The formulation of a new compound as a hyperpolarized imaging agent with the DNP method imposes several challenges. In order for the DNP process to be effective, the electron paramagnetic agent must be homogeneously distributed within the sample. Many compounds will be crystalline and have a tendency to crystallize as saturated aqueous solutions. This typically leads to poor polarization. To prevent crystallization and to produce an amorphous solid upon cooling of the sample, glass formers such as glycerol or DMSO can be added to the mixture of the compound and the electron paramagnetic agent. Since it is often a requirement for in vivo studies to achieve a high concentration of the compound after dissolution, it is necessary to be able to formulate the compound in a concentrated form. A solvent mixture with high solubility for the compound and good glassing properties is therefore chosen. Secondly, the solvent mixture has to be either biologically compatible or removed from the solution after polarization.

The source of the unpaired electron is typically an organic radical, but a few metal ions have been employed successfully for DNP, Cr(V) in particular (14–16). The choice of electron paramagnetic agent will depend on a number of factors. First, the electron paramagnetic agent needs to be chemically stable and dissolve readily in the matrix of interest. Second, the electron paramagnetic resonance (EPR) spectrum of the radical should have a width that allows DNP to be effective for the nucleus of interest, i.e., a linewidth that exceeds the Larmor frequency of the nuclear spin. In practice, the above criteria means that two classes of electron paramagnetic agents are available, namely, nitroxides (17, 18) and trityls (19–21). The nitroxides belong to a class of molecules that have been studied extensively by EPR, and which have been used for DNP in many systems. Nitroxides are characterized by having a broad EPR spectrum (16), covering the Larmor frequency of all nuclear spins. Some of them have reasonable chemical stability and come with different degrees of hydrophilicity. Another class of electron paramagnetic agents with superior properties for direct polarization of low-γ nuclei such as ¹³C, ¹⁵N, and ²H is the trityls (16, 22, 23). The trityls also exist with a range of hydrophilicities and some of them are chemically very stable. It was recently discovered that gadolinium can positively affect the solid-state DNP enhancement (24). Other paramagnetic ions and molecules (Mn²⁺ and O₂) can in part have the same effect. The physics is not understood, but Hu et. al. (23) have shown that the longitudinal relaxation time of the electron paramagnetic agent is shortened by the presence of the Gd ions. The effect of adding 1–2 mmol/L Gd³⁺ is a 50–100% improvement of the DNP. The effect seems to be general to most samples, but has to be optimized for each sample similarly to the concentration of the electron paramagnetic agent. There is no direct DNP effect of the Gd³⁺ by itself. Finally, Gd³⁺ may enhance the solid-state polarization by DNP, but care should be taken in eliminating relaxation in the liquid state. Free Gd-ions would cause detrimental liquid-state relaxation and pose an in vivo safety risk.

The method and instrumentation for hyperpolarization by DNP was first described by Ardenkjaer-Larsen et al. (6). The polarizer, which is depicted in Fig. 1, is built to a large extent on principles established in the polarized target community. Most solid-state DNP has been performed at magnetic fields between 0.35 T (25) and 16.5 T (26, 27), and at temperatures from below 1 K to room temperature. At temperatures below a few kelvin and magnetic field strengths above a few tesla, electron spins are almost fully polarized, and large nuclear polarizations can be obtained. Unlike in NMR spectroscopy applications where non-equilibrium polarizations can be regenerated by repeating the microwave irradiation and NMR acquisition (26), the polarization generated for in vivo applications will decay irreversibly after dissolution. Hence, the goal is to generate polarizations close to unity. It is therefore important to choose initial operating conditions that have been proven to provide high nuclear polarization, but, at the same time, that are easily achievable using standard instrumentation. Temperatures of ∼1 K can be achieved by pumping in liquid helium. In the original work, the liquid helium was supplied to the sample space through a needle valve from the magnet cryostat, but in a recent publication, an alternative arrangement that used a separate helium dewar was described (28). A magnetic field strength of 3.35 T was chosen since microwave sources are readily available at 94 GHz for irradiation of the electron spin. However, recently it has been demonstrated that for both nitroxides and trityls a significant improvement in polarization can be obtained by increasing the magnetic field strength (29, 30) or lowering the temperature (31). For the compound [1-¹³C]pyruvic acid the ¹³C polarization improved from 27% at 3.35 T to 60% at 4.64 T in the solid state. Typically, the literature has been limited and disagreeing on the magnetic field dependence of DNP (32). On the other hand, it has been well established that lowering the temperature is beneficial for DNP. Another recent development in DNP instrumentation for in vivo applications is the closed-cycle sorption pump cryostat (31). This system achieves a base temperature of less than 0.8 K for a pumped ⁴He bath.

