Oxidative Stress of Carbon Nanotubes on Proteins Is Mediated by

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Oxidative Stress of Carbon Nanotubes on Proteins is Mediated by Metals Originating from the Catalyst Remains Atsushi Hirano, Momoyo Wada, Takeshi Tanaka, and Hiromichi Kataura ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07936 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 27, 2019

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Oxidative Stress of Carbon Nanotubes on Proteins is Mediated by Metals Originating from the Catalyst Remains Atsushi Hirano,* Momoyo Wada, Takeshi Tanaka, Hiromichi Kataura Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8565, Japan Email: [email protected] ABSTRACT: Nanomaterials introduced into biological systems are immediately coated by proteins in vivo. They induce oxidative stress on adsorbed proteins and hence alter the protein structures, which determines the fate pathways and biological impacts of nanomaterials. Carbon nanotubes (CNTs) have been suggested to cause protein oxidation. In this work, we discovered that CNTs induce oxidative stress on proteins in cooperation with coexisting metals originating from catalyst remains. Protein sulfhydryl groups were readily oxidized by the coexistence of CNTs and metals. Numerical simulations of the reaction demonstrated that the metals effectively mediate electron transfer between the CNTs and protein sulfhydryl groups. Thus, the coexistence of CNTs and metals, even in low concentrations, generates oxidative stress on proteins with high reaction rates. Metal catalysts used for CNT growth, in turn, catalyze the oxidation reaction of proteins. The proposed protein oxidation mechanism will advance the fundamental understanding of the biological safety and toxicity of nanomaterials synthesized using metal catalysts. KEYWORDS: carbon nanotube, catalyst, disulfide bond, cysteine, iron, oxidation stress, protein Preparation of safe nanomaterials and accurate safety evaluation of nanomaterials play critical roles in ensuring sustainable development of nanotechnologies in industry and society. Biological impacts of nanomaterials can be ascribed not only to intrinsic nanomaterial properties in terms of morphology, physics and chemistry1 but also to extrinsic factors such as catalyst remains that are generated upon nanomaterial synthesis processes.2 To date, biological impacts of nanomaterials and catalyst remains have been individually reported, whereas their cooperative impacts remain unclear. Therefore, it is important to determine whether such cooperative reaction pathway is present or absent to obtain a full picture of nanomaterial behaviour in biological systems. In early stages of nanomaterial uptake into biological systems, the nanomaterial surfaces undergo protein adsorption, which leads to the formation of a protein layer called the protein corona.3 The stability, conformation and components of the protein corona are closely associated with various biological impacts, such as internalization of the nanomaterials into cells and hence cytotoxicity.4–6 Thus, it is necessary to clarify physical and chemical changes of protein structures on the nanomaterials during protein corona formation for understanding, predicting and controlling their biological impacts. As of yet, nothing is known about the cooperative impacts of nanomaterials and coexisting substances, e.g., catalyst remains, on protein corona formation. Carbon nanotubes (CNTs) are promising nanomaterials used for a wide range of applications, such as biosensing, bioimaging, and drug transport. Because CNTs are generally synthesized using transition metal catalysts, as-grown CNT samples principally contain transitional metal particles, i.e., catalyst remains, as impurities; the transition metal species include Fe, Y, Ni, Co, Mo and so on.7 Despite previous efforts to remove metal particles from the samples, these species remain in most cases because they are often encapsulated by graphitic shells.8 A portion of metals can be released from the shells, particularly though subsequent processes such as oxidation, mechanical grinding and sonication.9

Previous studies have demonstrated that metal particles induce cytotoxicity or oxidative stress on biomolecules through chemical reactions, producing reactive oxidative species.9,10 The CNTs themselves also have biological impacts; previous studies have reported the physical adsorption of various proteins onto CNTs, which undergo protein inactivation;11,12 while other studies have reported that the protein corona on the CNT surfaces modifies the intrinsic cytotoxicity of CNTs.13–16 Hydrophobic and – interactions are involved in protein adsorption.3,17,18 However, the effect of metal particles on protein corona formation has not been reported. In particular, the metal particles contain transition metals, so that they potentially participate in a redox reaction (including oxidation and reduction) between proteins and CNTs. In a recent work, CNTs were demonstrated to induce oxidative stress on proteins; specifically, cysteine (Cys) residues of the proteins are oxidized by CNTs, leading to the formation of cystine (Cys-Cys).19,20 Because the oxidative stress of CNTs on proteins is associated with charge transfer, the oxidative stress is expected to be mediated by metal ions dissociated from the metal particles. Such oxidative stress on proteins can principally affect the protein corona structure and homeostasis of biological systems.21 Here, we investigate the oxidation of proteins by CNTs in the presence of the remaining metal particles or extrinsic metal ions. Oxidation of the proteins, i.e., oxidation of Cys residues, by CNTs was demonstrated to be mediated by metal ions originating from the catalyst remains; such indirect redox mechanism between CNTs and Cys residues was confirmed by experiments using both a reduced protein and Cys and further by numerical simulations. RESULTS Determination of Concentration of Metals Dissociated from the Catalyst Particles. The CNTs used in this study were single-walled CNTs ca. 1 nm in diameter, which were synthesized by the high-pressure catalytic CO (HiPco) process. In this case, iron ions can dissociate from the remaining metal particle surfaces on the HiPco CNTs because the catalyst particle forms iron oxides, such as Fe2O3 (hematite) and Fe3O4

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(magnetite), after the synthesis process.22,23 The iron ion concentration contained in the CNT suspension was determined by high-performance liquid chromatography (HPLC) (see Fig. S1 in Supporting Information), where dissociated iron ions were captured using a chelating agent, i.e., ethylenediamineN,N,N',N'-tetraacetic acid (EDTA).24 The concentration of iron ions dissociated from 1 mg/mL CNTs was estimated to be approximately 60 M (Table S1). The major iron ion species was thought to be ferric ion (Fe3+) rather than ferrous ion (Fe2+) because the CNT samples mainly contain Fe2O3 and Fe3O4. Importantly, both Fe3+ and Fe2+ mediate the redox reaction between proteins and CNTs, as described below. Observation of Oxidation of Sulfhydryl Groups of a Reduced Protein by Carbon Nanotubes. Protein oxidation by CNTs primarily occurs at the cysteine (Cys) residues of proteins and simultaneously leads to CNT reduction.20 In the present study, protein oxidation by CNTs was examined using a reduced form of hen-egg lysozyme, which has 129 amino acid residues, as a model protein. The reduced lysozyme was prepared by the addition of dithiothreitol (DTT) to native lysozyme solution, which results in the breakage of its disulfide bonds and hence the production of free Cys residues (Fig. 1a);

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note that native lysozyme has a radius of ca. 2 nm. The reduced lysozyme principally has eight free Cys residues because native lysozyme has four disulfide bonds and no free Cys residues. The redox reaction of 70 M lysozyme with 1 mg/mL CNTs was induced by mixing them via ultrasonication in the presence and absence of 2 mM EDTA; the ultrasonication process was used to expose reactive CNT surfaces to the proteins. This sample contains 60 M iron on the basis of the HPLC analysis (see Fig. S1 in Supporting Information). The number of the remaining free Cys residues was determined using methoxypolyethylene glycol maleimide (MeO-PEG-Mal) (number-average molecular mass Mn = 5,000), which has the ability to confer the polyethylene glycol (PEG) moiety on a sulfhydryl group, i.e., PEGylation of Cys residues (Fig. 1b).20 Because the molecular weight of the PEG moiety is ca. 5 kDa, the molecular weight of the protein molecule increases by 5 kDa per free Cys residue (Fig. 1c). The number of free Cys residues of protein molecules was thus determined on the basis of their molecular weights estimated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS–PAGE) under nonreducing conditions.

