Econdary antibodies at room temperature for 2 hours. Specific protein bands were visualized by the enhanced chemiluminescence assay (Amersham Biosciences, Germany).Statistical analysisAll experiments, except long-term exposure to PPS, were repeated three times. At least triplicates were assessed for each sample. Due to the small sample size (n = 3), all data are described descriptively, as mean 6 standard deviation (SD). To investigate the effects of PPS on cell growth over time (n = 5), repeated measurements ANOVA was performed. For post-hoc analysis, a Bonferroni correction was conducted. A p-value ,0.05 was considered to indicate statistical significance. The statistical analysis was performed by using the statistical software SPSS Version 20.Microscopical evaluation of endothelial cells in microcarrier cultureCell attachment was recorded at regular intervals by staining the nuclei with 5 mg/ ml Hoechst 33342 staining solution (Invitrogen, Austria) for 15 minutes at room temperature. The viability of cells was determined by labelling different cell CB5083 organelles (nucleus, endoplasmic reticulum and mitochondria). An aliquot of the microcarrier cultures was stained with Hoechst 33342, 1 mM ER-Tracker green and 200 mM MitoTracker DeepRed 633 (Invitrogen). After incubation for 20 minutes at 37 uC and 5 CO2, staining was documented on a LSM510 Meta confocal laser scanning microscope (Zeiss, Germany). Changes in cell number and viability were recorded weekly. GEMTM were dissolved in pre-warmed trypsin/EDTA (PAA). Detachment of the cells was observed under a CKX41 light microscope (Olympus, Japan). Cell number and viability was determined using a CasyONEH cell counter (Inovatis, Germany) [36].ResultsPrior to the assessment of the long-term effects in 3D culture, NPs were characterized according to their physicochemical parameters and to their acute cytotoxic action in conventional culture.Particle characterizationThe hydrodynamic sizes of PPS were larger when diluted in medium, especially in the presence of FBS than indicated by the supplier. The differences between the indicated size and the hydrodynamic radius in medium with 10 FBS, were more pronounced for the 20 nm PPS than for the 200 nm PPS. All particles were negatively charged except 200 nm PPS when dissolved in DMEM with 10 FBS. Sizes of the .50 nm CNTs increased markedly when protein-free 
medium was used. A summary of the particle properties is presented in Table 1.Mode of NP action in microcarrier cell culturesInduction of apoptosis and/or necrosis of the long-term exposed cultures were assessed weekly and were compared to untreated controls. Mean values were normalized to total cell numbers in the culture vessels at each time point. To determine the main mode of cell death in PPS-exposed cultures, an CP21 web ApoTox-GloTM Triplex Assay (Promega) was performed. Thereby, the effects on viability, cytotoxicity, and apoptosis induction were assessed simultaneously at 24 hours and 7 days after exposure. The assay was performed according to manufacturer’s instructions.Acute cytotoxicityAfter exposure of cells to 20 nm PPS for 24 
hours, the cell viability was reduced in a dose-dependent manner (Fig. 1 A). Addition of serum to the medium decreased the cytotoxicity of the particles. The estimated IC50 was approximately 120 mg/ml in DMEM supplemented with 10 FBS and 30 mg/ml in medium without FBS. The viability of cells after exposure to 20 nm PPS did not decrease when cells were grown on GEMTM.Econdary antibodies at room temperature for 2 hours. Specific protein bands were visualized by the enhanced chemiluminescence assay (Amersham Biosciences, Germany).Statistical analysisAll experiments, except long-term exposure to PPS, were repeated three times. At least triplicates were assessed for each sample. Due to the small sample size (n = 3), all data are described descriptively, as mean 6 standard deviation (SD). To investigate the effects of PPS on cell growth over time (n = 5), repeated measurements ANOVA was performed. For post-hoc analysis, a Bonferroni correction was conducted. A p-value ,0.05 was considered to indicate statistical significance. The statistical analysis was performed by using the statistical software SPSS Version 20.Microscopical evaluation of endothelial cells in microcarrier cultureCell attachment was recorded at regular intervals by staining the nuclei with 5 mg/ ml Hoechst 33342 staining solution (Invitrogen, Austria) for 15 minutes at room temperature. The viability of cells was determined by labelling different cell organelles (nucleus, endoplasmic reticulum and mitochondria). An aliquot of the microcarrier cultures was stained with Hoechst 33342, 1 mM ER-Tracker green and 200 mM MitoTracker DeepRed 633 (Invitrogen). After incubation for 20 minutes at 37 uC and 5 CO2, staining was documented on a LSM510 Meta confocal laser scanning microscope (Zeiss, Germany). Changes in cell number and viability were recorded weekly. GEMTM were dissolved in pre-warmed trypsin/EDTA (PAA). Detachment of the cells was observed under a CKX41 light microscope (Olympus, Japan). Cell number and viability was determined using a CasyONEH cell counter (Inovatis, Germany) [36].ResultsPrior to the assessment of the long-term effects in 3D culture, NPs were characterized according to their physicochemical parameters and to their acute cytotoxic action in conventional culture.Particle characterizationThe hydrodynamic sizes of PPS were larger when diluted in medium, especially in the presence of FBS than indicated by the supplier. The differences between the indicated size and the hydrodynamic radius in medium with 10 FBS, were more pronounced for the 20 nm PPS than for the 200 nm PPS. All particles were negatively charged except 200 nm PPS when dissolved in DMEM with 10 FBS. Sizes of the .50 nm CNTs increased markedly when protein-free medium was used. A summary of the particle properties is presented in Table 1.Mode of NP action in microcarrier cell culturesInduction of apoptosis and/or necrosis of the long-term exposed cultures were assessed weekly and were compared to untreated controls. Mean values were normalized to total cell numbers in the culture vessels at each time point. To determine the main mode of cell death in PPS-exposed cultures, an ApoTox-GloTM Triplex Assay (Promega) was performed. Thereby, the effects on viability, cytotoxicity, and apoptosis induction were assessed simultaneously at 24 hours and 7 days after exposure. The assay was performed according to manufacturer’s instructions.Acute cytotoxicityAfter exposure of cells to 20 nm PPS for 24 hours, the cell viability was reduced in a dose-dependent manner (Fig. 1 A). Addition of serum to the medium decreased the cytotoxicity of the particles. The estimated IC50 was approximately 120 mg/ml in DMEM supplemented with 10 FBS and 30 mg/ml in medium without FBS. The viability of cells after exposure to 20 nm PPS did not decrease when cells were grown on GEMTM.
