Physicochemical Determinants of Multiwalled Carbon Nanotubes on

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Physicochemical Determinants of Multiwalled Carbon Nanotubes on Cellular Toxicity: Influence of a Synthetic Method and Post-treatment Ji-Eun Kim,†,‡ Seung-Hyon Kang,§ Youngmi Moon,⊥ Jin-Joo Chae,∥ Ah Young Lee,† Jae-Ho Lee,† Kyeong-Nam Yu,†,# Dae Hong Jeong,∥ Mansoo Choi,§ and Myung-Haing Cho*,†,‡,# †

Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea Graduate School of Convergence Science and Technology, Seoul National University, Suwon 443-270, Korea § Global Frontier Center for Multiscale Energy Systems, School of Mechanical and Aerospace Engineering, Seoul National University, Seoul 151-742, Korea ∥ Department of Chemistry Education, Seoul National University, Seoul 151-742, Korea ⊥ Interdisciplinary Program in Bioinformatics, College of Natural Science, Seoul National University, Seoul 151-742, Korea # Graduate Group of Tumor Biology, Seoul National University, Seoul 151-742, Korea ‡

S Supporting Information *

ABSTRACT: Since the discovery of carbon nanotubes (CNTs), scientists have performed extensive studies on nanotubes in the fields of materials science, physics, and electronic engineering. Because multiwalled CNTs (MWCNTs) are not homogeneous materials, and because it is not feasible to test every newly synthesized MWCNT, this study was aimed at investigating the physicochemical properties that primarily determine the cellular toxicity of MWCNTs. This study analyzed the relationship between cell viability and physicochemical characteristics following exposure to eight different MWCNTs. We generated eight different MWCNTs using various synthetic methods and post-treatments. From this analysis, we sought to identify the major physicochemical determinants that could predict the cellular toxicity of MWCNTs, regardless of the synthetic method and posttreatment conditions. Creation of binding sites on the tube walls by breaking C−C bonds played a pivotal role in increasing toxicity and was most clearly demonstrated by a Raman G peak shift and the ID/IG ratio. In addition, several factors were found to be strongly related to cellular toxicity: surface charge in the case of MWCNTs created by the chemical vapor deposition method and surface area and EPR intensity in the case of MWCNTs created by the arc discharge based method. The methods developed in this study could be applied to the prediction of the toxicity of newly synthesized MWCNTs.

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INTRODUCTION Polymer nanocomposites based on multiwalled carbon nanotubes (MWCNTs) have attracted great interest due to their remarkable mechanical, thermal, and electrical properties.1 Compared to conventional particles, such as carbon black or nanoclays, the high aspect ratio of CNTs allows for the properties of these CNTs to be enhanced at lower concentrations,2 demonstrating the lower percolation threshold for the application of polymer/CNT nanocomposites as conductive, strong, lightweight materials.3 However, prepared MWCNTs generally exist as highly entangled clusters because of their extremely long lengths, and large amounts of © XXXX American Chemical Society

unnecessary carbonaceous particles, such as fullerenes, nanoparticles, and amorphous phases, are always present in the soot.4 Since high purity and reproducible dispersity are general requirements for precise measurements and practical applications of MWCNTs as composite materials, it is necessary to disperse highly entangled MWCNTs uniformly in fluids or polymer melts without impurities. In general, there are largely two types of impurities found in prepared MWCNTs: catalytic metallic impurities and carbonaReceived: October 27, 2013

A

dx.doi.org/10.1021/tx400397g | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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Table 1. Characteristics of the Eight Types of MWCNTs As Determined by Raman Spectroscopya CVD

Arc

Raman shift (D) (cm−1)

Raman shift (G) (cm−1)

intensity (D)

intensity (G)