Fig. 1. a A reaction scheme of the reduction of lysozyme using DTT. The structure of native lysozyme was obtained from the Protein Data Bank (PDB 1HEL). The blue curved line indicates the peptide backbone of reduced lysozyme. The numerals on the peptide backbone indicate the positions of Cys residues. b A reaction scheme of PEGylation of a sulfhydryl group. The molecular weight of the PEG moiety is ca. 5 kDa. c A reaction scheme of the oxidation of lysozyme by CNTs and subsequent PEGylation of the remaining sulfhydryl groups. d SDS–PAGE under nonreducing conditions under the different conditions described in the upper table. The PEGylation gives multiple bands corresponding to lysozyme species with different numbers of free Cys residues (lane 9). The reduced lysozyme without PEGylation shows multiple bands for monomeric and oligomeric forms (lane 10). Fig. 1d shows the results of SDS–PAGE analyses. The reduced lysozyme sample without PEGylation, which was used as a control sample, principally has a molecular weight of 14.3 kDa; however, there were also small amounts of lysozyme oligomers (Fig. 1d, lane 10), which is ascribable to inevitable and irremovable oxidation of the protein under the present conditions. The brown bands for oligomers appeared at positions lower than expected from the molecular weights, which is attributed to the nonreducing conditions; specifically, partially folded structures of proteins lead to a decrease in the apparent molecular weight observed on the gel. For the other control sample that was treated by PEGylation, the three grey

bands were observed (Fig. 1d, lane 9), which were assigned to oxidized monomeric lysozyme with different numbers of free Cys residues. The paleness of the band for the species with no free Cys residues demonstrates that most of the monomeric lysozyme has one or two free Cys residues, which suggests that lysozyme reduced by DTT undergoes partial oxidation during the preparation process. In this lane, the bands for the dimeric and trimeric forms disappeared, which indicates that these forms had more than one free sulfhydryl group. In fact, there was a broad band (> 50 kDa) corresponding to PEGylated oligomers in this lane.

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The reduced lysozyme exposed to the CNTs in the absence of EDTA showed bands corresponding to monomeric, dimeric and other oligomeric species of lysozyme containing no free Cys residues, but it did not show clear bands corresponding to the monomeric lysozyme containing free Cys residues (Fig. 1d, lane 1); note that this band is similar to that without PEGylation (Fig. 1d, lane 5). In contrast, a distinct band corresponding to the monomeric lysozyme with one free Cys residue was observed in the presence of 2 mM EDTA (Fig. 1d, lane 2); the presence of the band corresponding to the monomeric lysozyme containing no free Cys residues is ascribed to inevitable oxidation of the reduced lysozyme during the sample preparation. This result unambiguously indicates that oxidation of the reduced lysozyme was facilitated by coexisting metal ions, i.e., iron ions originating from iron catalyst particles. EDTA used in the present study was disodium salt, and thus, oxidation might be ascribable to the cation. However, when 2 mM NaCl was used instead of 2 mM EDTA, it was almost inert (Fig. 1d, lanes 3 and 7). At 4 mM, though NaCl induced some oligomerization and aggregation (Fig. 1d, lanes 4 and 8), the amount of monomer containing a free Cys residue remained small even in this condition. Thus, it was concluded that EDTA suppresses oxidation of protein sulfhydryl groups by removing

the coexisting iron ions. In other words, iron ions can mediate the redox reaction in the absence of EDTA. Importantly, in the presence EDTA, PEGylation resulted in upshifts of the broad band for oligomers (> 50 kDa) (compare lanes 2 and 6 in Fig. 1d). This result demonstrates that free Cys residues were also retained for oligomeric forms of lysozyme in the presence of EDTA. Observation of Reduction of Carbon Nanotubes by a Reduced Protein. The observed oxidation of the reduced lysozyme should be coincident with the reduction of CNTs. In general, since CNTs are moderately oxidized at neutral pH through the O2,H+/H2O couple, their absorbance is relatively low compared with that at basic pH.25–27 Thus, reduction of CNTs, i.e., electron donation to the CNTs, is determined by their light absorption, especially for semiconducting CNTs.28 In the case of HiPco CNTs, light absorption by the semiconducting CNTs is detectable in the wavelength range of approximately 940–1350 nm and 620–940 nm, corresponding to their first optical transition energy (S11) and second optical transition energy (S22) (Fig. 2a). Absorption spectrum of SDS-dispersed CNT had relatively low intensities in the absence of lysozyme and EDTA (Fig. 2a, black solid line).

Fig. 2. Redox reactions of the CNTs with the reduced and native lysozyme. a. Representative absorption spectra of the CNTs in the S11 and S22 regions under different conditions more than 1800 s after sample preparation. The CNT and lysozyme concentrations were 0.008 mg/mL and 2 M, respectively. b Time courses of absorbance of the CNTs at 1276 nm under different conditions. The CNT and lysozyme concentrations were 0.008 mg/mL and 2 M, respectively. c Comparison of the absorbance values at peaks I-III depicted in panel a in the presence of 2 M reduced lysozyme with and without 2 mM EDTA. The peaks mainly correspond to (8,4), (7,6) and (9,4) tubes for peak I; (8,6) tubes for peak II; and (9,5), (8,7) and (10,5) tubes for peak III (see panel d). The solutions used in the experiments exhibited in panels a-c contained 5.5 M urea to inhibit protein aggregation. One-sided Student’s t-test, *P < 0.005. d Photoluminescence excitation (PLE) map of the CNTs dispersed using the reduced lysozyme. Inset, PLE map of the CNTs dispersed by native lysozyme in the same wavelength range. The dispersions were mixed with 1 wt% sodium deoxycholate prior to the measurements to reduce the influence of oxidation of the CNTs by dissolved oxygen.29 The addition of reduced lysozyme to a 0.008 mg/mL SDSdispersed CNTs that contains 0.5 M iron on the basis of HPLC analysis (see Fig. S1 in Supporting Information), increased the absorbance, especially in the S11 region (Fig. 2a, red solid line), indicating reduction of the CNTs; in other words, electron transfer from the reduced protein to the CNTs occurred. The increase in the absorbance at 1276 nm, which mainly corresponds to S11 of the (9,5), (8,7) and (10,5) tubes, reached a plateau in ca. 500 s (Fig. 2b, red solid line). In contrast, native lysozyme that has no free Cys residues showed a small increase in the absorbance (Fig. 2a,b, blue dashed lines); note that 2 mM EDTA itself, which was used for chelating the iron ions originating from the CNTs, marginally affected the absorbance (Fig. 2a,b, purple dot-dashed lines). The slight decreases at the

early time points observed for 2 mM EDTA and no additive were because of inevitable drift of the redox potential immediately after sample preparation. Thus, the absorbance increase by the reduced lysozyme is attributed to electron transfer from the free sulfhydryl groups of the Cys residues. Here, increases in the absorbance of peaks I–III were reduced by the addition of 2 mM EDTA to the reduced lysozyme solution (Fig. 2a,b green dotted lines and Fig. 2c). This result implies that the reduction of the CNTs is facilitated by the coexisting iron originating from the remaining metal particles, i.e., the catalyst remains. The differences in the absorbance values between the data for 0 mM EDTA and for 2 mM EDTA tend to be more pronounced in the order peak I < peak II < peak III, which is consistent with the redox mechanism; namely,

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CNTs with narrow band gaps, i.e., longer optical absorption wavelengths, are more readily oxidized through the redox reaction of the O2,H+/H2O couple, so that they can accept more electrons.30 However, as described above, the suppression of the absorption increase by EDTA was only partial, which is due to the physical adsorption of proteins that can exclude dissolved oxygen from the vicinity of the CNT surfaces, leading to partial reduction of the CNTs.29,31 In other words, the reduced lysozyme binds more strongly to the CNTs than does native lysozyme. CNTs debundled by adsorbates with higher affinity have been reported to exhibit higher photoluminescence.32 In fact, the CNTs dispersed using reduced lysozyme exhibited approximately four times higher photoluminescence than those dispersed using native lysozyme (Fig. 2d), which demonstrated the higher binding affinity of the reduced lysozyme for the CNTs. Such adsorption of the reduced lysozyme was also observed for the metallic species using Raman spectroscopy (see Fig. S2 in Supporting Information). Observation of Reduction of Carbon Nanotubes by Cysteine. We established a system containing one of the proteinogenic amino acids, i.e., Cys, instead of the reduced protein to examine the redox reaction between CNTs and a sulfhydryl group more simply. In this system, CNTs were dispersed by SDS. Fig. 3a shows the normalized absorption spectra of 0.005 mg/mL SDS-dispersed CNTs, which contains 0.3 M iron on the basis of HPLC analysis (see Fig. S1 in Supporting Information), in the presence of different solutes. The absorption spectral intensities were significantly increased by the addition of 100 M Cys (Fig. 3a, blue dashed line). A clear increase in absorbance by Cys was observed even at 0.1 M (see Fig. S3a in Supporting Information). In contrast, in the presence of 10 M EDTA, the increase in the spectral

