The Responsible Development of Nanotechnology:         Challenges and Perspectives Ne3LS Network International Conference ...
OUTLINE      Introductory remarks   Nanoparticle characteristics   Dosing the respiratory tract    Nanoparticle Biokinetic...
Introductory Remarks
Nanoscale Materials  Water   Glucose Antibody Virus    Bacteria   Cancer Cell   A Period         Tennis Ball   10-1      1...
NANOTECHNOLOGY        THE BRIGHT!              and                THE DARK?Multiple Applications/Benefits         Consumer...
Some Headlines in the Popular Press:Nanoparticles (NPs)              kill workers              cause cancer              d...
Nanotoxicology - Hype Cycle         All NPs are “toxic”                                               Realistic Assessment...
Conceptual Depiction of Factors for ConsideringDose-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         ...
MWCNT in Subpleural Tissues, Visceral Pleura and      Pleural Space of Mice Following Oro-Pharyngeal                     A...
Many Challenges in NanotoxicologyWhich testing strategy to assess environmental and      human safety (EHS) concerns?•   i...
Safety Assessment of Engineered Nanomaterials                                                       ENM Synthesis   Source...
Nanoparticle Characteristics
What is different: Nanoparticles vs Larger Particles (respiratory tract as portal-of-entry)                               ...
Fine, 250nmUltrafine, 25 nm
Physico-chemical NP Properties of Relevance for Toxicology   Size (aerodynamic, hydrodynamic)   Size distribution   Shape ...
Particle Number and Particle Surface Area per 10 pg/cm3 Airborne Particles                           (Unit density particl...
Surface Molecules as Function of                                 Particle Size                      60%                   ...
Which Dose-Metric?                           Percent of Neutrophils in Lung Lavage 24 hrs after                         In...
Which Dose-Metric?Percent of Neutrophils in BAL 24 hrs after Instillation of TiO        2 in Rats               Correlatio...
Dosing the Respiratory Tract   Impact of Dose-Rate
Fractional Deposition of Inhaled Particles in the Human Respiratory Tract                       (ICRP Model, 1994; Nose-br...
Highmore material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant
Highmore material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant
Intratracheal Instillation of Particle Suspension, Rat                                               From: Morello et al.,...
Comparing Responses when the same Lung Dose of TiO2 Nanoparticles       is administered by Inhalation or intratracheal Ins...
Dose-Rate matters as Determinant of Hazard
Biokinetics andTranslocation  of Inhaled Nanoparticles
Exposure and Biokinetics of Nanoparticles             Confirmed routes             Potential routes   Exposure Media      ...
Exposure and Biokinetics of Nanoparticles             Confirmed routes             Potential routes   Exposure Media      ...
Exposure and Biokinetics of Nanoparticles             Confirmed routes             Potential routes                       ...
FROM RESPIRATORY TRACT TO BRAIN:     POTENTIAL TRANSLOCATION PATHWAYS OF NANOPARTICLESCSF: Cerebrospinal FluidBBB: Blood-B...
Rat, Right Nostril Occlusion Model:                        Accumulation of Mn in Right and left Olfactory Bulb 24 Hours af...
Mn concentration in lung and brain regions of rats following                            12 days ultrafine Mn-oxide exposur...
Inflammation in the Brain Regions where                                                Mn Signal was Found                ...
Protein Adsorption on Nanoparticles
“Concept of Differential Adsorption”                       Modified from Müller and Heinemann, 1989  NP physicochemical pr...
Nano – Bio Interactions           Schematic drawing of the structure of NP-protein complexes in plasma:                 Tw...
Adsorption of Proteins (FBS) Is Inhibited by Surfactant Coating (Pluronic F127)                                         (S...
Cytotoxicity of 10 nm SiO2 in RAW264.7 Cells (18 hr. incubation)     Is Inhibited by Surfactant Treatment (Pluronic F127) ...
Hazard and Risk Characterization
Risk Assessment and Risk Management Paradigm                 For Engineered Nanoparticles (NPs)    Physico-chemical      P...
Risk Assessment and Risk Management Paradigm                 For Engineered Nanoparticles (NPs)    Physico-chemical      P...
Concepts of Nanomaterial Toxicity Testing:               Considering Exposure and Hazard for Risk AssessmentExposure – Dos...
Dosimetric Extrapolation of Inhaled Particles from Rats to Humans          Rat                                            ...
Two Subchronic MWCNT Inhalation Sudies in Rats
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Günter Oberdorster_How to assess the risks of nanotechnology?

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Günter Oberdorster_How to assess the risks of nanotechnology?

