Abstract:
A method for the preparation of a hyperpolarized solution of molecules comprises the steps: 1) suspending or coating of a micro-particulate matrix, which is comprising or consisting of the molecules, with a glass-forming solution or suspension comprising a DNP-suitable polarizing agent at a first temperature at which the micro-particulate matrix is not dissolving; 2) lowering the temperature leading to a frozen glassy DNP sample; 3) transferring the electron spin polarization of the polarizing agent in the glassy sample in a magnetic field to abundant nuclear spins of the glass-forming solution or suspension and/or the polarizing agent as well as to abundant nuclear spins of the molecules and cross-polarization from the abundant nuclear spins in the molecules to one different nuclear spin type in the molecules; and 4) increasing the temperature and dissolving the molecules which are hyperpolarized with respect of the different nuclear spins.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a method for the production of long-lived hyperpolarized samples, as well as to uses of such a method for measurement purposes. 
       PRIOR ART 
       [0002]    Dissolution dynamic nuclear polarization (D-DNP) allows one to increase the NMR polarization and hence the signals of molecules labeled with isotopes that have a nuclear magnetic moment such as  13 C,  15 N,  29 Si,  31 P,  6 Li and  89 Y, etc., by several orders of magnitude by performing low temperature DNP followed by rapid melting, dissolution and heating to room temperature. Molecules can be “hyperpolarized” by boosting the polarization of their nuclei by DNP. The production of hyperpolarized molecules of interest such as metabolites in bulk opens the way to a broad diversity of novel applications, such as the detection of intermediates in chemical reactions, the observation of protein folding in real time, or the monitoring of cancer in humans. In order to deliver hyperpolarized samples to the system under investigation (be it an NMR tube or a human), the melting or dissolution step is normally carried out in the magnetic field of the DNP polarizer, and the sample must be rapidly injected, within about 1 minute, depending on T 1 ( 13 C). If these two conditions are fulfilled, most of the hyperpolarized magnetization can be preserved. 
         [0003]    The need to melt or dissolve the sample within the polarizer is a major limitation. Because polarization in solution only lasts for a few minutes, it introduces the need for a polarizer at the point of use. Solid-state relaxation properties are very different from those in solution, so removal of the sample in the solid state might in principle allow the polarization to be preserved for long times. However, any attempts to take the hyperpolarized solid sample out of the polarizer and melt it in a remote place also appear inevitably doomed to fail. The reason for this limitation is that, as shown in the experimental section further below,  13 C nuclear spin-lattice relaxation times in DNP samples become prohibitively short in low magnetic fields because relaxation is exacerbated by the presence of the paramagnetic polarizing agents (PAs) that are indispensable for DNP. The problem therefore appeared unresolvable so far. 
         [0004]    In D-DNP experiments, the  13 C-labeled metabolites are usually polarized at very low temperatures (1.2&lt;T&lt;4.2 K) and moderate fields (usually B 0  ranges from 3.35 to 6.7 T) either directly, or indirectly by  1 H→ 13 C cross polarization. One feature of DNP is that it is usually done with a statistical distribution of PAs in a frozen glassy solution of the substrate of interest, which is normally achieved by shock-freezing aqueous mixtures of PAs and  13 C-labeled metabolites, possibly mixed with a glass-forming agent such as glycerol. For efficient DNP the frozen samples must be glassy and transparent. Samples turn opaque when crystallization occurs, leading to a partial separation of the metabolites and PAs, and DNP becomes very inefficient. Alternatively, the PAs can be covalently attached to the surface of mesostructured materials that are impregnated with metabolite solutions, or generated in-situ by UV irradiation. However, in all of these strategies paramagnetic relaxation in low fields precludes carrying the hyperpolarized samples over large distances for remote D-DNP. 
       SUMMARY OF THE INVENTION 
       [0005]    Hyperpolarization by dissolution DNP has enabled in vivo metabolic imaging of  13 C-labeled tracers by MRI. The NMR signals of metabolites can be enhanced by several orders of magnitude. During the limited lifetime of the hyperpolarized magnetization, a variety of observations can be performed, ranging from the detection of tumors in humans such as in prostate cancer, to the monitoring of the response to chemotherapy. Because of the limited lifetime of the hyperpolarized magnetization (typically T 1 &lt;50 s), the DNP process must be performed on-site, close to the MRI scanner, with sophisticated machinery that must be sterile for applications to humans. 
         [0006]    It is thus an object of the present invention to provide a solution to these obstacles and to propose for the first time a method allowing for remote production and long-distance transportation of hyperpolarized metabolites, or more generally hyperpolarized molecules of interest. This is achieved by suitable compartmentalization of the molecules of interest and the polarizing agents so that on the one hand cross-polarization between these two is possible, but on the other hand the effect of the polarizing agents on the relaxation of the molecules of interest can be reduced to a minimum. According to one embodiment this is for example achieved by impregnating microcrystals of molecules of interest, e.g. metabolites, with a glass-forming solvent that contains polarizing agents and cannot dissolve the microcrystals. The proton hyperpolarization produced in the component with polarizing agent (CPA) is then transported to the molecules of interest, e.g. metabolites, contained in the component with the molecule of interest (CMI) by proton spin diffusion, and converted into carbon-13 hyperpolarization by cross-polarization so that it cannot “leak” out of the CMI, which can be transported over long distances during extended times. 
         [0007]    To remove the polarized solid from the polarizer, the radicals and the substrate should preferably be physically separated from each other to prevent relaxation causing the decay of the polarization, yet close enough for the radicals to polarize the substrate on the DNP timescale. This seems like a paradox. However, here we show how this can be achieved by formulating the DNP sample with a suitable microarchitecture. The sample can be made of two matrices interleaved on a micrometer scale. 
         [0008]    More generally speaking, the present invention relates to a method for the preparation of a hyperpolarized solution or suspension of molecules of interest. 
         [0009]    The method for the preparation of a hyperpolarized solution or suspension of molecules of interest in particular comprises at least the following steps:
   1) providing a micro-particulate matrix, which can be crystalline or a non-porous aggregate, and which is comprising or consisting of the molecules of interest, or providing a porous aggregate which is impregnated with molecules of interest, and suspending, emulsifying or coating of said a micro-particulate matrix with a solution, emulsion or suspension comprising a DNP-suitable polarizing agent, or surface functionalizing the micro-particulate matrix with a DNP-suitable polarizing agent, at a first temperature at which the micro-particulate matrix is not dissolving; or providing a micro-particulate matrix, which is comprising or consisting of the DNP-suitable polarizing agent and suspending, emulsifying or coating of said a micro-particulate matrix with a solution, emulsion or suspension comprising the molecules of interest, at a first temperature at which the micro-particulate matrix is not dissolving;   2) lowering the temperature to a value of at most 15 K leading to a frozen DNP sample;   3) transferring the electron spin polarization of the polarizing agent in the DNP sample at this low temperature in a magnetic field of at least 2 T to abundant nuclear spins, allowing for spin diffusion to relay polarization to at least one of:
       abundant nuclear spins in the molecules of interest;   in case of the micro-particulate matrix comprising the molecules of interest abundant nuclear spins in the micro-particulate matrix comprising the molecules of interest;   in case of a micro-particulate matrix, which is comprising or consisting of the DNP-suitable polarizing agent, abundant nuclear spins in the solvent or emulsion or suspension agent comprising the molecules of interest;   and performing hetero-nuclear cross-polarization from the abundant nuclear spins of the molecules of interest and/or the abundant nuclear spins in the micro-particulate matrix comprising the molecules of interest or the abundant nuclear spins in the solvent or emulsion or suspension agent comprising the molecules of interest to at least one nuclear spin type different from the abundant nuclear spins in the molecules of interest;   
       4) increasing the temperature and melting, dissolving or suspending the molecules of interest where the nuclear spin type different from the abundant nuclear spins is hyperpolarized, in particular for use in a magnetic resonance imaging or nuclear magnetic resonance experiment.   
 
