NANO LETTERS
Cellular Toxicity of Carbon-Based Nanomaterials
2006 Vol. 6, No. 6 1121-1125
Arnaud Magrez,*,† Sandor Kasas,‡,§ Vale´rie Salicio,| Nathalie Pasquier,⊥ Jin Won Seo,† Marco Celio,| Stefan Catsicas,‡ Beat Schwaller,| and La´szlo´ Forro´† Institut de Physique de la Matie` re Complexe (IPMC), Ecole Polytechnique Fe´ de´ rale de Lausanne, 1015 Lausanne, Switzerland, Laboratoire de Neurobiologie Cellulaire, Faculte´ des Sciences de la Vie, Ecole Polytechnique Fe´ de´ rale de Lausanne, 1015 Lausanne, Switzerland, Institut de Biologie Cellulaire et de Morphologie, UniVersite´ de Lausanne, rue du Bugnon 9, 1005 Lausanne, Switzerland, UniVersity of Fribourg, DiVision of Histology, Department of Medicine, 14, Chemin du Muse´ e, CH-1705 Fribourg, Switzerland, and Laboratoire Cytopath, AVenue Cardinal-Mermillod 36, 1227 Carouge, Switzerland Received January 24, 2006; Revised Manuscript Received April 10, 2006
ABSTRACT The cellular toxicity of carbon-based nanomaterials was studied as a function of their aspect ratio and surface chemistry. These structures were multiwalled carbon nanotubes, carbon nanofibers, and carbon nanoparticles. Their toxicity was tested in vitro on lung tumor cells. Our work clearly indicated that these materials are toxic while the hazardous effect is size-dependent. Moreover, cytotoxicity is enhanced when the surface of the particles is functionalized after an acid treatment.
The discovery of numerous nanomaterials has added a new dimension to the rapid development of nanotechnology. Consequently, the professional and public exposure to nanomaterials is supposed to increase dramatically in the coming years. Especially, carbon-based nanomaterials (CBNs) are currently considered to be one of the key elements in nanotechnology. Their potential applications range from biomedicine through nanoelectronics to mechanical engineering. Thus, it is primordial to know the health hazards related to their exposure. Here, we performed studies on cultured cells exposed to three different types of CBNs, carbon nanotubes, carbon fibers, and carbon nanoparticles. Severe inhibition of cell proliferation and cell death have been observed, which become more pronounced as the aspect ratio of CBNs decreases and with the presence of chemically active functional groups on the graphene surfaces. These results indicate that more attention has to be paid to the health concerns of CBNs before pushing their application forward. * To whom correspondence should be addressed. E-mail:
[email protected]. † Institut de Physique de la Matie ` re Complexe (IPMC), Ecole Polytechnique Fe´de´rale de Lausanne. ‡ Laboratoire de Neurobiologie Cellulaire, Faculte ´ des Sciences de la Vie, Ecole Polytechnique Fe´de´rale de Lausanne. § Institut de Biologie Cellulaire et de Morphologie, Universite ´ de Lausanne. | University of Fribourg, Division of Histology, Department of Medicine. ⊥ Laboratoire Cytopath. 10.1021/nl060162e CCC: $33.50 Published on Web 05/20/2006
© 2006 American Chemical Society
Carbon-based nanomaterials are currently one of the most attractive nanomaterials with their different forms, such as fullerenes, single- and multiple-walled carbon nanotubes, carbon nanoparticles, nanofibers, and so forth. Although some of them are already in mass production, for carbon nanotubes only in the last two years have novel methods been able to considerably improve their synthesis and yield. Recent studies demonstrated that CBNs also aggregate in combustion streams of fuel gas and air commonly used in our everyday life, indicating that we are already strongly exposed to CBNs in the atmospheric environment both inand outdoor.1 Significant progress has been made in incorporating CBNs in potential applications. In particular, biological applications that employ CBNs for DNA, proteins, and drug delivery2,3 have attracted much attention. Unfortunately, the information concerning the potential hazards related to CBN exposure is rare and still under debate.4-10 In particular, the toxicity of water-soluble CNTs has been discussed.11,12 The scientific community is mostly concerned about the toxicity of carbon nanotubes because of their structural resemblance to asbestos. Inhalation of asbestos fibers is known to induce asbestosis (a progressive fibrotic disease of the lung), lung cancer, and malignant mesothelioma of the pleura.13 The role of asbestos in lung cancer is still under debate and, unfortunately, experiments performed on rats or guinea pigs14 are not conclusive because the
Figure 1. SEM images of (a) MWCNTs, (b) CNFs, and (c) carbon black. The aspect ratio of these nanoparticles is about 80-90, 30-40, and 1, respectively. The scale bars correspond to 2 µm.
