TEMPO-conjugated tobacco mosaic virus as a magnetic resonance imaging contrast agent for detection of superoxide production in the inflamed liver.

Superoxide, an anionic dioxygen molecule, plays a crucial role in redox regulation within the body but is implicated in various pathological conditions when produced excessively. Efforts to develop superoxide detection strategies have led to the exploration of organic-based contrast agents for magnetic resonance imaging (MRI). This study compares the effectiveness of two such agents, nTMV-TEMPO and kTMV-TEMPO, for detecting superoxide in a mouse liver model with lipopolysaccharide (LPS)-induced inflammation. The study demonstrates that kTMV-TEMPO, with a strategically positioned lysine residue for TEMPO attachment, outperforms nTMV-TEMPO as an MRI contrast agent. The enhanced sensitivity of kTMV-TEMPO is attributed to its more exposed TEMPO attachment site, facilitating stronger interactions with water protons and superoxide radicals. EPR kinetics experiments confirm kTMV-TEMPO's faster oxidation and reduction rates, making it a promising sensor for superoxide in inflamed liver tissue. In vivo experiments using healthy and LPS-induced inflamed mice reveal that reduced kTMV-TEMPO remains MRI-inactive in healthy mice but becomes MRI-active in inflamed livers. The contrast enhancement in inflamed livers is substantial, validating the potential of kTMV-TEMPO for detecting superoxide in vivo. This research underscores the importance of optimizing contrast agents for in vivo imaging applications. The enhanced sensitivity and biocompatibility of kTMV-TEMPO make it a promising candidate for further studies in the realm of medical imaging, particularly in the context of monitoring oxidative stress-related diseases.

Aggregation-induced emission luminogens for highly effective microwave dynamic therapy.

Aggregation-induced emission luminogens (AIEgens) exhibit efficient cytotoxic reactive oxygen species (ROS) generation capability and unique light-up features in the aggregated state, which have been well explored in image-guided photodynamic therapy (PDT). However, the limited penetration depth of light in tissue severely hinders AIEgens as a candidate for primary or adjunctive therapy for clinical applications. Coincidentally, microwaves (MWs) show a distinct advantage for deeper penetration depth in tissues than light. Herein, for the first time, we report AIEgen-mediated microwave dynamic therapy (MWDT) for cancer treatment. We found that two AIEgens (TPEPy-I and TPEPy-PF6) served as a new type of microwave (MW) sensitizers to produce ROS, including singlet oxygen (1O2), resulting in efficient destructions of cancer cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays reveal that the two AIEgens when activated by MW irradiation can effectively kill cancer cells with average IC-50 values of 2.73 and 3.22 µM, respectively. Overall, the ability of the two AIEgens to be activated by MW not only overcomes the limitations of conventional PDT, but also helps to improve existing MW ablation therapy by reducing the MW dose required to achieve the same therapeutic outcome, thus reducing the occurrence of side-effects of MW radiation.

Hyperpolarized 89Y-EDTMP complex as a chemical shift-based NMR sensor for pH at the physiological range.

Yttrium (III) complexes are interesting due to the similarity of their chemistry with gadolinium complexes that are used as contrast agents in nuclear magnetic resonance (NMR) spectroscopy or imaging (MRI). While most of the paramagnetic Gd3+-based MRI contrast agents are T1 or T2 relaxation-based sensors such as Gd3+-complexes for zinc or pH detection, a number of diamagnetic Y3+-complexes rely on changes in the chemical shift for potential quantitative MRI in biological milieu. 89Y, however, is a challenging nucleus to work with in conventional NMR or MRI due to its inherently low sensitivity and relatively long T1 relaxation time. This insensitivity problem in 89Y-based complexes can be circumvented with the use of dissolution dynamic nuclear polarization (DNP) which allows for several thousand-fold enhancement of the NMR or MRI signal relative to thermal equilibrium signal. Herein, we report on the feasibility of using hyperpolarized 89Y-complexes with phosphonated open-chain ligands, 89Y-EDTMP and 89Y-DTPMP, as potential chemical shift-based pH NMR sensors. Our DNP-NMR data show that hyperpolarized 89Y-DTPMP has an apparent pKa ~ 7.01 with a 4 ppm-wide chemical shift dispersion with the signal disappearing at pH below 6.2. On the other hand, pH titration data on hyperpolarized 89Y-EDTMP show that it has an apparent pKa of pH 6.7 and a 16-ppm wide chemical shift dispersion at pH 5-9 range. In comparison, the previously reported hyperpolarized pH NMR sensor 89Y-DOTP has a pKa of 7.64 and ~ 10-ppm wide chemical shift dispersion at pH 4-9 range. Overall, our data suggest that hyperpolarized 89Y-EDTMP is better than hyperpolarized 89Y-DOTP in terms of pH sensing capability at the physiological range.

