Examining Binding to Nanoparticle Surfaces Using Saturation Transfer

Oct 31, 2017 - The interaction of molecules with the surface of nanoparticles is important in many fields of study, including drug delivery and nanopa...
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Examining Binding to Nanoparticle Surfaces Using Saturation Transfer Difference (STD)-NMR Spectroscopy Yunzhi Zhang, Hui Xu, Austin M. Parsons, and Leah B. Casabianca* Department of Chemistry, Clemson University, Clemson, South Carolina 29634, United States S Supporting Information *

ABSTRACT: The interaction of molecules with the surface of nanoparticles is important in many fields of study, including drug delivery and nanoparticle toxicity. Solution-state NMR has the potential to provide structural as well as dynamic information regarding molecules adsorbed to the nanoparticle surface. Here, we use Saturation-Transfer Difference NMR (STD-NMR) to examine small molecules binding to the surface of polystyrene nanoparticles. Binding constants for this nonspecific adsorption are determined from the initial slope of the STD buildup curve at several ligand concentrations. We also use the STD-NMR technique to quantify the association of solvent water molecules with the nanoparticle surface. The results presented here will be useful to future studies involving peptides and proteins adsorbed on nanoparticle surfaces.



INTRODUCTION The interaction of nanoparticle surfaces with biological molecules such as peptides and proteins has importance in the fields of nanoparticle sensing, drug delivery, and nanoparticle toxicity. Pristine nanoparticle surfaces do not exist in a biological environment because nanoparticles are immediately surrounded by a corona of proteins as soon as they enter the body.1−14 It is this protein corona, and not the nanoparticle itself, that governs interactions of the nanoparticle with its environment.1,10,15−19 When corona proteins bind to a nanoparticle, they change their structure to maximize favorable interactions with the nanoparticle surface.20 When a protein changes structure, active sites or cell recognition segments may be hidden21,22 or previously sheltered hydrophobic groups may become exposed,14 leading to a loss or change of protein function.23 If we can develop a way to determine the structure of proteins or small molecules that are adsorbed on a nanoparticle surface in solution, we can tune these interactions of the nanoparticle, improving sensing and bioavailability and reducing nanoparticle toxicity.24−27 This will be especially important when investigating new size regimes such as ultrasmall nanoparticles (those with core diameters in the range of 1−3 nm).28 Since these nanoparticles are too small to be resolved by conventional techniques that are currently used to study nanoparticle−biomolecule interactions,28 new characterization techniques will be required. Nuclear Magnetic Resonance (NMR) Spectroscopy is one of the most powerful tools for molecular structure determination. The structure of molecules covalently adsorbed to nanoparticle surfaces can be determined using solid-state NMR29−47 and solution-state NMR when the ligand is flexible enough to be observed in solution.48−50 Solution-state NMR can also be used to study molecules that are noncovalently adsorbed on the © XXXX American Chemical Society

nanoparticle surface. NMR has the potential to provide not only structural information about adsorbed molecules but can also provide information about the dynamics of adsorption and desorption in an aqueous environment.8,51,52 One of the main difficulties in studying molecules adsorbed to the surface of nanoparticles by solution-state NMR is that once the molecule attaches to the nanoparticle, its motion in solution will resemble that of the large nanoparticle. This slow molecular tumbling leads to short nuclear T2 relaxation times and broad peaks in solution-state NMR. In the case of nanoparticles, this broadening can become so severe that the peaks are broadened into the baseline and essentially disappear. There are, however, several solution-state NMR techniques that rely on observing the free ligand that is in equilibrium with bound ligand in order to get information about the ligand in the bound state.53−59 These techniques, including measuring diffusion coeffieients60 and Diffusion Ordered Spectroscopy (DOSY), 61 Nuclear Overhauser Effect Spectroscopy (NOESY),62 hydrogen/deuterium exchange, and chemical shift analysis, are able to give information about molecules adsorbed to nanoparticle surfaces. One additional technique that has been suggested for studying nanoparticle-adsorbed molecules is the Saturation-Transfer Difference (STD) experiment.63−65 STD-NMR has traditionally been applied to study small-molecule ligands binding to protein receptors. As the name implies, STD-NMR relies on taking the difference of two spectra: an “on-resonance” spectrum and a reference “offresonance” spectrum. The on-resonance experiment is performed by saturating a region of the NMR spectrum in Received: September 5, 2017 Revised: October 10, 2017

