Friday, May 25, 2018

Multi-Frequency Pulsed Overhauser DNP at 1.2 Tesla

Schöps, P., E. Spindler Philipp, and F. Prisner Thomas, Multi-Frequency Pulsed Overhauser DNP at 1.2 Tesla, in Z. Phys. Chem. 2017. p. 561.


Dynamic nuclear polarization (DNP) is a methodology to increase the sensitivity of nuclear magnetic resonance (NMR) spectroscopy. It relies on the transfer of the electron spin polarization from a radical to coupled nuclear spins, driven by microwave excitation resonant with the electron spin transitions. In this work we explore the potential of pulsed multi-frequency microwave excitation in liquids. Here, the relevant DNP mechanism is the Overhauser effect. The experiments were performed with TEMPOL radicals in aqueous solution at room temperature using a Q-band frequency (1.2 T) electron paramagnetic resonance (EPR) spectrometer combined with a Minispec NMR spectrometer. A fast arbitrary waveform generator (AWG) enabled the generation of multi-frequency pulses used to either sequentially or simultaneously excite all three 14N-hyperfine lines of the nitroxide radical. The multi-frequency excitation resulted in a doubling of the observed DNP enhancements compared to single-frequency microwave excitation. Q-band free induction decay (FID) signals of TEMPOL were measured as a function of the excitation pulse length allowing the efficiency of the electron spin manipulation by the microwave pulses to be extracted. Based on this knowledge we could quantitatively model our pulsed DNP enhancements at 1.2 T by numerical solution of the Bloch equations, including electron spin relaxation and experimental parameters. Our results are in good agreement with theoretical predictions. Whereas for a narrow and homogeneous single EPR line continuous wave excitation leads to more efficient DNP enhancements compared to pulsed excitation for the same amount of averaged microwave power. The situation is different for radicals with several hyperfine lines or in the presence of inhomogeneous line broadening. In such cases pulsed single/multi-frequency excitation can lead to larger DNP enhancements.

Wednesday, May 23, 2018

(13)C Dynamic Nuclear Polarization Using a Trimeric Gd(3+) Complex as an Additive #DNPNMR

Niedbalski, P., et al., (13)C Dynamic Nuclear Polarization Using a Trimeric Gd(3+) Complex as an Additive. J. Phys. Chem. A, 2017. 121(27): p. 5127-5135.


Dissolution dynamic nuclear polarization (DNP) is one of the most successful techniques that resolves the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) by amplifying the signal by several thousand-fold. One way to further improve the DNP signal is the inclusion of trace amounts of lanthanides in DNP samples doped with trityl OX063 free radical as the polarizing agent. In practice, stable monomeric gadolinium complexes such as Gd-DOTA or Gd-HP-DO3A are used as beneficial additives in DNP samples, further boosting the DNP-enhanced solid-state (13)C polarization by a factor of 2 or 3. Herein, we report on the use of a trimeric gadolinium complex as a dopant in (13)C DNP samples to improve the (13)C DNP signals in the solid-state at 3.35 T and 1.2 K and consequently, in the liquid-state at 9.4 T and 298 K after dissolution. Our results have shown that doping the (13)C DNP sample with a complex which holds three Gd(3+) ions led to an improvement of DNP-enhanced (13)C polarization by a factor of 3.4 in the solid-state, on par with those achieved using monomeric Gd(3+) complexes but only requires about one-fifth of the concentration. Upon dissolution, liquid-state (13)C NMR signal enhancements close to 20000-fold, approximately 3-fold the enhancement of the control samples, were recorded in the nearby 9.4 T high resolution NMR magnet at room temperature. Comparable reduction of (13)C spin-lattice T1 relaxation time was observed in the liquid-state after dissolution for both the monomeric and trimeric Gd(3+) complexes. Moreover, W-band electron paramagnetic resonance (EPR) data have revealed that 3-Gd doping significantly reduces the electron T1 of the trityl OX063 free radical, but produces negligible changes in the EPR spectrum, reminiscent of the results with monomeric Gd(3+)-complex doping. Our data suggest that the trimeric Gd(3+) complex is a highly beneficial additive in (13)C DNP samples and that its effect on DNP efficiency can be described in the context of the thermal mixing mechanism.

