MATRESHCA: Microtesla Apparatus for Transfer of Resonance Enhancement of Spin Hyperpolarization via Chemical Exchange and Addition.

We present an integrated, open-source device for parahydrogen-based hyperpolarization processes in the microtesla field regime with a cost of components of less than $7000. The device is designed to produce a batch of 13C and 15N hyperpolarized (HP) compounds via hydrogenative or non-hydrogenative parahydrogen-induced polarization methods that employ microtesla magnetic fields for efficient polarization transfer of parahydrogen-derived spin order to X-nuclei (e.g., 13C and 15N). The apparatus employs a layered structure (reminiscent of a Russian doll "Matryoshka") that includes a nonmagnetic variable-temperature sample chamber, a microtesla magnetic field coil (operating in the range of 0.02-75 microtesla), a three-layered mu-metal shield (to attenuate the ambient magnetic field), and a magnetic shield degaussing coil placed in the overall device enclosure. The gas-handling manifold allows for parahydrogen-gas flow and pressure control (up to 9.2 bar of total parahydrogen pressure). The sample temperature can be varied either using a water bath or a PID-controlled heat exchanger in the range from -12 to 80 °C. This benchtop device measures 62 cm (length) × 47 cm (width) × 47 cm (height), weighs 30 kg, and requires only connections to a high-pressure parahydrogen gas supply and a single 110/220 VAC power source. The utility of the device has been demonstrated using an example of parahydrogen pairwise addition to form HP ethyl [1-13C]acetate (P13C = 7%, [c] = 1 M). Moreover, the Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) technique was employed to demonstrate efficient hyperpolarization of 13C and 15N spins in a wide range of biologically relevant molecules, including [1-13C]pyruvate (P13C = 14%, [c] = 27 mM), [1-13C]-α-ketoglutarate (P13C = 17%), [1-13C]ketoisocaproate (P13C = 18%), [15N3]metronidazole (P15N = 13%, [c] = 20 mM), and others. While the vast majority of the utility studies have been performed in standard 5 mm NMR tubes, the sample chamber of the device can accommodate a wide range of sample container sizes and geometries of up to 1 L sample volume. The device establishes an integrated, simple, inexpensive, and versatile equipment gateway needed to facilitate parahydrogen-based hyperpolarization experiments ranging from basic science to preclinical applications; indeed, detailed technical drawings and a bill of materials are provided to support the ready translation of this design to other laboratories.

Toward Ultra-High-Quality-Factor Wireless Masing Magnetic Resonance Sensing.

It has recently been shown that a bolus of hyperpolarized nuclear spins can yield stimulated emission signals similar in nature to maser signals, potentially enabling new ways of sensing hyperpolarized contrast media, including most notably [1-13C]pyruvate that is under evaluation in over 50 clinical trials for metabolic imaging of cancer. The stimulated NMR signal emissions lasting for minutes do not require radio-frequency excitation, offering unprecedented advantages compared to conventional MR sensing. However, creating nuclear spin maser emission is challenging in practice due to stringent fundamental requirements, making practical in vivo applications hardly possible using conventional passive MR detectors. Here, we demonstrate the utility of a wireless NMR maser detector, the quality factor of which was enhanced 22-fold (to 1,670) via parametric pumping. This active-feedback technique breaks the intrinsic fundamental limit of NMR detector circuit quality factor. We show the use of parametric pumping to reduce the threshold requirement for inducing nuclear spin masing at 300 MHz resonance frequency in a preclinical MRI scanner. Indeed, stimulated emission from hyperpolarized protons was obtained under highly unfavorable conditions of low magnetic field homogeneity (T2* of 3 ms). Greater gains of the quality factor of the MR detector (up to 1 million) were also demonstrated.

13C Radiofrequency Amplification by Stimulated Emission of Radiation Threshold Sensing of Chemical Reactions.