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intensities by the addition of Cys was somewhat small (Fig. 3a, green dotted line); such effect of EDTA appeared even at 1 M (see Fig. S3b in Supporting Information). Similar results were obtained for the CNTs dispersed by DNA (see Fig. S4 in Supporting Information), which indicating that the redox mechanism is independent of dispersants. These results plainly indicate that the reduction of the CNTs by Cys was mediated by iron on the catalyst particles, which is consistent with the results observed for the reduced lysozyme (Fig. 2a). Interestingly, the further addition of 50 M FeCl3 to the solution containing Cys and EDTA resulted in an increase in the absorption intensity (Fig. 3a, red solid line); that is, 50 M Fe3+ induced reduction of the CNTs even in the presence of 10 M EDTA. This result might seem inconsistent with the fact that Fe3+ is an oxidant; in fact, FeCl3 itself pronouncedly decreased the spectral intensities of the CNTs (Fig. 3a, black solid line), in contrast to FeCl2, i.e., a reductant, which reduces the CNTs (Fig. 3a, black dot-dashed line). Nevertheless, the reduction of the CNTs by FeCl3 is reasonable, considering the redox reaction of Fe3+ with Cys; specifically, oxidation of the CNTs by Fe3+ can be overwhelmed by reduction of the CNTs by Fe2+ generated through the redox reaction of Fe3+ with Cys, as described later. Iron was reported to form complexes with Cys, which in turn produces Cys-Cys;33 the production of Cys-Cys was confirmed by circular dichroism (CD) spectroscopy and mass spectrometry in the present study (see Fig. S5 in Supporting Information). The absorption peak positions of the CNTs were redshifted in the presence of Cys and in the absence of iron ions (Fig. 3a, blue dashed and green dotted lines). Because the peak positions depend on the environment, such as the dielectric constant, around the CNTs,34 the peak shifts imply the adsorption of Cys-Cys, which was produced in the vicinity of the CNTs, onto the CNTs.19

Fig. 3. Redox reaction of the CNTs with Cys. a Representative absorption spectra of the CNTs in the S11 and S22 regions under different conditions more than 1800 s after sample preparation. The concentrations of Cys, FeCl3, FeCl2 and EDTA used in this experiment were 100 M, 50 M, 50 M and 10 M, respectively. b Time courses of fractional concentrations of the reduced CNTs (CNT). The fractional concentration of the reduced CNTs (CNT) was defined as the equation CNT = [CNT] / ([CNT] + [CNT+]). The concentrations of the additives were identical to those used in the experiment described in panel a. c Time courses of the value of CNT in the different concentrations of Cys along with 50 M FeCl3. The trends observed in the spectra were more evident in the time course data of the redox reaction. Fig. 3b shows the time course data of the fractional concentration of the reduced CNTs (CNT), defined as the equation CNT=[CNT]/([CNT]+[CNT+]), where [CNT] and [CNT+] are the concentrations of reduced CNTs and oxidized CNTs, respectively, which were determined using the absorbance at approximately 1250 nm (For details of the definition, see Materials and Methods). In the absence of EDTA, 100 M Cys increased the value of CNT up to ca. 1 within ca. 500 s (Fig. 3b, blue dashed line), which is similar to

the data using the reduced lysozyme (Fig. 2b, red solid line). In contrast, 100 M Cys in the presence of 10 M EDTA results in a slower redox reaction, which likely requires more than 2,000 s to reach equilibrium (green dotted line). Importantly, the effect of EDTA on the suppression of CNT reduction was saturated at approximately 10 M (see Fig. S3b in Supporting Information); therefore, the observed slow redox reaction in the presence of 10 M EDTA is accounted for by the direct redox reaction between CNTs and Cys. This direct redox reaction process thus only slightly contributes to the overall redox

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reaction kinetics. Most importantly, a result obtained by the further addition of 50 M FeCl3 to the solution containing Cys and EDTA showed a characteristic profile in the time course data (Fig. 3b, red solid line); specifically, the value of CNT steeply decreased to ca. 0.1 at the early time points which was undeterminable, indicating fast oxidation of the CNTs, and subsequently, the value gradually increased to ca. 1 within ca. 1,000 s, indicating reduction of the CNTs. Under the present conditions, EDTA itself was inert to the CNTs, as there are no changes in the time course (Fig. 3b, black dotted line). In addition, 50 M FeCl3 and 50 M FeCl2 altered the values of CNT to approximately 0 and 1 within an unmeasurable time period in the presence of EDTA, respectively (Fig. 3b, black solid line and grey dashed line), indicating fast reaction rates of the redox reaction between the iron ions and the CNTs. Fig. 3c shows the Cys concentration dependence of the time courses in the presence of 10 M EDTA. Cys was found to be effective for CNT reduction at concentrations over 10 M. In addition, CNTs synthesized by CoMoCAT catalytic chemical vapor deposition (CVD) method also showed similar redox reactions (see Fig. S6 in Supporting Information), supporting the generality of the redox reaction; note that the CoMoCAT CNTs contain different metal catalysts including Co and Mo. Determination of Products from Ferric Ion and Cysteine by Redox Reaction with Carbon Nanotubes. As described above, the reduction of CNTs should be coincident with the oxidation of Cys, namely, the production of cystine (Cys-Cys). The production of Cys-Cys from Cys was confirmed by mixing 800 M Cys with 1 mg/mL CNT using ultrasonication (For

details, see Materials and Methods). Fig. 4a shows percentages of the yields of Cys-Cys and the remaining Cys in the absence of EDTA, which was determined by mass spectrometry. The yield of Cys-Cys was comparable to that of Cys under nitrogen atmosphere, whereas the yield of Cys-Cys was almost 100% in ambient air, indicating the CNTs oxidized by dissolved oxygen are active in the reaction with Cys. The marginal oxidation effect in the absence of CNTs suggests the contribution of both CNTs and coexisting iron ions to the oxidation of Cys. Fig. 4b shows the percentages in the presence of EDTA and the absence or presence of FeCl2. In the absence of FeCl2, the yield of Cys-Cys under nitrogen atmosphere was comparable to that in ambient air, supporting the suggestion of the contribution of the coexisting iron ions mentioned above. The product yield was still comparable to that in the presence of 4 mM FeCl2 under nitrogen atmosphere. In contrast, in ambient air, the yield of Cys-Cys increased with the addition of 4 mM FeCl2. This result is surprising because FeCl2 is a reductant; in other words, Cys is oxidized by the addition of a reductant. As already described, Fe2+ itself reduces the CNTs oxidized in ambient air (Fig. 3a,b black dot-dashed line); in turn, it becomes Fe3+, which is capable of oxidizing Cys, i.e., producing Cys-Cys. Thus, the formation of Cys-Cys can be greatly facilitated by Fe2+ with the coexistence of CNTs and dissolved oxygen, which is further supported by the fact that the production of Cys-Cys was marginal in the absence of CNTs, even in the presence of Fe2+ in ambient air (Fig. 4b). In contrast, FeCl3 readily produced Cys-Cys even in the absence of CNTs and oxygen (see Fig. S5 in Supporting Information).

