Developing a Yeast Cell Assay for Measuring the Toxicity of Inorganic Oxide Nanoparticles Citlali Garcia Saucedo Chemical & Environmental Engineering Department University of Arizona May 6 th, 2010 1
Outline Introduction Objectives Materials and Methods Toxicity Testing using Yeast Characterization of Nanoparticles Results Conclusions 2
Nanotechnology Understanding and control of matter at dimensions of roughly 100 nm where unique physical properties make novel applications possible Unusual Physicochemical properties Small size Chemical composition Surface structure Solubility Shape Aggregation http://www.fda.gov/consumer/updates/nanotech072507.html 3
Nanotechnology Applications Medicine Electronics Space Fuel Cells Solar Cells Batteries Cosmetics Nanotechnology has an important role to create many new devices and materials with an infinite range of applications 4
Organization for Economic Cooperation and Development (OECD) List of Nanoparticles (NPs) Fullerenes (C60) Carbon nanotubes Silver Iron Carbon black Titanium dioxide Aluminum oxide Cerium oxide Zinc oxide Silicon dioxide Polystyrene Dendrimers Nanoclays http://www.nanolawreport.com/2008/07/articles/oecd to begin testing nanoparticles/ 5
Engineered NPs in Semiconductor Industry Chemo Mechanical Planarization (CMP) slurries SiO 2 Al 2 O 3 CeO 2 Immersion Photolithography HfO 2 Nanowires Nanotubes Quantum dots 6
Examples of NP Toxicity Studies NPs SiO 2 TiO 2 ZnO CeO 2 Studies In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Cytotoxicity of titanium and silicon dioxide nanoparticles In vitro cytotoxicity assessment of selected nanoparticles using human skin fibroblasts Toxicity of cerium oxide nanoparticles in human lung cancer cells Reference Brunner et al. 2006 Wagner et al. 2009 Dechsakulthorn et al. 2007 Weisheng et al. 2006 7
Release of Nanomaterials to the Environment (Wiesner et al., 2009) 8
Methods to Evaluate Ecotoxicity of Nanoparticles 9
Ecotoxicity Testing of Nanoparticles Microorganisms Foundation of all known ecosystems Basis of food webs and the primary agents for global biogeochemical cycles Important components of soil health Microbial ecotoxicology test are used to study the toxicity of nano materials and to elucidate cytotoxicity mechanisms that could be extrapolated to eukaryotic cells 10
YEAST (Saccharomyces( cerevisiae) Unicellular eukaryotic model organisms Short generation time Used in the toxicological evaluation of chemicals 11
Concerns About the Evaluation of Nanoparticles Cytotoxicity Interferences of NPs on spectrophotometric based techniques Mitochondrial Toxicity Test (MTT) NPs agglomerate in biological medium complicating interpretation of data from toxicity studies Poor characterization of NPs 12
Objectives Develop a yeast based, O 2 uptake test to evaluate the toxicity of NPs To select non toxic dispersants to enhance the stability of NPs in biological media using in toxicity testing. To characterize some physicochemical properties of NPs in toxicity assay medium: particle size distribution and NP concentration. Apply the developed method to test the toxicity of NPS utilized in semiconductor manufacturing and in other important industries, in the presence and absence of dispersant. 13
ZrO 2 (20-30 nm) Materials Mn 2 O 3 (30-60 nm) HfO 2 CeO 2 (50 nm) (20 nm) Nanoparticles (NPs) TiO 2 (25 nm) ZnO (10-30 nm) Al 2 O 3 (30 nm) SiO2 (10-20 nm) 14
Toxicity Test with Yeast S. cerevisiae (0.1%) YEPD* medium, ph: 6.5 NPs + Dispersant (Dispex) 10:1 w/w Sonicated 5 min.70% amplitude DEX 130 20% O 2 O 2 (GC TCD) Incubation: 10 h at 30 C and 200 rpm *YEPD = Yeast Extract Peptone Dextrose 15
Experiment Design: Estimating O 2 Uptake from Theoretical Oxygen Demand (ThOD) 20% O 2 Glucose Head Space Volume Peptone ThOD x (1-cell yield) Liquid Volume [S] Yeast extract Predicted O 2 consumption ~ Amount O 2 in head space 25 ml of YEPD medium, 135 ml of head space and 5 g ThOD/L 16
Characterization of NPs 10 hrs shaking 30 min. statics Samples: Supernatant Total suspension Similar conditions to the toxicity test. Studies without yeast were carried out. Analytical methods: Particle size Zeta potential Concentration 17
Particle Size Distribution Zetasizer Nano ZS Malvern Instruments Dynamic Light Scattering Analyzes the velocity distribution of particle movement by measuring dynamic fluctuations of light scattering intensity caused by Brownian motion of the particle. 18
