Realizing Comparable Oxidative and Cytotoxic Potential of Single

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Realizing Comparable Oxidative and Cytotoxic Potential of Single- and Multi-Walled Carbon Nanotubes through Annealing Leanne Pasquini, Ryan Sekol, Andre Taylor, Lisa D. Pfefferle, and Julie Beth Zimmerman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es401786s • Publication Date (Web): 26 Jun 2013 Downloaded from http://pubs.acs.org on July 2, 2013

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Environmental Science & Technology

Realizing Comparable Oxidative and Cytotoxic Potential of Single- and Multi-Walled Carbon Nanotubes through Annealing Revised for Submission to Environmental Science and Technology June 24, 2013 LEANNE M. PASQUINIa, RYAN C. SEKOLa, ANDRÉ D. TAYLORa, LISA D. PFEFFERLEa, JULIE B. ZIMMERMAN*a,b a

Department of Chemical and Environmental Engineering, Yale University, New Haven, CT 06520-8286 b School of Forestry and Environmental Studies, Yale University, New Haven, CT 06520

*Corresponding author: Julie B. Zimmerman, Email: [email protected], Phone: (203) 4329703.

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Abstract The potential applications as well as the environmental and human health implications of carbon nanomaterials (CNMs) are well represented in the literature. There has been a recent focus on how specific physicochemical properties influence carbon nanotube (CNT) function as well as cytotoxicity. The ultimate goal is a better understanding of the causal relationship between fundamental physiochemical properties and cytotoxic mechanism in order to both advance functional design and to minimize unintended consequences of CNTs. This study provides characterization data on a series of multi-walled carbon nanotubes (MWNTs) that underwent acid treatment followed by annealing at increasing temperatures, ranging from 400 – 900 °C. These results show that MWNTs can be imparted with the same toxicity as singlewalled carbon nanotubes (SWNTs) by acid treatment and annealing. Further, we were able to correlate this toxicity to the chemical reactivity of the MWNT suggesting that it is a chemical rather than physical hazard. This informs the design of MWNT to be less hazardous or enables their implementation in antimicrobial applications. Given the reduced cost and ready dispersivity of MWNT as compared to SWNT, there is a significant opportunity to pursue the use of MWNT in novel applications previously thought reserved for SWNT.

Introduction In general, there are two distinct classes of carbon nanotubes (CNTs), single-walled (SWNTs) and multi-walled (MWNTs), which differ physically by the number of concentric cylinders comprising the nanotube. Both share similar attractive properties (high aspect ratio, tensile strength, thermal and electric conductivity) and have similar manufacturing processes, yet access to MWNTs is generally more facile [1-4]. The market price of MWNTs is much lower than SWNTs ranging from $1 to $25 per gram and $60 to $750 per gram, respectively [2, 5-7].

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In addition, MWNTs are inherently easier to disperse in aqueous and other solvents, an advantageous attribute for many applications in which high dispersivity is desired [8]. The current and proposed applications for purified and functionalized CNTs range from flexible conductive thin films to power your handheld devises [9] to drug delivery [10] and 3D scaffolds for tissue and cell (re)generation [11, 12]. The study of the antimicrobial properties of CNTs has led to promising applications in surface coatings [13] and water filtration [14, 15] to help prevent the spread of infection and provide safe driving water. However, this antimicrobial behavior can contribute to unintended consequences for human health and the environment. To this end, there are a number of studies examining the impact of carbon nanomaterials (CNMs) on bacterial growth [16-24]. The magnitude of MWNT cytotoxicity varies throughout the literature [25-29]. The observed differences in cytotoxic response are influenced by the physicochemical properties and preparation of the MWNT sample used in a given study [22, 25, 29-31]. As with SWNTs and other nanomaterials, the chosen test organism and cytotoxic endpoints will also impact the ultimate conclusion of MWNT toxicity for a given assay [32]. Yet, MWNTs are generally considered to be less cytotoxic than SWNTs [16, 33], which has been attributed to their larger tube diameters. While supporting the varied cytotoxic potential of MWNTs, the novel results presented here indicate that MWNTs can have equivalent cytotoxicity as SWNTs and that the antimicrobial property of MWNTs is not dependent solely on diameter. Further, the findings suggest that MWNT antimicrobial activity can potentially be tailored using standard treatment processes, such as strong acid and high temperature annealing, which are commonly used to purify CNTs and modify their surface properties [34-36]. High temperature treatment in the absence of air (usually under flowing inert gas such as helium or