         [0018]    In respect of the individual steps the following definitions and possible specifications apply: 
         [0019]    1) providing a micro-particulate matrix, which can be crystalline or a non-porous aggregate, and which is comprising or consisting of the molecules of interest, or a porous aggregate which is impregnated with molecules of interest, and suspending, emulsifying or coating of said a micro-particulate matrix with a glass-forming solution, emulsion or suspension comprising a DNP-suitable polarizing agent, or surface functionalizing the micro-particulate matrix with a DNP-suitable polarizing agent, at a first temperature at which the micro-particulate matrix is not dissolving; 
         [0020]    or providing a micro-particulate matrix, which is comprising or consisting of the DNP-suitable polarizing agent and suspending, emulsifying or coating of said a micro-particulate matrix with a solution, emulsion or suspension comprising the molecules of interest, at a first temperature at which the micro-particulate matrix is not dissolving. 
         [0021]    Preferably this step involves suspending or coating of a micro-particulate matrix, which is crystalline or a non-porous aggregate, and which is comprising or consisting of the molecules of interest, with a glass-forming solution, emulsion or suspension comprising a polarizing agent suitable for DNP at a first temperature at which the micro-particulate matrix is not dissolving. 
         [0022]    In an alternative approach it is however also possible to provide for a micro-particulate matrix which is a porous aggregate, and which is deeply impregnated with molecules of interest or with a mixture including the molecule of interest. This micro-particulate matrix can then be either suspended or coated as pointed out above. 
         [0023]    Such a micro-particulate matrix in the form of beads can also be surface functionalized with a polarizing agent that may be covalently bound to the surface. 
         [0024]    For example possible are experiments, where: 
         [0025]    (1) the polarizing agents are covalently attached to micro-beads of a polymer (e.g. polystyrene) or to a micro-porous polymer (e.g. polystyrene), or to a micro-drilled polymer (e.g. polystyrene). 
         [0026]    (2) the polymeric micro-particulate matrix is then impregnated with a solution containing the molecule of interest, or even the molecule of interest itself in a liquid form. One most interesting molecule can be pyruvic acid. 
         [0027]    (3) after dissolution, the polymer micro-particulate matrix beads can be separated from the solution by filtration. 
         [0028]    However in case of a crystalline or non-porous aggregate, functionalizing the surface with the polarizing agent is also possible, simplifying the separation process as pointed out above after hyperpolarization. In case of surface functionalizing, the micro-particulate matrix is simply suspended in a suitable solvent which preferably neither dissolves the aggregates nor the crystalline structures or, in case of a porous aggregate, neither dissolves the porous aggregate nor perturbs the impregnation with the molecule of interest. 
         [0029]    In yet another alternative approach it is also possible to provide a micro-particulate matrix (e.g. a polymer matrix, micro-beads), comprising or consisting of the DNP-suitable polarizing agent and suspending, emulsifying or coating of said a micro-particulate matrix with a solution, emulsion or suspension comprising the molecules of interest. 
         [0030]    The micro-particulate matrix is to be understood as being either essentially pure micro-crystallites of the molecule(s) of interest or, in the alternative approach, of the polarizing agent or micro-crystallites comprising the molecule of interest (or in the alternative approach the polarizing agent) together with one or several other components which then serve as a crystalline matrix. Also possible are, when talking about non-porous aggregates, micro-particles which are not crystalline but which are for example amorphous, in particular in this case it is further important that these aggregates are not open to being penetrated by the glass-forming solution or suspension comprising a DNP-suitable polarizing agent or in the alternative approach the molecules of interest in order not to allow the polarizing agent to get into close contact to essentially all the molecules of interest in the particles. 
         [0031]    It should further be noted that the micro-particulate matrix can be provided in the form of a powder, i.e., in the form of loose micro-particles, but it can also be provided in the form of a packed and coherent structure of these micro-particles which is however sufficiently open for inter-particle penetration by the glass-forming solution in step 1) and for release of the glass-forming solution in step 4) when separating the glass-forming solution/suspension. 
         [0032]    What is furthermore to be noted is that at least under the conditions chosen in step 1) the microparticles of the micro-particulate matrix are not dissolving in the glass-forming solution/suspension/emulsion. 
         [0033]    The microparticles can be suspended in the glass-forming solution/suspension/emulsion in step 1) but it can however also be sufficient to just coat or impregnate the microparticles in this step, as long as there is sufficient polarizing agent available on the surface for efficient polarization transfer in step 3). 
         [0034]    The polarizing agent, which is typically an organic or inorganic molecule with one or several unpaired electrons (inclusive of metal centers with unpaired electrons, possibly with suitable ligands), can be suspended, emulsified preferably dissolved, in a suitable solvent which, as pointed out above, should not, under the conditions chosen in step 1), dissolve the microparticles containing or consisting of the molecules of interest. 
         [0035]    In many situations the temperature chosen in step 1) will be room temperature, in any case the temperature in which the solvent comprising the polarizing agent is sufficiently liquid to mix with the microparticles comprising or consisting of the molecules of interest. One can also use lower temperatures or temperatures higher than room temperature. For example the PA can be dissolved in a liquid gas at −10°, or in a liquid polymer at 80°. 
