Method for the generation of radicals for dynamic nuclear polarization and uses thereof for NMR, MRS and MRI

Abstract

A method for the preparation of a sample comprising highly polarized nuclear spins is proposed, comprising at least the following steps: a) provision of molecules with 1,2-dione structural units and/or molecules with 2,5-diene-1,4-dione structural units in the solid state; b) generation of radicals from these molecules by photo induced electron transfer by a first electromagnetic irradiation in the visible or ultraviolet frequency range in the solid state; c) dynamic nuclear polarization in the presence of a magnetic field in the solid state by applying a second electromagnetic irradiation with a frequency adapted to transfer spin polarization from the electrons to the nuclear spins leading to a highly polarized state thereof. Furthermore uses of correspondingly prepared samples for NMR, MRS and MRI experiments are proposed.

Claims

1. A method for the preparation of a sample comprising highly polarized nuclear spins, comprising at least the following steps: a) provision of molecules in a solid state selected from the group consisting of: molecules with 1,2-dione structural units, molecules with 2,5-diene-1,4-dione structural units, and combinations thereof; b) generation of radicals from these molecules in the solid state by photo induced electron transfer by a first electromagnetic irradiation in the visible or ultraviolet frequency range; c) dynamic nuclear polarization in the presence of a magnetic field in the solid state by applying a second electromagnetic irradiation with a frequency adapted to transfer spin polarization from electrons to the nuclear spins leading to a highly polarized state thereof.

2. The method according to claim 1, wherein the first electromagnetic irradiation is applied with a frequency essentially equal to the difference or the sum of Larmor frequencies of the electron spins and the nuclear spins, respectively, in the presence of the applied magnetic field.

3. The method according to claim 1, wherein the first and second electromagnetic irradiation in steps b) and c) are applied sequentially.

4. The method according to claim 3, wherein for the first irradiation one, two or more light sources are used in pulsed mode operation forming trains of light pulses of different photon energies, different pulse intensities, different pulse lengths or combinations thereof, that either overlap or are separated in time in order to optically pump electronic transitions to reach a reactive molecular state.

5. The method according to claim 1, wherein the molecules with 1,2-dione structural units are selected from the molecules according to formula (1): ##STR00012## or wherein the molecules with 2,5-diene-1,4-dione structural unit are selected from the molecules according to formula (2) ##STR00013## wherein in both cases, independently, R.sub.1 and/or R.sub.2 are selected to be a non-reactive group, and in both cases, independently, R.sub.1 and/or R.sub.2 are selected to be a reactive functional group allowing for a linking to biomolecules and/or a crosslinking of biomolecules of interest.

6. The method according to claim 5, wherein the molecules are isotopically enriched.

7. The method according to claim 1, wherein the molecules are selected from the group consisting of: diacetyl, 2,3-pentanedione, 2,3-hexanedione, 2,3-heptanedione, pyruvic acid, oxaloacetatic acid, alpha-keto isocaproate, alpha-ketoisovaleric acid, 3-mercaptopyruvic acid, 2-oxoglutaric acid, 2-ketobutyric acid, 2-ketohexanoic acid, 2-oxo-4-methylthiobutanoic acid, 2-ketopalmitic acid, dehydroascorbic acid, fumaric acid, maleic acid, adrenochrome, cholesterol derivatives with 2,5-diene-1,4-dione structural unit, Coenzyme Q10, dopamine homologues with 1,2 dione structural units, dopa homologues with 1,2 dione structural units, and derivatives thereof and mixtures thereof.

8. The method according to claim 1, wherein the molecules or a mixture thereof are forming the sample for step a) entirely, or wherein the molecules are provided as a solvent for further molecules, and then cooled to the solid state for steps b) and c), or wherein the molecules are provided in another solvent, and then cooled to the solid state for steps b) and c).

9. The method according to claim 1, wherein subsequent to step c) the sample is warmed up until reaching liquid state, and used for liquid or solid nuclear magnetic resonance (NMR) or both, magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI), including in vitro or in vivo.

10. The method according to claim 1, wherein the molecules are peptides, proteins, or RNA or DNA comprising the structural units or with at least one structural unit attached either directly or via a spacer.

11. The method according to claim 1, in which the sample is photo-excited with light outside within step b) in a DNP polarizer or a cryo-DNP magic angle spinning (MAS) probe prior to be transferred inside a DNP polarizer or a Cryo-DNP MAS probe for step c) that will be inserted inside an NMR magnet, or in which the sample is photo-excited with the light inside a DNP polarizer or in which the sample is photo-excited with light inside a Cryo-DNP MAS probe inserted inside an NMR magnet.

