EFFECT OF PARTICLE DIMENSION ON CARBON NANOMATERIAL TOXICITY

Christin Grabinski, University of Dayton, 300 College Park, Dayton, OH 45469-0168, USA

Saber Hussain, AFRL/HEPB, 2729 R Street, Wright Patterson AFB, OH 45433, USA Khalid Lafdi, University of Dayton, 300 College Park, Dayton, OH 45469-0168, USA

Laura Braydich-Stolle, AFRL/HEPB, 2729 R Street, Wright Patterson AFB, OH 45433, USA John Schlager, AFRL/HEPB, 2729 R Street, Wright Patterson AFB, OH 45433, USA

Abstract

With various emerging applications in a broad range of fields, the risk of exposure to nanomaterials is rapidly increasing. Several routes of exposure to nanomaterials exist; the most important being dermal contact and inhalation. In this dermal toxicity study, the cellular effects of carbon materials with diameters ranging from micro- to nano- dimension were investigated using a mouse keratinocyte cell-line (HEL-30). The carbon materials tested included carbon fibers (CF; 10 um diameter), carbon nanofibers (CNF; 100 nm diameter), multi-walled carbon nanotubes (MWCNT; 10 nm diameter), and single-walled carbon nanotubes (SWCNT; 1 nm diameter). CF and CNF did not significantly affect cell viability; however, MWCNT and SWCNT reduced cell viability in a time-dependent manner up to 24 hours, with full recovery by the 72 hour time point. After a 24 hour exposure, cells exposed to MWCNT produced up to 3-fold higher increase in reactive oxygen species than those exposed to SWCNT. The morphology of MWCNT and SWCNT agglomerates in solution, as well as impurity content may explain this phenomenon. The results of this study suggest that carbon nanomaterial toxicity is dependent on dimension, agglomerate morphology, and impurity content.

Introduction The exploitation of properties inherent to materials at the nanoscale has initiated innovative

approaches to technologies which shape our world. In particular, carbon nanomaterials are a prime material for many novel applications because of their unique properties. Carbon exists in many allotropic forms, each containing high strength covalent carbon-carbon bonds, to which especially unique mechanical, thermal, and electrical properties are attributed.

Nanocarbons are being used in structural reinforcement of aircraft, optical devices, molecular lubricants, superconductors, electrical wires, quantum computers, biological warfare protection (e.g. protective skin, biosensors), reinforcement of biomaterials, drug delivery, among many more industrial, consumer, and medical products. Specifically, single-walled carbon nanotubes (SWCNT) have been proposed as infrared photosensitizers for cancer cells and as molecular transporters for protein delivery (Shi Kam et al., 2005, Shi Kam and Dai 2005). In addition, carbon nanofibers (CNF), multi-walled carbon nanotubes (MWCNT), and SWCNT have been shown to be potentially successful in bone implants and neural tissue regeneration (Xhao et al., 2005, Hu at al., 2004, Price et al., 2004, Zhang et al., 2005).

The growing use of carbon nanomaterials in a vast array of fields has researchers questioning the safety of these materials and has created the need to establish a paradigm for accurately predicting their toxicity. In order to establish this paradigm, researchers must compile information about the specific physicochemical properties unique to nanomaterials, which drive toxicity (Oberdoster et al., 2005). Such physicochemical properties include shape, surface chemistry, and dimension, and have been found to be of great importance in nanotoxicity.

Shape was shown to play a role in toxicity in a study where MWCNT where found to be more toxic than multi-walled nano-onions to human skin fibroblasts (Ding et al., 2005). Furthermore, studies examining the effect of surface chemistry on toxicity have shown that sidewall functionalized SWCNT were less toxic to human embryonic kidney cells than those without functionalization (Sayes et al., 2005), highly derivatized water-soluble fullerene species were less toxic than those which were not deriviatized (Sayes et al., 2004) and functionalized carbon nanotubes could cross the cell membrane and accumulate in the cytoplasm without being toxic for up to 10 μM (Pantarotto et al., 2004). In addition, the effect of

