The Cellular and Molecular
Toxicity of Low Solubility
Nanoparticles
Prof Vicki Stone
Centre for Health and Environment
Napier University, Edinburgh
http://www.lifesciences.napier.ac.uk/Research/CHE1.htm
Safety of nanomaterials Interdisciplinary Research Centre
http://www.snirc.org/
NAPIER UNIVERSITY
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PM10
Carbon based particles
•Traffic and industrial
*** •Many ultrafine ***
•Transition metals
500nm
Sulphates/Nitrates
•Traffic/photochemical
•Mainly ultrafine
Wind blown dust
•Mainly coarse
Biological components
•Spores
•Pollen
•Mainly fine and course
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Lung Defence Mechanisms
mucus
cilia
Particles trapped
and cleared on
mucociliary
escalator
Airway
Antioxidants
Gas
exchange
Alveolus
Particles
phagocytosed
and cleared
Blood vessel
•
••
Particles
••
••
Macrophage
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The Ultrafine Particle Hypothesis
• • ••
•• • •
•
•• • •
•
•
Particles ••
•
Blood vessel
•
Release of
mediators by
macrophages and
•
INFLAMMATION
•
• • epithelial cells
•
•
Impaired
•
Particles
•
•
movement
•
••• • •••• • • cross the
•
•
••
• • epithelial
•
•
•
• • • barrier
•
•
Seaton al. 1995 The Lancet 345: 176-178
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Ultrafine particle toxicology
Neutrophils in
bronchoalveolar
lavage fluid
X 105
21 nm TiO2
250 nmTiO2
100
80
60
40
20
0
0
10 20 30 40 50 60 70
Time from start of exposure (weeks)
Ferin et al 1992
Am.J.Respir.Cell.Mol.Biol. 6: 535-542
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‘Accidental’ versus ‘engineered’
nanoparticles
Nanoparticles Source
Exposure
Toxicology
Accidental
Fossil fuel combustion
E.g. Traffic, cooking.
Low exposure to
everyone
Lots e.g.
diesel, CB,
welding fume
etc
Purposeful
Bulk use of nanoparticles in High exposure to
industry e.g. carbon black
workers followed
by low exposure
to everyone
Nanotechnology
Virtually none
Adapted from Ken Donaldson
NAPIER UNIVERSITY
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Model nanoparticles
535nm
14 nm
254 m2/g
CB
202nm
100nm
260 nm
8 m2/g
64nm
100nm
Carbon black
500nm
Polystyrene beads
Low solubility, low toxicity materials.
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Factors involved in NP toxicity
1. SIZE
10μm 1 μm 0.1μm
Cilia 0.25μm diameter
.
1.0 μm
0.1μm
Bronchial epithelium
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Factors involved in NP toxicity
2. PARTICLE NUMBER
There are thousands more ultrafine particles / mg of
dust than larger, respirable particles.
3. SURFACE AREA
Particle surface area / mg is much greater for ultrafine
particles than for larger, respirable particles.
Fine carbon black
7.9 m2/g
Ultrafine carbon black
253.9 m2/g
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Particle surface area and surface reactivity
Duffin et al. (2002)
Ann.Occ.Hyg.46;242-245
10
Quartz
Neutrophils in 5
BAL (x106)
Relatively low toxicity particles
0
0
200
400 600 800 1000
Surface Area instilled (cm2)
Low toxicity low solubility particles
•straight line relationship between SA and neutrophils.
Highly pathogenic particles
•highly reactive surface (eg quartz).
•do not sit on same line as low toxicity particles.
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Nanoparticles, ROS and oxidative stress.
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Reactive oxygen species production by
carbon particles
Supercoiled
band intensity x 103
12
10
260 nm CB
14 nm CB
8
6
4
*
*
2
0
1
5
10
Particle dose / 290 μl plasmid
Stone et al. 1998 Toxicol In Vitro 12: 649-659.
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14 nm CB
150
***
100
***
50
**
0
0.5
260 nm CB
1.0
1.5
2.0 2.5
Particle dose (mg/ml)
Wilson et al. 2002 TAP 184: 172-179.
DCF Fluorescence Intensity
DCF Fluorescence Intensity
Reactive oxygen species produced by
nanoparticles
55
45
***
35
25
15
5
-5
64 nm
202 nm
535 nm
Polystyrene particle diameter
Brown et al. 2001 TAP 175: 191-199.
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Effect of carbon particles on intracellular
antioxidant content of A549 cells
120
110
14 nm CB
260 nm CB
Control
100
GSH (% of control) 90
80
70
60
*
50
40
0
2
4
6
0.78 μg/mm2
8 Time (hours)
Stone et al. 1998 TIV 12: 649-659.