Zeta potential The electrical potential that exists across the interface of all solids and liquids Zeta Potential [mv] from 0 to ±5, from ±10 to ±30 from ±30 to ±40 from ±40 to ±60 more than ±61 Stability behavior of the colloid Rapid coagulation or flocculation Incipient instability Moderate stability Good stability Excellent stability 19
Concentration of Nanoparticles ICP OES To determine the elemental composition of samples Microwave Assisted Digestions Optima 2100 DV Perkin Elmer To reduce interference by organic matter and to convert metals associated with particulates to a form (usually free metal) that can be determined with ICP 20
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Toxicity of dispersants Polyethylenimine (PEI) 0.0016 0.0014 0.0012 Control PEI dispersant was Oxygen (moles) 0.0010 0.0008 0.0006 0.0004 10 ppm 50 ppm 100 ppm 200 ppm toxic to S. cerevisiae. 0.0002 0.0000 0 2 4 6 8 Time (hrs) 22
Toxicity of dispersants Dispex (Ammonium polyacrylate) Oxygen (mol) Saccharomyces Cerevisiae at different concentrations of Dispex 0.0014 0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 0.0000 0 2 4 6 8 10 Time (hrs) 0 ppm 10 ppm 50 ppm 100 ppm Dispex is not toxic to yeast Dispex can be used in toxicity test to disperse NPs. Dispex:NPs ratio: 1:10 (w/w) 23
Stability of Mn 2 O 3 NP Dispersions in Demineralized Water ph= 6 Dispex stabilizes the NP dispersion in water (ph=6) 66 nm Average Particle Size (nm) Concentration (mg/l) 1400 1200 1000 800 600 400 200 0 300 250 200 150 100 50 0 Supernatant Total Supernatant + Dispex Total + Dispex Mn 2 O 3 (30 60 nm) No Dispex Dispex 24
3000 Stability of Mn 2 O 3 NPs in Yeast Medium ph= 6 Average Particle Size (nm) 2500 2000 1500 1000 500 Dispex increased the stability of NP dispersions in biological medium 66 nm Concentration (mg/l) 0 300 250 200 150 100 50 0 Supernatant Total Supernatant + Dispex Total + Dispex Mn 2 O 3 (30-60 nm) No Dispex Dispex 25
Mn 2 O 3 Nanoparticle with Dispex Saccharomyces Cerevisiae with Mn 2 O 3 + Dispex Oxygen (moles) 0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 Calculate slope Control 100 ppm 500 ppm 750 ppm 1000 ppm 0.0000 0 2 4 6 8 10 12 Time (hrs) Mn 2 O 3 nanoparticle are toxic to yeast at concn. > 500 ppm 26
Activity of Yeast Respiration Mn 2 O 3 Nanoparticles with Dispex 100.00 Activity (% of control) 75.00 50.00 25.00 0.00 IC 50 = 300 ppm 0 200 400 600 800 1000 C (ppm) 27
ZnO Nanoparticle with Dispex Oxygen (mol) 0.0014 0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 0.0000 Sacharomyces cerevisiae with ZnO + Dispex 0 2 4 6 8 10 Time (hrs) Calculate slope Control 50 ppm 75 ppm 100 ppm 250 ppm ZnO NPs toxic to yeast at > 50 ppm 28
Activity of Yeast respiration ZnO Nanoparticle with Dispex Activity (% control) 120 100 80 60 40 20 0 IC 50 =75 ppm 0 50 100 150 200 250 ZnO (ppm) ZnO + Dispex 29
Possible Mechanism of ZnO NP Toxicity 120 Activity (% control) 100 80 60 40 20 0 0 50 100 150 200 250 Zinc (ppm) ZnO + Dispex ZnO + Dispex ZnCl ZnCl 2 Toxicity observed with ZnO NPs could be associated to the Zn(II) ion Kasements et al. (2009) reported that ZnO toxicity was explained by soluble Zn(II) ion 30
Nanoparticles Toxicity NPs 50% inhibition* (ppm) ZnO 75 Mn 2 O 3 300 CeO 2 1000 SiO 2 >1000 HfO 2 >1000 Al 2 O 3 >1000 ZrO 2 >1000 *with Dispersant 31
Conclusions Monitoring of O 2 uptake by yeast cells is a reliable method to study the toxicity of NPs. The addition of the dispersant Dispex improved the stability of the NPs in yeast bioassay medium. Most NPs were not toxic to yeast at 1,000 mg/l. Only CeO 2, Mn 2 O 3 and ZnO displayed toxicity. ZnO was the most toxic compound tested. 32
Current and Future Work Complete the characterization of NPs and evaluation of their toxicity to yeast. Investigate the mechanisms of toxicity Membrane damage (Live/Dead assay, flow cytometry, microscopy) Production of reactive oxygen species (ROS) (Commercial kits) 33
Acknowledgements Dr. Reyes Sierra Dr. James Field Lila Otero, Antonia Luna, Angel Cobo Aida Tapia and Francisco Gomez All the students from the office OE 157 and Lab 168/314 Chemical & Environmental Engineering group Funding SRC/SEMATECH Engineering Research Center on Environmentally Benign Semiconductor Manufacturing (ERC) Thank you! 34
Questions? 35