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protein corona,107 in imaging studies of nanoparticles crossing in vitro blood-brain barriers,108 to investigate the mechanism of endocytotic uptake of nanoparticles into endothelial cells and macrophages,109 and to examine the effects of nanoscale plastic exposure on brine shrimp.110 We also examined the interaction of solvent water molecules with the nanoparticle bead surface. Water is often thought of as a passive solvent, but water molecules interact with the bead surface and receive some transfer of saturation from the large receptor. Additionally, in order for molecules of interest to adhere to a nanoparticle surface, solvent molecules must be displaced, so it is important to know the binding constant and kinetics of water− nanoparticle interactions. The work described here represents a first step toward examining larger molecules such as peptides and proteins binding to nanoparticle surfaces by the STD-NMR method.

which receptor protons are expected to resonate, even though they may not be visible due to the slow molecular tumbling of the large protein receptor. Saturation is then transferred through spin diffusion through the entire receptor, including the ligand binding pocket, and eventually to any bound ligands. These ligands will be observed in solution once they dissociate from the receptor, but with reduced peak intensity due to a partial transfer of saturation from the receptor. A proper onresonance saturation frequency must be chosen to be far from any of the ligand resonances, so that the ligand protons are not directly saturated. The off-resonance experiment is a reference experiment in which saturation is performed far from any ligand or receptor resonances. Ligands that do not bind to the receptor do not receive any saturation transfer, and their peaks have the same intensity in the on-resonance and off-resonance spectra. A difference spectrum, which is the off-resonance spectrum minus the on-resonance spectrum, clearly shows peaks from only those ligands that bind to the receptor. In addition to identifying molecules that bind to a receptor, the STD method can also give information about precise binding epitopes on the ligand.64,66 The COmplete Relaxation and Conformational Exchange MAtrix (CORCEMA) theory67−71 can be used to predict the magnitude of the STD effect as a function of saturation time, ligand and receptor correlation times, relaxation rates, and atomic distances between ligand and receptor protons. Results from CORCEMA analysis can then be used to evaluate and refine suggested protein−ligand structures.72−85 Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY)86,87 is another liganddetected solution-state NMR technique for observing binding to large, invisible receptors. WaterLOGSY relies on polarization transfer from bulk water to a bound ligand through bound water molecules at the receptor−ligand interface.86,87 The STD technique has been used extensively in drug screening studies, usually to detect binding of a small molecule ligand to a target receptor protein.64−66,88−101 It has also been used to study larger “receptors” including interactions between RNA and proteins102 and for a metal−organic-framework catalyst.103 Since STD-NMR is a ligand-detected technique, it should be an ideal method for studying binding with even larger receptors, including nanoparticles. STD-NMR has been suggested as an addition to the nanoparticle NMR “toolbox,”51 and has been used to examine the interactions between dispersant molecules and organic nanoparticles made of pigment red 122 molecules.104 A variation of the STD method, in which bound water molecules at the nanoparticle surface are saturated, has been used to determine the structure of Tibinding peptide bound to titanium nanoparticles.105 The STD technique relies on spin diffusion through a dipolar-coupled network of spins in the receptor. Thus, the STD technique should be applicable to any organic nanoparticle containing a dipolar-coupled network of protons.104 However, there are still many challenges to be addressed before STD-NMR can be widely used as a general technique for examining interactions at the surface of nanoparticles. In this work, we have employed the STD-NMR technique to observe small molecules binding to the carboxylate-modified surface of polystyrene nanoparticle beads. To our knowledge, this is the first time that the STD-NMR technique has been applied to widely-used organic nanoparticles that are relevant to biological nanoparticle research. The same kind of nanoparticle beads that we use here have been used as polarization agents for dynamic nuclear polarization,106 models for studying the