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argon), known as annealing, is commonly used to remove surface functional groups and repair tube defects [34, 37], thereby altering the point of zero charge as well as electronic properties. This study aims to characterize a variety of physiochemical properties of MWNTs as well as utilize multiple methods to evaluate cytotoxic potential. Overall, these findings contribute to a growing body of research aimed at understanding the relationship between specific CNT properties with the cytotoxic potential [19, 20, 22, 30] to enable the design of more effective CNTs for antimicrobial applications as well as the design of CNTs that minimize the unintended consequences to human health and the environment. Specifically, the MWNTs used in this study were modified from the same starting batch using both acid and annealing treatments to determine how these common purification techniques impact physicochemical properties and subsequently, cytotoxicity. Comprehensive physiochemical characterization includes oxidationreduction reaction experiments (electrochemical activity), X-Ray photoelectron spectroscopy (elemental composition), determination of the point of zero charge (surface charge), light scattering techniques (dispersed aggregate size and morphology), and thermogravimetric analysis (sample purity). Multiple assays are utilized here to comprehensively evaluate both the chemical and physical mechanisms of MWNT impact on bacterial cytotoxicity including acellular methods (i.e., glutathione oxidation) as well as in vitro studies with (filter-based and plating assays).

Experimental CNT Sample Preparation. MWNTs were obtained from Cheap Tubes (Brattleboro, VT, CCVD, >95 wt%, 10-20 nm diameter, 10-20 µm length) [7]. To remove residual metal and other carbon impurities, the as-Received MWNTs were refluxed in 70% nitric acid (15 M HNO3) for two hours and neutralized by successive DI water washes with. These AT-MWNT served as the

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starting material for samples S400 – S900, which were heat treated under inert conditions (He) at increasing maximum temperatures (400°C to 900°C, 5 °C/min) for 0.5 hour. Treatment conditions are tabulated in Table 1. In some experiments, a reference SWNT sample is used for impact comparison. Details on the preparation of this sample can be found in a previous study [19]. Bacterial Cytotoxicity Evaluation. Acellular Assay: MWNT-mediated Glutathione (GSH) Oxidation. The oxidation of GSH to GSH-disulfide can be mediated by CNTs and can therefore be used as an indicator to measure relative sample oxidative potential [38]. Assay details are outlined elsewhere [20]. Briefly, GSH (0.4 mM) was added to the reaction vials containing MWNT samples (0.025 mg/mL, buffered pH = 8.6), in triplicate. The amount of non-oxidized GSH remaining in the reaction vials over time was monitored spectrophotometrically using Ellman’s reagent (412 nm). MWNTs were filtered out of the solution (0.45 µm PES filter, Whatman) prior to adding Ellman’s to avoid any potential confounding interaction between the dye molecule and the MWNTs (confirmed by measuring the OD412 of the filtrate). The percent loss of GSH is calculated in reference to the absorbance measurements of a MWNT-free control sample. Cellular Assays: E.coli K12 (MG1655) was used as the model organism in the viability loss and reduction of CFU/mL experiments. Prior to commencing the experiment, appropriate dilutions of bacteria in exponential phase were prepared based on spectrophotometric estimation of the stock concentration (OD600). Filter-based MWNT Deposit Layer Assay. Method details were previously reported [19]. Briefly, MWNT deposit layers were prepared by dispersing MWNTs in DMSO (bath sonication, 30 minutes, VWR Model 150T, 135W, 10 mL total volume, 0.025 mg/mL). A portion of the

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dispersion, enough to sufficiently cover the surface (2.5 – 5.5 mL), was filtered onto a PTFE filter (Millipore JMWP 13 mm) using a vacuum pump. After washing with ethanol (60 mL) and DI water (100 mL), a final rinse with isotonic saline (0.9% NaCl) was used to remove any residual solvent detected by smell. If necessary, repeated rinses of ethanol and water were performed to remove all trace of the DMSO and ethanol, respectively. E.coli were gently filtered onto the prepared deposit layer by first turning the vacuum pump on for several seconds, then turning it off prior to filtering the bacterial solution. Propidium Iodide and DAPI (0.01 mg/mL) were used to enable visualization of the bacteria using an epifluorescence microscope. Cells were enumerated on 12-15 images per sample and viability loss calculated from the ratio of the number of viable cells over the total number of cells. Reduction CFU/mL by CFU Enumeration Assay. MWNTs were dispersed by bath sonication in 0.9% NaCl (30 minutes, VWR Model 150T, 135W, 1.4 mL sample volume, 0.20 mg/mL). The MWNT concentration was increased by a factor of 10 from the other assays to increase bacteria-MWNT contact events enabling an observable different in cytotoxic behavior. E.coli (1x106 cells/ml) were added to each experimental solution triplicate. Sample microcentrifuge tubes were continuously rotated (Elmeco Arma-Rotator, Model A-1) for one hour at 37 °C. Following the incubation period, 100 µL was removed from each experimental solution, serially diluted and plated (50 µL) on prepared nutrient agar plates. Plates were incubated overnight after which, colony forming units (CFUs) were enumerated. The percent reduction in CFU/mL for each sample compared with the control (no MWNTs) was calculated. MWNT Characterization. Electrochemical Measurements of Oxidation Reduction Reaction (ORR). Electrochemical measurements were conducted using the rotating disc electrode (RDE) technique in alkaline

