Abstract
Exposure of lung epithelia to aerosols is omnipresent. Chronic exposure to polluted air is a significant factor in the development of pulmonary diseases, which are among the top global causes of death, including COVID-19, chronic obstructive pulmonary disease, lung cancer, and tuberculosis. As efforts to prevent and treat lung diseases increase, the development of pulmonary drug delivery systems has become a major area of interest. In line with the '3 R' principles (Reduce, Refine, and Replace animal testing), we developed an in vitro aerosol exposure system, termed NAVETTA, which was designed to replicate lung conditions most realistically. This system exposes air-liquid interface-cultured lung epithelial cells to a low, laminar airflow, enabling efficient aerosol deposition within an electric field. The aim of this study was to test instrumental performance on different aerosols, with a focus on precision, reproducibility, and cellular response. Deposition of sodium fluorescein droplets, pristine, and fluorescently labeled silica nanoparticles was homogenous and reproducible across the different instrument positions and over several runs, hence, the coefficient of variance for run-to-run and position-to-position was below 15 % using reference aerosols. To showcase NAVETTA's versatile applicability, pristine silica nanoparticles and surface-functionalized fluorescently labeled silica nanoparticles were used. Various charging scenarios were studied, evidencing that deposition was enabled by and dependent on the applied electric field. Additional aerosol charging enhanced deposition compared to deposition achieved employing only the intrinsic charges of aerosol particles/droplets. In a second feasibility study two dry powder generators were tested for application with the NAVETTA system for testing deposition and cellular effects of nano-scale TiO2 aerosols. Cellular stress response was determined by interleukin-8 secretion, and viability post-exposure to TiO2 was monitored. Cells exhibited a trend to decreased viability and increased interleukin-8 secretion upon TiO2 deposition evidencing feasibility for application, however, more work is needed for optimizing reproducibility when using dry aerosol generators due to their discontinuous operation mode. Physiological conditions of 37°C and 98 % relative humidity within the NAVETTA resulted in 95 % viability over 24 h enabling longer-term exposure experiments. In summary, the market-ready NAVETTA presents a versatile exposure system for future in vitro pulmonary safety and efficacy studies by facilitating reliable and reproducible electrodeposition of various aerosols.