Once adequate solid-state polarization has been obtained, the sample needs to be dissolved in a suitable buffer. The dissolution may involve neutralization of the agent with acid or base, depending on the solid sample preparation. Buffering of the solution may be required to maintain control of pH and achieve a physiologically acceptable formulation. Physiological buffers such as tris(hydroxymethyl)aminomethane(TRIS) or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonate (HEPES) are commonly used. The dissolution has to be efficient and fast compared to nuclear T ₁ in order to preserve the nuclear polarization in this process. Formulating the solid sample as beads or powder may improve the dissolution (in terms of polarization and recovery of the solid sample), but understanding and optimizing the fluid dynamics (33) as well as providing the necessary heat is essential for optimal performance of more difficult agents. Relaxation during the dissolution process can depend on several factors. To minimize relaxation, dissolution is performed in the high field of the polarizer (e.g., 3 T in the case of a 3.35 T polarizer), but above the liquid helium surface. Any paramagnetic ions that could increase the relaxation rate are chelated by adding, for example, EDTA to the dissolution medium. To illustrate the severity of relaxation during dissolution [1-¹³C]pyruvic acid is chosen as an example. This molecule has been well studied with DNP and has high biological relevance. We assume a best case, i.e., relaxation by paramagnetic impurities is eliminated by having pure samples or chelating any remaining impurities. Chemical shift anisotropy is negligible due to the moderate field of the polarizer. Dipolar relaxation to the protons is unavoidable unless the sample is partially deuterated. In Fig. 2 the T ₁ of the C-1 of neat [1-¹³C]pyruvic acid at 9.4 T is given as a function of temperature (unpublished data). It can be seen that the minimum T ₁ is ∼1.6 s. With the trityl radical present (20 mmol/L), there will be an additional (dipolar) relaxation contribution. It can be seen that the contribution from the trityl is marginal and shifts the minimum to a different temperature (correlation time). The values in Fig. 2 agree well with theory based on the known distances between the spins. According to dipolar relaxation theory the minimum T ₁ scales approximately as B ₀, which means that a minimum T ₁ of 0.7 s should be expected during the dissolution (in the 3 T polarizer field). The data illustrates that the nuclear spin during the dissolution should pass through this T ₁ minimum on a much faster timescale to avoid a loss of polarization. The example illustrates that it is not unreasonable to expect that the loss of relaxation during dissolution can be overcome. The severity of the problem will depend on the target spin and sample properties, but several parameters can be controlled, e.g., the distance to other spins (labeling position), the abundance of other spins (full or partial deuteration), and the concentration of the electron paramagnetic agent (sacrificing DNP for longer T ₁).

Finally, the solution may undergo a filtration or chromatography step to remove the electron paramagnetic agent involved in the DNP process. In case a Gd chelate has been added, this agent may be removed as well. In most cases, the electron paramagnetic agent or Gd chelate do not cause significant relaxation after dissolution and may also be safe to inject into animals, but can often simply be removed by ion exchange or reverse phase chromatography. The filtration can either be in-line with the dissolution process or a subsequent step. In either case, the filtration is completed in a matter of a few seconds with insignificant loss of polarization or target molecule (unpublished work). However, significant research into optimal filtration materials will be required for new combinations of agents.