Monday, May 21, 2018

Improving Sensitivity of Solid-state NMR Spectroscopy by Rational Design of Polarizing Agents for Dynamic Nuclear Polarization #DNPNMR

Kubicki, D.J. and L. Emsley, Improving Sensitivity of Solid-state NMR Spectroscopy by Rational Design of Polarizing Agents for Dynamic Nuclear Polarization. Chimia (Aarau), 2017. 71(4): p. 190-194.


We review our recent efforts to optimize the efficiency of polarizing agents for Dynamic Nuclear Polarization (DNP) in solid-state MAS NMR spectroscopy. We elucidate the links between DNP performance, molecular structure and electronic relaxation properties of dinitroxide biradicals. We show that deuteration and increased bulkiness lead to slower electronic relaxation and in turn to higher DNP enhancements. We also show that the incorporation of solid dielectric particles into the sample is a general method of amplifying DNP enhancements by about a factor of two.

Friday, May 18, 2018

A radiofrequency system for in vivo hyperpolarized (13) C MRS experiments in mice with a 3T MRI clinical scanner

Giovannetti, G., et al., A radiofrequency system for in vivo hyperpolarized (13) C MRS experiments in mice with a 3T MRI clinical scanner. Scanning, 2016. 38(6): p. 710-719.


Hyperpolarized carbon-13 magnetic resonance spectroscopy (MRS) is a powerful tool to explore tissue metabolic state, by permitting the study of intermediary metabolism of biomolecules in vivo. However, a number of technological problems still limit this technology and need innovative solutions. In particular, the low molar concentration of derivate metabolites give rise to low signal-to-noise ratio (SNR), which makes the design and development of dedicated radiofrequency (RF) coils a fundamental task. In this article, the authors describe the simulation and the design of a RF coils configuration for MR experiments in mice, constituted by a (1) H whole body volume RF coil for imaging and a (13) C single circular loop surface RF coil for performing (13) C acquisitions. After the building, the RF system was employed in an in vivo experiment in a mouse injected with hyperpolarized [1-(13) C]pyruvate by using a 3 T clinical MR scanner. SCANNING 38:710-719, 2016. (c) 2016 Wiley Periodicals, Inc.

Wednesday, May 16, 2018

DNP-enhanced ultrawideline (207)Pb solid-state NMR spectroscopy: an application to cultural heritage science

Kobayashi, T., et al., DNP-enhanced ultrawideline (207)Pb solid-state NMR spectroscopy: an application to cultural heritage science. Dalton Trans, 2017. 46(11): p. 3535-3540. 


Dynamic nuclear polarization (DNP) is used to enhance the (ultra)wideline (207)Pb solid-state NMR spectra of lead compounds of relevance in the preservation of cultural heritage objects. The DNP SSNMR experiments enabled, for the first time, the detection of the basic lead carbonate phase of the lead white pigment by (207)Pb SSNMR spectroscopy. Variable-temperature experiments revealed that the short T'2 relaxation time of the basic lead carbonate phase hinders the acquisition of the NMR signal at room temperature. We additionally observe that the DNP enhancement is twice as large for lead palmitate (a lead soap, which is a degradation product implicated in the visible deterioration of lead-based oil paintings), than it is for the basic lead carbonate. This enhancement has allowed us to detect the formation of a lead soap in an aged paint film by (207)Pb SSNMR spectroscopy; which may aid in the detection of deterioration products in smaller samples removed from works of art.

Tuesday, May 15, 2018

[NMR] Postdoc in hyperpolarized NMR and MRI in the Theis lab at NC State University and close collaboration with Duke University



Dear colleagues,

A postdoctoral position is opening in the Theis lab at the North Carolina State University. The focus of the position will be on development and applications of hyperpolarization technology. In a highly interdisciplinary and collaborative environment we will be advancing parahydrogen induced polarization techniques towards applications in biomolecular structure elucidation, miniaturized NMR, and molecular imaging. Novel hyperpolarized markers and detection schemes will be developed, tested and applied. The position also involves a close collaboration with the Warren lab at Duke University.

