MATRESHCA: Microtesla Apparatus for Transfer of Resonance Enhancement of Spin Hyperpolarization via Chemical-exchange and Addition

Abstract

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 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 non-magnetic 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 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 °C to 80 °C. This benchtop device measures 62 cm (length) × 47 cm (width) × 47 cm (height), weights 30 kg, and requires only connections to 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 hyperpolarized ethyl [1-¹³C]acetate (P_13C = 7%, [c] = 1 M). Moreover, the 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 (P_13C = 14%, [c] = 27 mM), [1-¹³C]-α-ketoglutarate (P_13C = 17%), [1-¹³C]ketoisocaproate (P_13C = 18%), [¹⁵N₃]metronidazole (P_15N = 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 pre-clinical applications; indeed, detailed technical drawings and a bill of materials are provided to support the ready translation of this design to other laboratories.

Graphical Abstract

Although MRI has become one of the most useful imaging advances in modern medicine, the very low (10⁻⁶-10⁻⁵) spin polarization P of biomedically relevant spin-½ nuclei (¹H, ³¹P, ¹²⁹Xe, ¹³C, ¹⁵N) makes in vivo imaging of these nuclei challenging at clinically relevant conditions, with the exception of protons of abundant species, e.g., tissue water and fat.¹ However, it is possible to increase P up to the order of unity through the use of hyperpolarization approaches,¹ and produce exogenous hyperpolarized (HP) contrast agents for molecular imaging applications.^2–3 In such approaches, non-equilibrium spin polarization is transiently created through the use of NMR hyperpolarization techniques such as Spin Exchange Optical Pumping (SEOP),^4–5 dissolution Dynamic Nuclear Polarization (d-DNP),³ Parahydrogen-Induced Polarization (PHIP)^6–9 and other methods.^{1, 10} The d-DNP method is the leading hyperpolarization technique for the production of ¹³C¹¹ and ¹⁵N¹² HP contrast agents.¹³ This method relies on transfer of spin order from unpaired electrons. d-DNP requires complex and expensive (~$1M) equipment and typically long polarization times (~1 hour).¹⁴ In contrast, PHIP methods utilize parahydrogen (p-H₂) as a source of spin order, and can produce HP contrast agents using inexpensive (~$50-$100k), fast (~1 min), and relatively simple instrumentation.^15–21 The resulting p-H₂-hyperpolarized substrates can be employed for in vivo imaging^{6, 22–29} and other applications, e.g., for reaction monitoring.^30–33

The two major groups of p-H₂-based hyperpolarization techniques are non-hydrogenative Signal Amplification by Reversible Exchange (SABRE)^34–35 and hydrogenative PHIP.^{8–9, 36} SABRE technique is based on simultaneous chemical exchange of p-H₂ and to-be-hyperpolarized molecule on exchangeable metal center.³⁴ Both methods rely on p-H₂ gas as the source of spin order.⁶ Both techniques employ chemical catalysis to bring together a p-H₂ molecule and a target nucleus within a few chemical bonds in order to facilitate polarization transfer from p-H₂-derived protons to the target nucleus via spin-spin couplings. A wide range of transfer approaches can be envisioned for p-H₂-based polarization (PHIP and SABRE), but they all can be divided in two broad categories. One category of methods employs the application of radiofrequency (RF) pulses to enable polarization transfer,^37–39 which is typically performed in conventional high-field NMR spectrometers at a magnetic field of a few Tesla, or in a stand-alone hyperpolarizer, typically operating at a field of several mT. A number of PHIP devices employing this method have been reported with detailed description and bill of materials.^{16, 19–21, 40–42}

