DOI: 10.1002/chem.201400003
Full Paper
& Molecular Dynamics
Arginine Side Chains as a Dispersant for Individual Single-Wall Carbon Nanotubes Atsushi Hirano,[a] Takeshi Tanaka,[a] Hiromichi Kataura,[a] and Tomoshi Kameda*[b]
Chem. Eur. J. 2014, 20, 4922 – 4930
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Full Paper Abstract: Charged peptides and proteins disperse singlewall carbon nanotubes (SWCNTs) in aqueous solutions. However, little is known about the role of their side chains in their interactions with SWCNTs. Homopolypeptide–SWCNT systems are ideal for investigating the mechanisms of such interactions. In this study, we demonstrate that SWCNTs are individually dispersed by poly-l-arginine (PLA). The debundled SWCNTs exhibited a distinct fluorescence. The dispersibility of SWCNTs with PLA was greater than that of SWCNTs
Introduction Single-wall carbon nanotubes (SWCNTs) have useful optical and electrical properties. Because of these advantages, peptide and protein interactions with SWCNTs are important in medical technology and nanotechnology; in addition, the interaction is also taken up in nanotoxicology for understanding their biological impacts at the molecular level.[1–3] Understanding the mechanisms of such interactions is important for developing and improving SWCNT applications. Experiments and simulations have shown that peptides and proteins interact with the sidewalls of SWCNTs through various types of interactions, including hydrophobic, van der Waals, electrostatic, and p–p interactions.[1–3] For example, aromatic residues form van der Waals, p–p, and hydrophobic contacts with SWCNTs, aliphatic residues form hydrophobic contacts, and charged residues form electrostatic contacts. Peptide interactions with SWCNTs can also be used for dispersion of SWCNTs in aqueous solutions.[4–15] Applications of this property include the separation and purification of SWCNTs in addition to the other applications mentioned above.[16–18] The binding of peptides to SWCNTs confers colloidal stability, that is, dispersibility, on SWCNTs that have been debundled by using a sonication treatment. The colloidal stability depends on both the strength of binding between peptides and SWCNTs and the electrostatic repulsion between peptides on the sidewalls of the SWCNTs. Therefore, the dispersibility is readily reduced at the isoelectronic points.[4] Such properties are also relevant to SWCNT dispersion by proteins.[19, 20] Proteins can retain their intrinsic conformations even when bound to the sidewalls of SWCNTs.[21, 22] However, such proteins can be readily affected by solution conditions, such as the presence of solutes that reduce the binding forces and dis-
[a] Dr. A. Hirano, Dr. T. Tanaka, Dr. H. Kataura Nanosystem Research Institute National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan) [b] Dr. T. Kameda Computational Biology Research Center National Institute of Advanced Industrial Science and Technology (AIST), Koto, Tokyo 135-0064 (Japan) E-mail: [email protected]
with poly-l-lysine (PLL). Molecular dynamics simulations suggest that the side chains of PLA have stronger interactions with the sidewalls of SWCNTs compared with those of PLL. The guanidinium group at the end of the side chain of an arginine residue plays an important role in the interaction with SWCNTs, likely through hydrophobic, van der Waals, and p–p interactions. PLA can be useful as a tool for the dispersion of SWCNTs and can be used to non-covalently anchor materials to SWCNTs with strong binding.
persibility.[23] The resulting bundling and flocculation of SWCNTs deteriorates the optical and electrical properties of the SWCNTs[24] such that high colloidal stability, that is, high dispersibility, is required for their use in applications. Investigations of peptide–SWCNT or protein–SWCNT interactions often focus on the aromatic and hydrophobic side chains, which form hydrophobic and p–p interactions with SWCNTs.[1–3] However, based on our previous studies, we expect that charged side chains can also bind SWCNTs and have a dispersive effect. We recently reported that arginine, which is a basic amino acid that is positively charged at neutral pH, can have a solubilizing effect on aromatic compounds.[25–28] The results of experiments and molecular dynamics (MD) simulations indicate that the guanidinium group, which is at the end of the arginine side chain, interacts with the aromatic moieties of compounds through hydrophobic or p–p contacts. Thus, poly-l-arginine (PLA) should act as a dispersant to enable the stable dispersion of SWCNTs. To date, little has been reported about the dispersion of SWCNTs by using PLA. Lee et al. reported that SWCNTs did not disperse when using PLA;[12] however, the power density of their sonication system might explain these unsatisfactory results. In fact, they also found that SWCNTs did not disperse when using poly-l-lysine (PLL), whereas Wang and Chen reported the successful dispersion of SWCNTs by using PLL.[4] This discrepancy may depend on the sonication conditions used for dispersion.[29] In this study, we investigated the effects of PLA on the dispersion of SWCNTs by using a tip sonicator capable of debundling SWCNTs.[30] We observed remarkable dispersibility and fluorescent intensities, and we verified that the guanidinium group of PLA binds strongly to SWCNTs. This binding strength was supported by MD simulations. In contrast, exposure to PLL resulted in low dispersiblity. These results indicate that the ability of charged polypeptides to disperse SWCNTs depends on whether the power density of the sonication system is sufficient to debundle the SWCNTs. The binding properties of PLA show that it could be used to disperse SWCNTs or even to non-covalently anchor proteins or other materials to the sidewalls of SWCNTs, which could be useful for medicine and nanotechnology.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201400003. Chem. Eur. J. 2014, 20, 4922 – 4930
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Full Paper Results and Discussion Dispersibility of SWCNTs in polypeptide solutions SWCNTs in solution with PLA or PLL were subjected to ultrasonic homogenization with a flat tip in the absence of any buffers. The power of the homogenizer is sufficient to debundle SWCNTs (see Figure S1 in the Supporting Information). Subsequent ultracentrifugation was performed to obtain the dispersed SWCNT solution as the supernatant. In the experiments, the selected molecular weights of the polypeptides were > 70 000 (> 70 k) and 5000–15 000 (5–15 k) for PLA and > 30 000 (> 30 k) and 15 000–30 000 (15–30 k) for PLL. Figure 1 A presents the absorbance spectra of the SWCNTs dispersed by using PLA and PLL, which were normalized to the absorbance at 600 nm. The absorption peaks at approximately 940–1350 and 620–940 nm were assigned to the first and second optical transitions of the semiconducting species, which were designated as the S11 and S22 bands, respectively. The absorption peaks at approximately 400–620 nm were assigned to the first optical transition of metallic SWCNTs, which was designated as the M11 band.[31–33] Thus, both metallic and semiconducting SWCNTs were found among the dispersed SWCNTs. Absorption spectra in the UV region were excluded from the Figure because the p-plasmon of SWCNTs and peptide bonds exhibit absorption in the UV region. The spectrum of SWCNTs dispersed by 1 wt. % sodium dodecyl sulfate (SDS) was also measured after adding aliquots of NaOH to the SWCNT solutions to bring them to pH 12 to avoid absorbance reduction due to oxidation (Figure 1 A).[33] The SWCNTs dispersed by PLA displayed sharp peaks in the near-infrared region corresponding to the S11 and S22 bands, although these peaks had lower intensities than those in 1 wt. % SDS. Because the sharpness of the S11 and S22 bands relates to the dispersibility of the SWCNTs,[9] it appears that PLA is able to substantially disperse the SWCNTs. In contrast, the SWCNTs dispersed by PLL displayed attenuated intensities in the near-infrared region. The absorbance peaks of these solutions were shifted towards longer wavelengths by SDS, PLL, and PLA (in order of increasing shift). Such peak shifts depend on the effective dielectric constants around the sidewalls of the SWCNTs.[34] Figure 1 B presents the dispersibility of the SWCNTs in 0.2 and 1.0 mg mL 1 of the polypeptides. The dispersibility is defined as the concentration of the supernatant of the solution after ultracentrifugation. An absorbance at 600 nm was used to estimate the concentration of SWCNTs because absorbance in the UV region is affected by coexisting polypeptides (see also the Supporting Information, Figure S2). In addition, the absorbance at the S11 and S22 bands is also affected by oxidation depending on pH and temperature.[33, 35] Higher-molecular-weight polypeptides were found to increase the dispersibility of SWCNTs. PLA (> 70 k) was found to be the most effective at increasing the dispersibility. A 1.0 mg mL 1 solution of PLA exhibited greater dispersibility than a 0.2 mg solution, independent of molecular weight. Similarly, a 1.0 mg mL 1 solution of PLL exhibited greater dispersibility than a 0.2 mg mL 1 solution for PLL (> 30 k), whereas for PLL (15–30 k), a 0.2 mg mL 1 solution Chem. Eur. J. 2014, 20, 4922 – 4930
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Figure 1. (A) Absorption spectra of SWCNTs dispersed by polypeptides or 1 wt. % SDS. Absorbance values were normalized to the values at 600 nm. For 1 wt. % SDS, NaOH was added to the solution to raise the pH value to near 12, which avoids decreasing the absorbance due to oxidation.[33] (B) Dispersibility of SWCNTs in the presence of PLA or PLL at 0.2 and 1.0 mg mL 1.