MATERIALS AND METHODS All reagents and solvents were purchased from commercial suppliers and used as received. Carboxylate-modified (CML) polystyrene latex spheres in sizes of 20, 40, and 100 nm were purchased from ThermoFisher Scientific (Waltham, MA, USA). Isopropanol (laboratory grade) and deuterium oxide (99.8 atom % D, Acros Organics) were purchased from Fisher Scientific (Hampton, NH, USA). Samples were prepared by combining an appropriate amount of isopropanol (by weight), beads, and H2O or D2O. The polystyrene beads are supplied as suspensions in water and were exchanged into D2O when necessary by concentrating the sample through an Amicon centrifugal filter unit (100 kDa) and diluting the concentrated beads with the desired solvent. Final concentrations of isopropanol and water in each sample were determined by taking the integral of the 1D proton spectrum. The polystyrene beads are supplied as 4% w/v and were diluted to 2% w/v in the final samples. All NMR measurements were made on a Bruker Avance 500 MHz spectrometer operating at a proton frequency of 500.1324 MHz and equipped with an automatic tuning and matching BBO probe. For the STD experiments, the Bruker “stddiff” pulse sequence was used. Solvent suppression was not employed. Saturation was achieved with a train of Gaussian pulses, each 50 ms long at a power of approximately 0.8 mW. The total saturation time ranged from 0.5 to 12 s. On- and offresonance STD spectra were collected in an interleaved fashion. For the on-resonance spectrum, saturation was performed at 12 ppm, and for the off-resonance spectrum, saturation was performed at 40 ppm. A recycle delay of 15 s, acquisition time of 2 s, and spectral width of 10 ppm were used, and 16 scans (following 2 dummy scans) were averaged for each measurement. Measurements were carried out at 295.0 ± 0.2 K or 296.0 ± 0.2 K. Spectra were processed using Bruker Topspin version 2.1 Software. Processed spectra were read into MATLAB (MathWorks, Natick, MA, USA), and peak integrals were calculated using a custom-written MATLAB script.



RESULTS AND DISCUSSION An illustration of the potential of STD-NMR experiments for screening ligands binding to the functionalized surface of polystyrene nanoparticles is shown in Figure 1. This figure is a portion of the 1H STD-NMR spectrum of a mixture of glucose and ethanol in the presence of 20 nm carboxylate-functionalized polystyrene beads. Glucose and ethanol peaks are both B

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spectrum of this sample indicates that the STD effect seen in Figure 2a is indeed coming from saturation of the beads, followed by transfer of saturation from the beads to the isopropanol protons. Comparing the STD difference spectra in parts (a) and (b) in Figure 2 also indicates that 12 ppm is an appropriate on-resonance saturation frequency. The presence of peaks in the STD difference spectrum in Figure 2a indicates that 12 ppm is a region in which the polystyrene beads can be saturated, and the absence of peaks in the STD difference spectrum in Figure 2b indicates that saturating at 12 ppm does not lead to direct saturation of the isopropanol protons in the absence of polystyrene beads. Figure 3 shows the STD effect (defined as the STD difference intensity divided by the reference intensity66) as a function of saturation time for the methyl and CH protons of isopropanol in the presence of 20, 40, and 100 nm polystyrene beads. As expected, the STD effect builds up exponentially with saturation time before reaching a maximum at long saturation times. As the isopropanol concentration increases, the maximum STD effect decreases. The STD effect can be multiplied by the ligand excess to give the STD Amplification Factor66 (STD-AF):

