Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit Assessing the translocation of silver nanoparticles using an in vitro co-culture model of human airway barrier Fan Zhang, Grace V. Aquino, Amjad Dabi, Erica D. Bruce ⁎ Department of Environmental Science, Baylor University, Waco, TX 76798, United States ARTICLE INFO Keywords: Co-culture Nanotoxicity Inductively coupled plasma mass spectrometry Transepithelial electrical resistance (TEER) Calu-3 Thp-1 ABSTRACT The lung has been recognized as one of the main target organs for nanoparticles (NPs) exposure. Cellular uptake of nanoparticles into pulmonary components has been routinely evaluated in the conventional monoculture format, which lacks relevant cell to cell communications and interactions that are vital in the physiological environment. A more physiologically relevant co-culture model has thus been developed and described here to study the translocation of NPs across human airway barrier. The model consists of human bronchial epithelial cells (Calu-3), endothelial cells (EA.hy926) and macrophage-like cells (differentiated Thp-1) in a two-chamber system. Silver nanoparticles (AgNPs) coated with tannic acid were used as an example nanoparticle. These AgNPs were applied to the co-culture system where their movement and resultant toxicity were monitored. Cellular uptake and translocation of AgNPs through the modeled barrier were confirmed using analytical methods. Mild cytotoxicity at the given dosage levels was also observed, accompanied by reduced secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). This human airway model provides researchers with an alternative method for the quantitative evaluation of uptake, translocation and toxicity of aerosol contaminants or nano-sized drug delivery systems in a more relevant in vitro format. 1. Introduction Nanomaterials with significantly increased surface to volume ratio in the nanoscale display many unique physiochemical properties com- pared to their bulk counterpart. These properties have led to novel applications of engineered nanomaterials in energy production, medical therapeutics and diagnostics, food packaging, and cosmetics. A primary metal material used in nanotechnology is silver. Nanoscale silver comprises roughly one-quarter of the presently available commercial nano-enabled products and is mainly used as an antimicrobial agent (Vance et al., 2015). In light of their anti-inflammatory, optical and thermal properties, AgNPs have also been used in several platforms in nanomedicine, such as wound repair (Tian et al., 2007), tumor detec- tion (Braun et al., 2014), and drug targeting and delivery (Anandhakumar et al., 2012). With the increasing influx of silver-based nanomaterial into the commercial market and medical fields, it is im- perative that we evaluate whether contact with AgNPs will result in adverse effects in workers, consumers and patients. One major exposure route for AgNPs is inhalation. Animal studies have demonstrated pulmonary toxicity of inhaled AgNPs and their ac- cumulation in other organs. Treated rats experienced asthma-like symptoms including pulmonary eosinophilic and neutrophilic in- flammation, as well as bronchial hyper-responsiveness after a 7-day exposure to citrate-capped and PVP-capped AgNPs (Seiffert et al., 2015). Aggregated forms of AgNPs were found in the spleen and kidney in adult mice following intranasal exposure for 7 days (Genter et al., 2012). Multiple in vitro studies have reported cellular uptake of AgNPs into both normal and cancer lung cell lines (Gliga et al., 2014; Han et al., 2014). The internalized AgNPs can trigger generation of reactive oxygen species, and further cause membrane rupture and DNA damage (Gliga et al., 2014; Han et al., 2014). When NPs are inhaled, they can deposit in the conducting airway and alveolar region of the lung (Murgia et al., 2017). In order to limit the systemic circulation of foreign materials after inhalation, the re- spiratory tract is lined with epithelium cells that form tight junctions. Tight junctions fill up the space between two adjacent epithelial cells to avoid the paracellular passage of particles from crossing the epithelial barrier. It is difficult to access this region using in vivo methods due to the complexity of multilayered tissues and their relative location in the lung, which limits the insight into processes of site-specific particle-cell interaction (Rothen-Rutishauser et al., 2005). Also, it is impossible to test the vast diversity of existing nanomaterials through animal models https://doi.org/10.1016/j.tiv.2018.12.013 Received 28 July 2018; Received in revised form 29 November 2018; Accepted 18 December 2018 ⁎ Corresponding author. E-mail addresses: Fan_Zhang@baylor.edu (F. Zhang), Grace_Aquino@baylor.edu (G.V. Aquino), Amjad_Dabi@baylor.edu (A. Dabi), Erica_Bruce@baylor.edu (E.D. Bruce). Toxicology in Vitro 56 (2019) 1–9 Available online 27 December 2018 0887-2333/ © 2018 Published by Elsevier Ltd. T