Real-Time Metabolic Magnetic Resonance Spectroscopy of Pancreatic and Colon Cancer Tumor-Xenografts with Parahydrogen Hyperpolarized 1-13C Pyruvate-d3.

Parahydrogen-induced polarization (PHIP) is an emerging technique to enhance the signal of stable isotope metabolic contrast agents for Magnetic Resonance (MR). The objective of this study is to continue establishing 1-13C-pyruvate-d3, signal-enhanced via PHIP, as a hyperpolarized contrast agent, obtained in seconds, to monitor metabolism in human cancer. Our focus was on human pancreatic and colon tumor xenografts. 1-13C-vinylpyruvate-d6 was hydrogenated using parahydrogen. Thereafter, the polarization of the protons was transferred to 13C. Following a workup procedure, the free hyperpolarized 1-13C-pyruvate-d3 was obtained in clean aqueous solution. After injection into animals bearing either pancreatic or colon cancer xenografts, slice-selective MR spectra were acquired and analyzed to determine rate constants of metabolic conversion into lactate and alanine. 1-13C-pyruvate-d3 proved to follow the increased metabolic rate to lactate and alanine in the tumor xenografts.

Hyperpolarization of 15N-Pyridinium by Using Parahydrogen Enables Access to Reactive Oxygen Sensors and Pilot In Vivo Studies.

Magnetic resonance with hyperpolarized contrast agents is one of the most powerful and noninvasive imaging platforms capable for investigating in vivo metabolism. While most of the utilized hyperpolarized agents are based on 13C nuclei, a milestone advance in this area is the emergence of 15N hyperpolarized contrast agents. Currently, the reported 15N hyperpolarized agents mainly utilize the dissolution dynamic nuclear polarization (d-DNP) protocol. The parahydrogen enhanced 15N probes have proven to be elusive and have been tested almost exclusively in organic solvents. Herein, we designed a reaction based reactive oxygen sensor 15N-boronobenzyl-2-styrylpyridinium (15N-BBSP) which can be hyperpolarized with para-hydrogen. Reactive oxygen species plays a vital role as one of the essential intracellular signalling molecules. Disturbance of the H2O2 level usually represents a hallmark of pathophysiological conditions. This H2O2 probe exhibited rapid responsiveness toward H2O2 and offered spectrally resolvable chemical shifts. We also provide strategies to bring the newly developed probe from the organic reaction solution into a biocompatible injection buffer and demonstrate the feasibility of in vivo 15N signal detection. The present work manifests its great potential not only for reaction based reactive sensing probes but also promises to serve as a platform to develop other contrast agents.

Real-Time Pyruvate Chemical Conversion Monitoring Enabled by PHIP.

In recent years, parahydrogen-induced polarization side arm hydrogenation (PHIP-SAH) has been applied to hyperpolarize [1-13C]pyruvate and map its metabolic conversion to [1-13C]lactate in cancer cells. Developing on our recent MINERVA pulse sequence protocol, in which we have achieved 27% [1-13C]pyruvate carbon polarization, we demonstrate the hyperpolarization of [1,2-13C]pyruvate (∼7% polarization on each 13C spin) via PHIP-SAH. By altering a single parameter in the pulse sequence, MINERVA enables the signal enhancement of C1 and/or C2 in [1,2-13C]pyruvate with the opposite phase, which allows for the simultaneous monitoring of different chemical reactions with enhanced spectral contrast or for the same reaction via different carbon sites. We first demonstrate the ability to monitor the same enzymatic pyruvate to lactate conversion at 7T in an aqueous solution, in vitro, and in-cell (HeLa cells) via different carbon sites. In a second set of experiments, we use the C1 and C2 carbon positions as spectral probes for simultaneous chemical reactions: the production of acetate, carbon dioxide, bicarbonate, and carbonate by reacting [1,2-13C]pyruvate with H2O2 at a high temperature (55 °C). Importantly, we detect and characterize the intermediate 2-hydroperoxy-2-hydroxypropanoate in real time and at high temperature.

Metabolic Tumor Imaging with Rapidly Signal-Enhanced 1-13 C-Pyruvate-d3.

