Heavily Gd-Doped Non-Toxic Cerium Oxide Nanoparticles for MRI Labelling of Stem Cells.

Recently, human mesenchymal stem cells (hMSc) have attracted a great deal of attention as potential therapeutic agents in the treatment of socially significant diseases. Despite substantial advances in stem-cell therapy, the biological mechanisms of hMSc action after transplantation remain unclear. The use of magnetic resonance imaging (MRI) as a non-invasive method for tracking stem cells in the body is very important for analysing their distribution in tissues and organs, as well as for ensuring control of their lifetime after injection. Herein, detailed experimental data are reported on the biocompatibility towards hMSc of heavily gadolinium-doped cerium oxide nanoparticles (Ce0.8Gd0.2O2-x) synthesised using two synthetic protocols. The relaxivity of the nanoparticles was measured in a magnetic field range from 1 mT to 16.4 T. The relaxivity values (r1 = 11 ± 1.2 mM-1 s-1 and r1 = 7 ± 1.2 mM-1 s-1 in magnetic fields typical of 1.5 and 3 T MRI scanners, respectively) are considerably higher than those of the commercial Omniscan MRI contrast agent. The low toxicity of gadolinium-doped ceria nanoparticles to hMSc enables their use as an effective theranostic tool with improved MRI-contrasting properties.

Analytical solution for the inverting pulses with constant adiabaticity.

The exact solution was found for inverting pulses with constant adiabaticity for spin ½. The analytical relationship between the time-varying frequency of the microwave resonant field (or RF field in the case of NMR) and its amplitude time dependence such that the adiabaticity parameter remains constant for the single isochromat throughout the pulse is found. Comparison with EPR (hyperbolic tangent)-(hyperbolic secant) pulse method was carried out. On the basis of the analytical solution the pulses with different dependences of the microwave field amplitude conserving the constant adiabaticity have been constructed. The pulses exhibit rather sharp inversion selectivity that can be used in the field of EPR, NMR and MRI.

Floquet theory in magnetic resonance: Formalism and applications.

Floquet theory is an elegant mathematical formalism originally developed to solve time-dependent differential equations. Besides other fields, it has found applications in optical spectroscopy and nuclear magnetic resonance (NMR). This review attempts to give a perspective of the Floquet formalism as applied in NMR and shows how it allows one to solve various problems with a focus on solid-state NMR. We include both matrix- and operator-based approaches. We discuss different problems where the Hamiltonian changes with time in a periodic way. Such situations occur, for example, in solid-state NMR experiments where the time dependence of the Hamiltonian originates either from magic-angle spinning or from the application of amplitude- or phase-modulated radiofrequency fields, or from both. Specific cases include multiple-quantum and multiple-frequency excitation schemes. In all these cases, Floquet analysis allows one to define an effective Hamiltonian and, moreover, to treat cases that cannot be described by the more popularly used and simpler-looking average Hamiltonian theory based on the Magnus expansion. An important example is given by spin dynamics originating from multiple-quantum phenomena (level crossings). We show that the Floquet formalism is a very general approach for solving diverse problems in spectroscopy.

Nuclear singlet relaxation by chemical exchange.

The population imbalance between nuclear singlet states and triplet states of strongly coupled spin-1/2 pairs, also known as nuclear singlet order, is well protected against several common relaxation mechanisms. We study the nuclear singlet relaxation of 13C pairs in aqueous solutions of 1,2-13C2 squarate over a range of pH values. The 13C singlet order is accessed by introducing 18O nuclei in order to break the chemical equivalence. The squarate dianion is in chemical equilibrium with hydrogen-squarate (SqH-) and squaric acid (SqH2) characterized by the dissociation constants pK1 = 1.5 and pK2 = 3.4. Surprisingly, we observe a striking increase in the singlet decay time constants TS when the pH of the solution exceeds ∼10, which is far above the acid-base equilibrium points. We derive general rate expressions for chemical-exchange-induced nuclear singlet relaxation and provide a qualitative explanation of the TS behavior of the squarate dianion. We identify a kinetic contribution to the singlet relaxation rate constant, which explicitly depends on kinetic rate constants. Qualitative agreement is achieved between the theory and the experimental data. This study shows that infrequent chemical events may have a strong effect on the relaxation of nuclear singlet order.

Singlet to triplet conversion in molecular hydrogen and its role in parahydrogen induced polarization.

