Effects and uptake of gold nanoparticles deposited at the air–liquid interface of a human epithelial airway model

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Abstract

The impact of nanoparticles (NPs) in medicine and biology has increased rapidly in recent years. Gold NPs have advantageous properties such as chemical stability, high electron density and affinity to biomolecules, making them very promising candidates as drug carriers and diagnostic tools. However, diverse studies on the toxicity of gold NPs have reported contradictory results. To address this issue, a triple cell co-culture model simulating the alveolar lung epithelium was used and exposed at the air–liquid interface.

The cell cultures were exposed to characterized aerosols with 15 nm gold particles (61 ng Au/cm2 and 561 ng Au/cm2 deposition) and incubated for 4 h and 24 h. Experiments were repeated six times. The mRNA induction of pro-inflammatory (TNFα, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) was measured, as well as protein induction of pro- and anti-inflammatory cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFα, INFγ). A pre-stimulation with lipopolysaccharide (LPS) was performed to further study the effects of particles under inflammatory conditions. Particle deposition and particle uptake by cells were analyzed by transmission electron microscopy and design-based stereology.

A homogeneous deposition was revealed, and particles were found to enter all cell types. No mRNA induction due to particles was observed for all markers. The cell culture system was sensitive to LPS but gold particles did not cause any synergistic or suppressive effects.

With this experimental setup, reflecting the physiological conditions more precisely, no adverse effects from gold NPs were observed. However, chronic studies under in vivo conditions are needed to entirely exclude adverse effects.

Introduction

During the last years the impact of nanoparticles (NPs) in medicine and biology has increased rapidly, especially for bioimaging (Jain et al., 2008), biosensing (Olofsson et al., 2003) and drug delivery (Dhar et al., 2008, Joshi et al., 2006). Gold NPs are chemically stable, electron dense and posses an affinity to biomolecules, such as amino acids (Selvakannan et al., 2004), proteins (Wangoo et al., 2008) and DNA (Rosi et al., 2006), making them suitable as drug carriers and imaging reagents. Promising results have been obtained in cancer research where conjugated gold NPs have been used for cancer detection and treatment in vitro (Chen et al., 2007, Khaing Oo et al., 2008, Li et al., 2009).

Despite the potential medical benefits of gold NPs, it remains unclear whether their use in biological organisms and humans, in particular, will be safe. A variety of toxicity tests in vitro were reported for gold NPs providing controversial results. Most studies have excluded the toxicity of gold NPs (4 nm–18 nm in diameter) (Connor et al., 2005, Khan et al., 2007, Shukla et al., 2005), studying cell viability, pro-apoptotic effects, oxidative stress and inflammatory response. In contrast, cytotoxic and pro-apoptotic effects of gold particles ≤ 2 nm were reported (Pan et al., 2007, Tsoli et al., 2005), as well as the impact of the charge of surface coatings on 2 nm particles on the reduction of cell viability (Goodman et al., 2004). Furthermore, exposure of fibroblasts to 13 nm gold particles caused morphological changes in the cytoskeleton and a reduction in cell proliferation (Pernodet et al., 2006). A pro-inflammatory response was found after exposure to bovine serum albumin coated 25 nm gold particles in an epithelial airway model (Rothen-Rutishauser et al., 2007).

The reasons for these controversial results might include different experimental set ups and particle characteristics. Recent studies have identified that a number of important parameters influence the toxicity of NPs (Nel et al., 2006, Oberdörster et al., 2005), such as dose (Kwon et al., 2009), size (Kreyling et al., 2006), shape (Oberdörster et al., 2005), bulk material (Hussain et al., 2005), surface charge (Cho et al., 2009, Goodman et al., 2004), surface area (Brandenberger et al., 2009, Stoeger et al., 2006), as well as the composition of the exposure medium (Herzog et al., 2009b).