Fig. 4. a,b Residual amounts of Cys and amounts of cystine (Cys-Cys) produced after mixing 800 M Cys with 1 mg/mL CNTs (CNT (+)) or without CNTs (CNT (–)) in ambient air (Air) or under nitrogen atmosphere (N2) in the absence (a) and presence (b) of 2 mM EDTA. The amounts of Cys and Cys-Cys were determined by mass spectrometry. c Concentrations of the produced Fe2+ and the remaining Fe3+ after mixing 2 mM FeCl3 with 1 mg/mL CNTs using ultrasonication in the presence and absence of the CNTs in ambient air or under nitrogen atmosphere. One-sided Student’s t-test, *P < 0.005. As seen in Fig. 3, iron ions interact with CNTs in a redox reaction. In particular, the redox reaction of Fe3+ with the CNTs is expected to be the cause of the characteristic time course curve (Fig. 3b, red solid line). The redox reaction should be further confirmed by demonstrating the production of Fe2+ from Fe3+ by the reaction with CNTs. The concentrations of the produced Fe2+ and the remaining Fe3+ obtained after mixing 2 mM FeCl3 with or without CNTs were quantified using ionexchange chromatography (see Fig. S1b in Supporting Information); mixing was performed by ultrasonication in the presence of 2 mM EDTA in ambient air or under nitrogen

atmosphere (For details, see Materials and Methods). Fig. 4c shows the iron concentration data under those conditions. The concentration of Fe2+ obtained from the condition using CNTs in ambient air was found to be comparable to that obtained from the same conditions but under nitrogen atmosphere, i.e., ca. 0.2 mM. In contrast, the Fe2+ concentration was significantly higher than that obtained from the condition without CNTs, i.e., ca. 0.1 mM. Therefore, the oxidation of CNTs by Fe3+ leads to production of Fe2+. The total iron concentration was less than the initial concentration of 2 mM, especially for the data in

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ambient air, which is possibly associated with production of some oxides of Fe3+ during ultrasonication. Numerical Simulations of the Redox Reaction. Numerical simulations on the basis of reaction kinetics are useful for understanding the mechanism of the experimentally observed reactions. Because both the direct redox reaction and the indirect redox reaction mediated by iron are associated with the redox interaction between CNTs and Cys, as seen above, the reaction scheme shown in Fig. 5a was assumed in the simulations. The parameter k is the rate constant of each elementary reaction. The reactions governed by the parameters k0, k1 and k2 correspond to the indirect redox reaction between CNTs and Cys, whereas the reaction governed by the parameter k3 corresponds to the direct redox reaction. The reactions governed by the parameters k0 and k3 are virtually irreversible (see Fig. S3c and S5d in Supporting Information). Three differential rate equations are necessary to perform the numerical simulations (Scheme S1). The rate constants (k) and the initial concentrations of the reactants were set to the appropriate values so that each elementary process reproduces the experimental behaviour (see Fig. S7 in Supporting Information). The total concentration of electrons in the CNTs ([CNT]+[CNT+]) was set to 2 × 10-6 M on the basis of previous work, which reported that the electron density of CNTs was 0.2–0.4 electrons per 100 carbon atoms residing in the valence band,26 and the initial value of CNT was set to 0.6, similar to the value obtained from the experiments (For details, see Supporting Information, Section 1.6). Fig. 5b shows the time courses of the value of CNT under different conditions. Under the initial condition of 100 M Cys

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and 50 M Fe3+, the amount of reduced CNTs steeply decreases within 10 s and then gradually increases with time (Fig. 5b, inset). The value of CNT approaches 1.0 within ca. 1,000 s. Importantly, this profile is similar to the experimentally observed profile (Fig. 3b, red solid line); profiles for the other conditions also reproduce the experimental phenomena overall (compare Fig. 3b and Fig. 5b). Fig. 5c shows the initial Cys concentration dependence of the value of CNT. Steep decreases in the value of CNT at the early time points were observed in each condition, which also successfully reproduces the experimental phenomena (compare Fig. 3c and Fig. 5c). Fig. 5d shows the time courses of each reactant and product under the initial condition of 100 M Cys and 50 M Fe3+. Clearly, the formation of Cys-Cys is coincident with the production of Fe2+. Fig. 5e shows the Fe3+ concentration dependence of the value of CNT under the initial condition of 100 M Cys. The redox reaction is facilitated by the presence of Fe3+ even at a concentration of 0.5 M. Using different values of k, it was found that the steep decrease in the value of CNT at the early time points is ascribed to the reversible redox reaction between Fe3+ and CNTs, which are governed by the parameters k1 and k2 (Fig. 5f, inset). On the other hand, the subsequent gradual increase in the value of CNT is attributed to the irreversible redox reaction between iron ions and Cys, which is governed by the parameter k0 (Fig. 5f). Even when the parameter k3 is assumed to be zero, the profiles are almost identical, indicating the insignificant contribution of the direct redox reaction to the overall redox reaction. Taken together, the numerical model demonstrated that Fe3+ promotes the reduction of CNTs in the presence of Cys, even though it is an oxidant.

Fig. 5. Numerical simulation of the redox reaction of Cys with CNTs mediated by iron ions. The redox reaction of CNTs via the O2,H+/H2O couple was ignored in the simulations because the change in pH in the experiments was insignificant (for details, see Supporting Information, Sections 1.5 and 1.6). a A suggested reaction scheme described with the reaction rate parameters (k) of the elementary processes, where the values of k are as follows: k0 = 5 × 10 M-1 s-1, k1 = 1 × 104 M-1 s-1, k2 = 1 × 104 M-1 s-1, and k3 = 2 M-1 s-1. b Time courses of the values of CNT obtained from the numerical simulations under the different initial conditions described in the legend. The initial value of CNT was set to 0.6. c Time courses of the values of CNT under different initial conditions with the initial concentration of 50 M Fe3+, as described in the legend. d Time courses of the concentration of each component obtained from the numerical simulation under the initial condition of 100 M Cys and 50 M Fe3+. e Time courses of the values of CNT under different initial conditions with the initial concentration of 100 M Cys, as described in the legend. f Time courses of the values of CNT using the modified values of k described in the legend. Note that the data using k3 = 0 M-1 s-1 almost overlapped with the data using the default value throughout the time period.

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DISCUSSION To date, the biological impacts of nanomaterials have mainly been considered in terms of the chemical and physical properties of nanomaterial surfaces or the toxic properties of metal ions eluted from catalysts.10,35,36 The former is associated with the materials themselves, i.e., here, CNTs, and the latter is associated with coexisting catalyst remains, i.e., here, iron. In terms of the protein corona, it is important to clarify which factor primarily governs protein conformation onto the nanomaterials to understand the biological impacts of the nanomaterials, because cells see protein corona structures on the nanomaterial surfaces;37 in other words, the biological identity of the nanomaterials depends on the protein corona.6 Previous studies concerning the physical adsorption of proteins, peptides and amino acids onto CNT surfaces have showed that aromatic amino acids such as tryptophan and tyrosine and a basic amino acid containing a guanidinium group, i.e., arginine, have high affinity for CNT surfaces though hydrophobic and – interactions,3,17,18,38 leading to CNT dispersions, as shown in Fig. 2d. Such unspecific adsorption affinity of the amino acid side chains to CNTs generally causes protein denaturation and hence protein inactivation, except in some cases.39,40 Though such denaturation and inactivation are toxic in terms of protein function, the resulting protein corona structures have the ability to reduce the biological availability of the intrinsic nanomaterial surfaces in terms of cell response, in turn leading to a reduction of the cytotoxicity.3 Thus, proteinCNT interactions have Janus-like behaviour in terms of the biological impacts. In contrast, chemical reactions of proteins with CNTs have not fully been clarified. Since CNTs have oxidation potentials of approximately 5 V (vs. vacuum) or 0.5 V (vs. NHE),41 they can undergo redox reactions with various chemical compounds, including organic and inorganic compounds.26,27 For example, redox reaction of CNTs are governed by the O2,H+/H2O couple and depend on the pH values in aqueous solutions as follows19,20 4(e−h+)CNT + O2 + 4H+ ⇌ 4(h+)CNT + 2H2O (1) + − where h and e represent a hole and an electron in a CNT, respectively. Among proteinogenic amino acids, Cys has an apparent standard redox potential of −0.3 V (vs. NHE) at pH 7. Therefore, in principle, Cys reacts with CNTs via a redox reaction according to the following equation: 2(h+)CNT + 2RSH ⇌ 2(e−h+)CNT + RSSR + 2H+ (2) where RSH and RSSR depict the reduced and oxidized forms of the thiols, respectively.19,20 Simultaneously, Cys can react with coexisting metal ions, such as iron ions (see Fig. S5 in Supporting Information). Thus, Cys undergoes direct and indirect (iron-mediated) redox reactions with the CNTs (Fig. 5), and the apparent rate of the indirect redox reaction is substantially faster than that of the direct oxidation of Cys. In the indirect redox reaction, Cys molecules and CNTs act as electron donors and acceptors, respectively; therefore, even though the concentration of iron ions is low, the iron-mediated redox reaction between Cys and CNTs can proceed until the donors or acceptors are depleted. In fact, 0.5 M iron was effective in inducing the oxidation of 2 M reduced lysozyme (Fig. 2); in addition, 0.5 M iron was effective in facilitating the redox reaction between 100 M Cys and 0.005 mg/mL CNTs in the numerical simulation (Fig. 5e). CNTs purified by

