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Inactivation, Clearance and Functional Effects of LungInstilled Short and Long Silver Nanowires in Rats Kian Fan Chung, Joanna Seiffert, Shu Chen, Ioannis G. Theodorou, Angela Erin Goode, Bey Fen Leo, Catriona M. McGilvery, Farhana Hussain, Coen Wiegman, Christos Rossios, Jie Zhu, Jicheng Gong, Farid Tariq, Vladimir Yufit, Alexander J. Monteith, Teruo Hashimoto, Jeremy N. Skepper, Mary P. Ryan, Junfeng (Jim) Zhang, Teresa D Tetley, and Alexandra E Porter ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b07313 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017
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ACS Nano
Inactivation, Clearance and Functional Effects of Lung-Instilled Short and Long Silver Nanowires in Rats Kian Fan Chung1, Joanna Seiffert1, Shu Chen2, Ioannis G. Theodorou2, Angela Erin Goode2, Bey Fen Leo
2,3
, Catriona M. McGilvery2, Farhana Hussain1, Coen Wiegman1, Christos
Rossios1, Jie Zhu1, Jicheng Gong4, Farid Tariq5, Vladimir Yufit5, Alexander J. Monteith6, Teruo Hashimoto7, Jeremy N. Skepper8, Mary P. Ryan2, Junfeng (Jim) Zhang4, Teresa D Tetley1, Alexandra E. Porter2
1
Airways Disease, National Heart & Lung Institute, Imperial College, London SW3, UK;
2
Department of Materials and London Centre for Nanotechnology, Imperial College,
London SW7 2AZ, UK; 3Nanotechnology & Catalysis Research Centre (NANOCAT), University of Malaya, Kuala Lumpur 50603, MYS; 4Nicholas School of Environment & Duke Global Health Institute, Duke University, Durham, USA; 5Dept. Earth Science, Imperial College, London SW72AZ, The School of Materials; 6Department of Biological Sciences, Oxford Brookes University, Oxford, OX3 OBP, UK; 7The University of Manchester, Oxford Road, Manchester, M13 9PL, UK; 8Cambridge Advanced Imaging Centre, Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge, CB2 3DY UK.
Correspondence: Professor K F Chung, National Heart & Lung Institute Imperial College London, Dovehouse St, London SW3 6LY, UK
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ABSTRACT There is a potential for silver nanowires (AgNWs) to be inhaled but there is little information on their health effects and their chemical transformation inside the lungs in vivo. We studied the effects of short (S-AgNWs; 1.5µm) and long (L-AgNWs; 10 µm) nanowires instilled into the lungs of Sprague-Dawley rats. S- and L-AgNWs were phagocytosed and degraded by macrophages; there was no frustrated phagocytosis. Interestingly, both AgNWs were internalized in alveolar epithelial cells, with precipitation of Ag2S on their surface as secondary Ag2S nanoparticles.
Quantitative serial block face 3D scanning electron
microscopy showed a small, but significant, reduction of NW lengths inside alveolar epithelial cells. AgNWs were also present in the lung subpleural space where L-AgNWs exposure resulted in more Ag+ive macrophages situated within the pleura and sub-pleural alveoli, compared with the S-AgNWs exposure.
For both AgNWs, there was lung
inflammation at day 1, disappearing by day 21, but in bronchoalveolar lavage fluid (BALF), LAgNWs caused a delayed neutrophilic and macrophagic inflammation while S-AgNWs caused only acute transient neutrophilia.
Surfactant protein D (SP-D) levels in BALF
increased after S- and L-AgNWs exposure at day 7. L-AgNWs induced MIP-1α and S-AgNWs induced IL-18 at day 1. Large airway bronchial responsiveness to acetylcholine increased following L-AgNWs, but not S-AgNWs, exposure. The attenuated response to AgNW instillation may be due to silver inactivation after precipitation of Ag2S with limited dissolution. Our findings have important consequences for the safety of silver-based technologies to human health.
Key words: silver nanowires, alveolar epithelial cells, macrophages, silver sulfidation, surfactant protein D, bronchial hyperresponsiveness.
