HR-MAS NMR Spectroscopy - American Chemical Society

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HR-MAS NMR Spectroscopy: An Innovative Tool for the Characterization of Iron Oxide Nanoparticles Tracers for Molecular Imaging Céline Henoumont,† Sophie Laurent,† Robert N. Muller,†,‡ and Luce Vander Elst*,†,‡ †

Department of General, Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, 19 Avenue Maistriau, B-7000 Mons, Belgium ‡ Center for Microscopy and Molecular Imaging (CMMI), 8 Rue Adrienne Boland, 6041 Gosselies, Belgium S Supporting Information *

ABSTRACT: The development of molecular imaging by MRI requires the use of contrast agents able to recognize specifically a peculiar target at the molecular level. Iron oxide nanoparticles grafted with small organic molecules represent an interesting platform for molecular imaging. The characterization of the surface of these nanoparticles is an important step in the development of these molecular agents, and HR-MAS NMR spectroscopy appears to be a very interesting tool. The use of 1D and 2D NMR spectra is indeed very helpful to investigate the covalent grafting of organic molecules at the nanoparticle surface. DOSY spectra could also be very helpful, but we will show here that it is not possible to obtain accurate DOSY spectra on iron oxide nanoparticles.

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Infrared spectrocopy allows the identification of functional groups present on the nanoparticle surface. Neither of these techniques allow the precise identification of the organic molecules present on the nanoparticle surface and prove their covalent grafting. NMR could be a very interesting tool and was already used for the characterization of different types of diamagnetic nanoparticles.12−17 Unfortunately, the superparamagnetic character of iron oxide nanoparticles provides such broadened lines that it completely prevents the use of liquid NMR. Nevertheless, Polito et al. have published an article in 2008 showing the possibility to use high-resolution magic angle spinning (HR-MAS) spectroscopy to study organic molecules grafted on iron oxide nanoparticle surface.18 HR-MAS spectrocopy is usually used to study heterogeneous media, which has a liquid/ solid interface. The rotation of the sample at the so-called magic angle of 54.7° will indeed suppress the dipolar interactions, the chemical shift anisotropy and also the anisotropy due to magnetic susceptibility differences at the interface between the solid and the liquid phase. (Note: The magic angle (θ = 54.7°) is the solution of the eq 3cos2θ − 1 = 0. The dipolar interaction between two spins is directly proportional to this equation, and consequently, the fast rotation of a sample at this magic angle of 54.7° will average the dipolar interactions in the sample to 0, allowing the narrowing

edical imaging has evolved for several years toward molecular imaging. Molecular imaging aims at the noninvasive visualization of the molecular process at the origin of some diseases, in order to improve their diagnosis. It can also allow the observation of the specific delivery of drugs, in order to improve the treatment of pathologies. Molecular imaging has the potential to become an invaluable tool in the near future. Molecular probes for MRI have to be very efficient (i.e., able to highly modify the contrast in the pathological area) and very specific (i.e., able to recognize specifically a molecule or an entity expressed or overexpressed in pathological conditions). Iron oxide nanoparticles represent a very interesting platform for molecular imaging because they are endowed with a high relaxivity, and thus efficacy, and they offer the possibility of receiving a graft of several targeting molecules on their surface .1−8 (Note: The relaxivity of an MRI contrast agent is the increase of the water protons’ relaxation rate induced by 1 mmol per liter of the contrast agent. The higher the relaxivity, the higher the efficacy of the contrastophore.) However, an important challenge remains in the precise characterization of the surface of the particles, particularly examining the evidence of the covalent grafting of the organic molecules on the particle surface. The two more commonly used techniques to characterize iron oxide nanoparticle surface are X-ray photoelectron spectrometry (XPS) and Fourier transformed infrared spectroscopy (FTIR).9−11 The XPS technique allows an elemental analysis of the surface, providing information on the chemical environment and on the chemical bonds of the different atoms. © XXXX American Chemical Society