Conventional nuclear magnetic resonance (NMR) enables detection of chemicals and their transformations by exciting nuclear spin ensembles with a radio-frequency pulse followed by detection of the precessing spins at their characteristic frequencies. The detected frequencies report on chemical reactions in real time and the signal amplitudes scale with concentrations of products and reactants. Here, we employ Radiofrequency Amplification by Stimulated Emission of Radiation (RASER), a quantum phenomenon producing coherent emission of 13C signals, to detect chemical transformations. The 13C signals are emitted by the negatively hyperpolarized biomolecules without external radio frequency pulses and without any background signal from other, nonhyperpolarized spins in the ensemble. Here, we studied the hydrolysis of hyperpolarized ethyl-[1-13C]acetate to hyperpolarized [1-13C]acetate, which was analyzed as a model system by conventional NMR and 13C RASER. The chemical transformation of 13C RASER-active species leads to complete and abrupt disappearance of reactant signals and delayed, abrupt reappearance of a frequency-shifted RASER signal without destroying 13C polarization. The experimentally observed "quantum" RASER threshold is supported by simulations.

Nitrogen-15 and Fluorine-19 Relaxation Dynamics and Spin-Relayed SABRE-SHEATH Hyperpolarization of Fluoro-[15N3]metronidazole.

Efficient 15N-hyperpolarization of [15N3]metronidazole was reported previously using the Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) technique. This hyperpolarized FDA-approved antibiotic is a potential contrast agent because it can be administered in a large dose and because previous studies revealed long-lasting HP states with exponential decay constant T1 values of up to 10 min. Possible hypoxia-sensing applications have been proposed using hyperpolarized [15N3]metronidazole. In this work, we report on the functionalization of [15N3]metronidazole with a fluorine-19 moiety via a one-step reaction to substitute the -OH group. SABRE-SHEATH hyperpolarization studies of fluoro-[15N3]metronidazole revealed efficient hyperpolarization of all three 15N sites with maximum %P15N values ranging from 4.2 to 6.2%, indicating efficient spin-relayed polarization transfer in microtesla fields via the network formed by 2J15N-15N. The corresponding 15N to 19F spin-relayed polarization transfer was found to be far less efficient with %P19F of 0.16%, i.e., more than an order of magnitude lower than that of 15N. Relaxation dynamics studies in microtesla fields support a spin-relayed polarization transfer mechanism because all 15N and 19F spins share the same T1 value of ca. 16-20 s and the same magnetic field profile for the SABRE-SHEATH polarization process. We envision the use of fluoro-[15N3]metronidazole as a potential hypoxia sensor. It is anticipated that under hypoxic conditions, the nitro group of fluoro-[15N3]metronidazole undergoes electronic stepwise reduction to an amino derivative. Ab initio calculations of 15N and 19F chemical shifts of fluoro-[15N3]metronidazole and its putative hypoxia-induced metabolites clearly indicate that the chemical shift dispersions of all three 15N sites and the 19F site are large enough to enable the envisioned hypoxia-sensing approaches.

Parahydrogen-Induced Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation.

The feasibility of Carbon-13 Radiofrequency (RF) Amplification by Stimulated Emission of Radiation (C-13 RASER) is demonstrated on a bolus of liquid hyperpolarized ethyl [1-13 C]acetate. Hyperpolarized ethyl [1-13 C]acetate was prepared via pairwise addition of parahydrogen to vinyl [1-13 C]acetate and polarization transfer from nascent parahydrogen-derived protons to the carbon-13 nucleus via magnetic field cycling yielding C-13 nuclear spin polarization of approximately 6 %. RASER signals were detected from samples with concentration ranging from 0.12 to 1 M concentration using a non-cryogenic 1.4T NMR spectrometer equipped with a radio-frequency detection coil with a quality factor (Q) of 32 without any modifications. C-13 RASER signals were observed for several minutes on a single bolus of hyperpolarized substrate to achieve 21 mHz NMR linewidths. The feasibility of creating long-lasting C-13 RASER on biomolecular carriers opens a wide range of new opportunities for the rapidly expanding field of C-13 magnetic resonance hyperpolarization.