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media (1 M KOH) at room temperature as described elsewhere [39]. The standard three-cell setup comprised of a Pt-mesh counter electrode and Hg/HgO (MMO) reference electrode. The working electrode was made by depositing a prepared catalyst ink [40] (7.5 µg cm-2 MWNTs) onto the glassy carbon electrode (GCE) and allowing the solvent to evaporate. Further details on the data collection can be found in the SI. X-Ray Photoelectron Spectroscopy (XPS). X-ray photoelectron spectroscopy (XPS) was used to determine elemental composition. Data was collected using a ThermoScientific ESCALAB 250 instrument with a monochronized Al X-ray source (150 eV pass energy for survey scans, 20 eV for composition scans, 500 µm spot size) at the University of Oregon CAMCOR facility. The samples were prepared by sonication in ethanol, then drop cast onto a HF treated silicon coupon. The HF pretreatment minimized the evolution of background O in the sample spectra. The atomic percent O was still corrected for any O associated with Si, as SiOx, by collecting a background spectrum of the HF-treated coupon. Determination of Point of Zero Charge (PZC). PZC measurements were collected following a mass titration method outlined elsewhere [41, 42]. Dispersed aggregate state. Light scattering, both static (SLS) and dynamic (DLS) (ALV-GmbH, Germany), were utilized to characterize the dispersed MWNT aggregation state as previously described by Pasquini, et al. [19]. Samples were prepared in the same manner for both light scattering experiments (30 minute bath sonication in DI water and saline, VWR Model 150T, 135W, 0.025 mg/mL, 10 mL total volume) and diluted prior to measurement. UV-Vis measurements confirmed negligible absorbance at the wavelength of incident light used in the light scattering experiment (532 nm). A detailed description of the data collection method and results for each technique can be found in the SI.

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MWNT characterization results are compiled in Table 1. TABLE 1

Results and Discussion Proposed mechanisms of bacterial cytotoxicity involve a combination of physical and chemical interactions between CNTs and bacterial cells [16, 20, 22]. Kang, et al. [16] outline various pathways to disruption of cellular function, including physical puncture of the cell wall as well as CNT participation in redox reactions that form reactive species around and within the cell, that can ultimately lead to loss of cell viability. A study by Liu, et al. [22] supports the physical puncture mechanism by showing that individually dispersed SWNTs induce stronger antimicrobial activity than those that are aggregated or bundled. Vecitis, et al. [20] describe an additional chemical pathway related to the disruption of cellular function that is dependent on SWNT chirality (electronic properties) showing that metallic SWNTs exhibit greater cytotoxicity than semiconducting SWNTs. It may therefore be expected that MWNTs, which possess metallic characteristics, would demonstrate a high cytotoxicity equivalent to that of metallic SWNTs. Yet, both pristine and oxygen functionalized MWNTs have been reported to be less cytotoxic than SWNTs [16, 17], suggesting that the mechanism of MWNT cytotoxicity is more complex. A variety of methods have been used to evaluate the bacterial cytotoxicity of CNTs [17, 22, 43, 44]. The application of standard cell assays that rely on spectrophotometric or fluorescent indicators to evaluate CNT cytotoxicity is complicated due to the proposed interaction between the CNTs and dye molecules which can lead to quenching [31, 45]. Therefore, multiple methods are employed in this study to determine the relative cytotoxicity of MWNTs including the oxidation of glutathione (GSH), an acellular assay, to indicate oxidation potential. It was

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previously shown that there is a statistically significant correlation between CNT induced GSH oxidation and loss of bacteria cell viability (by filter-based assay) [20]. Data was collected for a subset of samples (acid treated (AT)- MWNT, S500 and S900) using the filter-based assay to confirm the correlation for this system. To extend the results of the observed GSH oxidation and the filter-based assay, traditional plating methods were employed where the percent reduction of colony forming units (CFUs) represents the relative cytotoxicity of MWNTs in suspension with E. coli. MWNT-Mediated Glutathione (GSH) Oxidation. The GSSG/2GSH redox couple serves as an indicator of MWNT bacterial cytotoxicity resulting from chemical mechanisms [20, 38, 46]. A potential contributing mechanism of carbon nanomaterial cytotoxicity is the ability to mediate harmful cellular oxidative stress, which can lead to disruption of routine cellular function [16, 20, 24]. The results from the GSH oxidation assay indicate that the annealing process has a significant impact on the MWNT oxidation potential and that under certain conditions (500 – 800 °C), MWNTs impart greater oxidation potential than that of SWNTs (Figure 1a). A stepwise increase in the oxidation potential is observed between 400 and 800 °C, followed by a significant decrease for the sample annealed at 900 °C. Data for the as-Received sample is included in Figure S1. FIGURE 1 In the time frame presented (1 – 6 hr), the GSH oxidation rate (percent loss of GSH over time) varies between samples. Relative sample reaction kinetics were investigated by collecting additional data for S500 and S900 (Figure 1b). S500 and S900 were chosen due to their relative difference in the observed percent loss of GSH during the initial 6-hour experimental period. Since S500 oxidizes GSH rapidly, additional measurements were taken at 10 and 30 minutes.