  1. 1. 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
  2. 2. OUTLINE Introductory remarks Nanoparticle characteristics Dosing the respiratory tract Nanoparticle BiokineticsNanoparticle – protein interactions Hazard/Risk characterization
  3. 3. Introductory Remarks
  4. 4. Nanoscale Materials Water Glucose Antibody Virus Bacteria Cancer Cell A Period Tennis Ball 10-1 1 10 102 103 104 105 106 107 108 NanometersBucky-ball Carbon Gold ZnO Nanorods Nano-onion Nanotube Nanoshell
  5. 5. NANOTECHNOLOGY THE BRIGHT! and THE DARK?Multiple Applications/Benefits Consumer Fears/Perceived Risks • Structural Engineering • Safety: Potential adverse effects • Electronics, Optics • Environmental Contamination • Food and Feed Industry • Inadvertent Exposure (inhalation, dermal, ingestion) • Consumer Products • Susceptible Subpopulation • Alternative Energy • Societal Implications • Soil/Water Remediation • Nanomedicine: – therapeutic • Nanotoxicology: – diagnostic – drug delivery Safety Assessment of – cancer engineered Nanomaterials and of – nanosensors Nanotechnology enabled Applications – nanorobotics
  6. 6. 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?
  7. 7. Nanotoxicology - Hype Cycle All NPs are “toxic” Realistic Assessment of Hazard and RiskHazard Increasing Insight of Nanotox Concepts Appreciation of Reality Effects and Biokinetics Time of UFP (1990s)
  8. 8. Conceptual Depiction of Factors for ConsideringDose-dependent Transitions in Determinants of Toxicity From: Slikker Jr., et al. 2004
  9. 9.
  10. 10. Nano TiO2 repeated bolus instillation into mouse:7.5 mg into mouse = 17.5 grams into human nose! …
  11. 11. 17.5 g TiO2 (P25)
  12. 12. DPPC/Alb-Dispersed Mitsui Multiwalled Carbon Nanotubes (MWCNTs)
  13. 13. 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, 2008Carbon nanotubes introduced into theabdominal cavity of mice show asbestos-likepathogenicity in a pilot studyCRAIG A. POLAND, RODGER DUFFIN, IAN KINLOCH, ANDREW MAYNARD,WILLIAM A. H. WALLACE, ANTHONY SEATON, VICKI STONE, SIMON BROWNWILLIAM 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
  14. 14. MWCNT in Subpleural Tissues, Visceral Pleura and Pleural Space of Mice Following Oro-Pharyngeal Aspiration (80 µg) From: Mercer et al., 2010 VP VP Mac Day 28 Day 28 Day 56 Day 56 VP PS Ly VP MoVP: visceral pleura; Mac: alveolar macrophage; Mo: monocyte; Ly: lymph vessel; PS: pleural space
  15. 15. Many Challenges in NanotoxicologyWhich 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
  16. 16. Safety Assessment of Engineered Nanomaterials ENM Synthesis Sources Characterization – Properties Life Cycle ReleasesExposure From Cradle to GravePathways Dispersion, Transformation Air, Water, Soil, Sediment ReceptorInteractions Exposure Assessment Hazard Characterization Environment; Human Eco/Human Toxicity Risk Assessment Risk Characterization Predictive Bioinformatics Models Predicting nanostructure- induced responses
  17. 17. Nanoparticle Characteristics
  18. 18. 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 effectsTranslocation 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 someCell entry/uptake yes (caveolae; clathrin; lip. rafts; diffusion) yes (primarily phagocytic cells) — mitochondria yes no — nucleus yes (<40 nm) noEffects (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
  19. 19. Fine, 250nmUltrafine, 25 nm
  20. 20. Physico-chemical NP Properties of Relevance for Toxicology Size (aerodynamic, hydrodynamic) Size distribution Shape Properties can change Agglomeration/aggregation -with: method of production Density (material, bulk) preparation process Surface properties: storage - area (porosity) - charge -when introduced into - reactivity physiol. media, organism - chemistry (coatings, contaminants) - defects Solubility/Sol-Rate (lipid, aqueous, in vivo) Crystallinity Biol. contaminants (e.g. endotoxin) Key parameter: Dose!
  21. 21. 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.
