Development of Physiologically Relevant In Vitro Models for Assessment of Nanoparticle-Induced Physicochemical Property–Dependent Cytotoxicity of Human Lung Cells

With a rapid increase in nanotechnology development and nanomaterial (NM) utilization, human health risk along a NMs’ lifecycle has become a public health concern. However, risk assessment of emerging NMs has been limited due to several technical challenges, including the lack of appropriate in vitro models that mimic real-life exposure scenarios and reliably predict in vivo responses. For example, the administered dose under normal cell culture conditions does not reflect the actual delivered dose due to NM’s slow settling rate. In addition, the artificial dissolution and/or instability of NMs under submerged conditions does not represent the real exposure condition of inhaled “dry” NMs. To address those issues, the objective of this study was to develop a physiologically relevant in vitro model for toxicity assessment of NMs with specific delivered dose of each NM, or using a 3D air-liquid interface (ALI) in combination with aerosol delivery system. These models allow us to evaluate and compare bioand toxic- potential of metal oxide nanoparticles (MONPs) and by-products from thermally decomposed nanocomposites at similar delivered doses in vitro. Following identification of targeted lung cells at the site of inhaled NM accumulation in vivo, specific lung cell types were selected for our in vitro models. The ALI model was constructed using the VitroCell system with cultured primary human bronchial epithelial cells. Cells were allowed to differentiate on ALI for 28 days, which exhibited a pseudostratified architecture with mucous production, as well as tight junction formation and ciliogenesis, resembling airway microenvironment. Delivered doses were calculated based on the sedimentation rate of each NM under each cell culture medium, and the dose/time course known to induce pulmonary toxicity in vivo. Well-characterized MONPs and two pairs of thermally decomposed pristine and carbon nanotube-containing polycarbonate (PC/PC-CNT) and polyurethane (PU/PU-CNT) ultrafine particles were evaluated for their cytotoxicity in this test model. Acute and subchronic exposure to MONPs induced property- and time-dependent cellular alterations, consistent with in vivo observations. NM type-dependent cytotoxicity was also observed with PC and PU, with CNT incorporation (PC-CNT and PU/ CNT) enhancing their bioactivity. Thermal decomposition of the nanocomposites resulted in the formation of particulate-bound polycyclic aromatic hydrocarbon (PAH), leading to enhanced cytotoxicity and mechanistic profiling. Data from this study will be used for structure-activity relationship and comparative toxicity studies to assess the health risks of NMs.