Future Treatment of Oxidative-Stress Related Disorders with Nanotechnology

1. Future Treatment of Oxidative-Stress Related Disorders with Nanotechnology Dr. Beverly A Rzigalinski Virginia College of Osteopathic Medicine Virginia Polytechnic & State University Blacksburg, VA

2. Cerium Oxide • Cerium - Rare earth element • Cerium atom surrounded by oxide lattice • Cerium has multiple valence states • Oxide lattice can have numerous “defects” • Micro sized particles used in coatings, particularly catalytic converters • Redox Reactions • NOX

3. What is Nano - Cerium Oxide? • Nanoscale materials behave differently from their micro-sized counterparts • Valence states of Cerium may change more quickly • Oxygen vacancies or “defects” increase dramatically at the nano scale • Better redox?

4. A Single Dose of Engineered Cerium Oxide Nanoparticles Increases Brain Cell Longevity Control Nano-Treated Days Average Life Span

5. 27 DIV Organotypic brain cell culture 27 DIV Organotypic brain cell Culture treated with 10 nM Ceria Nanoparticles at 10 DIV 123 DIV Organotypic brain cell Culture treated with 10 nM Ceria Nanoparticles at 10 DIV

6. Nanoparticle-Treated Cultures Maintain Normal Neuronal Signaling Throughout Their Extended Lifespan 400 28 DIV Nano-Treated 28 DIV Control 300 [Ca2+]i (nM) 200 68 DIV Nano-Treated - 100 Time (Seconds) 100 mM Glutamate

7. What’s Going On?

8. Ce+3 O2 Free Radical O2 • Regeneration: • Lattice Rearrangements? • Reaction with H2O? • Adsorption of OH or H+? • Other cellular reactants? Ce+4 O2--x?

9. Cerium Oxide Nanoparticles Scavenge Hydroxyl Radicals Generated by the Fenton Reaction Fenton Reaction +10 nm Ceria +7 nm Ceria +Fe-doped Ceria

10. Cerium Oxide Nanoparticles Scavenge Superoxide Radicals Irradiated riboflavin +7 nm Ceria +10 nm Ceria +Fe-doped Ceria

11. CeO2-Nanoparticles Have Activity SOD, GSH, And Catalase-Like Activity 10 nm 7 nm Activity (Units) SOD Glutathione Peroxidase Catalase

12. Cerium Oxide Nanoparticles Protect Cells from UV-Mediated Oxidative Stress

13. Cerium Oxide Nanoparticles Protect Cells Against Oxidative Stress Induced by H2O2 - Size Dependency Control Control 160 160 Control Control 50nm particles 50nm particles 50 nm Particles 50 nm Particles 20nm particles 20nm particles 10 nm Particles 10 nm Particles 140 140 7nm particles 7nm particles 7 nm Particles 7 nm Particles 120 120 4 100 100 80 80 Injured Cells x 10 60 60 40 40 4 20 20 Injured Cells x 10 0 0 m m m m Control 1nM 10nM 100nM 1 Control 1nM 10nM 100nM 1 M 10 M 10 M M Concentration Concentration

14. Brain Injury

15. Cerium Oxide Nanoparticles Protect Organotypic Brain Cell Cultures From In Vitro Trauma (10 nM, 10 DIV) Injured Cells/mg protein (x 106) n=16, in triplicate 7.5 mm Injury (Severe) 6.5 mm Injury (Moderate) Uninjured

16. Cerium Oxide Nanoparticles Protect Organotypic Brain Cell Cultures From In Vitro Trauma 4 _ Nano-Treated 1 hr Post Injury _ Control 3 Injured Cells/mg protein (x 106) _ 2 _ 1 7.5 mm Injury Uninjured 6.5 mm Injury

17. No Response No Response No Response D [Ca2+]i 100-265 nM D [Ca2+]i 100-265 nM D [Ca2+]i 100-265 nM D [Ca2+]i >265 nM D [Ca2+]i >265 nM D [Ca2+]i >265 nM Nanoparticles Preserve Normal Glutamate Signaling in Injured Neurons 120 Uninjured 100 24 Hr post Moderate Injury 80 24 Hr post Moderate Injury Nano-Treated Percentage Of Neurons Examined 60 40 20 0

18. AD Models

19. Ab Peptide + 10 nm Ceria Ceria Alone Ceria + Albumin Cerium Oxide Nanoparticles Block Amyloid-beta –Induced Free Radical Production

20. Cerium Oxide Nanoparticles Protect Neurons from Aβ(1-42) -Induced Toxicity 100 80 ) 4 60 Total injured cells(x10 40 20 0 Control 10CeONP 100CeONP Aβ Aβ+10 CeONP Aβ+100CeONP Treatment Groups

21. Control Ab Aβ+10nm Cerium Oxide NP (10 nM) Aβ+100nm Cerium Oxide NP ( 100 nM)

22. Drosophila ModelsLongevity & Parkinson’s Disease

23. Cerium Oxide Nanoparticles Increase the Life Span Of Female Drosophila and Maintain Normal Geotaxis with Aging 100 80 60 Percent Survival 40 Control 20 10nM CeO2 0 0 20 40 60 80 Days Geotactic Response

24. 120 Control m 100 1 M CeO2 NP 80 60 Percent Survival 40 20 0 1 3 5 7 24 48 72 Post-paraquat Time (in hours) Cerium Oxide Nanoparticles Decrease Paraquat Toxicity In Middle-Aged Female Drosophila 5 flies lived out Normal lifespan

25. What About Mammalian Models? • Mice – 90 nmols total, tail vein injection • Accumulation in brain, heart, lung • No metabolism? • Rats – Up to 150 mg injected. • No toxicity noted • No adverse tissue pathology

26. A New Generation of Nanopharmaceuticals • Cerium Oxide - A promising agent to treat diseases associated with oxidative stress. • More ground to cover….. • How much free radical scavenging is too much? • Controlled free radical scavenging? • Toxicity profile

27. Undergraduate Students Eric Amateis Matt Kurnick Julie Karafakis Clinical Dr. Jill Cramer Post Doctoral Dr. Shaadi Elswaifi Dr. Igor Danelison Research Associates Neeraj Singh Courie Cohen Kevin Hockey Chine Logan Collaborators Dr. Kathleen Meehan Dr. Rick Davis Dr. David Cox Nanophase Inc. Advanced Powder Tech. NanoNeuroLab at VCOM