  22. 22. Surface Molecules as Function of Particle Size 60% 50%% Surface Molecules 40% Ns/N Ns/N 30% 20% 10% 0% 1 10 100 1000 10000 Dp [nm] Diameter (nm) From Fissan, 2003
  23. 23. Which Dose-Metric? Percent of Neutrophils in Lung Lavage 24 hrs after Intratrachael Dosing of Ultrafine and Fine TiO2 in Rats ▲ Fine TiO2 (200nm) ■ Ultrafine TiO2 (25nm) ● Saline 50 UltrafineTiO 2 (~20nm) ultrafine TiO 2 (20nm) 40 pigment grade TiO 2 (~200nm) Fine TiO 2 (~250nm) saline 40 30% Neutrophils % Neutrophils 30 20 20 10 10 0 0 0 500 1000 1500 2000 108 10 9 1010 10 11 10 12 10 13 Particle Mass, ug Particle Number Particle Mass vs Particle Number
  24. 24. Which Dose-Metric?Percent of Neutrophils in BAL 24 hrs after Instillation of TiO 2 in Rats Correlation with Particle Surface Area 50 40 ultrafine TiO 2 (~25nm) fine TiO 2 (~200nm) saline% Neutrophils 30 20 10 0 0 50 100 150 200 250 2 Particle Surface Area, cm
  25. 25. Dosing the Respiratory Tract Impact of Dose-Rate
  26. 26. Fractional Deposition of Inhaled Particles in the Human Respiratory Tract (ICRP Model, 1994; Nose-breathing) 1.0 Total deposition Deposition Fraction 0.8 0.6 0.4 0.2 0.0 0.0001 0.001 0.01 0.1 1 10 100 Diameter (µm) Mechanisms Deposition Impaction Diffusion SedimentationInhalable Thoracic Respirable< 100um < 30um < 10um
  27. 27. Highmore material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant
  28. 28. Highmore material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant
  29. 29. Intratracheal Instillation of Particle Suspension, Rat From: Morello et al., 2009
  30. 30. 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
  31. 31. Dose-Rate matters as Determinant of Hazard
  32. 32. Biokinetics andTranslocation of Inhaled Nanoparticles
  33. 33. Exposure and Biokinetics of Nanoparticles Confirmed routes Potential routes Exposure Media Air, water, clothes Drug Delivery Air Food, water Deposition Injection Inhalation Ingestion Skin Respiratory Tract Uptake Pathways trach- GI-tract nasal bronchial alveolar Lymph CNS Blood PNS (platelets, monocytes, Liver endothelial cells) Translocation and Distribution Lymph Bone Marrow Other sites (e.g. muscle, placenta) Kidney Spleen Heart Excretory Sweat/exfoliation Urine Breast Milk Feces Pathways(Modified from Oberdörster et al., 2005)
  34. 34. Exposure and Biokinetics of Nanoparticles Confirmed routes Potential routes Exposure Media Air, water, clothes Drug Delivery Air Food, water Deposition Injection Inhalation Ingestion Skin Respiratory Tract Uptake Pathways trach- GI-tract nasal bronchial alveolar Lymph CNS Blood PNS (platelets, monocytes, Liver endothelial cells) Translocation and Distribution Lymph Bone Marrow Other sites (e.g. muscle, placenta) Kidney Spleen Heart Excretory Sweat/exfoliation Urine Breast Milk Feces Pathways Translocation rates are very low!(Modified from Oberdörster et al., 2005)
  35. 35. Exposure and Biokinetics of Nanoparticles Confirmed routes Potential routes Translocation and Elimination Pathways from Respiratory Tract Exposure Media Air, water, clothes Drug Delivery Air Food, water Deposition Injection Inhalation Ingestion Skin Respiratory Tract Uptake Pathways trach- GI-tract nasal bronchial alveolar ? Lymph CNS Blood PNS (platelets, monocytes, Liver endothelial cells) Translocation and Distribution Lymph Bone Marrow Other sites (e.g. muscle, placenta) Kidney Spleen Heart <~6 nm Excretory Sweat/exfoliation Urine Breast Milk Feces Pathways GI-tract and kidney as major excretory organs(Modified from Oberdörster et al., 2005)
  36. 36. FROM RESPIRATORY TRACT TO BRAIN: POTENTIAL TRANSLOCATION PATHWAYS OF NANOPARTICLESCSF: Cerebrospinal FluidBBB: Blood-Brain Barrier NoseCSBB: Cerebrospinal Brain Barrier Olfactory Mucosa Nasopharyngeal Mucosa Inhaled Nanoparticles Brain Olfactory Bulb Trigem. Ganglion BBB Blood Lymph CSF Lower Respirat. Tract
  37. 37. 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 1.0ng Mn/mg wet weight 0.8 0.6 0.4 0.2 0.0 Unexposed Exposed right nostril occluded Elder et al, 2006
  38. 38. Mn concentration in lung and brain regions of rats following 12 days ultrafine Mn-oxide exposure (mean +/- SD) (465 µg/m3; 17x106 part./cm3; CMD: 31 nm; GSD: 1.77) 2.0 * Control 12 days 1.5ng Mn/mg wet wt 1.0 * * * 0.5 * 0.0 Lung Olfactory Trigeminal Striatum Midbrain Frontal Cortex Cerebellum Cortex Elder et al, 2006
