Article pubs.acs.org/crt

Binding of Human Serum Albumin to Single-Walled Carbon Nanotubes Activated Neutrophils to Increase Production of Hypochlorous Acid, the Oxidant Capable of Degrading Nanotubes Naihao Lu,*,† Jiayu Li,‡ Rong Tian,‡ and Yi-Yuan Peng*,†,‡ †

Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life Science, and ‡Key Laboratory of Green Chemistry, Jiangxi Province and College of Chemistry and Chemical Engineering, Jiangxi Normal University, 99 Ziyang Road, Nanchang, Jiangxi 330022, China S Supporting Information *

ABSTRACT: Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl−) and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase (MPO). However, the importance of protein−SWCNT interactions in the biodegradation of SWCNTs was not stressed. Here, we used both experimental and theoretical approaches to investigate the interactions of SWCNTs with human serum albumin (HSA, one of the most abundant proteins in blood circulation) and found that the binding was involved in the electrostatic interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π−π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Compared with SWCNTs, the binding of HSA could result in a reduced effect for OCl− (or the human MPO system)-induced SWCNTs degradation in vitro. However, the HSA−SWCNT interactions would enhance cellular uptake of nanotubes and stimulate MPO release and OCl− generation in neutrophils, thereby creating the conditions favorable for the degradation of the nanotubes. Upon zymosan stimulation, both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was more for HSA-SWCNTs under these relevant in vivo conditions. Our findings suggest that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in human inflammatory cells and therefore shed light on the biomedical and biotechnological applications of safe carbon nanotubes by comprehensive preconsideration of their interactions with human serum proteins.

1. INTRODUCTION The presence of carbon nanotubes (CNTs) in biotechnological and biomedical applications has raised concerns about their possible adverse effects on human health and the environment.1−3 In vitro data have indicated that CNTs may be cytotoxic, largely by inducing oxidative stress.2,4−6 Mice exposed to nanotubes by aspiration, inhalation, or intraperitoneal injection develop an inflammatory response.7−9 CNTs are chemically inert and stable; thus, they are slowly cleared and accumulated in vivo. Biopersistence of CNTs resulting from their inherent durability is one of the major stumbling blocks in the way of their broad biomedical applications.9−11 Hence, studies on the biocompatibility, biodegradative mechanism of CNTs, and elimination from the body are of great importance in nanomedicine. Apart from the chemical degradation of CNTs by strong acids and oxidants (such as mixtures of sulfuric acid and nitric acid), enzymatic oxidative degradation of carbon nanostructures was only demonstrated in recent years using naturally occurring plant peroxidase, horseradish peroxidase (HRP),12,13 and human heme peroxidases such as myeloperoxidase (MPO),14 eosinophil peroxidase (EPO),15 and lactoperoxidase (LPO).16 The carboxylated single-walled CNTs (SWCNTs) © 2014 American Chemical Society

can be degraded by peroxidase enzyme activity of these heme proteins in the presence of low concentrations of hydrogen peroxide (H2O2).12−16 The interaction of H2O2 with the active heme leads to the formation of peroxidase reactive intermediates, which can oxidize a number of peroxidase substrates including CNTs. The destruction of SWCNTs is accompanied by the formation of multiple intermediate products (such as short-chain carboxylated alkanes and alkenes) with evolution of CO2 as a final product.12−18 Enzymatic degradation of SWCNTs proceeds to a high extent only if pristine CNTs have defect sites (such as heteroatoms and carboxylic groups) on the graphitic surface.17−19 In addition, the biodegradation of CNTs into short fragments can decrease their toxicity and accelerate their clearance from the body.12−16 As one of the major human peroxidases, MPO is abundantly expressed in neutrophils and to a lesser extent in monocytes and macrophages, the myeloid immune cells that play a crucial role in the principal defense mechanisms of innate immunity and are particularly important in mediating bacterial cell killing.20,21 In addition to oxidation of compounds in the Received: April 9, 2014 Published: May 28, 2014 1070

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Figure 1. Binding of HSA to SWCNTs on NaClO-mediated degradation of nanotubes in vitro: the reduced effects. (A) Photograph of different nanotube suspensions after 2 days. (B) Vis-NIR spectra of different nanotube suspensions after 2 days, showing the loss of M1 and S2 bands during the NaClO-mediated biodegradation of SWCNTs. (C) Raman spectra of different nanotube suspensions after 2 days. Insert: the radial breathing mode (RBM) region of the corresponding Raman spectra. (D) D/G intensity ratios obtained from Raman spectroscopy on SWCNTs at different time points, showing a trend of increasing defects on the walls of SWCNTs (*p < 0.05, groups versus untreated-nanotubes). (E) SEM analyses tracking the biodegradation of nanotubes after 3 days.

