Article pubs.acs.org/JPCB
BDPA-Doped Polystyrene Beads as Polarization Agents for DNP-NMR Yunzhi Zhang, Phillip J. Baker, and Leah B. Casabianca* Department of Chemistry, Clemson University, Clemson, South Carolina 29634, United States S Supporting Information *
ABSTRACT: The aromatic free radical BDPA (α,γ-bisdiphenylene-β-phenylallyl), which has been widely used as a polarizing agent for Dynamic Nuclear Polarization (DNP) of hydrophobic analytes, has been incorporated into nanometerscale polystyrene latex beads. We have shown that the resulting BDPA-doped beads can be used to hyperpolarize 13C and 7Li nuclei in aqueous environments, without the need for a glassing cosolvent. DNP enhancement factors of between 20 and 100 were achieved with overall BDPA concentrations of 2 mM or less. These Highly-Effective Polymer/Radical Beads (HYPR-beads) have potential use as an inexpensive polarizing agent for water-soluble analytes, and also have applications as model nanoparticles in DNP studies.
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INTRODUCTION Dynamic Nuclear Polarization (DNP) has been used in solidstate NMR for many years1 as a way to overcome the sensitivity limitations of NMR. DNP relies on the transfer of polarization from an unpaired electron to nearby nuclei, leading to “hyperpolarized” NMR signals with sensitivity comparable to that of Electron Paramagnetic Resonance (EPR). The unpaired electrons are usually introduced into the sample through addition of stable free radicals, although intrinsic paramagnetic centers such as metals or defects have been used as polarization agents as well.2−6 In order for the radical to be dispersed within the sample and remain in close proximity to the nuclei to be hyperpolarized, a glassing solvent is generally used. Solvents such as pure water form crystals, leading to phase separation between the radicals and the nuclei of interest and a consequent loss of DNP efficiency. More recently, dissolution DNP, in which a sample is polarized in the solid state at low temperature, then quickly melted and transferred to a high-field NMR spectrometer or MRI scanner for observation at room temperature,7 has become popular. Dissolution DNP has expanded the range of samples that can be polarized, and has even been used clinically.8 The potential for DNP-enhanced MRI in the fields of medicine, catalysis, fluid dynamics, and related fields is substantial. If a suitable polarized substrate can be found, the substrate could be polarized at low temperature, quickly melted by addition of hot solvent, injected into a material, animal, or person, and used to follow blood flow,9 metabolism,10 or other processes. Nanodiamonds, diamond particles with a characteristic length scale on the order of nanometers, have been suggested for use as polarized tracers in DNP studies. Nanodiamonds are nonreactive, consist of carbons in a rigid proton-free lattice, which leads to long carbon T1 relaxation times, and contain © 2015 American Chemical Society
naturally occurring paramagnetic defects that could potentially be used as polarizing agents. However, although the naturally occurring paramagnetic defects in diamond have been used to hyperpolarize diamond carbons through DNP, as the size of the nanodiamond sample decreases, coupling between these bulk defects and other paramagnetic defects such as dangling bonds on the nanodiamond surface leads to short nuclear and electron relaxation times, making the DNP process inefficient.3 Another reason why many groups are interested in hyperpolarizing nanodiamonds or other types of nanoparticles (NPs) is that this may provide a method by which to study the structure of surface groups on nanoparticles. DNP has been used to selectively enhance the signal of surface groups in microporous structures,11−15 and would be an excellent method by which to study functionalized nanoparticles. Nanoparticle toxicity is related to the functional groups on the surface. Cationic NH2-functionalized polystyrene (PS) NPs are more toxic to macrophage and epithelial cells than naked or COOHfunctionalized PS NPs.16 All PS nanoparticles induced pulmonary inflammation when inhaled, but the naked and COOH-functionalized NPs induced a much lower effect than the NH2-functionalized ones.16 Recently, Presti et al.17 attempted to use DNP to observe functional groups covalently bound to nanodiamonds, but they found that the intrinsic paramagnetic defects in the nanodiamonds could not be used as a DNP polarizing agent. Akbey et al.18 studied lysine- and arginine-functionalized silica nanoparticles by magic-angle spinning (MAS) DNP NMR. Using an extrinsic TOTAPOL radical dissolved in the solvent (10:90 H2O:D2O), they found that smaller nanoparticles could be hyperpolarized more Received: September 7, 2015 Revised: December 14, 2015 Published: December 30, 2015 18