12. The method according to claim 1 in which the sample is exposed to acoustic waves or an electrical field prior to be warmed up after step c) in order to force a scavenging of the radicals.

13. The method according to claim 1, wherein after step c) the sample is warmed up with a cold gas or a cold liquid to bring a temperature of a frozen solution between 77K and 273K in order to force a scavenging of the radicals while keeping the solution frozen, or wherein the samples are extracted as a solid and melted outside a polarizer.

14. The method according to claim 1, wherein the sample is dissolved outside a DNP polarizer in a solvent thermalized at a temperature above a melting temperature of the sample or wherein the sample is dissolved inside the DNP polarizer in a solvent thermalized at a temperature above the melting temperature of the samples.

15. Nuclear magnetic resonance (NMR), magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI) experiment, including in vitro or in vivo, using a hyperpolarized sample prepared using a method according to claim 1, for enhancing a signal-to-noise ratio.

16. The method according to claim 1, wherein the first electromagnetic irradiation is applied with a frequency essentially equal to the difference or the sum of Larmor frequencies of electron spins and the nuclear spins, respectively, in the presence of the applied magnetic field, wherein the magnetic field is a static, essentially homogeneous magnetic field with a strength of at least 3 T.

17. The method according to claim 1, wherein the first electromagnetic irradiation is applied with a frequency essentially equal to the difference or the sum of Larmor frequencies of electron spins and the nuclear spins, respectively, in the presence of the applied magnetic field, and wherein a temperature during the first or second electromagnetic irradiation or both is in the range of less than 120K.

18. The method according to claim 1, wherein the first electromagnetic irradiation is applied with a frequency essentially equal to the difference or the sum of Larmor frequencies of electron spins and the nuclear spins, respectively, in the presence of the applied magnetic field, and wherein a temperature during the first or second electromagnetic irradiation or both, in the range of less than 5K.

19. The method according to claim 1, wherein the first and second electromagnetic irradiation in steps b) and c) are applied sequentially, and wherein the first irradiation takes place in the absence of a magnetic field for at least 10 minutes.

20. The method according to claim 1, wherein the first and second electromagnetic irradiation in steps b) and c) are applied sequentially, and wherein the first irradiation takes place in the absence of a magnetic field for in the range of 40-300 minutes.

21. The method according to claim 1, wherein the first and second electromagnetic irradiation in steps b) and c) are applied sequentially, and wherein the frequency of the first irradiation is in the wavelength range between 200 and 800 nm, applied in continuous wave or pulsed, coherent or incoherent mode.

22. The method according to claim 1, wherein the molecules with 1,2-dione structural units are selected from the molecules according to formula (1): ##STR00014## or wherein the molecules with 2,5-diene-1,4-dione structural unit are selected from the molecules according to formula (2) ##STR00015## wherein in both cases, independently, R.sub.1 and/or R.sub.2 are selected to be a non-reactive group, selected from the group consisting of: halogen, OH, OR, CH.sub.2R, CH.sub.3, wherein R is selected from the group consisting of: H, alkyl, aryl or halogen, and in both cases, independently, R.sub.1 and/or R.sub.2 are selected to be a reactive functional group allowing for a linking to biomolecules or a crosslinking of biomolecules of interest.

23. The method according to claim 5, wherein the molecules are isotopically enriched, by at least one nuclear spin selected from the group consisting of: .sup.2H, .sup.6Li, .sup.13C, .sup.15N, .sup.17O.

24. The method according to claim 5, wherein the molecules are partly or completely carbon 13 enriched.

25. The method according to claim 1, wherein the molecule is selected to be pyruvic acid.

26. The method according to claim 1, wherein the molecules or a mixture thereof are forming the sample for step a) entirely, or wherein the molecules are provided as a solvent for further molecules, which further molecules are isotopically enriched in .sup.2H, .sup.6Li, .sup.13C, .sup.15N, .sup.17O, as a solvent, and then cooled to the solid state for steps b) and c), or wherein the molecules are provided in another solvent, selected from the group consisting of: water, ethanol, glycerol, 1, 2-propanediol, glycol, DMSO, Xenon or a mixture thereof, and then cooled to the solid state for steps b) and c).

27. The method according to claim 1, wherein subsequent to step c) the sample is warmed up until reaching liquid state, to a temperature above 273 K, and used for liquid or solid nuclear magnetic resonance (NMR) or both, magnetic resonance spectroscopy (MRS) or magnetic resonance imaging (MRI), including in vitro or in vivo.

