Insights into Interfacial Water Structuring at the Nafion Surface by T1-Weighted Magnetic Resonance Imaging.

T1-weighted magnetic resonance images of water in the surroundings of a Nafion surface allowed the identification of the presence of a low-mobility zone (LMZ), 60 µm thick, consisting of water molecules structured in a hydrogen-bonding network, promoted by the presence of the acidic protons on the surface of the sulphonated polymer. In parallel, the exclusion zone (EZ) was assessed by observing in optical microscopy the distribution of microspheres suspended in the medium in contact with the Nafion membrane. It was found that the LMZ and the EZ do not correspond: in fact, the former is thinner and more stable over time than the latter and they behave differently when ions are present in the medium in which Nafion is immersed.

1H-NMR and MALDI-TOF MS as metabolomic quality control tests to classify platelet derived medium additives for GMP compliant cell expansion procedures.

INTRODUCTION: Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples. AIMS: We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation. METHODS: We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS. RESULTS: Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro. DISCUSSION: MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products.

The Role of the Amino Protecting Group during Parahydrogenation of Protected Dehydroamino Acids.

A series of dehydroamino acids endowed with different protective groups at the amino and carboxylate moieties and with different substituents at the double bond have been reacted with parahydrogen. The observed ParaHydrogen Induced Polarization (PHIP) effects in the (1)H NMR spectra are strongly dependent on the amino protecting group. DFT calculations allowed us to establish a relationship between the structures of the reaction intermediates (whose energies depend on the amido substitution) and the observed PHIP patterns.

15N-permethylated amino acids as efficient probes for MRI-DNP applications.

The synthesis, NMR properties and preliminary polarization tests on protonated and perdeuterated forms of α-trimethylglutamine (NMe3Gln), α-trimethylglutamate (NMe3Glu) and ε-trimethyllysine (NMe3Lys) are reported. The (15)N-permethylated, perdeuterated amino acids display very long (15)N-T1 values, ranging between 190 and 330 s, are well polarized by the dynamic nuclear polarization (DNP) procedure, yielding good polarization levels (10%), and appear to be well tolerated by cells and mice. The obtained results make perdeuterated amino acids excellent candidates for innovative DNP (15)N-MRI applications such as perfusion or targeting studies.

15N magnetic resonance hyperpolarization via the reaction of parahydrogen with 15N-propargylcholine.

(15)N-Propargylcholine has been synthesized and hydrogenated with para-H(2). Through the application of a field cycling procedure, parahydrogen spin order is transferred to the (15)N resonance. Among the different isomers formed upon hydrogenation of (15)N-propargylcholine, only the nontransposed derivative contributes to the observed N-15 enhanced emission signal. The parahydrogen-induced polarization factor is about 3000. The precise identification of the isomer responsible for the observed (15)N enhancement has been attained through a retro-INEPT ((15)N-(1)H) experiment. T(1) of the hyperpolarized (15)N resonance has been estimated to be ca. 150 s, i.e., similar to that reported for the parent propargylcholine (144 s). Experimental results are accompanied by theoretical calculations that stress the role of scalar coupling constants (J(HN) and J(HH)) and of the field dependence in the formation of the observed (15)N polarized signal. Insights into the good cellular uptake of the compound have been gained.

Para-hydrogen induced polarization of Si-29 NMR resonances as a potentially useful tool for analytical applications.

Para-hydrogen-induced polarization effects have been observed in the (29)Si NMR spectra of trimethylsilyl para-hydrogenated molecules. The high signal enhancements and the long T(1) values observed for the (29)Si hyperpolarized resonances point toward the possibility of using (29)Si for hyperpolarization applications. A method for the discrimination of multiple compounds and/or complex mixtures of hydroxylic compounds (such as steroids), consisting of the silylization of alcoholic functionalities with an unsaturated silylalkyl moiety and subsequent reaction with para-H(2), is proposed.

Hyperpolarized (13)C-pyruvate magnetic resonance imaging in cancer diagnostics.

INTRODUCTION: The use of hyperpolarized molecules allows one to obtain information about metabolism in both cells and animals; such a task represents a tremendous advancement with respect to the results achieved so far with in vivo NMR techniques. Pyruvate appears an excellent tumor biomarker as it allows the attainment of early diagnosis, stadiation and monitoring of response to therapy. AREAS COVERED: As pyruvate conversion to lactate in the glycolytic pathway is highly enhanced in tumor cells, the 1-(13)C-lactate levels after administration of hyperpolarized 1-(13)C-pyruvate are markedly higher in tumor tissues and depend on the type and grade of the tumor. This review covers the most recent research results (both in vitro and in vivo) about the use of hyperpolarized 1-(13)C-pyruvate for tumor localization, stadiation and for monitoring the response to therapy. The technique may find application in clinics, especially when other imaging modalities are of difficult applicability. EXPERT OPINION: While (13)C-pyruvate has been shown to be the candidate of choice for metabolic imaging, high expectations are present in the scientific community to see if other hyperpolarized substrates could provide more specific and sensitive biomarkers. The use of hyperpolarized molecules will have a tremendous impact in the armory of diagnostic tools.