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dimension on toxicity was shown in a study where CNF were less detrimental to osteoblast viability compared to larger diameter CF (Price et al., 2004) and in another study where carbon nanomaterial toxicity was found to follow a mass basis with SWCNT being the most cytotoxic, followed by MWCNT, and then C60 (Jia et al., 2005). Nanomaterial exposure is likely to occur through skin absorption because skin is the first line of defense against the outside environment, and it provides a significant surface area for potential contamination. Unmodified MWCNT have been found to cause toxicity to human epidermal keratinocytes (Monteiro-Riviera et al., 2005), and unmodified SWCNT exposed to human embryonic kidney cells were found to play a role in the mechanisms that cause G1 phase arrest and cell apoptosis (Cui et al., 2005). Contrary to these findings, carbon nanotubes synthesized by catalytic chemical vapor deposition were found to be nontoxic to human umbilical vein endothelial cells (Flahaut et al., 2006). In an in vivo test, carbon nanotubes were administered to human volunteers with allergy susceptibility via skin patch and found to be nonirritant (Huczko et al., 2001). This study focuses on the role of dimension in dermal toxicity of carbon nanomaterials and uses a mouse keratinocyte cell line, HEL-30 cells, as a model for dermal exposure.

Materials and Methods

2.1 Carbon materials The four materials used in this study, carbon fiber (CF), CNF, MWCNT, and SWCNT, were

selected to test the effect of dimension on toxicity. The CF used was an IM7 fiber, which is polyacrylonitrile (PAN) based and about 10 µm in diameter. The CNF were produced through the pyrolysis of carbon-containing gases on an iron catalyst at 500-1500°C and were about 100 nm in diameter. As shown in figure 1, in some specific processing conditions the CNF consist of Dixie-cups (Baker and Harris, 1978, Inagaki, 2000). MWCNT are made up of multiple concentric graphite sheet cylinders and were about 10 nm in diameter, whereas SWCNT consist of a single cylinder of a graphite sheet and were about 1 nm in diameter. Materials were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to determine their dimension and structure (figure 1). The purity of each sample was determined using x-ray fluorescence (XRF) and an inductively coupled plasma-optical emission spectrometer (ICP-OES).

A B CA B CA B CA B C

Figure 1. Structure of CNF, MWCNT, and SWCNT imaged using electron microscopy.

A. CNF; B. MWCNT; C. SWCNT

2.2 Preparation and characterization of carbon materials in solution Continuous CF were cut into small lengths, and ground into shorter lengths using a mortar and pestle. All dry materials were weighed and suspended in culture media to stock concentrations of 0.1 mg/mL and kept for two weeks at 4°C. Prior to dilution, the stock solutions were sonicated using a probe sonicator at a power output of 9 watts until maximum dispersion was achieved with short breaks to avoid excessive heating. Diluted dosing solutions of established concentration ranging from 5 to 50 µg/ml were prepared in growth media on the day of exposure. Just prior to exposure, dosing solutions were vortexed, then pipette into 24-well plates or 2-chambered slides. 2.3 Cell Culture and Morphology

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Mouse keratinocyte cells, obtained from the Naval Health Research Center Detachment Environmental Health Effects Lab at Wright Patterson AFB, were cultured in flasks with a 1:1 mixture of Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DMEM/Ham's F-12, Sigma) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated in 5% CO2 at 37°C with 100% humidity. In preparation for in vitro experiments, keratinocytes were seeded in culture medium at 150,000 cells/mL in 24-well plates for assays or 75,000 cells/mL on 2-chambered slides for microscope observation.

Morphology was examined for each dose and time point using an ultra-resolution imaging (URI) system previously described in detail by Skebo et al. (2007). Cellular interaction of nanotubes was also examined using dual mode fluorescence (DMF), which is used in conjunction with the URI system for simultaneous imaging of fluorescent and non-fluorescent structures (Aetos Technologies, Inc.). For DMF microscopy, cells were stained with Alexa-Fluor 555 Phalloidin (Invitrogen/Molecular Probes) to tag the cellular actin and SYTOX Green Nuclear counterstain (Invitrogen) to label the nucleus. 2.4 Cell Viability Assay Cells were seeded, and the following day dosed with four concentrations of nanomaterials (5, 10, 25, and 50 μg/mL). After dosing, cell viability was assessed at 12, 24, 48, and 72 hours using the MTT assay, which was modified from Carmichael et al. (1987). In this assay the tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] was added to live cells after exposure, and the cells were incubated for 45 minutes while the mitochondria of healthy cells cleaved the ring of the MTT, reducing it to a blue formazan salt via succinate dehydrogenase. In the modified assay, the final test solution was transferred from well plates to individual eppendorf tubes and centrifuged at 10,000 rpm to pellet the carbon materials and reduce interference with spectrophotometer readings. Cadmium oxide (2.5 µg/mL), which exhibits an established toxic effect on cells (Hussain et al., 2005), was used as a positive control. 2.5 Reactive Oxygen Species The reactive oxygen species (ROS) assay was used to characterize what effects carbon materials might have on an irritation response to the cells marked by the production of oxygen radicals. Prior to dosing cells with nanomaterials, the fluorescent probe 2’,7’-dichlorohydrofluorescein diacetate (DCHF-DA, Sigma) was applied under a light controlled environment as described by Wang and Joseph (1999). Immediately following incubation with DCHF-DA, cells were washed and dosed with four concentrations of nanomaterials (5, 10, 25, and 50 μg/mL). The fluorescent intensity from each well was measured with a 485 nm excitation filter and a 530 nm emission filter after 24 hours on a SpectraMAX Gemini Plus microplate reader (Molecular Device). The positive control, hydrogen peroxide (30% H2O2, Fisher Scientific), was used to assess the reactivity of the probe. 2.6 Statistical Analysis

