Radical-free hyperpolarized MRI using endogenously occurring pyruvate analogues and UV-induced nonpersistent radicals.

It was recently demonstrated that nonpersistent radicals can be generated in frozen solutions of metabolites such as pyruvate by irradiation with UV light, enabling radical-free dissolution dynamic nuclear polarization. Although pyruvate is endogenous, the presence of pyruvate may interfere with metabolic processes or the detection of pyruvate as a metabolic product, making it potentially unsuitable as a polarizing agent. Therefore, the aim of the current study was to characterize solutions containing endogenously occurring alternatives to pyruvate as UV-induced nonpersistent radical precursors for in vivo hyperpolarized MRI. The metabolites alpha-ketovalerate (αkV) and alpha-ketobutyrate (αkB) are analogues of pyruvate and were chosen as potential radical precursors. Sample formulations containing αkV and αkB were studied with UV-visible spectroscopy, irradiated with UV light, and their nonpersistent radical yields were quantified with electron spin resonance and compared with pyruvate. The addition of 13 C-labeled substrates to the sample matrix altered the radical yield of the precursors. Using αkB increased the 13 C-labeled glucose liquid-state polarization to 16.3% ± 1.3% compared with 13.3% ± 1.5% obtained with pyruvate, and 8.9% ± 2.1% with αkV. For [1-13 C]butyric acid, polarization levels of 12.1% ± 1.1% for αkV, 12.9% ± 1.7% for αkB, 1.5% ± 0.2% for OX063 and 18.7% ± 0.7% for Finland trityl, were achieved. Hyperpolarized [1-13 C]butyrate metabolism in the heart revealed label incorporation into [1-13 C]acetylcarnitine, [1-13 C]acetoacetate, [1-13 C]butyrylcarnitine, [5-13 C]glutamate and [5-13 C]citrate. This study demonstrates the potential of αkV and αkB as endogenous polarizing agents for in vivo radical-free hyperpolarized MRI. UV-induced, nonpersistent radicals generated in endogenous metabolites enable high polarization without requiring radical filtration, thus simplifying the quality-control tests in clinical applications.

Hyperpolarization via dissolution dynamic nuclear polarization: new technological and methodological advances.

Dissolution-DNP is a method to boost liquid-state NMR sensitivity by several orders of magnitude. The technique consists in hyperpolarizing samples by solid-state dynamic nuclear polarization at low temperature and moderate magnetic field, followed by an instantaneous melting and dilution of the sample happening inside the polarizer. Although the technique is well established and the outstanding signal enhancement paved the way towards many applications precluded to conventional NMR, the race to develop new methods allowing higher throughput, faster and higher polarization, and longer exploitation of the signal is still vivid. In this work, we review the most recent advances on dissolution-DNP methods trying to overcome the original technique's shortcomings. The review describes some of the new approaches in the field, first, in terms of sample formulation and properties, and second, in terms of instrumentation.

Efficient Hyperpolarization of U-13 C-Glucose Using Narrow-Line UV-Generated Labile Free Radicals.

Free radicals generated by UV-light irradiation of a frozen solution containing a fraction of pyruvic acid (PA) have demonstrated their dissolution dynamic nuclear polarization (dDNP) potential, providing up to 30 % [1-13 C]PA liquid-state polarization. Moreover, their labile nature has proven to pave a way to nuclear polarization storage and transport. Herein, differently from the case of PA, the issue of providing dDNP UV-radical precursors (trimethylpyruvic acid and its methyl-deuterated form) not involved in any metabolic pathway was investigated. The 13 C dDNP performance was evaluated for hyperpolarization of [U-13 C6 ,1,2,3,4,5,6,6-d7 ]-d-glucose. The generated UV-radicals proved to be versatile and highly efficient polarizing agents, providing, after dissolution and transfer (10 s), a 13 C liquid-state polarization of up to 32 %.

Photogenerated Radical in Phenylglyoxylic Acid for in Vivo Hyperpolarized 13C MR with Photosensitive Metabolic Substrates.