Received: September 18, 2014 Accepted: December 28, 2014

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DOI: 10.1021/ac5035105 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry of the lines.19) HR-MAS spectroscopy obtains an NMR spectrum of organic molecules being at a solid/liquid interface as if they were in solution. The lines will nevertheless be thiner if the conformational mobility at the interface is isotropic. HRMAS spectroscopy is thus usually used to study tissues or cells,20−23 to follow chemical reactions on a solid support, like the peptidic synthesis,24−27 or also to study the mechanism of chromatographic separations.28,29 The use of HR-MAS to study the surface of iron oxide nanoparticles is quite new, and surprisingly, to the best of our knowledge, it has never been used in the literature to characterize iron oxide nanoparticles, except in the paper of Polito et al.18 HR-MAS spectroscopy has, however, the potential of becoming a very important and interesting tool to characterize the surface of iron oxide nanoparticles because, thanks to the fast rotation of the sample at the magic angle, it allows the investigator to average the magnetic heterogeneities around the nanoparticles, significantly narrowing the lines. The small molecules grafted on the nanoparticles can thus be easily detected and identified, by using 1D or even 2D NMR spectra. The covalent grafting of the molecules can also be easily demonstrated. To this aim, 1D spectra already provide substantial information, but DOSY spectra could also be very helpful. The diffusion coefficient of the small organic molecules should indeed drastically decrease when they are grafted on the iron oxide nanoparticles. Nevertheless, the recording of accurate DOSY spectra is not easy in HR-MAS because of the fast spinning of the sample, which can cause a centrifugallike effect and affect the diffusion coefficient measurement. Some precautions have to be taken. In 2008, Viel et al.30 showed that accurate diffusion coefficients can be obtained only if the rotor active volume is kept to a minimum (i.e., a rotor of 12 μL has to be used instead of a rotor of 50 μL) and if the spinning rate is kept between 3 and 6 kHz. If these two precautions are not fulfilled, the measurements are either overestimated or totally unreliable. This is well illustrated in the work of Polito et al.,18 where the authors present DOSY spectra of iron oxide nanoparticles recorded with an HR-MAS probe by using a spinning rate of 8 kHz and a rotor of 50 μL. The presented diffusion coefficients are completely overestimated and unreliable, because they obtained a diffusion coefficient higher than that of water for an organic molecule of approximately 550 g/mol grafted on an iron oxide nanoparticle. In this study, we have tried to record DOSY spectra on our iron oxide nanoparticles by taking into account the precautions cited by Viel et al. This study was performed on two types of iron oxide nanoparticles: the first ones are grafted with polyethylene glycol (PEG), and the second ones are grafted with a small peptide of six amino acids (Chart 1). PEG is needed to inject the nanoparticles in vivo in order to avoid a nonspecific capture by the reticuloendothelial system. The small peptide is the “scramble” of a peptide targeting apoptosis and selected in the laboratory by the phage display technique. 31 The “scramble” term means that the amino acids are the same as

the initial peptide but in a different order. These two types of nanoparticles represent thus a good model to evaluate the potential of HR-MAS and of DOSY spectra for the characterization of the surface of iron oxide nanoparticles.

