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ParaHydrogen Induced Polarization-Side Arm Hydrogenation (PHIP-SAH) is an inexpensive tool to obtain hyperpolarized pyruvate (and other metabolites) that can be applied to in vivo diagnostics, for the investigation of metabolic processes. This method is based on hydrogenation, using hydrogen enriched in the para-isomer, of unsaturated substrates, catalyzed, usually, by a homogeneous rhodium(I) complex. In this work, the effect of the solvent on the hydrogenation efficiency and the hyperpolarization level were investigated. Coordinating solvents, such as acetone and methanol, can increase significantly either the efficiency or the hyperpolarization level, but they are not compatible with the intended metabolic applications. The phase extraction of the hyperpolarized product (sodium pyruvate) in an aqueous solution was obtained carrying out the hydrogenation reaction in chloroform and toluene. The traces of the organic solvents in the water phase were removed, by means of filtration through a lipophilic resin, thus improving the biocompatibility of the aqueous solution of the hyperpolarized product.

One possibility for the non-invasive imaging of encapsulated cell grafts is to label the lumen of cell embedding capsules with a redox-responsive probe, as an increased extracellular reducing potential can be considered as a marker of hypoxia-induced necrosis. A Gd(III)-HPDO3A-like chelate has been conjugated to glycol-chitosan through a redox-responsive disulphide bond to obtain a contrast agent for Magnetic Resonance Imaging (MRI). Such a compound can be interspersed with fibroblasts within the lumen of alginate-chitosan capsules. Increasing reducing conditions within the extracellular microenvironment lead to the reductive cleavage of the disulphide bond and to the release of gadolinium in the form of a low molecular weight, non-ionic chelate. The efflux of such chelate from capsules is readily detected by a decrease of contrast enhancement in T 1 -weighted MR images.

An efficient synthesis of vinyl‐[1‐13C]pyruvate has been reported, from which 13C hyperpolarized (HP) ethyl‐[1‐13C]pyruvate has been obtained by means of ParaHydrogen Induced Polarization (PHIP). Due to the intrinsic lability of pyruvate, which leads quickly to degradation of the reaction mixture even under mild reaction conditions, the vinyl‐ester has been synthesized through the intermediacy of a more stable ketal derivative. 13C and 1H hyperpolarizations of ethyl‐[1‐13C]pyruvate, hydrogenated using ParaHydrogen, have been compared to those observed on the more widely used allyl‐derivative. It has been demonstrated that the spin order transfer from ParaHydrogen protons to 13C, is more efficient on the ethyl than on the allyl‐esterdue to the larger J‐couplings involved. The main requirements needed for the biological application of this HP product have been met, i. e. an aqueous solution of the product at high concentration (40 mM) with a good 13C polarization level (4.8 %) has been obtained. The in vitro metabolic transformation of the HP ethyl‐[1‐13C]pyruvate, catalyzed by an esterase, has been observed. This substrate appears to be a good candidate for in vivo metabolic investigations using PHIP hyperpolarized probes., 13C hyperpolarized ethyl‐[1‐13C]pyruvate by means of PHIP‐SAH: Vinyl‐[1‐13C]pyruvate has been prepared on laboratory scale through a new synthesis route. After its hydrogenation with p‐H2 and magnetization transfer (via MFC), polarized ethyl‐[1‐13C]pyruvate has been obtained in aqueous solution at high concentration (40 mM) and good polarization level (4.8 %). In the end, the in‐vitro conversion of HP ethyl‐[1‐13C]pyruvate into [1‐13C]pyruvate catalysed by an esterase, has been observed.