Another large category of PHIP methods relies on employing static or alternating μT magnetic fields operating to reach Level Anti-Crossings (LACs) regimes, where efficient polarization transfer can occur spontaneously.^43–44 For hydrogenative PHIP, a suitable unsaturated substrate is first hydrogenated with p-H₂ at a specific initial magnetic field. This step is typically accomplished at the Earth’s field for practical convenience. Next, a Magnetic Field Cycling (MFC)^45–46 procedure is applied as follows: the field is brought nonadiabatically (i.e., very fast change of magnetic field) to near zero, i.e., sub-μT. Finally, the magnetic field is increased adiabatically (i.e., gradually changing magnetic field, allowing the spin system to adapt its configuration⁴⁷) to establish a passage through LACs. This adiabatic field passage results in polarization transfer to the target nucleus (typically ¹³C or ¹⁵N for most biomedical applications) from nascent p-H₂ derived protons. For the SABRE technique, sub-μT static magnetic fields can be employed to reach LACs between p-H₂ derived hydrides and a to-be-hyperpolarized heteronucleus via spin-spin couplings established on a transiently formed polarization transfer complex (PTC) of a catalyst. This specialized variant of SABRE was called SABRE in SHield Enables Alignment Transfer To Heteronuclei (SABRE-SHEATH).^48–49 It has enabled the SABRE technique to efficiently hyperpolarize a wide range of biologically relevant molecules with ¹³C and ¹⁵N labels. This category of p-H₂-based hyperpolarization methods requires precise control of the magnetic fields experienced by a sample, which is conveniently achieved using mu-metal magnetic shields and electromagnets.^48–49 Additional requirements include p-H₂ gas delivery and temperature control for optimum polarization transfer with the goal of maximizing polarization gained by the heteronucleus. A number of experimental setups have been developed over the years for this second category of p-H₂ hyperpolarization, including the pioneering work from the Malmo group⁴⁶ and more recent work by TomHon and co-workers.⁵⁰ However, none of the previous literature reports have provided a complete design of the instrumentation with a bill of materials (BOM) that could be readily reproduced by others working in this rapidly expanding field. Moreover, many of these hyperpolarization setups have been designed with the vision of studying novel materials and novel detection approaches in ultra-low magnetic fields rather than with the goal of producing a batch of HP contrast agents for biomedical utilization.

Here, we present a complete design of the integrated hyperpolarizer for p-H₂-based hyperpolarization experiments in μT magnetic fields. The device chassis integrates the key components needed to perform hyperpolarization experiments including a mass flow controller for precise p-H₂ flow control, a pressure regulator (for p-H₂ gas pressure control), a mu-metal chamber with automated degaussing capability, non-magnetic temperature regulation, and precise magnetic field control in the μT magnetic field range (enabled by a solenoid magnet). We also provide examples of hyperpolarization experiments using this hyperpolarizer device for SABRE-SHEATH hyperpolarization of [¹⁵N₃]metronidazole (P_15N of 13%) and [1-¹³C]pyruvate (P_13C of 14%). The utility of the device is also demonstrated for PHIP-MFC hyperpolarization of ethyl [1-¹³C]acetate. While the vast majority of the studies have been performed in standard 5-mm NMR tubes (providing convenient means of spectroscopic detection using a wide range of NMR spectrometers), the device chamber can accommodate reaction vessels of over 1-liter size, providing a scalable platform for a wide range of studies.

MATERIALS AND METHODS

Design.

The main frame of the device is made from a 30-mm T-slot aluminum extrusion rail (McMaster Carr, P/N 5537T97 and 5537T98 or Misumi, P/N HFS6–3060 and HFS6–3030) chosen due to their non-magnetic and lightweight nature. This extrusion rail enables convenient mounting of other auxiliary components. The step-by-step construction of the polarizer skeleton is shown in Figures S4–S12 in the Supporting Information (SI). The front face of the polarizer chassis is divided into five sections for the attachment of 3-mm (or 1/8”) thick aluminum plates that are machined for mounting of device components, Figure 1. Each mounted plate on the front face is dedicated to a specific group of functions of the device: electrical switches, PID temperature control, magnetic field control and degaussing, p-H₂ gas flow control (using a mass flow controller) and p-H₂ line pressure control (using a pressure regulator). The inside of the chassis is divided into three major compartments, comprising a temperature control section, the magnetic shields with field control, and the electric wiring unit areas, Figure S13. The technical drawings for all polarizer panels are provided in Figures S21–S29.

Figure 1.

Annotated photograph of the integrated polarizer device outlining the key components. The variable-temperature manifold (heating components) is illustrated in detail in Figure 2 and in the SI (Figure S14).