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MATERIALS AND METHODS Chemicals. The used polyethylene glycol is O-(2-aminoethyl)-O′-methylpolyethylene glycol and is furnished by Fluka (Diegem, Belgium). This polyethylene glycol of molecular weight 750 has an amine-terminus group, which allows its covalent grafting on the surface of iron oxide nanoparticles (Chart 1). The peptide is furnished by PolyPeptide (Strasbourg, France) and will be called “scramble E3 peptide” in the following. As shown in Chart 1, it has a small PEG linker at the N-terminus moiety, with an amine-terminus group allowing its covalent grafting on the nanoparticle surface. The iron oxide nanoparticles are synthesized in the laboratory with a well-known protocol.6−8 An organosilane, (triethoxysilyl)propylsuccinic anhydride (TEPSA), is used to coat the nanoparticles with a thin polysiloxane shell exhibiting carboxylate groups. The nanoparticles have a mean diameter of ∼10 nm. Covalent Grafting at the Nanoparticle Surface. The carboxylate functions present at the nanoparticle surface are activated with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI) and react with the amine group of the PEG or of the peptide to form an amide bond, as described in ref 32. EDCI and PEG or peptide are added as a powder in the solution of iron oxide nanoparticles (for an iron concentration of approximately 200 mM, 20 μmol of PEG or peptide and 80 μmol of EDCI are added). The pH is adjusted to approximately 7, and the solution is stirred for 24 h. The excess of PEG and EDCI is eliminated by ultrafiltration (poly(ether sulfone) filters with a cutoff of 30 000, Merck Millipore). The iron concentration of the nanoparticle solutions before and after the grafting is determined by relaxometry after a digestion of the nanoparticles with acid.33 HR-MAS Measurements. The HR-MAS proton spectra are recorded on a AvanceII-500 spectrometer working at 500 MHz (Bruker, Karlsruhe, Germany), with a spinning rate of 5000 Hz, unless otherwise specified. The samples are prepared with a minimum of D2O (10%) and are inserted in a zirconium rotor of 50 μL. The water peak suppression is achieved with the noesypr1d sequence. The spectra of PEG and peptide alone in solution are recorded with the BBI probe, by using the same noesypr1d sequence. The DOSY spectra are collected on samples inserted in 50 or 12 μL rotors, with a spinning rate varying between 2000 and 5000 Hz. Bipolar gradient pulses with two spoil gradients are used to measure the diffusion coefficients (BPP-LED pulse sequence). The value of δ is 2 ms, whereas the value of Δ is set between 250 and 600 ms. The pulse gradients (g) are incremented in 16 steps from 2% to 95% of the maximum gradient strength (53.5 G/cm) in a linear ramp. The temperature is set at 25 °C. DOSY spectra were also recorded with a BBI probe by using the same sequence. The parameters are the same than in HR-MAS, except for the diffusion time, which is set between 250 ms and 4s. Unless otherwise specified, all the measurements are performed on PEG of 750 molecular weight, grafted or not on the nanoparticles. The results are

Chart 1. Structures of the Grafted PEG and Peptide

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Analytical Chemistry

Figure 1. (a) Proton NMR spectrum of PEG (pH = 6) recorded at 500 MHz with the BBI probe. The spectrum was recorded with the noesypr1d sequence in order to suppress the water peak. The attribution of the different peaks is indicated on the spectrum, as well as the integration of the different peaks. (b) Proton NMR spectra of PEG at different pH. From the bottom to the top, the pH is of 6, 8, 10, and 12. The integration of the different peaks is indicated in red. The frame draws attention to the A peak, which is shifting with the pH. All the spectra are recorded in water with 10% of D2O.

Figure 2. HR-MAS spectra recorded at 500 MHz with the noesypr1d sequence. All the spectra are recorded in water with 10% of D2O. (a) The bottom spectrum corresponds to the bare nanoparticles ([Fe] = 112 mM), the central one is that of the grafted nanoparticles ([Fe] = 112 mM), and the top spectrum corresponds to the free PEG. (b) The three bottom spectra are the same than those of Figure 2a, and the top spectrum corresponds to the grafted nanoparticles added with 1 mM of free PEG. A zoom on the area between 2.6 and 4.2 ppm is performed. The frames are there to draw attention to the peaks which are characteristic of free and grafted PEG.

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Figure 3. HR-MAS spectra recorded at 500 MHz with the noesypr1d sequence of nanoparticles grafted with PEG at two different pH. From bottom to top, the pH is of 6.5 and 9.5. The spectra are recorded in water with 10% of D2O.

