Pratip Bhattacharya

PASADENA Hyperpolarization of Succinic Acid for MRI and NMR Spectroscopy

We use the PASADENA (parahydrogen and synthesis allow dramatically enhanced nuclear alignment) method to achieve 13C polarization of approximately 20% in seconds in 1-13C-succinic-d2 acid. The high-field 13C multiplets are observed as a function of pH, and the line broadening of C1 is pronounced in the region of the pK values. The 2JCH, 3JCH, and 3JHH couplings needed for spin order transfer vary with pH and are best resolved at low pH leading to our use of pH approximately 3 for both the molecular addition of parahydrogen to 1-13C-fumaric acid-d2 and the subsequent transfer of spin order from the nascent protons to C1 of the succinic acid product. The methods described here may generalize to hyperpolarization of other carboxylic acids. The C1 spin-lattice relaxation time at neutral pH and 4.7 T is measured as 27 s in H2O and 56 s in D2O. Together with known rates of succinate uptake in kidneys, this allows an estimate of the prospects for the molecular spectroscopy of metabolism.

Real-Time Molecular Imaging of Tricarboxylic Acid Cycle Metabolism in Vivo by Hyperpolarized 1- 13 C Diethyl Succinate

The Krebs tricarboxylic acid cycle (TCA) is central to metabolic energy production and is known to be altered in many disease states. Real-time molecular imaging of the TCA cycle in vivo will be important in understanding the metabolic basis of several diseases. Positron emission tomography (PET) with FDG-glucose (2-[(18)F]fluoro-2-deoxy-d-glucose) is already being used as a metabolic imaging agent in clinics. However, FDG-glucose does not reveal anything past glucose uptake and phosphorylation. We have developed a new metabolic imaging agent, hyperpolarized diethyl succinate-1-(13)C-2,3-d(2) , that allows for real-time in vivo imaging and spectroscopy of the TCA cycle. Diethyl succinate can be hyperpolarized via parahydrogen-induced polarization (PHIP) in an aqueous solution with signal enhancement of 5000 compared to Boltzmann polarization. (13)C magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) were achieved in vivo seconds after injection of 10-20 μmol of hyperpolarized diethyl succinate into normal mice. The downstream metabolites of hyperpolarized diethyl succinate were identified in vivo as malate, succinate, fumarate, and aspartate. The metabolism of diethyl succinate was altered after exposing the animal to 3-nitropropionate, a known irreversible inhibitor of succinate dehydrogenase. On the basis of our results, hyperpolarized diethyl succinate allows for real-time in vivo MRI and MRS with a high signal-to-noise ratio and with visualization of multiple steps of the TCA cycle. Hyperpolarization of diethyl succinate and its in vivo applications may reveal an entirely new regime wherein the local status of TCA cycle metabolism is interrogated on the time scale of seconds to minutes with unprecedented chemical specificity and MR sensitivity.

In vivo magnetic resonance imaging of hyperpolarized silicon particles

Silicon-based micro- and nanoparticles have gained popularity in a wide range of biomedical applications due to their biocompatibility and biodegradability in vivo, as well as their flexible surface chemistry, which allows drug loading, functionalization and targeting. Here, we report direct in vivo imaging of hyperpolarized (29)Si nuclei in silicon particles by magnetic resonance imaging. Natural physical properties of silicon provide surface electronic states for dynamic nuclear polarization, extremely long depolarization times, insensitivity to the in vivo environment or particle tumbling, and surfaces favourable for functionalization. Potential applications to gastrointestinal, intravascular and tumour perfusion imaging at subpicomolar concentrations are presented. These results demonstrate a new background-free imaging modality applicable to a range of inexpensive, readily available and biocompatible silicon particles.

Parahydrogen-induced polarization (PHIP) hyperpolarized MR receptor imaging in vivo: a pilot study of 13C imaging of atheroma in mice.

