& Medicinal Chemistry

NMR Hyperpolarization Techniques for Biomedicine

Panayiotis Nikolaou,[a] Boyd M. Goodson,[b] and Eduard Y. Chekmenev*[a]

Chem. Eur. J. 2015, 21, 3156 – 3166 � 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim3156

ConceptDOI: 10.1002/chem.201405253

Abstract: Recent developments in NMR hyperpolarizationhave enabled a wide array of new in vivo molecularimaging modalities, ranging from functional imagingof the lungs to metabolic imaging of cancer. This Conceptarticle explores selected advances in methods for thepreparation and use of hyperpolarized contrast agents,many of which are already at or near the phase of theirclinical validation in patients.

Introduction

The first known attempts to split spectroscopic lines ofparticles using an applied magnetic field date back to MichaelFaraday in 1862.[1] However, the effect was not realized untilPieter Zeeman’s discovery in 1897 of field-induced broadeningof sodium spectra lines.[1] The Zeeman effect of energy-levelsplitting of nuclear spin states in a static magnetic field is fun-damental for nuclear magnetic resonance (NMR). For spin 1=2

nuclei, there are two such energy levels separated by DE =

�g�hB0, where g is the gyromagnetic ratio for the nuclear spinof a given isotope, �h = h/2p, h is Planck’s constant, and B0 isthe applied static magnetic field strength. Unlike in optical andother spectroscopic methods, wherein the entire ensemble (orlarge fractions of the ensemble) give rise to signal formation,conventional NMR represents a special case: here, the energylevel spacing is much lower than the ambient thermal energy(kT), and thus the different energy levels are nearly equallypopulated. Unfortunately, it is this minute difference amongthe populations of nuclear spin Zeeman energy levels—usuallyreferred to as nuclear spin polarization (P) or the degree of nu-clear spin alignment with the applied magnetic field—that infact contributes to the detectable NMR signal. As governed bythe Boltzmann distribution, it can be shown that P�DE/2kT orP�g�hB0/2kT (using the above equation) under thermal equilib-rium conditions. Biomedical or in vivo applications of NMRimply temperatures of greater than 300 K, and even when highmagnetic fields (up to tens of Tesla) are applied, P remains rela-tively low (10�6–10�4) for biomedically relevant nuclei such as1H, 13C, 15N, 129Xe, and others. This low value of P directly re-flects the fraction of the nuclear spin ensemble contributing tothe NMR signal (the rest are quite literally “radio-silent”), ex-plaining why NMR and MRI are frequently called low-sensitivitytechniques in comparison to other spectroscopic and imagingmodalities.

However, in some cases P can be artificially, albeit transiently,increased well above its low thermal equilibrium level. Thispossibility was demonstrated at the dawn of NMR by Carverand Slichter in 1953, when they increased P of 7Li nucleithrough polarization transfer from free electrons.[2] This signifi-cant (usually orders-of-magnitude) increase in nuclear spin po-larization above the thermal-equilibrium level was later calledhyperpolarization (HP). The increase in polarization may bequantified by the polarization enhancement factor e, defined asthe ratio of the nuclear spin polarization in HP state and thatobtained at thermal equilibrium. Because the NMR signal is di-rectly proportional to the nuclear spin polarization, the realizedpolarization enhancement manifests in the corresponding NMRsignal and corresponding gains in detection sensitivity, whichcan be approximately 4–5 orders of magnitude at high fieldand even greater at lower fields.[3] Despite these significantgains in sensitivity achieved through hyperpolarization, theenergy of RF (radiofrequency) quanta remains low, and theoverall sensitivity remains relatively low as compared, forexample, to optical methods.

In the more than 60 years since the first hyperpolarizationdemonstration, a number of hyperpolarization techniqueshave been developed and applied to pure elements, chemicalcompounds and complex mixtures with potential or realizedbiomedical relevance.[4] These HP compounds can be adminis-tered to patients via intravenous (IV) injection or inhalation totrace metabolism, and function in living organisms, as well asfor other biomedical applications. Because the produced HPsubstances (also referred to as HP contrast agents) generallycannot be rehyperpolarized after their administration, thehard-won HP state will exponentially decay back to equilibri-um. This decay ostensibly poses a fundamental limit on thetime scale of biochemical processes that can be probed by HPcontrast agents, and requires HP lifetimes that are sufficientlylong (� tens of seconds) for agent manipulation after itsproduction, administration (e.g. , inhalation or injection), in vivodelivery, and observation of the metabolic/functional event.The unrecoverable nature of the HP state additionally requireshighly specialized MR pulse sequences that are tailored to ex-tract as much information as possible from the HP contrastagents. However, such efficient sequences, combined with thebright HP-endowed signal, can enable high-quality clinical 3Dimages of HP contrast agents to be obtained in as little asa few seconds.[5] Moreover, because the HP nuclear state is nolonger endowed by the static magnetic field of the detectingMR magnet, high-field MRI scanners are no longer mandatory,and lower-cost, less-confining, low-field 3D MRI can be usedinstead,[6] with detection sensitivity potentially approaching oreven surpassing that of high-field MRI.[7]