CNTs,22−33 no previous efforts have been made to demonstrate the protein−SWCNT interaction that influences the biodegradation of nanotubes in vitro and in vivo. In this study, we used human serum albumin (HSA) as a common and wellcharacterized model of human serum proteins, and employed both experimental and theoretical approaches to investigate the HSA−SWCNTs interactions. Through the competitive binding of HSA and MPO to nanotubes, the binding of HSA could impair MPO-induced SWCNTs biodegradation in vitro. However, the binding of HSA to SWCNT could affect the cellular inflammatory responses and result in different biodegradation abilities. The HSA−SWCNTs interactions would enhance the cellular uptake of nanotubes and stimulate MPO release and OCl− generation in neutrophils, thereby promoting their own degradation. The current work demonstrates, for the first time, that the binding of HSA may be an important determinant for MPO-mediated SWCNT biodegradation in vivo, thus offering new opportunities for effective and safe regulation of SWNTs’ life-span in tissues and circulation.

peroxidase cycle, MPO possesses a unique chlorinating ability to catalyze the oxidation of Cl− to produce hypochlorous acid (HOCl).20,21 Both reactive radical intermediates of MPO and HOCl are the oxidants involved in the biodegradation process of SWCNTs in vitro and in vivo.14,16,22 A recent study showed that oxidation and clearance of SWCNTs from the lungs of MPO-deficient mice after pharyngeal aspiration was markedly less effective, whereas the inflammatory response was more robust than in that wild-type animals,22 providing direct evidence for the dominant participation of MPO in the in vivo biodegradation of SWCNTs. During the interactions with components of biofluids in vivo, noncovalent coating of SWCNTs with proteins and lipids will inevitably affect recognition patterns and metabolic pathways of the nanomaterials.22−27 In biomedical applications of CNTs, the blood is the important interaction organ exposed to these CNTs.28,29 Upon incubation with human plasma, CNTs have been shown to acquire a “corona” of proteins that may influence cellular uptake and the toxicity of CNTs.28,30 It has been reported that the binding of human serum proteins to SWCNTs strongly reduces their cytotoxicity.28 Generally, various weak interactions may contribute to protein adsorption on CNTs, such as electrostatic, hydrophobic, and π−π stacking interactions.30 In molecular dynamic modeling of protein binding to SWCNT surface, the adsorption of bovine serum albumin (BSA) molecules quickly reaches thermodynamic equilibrium in less than 10 min and simultaneously forms a stable “node-like” structure, and this intact structure is kept for a long time (5 h).28 Interactions of CNTs with BSA can enhance their biocompatibility and the BSA-dispersed SWCNTs can be taken up by HeLa cells and human mesenchymal stem cells without any apparent deleterious cellular effects.31 Hence, the nature of the nanomaterial coronation by human serum proteins may impact its degree of recognition and biodegradation by immune cells. Although the interactions between proteins and CNTs are believed to play an important role in the biological effects of

2. EXPERIMENTAL SECTION Detailed methods were provided in the Supporting Information. SWCNTs were prepared and used throughout the study unless specified otherwise. 2.1. Binding of HSA to SWCNTs. SWCNTs (200 μg) in 20 mM PBS (pH 7.4) were incubated with HSA in a 1:1 ratio (w/w) for 1 h at 37 °C with sonication for 1 min every 15 min. 2.2. Degradation of SWCNTs by NaClO. SWCNTs or HSASWCNTs (0.1 mg/mL) were incubated with sodium hypochlorite (NaClO, 1 mM) in 20 mM PBS (pH 7.4) at 37 °C. Because of the loss in the incubation system, HSA and NaClO were replenished every 8 h, and the reaction mixture was maintained for several days. Where indicated, HSA was incubated with SWCNTs 30 min prior to NaClO addition. 2.3. Molecular Modeling and Docking of SWCNTs to HSA. The 3-D structures of SWCNTs were generated using Nanotube Modeler software. SWCNTs were modified at the edge to contain carbonyl goups using Pymol visualization software. Then, SWCNTs 1071

