Andrea Capozzi

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

Significance Hyperpolarization is a significant development in MRI because it allows for imaging different metabolites in real time in vivo. There are no fundamental obstacles to rapid translation of this technique. Yet, to date, it has been necessary to use persistent radicals that need to be filtered out before injection and require pharmacological tests, which slow down the overall protocol, leading to reduced sensitivity. The demonstration that it is possible to prepare purely endogenous MRI agents to probe metabolism in vivo without using any potentially toxic compounds is a substantial step forward toward clinical radiology free of side effects. 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 13C 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 13C 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.

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.

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.

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

Hyperpolarised (HP) 13C 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 13C 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 [1‐13C]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. Copyright

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

To probe the cardiac metabolism of carbohydrates and short chain fatty acids simultaneously in vivo following the injection of a hyperpolarized 13C‐labeled substrate mixture prepared using photo‐induced nonpersistent radicals.

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.

Beyond Spin Exchange Optical Pumping: Hyperpolarization of 129Xe via Sublimation Dynamic Nuclear Polarization

Dynamic nuclear polarization (DNP) is a competitive alternative to the established spin exchange optical pumping method to hyperpolarize 129Xe. DNP consists of transferring the large electron spin polarization of incorporated free radicals to the surrounding nuclei in a frozen sample by continuous irradiation with microwaves at a frequency close to the electron spin resonance of the radicals. The introduction of a dissolution step to transform the frozen sample into a liquid while preserving the nuclear polarization opened new perspectives for metabolic and molecular imaging. Hyperpolarization via dissolution DNP is nowadays widespread in the biomedical community and it is therefore attractive to develop a method to prepare hyperpolarized 129Xe using the same instrumentation. In this chapter, the concept of sublimation DNP to hyperpolarize gases is presented. The physical and technical challenges associated with the preparation of an optimal frozen sample containing 129Xe are discussed. It is shown that photo-induced radicals and optimized hardware can boost the polarization level and the throughput. It is currently possible to produce 0.1 L of hyperpolarized 129Xe polarized at 25% in about 30 min and there are no fundamental barriers to rapidly scale up this method to the production of several litres per hour at the same polarization level.