Proteomic characterisation of zinc oxide nanoparticles and immunotoxic effects on A549 cells-implications for occupational exposure

Background: Engineered nanomaterials such as metal oxide nanoparticles (NPs) offer unique physico-chemical properties and are widely used in sunscreen formulations and personal care products. NPs become coated with proteins when exposed to biological fluids, forming the 'nanoparticle-protein corona' (NP-PC), which may affect its overall bioactivity in vivo. Information regarding the interaction of NPs with biomolecules and the associated risks of exposure to NPs is limited and needs further investigation. Exposure to NPs via inhalation is an important concern especially in the occupational setting. This study aimed to characterize the NP-PC formation on zinc oxide NPs (ZnO-NPs) with fetal bovine serum (FBS) proteins and the immunotoxic effects on human lung epithelial cells (A549). \ud \ud Method: NP-PC of pristine or surfactantdispersed (sZnO-NP) particles (30, 80 and 200 nm) were incubated in cell culture media (RPMI-1640 with 10% FBS). Proteins forming the corona were quantified using Bradford protein assay, separated by SDS gel electrophoresis and identified by MALDI-TOF mass spectrometry (MS). \ud \ud Result: Protein binding studies indicate that pristine ZnO-NP (30 > 80 > 200 nm) bind significantly more proteins then surfactantdispersed. Pristine ZnO-NP and sZnO-NP (30 and 80 nm) selectively bound proteins of either 11 or 14 kDa, respectively. MS analysis revealed most binding proteins to be derived from larger proteins (i.e. hemoglobin, albumin and histone). However, small intact proteins were also enriched on sZnONP surfaces, such as glucagon (3.4 kDa), apolipoprotein A-II (17 kDa) and apolipoprotein C-III (8.7 kDa), which are major constituents of high-density lipoproteins. Preliminary immunotoxicity data on human epithelial cells demonstrated increased IL-8 release for 30 nm ZnO-NP; with sZnO-NP causing a lesser stimulatory effect. \ud \ud Conclusion: In summary, we have demonstrated that, when compared to pristine ZnO-NP, the surfactant-dispersed NPs bind less protein and elicit a reduced proinflammatory IL-8 release in human lung epithelial cells. However, small physiological- important proteins such as apolipoproteins are enriched on sZnO-NP, which needs further investigation