Detailed experimental and comprehensive theoretical analysis of singlet-triplet conversion in molecular hydrogen dissolved in a solution together with organometallic complexes used in experiments with parahydrogen (the H2 molecule in its nuclear singlet spin state) is reported. We demonstrate that this conversion, which gives rise to formation of orthohydrogen (the H2 molecule in its nuclear triplet spin state), is a remarkably efficient process that strongly reduces the resulting NMR (nuclear magnetic resonance) signal enhancement, here of 15N nuclei polarized at high fields using suitable NMR pulse sequences. We make use of a simple improvement of traditional pulse sequences, utilizing a single pulse on the proton channel that gives rise to an additional strong increase of the signal. Furthermore, analysis of the enhancement as a function of the pulse length allows one to estimate the actual population of the spin states of H2. We are also able to demonstrate that the spin conversion process in H2 is strongly affected by the concentration of 15N nuclei. This observation allows us to explain the dependence of the 15N signal enhancement on the abundance of 15N isotopes.

Simultaneous 15 N polarization of several biocompatible substrates in ethanol-water mixtures by signal amplification by reversible exchange (SABRE) method.

Signal amplification by reversible exchange (SABRE) is a popular method for generating strong signal enhancements in nuclear magnetic resonance (NMR). In SABRE experiments, the source of polarization is provided by the nonthermal spin order of parahydrogen (pH2 , the H2 molecule in its nuclear singlet spin state). Polarization formation requires that both pH2 and a substrate molecule bind to an Ir-based complex where polarization transfer occurs. Subsequently, the complex dissociates and free polarized substrate molecules are formed. In this work, we present approaches towards biocompatible SABRE, meaning that several small biomolecules are simultaneously polarized by using the SABRE method in water-ethanol solutions at room temperature. We are able to demonstrate significant 15 N-NMR signal enhancements in water-ethanol solutions for biomolecules like nicotinamide, metronidazole, adenosine-5'-monophosphate, and 4-methylimidazole and found that the first three substrates are polarized at the same level as a well-known pyridine. We show that simultaneous polarization of several molecules is indeed feasible when the reactions are carried out at an ultralow field of about 400-500 nT. The achieved enhancements are between 100-fold and 15,000-fold. The resulting 15 N polarization (maximal value about 4% achieved for metronidazole and pyridine at 45°C) strongly depends on the sample temperature, pH2 bubbling pressure, and pH2 flow. One more parameter, which is important for optimizing the enhancement, is the solvent pH. Hence, this study presents a step in developing biocompatible SABRE polarization and gives a clue on how such SABRE experiments should be optimized to achieve the highest NMR signal enhancement.

Constant-adiabaticity pulse schemes for manipulating singlet order in 3-spin systems with weak magnetic non-equivalence.

Parahydrogen-induced polarization (PHIP) is a source of nuclear spin hyperpolarization, and this technique allows for the preparation of biomolecules for in vivo metabolic imaging. PHIP delivers hyperpolarization in the form of proton singlet order to a molecule, but most applications require that a heteronuclear (e.g. 13C or 15N) spin in the molecule is hyperpolarized. Here we present high field pulse methods to manipulate proton singlet order in the [1-13C]fumarate, and in particular to transfer the proton singlet order into 13C magnetization. We exploit adiabatic pulses, i.e., pulses with slowly ramped amplitude, and use constant-adiabaticity variants: the spin Hamiltonian is varied in such a way that the generalized adiabaticity parameter is time-independent. This allows for faster polarization transfer, and we achieve 96.2% transfer efficiency in thermal equilibrium experiments. We demonstrate this in experiments using hyperpolarization, and obtain 6.8% 13C polarization. This work paves the way for efficient hyperpolarization of nuclear spins in a variety of biomolecules, since the high-field pulse sequences allow individual spins to be addressed.

Photochemically Induced Dynamic Nuclear Polarization of Heteronuclear Singlet Order.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is a method to hyperpolarize nuclear spins using light. In most cases, CIDNP experiments are performed in high magnetic fields and the sample is irradiated by light inside a nuclear magnetic resonance (NMR) spectrometer. Here we demonstrate photo-CIDNP hyperpolarization generated in the Earth's magnetic field and under zero- to ultralow-field (ZULF) conditions. Irradiating a sample containing tetraphenylporphyrin and para-benzoquinone for several seconds with light-emitting diodes produces strong hyperpolarization of 1H and 13C nuclear spins, enhancing the NMR signals more than 200 times. The hyperpolarized spin states at the Earth's field and in ZULF are different. In the latter case, the state corresponds to the singlet order between scalar-coupled 1H-13C nuclear spins. This state has a longer lifetime than the state hyperpolarized at Earth's field. The method is simple and cost-efficient and should be applicable to many molecular systems known to exhibit photo-CIDNP, including amino acids and nucleotides.