The bare nanoparticles are characterized by several small peaks between 2 and 3 ppm, which correspond to the polysiloxane shell present at the surface of the particles. The highest peak at 3.2 ppm could correspond to tetramethyl ammoniun hydroxide (TMAOH), which is used during the synthesis to maintain the pH at a slightly basic level. On the HR-MAS spectrum of the nanoparticles grafted with PEG, the peaks of PEG are present in addition to those observed for the bare nanoparticles, which means that PEG is present on the nanoparticle surface. It has to be noted that the bare nanoparticle characteristic peaks seem to decrease in intensity when PEG is grafted at the particle surface. The presence of PEG induces probably a steric hindrance which causes a significant decrease of the mobility of the molecules present at the surface of the bare nanoparticles. This does not prove, however, that PEG is covalently grafted. To show its covalent grafting, free PEG at a concentration of 1 mM was added in the solution of nanoparticles grafted with PEG. The pH of the solution after the addition of free PEG is maintained constant at about 6.5. The HR-MAS spectrum recorded from this solution is added in Figure 2b, and a zoom is performed in the area between 2.6 and 4.2 ppm. The comparison of these four spectra shows that the peak which is characteristic of free PEG at 3.25 ppm is not present on the spectrum of the grafted nanoparticles, but rather, it appears on the spectrum of the solution with added free PEG. Moreover, a peak at 2.9 ppm is observed on the spectra of the grafted nanoparticles, but not on that of free PEG and of the bare nanoparticles. This peak could correspond to the CH2 beside the amide group of the grafted PEG. These observations seem to confirm that PEG is grafted covalently on the nanoparticle surface. As an additional proof of the covalent grafting of PEG on the nanoparticle surface, HR-MAS spectra were recorded at two

presented with the diffusion curves extracted from the DOSY spectra for the more intense peak of PEG at 3.6 ppm. These curves are represented by plotting the natural logarithm of the signal intensity versus the square of the gradient. According to the well-known diffusion equation (eq 1),34,35 it allows to obtain a linear curve for a monodiffusing species. I = Ioexp[−γ 2g 2Dδ 2(D − (δ /3) − (τ /2))]

(1)

where I0 is the intensity at 0% gradient, γ is the gyromagnetic ratio, g is the gradient strength, D is the diffusion coefficient, δ is the gradient pulse length, Δ is the diffusion time, and τ is the interpulse spacing in the BPP-LED pulse sequence.

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RESULTS AND DISCUSSION Characterization of Nanoparticles Grafted with PEG. Figure 1a shows the proton NMR spectrum of PEG, with the attribution of the different peaks and their respective integration. Among these signals, the A peak, corresponding to the CH2 beside the amine terminus group, is particularly interesting. Indeed, its chemical shift is sensitive to the pH, as shown in Figure 1b. Its chemical shift ranges from 3.2 at pH 6, corresponding to the protonated form, to 2.75 at pH 12, corresponding to the deprotonated form. Moreover, the PEG molecule is grafted on the nanoparticle surface by the formation of an amide bond. The formation of this amide bond will modify the chemical shift of the A peak and will allow to demonstrate the covalent grafting of PEG on the nanoparticle surface. Figure 2a shows a comparison between the HR-MAS spectra of the bare nanoparticles, of the nanoparticles grafted with PEG at the same iron concentration, and of free PEG in the absence of nanoparticles. The solutions of nanoparticles grafted with PEG and of free PEG are at the same pH of approximately 6.5. D


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Figure 4. Proton NMR spectrum of the scramble E3 peptide recorded at 500 MHz with the BBI probe. The peptide is solubilized in water with 10% of D2O at pH 7. The attribution of the different peaks is indicated on the spectrum.

