Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Fröhlich, M.
LONG-TERM CELLULAR EFFECTS OF CHRONIC EXPOSURE TO NANOMATERIALS
[ Dissertation ] Medical University of Graz; 2013. pp. 152
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Fröhlich Eleonore
- Prassl Ruth
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- Abstract:
- Nano-sized materials could find multiple applications in medical diagnosis and therapy. One main concern is that engineered nanoparticles, similar to combustion-derived nanoparticles, may cause adverse effects on human health by accumulation of entire particles or their degradation products. Chronic cytotoxicity must therefore be evaluated. In order to establish a model for chronic cytotoxicity testing, plain polystyrene nanoparticles (20 and 200 nm), the endothelial cell line EAhy 926, as representatives for epithelial cells, as well as THP-1 cells, representing immune cells, were used. Culturing was performed in a microcarrier cell culture system for anchorage-dependent cells (BioLevitatorTM) and a bioreactor culturing system for in suspension growing cells (CELLine CL350). Viability, mode of action assays, and cytokine secretion served as read-out parameters. The established system was used for cytotoxicity testing of > 50 nm short plain and carboxyl-functionalized multi-walled carbon nanotubes. Cells were cultured for four weeks and exposed to doses of polystyrene particles, which were not cytotoxic upon 24 hours of exposure. In addition, fluorescent polystyrene particles were applied in order to investigate their sub-cellular localization. For comparison, these particles were also studied in regularly sub-cultured cells, a method that has traditionally been used to assess chronic cellular effects. Culturing by using both, microcarrier and bioreactor culture methods, produced very high cell densities. After four weeks of exposure, the number of EAhy 926 cells exposed to 20 nm polystyrene particles decreased by 60% as compared to untreated controls. Fluorescent particles were mainly localized in the lysosomes of the exposed cells. When tested in sub-cultured cells, the same particles decreased cell numbers to 80% of the untreated controls. Dose-dependent decreases in cell numbers were also noted after exposure of microcarrier cultured cells to 50 nm short functionalized multi-walled carbon nanotubes, but not upon exposure to plain nanotubes of the same size. Our findings support that necrosis, but not apoptosis, contributed to cell death of the exposed cells in the microcarrier culture system. In contrast, exposure of THP-1 monocytes to polystyrene particles in bioreactor cultures, revealed unreliable findings. Not only the reaction to the particles, but also growth of untreated THP-1 cells in the bioreactor showed great variations between the experiments. The uptake of fluorescent particles was very poor; only one out of 1000 cells was found to contain particles. When sub-cultured cells were exposed to polystyrene particles, the cell number was reduced as expected in a dose- and size-dependent manner. Here, too, necrosis, following an inflammatory response, contributed to cell death of exposed monocytes. However, exposure to both types of carbon nanotubes showed no changes in cell proliferation, and induced no release of cytokines as compared to untreated cells. In conclusion, the established microcarrier model for anchorage-dependent cells appears to be more sensitive for the identification of cellular effects upon prolonged and repeated exposure to nanoparticles than traditional sub-culturing. In contrast, long-term effects on monocytes are superiorly assessed in sub-cultured cells than in bioreactor cultures. While polystyrene particles partially induce adverse effects on both cell types, carbon nanotubes seemed to be less harmful. These findings could prove short carbon nanotubes to be suitable for applications in biomedical applications.