1318 1317 1322* 1321* 1320 1332* 1322 1319

1572 1571* 1579 1574 1565 1578* 1568 1566

694.6 720.0 704.9 758.0 53.6 219.1 62.3 111.9

376.6 356.8 332.5 366.3 578.4 818.6 565.7 727.4

PMWCNT H-MWCNT A-MWCNT HA-MWCNT PMWCNT H-MWCNT A-MWCNT HA-MWCNT

ID/IG 1.83 2.04 2.14 2.07 0.09 0.28 0.11 0.16

± ± ± ± ± ± ± ±

0.19* 0.32 0.15 0.06 0.04 0.08* 0.03 0.03

a

Positive Raman shifts of D- and G-bands were predominant in Arc-H-MWCNTs, implying the splitting of the G-band. CVD-A-MWCNTs and CVD-HA-MWCNTs also showed positive Raman shifts of D- and G-peaks (*p < 0.05, ANOVA). sonicated for 25 min to disperse the MWCNTs and then refluxed at 120 °C for 90 min (A-MWCNTs). Following acid treatment, the carbon nanotubes were washed with deionized water and filtered using DM Metricel 800 filters (0.8 μm pore size; Pall Life Science, Port Washington, NY, USA). After treating the MWCNTs using these two processes, eight types of MWCNTs were prepared from the two different starting materials (CVD-PMWCNTs, CVD-A-MWCNTs, CVD-H-MWCNTs, CVD-HA-MWCNTs, Arc-PMWCNTs, Arc-AMWCNTs, Arc-H-MWCNTs, and Arc-HA-MWCNTs). Preparation of MWCNTs for in Vitro Application. The eight different MWCNTs were prepared as dry powders. Each type of MWCNT was weighed in a 10-mL glass vial in the fume hood on an analytical balance and was dry-heat sterilized at 200 °C for 1 h. MWCNTs were then suspended in a medium containing 10% fetal bovine serum (FBS) at a final concentration of 1 mg/mL in 10-mL glass vials. These suspensions were sonicated for 15 min in a water sonicator bath (5510-DTH; Branson, Danbury, CT, USA) and used as the stock solution for treatment in cell culture media (DMEM/F-12; Gibco BRL, Gaithersburg, MD, USA) for 16HBE14o- cells (immortalized human bronchial epithelial cells). An appropriate amount of each stock solution was added to the desired final concentration in cell culture flasks. To prevent morphological or physical changes in MWCNTs, the stock solution of each MWCNT was prepared immediately before each cell experiment. Transmission Electron Microscopy (TEM). MWCNTs were sonicated in ethanol for 3 min. A drop of the solution was placed on a Formvar/carbon-film-coated 400 mesh Cu TEM grid (Samchang Commercial Co., Ltd., Seoul, Korea), and the morphology and size of MWCNTs were analyzed using energy-filtering TEM (EF-TEM) on a LIBRA 120 instrument (Carl Zeiss, Oberkochen, Germany) with an operating voltage of 120 kV. Field-Emission Scanning Electron Microscopy (FE-SEM). After sputter coating samples with platinum, FE-SEM images of MWCNTs were obtained using a JSM-6700F (JEOL, Tokyo, Japan) at an acceleration voltage of 10 kV. The samples were prepared by loading a droplet of each MWCNT solution on a silicon wafer and then drying the droplet on a hot plate. Raman. Raman measurements were performed using a confocal microscope Raman system (LabRAM 300; JY-Horiba, Edison, NJ, USA) equipped with an optical microscope (Olympus, Tokyo, Japan). In this system, Raman scattering signals were collected in a 180° backscattering geometry and detected by a spectrometer equipped with a thermoelectrically cooled (−70 °C) charge-coupled device (CCD) detector. Focusing of the excitation laser and collection of the Raman signal were conducted using a 100× objective lens (NA 0.90; Olympus). The excitation source was a 647-nm Kr laser (Innova I-301; Coherent, CA, USA), and the power of the laser was approximately 1.2 mW at the samples. Raman signals were collected on the selected point for 1 s. Raman peak center wavenumber was analyzed by fullwidth-at-half-maximum (fwhm), and peak height was calculated as reported previously.13 Average characteristics of the Raman peaks are shown in Table 1. Electron Paramagnetic Resonance (EPR) Spectrometer. EPR signals were measured using a Bruker EMX/Plus spectrometer

ceous materials. Thus, the purification of raw products through various post-treatment processes, including chemical or thermal oxidation, is a prerequisite to various MWCNT applications, especially in the area of composite applications.5 In addition to removing impurities, improvement of dispersity can also be achieved by these post-treatments. Acid treatment of nanotubes is a well-known method for removing catalytic impurities and shortening the length of CNTs under simultaneous sonication.6 Acid treatment is also known to generate functional groups on the opened ends or side walls of CNTs to promote easy dispersion of CNTs in solution.7 Thermal oxidation of CNTs up to 400 °C will improve hydrophilicity by creating oxygenterminated surfaces,8 while thermal treatment above 1800 °C is generally used for annealing or enhancing CNT surface crystallization and removing amorphous carbon.8,9 As the temperature increases up to 400 °C, removal of smaller diameter tubes can occur through a thermal oxidation route.10 Single-walled CNTs (SWCNTs) are the first species to oxidize, followed by double- and triple-walled CNTs, and finally smalldiameter MWCNTs.10,11 In addition, the thermal oxidation of tubes generates binding sites on the tube walls by breaking C− C bonds.12 Through this process, composite materials may benefit greatly by utilizing tubes of a narrower diameter distribution that are free of amorphous carbon and contain more binding sites for better functionalization. The aim of the present work was to investigate the physicochemical characteristics of MWCNTs prepared using a synthetic method with either thermal or acid post-treatment and to elucidate the role of these physicochemical characteristics in mediating cell viability. Before their use in biological assays, all samples were thoroughly characterized in terms of diameter, length, surface chemistry, surface area, and metal impurities. Finally, the relationships between cellular toxicity and the analyzed physicochemical characteristics were evaluated by principal component analysis (PCA).