Figure 5. Author: Please verify that the changes made to improve the English still retain your original meaning.HR-MAS spectra recorded at 500 MHz with the sequence noesypr1d. All the spectra are recorded in water with 10% of D2O. (a) The bottom spectrum corresponds to the bare nanoparticles ([Fe] = 168 mM), the central one corresponds to the grafted nanoparticles ([Fe] = 168 mM), and the top spectrum corresponds to the free peptide. (b) The three bottom spectra are the same as those of panel (a), and the top spectrum corresponds to the grafted nanoparticles added with 0.5 mM of free peptide. A zoom on the area between 2.8 and 3.4 ppm is presented.

on the nanoparticle surface and that it is covalently grafted by the formation of an amide bond. This is an important information in order to allow the injection of the nanoparticles in vivo. Characterization of Nanoparticles Grafted with the Scramble E3 Peptide. The same study was performed on the nanoparticles grafted with the peptide. First, the NMR spectrum of the peptide alone is presented in Figure 4, with the attribution of the different peaks.

different pH, 6.5 and 9.5. Indeed, as shown in Figure 1b, a shift of the A peak (from 3.2 to 2.8 ppm) should be observed if the PEG is not grafted, whereas no shift will be observed in the opposite. This is illustrated in Figure 3, where no shift of the peaks situated in the interesting area between 2.8 and 4 ppm is observed. All these observations tend to confirm the covalent grafting of the PEG at the nanoparticle surface. This HR-MAS study on the iron oxide nanoparticles grafted with PEG has thus allowed to show that PEG is really present E


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Figure 6. COSY spectrum recorded at 500 MHz with the HR-MAS probe on the solution of grafted nanoparticles in water with 10% of D2O. The interesting correlation spots are surrounded.

Figure 7. HR-MAS spectra recorded at 500 MHz with the noesypr1d sequence on iron oxide nanoparticle solutions grafted with the scramble E3 peptide, at different pH. From bottom to top, the pH is 6.3, 8.3, 10.5, and 11.5. All the spectra are recorded in water with 10% of D2O. (a) Spectra between 0 and 4.5 ppm. The framed area correspond to the peptide peaks. (b) Zoom on the area between 2.7 and 3.5 ppm. The frame draws the attention to the peak which could correspond to the E peak, characteristic of the free peptide.

As shown for PEG, the E peak, which corresponds to the CH2 beside the amine-terminus group, is here very interesting because its chemical shift is sensitive to pH, and the replacement of the amine function by the amide group after the grafting will shift the E peak. Figure 5a shows a comparison of the HR-MAS spectra of the bare nanoparticles, of the nanoparticles grafted with the peptide at the same iron concentration, and of the free peptide. The solutions of the nanoparticles grafted with the peptide and of the free peptide are at the same pH of about 7. As mentionned

in the previous section, the small peaks of the bare nanoparticles, which are visible in the spectrum, are coming from the polysiloxane shell, whereas the more intense signal at 3.2 ppm is coming from TMAOH. Besides these peaks, some characteristic peaks of the peptide are present on the spectrum of the grafted nanoparticles, as highlighted by the frames. This proves thus that the peptide is present at the nanoparticle surface, but we do not know if it is grafted covalently. Again, in order to demonstrate the covalent grafting, free peptide at a concentration of 0.5 mM is added in the solution of F


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Figure 8. Diffusion curves extracted from the DOSY spectra for the more intense peak of PEG at 3.6 ppm. The left curve corresponds to the DOSY spectrum recorded with the 50 μL rotor, whereas the right one was obtained with the 12 μL rotor. These DOSY spectra were recorded at 25 °C, with a gradient pulse length of 2 ms and a diffusion time of 250 ms. The obtained diffusion coefficients are indicated on the graphs.