yielded a dispersion greater than 1.0 mg mL 1. The decrease in dispersibility at higher PLL concentrations may be attributed to non-covalent cross-linking of the SWCNTs by PLL; similar results were observed for poly-l-glutamate (the Supporting Information, Figure S3). The dispersion of SWCNTs using PLA or PLL has already been reported. Lee et al. demonstrated the high dispersibility of SWCNTs by using mixtures of PLA or PLL and poly-l-glutamate and no dispersibility of SWCNTs using the individual polypeptides.[12] Interestingly, their results are inconsistent with the results presented here. This inconsistency can be explained by differences between methods; they dispersed the SWCNTs by
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Full Paper using a bath sonicator, not a tip sonicator. Tip-sonicated SWCNTs display greater dispersibility than bath-sonicated SWCNTs.[29] The lack of dispersion when using PLA or PLL alone may be explained by the incompleteness of SWCNT debundling. In fact, broader spectral sharpness was observed in the previous study for the S11 and S22 bands, which suggests that the SWCNTs were insufficiently dispersed. Thus, differences in dispersion methods can lead to different conclusions about the effects of dispersants. The dispersibility of SWCNTs using peptides and proteins also depends on pH and salt concentration.[4, 19, 20, 23, 36] The dispersibility is reduced considerably at the isoelectric point or in the presence of salts. The solution used in this study was at neutral pH, without any other salts. The pKa values of side chains for arginine and lysine are 12.5 and 9.2, respectively. Thus, the positive charges of the side chains of the polypeptides on the SWCNTs generate electrostatic repulsion between SWCNTs, leading to dispersion. In fact, the addition of 100 mm NaCl caused the SWCNTs to aggregate (data not shown), which is accounted for by the electrostatic screening effect.[23] Fluorescence spectra of SWCNTs Debundled semiconducting SWCNTs are known to exhibit fluorescence in the near-infrared region.[37, 38] To demonstrate the debundling of SWCNTs in our polypeptide tests, we recorded fluorescence spectra for the SWCNTs treated with PLA (> 70 k) and PLL (> 30 k) at 1.0 mg mL 1. Figure 2 presents the fluorescence spectra of the SWCNTs dispersed by the polypeptides. These data were recorded by scanning the excitation wavelength from 500 to 850 nm and scanning the emission wavelength from 900 to 1400 nm. Distinctive peaks can be identified for each chirality based on the data for single-chirality semiconducting SWCNTs.[30] PLA (> 70 k) exhibited intense fluorescence, as expected based on the absorption spectra (Figure 1 A). The spectral profile was largely identical to that obtained with sodium deoxycholate, which is known as a good dispersant for SWCNTs;[39] thus, PLA disperses SWCNTs without any chirality bias. PLL (> 30 k) also displayed similar fluorescence spectra. As expected based on the absorbance spectra (Figure 1 A), fluorescence peaks corresponding to the S11 band were slightly shifted toward longer wavelengths with PLA (> 70 k) than with PLL (> 30 k), likely due to the effective dielectric constants of PLA and PLL around the sidewalls of the SWCNTs, as mentioned above. pH Dependence of the absorption spectra of SWCNTs As shown above, both PLA and PLL resulted in fluorescence due to SWCNT dispersion. However, the differences in absorption at S11 and S22 require further explanation (Figure 1 A). One possible cause of these differences is the formation of small bundles of SWCNTs. If the bundles were composed of semiconducting SWCNTs, then the absorbance and fluorescence would be reduced.[9, 24] Such low dispersibility by PLL may lead to the attenuation of absorbance at S11 and S22. Another possible cause is the oxidation of SWCNTs. SWCNTs are known to be Chem. Eur. J. 2014, 20, 4922 – 4930
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Figure 2. Fluorescence spectra of SWCNTs dispersed by (A) PLA (> 70 k) and (B) PLL (> 30 k) at 1 mg mL 1. The respective peaks were assigned to the corresponding chiralities.
oxidized by dissolved oxygen and protons in aqueous solution,[33, 40] and the oxidation depends on pH. The local pH around an SWCNT could induce the oxidation and resulting attenuation of the absorption spectra. Here, after the dispersion of SWCNTs using PLA (> 70 k) or PLL (> 30 k) at 1.0 mg mL 1, we added aliquots of HCl or NaOH to the SWCNT solutions to modify the pH before collecting spectra. Figure 3 A and B present the absorbance spectra of the SWCNTs at various pH
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Full Paper higher dispersibility by PLA is attributed to the high binding affinity and positive charges of its side chains.
MD Simulation of the interaction between a peptide and SWCNT Figure 3. Spectral changes of SWCNTs with (A) PLA (> 70 k) and (B) PLL (> 30 k) at 1 mg mL 1 that occurred when the pH was altered by HCl or NaOH. The spectra were normalized to the values at 600 nm. (C) pH dependence of absorbance at 1130 and 1270 nm for PLA (> 70 k) and PLL (> 30 k).
values, and the spectra were normalized to absorption at 600 nm. Because the pH was approximately 6 before the addition of an acid or base, the spectra were not significantly affected by the addition of NaOH, except when at extremely basic levels near pH 12; the small spectral changes at that pH may be caused by the deprotonation of the polypeptide side chains. Recall that the pKa values of the side chains of arginine and lysine are 12.5 and 9.2, respectively. Importantly, the spectral intensities were dramatically decreased at acidic pH values. The pH dependence of the intensities at 1130 and 1270 nm, which correspond to peaks in the S11 bands, are plotted in Figure 3 C. The intensities were found to decrease near pH 3 due to the oxidation. Thus, the SWCNTs dispersed by the polypeptides are not oxidized in the absence of HCl or NaOH. The attenuation of the absorption spectral intensities by PLL (> 30 k) is therefore attributable to the formation of small SWCNT bundles.[9] In addition, the existence of impurities such as amorphous carbon due to the low dispersibility by PLL may also affect the spectral intensity.[39] The lower attenuation of spectral intensity by PLA (> 70 k) suggests that PLA is tightly wrapped around individual SWCNTs[41]
Dispersibility of SWCNTs in oligopeptide solutions As seen in Figure 1, PLA (5–15 k) displayed higher dispersibility than PLL (> 30 k). Therefore, PLA is likely to be more effective for dispersion. However, these polypeptides have molecular weight distributions. Oligopeptides with specified lengths are more useful for an accurate estimation of the dispersive effect. Figure 4 presents the dispersibility of SWCNTs in the presence of 5-, 10-, and 20-residue oligopeptides, which are denoted by R5, R10, and R20 for oligoarginine and K5, K10, and K20 for oligolysine, respectively. Longer sequences resulted in higher dispersibility for oligoarginine, whereas only K20 exhibited dispersion for oligolysine, and this dispersion was very low. The fact that even R5 exhibits dispersion suggests tighter binding of the arginine side chains to the sidewalls of SWCNTs compared with the binding of the lysine side chains. The lower dispersibility when using shorter oligopeptides is expected because the binding affinity should be reduced by the shortening of the sequences. Incidentally, the monomers of arginine and lysine provide no dispersion of SWCNTs (data not shown). Thus, the Chem. Eur. J. 2014, 20, 4922 – 4930
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MD simulations were performed to understand the dynamics of the peptides on the SWCNT sidewalls and to determine how PLA
Figure 4. Dispersibility of SWCNTs in the presence of 5-, 10-, and 20-mer oligopeptides at 0.2 mg mL 1.
tightly binds to the sidewalls. A (7,6) SWCNT was chosen for the simulation because it was abundant in the sample, as shown in Figure 2. It is reasonable not to consider the chirality dependence of the dynamics, as previously reported,[42] because there is no chirality dependence for the dispersion (Figure 2). Figure 5 presents the representative conformations of 15-residue oligoarginine (R15) and 15-residue oligolysine (K15). The side chains of both peptides were found to bind to the sidewall of the SWCNT. The guanidinium group of R15 interacts with the sidewall of the SWCNT (Figure 5 A and B), which is consistent with the interaction of SWCNTs with lysozyme, an enzyme with many arginine residues (the Supporting Information, Figure S4).[43] K15 was found to interact with the sidewall of the SWCNT through the alkyl chains of its side chains (Figure 5 D and E). Interestingly, when the side chains face toward the solvent, the backbones close to the side chains tend to leave the sidewall of the SWCNT, as shown in Figure 5 B and E. Figure 5 C and F present the sphere representations of the backbones of R15 and K15, corresponding to Figure 5 A and B and Figure 5 D and E, respectively. The backbones also interact with the sidewall of the SWCNT; in particular, oxygen atoms tend to be located close to the sidewall.