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EXPERIMENTAL PROCEDURES

Post-treatment of MWCNTs and the Preparation of Eight Different Types of MWCNTs. Two different MWCNTs were used as starting materials for post-treatment procedures. The first was synthesized via chemical vapor deposition (CVD), and the second was synthesized via arc discharge. These two different commercially synthesized MWCNT powders were purchased from Hanwha Nanotech Inc. (Incheon, Korea), and each pristine MWCNT (PMWCNT) was treated using two processes: thermal treatment and acid treatment. In thermal treatment, MWCNTs were heated in air at 500 °C for 2 h to remove amorphous carbons in PMWCNTs (H-MWCNTs). In acid treatment, MWCNTs were mixed with HNO3 and H2SO4 (1:3 = v/v); approximately 10 mL of HNO3 and 30 mL of H2SO4 were used for 1 g of MWCNTs. Next, the mixture was B



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Figure 1. Flowchart of the treatment process for the preparation of MWCNTs. (A) As-purchased pristine MWCNTs (PMWCNTs) were used as the starting material and were treated by thermal treatment or acid treatment. (B) Two types of MWCNTs were used as starting materials; one was synthesized via chemical vapor deposition (CVD), and the other was synthesized via arc discharge (Arc). After these two post-treatment processes, eight MWCNTs were generated: CVD-PMWCNTs, CVD-A-MWCNTs, CVD-H-MWCNTs, CVD-HA-MWCNTs, Arc-PMWCNTs, Arc-AMWCNTs, Arc-H-MWCNTs, and Arc-HA-MWCNTs. (Bruker, Stuttgart, Germany). Measurements were performed at the following settings: temperature, 298 K; modulation amplitude, 5 G; modulation frequency, 100 kHz; microwave power, 0.94 mW; and microwave frequency, 9.6 GHz. For quantitative analysis, 5 mg of each of the eight different MWCNTs was placed into an EPR quartz tube and was analyzed by EPR. Thermo Gravimetric Analysis (TGA). The total metal content of MWCNTs was measured by TGA using a Q-5000IR instrument (TA Instruments, Brussels, Belgium). CVD-PMWCNTs were heated to 800 °C, and Arc-PMWCNTs were heated to 1000 °C at a heating rate of 10 °C/min in air atmosphere. Inductively Coupled Plasma−Atomic Emission Spectroscopy (ICP-AES). MWCNTs were exposed to the flame of an atomic emission spectrometer and subjected to nebulization, desolation, liquefaction, vaporization, atomization, excitation, and ionization using an Optima-4300 DV (PerkinElmer, Waltham, MA, USA). During the atomization and excitation stages, the emission wavelength was measured at the characteristic wavelength for the elements of interest. Samples were analyzed for the presence of the following transition metals: manganese, cobalt, nickel, copper, zinc, aluminum, iron, titanium, and platinum. Thirty milligrams of MWCNTs was dissolved in a mixture of 5 mL of aqua regia and 2 mL of hydrofluoric acid mixture for 5 h in 180 °C. The mixture was then centrifuged to separate the residual solids. Finally, the concentrations of metal elements in aqua regia were analyzed by ICP-AES after dilution with distilled water to a volume of 20 mL. X-ray Powder Diffraction (XRD). The crystallinity of MWCNTs was analyzed using an X-ray diffractometer (M18XHF-SRA, MAC

Science Co., Chiba, Japan) operating at 40 kV and 200 mA. The scan rate and angle were fixed at 4°/min and 5−90, respectively. Dynamic Light Scattering (DLS). The hydrodynamic diameter was obtained by dynamic light scattering (ELS-8000; Otsuka Electronics Co., Ltd., Osaka, Japan). MWCNTs were dispersed in dimethylformamide (DMF) and sonicated for 15 min for sufficient dispersion. The refractive index for DMF was used for the measurement. Manual Scaling of MWCNTs. The diameter and length of the eight different MWCNTs were calculated using 10 SEM microphotographs of each MWCNT with an average of three different measurements. The values were obtained by multiplying the magnification of the microphotograph, and the obtained values were applied to log-normal distributions. The geometric mean (GM) length, GM diameter, and geometric standard deviation (GSD) were calculated as previously described.14 Brunauer−Emmett−Teller (BET). BET surface area analysis of MWCNTs was performed by N2-adsorption−desorption isotherm nanoPOROSITY-XQ (MiraeSi, Gwangju, Korea). The samples were outgassed at 150 °C for 12 h before the adsorption test, reaching a final pressure of 10−6 mbar. Fourier Transform Infrared Absorption Spectroscopy (FTIR). Infrared absorption spectra were measured by FT-IR (Bruker IFS66/S, Bruker, Ettlingen, Germany) at room temperature. Samples were prepared by gently mixing 10 mg samples with 200 mg of KBr powder and compressed into discs at a force of 98 kN for 3 min using a manual tablet presser. C



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Figure 2. Images of eight different materials. TEM (A and B) and SEM (C and D) images of eight different MWCNTs. Compared to CVD-based MWCNTs (A and C), arc discharge-based MWCNTs (B and D) showed high crystallinity and sharp, straight fibers. After thermal treatment, CVDH-MWCNTs (A, lower left, and C, lower left) were shortened compared to CVD-PMWCNTs (A, upper left, and C, lower left), while Arc-HMWCNTs (B, lower left, and D, lower left) were not shortened. In both synthetic types of MWCNTs, acid post-treatment led to smaller diameters and shorted lengths of MWCNTs (B, upper right, compared to B, upper left; D, upper right, compared to D, upper left). CVD-HA-MWCNTs showed the shortest lengths among all CVD-based MWCNTs, while Arc-HA-MWCNTs did not exhibit significant differences in lengths.