While numerous hyperpolarization methods and strategieshave been developed, this Concept article describes fourmethods of hyperpolarization: dissolution dynamic nuclearpolarization (d-DNP),[8] spin exchange optical pumping(SEOP),[9] parahydrogen induced polarization (PHIP),[10] andsignal amplification by reversible exchange (SABRE).[11] TheseHP methods arguably have the greatest relevance to biomed-icine, given that they have each either already been tested in

[a] Dr. P. Nikolaou, Prof. E. Y. ChekmenevInstitute of Imaging Science (VUIIS)Department of Radiology, Department of Biomedical EngineeringDepartment of Physics and Astronomy and Department of BiochemistryVanderbilt-Ingram Cancer Center (VICC), Vanderbilt University1161 21st Ave South AA-1107, Nashville, Tennessee, 37232-2310 (USA)E-mail : eduard.chekmenev@vanderbilt.edu

[b] Prof. B. M. GoodsonDepartment of Chemistry and Biochemistry, Southern Illinois UniversityCarbondale, Illinois, 62901 (USA)

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patients or animals models of human diseases, or have greatpotential and will likely be tested in vivo soon. The fundamen-tal MR detection concepts of HP contrast are also discussedfrom the perspective of biomedical applications. Finally, exist-ing and potential biomedical applications of already availableand emerging HP contrast agents are discussed. Whilevalidated and emerging concepts are discussed here, thereader should additionally benefit from more comprehensivereviews on selected topics.[4, 9, 12]

Long-Lived Spin States

Values for the in vivo spin-lattice relaxation time constant (T1)for protons of water (~1.5 s)[13] and other biologically relevantand abundant molecules are usually too short to be useful forHP biomedical applications. However, T1 values of low-gspin 1=2 nuclei (e.g. , 13C, 15N, 129Xe, 29Si, etc.)[14] can significantlyexceed 1 min. From a molecular perspective, these long timestypically represent spin sites isolated from protons and para-magnetic O2 to reduce the relaxation contribution via dipolarmechanisms. Examples include 13C-carboxyl sites,[15] noblegases like 129Xe[5b] and 3He, 15N2O,[16] 29Si in silicon nanoparti-cles,[14] and quaternary and tertiary 15N-amines.[17] 13C-carboxylsites are of particular interest because of their wide distribu-tion in major metabolic pathways such as anaerobic glycolysis,the tricarboxylic acid cycle (TCA) cycle, glutaminolysis, andmany others.[4] Notably, the natural abundances of many bio-logically relevant isotopes such as 13C and 15N (1.1 and 0.36 %,respectively) are low, representing both an advantage (lowbackground signal) and a challenge (a requirement for isotopicenrichment to boost the detection sensitivity). An alternativeapproach for increasing the effective lifetime of HP agents in-troduced relatively recently involves the use of long-lived sin-glet states. In this approach, the high nuclear spin order of theHP state, which otherwise would be subject to the usual relax-ation mechanisms quantified by T1, is instead “encoded” andstored within a coupled spin pair’s singlet state of the form:jS0i/ (jabi- jbai) ; parahydrogen, discussed in greater detailbelow, contains perhaps the simplest example of a nuclearspin system in a singlet state. The decay of spin order“trapped” within a singlet state is governed by the singlet–trip-let interconversion time constant (TS), which in some circum-stances can surpass T1 values by orders of magnitude.[16, 18]

Regardless of the approaches chosen for hyperpolarizationpreparation and storage, their applicability is often limited toa narrow range of useful HP targets, particularly when one hasa given potential application in mind. This fact highlights thechallenges and opportunities for innovative concepts of thisrapidly emerging field of molecular imaging, where funda-mental chemistry, NMR spectroscopy, and spin physics havedirect and practical implications for biomedicine.

Dissolution Dynamic Nuclear Polarization(d-DNP)

d-DNP[8] relies on unpaired electrons as the source of largespin polarization in a manner similar to pioneering experi-

ments by Carver and Slichter,[2] because g(e)�660g(1H).Indeed, when electrons are subjected to sufficiently low tem-peratures in a static magnetic field of several Tesla, order-unityelectronic polarization can be attained.

To enable the d-DNP process, a compound containinga stable free radical (e.g. , the trityl radical (tris{8-carboxyl-2,2,6,6-tetra[2-(1-hydroxyethyl)]-benzo(1,2-d:4,5-d’)bis(1,3)di-thiole-4-yl}methyl sodium salt)[8] is first mixed with abiomolecule of interest (e.g. , 1-13C-pyruvic acid) in a solutionto form a glass matrix at low temperature inside the high-fieldmagnet (Figure 1 a).

