Pulmonary toxicity assessment following aerosolization of engineered nanomaterials using an in vitro air-liquid interface method

Although there are over 1,600 Engineered Nanomaterials (ENMs)-containing consumer products available, our understanding of ENM safety is still limited. Airborne ENMs can readily enter the human body through inhalation potentially leading to many adverse health effects such as cardiovascular and pulmonary diseases. The conventional in vitro submerged cell culture method was developed decades ago and has been widely used as a fast screening method to elucidate cellular toxicity upon exposure to hazardous materials; however, it has many limitations compared with the in vivo models. Our group has previously utilized and validated an integrated low flow system capable of generating and depositing airborne nanoparticles (NPs) directly onto cells at an air-liquid interface (ALI) condition, and our results confirmed that this exposure system produced reproducible toxicological data for ENMs including gold (Au), 16% silver coated onto silica (16% Ag-SiO2), and copper oxide (CuO). To further improve this ALI method for an even closer representation of the in vivo model, a co-culture model containing three cell lines (A549, THP-1 differentiated macrophages, and EA.hy 926) was established and validated for testing ENMs toxicity. The co-culture model was exposed to 16% Ag-SiO2 and CuO NPs under the same protocol (4 h ALI exposure with a concentration of 3.5 mg/m3) as monoculture (A549 only) for comparison. Toxicity was assessed by measuring cell viability, reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) release, and interleukin (IL) 8 level. Results showed that 16% Ag-SiO2 NPs induced higher ROS generation, and CuO NPs produced a significant level of proinflammatory response compared with monoculture. In addition, the co-culture model exhibited a similar response with the primary human bronchial epithelia cell line (HBEC) in terms of ROS and IL-8 responses after CuO NPs exposure, suggesting a more advanced refinement of the conventional model for in vitro inhalation study.