DOI: 10.1021/acs.jpcb.5b08741 J. Phys. Chem. B 2016, 120, 18−24
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The Journal of Physical Chemistry B
TEMPO were purchased from DyNuPol, Inc. (Newton, MA). C-urea (99 atom % 13C) was purchased from Sigma. All other reagents and solvents were purchased from commercial sources and used as received. BDPA was incorporated into the polystyrene beads following a protocol found in ref 32, with slight modifications. First, 0.5 mL of the aqueous beads suspension was mixed with 0.5 mL of methanol. To this mixture was added 25 μL of 38 mM BDPA in toluene, and the solution was stirred at room temperature for 3−6 h. The solution was then filtered through a 0.2 μm syringe filter (unless otherwise noted) to remove aggregated beads and dried at room temperature overnight in order to remove methanol. The final volume after drying was 0.5 mL. Control samples were prepared using the same method but for beads only without BDPA (25 μL of toluene alone was added to the beads sample in methanol:water) and for BDPA only without the beads (the same concentration of BDPA in toluene was added to methanol:water alone without beads present). The solubility of BDPA in water alone is very low, so most of the BDPA precipitated from solution during the drying step in this second control sample when the beads were not present. UV−vis spectra were collected with an Agilent 8453 UV−vis spectrophotometer in spectrum/peaks mode using ChemStation software version B.04.01. Samples were placed in a BrandTech ultramicro disposable spectrophometer cuvette (BrandTech Scientific, Inc., Essex, CT) with a sample volume of 70 μL and a path length of 10 mm. A blank containing only the appropriate solvent in the same cuvette was run before each set of samples and was subtracted from all reported spectra. Transmission Electron Microscopy (TEM) was performed using a Hitachi H-9500 high-resolution transmission electron microscope at 300 kV accelerating voltage. Samples were prepared by placing a 5-μL drop of each aqueous sample on a carbon-coated 300-mesh copper grid (Electron Microscopy Sciences, Hatfield, PA) for 30 seconds. Excess water was wicked from the grid using a kimwipe, and the sample was allowed to air-dry for at least 5 min before it was placed in the specimen holder. No stain was added. For the as-received beads sample, multiple drops were added and wicked dry in order to increase the amount of sample on the grid. Dissolution DNP experiments were performed using a commercial HyperSense polarizer (Oxford Instruments, Tubney Woods, U.K.) located at the Complex Carbohydrate Research Center at the University of Georgia. A microwave sweep was first performed on a sample of 13C-enriched urea with 50 mM BDPA in a 50:50 DMSO:sulfolane solvent mixture using the internal coil of the HyperSense. For each dissolution, approximately 12 mg of 13C-urea or 6 mg of LiCl was dissolved in 100 μL of either the aqueous HYPR-beads dispersion or a 60:40 mixture of DMSO-d6 and the aqueous HYPR-beads dispersion. Each sample was then polarized for 1 h at 1.4−1.6 K using 100 mW microwave power. Different microwave frequencies were used for each experiment, and are noted in the respective figure captions. After 1 h of polarization, the sample was quickly melted by addition of 4 mL of hot water and transferred to a Varian Inova 500 MHz NMR spectrometer with an 8 mm inverse-detection probe for observation at room temperature. The decay of polarization was followed using small tip-angle pulses every second for a time period of 200 s after the sample had arrived in the NMR tube and settled. For calculation of the DNP enhancement factor, thermal equilibrium spectra were collected for the same sample using the same small tip-angle pulse at least 10 min after completion