Whether for 13C magnetic resonance studies in chemistry, biochemistry, or biomedicine, hyperpolarization methods based on dynamic nuclear polarization (DNP) have become ubiquitous. DNP requires a source of unpaired electrons, which are commonly added to the sample to be hyperpolarized in the form of stable free radicals. Once polarized, the presence of these radicals is unwanted. These radicals can be replaced by nonpersistent radicals created by the photoirradiation of pyruvic acid (PA), which are annihilated upon dissolution or thermalization in the solid state. However, since PA is readily metabolized by most cells, its presence may be undesirable for some metabolic studies. In addition, some 13C substrates are photosensitive and therefore may degrade during the photogeneration of a PA radical, which requires ultraviolet (UV) light. We show here that the photoirradiation of phenylglyoxylic acid (PhGA) using visible light produces a nonpersistent radical that, in principle, can be used to hyperpolarize any molecule. We compare radical yields in samples containing PA and PhGA upon photoirradiation with broadband and narrowband UV-visible light sources. To demonstrate the suitability of PhGA as a radical precursor for DNP, we polarized the gluconeogenic probe 13C-dihydroxyacetone, which is UV-sensitive, using a commercial 3.35 T DNP polarizer and then injected this into a mouse and followed its metabolism in vivo.

Probing cardiac metabolism by hyperpolarized 13C MR using an exclusively endogenous substrate mixture and photo-induced nonpersistent radicals.

PURPOSE: To probe the cardiac metabolism of carbohydrates and short chain fatty acids simultaneously in vivo following the injection of a hyperpolarized 13 C-labeled substrate mixture prepared using photo-induced nonpersistent radicals. METHODS: Droplets of mixed [1-13 C]pyruvic and [1-13 C]butyric acids were frozen into glassy beads in liquid nitrogen. Ethanol addition was investigated as a means to increase the polarization level. The beads were irradiated with ultraviolet light and the radical concentration was measured by ESR spectroscopy. Following dynamic nuclear polarization in a 7T polarizer, the beads were dissolved, and the radical-free hyperpolarized solution was rapidly transferred into an injection pump located inside a 9.4T scanner. The hyperpolarized solution was injected in healthy rats to measure cardiac metabolism in vivo. RESULTS: Ultraviolet irradiation created nonpersistent radicals in a mixture containing 13 C-labeled pyruvic and butyric acids, and enabled the hyperpolarization of both substrates by dynamic nuclear polarization. Ethanol addition increased the radical concentration from 16 to 26 mM. Liquid-state 13 C polarization was 3% inside the pump at the time of injection, and increased to 5% by addition of ethanol to the substrate mixture prior to ultraviolet irradiation. In the rat heart, the in vivo 13 C signals from lactate, alanine, bicarbonate, and acetylcarnitine were detected following the metabolism of the injected substrate mixture. CONCLUSION: Copolarization of two different 13 C-labeled substrates and the detection of their myocardial metabolism in vivo was achieved without using persistent radicals. The absence of radicals in the solution containing the hyperpolarized 13 C-substrates may simplify the translation to clinical use, as no radical filtration is required prior to injection.

Hyperpolarization without persistent radicals for in vivo real-time metabolic imaging.

Hyperpolarized substrates prepared via dissolution dynamic nuclear polarization have been proposed as magnetic resonance imaging (MRI) agents for cancer or cardiac failure diagnosis and therapy monitoring through the detection of metabolic impairments in vivo. The use of potentially toxic persistent radicals to hyperpolarize substrates was hitherto required. We demonstrate that by shining UV light for an hour on a frozen pure endogenous substance, namely the glucose metabolic product pyruvic acid, it is possible to generate a concentration of photo-induced radicals that is large enough to highly enhance the (13)C polarization of the substance via dynamic nuclear polarization. These radicals recombine upon dissolution and a solution composed of purely endogenous products is obtained for performing in vivo metabolic hyperpolarized (13)C MRI with high spatial resolution. Our method opens the way to safe and straightforward preclinical and clinical applications of hyperpolarized MRI because the filtering procedure mandatory for clinical applications and the associated pharmacological tests necessary to prevent contamination are eliminated, concurrently allowing a decrease in the delay between preparation and injection of the imaging agents for improved in vivo sensitivity.

Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR.

Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor α-cyano-4-hydroxycinnamic acid.

129Xe Dynamic Nuclear Polarization Demystified: The Influence of the Glassing Matrix on the Radical Properties.