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Principal Component Analysis (PCA). PCA was carried out using the FactoMineR package (released from the Web site of the Institute for Statistics and Mathematics of the Vienna University of Wien) using the covariance matrix. A data frame was constructed with the average value of each physicochemical factor for the eight different MWCNT materials. For the factor length, results from the manual scaling method and lengths from SEM image analysis were used for PCA. Statistical Analysis. Results are shown as the mean ± standard deviation (SD) of repetitive experiments. Statistical analyses were performed using the SPSS 12K program. Factors including defect, surface charge, length, diameter, Raman shift (G), and Raman shift (D) were assumed to follow normality and were statistically analyzed. Equal variances were tested after finding significance using analysis of variance (ANOVA). The Duncan test was used in cases of equal variance, and Dunnett’s t test was performed in cases that did not satisfy equal variance. For the factor length, results by manual scaling method were used for ANOVA.

ATP ASSAY

Normal human bronchial epithelial 16HBE14o- cells were maintained in DMEM/F-12 (Gibco, Carlsbad, CA, USA) with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO2 incubator. For ATP assays, 16HBE14o- cells were seeded in 96-well white tissue culture plates at 1 × 104 cells/well. Twenty-four hours later, the culture medium was replaced with new medium, and MWCNTs were added at 2-fold serial dilutions (from 1000 μg/mL to 1 μg/mL). Cells were then incubated for 48 h. The medium was removed, and ATP was measured using CellTiter-Glo (Promega, Madison, WI, USA) on a luminometer (Titertek Berthold, Pforzheim, Germany) according to the manufacturer’s instructions. Half-maximal effective concentrations (EC50s) were determined by nonlinear regression analyses of sigmoidal dose−response curves using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). D



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Figure 3. Raman spectroscopy of the eight MWCNTs. (A) Raman spectra of each MWCNT showed two major peaks in the high frequency range: the tangential mode or so-called G-band at 1601 cm−1 and the D-band at 1289 cm−1 assigned to carbonaceous compounds or defects in MWCNTs. The inset shows the deconvolution of the D′-peak from the G-peak, which was observed in CVD-based MWCNTs and in Arc-H-MWCNT (redlined square). (B) The intensity ratio of the D-band to the G-band (ID/IG) in A-MWCNTs was higher than that in PMWCNTs, implying that PMWCNTs were less defective than A-MWCNTs. The ID/IG value of H-MWCNTs was higher than that of PMWCNTs, indicating that HMWCNTs contained more defects that could act as bonding sites for better functionalization on the side wall. *p < 0.05 was considered significant, and ***p < 0.001 was considered highly significant compared with the corresponding CVD-PMWCNT values. ###p < 0.001, @@@p < 0.001, and $$$p < 0.001 revealed highly significant differences compared to those of the corresponding Arc-PMWCNT, Arc-A-MWCNT, and Arc-H-MWCNT values, respectively.

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RESULTS Morphology of the Eight Different MWCNTs. There were obvious differences between CVD-PMWCNTs and ArcPMWCNTs as starting materials. The morphology of arc

discharge-based PMWCNTs (Figure 2B, upper left) was straight and sharp in shape compared to that of CVD-based PMWCNTs (Figure 2A, upper left). This tendency was found in all treated MWCNTs derived from the CVD method, as E



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Figure 4. FT-IR spectra of eight types of MWCNTs. (A) FT-IR spectra of CVD-PMWCNTs, CVD-H-MWCNTs, CVD-A-MWCNTs, and CVDHT-MWCNTs. (B) FT-IR spectra of Arc-PMWCNTs, Arc-H-MWCNTs, Arc-A-MWCNTs, and Arc-HA-MWCNTs.

the tangential mode, and a so-called G-band and at 1580 cm−1, which served as a measure of the structural defects in MWCNTs (Figure 3A). Similar to D-bands, the D′-peak at 1615 cm−1 was a double-resonance Raman feature induced by disorders and defects (Figure 3A). This D′-band was caused by a defect in the C−C bond and was observed for all CVD-based MWCNTs in addition to Arc-H-MWCNTs and Arc-HAMWCNTs (Figure 3A, square with red line). The intensity ratio of the D-band to the G-band (ID/IG) was higher in AMWCNTs than in PMWCNTs, implying that PMWCNTs were less defective than A-MWCNTs (Figure 3B). In addition, the ID/IG ratio of H-MWCNTs was higher than that of PMWCNTs, indicating that H-MWCNTs contained more defects for better functionalization on the side walls (Figure 3B).

opposed to those derived from the arc discharge method (Figure 2A and C versus Figure 2B and D). Sharp, clean XRD peaks of arc discharge-based MWCNTs also supported this (Figure S1, Supporting Information). After thermal posttreatment, there was no significant difference in morphology (Figure 2A−D, lower left panel). However, acid post-treatment of MWCNTs obtained by both synthetic routes resulted in AMWCNTs exhibiting smaller diameters and shorter lengths as compared to those of PMWCNTs (Figure 2A−D, upper right panel). Raman Spectroscopy of the Eight MWCNTs. The use of Raman spectroscopy allows for the identification of many characteristics of carbon materials, especially nanotubes. The Raman spectra of MWCNTs showed two major peaks: a Dband at 1320 cm−1 assigned to carbonaceous compounds and F