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Contrarily, S900 oxidizes GSH more slowly, so an additional measurement was collected at 8 hours at which time the percent loss of GSH had reached ~100%. These results indicate that the kinetics of GSH oxidation is potentially more relevant to differentiate cytotoxic potential between samples. The reactivity of the MWNT sample is a probable influence on GSH oxidation kinetics considering the necessary exchange of electrons that occurs in the GSH oxidation to the disulfide (GSSG) [20]: 2 GSH → GSSG 2 e 2H

O 2 e 2H → H O 2 GSH O → GSSG H O Given the significance of MWNT reactivity in mediating this reaction, the electrochemical properties of the MWNT were characterized. The correlation of these properties with oxidation potential (as measured by GSH oxidation) will be discussed further below. Loss of Cell Viability via Contact with MWNT Deposit Layer. The GSH oxidation method is an acellular assay and therefore, an indirect measure of bacterial cytotoxicity. To confirm the relative cytotoxic impact of each MWNT sample, bacterial loss of viability was measured utilizing a well-established filter-based assay for CNTs [16, 19, 20]. Previous studies have highlighted the importance of physical contact between CNTs and bacterial cells in the overall cytotoxicity assessment [16, 22]. This filter assay ensures contact between the bacterial cells and the MWNTs enabling the direct comparison of bacterial cytotoxicity between samples. The percent loss of cell viability for AT-MWNT, S500, and S900 quantified by the filter-based assay is consistent with the acellular assay yielding statistically significant results showing that S500 has a higher cytotoxic potential than both S900 and AT-MWNT (Figure 2a). Again, at certain annealing conditions (500 °C most prominently), MWNT exhibit comparable cytotoxic potential

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as has been reported for SWNTs using the same assay, ~95% (shown in Figure 2a) [19], ~85% [16], and ~25 – 80% [20]. FIGURE 2 Reduction of CFU/mL via Contact with Suspended MWNTs. To further probe a direct measure of cytotoxicity, dispersed MWNTs were contacted with E. coli in suspension and colony forming units (CFUs) were enumerated to indicate the relative cytotoxicity of MWNTs. AT-MWNT, S400, S600 and S900 were evaluated to represent the four major stages of point of zero charge (PZC) transitions as indicated in Table 1. In addition to these four dispersed MWNT samples, data was collected for a control (no MWNTs). Samples were incubated for 1 hour under constant rotation (37 °C), to minimize MWNT sedimentation. Upon enumeration of CFUs, the percent reduction in CFU/mL compared to the control was calculated (Figure 2b). The relative percent reduction in CFU/mL for the sample subset further supports the observed relative cytotoxicity of the samples trend established by both GSH oxidation and the filter-based assay. Overall, the percent loss of viability measured using the filter-based deposit layer assay indicates the largest cytotoxic potential (~70 – 85% mean loss of cell viability). This is expected due to the forced interaction between the MWNT deposit layer and the bacterial cells, which has been previously reported as being essential to observed cytotoxic outcomes [16]. In contrast, the CFU enumeration assay was used to evaluate a suspended system where interaction between the MWNTs and cells was induced by constantly rotating the samples. As such, the absolute percent reduction in CFU/mL (~30 – 61% mean reduction) is lower than the percent loss of viability observed for the deposit-layer method.

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The combined results from the three assays support the earlier findings that direct contact between the CNTs and the bacteria is important and that both chemical and physical mechanisms contribute to CNT bacterial cytotoxicity. More importantly, the results presented demonstrate that annealing has a clear and direct impact on MWNT cytotoxicity with 400°C, 600°C, and 900°C representing temperatures associated with significant transitions in antimicrobial behavior. This is most likely due to the influence that annealing at these temperatures has on the MWNT surface chemistry by reducing the oxygen containing functional groups. Further, under certain annealing conditions, MWNTs exhibit equivalent or greater cytotoxic potential, particularly related to chemical pathways (oxidative potential), as has been reported for SWNTs. Physicochemical Characterization to Determine the Governing Properties of MWNT Bacterial Cytotoxicity. The MWNTs assessed for cytotoxicity potential underwent common purification processes: strong acid treatment followed by annealing. As annealing temperatures are increased, surface functional groups added during the acid treatment are removed starting with the more labile carboxyl groups, at ~400°C, followed by hydroxyl and other ketone-like groups (for examples refer to Figure S2a), at ~700°C [42]. Finally, at annealing temperatures >900°C, the surface of the MWNT is fully reduced and defect healing can occur by carboncarbon bond restructuring [47, 48]. As the oxygen functional groups are sequentially removed under the increasing annealing temperature, the surface properties move from more acidic to more basic (Figure S2b) [49]. As expected, MWNT physicochemical properties are altered as the surface chemistry changes due to the strong acid and high temperature treatments. To evaluate these changes, properties of the MWNTs were characterized using chemical composition by X-ray photoelectron spectroscopy (XPS), dispersed aggregate state by light scattering (DLS and SLS),