         [0036]    2) lowering the temperature to a value of at most 15 K frozen-DNP-sample; 
         [0037]    Typically this temperature in step 2) is the same or close to the one used in step 3). The DNP sample is preferably a solid composite/solution/emulsion/suspension with the microparticles of the molecules of interest embedded in the solid composite/solution/emulsion/suspension with the polarizing agent, or in the alternative approach with the microparticles of the polarizing agent embedded in the solid composite/solution/emulsion/suspension with the molecules of interest. In order to make sure that the micro-particulate structures are sufficiently homogeneously distributed in the glassy structure it can be advantageous to stir or move the composite/solution/emulsion/suspension at least in an initial phase of step 2 to avoid agglomeration before freezing. 
         [0038]    3) transferring the electron spin polarization of the polarizing agent in the DNP sample e.g. by microwave irradiation at this low temperature in a magnetic field of at least 2 T to abundant nuclear spins, allowing for spin diffusion to relay polarization to at least one of: 
         [0039]    abundant nuclear spins in the molecules of interest; 
         [0040]    in case of the micro-particulate matrix comprising the molecules of interest abundant nuclear spins in the micro-particulate matrix comprising the molecules of interest; 
         [0041]    in case of the micro-particulate matrix, which is comprising or consisting of the DNP-suitable polarizing agent, abundant nuclear spins in the composite/solution/emulsion/suspension comprising the molecules of interest; and performing hetero-nuclear cross-polarization from the abundant nuclear spins of the molecules of interest and/or in the micro-particulate matrix to at least one different nuclear spin type in the molecules of interest; 
         [0042]    The step 3) can be repeated for stepwise polarization enhancement, e.g. 2-20 times, if necessary. 
         [0043]    In this 3 rd  step the high polarization of the electron spins shall be transferred to the different nuclear spins in the molecule of interest. This is normally not efficient directly but is done by 1 st  transferring the electron polarization to the abundant nuclear spins of the component containing the polarizing agent and/or of the solvent, or the composite/solution/emulsion/suspension agent, and by spin diffusion to the abundant nuclear spins of the component containing the molecule of interest or to the abundant nuclear spins of the molecule of interest itself. 
         [0044]    In the alternative approach the electron polarization of the polarizing agent contained in the microparticles is transferred to abundant nuclear spins of either the polarizing agent itself or the matrix in which the polarizing agent is embedded in the microparticles. Further the polarization is transferred to abundant spins of the composite/solution/emulsion/suspension agent with the molecules of interest and/or directly to the molecules of interest. 
         [0045]    Then subsequently hetero-nuclear cross-polarization transfer takes place, while polarization transfer can be cross-polarization at high field, or Nuclear Thermal Mixing by shuttling the sample at low field, from the abundant protons to the different nuclear spins, typically carbon-13 or nitrogen-15, or after initial start of DNP with microwave irradiation, the hetero-nuclear cross-polarization is restarted while maintaining the microwave irradiation transferring polarization from the electron spins to the abundant nuclear spins. This process may include continuous irradiation and/or pulsed irradiation for cross-polarization as well as flip back pulses and detection pulses for monitoring purposes. 
         [0046]    4) increasing the temperature and dissolving or suspending the molecules of interest which are hyperpolarized in respect of the different nuclear spins, in particular for use in a magnetic resonance imaging or nuclear magnetic resonance experiment. 
         [0047]    In this step 4) the actual making available of the hyperpolarized molecules of interest (hyperpolarized in terms of e.g. carbon-13 or nitrogen-15 or one of the other nuclei mentioned above) to an experimental setup typically at room temperature is realized. Preferably in order to avoid detrimental relaxation of the hyperpolarization during this step the polarizing agent is separated from the hyperpolarized molecules of interest. 
         [0048]    One gist of the present invention thus can be seen in that in one of the embodiments by impregnating microcrystals or essentially non-porous micro-aggregates of molecules of interest (MoI), e.g. metabolites, with a typically glass-forming, e.g. hydrophobic, DNP matrix that contains polarizing agents but cannot dissolve the microcrystals/non-porous micro-aggregates, proton hyperpolarization can be transported to the MoI contained in the microcrystals/non-porous micro-aggregates through spin diffusion, and converted into carbon-13 hyperpolarization by cross-polarization so that it cannot “leak” out of the microcrystals/non-porous micro-aggregates, which consequentially can be transported over long distances during extended times. 
         [0049]    One component that we call the DNP matrix according to one preferred embodiment contains the PAs and abundant spins such as protons, and the other component that we refer to as the micro-crystalline or non-porous micro-aggregate matrix contains the molecules of interest (e.g. metabolites) labeled with  13 C (and/or any of the other nuclei mentioned above) and containing the same abundant spins. The general idea, illustrated schematically in  FIG. 1A ,  FIG. 1B  and  FIG. 1C , is that abundant spins such as  1 H are directly and rapidly polarized by DNP in the glassy DNP matrix, and that this polarization is then relayed more slowly to the  1 H of the frozen liquid, micro-crystalline or non-porous micro-aggregate matrix by nuclear spin diffusion. The time needed for spin diffusion to propagate the  1 H polarization from the glassy DNP matrix to the micro-crystalline or non-porous micro-aggregate matrix depends on the dimensions of the microcrystals. We show that 10 μm diameter particles typically translate into a time-scale for spin diffusion of 10 minutes. Once the  13 C nuclei in the micro-crystalline or non-porous micro-aggregate matrix are surrounded by highly polarized  1 H spins, a  1 H→ 13 C cross-polarization sequence is applied to transfer the polarization to the  13 C spins of the metabolites in the micro-crystalline or non-porous micro-aggregate matrix. At this point, DNP is switched off, and the protons relax towards thermal equilibrium within minutes, while the  13 C spins, which are no longer in contact with the proton bath or the PAs, remain hyperpolarized for prolonged periods, typically for days. Indeed, at this point the  13 C hyperpolarization is locked in the micro-crystalline or non-porous micro-aggregate matrix where (i) there are no PAs that could act as relaxing agents, and (ii) the  13 C hyperpolarization cannot leak back from the micro-crystalline or non-porous micro-aggregate matrix to the glassy DNP matrix since  13 C spin diffusion is extremely inefficient. 
