Toxicology of high aspect ratio nanomaterials : how shape determines the biologically effective dose
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Nanotechnologies are the fastest growing industry sector ever recorded. The US budget for nanotechnology is predicted to reach the 1 trillion dollar threshold in 2015, meaning that nanotechnologies will indeed be larger than all other technologies combined. High aspect ratio nanomaterials (HARN) become increasingly important in the nanotechnology industries, and show great promise, offering many advantages and improvements to a significant range of products. The main feature of HARN is the ratio of the width of a nanomaterial to its height which can be up to 1000, making the material fibre or platelet- shaped. However, this feature leads to comparison between HARN and other high aspect ratio materials including fibre shaped materials, such as asbestos fibres. Due to the structural similarities between fibrous HARN and asbestos the question arises- do HARN pose the same risk as asbestos? This project aimed to assess the potential of a range of HARN to cause similar pathological effects as asbestos fibres. In order to address this aim a panel of HARN was tested against the fibre pathogenicity paradigm in vivo by examining the pulmonary and pleural responses as well as in vitro to reveal the mechanism of cell/HARN interaction. The first part of the study focused on fibre-shaped HARN, including a panel of distinct length classes of silver nanowires (AgNW) which were injected directly into the pleural space, a target tissue for asbestos related diseases. Injection of high aspect ratio AgNW into the pleural space of mice revealed a length dependent inflammatory response in line with the fibre pathogenicity paradigm which explains fibre pathogenicity. AgNW from 5 μm in length and above led to a significant increase in granulocytes in the pleural space which is similar to that seen after treatment with long amosite asbestos. The use of additional HARN with different compositions allowed us to identify a threshold length for fibre-induced pleural inflammation, which is 5 μm. Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. A novel technique, backscatter scanning electron microscopy (BSEM), was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. Using this technique we showed that the onset of inflammation does not correlate with the onset of frustrated phagocytosis, with a fibre length of ≥5 μm and ≥10 μm, respectively, leading to the conclusion that intermediate length fibres fully enclosed within macrophages as well as frustrated phagocytosis are associated with a proinflammatory state in the pleural space. We further showed that fibres compartmentalise in the mesothelial cells at the parietal pleura as well as in inflammatory cells in the pleural space. To investigate the mechanism of the lengthdependent inflammation caused by AgNW, the NALP3 inflammasome activation pathway was studied in vitro, however no clear correlation could be identified. We further aimed to investigate the threshold length of fibre-induced inflammation in the lung and the effect of fibre length on macrophage locomotion in an in vitro macrophage migration assay. Pharyngeal aspiration of AgNW resulted in a length dependent inflammatory response in the lungs with threshold at a fibre length of 14 μm. Shorter fibres including 3, 5 and 10 μm elicited no significant inflammation. This identified threshold length differs from that in the pleural space which may be explained by differences in clearance mechanism of deposited fibres from the airspaces compared to the pleural space. Particle clearance from the lung is partly performed by migration of particle-laden macrophages to the mucociliary escalator. We investigated if uptake of longer fibres leads to restricted mobility and showed that exposure to AgNW in the length of ≥ 5 μm resulted in impaired motility of macrophages in the wound closure assay. The second part of the study focused on HARN in the form of nanoplatelet-shaped particles since nanoplatelets may pose an unusual risk to the lungs and the pleural space because of their aerodynamic properties. We first derived the respirability of graphene nanoplatelets (GP) from the basic principles of the aerodynamic behaviour of plate-shaped particles which allowed us to calculate their aerodynamic diameter. This showed that the nanoplatelets, which were up to 25 μm in diameter, were respirable and so would deposit beyond the ciliated airways following inhalation. We therefore utilized models of pharyngeal aspiration and direct intrapleural installation of GP, as well as an in vitro model, to assess their inflammatory potential. These large but respirable GP were inflammogenic in both the lung and the pleural space at an acute timepoint although they decreased in their inflammatory potential over a 6 weeks period. Oxidation of GP in the lung tissue was investigated in order to identify if GP degraded over the 6 week period in the lung tissue and therefore showed reduced inflammogenicity. Raman spectroscopy was used to measure the oxidation state and revealed that no change occurred over the observed timeframe. The mechanism underlying acute GP inflammation was studied in THP-1 macrophages exposed to GP. These investigations showed that GP exposure led to significant expression of IL-1β, which could be blocked via a number of inhibitors related to the NALP3 inflammasome activation. This study highlights the importance of shape/length of HARN as a driver for in vivo and in vitro inflammogenicity by virtue of their respirable aerodynamic diameter, despite a considerable 2-dimensional size which leads to an inflammatory response when deposited in the distal lungs and the pleural space. The identification of the threshold length for nanofibre-induced pathogenicity in the pleura and the lung has important implications for the understanding of the structure–toxicity relationship for asbestos-induced mesothelioma. It also contributes to risk assessment by offering a template for production of safer synthetic nanofibres by the adoption of a benign-bydesign approach. The results of this work highlight the importance of testing new HARN to protect workers in nanotechnology industries and the public.