  39. 39. Inflammation in the Brain Regions where Mn Signal was Found mRNA Levels TNF- Protein 8 TNF-a 40 MIP-2 GFAP Neurogenin 7 Relative Intensity, fold increase MnSOD NCAM Relative Intensity, fold increase Na-K-Cl co-trans. 30 6 ATP-rel. K-channel 5 4 20 3 2 10Controls 1 0 0 Olfactory Frontal Midbrain Striatum Cerebellum Olfactory Frontal Midbrain Striatum Cerebellum Bulb Cortex Bulb Cortex Elder et al, 2006
  40. 40. Protein Adsorption on Nanoparticles
  41. 41. “Concept of Differential Adsorption” Modified from Müller and Heinemann, 1989 NP physicochemical properties: Body compartment media: •Size •Respiratory tract lining fluid •Surface size and chemistry •Gastrointestinal milieu •Charge + •Plasma proteins •Surface hydrophobicity •Other mucosal membranes •Redox activity •Intra/extracellular fluid •Others determine protein/lipid adsorption and desorption patterns determine biodispersion across barriers and in target tissues/cellsAltered plasma composition in disease state is likely to change adsorption pattern
  42. 42. Nano – Bio Interactions Schematic drawing of the structure of NP-protein complexes in plasma: 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
  43. 43. Adsorption of Proteins (FBS) Is Inhibited by Surfactant Coating (Pluronic F127) (SDS PAGE) kDa Albumin SWCNT Casein Proteins Silica NP (10 nm)BET surface 274 m2/g Hemoglobulins BET surface 212 m2/g Lactoglobulin (Dutta et al., 2007)
  44. 44. Cytotoxicity of 10 nm SiO2 in RAW264.7 Cells (18 hr. incubation) Is Inhibited by Surfactant Treatment (Pluronic F127) + From: Dutta et al., 2007
  45. 45. Hazard and Risk Characterization
  46. 46. Risk Assessment and Risk Management Paradigm For Engineered Nanoparticles (NPs) Physico-chemical Parameters! Inhalation Ingestion, Dermal Public health/social/ Adverse NP Effect: economical/political at portal of entry consequences Biological and remote organs Monitoring (markers of exposure) Regulations Expos. StandardsExperimental Humans Prevention/Intervention Animals Occupational/ Measures Environmental Biomed./Engineering Monitoring Biokinetics! Exposure-Dose- Risk Response Data Calculation In Vivo Studies Susceptibility (acute; chronic) Extrapolation Models (high low) (animal human) In Vitro Studies (non-cellular) (animal/human cells) (subcellular distribution) Mechanistic Data Dose-Metric! Modified from Oberdörster et al., 2005
  47. 47. Risk Assessment and Risk Management Paradigm For Engineered Nanoparticles (NPs) Physico-chemical Parameters! Inhalation Ingestion, Dermal Public health/social/ Adverse NP Effect: economical/political at portal of entry consequences Biological and remote organs Monitoring (markers of exposure) Regulations Expos. StandardsExperimental Humans Prevention/Intervention Animals Occupational/ Measures Environmental Biomed./Engineering Monitoring Biokinetics! Exposure-Dose- Risk Response Data Calculation In Vivo Studies Susceptibility (acute; chronic) Extrapolation Models (high low) (animal human) In Vitro Studies (non-cellular) (animal/human cells) (subcellular distribution) Mechanistic Data Dose-Metric! Modified from Oberdörster et al., 2005
  48. 48. Concepts of Nanomaterial Toxicity Testing: Considering Exposure and Hazard for Risk AssessmentExposure – Dose - Response Dose - Response Phys-chem. Properties in vivo Phys-chem. Properties in vivo Target Organs Inhal/ in vitro Humans Respirability Animals ( Bolus ) Endpoints; Ref. Material Bolus; ALI Hi-Lo Dose; Relevancy Workplace Biokinetics Target cells, NOAELs; OELs; (translocation; Mechanisms Laboratory corona formation) Tissues HECs Reproducibility Consumer Dose-Response Dose-Response Long-term In silico models Exposure (assessment) Hazard (characterization) Risk Assessment
  49. 49. Dosimetric Extrapolation of Inhaled Particles from Rats to Humans Rat Human Exposure [mg(m3)-1] Exposure (HEC) [mg(m3)-1] Breathing Minute Volume Inhaled Dose [mg(kg)-1] Inhaled Dose [mg(kg)-1] Tidal Volume, Resp. Rate Resp. Pause Particle characteristics Anatomy Deposited Dose µg(cm2)-1; Deposited Dose µg(cm2)-1; µg(g)-1 µg(g)-1 Clearance Retention Regional Uptake (Metabolism, T½) Retained (Accumulated) Dose [µg(g)-1; µg(cm2)-1] EffectsAssumption: If retained dose is the same as in rats and humans, then effects will be the same
  50. 50. Two Subchronic MWCNT Inhalation Sudies in Rats

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