The Responsible Development of Nanotechnology:

Challenges and Perspectives

Ne3LS Network International Conference 2012

November 1-2, 2012, Montréal, Canada

Plenary Session 1:

How to assess the risks of nanotechnology?

Analysis and discussion of the various risk facets

Günter Oberdörster Department of Environmental Medicine

University of Rochester

November 1, 2012

OUTLINE

Introductory remarks

Nanoparticle characteristics

Dosing the respiratory tract

Nanoparticle Biokinetics

Nanoparticle – protein interactions

Hazard/Risk characterization

Introductory Remarks

Nanoscale Materials

Gold

Nanoshell

Nanometers

10-1 1 10 102 103 104 105 106 107 108

Carbon

Nanotube

Bucky-ball ZnO Nanorods

Water Glucose Antibody Virus Bacteria Cancer Cell A Period Tennis Ball

Nano-onion

NANOTECHNOLOGY

THE BRIGHT! and THE DARK?

Multiple Applications/Benefits Consumer Fears/Perceived Risks

• Structural Engineering

• Electronics, Optics

• Food and Feed Industry

• Consumer Products

• Alternative Energy

• Soil/Water Remediation

• Nanomedicine: – therapeutic – diagnostic – drug delivery – cancer – nanosensors – nanorobotics

• Safety: Potential adverse effects

• Environmental Contamination

• Inadvertent Exposure (inhalation, dermal, ingestion)

• Susceptible Subpopulation

• Societal Implications

• Nanotoxicology:

Safety Assessment of engineered Nanomaterials and of Nanotechnology enabled Applications

Some Headlines in the Popular Press:

Nanoparticles (NPs)

kill workers

cause cancer

damage DNA

soften your brain

cause lung damage

cause genetic damage

in sunscreen cause Alzheimer’s?

are carbon nanotubes the next asbestos?

All NPs are “toxic”

Appreciation of Reality

Effects and Biokinetics

of UFP (1990s)

Nanotoxicology - Hype Cycle

Increasing Insight of

Nanotox Concepts

Time

Haza

rd Realistic Assessment of Hazard and Risk

From: Slikker Jr., et al. 2004

Conceptual Depiction of Factors for Considering

Dose-dependent Transitions in Determinants of Toxicity

…

…

Nano TiO2 repeated bolus instillation into mouse:

7.5 mg into mouse = 17.5 grams into human nose!

17.5 g TiO2 (P25)

DPPC/Alb-Dispersed Mitsui Multiwalled Carbon

Nanotubes (MWCNTs)

Induction of mesothelioma in p53+/- mouse by intraperitoneal

application of multi-wall carbon nanotube

Takagi et al., J. Toxicol. Sci. 33 (No. 1): 105-116, 2008

Carbon nanotubes introduced into the

abdominal cavity of mice show asbestos-like

pathogenicity in a pilot study CRAIG A. POLAND, RODGER DUFFIN, IAN KINLOCH, ANDREW MAYNARD, WILLIAM A. H. WALLACE, ANTHONY SEATON, VICKI STONE, SIMON BROWN WILLIAM MACNEE, AND KEN DONALDSON

Nature Nanotechnology/Vol. 3/July 2008

Induction of mesothelioma by a single intrascrotal administration

of multi-wall carbon nanotube in intact male Fischer 344 rats Sakamoto et al, J.Tox. Sci., 34, 65-76, 2009

MWCNT in Subpleural Tissues, Visceral Pleura and Pleural Space of Mice Following Oro-Pharyngeal

Aspiration (80 µg) From: Mercer et al., 2010

VP: visceral pleura; Mac: alveolar macrophage; Mo: monocyte; Ly: lymph vessel; PS: pleural space

Day 28

Day 56

Day 28

Day 56

VP VP Mac

VP

VP

Ly

Mo

PS

Many Challenges in Nanotoxicology

Which testing strategy to assess environmental and

human safety (EHS) concerns?

• in vitro – in vivo design/methodologies

• acute – chronic toxicities

• dose, dosimetry, dosemetric related issues

• correlations between phys.chem. properties and toxicity

• nano-bio interactions: corona formation

• risk extrapolation: laboratory animals to humans

• human exposure assessment

ENM Synthesis Sources

Exposure

Pathways

Receptor

Interactions

Risk Assessment

Predictive

Models

Characterization – Properties

Life Cycle Releases

From Cradle to Grave

Dispersion, Transformation

Air, Water, Soil, Sediment

Exposure Assessment

Environment; Human

Hazard Characterization

Eco/Human Toxicity

Risk Characterization

Bioinformatics

Predicting nanostructure- induced responses

Safety Assessment of Engineered Nanomaterials

Nanoparticle Characteristics

What is different: Nanoparticles vs Larger Particles (respiratory tract as portal-of-entry)