We offer access to parahydrogen hyperpolarizers (Duke and NCSU) and dissolution-DNP instrumentation (hypersense at Duke). The hyperpolarizers are installed next to imagers (7T and 1T) and NMR spectrometers (400 MHz). Schemes for optical detection of hyperpolarized signals with sensitive magnetometers will be designed and installed at NCSU. 

At NCSU METRIC (The Molecular Education, Technology and Research Innovation Center) gives access to the following NMR devices in addition to state-of-the-art mass spectrometry and X-Ray Crystallography instrumentation: 

  • Bruker Avance NEO 400 MHz NMR, RT BBO iProbe, VT, and SampleXpress Automatic Sample Changer
  • Bruker Avance NEO 500 MHz NMR, BBO PRODIGY LN2 -Cryoprobe, VT, and SampleCASE automatic Sample Changer
  • Bruker Avance NEO 600 MHz NMR, RT BBO Smart Probe and TXI 1H-13C/15N- 2H Probe, VT, and SampleXpress Automatic Sample Changer
  • Bruker Avance NEO 700 MHz NMR, TCI 1H/19F-13C/15N- 2H LHe Cryoprobe, TXI 1H-13C/15N- 2H Probe, VT, and SampleXPress Automatic Sample Changer
  • Bruker Avance III 700 MHz NMR, TCI 1H-13C/15N- 2H LHe CryoProbe, TXI 1H-13C/15N- 2HProbe, VT, and SampleXpress Automatic Sample Changer
  • Varian Mercury 400 MHz NMR, VT, 5mm ASW 4-nuclei 1H/19F/13C/31P Probe
  • Varian Inova 400 MHz NMR, VT, 5 mm PFG gradient 4-nuclei Probe (1H/19F/13C/31P)
  • Varian Mercury 300 MHz NMR, 5 mm ID Probe (1H/13C)
  • Varian Mercury Plus 300 MHz NMR, 5mm PFG 4-nuclei Probe
  • E500-10/12 EPR System with Digital High-Resolution Hall Field Controller and Dual Channel Signal Processing UnitFurthermore, we offer many collaborations across the Triangle (UNC, Duke, NC State), the US and Europe, and extended opportunities to travel for collaborations and attending conferences. 
The preferred candidate is a highly motivated and collaborative individual with expertise in magnetic resonance technologies and experimental design. Experience with hyperpolarization technologies, imaging and programming are a plus.

Candidates with a strong background in chemical synthesis that wish to broaden their skill-set by combining their strength in chemical design with innovative molecular imaging and spectroscopy approaches are also encouraged to apply. 


For more details please email ttheis@ncsu.edu or see:
The Theis lab is located on the modern centennial campus at NCSU. See: https://centennial.ncsu.edu/
To apply, please send CV/resume with contact information for 2 references to: ttheis@ncsu.edu

_________________________________
Thomas Theis, Ph.D.

Assistant Professor of Chemistry
North Carolina State University 

Adjunct at Duke University, Department of Chemistry 




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[NMR] PhD and Postdoctoral positions available to join the Emsley group at EPFL #DNPNMR

PhD and Postdoctoral positions available to join the Emsley group at EPFL, Lausanne

We are looking for highly motivated candidates to take up PhD and Postdoctoral positions developing new methods in NMR spectroscopy to address challenging problems in chemistry and materials science. In particular we will be working on extending dynamic nuclear polarization enhanced NMR crystallography to complex non-crystalline materials. Examples of our recent work and the application areas that we work on can be found on our website: http://lrm.epfl.ch

We are looking for highly motivated candidates with strong scientific background, independence, and who enjoy teamwork. You should hold a relevant qualification in chemistry, physics or related disciplines. Skills in one of the following fields of expertise are a plus:

• Experimental multi-dimensional nuclear magnetic resonance,
• 
Simulation, Theory, or Modelling of nuclear spin dynamics, NMR properties or chemical structures.
 

Our laboratory at EPFL is part of one the world’s leading chemistry departments, and is located Lausanne on the north shore of Lake Geneva. The laboratory is equipped with unique state of the art NMR spectrometers (including gyrotron DNP accessories at 400 and 900 MHz, a dissolution-DNP machine, and 100 kHz magic angle spinning probes).
 