The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1-13 C-pyruvate-d3 , which is rapidly enhanced via para-hydrogen, and thus, the signal is amplified by several orders of magnitudes in less than a minute. Using as a model, human melanoma xenografts injected with signal-enhanced 1-13 C-pyruvate-d3, we show that the conversion of pyruvate into lactate can be monitored along with its kinetics, which could pave the way for rapidly detecting and monitoring changes in tumor metabolism.

Molecular precursors to produce para-hydrogen enhanced metabolites at any field.

Enhancing magnetic resonance signal via hyperpolarization techniques enables the real-time detection of metabolic transformations even in vivo. The use of para-hydrogen to enhance 13 C-enriched metabolites has opened a rapid pathway for the production of hyperpolarized metabolites, which usually requires specialized equipment. Metabolite precursors that can be hyperpolarized and converted into metabolites at any given field would open up opportunities for many labs to make use of this technology because already existing hardware could be used. We report here on the complete synthesis and hyperpolarization of suitable precursor molecules of the side-arm hydrogenation approach. The better accessibility to such side-arms promises that the para-hydrogen approach can be implemented in every lab with existing two channel NMR spectrometers for 1 H and 13 C independent of the magnetic field.

In Vivo Metabolic Imaging of [1-13 C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra-fast and low-cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1-13 C]pyruvate-d3 for injection in 6 minutes. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13 C]pyruvate-d3 polarized to ≈11 % (residual 250 mM methanol and 20 µM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Bimodal Fluorescence/Magnetic Resonance Molecular Probes with Extended Spin Lifetimes.

Bimodal molecular probes combining nuclear magnetic resonance (NMR) and fluorescence have been widely studied in basic science, as well as clinical research. The investigation of spin phenomena holds promise to broaden the scope of available probes allowing deeper insights into physiological processes. Herein, a class of molecules with a bimodal character with respect to fluorescence and nuclear spin singlet states is introduced. Singlet states are NMR silent but can be probed indirectly. Symmetric, perdeuterated molecules, in which the singlet states can be populated by vanishingly small electron-mediated couplings (below 1 Hz) are reported. The lifetimes of these states are an order of magnitude longer than the longitudinal relaxation times and up to four minutes at 7 T. Moreover, these molecules show either aggregation induced emission (AIE) or aggregation caused quenching (ACQ) with respect to their fluorescence. In the latter case, the existence of excited dimers, which are proposed to use in a switchable manner in combination with the quenching of nuclear spin singlet states, is observed.

A Field-Independent Method for the Rapid Generation of Hyperpolarized [1-13 C]Pyruvate in Clean Water Solutions for Biomedical Applications.

Hyperpolarization methods in magnetic resonance enhance the signals by several orders of magnitude, opening new windows for real-time investigations of dynamic processes in vitro and in vivo. Here, we propose a field-independent para-hydrogen-based pulsed method to produce rapidly hyperpolarized 13 C-labeled substrates. We demonstrate the method by polarizing the carboxylic carbon of the pyruvate moiety in a purposely designed precursor to 24 % at ≈22 mT. Following a fast purification procedure, we measure 8 % polarization on free [1-13 C]pyruvate in clean water solutions at physiological conditions at 7 T. The enhanced signals allow real-time monitoring of the pyruvate-lactate conversion in cancer cells, demonstrating the potential of the method for biomedical applications in combination with existing or developing magnetic resonance technologies.

Localized singlet-filtered MRS in vivo.

MR is a prominent technology to investigate diseases, with millions of clinical procedures performed every year. Metabolic dysfunction is one common aspect associated with many diseases. Thus, understanding and monitoring metabolic changes is essential to develop cures for many illnesses, including for example cancer and neurodegeneration. MR methodologies are especially suited to study endogenous metabolites and processes within an organism in vivo, which has led to many insights about physiological functions. Advancing metabolic MR techniques is therefore key to further understand physiological processes. Here, we introduce an approach based on nuclear spin singlet states to specifically filter metabolic signals and particularly show that singlet-filtered glutamate can be observed distinctly in the hippocampus of a living mouse in vivo. This development opens opportunities to make use of the singlet spin phenomenon in vivo and besides its use as a filter to provide scope for new contrast agents.

Early Divergence in Misfolding Pathways of Amyloid-Beta Peptides.