Synthesis and testing of a p-H2 hyperpolarized 13C probe based on the pyrazolo[1,5-a]pyrimidineacetamide DPA-713, an MRI vector to target the peripheral benzodiazepine receptors.

DPA-713 is the lead compound of a recently developed 2-phenylpyrazolo[1,5-a]pyrimidineacetamide series that has been shown to display a good targeting capability toward peripheral benzodiazepine receptors, recently renamed translocator protein (18 kDa) or in short TSPO. On the basis of this structure, a novel derivative bearing a [(13)C]butynoate moiety has been designed and synthesized (three steps-42% overall yield) providing, upon rapid and quantitative para-hydrogenation, the corresponding hyperpolarized [(13)C]alkene. Para-hydrogen-induced polarization effects have been detected in both (1)H and (13)C-NMR spectra. Upon applying a field cycling procedure, the spin order of para-H(2) added hydrogens is transferred on the (13)C carboxylate moiety yielding a signal enhancement of approximately 4500 times. T(1) of the carboxylate carbon atom is approximately 21.9 s (at 9.37 T). A (13)C-MR image has been acquired by using the (13)C RARE (Rapid Acquisition by Relaxation Enhancement) acquisition protocol on a 10-mM solution. The main limitation to the in vivo use of this novel para-hydrogenated [(13)C]derivative is its relatively low solubility in aqueous systems.

How to design 13C para-hydrogen-induced polarization experiments for MRI applications.

The application of hyperpolarization techniques for MRI purposes is gathering increasing attention, especially for nuclei such as (13)C or (129)Xe. Among the different proposed methods, ParaHydrogen Induced Polarization requires relatively cheap equipment. The setup of an MRI experiment by means of parahydrogen requires the application of skills and methodologies that derive from different fields of knowledge. The basic theory and a practical insight of this method are presented here. Parahydrogenation of alkynes, having a labelled (13)CO group adjacent to the triple bond, catalyzed by Rh(I) complexes containing a chelating phosphine, represents the best choice for producing and maintaining high heteronuclear polarization effect. In order to transform anti-phase into in-phase (net) (13)C polarization for MRI application it is necessary to set up the described magnetic field cycle procedure. In vitro and in vivo images have been acquired using fast imaging sequences (RARE and trueFISP).

Para-hydrogenated glucose derivatives as potential 13C-hyperpolarized probes for magnetic resonance imaging.

A set of molecules in which a glucose moiety is bound to a hydrogenable synthon has been synthesized and evaluated for hydrogenation reactions and for the corresponding para-hydrogen-induced polarization (PHIP) effects, in order to select suitable candidates for an in vivo magnetic resonance imaging (MRI) method for the assessment of glucose cellular uptake. It has been found that amidic derivatives do not yield any polarization enhancement, probably due to singlet-triplet state mixing along the reaction pathway. In contrast, ester derivatives are hydrogenated in high yield and afford enhanced (1)H and (13)C NMR spectra after para-hydrogenation. The obtained PHIP patterns are discussed and explained on the basis of the calculated spin level populations in the para-hydrogenated products. These molecules may find interesting applications in (13)C MRI as hyperpolarized probes for assessing the activity of glucose transporters in cells.

Current concepts on hyperpolarized molecules in MRI.

Hyperpolarization provides a strong enhancement of the MR signal and allows the acquisition of images from nuclei other than protons. Besides vascular imaging and perfusion studies the use of hyperpolarized molecules has allowed the visualization of real-time substrate uptake and metabolism in vivo. Metabolic imaging is an outstanding field of research that aims at providing the clinicians with the possibility of interrogating real-time complex metabolic pathways that give a direct insight into the cellular state and activity.

New hyperpolarized contrast agents for 13C-MRI from para-hydrogenation of oligooxyethylenic alkynes.