This glass matrix is then irradiated with a high-power micro-wave source at the electron resonance frequency to transferelectron spin polarization to 13C nuclear spins in the solid state(Figure 2 b). The exponential build-up of 13C hyperpolarizationwas rather slow (~4900 s for 1-13C-pyruvic acid) in the originalDNP hyperpolarizer by Ardenkjaer-Larsen and co-workers.[8]

However, the 13C hyperpolarization build-up time can be signif-icantly minimized if the electron polarization is first transferredto the proton spin bath of the matrix, and then from 1H to 13Cspins by cross-polarization in the solid state. The latter conceptwas demonstrated for intramolecular[19] and intermolecular[20]

1H-to-13C hyperpolarization transfers, and the 13C exponentialbuild-up time was shortened to 810 s. The final steps of d-DNPis a rapid (on the time scale of a few seconds) dissolution andwarming of the cold HP sample, and the sample transfer fromthe DNP hyperpolarizer magnet to the imager or NMR magnet.The resulting HP 13C contrast agent is typically administered toa subject via IV injection within seconds after dissolution.

Many 13C- and 15N-enriched biomolecules have beenhyperpolarized with d-DNP, including 1-13C-pyruvic acid,[8, 21]

13C-bicarbonate,[22] 1-13C-fumarate,[23] 5-13C-glutamine,[24]

15N-choline,[17] and others. The use of long-lived singlet stateswas also demonstrated in a 13C–13C spin pair in diacetylhyperpolarized by d-DNP.[18b] Furthermore, recent advanced ef-forts of molecular deuteration enabled d-DNP and in vivo useof 13C-choline[25] and 13C-glucose,[26] which have significantlyshorter T1 values in their nondeuterated forms. 1-13C-pyruvicacid, which probes glycolysis in vivo,[21] is the leading 13C HP

Figure 1. The process of dynamic nuclear polarization (DNP) using high-power microwave irradiation of electron spins at low temperatures (fewKelvin) and high magnetic field (several Tesla). a) Glass matrix containing 13C-labeled metabolite and radicals with unpaired electron is formed inside thehigh-field DNP magnet at low temperature. b) High-power microwave irradi-ation at the electron resonance frequency enables 13C hyperpolarization bypolarization transfer from free electrons. c) A long-lived 13C hyperpolarizedstate is prepared.

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contrast agent, and is already being evaluated by FDA-ap-proved prostate cancer clinical trials in men,[4, 27] work that isalso fueled by the advent of a clinical-scale d-DNP hyperpolar-izer with a sterile path.[28] The DNP approach is also amenableto hyperpolarized silicon nanoparticles, where experimentshave exploited the high biocompatibility, surface “functionaliz-ability”, and ultralong 29Si T1 values (~40 min) of these sys-tems[14] to perform HP 29Si MRI in vivo.[14b] It should also bepointed out that the 29Si DNP process is conducted in the solid

state as a powder, without the need for the addition of radicals(because surface defects provide the unpaired electronsneeded for DNP) or glassing agents.[14b] Finally, we should addbriefly that potential clinical applications of DNP are not limit-ed strictly to d-DNP. For example, in ex situ Overhauser DNP(ODNP), target molecules can be flowed continuously throughbeds containing immobilized radical species, allowing thetarget spins to be partially hyperpolarized with microwaveapplication and subsequent delivery of the pure agent tothe subject. In one recent example, the potential utility ofODNP-polarized water for performing perfusion MRI wasdemonstrated in a rat model.[29]

Spin-Exchange Optical Pumping (SEOP)

Spin-exchange optical pumping can be used to generate largequantities of hyperpolarized noble gases (3He, 129Xe, 83Kr, etc.)with high nuclear spin polarization for biomedical applica-tions,[12b, c, f, 30] approaching unity for 129Xe[5b, 31] and 3He.[32] Crea-tion of hyperpolarized noble gases by SEOP generally requiresa high-power circularly polarized laser and an optical cell (Fig-ure 2 a) containing the target noble gas of interest, buffer gas(typically 4He and/or N2), and a small quantity of alkali metalthat is partially vaporized by heating. While one could arguethat the origins of the first step of SEOP—optical pumping ofthe alkali metal atoms (Figure 2 b)—began with Zeeman’s stud-ies of sodium vapor,[1] it was Kastler who found that the vaporof another alkali metal, rubidium, could become electronicallyspin-polarized when placed in a magnetic field and illuminatedwith resonant circularly polarized light.[33] Bouchiat, Carver, andothers later showed that the electronic spin polarization of thealkali metal vapor could be transferred to nuclear spins ofnoble gas atoms like 3He and 129Xe during gas-phasecollisions.[34] This second step of SEOP, termed spin exchange(Figure 2 c), allows the nuclear spin polarization of the noblegas to ultimately approach that of the alkali metal electrons,provided that the spin-exchange rate greatly exceeds thenoble gas spin-destruction rate (effectively, 1/T1). Variousstand-alone apparatus designs for SEOP have been developedover the years, particularly for 129Xe;[3, 5b, 35] such so-called “Xehyperpolarizers” are capable of generating clinically relevantquantities of HP 129Xe with polarizations now approachingunity.