decrease of the line widths, and the increase of the intensity of the different peaks. These two studies conducted on two different iron oxide nanoparticles highlight that grafting small organic molecules on nanoparticles can be problematic and that it is important to verify their covalent grafting on the nanoparticle surface. To that aim, 1D and 2D HR-MAS spectra appear to be very useful and efficient. Nevertheless, it requires the recording of several different spectra, at different pH, which can be very tedious. DOSY spectra could thus be very useful to simplify the demonstration of the covalent grafting of the small organic molecules and could also allow to simply confirm the results obtained with the 1D and 2D spectra. Evaluation of the Use of DOSY Spectra in HR-MAS for the Characterization of Iron Oxide Nanoparticles. As mentioned in the introduction, Viel et al.30 highly recommend the use of 12 μL rotors instead of 50 μL rotors in order to obtain accurate diffusion coefficients in HR-MAS. We have first verified this conclusion by measuring the diffusion coefficient of nongrafted PEG with a rotor of 50 and 12 μL. Both measurements were performed with a spinning rate of 5000 Hz, which is in the range of spinning rates recommended in ref 30. As shown in Figure 8, when the rotor of 50 μL is used, the diffusion curve extracted from the DOSY spectrum for the more intense peak at 3.6 ppm is not linear and could be fitted biexponentially, which does not have any sense for a solution of PEG. The extracted diffusion coefficient is thus totally erroneous. However, for the 12 μL rotor, the curve is monoexponential and a diffusion coefficient of 4.2 × 10−10 m2/s is extracted, which is coherent with the molecular weight of PEG and the used temperature. This value is indeed slightly higher than the value obtained on the same solution with our normal BBI probe (D = 3.6 × 10−10 m2/s) but this can be explained by the temperature, which is slightly higher in HRMAS because of the fast spinning of the sample. This causes indeed an increase of the sample temperature even if the temperature is controlled by the Bruker temperature unit. The measurements were also performed at spinning rates of 4000 and 3000 Hz with the two rotors, and similar diffusion

nanoparticles grafted with the peptide. This is represented in Figure 5b, where the spectrum corresponding to this solution is added. A zoom on the spectrum area between 2.8 and 3.4 ppm is performed. The frame shows that, quite logically, the E peak, which is characteristic of the free peptide, is present at 3.15 ppm on the spectra of the free peptide and of the grafted nanoparticles added with free peptide. On the spectrum corresponding to the grafted nanoparticles without free peptide, the situation is less clear, but a small shoulder at the left of the peak at 3.1 ppm could also suggest the presence of this E peak. A COSY spectrum recorded on this solution shows moreover a correlation between this small shoulder and one of the F peaks, strenghtening the hypothesis that this small shoulder corresponds to the E peak which is characteristic of the free peptide (Figure 6). Several spectra at different pH were also recorded in order to observe if this peak is shifting (Figure 7). A comparison of the spectra shows that the peptide characteristic peaks (see frame in Figure 7a) are becoming narrower and narrower as the pH is increasing, which results in an increase of the signals intensity. This is particularly true for the A, B, and C signals. Moreover, a zoom on the signal at 3.1 ppm (frame on Figure 7b) shows again a shoulder at the left of the more intense signal for the pH 6.3 and 8.3. That shoulder disappears however for the two higher pH of 10.5 and 11.5, and another peak appears at 3.05 ppm. This signal is particularly intense at the pH of 11.5. This strenghtens again the hypothesis that the shoulder, which is observed in Figure 5b, corresponds to the E peak of the free peptide, which is shifting to a lower chemical shift when the pH is increasing. The same trend is indeed observed for the peptide alone (data not shown). These observations can be explained by a chemical adsorption of the peptide at the nanoparticle surface for pH below 9, via the formation of an ionic bond between the carboxylate groups at the surface and the protonated amine functions of the peptide. As the pH is increasing, the amine functions are deprotonated, which leads to the desorption of the peptide from the nanoparticle surface. The conformational mobility of the peptide is thus enhanced, which explains the G


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Table 1. Values of Diffusion Coefficients Obtained for 20 kDa PEG with the BBI Probe and the HR-MAS Probe at Different Spinning Rates diffusion coefficient (× 10−11 m2/s) BBI probe

Δ = 250 ms

Δ=4s

1.04 (linear curve)

1.05 (linear curve)