The amyloid cascade hypothesis proposes that amyloid-beta (Aß) aggregation is the initial triggering event in Alzheimer's disease. Here, we utilize NMR spectroscopy and monitor the structural dynamics of two variants of Aß, Aß40 and Aß42, as a function of temperature. Despite having identical amino acid sequence except for the two additional C-terminal residues, Aß42 has higher aggregation propensity than Aß40. As revealed by the NMR data on dynamics, including backbone chemical shifts, intra-methyl cross-correlated relaxation rates and glycine-based singlet-states, the C-terminal region of Aß, especially the G33-L34-M35 segment, plays a particular role in the early steps of temperature-induced Aß aggregation. In Aß42, the distinct dynamical behaviour of C-terminal residues at higher temperatures is accompanied with marked changes in the backbone dynamics of residues V24-K28. The distinctive role of the C-terminal region of Aß42 in the initiation of aggregation defines a target for the rational design of Aß42 aggregation inhibitors.

Nuclear hyperpolarization of (1-13C)-pyruvate in aqueous solution by proton-relayed side-arm hydrogenation.

We employ Parahydrogen Induced Polarization with Side-Arm Hydrogenation (PHIP-SAH) to polarize (1-13C)-pyruvate. We introduce a new method called proton-relayed side-arm hydrogenation (PR-SAH) in which an intermediate proton is used to transfer polarization from the side-arm to the 13C-labelled site of the pyruvate before hydrolysis. This significantly reduces the cost and effort needed to prepare the precursor for radio-frequency transfer experiments while still maintaining acceptable polarization transfer efficiency. Experimentally we have attained on average 4.33% 13C polarization in an aqueous solution of (1-13C)-pyruvate after about 10 seconds of cleavage and extraction. PR-SAH is a promising pulsed NMR method for hyperpolarizing 13C-labelled metabolites in solution, conducted entirely in high magnetic field.

Exotic nuclear spin behavior in dendritic macromolecules.

Dendrimers are a class of branched, highly symmetric macromolecules that have been shown to be useful for a vast number of different applications. Potential uses as fluorescence sensors, in catalysis and perhaps most importantly in medical applications as drug delivery systems or cytotoxica have been proposed. Herein we report on an exotic behaviour of the nuclear spins in a dendritic macromolecule in the presence of different paramagnetic ions. We show that the stability of the long lived nuclear singlet state, is affected by the presence of Cu(II), whereas other ions did not have any influence at all. This effect could not be observed in the case of a simple tripeptide, in which the nuclear singlet stability was influenced by all investigated paramagnetic ions, a potentially useful effect in the development of Cu(II) selective probes. By adding a fluorescent marker to our molecule we could show that the nuclear singlet multimer (NUSIMER) is taken up by living cells. Furthermore we were able to show that nuclear singlet state NMR can be used to investigate the NUSIMER in the presence of living cells, showing that an application in in vivo NMR can be feasible.

High field parahydrogen induced polarization of succinate and phospholactate.

The signal enhancement provided by the hyperpolarization of nuclear spins of metabolites is a promising technique for diagnostic magnetic resonance imaging (MRI). To date, most 13C-contrast agents are hyperpolarized utilizing a complex or cost-intensive polarizer. Recently, the in situ parahydrogen-induced 13C hyperpolarization was demonstrated. Hydrogenation, spin order transfer (SOT) by a pulsed NMR sequence, in vivo administration, and detection was achieved within the magnet bore of a 7 Tesla MRI system. So far, the hyperpolarization of the xenobiotic molecule 1-13C-hydroxyethylpropionate (HEP) and the biomolecule 1-13C-succinate (SUC) through the PH-INEPT+ sequence and a SOT scheme proposed by Goldman et al., respectively, was shown. Here, we investigate further the hyperpolarization of SUC at 7 Tesla and study the performance of two additional SOT sequences. Moreover, we present first results of the hyperpolarization at high magnetic field of 1-13C-phospholactate (PLAC), a derivate to obtain the metabolite lactate, employing the PH-INEPT+ sequence. For SUC and PLAC, 13C polarizations of about 1-2% were achieved within seconds and with minimal equipment. Effects that potentially may explain loss of 13C polarization have been identified, i.e. low hydrogenation yield, fast T1/T2 relaxation and the rarely considered 13C isotope labeling effect.

