high throughput screening approach for nanomaterial
TRANSCRIPT
Tian Xia, PhD
Division of NanoMedicine
Department of Medicine
UCLA
High Throughput Screening Approach for
Nanomaterial Toxicity Testing
100’s/year 1000’s/year 10,000’s/day 100,000’s/day
High Throughput Bacterial, Cellular, Yeast, Zebrafish Embryo or Molecular Screening
Immediate Relevance
Prioritize in vivo testing
at increasing trophic levels
US National Academy of Science (NAS) Report
(2007): “Toxicity Testing in the 21st Century: A
Vision and a strategy”
Cellular or Bio-molecular Injury Endpoints
Up to 105 measurements per day
Maximum of 102 animals
per experiment (weeks to months)
In Vivo Adverse Outcomes
Material
physicochemical
properties
Validity of
predictions • mechanism of injury
• toxicological pathway
A predictive toxicological approach for
nanomaterial hazard testing
Meng et al. ASC Nano, 2009
NSF: DBI-0830117
Nel et al. Accounts in Chem Res, 2012
The Hierarchical Oxidative Stress Model
Nel et al. Science, 311, 622-627, 2006 Meng et al, ACS Nano, 2009
Establishment of a Multi-parameter HTS Assay based on
Oxidative Stress Paradigm
UCLA High Throughput Facility Selected cellular parameters for HTS
Nel et al. Accounts in Chem Res, 2012
Zhang et al. ACS Nano, 2012
George et al. ACS Nano. 4: 15-29, 2010
Xia et al. Small, 2012
Zhang et al, JACS, 2012
George et al. ACS Nano. 5: 1805-17, 2011
In vitro High Throughput platform for ENM Toxicity
Screening
Nucle
ar
area
Loss
of
MMP
Intra-
cellul
ar Ca
PI
Uptak
e
PI Uptake
0
10
20
30
40
50
60
70
80
90
100
TiO2 CeO2 ZnO NH2-PS
% c
ell
s p
osit
ive f
or P
I 0 ug/ml
6 ug/ml
12.5 ug/ml
25 ug/ml
50 ug/ml
Image acquisition Image analysis Data interpretation
Ta
rge
t c
ell
M
ult
iwe
ll p
late
E
pif
luo
res
ce
nce
mic
ros
co
pe
Zhang et al, JACS, 2012
George, Xia, et al. ACS Nano. 2011
Zhang et al, ACS Nano 2012
Xia et al. ACS Nano. 2011
K Ca
Rb Sr
Sc Ti
Y Zr
V Cr
Nb Mo
Mn Fe
Tc Ru
Co Ni
Rh Pd
Cu Zn
Ag Cd
Ga Ge
In Sn
As Se
Sb Te
Br Kr
I Xe
Cs Ba
Fr Ra
Ln Hf
An Rf
Ta W
Db Sg
Re Os
Bh Hs
Ir Pt
Mt Ds
Au Hg
Rg Cn
Tl Pb Bi Po At Rn
Li Be
Na Mg
B C
Al Si
N O
P S
F Ne
Cl Ar
H He
La
Ac
Ce Pr
Th Pa
Nd Pm
U Np
Sm Eu
Pu Am
Gd Tb
Cm Bk
Dy Ho
Cf Es
Er Tm
Fm Md
Yb Lu
No Lr
Metal OxideNanoparticle Library
HTS Hazard screening of a compositional library
of 24 MOx Nanoparticles
HTS
Zhang et al. ACS Nano, 2012
NiO
CeO2
50 nm
CoO
100 nm
HfO2
50 nm
SiO2
100 nm
Al2O3
50 nm
CuO
50 nm
Co3O4
50 nm
Cr2O3
200 nm
Fe3O4
50 nm
Fe2O3
50 nm
Gd2O3
100 nm
In2O3
100 nm
La2O3
