Safety and uptake assessment of nanostructured silica in an advanced intestinal in vitro model

Abstract

The food industry has identified the benefits of nanotechnology and exploited the unique properties of engineered nanomaterials (ENM) over the last decade. The number of products available containing ENM or nanostructured materials worldwide is expected to increase even further. Due to this presumed increase in number of available products and manufactur-ing quantities, authorities as well as consumers are concerned about potential adverse ef-fects of nano sized materials in food on public health. Materials directly added to food but also those leaking from the packaging into food might be ingested. Also nanostructured food processing agents, which are added to prevent caking, to improve flowing or to clarify and absorb, might be ingested. Considering the high oral exposure to all these food additives, a better understanding of the uptake, the accumulation and the biological effects of food rele-vant nano sized materials at the intestinal epithelium is needed. Ten differently produced synthetic amorphous silica (SAS) materials with different specific surface areas, different primary structure sizes and different surface charges have been characterised. Their biological impact has been screened in a cell line (Caco 2) representa-tive for the most common cell type in the small intestine, enterocytes. No acute impairment of viability or barrier integrity could be identified. Furthermore, the adhesion and internalization of one representative of fumed and precipitat-ed SAS have been investigated, exploiting flow cytometry, scanning electron microscope coupled with energy dispersive X ray spectroscopy, time of flight secondary ion mass spec-trometry, transmission electron microscopy micrographs, confocal, dark field and hyperspec-tral microscopy. Furthermore, also the impact of food grade titanium dioxide has been inves-tigated in the same setup. Titanium dioxide has been studied to identify if the restrictions in the detection of SAS, were due to the material or the cell environment. The SAS materials were only detected on the cell layer with scanning electron microscope coupled with energy dispersive X ray spectroscopy or the time of flight secondary ion mass spectrometry. It has been shown that for the detection of silica in cell environment a subsequent elemental analy-sis is needed to recognise the SAS materials. In the second part of this work an advanced co culture model has been established to better evaluate the impact of food grade materials in a more in vivo like setting. Caco 2 monocul-ture only presents one cell type of the very complex intestine. The newly established ad-vanced co culture model consists of Caco 2 cells and a mucus producing HT 29 cell line. The addition of B lymphocytes allowed the differentiation of one additional cell type: M cells. The exposition of the advanced co culture model to six different SAS selected due to the different production routes, specific surface areas and their different silanol content has led to no differences in the viability, barrier integrity, microvilli function and lipid uptake. Neverthe-less, the treatment has shown that the mucus production increases after the treatment with SAS materials with an aggregate size above 200 nm and which are highly negatively charged. A co effect has been found for the investigation of the iron cell type precipitated SAS with a small specific surface area decreased the iron uptake in the advanced co culture only in the ferritin uptake but not on the corresponding gene level. This newly established model also offers the possibilities to further investigated broader sci-entific questions. As one other scientific question the colloidal structural formation during milk digestion in the advanced co culture of Caco 2 and HT 29 has been compared with struc-tures formed in a cell free environment during milk digestion. The incorporation of cells has not resulted in different types of structural formations and has not increased the speed at which the colloidal structures are formed during the milk digestion compared to the setup without cells. The results show that the use of this advanced in vitro model can lead to an improved predic-tion on potential adverse outcomes of food components on the intestine. Mucus seems to be a very important protective barrier in the interaction of food components with the intestinal epithelium and should be studied in more detail. The advanced co culture model established in this thesis can be used for a first estimate of the interactions of food components with in-testinal epithelium and a further reduction of animal experiments in the future.