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Figure 2. Interactions between HSA and SWCNTs. (A−D) Molecular modeling demonstrating possible SWCNTs interaction sites on HSA. The two predicted interaction sites, site 1 (A) and site 2 (B), are shown. The residues that are in close proximity (with in 5 Å), stabilizing the binding sites 1 (C) and 2 (D). (E) AFM image confirming the binding of HSA with SWCNTs. (F) The far-UV CD spectra of HSA after incubation with SWCNTs, and the inset is near-UV CD spectra of HSA incubated with SWCNTs. were docked to the HSA-ray crystal structure (PDB ID: 1H9Z) by AutoDock 4.0 software, as described previously.13−15 2.4. Release of MPO and HOCl Production in Neutrophils. Cells were incubated with either serum-opsonized zymosan (SOZ) or nanotubes for 30 min. Then, neutrophils were centrifuged at 1,000g for 10 min. The supernatant was obtained and used for MPO and HOCl measurements according to a previous study.16

incubated with SWCNTs for 1 h to form HSA-absorbed SWCNTs (HSA-SWCNTs). Similar to a previous study on BSA,31 HSA could individually disperse SWCNTs while retaining SWCNTs’ optical properties (Figure 1A and B). After the binding of HSA to nanotubes, HSA-SWCNTs were also significantly oxidized by NaClO, and the degradation showed a slower rate than unbound SWCNTs (Figure 1). Moreover, the presence of HSA could also effectively impair MPO system-catalyzed SWCNTs degradation in vitro (Figure S1, Supporting Information). To further study the role of HSA binding in nanotube degradation, we changed the ways of adding HSA and SWCNTs and examined their effects on degradation. The premix of HSA and SWCNTs for 30 min before adding to the reaction mixture showed more significant inhibition on nanotube degradation than when adding HSA and SWCNTs to the reaction mixture one by one (Figure 1B). Taken together, these results herein demonstrated that the binding of HSA could mitigate MPO (or HOCl)-catalyzed biodegradation of SWCNTs in vitro. 3.2. Interactions of HSA with SWCNTs. To investigate the inhibitive mechanism of binding of HSA on SWCNT biodegradation, molecular docking studies were performed using AutoDock 4.0 software to structurally characterize possible SWCNT interaction sites on HSA. The docking of oxidized SWCNTs to HSA indicated two potential binding sites on HSA (Figure 2A−D). While the preference for each site was not identical, Arg160, Glu167, Ser435, and Tyr452 were located in both binding sites (Table S1, Supporting Information) and predicted to stabilize the interaction between SWCNTs and HSA. Specifically, the oxidized groups (carbonyl) on SWCNTs in both binding sites were stabilized by electrostatic interaction with a positively charged residue, Arg 160, on HSA (Table S1, Supporting Information, highlighted in bold). The similar electrostatic interactions with SWCNTs were observed for MPO, HRP, and EPO, and all of the interactions were mainly related to the positively charged Arg residues (such as Arg178 for HRP,13 Arg 294, Arg 307, and Arg 507 for MPO14). On the basis of previous studies13−15 and results herein, it can be demonstrated that the electrostatic interaction of positively charged arginine residues with

3. RESULTS AND DISCUSSION 3.1. Binding of HSA to SWCNTs on Biodegradation in Vitro: Reduced Effects. The degradation of SWCNTs was first studied. On incubation with NaClO, the nanotubes degraded over time, and the suspension appeared lighter in color (Figure 1A), indicating a decrease in graphitic material. After the chemical cleavage of pristine SWCNTs by H2SO4/ HNO3, carboxylated SWCNTs were formed with the significant loss of the characteristic metallic band (M1) and semiconducting (S2) transition band.12−14,17 Furthermore, visible−near-infrared (vis−NIR) absorbance spectra of NaClOtreated nanotubes showed that both M1 and S2 bands were suppressed, which was indicative of biodegradation (Figure 1B). Moreover, the D-band in the Raman spectra corresponded to defects in the CNT wall, and the ratio of this to the G-band (ID/IG ratio) was used to monitor the integrity of SWCNTs. The ID/IG ratio increased from 0.07 at the beginning to 0.21 at day 3 as a result of SWCNT biodegradation by NaClO (Figure 1C and D). In the presence of NaClO, these characteristic peaks of the radial breathing mode (RBM) region (150−250 cm−1) in Raman spectra were decreased, and the remaining peak was up shifted (from 216 to 222 cm−1, Figure 1C, insert), which might be attributed to the further oxidation of the larger diameter SWCNTs by NaClO.18 Changes in nanotube morphology were further confirmed by scanning electron microscopy (SEM). The fibrillar structure of intact nanotubes was lost, and the shortened nanotubes were present (Figure 1E). These results suggested that SWCNTs were oxidized and degraded during their incubation with NaClO, which was consistent with previous studies.14,16 To investigate the effect of human serum protein on nanotube biodegradation, HSA was used as one of the most abundant proteins in blood circulation and sufficiently 1072