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Full Paper Table 1. Positions of the highest peaks of the radial probability distributions for each atom of the peptide side chains. R15
Figure 5. Representative conformations of R15 and K15 on an SWCNT. Side chains and backbones of (A, B) R15 and (D, E) K15 are shown as stick and cartoon representations, respectively. Only the backbones for (C) R15 and (F) K15 are displayed by using sphere representations: red, oxygen; green, carbon; blue, nitrogen; light gray, hydrogen. SWCNTs are shown by using dark-gray sphere representations.
Estimating the radial distribution of the peptides is a useful way to identify the functional groups that contribute to the interaction. Figure 6 A and B present radial probability distributions for each atom of the side chains of R15 or K15, respectively, near the SWCNT; each atom is labelled as denoted in the insets. In addition, the peak positions of the atoms of the side chains are listed in Table 1. For R15, the probability of Cz exhib-
K15
Atom
Peak position []
Atom
Peak position []
Cb Cg Cd Ne Cz
7.9 0.1 7.8 0.0 7.9 0.0 7.8 0.1 7.7 0.0
Cb Cg Cd Ce Nz
7.9 0.0 7.9 0.0 8.0 0.0 7.8 0.0 8.8 0.1
ited the highest peak (at 7.7 ). Cb, Cg, Cd, and Ne have lower probabilities of being close to the sidewall, which indicates that the end of the side chain, that is, the guanidinium group, is the part that most frequently interacts with the sidewall of the SWCNT. In contrast, for K15, Cb exhibited its highest peak at 7.9 , and the distribution of Nz atoms was broadened. The peak position of Nz is at 8.8 , indicating that the end of the side chain of K15 is far away from the sidewall of SWCNT, as expected based on the conformation shown in Figure 5. Figure 6 C and D present the radial probability distributions of each atom in the peptide backbones for R15 and K15, respectively. R15 and K15 displayed similar profiles in the distribution; namely, the N and H atoms of the backbones were relatively far away from the sidewall, whereas the O atoms were closer to the sidewall than the Ca atoms. Interestingly, the peak position of O atoms is at 7.5 (Table 2), which is the closest position to the SWCNT sidewall among any of the highest peaks for different atoms (Tables 1 and 2). The characteristic profile of O atoms was found to be attributable to their close approach to the sidewall of the SWCNT (Figure 5 C and F). Such binding affinity is ascribable to strong van der Waals interactions between O atoms and the SWCNT sidewall. Steered MD simulation for estimating the binding force between a peptide and SWCNT
Figure 6. Radial probability distributions for each atom of the (A, B) side chains and (C, D) the peptide bonds for (A,C) R15 and (B,D) K15 on the sidewall of SWCNTs, as determined by MD simulations. Chem. Eur. J. 2014, 20, 4922 – 4930
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The conformations of the peptides on the sidewall of the SWCNT described above should affect the binding force of the peptides to the SWCNT. We used steered MD simulations with various constant forces to acquire the minimal force at which the peptides were stripped off the SWCNT sidewall. The steered MD
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Full Paper Table 2. Positions of the highest peaks of the radial probability distributions for each atom of the peptide backbones. R15
K15
Atom
Peak position []
Atom
Peak position []
Ca O C N H
7.9 0.0 7.5 0.1 8.5 0.1 8.5 0.3 8.9 0.6
Ca O C N H
8.1 0.2 7.5 0.1 8.5 0.0 8.6 0.2 9.3 0.3
simulations were executed for 50 ns and performed three times. The direction of the force between the center of mass of the peptide and an axis of the SWCNT is normal to the axis of the SWCNT. If a peptide was not stripped from the SWCNT in all three simulations, it was considered “unstripped”, and the next simulation was carried out with a force that was 25 pN larger. If a peptide was stripped in at least one of the simulations, it was considered “stripped”, and the force value was regarded as the minimum force needed to strip the polypeptide off the SWCNT (the Supporting Information, Figure S5). The minimum values for stripping were approximately 200– 225 pN for R15 and approximately 100–125 pN for K15. Importantly, the force value of R15 was larger than that of K15. The higher binding force for R15 should more effectively prevent it from coming off the sidewall of the SWCNT, which is associated with the fact that PLA leads to greater dispersibility of SWCNTs than PLL. Origin of the characteristic binding of the guanidinium group onto the sidewalls of SWCNTs Although several simulations of interactions between the guanidinium group of arginine and the sidewalls of SWCNTs have been performed,[1–3] few experimental studies have been reported. Some experimental studies using proteins and peptides with various sequences suggest that the arginine side chain is important for binding;[1, 2] however, none of these studies have specifically tested arginine alone. Guo et al. quantified the amount of arginine adsorbed onto SWCNTs in the cell culture medium.[44] However, no basic data were provided about the adsorbability of arginine or the guanidinium group onto SWCNTs. To our knowledge, the present study is the first to experimentally demonstrate that arginine side chains interact with SWCNTs and that the dispersibility with PLA is greater than that with PLL. Importantly, the characteristics of PLA that we describe are supported by MD simulations. The adsorption of the guanidinium group onto the sidewalls of SWCNTs depends on hydrophobic or p–p interactions. Previously, we reported that arginine interacts with aromatic compounds, such as nucleobases and polyphenols, increasing their solubilities.[25–27] MD simulations and binding free-energy calculations indicate that the arginine side chain, that is, the guanidinium group, interacts with aromatic moieties, which suggests a p–p interaction.[25, 26] These calculations were based on a classical force field, and thus, nonbonded interactions were Chem. Eur. J. 2014, 20, 4922 – 4930
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represented by the summation of Coulomb and van der Waals terms; the p–p interaction was thus implicitly considered by nonbonded interaction terms.[26] Our simulation was also performed by using the classical force field. The peak position of Cz for R15 is close to that of Ne (Table 1), indicating that the planes of the guanidinium groups are parallel to the SWCNT sidewall. This observation suggests the existence of p–p interactions between the two molecules. In contrast, K15 has no such interactions. The p–p interactions account for the strong binding force of PLA to the sidewalls of SWCNTs. In addition, various studies have demonstrated that hydrophobic interactions are also involved in the binding. Mason et al. reported the dynamics of guanidine, which is similar to the guanidinium group, in an aqueous solution using MD simulations. The simulations indicate that guanidine is poorly hydrated above and below its plane in aqueous solution.[45] Li et al. suggested that this property of guanidine is related to that of the guanidinium group; specifically, the guanidinium group of arginine possesses hydrophobicity, similar to guanidine.[46] The authors compared the dynamics of arginine to surfactant-like behavior. Characteristic interactions of arginine with SWCNTs were also demonstrated by using density functional theory.[47, 48] Mechanistic insight into the interactions between proteins and SWCNTs Interactions between proteins and SWCNTs have generally been accounted for by hydrophobic, van der Waals, electrostatic, and p–p interactions.[1–3] Some simulations of interactions between proteins and SWCNTs indicate binding of the guanidinium group of the arginine residues to the sidewalls of the SWCNTs.[43, 49] As mentioned above, arginine has a p-electron, a hydrophobic and positively charged moiety; therefore, the binding of arginine residues onto SWCNTs should be based on multimodal interactions consisting of the above interactions. The arginine residues of proteins can play a principal role in the binding to SWCNTs, as observed for PLA. In fact, basic proteins, that is, arginine-rich proteins, such as lysozyme and histones, were experimentally demonstrated to debundle or highly disperse SWCNTs.[19, 23, 50] This insight into the behavior of arginine on the sidewalls of SWCNTs will be useful for the application of SWCNTs in medicine, nanotechnology, and nanotoxicology.
Conclusion PLA has a distinct effect on the dispersion of SWCNTs. The SWCNT debundling caused by PLA generates fluorescence regardless of chirality. Unlike PLL, PLA has guanidinium groups that play a principal role in dispersion. MD simulations suggest that the guanidinium group favorably interacts with the sidewalls of SWCNTs. In addition to the strong binding of the guanidinium group, the positive charge of the group causes electrostatic repulsion between the SWCNTs, enhancing the dispersibility. PLA can be used to disperse SWCNTs without changing the optical properties, and these properties of PLA can be used to design new anchors to attach materials to SWCNTs.