Of the NMR-active noble gas isotopes, 3He has the greatestg value (and hence highest per atom detection sensitivity), anduntil recently it was the easiest to achieve the highest levels ofpolarization with this isotope. However, its natural abundanceis extremely low, and instead is typically obtained from tritiumdecay; thus its future in biomedicine may be limited by theworldwide shortage of this effectively non-renewable re-source.[36] The quadrupolar isotopes (I> 1=2, 21Ne, 83Kr, and 131Xe)can also be hyperpolarized (albeit not to the same extent) butgenerally suffer from inherently faster T1 relaxation; of these,83Kr arguably shows the greatest promise for biomedical appli-cations, where its quadrupolar interaction may embody a com-plementary, surface-sensitive source of contrast compared toits spin 1=2 brethren.[30, 35j] Nevertheless, 129Xe, being spin 1=2,

Figure 2. Spin-exchange optical pumping.[9] a) SEOP requires an optical cellcontaining a noble gas, buffer gases (e.g. , N2, as shown), and a small quanti-ty of vaporized alkali metal (typically Rb or Cs),[47] irradiated by laser lightresonant with an absorption line of the alkali metal (e.g. , 794.8 nm for Rb D1

transition). Collisions with N2 nonradiatively quench excited Rb states, effec-tively suppressing deleterious Rb fluorescence that can depolarize other Rbspins.[35a, 48] The first step of SEOP involves the absorption of photons of thesame circular polarization, which conserves angular momentum by selective-ly depleting population from one of two Rb ground electronic (mJ = �1/2)states (neglecting Rb nuclear spin for simplicity). Collisions with other gas-phase species tend to equalize the excited-state populations and theground states are repopulated at effectively equal rates. However, since onlyone ground state is depleted by the laser, ground-state population accumu-lates on the other mJ state, leaving the Rb electronically spin-polarized;a weak magnetic field along the direction of laser propagation (not shown)helps to maintain the electron spin polarization. Gas-phase collisions alsoallow spin exchange, c) between the polarized Rb electron spins and thenoble gas nuclear spins, a process mediated by Fermi-contact hyperfineinteractions. Constant laser illumination of the Rb vapor therefore allows thenuclear spin polarization to accumulate over time, thereby generating thehyperpolarized noble gas.

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relatively inexpensive, naturally abundant, and amenable tonear-unity hyperpolarization, while also possessing interestingproperties (see below), will be the primary focus of the presentdiscussion.

Xenon, while almost chemically inert, has a predilection fortransiently associating with a variety of substances andsurfaces, including host–guest forming “cage” compounds,nanoporous materials, membranes, and various proteins.[12b, 37]

Xe also exhibits a reasonable amount of solubility in blood andother living tissues.[38] Indeed, Xe’s general biomedical applica-tions are well-documented: it is a functional anesthetic;[39] re-cently, it has also been implicated as a possible performance-enhancing drug as well as a potential treatment for PTSD pa-tients.[40] Importantly here, 129Xe also possesses an extraordinar-ily sensitive chemical shift, causing its resonance frequency tovary over 200 ppm simply from being physically associatedwith different chemical environments, enabling HP 129Xe to bea sensitive “spin-spy” of its local surroundings. HP 129Xe canalso be a source of hyperpolarization for other systems usingvarious polarization-transfer techniques,[41] although such ap-proaches to date have not commonly been exploited for bio-medical applications. Instead, biomedical 129Xe MRS/MRI typi-cally exploits xenon location—as originally demonstrated inthe first biomedical application of HP Xe, void space imagingin excised mouse lungs[42]—density, motion (e.g. , apparentlocal diffusion or exchange), chemical shift, and/or spin relaxa-tion. One intriguing approach exploits Xe’s fondness for cagecompounds and its chemical shift and exchange properties inorder to directly encode specific biomolecular informationonto the 129Xe signal using so-called Xe biosensors.[43] Theseagents are comprised of a Xe-binding cage compound (typical-ly a cryptophane derivative)[44] covalently tethered to some bi-oanalyte; if the Xe biosensor binds to a targeted protein, thenthe chemical shift of the bound 129Xe changes, allowing it tobe resolved from both Xe in unbound cages and Xe freelyfloating in solution. The potential for using such agents in vivohas been strengthened by the “hyperCEST” (hyperpolarizedchemical exchange saturation transfer) approach, where thepresence of even a small amount of Xe-occupied cages boundto their biomolecular targets can be encoded onto the easier-to-detect signal from Xe in the bulk environment. As anotherexample, Branca and co-workers demonstrated the promise ofa different but clever approach to achieving molecular imagingusing HP gases:[45] with the goal of visualizing lung metastases,cancer cells are first targeted by superparamagnetic nanoparti-cles (SPIONs) surface-functionalized with cancer-binding mole-cules, and then the lung space is imaged with HP noble gases(here, 3He) to identify any regions where the HP signal hadbeen washed out by the local presence of SPIONs. Regardlessof the experiment and 129Xe detection modality, HP Xe can bedelivered to the subject by inhalation[12b, c, f, 30] or by the admin-istration of Xe-saturated biologically tolerable solutions.[46]