Motivated candidates should contact Lyndon Emsley by email to lyndon.emsley@epfl.ch


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[NMR] Postdoctoral position in Lewandowski group at University of Warwick, UK - solid-state & solution NMR of natural products megasynthesases


A 2-year ERC-funded postdoc position is available in the Lewandowski group (http://go.warwick.ac.uk/lewandowskigroup) in the Department of Chemistry at the University of Warwick, United Kingdom.

Duration: 2 years | Start: Aug-Sept 2018 | Application deadline: 22 June 2018


The applicant will work on a major ERC-funded project employing combination of chemical biology, solid-state and solution-state NMR to study the structures, dynamics and interactions of domains from within polyketide synthases in order to facilitate their rational engineering for synthetic biology applications. Polyketide synthases are multicomponent enzymatic assembly lines for a range of useful natural products, for example, antibiotics that are effective against certain multidrug resistant infections. 

Experience in applying solution and/or solid-state NMR spectroscopy to elucidating the structures and/or dynamics of proteins is advantageous and a good knowledge of techniques for the overproduction and purification of recombinant proteins is essential.

The applicant will join a highly interdisciplinary collaborative team (exposure to a wide range of expertise and high potential for picking up new skills) under the supervision of Józef Lewandowski (http://go.warwick.ac.uk/lewandowskigroup) and in collaboration with Greg Challis (https://warwick.ac.uk/fac/sci/chemistry/research/challis/challisgroup).

Facilities:
The Warwick Laboratory for Magnetic Resonance (http://www2.warwick.ac.uk/fac/sci/physics/research/condensedmatt/nmr/) is a world-class biological and material science magnetic resonance set up hosting 7 academic staff from Physics and Chemistry and housing a suite of 9 solid-state NMR spectrometers (from 100 to 850 MHz; the main spectrometers for the biomolecular applications are 500, 600 and 700 MHz + access to 850 MHz and soon 1 GHz through a national facility at Warwick; a ¾ of 700 MHz solid-state NMR spectrometer time is dedicated to the project), 2 dynamic nuclear polarization (DNP) spectrometers (94 and 200/400 GHz) and advanced EPR instrumentation. A wide range of magic angle spinning probes is available in the laboratory including several 1.3 mm Bruker probes (< 67 kHz spinning), 1 mm JEOL probe, 0.81 mm Samoson probe (100 kHz spinning) and 3x 0.7 mm Bruker probes (111 kHz spinning). Solution NMR facilities include 500, 600 and 700 MHz equipped with cryoprobe (https://warwick.ac.uk/fac/sci/chemistry/research/facilities/nmr/). We have access to the state of the art mass spec facilities (https://warwick.ac.uk/fac/sci/chemistry/research/facilities/massspec) and X-ray crystallography.
The Chemical Biology Research Facility at University of Warwick (https://warwick.ac.uk/fac/sci/chemistry/research/facilities/chembiolfacility/) provides cutting edge infrastructure for organic synthesis, molecular biology, protein chemistry, microbiology, radiochemistry, biophysical and biochemical analysis.

Informal inquiries should be directed to Józef Lewandowski (j.r.lewandowski@warwick.ac.uk) and formal applications submitted (deadline Jun 22 2018). at https://atsv7.wcn.co.uk/search_engine/jobs.cgi?owner=5062452&ownertype=fair&jcode=1727027&vt_template=1457&adminview=1

Another postdoctoral position on a related BBSRC-funded project will become available in June-July 2018.

==========================================================================

Józef R. Lewandowski
Associate Professor | Department of Chemistry | University of Warwick
j.r.lewandowski@warwick.ac.uk | External: +44 (0) 24 76151355 | Internal: 51355 

Chemistry: C519 | Millburn House: F07 | Coventry CV4 7AL | Find us on the interactive map







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Monday, May 14, 2018

Hyperpolarized 13C Metabolic MRI of the Human Heart: Initial Experience

Dissolution DNP has come a long way.


Cunningham, C.H., et al., Hyperpolarized 13C Metabolic MRI of the Human Heart: Initial Experience. Circ Res, 2016. 119(11): p. 1177-1182.