Supramolecular and biomacromolecular enhancement of metal-free magnetic resonance imaging contrast agents.

Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further overcame the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.

Hyperpolarized 15N-labeled, deuterated tris (2-pyridylmethyl)amine as an MRI sensor of freely available Zn2.

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.

Effects of glassing matrix deuteration on the relaxation properties of hyperpolarized 13C spins and free radical electrons at cryogenic temperatures.

Glassing matrix deuteration could be a beneficial sample preparation method for 13C dynamic nuclear polarization (DNP) when large electron paramagnetic resonance (EPR) width free radicals are used. However, it could yield the opposite DNP effect when samples are doped with small EPR width free radicals. Herein, we have investigated the influence of solvent deuteration on the 13C nuclear and electron relaxation that go along with the effects on 13C DNP intensities at 3.35 T and 1.2 K. For 13C DNP samples doped with trityl OX063, the 13C DNP signals decreased significantly when the protons are replaced by deuterons in glycerol:water or DMSO:water solvents. Meanwhile, the corresponding solid-state 13C T1 relaxation times of trityl OX063-doped samples generally increased upon solvent deuteration. On the other hand, 13C DNP signals improved by a factor of ∼1.5 to 2 upon solvent deuteration of samples doped with 4-oxo-TEMPO. Despite this 13C DNP increase, there were no significant differences recorded in 13C T1 values of TEMPO-doped samples with nondeuterated or fully deuterated glassing matrices. While solvent deuteration appears to have a negligible effect on the electron T1 relaxation of both free radicals, the electron T2 relaxation times of these two free radicals generally increased upon solvent deuteration. These overall results suggest that while the solid-phase 13C DNP signals are dependent upon the changes in total nuclear Zeeman heat capacity, the 13C relaxation effects are related to 2H/1H nuclear spin diffusion-assisted 13C polarization leakage in addition to the dominant paramagnetic relaxation contribution of free radical centers.

NMR Spectroscopy Unchained: Attaining the Highest Signal Enhancements in Dissolution Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) via the dissolution method is one of the most successful methods for alleviating the inherently low Boltzmann-dictated sensitivity in nuclear magnetic resonance (NMR) spectroscopy. This emerging technology has already begun to positively impact chemical and metabolic research by providing the much-needed enhancement of the liquid-state NMR signals of insensitive nuclei such as 13C by several thousand-fold. In this Perspective, we present our viewpoints regarding the key elements needed to maximize the NMR signal enhancements in dissolution DNP, from the very core of the DNP process at cryogenic temperatures, DNP instrumental conditions, and chemical tuning in sample preparation to current developments in minimizing hyperpolarization losses during the dissolution transfer process. The optimization steps discussed herein could potentially provide important experimental and theoretical considerations in harnessing the best possible sensitivity gains in NMR spectroscopy as afforded by optimized dissolution DNP technology.

Dynamic nuclear polarization of carbonyl and methyl 13C spins of acetate using 4-oxo-TEMPO free radical.