Two alkyne derivatives, which contain one and two oligooxyethylenic chains respectively, showed to be good substrates for para-hydrogenation reactions, yielding the corresponding hyperpolarized alkenes in good yields. A suitable theory has been developed to account for the observed results, fully explaining the different para-H 2 induced effects observed upon the para-hydrogenation of symmetrically and asymmetrically substituted alkynes in ALTADENA and PASADENA modes. The oligooxyethylenic substituent provides good water solubility to the para-hydrogenated symmetrical derivative. (13)C-MR in vitro images of the latter derivative were obtained both in acetone and in water solutions (130 mM), using the ALTADENA procedure and after application of the field cycling procedure which allows acquisition of an in-phase (13)C carbonyl resonance. The finding that the hydrogenated product is water-soluble in contrast to the parent alkyne which is not allows for the pursuit of a fast phase-transfer separation from the organic solvent, the unreacted substrate, and the catalyst to obtain a "ready-to-use" water solution suitable for further in vivo MRI applications.

Agents for polarization enhancement in MRI.

The intrinsic low sensitivity of the NMR phenomenon can be overcome thanks to hyperpolarization procedures that break the limits of the Boltzmann equilibrium and may increase the NMR signal by a factor of 10(5). Hyperpolarization procedures have been applied to enhance the signal from noble gases, such as 3He and 129Xe, and small 13C-containing molecules. For the latter class, attention has been focused on the use of methods based on dynamic nuclear polarization (DNP) and para-hydrogen induced polarization (PHIP). After discussion of the basics of the methods, an overview of the main applications with 13C-containing molecules is presented. This includes pre-clinical MR investigations of vascular imaging, perfusion and catheter tracking as well as molecular imaging protocols that allow the development of highly innovative studies in the field of metabolic imaging.

para-Hydrogenation of unsaturated moieties on poly(lysine) derived substrates for the development of novel hyperpolarized MRI contrast agents.

Four alkyne-functionalized poly(lysine) derivatives have been synthesized and characterized by 1H and 13C NMR spectroscopy. In the first poly(lysine) derivative, phenylpropiolate moieties are directly bound to the aminic arms, whereas in the other derivatives, propargylamine moieties are bound to the aminic poly(lysine) arms through glucaric acid and diethylene glycol (DG) chains, respectively. para-Hydrogenation of the alkyne-functionalized poly(lysine) compounds has been investigated and the results have been discussed in terms of spin lattice relaxation properties of the hydrogenated products. It is shown that the longer the mobile chain separating the unsaturation from the poly(lysine) backbone, the more intense the polarized signals when para-hydrogenation is carried out. This is due to (a) the maintenance of short reorientational times on the unsaturated ends, and therefore a sufficiently long T(1) of the protons added during hydrogenation, and (b) the minor effect of steric hindrance by the poly(lysine) backbone that decreases interaction of the unsaturation with the catalyst, allowing higher hydrogenation rates.

Spectroscopic and computational investigations of stable radical anions of triosmium benzoheterocycle clusters.

The variable temperature (1)H and (13)C NMR and EPR spectra of the stable radical anions [Os(3)(CO)(9)(micro(3)-eta(2)-L)(micro-H)] (LH=phenanthridine, 1; 5,6-benzoquinoline, 2), and [Os(3)(CO)(10)(micro(3)-eta(2)-L)(micro-H)] (LH=quinoxaline, 3) are reported. The radical anions 1(-), 2(-), and 3(-) can be prepared by both exhaustive electrolysis and partially by chemical reduction with cobaltocene and with sodium dispersion (only with sodium dispersion in the case of 3(-)). DFT calculations on 1-3 reveal that the LUMO for the electron-deficient compounds 1 and 2 involves significant contributions from both the heterocyclic ligand and the two metal atoms bridged by the ligand and the micro-hydride. The character of this orbital rationalizes the previously observed regioselective reactions of these complexes with nucleophiles. In contrast, the LUMO for the electron precise 3 involves only ligand-based orbitals. Partial chemical reduction of 1 and 2 requires an excess of either cobaltocene or sodium, and their (1)H and (13)C NMR spectra reveal selective line broadening of those proton resonances that are predicted by DFT calculations to bear the greatest amount of free spin density. The variable temperature behavior of the partially chemically reduced species of 1 and 2 indicates that electron transfer between the reduced/unreduced cluster pair and between the cobaltocene/cobaltocenium pair occurs on the NMR timescale. The radical anions of 1 and 2 prepared by exhaustive electrolysis show an EPR signal at room temperature, while the NMR signals are uniformly broadened. Compound 3 appears to be partially reduced by sodium at room temperature and shows uniformly broadened (1)H NMR resonances at room temperature that sharpen significantly at -80 degrees C. The temperature dependence of the spectra are discussed in terms of the effects of relative electron nuclear relaxation processes, chemical exchange, and the results of the DFT calculations.