RATIONALE: Altered cardiac energetics is known to play an important role in the progression toward heart failure. A noninvasive method for imaging metabolic markers that could be used in longitudinal studies would be useful for understanding therapeutic approaches that target metabolism. OBJECTIVE: To demonstrate the first hyperpolarized (13)C metabolic magnetic resonance imaging of the human heart. METHODS AND RESULTS: Four healthy subjects underwent conventional proton cardiac magnetic resonance imaging followed by (13)C imaging and spectroscopic acquisition immediately after intravenous administration of a 0.1 mmol/kg dose of hyperpolarized [1-(13)C]pyruvate. All subjects tolerated the procedure well with no adverse effects reported </=1 month post procedure. The [1-(13)C]pyruvate signal appeared within the chambers but not within the muscle. Imaging of the downstream metabolites showed (13)C-bicarbonate signal mainly confined to the left ventricular myocardium, whereas the [1-(13)C]lactate signal appeared both within the chambers and in the myocardium. The mean (13)C image signal:noise ratio was 115 for [1-(13)C]pyruvate, 56 for (13)C-bicarbonate, and 53 for [1-(13)C]lactate. CONCLUSIONS: These results represent the first (13)C images of the human heart. The appearance of (13)C-bicarbonate signal after administration of hyperpolarized [1-(13)C]pyruvate was readily detected in this healthy cohort (n=4). This shows that assessment of pyruvate metabolism in vivo in humans is feasible using current technology. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02648009.

Friday, May 11, 2018

Bastiaansen, J.A.M., M.E. Merritt, and A. Comment, Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-13C]butyrate and [1-13C]pyruvate. Scientific Reports, 2016. 6: p. 25573.


Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) 13C magnetic resonance spectroscopy (MRS) has the unique ability to detect realtime metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-13C] pyruvate and [1-13C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [13C]bicarbonate (−48%), [1-13C]acetylcarnitine (+113%), and [5-13C]glutamate (−63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-13C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-13C]acetoacetate and [1-13C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-13C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (−82%). Combining HP 13C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease.

Wednesday, May 9, 2018

Dynamic Nuclear Polarization as Kinetically Constrained Diffusion #DNPNMR

Karabanov, A., et al., Dynamic Nuclear Polarization as Kinetically Constrained Diffusion. Phys. Rev. Lett., 2015. 115(2): p. 020404. 


Dynamic nuclear polarization (DNP) is a promising strategy for generating a significantly increased nonthermal spin polarization in nuclear magnetic resonance (NMR) and its applications that range from medicine diagnostics to material science. Being a genuine nonequilibrium effect, DNP circumvents the need for strong magnetic fields. However, despite intense research, a detailed theoretical understanding of the precise mechanism behind DNP is currently lacking. We address this issue by focusing on a simple instance of DNP-so-called solid effect DNP-which is formulated in terms of a quantum central spin model where a single electron is coupled to an ensemble of interacting nuclei. We show analytically that the nonequilibrium buildup of polarization heavily relies on a mechanism which can be interpreted as kinetically constrained diffusion. Beyond revealing this insight, our approach furthermore permits numerical studies of ensembles containing thousands of spins that are typically intractable when formulated in terms of a quantum master equation. We believe that this represents an important step forward in the quest of harnessing nonequilibrium many-body quantum physics for technological applications.

Monday, May 7, 2018

Gadolinium based endohedral metallofullerene Gd2@C79N as a relaxation boosting agent for dissolution DNP at high fields

Wang, X., et al., Gadolinium based endohedral metallofullerene Gd2@C79N as a relaxation boosting agent for dissolution DNP at high fields. Chem Commun (Camb), 2018. 54(19): p. 2425-2428.


We show increased dynamic nuclear polarization by adding a low dosage of a S = 15/2 Gd based endohedral metallofullerene (EMF) to DNP samples. By adding 60 muM Gd2@C79N, the nuclear polarization of (1)H and (13)C spins from 40 mM 4-oxo-TEMPO increases by approximately 40% and 50%, respectively, at 5 T and 1.2 K. Electron-electron double resonance (ELDOR) measurements show that the high spin EMF shortens the electron relaxation times and increases electron spectral diffusion leading to the increased DNP enhancement.

