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Biokinetik und Toxikologie inhalierter Nanopartikel
Wolfgang G. Kreyling
Helmholtz Zentrum München – Forschungszentrum für Gesundheit & Umwelt
Institut für Inhalationsbiologie; Focus Netzwerk: Nanopartikel und Gesundheit
D-85758 Neuherberg / München
kreyling@helmholtz-muenchen.de; www.helmholtz-muenchen.de/en/ihb/; www.helmholtz-muenchen.de/aerosols/
6. BfR-Forum Verbraucherschutz: NanotechnologieBerlin, 10. November 2008
6. BfR-Forum Verbraucherschutz: NanotechnologieBerlin, 10. November 2008
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Nanotechnologien -Basis für zukünftige Wissenschaft + Technologien
WelcheRisiken?
Per senachhaltig:
Ressourcen+ Energiesparend
Medicine
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Paradigma der Risikoanalyse für NPexposure assessment hazard identification
hazard characterization risk characterization
NP Dosimetrie Toxikologische Reaktionen auf NP
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Nanoparticles (NP): Access to blood circulationNanoparticles (NP): Access to blood circulation
Alveolar epithelium
Heart
400 m²
~2 m²
Alveolar wallAlveolar wall
1 mm1 mm140 m140 m²², , ErythrozytErythrozyt, 7 , 7 µµmm
Gehr,1988
Daher liefern Biokinetikstudien mit inkorporierten NP:
- Fundierte Dosisabschätzung für jedes Organ
- Rationale Basis für toxikologische Studien mit relevantenDosen (dose-response, modes of action, mechanisms)
- Vermeidung von unsinnigen Überdosisstudien (Paracelcus)
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Nanoparticle (NP) translocation into circulationNanoparticle (NP) translocation into circulation
Alveolar epithelium
urine
kidneys
heart liver
brain
Y YImmunesystem
fetus
UFP
endoth. cell
vessel
UFP TiO2
Epith. cell
Geiser et al., 2005
Microscopic view: in which cells?
parenchymal immuno-competent
Where in cells?
cytoplasm vesicles/organelles nucleus
Macroscopic view
NP content in organs + tissues
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Application + analysis of radio-labeled NP
Inhalation (INH) Intravenous (IV) injection
Radio-NP
administration
t=0 t1 t2 t3
100% balanced sampling:
organs + tissues + remaining carcass + total excretion
no further sample preparation
Gamma-spectroscopic radio-analysis
Biokinetics
Intra-tracheal instillation (IT)
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Intratracheal intubation-ventilation of Ir-NP
-1500 Pa
85Kr-neutral
____50 nm
Ir
Kreyling et al.,2002
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Systemic translocation of Systemic translocation of IrIr NP towards NP towards 22ndnd target organstarget organs
Kreyling et al., 2002
days0 2 4 6 8
lung
cumm. feces
0.0
0.2
0.4
0.6
0.8
1.0
excr
eted
orr e
tain
edfr
actio
n
liver
WKY rat, 192Ir NP, 1 hr exposure15 nm CMD, 107 cm-3, 0.2 mg/m³
Semmler et al., 2004
Long-term translocation kineticssame exposure
Same for: spleen, heart, kidneys, brain
Tage nach Inhalation0 20 40 60 80 100 120 140 160 180
Ret
entio
n0,000
0,002
0,004
0,006
0,008
0,010
Leber Milz Gehirn Nieren Herz
WKY-Ratten, (n=4, je Messpunkt)LiverSpleenBrainKidneys Heart
WKY rats (n = 4)
Days after inhalation
There is little but persistent accumulation of Ir-NP in secondary target organsIr NP mass < 1 ng, but number > 108 NP per organ
0days
2 4 6 80.00
0.05
0.10
0.15
0.20
frac
tion
(*10
)-2
particulate Ir in liver excluding soluble Ir
192
192
Frac
tion
(* 1
0- ²)
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Systemic translocation of nanoparticles towards secondary targetSystemic translocation of nanoparticles towards secondary target organsorgans
WKY rat, 192Ir NP, 1 hr exposure15 nm CMD, 107 cm-3, 0.2 mg/m³
1,E+06
1,E+07
1,E+08
1,E+09
1,E+10
1,E+11
lungs liver spleen heart brain kidney
# of
Ir a
ggre
gate
s
six months afterinhalation
1,E-11
1,E-10
1,E-09
1,E-08
1,E-07
lungs liver spleen heart brain kidney
mas
s of
Ir a
ggre
gate
s (g
)
six months afterinhalation
Primary particles ~ 2 nm
Specific surface area 1200 m² / cm³
Zeta potential - 20 mV
SIMS-TOF surface analysis IrO2
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.
