the structure of HSA-NITs, and no oligomers were formed during modification. The cytotoxicity of HSA-NITs was comparable to that of native albumin. HSA-NITs were evaluated as potential "metal-free" organic radical relaxation-based contrast agents for 1H-MRI and as hyperpolarizing contrast agents for OMRI. Relaxivities (longitudinal and transversal relaxation rates r1 and r2) for HSA-NITs were measured at different magnetic field strengths (1.88, 3, 7, and 14 T). Phantoms were used to demonstrate the potential use of HSA-NIT as a T1- and T2-weighted relaxation-based contrast agent at 3 T and 14 T. The efficacy of 1H Overhauser dynamic nuclear polarization (ODNP) in liquids at an ultralow magnetic field (ULF, B0 = 92 ± 0.8 µT) was investigated for HSA-NIT conjugates. The HSA-NITs themselves did not show ODNP enhancement; however, under the proteolysis conditions simulating cancer tissue, HSA-NIT conjugates were cleaved into lower-molecular-weight (MW) protein fragments that activate ODNP capabilities, resulting in a maximum achievable enhancement |Emax| of 40-50 and a radiofrequency power required to achieve half of Emax, P1/2, of 21-27 W. The HSA-NIT with a higher degree of modification released increased the number of spin probes upon biodegradation, which significantly enhanced the Overhauser effect. Thus, HSA-NITs may represent a new class of MRI relaxation-based contrast agents as well as novel cleavable conjugates for use as hyperpolarizing contrast agents (HCAs) in OMRI.

An Overhauser-enhanced-MRI platform for dynamic free radical imaging in vivo.

Overhauser-enhanced MRI (OMRI) is an electron-proton double-resonance imaging technique of interest for its ability to non-invasively measure the concentration and distribution of free radicals. In vivo OMRI experiments are typically undertaken at ultra-low magnetic field (ULF), as both RF power absorption and penetration issues-a consequence of the high resonance frequencies of electron spins-are mitigated. However, working at ULF causes a drastic reduction in MRI sensitivity. Here, we report on the design, construction and performance of an OMRI platform optimized for high NMR sensitivity and low RF power absorbance, exploring challenges unique to probe design in the ULF regime. We use this platform to demonstrate dynamic imaging of TEMPOL in a rat model. The work presented here demonstrates improved speed and sensitivity of in vivo OMRI, extending the scope of OMRI to the study of dynamic processes such as metabolism.

Construction of 0.15 Tesla Overhauser Enhanced MRI.

Overhauser enhanced MRI (OMRI) is one of the free radical imaging technologies and has been used in biomedical research such as for partial oxygen measurements in tumor, and redox status in acute oxidative diseases. The external magnetic field of OMRI is frequently in the range of 5-10 mTesla to ensure microwave penetration into small animals, and the S/N ratio is limited. In this study, a 0.15 Tesla OMRI was constructed and tested to improve the S/N ratio for a small sample, or skin measurement. Specification of the main magnet was as follows: 0.15 Tesla permanent magnet; gap size 160 mm; homogenous spherical volume of 80 mm in diameter. The OMRI resonator was designed based on TE101 cavity mode and machined from a phosphorus deoxidized copper block for electron spin resonance (ESR) excitation and a solenoid transmission/receive resonator for NMR detection. The resonant frequencies and Q values were 6.38 MHz/150 and 4.31-4.41 GHz/120 for NMR and ESR, respectively. The Q values were comparable to those of conventional low field OMRI resonators at 15 mTesla. As expected, the MRI S/N ratio was improved by a factor of 30. Triplet dynamic nuclear polarization spectra were observed for 14N carboxy-PROXYL, along the excitation microwave sweep. In the current setup, the enhancement factor was ca. 0.5. In conclusion, the results of this preliminary evaluation indicate that the 0.15 Tesla OMRI could be useful for free radical measurement for small samples.

Quantification of regional CMRO2 in human brain using dynamic 17O-MRI at 3T.