         [0050]    In the alternative approach the roles of the compartments is so to speak reversed, i.e. the microparticles comprise the polarizing agent and the glassy matrix comprises the molecules of interest. One advantage of this alternative approach is that the polarizing agent is attached to the microparticles and upon heating of the corresponding sample separation is easily possible by filtering out the microparticles leaving the solution/suspension of the molecules of interest that are hyperpolarized. 
         [0051]    Nano-crystalline suspensions were first polarized by DNP assisted by spin diffusion by van der Wel et al., who used DNP to enhance NMR signals of amyloid fibrils in the vicinity of 100 K in combination with MAS (J. Am. Chem. Soc 2006, 128, 10840-10846). More recently, the idea was generalized to micro-particulate samples, and it was shown that this could lead to substantial polarization in ordinary organic solids or even provide information about domain structures in pharmaceutical formulations. All these studies were performed in magic-angle spinning (MAS) experiments around 100 K. 
         [0052]    Capitalizing on the concept of DNP relayed by spin diffusion, we show here inter alia how microcrystals of  13 C-labelled metabolites (such as [1- 13 C]urea and [1- 13 C]pyruvate) impregnated with a DNP matrix made of glass-forming solutions containing PA&#39;s, spin diffusion from the glassy DNP matrix to the microcrystals, followed by cross-polarization from  1 H to  13 C can lead to a “locked” state of  13 C polarization (P( 13 C)&gt;20%). We demonstrate that the hyperpolarization in these microcrystals or non-porous micro-aggregates can have lifetimes greater than 10 hours at 4.2 K. We also show that due to the special nature of these “locked” states, the sample can be removed from the DNP polarizer while preserving much of its polarization. 
         [0053]    This opens up possibilities, in opposition to conventional glassy DNP samples, to take the microcrystals out of the polarizer and even transport them to a remote location in a transport device consisting of an ordinary cryogenic Dewar equipped with an insert comprising a permanent magnetic field (here B transport &gt;0.8 T). In the transport device, the typical lifetime of  13 C hyperpolarization is &gt;30 minutes. The DNP sample can then be dissolved at a remote location. We show that if the DNP matrix is chosen to be non-miscible with water, the hyperpolarized metabolites (soluble in water) can be easily separated from the PAs and any other contaminants during the dissolution process, yielding a pure hyperpolarized solution without the need for further purification. 
         [0054]    For DNP to be efficient, the samples are typically doped with the PAs at concentrations of tens of mM, giving rise to intense electron-electron dipolar couplings, and thus to homogeneous line-widths on the order of tens of MHz. If the magnetic field is lowered to such an extent that the nuclear Larmor frequency of the  13 C spins falls below the homogeneous dipolar width, the nuclear spin system is said to be in ‘thermal contact’ with the electron dipolar spin bath, and relaxes with its dipolar relaxation time constant T 1D , which usually does not exceed a few tens of ms. The more intimate the contact of the PAs with the  13 C labelled metabolites, the shorter the relaxation times. 
         [0055]    According to a first preferred embodiment of the proposed method, the particles of the micro-particulate matrix have an average particle size in the range of 3-30 μm, preferably in the range of 5-20 μm, particularly preferably in the range of 8-15 μm. If the particle sizes are chosen in this range there is an optimum balance between the contact between the micro-particulate matrix and the polarizing agents during step 3) but also a sufficient separation between the molecules of interest and the polarizing agents to avoid detrimental relaxation of the polarization established in the molecules of interest during step 3) by the unpaired electrons of the polarizing agents. 
         [0056]    According to another preferred embodiment, the temperature in steps 2) and/or 3) is at most 10 K, preferably at most 5 K, particularly preferably at most 2 K. Typically the temperatures are in the range of 1-2 K. 
         [0057]    The static magnetic field in step 3) is preferably chosen at least 0.1 T, preferably at least 3 T or 4 T, particularly preferably at least 6 T. 
         [0058]    Transferring the electron spin polarization to the abundant nuclear spins according to a preferred embodiment includes transferring electron spin polarization to protons of the component containing the polarizing agent and other molecules by using microwave irradiation, which is relayed to the protons of the component containing the molecules of interest and/or of the molecule itself or other molecules by nuclear spin diffusion. 
         [0059]    The hetero-nuclear cross-polarization in step 3) according to another preferred embodiment includes pulsed and/or continuous radiofrequency irradiation essentially at the Larmor frequencies of both the abundant nuclear spins and the different nuclear spin type. 
         [0060]    The nuclear spin type different from the abundant nuclear spin is preferably selected from the group consisting of  13 C,  15 N,  29 Si,  31 P,  6 Li and  89 Y, and the molecules of interest can be correspondingly labeled. 