Δ = 300 ms

Δ = 600 ms

28 (nonlinear curve) 6.99 (linear curve) 6.23 (linear curve) 8.85 (nonlinear curve)

18 (nearly linear curve) 7.63 (linear curve) 5.91 (nonlinear curve) 4.05 (nonlinear curve)

spinning rate HR-MAS probe

2000 3000 4000 5000

Hz Hz Hz Hz

coefficients were obtained (data not shown). These observations confirm the conclusions of the article of Viel et al., and the subsequent measurements were performed with a 12 μL rotor. In ref 30, the authors also discussed the importance to use spinning rates not higher than 4000 Hz to analyze slowly diffusing species. For 750 molecular weight PEG, we have shown above that a spinning rate of 5000 Hz allows a correct measurement of its diffusion coefficient. However, when grafted on the nanoparticle surface, its diffusion coefficient will be smaller. In order to mimick it, we have measured the diffusion coefficient of a 20 kDa PEG, with the normal BBI probe and with the HR-MAS probe at different spinning rates (Table 1). With the BBI probe, a perfectly linear diffusion curve is obtained whatever the used diffusion time Δ. On the contrary, the diffusion coefficient measured with the HR-MAS probe depends strongly on the spinning rate and on the diffusion time Δ. Moreover, all HR-MAS measured diffusion coefficients are highly overestimated compared to the value obtained with the BBI probe, whatever the spinning rate or the diffusion time. The results also show that the spinning rates of 5000 and 2000 Hz have to be avoided, the first one because the obtained diffusion curves are biexponential for all the tested diffusion times, and the second one because the measured diffusion coefficients are dramatically overestimated. The more appropriate spinning rates seem thus to be 3000 and 4000 Hz, with, for the last one, a diffusion time that has to be kept to a minimum. A biexponential diffusion curve was indeed obtained with the longer diffusion time of 600 ms. These tests show that it becomes difficult to obtain a precise measurement of the diffusion coefficient for slowly diffusing molecules, even with a 12 μL rotor. The used spinning rate becomes very important but even with the more appropriate spinning rate of 3000 Hz, the obtained diffusion coefficients are overestimated. They remain however in the correct range of magnitude. It could thus be possible to demonstrate the grafting of small organic molecules with this technique by observing a decrease of their diffusion coefficient. On the basis of the above results, we have thus decided to perform the diffusion measurements on nanoparticles grafted with PEG with a spinning rate of 3000 Hz. The obtained diffusion curve is presented in Figure 9. It is linear, but the extracted diffusion coefficient of 8.13 × 10−10 m2/s is very high and completely incompatible with PEG grafted on nanoparticles. The obtained diffusion coefficient is indeed higher than that obtained for PEG alone in solution. Based on the Stokes−Einstein equation (eq 2), a diffusion coefficient of about 5 × 10−11 m2/s is expected for this type of nanoparticles with a mean diameter of 10 nm.

D = kT /6πηr0

Figure 9. Diffusion curve extracted from the DOSY spectrum recorded on iron oxide nanoparticles grafted covalently with PEG. This curve was extracted for the more intense peak of PEG at 3.6 ppm. The DOSY spectrum was recorded at 25 °C with a 12 μL rotor and a spinning rate of 3000 Hz. The gradient pulse length was 2 ms, and the diffusion time was 300 ms. The curve is composed of only 9 points even if 16 gradient steps were used, because from the tenth gradient step, no signal could be observed. The extracted diffusion coefficient is indicated on the graph.

where D is the diffusion coefficient, k is the Boltzmann constant, T is the temperature, η is the viscosity of the solution, and r0 is the mean radius of the particles. This very high diffusion coefficient seems to indicate the presence of an experimental artifact that leads to an enhanced apparent diffusion coefficient. As this problem was not so dramatic for PEG alone and for 20 kDa PEG, this could be due to the magnetic character of the particles. They are indeed inserted in a strong magnetic field to perform HR-MAS, and it is thus possible that the nanoparticles are no longer in a real colloidal solution; rather, they are partially or totally stuck on the walls of the rotor. Moreover, iron oxide nanoparticles can be seen as small magnets, generating magnetic field gradients which will perturb the DOSY experiment, which is based on the use of magnetic field gradients. This quite logically prevents the measurement of accurate diffusion coefficients.

