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68 translocation. In

68 translocation. In addition, Trans Epithelial Resistance was determined before and after exposure. No significant changes were observed. Nanoparticles tested Four groups of silver nanoparticles (Nanocomposix, San Diego, USA) were used: 30 ± 4 nm; 31 ± 5 nm; 69 ± 7 nm and 112 ± 9 nm as determined by TEM; while the hydrodynamic sizes in H20 as determined by DLS were 47 ± 5 nm; 70 ± 2 nm; 67 ± 4 and 115 ± 6 respectively. Methoxy (polyethylene glycol)-thiol (mPEG-SH) coated silver nanoparticles (4nm and 35nm) were a kind gift from Dr. P. Christian from the University of Manchester (UK). In addition, AgNO3 was used in a reference group. Concentrations used in the translocation experiment ranged from 5 to 25 µl/ml and were shown to be not cytotoxic to Caco-2 cells by WST-1 assay. We detected a considerable dissociation of silver ions from the silver nanoparticles in the exposure medium; 17% from the 30 and 31 nm sized nanoparticles and 6% from the 69 and 112 nm sized nanoparticles. Gene expression & Translocation The effects of the silver nanoparticles on gene expression of the Caco-2 and Mcells was assessed by transcriptomics. For this the monolayer was lysed using trizol, subsequently RNA was isolated, purified and Cy5-labeled. Isolates were hybridized on Agilent whole human genome microarrays. Briefly, both the groups exposed to silver nanoparticles and ionic silver resulted in an upregulation of 2 to 80 genes. Functional information could be attained for 79 genes, following Metacore analysis. The genes have been described being involved in processes such as proliferation (23), response to oxidative stress (20), metal ion binding (19), unfolded protein response or ER stress (9), apoptosis (9), cell structure and migration (8), other stress responses (6) and other functions (5). A proportion of the genes play a role in two processes. Specifically metallotheinins genes HSPA6 and HMOX1 were amongst the highest up-regulated genes. In a follow-up study we assessed the translocation of a selection of the uncoated and coated Ag nanoparticles. This was determined following an exposure of 4 hours. Silver translocation was determined by means of ICP-MS

in samples from the upper and lower compartments and subsequent calculation of the translocation (Paap). In this study we used both two smallest-sized uncoated Ag nanoparticles (20 and 30 nm) and two mPEG-SH coated Ag nanoparticles. In addition, we evaluated the translocation of AgNO3 as a control group. We observed significant overall differences in translocation rates (Paaps). Post-hoc analysis revealed that the coated nanoparticles were significantly more translocated than the uncoated (p=0.006). The coated Ag nanoparticles were also translocated significantly more (p=0.03) than the Ag + from the AgNO3 control group, while no differences between the uncoated nanoparticles and Ag + -group were observed. Furthermore, translocation of either the coated or uncoated nanoparticles were not size and concentration dependent. TEER was measured before and after exposure to Ag nanoparticles. Conclusion In conclusion, for our microarray study we were mainly interested in the primary effects of the silver NPs, and therefore selected a short exposure time of 4 h. Using SAM analysis, 97 genes were found to be significantly up-regulated by at least one treatment. No gene was down-regulated, which is likely due to the short exposure time. An important aim from the microarray experiments was to detect possible nanoparticle specific effects. However, genes affected by any of the nanoparticles were at least to some extent affected by AgNO3 as well. We detected a considerable dissociation of silver ions from the silver nanoparticles in the exposure medium around the silver ion concentrations of the AgNO3 (1.5 µg/ml) group. It is, therefore, most likely that the gene expression changes are completely caused by the effect of silver ions. This implies that at least in the present experimental setting, nanoparticles themselves have no effect on the gene expression in Caco-2 cells. The results of our translocation experiment clearly shows the importance of coating of nanoparticles on the translocation. 69

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