Figure 1. 1H Saturation transfer difference (STD)-NMR results for a mixture of 1 M glucose and 1 M ethanol in the presence of 2% w/v 20 nm carboxylate-modified polystyrene beads. Ethanol binds to the beads, leading to peaks in the STD difference spectrum (red) at 3.52 and 1.05 ppm. The remaining peaks in the reference spectrum (blue) are attributed to glucose. Glucose does not interact with the beads, so the glucose peaks are not present in the STD difference spectrum.

seen in the reference spectrum, shown in blue in this figure. In the red difference spectrum, however, only the ethanol peaks appear. This indicates that ethanol binds to the nanoparticles and receives some of the saturation, while glucose does not interact with the nanoparticle surface. This figure clearly shows the ease with which the STD experiment can be used to quickly screen for ligands that bind to a receptor by highlighting only those resonances corresponding to binding ligands. Here, we have shown that the STD-NMR technique can be used to screen for small molecules binding to organic nanoparticles. Through experiments similar to those shown in Figure 1, we found that ethanol and isopropanol bind to the nanoparticle surface, while glucose and glycine do not exhibit any measurable interactions with the nanoparticle surface. To confirm that we are actually observing transfer of saturation from the nanoparticle to the ligand and not some other effect, we performed the same experiment for isopropanol in the presence and absence of beads. The reference and difference STD-NMR spectra of isopropanol in the presence and absence of 20 nm CML polystyrene beads are shown in Figure 2. In

ISTD I0

(1)

ISTD [L] I0 [R]

(2)

STD Effect =

STD‐AF =

In the above equations, ISTD is the integral of a given peak in the STD difference spectrum, I0 is the integral of the same peak in the reference spectrum, [L] is the ligand concentration, and [R] is the receptor or nanoparticle concentration. It should be noted that, technically, [R] should refer to the concentration of ligand binding sites. At present, we have no knowledge of the number of isopropanol binding sites per nanoparticle. However, we can calculate the relative STD-AF for each ligand concentration by dividing by a constant proportional to the surface area for each size bead. This amounts to determining a value that is proportional to the actual STD-AF. By examining the STD-AF as a function of isopropanol concentration and fitting the results to a binding isotherm, we can determine the dissociation constant, KD , between isopropanol and the carboxylate-modified beads. Although KD is generally used to refer to specific binding, here we will use it to describe the nonspecific adsorption that occurs between a small molecule and a functionalized nanoparticle surface. Angulo et al.111 have shown that ligand rebinding can contribute to an overestimation of the KD when long saturation times and large ligand concentrations are used. In order to alleviate these effects, they recommend using the initial slope of the STD buildup curve, STD-AF(0), to fit to a binding isotherm, and this is what we have done here. The initial growth rate of the STD-AF can be calculated by fitting the STD-AF buildup curve to an exponential equation and taking the derivative at time zero:111

Figure 2. (a) STD reference (black) and difference (red) spectra for 1.3 M isopropanol in the presence of 2% w/v 20 nm carboxylatemodified polystyrene beads. (b) Same as in (a) but for a sample of 1.3 M isopropanol in the absence of beads.

Figure 2a, interaction of isopropanol with the bead surface is clearly seen from the appearance of both the CH3 and CH groups of isopropanol in the STD difference spectrum. Figure 2b contains STD reference and difference spectra for an identical sample of isopropanol, but without polystyrene beads. The absence of isopropanol peaks in the STD difference

STD‐AF(t ) = STD‐AF(max)[1 − exp(−kt )]

(3a)

STD‐AF(0) = STD‐AF(max)[k]

(3b)

where t is the saturation time and k is a buildup time constant. Figure 4 shows plots of STD-AF(0) versus isopropanol concentration. These curves are fit to a Langmuir isotherm112 C

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Figure 3. Buildup of the STD effect with increasing saturation time for various concentrations of isopropanol in the presence of polystyrene nanoscale beads. (a−c) CH3 protons of isopropanol, (d−f) CH proton of isopropanol: (a, d) 20 nm beads, (b, e) 40 nm beads, (c, f) 100 nm beads. The concentration of isopropanol is shown in the legend. Polystyrene beads were 2% w/v in all samples.