nitric acid, which removes coexisting metals,42 also showed a similar redox reaction (data not shown), indicating that a small amount of irremovable iron ions is reactive in the redox chemistry. Serum iron concentration is typically several tens of micromolar and the iron is bound to proteins such as transferrin and ferritin. Considering the effective concentration range of iron, such bound iron ions can mediate the redox reaction between protein Cys residues and CNTs if it is dissociated from the proteins in blood. The generality of the iron-mediated redox reaction was confirmed using oxidized forms of glutathione (L-γ-glutamylL-cysteinyl-glycine (GSH)) (see Fig. S8 in Supporting Information). The degree of CNT reduction by GSH was lower than that of Cys, which is concordant with the previous data showing the effectiveness of thiols in CNT reduction in the absence of EDTA.19 In addition, DTT containing two sulfhydryl groups also showed the iron-mediated redox reaction (data not shown). These results indicate that the iron-mediated redox reactions between thiols and CNTs is a general phenomenon. Since oxidized proteins and peptides such as GSH can have additional physical interactions with the CNT surfaces, their adsorption tends to be irreversible, which makes the redox reaction rate lower than expected for reversible adsorption.19 Such irreversible adsorption can also lead to the accumulation of oxidized protein molecules on the CNTs (Fig. 1d). Thus, the combinatorial effects of the CNTs and coexisting metals lead to protein corona structures different from those expected only from physical interactions. In other words, the biological identity of the CNTs is affected by the metals. In general, iron itself has the ability to induce oxidative stress on cells through the Fenton reaction, where H2O2 reacts with iron ions and then transforms to hydroxyl radicals, leading to oxidative stress.10 The GSH concentration in murine macrophages was reported to decrease in the presence of nonpurified CNTs, which was ascribed to oxidative stress by reactive oxidative species (ROS) generated from H2O2 via the Fenton reaction.43 However, on the basis of the present study (see Fig. S8 in Supporting Information), iron-mediated oxidation will be possible for GSH in the macrophage cell culture model system; note that this process needs no H2O2. Thus, the oxidative stress of CNTs in cells can be ascribed not only to the ROS but also to the non-purified CNTs themselves that contain metal particles. Such oxidative stress on proteins also possibly affects intravascular homeostasis.21 The present redox reaction model can explain the dependence of CNT production methods on the oxidative stress. A previous report regarding the oxidation of thiols showed that the oxidation efficiency of thiols by CNTs highly depends on the CNT production method;19 specifically, the CNT oxidation efficiency is in the following order: HiPco > arc plasma jet (APJ) > enhanced direct injection pyrolytic synthesis (eDIPS). Importantly, this order is inconsistent with the order of their oxidation potentials. Those methods include the use of different metal catalysts in different amounts, e.g., Fe for HiPco,44 Ni for APJ,45 and Fe for eDIPS.46 In particular, the CNTs produced by the eDIPS method have significantly smaller amounts of iron catalysts than do those produced by the HiPco method; therefore, the higher oxidation effect of HiPco CNTs is reasonable based on the iron concentration. Accordingly, the protein oxidation efficiency depending on the CNT production method is attributable to coexisting metal catalyst species and

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amounts. In addition, because CNTs synthesized by CoMoCAT catalytic chemical vapor deposition (CVD) method, which include the use of different metal catalysts including Co and Mo, also showed similar redox reactions (see Fig. S6 in Supporting Information), the redox reaction scheme will be applied to various metals. Roles of other metal species in oxidative stress will need to be validated in future studies.10 CONCLUSIONS Previous studies have focused on the biological impacts of CNTs or coexisting catalyst remains independently. The present study demonstrated that both factors are simultaneously involved in protein oxidation. Metal catalysts used for CNT growth, in turn, catalyze the redox reaction between CNTs and proteins; this scheme should be applied to other nanomaterials synthesized using metal catalysts. The biological impacts of nanomaterials are thus governed not only by their morphology, physics and chemistry but also by the catalyst remains, cooperatively. One should consider both CNTs and coexisting metal impurities to advance the fundamental understanding of their biological impacts, at least in terms of oxidative stress on proteins and protein corona formation.

METHODS Chemicals. Carbon nanotubes (CNTs) synthesized by the highpressure catalytic CO (HiPco) decomposition method (HiPco CNTs) were purchased from Nano-Integris. The following chemicals were obtained from Wako Pure Chemical Industries. Ltd: lysozyme from egg white, L-cysteine, L(−)-cystine, iron (III) chloride hexahydrate (FeCl3·6H2O), iron (II) chloride tetrahydrate·(FeCl2·4H2O), sodium dodecyl sulfate (SDS), sodium chloride (NaCl), sodium hydroxide, sodium deoxycholate, sodium thiosulfate, sodium carbonate, methanol, ethanol, dimethyl sulfoxide, acetonitrile, urea, dodecyltrimethylammonium bromide, (±)-dithiothreitol, glutathione, 2-amino-2-hydroxymethl-1,3-propanediol, sucrose, bromophenol blue, glycine, guanidine hydrochloride, L(+)ascorbic acid, L-serine, formic acid, lactic acid, silver nitrite, acetic acid, trifluoroacetic acid, formaldehyde solution (ca. 37%) and deoxyribonucleic acid, from salmon sperm. CNTs synthesized by CoMoCAT catalytic chemical vapor deposition (CVD) method (SG65), ethylenediaminetetraacetic acid diammonium salt hydrate (2NH4(EDTA·2NH4)) and methoxypolethyleneglycol maleimide (Meo-PEG-Mal) were obtained from Sigma Aldrich. Ethylenediamine-N,N,N',N'tetraacetic acid, disodium salt, dehydrate (2NA(EDTA·2Na)) was obtained from Dōjindo Laboratories. Hydrochloric acid was obtained from Kanto Chemical Co., Inc. Precision Plus ProteinTM Dual Xtra Standards was obtained from BIO-RAD Laboratories, Inc. e-PAGEL E-T520L was obtained from ATTO Corporation. Determination of concentration of iron adsorbed on the CNTs. A 30-mg aliquot of HiPco CNTs was suspended in 30 mL of 1 wt% SDS along with 0 or 2 mM 2NA(EDTA·2Na) (hereafter referred to as EDTA) using an ultrasonic processor (Nanoruptor NR-350, Cosmo Bio Co., Ltd.) for 1 min at a

