Elsevier

Toxicology in Vitro

Volume 20, Issue 7, October 2006, Pages 1202-1212
Toxicology in Vitro

Cytotoxicity of single-wall carbon nanotubes on human fibroblasts

https://doi.org/10.1016/j.tiv.2006.03.008 Get rights and content

Abstract

We present a toxicological assessment of five carbon nanomaterials on human fibroblast cells in vitro. We correlate the physico-chemical characteristics of these nanomaterials to their toxic effect per se, i.e. excluding catalytic transition metals. Cell survival and attachment assays were evaluated with different concentrations of refined: (i) single-wall carbon nanotubes (SWCNTs), (ii) active carbon, (iii) carbon black, (iv) multi-wall carbon nanotubes, and (v) carbon graphite. The refined nanomaterial that introduced the strongest toxic effect was subsequently compared to its unrefined version. We therefore covered a wide range of variables, such as: physical dimensions, surface areas, dosages, aspect ratios and surface chemistry. Our results are twofold. Firstly, we found that surface area is the variable that best predicts the potential toxicity of these refined carbon nanomaterials, in which SWCNTs induced the strongest cellular apoptosis/necrosis. Secondly, we found that refined SWCNTs are more toxic than its unrefined counterpart. For comparable small surface areas, dispersed carbon nanomaterials due to a change in surface chemistry, are seen to pose morphological changes and cell detachment, and thereupon apoptosis/necrosis. Finally, we propose a mechanism of action that elucidates the higher toxicity of dispersed, hydrophobic nanomaterials of small surface area.

Introduction

There is a clear gap in our current knowledge about the potential health effects of carbon nanotubes. The carbon nanotubes (CNTs) are seen as having a huge potential in many areas of research and application. These nanomaterials are therefore attracting investments from governments and industry in many parts of the world. The industrialization of engineered nanomaterials is advancing at a fast pace, but the risk assessment lags far behind this development. It is recently recognized that the use of nanotechnologies may raise new challenges in the safety, regulatory and ethical domains that will require scientific debate (RS/RAEng, 2004, DEFRA, 2005). In fact, the limited information available in the peer-reviewed literature suggests that CNTs possess a potential toxicity.

There are only a handful of papers about the health effects of CNTs, basically organized in two fields: exposure toxicity of CNTs to the respiratory tract (Huczko et al., 2001, Lam et al., 2004, Warheit et al., 2004, Jia et al., 2005) and dermal/epidermal toxicity (Huczko and Lange, 2001, Shvedova et al., 2003, Pantarotto et al., 2004, Ding et al., 2005, Monteiro-Riviere et al., 2005). Actually, a positive association between exposure to single-wall carbon nanotubes (SWCNTs) and pulmonary and dermal toxicity was observed from the first studies on animals. Therefore, regulatory agencies started to pay attention to the risk assessment of these novel nanomaterials. Recently, the Royal Society and the Royal Academy of Engineering, commissioned by the UK Government, issued a report (RS/RAEng, 2004) on nanotechnologies in which is admitted the many uncertainties around health, safety and environmental impact of engineered nanomaterials. A follow up report (DEFRA, 2005) identified gaps in knowledge needed to measure and characterize their risk. We are quite certain that the data currently available is insufficient for any conclusive risk assessment of nanomaterials, and further research has to be done on their toxicity in vivo, as well as their persistence and bioaccumulation (RS/RAEng, 2004, DEFRA, 2005, Stoeger et al., 2006).

