Wolfgang Kreyling - NanoImpactNet

NanoImpactNet: Workshop on on Protocols for Assessing 

Biological Hazards 

Lausanne, Switzerland, 24 24 March 2009 

Introduction to Most Pressing Issues of 

in vivo Animal Testing 

Wolfgang G. Kreyling 

Helmholtz Center Munich – Research Center for Environmental Health 

Institut of Lung Biology and Disease; Focus Network: Nanoparticles and Health 

D-85764 Neuherberg / Munich, Germany 

kreyling@helmholtz-muenchen.de; www.helmholtz-muenchen.de/en/ilbd/; 

www.helmholtz-muenchen.de/aerosols/ 

HelmholtzZentrum münchen 

German Research Center for EnvironmentalHealth 

Institute of Lung 

Biology & Disease 

Focus Network 

Nanoparticles and Health

Outline of of pressing issues from my point of of view 

• Routes of Nanoparticle intake 

• Exposure and dosimetry 

• Long-term NP dose estimates 

• Controlled NP administration 

• NP trafficking back + forth across lung membrane 

• Long-term NP translocation 

• Mechanisms of NP accumulation in 2 nd target organs (role NP- 

protein-complex formation from our biokinetic point of view) 

• Toxicological testing in primary vs. secondary target organs 





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NP in human lungs 

Gehr,1988 

1 mm 

Alveolar wall 



Nanoparticles (NP): Routes of of Intake 

140 m², m , erythrocyte, erythrocyte, 

7 µm 

Institute for 

Inhalation Biology 

Focus Network 

Nanoparticles and Health 

~2 m² 

400 m²

Systemic translocation of nanoparticles towards 

secondary target organs 

Human dose estimate during continuous exposure applying rat 

translocation dynamics determined for Ir UFP: 

NC (UFP) (10 5 p/cm³) 10 11 p/m³ 

Daily inhaled volume 10 m³ 

Deposition fraction 0.3 

Insoluble UFP fraction 0.1 

Translocated fraction to brain, heart 0.001 (of lung deposit) 

(From 6-months 20 nm Ir NP retention study; Semmler et al., 2004) 

Retained UFP number in brain, heart ~10 10 UFP/year 

Estimated surface area ~1 mm² 

(based 15 nm NP assumption) 

Estimated mass in brain, heart (lungs) ~10 ng/year ~10 µg/year 





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Lomer et al. J. Nutr., 2004 



Particle uptake from GI-Tract 

pigment TiO 2 



SiO 2 

100 - 250 nm, NP?? nm ?? (SSA > 100 m²/g) 

Daily consumption (mg) 4.5 45 

Intestinal absorption 0.001 - 0.01 

Absorbed PM per day (µg) 4.5 – 45 45 – 450 

Absorbed PM per year (mg) 1.6 – 16 16 – 160 

Assume 0.5% mass fraction NP 

Absorbed NP per year (µg) 8 – 80 80 – 800 

Lung deposit of insoluble NP 

per year after inhalation (µg) ~10 

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Inhalation vs. Instillation +Aspiration 

inhalation instillation 

head 

abdomen 



head 

head head head 

head 

• NP inhalation is the gold standard, but NP 

dosimetry remains difficult 

• NP instillation does not reach all lung areas 

aspiration 

but NP dosimetry is rather accurate 

• NP aspiration does not reach all lung areas 

and shows large variable airway deposition 

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Exposure and Dosimetry 

NP Trafficking across Lung Epithelium 



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inhalation 

AM 



Airway 

epithelium 

Acinus 

Rapid translocation from surface 



NP 

clearance 

Airways 

NP re-entrain- 

ment 

Alveolar 

epithelium 

Semmler-Behnke et al., EHP 2007 

Focus Network 



interstitium 

Epithelial lining layer 

blood erythrocyte 

• Translocation into blood is a minor transport mechanism 

Courtesy P. Gehr 

• But relocation from the epithelial surface to into the epithelium and interstitium 

is a prominant transport mechanism including re-entrainment of NP back to the 

epithelial surface + macrophage-mediated clearance towards the larynx 



Institute for 

Inhalation Biology 

Ir NP: Semmler-Behnke et al., 2007 

Focus Network 




Exposure and Dosimetry 

Short-term + long-term NP translocation + accumulation 

in 2 nd target organs 



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Nanoparticle (NP) translocation into circulation 