Hyperpolarization‐enhanced magnetic resonance imaging can be used to study biomolecular processes in the body, but typically requires nuclei such as 13C, 15N, or 129Xe due to their long spin‐polarization lifetimes and the absence of a proton‐background signal from water and fat in the images. Here we present a novel type of 1H imaging, in which hyperpolarized spin order is locked in a nonmagnetic long‐lived correlated (singlet) state, and is only liberated for imaging by a specific biochemical reaction. In this work we produce hyperpolarized fumarate via chemical reaction of a precursor molecule with para‐enriched hydrogen gas, and the proton singlet order in fumarate is released as antiphase NMR signals by enzymatic conversion to malate in D2O. Using this model system we show two pulse sequences to rephase the NMR signals for imaging and suppress the background signals from water. The hyperpolarization‐enhanced 1H‐imaging modality presented here can allow for hyperpolarized imaging without the need for low‐abundance, low‐sensitivity heteronuclei., A proton singlet state on fumarate derived from parahydrogen can be broken by enzymatic conversion to malate, which releases hyperpolarized NMR signals. We demonstrate two “out‐of‐phase echo” pulse sequences to convert the I 1z I 2z state into net proton magnetization, and show how this can be used for hyperpolarized 1H imaging.

Eu(HP-DO3A) is present in solution as a mixture of two diastereoisomers whose alcoholic groups are the source of the mobile protons for the CEST effect. The exchange is base catalyzed. Two novel EuIII complexes of HP-DO3A-like ligands containing an amino or a carboxylate functionality in the proximity of the -OH groups showed the occurrence of intramolecular catalysis of the prototropic exchange. New insights into the role of the intramolecular proton exchange on the CEST properties have been gained.

ParaHydrogen induced polarization (PHIP) is an efficient and cost-effective hyperpolarization method, but its application to biological investigations has been hampered, so far, due to chemical challenges. PHIP is obtained by means of the addition of hydrogen, enriched in the para-spin isomer, to an unsaturated substrate. Both hydrogen atoms must be transferred to the same substrate, in a pairwise manner, by a suitable hydrogenation catalyst; therefore, a de-hydrogenated precursor of the target molecule is necessary. This has strongly limited the number of parahydrogen polarized substrates. The non-hydrogenative approach brilliantly circumvents this central issue, but has not been translated to in-vivo yet. Recent advancements in hydrogenative PHIP (h-PHIP) considerably widened the possibility to hyperpolarize metabolites and, in this review, we will focus on substrates that have been obtained by means of this method and used in vivo. Attention will also be paid to the requirements that must be met and on the issues that have still to be tackled to obtain further improvements and to push PHIP substrates in biological applications.

The properties of LnIII -HPDO3A complexes as relaxation enhancers and paraCEST agents are essentially related to the hydroxylpropyl moiety. A series of three HPDO3A derivatives, with small modifications to the hydroxyl arm, were herein investigated to understand how heightened control can be gained over the parameters involved in the design of these agents. A full 1 H and 17 O-NMR relaxometric analysis was conducted and demonstrated that increasing the length of the OH group from the lanthanide centre significantly enhanced the water exchange rate of the gadolinium complex, but with a subsequent reduction in kinetic stability. Alternatively, the introduction of an additional methyl group, which increased the steric bulk around the OH moiety, resulted in the formation of almost exclusively the TSAP isomer (95 %) as identified by 1 H-NMR of the europium complex. The gadolinium analogue of this complex also exhibited a very fast water exchange rate, but with no detectable loss of kinetic stability. This complex therefore demonstrates a notable improvement over Gd-HPDO3A.

Hyperpolarized fumarate can be used as a probe of real-time metabolism in vivo, using carbon-13 magnetic resonance imaging. Dissolution dynamic nuclear polarization is commonly used to produce hyperpolarized fumarate, but a cheaper and faster alternative is to produce hyperpolarized fumarate via PHIP (parahydrogen induced polarization). In this work we trans-hydrogenate [1-13C]acetylene dicarboxylate with para-enriched hydrogen using a commercially available Ru catalyst in water to produce hyperpolarized [1-13C]fumarate. We show that fumarate is produced in 89% yield, with succinate as a side product in 11% yield. The proton polarization is converted into 13C magnetization using a constant adiabaticity field cycle, and a polarization level of 25% is achieved using 86% para-enriched hydrogen gas. We inject the hyperpolarized [1-13C]fumarate into cell suspensions and track the metabolism. This work opens the path to greatly accelerated preclinical studies using fumarate as a biomarker.