efficiently than larger nanoparticles. This is in contrast to what was found for diamond particles, but may be attributed to the difference between using external added radicals versus intrinsic defects as the polarization agents. Nanoparticles also have potential as attractive polarization agents for polarizing bulk solvent, or for polarizing analytes dissolved in a solvent. Their advantage lies in the ease of separating the nanoparticle from the solvent (and analyte) after the polarization step. The presence of radical in the solution during the dissolution and sample shuttling step leads to rapid T1 relaxation, and it is best to avoid introducing contaminating radicals into an in vivo system. Intrinsic defects in silicon nanoparticles, namely, dangling bonds near the silicon surface, have been used to hyperpolarize 1H nuclei in the solvent.5 Silicon nanoparticles can have extremely long T1 relaxation times,19 which has allowed for in vivo hyperpolarized silicon MRI with long hyperpolarization decay times, allowing imaging for as long as 60 min after dissolution.6,20 Dutta et al.21 recently showed that intrinsic defects in nanodiamonds can be used as polarization agents for carbons in the nanodiamond when the nanodiamonds are dispersed in a 1:1 mixture of DMSO-d6 and D2O. In this work, rather than using intrinsic paramagnetic defects in nanodiamonds as DNP polarizing agents, we take a different approach. Here we introduce a known DNP radical, α,γbisdiphenylene-β-phenylallyl (BDPA), into organic nanoparticles. BDPA is one of the most effective DNP radicals used as an extrinsic polarizing agent. The disadvantage of BDPA, however, is that it is not water-soluble and therefore cannot be used to hyperpolarize water-soluble analytes. Many groups have proposed ways to overcome this difficulty, including synthesizing water-soluble analogues of BDPA22 and using sulfolane as a glassing cosolvent.23 Similar hydrophobic radicals have been solubilized by encapsulating them in surfactant micelles.24−26 BDPA is a large aromatic molecule, and has been previously incorporated into thin films of polystyrene/polycarbonate and used for DNP studies of these films.27−30 More recently, Can et al.31 have shown that BDPA dispersed in polystyrene exhibits an Overhauser DNP effect in addition to the usual solid effect that is typically seen in solids. The advantages of our approach over using intrinsic defects in silicon nanoparticles are the following: (a) the amount of BDPA incorporated into the beads could potentially be tunable, and (b) the polarization buildup times of analytes using these beads as polarizing agents are much shorter. We found that DNP enhancements for watersoluble analytes of between 20- and 100-fold could be attained using these BDPA-doped beads as polarizing agents, at much lower BDPA concentrations than are used in typical dissolution DNP experiments. Additionally, the beads we used have hydrophilic surfaces, and form a stable colloidal dispersion in water. We found that hyperpolarization could be achieved in 60:40 mixtures of DMSO-d6:H2O, but also in water alone without the addition of a glassing cosolvent. This allows us to polarize water-soluble compounds using BDPA, an efficient, but water-insoluble, polarizing agent. Our Highly-Effective Polymer/Radical Beads (HYPR-beads) are easy to make, and are an economical alternative to ox-063, the radical of choice that is currently used for polarizing hydrophilic compounds.
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MATERIALS AND METHODS Carboxylate-modified (CML) latex particles (4% w/v suspension in water, 27 nm) were purchased from Life Technologies. α,γ-bisdiphenylene-β-phenylallyl (BDPA) and 4-hydroxy19
DOI: 10.1021/acs.jpcb.5b08741 J. Phys. Chem. B 2016, 120, 18−24
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Figure 1. UV−vis spectra of BDPA, HYPR-beads, and control samples without BDPA and without beads. The structure of BDPA is shown in the inset.
of the dissolution. All NMR spectra were acquired using Varian VNMR software and processed offline using custom-written MATLAB scripts.