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CONCLUSION This study has allowed us to show the importance of evaluating the covalent grafting of small organic molecules on iron oxide nanoparticle surface and the potential of 1D and 2D spectra in

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Analytical Chemistry HR-MAS to achieve this goal. Indeed, for particles grafted with PEG, the covalent grafting was confirmed, but for the particles grafted with the peptide, most of the small organic molecules seem to be simply chemisorbed on the nanoparticle surface. Nevertheless, it has to be noted that the results do not preclude that a small quantity of peptide is covalently grafted, the corresponding characteristic peak of the CH2 beside the amide group being too small and/or too broad to be detectable. DOSY is the method of choice to prove the covalent grafting of small organic molecules on nanoparticles and is used extensively in the literature12−17 on diamagnetic nanoparticles. DOSY spectra could thus have here a high interest to confirm the results obtained with the 1D spectra. We have shown that the best experimental conditions to measure the diffusion coefficient of a slowly diffusing species are the use of a 3000 Hz spinning rate in a 12 μL rotor. A faster spinning rate has to be avoided because, as previously described in 30, the centrifugallike effect caused by the sample spinning becomes more dramatic for low diffusing molecules. A lower spinning rate of 2000 Hz has also to be avoided, which is less obvious. It could probably be explained by an unstable spinning of the sample, which causes a dramatic increase of the measured diffusion coefficient. A spinning rate of 2000 Hz is indeed far below the limit recommended by Bruker for HR-MAS measurements. Nevertheless, even with these best experimental conditions, and contrary to what was presented in the work of Polito et al.,18 the superparamagnetic character of the iron oxide nanoparticles seems to prevent an accurate measurement of their diffusion coefficient, making the DOSY technique inefficient to demonstrate the covalent grafting of organic molecules. Each iron oxide nanoparticle can indeed be seen as a small magnet, characterized by a saturation magnetization of about 50 A.m2/ kg or 2.6 × 105 A/m, as previously published.8 This magnetization can be approximated as a magnetic field of 0.3 T and will generate some magnetic field gradients around the nanoparticles. These will be added to the magnetic field gradients used during the DOSY experiment, giving erroneous diffusion coefficients. Even without the help of DOSY spectra, HR-MAS remains however a good alternative and complementary technique to XPS and FTIR in order to characterize iron oxide nanoparticles grafted with small organic molecules. In Supporting Information, FTIR spectra of the above nanoparticles were indeed recorded and clearly show that FTIR is not sufficient to demonstrate the presence of the grafted organic molecules at the nanoparticle surface.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Prof. Rudi Willem and Ingrid Verbruggen from VUB (Vrije Universiteit of Brussel) for their help in the implementation of the HR-MAS technique in their laboratory of Mons. The authors also thank Dr. Dimitri Stanicki for the synthesis of the bare iron oxide nanoparticles. This work was supported by the Walloon Region (program First spin-off), the FNRS (Fond National de la Recherche Scientif ique), the UIAP VII and ARC Programs (AUWB-2010-10/15-UMONS-5) of the French Community of Belgium. The authors thank the Center for Microscopy and Molecular Imaging (CMMI, supported by the European Regional Development Fund and the Walloon Region).

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ASSOCIATED CONTENT

S Supporting Information *

FTIR spectra of the nanoparticles described in this article were recorded as a comparison with the results obtained by HRMAS. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +32-65-373518. Fax: +32-65-373533. Notes

The authors declare no competing financial interest. I


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HR-MAS NMR Spectroscopy - American Chemical Society

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