Biochemical assays (MTT and ROS) were performed in triplicate and expressed as an average ± standard deviation. Data were evaluated using one way analysis of variance (ANOVA) and the Tukey-Kramer multiple comparisons test with statistical significance assessed at p < 0.05 using PHStat2 software.

Results 3.1 Material characterization The structure and diameter of each material was verified using SEM and TEM (figure 1). The average diameter of CF, CNF, MWCNT, and SWCNT were 10 µm, 100 nm, 10 nm, and 1 nm, respectively. The impurity content was limited to iron, found using XRF, and the percent in each carbon sample was determined as follows: CF-0%, CNF-0.25%, MWCNT-3.75%, and SWCNT-1.13%. The carbon nanotubes formed agglomerates in cell growth media due to their hydrophobic nature. In images with cells, MWCNT seem to form clusters in cell growth media, whereas SWCNT form long bundles (figure 3). 3.2 Cell Morphology

Confluency and overall morphology of HEL-30 cells were examined using the URI system (figure 2). The tendency for growth of cells along CF was shown at this magnification. The cells exposed to CNF

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did not have any morphological changes when compared to the control. Cells exposed to CNTs became round, but remained attached to the slide. In an image of one isolated cell, SWCNT were bundled along the cell membrane (figure 2). Interaction of carbon nanotubes with the cell membrane is also shown in figure 3.

Figure 2. Cell morphology observed via URI system.

Each measure bar represents 20 μm. A. Control; B. CF, 10 µg/mL; C. CNF, 10 µg/mL; D. SWCNT, 10 µg/mL.

20 µm 20 µm 20 µm20 µm 20 µm 20 µm

Figure 3. Cell and carbon nanotube agglomerate morphology observed via DMF microscopy. Each measure bar represents 20 μm. A. Control; B. MWCNT, 10 µg/mL; C. SWCNT, 10 µg/mL.

3.3 Cell viability MTT data were used to assess cell viability after exposure to each material at four concentrations (5, 10, 25, and 50 µg/mL) after four time points (12, 24, 48, and 72 hours). After 24 hours, CF and CNF did not cause significant toxicity, but cells exposed to MWCNT and SWCNT were significantly toxic compared to control (figure 4A). After 72 hours, all four materials presented no toxicity and no dose-dependence (figure 4B). This data was verified using a control experiment to test for adsorption of the formazan crystal produced in the MTT assay onto the nanotube surface, which has been shown to cause false positives for cytotoxicity (Worle-Knirsch et al., 2006). However, the control experiment provided little evidence of false reduction of cell viability, with significant reduction of cell viability to 80% of

A B

C D

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control occurring only for the highest concentration of MWCNT (data not shown).

Figure 4. Cell viability at 24 and 72 hour time points determined via MTT Assay

Cell Viability after exposure to CF, CNF, MWCNT, and SWCNT at four concentrations (5, 10, 25, and 50 µg/mL) after 24 hours (Figure 4A) and 72 hours (Figure 4B).

3.4 Reactive Oxygen Species

ROS data were used to show reactive oxygen species generation after exposure to each material at four concentrations (5, 10, and 25 µg/mL) after 24 hours. CF and CNF did not increase ROS significantly from control. MWCNT generated 3-7 fold ROS, while SWCNT in increased generation of ROS about 1-4 fold in a dose-dependent manner. Both MWCNT and SWCNT produced higher-fold ROS than the positive control, hydrogen peroxide (figure 5).