Spontaneous Enhancement of Magnetic Resonance Signals Using a RASER.

Nuclear magnetic resonance is usually drastically limited by its intrinsically low sensitivity: Only a few spins contribute to the overall signal. To overcome this limitation, hyperpolarization methods were developed that increase signals several times beyond the normal/thermally polarized signals. The ideal case would be a universal approach that can signal enhance the complete sample of interest in solution to increase detection sensitivity. Here, we introduce a combination of para-hydrogen enhanced magnetic resonance with the phenomenon of the RASER: Large signals of para-hydrogen enhanced molecules interact with the magnetic resonance coil in a way that the signal is spontaneously converted into an in-phase signal. These molecules directly interact with other compounds via dipolar couplings and enhance their signal. We demonstrate that this is not only possible for solvent molecules but also for an amino acid.

Hyperpolarization of Amino Acids in Water Utilizing Parahydrogen on a Rhodium Nanocatalyst.

NMR offers many possibilities in chemical analysis, structural investigations, and medical diagnostics. Although it is broadly used, one of NMR spectroscopies main drawbacks is low sensitivity. Hyperpolarization techniques enhance NMR signals by more than four orders of magnitude allowing the design of new contrast agents. Parahydrogen induced polarization that utilizes the para-hydrogen's singlet state to create enhanced signals is of particular interest since it allows to produce molecular imaging agents within seconds. Herein, we present a strategy for signal enhancement of the carbonyl 13 C in amino acids by using parahydrogen, as demonstrated for glycine and alanine. Importantly, the hyperpolarization step is carried out in water and chemically unmodified canonical amino acids are obtained. Our approach thus offers a high degree of biocompatibility, which is crucial for further application. The rapid sample hyperpolarization (within seconds) may enable the continuous production of biologically useful probes, such as metabolic contrast agents or probes for structural biology.

Production of highly concentrated and hyperpolarized metabolites within seconds in high and low magnetic fields.

Hyperpolarized metabolites are very attractive contrast agents for in vivo magnetic resonance imaging studies enabling early diagnosis of cancer, for example. Real-time production of concentrated solutions of metabolites is a desired goal that will enable new applications such as the continuous investigation of metabolic changes. To this end, we are introducing two NMR experiments that allow us to deliver high levels of polarization at high concentrations (50 mM) of an acetate precursor (55% 13C polarization) and acetate (17% 13C polarization) utilizing 83% para-state enriched hydrogen within seconds at high magnetic field (7 T). Furthermore, we have translated these experiments to a portable low-field spectrometer with a permanent magnet operating at 1 T. The presented developments pave the way for a rapid and affordable production of hyperpolarized metabolites that can be implemented in e.g. metabolomics labs and for medical diagnosis.

Thermal maps of gases in heterogeneous reactions.

More than 85 per cent of all chemical industry products are made using catalysts, the overwhelming majority of which are heterogeneous catalysts that function at the gas-solid interface. Consequently, much effort is invested in optimizing the design of catalytic reactors, usually by modelling the coupling between heat transfer, fluid dynamics and surface reaction kinetics. The complexity involved requires a calibration of model approximations against experimental observations, with temperature maps being particularly valuable because temperature control is often essential for optimal operation and because temperature gradients contain information about the energetics of a reaction. However, it is challenging to probe the behaviour of a gas inside a reactor without disturbing its flow, particularly when trying also to map the physical parameters and gradients that dictate heat and mass flow and catalytic efficiency. Although optical techniques and sensors have been used for that purpose, the former perform poorly in opaque media and the latter perturb the flow. NMR thermometry can measure temperature non-invasively, but traditional approaches applied to gases produce signals that depend only weakly on temperature are rapidly attenuated by diffusion or require contrast agents that may interfere with reactions. Here we present a new NMR thermometry technique that circumvents these problems by exploiting the inverse relationship between NMR linewidths and temperature caused by motional averaging in a weak magnetic field gradient. We demonstrate the concept by non-invasively mapping gas temperatures during the hydrogenation of propylene in reactors packed with metal nanoparticles and metal-organic framework catalysts, with measurement errors of less than four per cent of the absolute temperature. These results establish our technique as a non-invasive tool for locating hot and cold spots in catalyst-packed gas-solid reactors, with unprecedented capabilities for testing the approximations used in reactor modelling.