Figure 4. STD-AF(0) as a function of isopropanol concentration. STD-AF(0) was calculated from eq 3b. Black diamonds are experimental data points and red lines are fits to Langmuir isotherms as in eq 4. The value of KD obtained from the fit is shown on each plot. (a−c) CH3 protons of isopropanol, (d−f) CH proton of isopropanol: (a, d) 20 nm beads, (b, e) 40 nm beads, (c, f) 100 nm beads.

STD‐AF(0) =

STD‐AF(0)max [L] KD + [L]

dissociation constant for the 40 nm beads is the smallest. Although all three binding constants represent weak nonspecific binding, and the differences between the binding constants are small, this could reflect a slight preference for the specific shape and curvature of the 40 nm diameter beads. In all cases, the STD effect for the CH proton of isopropanol is greater than the STD effect for the CH3 protons of isopropanol, indicating that the CH group interacts more strongly with the nanoparticle surface and that the isopropanol molecule binds to the carboxylate-modified nanoparticles through its OH group. Similar studies with larger, more complicated molecules are currently underway. In the case of larger molecules, the different STD effects for protons on different parts of the molecule may be compared to determine which part of the molecule is most strongly involved in binding.

(4)

and the dissociation constant KD is found from a best fit. According to eq 4, plotting values that are proportional to the real STD-AF instead of the real STD-AF will affect the resulting value of STD-AF(0)max, but not the value of KD that is determined from this fit. Dynamic light scattering experiments, described in the Supporting Information, indicate that these concentrations of isopropanol did not lead to swelling of the beads. When comparing interactions between isopropanol and 20, 40, and 100 nm beads, it appears that isopropanol binds the 40 nm beads most strongly, followed by the 100 nm beads and then the 20 nm beads. The STD effects are larger for the 40 nm beads than for the other two bead sizes, and the calculated D

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Figure 5. Buildup of the STD effect for the residual water peak of D2O in the presence of isopropanol and polystyrene nanobeads. (a) 20 nm beads, (b) 40 nm beads, (c) 100 nm beads. The concentration of isopropanol is shown in the legend of each plot. Polystyrene beads were 2% w/v in all samples.

Figure 6. Effect of H2O:D2O ratio on the buildup of the STD effect for isopropanol and water protons in the presence of 20 nm polystyrene nanobeads. (a) isopropanol CH3 peak, (b) isopropanol CH peak, (c) water peak. The percent H2O is shown in the legend of each plot.

hydrogen nuclei in H2O solvent leads to enhanced relaxation, preventing the saturation transfer from reaching its maximum due to spin diffusion. Thus, STD enhancements in H2O are expected to reach a lower maximum and to reach the maximum more quickly than experiments done in D2O.113 However, using the initial slope of the STD buildup curve should reduce the effect of different relaxation times on the KD values obtained.111 Figure 6 shows STD buildup curves for the isopropanol and water protons for samples of isopropanol with 20 nm beads at different H2O:D2O ratios. Since the isopropanol concentration was kept constant in all of these samples, the initial slope of the isopropanol buildup curve does not depend on the water concentration. The initial slope of the buildup curve for the water peak, however, decreases with increasing amount of H2O present. These initial slopes can be fit to a Langmuir isotherm to determine a KD for water binding to the surface of the nanoparticles, as shown in Figure 7.