Temperature Cycling Enables Efficient 13C SABRE-SHEATH Hyperpolarization and Imaging of [1-13C]-Pyruvate.

Molecular metabolic imaging in humans is dominated by positron emission tomography (PET). An emerging nonionizing alternative is hyperpolarized MRI of 13C-pyruvate, which is innocuous and has a central role in metabolism. However, similar to PET, hyperpolarized MRI with dissolution dynamic nuclear polarization (d-DNP) is complex costly, and requires significant infrastructure. In contrast, Signal Amplification By Reversible Exchange (SABRE) is a fast, cheap, and scalable hyperpolarization technique. SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) can transfer polarization from parahydrogen to 13C in pyruvate; however, polarization levels remained low relative to d-DNP (1.7% with SABRE-SHEATH versus ≈60% with DNP). Here we introduce a temperature cycling method for SABRE-SHEATH that enables >10% polarization on [1-13C]-pyruvate, sufficient for successful in vivo experiments. First, at lower temperatures, ≈20% polarization is accumulated on SABRE catalyst-bound pyruvate, which is released into free pyruvate at elevated temperatures. A kinetic model of differential equations is developed that explains this effect and characterizes critical relaxation and buildup parameters. With the large polarization, we demonstrate the first 13C pyruvate images with a cryogen-free MRI system operated at 1.5 T, illustrating that inexpensive hyperpolarization methods can be combined with low-cost MRI systems to obtain a broadly available, yet highly sensitive metabolic imaging platform.

Rapid 13C Hyperpolarization of the TCA Cycle Intermediate α-Ketoglutarate via SABRE-SHEATH.

α-Ketoglutarate is a key biomolecule involved in a number of metabolic pathways─most notably the TCA cycle. Abnormal α-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-13C,5-12C,D4]α-ketoglutarate with the future goal of utilizing its [1-13C]-hyperpolarized state for real-time metabolic imaging of α-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-13C] polarization in 1 minute in this isotopologue. The efficient 13C hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 µT field (the optimal polarization transfer field): the exponential 13C polarization buildup constant Tb is 11.0 ± 0.4 s whereas the 13C polarization decay constant T1 is 18.5 ± 0.7 s. An even higher 13C polarization value of 17.3% was achieved using natural-abundance α-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 µT field, indicating that substrate deuteration leads only to a slight increase (∼1.2-fold) in the relaxation rates for 13C nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1-13C]-labeled) is rationalized through the smaller heat capacity of the "spin bath" comprising available 13C spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous 15N SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE-SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in α-ketoglutarate differ from that in pyruvate. We also demonstrate that 13C spectroscopic characterization of α-ketoglutarate and pyruvate analytes can be performed at natural 13C abundance with an estimated detection limit of 80 micromolar concentration × *%P13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases.

Order-Unity 13 C Nuclear Polarization of [1-13 C]Pyruvate in Seconds and the Interplay of Water and SABRE Enhancement.

Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) is investigated to achieve rapid hyperpolarization of 13 C1 spins of [1-13 C]pyruvate, using parahydrogen as the source of nuclear spin order. Pyruvate exchange with an iridium polarization transfer complex can be modulated via a sensitive interplay between temperature and co-ligation of DMSO and H2 O. Order-unity 13 C (>50 %) polarization of catalyst-bound [1-13 C]pyruvate is achieved in less than 30 s by restricting the chemical exchange of [1-13 C]pyruvate at lower temperatures. On the catalyst bound pyruvate, 39 % polarization is measured using a 1.4 T NMR spectrometer, and extrapolated to >50 % at the end of build-up in situ. The highest measured polarization of a 30-mM pyruvate sample, including free and bound pyruvate is 13 % when using 20 mM DMSO and 0.5 M water in CD3 OD. Efficient 13 C polarization is also enabled by favorable relaxation dynamics in sub-microtesla magnetic fields, as indicated by fast polarization buildup rates compared to the T1 spin-relaxation rates (e. g., ∼0.2 s-1 versus ∼0.1 s-1 , respectively, for a 6 mM catalyst-[1-13 C]pyruvate sample). Finally, the catalyst-bound hyperpolarized [1-13 C]pyruvate can be released rapidly by cycling the temperature and/or by optimizing the amount of water, paving the way to future biomedical applications of hyperpolarized [1-13 C]pyruvate produced via comparatively fast and simple SABRE-SHEATH-based approaches.