To avoid paramagnetic interference of the heating equipment with the mu-metal shields, a non-magnetic heating/cooling solution was adopted, Figures 1 and 2a. Specifically, a 220-mm-long T-slot aluminum rail (25-mm size) with a 5-mm-diameter central hole was employed for heat exchange between the aluminum rail and a 5-mm NMR tube placed inside. The temperature of the aluminum T-slot rail block is maintained by the recirculating-water-filled PVC tubing uniformly coiled around it, Figure 3 and SI. The ends of the coiled PVC tubing are connected to a heat exchanger, and the water flow inside the tubing is maintained via a miniature water pump (Figure 2b and SI), with a flow rate of 8 L/min. The heating elements of the heat exchanger are controlled by a PID controller, Figure 1, Figure 2a and Figure 2c. Water media inside the PVC tubing was doped with ethylene glycol to prevent foaming and air bubble formation. Our setup can maintain a sample temperature of up to 80 °C. Finally, the thermoelectric cooler (TEC) can be switched on in case that temperature control below room temperature is needed, Figure 3. In this case, the TEC is constantly turned on to maximum power for chilling, while the PID with heater provide control for temperatures between 8 °C and room temperature.

Figure 2.

Hyperpolarizer 3D rendering of a) external view of the device, b) exploded view of the device, c) side view with two panels removed and back panel transparent. Note that gas tubes and electric wires are not displayed to improve visibility. The minute differences between the 3D renderings presented in this figure versus the annotated photograph shown in Figure 1 is due to the fact that eight copies of the hyperpolarizer have been built and installed so far with small differences in the component layout in the front and the back panels.

Figure 3.

(left) Schematic of the internal components of the device, showing the electrical connections. (right) Schematic of the fluid path of the hyperpolarizer, using the example of p-H₂ gas bubbling via a standard 5-mm NMR tube.

Parahydrogen gas pressure and flow regulation is integrated by using a brass pressure regulator (Concoa, P/N COA4025301) and mass flow controller (Sierra Instruments, C100L-DD-1-OV1-SV1-PV2-V1-S0-C0) respectively, Figure 3. Parahydrogen enters the device via a through-wall push-to-connect 1/8” outer-diameter (OD) Teflon line at the back of the device, flows through the regulator (set to the desired pressure) before it passes through the mass-flow controller and delivered to the sample for bubbling. A pressure gauge positioned immediately after the flow controller along the way of the manifold reports on the operating pressure, whereas the mass flow controller display reports on the p-H₂ flow (5–150 standard cubic centimeters per minute, sccm). Since this mass-flow controller is limited to 130 psi maximum pressure, the total operational pressure range is limited to 100 psi p-H₂ gas overpressure, which is controlled by a safety valve, Figure 3. If a less expensive setup is desired with higher dynamic p-H₂ gas flow rate and pressure, the mass-flow controller is readily replaced by a brass high-precision threaded flow-adjustment valve (McMaster-Carr, P/N 7832K21), Figure S31. Parahydrogen gas continuously flows through the hyperpolarizer to ensure positive pressure and removal of any trapped air. When the bypass valve is in the open position, the gas flow is directed via the bypass and no p-H₂ gas bubbling through the sample occurs. When the bypass valve is closed, the p-H₂ gas is directed to the NMR tube (or other vessel) for bubbling through a liquid sample. Safety and vent valves are connected in series with the bypass valve for pressure relief, Figure 3. Optionally, the gas manifold can be modified by the addition of a saturator vessel (for example, another NMR tube with pure solvent) in-line before the NMR tube. The saturator is important for continuous hours-long SABRE-SHEATH experiments, as it allows for compensating the gradual solvent evaporation from the sample as a result of gas bubbling.⁵¹ This saturator system should have its own bypass valve: in this way, one can start and stop p-H₂ gas bubbling through the saturator and the sample independently from each other. One can either use the vent valve for these purposes (i.e., change the gas line connections to the vent valve and use the safety valve for pressure relief) or incorporate an additional (third) valve in the setup.