28. The method according to claim 1 in which the sample is exposed to acoustic waves or an electrical field prior to be warmed up after step c) in order to force a scavenging of the radicals, wherein the acoustic waves are produced by piezoelectric transducers located inside a DNP polarizer.

29. The method according to claim 1, wherein after step c) the sample is warmed up with a cold gas or a cold liquid to bring a temperature of a frozen solution between 77K and 273K in order to force a scavenging of the radicals while keeping the solution frozen, wherein the cold liquid is an alcohol, or wherein the samples are extracted as a solid and melted outside a polarizer, inside a magnetic field.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

(2) FIG. 1 shows a schematic cross-sectional view of an embodiment of the ex situ setup to create radicals in the frozen samples containing maleic acid, fumaric acid or molecules with alpha-diketone functional groups via the creation of photo-excited reactive states;

(3) FIG. 2 shows schematically an embodiment of a setup to produce the radicals in situ by bringing the light inside a DNP polarizer through an optical fiber;

(4) FIG. 3 shows schematically an embodiment of a setup to produce the radicals in situ by bringing the light inside a NMR magnet with a cryo-MAS probe. (1) Cryo-MAS NMR probe;

(5) FIG. 4 shows the radical concentration in pure [1-.sup.13C]PA and in a [1-.sup.13C]PA:H.sub.2O 1:1 (vol./vol.) solution as a function of UV irradiation time as measured by X-band ESR at 77K;

(6) FIG. 5 shows the integral of the X-band ESR spectrum measured at 77K in UV-irradiated (a) natural abundance PA and (b) [1-.sup.13C]PA;

(7) FIG. 6 shows a comparison of the .sup.13C microwave spectrum measured in UV-irradiated [1-.sup.13C]pyruvic acid, [1-.sup.13C]pyruvic acid doped with 20 mM trityl radicals, and a 3M aqueous solution of [1-.sup.13C]pyruvate doped with 50 mM TEMPO;

(8) FIG. 7 shows a high-resolution NMR spectrum of UV-irradiated [1-.sup.13C]pyruvic acid melted in D.sub.2O (0.9M); and

(9) FIG. 8 shows in vivo .sup.13C spectra recorded in a mouse brain following the injection of 300 uL of hyperpolarized [1-.sup.13C]pyruvate solution prepared with UV-irradiated pure [1-.sup.13C]pyruvic acid.

DESCRIPTION OF PREFERRED EMBODIMENTS

(10) An example of an experimental set-up is schematically shown in FIG. 1. The radical concentration can be measured by ESR prior to inserting the sample inside the DNP polarizer (see FIGS. 4 and 5). In the FIG. 1, 1 designates a cryostat transparent to irradiating light, 2 liquid nitrogen, 3 a light source and 4 is a frozen sample containing polarizable molecules e.g. with maleic acid, fumaric acid or one or more alpha-diketone functional group.

(11) In a second example of an experimental set-up schematically shown in FIG. 2, the radicals are produced inside the polarizer by irradiation through an optical fiber. In this case 11 designates a warm or cold fluid to thermalize or dissolve the frozen sample in order to force the scavenging of the radicals prior to sample extraction, 12 a microwave source tuned to the frequency corresponding to the absorption of the electron and nuclear spins system in the frozen sample, 13 a light source, 14 a frozen sample containing polarizable molecules, 15 an optical fiber, 16a superconducting magnet, 17 a liquid helium or cold helium gas, 18 a piezoelectric actuator and 19 an acoustic wave source.

(12) The set-ups can generally be adapted to improve the radical production via electro-assisted electron capture by placing electrodes across the sample and produce an electric field during light irradiation.

(13) The set-ups can also be adapted to force the scavenging of the radical in the solid state by applying acoustic waves produced by piezoelectric transducers or other sources outside the polarizer and brought to the samples inside the polarizer through a waveguide (see FIG. 2). The radicals can also be scavenged via electro-oxidation or electro-reduction (pulsed or continuous).

(14) Another way to scavenge the radicals in the solid state is to exposing the frozen samples to a cold gas or a cold liquid, such as an alcohol, in order to bring the temperature of the frozen samples between 77K and 273K and force the scavenging of the radicals while keeping the samples in a solid state. The sample can then be extracted as a solid to be melted outside the DNP polarizer in an external magnet.

