Elsevier

Carbon

Volume 45, Issue 9, August 2007, Pages 1743-1756
Carbon

An in vitro study of the potential of carbon nanotubes and nanofibres to induce inflammatory mediators and frustrated phagocytosis

https://doi.org/10.1016/j.carbon.2007.05.011Get rights and content

Abstract

There has been little information to date based on the potential health effects and hazards associated with the inhalation of carbon nanofibrous materials by workers despite their growing use in industry. This study examines the in vitro effects of a range of nanofibres and nanotubes for their ability to stimulate the release of the pro-inflammatory cytokine TNF-α and reactive oxygen species (ROS) from monocytic cells. Also assessed were the toxic effect of the nanomaterials on the cells and the phagocytic ability of the cells after exposure. Our studies showed that the cellular response varied with fibre morphology and state of aggregation; long, straight, well-dispersed nanofilaments produced significantly more TNF-α and ROS in monocytic cells compared with highly curved and entangled materials. We also demonstrated that monocytic cell phagocytic ability was reduced after exposure to all of the nanotubes used in this study. Microscopic examination of the cells after treatment with the nanotubes showed ‘frustrated phagocytosis’, The frustrated phagocytosis suggests that clearance of nanotubes from the lungs by macrophages may be impaired. There was no evidence of a toxic effect at any of the doses or time points used. These considerations may have important consequences for workers exposed to these nanomaterials.

Introduction

Nanoparticles are defined as particles with at least one dimension less than 100 nm and include quantum dots, nanotubes and exfoliated clays (confinement in 3, 2 and 1 dimensions, respectively). A number of reports [1] have suggested that risks associated with nanoparticles exposure require investigation due to evidence that these particles can be more inflammogenic and toxic than larger particles comprising of the same material. This study focuses on carbon nanofibres, which typically possess diameters below 100 nm and lengths of the order of tens of microns. There is broad range of different carbon nanofibre types, depending on the size and orientation of the graphene layers within their structure. It is generally accepted that the major types of nanofibre are nanotubes consisting of hollow tubes of graphene sheets with the graphene planes parallel to the long axis of the fibre, herringbone fibres which consist of stacked graphene cones with the planes typically ∼30–60° to the long fibre axis and platelet fibres with the graphene planes perpendicular to the long axis of the fibre. Of these, nanotubes have received the majority of the scientific interest due to their intriguing combination of electrical, thermal and mechanical properties. However all types of nanofibres are used in scientific studies and commercially, with applications including fillers in composites for anti-static applications, reducing surface wear, catalyst supports, and components within rechargeable battery electrodes [2], [3]. The implications for the increasing use and production of nanotubes include the potential increases in health risks to workers exposed to these materials. Until now, there is little information on the potential health effects and in particular, the hazards associated with the inhalation of nanotubes and nanofibres. All types of carbon nanofibres can exist as individual entities, however typically they are aggregated into micron-sized agglomerates. If these aggregates are formed during nanotube growth, then the nanotubes are highly entangled and the aggregates can be very hard to separate [4]. However, particles which are respirable can be generated from these aggregates, and it is these fine particles which are the main risk if inhaled into the lungs [5].