As reported previously, one challenge frequently encountered in microtesla field experiments is the eventual magnetization of magnetic shields used, which can lead to substantial undesirable dephasing effects.⁵² In this hyperpolarizer, we have integrated a previously developed automated degaussing circuit, Figure 2b and Figure 3.⁵³ It is based on adiabatic attenuation of input current using thermistors in parallel with a capacitor and the degaussing coil.⁵³ The circuit allows degaussing the shield to a residual field of less than 20 nT in less than 3 seconds by pressing the degauss button, Figure 1.

Operation.

The device is initiated by a main power switch at the back. Once it is turned on, the PID temperature controller begins temperature regulation, which is usually achieved in 10–15 minutes. The recirculating pump must be on for temperature-controlled experiments. Next, the field inside the mu-metal shield is measured using a three-axis fluxgate magnetometer (Bartington Instruments, Oxford, UK). If the residual field is not within the desired range, the degaussing button is pressed and released after approximately 2 seconds (the button is released when the degauss LED light dims off) to demagnetize the shield. It is important to note that the degauss switch is turned on first before pressing the degauss button. This procedure prevents the accidental magnetization of the mu-metal shield due to unintentional consecutive pressing of the degauss button.

For typical operation, the regulator is set to 130 psi p-H₂ pressure, and the p-H₂ input line is pressurized. In our lab, this is established using the p-H₂ distribution system that routes p-H₂ gas via 1/8” OD line to the back of the hyperpolarizer.⁵⁴ Alternatively, p-H₂ gas is fed into the device from a p-H₂ tank via 1/8” OD PTFE tubing with a push-to-connect through-wall connector on the back panel. Next, the sample in 5-mm NMR tube is connected using a Teflon jacket (made from 0.25”-OD tubing) via non-magnetic push-connect wye adapter (McMaster Carr 5779K262) shown in Figure 3 and Figure S18.⁵³ Next, the mass-flow controller is turned on using the power switch located on the front panel, and the p-H₂ gas flow rate is set using the control buttons. At this point, the pressure reading on the gauge (Figure 3) starts rising to the set the pressure of the safety valve (typically 100 psi). The device is usually purged with incoming p-H₂ for several minute to remove any trapped air in gas lines. Once the desired p-H₂ pressure is reached, and the p-H₂ now exits via the safety valve (with relief valve closed), the sample is fully pressurized and ready for bubbling. Finally, when the desired temperature is reached, and the sample is pressurized, the magnetic field required for the experiment is set either using internal PSU or external AFG, and the hyperpolarization experiment can proceed.

For hyperpolarization experiments (PHIP or SABRE), the liquid sample is bubbled with p-H₂ gas by closing the bypass valve for the desired period of time. When polarization is completed, the valve is switched to the open position to cease the flow of p-H₂ gas (and direct the flow via the bypass). The sample is then transferred to the NMR spectrometer for the detection. The p-H₂ bubbling is terminated because there is no more gain in polarization build-up (after the sample is removed from the μT field), and to minimize susceptibility-induced magnetic field gradients to prevent otherwise undesirable NMR line broadening.

RESULTS AND DISCUSSION

The performance of the hyperpolarizer has been tested in several recently reported PHIP and SABRE-SHEATH hyperpolarization studies.^{52, 54–61} Provided below are illustrative examples of the hyperpolarizer utility for producing ¹³C and ¹⁵N HP solutions of biologically relevant molecules using SABRE and PHIP hyperpolarization techniques. Approximately 95–98.5% p-H₂ gas was employed for all experiments described here.⁶²

SABRE-SHEATH hyperpolarization of [¹⁵N₃]metronidazole.