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RESULTS AND DISCUSSION The HYPR-beads were characterized by UV−vis spectroscopy and electron microscopy (EM) prior to using them for dissolution DNP experiments. The UV−vis spectra of the HYPR-beads are shown in Figure 1. The HYPR-beads have a strong absorption in the visible region at 495 nm. This peak is due to the absorbance of the BDPA, a large conjugated molecule, since beads alone do not have any UV−vis absorption. The control sample with beads only does not show any absorbance in the visible region. Because of the low solubility of BDPA in water alone or 50:50 water:methanol, the control sample containing BDPA alone also did not show a noticeable absorption peak in the visible region. All attempts to dissolve BDPA in water or water: methanol resulted in concentrations too low to be observed by UV−vis. For this reason, we have also included for comparison the UV−vis spectrum of approximately 50 μM BDPA in sulfolane. The maximum of this absorption peak is 488 nm. These results suggest that BDPA is incorporated into the beads, since neither BDPA in the same solvent nor beads alone show any UV−vis absorption near 500 nm. Both the HYPR-beads and the control sample with beads only exhibit a Tyndall effect: wavelength-dependent apparent absorption that is due to light scattering by suspended nanoscale particles, rather than true UV−vis absorption that is due to excitation of electrons.33,34 Figure 2 shows transmission electron microscopy images of as-received polystyrene beads and HYPR-beads. Figure 2a is an image of the beads as received. This sample was imaged after taking a sample directly from the bottle. The beads have a spherical morphology with diameters ranging from 50 to 100 nm. Figure 2b is an image of HYPR-beads, which are also spherical with diameters ranging from 50 to 100 nm. The TEM images in Figure 2c,d indicate that neither flash-freezing the HYPR-beads in liquid helium nor combining them with DMSO followed by flash-freezing disturbs the morphology of the HYPR-beads. Figure 3 shows the results of hyperpolarizing 13C carbons in 13 C-labeled urea using the BDPA-doped beads as polarizing agents. Figure 3a shows a microwave sweep performed on BDPA with dissolved 13C-urea in 50:50 DMSO:sulfolane.
Figure 2. Transmission electron microscopy images of polystyrene beads with and without BDPA. (a) Commercial beads as received (scale bar = 100 nm), (b) HYPR-beads (scale bar = 50 nm), (c) HYPR-beads after freezing in liquid helium (scale bar = 50 nm), (d) HYPR-beads that were mixed in a 60:40 ratio of DMSO:aqueous beads sample, then frozen in liquid helium (scale bar = 50 nm).
Figure 3b,c shows the results of a dissolution DNP experiment using HYPR-beads as polarizing agent in 60:40 DMSO-d6:H2O. In these figures, a time series of spectra resulting from a series of small tip-angle pulses following sample polarization and dissolution with 4 mL of water are shown. Spectra resulting from polarizing for 1 h at the low microwave frequency (ωe − ωn, 93.965 GHz, Figure 3b) increase in intensity for the first approximately 20 s, and then gradually decay to the equilibrium value. Spectra resulting from polarizing for 1 h at the high frequency (ωe + ωn, 94.060 GHz, Figure 3c) are initially negative, and then go through zero before increasing to their equilibrium value. As shown in Figure 3d, the initial increase in peak intensity may be misleading as the peaks in the first few spectra are broad. In fact, Figure 3e shows the time series of integrated intensity of the absolute value of the 13C-urea carbon 20
DOI: 10.1021/acs.jpcb.5b08741 J. Phys. Chem. B 2016, 120, 18−24
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Figure 3. (a) Microwave sweep for a sample of 13C-urea dissolved in 50:50 sulfolane:DMSO with 50 mM BDPA (1.4 K, 100 mW, 5 min polarization at each frequency). (b) Series of 13C NMR spectra of 13C-labeled urea generated by a succession of small tip-angle pulses following a dissolution DNP experiment. A total of 200 spectra were acquired, but for clarity only every fourth spectrum is shown. The sample was 13C-urea dissolved in 60:40 DMSO-d6:H2O containing HYPR-beads. A 100 μL portion of the sample was polarized at 93.965 GHz at 100 mW power and 1.54 K for 1 h, followed by dissolution with 4 mL of water. Successive spectra were acquired 1 s apart. (c) Same as in part b, but the sample was polarized at 94.060 GHz, leading to a negative signal enhancement. (d) Stacked plot showing that the initial spectra (during the first approximately 20 s following dissolution) are broadened, leading to an initial increase in magnitude of the signal height in parts b and c before the signal decays to that representing thermal equilibrium. For clarity, only every fourth spectrum is shown. (e) Peak integral of the absolute value spectra vs time after dissolution for the two sets of spectra shown in parts b and c. Both signals can be fit to an exponential decay with a T1 of 53 and 41 s for the positive and negative signal enhancements, respectively. These particular samples, used for the dissolutions in parts b−e, were not filtered.