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surface charge properties by point of zero charge (PZC), and electrochemical activity by oxidation-reduction reaction (ORR) experiments. Sample purity data, evaluated by thermogravimetric analysis (TGA) is included in the SI (Figure S3). In some cases, a representative subset of samples was chosen for characterization to evaluate the relative trend for a given property. X-Ray Photoelectron Spectroscopy (XPS). XPS was used to determine the elemental composition of the MWNTs after acid and/or annealing treatments. XPS results (Table 1) indicate that the purchased MWNT batch (as-Received) contained 7.43% oxygen. This could be in the form of oxygen functional groups resulting from manufacturer purification treatment or as oxides associated with the metal catalyst particles or other carbonaceous material (amorphous and graphitic carbon). Acid treatment is known to remove metal catalyst contamination as well as introduce oxygen functional groups to the surface of MWNTs [36, 50]. Therefore, the oxygen content remained relatively high (5.76%) in the AT-MWNT sample. Upon annealing, the amount of tube surface is reduced with increased treatment temperature due to evolution of oxygen functional groups. The loss of carboxyl groups is demonstrated by inspection of the C1s scan in the XPS spectra. The peak near 289 eV is associated with the C–O bond in carboxylic acid groups and is no longer present after treatment at 400 °C (Figure S4) [36, 50]. The peak at 289 eV remains absent from the C1s scan in all subsequent samples. As the maximum annealing temperature increases, the percent oxygen present in the sample decreases. Since oxygen functional groups are known to influence the material surface charge properties [42, 51], the point of zero charge (PZC) is expected to change with the evolution of these groups. Dispersed Aggregate State. The dispersed aggregate state has been shown to be a potentially important factor influencing SWNT impact on bacteria viability [19]. Light scattering can be

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used to determine both the dispersed aggregate size distribution and the aggregate morphology as described previously [19, 52, 53]. Probability distributions of diffusion time, P(τ), were determined by a post processing procedure of the raw DLS data using the CONTIN algorithm [54, 55] (Figure S5). Using a previously described method [19], the compiled MWNT aggregate radii fall in the associated range of 4.3 nm to 378.7 nm, with the majority of samples between 10.8 nm and 108 nm. Estimated aggregate radii were calculated from the peak location in the distribution, representing the most probable aggregate size and are compiled in Table 1. The size range for each sample is also noted (Table 1) and was calculated from the associated location (τ) of the half maxima. Functional groups, particularly oxygen functional groups, have been shown to influence CNT aggregation and dispersion in polar solvents like DI water [19, 51]. Therefore, the aggregate radius calculated from the distribution curve is expected to differ among the samples studied here. Accordingly, there is an observed decrease in aggregate radius upon acid treatment (Table 1). The aggregate radius varies upon annealing at different temperatures (S400-S900), indicating changes in the material surface chemistry, though there is no notable trend in the observed fluctuations. Dispersed aggregate morphology is also an important parameter to consider and is characterized by the sample’s fractal dimension (Df). Details related to the determination of Df values from SLS are outlined elsewhere [19, 52]. The compiled Df,DI values fall between 1.82 and 2.18, and Df,saline between 1.91 and 2.43, indicating a range of dispersed aggregate compactness in each solvent (Table 1). Since a Df value of 3 is associated with a perfect sphere, the closer the Df is to 1, the less compact the aggregate [56]. Except for S900, the Df,saline > Df,DI for each sample, indicating increased aggregate compactness for samples dispersed in saline.