The assessment is complicated because little is known about how do factors like surface chemistry, surface area, aggregation and catalytic metals in CNTs affect the cell cycle. It is assumed that these factors have a different impact on different cell types. There is, however, no systematic study that correlates toxicity to physico-chemical properties of carbon nanomaterials. The present systematic study in vitro is a step forward to fill this gap. It is known (Lam et al., 2004, Warheit et al., 2004) about effects, ranging from transient adverse reactions to granulomas formation in animal lungs, depending upon the dosage of SWCNTs. One of those works (Lam et al., 2004) studied SWCNTs with and without catalytic transition materials, e.g. with different percentage of nickel, yttrium and iron. In another study (Ding et al., 2005), the toxicological effect of two carbon nanomaterials, with different aspect ratio, were compared. It was found that multi-wall carbon nanotubes (MWCNTs) are about ten times more toxic to fibroblast cells than multi-wall carbon onions. Two studies that used SWCNTs with these catalytic metals are found in Shvedova et al., 2003, Ding et al., 2005, in which a dose-depended toxicity of SWCNTs was used, in concentrations ranging from 0.06 μg/ml to 0.6 mg/ml. To make our results comparable to the existing literature we adopted this dose-dependent approach. It is interesting to remark that (Shvedova et al., 2003) showed that unrefined SWCNTs generate reactive oxygen species (ROS) and oxidative stress. It is thus hard to draw conclusions of the potential toxicity in vivo, generalize their results to other cell types in vitro or understand the role of catalytic transition metals.

In this paper we correlate the toxic effect of five engineered carbon nanomaterials to their physico-chemical characteristics. We show how surface area and surface chemistry impact the cell survival of human fibroblast in vitro. The refined nanomaterial showing the strongest toxic effect is taken a step further: we compare it to its unrefined version as to test whether or not catalytic transition metals enhance or decrease the toxic effect of the given nanomaterial. It is shown that catalytic transition metals, used in the production of carbon nanomaterials, influence the cell cycle of human fibroblast cells in vitro. The nanomaterials used in this research are: (i) SWCNT, (ii) active carbon (AC), (iii) carbon black (CB), (iv) MWCNT, and (v) carbon graphite (CG). A dosage-dependant analysis on human fibroblast cells was employed to be comparable to the existing literature. Fibroblast cells are important for in vitro models because one way in which these engineered nanomaterials can enter the human vascular system is through open wounds. Moreover, dermis fibroblasts cells play an important role in the cell renewing system and in maintaining the skin integrity. The in vitro model helps to assess potential toxic effects of dermal exposure to carbon nanomaterials. Since the cell cycle of fibroblast is about 24 h, we selected an exposure time of 1/2 to 2 times its cell cycle. We observed detaching and morphological changes in fibroblast cells, and analyzed these phenomena by monitoring the expression levels of extracellular matrix and adhesion-related proteins.

Section snippets

Nanomaterials

Fig. 1 shows high-resolution transmission electron microscopy (HR-TEM) images of the five carbon nanomaterials: CG, MWCNT, CB, AC and SWCNT. Table 1 shows their physical dimensions, providers and surface area. The surface area was calculated as either cylinders (SWCNT and MWCNT) or spheres (CG, CB and AC). Each nanomaterial was refluxed at 120 °C in 4 M hydrochloric acid (HCl) for 19 h. The catalytic metals, i.e. iron, was removed from a group of SWCNTs by HCl. The nanomaterials were washed with

Purification of nanomaterials

Before the purification process, the spectrum of SWCNTs shows two peaks: C 1s and Fe 2p 3/2, in Fig. 2A. The peak C 1s means carbon at 282 eV of binding energy, and Fe 2p 3/2 represents iron at 740 eV of binding energy. This result means that iron is contained in unrefined SWCNTs. On the other hand, only one peak, C 1s in Fig. 2B, appears in the binding energy intensity of SWCNTs after the purification. There is no iron in the refined SWCNTs. No other catalytic metals are seen in the spectra, e.g.

Discussion

There are two main results to be discussed. Firstly, SWCNT induces the strongest adverse effect, apoptosis and necrosis, amid five refined carbon nanomaterials (Fig. 3, Fig. 4). Secondly, refined SWCNTs are also more toxic that unrefined SWCNTs. We organize the results to explain why surface area and surface chemistry are the main variables involved in this toxic effect. While doing so, we show how our hypotheses can support a substantial body of findings on the subject.

Our results are in stark

Acknowledgements

We wish to thank Ms. M. Kelsch and Dr. F. Phillipp, at the Max Planck Institute in Stuttgart, for their help with HRTEM measurements; Mr. J. Berger, at the Max Planck Institute in Tübingen, for his technical assistance with SEM; as well as Prof. H. Gao and Mr. S. Coyer for their useful comments on previous versions of this manuscript.

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