Toxicity of nanofillers for polymer composites – in vitro studies

Introduction
Nanoclays, especially layered silicates (bentonite, halloysite) belong to the most popular nanofillers used in polymer nanocomposites. This study was carried out to assess the cytotoxic potency of bentonite and halloysite on commonly used in vitro model for cytotoxicity research.

Methods
Cytotoxicity of bentonite (HB) and halloysite (HN) (<25 µm particle size) was evaluated on CHO and A549 cells after 24-hour exposure. The MTT reduction assay, which assesses the metabolic activity of cells, the NRU assay measuring cell membrane permeability and CFA assay, which assesses the proliferation ability of the cells, were used. The IC50 values (i.e. the concentration required for a 50% cell density/viability reduction) were used as the main measure for comparing the cytotoxicities of the tested compounds.

Results
The tested compounds showed a dose-dependent cytotoxicity evaluated on CHO and A549 cells on the basis of the MTT, NRU and CFA assays. Both compounds showed a similar toxicity in all tests performed on CHO cells, whereas in the case of the studies on A549 cells, the HN showed greater toxicity than HB. There were great differences in the reactivity of A549 and CHO cells to the tested compounds. The IC50 values determined in the three assays in studies performed on CHO cells were over hundred times lower than in the studies performed on A549 cells.

Discussion
A549 cells are largely acknowledged as a suitable model to study the interaction of particulates with lung epithelial cells. The experimental results indicated that A549 cells were a less sensitive experimental model for testing the overall cytotoxic effect of nanoclays (IC50 values were over 100 times higher in comparison with the CHO cells). On the other hand, studies on A549 cells have shown differences in the toxicity of tested compounds. Halloysite, which has a tubular structure resembles that of carbon nanotubes, was more toxic than bentonite.