development of the asbestos-induced disease takes longer than the lifetime of these test animals. In the case of asbestos, where a benign silicate mineral becomes carcinogenic in its fibrous form, the size, aspect ratio, and surface charges have proven to have a strong influence on their toxicity.15 How these parameters affect the biotoxicity of CBNs is totally unknown, although it is generally expected that they play a significant role. To explore the shape influence of CBNs on cell toxicity, we exposed different cell types to CBNs in vitro with different aspect ratios, namely, multiwalled carbon nanotubes (MWCNTs, produced by chemical vapor deposition16), with an average diameter of 20 nm and aspect ratios ranging from 80 to 90 (Figure 1a); carbon nanofibers, (CNFs, obtained from Pyrograf Products, Inc.) with a mean diameter of 150 nm and aspect ratios of 30-40 (Figure 1b); and finally flakelike-shaped carbon nanoparticles (carbon black, obtained after grinding graphite, used as electrodes for the arc synthesis of carbon nanotubes) with aspect ratios of about 1 and size distributions ranging in the submicrometer range (Figure 1c). All CBNs were suspended in a strongly diluted gelatin solution to avoid aggregation. The effect on the cell proliferation and cytotoxicity of the CBNs was evaluated by the widely established MTT assay17 performed with three different human lung-tumor cell lines, H596, H446, and Calu-1. The assay is based on the accumulation of dark-blue formazan crystals inside living cells (but not in dead cells) after their exposure to MTT (for details, see the Supporting Information). For each cell type, a linear relationship between the number of living cells and the optical density was established, and this allowed us an accurate quantification of cell numbers. At first we approved the suitability of different lung cancer cell lines for the cytotoxicity assay by treating them with MWCNTs at concentrations ranging from 0.002 to 0.2 µg/mL for 4 days (Figure 2). The number of viable cells showed a CBN concentration-dependent decrease in all cell lines. Nevertheless, because H596 cells showed the highest sensitivity as well as the best reproducibility, H596 cells were used for the principal experiments. We also validated that the cell growth in a standard medium and in a medium containing the same gelatin concentration as the CBN-treated cells was not significantly different, indicating that addition of gelatin at this concentration does not affect the proliferation. An average growth curve of H596 cells in a standard medium is shown in Figure 3a (control). Although growth curves of cells grown in a medium with the same gelatin concentration but without exposure to CBNs were not different from the control conditions, the number of viable 1122
Figure 2. Representative growth curve for three different human lung tumor cells grown in normal medium (control), medium containing gelatin, or medium containing 0.02 and 0.2 µg/mL of MWCNTs (see the Supporting Information for details). In control cells, the increase in optical density, which is proportional to the number of viable cells, is due to cell division. In MWCNT-treated samples, the number of viable cells is lower at all time points.
cells in all CBN-treated samples was decreased. The toxic action on living cells already appeared within 24 h after Nano Lett., Vol. 6, No. 6, 2006
Figure 3. (a) Representative growth curve for H596 cells grown in normal medium (control), medium containing gelatin (see the Supporting Information for details), or 0.02 µg/mL of CBNs. MWCNTs, CNFs, and carbon black are dispersed in the same gelatin-containing medium. (b) Dose-dependent toxicity of H596 cells exposed to CBNs for 2 days. In all cases, the toxicity order was carbon black > CNFs > MWCNTs.