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Figure 3. HSA-functionalized SWCNTs induce the release of MPO and the generation of HOCl in human blood neutrophils. In response to CNT and HSA-SWCNTs in the absence or presence of serum-opsonized zymosan (SOZ), extracellular release of MPO (A), generation of extracellular HOCl (B), superoxide radicals (C), and H2O2 (D) were assessed in neutrophils (1 × 106 cells per mL) (*p < 0.05 and **p < 0.01, groups versus the control).

that the structures of HSA-SWCNTs were about 3.5−20 nm in height; and that (2) the length of HSA-SWCNTs was about 0.5−1 μm (Figures 2E and S4, Supporting Information). Compared with HSA-unbound SWCNTs (outer diameter, 1−2 nm; length, 0.5−2 μm), HSA molecules could accumulate on the SWCNT surface layer by layer, thus resulting in larger diameter (not length) distribution of nanotubes. After an incubation time of 30 min, the far- and near-UV CD spectrum of HSA showed no obvious changes in the protein secondary and tertiary structures (Figure 2F and Table S2, Supporting Information). These findings indicated that some of the protein structures had fewer changes after the adsorption to SWCNTs surfaces and remained intact, which was similar to the binding of BSA to pristine SWCNTs.28 To confirm the findings mentioned above, we attempted to quantify the binding of HSA to SWCNTs by the Folin−Lowry method (Figure S5, Supporting Information) and semiquantitatively analyze protein adsorption by SDS−PAGE (Figure S6, Supporting Information), and further confirmed the strong binding of HSA to SWCNTs. The protein-binding capacity of SWCNTs at 60 min was 0.48 ± 0.02 mg of HSA per mg of SWCNTs (Figure S5, Supporting Information), much higher than the MPO-binding ability of SWCNTs (0.18 ± 0.02 mg per mg of nanotubes)16,38 and the BSA-binding ability of pristine SWCNTs (about 0.3 mg per mg of nanotubes).28 The differences were possibly related to the characteristics of protein (the sizes of HSA and BSA (about 2 nm, ∼66 kDa) were much smaller than that of MPO (>10 nm, ∼144 kDa)14) and SWCNTs (carboxylated not pristine SWCNTs showed an electrostatic interaction with positively charged residues in protein). Moreover, previous studies have suggested that carboxyl groups on SWCNTs are found to play a critical role in biodegradation and that the interactions of basic amino acids

carboxyls on SWCNTs may be the crucial factor in stabilizing the binding of carboxylated SWNTs with various proteins. Besides the critical contribution of Arg residues to the binding, Tyr452 (Table S1, Supporting Information, highlighted in bold) was found to be located in both binding sites. Further, the binding could cause a red shift in the UV spectra of SWCNTs (Figure S2, Supporting Information) and result in a decrease in the intrinsic fluorescence spectra of HSA (Figure S3, Supporting Information), which was indicative of protein environmental changes of Trp and Tyr residues.34,35 A previous report by Ge et al. found that the π−π stacking interactions were the driving forces for the binding of aromatic residues (including Trp, Phe, and Tyr) in human serum proteins (including BSA) onto pristine SWCNTs.28 Nevertheless, both our experiments and molecular modeling showed that the π−π stacking interactions between SWCNTs and HSA were mainly ascribed to aromatic Tyr residues (e.g., Tyr452) and could play a critical role in the binding process, relating to the fact that there were 18 Tyr residues and only 1 Trp residue in HSA.35 Therefore, the mechanism for the interactions of carboxylated SWCNTs with HSA in the present study was different from the binding of BSA (or HSA) to pristine SWCNTs28,36,37 and the interactions of carboxylated SWCNTs with human heme peroxidases (such as MPO, EPO, and LPO).13−15 Also, the binding might be related to the hydrophobic interactions since the SWNT side walls were not fully oxidized and contained hydrophobic regions. Atomic force microscopy (AFM) and circular dichroism (CD) spectra were further applied to monitor interactions between HSA and SWCNTs. The HSA molecule could spontaneously and rapidly bind to the surface of SWCNTs. The section analysis of HSA binding to SWCNTs demonstrated that (1) a single protein molecule was about 2.0 nm and 1073