Metronidazole is an FDA-approved antibiotic. Moreover, the detection of HP [¹⁵N₃]metronidazole in brain has been recently demonstrated, and the potential utility for hypoxia sensing has been proposed.⁶³ For [¹⁵N₃]metronidazole hyperpolarization, the to-be-hyperpolarized sample employing 2 mM of [IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl) imidazole-2-ylidene and COD = cyclooctadiene)⁶⁴ polarization transfer pre-catalyst and 20 mM of [¹⁵N₃]metronidazole⁶⁵ in CD₃OD (0.6 mL aliquot) was placed into a medium-wall 5-mm NMR tube. The sample was bubbled with p-H₂ at room temperature for 1 hour at 100 psi using a flow rate of 40 sccm to activate the sample.⁶⁶ After activation was achieved, the sample was bubbled with p-H₂ for 1 min (at room temperature, 70 sccm and 100 psi p-H₂ overpressure) to perform SABRE-SHEATH hyperpolarization (Figure 4a) and quickly transferred into a 1.4 T NMR spectrometer (NMR Pro60, Nanalysis) situated right next to the hyperpolarizer for ¹⁵N detection, Figure 4c. A corresponding ¹⁵N spectrum of thermally polarized neat [¹⁵N]pyridine (obtained using 64 scans with repetition time of 10 min) was employed as a signal reference, Figure 4b.^{59, 66} Following the detection of the HP signal, the sample was returned back to the hyperpolarizer, where the sample temperature was stabilized for 1 min, and the experiment was repeated (on average, it was possible to repeat SABRE-SHEATH re-hyperpolarization of the sample every 4–5 minutes for 4–5 hours). Repeating the experiment at various microtesla fields allows performing a field sweep experiment, Figure 4d, that identifies the optimal hyperpolarization conditions for SABRE-SHEATH, i.e., where the magnetic field matches the LACs⁴⁴ position of the spin system participating in the polarization transfer from p-H₂-derived hydrides to the ¹⁵N₃ nucleus, Figure 4a.^48–49 The spin-relayed polarization transfer mechanism⁶⁷ facilitates the polarization transfer from the ¹⁵N₃ site to ¹⁵N₁ site and ¹⁵NO₂ site, Figure 4a.⁶⁵ Moreover, performing the experiment at the optimal field at various temperatures⁶⁸ (i.e., temperature sweep) shown in Figure 4e allows identifying the optimal temperature, corresponding to the optimal rate of chemical exchange of [¹⁵N₃]metronidazole on the PTC, Figure 4a. Using the combination of the optimized temperature (~18 °C) and the field (~0.6 μT) in the SABRE-SHEATH experiment allowed reaching P_15N of 13% on all three ¹⁵N sites, Figure 4c. All in all, we conclude that the device provides sufficient reproducibility to enable systematic optimization studies of temperature and magnetic field, Figure 4.

Figure 4.

a) Schematic of spin-relayed SABRE-SHEATH hyperpolarization of [¹⁵N₃]metronidazole. b) ¹⁵N spectrum of thermally polarized neat [¹⁵N]pyridine (obtained using 64 scans with repetition time of 10 min) used as a signal reference for computing ¹⁵N signal enhancement and P_15N values. c) ¹⁵N spectrum of HP [¹⁵N₃]metronidazole obtained using 1 scan and otherwise the same acquisition parameters. d) Magnetic field sweep of SABRE-SHEATH process using temperature of ~18 °C. e) Temperature sweep of SABRE-SHEATH process at a field of 0.6 μT.

SABRE-SHEATH hyperpolarization of [1-¹³C]pyruvate.

HP [1-¹³C]pyruvate is the emerging molecular probe for imaging of elevated anaerobic glycolysis, a pathway upregulated in many cancers and other metabolically challenged diseases.^{13, 69} Over 50 clinical trials are now pending using HP [1-¹³C]pyruvate as an injectable contrast agent produced via d-DNP technology according to clinicaltrials.gov.⁷⁰ The SABRE-SHEATH hyperpolarization method presents a cheaper and faster approach to hyperpolarize [1-¹³C]pyruvate, Figure 5a.^71–72 We have recently demonstrated that [1-¹³C]pyruvate can be hyperpolarized via SABRE-SHEATH up to 14.8%,^{52, 55, 73} using the following experimental parameters of our hyperpolarizer: 27 mM [1-¹³C]pyruvate, 18 mM DMSO, and 6 mM IMes PTC catalyst in 0.6 mL CD₃OD, activation time of 30 minutes, polarization time of 1 min with 120 psi p-H₂ overpressure and 120 sccm p-H₂ flow rate and pulsed SABRE-SHEATH approach.⁵² Other structurally-similar α-ketocarboxylates have been also successfully ¹³C-hyperpolarized using the hyperpolarizer and similar experimental conditions, including α-ketoglutarate (P_13C of up to 17%)⁵⁴ and ketoisocaproate (P_13C of up to 18%).⁵⁸ Moreover, the hyperpolarizer has also been employed for redissolution (Re-D) SABRE-SHEATH method that yields biocompatible aqueous solution of HP [1-¹³C]pyruvate with P_13C of 9%.⁵⁷

