Synopsis

Keywords: High-Field MRI, Hyperpolarized MR (Non-Gas), parahydrogen cancer spectroscopy

Parahydrogen-induced Polarization (PHIP) by Synthesis Amid the Magnet Bore Allows Dramatically Enhanced Nuclear Alignment (SAMBADENA) of agents in situ within the MRI magnet and requires very little hardware in addition to the MRI system. However, side-arm hydrogenation (PHIP-SAH), fast side-arm cleavage and purification have not been attempted yet.
Here, we present a SAMBADENA setup made from commercially-available components for future preclinical applications. Using this setup, we demonstrate high ¹³C polarizations of 17% for 25mM ethyl-[1-¹³C]-acetate. In addition, for ethyl-pyruvate, fast side-arm cleavage and removal of 96% of the organic solvent within 10s to obtain pyruvate in neat water is demonstrated.

INTRODUCTION

Hyperpolarized (HP) ¹³C MRI has enabled unprecedented real-time imaging of metabolism in patients with great potential for diagnostics and personalized therapy^1,2. Parahydrogen (pH₂) induced polarization (PHIP) by side-arm hydrogenation (PHIP-SAH) has opened a low-cost, high-throughput pathway to produce hyperpolarized metabolites^3,4. Recently, we demonstrated Synthesis Amid the Magnet Bore that Allowed a Dramatically Enhanced Nuclear Alignment (SAMBADENA)⁵. Using this approach, PHIP agents have been produced quasi-continuously in situ inside the MRI system every 15s and fast administration and ¹³C-imaging in vivo within 15s after hyperpolarization has been demonstrated^6,7. However, the purification to obtain neat aqueous metabolite solutions has not been attempted yet.

METHODS

Setup: The SAMBADENA setup comprised a preclinical 7T MRI system, ¹H-¹³C volume coil for excitation, ¹³C receive surface coil, and the pH₂-bubbling setup (Fig. 1). The latter comprised two gas cylinders (N₂ and pH₂), pressure regulators, a digital manometer, needle valves to regulate gas-flow, tubing and fittings, magnetic valves to gate the flow of gases, a custom power-relay box to control the valve actuation from the MRI pulse program, and a standard 5-mm NMR tube to host the hydrogenation reaction. An ultra-fine 1/32” PEEK catheter was used to guide pH₂ to the bottom of the NMR tube and through the reaction solution, to reduce magnetic field inhomogeneities⁸. The NMR tube was centered in the MRI system using a custom 3D-printed holder (Fig. 1A). Apart from the holder and power relay, all materials are commercially available (Table 1). Total cost of the pH₂-bubbling system is ≈1000€.
Samples: pH₂ was enriched to ≈85% using a helium-based cryostat and an iron-oxide catalyst⁹. For the reaction solutions, 25mM vinyl-[1-¹³C]-acetate-d₆ (VA, 1.1% naturally abundant ¹³C; CAS: 189765-98-8) and 3mM rhodium-based hydrogenation catalyst ([Rh(dppb)(COD)]BF4, CAS: 79255-71-3) were mixed in acetone-d₆ (CAS: 666-52-4) and oxygen was removed by guiding N₂ through the solution for 1min prior to hydrogenation.
Hyperpolarization: 200µL of this solution was filled into the NMR tube, which was then connected to the pH₂-bubbling system. The hyperpolarization pulse program was started and the NMR tube was back-pressurized with 2.5bar N₂ to prevent evaporation before heating. The sample was heated for 20s in an ≈80°C water bath. Subsequently, the tube was positioned at the MRI isocenter and a manual trigger button was actuated to start the hydrogenation of the precursor (Fig. 1B): 8bar pH₂ were guided through the sample for 12s under continuous-gas-flow condition. Next, the NMR tube was bypassed for 1s to stop the pH₂ flow and the spin-order transfer (SOT) from pH₂-derived protons to the [1-¹³C]-acetate nucleus was conducted (ESOTHERIC sequence¹⁰, (Fig. 1C), total length of 388ms). Hyperpolarized ¹³C signal was detected at the end of SOT and quantified by comparing it with the signal of a thermally-polarized reference solution (4M [1-¹³C]sodium acetate (CAS:23424-28-4) in 250µL H₂O).
Hydrolysis and purification: Similar to a recent report¹¹, the NMR tube connected to the SAMBADEMA setup was filled with a thermally-polarized 400µL solution of 50mM ethyl pyruvate (CAS:617-35-6) in acetone-d₆. Note that ethyl pyruvate was used here instead of ethyl acetate used for the HP experiments. Note that the precursor was not ¹³C-enriched. To perform side-arm cleavage, 200µL of 150mM sodium carbonate (CAS:144-55-8) in D₂O (CAS:7789-20-0) was injected into the tube and mixed with N2 for 10s. The tube was then exposed to vacuum while being heated in a 50°C water bath to remove acetone from the solution. The vacuum pump was connected to the SAMBADENA near the outlet. (Fig. 1A) NMR spectra of the solution were acquired after each of these steps using a 1T benchtop NMR spectrometer (43MHz, Magritek) and integrated with a custom routine (Matlab R2020a, MathWorks, USA). (Fig. 3)

RESULTS

Using this setup made from commercially-available materials, ¹³C polarization of 16.7% was achieved for 25mM ethyl-acetate-d₆ (EA), the hydrogenation product of VA (Fig. 2). With the described protocol for hydrolysis and purification, fast side-arm cleavage was observed (<10s). Only 4.3% of the final solution volume was acetone-d₆, and 56.6% of the starting amount of pyruvate was successfully reconstituted in D₂O. (Fig. 3).

DISCUSSION

The observed ¹³C polarizations are already high and can likely be improved considering that >50% has been demonstrated for ethyl-[1-¹³C]-acetate using an NMR setup¹². A higher pH₂ enrichment and faster hydrogenation, which seems feasible by increasing the pH₂ flow and temperature during the reaction, are expected to be constructive. Also, it has been shown that SOT in the MRI system can be compromised by B₀ inhomogeneity and B₁ ¹H-excitation bandwidth^8,13. Hence, more sophisticated RF pulse schemes like composite or adiabatic pulses should be investigated¹⁴. The successful extraction of purified pyruvate in water is most promising and next needs to be refined for faster purification procedure and removal of hydrogenation catalyst.

CONCLUSION

For the first time, SAMBADENA was demonstrated using mostly commercially available parts. While polarization and agent concentration can likely be improved, they are already sufficient for preclinical studies. By applying the cleavage and purification protocol to hyperpolarized samples, first pilot biomedical applications using SAMBADENA appear to be on the horizon¹⁵. This technique and setup are available to many groups with the promise to accelerate the translation of PHIP and wide use of HP metabolic MRI.

Acknowledgements

This work was supported by the German Cancer Consortium (DKTK), B.E.S.T. Fluidsysteme GmbH I Swagelok Stuttgart, the German Research Foundation (SCHM 3694/1-1, SCHM 3694/2-1, SFB1479, HO-4604/2, HO-4604/3), and the Federal Ministry of Education and Research (BMBF) lighthouse project QuE-MRT and Juniorverbund 01ZX1915C (AP). ABS and EYC thank Wayne State University for Postdoctoral Fellow award.

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