Comparison of silver nanoparticles stored under air or argon with respect to the induction of intracellular free radicals and toxic effects toward keratinocytes.

Bacterial infections decreased considerably after the discovery of antibiotics. Nevertheless, because of the rising rate of infections caused by antibiotic-resistant bacteria strains, the search for new bactericidal agents has again become a crucial topic in clinical medicine. Silver nanoparticles (AgNP) have a huge potential in dermatology and wound care management because of their ability to release silver ions (Ag(+) ions) in a prolonged and sustained way. However, negative effects of silver on the patient's cells should not be underestimated. Furthermore, it has been controversially discussed whether AgNP are responsible for nanoparticle-specific outcomes or not. In this study, we investigated the effects of AgNP on human skin keratinocytes (HaCaT) in order to better understand the mechanisms of cytotoxicity and to improve the use of this highly reactive biocide in wound healing. We found that most of the cells with internalized AgNP displayed the typical morphological signs of apoptosis. The cell viability assay (XTT) showed concentration-dependent toxic effects of the AgNP toward HaCaT cells. The generation of reactive oxygen species (ROS) induced by AgNP was investigated in cell suspensions by means of electron paramagnetic resonance (EPR) spectroscopy. In order to distinguish between the effects of Ag(+) ions released during AgNP storage and those of Ag(+) ions released after nanoparticle application, we compared AgNP stored under air (O2) with AgNP stored under argon (Ar). Dispersions of AgNP stored under Ar have a low content of Ag(+) ions because of the absence of oxygen which is needed for oxidative dissolution. The results show that Ag(+) ions released during particle storage are responsible for most of the ROS produced during 1h incubation with the cells. AgNP (Ar) also induced intracellular ROS but to a much smaller extent compared to AgNP (O2). These findings highlight the complexity of experiments to assess the toxicity of AgNP and suggest the possibility of reducing AgNP toxic effects by storing AgNP formulations and even silver-containing wound dressing under an inert gas atmosphere.

Upregulation of metallothioneins after exposure of cultured primary astrocytes to silver nanoparticles.

To test for the prolonged consequences of a short transient exposure of astrocytes to silver nanoparticles (AgNP), cultured primary astrocytes were incubated for 4 h in the presence of AgNP and the cell viability as well as various metabolic parameters were investigated during a subsequent incubation in AgNP-free medium. Acute exposure of astrocytes to AgNP led to a concentration-dependent increase in the specific cellular silver content to up to 46 nmol/mg protein, but did not compromise cell viability. During a subsequent incubation of the cells in AgNP-free medium, the cellular silver content of AgNP-treated astrocytes remained almost constant for up to 7 days. The cellular presence of AgNP did neither induce any delayed cell toxicity nor were alterations in cellular glucose consumption, lactate production or in the cellular ratio of glutathione to glutathione disulfide observed. However, Western blot analysis and immunocytochemical staining revealed that AgNP-treated astrocytes strongly upregulated the expression of metallothioneins. These results demonstrate that a prolonged presence of accumulated AgNP does not compromise the viability and the basal metabolism of cultured astrocytes and suggest that the upregulation of metallothioneins may help to prevent silver-mediated toxicity that could be induced by AgNP-derived silver ions.

Accumulation of silver nanoparticles by cultured primary brain astrocytes.

Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO(3) already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.