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power of 350 W as a preliminary dispersion treatment. The suspension, in a glass vial, was further dispersed for 1 h at a power density of 20 W cm-2 using an ultrasonic homogenizer (Sonifire 250D, Branson Ultrasonics, Emerson Japan, Ltd.) equipped with a 0.5-in flat tip, during which the glass vial containing the dispersion was immersed in a water bath at 18°C to prevent an increase in temperature. The dispersion (3 mL) was then centrifuged at 571,000 × g at 25°C for 15 h using an ultracentrifuge (Himac CS100GXII with S110AT rotor, Hitachi Koki Co., Ltd.) to precipitate the CNTs. The upper ca. 67% of the transparent supernatant was collected and then diluted twofold into 4 mM EDTA for the non-EDTA-containing sample or into 2 mM EDTA for the EDTA-containing sample; hence, the respective diluted samples contained iron ions, 0.5 wt% SDS and 2 mM EDTA. These samples were used for determination of the soluble iron concentration. Since the soluble iron ions are complexed with EDTA under the present conditions, they were spectroscopically detected as iron-EDTA complexes. Thus, to determine the soluble iron concentration, the iron-EDTA complexes were detected using reversed-phase high-performance liquid chromatography (HPLC) on an ODS column (TSKgel ODS-80Ts, 4.6 × 250 mm, Tosoh Corp.) at 40°C; specifically, the diluted samples prepared above were applied to the column and then detected by monitoring the absorbance at 257 nm. The mobile phase used was 2 mM EDTA, and the flow rate was 1 mL/min. The iron concentration was quantified using a standard curve for an Fe3+EDTA complex in the presence of 0.5 wt% SDS and 2 mM EDTA. Preparation of reduced lysozyme. A 100-mg aliquot of native lysozyme was dissolved in 2 mL of solution containing 2 mM EDTA, 30 mM DTT and 6 M guanidine hydrochloride. The lysozyme solution was incubated overnight at room temperature to reduce the disulfide bonds of the Cys residues of lysozyme. The reduced lysozyme solution was dialyzed against 2 mM EDTA solution using dialysis tubing (Visking tubing, MWCO 12,000–14,000, Nihon Medical Science, Inc.) for 4 days, during which it gradually showed protein precipitation. The lysozyme precipitate was washed with 2 mM EDTA solution to remove the other solutes and then stored at ca. 12°C. The precipitate was dissolved into 6 M urea immediately before experiments. Complete removal of DTT was confirmed using HPLC on the ODS column (TSKgel ODS-80Ts),20 where absorbance was monitored at 210 nm. The concentration of lysozyme in this solution was quantified with a spectrophotometer (NanoDrop ND-2000, Thermo Fisher Scientific Inc.). Pretreatment of CNTs. HiPco CNTs were used to examine the redox reaction of CNTs. A 30-mg aliquot of HiPco CNTs was suspended in 30 mL of 1 wt% SDS using an ultrasonic processor (Nanoruptor) for 1 min at a power of 350 W as a preliminary dispersion treatment. The suspension was further dispersed for 1 h at a power density of ca. 20 W cm-2 using an ultrasonic homogenizer (Sonifire 250D) equipped with a 0.5-in flat tip. The glass vial containing the dispersion was immersed in a water bath at 18°C to prevent an increase in temperature during ultrasonication treatment. The dispersion was then centrifuged at 210,000 × g for 1 h using an ultracentrifuge (Himac CS100GXII with S80AT3 rotor, Hitachi Koki Co.,

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Ltd.) to remove the residue of catalytic metal particles, CNT bundles, and other impurities. The upper 70% of the supernatant was collected as a highly dispersed CNT sample. In addition, DNA-dispersed CNTs was also prepared by the same procedure. The SDS-dispersed CNT sample was then diluted into a 1 wt% SDS solution to obtain different concentrations (0.1 mg/mL or 0.2 mg/mL) of CNTs, where the CNT concentration was determined using the relationship in which 2.8 units of absorbance at 600 nm for a 1-cm path length corresponds to 0.1 mg/mL. This SDS-dispersed CNT sample (hereafter referred to as stock CNT dispersion) was then used to examine the effect of the CNTs under various conditions, as described below. SDS–PAGE under nonreducing conditions. Sample preparation. The stock CNT dispersion (0.2 mg/mL) in 1 wt% SDS, which was prepared by the method described in the section “Preparation of CNT dispersion”, was mixed in a 1:1 ratio with methanol to precipitate the CNTs. The CNT precipitants were subjected to suction filtration to collect the CNTs on a polytetrafluoroethylene (PTFE) polymer membrane (Omnipore™ membrane filter, 0.2 μm JGWP, Millipore). Subsequently, the CNTs were sufficiently washed with water to remove SDS molecules. Approximately 4 mg of the CNTs was transferred into a glass vial. EDTA, NaCl or urea was then added to the glass vial to obtain a solution of 3.8 mL of 6 M urea, 3.8 mL of 2.1 mM EDTA and 6 M urea, 3.8 mL of 2.1 mM NaCl and 6 M urea, or 3.8 mL of 4.2 mM NaCl and 6 M urea. Then, a 200-L aliquot of 20 mg/mL reduced lysozyme dissolved in 6 M urea, which was prepared by the method described in the section “Preparation of reduced lysozyme”, was added to each glass vial containing the above solution to obtain a final concentration of 70 M reduced lysozyme. These samples were then mixed using an ultrasonic homogenizer (Sonifire 250D) equipped with a 0.25-in flat tip for 30 min at a power density of 16 W cm-2, during which the glass vial containing the mixtures was immersed in a water bath at 18°C to prevent an increase in temperature. A similar mixing process (but for 10 min) was performed again after the addition of 200 mg of dodecyltrimethylammonium bromide to desorb the lysozyme molecules from the CNT surfaces.20 The mixtures were then centrifuged at 16,100 × g for 2 min using a bench-top centrifuge (Centrifuge 5415R, Eppendorf). The supernatant was subsequently dialyzed against water, 2 mM EDTA solution, 2 mM NaCl solution or 4 mM NaCl solution using dialysis tubing (Visking tubing). Each dialyzed solution was centrifuged at 16,100 × g for 2 min again using the bench-top centrifuge (Centrifuge 5415R), and then, the supernatant was collected. The protein samples were diluted into the respective dialysis solution (water, 2 mM EDTA, 2 mM NaCl or 4 mM NaCl) to a concentration of 0.1 mg/mL. The reduced lysozyme concentration was quantified with the spectrophotometer (NanoDrop ND-2000). In the preparation of the control sample both without the addition of CNTs and without the mixing process using the ultrasonic homogenizer, 200 L of 20 mg/mL reduced lysozyme dissolved in 6 M urea was diluted into 3.8 mL of 6 M urea in a glass vial. After the addition of 200 mg of dodecyltrimethylammonium bromide, the mixtures were then centrifuged at 16,100 × g for 2 min using a bench-top centrifuge (Centrifuge 5415R). The solution was subsequently dialyzed

against water using dialysis tubing (Visking tubing). The dialyzed solution was centrifuged at 16,100 × g for 2 min again using the bench-top centrifuge (Centrifuge 5415R), and then, the supernatant was collected. The protein samples were diluted into water to a concentration of 0.1 mg/mL. The reduced lysozyme concentration was quantified with the spectrophotometer (NanoDrop ND-2000). SDS–PAGE. The molecular weights of the protein samples with and without PEGylation were estimated using SDS–PAGE under nonreducing conditions. MeO-PEG-Mal was dissolved in DMSO to obtain a concentration of 10 mM and used within 1 day. A 9-L aliquot of the protein sample (0.1 mg/mL) prepared above was mixed with 1 L of 10 mM MeO-PEG-Mal dissolved in DMSO or with 1 L of DMSO to obtain the PEGylated or non-PEGylated sample, respectively. These sample solutions were then incubated at 37°C for 30 min. After incubation, the 10-L sample solutions were mixed with an equal volume of a sample buffer solution containing 0.125 M Tris-HCl, 4 wt% SDS, 10 wt% sucrose and 0.004 wt% bromophenol blue prior to SDS–PAGE. A 10-L aliquot of each sample was applied to a precast polyacrylamide gel (ePAGEL E-T520L, ATTO Corp.), where electrophoresis was conducted at 20 mA for 75 min using a mini-slab size electrophoresis system (AE-6530, ATTO Corp.) and an electrophoresis power supply (WSE-3200 PowerStation III, ATTO Corp.). The protein molecular weight was determined using a molecular weight marker (Precision Plus Protein™ Dual Xtra Prestained Protein Standards). The electrophoresis buffer solution contained 25 mM Tris, 192 mM Glycine, and 0.1 wt% SDS. Silver staining of the electrophoresed lysozyme samples was performed as follows. The gel was immersed in a solution containing 25% (v/v) ethanol and 5% (v/v) acetic acid for 24 h with shaking, which was followed by two 30-min washes with distilled water. After the gel was pretreated with 0.03% sodium thiosulfate for 2 min, which was followed by two 15-s washes with distilled water. Subsequently, the gel was treated with 0.1 wt% silver nitrite for 30 min. After silver impregnation, the gel was rinsed twice with distilled water for 30 s and then developed with a 100-mL solution containing ca. 0.022% (v/v) formaldehyde and 2 wt% sodium carbonate. A 2-mL aliquot of acetic acid was added to the solution, and then, the solution was shaken with the gel for 15 min to stop the reaction, which was followed by three 5-min washes with distilled water. Measurement of absorption spectra of CNTs in the presence and absence of lysozyme. Reduced and native lysozyme were mixed with the CNT dispersion, as described below, to examine their redox reactions. A 100-M aliquot of reduced lysozyme solution dissolved in 6 M urea prepared by the method described in the section “Preparation of reduced lysozyme” was mixed with a solution containing EDTA, urea and the stock CNT dispersion (0.1 mg/mL) that was prepared by the method described in the section “Preparation of CNT dispersion” to make solutions containing 0 or 2 mM EDTA, 5.5 M urea, 0 or 2 M reduced lysozyme and 0.008 mg/mL CNTs. As a control sample, native lysozyme was used according to the above method. Absorption spectra of the CNTs were collected with a UV-vis-NIR spectrophotometer (UV-3600, Shimadzu Corp.) using a quartz cell with a path length of 10 mm. Time courses of absorbance at 1276 nm of the CNTs were collected with a

