Assessing the translocation of silver nanoparticles using an in vitro co-culture model of human airway barrier
Graphical abstract
Introduction
Nanomaterials with significantly increased surface to volume ratio in the nanoscale display many unique physiochemical properties compared 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 detection (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 imperative 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 accumulation in other organs. Treated rats experienced asthma-like symptoms including pulmonary eosinophilic and neutrophilic inflammation, 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 respiratory 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 due to ethical concerns (George et al., 2015). Conventional monoculture systems, however, lack the insight and understanding of efflux or translocation of NPs through a biological barrier involving the interplay among several cell types. Therefore, the field calls for non-animal alternative approaches that represent the human respiratory system and permit cost- and time-efficient evaluation for acute systemic toxicity (Hamm et al., 2017).
Primary human epithelial cells isolated from donors are most representative of the in vivo situation in terms of phenotype characteristics; however, they are not widely used due to limited availability and challenges maintaining functional properties upon passaging (Ren et al., 2016). Recently, several in vitro models using human pulmonary epithelial cell lines such as NCI-H441and A549 have been developed for alveolar epithelial transport. In these models, cells are in co-culture with endothelial cells or inflammatory-responding cells (Hermanns et al., 2004; Ren et al., 2016; Rothen-Rutishauser et al., 2005). However, despite their epithelium alveolar origin, the co-culture models fail to form tight junctions as indicated by low TEER values, which hampers their ability to provide the most physiologically-relevant cellular response to translocation of test particles. Several new studies reported use of Calu-3, a well-differentiated and characterized cell line derived from human bronchial submucosal gland, able to develop high TEER values on porous membrane, to achieve the experimental conditions required for tight junction complexes (Clippinger et al., 2016; Cohen et al., 2014; Dekali et al., 2014; Derk et al., 2015; George et al., 2015). Lung barrier simulation was also attempted at the air-liquid interface (Holder and Marr, 2013). While advantages of this method include more realistic responses to inhalation exposure and limited interference from culture medium, researchers experience problems maintaining constant temperature and humidification of the system, resulting in less reproducible results (Braakhuis et al., 2015).
In the present study, the authors introduce an in vitro co-culture model that mimics the human airway barrier by assembling pulmonary epithelial cells (Calu-3), endothelial cells (EA.hy926), and macrophage cells (differentiated Thp-1) in a two-chamber system. Cellular uptake of AgNPs, their translocation through the barrier, and the resultant acute toxicity were examined using this model. Tannic-coated AgNPs were used as a model particle and tested on established tri-culture systems.
Section snippets
Silver nanoparticles
Spherical AgNPs (with a core diameter of 50 nm ± 4 nm, based on TEM measurements by the manufacturer) capped with tannic acid in a stock concentration of 1.0 mg/mL (NanoComposix Inc., San Diego, CA, US) were used in this study. The choice of this type of AgNPs was based on our previous study where tannic acid-AgNPs of the same production line exhibited the most efficient penetration efficacy through cell membranes compared to two other coated AgNPs used in that study (Zhang et al., 2015).
TEER measurements
In order to evaluate the role of Calu-3 in the development of TEER in the barrier model, membrane inserts seeded with EA.hy926 only, Calu-3 only and both cell lines together were prepared on the same day. Post-seeding, beginning at 24 h, the TEER of each culture was documented until it reached 1000 Ω·cm2. The development of TEER in these individual cultures is illustrated in Fig. 2A. Endothelial cells EA.hy926 did not gain much electrical resistance in 7 days, showing rather stable values
Barrier properties
In an effort to establish a reliable and realistic barrier to in vivo, the current lung model employed Calu-3, a type of bronchial epithelial cells, that have demonstrated formation of intracellular tight junction under submerged condition by previous study (Wan et al., 2000). Transepithelial electrical resistance (TEER) is a common measurement of epithelial cells for membrane integrity and tight junction dynamics. It is a real-time and non-invasive approach, and is widely used in barrier model
Conclusion
To conclude, this is the first time that Calu-3, Thp-1 and EA.hy926 cells were cultivated together to develop a human airway model and applied to study the translocation and toxicity of nano-sized particles. The model is generally easy to build, replicate, and can be used in multiple capacities in nano-biotechnologies and environmental or human health fields. With its unique three-dimensional setup, the authors not only confirmed the AgNPs translocation through the airway barrier, but also
Acknowledgement
The authors would like to thank Baylor University for the availability of core laboratory facilities made available to Dr. Bruce and her colleagues during the time of data collection for this manuscript. Dr. Bruce reports grants from Hemotek, LLC, (32370181) during the conduct of the study.
References (38)
- et al.
Silver nanoparticles modified nanocapsules for ultrasonically activated drug delivery
Mater. Sci. Eng. C
(2012) - et al.
Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles
Toxicol. Rep.
(2014) - et al.
Potential in vitro model for testing the effect of exposure to nanoparticles on the lung alveolar epithelial barrier
Sens. Biosens. Res.
(2015) - et al.
Development of an in vitro model of human bronchial epithelial barrier to study nanoparticle translocation
Toxicol. in Vitro
(2015) - et al.
In vitro study of the pulmonary translocation of nanoparticles: a preliminary study
Toxicol. Lett.
(2006) - et al.
Alternative approaches for identifying acute systemic toxicity: moving from research to regulatory testing
Toxicol. in Vitro
(2017) - et al.
Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro
Lab. Investig.
(2004) - et al.
A coculture model of the lung-blood barrier: the role of activated phagocytic cells
Toxicol. in Vitro
(2015) - et al.
Modelling the bronchial barrier in pulmonary drug delivery: a human bronchial epithelial cell line supplemented with human tracheal mucus
Eur. J. Pharm. Biopharm.
(2017) - et al.
TEER measurement techniques for in vitro barrier model systems
Jala
(2015)