Elsevier

Toxicology in Vitro

Volume 56, April 2019, Pages 1-9
Toxicology in Vitro

Assessing the translocation of silver nanoparticles using an in vitro co-culture model of human airway barrier

https://doi.org/10.1016/j.tiv.2018.12.013Get rights and content

Highlights

  • This study developed a novel in vitro co-culture model of the human airway barrier.

  • Silver nanoparticles were introduced to co-culture models and translocation and cytotoxicity were quantitatively evaluated.

  • Results revealed the ability of AgNPs to translocate through barriers at 37ºC in a concentration dependent manner.

  • Mild cytotoxicity of AgNPs were confirmed by several bioassays.

  • Cellular expression of interlukin-6, interlukin-8 and tumor necrosis factor-alpha were suppressed after AgNPs treatment.

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.

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)

  • N.R. Yacobi et al.

    Polystyrene nanoparticle trafficking across alveolar epithelium

    Nanomedicine

    (2008)
  • H.M. Braakhuis et al.

    Progress and future of in vitro models to study translocation of nanoparticles

    Arch. Toxicol.

    (2015)
  • G.B. Braun et al.

    Etchable plasmonic nanoparticle probes to image and quantify cellular internalization

    Nat. Mater.

    (2014)
  • A.J. Clippinger et al.

    Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials

    Arch. Toxicol.

    (2016)
  • J.M. Cohen et al.

    Tracking translocation of industrially relevant engineered nanomaterials (ENMs) across alveolar epithelial monolayers in vitro

    Nanotoxicology

    (2014)
  • Corning

    Permeable Supports Selection Guide Including Transwell® and Falcon® Cell Culture Inserts

    (2014)
  • M. Daigneault et al.

    The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages

    PLoS One

    (2010)
  • C. Ehrhardt et al.

    In vitro models of the alveolar epithelial barrier

  • L.P. Ge et al.

    Nanosilver particles in medical applications: synthesis, performance, and toxicity

    Int. J. Nanomedicine

    (2014)
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