Silver nanoparticles (Ag-NPs) are widely used as biocides in everyday products including sanitization products, textiles, electronics, paints, food packaging and personal care products. This extensive use leads to contamination of the environment and possible adverse effects on humans. The toxicity of silver nanoparticles is known for long to be related to their dissolution, which releases some toxic Ag(I) ions. The objective of the project was to characterize the kinetics of dissolution of Ag-NPs and to correlate it with toxic properties, in particular the impact of Ag-NPs on the DNA of human cells. Moreover, it aimed at identifying the transformation products of Ag-NPs, i.e., the intracellular ligands that complex the Ag(I) ions released from Ag-NPs, which would inform on the mechanisms of their toxicity.


Using X-ray absorption spectroscopy (XAS) on the FAME beamline (BM30B) at ESRF, we analyzed the chemical speciation of Ag in A549 human lung alveolar cells exposed to two Ag-NPs, known to have distinct dissolution rates. The first Ag-NPs (NP1) is supposed to rapidly dissolve, while the surface of the second Ag-NP (NP2) is coated with polyvinylpyrrolidone (PVP), which is supposed to limit its dissolution. We proved that intracellular dissolution of NP1 was much more rapid than that of NP2, with 54% of NP1 being dissolved after 24 h of cell exposure, while only 13% of NP2 was dissolved in the same condition (Figure 1A, B). This was correlated to a stronger toxicity of NP1, compared to NP2.


Figure 1. Ag-NP dissolution inside A549 alveolar lung cells.
Ag K-edge XANES analysis of cells exposed for 24, 48 or 96 h to NP1 or NP2 (A, B). Transmission electron microscopy images (C, D) and energy dispersive spectroscopy analysis (F-G) of Ag deposits in A549 cell cytoplasm. E is a HAADF image of the Ag deposit, F and G are the images of Ag and S distributions in the deposit, respectively.
n.: nucleus, c.: cytoplasm, m.: mitochondria.

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Due to their abundance in the cell, the most likely ligands for Ag(I) ions are glutathione (GSH), cysteine and thiolated proteins, including metallothioneins and Zn-finger proteins.
GSH is involved in the maintenance of cellular oxidative balance, while metallothioneins are chelators of metal ions, involved in their detoxification but also in the maintenance of the oxidative balance. Zn-finger proteins have a wide range of molecular functions and among them some transcription factors are Zn-finger proteins, because Zn-fingers have ability to bind to DNA. Results from this analysis suggest that the preferred ligands for Ag(I) ions in this lung cell line are proteins rather than the low molecular weight GSH molecule, as the distance between Ag atoms and S atoms in the complex is > 2.45Å, while Ag-S distance when Ag(I) is complexed with GSH is rather close to 2.40Å (Veronesi et al., Inorganic Chemistry, 2015). Interestingly, the same experiment had been performed earlier in mouse macrophages and had revealed that, in this specific cell line, Ag(I) released from Ag-NPs rather formed complexes with GSH (Veronesi et al., Nanoscale, 2015). This suggests that the cell type, due to its specific GSH content, plays a major role in the intracellular chemical speciation of Ag and therefore in its toxicity. Indeed, disturbing the GSH function leads to impaired redox balance in human cells, as this small molecule is a major cellular antioxidant. This, in turn, would lead to oxidative stress. In lung cells, Ag(I) ions are rather scavenged by metallothioneins and/or Zn-finger proteins, suggesting toxicological impact on metal homeostasis, on gene expression but also oxidative stress.

In terms of toxicity, this progressive dissolution of Ag-NPs inside A549 cells was correlated with the occurrence of cell death, of increased reactive oxygen species (ROS) content inside cells and of oxidative damage to the DNA. Moreover, it impaired the cell’s ability to repair damaged DNA, via mechanisms that are currently unknown. The cell cycle was also affected, with an overall cell cycle blockade in conditions of high Ag-NP dissolution. Cell cycle blockade is known to occur in cells with highly damaged DNA, it is supposed to provide the cell enough time and resource for DNA repair. Therefore, all the observed events are closely intricated: Ag-NPs dissolve and release Ag(I) ions, which combine with thiolated proteins inside the cells. This leads to impaired antioxidant activity, causing the cell’s inability to eliminate the endogenous ROS, which subsequently attack DNA and other biomolecules. DNA repair is also affected, causing the persistence of DNA damage, which leads to cell cycle blockade.