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Functional Group Contents of MWCNTs. The FT-IR signals of MWCNTs indicated their functional group contents (Figure 4). Carboxyl groups, which are indicated by a peak at 3428−3436 cm−1, were most abundant in acid-treated MWCNTs (both CVD- and Arc-MWCNTs; Figure 4A and B, green lines), followed by HA-MWCNTs and H-MWCNTs (Figure 4A and B, purple and red lines, respectively). Singlet Electrons on the Surfaces of the Eight Types of MWCNTs. The EPR signals of MWCNTs in Figure 4 show quantitative EPR signal detection. In CVD-based MWCNTs, singlet electrons were not detected (Figure 5A). However,

Table 2. Metal Catalyst Remnant Percentages and Burn out Temperatures after TGA Analysis

CVD

Arc

catalyst remnants (%) burn out temperature (°C) catalyst remnants (%) burn out temperature (°C)

PMWCNT

HMWCNT

AMWCNT

HAMWCNT

14.75

15.05

0.98

0.92

600.25

597.57

528.18

643.72

0.32

0.87

0.19

0.36

803.65

804.41

727.88

777.12

H-MWCNTs showed 14.75% and 15.05% metal catalyst remnants, respectively. Acid post-treatment removed metal catalyst remnants in PMWCNTs, while thermal treatment concentrated the amount of metal catalysts due to the removal of carbonaceous particles. After thermal treatment, the residual mass increased because heating in air at 500 °C for 2 h did not remove metal contents but instead burned amorphous carbon in PMWCNTs. Consequently, for MWCNTs of the same weight, H-MWCNTs contained more metal contents than PMWCNTs. The burn-out temperature of arc discharge-based MWCNTs was higher than that of CVD-based MWCNTs, indicating that arc discharge-based MWCNTs were more effectively crystallized than were CVD-based MWCNTs (Table 2). The concentrations of metal elements in MWCNTs are shown in Table 3, as determined by ICP-AES. Using this, the quantity of each metal element was estimated by multiplication of the metal catalyst remnants resulting from TGA (Tables 2 and 3). Changes in the Length, Diameter, and Surface Area of the Eight Types of MWCNTs after Post-treatment. In CVD-based MWCNTs, PMWCNTs were the longest, followed by H-PMWCNTs, A-PMWCNTs, and HA-PMWCNTs (Table 4). arc discharge-based MWCNTs did not show large differences in lengths after post-treatment (Table 4). DLS and manual scaling method results showed the same patterns, although the actual length appeared longer when measured using the manual scaling method (Figure S3, Supporting Information). For correlation, PCA and ANOVA tests between the physicochemical factors and EC50s, GMs calculated using the manual scaling method were used. Diameters measured from SEM images did not show any significant changes (Table 4). However, changes in the surface area after acid treatment in arc discharge-based MWCNTs and CVD-based MWCNTs yielded conflicting results; several previous reports have also shown contradictory results after acid treatment.16,17 The overall characteristics of length, diameter, and surface area are summarized in Table 4. Intracellular ATP Levels in Cells Treated with the Eight Types of MWCNTs. ATP levels of MWCNT-treated cell lines were assessed using the CellTiter-Glo luciferase assay (Figure 6A and B), and the EC50 (Table 5) was calculated from data presented in Figure 6A and B. In general, the EC50 of CVD-MWCNT-treated cells was lower than that of ArcMWCNT-treated cells (Table 5). The effects of acid treatment on intracellular ATP levels were clear in 16HBE14o- cells treated with CVD-MWCNTs; the EC50 of cells treated with CVD-PMWCNTs was 195.74 μg/mL, while that of CVD-AMWCNTs was 28.25 μg/mL (Table 5). However, this toxicity potentiation effect by acid treatment was not observed in arc

Figure 5. Quantitative electron paramagnetic resonance (EPR) signals of MWCNTs. (A) CVD-based MWCNTs did not show positive EPR signals. (B) MWCNTs synthesized using the arc discharge method showed positive EPR signals. Arc-HA-MWCNTs (peak intensity: 117.0) showed the strongest signal, followed by Arc-A-MWCNTs (peak intensity: 39.3), Arc-H-MWCNTs (peak intensity: 33.6), and Arc-PMWCNTs (peak intensity: 17.9), in decreasing order.

MWCNTs synthesized using the arc discharge method showed positive EPR signals (Figure 5B). EPR results strongly depended both on the degree of purity of the sample and the method of preparation.15 Amount of Metal Catalyst Remnants for the Eight Types of MWCNTs. Sample purity was tabulated as residual mass after TGA (Table 2 and Figure S2, Supporting Information). In TGA analysis, CVD-PMWCNTs and CVDG



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Table 3. Concentration of Each Metal Element in MWCNT Samples, As Measured by ICP-AESa CVD (CM-100) Mn Co Ni Cu Zn Al Fe Ti Pt a

Arc-discharge

PMWCNT

H-MWCNT

A-MWCNT

HA-MWCNT

PMWCNT

H-MWCNT

A-MWCNT

HA-MWCNT

nd 568.47 nd nd 14.59 4220.04 721.76 nd nd

nd 4927.68 6.56 nd 15.85 9572.47 4648.10 nd nd

nd nd nd nd 6.73 21.51 363.92 8.96 nd

nd 1027.70 nd nd 56.03 7482.92 862.66 nd nd

nd nd nd nd 35.65 53.98 86.47 nd nd

nd nd nd 6.60 15.32 103.03 98.10 nd nd

nd nd nd nd nd 51.40 51.71 nd nd

nd nd nd nd 9.20 11.91 4.74 nd nd

Unit, parts per billion (ppb); nd, not detected.