         [0061]    Within step 4), preferably before solution/suspension, the molecules of interest are separated from the polarizing agent. This can be done in that first the temperature is raised in the sample until the glass-forming solution or suspension comprising the polarizing agent is in the liquid state but the micro-particulate matrix is still in the solid state, separating the micro-particulate matrix from the glass-forming solution or suspension or at least from the polarizing agent contained therein, preferably using filtering, centrifugation, sedimentation, chromatography or a combination thereof, and dissolving the isolated micro-particulate matrix and/or the molecules of interest. 
         [0062]    In the alternative approach where the polarizing agent is contained in the microparticles, separation can be carried out by heating the samples until the solution/suspension of the molecules of interest is sufficiently liquid to filter out the microparticles containing the polarizing agent, making a solution/suspension of hyperpolarized molecules of interest directly available. 
         [0063]    In this manner, essentially pure hyperpolarized molecules of interest can be obtained, well separated from the relaxation-active polarizing agents allowing for longer storage and/or more extended experiments. 
         [0064]    According to yet another preferred embodiment of the proposed method, after step 3) and before step 4) the sample is transported to a remote location, said transport involving taking the sample temporarily out of the magnetic field of step 3). 
         [0065]    This is one of the key advantages of the proposed method, namely due to the shielding of the hyperpolarized molecules in the microparticles from the relaxing effect of the polarizing agents in the component containing the PA, the hyperpolarization is much more stable over time, since relaxation is much slower. Due to this it is even possible to transport the sample to a remote location and/or take it briefly (typically for not more than 10 seconds) out of the magnetic field. 
         [0066]    Another preferred embodiment of the proposed method is characterized in that the solution or suspension comprising a DNP-suitable polarizing agent comprises the DNP-suitable polarizing agent at a concentration of at least 5 mM, preferably at least 10 mM, more preferably in the range of 10-50 mM. 
         [0067]    The DNP-suitable polarizing agent can be selected from nitroxide derivatives, preferably from the group consisting of TEMPO, TEMPOL, TEMPO-benzoate, or a mixture thereof, but also other radicals such as BDPA, DPPH, galvinoxyl and other polarizing agents containing rare earth elements. 
         [0068]    The solvent and/or suspending liquid of the solution or suspension comprising a DNP-suitable polarizing agent can be selected from a group comprising or consisting of tetrahydrofurane, dioxane, hexane, heptane, octane, nonane, benzene, toluene, DMSO, water, glycerol or a mixture thereof. 
         [0069]    Furthermore the molecule of interest can be selected from the group comprising or consisting of urea, pyruvate, fumarate, malate, glucose, acetate, alanine, or a mixture thereof. 
         [0070]    In step 4) the molecule of interest can for example be dissolved in water or in a physiological liquid. Also possible is however a different liquid. 
         [0071]    Furthermore the present invention relates to the use of a sample produced in a method as outlined above in a magnetic resonance imaging or a nuclear magnetic resonance experiment at room temperature, in vivo or in vitro. 
         [0072]    Last but not least the present invention relates to a sample produced using a method as outlined above. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0073]    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, 
           [0074]      FIGS. 1A -IC shows an illustration of the DNP mechanism in  FIG. 1A  a conventional glassy DNP solution containing both metabolites (small dots) and polarizing agents (filled circles);  FIG. 1B  micro-crystals containing only metabolites but no polarizing agents, coated or covered by a DNP solution containing only polarizing agents but no metabolites;  FIG. 1C  polymer containing the polarizing agents, covered by a solution containing the metabolites; 
           [0075]      FIGS. 2A-2B  shows in  FIG. 2A  a microscope image of micro-crystals of [1- 13 C]urea after manual grinding, and in  FIG. 2B  a distribution of the particle size of these micro-crystals; 
           [0076]      FIGS. 3A-3B  shows the DNP build-up of the proton NMR signal intensity that is proportional to the proton polarization P( 1 H) in  FIG. 3A  a glass-forming DNP solution and  FIG. 3B  in microcrystals that are impregnated in a glass-forming DNP solution, where the fast build-up is associated with the glass-forming DNP solution, and the slow component is associated with the microcrystals; 
           [0077]      FIG. 4  shows the DNP build-up of  13 C polarization P( 13 C) after cross-polarization (CP) in (empty circles) a glassy DNP solution containing [1- 13 C]urea, and (filled circles) in microcrystals of [1- 13 C]urea that are impregnated in a glass-forming DNP solution; 
           [0078]      FIG. 5  shows  13 C nuclear spin lattice relaxation at 4.2 K and 6.7 T in (empty circles) a glassy DNP solution containing [1- 13 C]urea, and (filled circles) in microcrystals of [1- 13 C]urea that are impregnated in a glass-forming DNP solution; 
           [0079]      FIG. 6  shows  13 C nuclear signals in microcrystals of pure pyruvate that are impregnated in a glass-forming DNP solution measured at 4.2 K and 6.7 T before and after brief transport to low fields in the stray field of the NMR magnet; 
           [0080]      FIG. 7  shows a transportation device formed by eight permanent magnets assembled in a Halbach configuration to give a field of 1 T, inserted into a standard liquid helium transport Dewar; the polarized DNP sample can be lowered manually into the Dewar to be transported to a remote location, since T 1 ( 13 C)&gt;30 min; and 
           [0081]      FIG. 8  shows a hyperpolarized signal of 20 mg of [1- 13 C]urea after dissolution of the impregnated microcrystals with 5 mL of water in the DNP polarizer, transport through a magnetic tunnel, and injection into a 300 MHz NMR spectrometer; the  13 C signal was measured every 5 s with 50 pulses; the hyperpolarization decay was fitted with a mono-exponential curve with T 1 ( 13 C)=60 s, typical for a radical-free solution. 
       