In the case of nanoparticles in general, the various geometries and sizes which are produced in the manufacturing process provide a range of samples which suggest that potentially these materials may present a health risk. A relationship between increased exposure to nanoparticles and adverse health effects has been described [6] and in individuals with pre-existing lung disease, inhalation of nanoparticles may induce inflammation and exacerbate respiratory and cardiovascular effects through the induction of oxidative stress and inflammation [7], [8], [9]. Nanoparticles of various types have been used in inhalation studies and have demonstrated various conditions such as pulmonary fibrosis, lung tumours, epithelial cell hyperplasia, inflammation and increased cytokine expression [10], [11], [12], [13]. It is widely recognized that the mechanisms of fibre-induced lung injury with mineral fibres such as asbestos depend on several factors, for example, length [14], [15], diameter, chemical nature [16], [17] and biopersistence [18], [19]. Particles which enter the lung become coated with lung lining material, which is likely to modify the surface reactivity and hence the oxidant generating ability and phagocytosis of the particles. Interaction between lung phagocytic cells such as macrophages, by their surface receptor leads to phagocytosis of foreign particles and possibly a secretory response which is enhanced in the presence of opsonins such as IgG [20], [21], [22], [23], [24]. Phagocytosis is a stimulus for superoxide anion release and it has been shown that when macrophages attempt to phagocytose long fibres such as crocidolite asbestos the process of phagocytosis is frustrated and, superoxide is released to the outside of the cell [25], [24]. The release of reactive oxygen species may be the initiating factor in the pathogenesis of lung disease after exposure to respirable fibres [26], [27].

Phagocytic cells play a key role in the removal of deposited material in the lung. However, cells may become overloaded, phagocytic ability impaired and consequently clearance from the lung is reduced. Impaired macrophage function has been described after instillation of nanoparticles into rat lungs [28]. Macrophages demonstrated increased sensitivity, with regard to their ability to migrate towards a chemoattractant, and impaired phagocytic ability after exposure. Impaired clearance can result in damage to macrophages and the lung epithelium and it has been suggested that translocation of spherical nanoparticles into the cardiovascular system from the lungs could take place [29], [30]. Translocation for nanofibres and nanotubes has not as yet been investigated. Finally, exposure of macrophages to nanoparticles has previously been shown to stimulate release the pro-inflammatory cytokine TNF-α[31], [32], and TNF-α can stimulate lung epithelial cells to produce IL-8, a potent chemotactic cytokine for neutrophils. Prolonged release of TNF-α may increase the inflammatory response with resulting pathological consequences.

The purpose of the present study has been to investigate the ability of various nanofibrous materials of different morphologies to stimulate the production of superoxide anions and release of the pro-inflammatory mediator TNF-α in human monocytes. Furthermore, the phagocytic ability of a human macrophage cell line after exposure to nanomaterials was assessed.

Section snippets

Methods

The methods are given in detail below but in summary, a variety of different nanotube and nanofibre samples were either synthesised by the authors or purchased and analysed by BET surface area, electron microscopy and elemental dispersive X-ray (EDX). These samples were the dispersed in a RPMI medium and introduced to cells at different concentrations. Two different cell types were used, human mononuclear cells derived from donor’s blood and cells from an immortalised THP-1 cell line. The

Characterisation of nanofibre (CNF) and nanotube (CNT) materials

The nanofibres used in this study were obtained from the University of Cambridge [33], University of Nottingham and Applied Sciences Incorporated (ASI) and are summarised in Table 1. These samples can be split into carbon nanotubes (NTs) where the graphene planes are parallel to the fibre axis and nanofibres (NFs). The samples used were analysed by transmission electron microscopy (TEM, Jeol 200CX and FEI Tecnai), using bright field imaging, dark field imaging, electron diffraction and high

Discussion

Short-term in vitro studies have been a focus of testing, in the case of asbestos and man-made mineral fibres, in the hope that these tests may give a clear indication of the potential pathogenicity of different fibres and particles [37]. The purpose of the present study was to investigate a range of nanomaterials in order to gain an understanding of the mechanisms by which nanoparticles of varying dimensions and composition interact with phagocytic cells of the lung. Apart from dimension,

Conclusions

We have demonstrated here that the ability of nanomaterials to stimulate the release of the pro-inflammatory mediator TNF-α and the release of ROS in monocytic cells in vitro may depend to a large extent of the geometry and surface characteristics of the nanomaterial. We have also shown that all the materials used here had a negative impact on the phagocytic ability of cells, which may in turn be the reason for ‘frustrated phagocytosis’. These important considerations may have important

Acknowledgements

This study was generously funded by the COLT Foundation, the Royal Academy of Engineering and the EPSRC.

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