Nanoparticles (<100 nm) Larger Particles (>500 nm) General characteristics:

Ratio: number/surface area/volume high low

Agglomeration in air, liquids likely (dependent on medium; surface) less likely

Deposition in respiratory tract diffusion; throughout resp. tract sedimentation, impaction, inter- ception; throughout resp. tract

Protein/lipid adsorption in vitro very effective and important for bio- less effective kinetics and effects

Translocation to secondary target organs: yes generally not (to liver under “overload”)

Clearance

— mucociliary probably yes efficient

— alv. macrophages poor efficient

— epithelial cells yes mainly under overload

— lymphatic yes under overload

— blood circulation yes under overload

— sensory neurons (uptake + transport) yes not likely

Proteinl/lipid adsorption in vivo yes some

Cell entry/uptake yes (caveolae; clathrin; lip. rafts; diffusion) yes (primarily phagocytic cells)

— mitochondria yes no

— nucleus yes (<40 nm) no

Effects (caveat: dose!) :

at secondary target organs yes (no)

at portal of entry (resp. tract) yes yes

— inflammation yes yes

— oxidative stress yes yes

— activation of signaling pathways yes yes

— genotoxicity, carcinogenicity probably yes some

Fine, 250nm

Ultrafine, 25 nm

Physico-chemical NP Properties of Relevance for Toxicology

Size (aerodynamic, hydrodynamic)

Size distribution

Shape

Agglomeration/aggregation

Density (material, bulk)

Surface properties:

- area (porosity)

- charge

- reactivity

- chemistry (coatings, contaminants)

- defects

Solubility/Sol-Rate (lipid, aqueous, in vivo)

Crystallinity

Biol. contaminants (e.g. endotoxin)

Properties can change

-with: method of production

preparation process

storage

-when introduced into

physiol. media, organism

Key parameter: Dose!

Particle Number and Particle Surface Area per 10 pg/cm3 Airborne Particles

(Unit density particles)

Particle Diameter Particle Number Particle Surface Area

nm N/cm3 µm2/cm3

5 153,000,000 12,000

20 2,400,00 3,016

250 1,200 240

5,000 0.15 12

Small size, high number per mass, and surface chemistry

confer both desirable and undesirable properties.

Detailed Physico-chemical characterization of NP is essential.

0%

10%

20%

30%

40%

50%

60%

1 10 100 1000 10000

Dp [nm]

Ns/NNs/N

Surface Molecules as Function of

Particle Size

From Fissan, 2003

Diameter (nm)

% S

urf

ace

Mo

lecu

les

0 500 1000 1500 20000

10

20

30

40

50ultrafine TiO2 (20nm)pigment grade TiO2 (~200nm)saline

Particle Mass, ug

% N

eu

tro

ph

ils

0

10

20

30

40Fine TiO2 (~250nm)

UltrafineTiO2 (~20nm)

108 10 9 1010 1011 10 12 1013

Particle Number

% N

eu

tro

ph

ils

Percent of Neutrophils in Lung Lavage 24 hrs after Intratrachael Dosing of Ultrafine and Fine TiO2 in Rats

Particle Mass vs Particle Number

▲ Fine TiO2 (200nm) ■ Ultrafine TiO2 (25nm) ● Saline

Which Dose-Metric?

0 50 100 150 200 250 0

10

20

30

40

50

ultrafine TiO 2 (~25nm) fine TiO 2 (~200nm) saline

Percent of Neutrophils in BAL 24 hrs after Instillation of TiO 2 in Rats

Correlation with Particle Surface Area

Particle Surface Area, cm 2

% N

eu

tro

ph

ils

Which Dose-Metric?

Dosing the Respiratory Tract

Impact of Dose-Rate

Tracheobronchial

Nasal Pharyngeal

Laryngeal

Alveolar

TOTAL

0.0001 0.001 0.01 0.1 1 10 100 0.0

0.2

0.4

0.6

0.8

1.0

Diameter (µm) D

ep

osi

tio

n F

racti

on

Dep

osi

tion

Mech

an

ism

s

Diffusion

Sedimentation

Impaction

Total deposition

Fractional Deposition of Inhaled Particles in the Human Respiratory Tract (ICRP Model, 1994; Nose-breathing)

Thoracic Respirable Inhalable

< 100um < 30um < 10um

High

more material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant

High

more material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant

From: Morello et al., 2009

Intratracheal Instillation of Particle Suspension, Rat

Comparing Responses when the same Lung Dose of TiO2 Nanoparticles

is administered by Inhalation or intratracheal Instillation

4 Groups of Rats:

1. Controls (instillation of saline)

2. 200 µg TiO2 (25 nm) instillation into lungs

3. Four hour inhalation to deposit 200µg TiO2 into lungs

4. Four days of 4 hour daily inhalation to deposit 200µg TiO2 into lungs

24 hours after dosing lungs were lavaged and inflammatory cellular and biochemical parameters determined