Hyperpolarization of cis-15 N2 -Azobenzene by Parahydrogen at Ultralow Magnetic Fields*.

The development of nuclear spins hyperpolarization, and the search for molecules that can be efficiently hyperpolarized is an active area in nuclear magnetic resonance. In this work we present a detailed study of SABRE SHEATH (signal amplification by reversible exchange in shield enabled alignment transfer to heteronuclei) experiments on 15 N2 -azobenzene. In SABRE SHEATH experiments the nuclear spins of the target are hyperpolarized through transfer of spin polarization from parahydrogen at ultralow fields during a reversible chemical process. Azobenzene exists in two isomers, trans and cis. We show that all nuclear spins in cis-azobenzene can be efficiently hyperpolarized by SABRE at suitable magnetic fields. Enhancement factors (relative to 9.4 T) reach up to 3000 for 15 N spins and up to 30 for the 1 H spins. We compare two approaches to observe either hyperpolarized magnetization of 15 N/1 H spins, or hyperpolarized singlet order of the 15 N spin pair. The results presented here will be useful for further experiments in which hyperpolarized cis-15 N2 -azobenzene is switched by light to trans-15 N2 -azobenzene for storing the produced hyperpolarization in the long-lived spin state of the 15 N pair of trans-15 N2 -azobenzene.

15 N SABRE Hyperpolarization of Metronidazole at Natural Isotope Abundance.

Signal Amplification By Reversible Exchange (SABRE) is gaining increased attention as a tool to enhance weak Nuclear Magnetic Resonance (NMR) signals. In SABRE, spin order is transferred from parahydrogen (H2 in its nuclear singlet spin state) to a substrate molecule in a transient Ir-based complex. In recent years, SABRE polarization of biologically active substrates has been demonstrated, notably of metronidazole - an antibiotic and antiprotozoal drug. In this work, we study 15 N SABRE polarization of metronidazole at natural isotope abundance. We are able to demonstrate significant 15 N polarization reaching 15 %, which corresponds to a signal enhancement of 46,000 at 9.4 T for the nitrogen atom with lone electron pair. Additionally, the other two N-atoms can be polarized, although less efficiently. We present a detailed study of the field dependence of polarization and explain the maxima in the field dependence using the concept of coherent polarization transfer at level anti-crossings in the SABRE complex. A study of spin relaxation phenomena presented here enables optimization of the magnetic field for efficient storage of non-thermal polarization.

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with zero- and ultra-low-field total correlation spectroscopy (ZULF-TOCSY).

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy (TOCSY). ZULF-TOCSY is a new building block for NMR methods, which has the unique property that the polarization is evenly distributed among all NMR-active nuclei such as 1H, 13C, 15N, 31P, etc., provided that they belong to the same coupling network, and provided that their relaxation is not too fast at low fields, as may occur in macromolecules. Here, we show that ZULF-TOCSY correlations can be observed for peptides at natural isotopic abundance, such as the protected hexapeptide Boc-Met-enkephalin. The analysis of ZULF-TOCSY spectra readily allows one to make sequential assignments, thus offering an alternative to established heteronuclear 2D experiments like HMBC. For Boc-Met-enkephalin, we show that ZULF-TOCSY allows one to observe all expected cross-peaks between carbonyl carbons and α-CH protons, while the popular HMBC method provides insufficient information.

Correlation of high-field and zero- to ultralow-field NMR properties using 2D spectroscopy.

The field of zero- to ultralow-field (ZULF) nuclear magnetic resonance (NMR) is currently experiencing rapid growth, owing to progress in optical magnetometry and attractive features of ZULF-NMR such as low hardware cost and excellent spectral resolution achieved under ZULF conditions. In this work, an approach is proposed and demonstrated for simultaneous acquisition of ZULF-NMR spectra of individual 13C-containing isotopomers of chemical compounds in a complex mixture. The method makes use of fast field cycling such that the spin evolution takes place under ZULF conditions, whereas signal detection is performed in a high-field NMR spectrometer. This method has excellent sensitivity, also allowing easy assignment of ZULF-NMR spectra to specific analytes in the mixture. We demonstrate that the spectral information is the same as that given by ZULF-NMR, which makes the method suitable for creating a library of ZULF-NMR spectra of various compounds and their isotopomers. The results of the field-cycling experiments can be presented in a convenient way as 2D-NMR spectra with the direct dimension giving the high-field 13C-NMR spectrum (carrying the chemical-shift information) and the indirect dimension giving the ZULF-NMR spectrum (containing information about proton-carbon J-couplings). Hence, the method can be seen as a variant of heteronuclear J-resolved spectroscopy, one of the first 2D-NMR techniques.