Do these many NP cause harm?
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Human dose estimate during continuous exposure applying rat translocation dynamics determined for Ir UFP:
NC (UFP) (3 104 p/cm³) 3 1010 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)
Retained UFP number in brain, heart ~3 109 UFP / year
Systemic translocation of nanoparticles towards secondary targetSystemic translocation of nanoparticles towards secondary target organsorgans
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Translocation of iridium + carbon NP towards Translocation of iridium + carbon NP towards 22ndnd target organstarget organs
Ar
192Ir 192Ir
sparkgenerator 192Ir
agglom.192Ir labeled carb. agglom.
carbon
WKY rat, Ir or carbon UFP + 192Ir label, 1-hr inhalation exposure20 or 80 nm CMD, 107 cm-3, 0.1 mg/m³
Szymczak et al. 2006
0.0001
0.001
0.01
0.1
liver een
neys
heart
brain
lood
24-h
rete
ntio
n
spl
kid b
192-Ir NP, 20nm
EC + 192-Ir NP, 25 nm
* ***
* p<0.05
0.001
0.01
0.1
2nd organs skeleton soft tissue
24-h
rete
ntio
n
192-Ir NP 20 nmEC + 192-Ir NP 25 nm192-Ir NP 80 nm
• Strong dependency of translocation on NP material: iridium vs. carbon NP of same size
• Size dependency of translocation of iridium NP (20 vs. 80 nm)
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
- 1500 Pa
O2 N2
sample
Ar
Ti Ti
sparkgenerator
CPC DMPS
-30 Pa
HU
M
Control
anesthetized intubated ratsventilated in
plethysmographs
Kr-85
O (0.1%)2
Ventilation-inhalation system of TiO2 NP
Morphological characterisation of NP distribution in the lungs
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
M. Geiser et al., EHP 2005
Morphometry of inhaled TiO2 nanoparticles in rat lungsMorphometry of inhaled TiO2 nanoparticles in rat lungs
tissue
V(v) 1h 24h mean
Air spaceson epithelium
Intracellular,epithelium
Connective Capillarytissue
05
1015205060708090
Part
icle
prof
iles
(% o
f tot
al lu
ngs)
Volu
me
dens
ityV(
v) o
f lun
gco
mpa
rtm
ent
Intubated ventilated WKY rat, TiO2 NP, 1 hr exposure22 nm CMD, 107 cm-3, 0.1 mg/m³morphometry 0 + 24 h after inhalation
Rapid translocation of ~ 20% NP fraction
Volume proportional re-distribution: by which mechanism?
UFP
endoth. cell
vessel
UFP TiO2
Epith. cell
Microscopic view
500 nm
Red blood cell in lung capillary
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
198Au55 cluster1.4 nm
Translocation of gold nanoparticles: Effect of particle size
Intratracheal instillation or intravenous injection in WKY rats
1-10 µg 198Au particles in 50 µL saline, negative ionic surface charge
# of particles: 1 1014 (1.5 nm cluster) 2 1011 (18 nm colloid)G. Schmid, Univ of Essen, Germany
Mass fractions of gold nanoparticles in different organs after 24 h
Indeed, size matters!!
