Stable electron-irradiated [1-$^{13}$C]alanine radicals for clinically viable metabolic imaging with Dynamic Nuclear Polarization

TL;DR

This work introduces a clinically viable route to hyperpolarised metabolic imaging by generating stable, endogenously produced radicals in alanine via ultra-high dose-rate electron irradiation. The irradiated alanine–glycerol system achieves substantial solid-state polarisation (~20%) at 6.7 T and supports dissolution with radical quenching, enabling safe in vivo imaging of alanine metabolism with comparable performance to conventional trityl-based methods. The study combines extensive experimental data (EPR, DNP, XRD, in vivo MRS/MRSI) with molecular dynamics and quantum simulations to reveal a partially ordered, cooperative spin regime that is not captured by standard DNP theories, and demonstrates the potential for centralized, sterile production and transport of hyperpolarised agents. If further optimized, this approach could broaden access to dDNP-based metabolic imaging by reducing sterility, regulatory, and logistical barriers while enabling sterile, room-temperature sample handling and broader substrate applicability.

Abstract

Dissolution Dynamic Nuclear Polarisation (dDNP) increases the sensitivity of magnetic resonance experiments by $>10^4$-fold, permitting isotopically-labelled molecules to be transiently visible in MRI scans. dDNP requires a source of unpaired electrons in contact with labelled nuclei, cooled to $\sim$1K, and spin-pumped into a given state by microwaves. These electrons are usually chemical radicals, requiring removal by filtration prior to injection into humans. Alternative sources, such as UV irradiation, generate lower polarisation and require cryogenic transport. We present ultra-high-dose-rate electron irradiation as a novel alternative for generating non-persistent radicals in alanine/glycerol mixtures. These are stable for months at room temperature, quench spontaneously upon dissolution, are present in dose-dependent concentrations, and generate comparable nuclear polarisation to trityl radicals used clinically (20\%) through a novel mechanism. This process is inherently sterilising, permitting imaging of alanine metabolism \textit{in vivo}. As well as scientific novelty, this overcomes the biggest barrier to clinically translating dDNP.

Figures (14)

• Figure 1: Radical generation. (A) An ultra-high dose rate optimised in-house developed linear accelerator was used to bombard (B) a target containing polycrystalline [1-13C]alanine with 6 MeV electrons. (C) These generate Stable Alanine Radicals (SAR) detectable by EPR in a dose-dependent fashion compared to (D) TEMPO as a concentration standard. (E) The 13C-hyperfine interaction is strong for the SARs, significantly altering the EPR spectrum compared to natural abundance (NA) alanine, which we quantified (F) via simulations in EasySpin.
• Figure 2: (A) DNP build-up profiles of different high dose rate irradiated samples at 3.35 T; we found that dispersing irradiated polycrystalline alanine powder in glycerol produced substantially higher polarisation. (B) Longitudinal measurements of the spin counts showed that there was no significant effect on the endogenous radical concentration with increased duration of storage in anhydrous glycerol up to 4 months, in sharp contrast to pyruvic acid/trityl radical mixtures which quench within hours (mean $\pm$ SD of three technical replicates). (C) Alanine/glycerol mixtures did not significantly alter acquired EPR data, but at (D) 5 K the electronic environment altered considerably
• Figure 3: (A) The frequency sweep profile of DNP with alanine/glycerol mixtures showed qualitative differences with increasing radiation dose, which we hypothesise is due to cooperativity. (B) Polarisation build-up as a function of time showed profound biexponential behaviour under these conditions, distinct from narrow linewidth radicals such as trityl. (C, Di) X-ray diffractometery measurements of both samples were able to demonstrate that polycrystalline alanine remained dispersed (not dissolved) in glycerol and was well described by (Dii) existing crystal structures with slight refinement but had profound crystallographic texture, indicative of an ordered crystalline arrangement within the glycerol matrix. (E) The efficiency of DNP under these conditions is surprising, comparable to narrow linewidth trityl radicals.
• Figure 4: (A) At 6.7 T, a unimodal, always-positive frequency sweep curve was observed. Pulsed modulation with a swept-frequency microwave pulse as illustrated obtained a significant increase in nuclear polarisation. (B) The molecular orientation of the alanine radical matters significantly for the spectral density function $g(\omega)$ as defined by Wenckebach,Wenckebach2017a simulated here using parameters from powder 13C alanine EPR data and assuming a partial ordering parameter $\lambda$. (C) The resultant limiting polarisation obtained was high, and comparable to the use of trityl radicals with alanine. (D) After dissolution, spontaneous quenching of endogeneous radicals formed by electron irradiation resulted in a lengthened $T_1$ (77 s) compared to that with trityl (40 s) at 1.4 T.
• Figure 5: (A, B) Stacked and (C, D) summed spectra from slice-selective spectroscopic acquisitions following separate tail-vein injections of [1-13C]alanine polarised via 38 mM OX063 trityl radical and hyperpolarised irradiated 13C-alanine. (C, D) Inset: a magnification of the spectra presented in the panels to highlight the intensities and positions of the downstream metabolites, whose chemical shift values match those reported in the literature for 13C-lactate and 13C-pyruvate. (E, F) After quantification by AMARES, metabolite dynamics were comparable between the two methods of preparation. (G) MR Spectroscopic Imaging (MRSI) images obtained in a separate rat were likewise near identical, validating the use of the technique for in vivo imaging of redox potential, alanine images of which are shown concatenated over time.