Initializing 214 Pure 14-Qubit Entangled Nuclear Spin States in a Hyperpolarized Molecular Solid.

Quantum entanglement has been realized on a variety of physical platforms such as quantum dots, trapped atomic ions, and superconductors. Here we introduce specific molecular solids as promising alternative platforms. Our model system is triplet pentacene in a host single crystal at level anticrossing (LAC) conditions. First, a laser pulse generates the triplet state and initiates entanglement between an electron spin and 14 hyperfine coupled proton spins (quantum bits or qubits). This gives rise to large nuclear spin polarization. Subsequently, a resonant high-power microwave (mw) pulse disentangles the electron spin from the nuclear spins. Simultaneously, high-dimensional multiqubit entanglement is formed among the proton spins. We verified the initialization of 214 pure 14-qubit entangled nuclear spin states with an average degree of entanglement of Eav = 0.77 ± 0.03. These results pave the way for large-scale quantum information processing with more than 10 000 multiqubit entangled states corresponding to computational (Hilbert) space dimensions of dim >1053.

Mixed-Valence Compounds as Polarizing Agents for Overhauser Dynamic Nuclear Polarization in Solids*.

Herein, we investigate a novel set of polarizing agents-mixed-valence compounds-by theoretical and experimental methods and demonstrate their performance in high-field dynamic nuclear polarization (DNP) NMR experiments in the solid state. Mixed-valence compounds constitute a group of molecules in which molecular mobility persists even in solids. Consequently, such polarizing agents can be used to perform Overhauser-DNP experiments in the solid state, with favorable conditions for dynamic nuclear polarization formation at ultra-high magnetic fields.

Constant-adiabaticity ultralow magnetic field manipulations of parahydrogen-induced polarization: application to an AA'X spin system.

The field of magnetic resonance imaging with hyperpolarized contrast agents is rapidly expanding, and parahydrogen-induced polarization (PHIP) is emerging as an inexpensive and easy-to-implement method for generating the required hyperpolarized biomolecules. Hydrogenative PHIP delivers hyperpolarized proton spin order to a substrate via chemical addition of H2 in the spin-singlet state, but it is typically necessary to transfer the proton polarization to a heteronucleus (usually 13C) which has a longer spin lifetime. Adiabatic ultralow magnetic field manipulations can be used to induce the polarization transfer, but this is necessarily a slow process, which is undesirable since the spins continually relax back to thermal equilibrium. Here we demonstrate two constant-adiabaticity field sweep methods, one in which the field passes through zero, and one in which the field is swept from zero, for optimal polarization transfer on a model AA'X spin system, [1-13C]fumarate. We introduce a method for calculating the constant-adiabaticity magnetic field sweeps, and demonstrate that they enable approximately one order of magnitude faster spin-order conversion compared to linear sweeps. The present method can thus be utilized to manipulate nonthermal order in heteronuclear spin systems.

Exchange interaction in short-lived flavine adenine dinucleotide biradical in aqueous solution revisited by CIDNP (chemically induced dynamic nuclear polarization) and nuclear magnetic relaxation dispersion.

Flavin adenine dinucleotide (FAD) is an important cofactor in many light-sensitive enzymes. The role of the adenine moiety of FAD in light-induced electron transfer was obscured, because it involves an adenine radical, which is short-lived with a weak chromophore. However, an intramolecular electron transfer from adenine to flavin was revealed several years ago by Robert Kaptein by using chemically induced dynamic nuclear polarization (CIDNP). The question of whether one or two types of biradicals of FAD in aqueous solution are formed stays unresolved so far. In the present work, we revisited the CIDNP study of FAD using a robust mechanical sample shuttling setup covering a wide magnetic field range with sample illumination by a light-emitting diode. Also, a cost efficient fast field cycling apparatus with high spectral resolution detection up to 16.4 T for nuclear magnetic relaxation dispersion studies was built based on a 700 MHz NMR spectrometer. Site-specific proton relaxation dispersion data for FAD show a strong restriction of the relative motion of its isoalloxazine and adenine rings with coincident correlation times for adenine, flavin, and their ribityl phosphate linker. This finding is consistent with the assumption that the molecular structure of FAD is rigid and compact. The structure with close proximity of the isoalloxazine and purine moieties is favorable for reversible light-induced intramolecular electron transfer from adenine to triplet excited flavin with formation of a transient spin-correlated triplet biradical F⚫--A⚫+. Spin-selective recombination of the biradical leads to the formation of CIDNP with a common emissive maximum at 4.0 mT detected for adenine and flavin protons. Careful correction of the CIDNP data for relaxation losses during sample shuttling shows that only a single maximum of CIDNP is formed in the magnetic field range from 0.1 mT to 9 T; thus, only one type of FAD biradical is detectable. Modeling of the CIDNP field dependence provides good agreement with the experimental data for a normal distance distribution between the two radical centers around 0.89 nm and an effective electron exchange interaction of -2.0 mT.