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0082]    Metabolite Component. 
         [0083]    [1- 13 C]urea and [1- 13 C]pyruvate were chosen as test substances (molecules of interest) since urea was the first hyperpolarized metabolite produced by Dissolution DNP, and because pyruvate has become the metabolite of reference for in-vivo clinical studies as it provides a reliable and efficient means to detect prostate tumors. However, any metabolite or molecule of interest that can be provided in the form of a powder can be equally suitable for the proposed method. 
         [0084]    Here powdered samples of micro-crystalline  13 C-labelled metabolites were ground by hand down to an average particle diameter of 13 μm with a distribution of ±5 μm (see  FIGS. 2A and 2B ). This protocol was used for small quantities of metabolites, less than 1 g. For larger-scale production, we have obtained good results by e.g. zirconia ball milling. Of course, many other preparation methods are possible, such as precipitation/crystallization from a saturated solution, co-crystallization, spray-drying, or re-crystallization by solvent evaporation, etc. for the production of the microcrystalline or non-porous micro-aggregate forms of the molecules of interest. The molecules of interest can essentially be forming this micro-matrix, or the molecules of interest can be embedded in such a micro-matrix, so the micro-matrix does not necessarily consist of the molecules of interest but can also be a mixture of different molecules of interest or can also be a mixture of a carrier with molecules of interest. 
         [0085]    Polarizing Component. 
         [0086]    The microcrystals are impregnated by a glass-forming DNP matrix containing PAs chosen such that the microcrystals are not soluble in the DNP matrix under the chosen conditions. Here we used a DNP matrix consisting of Toluene-d6:THF (8:2) doped with 50 mM TEMPOL-benzoate (4-Hydroxy-2,2,6,6-tetramethylpiperidine-oxyl-benzoate), a hydrophobic derivative of TEMPO. This DNP medium was chosen because: (i) a proton polarization P( 1 H)&gt;50% can be achieved in less than 5 minutes at 1.2 K and 6.7 T (see  FIG. 2A ); (ii) none of the metabolites that we have tested (urea, pyruvate, acetate, fumarate, glycine, glucose) are soluble in this mixture; and (iii) the mixture is not miscible with water, and can be extracted on-line after dissolution using a suitable resin filter such as Amberlite® XAD4. Of course, many other combinations of glass-forming solvents and PA&#39;s are possible, and may turn out to be effective. 
         [0087]    Among other candidates for DNP matrices, the use of butane is possible. The microcrystals of metabolites can be immersed at −50 C in liquid butane doped with TEMPOL-benzoate. During the dissolution process the butane is sublimed and flushed out with helium gas. The PAs precipitate as a powder and can be retained by an in-line 0.22 μm syringe filter (Merck Millipore) 
         [0088]    DNP Build-Up Curves. 
         [0089]      FIG. 3A  shows the  1 H DNP build-up at 1.2 K and 6.7 T for 60 μL of a pure glass-forming DNP matrix consisting of Toluene-d6:THF doped with 50 mM of TEMPOL-benzoate, without any microcrystals of the molecules of interest, i.e., without any metabolites. The polarization reaches P( 1 H)&gt;50% after a mono-exponential build-up with a characteristic time constant τ DNP ( 1 H)=50 s.  FIG. 3B  shows the  1 H DNP build-up curve under the same conditions by impregnating 15 mg of the micro-crystalline [1- 13 C]urea described above with the 60 μL of the Toluene-d6:THF:TEMPOL-benzoate P-matrix. The  1 H DNP build-up curve is now bi-exponential with a fast component τ DNP   fast ( 1 H)=56 s arising from the direct polarization of the glassy DNP matrix as in  FIG. 3A , and a slow component τ DNP   slow ( 1 H)=600 s corresponding to the polarization of the microcrystals which builds up through proton spin diffusion from the glassy DNP matrix. This clearly shows that the polarization enhanced by DNP propagates, albeit slowly, from the glassy DNP matrix to the microcrystals. Polarization can be transferred from the protons to the  13 C spins by cross-polarization at low temperature, e.g. using the methods disclosed by Jannin et al (Chem. Phys. Lett 517 (2011) 234-236). 
         [0090]      FIG. 4  shows the  13 C build-up of the magnetization of [1- 13 C]urea obtained by multiple-contact  1 H→ 13 C cross-polarization at 1.2 K, as compared with a similar  1 H→ 13 C cross-polarization build-up measured on the same amount of [1- 13 C]urea, but dissolved in the conventional glassy matrix H 2 O:D 2 O:glycerol-d 8  (2:3:5) doped with 40 mM TEMPOL, known as “DNP juice”. Although these two  1 H→ 13 C cross-polarization build-up curves might look similar at first glance, it is worth stressing two major differences, (i) the time between two consecutive cross-polarization contacts should be extended (from Δt CP =5 minutes to 20 minutes) to ensure that  1 H spin diffusion spreads polarization uniformly inside the crystals between cross-polarization steps, and (ii)  13 C relaxation between cross-polarization contacts, which usually limits the  13 C build-up, is strongly attenuated in the micro-crystals because of the absence of PAs. These two new features imply that  13 C polarization is slower to build up but can achieve higher final polarization levels, theoretically up to P( 13 C)═P( 1 H). 
         [0091]    Extended Relaxation Times. 
         [0092]    Although our micro-crystalline suspension in the glassy DNP matrix is different from the usual glassy solutions,  13 C CP-DNP can be performed essentially in the same way. One primary novelty associated with this new sample formulation resides in the dramatically different  13 C spin-lattice relaxation properties. The  13 C spins of metabolites dispersed in glassy matrices are inevitably in dipolar contact with the PAs that lead to paramagnetic relaxation. In the microcrystalline formulation on the other hand, the  13 C spins of the metabolites are physically separated from the PAs on a length scale much larger than that of the electron-nuclear dipolar interaction. 