Nuclear Spin Singlet States in Photoactive Molecules: From Fluorescence/NMR Bimodality to a Bimolecular Switch for Spin Singlet States.

Nuclear spin singlet states are silent states in nuclear magnetic resonance (NMR). However, they can be probed indirectly and offer great potential for the development of contrast agents for magnetic resonance imaging (MRI). Introduced here are two novel concepts: Firstly, the bimodal NMR/fluorescence properties of 13 C2 -tetraphenylethylene. It possesses a long-lived singlet state in organic solvents, and it shortens upon the addition of water. This simultaneously increases the aggregation-induced emission (AIE) of the molecule, resulting in a substantial enhancement of fluorescence. Secondly, introduced is a bimolecular switch for singlet states based on 3-2 H-coumarin containing an isolated proton. Upon UV-light exposure, a dimer forms, leading to a coupling between two previously isolated protons. A nuclear spin singlet state can now be populated. Excitation with a wavelength of 254 nm results in partial ring cleavage of the molecule back to its monomer.

Online 1 H-MRS measurements of time-varying lactate production in an animal model of glioma during administration of an anti-tumoral drug.

The aims of this study were to implement a magnetic resonance spectroscopy (MRS) protocol for the online profiling of subnanomolar quantities of metabolites sampled from the extracellular fluid using implanted microdialysis and to apply this protocol in glioma-bearing rats for the quantification of lactate concentration and the measurement of time-varying lactate concentration during drug administration. MRS acquisitions on the brain microdialysate were performed using a home-built, proton-tuned, microsolenoid with an active volume of 2 µL. The microcoil was placed at the outlet of the microdialysis probe inside a preclinical magnetic resonance imaging (MRI) scanner. C6-bearing rats were implanted with microdialysis probes perfused with artificial cerebrospinal fluid solution and the lactate dehydrogenase (LDH) inhibitor oxamate. Microcoil magnetic resonance spectra were continuously updated using a single-pulse sequence. Localized in vivo spectra and high-resolution spectra on the dialysate were also acquired. The limit of detection and limit of quantification per unit time of the lactate methyl peak were determined as 0.37 nmol/√min and 1.23 nmol/√min, respectively. Signal-to-noise ratios (SNRs) of the lactate methyl peak above 120 were obtained from brain tumor microdialysate in an acquisition time of 4 min. On average, the lactate methyl peak amplitude measured in vivo using the nuclear magnetic resonance (NMR) microcoil was 193 ± 46% higher in tumor dialysate relative to healthy brain dialysate. A similar ratio was obtained from high-resolution NMR spectra performed on the collected dialysate. Following oxamate addition in the perfusate, a monotonic decrease in the lactate peaks was observed in all animals with an average time constant of 4.6 min. In the absence of overlapping NMR peaks, robust profiling of extracellular lactate can be obtained online using a dedicated sensitive NMR microcoil. MRS measurements of the dynamic changes in lactate production induced by anti-tumoral drugs can be assessed accurately with temporal resolutions on the order of minutes. The MRS protocol can be readily transferred to the clinical environment with the use of suitable clinical microdialysis probes.

Nuclear spin singlet states as magnetic on/off probes in self-assembling systems.

Self-assembling processes occur in a variety of compounds such as peptides, proteins and DNA. These processes have been linked to pathologies and have as well been exploited for designing responsive contrast agents for disease detection. Novel methods to investigate and detect self-assembly therefore hold promise to obtain more insights into disease progression or open pathways to the design of novel self-assembling materials. In this article we are introducing nuclear singlet states to probe self-assembly in the dipeptide isoleucine-phenylalanine (IF) as a thermoresponsive on/off switch for nuclear magnetic resonance (NMR). We have investigated the relaxation and singlet state properties of the ß-protons of phenylalanine in the IF dipeptide in aqueous solutions. At IF concentrations of 2 wt% and above 308 K, a long lived nuclear singlet state, as compared to the longitudinal relaxation, was observed. At 308 K the dipeptide starts forming a gel and no singlet state is accessible at lower temperatures. Upon heating, the gel disassembles and an isotropic liquid forms making the singlet state accessible again. This demonstrates the thermoresponsive on-off character of the nuclear spin singlet state in the IF dipeptide.