198Au colloid18 nm
IV injection
1,E-06
1,E-05
1,E-04
1,E-03
1,E-02
1,E-01
1,E+00liv
ersp
leen
lungskid
neybloodurin
ebrai
nGIT+fe
ces
24-h
rete
ntio
n
1.5nm cluster 18nm colloidlung instillation
1,E-06
1,E-05
1,E-04
1,E-03
1,E-02
1,E-01
1,E+00
lungsGIT+fe
ces
liver
splee
nkid
ney
blood
urine
brain
24-h
rete
ntio
n
1.5nm cluster 18nm colloidlung instillation
1,E-06
1,E-05
1,E-04
1,E-03
1,E-02
1,E-01
1,E+00
lungsGIT+fe
ces
liver
splee
nkid
ney
blood
urine
brain
24-h
rete
ntio
n
1.5nm cluster 18nm colloidlung instillation
1.E-06
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
lungsGIT+fe
ces
liver
splee
nkid
ney
blood
urine
brain
24-h
rete
ntio
n
1.5nm cluster 18nm colloid IV injection
1,E-06
1,E-05
1,E-04
1,E-03
1,E-02
1,E-01
1,E+00liv
ersp
leen
lungskid
neybloodurin
ebrai
nGIT+fe
ces
24-h
rete
ntio
n
1.5nm cluster 18nm colloidIV injection
1,E-06
1,E-05
1,E-04
1,E-03
1,E-02
1,E-01
1,E+00liv
ersp
leen
lungskid
neybloodurin
ebrai
nGIT+fe
ces
24-h
rete
ntio
n
1.5nm cluster 18nm colloid
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Semmler-Behnke, Small, 2008
Administered Gold NP
198Au55 cluster
198Au cluster
198Au cluster
198Au colloid
198Au colloid
198Au-colloid
ionic ionic ionic ionic ionic ionic
diameter (nm) 1.4 2.5 2.5 5 18 80 + 200
ligandphosphi
ne carboxyl aminophosphi
nephosphi
ne phosphine
surface charge negative negative positive negative negative negativeadmin. suspension
vol. (µL) 50 50 50 50 50 50NP mass admin. (µg) 2 1 1 10 5 2
NP number admin. 1014 1013 1013 1013 1011 109 + 108
NP surface admin. (cm²) 5 2 2 7 1 0.1 + 0.01
All gold NP were neutron activated prior to administration to achieve 198Au radio-label
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Toxikologische Reaktionen auf NP:
Rolle verschiedener NP Parameter
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Influx of neutrophils (PMN) : indicator of inflammation
Instillation of ultrafine UF-TiO2 (20 nm) or fine F-TiO2 (250 nm) into rat lungs
Particle surface area (cm2/g lung tissue)
Oberdörster et al., HEI 2000
002000100 300 4000Particle mass (µg)
F-TiO2
UF-TiO2
PMN
influ
x(%
BA
LF c
ells
)Surface area of NP is associated with inflammatory response
250 nm
20 nm
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
contr.
Asbestlong
CNTtang1
CNTtang1
Nanopart. carbon black
CNTlong1
Asbesshort
CNToang2
Alv. Macroph: frustriertePhagocytose von CNTlang
Inflammatory parameters only Asbestlong und CNTlong1+2 :
Neutrophil cell influx
increased total protein
Formation of granuloma :
only Asbestlang und CNTlong1+2
Life shortening by mesothelioma
Long, biopersistent carbon nanotubes (CNT) lead to similar pathogenesise as asbestos fibers
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Takagi et al., J. Toxicol Sci. 33:105-116, 2008
Poland et al. Nature Nanotechnology, 2008
SWNT in urinary supernatant
Virtually no organ & tissue retention after 3 hours
No signs of inflammation
1,3-dipolar cyclo-addition of azomethineylides at oxidized NT sides
Functionalized SWNT & MWNT lead to high solubilization + no toxic response
Singh et al. PNAS (2006) MWNT in urinary supernatant
Original SWNT and MWNT were not excreted in urine and showed
inflammatory responses
Less toxicity of functionalized nanotubes
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Relating particle characteristics to toxicity reduction:
Paradigm of toxicity
Free radicals / Reactive oxygen species
Oxidative stress
Activation of cell signalling pathways
Activation of pro-inflammatory reactionse.g. cytokine expression
at site of exposure and in target organs
Dimensionse.g. Nanotubes
vs spherical nanoparticles
Compositione.g. metals, organics
Surface area Surface reactivitySurface charge
Bioper-sistence
Toxico-kinetics
Mechanisms
Health effects
Search for Safe Nanomaterials
NP + ihre Oberflächenmüssen + können so gestaltet + modifiziertwerden, dass toxische
Reaktionen biologischerSysteme und
Gesundheitsproblemevermieden werden.
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology
Acknowledgements
HMGU-Inst. f. Inhalation Biology: University of Essen:
Manuela Semmler-Behnke Günter Schmid
Steffi Fertsch Wolfgang Brandau
Jens Lipka
Winfried Möller University of Aachen:
Shinji Takenaka Ulrich Simon
Tobias Stöger
Holger Schulz LM University of Munich:
Wolfgang Parak
University of Berne: Ralph Sperling
Marianne Geiser
Peter Gehr University of Rochester:
Günter Oberdörster
HelmholtzZentrum münchenGerman Research Center for Health and Environment
Focus NetworkNanoparticles and Health
Institute forInhalation Biology