The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles
Introduction
Nanotechnology allows for an efficient exploitation of the antimicrobial properties of silver by using silver in the form of nanoparticles. These are used in applications such as preservatives in cosmetics, textiles, water purification systems, coatings in catheters and wound dressings. The anticipated widespread exposure to silver nanoparticles in the near future has prompted governmental bodies and the public to raise questions about the safety of such applications [1], [2], [3]. In response, many studies investigating the effects of various types of silver nanoparticles in different test systems are now emerging in the scientific literature.
A recent kinetic and distribution study in rats with silver nanoparticles of three different sizes demonstrated that after 28 days of intravenous administration, all particles mainly distributed to organs containing high numbers of phagocytosing cells, such as liver, spleen, and lung [4]. This is not surprising, as these cell types have an important function in clearing the body of particulate matter. At the same time, a number of studies have shown that silver nanoparticles may induce cytotoxicity in phagocytosing cells, such as mouse peritoneal macrophages, but also human monocytes [5], [6], [7], [8]. It has been suggested that the cytotoxic effects were induced by reactive oxygen species (ROS) resulting in cellular apoptosis, at least at low concentrations and short incubation times [5], [9], [10], [11], [12]. The production of ROS has also been implicated in DNA damage caused by silver nanoparticles, which was reported in a number of in vitro studies [13], [14], [15], [16], [17].
The effects on markers of inflammation are much less understood, with some studies reporting an anti-inflammatory effect of silver nanoparticles by suppressing cytokine production in post-operative adhesion or burn wound models and in stimulated human peripheral blood mononuclear cells [18], [19], [20], [21] while others report the induction of pro-inflammatory cytokines in rat peritoneal and alveolar macrophages, human mesenchymal stem cells and in an in vitro blood brain barrier model [6], [9], [11], [16], [22].
Another reported effect of silver nanoparticles is the induction of various morphological malformations in developing zebrafish embryos [23], [24], [25] and impairing development and implantation of mouse blastocysts [26].
For risk assessment purposes, it is useful to know which of these reported effects of silver nanoparticles is the most relevant for further investigation, i.e. whether one effect is more pronounced than another at similar exposure concentrations. Comparison across studies is complicated, as materials of different sources, sizes, coatings and manufacturing processes have been studied. Another important question from the view of risk assessors is: How do the effects of silver nanoparticles compare to those of other forms of silver such as ionic silver?
The aim of this study was to investigate the role of nanoparticle size in these effects, using well-characterized silver nanoparticles of the same source in assays for various toxicity endpoints and compare these findings to effects reported in the literature. Cytotoxicity was investigated in two commonly used cell types that may be exposed to nanoparticles: L929 murine fibroblasts and RAW 264.7 murine macrophages, and effects of silver nanoparticles were compared to those of silver in ionic form. In addition, in view of the phagocytic and immune-related function of macrophages, we investigated the role of silver nanoparticle size on the generation of ROS and on parameters of inflammation. Lastly, the role of silver nanoparticle size was investigated in assays for developmental toxicity and genotoxicity in mouse embryonic stem cells and embryonic fibroblasts, respectively.
Section snippets
Silver nanoparticles
Silver nanoparticles of average nominal diameters of 20, 80, and 110 nm were provided by nanoComposix, Inc. (San Diego, CA, USA). The nanoparticles were synthesized by aqueous reduction synthesis from silver salts and then purified 20× in a 4 mm phosphate buffer (pH 7.4, mono- and di-sodium phosphate) and then concentrated. Nanoparticles were characterized for physicochemical properties, purity of residual synthesis products, and concentration prior to synthesis and after dilution in phosphate
Metabolic activity (WST-1) in fibroblasts and macrophages
In both L929 fibroblasts and RAW 264.7 macrophages, metabolic activity was decreased concentration-dependently by silver nanoparticles as well as by ionic silver, as measured by reduction of WST-1 (Fig. 2, Table 3). For ionic silver, the decrease in metabolic activity was similar between the cell types (EC20 values of 7.1 and 6.7 μg/ml for L929 and RAW 264.7, respectively) and their confidence intervals overlapped, implying there were no statistically significant differences between the two
Discussion
We compared the effects of silver nanoparticles of different sizes in assays for cellular metabolic activity, membrane integrity, generation of ROS, markers of inflammation, developmental toxicity and genotoxicity. Of all toxicity endpoints studied, the most pronounced effect was observed in L929 fibroblasts, where silver nanoparticles of all tested sizes compromised cell membrane integrity and, at higher concentrations, metabolic activity. The reverse was observed in RAW 264.7 macrophages,
Conclusions
Our collective results suggest that the most pronounced effect of silver nanoparticles is inflicting damage towards a range of different cell types, potentially resulting in a myriad of secondary effects, such as generation of ROS, DNA damage and inhibiting stem cell differentiation. Despite the likelihood that macrophages will be amongst the highest exposed cell types, our studies showed that they may not be the most sensitive to the effects of silver nanoparticles, indicating that other cell
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Prof. Wout Slob for his assistance with the data analysis.
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