Hyperpolarization of 13C-enriched biomolecules via dissolution dynamic nuclear polarization (DNP) has enabled real-time metabolic imaging of a variety of diseases with superb specificity and sensitivity. The source of the unprecedented liquid-state nuclear magnetic resonance spectroscopic or imaging signal enhancements of >10 000-fold is the microwave-driven DNP process that occurs at a relatively high magnetic field and cryogenic temperature. Herein, we have methodically investigated the relative efficiencies of 13C DNP of single or double 13C-labeled sodium acetate with or without 2H-enrichment of the methyl group and using a 4-oxo-TEMPO free radical as the polarizing agent at 3.35 T and 1.4 K. The main finding of this work is that not all 13C spins in acetate are polarized with equal DNP efficiency using this relatively wide electron spin resonance linewidth free radical. In fact, the carbonyl 13C spins have about twice the solid-state 13C polarization level of methyl 13C spins. Deuteration of the methyl group provides a DNP signal improvement of methyl 13C spins on a par with that of carbonyl 13C spins. On the other hand, both the double 13C-labeled [1,2-13C2] acetate and [1,2-13C2, 2H3] acetate have a relative solid-state 13C polarization at the level of [2-13C] acetate. Meanwhile, the solid-state 13C T1 relaxation times at 3.35 T and 1.4 K were essentially the same for all six isotopomers of 13C acetate. These results suggest that the intramolecular environment of 13C spins plays a prominent role in determining the 13C DNP efficiency, while the solid phase 13C T1 relaxation of these samples is dominated by the paramagnetic effect due to the relatively high concentration of free radicals.

Magnetic-Field-Dependent Lifetimes of Hyperpolarized 13C Spins at Cryogenic Temperature.

Using a home-built cryogen-free dynamic nuclear polarization (DNP) system with a variable magnetic field capability, 13C spin-lattice T1 relaxation times of hyperpolarized [1-13C] carboxylates (sodium acetate, glycine, sodium pyruvate, and pyruvic acid) doped with trityl OX063 free radical were systematically measured for the first time at different field strengths up to 9 T at T = 1.8 K. Our data reveal that the 13C T1 values of these frozen hyperpolarized 13C samples vary drastically with the applied magnetic field B according to an apparent empirical power-law dependence (13C T1 ∝ Bα, 2.3 < α < 3.1), with relaxation values ranging from a few hundred seconds at 1 T to over 200,000 s at fields close to 9 T. This low temperature relaxation behavior can be ascribed approximately to a model that accounts for the combined effect of 13C-1H intramolecular dipolar interaction and the relaxation contribution from the paramagnetic impurities present in the DNP sample. Since the lifetime or T1 storage of the hyperpolarized state is intimately linked to DNP efficiency, these 13C relaxation data at cryogenic temperature have important theoretical and experimental implications as the DNP of 13C-labeled biomolecules is pushed to higher magnetic fields.

Transition Metal Doping Reveals Link between Electron T1 Reduction and 13C Dynamic Nuclear Polarization Efficiency.

Optimal efficiency of dissolution dynamic nuclear polarization (DNP) is essential to provide the required high sensitivity enhancements for in vitro and in vivo hyperpolarized 13C nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI). At the nexus of the DNP process are the free electrons, which provide the high spin alignment that is transferred to the nuclear spins. Without changing DNP instrumental conditions, one way to improve 13C DNP efficiency is by adding trace amounts of paramagnetic additives such as lanthanide (e.g., Gd3+, Ho3+, Dy3+, Tb3+) complexes to the DNP sample, which has been observed to increase solid-state 13C DNP signals by 100-250%. Herein, we have investigated the effects of paramagnetic transition metal complex R-NOTA (R = Mn2+, Cu2+, Co2+) doping on the efficiency of 13C DNP using trityl OX063 as the polarizing agent. Our DNP results at 3.35 T and 1.2 K show that doping the 13C sample with 3 mM Mn2+-NOTA led to a substantial improvement of the solid-state 13C DNP signal by a factor of nearly 3. However, the other transition metal complexes Cu2+-NOTA and Co2+-NOTA complexes, despite their paramagnetic nature, had essentially no impact on solid-state 13C DNP enhancement. W-band electron paramagnetic resonance (EPR) measurements reveal that the trityl OX063 electron T1 was significantly reduced in Mn2+-doped samples but not in Cu2+- and Co2+-doped DNP samples. This work demonstrates, for the first time, that not all paramagnetic additives are beneficial to DNP. In particular, our work provides a direct evidence that electron T1 reduction of the polarizing agent by a paramagnetic additive is an essential requirement for the improvement seen in solid-state 13C DNP signal.

