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 <i>T</i>₁ 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

Preclinical MRI to quantify pulmonary disease severity and trajectories in poorly characterized mouse models: A pedagogical example using data from novel transgenic models of lung fibrosis

Structural remodeling in lung disease is progressive and heterogeneous, making temporally and spatially explicit information necessary to understand disease initiation and progression. While mouse models are essential to elucidate mechanistic pathways underlying disease, the experimental tools commonly available to quantify lung disease burden are typically invasive (e.g., histology). This necessitates large cross-sectional studies with terminal endpoints, which increases experimental complexity and expense. Alternatively, magnetic resonance imaging (MRI) provides information noninvasively, thus permitting robust, repeated-measures statistics. Although lung MRI is challenging due to low tissue density and rapid apparent transverse relaxation (T2* <1 ms), various imaging methods have been proposed to quantify disease burden. However, there are no widely accepted strategies for preclinical lung MRI. As such, it can be difficult for researchers who lack lung imaging expertise to design experimental protocols—particularly for novel mouse models. Here, we build upon prior work from several research groups to describe a widely applicable acquisition and analysis pipeline that can be implemented without prior preclinical pulmonary MRI experience. Our approach utilizes 3D radial ultrashort echo time (UTE) MRI with retrospective gating and lung segmentation is facilitated with a deep-learning algorithm. This pipeline was deployed to assess disease dynamics over 255 days in novel, transgenic mouse models of lung fibrosis based on disease-associated, loss-of-function mutations in Surfactant Protein-C. Previously identified imaging biomarkers (tidal volume, signal coefficient of variation, etc.) were calculated semi-automatically from these data, with an objectively-defined high signal volume identified as the most robust metric. Beyond quantifying disease dynamics, we discuss common pitfalls encountered in preclinical lung MRI and present systematic approaches to identify and mitigate these challenges. While the experimental results and specific pedagogical examples are confined to lung fibrosis, the tools and approaches presented should be broadly useful to quantify structural lung disease in a wide range of mouse models

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

The 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 the 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 three-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 <i>T</i>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 three times the liquid-state NMR signal enhancement of the control sample. While the presence of SPION in hyperpolarized solution drastically reduces ¹³C <i>T</i>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, and 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

Transition Metal Doping Reveals Link between Electron <i>T</i>₁ 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 <i>in vitro</i> and <i>in vivo</i> 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 <i>T</i>₁ 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 <i>T</i>₁ reduction of the polarizing agent by a paramagnetic additive is an essential requirement for the improvement seen in solid-state ¹³C DNP signal