         [0093]      FIG. 5  shows how the  13 C nuclear spin lattice relaxation at 4.2 K and 6.7 T in [1- 13 C]urea is dramatically extended by switching from the glassy solvent where T 1 ( 13 C)=20 minutes to the microcrystalline approach where it becomes 37 hours. 
         [0094]    Transport. 
         [0095]    This extension of the nuclear spin lattice relaxation time T 1 ( 13 C) is remarkable. The comparison is likely to be even more favorable in solutions at low fields where solid-state paramagnetic relaxation is exacerbated. The extension of T 1 ( 13 C) enables transport to remote locations using a simple transport device that maintains a low magnetic field to preserve the polarization. 
         [0096]      FIG. 6  shows how the  13 C polarization can even be transported to a low magnetic field (B 0 &lt;10 mT) at room temperature for brief (t&lt;10 s) periods of time, whereas the polarization vanishes entirely in glassy samples that contain PAs. Obviously, this feature enables facile manual handling and transportation of a polarized sample from the polarizer (in our case at 6.7 T) to a transport device. 
         [0097]      FIG. 7  shows a drawing of a transport device equipped with a sample insert with permanent magnets that give a field of 0.8 T, and its accompanying dissolution insert. 
         [0098]    Dissolution. 
         [0099]    The thermodynamics of dissolution is always advantageous since (i) the heat capacity C P  and latent heat of fusion L V  of organic solvents are significantly smaller than those of water, and (ii) the enthalpy of solution ΔH soln  of most organic crystals is negative, so that dissolution is exothermic. Dissolution can therefore be performed by conventional means, without modification of existing devices, and possibly with a smaller dilution factor. The resulting hyperpolarized solutions were pushed through a magnetic tunnel and injected into a 300 MHz liquid-state NMR spectrometer. The dissolution was performed either directly in the DNP polarizer or in the transport device with permanent magnets that give a field of 0.8 T in a helium Dewar. 
         [0100]      FIG. 8  shows the hyperpolarization decay measured in [1- 13 C]urea, featuring a polarization P( 13 C)=4%, corresponding to an enhancement factor ε DNP =8000 with respect to thermal equilibrium. Interestingly, the  13 C spin-lattice relaxation time T 1 ( 13 C)=60 s is typical for a radical-free solution. This is evidence that the TEMPO-benzoate remains mostly in the organic phase, while the urea dissolves as expected in the aqueous phase. 
         [0101]    Conclusions: We have demonstrated a novel approach to Dissolution DNP (D-DNP) dubbed Remote-DNP (R-DNP) that can produce transportable hyperpolarized micro-crystalline samples of pure metabolites free of paramagnetic polarizing agents. The lifetime of the hyperpolarization in the micro-crystalline samples studied here lies in the range between 30 minutes and 30 hours, thus enabling transportation of hyperpolarized micro-crystalline samples to remote locations in suitable transport devices, here an assembly of permanent magnets immersed in liquid helium. 
         [0102]    The R-DNP method comprises: 
         [0103]    (i) the embedding of hydrophilic microcrystals of pure metabolites in hydrophobic glass-forming DNP solutions that do not dissolve the micro-crystals, 
         [0104]    (ii) freezing to ca. 1.2 K, 
         [0105]    (iii) proton DNP followed by spin diffusion of proton polarization from the frozen glassy DNP solutions to the microcrystals of metabolites 
         [0106]    (iv)  1 H→ 13 C cross-polarization (CP) in the microcrystals, 
         [0107]    (v) optional transportation to a remote location in suitable transport device, and finally 
         [0108]    (vi) dissolution to the liquid state and injection in an MRI or NMR system. 
         [0109]    Methods: 
         [0110]    Sample preparation: the metabolites as well as the solvents and polarizing agents were purchased from Sigma Aldrich. The metabolites powders were ground by hand in a ceramic mortar during ca. 5 minutes. 
         [0111]    Microscope and Particle distribution analysis: Pictures of the ground metabolites were taken with a suitable microscope, and the particle size distribution analysis was performed with the freeware ImageJ. 
         [0112]    Low-temperature DNP and  1 H→ 13 C cross polarization: Samples were polarized at liquid helium temperatures (1.2 to 4.2 K) and in a magnetic field of 6.7 T in a home-built DNP polarizer. The microwave frequency used was 188.3 GHz with a power of about 80 mW. 
         [0113]    Cross-polarization sequences were applied with dedicated NMR coils coupled to a Bruker NMR spectrometer. The NMR coils were tuned and matched to 285.2 MHz for protons and 71.7 MHz for carbon-13. The CP pulse sequence consisted in two 180° WURST pulses swept through 100 kHz in 1 milliseconds, and with a B 1  strength of about 20 kHz, simultaneously applied to the proton and carbon-13 channels. These CP sequences were repeated several times, at intervals typically varying from 5 to 20 minutes. 
         [0114]    Dissolution experiment: the polarized samples were dissolved with 5 mL of preheated deuterated water (at about 450 K at a pressure of 1 MPa). The water was then sprayed onto the sample and the resulting solution was pushed by helium gas at a pressure 0.6 MPa through a 1.5 mm tube through a magnetic tunnel towards the NMR spectrometer, and injected into the NMR tube. 
         [0115]    Liquid-state NMR: The  13 C signals of the hyperpolarized sample were measured by applying 5° nutation angle pulses every 5 seconds. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 LIST OF REFERENCE SIGNS AND ABBREVIATIONS 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 NMR 
                 Nuclear magnetic resonance 
               