13C Dynamic Nuclear Polarization Using a Trimeric Gd3+ Complex as an Additive.

Dissolution dynamic nuclear polarization (DNP) is one of the most successful techniques that resolves the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) by amplifying the signal by several thousand-fold. One way to further improve the DNP signal is the inclusion of trace amounts of lanthanides in DNP samples doped with trityl OX063 free radical as the polarizing agent. In practice, stable monomeric gadolinium complexes such as Gd-DOTA or Gd-HP-DO3A are used as beneficial additives in DNP samples, further boosting the DNP-enhanced solid-state 13C polarization by a factor of 2 or 3. Herein, we report on the use of a trimeric gadolinium complex as a dopant in 13C DNP samples to improve the 13C DNP signals in the solid-state at 3.35 T and 1.2 K and consequently, in the liquid-state at 9.4 T and 298 K after dissolution. Our results have shown that doping the 13C DNP sample with a complex which holds three Gd3+ ions led to an improvement of DNP-enhanced 13C polarization by a factor of 3.4 in the solid-state, on par with those achieved using monomeric Gd3+ complexes but only requires about one-fifth of the concentration. Upon dissolution, liquid-state 13C NMR signal enhancements close to 20 000-fold, approximately 3-fold the enhancement of the control samples, were recorded in the nearby 9.4 T high resolution NMR magnet at room temperature. Comparable reduction of 13C spin-lattice T1 relaxation time was observed in the liquid-state after dissolution for both the monomeric and trimeric Gd3+ complexes. Moreover, W-band electron paramagnetic resonance (EPR) data have revealed that 3-Gd doping significantly reduces the electron T1 of the trityl OX063 free radical, but produces negligible changes in the EPR spectrum, reminiscent of the results with monomeric Gd3+-complex doping. Our data suggest that the trimeric Gd3+ complex is a highly beneficial additive in 13C DNP samples and that its effect on DNP efficiency can be described in the context of the thermal mixing mechanism.

Assembly and performance of a 6.4 T cryogen-free dynamic nuclear polarization system.

We report on the assembly and performance evaluation of a 180-GHz/6.4 T dynamic nuclear polarization (DNP) system based on a cryogen-free superconducting magnet. The DNP system utilizes a variable-field superconducting magnet that can be ramped up to 9 T and equipped with cryocoolers that can cool the sample space with the DNP assembly down to 1.8 K via the Joule-Thomson effect. A homebuilt DNP probe insert with top-tuned nuclear magnetic resonance coil and microwave port was incorporated into the sample space in which the effective sample temperature is approximately 1.9 K when a 180-GHz microwave source is on during DNP operation. 13 C DNP of [1-13 C] acetate samples doped with trityl OX063 and 4-oxo-TEMPO in this system have resulted in solid-state 13 C polarization levels of 58 ± 3% and 18 ± 2%, respectively. The relatively high 13 C polarization levels achieved in this work have demonstrated that the use of a cryogen-free superconducting magnet for 13 C DNP is feasible and in fact, relatively efficient-a major leap to offset the high cost of liquid helium consumption in DNP experiments.

Construction and 13 C hyperpolarization efficiency of a 180 GHz dissolution dynamic nuclear polarization system.