Figure 4. Cytopathological analyses of H596 cells (a) A typical control image of H596 cells is depicted; cells were stained with hematoxylineeosine; the nuclei appear purple and patchy; the cytoplasm is weekly stained (pink). Clusters of cells are characterized by close cell/cell contacts, and individual cells are polygonal-shaped. (b) H596 cells after 1 day treatment with 0.02 µg/mL of MWCNT. Cells have lost their mutual attachments, retracted their cytoplasm (arrows) such that the pink color appears stronger, and the nuclei are smaller and more condensed (picnotic) also evidenced by the stronger purple staining.
exposure, and differences between control and CBN-treated become even more pronounced after culturing the cells for 5 days. Analysis of the dose-dependent toxicity after 2 days of CBN exposure (Figure 3b) revealed that the number of viable cells decreases as a function of the exposed CBN dose for all CBNs tested. Moreover, a clear CBN morphological dependence on the cell toxicity was observed: Contrary to our expectations, carbon black particles exhibited the highest cytotoxicity evidenced by the lowest number of viable cells at all concentrations and time points tested (Figure 3 and also by the large amount of cell debris in the culture medium (not shown)). In particular, at low CBNs concentrations (0.002 and 0.02 µg/mL) the number of viable cells decreased in the following sequence: carbon black > CNFs > carbon nanotubes. Thus, filaments were less toxic than particles in our experiments. At a high CBNs concentration of 0.2 µg/ mL, differences diminished especially for CNFs and carbon black. However, MWCNTs always appeared to be less toxic Nano Lett., Vol. 6, No. 6, 2006
than the other two materials. Thus, surprisingly, filaments were less toxic than particles in our experiments. Light microscopic examination of the cells exposed to the different CBNs revealed several morphological alterations compared to the control cells grown in standard medium (Figure 4). Already after 1 day of exposure, a fraction of cells were only loosely attached to the culture dishes or even completely detached. In the remaining cells cytoplasm retraction with eosinophilia and shrunken (picnotic) nuclei were observed, which are typical for irreversible cell injuries and cell death. It has to be pointed out that no specific differences could be observed at the light microscopic level with respect to the cell morphology between cells exposed to the different CBNs. Thus, differences in the number of viable cells determined from the MTT assays do merely reflect the sensitivity of cells to different CBNs, while the morphological alterations obtained from light microscopy indicate a common final cell death pathway for all CBNs. 1123
Figure 5. Effect of filament decoration on cell toxicity in H596 cells. The growth curves obtained from chemically decorated MWCNTs and CNFs are denoted De-MWCNTs and De-CNFs, respectively. The filament concentration to which all samples were exposed to was 0.02 µg/mL. In both cases, the number of viable cells is lower in the decorated samples, indicative of increased toxicity.
A possible explanation for the observed aspect-ratio dependence of the CBN toxicity is the presence of dangling bonds, which are highly reactive sites. In general, they are present in carbon black with a high density, whereas in carbon nanotubes they preferentially occur at lattice defects or end caps. To explore the effect of surface chemical properties on the toxicity, we performed another set of experiments in which we modified the surface chemistry of the filaments. The surface of MWCNTs and CNFs has been decorated according to the method reported by H. Hiura et al.18 that involved a chemical modification of the outer layer of the carbon nanotube after acid treatment. This procedure results in adding carbonyl (CdO), carboxyl (COOH), and/or hydroxyl (OH) groups onto the nanotube and nanofiber surfaces. H596 cells were grown in a gelatin-containing medium containing 0.02 µg/mL of dispersed CBNs. The MTT assay was carried out between one and 4 days after exposure to CBNs. Cells grown in a plain gelatin-containing medium served as reference. The chemical decoration effect on cytotoxicity is displayed in Figure 5. When