Sunday, May 6, 2018

[NMR] PhD position available in Grenoble on DNP-enhanced ssNMR

Funded PhD position available in Grenoble, France, on DNP-enhanced solid-state NMR

Project: “Improving DNP for the advanced study of glycopolymers”

A 3-year funded PhD position is available at CEA Grenoble (twinned with the University Grenoble Alpes) in the field of Dynamic Nuclear Polarization (DNP) NMR applied to glycopolymers. 

The PhD will deal with the development and application of a new and emerging hyperpolarization technique: high-magnetic field MAS-DNP (Magic Angle Spinning Dynamic Nuclear Polarization). This approach is used to hyperpolarize nuclei such that high-sensitivity and high-resolution solid-state NMR (Nuclear Magnetic Resonance) spectra can be obtained and used to extract important structural information at the atomic scale, such as surface functionalization and internuclear proximities/distances, as well as crystallographic data, etc. Since the potential of this technique is beginning to be realized, the aim of this PhD is to further develop the methodology (sample preparation, new polarizing agents and radio-frequency pulse schemes) for the study of complex glycopolymers. This will be achieved through the combined use of EPR, DNP and MAS-DNP numerical simulation as well as developing advanced NMR experiments.

The thesis will mainly be performed in the DNP group of the Institute for Nanosciences and Cryogenics (CEA/University Grenoble Alpes) (http://www.dnpgrenoble.net/) in the context of an international collaboration. The PhD studentship will be funded for a duration of 3 years, starting October 1st 2018. 

Grenoble is one of the major cities in Europe for research with a large international scientific community. In addition, Grenoble has a large international student population, is a very pleasant city to live in, and is known as the “Capital of the Alps” with easy access to great skiing and hiking. It’s also only 2 hours’ drive to the Mediterranean Sea, Italy, or Switzerland. Grenoble, Lyon, and Geneva airports are nearby and permit straightforward international travel. 

The candidate should have a Master degree in Chemistry or Physics. Previous experience in NMR spectroscopy and/or computational methods is an advantage. He/she should have no issues with mobility and have a good comment (written/spoken) of English.

Motivated candidates should send a detailed CV, a letter of motivation, Master grades, and at least two names for recommendation to Sabine Hediger (sabine.hediger@cea.fr) and Gaël De Paëpe(gael.depaepe@cea.fr).
--
_____________________________________________________________
Dr. Sabine Hediger
INAC/MEM/LRM
CEA Grenoble
17 rue des Martyrs
38054 Grenoble Cedex 9
France
Tel.: +33 4 38 78 65 79
Fax : +33 4 38 78 50 90
_____________________________________________________________
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Friday, May 4, 2018

Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy #DNPNMR

Viger-Gravel, J., et al., Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy. The Journal of Physical Chemistry B, 2018. 122(7): p. 2073-2081.


Here, we show how dynamic nuclear polarization (DNP) NMR spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs). The LNPs used here were synthesized by the microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (distearoylphosphatidylcholine, DSPC), cholesterol, and poly(ethylene glycol) (PEG) (dimyristoyl phosphatidyl ethanolamine (DMPE)–PEG 2000), as well as encapsulated cargoes that are either phosphorothioated siRNA (50 or 100%) or mRNA. We show that LNPs form physically stable complexes with bioactive drug siRNA for a period of 94 days. Relayed DNP experiments are performed to study 1H–1H spin diffusion and to determine the spatial location of the various components of the LNP by studying the average enhancement factors as a function of polarization time. We observe a striking feature of LNPs in the presence and in the absence of encapsulating siRNA or mRNA by comparing our experimental results to numerical spin-diffusion modeling. We observe that LNPs form a layered structure, and we detect that DSPC and DMPE–PEG 2000 lipids form a surface rich layer in the presence (or absence) of the cargoes and that the cholesterol and ionizable cationic lipid are embedded in the core. Furthermore, relayed DNP 31P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined. On the basis of the results, we propose a new structural model for the LNPs that features a homogeneous core with a tendency for layering of DSPC and DMPE–PEG at the surface.

