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

Abstract

croscope. 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.© 2014 Baishideng Publishing Group Inc. All rights reserved.Key words: 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. 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 mi-

Silver nanoparticles-induced cytotoxicity requires ERK activation in human bladder carcinoma cells

“…Several studies have reported the toxicity of Ag NP in different cell systems both in vitro and in vivo [6][7][8]. Consequently, the possibility to exploit their cytotoxic activity to treat proliferative diseases is particularly challenging.…”

“…Ag and AgPVP NP (NanoAmor, Houston, USA) were previously used [8]. Briefly, Ag NP are 99.5% pure with an average particle size of 35 nm.…”

“…Cells were seeded into black bottomed 96 plates (Greiner bio-one, Frickenhausen, Germany) and 24 h later exposed for 30 min to different concentrations of NP prepared in a 20 µM DCFH solution. The rate of intracellular oxidative stress was evaluated by monitoring the emission at 529 nm of the DCFH dye using Promega Glomax Multi Detection System [8].…”

Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis

“…It could be that these observations are influenced by AuNP concentrations and culture time. Most studies focus on the short term acute effects of AuNP exposure but long term culture with AuNPs could change gene expressions levels and cell behavioral responses [30,31]. Hence it is also important to investigate such exposure especially as AuNPs are being put to use for long term clinical applications [32,33].…”

Regeneration of Limbal Stem Cells in the Presence of Silver and Gold Nanoparticles

“…The idea of reversibility is in conformity with the observation of others that upon exposure of sublethal concentrations of AgNPs for long times, cells remained viable and their growth retarded. Similarly, genotoxicity was found to be reversible upon removal of AgNPs from cell culture [30], suggesting that chromatin changes are not permanent. However, extended regeneration time associated with the treatment of higher concentrations (160, 320 ppm) of large AuNP particles could raise concern about smaller (<100 nm) nanogold particles that could be toxic alone or in combination with experimental drugs.…”