ParaHydrogen Induced Polarization (PHIP)

The PHIP method of hyperpolarization relies on fast chemicalreactions that enable pairwise addition of p-H2 across unsatu-

rated chemical bonds, typically C=C or C�C bonds adjacent to13C carboxyl or 15N[49] nuclei. Bowers and Weitekamp demon-strated that the nuclear spin singlet state of parahydrogenmolecules can be converted into observable magnetizationarising from the magnetically inequivalent positions at the siteof hydrogenation with p-H2

[50] (Figure 3 c). The chemical reac-tion of pairwise p-H2 addition should be performed faster thanthe nuclear spin relaxation of the nascent protons. Goldman,Spiess, Bargon and Golman later demonstrated that the nas-cent proton hyperpolarization can be transferred to carboxyl13C nuclei to yield HP 13C contrast agents in seconds usingeither RF-based or field-cycling hyperpolarization-transfer ap-proaches, respectively (Figure 3 c).[51] The speed of the PHIPmethod is one of its main advantages, while the requirementfor an unsaturated molecular PHIP precursor with appropriateasymmetry is a significant limitation for biological applications.Moreover, RF-based PHIP polarization transfer (Figure 3 c) bene-fits from deuteration of the molecular precursor and also fur-ther increases the design complexity for the molecular PHIPprecursor.[52] Nevertheless, a number of potentially usefulbiomolecules were successfully hyperpolarized using PHIP,including 1-13C-succinic acid,[53] 1-13C-phospholactate,[52, 54]

tetrafluoropropyl 1-13C-propionate,[55] 15N-propargylglycine,[49]

13C-glucose derivative,[56] and others. Despite the syntheticchallenges, new concepts of using �OH protection in �C=C�O�R motifs have significantly expanded the reach of moleculartargets for PHIP,[54, 57] because the protecting group can be re-moved after PHIP either metabolically[52, 54, 58] or synthetically.[57]

The PHIP hydrogenation procedure has often been con-ducted conventionally with homogeneous RhI-based catalysts(e.g. , Wilkinson’s catalyst),[59] which more recently have beenreplaced by water-soluble bisphosphine RhI catalysts, enablingbiomedical use in vivo.[51a, 53a, 55] However, Koptyug and co-work-ers[60] demonstrated that heterogeneous catalysts includingsupported metal nanoparticles of Rh and other metals,[12e]

which despite ostensibly relying on predominantly non-molec-ular mechanisms of hydrogenation, can in fact yield a signifi-cant fraction of the hydrogenated product by effectively “pair-wise” addition of p-H2, resulting in HP product. The “HET-PHIP”concept was demonstrated in the creation of HP propane andother gases,[12e] substances which in principle can be used di-rectly in biomedical applications like gas MRI (similar to HPnoble gases discussed above). HP propane with PH>0.01 hasalready been demonstrated to be useful for high-resolution 3DMRI in phantoms.[61] These new supported metal catalysts canalso be applied for heterogeneous PHIP at the liquid–solid in-terface,[12e] and may enable the production of pure aqueousHP contrast agents—agents that have already been previouslyprepared by homogeneous catalysis and shown to bepromising targets for biomedical applications.[52, 53b, c, 54, 55, 58] Thepotential realization of this emerging concept wouldadditionally allow recycling the catalyst, making PHIP a trulylow-cost, high-throughput, sustainable, and scalable HPtechnique.

As stated above, PHIP relies on the pure spin order of thesinglet state of p-H2 ;[50] but first, the spin order must be gener-ated. H2 molecules can exist in both para- (spin-0) and ortho-