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0 10 20 30Concentration (μg/ml)

Fold

Incr

ease

in R

elat

ive

Fluo

resc

ence

Uni

ts

CF NF MWCNT SWCNT H2O2

*

*

*

* *

Figure 5. Reactive oxygen species generation after 24h exposure to CF, CNF, MWCNT, and SWCNT

determined via ROS Assay.

Discussion

When evaluating the role of particle dimension in this study, we found that there is an observable change in morphology in cells near the surface of CF, but this does not seem to affect viability according to MTT data. CNF demonstrated very minor toxicity at any of the time points, and the morphology is unchanged from control. Neither CF or CNF caused significant oxidative stress. Therefore, it is determined that the two larger dimension materials do not cause toxicity to HEL-30 cells.

After 24 hours, MWCNT and SWCNT showed decreased cellular viability when compared to CF and CNF. The mechanism involved in the toxicity trend of recovery of viability after 72 hours of exposure to MWCNT and SWCNT is not completely understood at this time. Interaction of carbon nanotubes with the cellular membrane (figures 2D, 3B and 3C) may initiate an irritation response from the cells, leading to the release of protective proteins from cellular enzymes, causing the membrane to become impermeable to nanotubes (Beyersmann and Hechtenberg, 1997, Montiero-Riviere and Inman, 2005). Also, proteins that have been affected may be repaired after initial injury (Hall, 2002, Ernest et al., 2005). Furthermore, it is

A. B. Concentration (µg/mL) Concentration (µg/mL)

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hypothesized that resistant cells release growth factors to help other cells proliferate or use energy from the nanoparticles to proliferate (Hashimoto, 2000). If the nanotubes are taken up into the cells, it is possible that enzymes signal cells to exclude particles from major organelles and trap materials within vacuoles (Montiero-Riviere and Inman, 2005, Hall, 2002, Hashimoto, 2000), allowing cells to recover their ability to proliferate after the nanotubes have been removed from the medium. Cellular uptake of carbon nanomaterials has been previously demonstrated. In one study, MWCNT accumulated within the free cytoplasm and cytoplasmic vacuoles of human epidermal keratinocytes with time and dose (Monteiro-Riviere et al., 2005), and in a separate study, CNF (diameter=60-100 nm) were found to be taken up into cells and enclosed in vacuoles (Price et al., 2004). Currently studies to determine the mechanism of the trend in cell viability are in progress.

In a previous study, ROS generation was verified as a valid test to compare nanomaterial toxicity (Xia et al., 2006). Nel et al. (2006) found that when they treated keratinocytes and bronchial epithelial cells with high dose of SWCNTs, this resulted in ROS generation and oxidative stress. Similarly, here we show that HEL-30 cells treated with MWCNT and SWCNT exhibit ROS generation. Furthermore, the ROS generation increased as the concentration of MWCNT and SWCNT increased implying this was a dose-dependent effect. However, results from the cell viability assay show no dose-dependence and similar toxicity between MWCNT and SWCNT.

This phenomenon can be explained by sample composition and agglomerate morphology. The increase in ROS after exposure to MWCNT versus SWCNT corresponds with iron content determined using ICP-OES, where MWCNT contained about 2.5% higher iron impurities than SWCNT. This suggests iron impurities in the carbon nanotube samples may be directly related to the difference in oxidative stress. In addition to iron content, the morphology of the nanotube agglomerates in solution can be investigated for further explanation. As shown in figure 3, the MWCNT used in this study formed clusters, whereas the SWCNT formed long bundles or ropes, and these different morphologies likely affect the way in which the two materials interact with the cell. For example, large MWCNT agglomerates could be blocking important receptors on the cell membrane in a more significant manner than thin SWCNT bundles, which could contribute to differences in ROS.

Conclusion

Since carbon materials below 100 nm show interesting trends in toxicity, further studies are necessary before these materials are used in applications such as drug delivery and biosensors. Carbon nanotubes produced under different processing conditions can vary in length and other properties, which could affect their agglomerate morphology and impurity content. Therefore, future studies should evaluate these effects on toxicity. Furthermore, when decreasing the dimension of a material, the surface area per volume and surface energy increase. Therefore, it must be determined whether MWCNT and SWCNT exhibit toxicity due to the increase in one of these properties, and future studies will focus on the effects of surface energy.

Acknowledgements

We would like to thank Col. James Riddle at the AFRL for his support of this project, as well as ChemSys Incorporated for help with material characterization. We would also like to thank the Dayton Area Graduate Studies Institute and the Consortium Research Fellows Program for supporting Grabinski with a fellowship and the National Research Council for supporting Braydich-Stolle with a post-doctoral fellowship.

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