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Figure 4. Biodegradation of HSA-functionalized SWCNTs in neutrophils upon stimulation with serum-opsonized zymosan. (A) Vis−NIR spectra showing biodegradation of SWCNTs and HSA−SWCNTs by human neutrophils after 12 h. (B) D/G intensity ratios obtained from Raman spectroscopy showing biodegradation of nanotubes inside human neutrophils after 0, 6, and 12 h (*p < 0.05, groups versus nanotubes at 0 h).

MPO and generation of HOCl, O2•−, and H2O2 were observed in human neutrophils (Figure 3). Under these stimulated conditions (relevant to the in vivo situation: at inflammatory sites), HSA-SWCNTs underwent more significant degradation than non-HSA-functionalized nanotubes in neutrophil suspensions (Figure 4A). Inhibitors of NADPH oxidase (apocynin) and MPO (4-aminobenzoic acid hydrazide)14 attenuated the oxidative degradation process (Figure S8, Supporting Information). This result suggested that both NADPH oxidase and MPO were essential for SWCNTs biodegradation by human neutrophils. This discovery of enzymatic SWCNT degradation in neutrophils will also open new opportunities for the regulation of CNT’s fate in vivo by controlling inflammatory response and employing nanotube-metabolizing enzymes. Raman spectra of HSA-absorbed SWCNTs inside neutrophils showed an increase in the ID/IG ratio (Figure 4B), which was characteristic of oxidatively modified nanotubes. Furthermore, we found an increased cellular uptake of HSA-SWCNTs by neutrophils compared to that of nonfunctionalized nanotubes (Figure S9, Supporting Information), which was consistent with (1) HSA possibly functioning as a carrier protein for nanotube transport and internalization,24,31 (2) the higher stimulatory effect of HSA-SWCNTs on these inflammatory cells (Figure 3), and (3) the higher degree of oxidative degradation on HSA-SWCNTs inside and outside of neutrophils (Figure 4). Therefore, these results herein demonstrated that the uptake of HSA-absorbed SWCNTs by neutrophils might result in a MPO-driven biodegradation process in activated inflammatory cells. After HSA binding, the cellular uptake of SWCNTs by neutrophils was about 2.5-fold higher than that of nonfunctionalized SWCNTs, which was similar to the higher cellular uptake of BSA-SWCNTs by macrophage cells.39 In J774A.1 macrophage-like cells, the majority of naked and BSA-modified SWCNTs were preferentially localized in the perinuclear space and regions along the cell periphery, which was consistent with normal cell processing from phagocytosis and pinocytosis accumulation.39 In future studies, it will be of interest to investigate the subcellular localization of naked and HSA-modified SWCNTs inside neutrophils. The presence of BSA or IgG coating markedly enhanced the uptake of SWCNTs by macrophages or neutrophils, respectively,14,39,40 but without the focus on the stimulatory effect and degradation of protein-absorbed CNTs. The oxidative degradation of HSA-SWCNTs was observed in macrophages, and the degree of biodegradation was lower than that in neutrophils (data not shown), which was consistent