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(spin-1) states, with an energy difference of approximately170 K,[63] resulting in a statistical 3:1 (ortho/para) spin isomerdistribution at normal “high-temperature” conditions. However,because the p-H2 singlet corresponds to the lower-energystate of these two spin isomers, “normal” H2 gas can be con-verted to preferentially favor the p-H2 spin isomer by coolingthe gas to cryogenic temperatures (Figure 3 b). The process ofpara,ortho interconversion is normally very slow (on the timescale of months if not longer)[64] but can be significantly accel-erated through the use of a catalyst in the p-H2-generatingequipment (Figure 3 a).[65] When H2 gas passes through thecold catalyst-filled chamber (Figure 3 a), it is rapidly converted

to the local equilibrium p-H2 fraction determined by the lowtemperature of the cryogenically cooled chamber (Figure 3 b).The exiting p-H2 is then warmed to room temperature (RT),and is typically stored in an aluminum tank for PHIP andSABRE applications. While the enriched p-H2 is thermodynami-cally less favorable at RT, it is effectively kinetically trapped inthe para-state, and can be stored for weeks or months in theabsence of paramagnetic impurities such as O2. It should alsobe noted that p-H2 is NMR-invisible, because its net spin iszero; perhaps ironically, it is the o-H2 spin isomer that providesthe NMR signal for H2 gas. This property is conveniently usedto quantify the percentage of p-H2 enrichment usingconventional high-resolution NMR.[64, 66]

Signal Amplification by Reversible Exchange(SABRE)

SABRE[11, 62] is a relatively new PHIP-based hyperpolarizationtechnique pioneered by Duckett and co-workers in 2009. How-ever, while SABRE relies on p-H2 exchange on a metal complex,it differs from traditional PHIP in that the p-H2 does not chemi-cally react with the substrate molecule to be hyperpolarized.Indeed, in SABRE the substrate also exchanges rapidly betweenthe bulk solution environment and the organometallic catalystcomplex, allowing spin order to be spontaneously transferredfrom exchangeable (“para-”)hydride to the substrate moleculeat low[62] and high[67] magnetic fields (Figure 3 d; believed to bemediated by scalar couplings, applied fields, and dipolar cross-relaxation). The exchangeable organometallic catalyst is thekey element of SABRE hyperpolarization method,[68] and themost effective iridium N-heterocyclic carbene complexes[69]

have been used to achieve substrate hyperpolarization with Pas high as approximately 8 %. This method, previously demon-strated on pyridine and nicotinamide, has already been effi-ciently expanded to other biologically relevant molecules suchas the tuberculosis drugs pyrazinamide and isoniazid.[70] Whilethe vast majority of SABRE studies are carried out in organicsolvents, the first efforts to achieve SABRE hyperpolarization inaqueous/organic[71] and purely aqueous media[72] are promis-ing, particularly given the fact that SABRE—barely five yearsold—is still very much in its infancy. Moreover, the first demon-stration of heterogeneous SABRE was recently reported,[73]

ultimately paving the way to applications where the SABREcatalysts, consisting of metal complexes covalently tethered tosolid supports, may be recycled, and pure solutions of HP con-trast agent may be produced. Once combined with SABRE inaqueous media, this method may in fact embody a cheap, effi-cient, and scalable approach to quickly produce large quanti-ties of pure HP contrast agents on demand. If realized, thiswould provide a clear strength of the SABRE approach.

Detection of Hyperpolarized Contrast Media

Because HP magnetization for a given “batch” of HP agent istransient and cannot be regenerated in biomedical applica-tions, it irrevocably decays to its equilibrium value, typicallyseveral orders of magnitude weaker than that embodied by

Figure 3. a, b) Conversion of “normal” H2 gas (75 % ortho- and 25 % para-iso-mers) into parahydrogen (p-H2). a) Schematic of a p-H2 generator, where theentering room temperature (“normal”) H2 gas is cryo-cooled to �77 K andcatalytically converted under local equilibrium to a mixture of spin isomersthat preferentially favors p-H2. Because the catalyst is confined to a cryogeni-cally cooled chamber of the polarizer, once the p-H2 leaves the chamber it iskinetically trapped in the para-state. b) Temperature dependence of theequilibrium p-H2 percentage. Liquid N2 temperature (77 K) allows for prepa-ration of 50 % p-H2, whereas temperatures below 20 K enable production of>97 % p-H2 fraction. c) The process of parahydrogen induced polarization(PHIP). The pairwise addition of a p-H2 molecule to a molecular precursor,which is typically accomplished across a C=C or C�C bond adjacent toa 13C nucleus using a hetero- or homogeneous catalyst. The resulting chemi-cally “unlocked” nuclear spin hyperpolarization of the nascent, magneticallyinequivalent protons can be used as-is, or transferred to the typically longer-lived (greater T1) 13C site using either an RF pulse sequence or a field-cyclingmethod. d) The process of signal amplification by reversible exchange(SABRE). A metal complex [M] enables p-H2 and a substrate to be transientlyco-located under conditions of dynamic exchange, resulting in spontaneouspolarization transfer[11, 62] from p-H2 to the substrate.