Since no solvent suppression was employed in these experiments, we were able to observe the water proton peak in the STD difference spectrum. Water and other solvents are often considered passive observer molecules, but in this case, the solvent does interact with the nanoparticle surface and receives some transfer of saturation from the nanoparticles. Figure 5 shows the STD buildup curves of the water proton peak as a function of saturation time for the same samples that were used to create Figure 3. The water STD effect builds up with increasing saturation time, eventually reaching a plateau. Similar to the behavior of the STD effects for isopropanol protons in Figure 3, the STD effects for the water protons reach a lower maximum value more quickly as the isopropanol concentration increases. However, the case of the water protons is different from that of the isopropanol protons in that the initial slope of the STD buildup curve is independent of the concentration of isopropanol. This is expected since the residual water concentration in all of these samples is nearly the same. The water STD buildup curves shown in Figure 5 are an excellent illustration of the effect described by Angulo et al.111 in which the initial slope of the STD buildup curve is a more accurate number to use for calculating the dissociation constants than the STD effect at a particular saturation time. The STD effect of the water peak in the presence of different amounts of isopropanol builds up with the same initial growth rate, but then reaches a different maximum depending on the isopropanol concentration. Since water was seen to interact with the surface of the nanoparticles as observed by the presence of the water peak in the STD difference spectrum, we considered water as a ligand and performed STD experiments at different values of water concentration in order to determine the binding constant between water and the polystyrene beads. The water “concentration” was varied by changing the H2O:D2O ratio of the solvent. Changing the H2O:D2O ratio, however, will also affect the STD results due to the different relaxation times of the ligand protons in H2O and D2O. The large number of



CONCLUSIONS In the current work, we have shown that the STD-NMR experiment can be used to detect small molecules binding to

Figure 7. STD-AF(0) as a function of water percent. STD-AF(0) was calculated from eq 3b using the initial slopes of the STD buildup curves in Figure 6c. Black diamonds are experimental data points and the red line is a fit to a Langmuir isotherm as in eq 4. E

