Real Time Cell Electronic Sensing (RT-CES) for Nanotoxicity Evaluation

Transcription

1 Real Time Cell Electronic Sensing (RT-CES) for Nanotoxicity Evaluation Reyes Sierra 1, Lila Otero 1, Scott Boitano 2 & Jim Field 1 1 Dept Chemical & Environmental Engineering 1 2 Dept Cell Physiology The University of Arizona RSIERRA@ .ARIZONA.EDUARIZONA EDU SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

2 NANOPARTICLES Nanoparticles (NPs) are particles sized in less than 100 nm. 100,000 smaller Nano = Dwarf (Greek) = SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

3 INTRODUCTION Conventional Filtration Microfiltration Ultrafiltration RO Nanoparticles 3 3 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

4 Unique Properties of Nanoscale Materials Small size High specific surface area (> 100 m 2 /g) Quantum effects (dual behavior, wave- and particle-like) unique mechanical, electronic, photonic, and magnetic properties 4 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

5 Increase of Surface Area with Decreasing Particle Size Nel et al. Science, 2006, 311: SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

6 NPs - Applications Increasing industrial i / commercial applications Catalysis Medicine Environmental technology Cosmetics Semiconductors Microelectronics Nanotechnology 1 trillion US $ market by SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

7 NPs in Semiconductor Manufacturing CMP slurries SiO 2 Al 2 O 3 CeO 2 NPs for immersion lithography Carbon nanotubes Colloidal silica ( nm) (Source: 7 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

8 NPs in Chemo-Mechanical Planarization Untreated CMP effluent ( mg/l) 8 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

9 Nanomaterials ESH Concerns Concern about the adverse effects of NPs on biological systems - ENM: unusual properties due to their small size - Increasing evidence that some NP cause toxicity Poor understanding of nanotoxicity - Uncertainty about the real-life hazards of engineered NPs Need for improved bioassays to evaluate nanotoxicity SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

10 Problems Assessing Nanotoxicity Quenching of fluorescence Reduction of MTT dye by NP surface Sequestration of insoluble MTT dye by CNT Interference in classical methods dependent on colorimetric or fluorimetric measurements. NP characterization most studies do not include characterization of NPs in biological medium. 10 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

11 Real Time Cell Electronic Sensing (RT-CES) E-Plate 96 The RT-CES system measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of culture plates. SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing 11

12 E-plate 12 Chem. Res. Toxicol. 2005, 18,

13 Cell Index CI- Quantitative measure of the overall status of the cells: Cell number Cell adhesion and spreading Cell morphology Increase in Cell Index with cell number Chem. Res. Toxicol. 2005, 18, Cell morphology before (B) and after 3h treatment t t with As(III) (C) Chem. Res. Toxicol. 2005, 18,

14 Impedance-based Real Time Cell Analysis Advantages (Fluorescent) label free Dynamic data of the biological status of the cells Noninvasive High throughput technique Limitations Requires adherent cells Correlation analysis between RT-CES and classical toxicity endpoints performed on a limited number of compounds Limited information of its applicability to NPs. 14 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

15 OBJECTIVES Assess the applicability of a real time cell electronic sensing (RT-CES) technique based on impedance measurements to evaluate the cytotoxicity t it of nanosized inorganic i oxides used in semiconductor manufacturing Validation of the RT-CES assay results: RT-CES vs. MTT Characterizing the aggregation g of nanomaterials in the biological medium. 15 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

16 RT-CES VS. MTT BIOASSAYS Human bronchial epithelial l cells (16HBE14o-) RT-CES Impedance based RT-CES assay: (xcelligence, Roche) MTT O 2 uptake Cell membrane integrityi 16 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

17 IMPEDANCE-BASED RT-CES Impedance-based d Real Time Cell Analysis (RTCA) (RT-CES, xcelligence, Roche) - Lung epithelial cells: 16HBE14o- 16 HBE culture E-Plate 96 MEM 1 (10% FBS 2 ) 37ºC MEM (3.75% FBS) 100,000 cells/well 37ºC RT-CES system 37ºC o o 1 Minimum Essential Medium 2 Fetal Bovine Serum Cells are transferred to the E-Plate at 100,000 cells/well. NPs are dosed after 16 h of incubation. o Cells are monitored for at least 48 h. 17 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

18 RT-CES Bioassay Experiment stages nano-fe Cell Inde ex Washing Control 10 mg/l 100 mg/l 1,000 mg/l Time (hours) Overnight growth Exposure to nanoparticles Response to nanoparticles 18 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

19 RT-CES Bioassay CELL INDEX R cell = Electrode impedance with cells present R b = Electrode impedance without cells N= Number of points measured = 3 19 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

20 Example Output RT-CES with As(III) 8 7 Dosing of As(III) Control 6 Ce ell Index mg/l mg/l -> 100 mg/l -> 10 mg/l mg/l Time (hours) Control As 500 ppb As 2,500 ppb As 10,000 ppb As 30,000 ppb As 100,000 ppb 20 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

