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Translocation of Functionalized Multi-Walled Carbon Nanotubes across Human Pulmonary Alveolar Epithelium: Dominant Role of Epithelial Type 1 Cells Pakatip Ruenraroengsak, Shu Chen, Sheng Hu, Jodie Melbourne, Sinbad Sweeney, Andrew J Thorley, Jeremy N. Skepper, Milo Sebastian Peter Shaffer, Teresa D Tetley, and Alexandra E Porter ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b08218 • Publication Date (Web): 01 Apr 2016 Downloaded from http://pubs.acs.org on April 5, 2016
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ACS Nano
Translocation of Functionalized Multi-Walled Carbon Nanotubes across Human Pulmonary Alveolar Epithelium: Dominant Role of Epithelial Type 1 Cells Pakatip Ruenraroengsak1,2*, Shu Chen1, Sheng Hu3, Jodie Melbourne1, Sinbad Sweeney2, Andrew J. Thorley2, Jeremy N. Skepper4, Milo S. P. Shaffer3, Teresa D. Tetley2* and Alexandra E. Porter1* 1
Department of Materials and London Centre for Nanotechnology, Imperial College London,
Exhibition Road, London, UK, SW7 2AZ. 2
Lung Cell Biology, Airways Disease, National Heart and Lung Institute, Imperial College
London, Dovehouse Street, London, UK, SW3 6LY. 3
Department of Chemistry and London Centre for Nanotechnology, Imperial College London,
Exhibition Road, London, UK. 4
Cambridge Advanced Imaging Centre, Department of Physiology, Development and
Neuroscience, University of Cambridge, UK, CB2 3DY. KEYWORDS
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Functionalized multi-walled carbon nanotubes, uptake, translocation, alveolar epithelial barrier, electron microscopy, quantitative imaging technique.
ABSTRACT
Uptake and translocation of short functionalised multi-walled carbon nanotubes (shortfMWCNTs) through the pulmonary respiratory epithelial barrier depend on physicochemical property and cell type. Two mono-culture models, immortalised human alveolar epithelial type 1 (TT1) cells and primary human alveolar epithelial type 2 cells (AT2), which constitute the alveolar epithelial barrier, were employed to investigate the uptake and transport of 300 and 700nm in length, poly(4-vinyl pyridine)-functionalized, multi-walled carbon nanotubes (p(4VP)MWCNTs) using quantitative imaging and spectroscopy techniques. The p(4VP)-MWCNT exhibited no toxicity on TT1 and AT2 cells, but significantly decreased barrier integrity (*p0.001) release of LDH by either cell type (Figure S2b-c), indicating that the non-fMWCNTs (p-MWCNTs) and p(4VP)-MWCNTs did not damage the cell membrane integrity. The effects of p(4VP)MWCNTs on release of inflammatory mediators, IL-6 and IL-8, were also investigated (Figure S3); neither length of the MWCNTs caused significant (p>0.001, n=3(TT1) and n=6(AT2)) release of IL-6 and IL-8 from either TT1 or AT2 cells. Effect of p(4VP)-MWCNTs on TEER, Dextran Permeability and Tight Junctions TEER assesses the level of tight junction formation by measuring the Ohmic resistance of the cell barrier. Both cell types formed tight junctions (Figure 1a-d). The baseline TEER value trends measured from the TT1 and AT2 cells were consistent with previous studies20,21 with TEER values for TT1 and AT2 of 56±3 (number of experiments, n=3) and 639±30 Ωcm2 (number of subject samples and number of experiments, n=6) respectively (Figure 1a-b); the order of magnitude higher value for AT2 cells reflects the very deep, strong tight junctions developed by AT2 cells in vitro, approximately twice as deep TT1 cell tight junctions.12,14 p(4VP) alone induced a sharp significant increase in TEER, *p