MR techniques using hyperpolarized 13C have successfully produced examples of angiography and intermediary metabolic imaging, but, to date, no receptor imaging has been attempted. The goal of this study was to synthesize and evaluate a novel hyperpolarizable molecule, 2,2,3,3‐tetrafluoropropyl 1‐13C‐propionate‐d2,3,3 (TFPP), for the detection of atheromatous plaques in vivo. TFPP binds to lipid bilayers and its use in hyperpolarized MR could prove to be a major step towards receptor imaging. The precursor, 2,2,3,3‐tetrafluoropropyl 1‐13C‐acrylate‐d2,3,3 (TFPA), binds to 1,2‐dimyristoylphosphatidylcholine lipid bilayers with a 1.6‐ppm chemical shift in the 19F MR spectrum. This molecule was designed to be hyperpolarized through the addition of parahydrogen to the 13C‐acrylate moiety by parahydrogen‐induced polarization. TFPA was hyperpolarized to TFPP to an extent similar to that of the hydroxyethylacrylate to hydroxyethylpropionate transition: 17 ± 4% for TFPP versus 20% for hydroxyethylpropionate; T1 relaxation times (45 ± 2 s versus 55 ± 2 s) were comparable and the hyperpolarized properties of TFPP were characterized. Hydroxyethylacrylate, like TFPA, has a chemical structure with an acrylate moiety, but does not contain the lipid‐binding tetrafluoropropyl functional group. Hyperpolarized TFPP binds to the lipid bilayer, appearing as a second, chemically shifted 13C hyperpolarized MR signal with a further reduction in the longitudinal relaxation time (T1 = 21 ± 1 s). In aortas harvested from low‐density lipoprotein receptor knock‐out mice fed with a high‐fat diet for 9 months, and in which atheroma is deposited in the aorta and heart, TFPP showed greater binding to lipid on the intimal surface than in control mice fed a normal diet. When TFPP was hyperpolarized and administered in vivo to atheromatous mice in a pilot study, increased binding was observed on the endocardial surface of the intact heart compared with normally fed controls. Hyperpolarized TFPP has bio‐sensing specificity for lipid, coupled with a 42 000‐fold sensitivity gain in the MR signal at 4.7 T. Binding of TFPP with lipids results in the formation of a characteristic second peak in MRS. TFPP therefore has the potential to act as an in vivo molecular probe for atheromatous plaque imaging and may serve as a model of receptor‐targeted bio‐imaging with enhanced MR sensitivity. Copyright

Hyperpolarized MR Imaging: Neurologic Applications of Hyperpolarized Metabolism

SUMMARY: Hyperpolarization is the general term for a method of enhancing the spin-polarization difference of populations of nuclei in a magnetic field. No less than 5 distinct techniques (dynamic nuclear polarization [DNP]; parahydrogen-induced polarization−parahydrogen and synthesis allow dramatically enhanced nuclear alignment [PHIP-PASADENA]; xenon/helium polarization transfer; Brute Force; 1H hyperpolarized water) are currently under exhaustive investigation as means of amplifying the intrinsically (a few parts per million) weak signal intensity used in conventional MR neuroimaging and spectroscopy. HD-MR imaging in vivo is a metabolic imaging tool causing much of the interest in HD-MR imaging. The most successful to date has been DNP, in which carbon-13 (13C) pyruvic acid has shown many. PHIP-PASADENA with 13C succinate has shown HD-MR metabolism in vivo in tumor-bearing mice of several types, entering the Krebs−tricarboxylic acid cycle for ultrafast detection with 13C MR imaging, MR spectroscopy, and chemical shift imaging. We will discuss 5 promising preclinical studies: 13C succinate PHIP in brain tumor; 13C ethylpyruvate DNP and 13C acetate; DNP in rodent brain; 13C succinate PHIP versus gadolinium imaging of stroke; and 1H hyperpolarized imaging. Recent developments in clinical 13C neurospectroscopy encourage us to overcome the remaining barriers to clinical HD-MR imaging.

Hyperpolarized 1H NMR Employing Low γ Nucleus for Spin Polarization Storage

Here, we demonstrate the utility of low gamma nuclei for spin storage of hyperpolarization followed by proton detection, which theoretically can provide up to approximately (gamma[1H]/gamma[X])(2) gain in sensitivity in hyperpolarized biomedical MR. This is exemplified by hyperpolarized 1-(13)C sites of 2,2,3,3-tetrafluoropropyl 1-(13)C-propionate-d(3) (TFPP), (13)C T(1) = 67 s in D(2)O, and 1-(13)C-succinate-d(2), (13)C T(1) = 105 s in D(2)O, pH 11, using PASADENA. In a representative example, the spin polarization was stored on (13)C for 24 and 70 s, respectively, while the samples were transferred from a low magnetic field polarizer operating at 1.76 mT to a 4.7 T animal MR scanner. Following sample delivery, the refocused INEPT pulse sequence was used to transfer spin polarization from (13)C to protons with an efficiency of 50% for TFPP and 41% for 1-(13)C-succinate-d(2) increasing the overall NMR sensitivity by a factor of 7.9 and 6.5, respectively. The low gamma nuclei exemplified here by (13)C with a T(1) of tens of seconds acts as an efficient spin polarization storage, while J-coupled protons are better for NMR detection.