(15) In a third example of an experimental set-up schematically shown in FIG. 3, the radicals are produced in frozen samples, f.i. cells or cell membranes containing proteins tagged with one or more alpha-diketone groups, placed inside the rotor of a cryo-MAS probe coupled to an optical fiber that can photo-excite the alpha-diketone groups prior to performing solid-state DNP-NMR experiments. In this case, 21 designates a cryo-MAS NMR probe, 22 a microwave source tuned to the frequency corresponding to the absorption of the electron and nuclear spins system in the frozen sample; 23 a light source, 24 a frozen sample containing polarizable molecules; 25 an optical fiber, 26 a superconducting magnet, 27 cold nitrogen or helium gas and 28 rf cables.

(16) A preferred sample is frozen pure [1-.sup.13C]pyruvic acid irradiated at 365 nm using the setup described in FIG. 1 (see FIGS. 4 and 5).

(17) As a consequence of the rather narrow solid-state ESR line width (see FIG. 5), the microwave spectrum measured via .sup.13C NMR at 1.2K by varying the ESR frequency is narrower than the one measured in samples doped with TEMPO nitroxyl radicals (see FIG. 6). Although the observed line width is substantially wider than the one obtained with trityl radicals (see FIG. 6), the so-called solid effect is expected to participate in the DNP process and the final .sup.13C polarization is larger than the polarization that can be obtained with nitroxyl radicals for which the thermal mixing is largely dominant. A .sup.13C polarization of 15% could be achieved in a sample containing 15 mM of radical within 1.5 h at 5 T and 1K using 30 mW microwave power.

(18) One compelling feature of the radicals created by UV illumination of frozen PA in the framework of hyperpolarized MR is that they recombine to biocompatible non-radical species within a fraction of second upon dissolution. One observes by .sup.13C high-resolution room-temperature NMR measurements performed on UV irradiated frozen [U-.sup.13C]PA dissolved in D.sub.2O at 900 mM that the only products of recombination are CO.sub.2 and the biomolecule acetic acid (see FIG. 7), a biomolecule present in human blood at a concentration 0.05-0.2 mM in healthy subjects. Upon dissolution, the carboxyl carbon of pyruvic acid is cleaved from the molecule to become carbon dioxide and the other two carbons react with H.sub.2O to become acetic acid (see FIG. 7). The concentration of acetic acid in the hyperpolarized PA solution is about 1000 times lower than the amount of PA, which means that a 50 mM pyruvate solution will contain around 0.05 mM of acetate and would thus not lead to an increase in acetate blood concentration by more than 10%. In addition, with the most widely used substrate [1-.sup.13C]PA, the resulting acetate molecule is unlabeled and its contribution to the .sup.13C signal will thus be negligible at such low concentrations. In fact, the label ends up in .sup.13CO.sub.2 gas, which is expelled from the solution during the extraction of the dissolved frozen sample from the hyperpolarizer with helium gas (one measures .sup.13CO2 in the experiment shown in FIG. 7 because the frozen sample is simply melted in D.sub.2O and not degassed or flushed with helium gas). As a consequence, not only the solution is free of paramagnetic impurities as demonstrated by the observed relaxation time equivalent to the T.sub.1 measured in a degassed pure aqueous PA solution, but also all potential toxicity issues are alleviated.

(19) To demonstrate the potential of the method, contrast agents for in vivo MR are prepared, namely a 50 mM hyperpolarized pyruvate solution from a UV irradiated PA sample using the hardware described above, and 300 uL of the solution were injected in a mouse femoral vein prior to performing real-time metabolic measurements in the brain with a 3 s time resolution. The lactate, alanine and bicarbonate signals were recorded as a function of time in the mouse brain (see FIG. 8). This is the first in vivo hyperpolarized MR study of cerebral metabolism ever reported in mice.

(20) So a MR contrast agent can be obtained via dissolution DNP from a frozen pure endogenous substance that is simply exposed to a commercially available LED UV light source for less than an hour. The solution containing the hyperpolarized .sup.13C-labeled contrast agent is uncontaminated by paramagnetic impurities such as free stable radicals or any other non-endogenous substance and in vivo real-time metabolic measurements with a three second time resolution could be recorded in a 9.4 T MR imager following its injection thanks to the four-orders of magnitude .sup.13C SNR enhancement. So the novel method was applied to prepare hyperpolarized PA, the clinical potential of which has been already demonstrated in oncology and cardiology.