Figure 4. Representative DNP enhancements. (a) Hyperpolarized 13C-urea, HYPR-beads in water only, microwave frequency = 94.060 GHz, enhancement factor = 112. (b) Hyperpolarized 7Li in LiCl, HYPR-beads in water only, microwave frequency = 93.939 GHz, enhancement factor = 21. (c) Control sample of 13C-urea with the same concentration of BDPA but without PS beads, water only, microwave frequency = 93.965 GHz, enhancement factor = 5.
peak in the two dissolutions. Although the initial peaks are broad, the integrated intensity indicates that maximum signal enhancement is achieved in the first spectrum, acquired 0.7 s after dissolution and settling, and that the signal integral decreases exponentially over time. The two integral time series were each fit to an exponential decay with a T1 of 53 and 41 s for the positive and negative signal enhancements, respectively. The broadness of peaks for the first 20 s after dissolution merits discussion. This broadness of peaks in the initial spectra is seen in all experiments using beads as the polarizing agent, but is not seen in corresponding experiments, for example, when BDPA is used to hyperpolarize carbons in toluene. This initial line broadening may be due to the presence of large bead particles in the solution, which may increase the time required for the sample to fully settle within the NMR tube after the dissolution, resulting in bad shimming in initial experiments.
Since the BDPA is incorporated into the polystyrene beads and not dispersed directly in the solvent, we wanted to test whether HYPR-beads could be used to hyperpolarize a watersoluble analyte in pure water, without the addition of a glassing cosolvent such as DMSO or glycerol. We thus performed the same experiment dissolving 13C-urea in an aqueous sample of HYPR-beads, without the addition of a glassing cosolvent. Polarization and dissolution were done as described above. This lead to even higher enhancement factors than the experiment in which the sample was dissolved in 60:40 DMSO-d6:H2O. Representative enhancements are shown in Figure 4. In Figure 4a, the result of polarizing 13C-urea in water alone using HYPRbeads is shown. The enhancement factor is 112, compared to enhancements of 20−22 for similar experiments in DMSO:water. The higher enhancement factor in pure water may be due to the increased overall concentration of BDPA in the 21
DOI: 10.1021/acs.jpcb.5b08741 J. Phys. Chem. B 2016, 120, 18−24
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enhancement factors that are about half of that which is attainable with TEMPO under similar conditions. Enhancement factors are heavily dependent on factors such as the time required for sample shuttling between the DNP polarizer and NMR magnet, and small fluctuations in the minimum temperature that the HyperSense is able to reach on that particular day, so should not be considered reproducible in general. Nonetheless, the fact that positive and negative enhancements are seen at microwave frequencies ωe − ωn and ωe + ωn, respectively, indicates that the signal enhancement seen in dissolution DNP experiments using HYPR-beads as polarization agents is in fact coming from DNP, and not from a difference in thermal equilibrium polarization. The tunable nature of HYPR-beads will presumably allow higher enhancement factors to be achieved in subsequent generations of HYPR-beads.