Continuous flow Overhauser dynamic nuclear polarization of water in the fringe field of a clinical magnetic resonance imaging system for authentic image contrast

We describe and demonstrate a system to generate hyperpolarized water in the 0.35 T fringe field of a clinical 1.5 T whole-body magnetic resonance imaging (MRI) magnet. Once generated, the hyperpolarized water is quickly and continuously transferred from the 0.35 T fringe to the 1.5 T center field of the same magnet for image acquisition using standard MRI equipment. The hyperpolarization is based on Overhauser dynamic nuclear polarization (DNP), which effectively and quickly transfers the higher spin polarization of free radicals to nuclear spins at ambient temperatures. We visualize the dispersion of hyperpolarized water as it flows through water-saturated systems by utilizing an observed -15-fold DNP signal enhancement with respect to the unenhanced (1)H MRI signal of water at 1.5 T. The experimental DNP apparatus presented here is readily portable and can be brought to and used with any conventional unshielded MRI system. A new method of immobilizing radicals to gel beads via polyelectrolyte linker arms is described, which led to superior flow Overhauser DNP performance compared to previously presented gels. We discuss the general applicability of Overhauser DNP of water and aqueous solutions in the fringe field of commercially available magnets with central fields up to 4.7 T.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Hyperpolarized Water as an MR Imaging Contrast Agent: Feasibility of in Vivo Imaging in a Rat Model

PURPOSE To assess the feasibility of a perfusion magnetic resonance (MR) imaging technique that uses Overhauser dynamic nuclear polarization (DNP) to provide contrast during the continuous delivery of hyperpolarized water in rats. MATERIALS AND METHODS Protocols approved by the local institutional animal care and use committees were followed. Twelve male Wistar rats were anesthetized and prepared by placing injection tubing in the subcutaneous layer (n=3), peritoneum (n=3), aorta (n=3), or carotid artery (n=3). Water was hyperpolarized by means of Overhauser DNP in the 0.35-T fringe field of a 1.5-T MR imaging magnet by using a custom-built system to continuously deliver radical-free hyperpolarized water to the subject. Fast gradient-echo and spoiled gradient-recalled-echo MR imaging sequences were used. The signal-to-noise ratio (SNR) of the images was calculated and compared. RESULTS Images showed greatly altered SNR and enhanced flow contrast at all injection locations. For subcutaneous and intraperitoneal injections, the water perfusion trajectory was observed for approximately 5 seconds after injection. Flow through a 4.2-cm length of artery was seen during intra-aortic injection. The right hemisphere of the brain was seen during injection into the right carotid artery. Images with hyperpolarized water had greatly altered SNR compared with images without injection or with the injection of nonhyperpolarized water, with a range of 13%-27% for the carotid and 444%-2900% for the other regions. CONCLUSION Perfusion contrast for MR imaging can be obtained by continuously infusing hyperpolarized water, providing localized angiography or brain perfusion information in vivo for rat models.

Cardiovascular Applications of Hyperpolarized Contrast Media and Metabolic Tracers

Modern hyperpolarization technology enhances the recordable magnetic resonance signal four to five orders of magnitude, making in vivo assessments of tracer pathways and metabolic compartments feasible. Existing hyperpolarization instrumentation and previous tracer studies using hydroxyethylpropionate (HEP) as an extracellular marker and 14-carbon label pyruvate as examples are described and reviewed as applicable to the working heart. Future metabolic imaging based on the use of hyperpolarized pyruvate needs to consider extra- and intra-cellular label dilution due to glycolysis, lactate oxidation and protein degradation. This dilution can substantially decrease the recordable signals from PDH flux (oxidative decarboxylation of pyruvate) and other pyruvate pathways. The review of previous literature and data suggests that the 13C-alanine signal is a better index of mitochondrially oxidized pyruvate than L-lactate. These facts and considerations will help in the interpretation of the in vivo recorded hyperpolarization signals of metabolic tracers and contrast media.