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Full Paper Experimental Section Preparation of debundled SWCNTs Raw SWCNTs produced by high-pressure catalytic CO (HiPco) decomposition were purchased from Nano-Integris and were used as the starting materials. Polypeptides with molecular weight distributions were purchased from Sigma–Aldrich; pure 20-mers (purity > 95 %) were synthesized by Sigma–Aldrich; 5- and 10-mers (purity > 98 %) were synthesized by GenScript. Aliquots of 5 mg HiPco SWCNTs were dispersed at 0.2 mg mL 1 in purified water (5 mL) with 0.2 or 1.0 mg mL 1 polypeptide by using an ultrasonic homogenizer (Sonifire 250D, Branson) equipped with a 0.25-inch flat-tip for 0.5 h at a power density of about 20 W cm 2. To prevent heating during sonication, the bottle containing the sample solution was immersed in a water bath at 18 8C. The dispersed sample solution was centrifuged at 210 000 g for 0.5 h using an ultracentrifuge (S80AT3 rotor, Hitachi Koki) to remove the residue of the catalytic metal particles, nanotube bundles, and other impurities. The upper 70 % of the supernatant was collected, and it was a highly dispersed SWCNT solution.
Measurement of absorption spectra The absorption spectra of the SWCNTs were recorded over a wavelength range of 200–1350 nm by using a UV/Vis-NIR spectrophotometer (UV-3600, Shimadzu) with a quartz cell that has a path length of 10 mm.
Measurement of fluorescence spectra Fluorescence spectra of the SWCNTs were measured with a spectrofluorometer (Nanolog, Horiba) equipped with a liquid-nitrogencooled InGaAs near-IR alloy detector. The spectra can be used to assign SWCNTs to their corresponding chiralities based on data for single-chirality semiconducting SWCNTs.[30]
pH Dependence of absorption spectra The pH dependence of the absorption spectra of the SWCNTs was measured by adding HCl or NaOH. A dispersed SWCNT sample (300 mL) was treated by adding 5 mL of various concentrations of HCl or NaOH.
MD Simulation of interaction between a polypeptide and SWCNT The details of the MD system setup and simulations are described in the Supporting Information. Briefly, we used GROMACS 4.5.5[51] to perform the MD simulations. We used the AMBER force field (parm99SB)[52] to describe the protein energy function and the general AMBER force field (GAFF)[53] to describe the SWCNT. The SWCNT was modelled as a (7,6) tube with a length of 46 . The simulations were performed in an NPT ensemble with a temperature of 300 K and a pressure of 1 atm. The particle mesh Ewald method[54] was used to calculate long-range electrostatic interactions. The cut-off for van der Waals interactions was set to 10 . All systems were simulated with a time step of 2 fs.
Acknowledgements This work was partially supported by the Hirosawa Foundation for Technology, the Foundation Advanced Technology Institute, Chem. Eur. J. 2014, 20, 4922 – 4930
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and JSPS KAKENHI Grant Number 24750172. The theoretical calculations were performed using the Research Center for Computational Science, Okazaki, Japan, and the supercomputing resources were provided by the Human Genome Center (University of Tokyo) and the National Institute of Genetics (NIG), Research Organization of Information and Systems (ROIS). Keywords: dispersion · molecular dynamics · nanotubes · peptides · proteins [1] M. Calvaresi, F. Zerbetto, Acc. Chem. Res. 2013, 46, 2454 – 2463. [2] G. Zuo, S. G. Kang, P. Xiu, Y. Zhao, R. Zhou, Small 2013, 9, 1546 – 1556. [3] N. Yanamala, V. E. Kagan, A. A. Shvedova, Adv. Drug Delivery Rev. 2013, 65, 2070 – 2077. [4] D. Wang, L. Chen, Nano Lett. 2007, 7, 1480 – 1484. [5] V. Zorbas, A. L. Smith, H. Xie, A. Ortiz-Acevedo, A. B. Dalton, G. R. Dieckmann, R. K. Draper, R. H. Baughman, I. H. Musselman, J. Am. Chem. Soc. 2005, 127, 12323 – 12328. [6] M. J. Pender, L. A. Sowards, J. D. Hartgerink, M. O. Stone, R. R. Naik, Nano Lett. 2006, 6, 40 – 44. [7] H. Xie, A. Ortiz-Acevedo, V. Zorbas, R. H. Baughman, R. K. Draper, I. H. Musselman, A. B. Dalton, G. R. Dieckmann, J. Mater. Chem. 2005, 15, 1734 – 1741. [8] G. R. Dieckmann, A. B. Dalton, P. A. Johnson, J. Razal, J. Chen, G. M. Giordano, E. Munoz, I. H. Musselman, R. H. Baughman, R. K. Draper, J. Am. Chem. Soc. 2003, 125, 1770 – 1777. [9] R. Haggenmueller, S. S. Rahatekar, J. A. Fagan, J. Chun, M. L. Becker, R. R. Naik, T. Krauss, L. Carlson, J. F. Kadla, P. C. Trulove, D. F. Fox, H. C. Delong, Z. Fang, S. O. Kelley, J. W. Gilman, Langmuir 2008, 24, 5070 – 5078. [10] D. A. Tsyboulski, E. L. Bakota, L. S. Witus, J. D. Rocha, J. D. Hartgerink, R. B. Weisman, J. Am. Chem. Soc. 2008, 130, 17134 – 17140. [11] E. J. Becraft, A. S. Klimenko, G. R. Dieckmann, Biopolymers 2009, 92, 212 – 221. [12] G. K. C. Lee, C. Sach, M. L. H. Green, L. L. Wong, C. G. Salzmann, Chem. Commun. 2010, 46, 7013 – 7015. [13] H. Xie, E. J. Becraft, R. H. Baughman, A. B. Dalton, G. R. Dieckmann, J. Pept. Sci. 2008, 14, 139 – 151. [14] Z. Li, T. Uzawa, T. Tanaka, A. Hida, K. Ishibashi, H. Kataura, E. Kobatake, Y. Ito, Biotechnol. Lett. 2013, 35, 39 – 45. [15] D. R. Samarajeewa, G. R. Dieckmann, S. O. Nielsen, I. H. Musselman, Nanoscale 2012, 4, 4544 – 4554. [16] L. S. Witus, J. D. R. Rocha, V. M. Yuwono, S. E. Paramonov, R. B. Weisman, J. D. Hartgerink, J. Mater. Chem. 2007, 17, 1909 – 1915. [17] A. Ortiz-Acevedo, H. Xie, V. Zorbas, W. M. Sampson, A. B. Dalton, R. H. Baughman, R. K. Draper, I. H. Musselman, G. R. Dieckmann, J. Am. Chem. Soc. 2005, 127, 9512 – 9517. [18] T. Yu, Y. X. Gong, T. T. Lu, L. Wei, Y. Q. Li, Y. G. Mu, Y. Chen, K. Liao, RSC Adv. 2012, 2, 1466 – 1476. [19] D. Nepal, K. E. Geckeler, Small 2007, 3, 1259 – 1265. [20] D. Nepal, K. E. Geckeler, Small 2006, 2, 406 – 412. [21] P. Asuri, S. S. Karajanagi, H. Yang, T. J. Yim, R. S. Kane, J. S. Dordick, Langmuir 2006, 22, 5833 – 5836. [22] S. S. Karajanagi, A. A. Vertegel, R. S. Kane, J. S. Dordick, Langmuir 2004, 20, 11594 – 11599. [23] A. Hirano, Y. Maeda, X. Yuan, R. Ueki, Y. Miyazawa, J. Fujita, T. Akasaka, K. Shiraki, Chem. Eur. J. 2010, 16, 12221 – 12228. [24] T. J. McDonald, C. Engtrakul, M. Jones, G. Rumbles, M. J. Heben, J. Phys. Chem. B 2006, 110, 25339 – 25346. [25] A. Hirano, T. Kameda, D. Shinozaki, T. Arakawa, K. Shiraki, J. Phys. Chem. B 2013, 117, 7518 – 7527. [26] A. Hirano, T. Kameda, T. Arakawa, K. Shiraki, J. Phys. Chem. B 2010, 114, 13455 – 13462. [27] A. Hirano, H. Tokunaga, M. Tokunaga, T. Arakawa, K. Shiraki, Arch. Biochem. Biophys. 2010, 497, 90 – 96. [28] A. Hirano, T. Arakawa, K. Shiraki, J. Biochem. 2008, 144, 363 – 369. [29] A. J. Blanch, C. E. Lenehan, J. S. Quinton, Carbon 2011, 49, 5213 – 5228.