sample, since adding DMSO effectively dilutes the sample. A similar dissolution experiment using the control sample with BDPA only without beads led to an enhancement factor of 5 (Figure 4c), which could be attributed to the difference between the thermal equilibrium polarization at 1.54 K and at room temperature. We also wanted to see whether our HYPR-beads could be used to polarize other water-soluble samples as well as nuclei other than 13C. For this we dissolved LiCl in the aqueous sample of HYPR-beads (again no glassing cosolvent was used) and observed the enhancement of the 7Li nucleus. As can be seen in Figure 4b, good DNP enhancements were seen for this nucleus as well. This is an important nucleus in clinical applications, as lithium is used as a treatment in bipolar disorder. Lithium has two NMR-active nuclei, both quadrupolar. 7Li (S = 3/2) is the most abundant (92.58%) while 6Li (S = 1) is often difficult to observe due to its low γ and low natural abundance (7.42%), but would be an ideal target for hyperpolarized studies due to its relatively long T1 relaxation time for a quadrupolar nucleus. Indeed, hyperpolarized 6Li has been observed in the brain.35 No signals were observed from the polystyrene carbons in the HYPR-beads. The close proximity of the BDPA radicals to the PS carbons is expected to decrease the PS carbon T1 relaxation times, so that much of the hyperpolarization may be lost during the sample transfer from the polarizer to the NMR magnet. Additionally, in the absence of hyperpolarization, we observed only very broad 13C NMR signals from the beads, both with or without BDPA. Because of their large size, PS nanoparticles are expected to tumble slowly in solution, leading to incomplete averaging of dipolar interactions and shorter T2 relaxation times. This may also contribute to a lack of observable signal from the PS beads themselves. Both this difficulty and the increased line width in spectra immediately after dissolution are expected to be worse with larger PS particles. The fact that BDPA is dispersed throughout the polystyrene beads allows for efficient hyperpolarization, even though the net concentration of BDPA in the final sample is low. The final concentration of BDPA in the aqueous HYPR-beads dispersion is expected to be no more than 2 mM, considering that a total of 0.95 μmol of BDPA is added during the sample preparation stage, and the final overall volume is 0.5 mL. This concentration is an overestimation, assuming that no BDPA is lost during the filtering step. By contrast, BDPA concentrations of 40 mM are generally used in DNP studies.23 The enhancement factors we obtained using HYPR-beads are notable, but are far from enhancement factors in the 10 000fold range that are obtainable7 using dissolution DNP. In order to compare the performance of HYPR-beads with that of known water-soluble polarization agents, the results of several control dissolution DNP experiments are presented in the Supporting Information. The water-soluble radical 4-hydroxyTEMPO was used to hyperpolarize 13C-urea in water alone and in water with DMSO as an added glassing cosolvent. An enhancement factor of ∼2000 was found using 40 mM 4hydroxy-TEMPO in 60:40 DMSO−D2O, which represents the optimized conditions for use of the TEMPO radical. When the glassing cosolvent was removed, the enhancement dropped to 211, and when a concentration of 10,000 Times in Liquid-State NMR. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 10158−10163. (8) Nelson, S. J.; Kurhanewicz, J.; Vigneron, D. B.; Larson, P. E. Z.; Harzstark, A. L.; Ferrone, M.; van Criekinge, M.; Chang, J. W.; Bok, R.; Park, I.; Reed, G.; Carvajal, L.; et al. Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-C-13]Pyruvate. Sci. Transl. Med. 2013, 5, 198ra108. (9) Johansson, E.; Månsson, S.; Wirestam, R.; Svensson, J.; Petersson, J. S.; Golman, K.; Ståhlberg, F. Cerebral Perfusion Assessment by Bolus Tracking Using Hyperpolarized 13C. Magn. Reson. Med. 2004, 51, 464−472. (10) Brindle, K. M. Imaging Metabolism with Hyperpolarized 13CLabeled Cell Substrates. J. Am. Chem. Soc. 2015, 137, 6418−6427. (11) Rossini, A. J.; Zagdun, A.; Lelli, M.; Lesage, A.; Copéret, C.; Emsley, L. Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy. Acc. Chem. Res. 2013, 46, 1942−1951. (12) Grüning, W. R.; Rossini, A. J.; Zagdun, A.; Gajan, D.; Lesage, A.; Emsley, L.; Copéret, C. Molecular-Level Characterization of the Structure and the Surface Chemistry of Periodic Mesoporous Organosilicates Using DNP-Surface Enhanced NMR Spectroscopy. Phys. Chem. Chem. Phys. 2013, 15, 13270−13274. 23
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