High concentrations of Sicastar Red (300 μg/ml) exhibited minimal assay interferences (assay reagent in cell culture medium with NPs without cells), which was negligible compared to the respective Epacadostat mouse lysis control (H441: 0.95 ± 0.34% and ISO-HAS-1: 4.4 ± 1.6% of lc). After 4 h NP exposure, the NP

suspension was removed, and the cells were cultured for a further 20 h period to examine IL-8 and soluble sICAM release after NP exposure. Corresponding to the MTS and LDH assay, AmOrSil did not result in any toxic effects on H441 and ISO-HAS-1 concerning IL-8 and sICAM (Fig. 1C). By contrast, Sicastar Red resulted in an IL-8 release in both cell types (H441 and ISO-HAS-1) at 60 μg/ml (H441: 2.1 ± 0.22% and ISO-HAS-1: 2.3 ± 0.1% of uc). Due to the high cytotoxic effects and cell death, which was also observed in the MTS and LDH assay, lower IL-8 levels were measured at higher NP concentrations (100 and 300 μg/ml) compared MEK inhibitor drugs to 60 μg/ml in both cell types. A significant sICAM release was also observed for Sicastar Red at a concentration of 60 μg/ml (H441: 1.8 ± 0.14% and ISO-HAS-1: 1.6 ± 02% of uc). With increasing concentrations (100 and 300 μg/ml), the sICAM level still remained significantly high for H441 (100 μg/ml: 1.3 ± 0.17%, 300 μg/ml: 1.5 ± 0.3% of uc) and was further augmented for ISO-HAS-1 (100 μg/ml: 1.8 ± 0.32%, 300 μg/ml: 2.6 ± 0.4% of uc). Colocalisation of NPs with endosomal marker

proteins belonging to the clathrin-mediated (clathrin heavy chain) or caveolae-mediated (caveolin-1) endocytosis pathways were performed in H441 and ISO-HAS-1 by means of immunofluorescence staining procedures (Fig. 2, only Sicastar Red is depicted, AmOrSil yielded similar results). Neither Sicastar Red nor AmOrSil exhibited an uptake in such organelles after 20 min, 4 h or 4 h incubation followed by further cultivation Vasopressin Receptor for 20 h in fresh serum-containing media. Thus, an early endosomal uptake via this method could not be identified

at the three time points investigated. However, after 4 h incubation followed by 20 h of further cultivation, the fluorescence signals of both NPs were clearly colocalised with flotillin-1 and -2 signals in H441 and ISO-HAS-1 (Fig. 3). The NPs were clearly enclosed by flotillin-1 and -2 containing vesicles. In ISO-HAS-1, colocalisation of NPs with flotillin-1/2 was already observed after 4 h, indicating a faster uptake mechanism in these cells (data not shown). TEM was used to define at higher magnification the cellular uptake of AmOrSil in endosomes of H441 (Fig. 4). The iron oxide core and its poly(organosiloxane) shell were clearly visible, and the NPs were incorporated into endosomal structures. Sicastar Red NPs were not visible via TEM due to its low electron density, which resulted in a low contrast. Thus, this method was not applicable to associate these NPs to a particular subcellular compartment.