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Received: January 1, 2014 Published online on April 7, 2014
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& Molecular Dynamics
Arginine Side Chains as a Dispersant for Individual Single-Wall Carbon Nanotubes Atsushi Hirano,[a] Takeshi Tanaka,[a] Hiromichi Kataura,[a] and Tomoshi Kameda*[b]
Chem. Eur. J. 2014, 20, 4922 – 4930
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Full Paper Abstract: Charged peptides and proteins disperse singlewall carbon nanotubes (SWCNTs) in aqueous solutions. However, little is known about the role of their side chains in their interactions with SWCNTs. Homopolypeptide–SWCNT systems are ideal for investigating the mechanisms of such interactions. In this study, we demonstrate that SWCNTs are individually dispersed by poly-l-arginine (PLA). The debundled SWCNTs exhibited a distinct fluorescence. The dispersibility of SWCNTs with PLA was greater than that of SWCNTs
Introduction Single-wall carbon nanotubes (SWCNTs) have useful optical and electrical properties. Because of these advantages, peptide and protein interactions with SWCNTs are important in medical technology and nanotechnology; in addition, the interaction is also taken up in nanotoxicology for understanding their biological impacts at the molecular level.[1–3] Understanding the mechanisms of such interactions is important for developing and improving SWCNT applications. Experiments and simulations have shown that peptides and proteins interact with the sidewalls of SWCNTs through various types of interactions, including hydrophobic, van der Waals, electrostatic, and p–p interactions.[1–3] For example, aromatic residues form van der Waals, p–p, and hydrophobic contacts with SWCNTs, aliphatic residues form hydrophobic contacts, and charged residues form electrostatic contacts. Peptide interactions with SWCNTs can also be used for dispersion of SWCNTs in aqueous solutions.[4–15] Applications of this property include the separation and purification of SWCNTs in addition to the other applications mentioned above.[16–18] The binding of peptides to SWCNTs confers colloidal stability, that is, dispersibility, on SWCNTs that have been debundled by using a sonication treatment. The colloidal stability depends on both the strength of binding between peptides and SWCNTs and the electrostatic repulsion between peptides on the sidewalls of the SWCNTs. Therefore, the dispersibility is readily reduced at the isoelectronic points.[4] Such properties are also relevant to SWCNT dispersion by proteins.[19, 20] Proteins can retain their intrinsic conformations even when bound to the sidewalls of SWCNTs.[21, 22] However, such proteins can be readily affected by solution conditions, such as the presence of solutes that reduce the binding forces and dis-
[a] Dr. A. Hirano, Dr. T. Tanaka, Dr. H. Kataura Nanosystem Research Institute National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan) [b] Dr. T. Kameda Computational Biology Research Center National Institute of Advanced Industrial Science and Technology (AIST), Koto, Tokyo 135-0064 (Japan) E-mail: [email protected]
with poly-l-lysine (PLL). Molecular dynamics simulations suggest that the side chains of PLA have stronger interactions with the sidewalls of SWCNTs compared with those of PLL. The guanidinium group at the end of the side chain of an arginine residue plays an important role in the interaction with SWCNTs, likely through hydrophobic, van der Waals, and p–p interactions. PLA can be useful as a tool for the dispersion of SWCNTs and can be used to non-covalently anchor materials to SWCNTs with strong binding.
persibility.[23] The resulting bundling and flocculation of SWCNTs deteriorates the optical and electrical properties of the SWCNTs[24] such that high colloidal stability, that is, high dispersibility, is required for their use in applications. Investigations of peptide–SWCNT or protein–SWCNT interactions often focus on the aromatic and hydrophobic side chains, which form hydrophobic and p–p interactions with SWCNTs.[1–3] However, based on our previous studies, we expect that charged side chains can also bind SWCNTs and have a dispersive effect. We recently reported that arginine, which is a basic amino acid that is positively charged at neutral pH, can have a solubilizing effect on aromatic compounds.[25–28] The results of experiments and molecular dynamics (MD) simulations indicate that the guanidinium group, which is at the end of the arginine side chain, interacts with the aromatic moieties of compounds through hydrophobic or p–p contacts. Thus, poly-l-arginine (PLA) should act as a dispersant to enable the stable dispersion of SWCNTs. To date, little has been reported about the dispersion of SWCNTs by using PLA. Lee et al. reported that SWCNTs did not disperse when using PLA;[12] however, the power density of their sonication system might explain these unsatisfactory results. In fact, they also found that SWCNTs did not disperse when using poly-l-lysine (PLL), whereas Wang and Chen reported the successful dispersion of SWCNTs by using PLL.[4] This discrepancy may depend on the sonication conditions used for dispersion.[29] In this study, we investigated the effects of PLA on the dispersion of SWCNTs by using a tip sonicator capable of debundling SWCNTs.[30] We observed remarkable dispersibility and fluorescent intensities, and we verified that the guanidinium group of PLA binds strongly to SWCNTs. This binding strength was supported by MD simulations. In contrast, exposure to PLL resulted in low dispersiblity. These results indicate that the ability of charged polypeptides to disperse SWCNTs depends on whether the power density of the sonication system is sufficient to debundle the SWCNTs. The binding properties of PLA show that it could be used to disperse SWCNTs or even to non-covalently anchor proteins or other materials to the sidewalls of SWCNTs, which could be useful for medicine and nanotechnology.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201400003. Chem. Eur. J. 2014, 20, 4922 – 4930
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Full Paper Results and Discussion Dispersibility of SWCNTs in polypeptide solutions SWCNTs in solution with PLA or PLL were subjected to ultrasonic homogenization with a flat tip in the absence of any buffers. The power of the homogenizer is sufficient to debundle SWCNTs (see Figure S1 in the Supporting Information). Subsequent ultracentrifugation was performed to obtain the dispersed SWCNT solution as the supernatant. In the experiments, the selected molecular weights of the polypeptides were > 70 000 (> 70 k) and 5000–15 000 (5–15 k) for PLA and > 30 000 (> 30 k) and 15 000–30 000 (15–30 k) for PLL. Figure 1 A presents the absorbance spectra of the SWCNTs dispersed by using PLA and PLL, which were normalized to the absorbance at 600 nm. The absorption peaks at approximately 940–1350 and 620–940 nm were assigned to the first and second optical transitions of the semiconducting species, which were designated as the S11 and S22 bands, respectively. The absorption peaks at approximately 400–620 nm were assigned to the first optical transition of metallic SWCNTs, which was designated as the M11 band.[31–33] Thus, both metallic and semiconducting SWCNTs were found among the dispersed SWCNTs. Absorption spectra in the UV region were excluded from the Figure because the p-plasmon of SWCNTs and peptide bonds exhibit absorption in the UV region. The spectrum of SWCNTs dispersed by 1 wt. % sodium dodecyl sulfate (SDS) was also measured after adding aliquots of NaOH to the SWCNT solutions to bring them to pH 12 to avoid absorbance reduction due to oxidation (Figure 1 A).[33] The SWCNTs dispersed by PLA displayed sharp peaks in the near-infrared region corresponding to the S11 and S22 bands, although these peaks had lower intensities than those in 1 wt. % SDS. Because the sharpness of the S11 and S22 bands relates to the dispersibility of the SWCNTs,[9] it appears that PLA is able to substantially disperse the SWCNTs. In contrast, the SWCNTs dispersed by PLL displayed attenuated intensities in the near-infrared region. The absorbance peaks of these solutions were shifted towards longer wavelengths by SDS, PLL, and PLA (in order of increasing shift). Such peak shifts depend on the effective dielectric constants around the sidewalls of the SWCNTs.[34] Figure 1 B presents the dispersibility of the SWCNTs in 0.2 and 1.0 mg mL 1 of the polypeptides. The dispersibility is defined as the concentration of the supernatant of the solution after ultracentrifugation. An absorbance at 600 nm was used to estimate the concentration of SWCNTs because absorbance in the UV region is affected by coexisting polypeptides (see also the Supporting Information, Figure S2). In addition, the absorbance at the S11 and S22 bands is also affected by oxidation depending on pH and temperature.[33, 35] Higher-molecular-weight polypeptides were found to increase the dispersibility of SWCNTs. PLA (> 70 k) was found to be the most effective at increasing the dispersibility. A 1.0 mg mL 1 solution of PLA exhibited greater dispersibility than a 0.2 mg solution, independent of molecular weight. Similarly, a 1.0 mg mL 1 solution of PLL exhibited greater dispersibility than a 0.2 mg mL 1 solution for PLL (> 30 k), whereas for PLL (15–30 k), a 0.2 mg mL 1 solution Chem. Eur. J. 2014, 20, 4922 – 4930
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Figure 1. (A) Absorption spectra of SWCNTs dispersed by polypeptides or 1 wt. % SDS. Absorbance values were normalized to the values at 600 nm. For 1 wt. % SDS, NaOH was added to the solution to raise the pH value to near 12, which avoids decreasing the absorbance due to oxidation.[33] (B) Dispersibility of SWCNTs in the presence of PLA or PLL at 0.2 and 1.0 mg mL 1.
