Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release

Abstract

Background: Silver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity.

Methods: BEAS-2B cells were exposed to citrate coated AgNPs of different primary particle sizes (10, 40 and 75 nm) as well as to 10 nm PVP coated and 50 nm uncoated AgNPs. The particle agglomeration in cell medium was investigated by photon cross correlation spectroscopy (PCCS); cell viability by LDH and Alamar Blue assay; ROS induction by DCFH-DA assay; genotoxicity by alkaline comet assay and γH2AX foci formation; uptake and intracellular localization by transmission electron microscopy (TEM); and cellular dose as well as Ag release by atomic absorption spectroscopy (AAS).

Results: The results showed cytotoxicity only of the 10 nm particles independent of surface coating. In contrast, all AgNPs tested caused an increase in overall DNA damage after 24 h assessed by the comet assay, suggesting independent mechanisms for cytotoxicity and DNA damage. However, there was no γH2AX foci formation and no increased production of intracellular reactive oxygen species (ROS). The reasons for the higher toxicity of the 10 nm particles were explored by investigating particle agglomeration in cell medium, cellular uptake, intracellular localization and Ag release. Despite different agglomeration patterns, there was no evident difference in the uptake or intracellular localization of the citrate and PVP coated AgNPs. However, the 10 nm particles released significantly more Ag compared with all other AgNPs (approx. 24 wt% vs. 4-7 wt%) following 24 h in cell medium. The released fraction in cell medium did not induce any cytotoxicity, thus implying that intracellular Ag release was responsible for the toxicity.

Conclusions: This study shows that small AgNPs (10 nm) are cytotoxic for human lung cells and that the toxicity observed is associated with the rate of intracellular Ag release, a 'Trojan horse' effect.

Figure 1

AgNPs characterization by TEM (A), PCCS (B) and UV–vis (C). TEM images of the AgNPs confirmed the primary size stated by the manufacturers (A). The hydrodynamic size distribution was performed in cell medium (BEGM) using PCCS. Data is presented as density distribution by volume together with the corresponding scattered light intensity (B). All the AgNPs agglomerated in cell medium as seen by the size and position of the density distribution peaks. All but the 10 nm PVP coated AgNPs sedimented significantly over time as indicated by the reduction of the scattered light intensity. The UV–vis spectra of the 10 nm citrate (C right) and 10 nm PVP (C left) coated AgNPs indicated particle sedimentation over time as seen from the 400 nm peak reduction.

Figure 2

Cell viability of BEAS-2B cells after exposure to AgNPs. Cell viability of BEAS-2B cells after 24 h exposure to AgNPs (5–50 μg/mL) was assessed using Alamar Blue assay (A) and LDH assay (B). Results are expressed as % mitochondrial activity for the Alamar Blue assay and % membrane integrity for the LDH assay. Results are presented as mean ± standard deviation of 3 independent experiments. Significant results as compared to the control are marked with asterisks (* for P-value < 0.05, ** for P-value < 0.01, *** for P-value < 0.001).

Figure 3

Genotoxicity of AgNPs in BEAS-2B cells. Comet assay was performed after 4 h (A) and 24 h (B) exposure to AgNPs. The induction of DNA damage was evaluated after 4 and 24 h incubation with 10 μg/mL AgNPs. DNA damage was expressed as % DNA in tail. Results are presented as mean ± standard deviation of at least 3 independent experiments. Significant results as compared to the control are marked with asterisks (* for P-value < 0.05, ** for P-value < 0.01). Immunocytochemistry of γH₂AX foci in BEAS-2B cells was performed after exposure to AgNPs for 24 h (C). Cells were treated for 24 h with 10 μg/mL AgNPs (10 nm and 75 nm citrate coated) and etoposide 10 μM (positive control). After exposure, cells were fixed and stained for γH₂AX foci (FITC conjugated anti-phospho-histone H₂AX-Ser 139 antibody, green) and nucleus (DAPI, blue). Pictures were taken with a confocal laser scanning microscope, 40X, oil objective. Images are representative for 3 independent experiments.

Figure 4

ROS levels in BEAS-2B cells after exposure to AgNPs. ROS formation after exposure to AgNPs was investigated using the DCFH-DA assay. Cells were incubated with AgNPs (5, 10, 20 μg/mL) for 24 h and then with 20 μM DCF-DA probe for 40 min. Readings (Ex485 nm/Em535 nm) were performed every 5 min over 30 min. Tert-butyl hydroperoxide (TBP, 15 μM) was used as positive control. ROS increase was calculated as mean slope per min and normalized to the unexposed control. Results are presented as mean ± standard deviation of 4 independent experiments.

Figure 5

Intracellular localization of AgNPs in BEAS-2B cells. Intracellular localization of AgNPs was investigated by TEM (A-F with corresponding inserts a-f).TEM images of untreated BEAS-2B cells show no morphological modifications (A, a). After 24 h exposure to 10 μg/mL 10 nm citrate coated (B, b), 10 nm PVP coated (C, c), 40 nm citrate coated (D, d), 75 nm citrate coated (E, e), and 50 nm uncoated (F, f) AgNPs, particles were taken up and contained mainly within membrane-bound structures (b, d, e, f). Vesicular structures consistent with autophagy were detected for the 10 nm PVP coated AgNPs (c).

Figure 6

Uptake of AgNPs by BEAS-2B cells. The cellular Ag dose was quantified by AAS (A). BEAS-2B cells were exposed to 10 μg/mL AgNPs for 4 h and the total cellular Ag content was analyzed by AAS. The Ag dose was expressed as pg per cell. Results are presented as mean ± standard deviation of 2 replicates. The uptake mechanisms were investigated using pharmacological inhibitors and 4°C exposure (B). BEAS-2B cells were pre-incubated with different pharmacological inhibitors at 37°C (clathrin-mediated endocytosis: amantadine 200 μM for 30 min, caveolin/lipid raft mediated endocytosis: nystatin 25 μM for 30 min, macropinocytosis: amiloride-HCl 100 μM for 30 min, general fluid-phase endocytosis: wortmannin 400 nM for 30 min, actin-dependent phagocytosis: cytochalasin D 1 μM for 1 h). For energy dependent inhibition of uptake the cells were pre-incubated at 4°C for 30 min. Following the pre-incubations, cells were exposed to 10 μg/mL 10 nm citrate coated or 75 nm citrate coated AgNPs for 2 h in the presence of the inhibitors or at 4°C. The total Ag content was determined using AAS. The results are expressed as % of the corresponding controls and presented as mean ± standard deviation of 2 replicates.

Figure 7

Quantification of the Ag release in cell medium. The amount of released Ag in cell medium (BEGM) after 4 and 24 h at 37°C was quantified by means of AAS and expressed as percentage from the total added Ag (10 μg/mL). Results are presented as mean ± standard deviation of 2 replicates.

Figure 8

Cytotoxicity of the extracellular release Ag fraction. The Ag released fraction in cell medium was obtained by incubating 50 μg/mL AgNPs in BEGM at 37°C for 24 h. BEAS-2B cells were incubated with the respective supernatants for 24 h and cytotoxicity was measured with the Alamar Blue assay. Results are presented as mean ± standard deviation of 2 independent experiments.