Interplay of Near-Zero-Field Dephasing, Rephasing, and Relaxation Dynamics and [1-13C]Pyruvate Polarization Transfer Efficiency in Pulsed SABRE-SHEATH.

Hyperpolarized [1-13C]pyruvate is a revolutionary molecular probe enabling ultrafast metabolic MRI scans in 1 min. This technology is now under evaluation in over 30 clinical trials, which employ dissolution Dynamic Nuclear Polarization (d-DNP) to prepare a batch of the contrast agent; however, d-DNP technology is slow and expensive. The emerging SABRE-SHEATH hyperpolarization technique enables fast (under 1 min) and robust production of hyperpolarized [1-13C]pyruvate via simultaneous chemical exchange of parahydrogen and pyruvate on IrIMes hexacoordinate complexes. Here, we study the application of microtesla pulses to investigate their effect on C-13 polarization efficiency, compared to that of conventional SABRE-SHEATH employing a static field (∼0.4 µT), to provide the matching conditions of polarization transfer from parahydrogen-derived hydrides to the 13C-1 nucleus. Our results demonstrate that using square-microtesla pulses with optimized parameters can produce 13C-1 polarization levels of up to 14.8% (when detected, averaging over all resonances), corresponding to signal enhancement by over 122,000-fold at the clinically relevant field of 1.4 T. We anticipate that our results can be directly translated to other structurally similar biomolecules such as [1-13C]α-ketoglutarate and [1-13C]α-ketoisocaproate. Moreover, other more advanced pulse shapes can potentially further boost heteronuclear polarization attainable via pulsed SABRE-SHEATH.

Novel cross-linked melamine based polyamine/CNT composites for lead ions removal.

A novel series of polyamine/CNT composites were synthesized via a single step polycondensation reaction of melamine, paraformaldehyde, various alkyldiamines and chlorinated carbon nanotubes (CNT) at optimized reaction conditions in the presence of N, N-Dimethyl formamide as a solvent. Chlorinated carbon nanotubes synthesized by reacting acidified CNT and thionyl chloride was used. The pure polymer (MFDH) and the functionalized composites (MFDH1, MFDH2, MFDH3 and MFDH4) having 0.01, 0.02, 0.05 and 0.1% weight of the starting precursors were used. The morphology, surface area, molecular structures and overall properties of the new series of polymers were characterized using Raman Spectroscopy, FT-IR, 13C NMR, X-ray diffraction experiments, BET and TGA. A comprehensive design was set up in order to evaluate the effects of pH, temperature, Lead ion initial concentrations and contact time on the ability of the new series of functionalized polymers for Lead (II) ion removal. Wastewater treatment revealed the high efficacy of the synthesized polyamine/CNT composite in the removal of ∼99% of Lead ions in wastewater samples.

Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation of the Hyperpolarized Ketone and Hemiketal Forms of Allyl [1-13C]Pyruvate.