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This is expected as the increase in ionic concentration has been shown to induce CNT aggregation [57]. Within Df,DI, there is a trend towards decreased compactness upon acid treatment that is maintained at low temperature (400 °C) treatment (Df, DI, as-Received > Df, DI, AT ≅ Df, DI, S400). In general, the Df,DI remains fairly constant ~2 at temperatures greater than 400 °C; however, the lack of overall trend in aggregate state suggests that another property is of greater significance in relation to the observed trend in bacterial cytotoxicity unlike previous reports for SWNT where aggregate state dominated [19]. Point of Zero Charge (PZC). The PZC is the pH at which the charge on the surface of the MWNT is neutral. Due to the charged nature of MWNTs and bacterial cells, the sample surface charge will influence both tube-tube interactions and tube-cell interactions [51]. PZC was determined for each sample using mass titration method [42]. Transitions in sample PZC indicate significant changes in surface chemistry. The significant decrease in PZC value (Table 1) from the as-Received to AT-MWNT indicates that acidic oxygen functional groups are added to the nanotube surface as a result of acid treatment. In addition to this significant decrease in PZC, there are three other notable transitions between AT-MWNT and S400, between S500 and S600, and between S800 and S900 as the PZC increases with increased annealing temperature. This step-wise increase in PZC indicates a fundamental change in the MWNT surface charge properties, which can be attributed to the evolution of more acidic functional groups (carboxyl groups with lower pKa values than hydroxyl and other carbonyl groups), reformation of oxygen functional groups upon atmospheric exposure after annealing, and restructuring of the C–C bonds at high enough temperatures [47-49]. These surface changes are expected to significantly influence electrochemical activity of the MWNTs, which may be related to cytoxicity potential.

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Electrochemical Activity. Since CNTs can function as the electron acceptor or donor to facilitate oxidation-reduction reactions around and within cells [16, 20, 58], it is important to consider the relative reactivity of the MWNT samples. MWNT reactivity is represented here by the sample electrochemical activity measured by ORR. There are a number of factors that contribute to the electrochemical activity of CNTs, including presence of impurities, surface functional groups, and tube defects [59]. It is generally accepted that tube ends and defect sites on the tube wall are the primary sites of electrochemical activity [47, 48]. In particular, oxygen functional groups are shown to differentially influence electron transfer mechanisms involving carbon materials, including CNTs [60]. Strelko, et al. describes how different C-O surface chemistry in active carbon compounds influences the electron donor ability, noting that furanand pyrone-type (including carbonyl-types) offer the greatest reductive ability for samples with 4 – 6% oxygen [48]. Furthermore, Jurmann, et al. suggest that quinone-type groups on MWNT edges are the most active sites for O2 reduction [60]. One indication of the presence of different pyrone-type bonding orientations on the MWNTs is the observed change in PZC as it has been shown that the pKa varies with different pyrone-like bonding conformations [49]. The electrochemical activity of MWNTs was determined by ORR experiments using a rotating disc electrode (RDE) technique [40, 60, 61]. The current produced during the MWNT mediated oxygen reduction was measured at varying rotational disc speeds producing polarization curves (compiled curves collected at 1600 rpm shown in Figure 3). Increasing rotational disc speeds (400, 625, 900, 1600 and 2500 rpm) enable characterization through the three different transport regimes: kinetic, mixed and diffusion limited [62]. ORR experiments were run in quadruplicate and the average of the four runs was used to compare the reactivity of the as-Received, AT-MWNT, S400, S600 and S900.

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FIGURE 3 There are several important points in the polarization curves that indicate the relative sample electrochemical activity [62]. First, a more reactive sample will have a more positive measured onset potential (the energy required to initiate the ORR) (Table 1). Second, a more reactive sample will produce a greater current at -0.05 and -0.1 V. The current is typically measured at these two points due to their location in the mixed regime, which involves both kinetic and mass transfer limited ORR [62]. The half-wave potential (E1/2) is another commonly reported value and, as with the onset potential, the more positive the E1/2, the more reactive the sample. The data for each sample associated with these points of interest on the curve is compiled in Table 2. TABLE 2 Observation of the combined data reveals several important transitions in the sample reactivity. The initial increase in electrochemical activity from the as-Received to AT-MWNT, observed most prominently in the increased current produced at -0.1 V, is likely due to the removal of residual carbon impurities, opening of closed tube ends and the introduction of active oxygen functional groups [47]. Upon annealing at increasing maximum temperature, the surface is being reduced leaving bare, highly reactive defect sites [47]. While annealing at 400 °C does not significantly impact the sample reactivity, there is a significant positive shift upon treatment at 600 °C as indicated by significant changes in all values (E1/2, I-0.05V, I-0.1V). This trend reverses at 900 °C where there is a significant negative shift indicative of a decrease in the electrochemical activity. Notably, the trend observed for electrochemical activity mirrors the trend in bacterial cytotoxicity, with greater cytotoxicity associated with greater electrochemical activity, and corresponds to major PZC transitions likely resulting from changes in MWNT surface chemistry.

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The electron transfer efficiency (ETE) is an additional parameter obtained from the ORR measurements and is used to determine the oxygen reduction pathway via either a two- or fourelectron exchange [63]. Though the two-electron pathway is common for CNTs, the ETE has been shown to change as a function of pH and the presence of certain oxygen-containing functional groups [60, 61]. ETEs for each sample were calculated as previously reported [60-62] using the Koutecky-Levich equation ω⁄

(1)[64]

where iL is the mass transport limited current, ω is the rotation rate, ⁄

0.62 υ ⁄ !