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the HP state; moreover, each acquisition RF pulse “uses up”some of the hard-won HP magnetization. On the other hand,the bright signal provided by the HP agent obviates the needfor built-in delays that may otherwise be required to allowequilibrium spin polarization to refresh after each acquisition.As a result of these facts, most conventional NMR and MRIpulse sequences cannot be readily used for HP media withoutat least some modification. Importantly, an additional source ofsignal decay for a HP contrast agent in living tissues is its me-tabolism. For example, in vivo injection of HP 1-13C-pyruvateleads to its rapid enzymatic conversion to HP 1-13C-lactate and1-13C-alanine (Figure 4 a).[21a] Such processes highlight a clearstrength of HP MRI, as both contrast agent uptake and its me-tabolism can be detected and quantified, thereby providing anadditional layer of molecular information. However, thebiochemical change of the signal source also represents achallenge for MRI pulse sequence development, because thesimultaneous presence of HP 13C signals from multiple speciescomplicates image encoding and reconstruction. This problemis resolved through the addition of the spectroscopic dimen-sion to the spatial dimensions of MRI pulse sequences throughan established method called chemical shift imaging (CSI),which enables detection of spectra from individual voxels[21a, 74]

that can later be used to reconstruct metabolic maps of HPspecies, that is, originally injected HP contrast agent (pyruvate)

and its products (lactate and alanine), as shown in Figure 4 a.Furthermore, more advanced pulse sequences with frequency-selective RF pulses can be designed to sample only metabo-lites (the products of interest) rather than the original contrastagent, which also serves to minimize polarization losses due toRF excitation.[75]

HP RF pulse sequences rarely use full (908 flip-angle) ornearly full NMR signal excitation (with an exception of state–state free precession (ssfp) sequences, where gradient refocus-ing is utilized),[66, 75c, 76] because the HP magnetization is finiteand nonrenewable; the detected magnetization is proportionalto the sine of the applied RF pulse angle, whereas the residualmagnetization is proportional to the cosine of the applied RFpulse angle (Figure 4 c). As a result, the use of low-tipping-angle excitation RF pulses (i.e. , just a few degrees) is highly ad-vantageous for HP MRI sequences, because doing so retainsmost of the available HP magnetization for subsequent acquis-itions (allowing the HP but finite magnetization to be “meteredout” as efficiently as possible). Moreover, unlike conventionalMR, where a recovery time interval in the pulse sequence isneeded to re-establish the equilibrium nuclear spin polariza-tion, HP MR pulse sequences no longer need this recovery in-terval because HP magnetization is created outside of, and notby, the imaging magnet (Figure 4 d). Thus, a HP pulse se-quence train needs only to contain the encoding and detect-

Figure 4. MR detection concepts of HP contrast agents. a) In vivo administration of HP 1-13C-pyruvate leads to its in vivo uptake as well as its subsequent me-tabolism to 1-13C-alanine (using alanine transaminase [ALT]), 1-13C-lactate (using lactate dehydrogenase [LDH]), which can be differentiated using 13C chemicalshifts, and b) detected using chemical shift imaging (CSI) to simultaneously produce multiple metabolic maps. Note the red (lactate), blue (alanine) and green(pyruvate) color coding in a) and b). c) Trigonometric dependence of observed (sine) and remaining (cosine) magnetization as a function of RF pulse excita-tion (or “tipping”) angle. d) Fundamental blocks of RF pulse sequences for conventional MR (top); hyperpolarized (HP) MR with fixed small-angle encodingpulses (middle), and HP MR with variable-angle encoding pulses (bottom).

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ing elements, thereby providing significant imaging accelera-tion, with repetition times on the order of a few ms, and entire3D scanning times on the order of seconds.[5b] However, evenwith small RF tipping angles, if the excitation RF pulse width iskept fixed, the decaying HP magnetization will a yield progres-sively reduced MR signal during the pulse sequence train dueto the previous pulsing, as well as T1 and other polarizationlosses. This shortcoming can be mitigated through the use ofpulse sequences with progressively increasing RF pulsetipping-angle to maintain (ideally) the same induced MRsignal throughout the progression of the sequence train(Figure 4 d).

Many HP imaging applications require fast scanning speed.For example, performing a 3D MRI lung scan on a singlepatient breath hold restricts the scanning duration to justa few seconds. Imaging scanning speed can be additionallysignificantly accelerated through the use of compressedsensing,[75a, 76] where only a fraction of k-space is sampled,which can lead to a corresponding several-fold reduction intotal scan time.

