Pe (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 GFT505 site 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. 200 nm PPS exhibited no cytotoxicity, irrespective of the medium used (Fig. 1 A ). Exposure of cells to CNT dissolved in serum-containing DMEM decreased cell viability similarly to 20 nm PPS (Fig. 1 B); the estimated IC50 was approximately 125 mg/ml. No changes in cell viability were detected when cells were seeded onto GEMTM (Fig. 1 C).Western blot analysis for PARP-In order to discriminate between apoptotic and necrotic cell death, a Western blot for poly (ADP-ribose) polymerase (PARP-1) was performed 7 days after exposing microcarrier cultures to both, PPS and CNTs. To obtain a positive control, cells were treated with 1 mg/ml staurosporine for 5 hours. Upon washing cells with ice-cold PBS, cell lysates were prepared in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 Triton X-100, 0.5 sodiumLong-Term Effects of NanoparticlesTable 1. Characterization of tested NMs.EED226 web ParticleSize (nm) Aqua bidest. DMEM DMEM+10 FBS 73.59*/12.37 224.8 58.26 50.f-pot.(mV) Aqua bidest. DMEM 237.1 257.6 211.1 n.d.{ 213.4 211.0 228.2 216.9 DMEM+10 FBS 211.7 8.08 210.3 211.PPS 20 nm PPS 200 nm Fluoro-Max 1081537 20 nm MWCNT .50 nm22.76 211.6 22.22 n.d.{28.89 211.5 44.56 602.*Predominant peak is indicated first, {Not determined due to aggregates. doi:10.1371/journal.pone.0056791.tMode of actionLow concentrations (,200 mg/ml) of 20 nm PPS had no influence on membrane integrity or on activation of caspase.Pe (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. 200 nm PPS exhibited no cytotoxicity, irrespective of the medium used (Fig. 1 A ). Exposure of cells to CNT dissolved in serum-containing DMEM decreased cell viability similarly to 20 nm PPS (Fig. 1 B); the estimated IC50 was approximately 125 mg/ml. No changes in cell viability were detected when cells were seeded onto GEMTM (Fig. 1 C).Western blot analysis for PARP-In order to discriminate between apoptotic and necrotic cell death, a Western blot for poly (ADP-ribose) polymerase (PARP-1) was performed 7 days after exposing microcarrier cultures to both, PPS and CNTs. To obtain a positive control, cells were treated with 1 mg/ml staurosporine for 5 hours. Upon washing cells with ice-cold PBS, cell lysates were prepared in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 Triton X-100, 0.5 sodiumLong-Term Effects of NanoparticlesTable 1. Characterization of tested NMs.ParticleSize (nm) Aqua bidest. DMEM DMEM+10 FBS 73.59*/12.37 224.8 58.26 50.f-pot.(mV) Aqua bidest. DMEM 237.1 257.6 211.1 n.d.{ 213.4 211.0 228.2 216.9 DMEM+10 FBS 211.7 8.08 210.3 211.PPS 20 nm PPS 200 nm Fluoro-Max 1081537 20 nm MWCNT .50 nm22.76 211.6 22.22 n.d.{28.89 211.5 44.56 602.*Predominant peak is indicated first, {Not determined due to aggregates. doi:10.1371/journal.pone.0056791.tMode of actionLow concentrations (,200 mg/ml) of 20 nm PPS had no influence on membrane integrity or on activation of caspase.