               
                   
                 MRI 
                 Magnetic resonance imaging 
               
               
                   
                 DNP 
                 Dynamic Nuclear Polarization 
               
               
                   
                 D-DNP 
                 Dissolution DNP 
               
               
                   
                 R-DNP 
                 Remote DNP 
               
               
                   
                 CP 
                 Cross Polarization 
               
               
                   
                 CPA 
                 Component with polarizing agent 
               
               
                   
                 CMI 
                 Component with Molecule of 
               
               
                   
                   
                 Interest 
               
               
                   
                 PA 
                 polarizing agent (e.g. stable 
               
               
                   
                   
                 mono- or biradical) 
               
               
                   
                 Micro- 
                 (“microcrystals”) that 
               
               
                   
                 crystalline 
                 contains the MOI with nuclei 
               
               
                   
                 matrix 
                 to be polarized (e.g. carbon- 
               
               
                   
                   
                 13, enriched or in natural 
               
               
                   
                   
                 abundance) and abundant 
               
               
                   
                   
                 spins (e.g., protons that allow 
               
               
                   
                   
                 spin diffusion to occur.) 
               
               
                   
                 DNP 
                 (“DNP juice”) glassy matrix 
               
               
                   
                 matrix 
                 that contains the PAs and 
               
               
                   
                   
                 abundant spins such as 
               
               
                   
                   
                 protons for spin diffusion) 
               
               
                   
                 MOI 
                 Molecule of Interest 
               
               
                   
                   
                 (metabolite, etc) 
               
               
                   
                 HYPSO 
                 Hybrid Polarizing Solid 
               
               
                   
                 FDA 
                 Federal Drug Administration 
               
               
                   
                 TEMPOL 
                 4-hydroxy-2,2,6,6- 
               
               
                   
                   
                 tetramethylpiperidin-1-oxyl 
               
               
                   
                 TEMPO 
                 (2,2,6,6- 
               
               
                   
                   
                 Tetramethylpiperidin-1-yl)oxyl