Table 4. Overall Characteristics of the Eight Types of MWCNTs CVD (CM-100) PMWCNT diameter (nm) (GSDa) length (μm) (GSDa) surface charge (mV) purity (by TGA) Raman ID/IG surface area (m2/g) a

H-MWCNT

arc discharge

A-MWCNT

HA-MWCNT

PMWCNT

H-MWCNT

A-MWCNT

HA-MWCNT

47.87 nm (1.18) 1.33 μm* (1.62) −14.55

28.26 nm (1.18) 1.10 μm* (2.03) −22.93

46.02 nm (1.24) 0.85 μm (1.95)

36.53 nm (1.36) 0.70 μm (1.91)

46.76 nm (1.20) 1.84 μm (1.73)

41.48 nm (1.32) 1.95 μm (1.54)

58.66 nm (1.28) 1.71 μm (1.53)

56.24 nm (1.35) 1.66 μm (1.72)

−26.22

−16.14

−24.85

−21.58

−33.18

−31.70

85.25% 1.83* 243.38

84.95% 2.04 235.78

99.02% 2.14 139.84

99.08% 2.07 179.28

99.68% 0.09 11.31

99.13% 0.28* 47.22

99.81% 0.11 18.89

99.64% 0.16 36.15

GSD: geometric standard deviation. (*p < 0.05, ANOVA.)

Figure 6. Effects of the eight types of MWCNTs on cellular ATP amounts. 16HBE14o- cells were seeded in 96-well plates and maintained in DMEM/F-12 media supplemented with 10% fetal bovine serum. Cell viability was measured after 48 h of treatment with CVD-based MWCNTs (A) or arc discharge-based MWCNTs (B) at the indicated concentrations using the CellTiter-Glo luminescent cell viability assay. The half-maximal effective concentration (EC50) in Table 5 was calculated from the data presented in A and B.

Table 5. EC50 Values for the Eight Types of MWCNTs, As Measured Using ATP Assays in 16HBE14o- Cells EC50 (μg/mL) (95% confidence limits) R2

ATP assay PMWCNT 16HBE14o-

CVD Arc

195.74 (164.0−251.4) R2 = 0.9856 179.67 (136.4−265.7) R2 = 0.967

H-MWCNT

A-MWCNT

91.36 (78.14−111.0) R2 = 0.989 129.81 (106.0−172.2) R2 = 0.9801

discharge-based MWCNTs (Table 5). The toxicity of heattreated MWCNTs in 16HBE14o- cells was also higher than that of PMWCNTs for MWCNTs prepared using both the CVD and arc discharge methods, although the potentiation effect was not as substantial as that of the acid treatment of

28.25 (25.24−31.59) R2 = 0.9953 182.39 (132.5−235.1) R2 = 0.9735

HA-MWCNT 18.94 (16.88−21.40) R2 = 0.9948 114.83 (93.91−140.9) R2 = 0.9857

CVD-MWCNTs (Table 5). For both heat and acid treatment, the EC50 correlated with the treatment that endowed higher toxicity (Table 5). Correlation Analysis between the Physicochemical Factors and the Cellular Toxicity of MWCNTs. CorrelaH



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Figure 7. Correlations of physicochemical factors of the eight different MWCNTs using PCA. (A) Individual factor map showing different clusters of MWCNTs. (B) Variable factor map showing correlations between physicochemical factors and the factor EC50. The factor Raman shift (G), which projected to the side opposite the factor EC50, had the greatest association with the cellular toxicity of MWCNTs because EC50 and toxicity were inversely related. I



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Figure 8. Correlations of physicochemical factors of the four different CVD-based MWCNTs using PCA. (A) Individual factor map showing different clusters of CVD-based MWCNTs. (B) Variable factor map showing correlations between physicochemical factors and the factor EC50. The factor defect (ID/IG), which projected to the side opposite the factor EC50, had the greatest association with the cellular toxicity of CVD-basedMWCNTs. J



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Figure 9. Correlations of physicochemical factors of the four different arc discharge-based MWCNTs using PCA. (A) Individual factor map showing different clusters of arc discharge-based MWCNTs. (B) Variable factor map showing correlations between physicochemical factors and the factor EC50. The factors surface area, EPR peak intensity, and defect (ID/IG), which projected to the side opposite the factor EC50, had the greatest associations with the cellular toxicity of arc discharge-based MWCNTs. K



