Nanotoxicology: Assessing the Health Hazards of Engineered Nanomaterials

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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

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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

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

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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

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How do you assess safety?
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Safety lack of risk Risk hazard x exposure

• Exposure assessment
• Hazard identification
• Hazard characterisation
• Dose-response

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All nanomaterials are not the same
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Nano-sized is already part of our knowledge base
Atomic
Physical
1nm
10nm
100nm
1um
100 pm
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
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Diversity of size and shape of nanomaterials
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Diversity of nanomaterials
Anatase Ti02
Fullerene C60 aggregates
Multiwalled Carbon Nanotubes
Rutile Ti02
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Why would nanomaterials be different?
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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 ?

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Routes of exposure and kinetics may differ
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Contexts for use and exposure to nanoscale
materials

• Materials may be nano in only certain contexts
  for exposure or applications
• The nanocontext 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
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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

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Size determines sites of deposition within the
lung
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Mass-based dose may be inadequate
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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

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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

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The importance of characterization
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Nanomaterial characterization requires new skills
sets

• 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 lt 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

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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
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New properties lead to new mode of action
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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

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Strategies and pitfalls
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Biological levels and hazard evaluation strategies
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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
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• Use of both in vivo and in vitro approaches
• Need comprehensive physical/chemical
  characterizations

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Carbon-based NSMs

• Fullerenes
• eg C60 Buckyballs
• Nanotubes
• Single walled (SWNT)
• Multi walled (MWNT)
• Nanofibres/nanofibrils

Source J Nucl Med 48 1039
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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 381327-1333

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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

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Multiwalled nanotubes

• Ma-Hock et al 2009
• Nanocyl NC 7000
• 515 nm x 0.110 µm, 250300 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.

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Asbestos like activity of long MWCNT

• Poland et al 2008
• Nature Nanotech 3423
• 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

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No asbestos like activity of short/tangled MWCNT

• Muller et al 2009
• Toxicol Sci 110 442448
• 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

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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

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An Englishmans never so natural as when hes
holding his tongue. Henry James