13C hyperpolarized pyruvate is an emerging MRI contrast agent for sensing molecular events in cancer and other diseases with aberrant metabolic pathways. This metabolic contrast agent can be produced via several hyperpolarization techniques. Despite remarkable success in research settings, widespread clinical adoption faces substantial roadblocks because the current sensing technology utilized to sense this contrast agent requires the excitation of 13C nuclear spins that also need to be synchronized with MRI field gradient pulses. Here, we demonstrate sensing of hyperpolarized allyl [1-13C]pyruvate via the stimulated emission of radiation that mitigates the requirements currently blocking broader adoption. Specifically, 13C Radiofrequency Amplification by Stimulated Emission of Radiation (13C RASER) was obtained after pairwise addition of parahydrogen to a pyruvate precursor, detected in a commercial inductive detector with a quality factor (Q) of 32 for sample concentrations as low as 0.125 M with 13C polarization of 4%. Moreover, parahydrogen-induced polarization allowed for the preparation of a mixture of ketone and hemiketal forms of hyperpolarized allyl [1-13C]pyruvate, which are separated by 10 ppm in 13C NMR spectra. This is a good model system to study the simultaneous 13C RASER signals of multiple 13C species. This system models the metabolic production of hyperpolarized [1-13C]lactate from hyperpolarized [1-13C]pyruvate, which has a similar chemical shift difference. Our results show that 13C RASER signals can be obtained from both species simultaneously when the emission threshold is exceeded for both species. On the other hand, when the emission threshold is exceeded only for one of the hyperpolarized species, 13C stimulated emission is confined to this species only, therefore enabling the background-free detection of individual hyperpolarized 13C signals. The reported results pave the way to novel sensing approaches of 13C hyperpolarized pyruvate, potentially unlocking hyperpolarized 13C MRI on virtually any MRI system─an attractive vision for the future molecular imaging and diagnostics.

Delivering Robust Proton-Only Sensing of Hyperpolarized [1,2-13C2]-Pyruvate Using Broad-Spectral-Range Nuclear Magnetic Resonance Pulse Sequences.

Hyperpolarized [1-13C]pyruvate is the leading hyperpolarized injectable contrast agent and is currently under evaluation in clinical trials for molecular imaging of metabolic diseases, including cardiovascular disease and cancer. One aspect limiting broad scalability of the technique is that hyperpolarized 13C MRI requires specialized 13C hardware and software that are not generally available on clinical MRI scanners, which employ proton-only detection. Here, we present an approach that uses pulse sequences to transfer 13C hyperpolarization to methyl protons for detection of the 13C-13C pyruvate singlet, employing proton-only excitation and detection only. The new pulse sequences are robust to the B1 and B0 magnetic field inhomogeneities. The work focuses on singlet-to-magnetization (S2M) and rotor-synchronized (R) pulses, both relying on trains of hard pulses with broad spectral width coverage designed to effectively transform hyperpolarized 13C2-singlet hyperpolarization to 1H polarization on the CH3 group of [1,2-13C2]pyruvate. This approach may enable a broader adoption of hyperpolarized MRI as a molecular imaging technique.

Efficient SABRE-SHEATH Hyperpolarization of Potent Branched-Chain-Amino-Acid Metabolic Probe [1-13C]ketoisocaproate.