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1800−2000 °C increases the crystallinity of MWCNTs by annealing them; however, raising the temperature up to 500 °C has the opposite effect by creating defective sites on the side walls.8 We applied the latter method; therefore, the effects of thermal treatment were expected to decrease amorphous carbon and the creation of binding sites by breaking C−C bonds. Because thermal treatment reduced amorphous carbon, which can be functionalized by acid treatment, in FT-IR data, we observed fewer functional groups in HA-MWCNTs than in A-MWCNTs (Figure 4A,B). For HA-MWCNTs, heat treatment would usually be expected to increase functionalization due to resulting defects, which functional groups easily attack; however, heat treatment of these HA-MWCNTs resulted in decreased functional groups due to the removal of amorphous carbon, offsetting the effects of heat-induced generation of defects. A reduction in amorphous carbon was also observed by analyzing TGA patterns (Figure S2, Supporting Information). In the TGA graph, H-CVD-MWCNTs showed a gradual decrease in amorphous carbon before exhibiting characteristics of burn out, as compared to A- or HA-CVD-MWCNTs. Creation of binding sites by breaking C−C bonds was apparent in Arc-H-MWCNTs and was well characterized by the Raman G-peak shift (Figure 3A,B, and Table 1). This phenomenon was not noticeable in CVD-H-MWCNTs because CVDPMWCNTs tended to have many innate defects. In PCA analysis (Figure 7B), broken C−C bonds represented by this Raman G-peak shift revealed the major determinant, and subsequent particle characterization results did not have any correlation with the EC50 of MWCNTs. Broken C−C bonds may attack important molecules in the cell since π-orbital misalignment between adjacent carbon atoms has an influence on changes in overall reactivity.21,22 The same pattern observed in PCA analysis was further confirmed with different cell lines, i.e., human hepatic stellate cells (LX2) and human embryonic kidney cells (HEK293), using ATP assays (Figures S4−S6 and Table S1, Supporting Information) and WST assays (Figures S7−S9 and Table S2, Supporting Information). Our results also showed that factors related to cell wall defects and surface reactivity, i.e., defect and Raman shift (G), were highly correlated with cellular EC50s when compared with all of the eight different types of MWCNTs (Table S3, Supporting Information). Through acid treatment, MWCNTs are decapped, and side wall defects are functionalized to form functional groups. Consequently, the physicochemical characteristics of PMWCNTs are changed.20,23,24 In our study, A-MWCNTs showed enhanced dispersion in water because of the introduction of functional groups to the defects (Figures 3B and 4A,B) and to amorphous carbon. Solubility is determined not only by the functional groups on MWCNTs, but also by functionalized amorphous carbon, which is generated during nanotube destruction.25 FT-IR spectra also showed the presence of a very broad O−H stretching peak of the −COOH functional group from 3000 to 3600 cm−1 for both CVD- and Arc-A-MWCNTs (Figure 4A,B). Additionally, AMWCNTs were shorter in length (Table 4) and exhibited negative charge shifts of zeta potential as the ionization reaction of carboxylic acids in water was sufficiently effective (Table 4).23 Metal catalysts were also removed (Table 2), and TMWCNTs exhibited reduced surface areas compared to that of PMWCNTs (Table 4); this was caused by rebundling of the tube walls.16 These features were observed in CVD-AMWCNTs but were not apparent in Arc-A-MWCNTs. Since

tions were analyzed three ways. First, to identify general factors correlated with toxicity, integrated data of eight different MWCNTs were applied to PCA (Figure 7). In addition, correlations were analyzed using the data of CVD-based MWCNTs (Figure 8) and arc discharge-based MWCNTs (Figure 9). In integrated analysis using data from all eight MWCNTs, the factor Raman shift (G), which projected to the side opposite the factor EC50, had the greatest relevance because EC50 and toxicity were inversely related (Figure 7B). The graph shows different clusters of the eight different MWCNTs (Figure 7A). The first dimension (52.3% of the total inertia) opposed Arc-H-MWCNTs (high intensity ratings in Raman shift (D)) to CVD-HA-MWCNTs and CVD-AMWCNTs (high intensity ratings in Raman shift (G) and defect); the second dimension (21.03% of the total inertia) opposed Arc-PMWCNTs, Arc-A-MWCNTs, and Arc-HAMWCNTs (high intensity ratings in diameter, length, Raman shift (D), EPR intensity, EC50, and oxidation temperature) to CVD-PMWCNTs and CVD-H-MWCNTs (high intensity ratings in iron, aluminum, TGA metal catalyst, and surface area). In PCA using data from CVD-based-MWCNTs, the factor defect (ID/IG), which projected to the side opposite the factor EC50, had the greatest relevance with the cellular toxicity of CVD-based-MWCNTs (Figure 8A and B). In addition, surface charge, which stretched in the same direction as EC50, was also positively correlated with cellular toxicity because it had negative value (Figure 8B). As the surface charge became larger, i.e., the value of the surface charge approaches a positive value, the EC50 increases. Consequently, as the surface charge becomes larger, the cellular toxicity decreases. However, iron content and length were negatively correlated with cellular toxicity. For arc discharge-based MWCNTs, the factors surface area, EPR peak intensity, and defect (ID/IG), which projected to the side opposite the factor EC50, had great relevance (Figure 9A and B).