21 RT-CES: Al 2 O 3 Nanoparticles Al 2 O 3 Nanoparticles (50 nm) - IC 50 = 300 mg/l control 250 ppm Al 2 O ppm Al 2 O ppm Al 2 O 3 21 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

22 RT-CES: SiO 2 nanoparticles SiO 2 Nanoparticles (10-20 nm) - IC 50 = 225 mg/l Control 100 mg/l 200 mg/l 600 mg/l 300 mg/l 22 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

23 RT-CES: CeO 2 nanoparticles CeO 2 Nanoparticles (50 nm) - IC 50 > 1,000 mg/l 23 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

24 MTT Bioassay Assay relies on the reduction by live cells of the watersoluble tetrazolium MTT salt to a colored formazan dye. Indicator of cell redox activity and viability 24 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

25 MTT BIOASSAY Cells MEM (10% FBS) 37 C, 5% CO 2 24 wells plate MEM (3.75% FBS) 500,000 cells/well Incubation 37 C, 5% CO 2 Staining MTT reagent 0.4 mg/ml Measurement Plate reader 550 nm o o o Cells are transferred to a 24-well plate ( cells/well) NPs dosedd after 24 h of incubation 25 After 48 h, cells are washed and stained with MTT reagent

26 MTT BIOASSAY: NANO- AL 2 O Al 2O 3 NP (IC 50 = 980 mg/l) 0.8 Ab bsorbance ,000 1,000 Blank (no cells) (DMSO) Concentration (mg/l) NP did not interfere with the MTT analysis. Cell-free controls with the highest NP level caused a marginal increase of the absorbance relative to the DMSO control (2-3% of max. absorbance, depending on the NP used) 26

27 RT-CES & MTT BIOASSAY: NANO- SIO 2 SiO 2 NPs- IC 50 = 225 mg/l (RT-CES), 172 mg/l (MTT) 120 control) Activi ity (% of MTT RT-CES Conc. (mg/l) 27 xcelligence SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing MTT

28 COMPARISON RT-CES vs MTT RESULTS IC50 > 1,000 mg/l 50 % Inhibito ry Conc (m mg/l) SiO 2 Al 2 O 3 CeO 2 ZrO 2 ZnO Mn2O3 Ag0 SiO2 Al2O3 CeO2 Fe0 Fe2O3 TiO2 ZrO2 Nanomaterial RT-CES RTCA MTT Conclusions: Good correlation between RT-CES and MTT results Al 2 O 3 and SiO 2 moderate toxicity, CeO 2 not toxic 28

29 Aggregation of NPs in Biological Medium Particle size distribution (PSD) & chemical analysis Same conditions as in toxicity assays PSD & Zeta Potential (DLS) Sampling Concentration (ICP-OES) 29 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

30 AGGREGATION OF NPS IN BIOLOGICAL MEDIUM Dispersion Aggregation Sedimentation supernatant total 30 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

31 AGGREGATION OF NPS IN BIOLOGICAL MEDIUM % CeO2 (in super rnatant) CeO 2 0 H2O ph4.5 RT-CES PBS YEDP Microtox Medium In bioassay media, CeO 2 NP agglomerated and the concentration that is 31 effectively dispersed decreased many-fold

32 PROTEIN ADDITION (FBS)- Effect on Stability of Al 2 O 3 NPs Particle size (nm) Particle size at different %FBS 3, % FBS 2% FBS 5% FBS 10% FBS % Rec overy Al 2 O 3 in the supernatant % FBS 2% FBS 5% FBS 10% FBS FBS (%) FBS (%) FBS in the growth medium (MEM) stabilizes the Al 2 O 3 NP dispersions 32 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

33 CONCLUSIONS RT-CES is a useful, high throughput technique for dynamic monitoring of NP cytotoxicity. The test relies on impedance measurements, avoiding interference problems often associated with colorimetric/fluorimetric tests. The inhibitory concentrations determined for NPs using the RT-CES technique correlated well with those generated by a commonly used cytotoxicity t it assay (MTT). Al 2 O 3 and SiO 2 NPs showed moderate toxicity in the MTT and RT-CES assays, CeO 2 was not toxic at very high concentrations (1,000 mg/l) Most of the nanoscale inorganic oxides tested showed a high tendency to aggregate g in the RT-CES & MTT medium resulting in micron-size aggregates that settled out of the dispersion. 33 SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

34 ONGOING RESEARCH: CYTOXICITY MECHANISMS OF INORGANIC OXIDE NPS Release of toxic products Disruption of cell membrane (flow cytometry) ROS formation (intra- and extracellular generation) Protein damage (ELISA bioassay) 34 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing 34

35 ONGOING RESEARCH: REDUCING AGGREGATION OF NPS INBIOLOGICAL MEDIUM Inte ensity (% %) CeO2+Dispex ph CeO2 ph Particle Size Distribution (d.nm) 35 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

36 ACKNOWLEDGEMENTS SEMATECH / SRC UA Nanotoxicity group SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing 36

37 SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing 37