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UV-vis-NIR spectrophotometer (V-780, Jasco Corp.) using a quartz cell with a path length of 10 mm. The absorbance values of a 5.5 M urea solution were subtracted from each absorbance of the samples because urea affects the absorption spectra. The absorption peaks of the CNTs at approximately 940–1350 nm and 620–940 nm were assigned to the first optical transition (S11) and the second optical transition (S22) of the semiconducting species, respectively, for the HiPco CNTs. The absorption peak at approximately 400–620 nm was assigned to the first optical transition of the metallic species (M11). Measurement of photoluminescence spectra of CNTs dispersed by reduced and native lysozyme. The stock CNT dispersion (0.1 mg/mL) in 1 wt% SDS, which was prepared by the method described in the section “Preparation of CNT dispersion”, was mixed in a 1:1 ratio with methanol to precipitate the CNTs. The CNT precipitants were subjected to suction filtration to collect the CNTs on a polytetrafluoroethylene (PTFE) polymer membrane (Omnipore™ membrane filter, 0.2 μm JGWP, Millipore). Subsequently, the CNTs were sufficiently washed with water to remove SDS molecules. A 320-L aliquot of 1.25 mg/mL CNT suspension was transferred into a glass vial, and then, it was mixed with 3,280 L of a 6 M urea solution and a 400-L solution containing 100 M reduced lysozyme dissolved with 6 M urea, prepared by the method described in the section “Preparation of reduced lysozyme”, or with 3,280 L of a 6 M urea solution and a 400-L solution containing 100 M native lysozyme dissolved in 6 M urea in the glass vial to obtain final concentrations of 0.1 mg/mL CNTs, 5.5 M urea, and 10 M reduced lysozyme or native lysozyme. The 4-mL mixture was suspended using the ultrasonic processor(Nanoruptor NR-350) for 1 min at a power of 350 W as a preliminary dispersion treatment and then dispersed for 30 min at a power density of 16 W cm-2 using an ultrasonic homogenizer (Sonifire 250D) equipped with a 0.25-in flat tip, during which the glass vial containing the dispersion was immersed in a water bath at 18°C to prevent an increase in temperature. The dispersion was then centrifuged at 210,000 × g at 25°C for 10 min using an ultracentrifuge (Himac CS100GXII with S80AT3 rotor), and then, the upper ca. 70% of the supernatant was collected. A 700L aliquot of the collected CNT dispersions containing reduced or native lysozyme was mixed with 100 L of an 8 wt% sodium deoxycholate solution; namely, the solution contains dispersed CNTs with lysozyme, 4.8 M urea and 1 wt% sodium deoxycholate, where sodium deoxycholate was used to reduce the influence of CNT oxidation by dissolved oxygen.29 Finally, the CNT dispersions containing the reduced or native lysozyme were diluted using a solution containing 4.8 M urea and 1 wt% sodium deoxycholate to obtain identical CNT concentrations and then subjected to photoluminescence spectroscopy. Photoluminescence excitation (PLE) maps of the SWCNTs were obtained by a spectrofluorometer (Nanolog, HORIBA, Ltd.) equipped with a liquid-nitrogen-cooled InGaAs near-IR alloy detector. Measurement of Raman spectra of CNTs in the presence and absence of reduce lysozyme. The CNT samples containing the reduced lysozyme, which were prepared by the method described in the section “Measurement of absorption spectra of CNTs in the presence and absence of lysozyme”,

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were subjected to Raman spectroscopy. Raman spectra of the samples were measured with an excitation wavelength of 532 nm using a confocal Raman microscope (XploRA, HORIBA, Ltd.). Measurements of absorption spectra of CNTs in the presence and absence of thiols or EDTA. Sample preparation. Cys was mixed with the CNT dispersion as described below to examine their redox reactions. An aliquot of Cys solution was mixed with a solution containing iron ions, EDTA and the stock CNT dispersion that was prepared by the method described in the section “Preparation of CNT dispersion” to give final concentrations of 0–100 M Cys, 0–100 M FeCl3 or FeCl2, 0–20 M EDTA and 0.005 mg/mL CNTs. Similarly, solutions containing 0–200 M Cys-Cys, 10 M EDTA and 0.005 mg/mL CNTs were also prepared to examine the redox reactions of the CNTs with Cys-Cys. For DNA-dispersed CNTs, an aliquot of Cys solution was mixed with a solution containing iron ions, EDTA and the CNT dispersion give final concentrations of 100 M Cys, 10 M EDTA and 0.005 mg/mL CNTs. In addition, solutions containing 0–100 M Cys or glutathione (GSH), 10 M EDTA, 50 M FeCl3 and 0.005 mg/mL CNTs were also prepared to compare the redox reaction of the CNTs with Cys and that with GSH, where the pH of the CNT dispersion containing GSH was adjusted to the same value as that of the CNT dispersion containing Cys using NaOH. The pH was measured using a pH meter (LAQUAtwin, AS-712, Horiba, Ltd.). Absorption spectroscopy. Absorption spectra of the CNTs were collected with a UV-vis-NIR spectrophotometer (UV3600) using a quartz cell with a path length of 10 mm. Time courses of absorbance at 1250 nm of the HiPco CNTs or at 984 nm of the CoMoCAT CNTs were collected with the UV-visNIR spectrophotometer (V-780) using a quartz cell with a path length of 10 mm. As reported in a previous analysis of the redox chemistry of CNTs,26 the fractional concentration of reduced CNTs (CNT) can be defined by the equation CNT = [CNT]/([CNT] + [CNT+]), where [CNT] and [CNT+] are the concentrations of reduced and oxidized CNTs, respectively. The value of CNT was experimentally determined because it can be expressed as a function of time (t) by the following equation CNT(t) = (A(t) − ACNT+)/(ACNT − ACNT+), where A is the absorbance of a sample at 1250 nm at arbitrary time periods and ACNT+ and ACNT correspond to the absorbance values at1250 nm for the samples obtained in the presence of 50 M Fe3+ and 50 M Fe2+ at equilibrium (at 1,800 s), respectively; note that Fe3+ and Fe2+ at 50 M induced sufficient oxidation and reduction of the CNTs in the absence of EDTA. However, for the samples without the addition of iron ions, the peak positions were redshifted (Fig. 3a); therefore, to determine the value of CNT of those samples, ACNT was corrected to the absorbance value of the sample obtained in the presence of 100 M Cys and the absence of EDTA at equilibrium (at 1,800 s). Measurement of circular dichroism spectra of cysteine and cystine. Oxidation of Cys was observed using circular dichroism (CD) spectrometer in a quartz cell having a cap with an 8.75-mm path length. Samples were measured in ambient air or under nitrogen atmosphere. An aqueous solution containing Cys was mixed with an aqueous solution containing different