OBJECTIVE: To investigate the feasibility of cerebral metabolic rate of oxygen consumption (CMRO2) measurements with MRI at 3 Tesla in different brain regions. METHODS: CMRO2 represents a key indicator of the physiological state of brain tissue. Dynamic 17O-MRI with inhalation of isotopically enriched 17O gas has been used to quantify global CMRO2 in brain white (WM) and gray matter (GM). However, global CMRO2 can only reflect the overall oxygen metabolism of the brain and cannot provide enough information on local tissue oxygen metabolism. To investigate the feasibility of determination of regional CMRO2 at a clinical 3 T MRI system, CMRO2 values in frontal, parietal and occipital WM and GM were determined in 5 healthy volunteers and compared to evaluate the regional differences of oxygen metabolism in WM and GM. Additionally, regional CMRO2 values were determined in deep brain structures including thalamus, dorsal striatum, caudate nucleus and insula cortex and in the cerebella, and compared with literature values from 15O-PET studies. RESULTS: In cortical GM the determined CMRO2 values were in good agreement with the literature, whereas values in WM were about 32-48% higher than literature values. Regional analysis revealed a significantly higher CMRO2 in the occipital GM compared to the frontal and parietal GM. By contrast, no significant difference of CMRO2 was observed across the WM. In addition, CMRO2 in deep brain structures was lower compared to literature values and in the cerebella a good hemispheric symmetry of the tissue oxygen metabolism was found. CONCLUSION: Dynamic 17O-MRI enables direct, non-invasive determination of regional CMRO2 in brain structures in healthy volunteers at 3T.

Research and Development of Preclinical Overhauser-Enhanced Magnetic Resonance Imaging.

Significance: Imaging free radicals, including reactive oxygen species and reactive nitrogen species, can be useful for understanding the pathology of diseases in animal disease models, as they are related to various physiological functions or diseases. Among the methods used for imaging free radicals, Overhauser-enhanced magnetic resonance imaging (OMRI) has a short image acquisition time and high spatial resolution. Therefore, OMRI is used to obtain various biological parameters. In this study, we review the methodology for improving the biological OMRI system and its applications. Recent Advances: The sensitivity of OMRI systems has been enhanced significantly to allow the visualization of various biological parameters, such as redox state, partial oxygen pressure, and pH, in different body parts of small animals, using spin probes. Furthermore, both endogenous free radicals and exogenous free radicals present in drugs can be visualized using OMRI. Critical Issues: To acquire accurate biological parameters at a high resolution, it is essential to increase the electron paramagnetic resonance (EPR) excitation efficiency and achieve a high enhancement factor. In addition, the size and magnetic field strength also need to be optimized for the measurement target. Future Directions: The advancement of in vivo OMRI techniques will be useful for understanding the pathology, diagnosis, and evaluation of therapeutic effects of drugs in various disease models. Antioxid. Redox Signal. 37, 1094-1110.

In Vivo Application of Proton-Electron Double-Resonance Imaging.

SIGNIFICANCE: 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 (pO2), tissue permeability, redox status, and acid-base balance. Recent Advances: Local acid-base balance can be imaged by exploiting the different resonance frequency of radical probes between R and RH+ forms. Redox status can also be imaged by using the loss of radical-related signal after reduction. These methods require optimized radical probes and pulse sequences. CRITICAL ISSUES: High-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. FUTURE DIRECTIONS: 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.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.

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.

In vivo Overhauser-enhanced MRI of proteolytic activity.

There is an increasing interest in developing novel imaging strategies for sensing proteolytic activities in intact organisms in vivo. Overhauser-enhanced MRI (OMRI) offers the possibility to reveal the proteolysis of nitroxide-labeled macromolecules thanks to a sharp decrease of the rotational correlation time of the nitroxide moiety upon cleavage. In this paper, this concept is illustrated in vivo at 0.2 T using nitroxide-labeled elastin orally administered in mice. In vitro, this elastin derivative was OMRI-visible and gave rise to high Overhauser enhancements (19-fold at 18 mm nitroxide) upon proteolysis by pancreatic porcine elastase. In vivo three-dimensional OMRI detection of proteolysis was carried out. A keyhole fully balanced steady-state free precession sequence was used, which allowed 3D OMRI acquisition within 20 s at 0.125 mm(3) resolution. About 30 min after mouse gavage, proteolysis was detected in the duodenum, where Overhauser enhancements were 7.2 ± 2.4 (n = 7) and was not observed in the stomach. Conversely, orally administered free nitroxides or pre-digested nitroxide-labeled elastin were detected in the mouse's stomach by OMRI. Combined with specific molecular probes, this Overhauser-enhanced MRI technique can be used to evaluate unregulated proteolytic activities in various models of experimental diseases and for drug testing.

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping.

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-gadolinium-diethylene triamine pentaacetic acid-biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T1 relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.