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ACS Nano
Silver nanomaterials are increasingly used in industrial and domestic products ranging from odour-resistant socks, personal care products, respiratory devices, food storage boxes, computers and cleaning sprays. Silver nanowires (AgNWs) with a high aspect ratio are also becoming important in the field of optoelectronics, where they are used in the production of transparent, conductive thin-film electrodes for touchscreens, smart phones and computers.1,
2
Potentially, there is a high risk of aerosolisation
associated with industrial processes that manufacture nanolayer films, involving the deposition of tiny droplets of suspensions of AgNWs onto surfaces,3 and therefore of increased risk of inhalation by such workers.4 Inhalation and instillation of silver nanoparticles (AgNPs) mainly as nanospheres have been reported to induce dose-dependent inflammation5, 6 and alterations in lung function, including bronchial hyperresponsiveness.7-9 A very limited number of studies have investigated the toxicity of AgNWs in vivo. AgNWs ≥5 µm in length instilled directly into the pleural cavity induced pulmonary inflammation, and those >10 µm induced ‘frustrated phagocytosis’.10, 11 Intratracheally-instilled AgNWs produced similar lengthdependent frustrated phagocytic responses, including macrophage clumping and ineffective clearance from the lungs, together with a neutrophilic and eosinophilic response induced by the long (20 µm) and short (2µm) AgNWs.12 With these very limited available studies, little is known about how the shape or aspect ratio of AgNWs can affect uptake, processing and biopersistence by lung cells, or if the release of Ag+ ion in vivo alters signalling pathways, or leads to alterations in pulmonary function. There is growing evidence that AgNPs and AgNWs can enter epithelial cells, interstitial sites, airway smooth muscle cells, the vascular endothelium and the pleural membrane.10-12 Another aspect is that interaction of nanoparticles with lung lining fluid containing pulmonary surfactant is known to influence uptake of nanowires by alveolar epithelial cells13 and also the aggregation of nanowires for clearance by alveolar macrophages.14 Moreover, the effect of AgNWs on the trafficking or production of surfactant proteins such as surfactant protein D (SP-D) is unclear. We hypothesized that AgNWs could undergo oxidative dissolution as silver ions (Ag+), and so the chemical nature of the AgNWs in the local environment would change with time. Therefore, both size-aspect ratio and Ag+ ion release and transformation to Ag salts could be critical in determining the biopersistence and cytotoxicity of AgNWs. This 3 ACS Paragon Plus Environment
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process of intracellular transformation has been demonstrated in our previous work for a single AgNW length in in vitro cell cultures15 and a recent study demonstrated the intracellular transformation of spherical AgNPs into nano-Ag2S on the surface.16 There is no evidence so far that this process occurs in vivo. There is also no understanding of how this high aspect ratio nanomaterial is distributed, internalised and transformed inside lung cells in vivo, how this process is influenced by AgNW length, and whether these processes could be correlated to inflammatory responses, pulmonary function and responsiveness, and the persistence of silver in the lungs. Therefore, in this study, we use backscattered and serial block face scanning electron microscopy to map and generate three-dimensional volume reconstruction of the morphology of the AgNWs inside the alveoli. Transmission electron microscopy (TEM) combined with energy dispersive–x-ray spectroscopy (EDX) were used to characterise the size, bulk and surface chemistry and any chemical and morphological transformations of instilled short and long silver nanowires (1 and 10 µm length) in vivo.17 This information was correlated to lengthdependent biological responses of AgNWs in the lung by measuring inflammation, chemokine and cytokine profiles, alterations in surfactant composition and pulmonary function.