Figure 5.

a) Schematic of simultaneous chemical exchange of p-H₂ and [1-¹³C]pyruvate on the activated IrIMes PTC complex 3b to yield “free” HP [1-¹³C]pyruvate. The complex 3a undergoes only p-H₂ exchange. b) ¹³C NMR spectrum of 27 mM HP sodium [1-¹³C]pyruvate hyperpolarized via SABRE- SHEATH; c) Corresponding ¹³C spectrum of signal reference compound (neat [1-¹³C]acetic acid). Reprinted with permission from ref⁵². Copyright 2022 ACS.

PHIP-MFC hyperpolarization of ethyl [1-¹³C]acetate.

Due to the ease with which it is taken up by brain cells, ¹³C-labeled acetate has been used for brain metabolic tracing in rats and humans by magnetic resonance spectroscopy (MRS).^74–75 HP [1-¹³C]acetate (produced by d-DNP) has also been used for the investigation of other metabolic transformations in the TCA cycle.⁷⁶ Hyperpolarizing acetate and pyruvate via side-arm (SAH) PHIP has been demonstrated by Reineri and co-workers.⁷⁷ In the PHIP-SAH method, a precursor molecule with an unsaturated side-arm is hydrogenated with p-H₂ and then the singlet order obtained on the product protons is converted into magnetization on ¹³C using magnetic field cycling.^{46, 77–79} Following the ¹³C hyperpolarization of the unsaturated precursor, e.g., using vinyl arm hydrogenation shown in Figure 6a, the arm can be cleaved by hydrolysis for production of HP [1-¹³C]acetate or [1-¹³C]pyruvate.⁷⁷ The hyperpolarizer was employed for production of HP ethyl [1-¹³C]acetate using PHIP-SAH method, Figure 6a, via magnetic field cycling procedure, Figure 6b. Briefly, 0.6 mL of 0.5–1 M of vinyl [1-¹³C]acetate in acetone (with 10 mM rhodium-based catalyst (1,4-bis-(diphenylphosphino)butane(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate ([Rh(dppb)(COD)]BF₄), Strem 45–0190, CAS 79255- 71-3) was measured into a regular-wall 5-mm NMR tube and heated for 15 s at 80 °C in the Earth’s field.^60–61 The sample was then shuttled into the hyperpolarizer’s mu-metal shield and then slowly pulled out in 5 s before being transferred for ¹³C detection with a 1.4 T benchtop NMR spectrometer, Figure 6c.^60–61 Comparison of HP signal intensity with the signal intensity obtained from thermally polarized reference (neat [1-¹³C]acetic acid, Figure 6c) allowed determining P_13C of up to 7.33%, Figure 6c.⁶⁰ The robust operation of the device enabled optimization of hyperpolarization kinetics (Figure 6d) and reaction temperature (Figure 6e) with the goal of maximizing the degree of ¹³C polarization.^60–61 Achieving the relatively high level of ¹³C polarization at high substrate concentration was useful to demonstrate the feasibility of creating ¹³C Radio Amplification by Stimulated Emission of Radiation (RASER).^60–61 While the manual MFC approach was used for hyperpolarization of ethyl [1-¹³C]acetate, Figure 6, the AFG can be programmed for automated operation (see SI for details). Indeed, the automated procedure has been implemented recently using the polarizer prototype, demonstrating the feasibility of performing PHIP-SAH hyperpolarization studies using natural ¹³C abundance and a benchtop spectrometer.⁸⁰ We envision that other biomolecules⁸¹ can also be amenable to PHIP-SAH hyperpolarization using our device.