Alveolar epithelium 





Y Y 

Immune 

system 

brain 

heart liver 

kidneys 

Focus Network 


fetus 

urine

excreted or retained fraction 

Systemic Translocation of Ir NP towards 2 nd Target Organs 

1.0 

0.8 

0.6 

0.4 

0.2 

WKY rat, 192 Ir NP, 1 hr exposure 

15 nm CMD, 10 7 cm -3 , 0.2 mg/m³ 

Fraction (* 10-2 fraction (*10 ) 

- ²) 

lung 

0.20 

0.15 

0.10 

0.05 

0.00 

0 

Kreyling et al., 2002 

2 4 6 8 

days 

cumm. feces Same for: 

spleen, heart, 

kidneys, brain 

Long-term translocation kinetics 

same exposure 

Semmler et al., 2004 

0 20 40 60 80 100 120 140 160 180 

liver 

Days Tage after nach inhalation Inhalation 

0.0 

0 2 4 6 8 

days 

There is little but persistent accumulation of Ir-NP in secondary target organs 



192 

particulate Ir in liver 

192 

excluding soluble Ir 

Retention 



0,010 

0,008 

0,006 

0,004 

0,002 

0,000 

WKY-Ratten, (n=4, WKY je Messpunkt) rats (n = 4) 

Focus Network 


Liver 

Leber 

Milz Spleen 

Gehirn Brain 

Nieren Kidneys 

Heart Herz

Systemic translocation of nanoparticles towards secondary target organs 

mass of Ir aggregates (g) 

WKY rat, 192 Ir NP, 1 hr exposure 

15 nm CMD, 10 7 cm -3 , 0.2 mg/m³ 

1,E-07 

1,E-08 

1,E-09 

1,E-10 

1,E-11 

six months after 

inhalation 

lungs liver spleen heart brain kidney 

# of Ir aggregates 

1,E+11 

1,E+10 

1,E+09 

1,E+08 

1,E+07 

1,E+06 

six months after 

inhalation 

lungs liver spleen heart brain kidney 

Unexpected high NP numbers in secondary target organs including heart and brain after 

six months which are not considered to be exposed to particulate foreign bodies. 

Lack of long-term studies with toxicological endpoints 

From epidemiology we know the association between UFP air pollution and cardio- 

vascular failure (pro-inflammation, pro-thrombosis, cardiac malfunctions) 

There is first evidence of similar effects associated with the central nervous system. 





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Health Effects in 2nd Health Effects in 2 Target Organs Mediated by NP 

nd Target Organs Mediated by NP 

Alveolar epithelium 

• No health effect after short-term NP exposure 





Y Y 

Immune 

system 

brain 

heart liver 

kidneys 

Focus Network 


fetus 

urine 

• Possibly sufficient NP accumulation in 2 nd target organs after chronic exposure 

• Mediators released from lungs into blood affecting 2 nd target organs



Toxicological Testing 

• NP toxicology in primary organs of intake 

• respiratory tract, cardio-vascular system and skin are under current 

scrutiny 

• but long-term studies are lacking 

• GIT almost neglected although probably the most prominant route of 

intake of some mass production NP in food and consumer products 

(SiO2 ,TiO2 , ZnO, etc.) 

• NP toxicology in secondary target organs 

• NP toxicity only likely if there is sufficient NP accumuation during 

chronic exposure 

• Mediator modulated toxicity is not yet studied using engineered NP 



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The end 

Thanks very much for your attention! 



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Wolfgang Kreyling - NanoImpactNet

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