Dynamic nuclear polarization (DNP) via the dissolution method has become one of the rapidly emerging techniques to alleviate the low signal sensitivity in nuclear magnetic resonance (NMR) spectroscopy and imaging. In this paper, we report on the development and 13 C hyperpolarization efficiency of a homebuilt DNP system operating at 6.423 T and 1.4 K. The DNP hyperpolarizer system was assembled on a wide-bore superconducting magnet, equipped with a standard continuous-flow cryostat, and a 180 GHz microwave source with 120 mW power output and wide 4 GHz frequency tuning range. At 6.423 T and 1.4 K, solid-state 13 C polarization P levels of 64% and 31% were achieved for 3 M [1-13 C] sodium acetate samples in 1 : 1 v/v glycerol:water glassing matrix doped with 15 mM trityl OX063 and 40 mM 4-oxo-TEMPO, respectively. Upon dissolution, which takes about 15 s to complete, liquid-state 13 C NMR signal enhancements as high as 240 000-fold (P=21%) were recorded in a nearby high resolution 13 C NMR spectrometer at 1 T and 297 K. Considering the relatively lower cost of our homebuilt DNP system and the relative simplicity of its design, the dissolution DNP setup reported here could be feasibly adapted for in vitro or in vivo hyperpolarized 13 C NMR or magnetic resonance imaging at least in the pre-clinical setting. Copyright © 2017 John Wiley & Sons, Ltd.

Influence of 13C Isotopic Labeling Location on Dynamic Nuclear Polarization of Acetate.

Dynamic nuclear polarization (DNP) via the dissolution method has alleviated the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy by amplifying the signals by several thousand-fold. This NMR signal amplification process emanates from the microwave-mediated transfer of high electron spin alignment to the nuclear spins at high magnetic field and cryogenic temperature. Since the interplay between the electrons and nuclei is crucial, the chemical composition of a DNP sample such as the type of free radical used, glassing solvents, or the nature of the target nuclei can significantly affect the NMR signal enhancement levels that can be attained with DNP. Herein, we have investigated the influence of 13C isotopic labeling location on the DNP of a model 13C compound, sodium acetate, at 3.35 T and 1.4 K using the narrow electron spin resonance (ESR) line width free radical trityl OX063. Our results show that the carboxyl 13C spins yielded about twice the polarization produced in methyl 13C spins. Deuteration of the methyl 13C group, while proven beneficial in the liquid-state, did not produce an improvement in the 13C polarization level at cryogenic conditions. In fact, a slight reduction of the solid-state 13C polarization was observed when 2H spins are present in the methyl group. Furthermore, our data reveal that there is a close correlation between the solid-state 13C T1 relaxation times of these samples and the relative 13C polarization levels. The overall results suggest the achievable solid-state polarization of 13C acetate is directly affected by the location of the 13C isotopic labeling via the possible interplay of nuclear relaxation leakage factor and cross-talks between nuclear Zeeman reservoirs in DNP.

Influence of Dy3+ and Tb3+ doping on 13C dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) is a technique that uses a microwave-driven transfer of high spin alignment from electrons to nuclear spins. This is most effective at low temperature and high magnetic field, and with the invention of the dissolution method, the amplified nuclear magnetic resonance (NMR) signals in the frozen state in DNP can be harnessed in the liquid-state at physiologically acceptable temperature for in vitro and in vivo metabolic studies. A current optimization practice in dissolution DNP is to dope the sample with trace amounts of lanthanides such as Gd3+ or Ho3+, which further improves the polarization. While Gd3+ and Ho3+ have been optimized for use in dissolution DNP, other lanthanides have not been exhaustively studied for use in C13 DNP applications. In this work, two additional lanthanides with relatively high magnetic moments, Dy3+ and Tb3+, were extensively optimized and tested as doping additives for C13 DNP at 3.35 T and 1.2 K. We have found that both of these lanthanides are also beneficial additives, to a varying degree, for C13 DNP. The optimal concentrations of Dy3+ (1.5 mM) and Tb3+ (0.25 mM) for C13 DNP were found to be less than that of Gd3+ (2 mM). W-band electron paramagnetic resonance shows that these enhancements due to Dy3+ and Tb3+ doping are accompanied by shortening of electron T1 of trityl OX063 free radical. Furthermore, when dissolution was employed, Tb3+-doped samples were found to have similar liquid-state C13 NMR signal enhancements compared to samples doped with Gd3+, and both Tb3+ and Dy3+ had a negligible liquid-state nuclear T1 shortening effect which contrasts with the significant reduction in T1 when using Gd3+. Our results show that Dy3+ doping and Tb3+ doping have a beneficial impact on C13 DNP both in the solid and liquid states, and that Tb3+ in particular could be used as a potential alternative to Gd3+ in C13 dissolution DNP experiments.