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DISCUSSION This study was aimed at describing which physicochemical properties were involved in determining the cellular toxicity of MWCNTs. Generally, the toxicity of certain nanoparticles, such as CNTs, is known to be related to factors such as surface area, length, degree of agglomeration, water solubility, metal catalyst remnants, and surface charge.18 However, studies seeking mainly to identify physicochemical determinants that have dominant roles in toxicity according to the synthetic method and acid and thermal post-treatments have not provided detailed descriptions of these factors.19 In fact, studying the toxicity of MWCNTs is difficult in terms of restricting particle conditions because it is difficult to control the lengths of MWCNTs during synthesis, and prepared MWCNTs tend to be contaminated with impurities, such as metal catalyst particles, amorphous carbon, broad diameters, and chirality distribution.20 Therefore, we used eight types of MWCNTs, prepared using different synthetic methods and post-treatments, and analyzed the different characteristics of these MWCNTs, including cellular toxicity (Figure 1). By this simple comparison, we observed which physicochemical properties had the greatest impact on cellular toxicity and which characterization methods were useful to predict the toxicity results. Physicochemical changes in thermal-treated MWCNTs vary according to temperature.8 Raising the temperature up to L



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Arc-PMWCNTs originally showed high crystallinity and had few defects,26 acid oxidation through defective walls may occur, albeit in a limited fashion. Consequently, Arc-A-MWCNTs did not show significant differences in cell viability compared to Arc-PMWCNTs, as determined by cellular ATP amount and lactate dehydrogenase activity in 16HBE14o- cells (Figure 6B and Table 5) and HEK293 cells (Figures S4 and S7 and Tables S1 and S2, Supporting Information). Generally, among several physicochemical characteristics, length and surface area are believed to be positively correlated with toxicity.27 Metal remnants and negative surface charge are also known to enhance the toxicity of reactive oxygen species.18 However, in correlation analysis using EC50 and physicochemical factors with the data obtained from our eight types of MWCNTs, the factor Raman shift (G), which projected to the side opposite the factor EC50, had the greatest relevance (Figure 7B). In PCA using data obtained only for CVD-based-MWCNTs, the factors defect (ID/IG), which projected to the side opposite the factor EC50, and surface charge had significant associations with cellular toxicity (Figure 8B). It is interesting that the factors iron and length were negatively associated with cellular toxicity in the analysis of CVD-based MWCNTs (Figures 7B and 8B). Because this study showed cell viability based on a 48h treatment, our result cannot explain long-term tumorigenic studies of MWCNTs, and further studies using longer treatment times are needed to show whether these factors may have long-term effects on the tumorigenicity of CVDbased MWCNTs. For arc discharge-based MWCNTs, the factors surface area, EPR peak intensity, and defect (ID/IG), which projected to the side opposite the factor EC50, had the most relevance (Figure 9B). All of these factors were correlated with the surface reactivity of arc discharge-based MWCNTs. These three factors also had significant associations with the cytotoxicity of arc discharge-based MWCNTs in LX2 and HEK293 cells, as determined by ATP assays (Figures S5F and S6F, Supporting Information) and WST assays (Figures S8F and S9F, Supporting Information). From this comparative study, we identified some important determinants of the physicochemical characteristics of MWCNTs according to a synthetic method and posttreatments. In both Arc- and CVD-MWCNTs, the creation of binding sites on the tube walls by breaking the C−C bonds after thermal oxidation or introducing functional groups by acid treatment played a pivotal role in increased toxicity. This broken C−C bond feature was most clearly demonstrated by the Raman shift (G), which is related to the generation of the D′-peak at 1615 cm−1, a double resonance Raman feature.8,28 Furthermore, in cases where particles showed an EPR peak, that EPR peak played a pivotal role in mediating the cellular toxicity of MWCNTs, as shown in PCA with arc dischargebased MWCNTs. Our results indicated that increased surface reactivity, which could be identified by changes in the Raman and EPR peak intensity, may be widely used for predicting the toxicity of MWCNTs, regardless of the synthesis method and posttreatment conditions, and this factor was a more effective predictor than other factors known to be related to cellular toxicity, such as length, diameter, surface charge, and metal catalyst remnants. Further studies to identify correlations between the cellular and long-term systemic toxicities of these different MWCNTs may be required for predicting the safety of MWCNTs.

Article

ASSOCIATED CONTENT

S Supporting Information *

XRD, TGA graph, length comparison pattern, PCA analysis, and related results using EC50 values of two different cell lines (LX2 and HEK293) by ATP and WST assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-880-1276. Fax: +82-2-873-1268. E-mail: [email protected]. Funding

This study was partly supported by the Korean Ministry of Science, ICT and Future Planning (2009-0082677). J.-E.K. is the recipient of a Brain Korea 21 Program for Veterinary Science of Seoul National University. M.-H.C. was also partially supported by the Research Institute for Veterinary Science, Seoul National University. M.C. was supported by the Global Frontier Center for Multiscale Energy Systems under the Korean Ministry of Science, ICT and Future Planning (20110031561). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ATP, adenosine triphosphate; BET, Brunauer−Emmett−Teller; DLS, dynamic light scattering; CNT, carbon nanotubes; CVD, chemical vapor deposition; EC50, effective concentration 50; EF-TEM, energy-filtering transmission electron microscope; EPR, electron paramagnetic resonance; FBS, fetal bovine serum; FE-SEM, field emission scanning electron microscopy; ID/IG ratio, peak intensity of defect/graphite ratio; HMWCNT, heat-treated multiwalled carbon nanotube; HAMWCNT, heat- and acid-treated multiwalled carbon nanotube; ICP-AES, inductively coupled plasma−atomic emission spectroscopy; MWCNT, multiwalled carbon nanotube; PCA, principal component analysis; PMWCNT, pristine multiwalled carbon nanotube; TGA, thermo gravimetric analysis; TMWCNT, acid-treated multiwalled carbon nanotube; XRD, X-ray powder diffraction

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REFERENCES

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