Nanotoxicology: Assessing the Health Hazards of Engineered Nanomaterials

1. Nanotoxicology: Assessing the Health Hazards of Engineered Nanomaterials Nigel Walker, PhD DABT National Toxicology Program National Institute of Environmental Health Sciences, NIH Research Triangle Park, North Carolina, USA Nanomedicine and Molecular Imaging Summit Society of Nuclear Medicine Midwinter Meeting - Albuquerque, NM January 31-February 1, 2010

2. Outline • Early fears over nanotechnology and nanomaterials • How do you assess safety? • Are all nanomaterials the same? • Why would nanomaterials be different? • Importance of characterization • Strategies and pitfalls • Examples: Carbon based nanomaterails • Take home key issues

3. Desirable Applications of Nanotechnology 1. “Smart” therapeutics 2. Targeted molecular imaging agents 3. Biological sensors/ diagnostic tools 4. Tissue engineering 5. Nano-enabled products

4. Nano at NIEHS • Funded by NIEHS • Division of Extramural Research and Training (DERT) • Grants • Training • Research at NIEHS • Division of Intramural Research (DIR) • National Toxicology Program (NTP) • Contract based research and testing • DIR Investigator Initiated • Application of nanotechnology in EHS Dept of Health and Human Services (DHHS) NIH CDC FDA NIEHS NIOSH NCTR DERT DIR NTP

5. Early fears • Self replicating nanobots • “Grey goo” scenario • Past examples of “technology gone wrong” • Genetically Modified Organisms (GMO) • Ethyl lead • Asbestos • “Fear of the unknown”

6. “Early” studies on showing toxicity of nanotubes • Carbon nanotubes • Lung granulomas after intratracheal instillation in rats and mice • Warheit et al 2003 • Lam et al 2003 • Reaction to foreign particulate • Supported by later studies • Mueller et al 2005 • MWCNT • Shvedova et al 2006

7. How do you assess safety?

8. Safety = lack of risk Risk = hazard x exposure • Exposure assessment • Hazard identification • Hazard characterisation • Dose-response

9. All nanomaterials are not the same

10. “Nano-sized” is already part of our knowledge base Physical Atomic 100 pm 1nm 10nm 100nm 1um 10um 100um Dendrimers Metal oxides H2 C60 Nanosilver H20 Quantum dots Organic molecules Gold Nanoshells Grain of salt Nanotubes Proteins Human cell polymers Dust Particles Thickness of a cell membrane Bacteria Viruses

11. Diversity of size and shape of “nanomaterials”

12. Diversity of nanomaterials Anatase Ti02 Fullerene C60 aggregates Multiwalled Carbon Nanotubes Rutile Ti02

13. Why would nanomaterials be different?

14. General concerns over nanoscale vs microscale materials • Routes of exposure may differ • Different portal of entry and target cell populations • Different kinetics and distribution to tissues • Due to size or surface coating/chemistry • Higher exposure per unit mass • Biological effects may correlate more closely a surface area dose metric • Unique properties = unique modes of action ?

15. Routes of exposure and kinetics may differ

16. Contexts for use and exposure to nanoscale materials • Materials may be “nano” in only certain contexts for exposure or applications • The “nano”context may change through the materials life-cycle • Bulk production • Incorporation into products • Use • Disposal • Environmental cycling • Nanomaterials as “particles” in dispersed applications are likely to be of high initial concern than in “closed” or embedded applications Hansen et al 2007

17. Increased uptake of nanoscale vs microscale particles • Jani et al 1990. • Uptake of polystyrene microspheres • 50, 100, 300, 500, 1000 and 3000 nm • Oral administration to female SD rats • Size dependent increase in uptake • As particle size changes so does the bioavailability

18. Size determines sites of deposition within the lung

19. Mass-based “dose” may be inadequate

20. Effects may be related to surface area based “dose” • 1um cube • e.g. respirable particle • Surface area of = 6um2 • 100nm cube • 1000 cubes is equivalent volume • Surface area = 60 um2 • 10x more surface area for the same mass

21. Surface area metrics: A key consideration Mass-based Surface area-based • Particle number-based and surface area-based metrics increase with decreasing particle size • Mass-based potency may differ, but surface area-based potency may not • Requires studying particles of similar composition but varying particle size, coatings, shape or other physicochemical parameter