Dose-Rate matters as Determinant of Hazard

Biokinetics andTranslocation

of Inhaled Nanoparticles

Exposure Media

Uptake Pathways

Translocation

and Distribution

Excretory

Pathways

Air, water, clothes Air Food, water

Skin

Respiratory Tract

GI-tract

Blood Liver

Lymph

Bone Marrow Spleen

Urine Feces

Deposition Ingestion Inhalation

Other sites (e.g. muscle, placenta)

(platelets, monocytes, endothelial cells)

Breast Milk

Heart

Sweat/exfoliation

Drug Delivery

Injection

Confirmed routes

Potential routes

Lymph

trach- nasal bronchial alveolar

CNS

PNS

Exposure and Biokinetics of Nanoparticles

Kidney

(Modified from Oberdörster et al., 2005)

Exposure Media

Uptake Pathways

Translocation

and Distribution

Excretory

Pathways

Air, water, clothes Air Food, water

Skin

Respiratory Tract

GI-tract

Blood Liver

Lymph

Bone Marrow Spleen

Urine Feces

Deposition Ingestion Inhalation

Other sites (e.g. muscle, placenta)

(platelets, monocytes, endothelial cells)

Breast Milk

Heart

Sweat/exfoliation

Drug Delivery

Injection

Confirmed routes

Potential routes

Lymph

trach- nasal bronchial alveolar

CNS

PNS

Exposure and Biokinetics of Nanoparticles

Kidney

(Modified from Oberdörster et al., 2005) Translocation rates are very low!

Exposure Media

Uptake Pathways

Translocation

and Distribution

Excretory

Pathways

Air, water, clothes Air Food, water

Skin

Respiratory Tract

GI-tract

Blood Liver

Lymph

Bone Marrow Spleen

Urine Feces

Deposition Ingestion Inhalation

Other sites (e.g. muscle, placenta)

(platelets, monocytes, endothelial cells)

Breast Milk

Heart

Sweat/exfoliation

Drug Delivery

Injection

Confirmed routes

Potential routes

Lymph

trach- nasal bronchial alveolar

CNS

PNS

Exposure and Biokinetics of Nanoparticles

Kidney

GI-tract and kidney as major excretory organs (Modified from Oberdörster et al., 2005)

?

Respiratory Tract

GI-tract

Blood Liver

Urine

(platelets, monocytes, endothelial cells)

Lymph

trach- nasal bronchial alveolar

CNS

PNS

Kidney

Translocation and Elimination Pathways from Respiratory Tract

<~6 nm

GI-tract and kidney as major excretory organs

FROM RESPIRATORY TRACT TO BRAIN:

POTENTIAL TRANSLOCATION PATHWAYS OF NANOPARTICLES

CSF: Cerebrospinal Fluid

BBB: Blood-Brain Barrier

CSBB: Cerebrospinal Brain Barrier

Inh

aled

Na

no

pa

rticles

Nose Olfactory Mucosa Nasopharyngeal Mucosa

Lymph CSF

Brain Olfactory Bulb Trigem. Ganglion

Blood BBB

Lower Respirat.

Tract

0.0

0.2

0.4

0.6

0.8

1.0

Unexposed Exposedright nostril occluded

Rat, Right Nostril Occlusion Model: Accumulation of Mn in Right and left Olfactory Bulb 24 Hours after Exposure to Ultrafine (~30nm) Mn Oxide Particles (n=3-5, mean+/-SD)

left olfactory bulb right olfactory bulb

ng

Mn

/mg

w

et

weig

ht

Elder et al, 2006

Mn concentration in lung and brain regions of rats following 12 days ultrafine Mn-oxide exposure (mean +/- SD)

0.0

0.5

1.0

1.5

2.0

Control 12 days

Lung Olfactory Trigeminal Striatum Midbrain Frontal Cortex Cerebellum Cortex

*

*

* *

*

ng

Mn

/mg

we

t w

t

(465 µg/m3; 17x106 part./cm3; CMD: 31 nm; GSD: 1.77)

Elder et al, 2006

Inflammation in the Brain Regions where

Mn Signal was Found

0

1

2

3

4

5

6

7

8

Neurogenin

MnSOD

NCAM

Na-K-Cl co-trans.

ATP-rel. K-channel

TNF-a

MIP-2

GFAP

Olfactory

Bulb

CerebellumStriatumMidbrainFrontal

Cortex

Controls

Rel

ati

ve

Inte

nsi

ty,

fold

in

crea

se

0

10

20

30

40

Rel

ati

ve

Inte

nsi

ty, fo

ld i

ncr

ease

Olfactory

Bulb

CerebellumStriatumMidbrainFrontal

Cortex

mRNA Levels TNF- Protein

Elder et al, 2006

Protein Adsorption on Nanoparticles

NP physicochemical properties: •Size

•Surface size and chemistry

•Charge

•Surface hydrophobicity

•Redox activity

•Others

Body compartment media: •Respiratory tract lining fluid

•Gastrointestinal milieu

•Plasma proteins

•Other mucosal membranes

•Intra/extracellular fluid

protein/lipid adsorption and desorption patterns

biodispersion across barriers and in target tissues/cells

determine

determine

“Concept of Differential Adsorption” Modified from Müller and Heinemann, 1989

Altered plasma composition in disease state is likely to change adsorption pattern

+

Two Layers of Proteins of the “Core” Nanoparticle :

An outer “weak” layer of protein corona And an inner “hard”layer of stable, rapidly exchanging with free proteins slowly exchanging corona of proteins (red arrows)

From: Walczyk et al., 2010

Nano – Bio Interactions Schematic drawing of the structure of NP-protein complexes in plasma:

kDa

Albumin

Adsorption of Proteins (FBS) Is Inhibited by Surfactant Coating (Pluronic F127) (SDS PAGE)

(Dutta et al., 2007)

Casein Proteins

Hemoglobulins

Lactoglobulin

SWCNT BET surface 274 m2/g

Silica NP (10 nm) BET surface 212 m2/g

From: Dutta et al., 2007

Cytotoxicity of 10 nm SiO2 in RAW264.7 Cells (18 hr. incubation)

Is Inhibited by Surfactant Treatment (Pluronic F127)

+

Hazard and Risk Characterization

Adverse NP Effect: at portal of entry and remote organs

Experimental Animals

Humans

Biological Monitoring (markers of exposure)

Occupational/ Environmental Monitoring

Public health/social/ economical/political consequences

Regulations Expos. Standards

Prevention/Intervention Measures Biomed./Engineering

Exposure-Dose- Response Data

In Vivo Studies (acute; chronic)

In Vitro Studies (non-cellular) (animal/human cells) (subcellular distribution)

Risk Calculation

Susceptibility Extrapolation Models (high low) (animal human)

Mechanistic Data

Risk Assessment and Risk Management Paradigm For Engineered Nanoparticles (NPs)

Inhalation Ingestion, Dermal

Biokinetics!

Dose-Metric!

Physico-chemical

Parameters!

Modified from Oberdörster et al., 2005

Adverse NP Effect: at portal of entry and remote organs

Experimental Animals

Humans

Biological Monitoring (markers of exposure)

Occupational/ Environmental Monitoring

Public health/social/ economical/political consequences

Regulations Expos. Standards

Prevention/Intervention Measures Biomed./Engineering

Exposure-Dose- Response Data

In Vivo Studies (acute; chronic)

In Vitro Studies (non-cellular) (animal/human cells) (subcellular distribution)

Risk Calculation

Susceptibility Extrapolation Models (high low) (animal human)

Mechanistic Data

Risk Assessment and Risk Management Paradigm For Engineered Nanoparticles (NPs)

Inhalation Ingestion, Dermal

Biokinetics!

Dose-Metric!

Physico-chemical

Parameters!

Modified from Oberdörster et al., 2005

in vivo Humans Workplace Laboratory Consumer

in vitro Bolus; ALI Target cells,

Tissues Dose-Response

in vivo Animals

Biokinetics (translocation;

corona formation) Dose-Response

Phys-chem. Properties Target Organs Respirability

NOAELs; OELs; HECs

Phys-chem. Properties Endpoints; Ref. Material Hi-Lo Dose; Relevancy

Mechanisms Reproducibility

Exposure (assessment) Hazard (characterization)

Risk Assessment

Considering Exposure and Hazard for Risk Assessment

Concepts of Nanomaterial Toxicity Testing:

Inhal/ Bolus ( )

Long-term

In silico models

Dose - Response Exposure – Dose - Response

Dosimetric Extrapolation of Inhaled Particles from Rats to Humans

Rat Human

Exposure [mg(m3)-1] Exposure (HEC) [mg(m3)-1]

Inhaled Dose [mg(kg)-1] Inhaled Dose [mg(kg)-1]

Deposited Dose µg(cm2)-1;

µg(g)-1

Deposited Dose µg(cm2)-1;

µg(g)-1

Retained (Accumulated) Dose [µg(g)-1; µg(cm2)-1]

Effects

Assumption: If retained dose is the same as in rats and humans, then effects will be the same

Breathing

Minute

Volume

Tidal Volume, Resp. Rate Resp. Pause

Particle characteristics Anatomy

Clearance Retention

Regional Uptake (Metabolism, T½)

Two Subchronic MWCNT Inhalation Sudies in Rats