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Effects of Nanoparticles and PM10 on
Intracellular Signalling Pathways
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Effect of particles on Ca2+ signalling in
human MonoMac 6 cells
[Ca2+]c
160
Stone et al. 2000
Eur Resp J
15: 297-303.
**
140
120
100
80
60
40
20
0
Control
14 nm
CB
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260 nm
CB
2000 sec
The response to thapsigargin in human MM6
cells in the presence of CB and verapamil
Ca 2+
260 nm
CB
Ca 2+
Ca 2+
THAPSIGARGIN
14 nm
CB
VERAPAMIL
***
Control
0
Particles
Particles
+ verapamil
200 400 600 800 1000 1200
[Ca2+]c nM after treatment with thapsigargin
Stone et al. 2000 Eur Resp J 15: 297-303.
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2000 sec
Polystyrene bead diameter
The effect of polystyrene nanoparticles on
calcium signalling in macrophages
535 nm
202 nm
64 nm
***
Control
0
100 200 300 400 500 600 700 800 900
[Ca2+]c nM after treatment with thapsigargin
1500 sec
Stone et al. 2000 Inhal Tox 12 (suppl 3): 345-351.
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Calcium Signalling – Inhibitors
Calcium
channel
Ca 2+
BAPTA
Ca chellator
Endoplasmic
reticulum
[mM] [nM]
Ca 2+
Ca 2+
Calmodulin
W-7
Signalling
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Verapamil
Ca2+
channel
blocker
The role of Ca2+ in the induction of TNFα
expression by carbon nanoparticles
TNFα pg/ml
250
200
***
Rat alveolar macrophages
4 hour treatments
150
100
50
0
Control 14nm 260nm 14nm Verap 14nm BAPTA
CB
CB
CB
CB
Verap
BAPTA
Brown et al. 2004 AJP 286; L344-L353
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The role of Ca2+ in the induction of TNFα
mRNA expression by CB nanoparticles
GAPDH
TNFα
Control 14nm 260nm 14 CB Verap 14 CB BAPTA LPS
CB
BAPTA
CB Verap
30
**
25
TNFα mRNA 20
(% GAPDH)
15
***
10
5
0 Control 14nm 260nm 14nm CB Verap 14nm CB BAPTA
CB
CB
Verap
BAPTA
Brown et al. 2004 AJP 286; L344-L353
NAPIER UNIVERSITY
EDINBURGH
The effect of antioxidants on CB induced
TNFα expression
TNFα mRNA
(% GAPDH)
30
TNFα pg/ml
250
200
**
***
25
20
150
***
15
100
10
50
0
5
Control 14nm 14nm Nacystelin
CB
CB
Nacystelin
0
Control14nm 14nm Nacystelin
CB
CB
Nacystelin
Brown et al. 2004 AJP 286; L344-L353
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Particle induced TNFα expression – role
of calcium and oxidants
TNFα pg/ml
200
**
Human monocyte derived macrophages
4 hour treatments
150
100
50
0
Control
14 nm
CB
CB + verap
CB
CB
+ BAPTA + W-7
CB
+ Trolox
Brown et al. 2004 AJP 286; L344-L353
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Activation of NFκB and promoter binding
Degraded
P
IκB
PP
U
IκB
NFκB
DNA
IκB
NFκB
Phosphorylation
NFκB
NFκB
Promoter
mRNA
TRANSCRIPTION
Nuclear membrane
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Immunofluorescent staining of the p65 subunit
of NFκB in human monocyte/macrophages
14 nm CB
Control
Brown et al. 2004 AJP 286; L344-L353
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Nuclear localisation of the p65 subunit of
NFκB in human monocytes
Nuclear p65 intensity
(% control)
160
*
120
80
40
Control
CB
CB
CB
CB
CB
CB
+ verap + W-7 + BAPTA + Nac + Trolox
Brown et al. 2004 AJP 286; L344-L353
NAPIER UNIVERSITY
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Nanoparticles, calcium and oxidants
Ultrafine, nanoparticles
Macrophages
Oxidative
stress
Calcium
signalling
Activation of NF-κB
Production of
pro-inflammatory proteins
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J774 macrophages exposed to 14nm CB
CONTROL
14 nm CB
Wilson et al., manuscript in prep
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Measurement of cytoskeletal stiffness using
ferromagnetic particles
Step twist
Twisting field BTW
Θ
Magnetic particles
within phagosomes
Fluxgate sensor
6 Hz
Macrophage
•Magnetic twisting device
•Aligned ferromagnetic microparticles ingested by macrophages,
•Detection by an array of magnetic fluxgate sensors (Förster device).