All the studies on gold NPs, discussed previously, were performed in cell cultures under submerged conditions with the particles suspended in different media. Besides the effects of the media on agglomeration status, the movements of single NPs in fluids are mainly driven by diffusion and not by sedimentation (Limbach et al., 2005, Teeguarden et al., 2007). As a consequence from the latter the probability of agglomerates to settle down onto the cell culture is higher than that for single NPs, thus increasing the possibility of attributing the observations to the wrong kind of particles (Limbach et al., 2005). A direct deposition of the particles on the cells at the air–liquid interface has the advantage of minimizing these effects, resulting in a more reliable dosimetry and less particle agglomeration. The air–liquid interface exposure scenario not only shows ideal characteristics to study particle-cell interactions but also mimics particle deposition in the respiratory tract after inhalation and it is conceivable that the cellular response to particle exposure is different for submerged and air–liquid interface culture conditions.

Lung epithelial cells represent, along with alveolar macrophages, the first line of cellular defense against inhaled particles. These cells participate in the initiation and modulation of the inflammatory response by the production of chemokines. Of particular interest is the involvement of dendritic cells, which are present at the base of the epithelium and are the most competent antigen presenting cells in the lung (Holt et al., 1990). Therefore, in this study an in vitro co-culture model of the human epithelial airway barrier was used (Blank et al., 2007, Rothen-Rutishauser et al., 2005), consisting of human blood monocyte derived macrophages and dendritic cells as well as the widely used A549 human alveolar epithelial cell line.

The present study aimed to analyze the inflammatory and oxidative potential of 15 nm gold NPs in the triple cell co-culture model exposed at the air–liquid interface with a newly developed exposure system (Schmid et al., 2009), which allows for efficient, spatially uniform and dose-controlled exposure of cells to NPs. In addition, it was investigated whether the gold NPs aggravate or suppress the response to a pro-inflammatory stimulus, a finding previously observed by others (Chan et al., 2006, de Haar et al., 2006, Herzog et al., 2009a, Hofer et al., 2004).

Section snippets

Air–liquid interface exposure system

The cell cultures were exposed to the particles at the air–liquid interface with an exposure system (ALICE), described by Schmid et al. (2009). Briefly, the ALICE consists of three main components, a droplet generator (nebulizer), an exposure chamber and a flow system with an incubation chamber, which provides temperature and humidity conditions suitable for cell cultivation (temperature: 37 °C; humidity: nearly saturated). A dense cloud of micron-sized droplets (mass median diameter

Particle exposure

The particle exposure was performed at the air–liquid interface of the triple cell co-cultures. Using 1 mL of stock solution (40 μg/mL) a deposition of 61 ng/cm2 ± 5.5 ng/cm2 was obtained, as determined by gamma spectroscopy, and with the 10 fold higher concentration of the stock solution a deposition of 561 ng/cm2 ± 48.5 ng/cm2 was reached. This corresponds to a deposition efficiency of 61% and 55%, respectively. The quality of the particle suspension prior to the exposure was assessed by

Discussion

In this study the uptake and possible adverse effects of 15 nm gold particles were analyzed in a triple cell co-culture model exposed at the air–liquid interface. The particles were found to enter the cells in a concentration dependent way and were translocated through the epithelium to dendritic cells located at the basal side. No significant induction of oxidative stress or inflammatory response due to the gold NPs was observed, neither any synergistic or suppressive effect in an inflammatory

Acknowledgments

The authors would like to thank Mr. Bukalis and Mrs. Alber for performing the neutron-activated gamma spectroscopic determination of the mass of the gold nanoparticles, Alexander Wenk, Helga Hinze-Heyn, Barbara Krieger, Andrea Stokes and Mohamed Ouanella for their excellent technical assistance and Kirsten Dobson for proof reading the manuscript.

This study was supported by grants of the AnimalFreeResearch Foundation, the Doerenkamp-Zbinden-Foundation, the Deutsche Forschungs Gemeinschaft (DFG),

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