of MPO with the carboxyls on SWCNTs are near the catalytic site.14 Taken together, the information above, the binding of HSA, would impair MPO-induced SWCNT biodegradation in vitro (Figures 1 and S1, Supporting Information) mainly through the competitive binding of HSA and MPO to nanotube surfaces. However, the presence of the MPO product, NaClO, would impair the attachment of HSA to the nanotube surface and successively strip the protein from SWCNTs, up to nearly complete at 60 min (Figure S6B, Supporting Information). Moreover, the CD spectrum of SWCNT-absorbed HSA after the addition of NaClO for 5 min had noticeable changes with reduced α-helix and increased β-sheets, similar to changes of HSA alone upon NaClO treatment (Figure 2F and Table S2, Supporting Information). Therefore, the NaClO or MPO system could rapidly damage and peel off HSA from the surface of SWCNTs and subsequently degrade nanotubes. However, HSA could be considered as a competitive substrate for MPOinduced oxidants and effectively protect SWCNT surfaces from the direct exposure of oxidants, thus exhibiting a protective effect on oxidative biodegradation of SWCNTs. 3.3. Binding of HSA to SWCNTs on Biodegradation in Neutrophils: Enhanced Effects. The present results above provided the direct evidence for the binding of HSA to carboxylated SWCNTs. Then, we carried out an additional experiment exploring whether the binding of HSA influenced the biological action of SWCNTs in human blood neutrophils. MPO and HOCl are generated upon activation of neutrophils.20,21 As shown in Figure 3A and B, SWCNTs exhibited no significant effect on neutrophils with respect to the generation of MPO and HOCl. In contrast, a slightly stimulatory effect of HSA-SWCNTs on these cells was observed, which was based on the elevated MPO level, HOCl, superoxide radicals (O2•−), and H2O2 production from neutrophils. Consistent with the slightly stimulatory effect of HSA-SWCNTs on neutrophil activation, the binding of HSA to SWCNTs could facilitate these inflammatory cells to secrete higher levels of proinflammatory cytokines, such as TNF-α and IL-6 (data not shown). Probably due to the low levels of MPO and HOCl, biodegradation of HSA-functionalized nanotubes did not occur during their incubation in neutrophils (Figure S7, Supporting Information). Next, we evaluated whether SWCNT biodegradation could be executed by MPO-rich neutrophils upon activation. Stimulation of neutrophils was then performed using serumopsonized zymosan (SOZ), which was known to effectively trigger MPO release in human neutrophils.16 During neutrophil activation by SOZ, these marked elevations on the release of 1074

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with the fact that macrophages contained lower levels of MPO than neutrophils.14,20,21 These results further suggest that MPO is an important determinant of the biodegradation of nanotubes in inflammatory responses of immune cells. Furthermore, recent work has shown that the BSA-stabilized SWCNTs can be taken up by various cells without apparent deleterious effects,31,39 demonstrating the efficacy of BSA as a biocompatible dispersant and a mediator of bioactivity. In vivo, mice exposed to nanotubes by pharyngeal aspiration, inhalation, or intraperitoneal injection developed an acute inflammatory response with increased amounts of circulating neutrophils and activation of neutrophils.2,4−9,14,16,22 Meanwhile, activated neutrophils would produce MPO and OCl− (Figure 3), and the binding of HSA would enhance cellular uptake of nanotubes (Figure S9, Supporting Information). Then, both oxidants (reactive radical intermediates of MPO, OCl−) and SWCNTs coexisted in vivo and thereby created the conditions favorable for degradation of the nanotubes. Collectively, the interaction of HSA with SWCNTs herein would influence their delivery, uptake, and toxicity, and hence offer new opportunities to modulate MPO-driven nanotube biodegradation and clearance at the sites of inflammation and in phagosomes. Further, we investigated whether SWCNTs could activate neutrophils and degrade in the more physiologic milieu of whole blood. Different from the insignificant effect on isolated neutrophils, SWCNTs stimulated neutrophils in whole blood, as evidenced by the increase of release of MPO and HOCl formation in plasma (Figure S10A and B, Supporting Information). The stimulatory effect of SWCNTs was similar to that of SOZ. These results suggested that as the most abundant proteins in blood circulation, binding of human serum proteins to SWCNTs would be favorable for the cellular uptake of nanotubes and might play a potentially important role in neutrophil activation and subsequently stimulate MPO release and OCl− generation in whole blood. Moreover, human blood-incubated nanotubes underwent degradation in neutrophil cells under SOZ stimulated conditions (Figure S10C, Supporting Information), which was supported by the results that (1) nanotubes could be internalized in the cell and taken up to neutrophils by HSA functionalization (Figure S9, Supporting Information), (2) SWCNTs can activate neutrophils and stimulate MPO release and OCl− generation in human blood (Figure S10A and B, Supporting Information), thereby providing necessary conditions for the biodegradation of the nanotubes in vivo, and that (3) MPO were mainly formed inside the cells (1 μg/106 cells) and could bind nanotubes without the highly competitive absorption of blood protein. Hence, the binding of HSA to SWCNTs may reveal previously unrecognized physiologic effects of human serum proteins in connection with nanotube biodegradation (Figure 5). However, total protein concentrations in the plasma are very high (67−86 mg/mL, whereas serum albumin concentration in plasma from adults is 35−55 mg/mL),28 and levels of plasma MPO are very low (less than 2 μg/mL (14 nM)) even in patients with severe sepsis.16,41 Thus, the attachment of the MPO active site to SWCNTs and the degradation of SWCNTs through peroxidase enzyme activity seem to be difficult in human blood, whereas the in vivo degradation of SWCNTs can occur inside neutrophil cells (Figures 4B and S10C, Supporting Information).