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Results/Discussion Characteristics of long and short silver nanowires The PVP-capped short silver nanowires (S-AgNWs) have been characterised previously.15, 17
S-AgNWs (Fig1a &b) had an average length of 1.6 ± 1.4 μm (Fig 1c) and an average
diameter of 72 ± 36 nm (Fig 1d), as measured by TEM. High-aspect ratio long silver nanowires (L-AgNWs) (Fig 1e & f) had a similar average diameter of 73 ± 37 nm (Fig 1h), but a longer average length of 10.5 ± 8.5 μm (Fig 1g). From dynamic light scattering (DLS) measurements, the average hydrodynamic sizes of the S-AgNWs and L-AgNWs were 807 ± 66 nm and 1447 ± 94 nm, respectively. For both types of AgNWs, the measured hydrodynamic sizes were not representative of the sizes measured by transmission electron microscopy (TEM). This is because NW size information in DLS is obtained by measuring the particle’s diffusion coefficient, which in the case of high-aspect ratio NWs has both a translational and rotational component. The measured sizes may therefore be interpreted only as the sizes of spherical Ag particles that would have the same diffusion coefficient as S-AgNWs and L-AgNWs. However, using light microscopy, we have previously shown that in the absence of pulmonary surfactant components, AgNWs did not agglomerate during up to 7 days of incubation.14 In the case of L-AgNWs, the interplanar spacing’s measured from selected area electron diffraction (SAED) patterns were 0.235 nm, 0.204 nm and 0.144 nm, corresponding to the (111), (200) and (220) planes of metallic silver (ref. # 01-087-0597). TEM energy dispersive X-Ray (EDX) spectra collected from several L-AgNWs showed that they consisted of pure Ag, confirming the absence of impurities from the synthesis product and that no sulfidation had occurred on the material. A summary of the properties of the AgNWs used is given in Table 1.
2D and 3D imaging of nanowires in the lung Since we had seen various types of staining in cells and the lung in response to the silver nanowires, we were interested in the chemical speciation of silver in the lung tissue alveolar Type II (ATII) cells and the macrophages – especially due to the possibility of transformative processes such as dissolution and sulfidation, which have so far only been seen in in vitro exposures of lung cells for both AgNWs, as well as for AgNPs.13, 18 We used a combination of 2-D and 3-D scanning electron microscopy (SEM), high resolution phase contrast imaging (HRTEM) and energy dispersive X-Ray spectroscopy (EDX) to 5 ACS Paragon Plus Environment
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visualise the location of the AgNWs within the cells, and to characterise their chemistry. Imaging was performed at 24 hours after instillation of the S-AgNWs and L-AgNWs. The data are shown in Figs 2 and 3.
Ag Nanowires Both the S-AgNWs and L-AgNWs were found in the alveoli using SEM and TEM; the backscattered electron detector was used in the SEM to generate contrast from the AgNWs (Fig. 2a, b, g & h). The AgNW samples were analysed by spatially resolved analytical EM techniques to study whether the AgNWs had dissolved or transformed to other compounds in the alveoli. Changes to the morphology of the AgNWs were observed at the end of the SAgNWs located inside an ATII cell (Fig 2c & d). The interplanar spacing measured from a high resolution (HR-) TEM image acquired from the tip of the S-NW (Fig 2e) was 0.331 ± 0.010 nm, corresponding to the (012) plane of monoclinic silver sulfide Ag2S (ref. # 00-0140072). STEM-EDX analysis showed that Ag2S was detected on the surface of the S-AgNWs by STEM-EDX (Fig. 2f, spectrum 2); the core of the S-AgNWs consisted of pure Ag (Fig. 2f, spectrum 1). Only carbon was detected from the background (Fig. 2f, spectrum 3). Partiallysulfidised AgNWs were also found with the L-AgNWs inside the ATII cells (Fig 2 g-l). In 2D projection images, it was not possible to measure the length of the S-AgNWs, therefore we used serial block face SEM (Fig.3a-e) to generate 3D volume reconstructions of the AgNW inside the structural cells (Fig. 3d, e; Suppl movie). Since dissolution and transformation were the same for S-AgNWs and L-AgNWs, their morphological and chemical evolution inside the lung in vivo, was examined by 3D SEM for the S-AgNWs only. Figs 3a-c show selected SEM images from a serial block face dataset taken over a volume of 40 × 40 × 11 µm, as well as a visualisation of the two cells within this region containing S-AgNW. After segmentation of individual S-AgNW, their length distribution was quantified. The histogram of internalised S-AgNW lengths revealed that the S-NW contained within the cells were significantly shorter compared to the instilled material (p