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concentrations of FeCl3 to obtain a final composition of 100 M Cys and 0–100 M FeCl3, where dissolved oxygen concentrations of the Cys and FeCl3 solutions prepared under nitrogen atmosphere were less than 0.1 mg/L (not detected), whereas those prepared in ambient air were approximately 7–8 mg/L. The dissolved oxygen concentration was determined using a dissolved oxygen electrode (9520-10D, HORIBA, Ltd.) connected to a dissolved oxygen meter (OM-51, HORIBA, Ltd.). In addition, CD signals of Cys-Cys at 50 M in the absence of iron ions was also measured in a quartz cell in ambient air. In the oxidation reaction of Cys, the fractional concentrations of Cys (Cys) were defined by the equation Cys = [Cys]/[Cys]0 = [Cys]/([Cys] + 2[Cys-Cys]), where [Cys] and [Cys-Cys] are the concentrations of Cys and Cys-Cys, respectively. The value of Cys was experimentally determined because it can be expressed by the equation Cys(t) = (CD(t) − CDCys-Cys)/(CDCys − CDCys-Cys), where CD, CDCys and CDCys-Cys were the CD signals of the sample, 100 M Cys and 50 M Cys-Cys at 201 nm, respectively. To examine the effect of Fe2+ on the reduction of Cys-Cys, the CD signals of Cys-Cys at 50 M were measured in the presence of 0 and 100 M FeCl2 in ambient air using a quartz cell with a 10-mm path length. Measurement of electrospray ionization mass spectra of cysteine and cystine. Sample preparation. Cys was reacted with the CNTs in the presence and absence of iron ions by the following methods. The stock CNT dispersion at 0.1 mg/mL prepared in the section “Preparation of CNT dispersion” was mixed 1:1 with methanol to induce aggregation of the CNTs. The mixture was subjected to suction filtration to collect the CNTs on a polytetrafluoroethylene (PTFE) polymer membrane (Omnipore™ membrane filter, 0.2 μm JGWP). Subsequently, the CNTs were sufficiently washed with water to remove SDS molecules. A 1-mg aliquot of the CNTs was transferred into a glass vial with a cap. Cys was then added to obtain a 1-mL sample solution containing 1 mg/mL CNTs and 800 M Cys in ambient air or under nitrogen atmosphere. A 1-mL sample solution containing 800 M Cys without CNTs was also made in a glass vial in ambient air as control samples. The mixtures were suspended 15 times by 1-min ultrasonication using an ultrasonic processor (Nanoruptor) at a power of 350 W as a preliminary dispersion treatment. After the centrifugation of the sonicated mixtures using a centrifuge (H-103n, KOKUSAN Co., Ltd.) at 3,000 rpm for 10 min, the supernatants were filtered using a syringe filters with a pore size of 0.2 m (WhatmanTM SPARTANTM 13/0.2RC, GE Healthcare Life Sciences). A similar method was used for the detection and quantification of Cys and Cys-Cys after mixing Cys and CNTs in the presence of 2 mM 2NH4(EDTA·2NH4) with or without Fe2+; note that the form of EDTA used for mass spectrometry was ammonium salt. Briefly, 1-mL sample solutions containing 0 or 1 mg/mL CNTs, 800 M Cys, 2 mM 2NH4(EDTA·2NH4) and 0 or 4 mM FeCl2 were prepared in ambient air or under nitrogen atmosphere. The sample solutions prepared above were then subject to electrospray ionization mass spectrometry (ESI-MS). In addition, Cys was reacted with FeCl3 by the following method. Cys was mixed with FeCl3 to make a solution containing 100 M Cys and 0 or 100 M FeCl3 in ambient air

without ultrasonication. The solutions were then subjected to ESI-MS 1200 s after mixing. Electrospray ionization mass spectrometry (ESI-MS). Detection and quantification of Cys and Cys-Cys contained in the above samples were performed by ESI-MS (ZQ 2000, Nihon Waters K.K.) operated in positive-ion mode. For the samples prepared by ultrasonication mixing, a 20-L aliquot of the sample solution was mixed with 180 L of a solution containing 2NH4(EDTA·2NH4) and L-serine and with an 800L aliquot of 100:0.3 methanol:formic acid mixture to obtain a solution containing 120 M 2NH4(EDTA·2NH4) and 100 M L-serine; alternatively, for the samples without ultrasonication mixing, a 150-L aliquot of the sample solution was mixed with 50 L of a solution containing 2NH4(EDTA·2NH4) and Lserine and with an 800-L aliquot of 100:0.3 methanol:formic acid mixture to obtain a solution containing 120 M 2NH4(EDTA·2NH4) and 100 M L-serine. Solutions used for making standard curves of Cys and Cys-Cys contained the same concentrations of 2NH4(EDTA·2NH4), L-serine, methanol and formic acid as the above samples. Each solution was filtered using a syringe filter with a pore size of 0.2 m (WhatmanTM SPARTANTM 13/0.2RC) and then subjected to ESI-MS. Mass spectra were collected under MS mode, while quantification of the amount of Cys and Cys-Cys was performed under SIR mode. L-serine was added to the solutions as an internal standard for quantification; specifically, the signal intensity of each Cys and Cys-Cys peak was corrected by that of the internal standard. The standard curves of Cys and Cys-Cys, which were also corrected by the internal standard, were used for determination of their concentration. Determination of the concentration of iron ions produced by the redox reaction of ferric ions with the CNTs. Sample preparation. The stock CNT dispersion (0.2 mg/mL) in 1 wt% SDS, which was prepared by the method described in the section “Preparation of CNT dispersion”, was mixed in a 1:1 ratio with methanol to precipitate the CNTs. The CNT precipitates were subjected to suction filtration to collect the CNTs on a polytetrafluoroethylene (PTFE) polymer membrane (Omnipore™ membrane filter, 0.2 μm JGWP). Subsequently, the CNTs were washed with water to remove the SDS molecules. Approximately 1 mg of the CNTs was transferred into a glass vial with a cap. Water or 200 mM FeCl3 was added to the glass vial in ambient air or under nitrogen atmosphere to obtain a 1-mL solution containing 1 mg/mL CNT and 2 mM FeCl3. A 1-mL solution containing 2 mM FeCl3 without CNTs was also prepared as a control sample in ambient air. The mixtures were subjected 15 times to 1-min sonication at 15°C and a power of 350 W using an ultrasonic processor (Nanoruptor). After centrifugation of the sonicated mixtures using a centrifuge (H-103n) at 3,000 rpm for 10 min, the supernatants were filtered using syringe filters with a pore size of 0.2 m (WhatmanTM SPARTANTM 13/0.2RC) and then subjected to HPLC on an ion-exchange column, as described below. HPLC. Determination of Fe3+ and Fe2+ contained in the filtered samples was performed by HPLC on an ion-exchange column (Shim-pack IC-C1 PEEK, 4.6 × 150 mm, Shimadzu Corp.) at 40°C. A 10-L aliquot of each sample was mixed with 490 L of water, 250 L of 1.6 M sodium L-lactate (pH 2.8)

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and 250 L of 0.2 N HCl. Each solution was filtered using a syringe filter with a pore size of 0.2 m (WhatmanTM SPARTANTM 13/0.2RC) and then subjected to HPLC. Eluted Fe3+ and Fe2+ were reacted with 2 mM EDTA in a reaction coil and subsequently detected by monitoring the absorbance at 257 nm. The flow rate of the mobile phase (0.4 M sodium L-lactate (pH 2.9)) was 1 mL/min, and that of the reaction solution (2 mM EDTA) was 0.3 mL/min. Fe3+ and Fe2+ concentrations were quantified using standard curves for FeCl3 and FeCl2, respectively. In the sample preparation of the standard curve for FeCl2, ascorbic acid was added to prevent the oxidation of Fe2+, and then, those samples were immediately subjected to HPLC measurements. Numerical Simulations of Redox Reactions. Numerical simulations of the redox reactions were performed with a time difference (t) of 0.5 s using Igor Pro 8 (WaveMetrics, Inc.).

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ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Materials and methods; determination of iron ions using liquid chromatography; examination of reduction CNTs by lysozyme using Raman spectroscopy; observation of times course of fractional concentrations of the reduced CNTs at different concentrations of Cys, EDTA and CysCys; observation of cysteine oxidation by ferric ion using circular dichroism spectroscopy and mass spectroscopy; a suggested reaction model for numerical simulations; determination of reaction rate constants used for the numerical simulations by comparing with the experimental data; examination of reduction of CNTs by glutathione using absorption spectroscopy. The authors declare no competing interests.

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AUTHOR INFORMATION Corresponding Author

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*E-mail: [email protected]

Author Contributions A.H. wrote the manuscript; M.W. performed the experiments; A.H. and M.W. analysed the data; All authors discussed the results and commented on the manuscript.

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ACKNOWLEDGMENT This work was supported by JSPS KAKENHI Grant Numbers 18H01809,18K19009,25220602 and the Hosokawa Powder Technology Foundation.

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