yielded a dispersion greater than 1.0 mg mL 1. The decrease in dispersibility at higher PLL concentrations may be attributed to non-covalent cross-linking of the SWCNTs by PLL; similar results were observed for poly-l-glutamate (the Supporting Information, Figure S3). The dispersion of SWCNTs using PLA or PLL has already been reported. Lee et al. demonstrated the high dispersibility of SWCNTs by using mixtures of PLA or PLL and poly-l-glutamate and no dispersibility of SWCNTs using the individual polypeptides.[12] Interestingly, their results are inconsistent with the results presented here. This inconsistency can be explained by differences between methods; they dispersed the SWCNTs by
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Full Paper using a bath sonicator, not a tip sonicator. Tip-sonicated SWCNTs display greater dispersibility than bath-sonicated SWCNTs.[29] The lack of dispersion when using PLA or PLL alone may be explained by the incompleteness of SWCNT debundling. In fact, broader spectral sharpness was observed in the previous study for the S11 and S22 bands, which suggests that the SWCNTs were insufficiently dispersed. Thus, differences in dispersion methods can lead to different conclusions about the effects of dispersants. The dispersibility of SWCNTs using peptides and proteins also depends on pH and salt concentration.[4, 19, 20, 23, 36] The dispersibility is reduced considerably at the isoelectric point or in the presence of salts. The solution used in this study was at neutral pH, without any other salts. The pKa values of side chains for arginine and lysine are 12.5 and 9.2, respectively. Thus, the positive charges of the side chains of the polypeptides on the SWCNTs generate electrostatic repulsion between SWCNTs, leading to dispersion. In fact, the addition of 100 mm NaCl caused the SWCNTs to aggregate (data not shown), which is accounted for by the electrostatic screening effect.[23] Fluorescence spectra of SWCNTs Debundled semiconducting SWCNTs are known to exhibit fluorescence in the near-infrared region.[37, 38] To demonstrate the debundling of SWCNTs in our polypeptide tests, we recorded fluorescence spectra for the SWCNTs treated with PLA (> 70 k) and PLL (> 30 k) at 1.0 mg mL 1. Figure 2 presents the fluorescence spectra of the SWCNTs dispersed by the polypeptides. These data were recorded by scanning the excitation wavelength from 500 to 850 nm and scanning the emission wavelength from 900 to 1400 nm. Distinctive peaks can be identified for each chirality based on the data for single-chirality semiconducting SWCNTs.[30] PLA (> 70 k) exhibited intense fluorescence, as expected based on the absorption spectra (Figure 1 A). The spectral profile was largely identical to that obtained with sodium deoxycholate, which is known as a good dispersant for SWCNTs;[39] thus, PLA disperses SWCNTs without any chirality bias. PLL (> 30 k) also displayed similar fluorescence spectra. As expected based on the absorbance spectra (Figure 1 A), fluorescence peaks corresponding to the S11 band were slightly shifted toward longer wavelengths with PLA (> 70 k) than with PLL (> 30 k), likely due to the effective dielectric constants of PLA and PLL around the sidewalls of the SWCNTs, as mentioned above. pH Dependence of the absorption spectra of SWCNTs As shown above, both PLA and PLL resulted in fluorescence due to SWCNT dispersion. However, the differences in absorption at S11 and S22 require further explanation (Figure 1 A). One possible cause of these differences is the formation of small bundles of SWCNTs. If the bundles were composed of semiconducting SWCNTs, then the absorbance and fluorescence would be reduced.[9, 24] Such low dispersibility by PLL may lead to the attenuation of absorbance at S11 and S22. Another possible cause is the oxidation of SWCNTs. SWCNTs are known to be Chem. Eur. J. 2014, 20, 4922 – 4930
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Figure 2. Fluorescence spectra of SWCNTs dispersed by (A) PLA (> 70 k) and (B) PLL (> 30 k) at 1 mg mL 1. The respective peaks were assigned to the corresponding chiralities.
oxidized by dissolved oxygen and protons in aqueous solution,[33, 40] and the oxidation depends on pH. The local pH around an SWCNT could induce the oxidation and resulting attenuation of the absorption spectra. Here, after the dispersion of SWCNTs using PLA (> 70 k) or PLL (> 30 k) at 1.0 mg mL 1, we added aliquots of HCl or NaOH to the SWCNT solutions to modify the pH before collecting spectra. Figure 3 A and B present the absorbance spectra of the SWCNTs at various pH
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Full Paper higher dispersibility by PLA is attributed to the high binding affinity and positive charges of its side chains.
MD Simulation of the interaction between a peptide and SWCNT Figure 3. Spectral changes of SWCNTs with (A) PLA (> 70 k) and (B) PLL (> 30 k) at 1 mg mL 1 that occurred when the pH was altered by HCl or NaOH. The spectra were normalized to the values at 600 nm. (C) pH dependence of absorbance at 1130 and 1270 nm for PLA (> 70 k) and PLL (> 30 k).
values, and the spectra were normalized to absorption at 600 nm. Because the pH was approximately 6 before the addition of an acid or base, the spectra were not significantly affected by the addition of NaOH, except when at extremely basic levels near pH 12; the small spectral changes at that pH may be caused by the deprotonation of the polypeptide side chains. Recall that the pKa values of the side chains of arginine and lysine are 12.5 and 9.2, respectively. Importantly, the spectral intensities were dramatically decreased at acidic pH values. The pH dependence of the intensities at 1130 and 1270 nm, which correspond to peaks in the S11 bands, are plotted in Figure 3 C. The intensities were found to decrease near pH 3 due to the oxidation. Thus, the SWCNTs dispersed by the polypeptides are not oxidized in the absence of HCl or NaOH. The attenuation of the absorption spectral intensities by PLL (> 30 k) is therefore attributable to the formation of small SWCNT bundles.[9] In addition, the existence of impurities such as amorphous carbon due to the low dispersibility by PLL may also affect the spectral intensity.[39] The lower attenuation of spectral intensity by PLA (> 70 k) suggests that PLA is tightly wrapped around individual SWCNTs[41]
Dispersibility of SWCNTs in oligopeptide solutions As seen in Figure 1, PLA (5–15 k) displayed higher dispersibility than PLL (> 30 k). Therefore, PLA is likely to be more effective for dispersion. However, these polypeptides have molecular weight distributions. Oligopeptides with specified lengths are more useful for an accurate estimation of the dispersive effect. Figure 4 presents the dispersibility of SWCNTs in the presence of 5-, 10-, and 20-residue oligopeptides, which are denoted by R5, R10, and R20 for oligoarginine and K5, K10, and K20 for oligolysine, respectively. Longer sequences resulted in higher dispersibility for oligoarginine, whereas only K20 exhibited dispersion for oligolysine, and this dispersion was very low. The fact that even R5 exhibits dispersion suggests tighter binding of the arginine side chains to the sidewalls of SWCNTs compared with the binding of the lysine side chains. The lower dispersibility when using shorter oligopeptides is expected because the binding affinity should be reduced by the shortening of the sequences. Incidentally, the monomers of arginine and lysine provide no dispersion of SWCNTs (data not shown). Thus, the Chem. Eur. J. 2014, 20, 4922 – 4930
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MD simulations were performed to understand the dynamics of the peptides on the SWCNT sidewalls and to determine how PLA
Figure 4. Dispersibility of SWCNTs in the presence of 5-, 10-, and 20-mer oligopeptides at 0.2 mg mL 1.
tightly binds to the sidewalls. A (7,6) SWCNT was chosen for the simulation because it was abundant in the sample, as shown in Figure 2. It is reasonable not to consider the chirality dependence of the dynamics, as previously reported,[42] because there is no chirality dependence for the dispersion (Figure 2). Figure 5 presents the representative conformations of 15-residue oligoarginine (R15) and 15-residue oligolysine (K15). The side chains of both peptides were found to bind to the sidewall of the SWCNT. The guanidinium group of R15 interacts with the sidewall of the SWCNT (Figure 5 A and B), which is consistent with the interaction of SWCNTs with lysozyme, an enzyme with many arginine residues (the Supporting Information, Figure S4).[43] K15 was found to interact with the sidewall of the SWCNT through the alkyl chains of its side chains (Figure 5 D and E). Interestingly, when the side chains face toward the solvent, the backbones close to the side chains tend to leave the sidewall of the SWCNT, as shown in Figure 5 B and E. Figure 5 C and F present the sphere representations of the backbones of R15 and K15, corresponding to Figure 5 A and B and Figure 5 D and E, respectively. The backbones also interact with the sidewall of the SWCNT; in particular, oxygen atoms tend to be located close to the sidewall.
