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Sigma Xi Student Research Presentation
- 1. Effect of the Nano-Bio Interface on the Genotoxicity of Titanium Dioxide Nanoparticles and Associated Cellular Response Raju Y. Prasad Ph.D Candidate Environmental Science and Engineering Department UNC Gillings’ Global School of Public Health GPSF University Research Day February 26, 2012 1
- 2. Brief History of Engineered Nanoparticles “There’s Plenty of Room at the Bottom” – Richard Feynman, Nobel Laureate Physicist, 1959 1985 – First nanoparticle 2001 – President Clinton Development of synthesized (carbon establishes National Nanoparticles for: fullerenes) Nanotechnology Initiative • Drug Delivery • Consumer Products • Industrial Products “The 2013 Federal Budget provides $1.8 billion for the National Nanotechnology Initiative (NNI)” 2
- 3. Definition of Engineered Nanoparticles ~1.6 m ~2 nm width 100 10-1 10-2 10-3 10-6 10-9 1-100 nm meter decimeter centimeter millimeter micrometer nanometer Particles with a size dimension between 1 and 100 nm that show novel properties that are not found in bulk samples of the same material 3
- 4. Titanium Dioxide Nanoparticles Crystal Structures: Anatase, Rutile and Brookite High Refractive Index, White Pigmentation 4
- 5. Use of Titanium Dioxide Nanoparticles in Consumer Products Concentration of nano Ti in consumer products Sunscreens : ~10-100 µg Ti/mg product (1-10%) Sunscreen w/o Sunscreen w/ TiO2 nanoparticles TiO2 nanoparticles Weir et al. Environ Sci Technol 2012 5
- 6. Association of Physicochemical Properties with Toxicity Due to small size dimensions, large surface area to volume ratio, novel properties, and potential exposure, it is important to determine effects on human health Sayes et al. Tox Sci 2006 There is a need to determine physicochemical characteristics of nanoparticles that can lead to adverse effects 6
- 7. Nano-Bio Interface Formation of Protein Corona Interaction with Cells Nel et al. Nature Materials 2009 7
- 8. Impact of Nano-Bio Interface on Genotoxicity Degussa P25 TiO2 nanoparticles Cell Culture Medium: Serum-free KGM KB DM KF • Cell Culture medium • PBS + 0.6% BSA + 0.01% • Cell Culture medium (KGM) + 0.1% BSA DSPC (surfactant) (KGM) + 10% FBS • 2 min probe sonication • 20 min cup sonication of • 2 min probe sonication of stock solution stock solution of stock solution • Centrifuged and re- suspended in cell culture medium (KGM) Effects on Human Lung Epithelial Cells (BEAS-2B) Prasad et al., 2013 ACS Nano 8
- 9. Agglomeration: Physicochemical Characteristic Impacted by the Nano-Bio Interface In liquid medium, TiO2 nanoparticles can agglomerate to form larger particles. The agglomeration profile is affected by the composition of the medium, therefore must be measured to determine its role in genotoxicity Two different media show different agglomeration profiles
- 10. Study Hypothesis The treatment medium that elicits the smallest nanoparticle agglomerates will be associated with cellular interaction and genotoxicity
- 11. Identification of the Protein Adsorbed to the Surface of the Nanoparticles KB DM KF TiO2 in KB TiO2 in DM TiO2 in KF ladder kDa 250 130 95 72 55 36 28 Tedja et al., 2012 ACS Nano, Lundqvist et al., 2008 PNAS 11
- 12. Size Measurements in Treatment Media 0h KB 24 h DM KF Prasad et al., 2013 ACS Nano 12
- 13. No Significant Cytotoxicity in BEAS-2B cells 0 µg/ml 10 µg/ml 20 µg/ml 50 µg/ml 100 µg/ml MMS (100 µM) Li ve: C cei n- al AM D ead: Pr opi di um I odi de Prasad et al., 2013 ACS Nano 13
- 14. Methods: Cellular Uptake Determined by Flow Cytometry Flow Cytometry Histograms ↑uptake in KF blue green red Prasad et al., 2013 ACS Nano 14
- 15. Methods for Evaluation of Genotoxicity DNA Damage Chromosomal Damage Binucleated cell (BN) treat 24 h 48 h Add CytoSpin cells cytochalasin B on glass slides Micronucleated Binucleated cell (MN) Fenech et al. Mutat Res-Fund Mol M 2000 , Kirsch-Volders et al. Mutat Res-Fund Mol M 2003, Singh et al. Mutat Res 1991 15
- 16. TiO2 Nanoparticles Induce DNA Damage in All Media C r ol ont D aged am Prasad et al., 2013 ACS Nano 16
- 17. Chromosomal Damage Only in KF Medium Prasad et al., 2013 ACS Nano 17
- 18. Difference between DNA and Chromosomal Damage: Role of the Cell Cycle Cell Cycle Increased % S phase cells in KF Prasad et al., 2013 ACS Nano 18
- 19. Summary Medium that elicited smallest agglomerates (KF) was associated with highest cellular uptake, micronuclei formation, and increased % of cells in S phase. In contrast, DNA damage was induced regardless of agglomeration and cellular uptake Inhalation exposure to titanium dioxide nanoparticles may not induce chromosomal damage in lung cells, whereas, serum proteins in “blood” (for example, ingestion exposure) may act to lower agglomeration and induce micronuclei formation. Prasad et al., 2013 ACS Nano 19
- 20. Thank you Dr. Rebecca Fry Dr. David DeMarini Dr. Ivan Rusyn Dr. Jim Swenberg Dr. Bill Kaufmann Kathleen Wallace Dr. Carl Blackman Dr. Nana Nikolaishvili-Feinberg Kaitlin Daniel Dr. Jorge Muniz Ortiz Dr. Stephanie Cohen Alan Tennant Dr. Andrew Kligerman Mark Olorvida Jim Campbell Dr. Robert Zucker Bentley Midkiff Micaela Killius Dr. Steve Simmons Mervi Eeva Sharon Leavitt Dr. Kevin Dreher Dr. Stephanie Smith-Roe Ben Castellon Dr. Bellina Veronesi Lisa Smeester Jenna Strickland Dr. Brian Chorley Dr. Kathryn Bailey Danielle Ackerman Dr. Thomas Knudsen Dr. Bhavesh Ahir Peggy Matthews Dr. Kirk Kitchin Dr. Julia Rager Sarah Warren Dr. Will Boyes Alison Sanders Nancy Hanley Dr. Bill Lefew Fry lab Laura Degn Keith Tarpley Kristen Sanders U.S. EPA NHEERL Questions?