129Xe dissolution dynamic nuclear polarization (DNP) is a controversial topic. The gold standard technique for hyperpolarized xenon magnetic resonance imaging (MRI) is spin exchange optical pumping, which received FDA approval in 2022. Nevertheless, the versatility of DNP for enhancing the signal of any NMR active nucleus might provide new perspectives for hyperpolarized 129Xe NMR/MRI. Initial publications about 129Xe DNP underlined the increased complexity in the sample preparation and lower polarization levels when compared to more conventional 13C-labeled molecules, at same experimental conditions, despite very close gyromagnetic ratios. Herein, we introduce, using a Custom Fluid Path system, a user-friendly and very robust sample preparation method. Moreover, investigating the radical properties at real DNP conditions by means of LOngitudinal Detected Electron Spin Resonance, we discovered a dramatic shortening of the electron spin longitudinal relaxation time (T1e) of nitroxyl radicals in xenon DNP samples' matrices, with respect to more commonly used water:glycerol ones. Mitigating those challenges through microwave frequency modulation, we achieved over 20% 129Xe polarization without employing any deuterated solvent.

Over 35% liquid-state 13C polarization obtained via dissolution dynamic nuclear polarization at 7 T and 1 K using ubiquitous nitroxyl radicals.

The most versatile method to increase liquid-state (13)C NMR sensitivity is dissolution dynamic nuclear polarization. The use of trityl radicals is usually required to obtain very large (13)C polarization via this technique. We herein demonstrate that up to 35% liquid-state (13)C polarization can be obtained in about 1.5 h using ubiquitous nitroxyl radicals in (13)C-labeled sodium salts by partially deuterating the solvents and using a polarizer operating at 1 K and 7 T.

Multi-sample/multi-nucleus parallel polarization and monitoring enabled by a fluid path technology compatible cryogenic probe for dissolution dynamic nuclear polarization.

Low throughput is one of dissolution Dynamic Nuclear Polarization (dDNP) main shortcomings. Especially for clinical and preclinical applications, where direct 13C nuclei polarization is usually pursued, it takes hours to generate one single hyperpolarized (HP) sample. Being able to hyperpolarize more samples at once represents a clear advantage and can expand the range and complexity of the applications. In this work, we present the design and performance of a highly versatile and customizable dDNP cryogenic probe, herein adapted to a 5 T "wet" preclinical polarizer, that can accommodate up to three samples at once and, most importantly, it is capable of monitoring the solid-state spin dynamics of each sample separately, regardless of the kind of radical used and the nuclear species of interest. Within 30 min, the system was able to dispense three HP solutions with high repeatability across the channels (30.0 ± 1.2% carbon polarization for [1-13C]pyruvic acid doped with trityl radical). Moreover, we tested multi-nucleus NMR capability by polarizing and monitoring simultaneously 13C, 1H and 129Xe. Finally, we implemented [1-13C]lactate/[1-13C]pyruvate polarization and back-to-back dissolution and injection in a healthy mouse model to perform multiple-substrate HP Magnetic Resonance Spectroscopy (MRS) at 14.1 T.

Design and performance of a small bath cryostat with NMR capability for transport of hyperpolarized samples.

As of today, dissolution Dynamic Nuclear Polarization (dDNP) is the only clinically available hyperpolarization technique for 13C-MRI. Despite the clear path towards personalized medicine that dDNP is paving as an alternative and/or complement to Positron Emission Tomography (PET), the technique struggles to enter everyday clinical practice. Because of the minute-long hyperpolarization lifetime after dissolution, one of the reasons lies in the need and consequent complexities of having the machine that generates the hyperpolarization (i.e. the dDNP polarizer) on site. Since some years, research groups are working to make hyperpolarization transportable. Two different methods have been developed that allow "freezing" of the nuclear spin state prior to samples extraction from the polarizer. Nevertheless, so far, all attempts of transport have been limited to a very small scale and to the level of proof-of-principle experiments. The main reason for that is the lack of adequate hardware, strategy, and control on most of the crucial parameters. To bridge the technical gap with PET and provide MRI facilities with hours long relaxing hyperpolarized compounds at controlled conditions, a new generation of low cost/small footprint liquid He cryostats equipped with a magnetically enforced cryogenic probe is needed. In this paper, we detail the theoretical and practical construction of a hyperpolarized samples transportation device small enough to fit in a car and able to hold a sample at 4.2 K for almost 8 h despite the presence of a cryogenically-demanding purpose-built probe that provides enough magnetic field upon insertion of the sample and NMR quality homogeneity at storage position. Should transportable hyperpolarization via DNP become a reality, we herein provide important details to make it possible.

