Toxicological Effects of Metal Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 195

from the still incomplete data, the studies presented here suggest that the biologi-
cal effects of platinum nanoparticles are generally low compared to other nano-
sized metals such as silver and copper.
In the nanoGEM project, a number of different silver nanoparticles were inves-
tigated regarding their cytotoxic potential. Ag50.EO and Ag50.Citrate nanopar-
ticles were selected to address the influence of particle surface modifications
whereas Ag50.PVP and Ag200.PVP nanoparticles should illustrate the role of
particle size (see Table 1.2). As the sensitivity toward nanomaterial exposure not
only is cell type-specific but also depends on the particle type as well as on the
addressed end point and method, to determine cytotoxicity we applied a multi-
plex in vitro testing strategy. This approach is based on a test matrix that includes
both a series of suitable cell lines and a set of standardized cytotoxicity assays
to obtain a more comprehensive view of the biological impact of nanomaterials.
The well characterized standard cell lines representing various routes of par-
ticle exposure (see Table 8.1) and three in vitro toxicity assays were selected and
adapted specifically for the application of nanomaterials as already described in
more detail earlier (see Chapter 8).
The LDH assay was applied to detect cellular necrosis and displayed a cyto-
toxic effect of Ag50.EO nanoparticles for the lung epithelial cell line RLE-6TN
at a concentration of 32 μg/mL. These cells were also affected during the expo-
sure of surface-modified nanomaterial Ag50.citrate; however, the detected necrosis
was less pronounced compared to the effect of Ag50.EO. The silver nanoparticles
Ag50.PVP and Ag200.PVP exhibit a size distribution around 50 nm and 200 nm,
respectively, and were not found to induce LDH release by cell death when applied
in concentrations up to 32 μg/mL. Intracellular reactive oxygen species (ROS) were
not significantly enhanced during the exposure of any tested silver nanomaterials, as
determined by the DCF in vitro assays.
When using the tetrazolium salt WST-8 to determine the metabolic activity of
proliferating tissue culture cells, clear adverse effects on cellular functions could
be demonstrated for the application of each of the silver nanoparticles. However,
not all of the used cell lines were sensitive to these metal nanomaterials. Alveolar
epithelium-like RLE-6TN, and kidney epithelium-like NRK-52E, as well as mouse
embryo fibroblasts NIH-3T3, cells were more severely affected by Ag50.EO at
0.32 μg/mL, compared to Ag50.Citrate at concentrations of 3.2 μg/mL. The test
cell lines HaCaT (human skin keratinocytes) and MDCK-NBL2 (kidney epithe-
lium-like cells) were affected by Ag50.EO at 32 μg/mL, but no measurable adverse
effects could be determined for Ag50.citrate with these cells. In a similar manner,
the silver ENMs Ag50.PVP and Ag200.PVP displayed differing cytotoxic poten-
tial with these cell lines. At the concentration of 3.2 μg/mL or 32 μg/mL nanosilver
Ag50.PVP was shown to impair proliferation of NRK-52E, MKDCK-NBL2, and
NIH-3T3 or RLE-6TN and HaCaT cell lines, respectively. Instead, 160 μg/mL
of Ag200.PVP was needed to affect the RLE-52E cell line, and other cells lines
were less severely affected by Ag200.PVP compared to Ag50.PVP at the same
concentration.
In parallel, we could establish an indirect assessment of oxidative stress via
the detection of protein modification (i.e., protein carbonylation), which proved