Toxicity of nanoparticles on lung cells and pulmonary immune defences

Background: Nanoparticles (NPs) are a large area of research due to their unique properties. On the one hand, NPs are actively developed as drug delivery systems and diagnostic tools. On the other hand, their excessive use in modern products and inappropriate utilisation can increase existing air pollution. The most common route of NP exposure is via the pulmonary system. Once the particles reach the alveoli, they encounter pulmonary surfactant, rapidly adsorbing biomolecules. Surfactant proteins A (SP-A) and D (SP-D) are innate immunity proteins and are essential in the resolution and clearance of allergens and microorganisms. They are also thought to play an essential role in opsonisation and clearance of engineered NPs and particulate matter. This research aimed to explore the role of SP-A and SP-D in the uptake, clearance, and translocation of 20 nm gold NPs (AuNPs) and to understand potential hazards that can be encountered in individuals deficient in these proteins.Methods: The research employed a combination of in vitro and in vivo methods. Advanced multimodal microscopy was used for the semi-quantitative three-dimensional imaging of AuNPs and analysis within the cells and tissue. AuNPs were used to study their behaviour in physiological-like buffers and bio-nano interactions with SP-A and SP-D, applying spectrophotometric and protein analysis methods. The cell culture model, RAW264.7 cells, were exposed to AuNPs, and the impact of SP-A and SP-D on the viability was measured. Genetic knockout mice for SP-A and SP-D, respectively, and wild-type mice were nose-only exposed to 20 nm AuNPs aerosol, followed by the quantitative analysis of the clearance and translocation of AuNPs to secondary organs. Finally, environmental particles were collected from Southampton’s harbour and analysed for their elemental composition, morphology, and nanotoxicity on RAW264.7 cells.Results: Label-free multimodal microscopy was optimised for AuNPs imaging within single cells, but not tissue. This technique allowed semi-quantitative AuNPs analysis. The presence of proteins in physiological-like buffers was essential to prevent aggregation. The results showed tentativeevidence of selective SP-A and SP-D adsorption to AuNPs; no opsonisation could be detected. SP-A and SP-D had no or little impact on the viability of the cells in the presence of AuNPs, but suppressed uptake of AuNPs. The in vivo results were inconclusive. The SP-D knockout mice showed altered clearance of AuNPs from the lungs compared to the SP-A knockout and wild-type mice. Both SP-A and SP-D knockout mice macrophages showed suppressed AuNPs uptake. Environmental particles were highly heterogeneous in composition and morphology, and potentially more toxic than AuNPs. No alteration on cell viability was found in the presence of SP-A or SP-D.Conclusion: Multimodal microscopy was shown to be a powerful tool for semi-quantitative analysis; more research is required to shift into the fully quantitative analysis. In vitro experiments partially supported the hypothesis, as proteins semi-selectively adsorbed to AuNPs and suppressed the phagocytosis. However, additional verification techniques are required to overcome the intrinsic limitations of AuNPs. It can be speculated that SP-D is essential for clearance of AuNPs, although it remains unclear whether this is a phenotype induced effect or if it is protein-mediated