How to improve the efficiency of a traditional dissolution dynamic nuclear polarization (dDNP) apparatus: Design and performance of a fluid path compatible dDNP/LOD-ESR probe.

Dissolution Dynamic Nuclear Polarization (dDNP) was invented almost twenty years ago. Ever since, hardware advancement has observed 2 trends: the quest for DNP at higher field and, more recently, the development of cryogen free polarizers. Despite the DNP community is slowly migrating towards "dry" systems, many "wet" polarizers are still in use. Traditional DNP polarizers can use up to 100 L of liquid helium per week, but are less sensitive to air contamination and have higher cooling power. These two characteristics make them very versatile when it comes to new methods development. In this study we retrofitted a 5 T/1.15 K "wet" DNP polarizer with the aim of improving cryogenic and DNP performance. We designed, built, and tested a new DNP insert that is compatible with the fluid path (FP) technology and a LOgitudinal Detected Electron Spin Resonance (LOD-ESR) probe to investigate radical properties at real DNP conditions. The new hardware increased the maximum achievable polarization and the polarization rate constant of a [1-13C]pyruvic acid-trityl sample by a factor 1.5. Moreover, the increased liquid He holding time together with the possibility to constantly keep the sample space at low pressure upon sample loading and dissolution allowed us to save about 20 L of liquid He per week.

Measuring Glycolytic Activity with Hyperpolarized [2H7, U-13C6] D-Glucose in the Naive Mouse Brain under Different Anesthetic Conditions.

Glucose is the primary fuel for the brain; its metabolism is linked with cerebral function. Different magnetic resonance spectroscopy (MRS) techniques are available to assess glucose metabolism, providing complementary information. Our first aim was to investigate the difference between hyperpolarized 13C-glucose MRS and non-hyperpolarized 2H-glucose MRS to interrogate cerebral glycolysis. Isoflurane anesthesia is commonly employed in preclinical MRS, but it affects cerebral hemodynamics and functional connectivity. A combination of low doses of isoflurane and medetomidine is routinely used in rodent fMRI and shows similar functional connectivity, as in awake animals. As glucose metabolism is tightly linked to neuronal activity, our second aim was to assess the impact of these two anesthetic conditions on the cerebral metabolism of glucose. Brain metabolism of hyperpolarized 13C-glucose and 2H-glucose was monitored in two groups of mice in a 9.4 T MRI system. We found that the very different duration and temporal resolution of the two techniques enable highlighting the different aspects in glucose metabolism. We demonstrate (by numerical simulations) that hyperpolarized 13C-glucose reports on de novo lactate synthesis and is sensitive to CMRGlc. We show that variations in cerebral glucose metabolism, under different anesthesia, are reflected differently in hyperpolarized and non-hyperpolarized X-nuclei glucose MRS.

Metabolic contrast agents produced from transported solid 13C-glucose hyperpolarized via dynamic nuclear polarization.

Magnetic Resonance Imaging combined with hyperpolarized 13C-labelled metabolic contrast agents produced via dissolution Dynamic Nuclear Polarization can, non-invasively and in real-time, report on tissue specific aberrant metabolism. However, hyperpolarization equipment is expensive, technically demanding and needs to be installed on-site for the end-user. In this work, we provide a robust methodology that allows remote production of the hyperpolarized 13C-labelled metabolic contrast agents. The methodology, built on photo-induced thermally labile radicals, allows solid sample extraction from the hyperpolarization equipment and several hours' lifetime of the 13C-labelled metabolic contrast agents at appropriate storage/transport conditions. Exemplified with [U-13C, d7]-D-glucose, we remotely produce hyperpolarized 13C-labelled metabolic contrast agents and generate above 10,000-fold liquid-state Magnetic Resonance signal enhancement at 9.4 T, keeping on-site only a simple dissolution device.

Hyperpolarized water through dissolution dynamic nuclear polarization with UV-generated radicals.

In recent years, hyperpolarization of water protons via dissolution Dynamic Nuclear Polarization (dDNP) has attracted increasing interest in the magnetic resonance community. Hyperpolarized water may provide an alternative to Gd-based contrast agents for angiographic and perfusion Magnetic Resonance Imaging (MRI) examinations, and it may report on chemical and biochemical reactions and proton exchange while perfoming Nuclear Magnetic Resonance (NMR) investigations. However, hyperpolarizing water protons is challenging. The main reason is the presence of radicals, required to create the hyperpolarized nuclear spin state. Indeed, the radicals will also be the main source of relaxation during the dissolution and transfer to the NMR or MRI system. In this work, we report water magnetizations otherwise requiring a field of 10,000 T at room temperature on a sample of pure water, by employing dDNP via UV-generated, labile radicals. We demonstrate the potential of our methodology by acquiring a 15N spectrum from natural abundance urea with a single scan, after spontaneous magnetization transfer from water protons to nitrogen nuclei.

