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Title: Investigation of the effects of nano-sized materials at the blood-gas barrier of the lung : implications for cardiovascular reactivity
Author: Smith, Elizabeth
ISNI:       0000 0004 6423 4340
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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Carbon nanotubes (CNTs) possess a number of unique properties and play a pivotal role in the nanotechnology industry with widespread applications in construction, medicine and electronics. Previous studies have demonstrated an association between CNT inhalation and adverse cardiovascular effects, however, it is currently unclear whether this is due to the release of pulmonary inflammatory mediators or particle translocation across the blood-gas barrier. A better understanding of CNT interactions at this barrier is therefore essential for developing safer nanomedicines and reducing occupational health risks associated with carbon nanotube manufacture. The working hypothesis of this study was that the physicochemical properties of multi-walled carbon nanotubes (MWNTs) would determine their toxicity and reactivity at the blood-gas barrier. To test this hypothesis, primary alveolar type-2 epithelial cells (AT2) and microvascular endothelial cells (HPMVEC) were isolated from resected human lung and employed alongside a unique immortalised human type-1 alveolar cell line (TT1). The cells were then exposed to functionalised MWNT to determine the cellular reactivity of the particles. Using this model, it was shown that MWNT exposure induced endothelial cell activation, characterised by a pro-inflammatory and pro-thrombotic response, and MWNT functionalisation was a key factor in determining the nature of this response. Acid-oxidised MWNTs induced the greatest endothelial activation, however as-received and thermochemically grafted MWNTs induced a lesser pro-inflammatory response that also coincided with an increase in ROS formation. Of the thermochemically grafted MWNTs, P(M4-VP) and P(PEGMA) MWNTs induced the greatest and smallest response respectively. At the epithelium, MWNTs were far less reactive. All MWNTs were non-toxic to AT2 cells, however a small cytotoxic and inflammatory response was observed in TT1s following chronic exposures (72h) and MWNTs were present in both vesicles and the cytoplasm of TT1s following 24h exposure. The thermochemically grafted P(M4-VP) MWNTs were shown to induce the greatest pro-inflammatory response during these chronic exposures. In vivo, disruption of cell-cell junction could have implications for particle translocation, inflammation and atherosclerotic lesion formation. Exposure to MWNTs was shown to increase the permeability of type 1, but not type 2 alveolar epithelial barriers. The greatest disruption was seen at the endothelial cell junctions, which was accompanied by depletion of adherens junction protein and rearrangement of the actin cytoskeleton. Finally, to assess whether epithelial-endothelial crosstalk could affect particle toxicity a co-culture model of the alveolar blood-gas barrier was constructed using a transwell system where alveolar type 1 and type 2 epithelial cells were separated from pulmonary microvascular cells by a porous membrane. There were disparities between the mono and co-culture findings and acid oxidised MWNTs applied to the epithelial surface of the co-culture induced a significant release of inflammatory mediator into both the apical and basolateral chambers of the co-culture system, indicative of inflammation in both the pulmonary and vascular systems. This highlighted the need to incorporate endothelial cells into NP toxicity assays, especially when considering the cardiovascular reactivity of a particle. This thesis has demonstrated that MWNT toxicity is both cell and MWNT surface functionalisation dependent, and that endothelial activation could be responsible for the adverse cardiovascular effects observed following CNT inhalation in vivo. These findings will aid the generation of safer nanomaterials and further the understanding of CNT induced cardiovascular reactivity.
Supervisor: Tetley, Terry ; Thorley, Andrew Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral