Transcript NTP activities evaluating the safety of nanoscale materials.

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
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
Desirable Applications of Nanotechnology
1. “Smart” therapeutics
2. Targeted molecular imaging agents
3. Biological sensors/
4. Tissue engineering
diagnostic tools
5. Nano-enabled products
Nano at NIEHS
• Funded by NIEHS
– Division of Extramural Research and
Training (DERT)
• Grants
• Training
Dept of Health
and Human Services (DHHS)
NIH
CDC
FDA
NIEHS
NIOSH
NCTR
• 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
DERT
DIR
NTP
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”
“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
How do you assess safety?
Safety = lack of risk
Risk = hazard x exposure
• Exposure assessment
• Hazard identification
• Hazard characterisation
• Dose-response
All nanomaterials are not the same
“Nano-sized” is already part of our knowledge base
Physical
Atomic
100 pm
1nm
10nm
100nm
Dendrimers
H2
C60
1um
10um
100um
Metal oxides
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
Diversity of size and shape of “nanomaterials”
Diversity of nanomaterials
Multiwalled
Carbon Nanotubes
Anatase Ti02
Fullerene C60
aggregates
Rutile Ti02
Why would nanomaterials be different?
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 ?
Routes of exposure and kinetics may differ
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
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
Size determines sites of deposition within the lung
Mass-based “dose” may be inadequate
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
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
The importance of characterization
Nanomaterial characterization requires new skills sets
• Chemical:
– Unequivocal Identity
• Spectroscopic techniques
• Nanomaterial:
– Size, shape and size distribution
• Electron microscopy
– Physical Constants
• Atomic force microscopy
– Purity Determination
• Dynamic light scattering
• 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
%,
– XRD-Crystalline state
– Surface area
• BET analysis
– Charge
• Zeta potential
– Surface chemistry
• Stoichiometry of targeting
molecules on surface
“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
New properties lead to new mode of action
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
Strategies and pitfalls
Biological levels and hazard evaluation strategies
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
Carbon-based NSMs
• Fullerenes
–
eg C60 “Buckyballs”
• Nanotubes”
– Single walled (SWNT)
– Multi walled (MWNT)
• Nanofibres/nanofibrils
Source: J Nucl Med 48: 1039
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
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
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.
“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
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
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
“An Englishman’s never so natural as when he’s
holding his tongue.”
Henry James