(2)[64],

n is the number of electrons transferred, F is the Faraday constant, A is the electrode active area (0.1963 cm2), oxygen diffusion coefficient (1.65 x 10-5 cm2 s-1 at pH = 14)[61], ν is the kinematic viscosity of the solution, and ! is the dissolved oxygen concentration (0.84 x 10-6 mol cm-3 at pH = 14) [61]. The compiled ETE values can be found in Table 2. The ETE values are fairly similar and as expected ~2 with some noteworthy slight variations. First, there is an increase in ETE with acid treatment. This is likely due to removal of residual contaminants in the as-Received sample in combination with the addition of oxygen-containing functional groups. The ETE reaches a local maximum after treatment at 600 °C followed by a significant decrease at 900 °C. This same trend is observed in the cytotoxicity results for all three assays. Like reactivity, cytotoxicity increases with increasing annealing temperature until 900°C when reactivity, GSH oxidation, and loss of cell viability (measured by two cellular assays) decrease. It is likely that these are all related through the quantity and type of oxygen-containing functional group on the surface.

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Implications. The results presented here, in combination with previous studies [19, 20, 22], suggest that MWNT bacterial cytotoxicity is governed by specific material physicochemical properties. Acid treatment and annealing between 400 – 900 °C induces changes in MWNT surface properties, influences the MWNT physiochemical properties, and subsequently the bacterial cytotoxicity (measured here by the acellular GSH oxidation assay and the direct loss of viability upon contact with deposited and suspended MWNTs). The trend in PZC suggests changes in the type of oxygen-containing surface groups with acid and annealing treatments [49], where certain conditions lead to high reactivity as well as cytotoxicity that is the on par with that reported for SWNTs. It is suggested that the sample reactivity, measured by ORR, is the main correlating factor to observed trends in bacterial cytotoxicity, and is directly influenced by acid and annealing treatment via the type of oxygen-containing surface functional groups. Of particular note, this study offers insights into the potential ability to control the antimicrobial properties of MWNTs using common purification treatments. MWNT reactivity is an intrinsic property and can potentially be controlled during or post manufacture by varying the synthesis and treatment conditions. This enables potential manipulation of MWNT antimicrobial properties that could be enhanced when it is advantageous to the product application, such as in antimicrobial surface coatings and water treatment and purification processes. When antimicrobial activity is not necessary or undesirable, as in many other MWNT applications, the reactivity of the sample can be minimized by controlling surface chemistry, particularly oxygencontaining groups, to minimize unintended implications to human health and the environment. In addition to enhanced dispersivity and lower cost of MWNTs, the range and control of antimicrobial properties, from low to comparable to SWNT, may make MWNT more desirable for a number of applications.

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Acknowledgements We acknowledge the generous support of the National Science Foundation under the Research Grant CBET-0854373. LMP recognizes support from the U.S. Environmental Protection Agency (EPA) STAR Fellowship Assistance Agreement no. FP91716701-0 and the NSF Graduate Research Fellowship Program (GRFP). This publication has not been formally reviewed by EPA. The views expressed in this publication are solely those of the authors, and EPA does not endorse any products or commercial services mentioned in this publication. We gratefully acknowledge the use of CAMCOR facilities at the University of Oregon, which have been purchased with a combination of federal and state funding. The authors thank Zhiteng Zhang for preparing the MWNTs and compiling PZC measurements and Gayatri Nangia for assisting with cell enumeration.

Supporting Information Available: Additional methodological details and figures can be found in the supplemental information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table 1. Compiled treatment conditions and physicochemical characteristics of the seven MWNT samples carefully treated from the same starting batch (as-Received).a Anneal Temp (°C)

Onset Potential (V vs MMO)

%C

%O

Point of Zero Charge (PZC)b

Dispersed Aggregate Radius (nm)c

Range at Half Maximum (nm)d

Df,DI

Df,saline

None

-0.1451

89.2

7.43

7.84

74.52

17.0 – 214.9

2.18

2.32

None

-0.1387

92.0

5.76

3.08

37.48

14.5 – 82.8

1.85

2.12

S400

400

-0.1505

88.4

3.23

4.16

48.82

19.9 – 102.4

1.84

2.22

S500

500

4.73

57.24

22.0 – 133.9

2.05

S600

600

6.11

37.48

17.8 – 69.0

2.07

S700

700

6.35

39.53

19.9 – 78.6

1.82

S800

800

6.43

92.12

46.3 – 173.9

2.07

S900

900

9.42

48.82

19.9 – 108.2

2.14

MWNT Sample asReceived AcidTreated

-0.0532

-0.1787

92.4

92.7

2.24

1.32

a

Samples were acid treated (HNO3) for 2 hours and annealed for 1 hour at the maximum temperature indicated. Characteristics include electrochemical activity (onset potential), elemental composition (XPS), surface charge (PZC), aggregate size distribution, and aggregate compactness (fractal dimension, Df). b Significant transitions in PZC are indicated by the darker shaded lines in the table. c Dispersed aggregate radii estimates were determined from P(τ) distribution curves using Stokes-Einstein equation. The value is associated with the peak in the distribution curve representing the most probable aggregate size. d The range at half maximum refers to the estimated size associated with the half maximum values for each curve and provides the distribution in aggregate sizes for each sample.