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(4) Zanganeh, S.; Spitler, R.; Erfanzadeh, M.; Alkilany, A. M.; Mahmoudi, M. Protein Corona: Opportunities and Challenges. Int. J. Biochem. Cell Biol. 2016, 75, 143−147. (5) Cedervall, T.; Lynch, I.; Foy, M.; Berggård, T.; Donnelly, S. C.; Cagney, G.; Linse, S.; Dawson, K. A. Detailed Identification of Plasma Proteins Adsorbed on Copolymer Nanoparticles. Angew. Chem., Int. Ed. 2007, 46, 5754−5756. (6) Docter, D.; Westmeier, D.; Markiewicz, M.; Stolte, S.; Knauer, S. K.; Stauber, R. H. The Nanoparticle Biomolecule Corona: Lessons Learned − Challenge Accepted? Chem. Soc. Rev. 2015, 44, 6094−6121. (7) Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the NanoparticleProtein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 2050−2055. (8) Ceccon, A.; Tugarinov, V.; Bax, A.; Clore, G. M. Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy. J. Am. Chem. Soc. 2016, 138, 5789−5792. (9) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14265−14270. (10) Lundqvist, M.; Stigler, J.; Cedervall, T.; Berggård, T.; Flanagan, M. B.; Lynch, I.; Elia, G.; Dawson, K. The Evolution of the Protein Corona around Nanoparticles: A Test Study. ACS Nano 2011, 5, 7503−7509. (11) Milani, S.; Baldelli Bombelli, F.; Pitek, A. S.; Dawson, K. A.; Rädler, J. Reversible versus Irreversible Binding of Transferrin to Polystyrene Nanoparticles: Soft and Hard Corona. ACS Nano 2012, 6, 2532−2541. (12) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; et al. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol. 2013, 8, 772−781. (13) Shao, Q.; Hall, C. K. Allosteric Effects of Gold Nanoparticles on Human Serum Albumin. Nanoscale 2017, 9, 380−390. (14) Lynch, I.; Dawson, K. A.; Linse, S. Detecting Cryptic Epitopes Created by Nanoparticles. Sci. Signaling 2006, 2006, pe14. (15) Monopoli, M. P.; Åberg, C.; Salvati, A.; Dawson, K. A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials. Nat. Nanotechnol. 2012, 7, 779−786. (16) Lesniak, A.; Fenaroli, F.; Monopoli, M. P.; Åberg, C.; Dawson, K. A.; Salvati, A. Effects of the Presence or Absence of a Protein Corona on Silica Nanoparticle Uptake and Impact on Cells. ACS Nano 2012, 6, 5845−5857. (17) Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Baldelli Bombelli, F.; Hristov, D. R.; Kelly, P. M.; Åberg, C.; Mahon, E.; Dawson, K. A. Transferrin-Functionalized Nanoparticles Lose Their Targeting Capabilities When a Biomolecule Corona Adsorbs on the Surface. Nat. Nanotechnol. 2013, 8, 137−143. (18) Hoshino, Y.; Koide, H.; Furuya, K.; Haberaecker, W. W.; Lee, S.-H.; Kodama, T.; Kanazawa, H.; Oku, N.; Shea, K. J. The Rational Design of a Synthetic Polymer Nanopartide that Neutralizes a Toxic Peptide In Vivo. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 33−38. (19) O’Connell, D. J.; Baldelli Bombelli, F.; Pitek, A. S.; Monopoli, M. P.; Cahill, D. J.; Dawson, K. A. Characterization of the Bionano Interface and Mapping Extrinsic Interactions of the Corona of Nanomaterials. Nanoscale 2015, 7, 15268−15276. (20) Burkett, S. L.; Read, M. J. Adsorption-Induced Conformational Changes of α-Helical Peptides. Langmuir 2001, 17, 5059−5065. (21) Lo Giudice, M. C.; Herda, L. M.; Polo, E.; Dawson, K. A. In Situ Characterization of Nanoparticle Biomolecular Interactions in Complex Biological Media by Flow Cytometry. Nat. Commun. 2016, 7, 13475. (22) Herda, L. M.; Hristov, D. R.; Lo Giudice, M. C.; Polo, E.; Dawson, K. A. Mapping of Molecular Structure of the Nanoscale Surface in Bionanoparticles. J. Am. Chem. Soc. 2017, 139, 111−114.

the surface of organic nanoparticles composed of a polystyrene latex. Alcohols such as ethanol and isopropanol interact with the carboxylate surface of the nanobeads and receive transfer of saturation from the protons in the bead, so that their proton peaks appear in the STD difference spectrum. Other molecules, such as glucose and glycine, do not interact with the bead surface. Dissociation constants for isopropanol binding to carboxylate modified beads were found to be between 2 and 6 M, indicating weak nonspecific binding, but binding that is substantial enough to result in an STD effect. By fitting to Langmuir isotherms, these binding constants could be determined even without knowledge of the number of binding sites per nanoparticle. We have also considered the interaction of solvent water molecules with the surface of these organic nanoparticles, and the transfer of saturation from the nanoparticles to water at the same time that saturation is being transferred to a small molecule ligand. We treated water as a ligand by changing the H2O:D2O ratio of solvent in the samples and found that the resulting initial slopes of the STD-AF buildup curves could be fit to a Langmuir isotherm with a dissociation constant of 40 M. To our knowledge, this is the first time water binding to the surface of a nanoparticle has been studied by the STD-NMR technique, and the methods described here could be useful in interpreting STD-NMR results relating to exchangeable hydrogen atoms.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b08828. Experimental details and results of dynamic light scattering experiments, and full refs 12, 77, 79, 83−85, 96, and 101 (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Leah B. Casabianca: 0000-0001-9447-3236 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS A.M.P. was supported by the NSF Research Experiences for Undergraduates program (Grant No. CHE-1560300). Clemson University and the Department of Chemistry are thanked for startup funding. We thank Prof. Jason McNeill for helpful suggestions.



REFERENCES

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