(21) Experimental Part:

(22) In summary, [1-.sup.13C] PA was frozen in the form of 2 uL beads by plunging droplets in liquid nitrogen. The frozen beads were irradiated for 1 h with UV light using a LED source (Hammamatsu Photonics LC-L2) and consecutively placed inside a 5 T and 10.05 K custom-designed DNP polarizer. Following polarization, the PA samples were rapidly dissolved and transferred into an infusion pump placed inside the bore of a 9.4 T imager. The pump was programmed to automatically inject 300 uL of the hyperpolarized solution (pyruvate concentration 50 mM) into a mouse femoral vein. In vivo .sup.13C spectra and images were acquired with a custom-designed surface coil placed on top of the mouse head using adiabatic RF pulses to compensate for B1 inhomogeneities. Localization was achieved using an outer volume suppression scheme. Anesthetized animal physiology was monitored (respiration rate 100 min-1) and body temperature was kept between 37-38 C.

(23) Experimental Details:

(24) Dynamic Nuclear Polarization of [1-.sup.13C]PA.

(25) The carbon-13 nuclear spins in frozen[1-.sup.13C]PA (Sigma-Aldrich) were dynamically polarized using a custom-designed DNP polarizer operating at 5 T and 1+0.05 K. Droplets of pure [1-.sup.13C]PA were plunged in liquid nitrogen to form frozen beads of about 2 mm diameter. Ten frozen beads were placed inside the DNP polarizer sample holder, which was then inserted into the microwave cavity located within the cryostat filled with about 0.5 L of liquid helium at atmospheric pressure (4.2 K). The vacuum pump system was then turned on to lower the temperature of the sample space such as to maintain the frozen sample under superfluid helium at 10.05 K.

(26) The microwave power at the output of the source was set to 30 mW and the irradiation frequency was set to 139.85 GHz. The nuclear polarization was monitored as a function of time by means of pulsed NMR using 5 degree tipping pulses. Once the polarization on the .sup.13C nuclei had reached a targeted value (the .sup.13C solid-state polarization reached 152% after 1.50.2 h in [1-.sup.13C]PA samples, the frozen solution was dissolved in 5 ml of superheated D.sub.2O (170 C.) by means of a dissolution apparatus. In the system used for the present experiments, a helium gas stream drives the resulting solution out of the polarizer magnet through a 6 m long capillary into the bore of an animal imager, where the sample is collected in a remotely controlled infusion pump that separates the liquid solution from the gas, and infuses a chosen amount of liquid solution into an animal. The delay between dissolution and infusion was set to 3 s.

(27) Animal Preparation.

(28) In vivo experiments were performed on NMRI mice. All experiments were approved by the local ethics committee. Animals were anesthetized with 1.5% isoflurane in a 30% O.sub.2/70% N.sub.2O mixture. A femoral vein was catheterized for PA infusion. The mouse was placed on a holder along with the infusion pump and the femoral vein catheter was connected to the outlet of the pump. The holder was then inserted inside the scanner. A bolus of about 0.3 ml of hyperpolarized solution containing approximately 50 mM of the .sup.13C labeled PA was infused within 5 s. Mouse physiology was monitored and kept stable during the experiments, body temperature was kept between 37-38 C., while respiration rate was maintained at around 100 min.sup.1 by adjustment of the isoflurane dose. The rate and dose of the infusion was determined in bench experiments to ensure that the bolus-like infusion is not lethal for mice. After the experiment animals were euthanized with an overdose of pentobarbital.

(29) MRI and MRS experiments. All measurements were performed on a Varian INOVA spectrometer (Varian, Palo Alto, Calif., USA) interfaced to a 31 cm horizontal-bore actively-shielded 9.4 T magnet (Magnex Scientific, Abingdon, UK). RF transmission and reception was done using a home-built hybrid surface coil consisting of a proton quadrature coil and a three-loop 10 mm diameter carbon coil. This coil was placed on top of the mouse head. The .sup.13C spectra were acquired through single pulse experiments with adiabatic radiofrequency pulses. The acquisition time was set to 200 ms. To generate an external reference signal a small sphere filled with 99% .sup.13C-labeled formic acid was placed in the center of the carbon coil. High order shimming was performed using the FASTESTMAP algorithm.

(30) TABLE-US-00001 LIST OF REFERENCE SIGNS 1 cryostat transparent to irradiating light 2 liquid nitrogen 3 light source 4 frozen sample containing polarizable molecules 11 warm or cold fluid 12 microwave source 13 light source 14 frozen sample containing polarizable molecules 15 optical fiber 16 superconducting magnet 17 liquid helium or cold helium gas 18 piezoelectric actuator 19 acoustic wave source 21 cryo-MAS NMR probe 22 microwave source 23 light source 24 frozen sample containing polarizable molecules 25 optical fiber 26 superconducting magnet 27 cold nitrogen or helium gas 28 rf cables