20


2.43

1.91


Loss of Glutathione (%)

a.

AT-MWCNT S400 S500

S600 S700 S800

100 ** ** 80

Page 22 of 30

S900 SWNT Reference

* *

**

*

* ** *

**

**

** *

60

* *

40

*

*

* 20 0

t=0

t=1

t=4

t=6

Time (hr)

Loss of Glutathione (%)

b. 100

100

S500

80

80

60

60

40

40

20

20

0

S900

0

0

10 min 30 min

1 hr

4 hr

6 hr

0

Time

1 hr

4 hr

6 hr

8 hr

Time

Figure 1. a) Percent loss of glutathione (GSH) for all samples, compared to the control (no MWNT), over time via MWNT-mediated oxidation. As a reference point, data is presented here for an acid treated SWNT sample used in a previous study [19]. Asterisk (*) indicates statistically significant differences in means compared with AT-MWNT. Double asterisk (**) indicate additional statistically significant differences in mean compared with the SWNT reference. Statistical differences were determined by two-sample t tests (95% CI, α = 0.05) b) Percent loss of glutathione for S500 and S900 over an extended time period demonstrating the difference in the oxidation kinetics for these samples. Error bars represent the standard deviation of the sample triplicates.

21



Percent Loss of Viability (%)

a.

*

100

*

90

*

80 70 60 50 0 AT

S500

S900

b. Reduction of CFU/mL compared to control (%)

Page 23 of 30

100 80 60 40 20 0 AT

SWNT Reference

S400

S600

S900

Figure 2. a) Percent loss of cell viability when E.coli were filtered onto a prepared MWNT deposit layer and incubated for 45 minutes total. Fluorescent indicator dyes (Propidium Iodide and DAPI) were utilized to identify viable cells from cells with compromised cell walls. Percent loss of viability was calculated by enumeration of the cells in ~15 images per sample (unviable cells/total cells) and the error bars represent the standard deviation of the 15 images. The percent loss of viability of the control calculated for all sample sets of data collection 3.86 ± 0.31%. The samples’ mean cell viability loss are significantly different from one another as determined by a two-sample unpaired t test (95% CI, α = 0.05, Welch’s correction). Data collected for an acid treated SWNT sample was previously reported [19] and is included here as a reference point. b) Reduction of CFU/mL compared to the control (no MWNT) determined by enumeration of colony forming units on prepared agar plates. E.coli were exposed to suspended MWNT for 1 hour under constant rotation at 37 °C. The error bars represent the standard deviation of the sample triplicates.

22



as-Received

S400

Acid Treated

S600

Page 24 of 30

S900

0.0

Current (mA)

-0.1 -0.2 -0.3 -0.4 -0.5 -1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

Potential (V vs MMO) Figure 3. Polarization curves (at 1600 rpm), from oxidation reduction reaction (ORR) experiments, for a representative subset of samples. Several points on the curve are used to determine the relative sample electrochemical activity, including the onset potential, half-wave potential (E1/2), and current measured at -0.05 and -0.1 V. In general, a positive shift in the polarization curve is representative of a transition towards more electrochemically active material.

23


Page 25 of 30


Table 2. Associated values used to determine the relative electrochemical activity of the MWNT samples. Sample

E1/2 (V)a

Current (mA) at -0.05 Va

Current (mA) at -0.1 Va

Electron Transfer Efficiency (ETE)b

as-Received

-0.215

-0.0014

-0.0056

2.25

Acid-Treated S400

-0.219 -0.221

-0.0019 -0.0017

-0.0081 -0.0084

2.65 2.28

S600

-0.127

-0.0272

-0.0993

2.47

S900

-0.244

-0.0011

-0.0026

2.12

a

A more positive E1/2 and greater absolute value of the current produced at -0.05 and -0.1 V are indicative of a more electrochemically active sample. b The electron transfer efficiency (ETE) represents the 2 or 4-electron oxygen reduction pathway. Positive deviations from the typical 2-electron pathway for CNTs indicate increasing contribution of the 4-electron pathway.

24



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28



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O

HO

OH

O H O

O

HO

Increase

Reactivity

O O

HO

H

O OH

O

O OH

O

O HO

HO O

O O

O

OH

H

O

O O

Labile groups O

0

300

600

Annealing Temperature (°C) Increase PZC ACS Paragon Plus Environment

O

O

900

Realizing Comparable Oxidative and Cytotoxic Potential of Single

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