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Full Paper Table 1. Positions of the highest peaks of the radial probability distributions for each atom of the peptide side chains. R15
Figure 5. Representative conformations of R15 and K15 on an SWCNT. Side chains and backbones of (A, B) R15 and (D, E) K15 are shown as stick and cartoon representations, respectively. Only the backbones for (C) R15 and (F) K15 are displayed by using sphere representations: red, oxygen; green, carbon; blue, nitrogen; light gray, hydrogen. SWCNTs are shown by using dark-gray sphere representations.
Estimating the radial distribution of the peptides is a useful way to identify the functional groups that contribute to the interaction. Figure 6 A and B present radial probability distributions for each atom of the side chains of R15 or K15, respectively, near the SWCNT; each atom is labelled as denoted in the insets. In addition, the peak positions of the atoms of the side chains are listed in Table 1. For R15, the probability of Cz exhib-
K15
Atom
Peak position []
Atom
Peak position []
Cb Cg Cd Ne Cz
7.9 0.1 7.8 0.0 7.9 0.0 7.8 0.1 7.7 0.0
Cb Cg Cd Ce Nz
7.9 0.0 7.9 0.0 8.0 0.0 7.8 0.0 8.8 0.1
ited the highest peak (at 7.7 ). Cb, Cg, Cd, and Ne have lower probabilities of being close to the sidewall, which indicates that the end of the side chain, that is, the guanidinium group, is the part that most frequently interacts with the sidewall of the SWCNT. In contrast, for K15, Cb exhibited its highest peak at 7.9 , and the distribution of Nz atoms was broadened. The peak position of Nz is at 8.8 , indicating that the end of the side chain of K15 is far away from the sidewall of SWCNT, as expected based on the conformation shown in Figure 5. Figure 6 C and D present the radial probability distributions of each atom in the peptide backbones for R15 and K15, respectively. R15 and K15 displayed similar profiles in the distribution; namely, the N and H atoms of the backbones were relatively far away from the sidewall, whereas the O atoms were closer to the sidewall than the Ca atoms. Interestingly, the peak position of O atoms is at 7.5 (Table 2), which is the closest position to the SWCNT sidewall among any of the highest peaks for different atoms (Tables 1 and 2). The characteristic profile of O atoms was found to be attributable to their close approach to the sidewall of the SWCNT (Figure 5 C and F). Such binding affinity is ascribable to strong van der Waals interactions between O atoms and the SWCNT sidewall. Steered MD simulation for estimating the binding force between a peptide and SWCNT
Figure 6. Radial probability distributions for each atom of the (A, B) side chains and (C, D) the peptide bonds for (A,C) R15 and (B,D) K15 on the sidewall of SWCNTs, as determined by MD simulations. Chem. Eur. J. 2014, 20, 4922 – 4930
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The conformations of the peptides on the sidewall of the SWCNT described above should affect the binding force of the peptides to the SWCNT. We used steered MD simulations with various constant forces to acquire the minimal force at which the peptides were stripped off the SWCNT sidewall. The steered MD
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Full Paper Table 2. Positions of the highest peaks of the radial probability distributions for each atom of the peptide backbones. R15
K15
Atom
Peak position []
Atom
Peak position []
Ca O C N H
7.9 0.0 7.5 0.1 8.5 0.1 8.5 0.3 8.9 0.6
Ca O C N H
8.1 0.2 7.5 0.1 8.5 0.0 8.6 0.2 9.3 0.3
simulations were executed for 50 ns and performed three times. The direction of the force between the center of mass of the peptide and an axis of the SWCNT is normal to the axis of the SWCNT. If a peptide was not stripped from the SWCNT in all three simulations, it was considered “unstripped”, and the next simulation was carried out with a force that was 25 pN larger. If a peptide was stripped in at least one of the simulations, it was considered “stripped”, and the force value was regarded as the minimum force needed to strip the polypeptide off the SWCNT (the Supporting Information, Figure S5). The minimum values for stripping were approximately 200– 225 pN for R15 and approximately 100–125 pN for K15. Importantly, the force value of R15 was larger than that of K15. The higher binding force for R15 should more effectively prevent it from coming off the sidewall of the SWCNT, which is associated with the fact that PLA leads to greater dispersibility of SWCNTs than PLL. Origin of the characteristic binding of the guanidinium group onto the sidewalls of SWCNTs Although several simulations of interactions between the guanidinium group of arginine and the sidewalls of SWCNTs have been performed,[1–3] few experimental studies have been reported. Some experimental studies using proteins and peptides with various sequences suggest that the arginine side chain is important for binding;[1, 2] however, none of these studies have specifically tested arginine alone. Guo et al. quantified the amount of arginine adsorbed onto SWCNTs in the cell culture medium.[44] However, no basic data were provided about the adsorbability of arginine or the guanidinium group onto SWCNTs. To our knowledge, the present study is the first to experimentally demonstrate that arginine side chains interact with SWCNTs and that the dispersibility with PLA is greater than that with PLL. Importantly, the characteristics of PLA that we describe are supported by MD simulations. The adsorption of the guanidinium group onto the sidewalls of SWCNTs depends on hydrophobic or p–p interactions. Previously, we reported that arginine interacts with aromatic compounds, such as nucleobases and polyphenols, increasing their solubilities.[25–27] MD simulations and binding free-energy calculations indicate that the arginine side chain, that is, the guanidinium group, interacts with aromatic moieties, which suggests a p–p interaction.[25, 26] These calculations were based on a classical force field, and thus, nonbonded interactions were Chem. Eur. J. 2014, 20, 4922 – 4930
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represented by the summation of Coulomb and van der Waals terms; the p–p interaction was thus implicitly considered by nonbonded interaction terms.[26] Our simulation was also performed by using the classical force field. The peak position of Cz for R15 is close to that of Ne (Table 1), indicating that the planes of the guanidinium groups are parallel to the SWCNT sidewall. This observation suggests the existence of p–p interactions between the two molecules. In contrast, K15 has no such interactions. The p–p interactions account for the strong binding force of PLA to the sidewalls of SWCNTs. In addition, various studies have demonstrated that hydrophobic interactions are also involved in the binding. Mason et al. reported the dynamics of guanidine, which is similar to the guanidinium group, in an aqueous solution using MD simulations. The simulations indicate that guanidine is poorly hydrated above and below its plane in aqueous solution.[45] Li et al. suggested that this property of guanidine is related to that of the guanidinium group; specifically, the guanidinium group of arginine possesses hydrophobicity, similar to guanidine.[46] The authors compared the dynamics of arginine to surfactant-like behavior. Characteristic interactions of arginine with SWCNTs were also demonstrated by using density functional theory.[47, 48] Mechanistic insight into the interactions between proteins and SWCNTs Interactions between proteins and SWCNTs have generally been accounted for by hydrophobic, van der Waals, electrostatic, and p–p interactions.[1–3] Some simulations of interactions between proteins and SWCNTs indicate binding of the guanidinium group of the arginine residues to the sidewalls of the SWCNTs.[43, 49] As mentioned above, arginine has a p-electron, a hydrophobic and positively charged moiety; therefore, the binding of arginine residues onto SWCNTs should be based on multimodal interactions consisting of the above interactions. The arginine residues of proteins can play a principal role in the binding to SWCNTs, as observed for PLA. In fact, basic proteins, that is, arginine-rich proteins, such as lysozyme and histones, were experimentally demonstrated to debundle or highly disperse SWCNTs.[19, 23, 50] This insight into the behavior of arginine on the sidewalls of SWCNTs will be useful for the application of SWCNTs in medicine, nanotechnology, and nanotoxicology.
Conclusion PLA has a distinct effect on the dispersion of SWCNTs. The SWCNT debundling caused by PLA generates fluorescence regardless of chirality. Unlike PLL, PLA has guanidinium groups that play a principal role in dispersion. MD simulations suggest that the guanidinium group favorably interacts with the sidewalls of SWCNTs. In addition to the strong binding of the guanidinium group, the positive charge of the group causes electrostatic repulsion between the SWCNTs, enhancing the dispersibility. PLA can be used to disperse SWCNTs without changing the optical properties, and these properties of PLA can be used to design new anchors to attach materials to SWCNTs.