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Impact of particles on the macrophage
phagosome transport
Relaxation b5 (drug/control)
*
1.6
1.4
1.2
Relaxation b5 (drug/control)
1.6
$
1.4
*
$
$
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
14nm EC90
CB
320 μg/million cells EC90
*
DEP
UD
EC90 Verap BAPTA W7
Moeller et al., 2003 Manuscript submitted
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Effects of nanoparticles and PM10 on
migration and phagocytosis
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Chemotaxis Chamber
Macrophages (J774.2)
Filter (5 µm pore)
Direction of migration
Chemoattractant
Actual Filter
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Chemotaxis of J774 macrophages in response
to epithelial cell conditioned medium
Chemotaxis
% of Control
200
Particles &
Cells
Particles
Only
*
150
100
50
ZAS
250 nm
TiO2
20 nm
TiO2
260 nm
CB
Barlow et al. 2005 Particle Fibre Toxicol 2:11.
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14 nm
CB
Chemotactic response of J774 macrophages to
epithelial cell conditioned medium
% of Control
200
180
160
140
120
100
ZAS
>30
10-30
5-10
<5
Molecular Weight Fraction of Conditioned Medium kDa
Barlow et al. 2005 Particle Fibre Toxicol 2:11.
NAPIER UNIVERSITY
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Chemotactic response of J774 macrophages to
serum treated with particles
210
% of Negative Control
190 ***
***
170
150
130
110
90
ZAS
1 1.5 5
14nm CB
1 1.5 5
260nm CB
1 1.5 5
21nm TiO2
Barlow et al. 2005 Toxicol Lett 155:397-401.
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1 1.5 5 mg/ml
250nm TiO2
Nanoparticle uptake by macrophages
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Phagocytosis
Barlow et al. 2006 Submitted.
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Effect of TiO2 and CB on phagocytosis
% of cells that phagocytose >2 beads
8h exposure
Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127
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Non-phagocytic cells
Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127
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Cambridge multiwalled carbon nanotube
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Nottingham Multiwalled carbon nanotubes
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Carbon nanotubes
Control
Cambridge
Commercial
Nottingham
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Effects of nanotubes on cell viability
15.625ug/m l Nanotubes
4040 % total cells
35
25
Necrotic
2020
Apoptotic
15
16μg/ml
10
10
5
00
Control
E21
3-01
PR19
1-03
9-01
UfCB
LFA
Triton
TNF
CB
LFA
Triton
TNFα
Control Nanotubes/fibres
Nanotube Treatm ent
31.25ug/m l nanotubes
4040 % total cells
35
3030
% Total Cells
Apoptosis
Necrosis
% Total Cells
3030
25
Necrotic
2020
Apoptotic
15
1010
5
00
Control
E21
3-01
PR19
1-03
9-01
UfCB
LFA
Triton
TNF
CB
LFA
Triton
TNFα
Control Nanotubes/fibres
Nanotube treatm ent
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31μg/ml
Effect of nanotubes and nanofibres on
phagocytosis
Phagocytosis (% Total)
90
80
70
60
15.625ug/m l
50
31.25ug/m l
40
62.5ug/m l
30
20
10
A
LF
B
fC
U
103
pl
e
301
Sa
m
Sa
m
pl
e
Sa
m
pl
e
901
19
PR
E2
1
M
ed
iu
m
0
Treatm ent
All treatments induced a significant (p<0.05) inhibition
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Summary
Low toxicity, low solubility NP:
•Inflammation is related to surface area dose.
•Generate ROS leading to oxidative stress in epithelial
and macrophage cells.
•Induce Ca2+ influx in macrophages in vitro resulting in;
•activation of NFκB and AP1
•up-regulation of pro-inflammatory cytokine
expression
•Inhibition of phagosome transport
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Summary
Low toxicity, low solubility NP:
•Stimulate production of chemotaxins by epithelial cells.
•Rapidly taken up into macrophages.
•Inhibit subsequent phagocytosis of larger particles
MWCNT:
•Morphology determines uptake by macrophages.
•Inhibit subsequent phagocytosis of E.Coli.
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Napier University
UIF Dusseldorf
Dr David Brown
Dr Martin Wilson
Marieke Tuinman
Dr Suzanne Patterson
Janis Vamvakopolous
Dr Peter Barlow
Martin Clift
Dr Rodger Duffin
Dr Roel Schins
Dr Paul Borm
Dr Markus Denhart
University of Edinburgh
Nottingham University
GSF Munich
Dr Winfried Moeller
Prof Ken Donaldson
Dan Walters
University of Cambridge
THE COLT FOUNDATION
Prof Alan Windle
Dr Ian Kinloch
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