22. The importance of characterization

23. Chemical: Unequivocal Identity Spectroscopic techniques Physical Constants Purity Determination Chromatographic Analyses – (Organics) Inductively Coupled Plasma/AES or MS, XRD - (Inorganics) Water Determination Elemental Analysis Constituents identified when at < 1 %, (primary and byproducts) Byproducts when between 0.1 and 1 %, Nanomaterial: Size, shape and size distribution Electron microscopy Atomic force microscopy Dynamic light scattering XRD-Crystalline state Surface area BET analysis Charge Zeta potential Surface chemistry Stoichiometry of targeting molecules on surface Nanomaterial characterization requires new skills sets

24. “Indeed, in the absence of a careful and complete description of the nanoparticle-type being evaluated (as well as the experimental conditions being employed), the results of nanotoxicity experiments will have limited value or significance.” David Warheit, Toxicological Sciences , 2008

25. New properties lead to new mode of action

26. Protein fibrillation in vitro induced by nanoparticles • Linse et al 2007, PNAS 104,8691 • Induction of b2-microglubulin protein fibril formation in vitro • Surface assisted nucleation • Observed with multiple NPs • 70, 200 nm NIPAM/BAM NPs • 16nm Cerium oxide NPs • 16nm quantum dots • 6nm dia MWCNTs • Fibril formation is implicated in development of human disease • Alzheimer's • Creutzfeldt-Jakob disease • Dialysis related amyloidosis

27. Strategies and pitfalls

28. Biological levels and hazard evaluation strategies

29. We have experimental strategies to detect hazards • In vivo toxicity testing models can detect manifestations of novel mechanisms of action if there are any. • Based on apical endpoints • Several workshops/reports with common issues/recommendations • NTP workshop on Experimental strategies • University of Florida-Nov 2004 • http://ntp.niehs.nih.gov/go/100 • ILSI-RSI report • Oberdorster et al 2005, Particle Fibre Toxicol 2:8 • Use of both in vivo and in vitro approaches • Need comprehensive physical/chemical characterizations

30. Carbon-based NSMs • Fullerenes • eg C60 “Buckyballs” • Nanotubes” • Single walled (SWNT) • Multi walled (MWNT) • Nanofibres/nanofibrils Source: J Nucl Med 48: 1039

31. Technegas • Diagnostic radio-aerosol used in lung ventilation scintigraphy • Technegas is comprised of nanoparticles • Mesoscopic fullerenes • Hexagonal platelets of metallic technetium, each closely encapsulated with a thin layer of graphitic carbon. • Size: 30-60nm X 5nm • Selden et al J Nucl Med 1997; 38:1327-1333

32. Pulmonary toxicity evaluation of Fullerene-C60 • NTP inhalation study conducted under GLP • 90 days-nose only exposure, 3hrs/day, 5d/wk • B6C3F1 mice and Wistar-Han rats, • 50nm (0.5 and 2 mg/m3) • 1um (2, 15 and 30 mg/m3 ) • Preliminary findings • Shorter clearance in mouse vs rat • Not different by size • No biologically significant toxic responses • Expected response to particles • Comparable surface area-based doses between 50nm and 1um study

33. Multiwalled nanotubes • Ma-Hock et al 2009 • Nanocyl NC 7000 • 5–15 nm x 0.1–10 µm, 250–300 m2/g • Exposure: head-nose exposed for 6 h/day, 5 days/week, 13 weeks • No systemic toxicity. • Increased lung weights, multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis in lung and lung-associated lymph nodes • 0.5 and 2.5 mg/m3.

34. “Asbestos like” activity of “long” MWCNT • Poland et al 2008 • Nature Nanotech 3:423 • Injection to C57Bl6 mice • 50ug or vehicle into peritoneal cavity • Evaluation at 7 days • Pathology • Inflammation • Foreign body Giant Cells • Granulomas • Long MWCNTs and long fibre amosite (LFA) gave similar responses • Tangled MWCNT gave different responses

35. No “asbestos like” activity of short/tangled MWCNT • Muller et al 2009 • Toxicol Sci 110; 442–448 • 20 mg IP injection – male Wistar rats • 24 month followup • MWCNT +, MWCNT-, 11nm x 0.7um • Crocidolite asbestos 330 nm x 2.5um • Clear carcinogenic response with crocidolite but not MWCNT • Authors note • Model may not be responsive to short fibres • Consistent with Poland et al 2008

36. Key issues for the field of “nanotoxicology” • “Are nanomaterials safe?” = “Are chemicals safe?” • There is no single type of nanomaterial • Effects can scale with surface area • Paradigm shift in how we estimate “dose” for assessing risks relative to other agents. • Lack of adequate characterization of what a given “test article” is • Major obstacle to developing structure-activity relationships • Nanoscale phenomena occurs at the interface between chemical space and physical space. • Very limited information on exposures

37. “An Englishman’s never so natural as when he’s holding his tongue.” Henry James