13C Dynamic Nuclear Polarization using SA-BDPA at 6.7 T and 1.1 K: Coexistence of Pure Thermal Mixing and Well-Resolved Solid Effect.

SA-BDPA is a water-soluble, narrow-line width radical previously used for dynamic nuclear polarization (DNP) signal enhancement in solid-state magic angle spinning NMR spectroscopy. Here, we report the first study using SA-BDPA under dissolution DNP conditions (6.7 T and 1.15 K). Longitudinal-detected (LOD)-electron spin resonance (ESR) and 13C DNP measurements were performed on samples containing 8.4 M [13C]urea dissolved in 50:50 water:glycerol (v/v) doped with either 60 or 120 mM SA-BDPA. Two distinct DNP mechanisms, both "pure" thermal mixing and a well-resolved solid effect could clearly be identified. The radical's ESR line width (30-40 MHz), broadened predominantly by dipolar coupling, excluded any contribution from the cross effect. Microwave frequency modulation increased the enhancement by DNP at the lower radical concentration but not at the higher radical concentration. These results are compared to data acquired with trityl radical AH111501, highlighting the unusual 13C DNP properties of SA-BDPA.

Gadolinium Effect at High-Magnetic-Field DNP: 70% 13C Polarization of [U-13C] Glucose Using Trityl.

We show that the trityl electron spin resonance (ESR) features, crucial for an efficient dynamic nuclear polarization (DNP) process, are sample-composition-dependent. Working at 6.7 T and 1.1 K with a generally applicable DNP sample solvent mixture such as water/glycerol plus trityl, the addition of Gd3+ leads to a dramatic increase in [U-13C] glucose polarization from 37 ± 4% to 69 ± 3%. This is the highest value reported to date and is comparable to what can be achieved on pyruvic acid. Moreover, performing ESR measurements under actual DNP conditions, we provide experimental evidence that gadolinium doping not only shortens the trityl electron spin-lattice relaxation time but also modifies the radical g-tensor. The latter yielded a considerable narrowing of the ESR spectrum line width. Finally, in the frame of the spin temperature theory, we discuss how these two phenomena affect the DNP performance.

Optimized microwave delivery in dDNP.

Dissolution dynamic nuclear polarization (dDNP) has permitted the production of highly polarized liquid-state samples, enabling real-time imaging of metabolic processes non-invasively in vivo. The desire for higher magnetic resonance sensitivity has led to the development of multiple home-built and commercial dDNP polarizers employing solid-state microwave sources. Providing efficient microwave delivery that avoids unwanted heating of the sample is a crucial step to achieve high nuclear polarization. Consequently, a process is described to reduce waveguide attenuation due to resistive loss thereby doubling the delivered power. A mirror and reflector are designed and tested to increase the microwave field density across the sample volume resulting in a 2.3 dB increase of delivered power. Thermal considerations with regards to waveguide geometry and dDNP probe design are discussed. A thermal model of the dDNP probe is computed and experimentally verified.

Thermal annihilation of photo-induced radicals following dynamic nuclear polarization to produce transportable frozen hyperpolarized 13C-substrates.

Hyperpolarization via dynamic nuclear polarization (DNP) is pivotal for boosting magnetic resonance imaging (MRI) sensitivity and dissolution DNP can be used to perform in vivo real-time 13C MRI. The type of applications is however limited by the relatively fast decay time of the hyperpolarized spin state together with the constraint of having to polarize the 13C spins in a dedicated apparatus nearby but separated from the MRI magnet. We herein demonstrate that by polarizing 13C with photo-induced radicals, which can be subsequently annihilated using a thermalization process that maintains the sample temperature below its melting point, hyperpolarized 13C-substrates can be extracted from the DNP apparatus in the solid form, while maintaining the enhanced 13C polarization. The melting procedure necessary to transform the frozen solid into an injectable solution containing the hyperpolarized 13C-substrates can therefore be performed ex situ, up to several hours after extraction and storage of the polarized solid.