Figure 5. Binding of HSA to SWCNTs enhanced the cellular uptake of nanotubes and activated neutrophils to increase the release of MPO and the production of HOCl, the oxidant capable of degrading nanotubes.

4. CONCLUSIONS AND PERSPECTIVES Since the blood circulation system is the important site of entry of CNTs in the human body, it is critical to understand how adsorption of blood proteins onto CNTs alters the cellular inflammatory responses and CNT biodegradation. We used both experimental and theoretical approaches to investigate the interactions of SWCNTs with HSA (one of the most abundant proteins in blood circulation) and found that the binding was involved in the interactions of positively charged Arg residues of HSA with the carboxyls on the nanotubes, along with the π−π stacking interactions between SWCNTs and aromatic Tyr residues in HSA. Although the binding of HSA could impair MPO-induced SWCNTs degradation in vitro, the HSA− SWCNTs interactions could affect the cellular inflammatory responses and result in different biodegradation abilities. The binding of HSA to SWCNTs would enhance cellular uptake of nanotubes and stimulate MPO release and OCl− generation in neutrophils, thereby facilitating their own enzymatic degradation process (Figure 5), which was rarely focused on in previous studies. Upon SOZ stimulation (i.e., under natural inflammatory response), both SWCNTs and HSA-SWCNTs were significantly biodegraded in neutrophils, and the degree of biodegradation was higher for HSA-SWCNTs. These findings suggest that the binding of HSA may be an important determinant for SWCNT biodegradation in human inflammatory cells or biofluids and therefore may provide more insight into the safe design of nanomaterials for future biomedical applications. Apart from HSA, other human blood proteins, such as fibrinogen, immunoglobulin, transferrin, and ferritin, can competitively bind to the surface of SWCNTs and may exhibit different binding capabilities.28,29 Since the binding of proteins to CNTs and the immune system are dependent on nanotube characteristics and surface functionalization,14,39,40,42,43 the characterization of different human blood protein-bindings of CNTs to induce an inflammatory response and to activate neutrophils may serve as an important study before applying nanoparticles in biomedical living systems. The binding of CNTs to human blood proteins aimed to target them to inflammatory cells may provide a good platform to improve the 1075

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biocompatibility and biodegradability of CNTs. This relevant research is being done in our laboratory.



ASSOCIATED CONTENT

S Supporting Information *

Detailed methods; inhibitive effect of HSA on MPO-mediated SWCNTs biodegradation; UV-vis spectra and fluorescence analysis of the interaction between SWCNTs and HSA; section analysis of HSA incubated with SWCNTs; kinetics curves of protein adsorption on SWCNTs; SDS−PAGE analysis of interaction between SWCNTs and HSA; uptake of SWCNTs and HSA-functionalized SWCNTs; SWCNTs induce the release of MPO and the generation of HOCl in human blood plasma; and molecular docking. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*(N.L.) Tel: 86-791-8120391. Fax: 86-791-8120391. E-mail: [email protected]. *(Y.-Y.P.) Tel: 86-791-8120391. Fax: 86-791-8120391. E-mail: [email protected]. Funding

Financial support was from the National Natural Science Foundation of China (Nos. 31100608 and 31260216), the Natural Science Foundation of Jiangxi province (No. 20114BAB204013), Education Department of Jiangxi Province (No. GJJ12183), and the Sponsored Program for Cultivating Youths of Outstanding Ability in Jiangxi Normal University. Notes

The authors declare no competing financial interest.



ABBREVIATIONS MPO, myeloperoxidase; HOCl, hypochlorous acid; HSA, human serum albumin; SOZ, serum-opsonized zymosan; SWCNTs, single-walled carbon nanotubes



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Binding of human serum albumin to single-walled carbon nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes.

Previous studies have shown that carboxylated single-walled carbon nanotubes (SWCNTs) can be catalytically biodegraded by hypochlorite (OCl-) and reac...
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