Hyperpolarization of the 13C magnetic resonance signal of L-[1-13C]Lactate has been obtained using the chemically based, cost-effective method named ParaHydrogen Induced Polarization by means of Side Arm Hydrogenation (PHIP-SAH). Two ester derivatives of lactate have been tested and the factors that determine the polarization level on the product have been investigated into details. The metabolic conversion of hyperpolarized L-[1-13C]Lactate into Pyruvate has been observed in vitro using lactate dehydrogenase (LDH) and in a cells lysate. From the acquisition of a series of 13C-NMR spectra, the metabolic build-up of the [1-13C]Pyruvate signal has been observed. These studies demonstrate that, even if the experimental set-up used for these PHIP-SAH hyperpolarization studies is still far from optimal, the attained polarization level is already sufficient to carry out in vitro metabolic studies.

The redox microenvironment within a cell graft can be considered as an indicator to assess whether the graft is metabolically active or hypoxic. We present a redox-responsive MRI probe based on porous silica microparticles whose surface has been decorated with a Gd-chelate through a disulphide bridge. Such microparticles are designed to be interspersed with therapeutic cells within a biocompatible hydrogel. The onset of reducing conditions within the hydrogel is paralleled by an increased clearance of Gd, that can be detected by MRI.

We report the synthesis and characterization of a water-soluble, star-shaped macromolecular platform consisting of eight β-cyclodextrin (β-CD) units anchored to the narrower rim of a γ-CD core through bis(triazolyl)alkyl spacers. The efficient synthetic protocol is based on the microwave (MW)-promoted Cu-catalyzed 1,3-dipolar cycloaddition of CD monoazides to CD monoacetylenes. The ligand-hosting capability of the construct has been assessed by relaxometric titration and nuclear magnetic relaxation dispersion (NMRD) profiling, which showed it to be good, and this was supported by molecular dynamics simulations. To demonstrate the feasibility of obtaining supramolecular structures with high hosting ability, we designed a dimeric platform, formed by joining two nonamers through the γ-CD cores through a bis(lithocholic acid) linker. With a view to the potential biological applications, cytotoxicity and extent of binding to human serum albumin were assessed. The properties of this dendrimeric multicarrier make it suitable for pharmaceutical and diagnostic purposes, ranging from targeted drug delivery to molecular imaging.

A novel class of paramagnetic liposome-based systems acting as dual T(1) and CEST (1)H-MRI contrast agents is described. The vesicles contain a shift reagent in the aqueous core and a Gd-complex on the external surface conjugated through a biodegradable linker. As such, the probe can generate T(1) contrast only, but after the cleavage and removal of the Gd-coating, the CEST contrast is switched on.

Hyperpolarized fumarate can be used as a probe of real-time metabolism in vivo, using carbon-13 magnetic resonance imaging. Dissolution dynamic nuclear polarization is commonly used to produce hyperpolarized fumarate, but a cheaper and faster alternative is to produce hyperpolarized fumarate via PHIP (parahydrogen-induced polarization). In this work, we trans-hydrogenate [1-13C]acetylene dicarboxylate with para-enriched hydrogen using a commercially available Ru catalyst in water to produce hyperpolarized [1-13C]fumarate. We show that fumarate is produced in 89% yield, with succinate as a side product in 11% yield. The proton polarization is converted into 13C magnetization using a constant adiabaticity field cycle, and a polarization level of 24% is achieved using 86% para-enriched hydrogen gas. We inject the hyperpolarized [1-13C]fumarate into cell suspensions and track the metabolism. This work opens the path to greatly accelerated preclinical studies using fumarate as a biomarker.