Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells

Abstract

AIM: To study the response to silver nanoparticles (Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis.

METHODS: We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan (MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2’-7’-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope.

RESULTS: Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent modifications occur. It is noteworthy that Ag NP are cytotoxic and genotoxic also for endothelial progenitors, in particular for endothelial colony-forming cells, which participate to angiogenesis.

CONCLUSION: Silver nanoparticles are cytotoxic and genotoxic for human microvascular endothelial cells and might become a useful tool to control excessive angiogenesis.

Keywords: Endothelial cells, Nanoparticles, Genotoxicity, Cytotoxicity, Angiogenesis

Core tip: We studied the sensitivity to silver nanoparticles of microvascular endothelial cells, which are responsible for tissue homeostasis and fundamental in angiogenesis. Silver nanoparticles are cytotoxic and lead to membrane leakage. Cytotoxicity is not prevented by the antioxidants Trolox, N-acetyl-L-cysteine or apocynin. Silver nanoparticles also induce DNA damage as demonstrated by comet assay. When exposed to sublethal concentrations of silver nanoparticles for long times, the cells remain viable but are growth retarded. Interestingly, removal of silver nanoparticle rescue cell growth, suggesting that no permanent modifications occur. Silver nanoparticles are cytotoxic and genotoxic also for endothelial progenitors, which contribute to angiogenesis.