Staining for mitochondria and endoplasmic reticulum (ER) demonstrated the viability of the cells (Fig. 4 B).Long-term effects of NPs in microcarrier cultureCells were cultured according to the established protocol (basal membrane coated GEMTM, and incubation protocol for endothelial cells) for four weeks with a medium change performed once per week. After inoculation, NPs were added at a concentration 250 times lower than the concentration where cytotoxicity was seen in the acute cytotoxicity setting (24 hours). Exposure of the cells to 20 mg/ml of 20 nm PPS resulted in a 
significantly reduced cell number already after 7 days, showing a decrease in cell numbers of approximately 50 . Even stronger effects were observed at later time-points. No decrease in cell number was observed when the cells were exposed to 20 mg/ml of 200 nm PPS (Fig. 5 A). Exposure to concentrations of 5?0 mg/ml of CNT decreased cell numbers in a 1326631 dose-dependent manner at day 7 to approximately 75 ?0 of control cells, respectively. However, with prolonged contact the cell populations recovered. The recovery rate was more rapid for cells exposed to 20 mg/ml than for cells that were exposed to 5 mg/ml and the values reached 90 ?0 of the control after 4 weeks (Fig. 5 B). Cell viability was not impaired at any time-point.Intracellular localization of PPSCells were exposed to 20 mg/ml of the red fluorescent PPS in order to study cellular localization. R25 were observed within the cells, order BIBS39 mainly localized in lysosomes (Fig. 4 C).Mode of action in microcarrier culturesLong-term exposure to 20 nm PPS induced an 80 higher activation of the effector caspases 3 and 7 after 7 days as compared to the control cells. Over time, the induction of apoptosis decreased to about 30 of the untreated control (Fig. 6 A). 20 nm PPS induced necrosis with a maximum of LDH release, about 65 higher than in control cells, after 7 days of culturing (Fig. 6 C). At later time-points, LDH release Mirin custom synthesis slightly dropped by about 15 . However, exposure to 20 nm PPS resulted in a 2.5- to 5-fold increase in cytotoxicity, while the viability was slightly reduced, as detected by the ApoTox-GloTM Triplex Assay. No induction of apoptosis was detected (Fig. 6 E). Exposure to 200 nm PPS induced neither apoptosis nor necrosis at any time-point (Fig. 6 A, C, E). CNT induced both, apoptosis and necrosis, in a doseFigure. 3. Growth curve of EAhy 926 cultured on basal membrane coated GEMTM. Two pre-installed protocols for cell culturing epithelial (HEK 293) and endothelial (HUVEC) cells were compared. (d), days.Long-Term Effects of NanoparticlesFigure. 4. EAhy 926 attached to GEMTM. Nuclear staining with 5 mg/ml Hoechst 33342 was performed 1, 5, 7, and 14 days after inoculation (A). Vital dye staining for ER and mitochondria five days after inoculation. Hoechst 33342 dye was used as nuclear counterstain (B). Internalized red fluorescent NPs co-localize with the lysosomal dye LysoSensorTM Green DND-189 but not with the nucleus (blue) (C). doi:10.1371/journal.pone.0056791.gdependent manner, reaching a 2.5-fold increase as compared to the control upon exposure to a concentration of 20 mg/ml (Fig. 6 B and D). Similar to PPS, the strongest induction of caspases andthe highest release of LDH occurred after 7 days of exposure, after which the levels approached those of control cells.Long-Term Effects of NanoparticlesFigure. 5. Long-term cytotoxic effects of NPs on EAhy 926. Cultures were exposed to PPS over a period of 28 d.Staining for mitochondria and endoplasmic reticulum (ER) demonstrated the viability of the cells (Fig. 4 B).Long-term effects of NPs in microcarrier cultureCells were cultured according to the established protocol (basal membrane coated GEMTM, and incubation protocol for endothelial cells) for four weeks with a medium change performed once per week. After inoculation, NPs were added at a concentration 250 times lower than the concentration where cytotoxicity was seen in the acute cytotoxicity setting (24 hours). Exposure of the cells to 20 mg/ml of 20 nm PPS resulted in a significantly reduced cell number already after 7 days, showing a decrease in cell numbers of approximately 50 . Even stronger effects were observed at later time-points. No decrease in cell number was observed when the cells were exposed to 20 mg/ml of 200 nm PPS (Fig. 5 A). Exposure to concentrations of 5?0 mg/ml of CNT decreased cell numbers in a 1326631 dose-dependent manner at day 7 to approximately 75 ?0 of control cells, respectively. However, with prolonged contact the cell populations recovered. The recovery rate was more rapid for cells exposed to 20 mg/ml than for cells that were exposed to 5 mg/ml and the values reached 90 ?0 of the control after 4 weeks (Fig. 5 B). Cell viability was not impaired at any time-point.Intracellular localization of PPSCells were exposed to 20 mg/ml of the red fluorescent PPS in order to study cellular localization. R25 were observed within the cells, mainly localized in lysosomes (Fig. 4 C).Mode of action in microcarrier culturesLong-term exposure to 20 nm PPS induced an 80 higher activation of the effector caspases 3 and 7 after 7 days as compared to the control cells. Over time, the induction of apoptosis decreased to about 30 of the untreated control (Fig. 6 A). 20 nm PPS induced necrosis with a maximum of LDH release, about 65 higher than in control cells, after 7 days of culturing (Fig. 6 C). At later time-points, LDH release slightly dropped by about 15 . However, exposure to 20 nm PPS resulted in a 2.5- to 5-fold increase in cytotoxicity, while the viability was slightly reduced, as detected by the ApoTox-GloTM Triplex Assay. No induction of apoptosis was detected (Fig. 6 E). Exposure to 200 nm PPS induced neither apoptosis nor necrosis at any time-point (Fig. 6 A, C, E). CNT induced both, apoptosis and necrosis, in a doseFigure. 3. Growth curve of EAhy 926 cultured on basal membrane coated GEMTM. Two pre-installed protocols for cell culturing epithelial (HEK 293) and endothelial (HUVEC) cells were compared. (d), days.Long-Term Effects of NanoparticlesFigure. 4. EAhy 926 attached to GEMTM. Nuclear staining with 5 mg/ml Hoechst 33342 was performed 1, 5, 7, 
and 14 days after inoculation (A). Vital dye staining for ER and mitochondria five days after inoculation. Hoechst 33342 dye was used as nuclear counterstain (B). Internalized red fluorescent NPs co-localize with the lysosomal dye LysoSensorTM Green DND-189 but not with the nucleus (blue) (C). doi:10.1371/journal.pone.0056791.gdependent manner, reaching a 2.5-fold increase as compared to the control upon exposure to a concentration of 20 mg/ml (Fig. 6 B and D). Similar to PPS, the strongest induction of caspases andthe highest release of LDH occurred after 7 days of exposure, after which the levels approached those of control cells.Long-Term Effects of NanoparticlesFigure. 5. Long-term cytotoxic effects of NPs on EAhy 926. Cultures were exposed to PPS over a period of 28 d.