Theoretical description of hyperpolarization formation in the SABRE-relay method.

SABRE (Signal Amplification By Reversible Exchange) has become a widely used method for hyper-polarizing nuclear spins, thereby enhancing their Nuclear Magnetic Resonance (NMR) signals by orders of magnitude. In SABRE experiments, the non-equilibrium spin order is transferred from parahydrogen to a substrate in a transient organometallic complex. The applicability of SABRE is expanded by the methodology of SABRE-relay in which polarization can be relayed to a second substrate either by direct chemical exchange of hyperpolarized nuclei or by polarization transfer between two substrates in a second organometallic complex. To understand the mechanism of the polarization transfer and study the transfer efficiency, we propose a theoretical approach to SABRE-relay, which can treat both spin dynamics and chemical kinetics as well as the interplay between them. The approach is based on a set of equations for the spin density matrices of the spin systems involved (i.e., SABRE substrates and complexes), which can be solved numerically. Using this method, we perform a detailed study of polarization formation and analyze in detail the dependence of the attainable polarization level on various chemical kinetic and spin dynamic parameters. We foresee the applications of the present approach for optimizing SABRE-relay experiments with the ultimate goal of achieving maximal NMR signal enhancements for substrates of interest.

Tailored flavoproteins acting as light-driven spin machines pump nuclear hyperpolarization.

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-Boltzmann nuclear spin magnetization, referred to as hyperpolarization, allowing for high gain of sensitivity in nuclear magnetic resonance (NMR). Well known to occur in photosynthetic reaction centers, the effect was also observed in a light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin, in which the functional cysteine was removed to prevent photo-chemical reactions with the cofactor, a flavin mononucleotide (FMN). Upon illumination, the FMN abstracts an electron from a tryptophan to form a transient spin-correlated radical pair (SCRP) generating the photo-CIDNP effect. Here, we report on designed molecular spin-machines producing nuclear hyperpolarization upon illumination: a LOV domain of aureochrome1a from Phaeodactylum tricornutum, and a LOV domain named 4511 from Methylobacterium radiotolerans (Mr4511) which lacks an otherwise conserved tryptophan in its wild-type form. Insertion of the tryptophan at canonical and novel positions in Mr4511 yields photo-CIDNP effects observed by 15N and 1H liquid-state high-resolution NMR with a characteristic magnetic-field dependence indicating an involvement of anisotropic magnetic interactions and a slow-motion regime in the transient paramagnetic state. The heuristic biomimetic design opens new categories of experiments to analyze and apply the photo-CIDNP effect.

Spin dynamics in experiments on orthodeuterium induced polarization (ODIP).

A comprehensive description of the spin dynamics underlying the formation of Ortho-Deuterium Induced Polarization (ODIP) is presented. ODIP can serve as a tool for enhancing Nuclear Magnetic Resonance (NMR) signals of 2H nuclei, being important probes of molecular structure and dynamics. To produce ODIP, in the first step, the D2 gas is brought to thermal equilibrium at low temperature, here 30 K, so that the ortho-component, corresponding to the total spin of the 2H nuclei equal to 0 and 2, is enriched, here to 92%. In the second step, the orthodeuterium molecule is attached to a substrate molecule using a suitable hydrogenation catalyst such that the symmetry of the two 2H nuclei is broken. As a result, the non-thermal spin order of orthodeuterium is converted into enhancement of observable NMR signals. In this work, we perform a theoretical study of ODIP and calculate the shape of ODIP spectra and their dependence on the magnetization flip angle. These results are compared with experiments performed for a number of substrates; good agreement between experimental and calculated ODIP spectra is found. We also discuss the performance of NMR techniques for converting anti-phase ODIP spectral patterns into in-phase patterns, which are more suitable for signal detection and for transferring ODIP to heteronuclei, here to 13C spins. Experimental procedures reported here allowed us to reach signal enhancement factors of more than 1000 for 2H nuclei in the liquid phase. These results are useful for extending the scope of spin hyperpolarization to the widely used 2H nuclei.