Wednesday, May 2, 2018

Verdazyl-ribose: A new radical for solid-state dynamic nuclear polarization at high magnetic field #DNPNMR



Thurber, K.R., et al., Verdazyl-ribose: A new radical for solid-state dynamic nuclear polarization at high magnetic field. J Magn Reson, 2018. 289: p. 122-131.


Solid-state dynamic nuclear polarization (DNP) using the cross-effect relies on radical pairs whose electron spin resonance (ESR) frequencies differ by the nuclear magnetic resonance (NMR) frequency. We measure the DNP provided by a new water-soluble verdazyl radical, verdazyl-ribose, under both magic-angle spinning (MAS) and static sample conditions at 9.4T, and compare it to a nitroxide radical, 4-hydroxy-TEMPO. We find that verdazyl-ribose is an effective radical for cross-effect DNP, with the best relative results for a non-spinning sample. Under non-spinning conditions, verdazyl-ribose provides roughly 2x larger (13)C cross-polarized (CP) NMR signal than the nitroxide, with similar polarization buildup times, at both 29K and 76K. With MAS at 7kHz and 1.5W microwave power, the verdazyl-ribose does not provide as much DNP as the nitroxide, with the verdazyl providing less NMR signal and a longer polarization buildup time. When the microwave power is decreased to 30mW with 5kHz MAS, the two types of radical are comparable, with the verdazyl-doped sample having a larger NMR signal which compensates for its longer polarization buildup time. We also present electron spin relaxation measurements at Q-band (1.2T) and ESR lineshapes at 1.2 and 9.4T. Most notably, the verdazyl radical has a longer T1e than the nitroxide (9.9ms and 1.3ms, respectively, at 50K and 1.2T). The verdazyl electron spin lineshape is significantly affected by the hyperfine coupling to four (14)N nuclei, even at 9.4T. We also describe 3000-spin calculations to illustrate the DNP potential of possible radical pairs: verdazyl-verdazyl, verdazyl-nitroxide, or nitroxide-nitroxide pairs. These calculations suggest that the verdazyl radical at 9.4T has a narrower linewidth than optimal for cross-effect DNP using verdazyl-verdazyl pairs. Because of the hyperfine coupling contribution to the electron spin linewidth, this implies that DNP using the verdazyl radical would improve at lower magnetic field. Another conclusion from the calculations is that a verdazyl-nitroxide bi-radical would be expected to be slightly better for cross-effect DNP than the nitroxide-nitroxide bi-radicals commonly used now, assuming the same spin-spin coupling constants.



Tuesday, May 1, 2018

[NMR] Post-doctoral position in preclinical MRI/NMR spectroscopy

Two post-doctoral fellowships are available in the Department of Radiology and Biomedical Imaging at UCSF, in Dr. John Kurhanewicz’s Laboratory. The fellow will be involved in hyperpolarized 13C MR metabolism and physiology studies involving cutting edge preclinical models of prostate and renal cancer. The studies will utilize living cells in bioreactors, patient derived tissue slices as well as transgenic mouse models. These biologically relevant animal models will be used to identify and validate imaging markers of disease presence, severity and treatment response.
The ideal candidate should have a strong background in MR spectroscopic imaging. Familiarity with dissolution dynamic nuclear polarization 13C imaging is an added bonus, but not required. Candidates with a broad experience in animal and biologic tissue and cell handling will be preferred. Candidates with fervent interest in metabolism and its implication in diseases like cancer are encouraged to apply.

The Biomedical NMR Laboratory within the NMR Lab on the Mission Bay Campus of UCSF occupies 1660 sq. ft. and houses two high field (500 and 600 MHz) Varian NMR spectrometers, and a low field (3T) animal imaging system and 1.5T bench top NMR (PulsarTM, Oxford Instruments) uniquely integrated with two HyperSenseTM (Oxford Instruments) DNP polarizers enabling cell and tissue culture bioreactor and animal studies. The high filed magnets have complimentary features, including high-resolution magic angle spinning (HR-MAS) spectroscopy capabilities, and micro-imaging capabilities. The department also has facilities for cell and tissue molecular biology and RF coil fabrication.

If interested, please contact Dr. John Kurhanewicz (John.Kurhanewicz@ucsf.edu) or Dr. Renuka Sriram (renuka.sriram@ucsf.edu)

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