Many nuclei have been hyperpolarized by the DNP method: ¹H (34), ⁶Li (35), ¹⁹F (36), ¹³C, ¹⁵N (37), ³¹P (38), ⁸⁹Y (39), and ¹²⁹Xe (40). Nuclei with spin greater than ½ other than ⁶Li will have too short relaxation times and are unattractive for hyperpolarization studies in vivo. High gyromagnetic ratio nuclei such as ¹H, ¹⁹F, and ³¹P will also typically have relatively short T ₁. The obvious nucleus of choice is ¹³C, since many cellular metabolites can be labeled at positions in which the nucleus has a long relaxation time. The signal (polarization) that is created in the polarization process decays to thermal equilibrium with a rate (relaxation time) that depends on a number of factors. Some factors can be controlled in the dissolution process e.g., temperature, magnetic field, pH, and paramagnetic impurities. Others, such as intramolecular dipolar, chemical shift and spin rotation contributions depend on the ¹³C position in a specific molecule and cannot be controlled as easily (with the possible exception of deuteration in some cases to avoid ¹³C–¹H dipolar relaxation).

The range of compounds that have been polarized by DNP is extensive and demonstrates the generality of the method: [¹³C]urea (6), [1-¹³C]pyruvate (41), [2-¹³C]pyruvate (42), [1-¹³C]lactate (43), [¹³C]bicarbonate (44), [1,4-¹³C₂]fumarate (45), [1-¹³C]acetate (46), [5-¹³C]glutamine (47), [1-¹³C]glutamate (48), amino acids in general (49), [1,1-¹³C]acetic anhydride (50), and [2-¹³C]fructose (51). The choice of compound is motivated not only by formulation and relaxation considerations but also by the imaging application; examples of which will be reviewed in Chapter 33. The list above is not exhaustive, but illustrates the versatility of the DNP dissolution method. The listed compounds have been imaged in vivo and are examples of potentially useful biomarkers for the hyperpolarized MR.

The hyperpolarization of small organic molecules by DNP and dissolution for use as MR agents have been successfully demonstrated for a range of compounds of biological interest. Whereas pre-clinical research applications are now pursued at a number of academic institutions, the development of the technology to clinical routine application is a challenge. Partly, this is due to the need for a relatively complex instrumentation in a hospital environment, but also due to the fact that pharmaceutical requirements of sterility and quality control need to be assured.

The nuclear spin states of the hydrogen molecule exist in two varieties; a triplet state of total spin S = 1 called ortho-hydrogen, and a singlet state of total spin S = 0 called para-hydrogen. The spin states of ortho- and para-hydrogen are symmetric and anti-symmetric respectively, and are given by
where + and – refer to the sign of the two protons spin values along a given axis, i.e., “spin up” and “spin down.”

According to the Pauli principle, the total wave function of two identical fermions must be anti-symmetric. The implication of this for the hydrogen molecule is that symmetric rotational quantum states with even rotational quantum numbers are only allowed for the nuclear singlet state and that the anti-symmetric rotational quantum states with odd quantum numbers are only allowed for the triplet states. In the high-temperature limit all four nuclear states are equally populated resulting in ¼ of the states being para-hydrogen, whereas in the low temperature limit only the ground state, the singlet state with J = 0, is populated and we have 100% para-hydrogen. For intermediate temperatures the fraction of para-hydrogen at thermal equilibrium is given by

where is the energy of the rotational state with quantum number J, k _B is the Boltzmann constant, and . At liquid hydrogen temperatures (≈14–20 K), we have essentially pure para-hydrogen, and at room temperature, we are in the high-temperature regime (Fig. 3). The conversion between the ortho and para states is however very slow, so for practical purposes an efficient catalyst must be used.

The spin density operators for ortho- and para-hydrogen are respectively equal to

and , where I ₁ and I ₂ are the spin operators of the two protons. For a mixture of ortho- and para-hydrogen, with a molar fraction x _p of para-hydrogen, the total spin density operator is , with .

The basic principle behind the PHIP phenomenon is the transfer of order from the para-hydrogen proton spin state to nuclear spins of other molecules. This requires a coupling between the para-hydrogen protons and the other nuclear spins. The most common method to obtain this coupling is to hydrogenate the molecule of interest with para-hydrogen, but also the possibility of using a transient coupling, without hydrogenation, has been demonstrated (61). The hydrogenation requires a catalyst that preserves the spin correlation between the protons, i.e., the hydrogen molecules are transferred to the substrate without scrambling of protons between different para-hydrogen molecules. It has recently been demonstrated that not only homogeneous catalysts can be used but also heterogeneous ones (62, 63).