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Full Paper Experimental Section Preparation of debundled SWCNTs Raw SWCNTs produced by high-pressure catalytic CO (HiPco) decomposition were purchased from Nano-Integris and were used as the starting materials. Polypeptides with molecular weight distributions were purchased from Sigma–Aldrich; pure 20-mers (purity > 95 %) were synthesized by Sigma–Aldrich; 5- and 10-mers (purity > 98 %) were synthesized by GenScript. Aliquots of 5 mg HiPco SWCNTs were dispersed at 0.2 mg mL 1 in purified water (5 mL) with 0.2 or 1.0 mg mL 1 polypeptide by using an ultrasonic homogenizer (Sonifire 250D, Branson) equipped with a 0.25-inch flat-tip for 0.5 h at a power density of about 20 W cm 2. To prevent heating during sonication, the bottle containing the sample solution was immersed in a water bath at 18 8C. The dispersed sample solution was centrifuged at 210 000 g for 0.5 h using an ultracentrifuge (S80AT3 rotor, Hitachi Koki) to remove the residue of the catalytic metal particles, nanotube bundles, and other impurities. The upper 70 % of the supernatant was collected, and it was a highly dispersed SWCNT solution.
Measurement of absorption spectra The absorption spectra of the SWCNTs were recorded over a wavelength range of 200–1350 nm by using a UV/Vis-NIR spectrophotometer (UV-3600, Shimadzu) with a quartz cell that has a path length of 10 mm.
Measurement of fluorescence spectra Fluorescence spectra of the SWCNTs were measured with a spectrofluorometer (Nanolog, Horiba) equipped with a liquid-nitrogencooled InGaAs near-IR alloy detector. The spectra can be used to assign SWCNTs to their corresponding chiralities based on data for single-chirality semiconducting SWCNTs.[30]
pH Dependence of absorption spectra The pH dependence of the absorption spectra of the SWCNTs was measured by adding HCl or NaOH. A dispersed SWCNT sample (300 mL) was treated by adding 5 mL of various concentrations of HCl or NaOH.
MD Simulation of interaction between a polypeptide and SWCNT The details of the MD system setup and simulations are described in the Supporting Information. Briefly, we used GROMACS 4.5.5[51] to perform the MD simulations. We used the AMBER force field (parm99SB)[52] to describe the protein energy function and the general AMBER force field (GAFF)[53] to describe the SWCNT. The SWCNT was modelled as a (7,6) tube with a length of 46 . The simulations were performed in an NPT ensemble with a temperature of 300 K and a pressure of 1 atm. The particle mesh Ewald method[54] was used to calculate long-range electrostatic interactions. The cut-off for van der Waals interactions was set to 10 . All systems were simulated with a time step of 2 fs.
Acknowledgements This work was partially supported by the Hirosawa Foundation for Technology, the Foundation Advanced Technology Institute, Chem. Eur. J. 2014, 20, 4922 – 4930
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and JSPS KAKENHI Grant Number 24750172. The theoretical calculations were performed using the Research Center for Computational Science, Okazaki, Japan, and the supercomputing resources were provided by the Human Genome Center (University of Tokyo) and the National Institute of Genetics (NIG), Research Organization of Information and Systems (ROIS). Keywords: dispersion · molecular dynamics · nanotubes · peptides · proteins [1] M. Calvaresi, F. Zerbetto, Acc. Chem. Res. 2013, 46, 2454 – 2463. [2] G. Zuo, S. G. Kang, P. Xiu, Y. Zhao, R. Zhou, Small 2013, 9, 1546 – 1556. [3] N. Yanamala, V. E. Kagan, A. A. Shvedova, Adv. Drug Delivery Rev. 2013, 65, 2070 – 2077. [4] D. Wang, L. Chen, Nano Lett. 2007, 7, 1480 – 1484. [5] V. Zorbas, A. L. Smith, H. Xie, A. Ortiz-Acevedo, A. B. Dalton, G. R. Dieckmann, R. K. Draper, R. H. Baughman, I. H. Musselman, J. Am. Chem. Soc. 2005, 127, 12323 – 12328. [6] M. J. Pender, L. A. Sowards, J. D. Hartgerink, M. O. Stone, R. R. Naik, Nano Lett. 2006, 6, 40 – 44. [7] H. Xie, A. Ortiz-Acevedo, V. Zorbas, R. H. Baughman, R. K. Draper, I. H. Musselman, A. B. Dalton, G. R. Dieckmann, J. Mater. Chem. 2005, 15, 1734 – 1741. [8] G. R. Dieckmann, A. B. Dalton, P. A. Johnson, J. Razal, J. Chen, G. M. Giordano, E. Munoz, I. H. Musselman, R. H. Baughman, R. K. Draper, J. Am. Chem. Soc. 2003, 125, 1770 – 1777. [9] R. Haggenmueller, S. S. Rahatekar, J. A. Fagan, J. Chun, M. L. Becker, R. R. Naik, T. Krauss, L. Carlson, J. F. Kadla, P. C. Trulove, D. F. Fox, H. C. Delong, Z. Fang, S. O. Kelley, J. W. Gilman, Langmuir 2008, 24, 5070 – 5078. [10] D. A. Tsyboulski, E. L. Bakota, L. S. Witus, J. D. Rocha, J. D. Hartgerink, R. B. Weisman, J. Am. Chem. Soc. 2008, 130, 17134 – 17140. [11] E. J. Becraft, A. S. Klimenko, G. R. Dieckmann, Biopolymers 2009, 92, 212 – 221. [12] G. K. C. Lee, C. Sach, M. L. H. Green, L. L. Wong, C. G. Salzmann, Chem. Commun. 2010, 46, 7013 – 7015. [13] H. Xie, E. J. Becraft, R. H. Baughman, A. B. Dalton, G. R. Dieckmann, J. Pept. Sci. 2008, 14, 139 – 151. [14] Z. Li, T. Uzawa, T. Tanaka, A. Hida, K. Ishibashi, H. Kataura, E. Kobatake, Y. Ito, Biotechnol. Lett. 2013, 35, 39 – 45. [15] D. R. Samarajeewa, G. R. Dieckmann, S. O. Nielsen, I. H. Musselman, Nanoscale 2012, 4, 4544 – 4554. [16] L. S. Witus, J. D. R. Rocha, V. M. Yuwono, S. E. Paramonov, R. B. Weisman, J. D. Hartgerink, J. Mater. Chem. 2007, 17, 1909 – 1915. [17] A. Ortiz-Acevedo, H. Xie, V. Zorbas, W. M. Sampson, A. B. Dalton, R. H. Baughman, R. K. Draper, I. H. Musselman, G. R. Dieckmann, J. Am. Chem. Soc. 2005, 127, 9512 – 9517. [18] T. Yu, Y. X. Gong, T. T. Lu, L. Wei, Y. Q. Li, Y. G. Mu, Y. Chen, K. Liao, RSC Adv. 2012, 2, 1466 – 1476. [19] D. Nepal, K. E. Geckeler, Small 2007, 3, 1259 – 1265. [20] D. Nepal, K. E. Geckeler, Small 2006, 2, 406 – 412. [21] P. Asuri, S. S. Karajanagi, H. Yang, T. J. Yim, R. S. Kane, J. S. Dordick, Langmuir 2006, 22, 5833 – 5836. [22] S. S. Karajanagi, A. A. Vertegel, R. S. Kane, J. S. Dordick, Langmuir 2004, 20, 11594 – 11599. [23] A. Hirano, Y. Maeda, X. Yuan, R. Ueki, Y. Miyazawa, J. Fujita, T. Akasaka, K. Shiraki, Chem. Eur. J. 2010, 16, 12221 – 12228. [24] T. J. McDonald, C. Engtrakul, M. Jones, G. Rumbles, M. J. Heben, J. Phys. Chem. B 2006, 110, 25339 – 25346. [25] A. Hirano, T. Kameda, D. Shinozaki, T. Arakawa, K. Shiraki, J. Phys. Chem. B 2013, 117, 7518 – 7527. [26] A. Hirano, T. Kameda, T. Arakawa, K. Shiraki, J. Phys. Chem. B 2010, 114, 13455 – 13462. [27] A. Hirano, H. Tokunaga, M. Tokunaga, T. Arakawa, K. Shiraki, Arch. Biochem. Biophys. 2010, 497, 90 – 96. [28] A. Hirano, T. Arakawa, K. Shiraki, J. Biochem. 2008, 144, 363 – 369. [29] A. J. Blanch, C. E. Lenehan, J. S. Quinton, Carbon 2011, 49, 5213 – 5228.
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Received: January 1, 2014 Published online on April 7, 2014
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