Figure 6.

a) Hyperpolarization reaction of [1-¹³C]vinyl acetate with p-H₂ to form ¹H HP ethyl [1-¹³C]acetate, which is field-cycled to yield ¹³C hyperpolarized ethyl [1-¹³C]acetate. b) Overall scheme of magnetic field cycling. c) ¹³C spectrum of HP ethyl [1-¹³C]acetate using manual field cycling (0.5 M, red trace) and corresponding ¹³C spectrum of thermally polarized signal reference of neat [1-¹³C]acetic acid (17.5 M, blue trace). All spectra are acquired using a 9° flip angle of RF excitation pulse. d) p-H₂-based hyperpolarization kinetics of 0.5 M vinyl acetate (VA) conversion to HP ethyl acetate at 80 °C with 10 mM catalyst concentration. e) Temperature dependence of P_13C at 1 M VA and 10 mM catalyst concentrations and hydrogenation time (T_hyd) of 10 seconds. Reproduced with permission from ref⁶⁰. Copyright 2023 Wiley.

The key limitation of the reported hyperpolarizer device is the lack of automation and the focus on hyperpolarization of small-size samples intended for detection in 5-mm NMR tubes. At the same time, the simplicity and the low cost of the device clearly highlight the benefits of the reported design. We envision the relatively large chamber of the solenoid magnet can in principle enable the use of a substantially larger reaction chamber, and sample sizes over 1 liter can in principle be accommodated. At the same time, the small scale of polarization in 5-mm NMR tubes reported here is large enough to produce a dose of the ¹³C contrast agent for its utility in mice and rats.^{24–25, 58} We also envision that other biologically relevant molecules can be hyperpolarized using the reported hyperpolarized via SABRE-SHEATH and pulsed-SABRE-SHEATH because all enabling functionalities are indeed available in the reported hyperpolarizer.^{35, 82} Moreover, while the feasibility demonstrations of hyperpolarization processes have been performed using a large (ca. 10 L size) aluminum cylinder⁶² containing compressed p-H₂ gas, the reported device can potentially utilize p-H₂ gas packed in cheap and disposable pressurized aluminum cans reported recently⁸³—an approach that may further reduce the cost and complexity of hyperpolarizer operation, especially in the proximity of MRI scanners.

It should also be noted that a number of approaches have been recently demonstrated to enable the production of biocompatible aqueous solutions of HP [1-¹³C]pyruvate using the gas manifold and other components employed in the reported hyperpolarizer design.^{24–25, 57, 84} Two of these recent advances reported on HP [1-¹³C]pyruvate production via SABRE-SHEATH in 5-mm NMR tubes and its utilization for metabolic imaging of HP [1-¹³C]pyruvate in mice and rats.²⁴ Therefore, the reported hyperpolarizer design can be readily employed for preclinical studies using the emerging new protocols for production of biocompatible formulations of HP [1-¹³C]pyruvate already. We envision that these methods for production of biocompatible solutions of HP media can be expanded to other biologically relevant HP molecular probes for their in vivo utilization in the near future. However, it should be noted that the reported hyperpolarizer design is not approved for clinical utilization.

CONCLUSIONS

We have presented a hyperpolarizer for performing p-H₂-based hyperpolarization experiments in microtesla magnetic fields. The device has been designed with the emphasis on simplicity and low cost (less than $7,000 in components, ca. 2021, when it was developed and first constructed). The device can be conveniently sited near an NMR spectrometer^{52, 54–58} for high-resolution characterization and optimization of the hyperpolarization processes or in the proximity of an MRI scanner⁵⁸ to perform rapid hyperpolarization of substrates for utilization in small animals. Moreover, the hyperpolarizer can easily be relocated between different sites due to its compact footprint and quick start time. The utility of the hyperpolarizer has been demonstrated for hyperpolarization of a wide range of biologically relevant molecules, including most notably [1-¹³C]pyruvate (P_13C = 14%). Future improvements of the reported hyperpolarizer are envisioned in the area of automation, process optimization (i.e., the development of high-pressure and high-flow reaction vessels for p-H₂ bubbling), and demonstration of larger production volumes and quantities of HP biomolecules (all the way to clinical scale), and implementation of new approaches and chemistries to mitigate the toxicity of p-H₂-hyperpolarized solutions that frequently contain catalyst and organic solvents.^{25, 57} All in all, the reported open-source design bodes well for future development and widespread use of p-H₂-hyperpolarized injectable contrast agents.