Biomedical Translation

To date, a number of HP contrast agents have been developedand tested in animal models of human diseases, humansubjects, or both, including: HP 1-13C-pyruvate, reporting onupregulated glycolysis in cancers ;[21b, 77] HP 129Xe and 3He,reporting on pulmonary function through images of lungventilation, diffusion maps, and gas perfusion as well as Xedistribution in vivo;[12c, f, 78] HP 1-13C-succinate[53c] and 1-13C-fu-marate,[23] reporting on TCA cycle metabolism; HP 5-13C-gluta-mine, reporting on elevated glutaminolysis in cancer; HP 29Sinanoparticles[14] and others, with more on the way. However,clinical biomedical translation of the fundamental advances inthe chemistry and physics of HP MR requires several compo-nents: 1) a clinical-scale hyperpolarizer capable of producingHP agent doses suitable (quantity, purity, sterility, etc.) for ad-ministration to patients ; 2) regulatory approvals from govern-ment agencies and local institutions; and 3) an MRI scanner ca-pable of multinuclear (1H as well as 13C, 129Xe, etc.) detectingcapabilities, including specialized RF coils, custom RF pulsesequences, etc. ; and of course 4) trained, dedicated personnelcapable of navigating/operating 1–3 above. Despite these re-quirements and challenges, a number of clinical or preclinicalhuman trials are underway, or already completed, with HP129Xe (produced via SEOP) and HP 13C (produced by d-DNP). Ex-amples of clinical research HP MRI are provided in Figure 5.Figure 5 a shows two selected slices of 3D functional pulmon-ary ventilation imaging scan using HP 129Xe. HP 129Xe wasinhaled by a healthy volunteer, and the entire 3D MRI examwas conducted in less than five seconds on a single breathhold.[5b, 35e] Bright signals are obtained from the void spaces ofthe lungs, as the enormous signal provided by the HP 129Xemore than outweighs the low-spin density of the gas phase.Moreover, the background signal from surrounding tissues isnon-existent due to the virtual absence of Xe naturally occur-ring in the body. The potential utility of two other features of

129Xe—its highly sensitive chemical shift and its tendency to berapidly absorbed by living tissues—is demonstrated in Fig-ure 5 b. First, a HP 129Xe NMR spectrum from the brain ofa healthy human subject after inhaling HP Xe (left) is shown toexhibit five different peaks, tentatively assigned to red bloodcells, blood plasma, grey matter, white matter, and lipid, re-spectively.[79] The ability to selectively create a spatial map ofone of these resonances/compartments, here, that of 129Xe re-siding in red blood cells, is demonstrated in the correspondingimage (right), where the strongest 129Xe signals likely originat-ing from the brain’s Circle of Willis.[79] Figure 5 c shows exam-ples taken from a magnetic resonance spectroscopic imaging(MRSI) study using intravenous injection of HP 1-13C-pyru-vate.[27] The injected HP 1-13C-pyruvate is rapidly converted toHP 1-13C-lactate by LDH (Figure 4 a), and the ratio 1-13C-lactate/1-13C-pyruvate[77b] (measured as the intensity ratio of HP signalsof HP 1-13C-lactate and 1-13C-pyruvate in the spectroscopicdomain of MRSI; see Figure 4 a) serves as a metabolic imagingbiomarker of prostate cancer. This ratio of HP signals is pre-sented as a false-color overlay image co-registered with a con-ventional T2-weighted MRI of prostate (greyscale), and isshown for three prostate cancer subjects. Note that the tumorsare very difficult to detect on conventional proton MRI images(top row of Figure 5 b), while the metabolic molecular imagingoffers additional contrast mechanism that can delineate dis-eased tissues from healthy ones by “lighting up” regions withpotentially pathological metabolism.

Conclusion

HP contrast agents with biomedical relevance can be nowprepared by several hyperpolarization techniques, including:d-DNP, SEOP, PHIP, and SABRE. These techniques significantlyincrease nuclear spin polarization and hence MR sensitivity byorders of magnitude, permitting new biomedical applicationsincluding functional and metabolic molecular imaging. HP con-trast agents hold great promise to enable a broad range ofnew imaging biomarkers for molecular imaging of deadly dis-eases such as cancer, COPD, emphysema, and others. Com-pared to positron emission tomography (PET), which is theleading molecular imaging modality, HP MRI offers advantagesof: 1) being nonradioactive and nonionizing, and 2) potentiallyproviding more metabolic information rather than only report-ing on contrast-agent uptake, and 3) very short (few seconds)imaging scan with possibility of multiple same-day examina-tions.[80] Furthermore, some hyperpolarization techniques (PHIPand SABRE) and detection methods (low-field MRI) potentiallyoffer additional advantages of speed, throughput and cost.While the seeds of most hyperpolarization techniques wereplanted decades ago, most have matured for biomedical appli-cations only during the last decade. Indeed, there is a tremen-dous room for innovation in fundamental physics, chemistry,biochemistry, and engineering that could significantly improveor even revolutionize this emerging area of molecular imaging.

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Acknowledgements

We thank for funding support NIH 1R21EB018014, National Sci-ence Foundation CHE-1416268, and DoD CDMRP Breast CancerProgram Era of Hope Award W81XWH-12-1-0159/BC112431.

Keywords: dynamic nuclear polarization · hyperpolarization ·medicinal chemistry · molecular imaging · spin exchangeoptical pumping

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