Efficient 13C hyperpolarization of ketoisocaproate is demonstrated in natural isotopic abundance and [1-13C]enriched forms via SABRE-SHEATH (Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei). Parahydrogen, as the source of nuclear spin order, and ketoisocaproate undergo simultaneous chemical exchange with an Ir-IMes-based hexacoordinate complex in CD3OD. SABRE-SHEATH enables spontaneous polarization transfer from parahydrogen-derived hydrides to the 13C nucleus of transiently bound ketoisocaproate. 13C polarization values of up to 18% are achieved at the 1-13C site in 1 min in the liquid state at 30 mM substrate concentration. The efficient polarization build-up becomes possible due to favorable relaxation dynamics. Specifically, the exponential build-up time constant (14.3 ± 0.6 s) is substantially lower than the corresponding polarization decay time constant (22.8 ± 1.2 s) at the optimum polarization transfer field (0.4 microtesla) and temperature (10 °C). The experiments with natural abundance ketoisocaproate revealed polarization level on the 13C-2 site of less than 1%-i.e., one order of magnitude lower than that of the 1-13C site-which is only partially due to more-efficient relaxation dynamics in sub-microtesla fields. We rationalize the overall much lower 13C-2 polarization efficiency in part by less favorable catalyst-binding dynamics of the C-2 site. Pilot SABRE experiments at pH 4.0 (acidified sample) versus pH 6.1 (unaltered sodium [1-13C]ketoisocaproate) reveal substantial modulation of SABRE-SHEATH processes by pH, warranting future systematic pH titration studies of ketoisocaproate, as well as other structurally similar ketocarboxylate motifs including pyruvate and alpha-ketoglutarate, with the overarching goal of maximizing 13C polarization levels in these potent molecular probes. Finally, we also report on the pilot post-mortem use of HP [1-13C]ketoisocaproate in a euthanized mouse, demonstrating that SABRE-hyperpolarized 13C contrast agents hold promise for future metabolic studies.

Proton-Only Sensing of Hyperpolarized [1,2-13C2]Pyruvate.

Hyperpolarized MRI is emerging as a next-generation molecular imaging modality that can detect metabolic transformations in real time deep inside tissue and organs. 13C-hyperpolarized pyruvate is the leading hyperpolarized contrast agent that can probe cellular energetics in real time. Currently, hyperpolarized MRI requires specialized "multinuclear" MRI scanners that have the ability to excite and detect 13C signals. The objective of this work is the development of an approach that works on conventional (i.e., proton-only) MRI systems while taking advantage of long-lived 13C hyperpolarization. The long-lived singlet state of [1,2-13C2]pyruvate is hyperpolarized with parahydrogen in reversible exchange, and subsequently, the polarization is transferred from the 13C2 spin pair to the methyl protons of pyruvate for detection. This polarization transfer is accomplished with spin-lock induced crossing pulses that are only applied to the methyl protons yet access the hyperpolarization stored in the 13C2 singlet state. Theory and first experimental demonstrations are provided for our method, which obviates 13C excitation and detection for proton sensing of 13C-hyperpolarized pyruvate with an overall experimental-polarization transfer efficiency of ∼22% versus a theoretically predicted polarization transfer efficiency of 25%.

Catalyst-Free Aqueous Hyperpolarized [1-13C]Pyruvate Obtained by Re-Dissolution Signal Amplification by Reversible Exchange.

Despite great successes in oncology, patient outcomes are often still discouraging, and hence the diagnostic imaging paradigm is increasingly shifting toward functional imaging of the pathology to better understand individual disease biology and to personalize therapies. The dissolution Dynamic Nuclear Polarization (d-DNP) hyperpolarization method has enabled unprecedented real-time MRI sensing of metabolism and tissue pH using hyperpolarized [1-13C]pyruvate as a biosensor with great potential for diagnosis and monitoring of cancer patients. However, current d-DNP is expensive and suffers from long hyperpolarization times, posing a substantial translational roadblock. Here, we report the development of Re-Dissolution Signal Amplification By Reversible Exchange (Re-D SABRE), which relies on fast and low-cost hyperpolarization of [1-13C]pyruvate by chemical exchange with parahydrogen at microtesla magnetic fields. [1-13C]pyruvate is precipitated from catalyst-containing methanol using ethyl acetate and rapidly reconstituted in aqueous media. 13C polarization of 9 ± 1% is demonstrated after redissolution in water with residual iridium mass fraction of 8.5 ± 1.5 ppm; further improvement is anticipated via process automation. Re-D SABRE makes hyperpolarized [1-13C]pyruvate biosensor available at a fraction of the cost (<$10,000) and production time (≈1 min) of currently used techniques and makes aqueous hyperpolarized [1-13C]pyruvate "ready" for in vivo applications.