the number of viable cells is compared after the treatment with CBNs, it becomes evident that the toxicity increases with the chemical surface treatment. This is significant in the case of MWCNTs and moderate for CNFs. The latter observation might be somewhat obscured by the fact that a relatively high toxicity already occurred with unmodified CNFs. Nevertheless, these results clearly demonstrate that grafting additional putatively “toxic” chemical groups on the surface of MWCNTs reduces the number of viable cells significantly. In conclusion, our experiments demonstrate that CBNs generally lead to proliferation inhibition and cell death. Although carbon nanotubes are less toxic than carbon fibers 1124
and nanoparticles, the toxicity of carbon nanotubes increases significantly when carbonyl (CdO), carboxyl (COOH), and/ or hydroxyl (OH) groups are present on their surface. The exact mechanisms that lead to cell death are still unclear, but CBNs can induce cell death either after contact with cell membranes or after their internalization. One has to consider that the toxicity of the investigated CBNs could be specific to the processing methods applied. Moreover, it has to be emphasized that this study does not address the carcinogenicity of CBNs, that is, the potential to transform a normal cell to a tumor cell, which requires a detailed follow-up investigation on that specific topic. In the last five years, the question about potential toxicity of carbon nanotubes was raised steadily. Although our study shows a lower toxicity of carbon nanotubes compared to carbon black or filaments, precautions in their manipulation need to be taken. In particular, in applications where carbon nanotubes are injected into human body for drug delivery,19 for example, as contrast agent carrying entities for MRI,20 the toxicity issue must be carefully addressed. Acknowledgment. The work in Lausanne was supported by the Swiss National Science Foundation and its NCCR “Nanoscale Science”. The research of B.S. is supported by the Swiss National Science Foundation (grant no. 3100A0100400/1). Supporting Information Available: Experimental methods as well as additional time and dose dependence plots of CBNs toxicity on H596 cells. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Murr, L. E.; Bang, J. J.; Esquivel, E. V.; Guerrero, P. A.; Lopez, A. J. Nanopart. Res. 2004, 6, 241-251. (2) Wong Shi Kam, N.; O’Connell, M.; Wisdom, J. A.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11600. (3) Wu, W.; Wieckoski, S.; Pastorin, G.; Benincasa, M.; Klumpp, C.; Briand, J. P.; Gennaro, R.; Prato, M.; Bianco, A. Angew. Chem., Int. Ed. 2004, 43, 5242-5246. (4) Sayes, C. M.; Fortner, J. D.; Guo, W.; Lyon, D.; Boyd, A. M.; Ausman, K. D.; Tao, Y. J.; Sitharaman, B.; Wilson, L. J.; Hughes, J. B.; West, J. L.; Colvin, V. L. Nano Lett. 2004, 4, 1881-1887. (5) Oberdorster, E. EnViron. Health Perspect. 2004, 112, 1058-1062. (6) Shvedova, A. A.; Castranova, V.; Kisin, E. R.; Schwegler-Berry, D.; Murray, A. R.; Gandelsman, V. Z.; Maynard, A.; Baron, P. J. Toxicol. EnViron. Health 2003, 66, 1909-1926. (7) Lam, C. W.; James, J. T.; McCluskey, R.; Hunter, R. L. Toxicol. Sci. 2004, 77, 126-134. (8) Warheit, D. B.; Laurence, B. R.; Reed, K. L.; Roach, D. H.; Reynolds, G. A. M.; Webb, T. R. Toxicol. Sci. 2004, 77, 117-125. (9) Ding, L.; Stilwell, J.; Zhang, T.; Elboudwarej, O.; Jiang, H.; Selegue, J. P.; Cooke, P. A.; Gray, J. W.; Chen, F. F. Nano Lett. 2005, 5, 2448-2464. (10) Hurt, R. H.; Monthioux, M.; Kane, A. Carbon 2006, 44, 10271120. (11) Singh, R.; Pantarotto, D.; Lacerda, L.; Pastorin, G.; Klumpp, C.; Prato, M.; Bianco, A.; Kostarelos, K. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3357-3362. (12) Chen, X.; Chong Tam, U.; Cziapinski, J. L.; Lee, G. S.; Rabuka, D.; Zettl, A.; Bertozzi, C. R. J. Am. Chem. Soc., in press, 2006. (13) LaDou, J. EnViron. Health Perspect. 2004, 112, 285-290. (14) Huczko, A.; Lange, H.; Calko, E.; Grubek-Jaworska, H.; Droszcz, P. Fullerenes, Nanotubes, Carbon Nanostruct. 2001, 9, 253. (15) Rogers, R. A.; Antonini, J. M.; Brismar, H.; Lai, J.; Hesterberg, T. W.; Oldmixon, E. H.; Thevenaz, P.; Brain, J. D. EnViron. Health Perspect. 1999, 107, 367-375.
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