Enhanced Efficiency of 13C Dynamic Nuclear Polarization by Superparamagnetic Iron Oxide Nanoparticle Doping.

Attainment of high NMR signal enhancements is crucial to the success of in vitro or in vivo hyperpolarized NMR or imaging (MRI) experiments. In this work, we report on the use of a superparamagnetic iron oxide nanoparticle (SPION) MRI contrast agent Feraheme (ferumoxytol) as a beneficial additive in 13C samples for dissolution dynamic nuclear polarization (DNP). Our DNP data at 3.35 T and 1.2 K reveal that addition of 11 mM elemental iron concentration of Feraheme in trityl OX063-doped 3 M [1-13C] acetate samples resulted in a substantial improvement of 13C DNP signal by a factor of almost 3-fold. Concomitant with the large DNP signal increase is the narrowing of the 13C microwave DNP spectra for samples doped with SPION. W-band electron paramagnetic resonance (EPR) spectroscopy data suggest that these two prominent effects of SPION doping on 13C DNP can be ascribed to the shortening of trityl OX063 electron T 1 as explained within the thermal mixing DNP model. Liquid-state 13C NMR signal enhancements as high as 20,000-fold for SPION-doped samples were recorded after dissolution at 9.4 T and 297 K, which is about 3 times the liquid-state NMR signal enhancement of the control sample. While the presence of SPION in hyperpolarized solution drastically reduces 13C T 1, this can be mitigated by polarizing smaller aliquots of DNP samples. Moreover, we have shown that Feraheme nanoparticles (~30 nm in size) can be easily and effectively removed from the hyperpolarized liquid by simple mechanical filtration, thus one can potentially incorporate an in-line filtration for these SPIONS along the dissolution pathway of the hyperpolarizer-a significant advantage over other DNP enhancers such as the lanthanide complexes. The overall results suggest that the commercially-available and FDA-approved Feraheme is a highly efficient DNP enhancer that could be readily translated for use in clinical applications of dissolution DNP.

13 C dynamic nuclear polarization using isotopically enriched 4-oxo-TEMPO free radicals.

The nitroxide-based free radical 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) is a widely used polarizing agent in NMR signal amplification via dissolution dynamic nuclear polarization (DNP). In this study, we have thoroughly investigated the effects of 15 N and/or 2 H isotopic labeling of 4-oxo-TEMPO free radical on 13 C DNP of 3 M [1-13 C] sodium acetate samples in 1 : 1 v/v glycerol : water at 3.35 T and 1.2 K. Four variants of this free radical were used for 13 C DNP: 4-oxo-TEMPO, 4-oxo-TEMPO-15 N, 4-oxo-TEMPO-d16 and 4-oxo-TEMPO-15 N,d16 . Our results indicate that, despite the striking differences seen in the electron spin resonance (ESR) spectral features, the 13 C DNP efficiency of these 15 N and/or 2 H-enriched 4-oxo-TEMPO free radicals are relatively the same compared with 13 C DNP performance of the regular 4-oxo-TEMPO. Furthermore, when fully deuterated glassing solvents were used, the 13 C DNP signals of these samples all doubled in the same manner, and the 13 C polarization buildup was faster by a factor of 2 for all samples. The data here suggest that the hyperfine coupling contributions of these isotopically enriched 4-oxo-TEMPO free radicals have negligible effects on the 13 C DNP efficiency at 3.35 T and 1.2 K. These results are discussed in light of the spin temperature model of DNP. Copyright © 2016 John Wiley & Sons, Ltd.

