Silver nanoparticle-induced cytotoxicity in rat brain endothelial cell culture
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The emerging field of nanotechnology introduces a wide range of nanoparticles in our daily life. Nanoparticles possess unique physicochemical properties compared to their larger-sized counterparts, which make them attractive for many applications. Silver nanoparticles (AgNPs) are among those nanoparticles which are highly commercialised and they are included in many household products and medical devices because of their antimicrobial properties. However, increasing exposure of humans to AgNPs has raised concerns about adverse health effects. The present study examined the cytotoxic effects of different-sized AgNPs (10, 50 and 100 nm) on rat brain endothelial (RBE4) cells and investigated whether the observed cytotoxicity can be explained by the intrinsic toxicity of the silver nanoparticles themselves, the silver-ions released from the silver nanoparticles or a combined effect of silver nanoparticles and silver-ions. In vitro assays measuring metabolic activity, membrane function and a colony formation assay were employed to study the cytotoxic effects of AgNPs and Ag+-ions. The silver content (total Ag and Ag+-ions) in the samples was measured using high-resolution inductively coupled plasma-mass spectrometry (HR-ICP-MS). C6 Glioma cells were used for initial studies with AgNO3, which served as a source of Ag+-ions. RBE4 cells were exposed to AgNPs and Ag+-ions. The results showed that metabolic activity (MTT assay) of RBE4 cells after exposure to 1 to 25 μg/ml AgNPs was unchanged or increased compared to unexposed cells and was found to be dependent on dose and exposure time (1h to 24h). No clear particle size-dependence of the effects was observed. Interestingly, corresponding concentrations of AgNO3 reduced metabolic activity after 24h. On the other hand, exposure of RBE4 cells to AgNPs led to severe impairment of membrane function, tested with the Neutral red assay, which was found to be particle size- and surface area-, dose- and timedependent. A role of Ag+-ions in the observed effects is questionable as corresponding concentrations of AgNO3 resulted in less membrane damage than the smallest AgNPs. Most intriguingly, colony formation of RBE4 cells was completely inhibited by AgNPs (1 μg/ml), whereas colonies were observed after exposure to AgNO3 (up to 7.5 μM). All these findings suggest that Ag+-ions are not solely responsible for the observed AgNP toxicity on RBE4 cells. Instead, other mechanisms of toxicity, attributed to different properties of the AgNPs, are likely to have an important contribution to the observed effects.