Impact of Ho(3+)-doping on (13)C dynamic nuclear polarization using trityl OX063 free radical.

We have investigated the effects of Ho-DOTA doping on the dynamic nuclear polarization (DNP) of [1-(13)C] sodium acetate using trityl OX063 free radical at 3.35 T and 1.2 K. Our results indicate that addition of 2 mM Ho-DOTA on 3 M [1-(13)C] sodium acetate sample in 1 : 1 v/v glycerol : water with 15 mM trityl OX063 improves the DNP-enhanced (13)C solid-state nuclear polarization by a factor of around 2.7-fold. Similar to the Gd(3+) doping effect on (13)C DNP, the locations of the positive and negative (13)C maximum polarization peaks in the (13)C microwave DNP sweep are shifted towards each other with the addition of Ho-DOTA on the DNP sample. W-band electron spin resonance (ESR) studies have revealed that while the shape and linewidth of the trityl OX063 ESR spectrum was not affected by Ho(3+)-doping, the electron spin-lattice relaxation time T1 of trityl OX063 was prominently reduced at cryogenic temperatures. The reduction of trityl OX063 electron T1 by Ho-doping is linked to the (13)C DNP improvement in light of the thermodynamic picture of DNP. Moreover, the presence of Ho-DOTA in the dissolution liquid at room temperature has negligible reduction effect on liquid-state (13)C T1, in contrast to Gd(3+)-doping which drastically reduces the (13)C T1. The results here suggest that Ho(3+)-doping is advantageous over Gd(3+) in terms of preservation of hyperpolarized state-an important aspect to consider for in vitro and in vivo NMR or imaging (MRI) experiments where a considerable preparation time is needed to administer the hyperpolarized (13)C liquid.

Hyperpolarized (13)C Magnetic Resonance and Its Use in Metabolic Assessment of Cultured Cells and Perfused Organs.

Diseased tissue is often characterized by abnormalities in intermediary metabolism. Observing these alterations in situ may lead to an improved understanding of pathological processes and novel ways to monitor these processes noninvasively in human patients. Although (13)C is a stable isotope safe for use in animal models of disease as well as human subjects, its utility as a metabolic tracer has largely been limited to ex vivo analyses employing analytical techniques like mass spectrometry or nuclear magnetic resonance spectroscopy. Neither of these techniques is suitable for noninvasive metabolic monitoring, and the low abundance and poor gyromagnetic ratio of conventional (13)C make it a poor nucleus for imaging. However, the recent advent of hyperpolarization methods, particularly dynamic nuclear polarization (DNP), makes it possible to enhance the spin polarization state of (13)C by many orders of magnitude, resulting in a temporary amplification of the signal sufficient for monitoring kinetics of enzyme-catalyzed reactions in living tissue through magnetic resonance spectroscopy or magnetic resonance imaging. Here, we review DNP techniques to monitor metabolism in cultured cells, perfused hearts, and perfused livers, focusing on our experiences with hyperpolarized [1-(13)C]pyruvate. We present detailed approaches to optimize the DNP procedure, streamline biological sample preparation, and maximize detection of specific metabolic activities. We also discuss practical aspects in the choice of metabolic substrates for hyperpolarization studies and outline some of the current technical and conceptual challenges in the field, including efforts to use hyperpolarization to quantify metabolic rates in vivo.

Hyperpolarized 15N-pyridine derivatives as pH-sensitive MRI agents.

Highly sensitive MR imaging agents that can accurately and rapidly monitor changes in pH would have diagnostic and prognostic value for many diseases. Here, we report an investigation of hyperpolarized (15)N-pyridine derivatives as ultrasensitive pH-sensitive imaging probes. These molecules are easily polarized to high levels using standard dynamic nuclear polarization (DNP) techniques and their (15)N chemical shifts were found to be highly sensitive to pH. These probes displayed sharp (15)N resonances and large differences in chemical shifts (Δδ > 90 ppm) between their free base and protonated forms. These favorable features make these agents highly suitable candidates for the detection of small changes in tissue pH near physiological values.