50 nm
SnO2
100 nm
Sb2O3
100 nm
Ni2O3
200 nm 50 nm
Mn2O3
100 nm
WO3
50 nm
TiO2
50 nm
Y2O3
50 nm
Yb2O3
200 nm
ZnO
50 nm
ZrO2
50 nm
TEM images for 24 metal oxide nanoparticles
1h 24h Time
CuO
Co3O4
Cr2O3
NiO
WO3
Fe2O3
Fe3O4
ZnO
CoO
Mn2O3
Ni2O3
La2O3
-3 3 PI Fluo-4 MitoSox DCF JC-1
CeO2
Gd2O3
TiO2
Y2O3
Yb2O3
In2O3
Sb2O3
ZrO2
Al2O3
HfO2
SiO2
SnO2
1h 24h Time
-3 3
0.4 µg/mL
200 µg/mL
Dose
PI Fluo-4 MitoSox DCF JC-1
Heatmap for metal oxides in epithelial cell BEAS-2B
CeO2
Gd2O3
TiO2
Y2O3
Yb2O3
In2O3
Sb2O3
ZrO3
Al2O3
HfO2
SiO2
SnO2
PI Fluo-4 MitoSox DCF JC-1 PI Fluo-4 MitoSox DCF JC-1
Heatmap for metal oxides in macrophages RAW 264.7
1h 24h Time
1h 24h Time
CuO
Co3O4
Cr2O3
NiO
WO3
Fe2O3
Fe3O4
ZnO
CoO
Mn2O3
Ni2O3
La2O3
-3 3 -3 3
200 µg/mL
0.4 µg/mL
Dose
In vivo acute pulmonary inflammation in mice confirmed
in vitro screening results
MC
P-1
le
ve
l(×
10
3p
g/m
L)
0
1.5
1.0
0.5
*
*
**
*
*
IL-6
(m
g/m
L)
0
250
200
150
100
50
**
*
*
**
Ne
utro
ph
il c
ou
nts
(×
10
4ce
lls)
0
160
120
80
40
*
*
*
**
*
NiO
Fe
2O
3
Fe
3O
4
Co
3O
4
Cr 2
O3
Cu
O
Co
ntr
ol
HO-1
b-actin
MCP-1
IL-6
Neutrophil
Tier 1
C57BL/6
Zhang et al. ACS Nano, 2012
Toxicity results of MOx were reproduced in E. Coli bacteria
0 50 100 150 200 250
0
20
40
60
80
100
120
Gro
wth
in
hib
itio
n r
ate
(%
)
Dose (ug/mL)
Co3O4 Cr2O3 Ni2O3 CuO Mn2O3 CoO ZnO
0 50 100 150 200 250
0
20
40
60
80
100
120
140
Dose (ug/mL)
Gro
wth
in
hib
itio
n r
ate
(%
) Al2O3 CeO2 Fe2O3 Fe3O4 Gd2O3 HfO2 In2O3 NiO SiO2 SnO2 TiO2 WO3 Y2O3 Yb2O3 ZrO2 La2O3 Sb2O3
E. Coli
HTS
Growth inhibition curve 96 well plates
Toxicity based on the bandgap energy to the cellular redox potential
-12.00
-11.00
-10.00
-9.00
-8.00
-7.00
-6.00
-5.00
-4.00
-3.00
-2.00
-1.00
E to
Vac
uu
m (e
V)
Χoxide: electronegativity of
constituent atoms;
Eg: band gap value of oxide;
PZZP: pH of zero zeta potential.
Ec= -Χoxide + 0.5 Eg + 0.059 (PZZP-pH)
Ev= -Χoxide - 0.5 Eg + 0.059 (PZZP-pH)
Χoxide: electronegativity of
constituent atoms;
Eg: band gap value of oxide;
PZZP: pH of zero zeta potential.
Ec= -Χoxide + 0.5 Eg + 0.059 (PZZP-pH)
Ev= -Χoxide - 0.5 Eg + 0.059 (PZZP-pH)
Reduced cytochrome C Oxidized cytochrome C
- e
+ e
Fe 2+ Fe 3+
0.00
0.02
0.04
0.06
0.08
0.10
Ab
s.
Wavelength (nm)
450 500 550 600 650
Oxidized
Reduced
Cytochrome C
0.00
0.02
0.04
0.06
0.08
0.10
Ab
s.
Wavelength (nm)
450 500 550 600 650
Mn2O3
Ctrl.
6.3
12.5
25
50
µg/mL
0 10 20 30 40 50
Dose (µg/mL)
(%)
Re
du
ce
d c
yto
ch
rom
e c
50
60
70
80
90
100
110
Co3O4
Mn2O3
Ni2O3
CoO
CuO
ZnO
Cr2O3
TiO2
Abiotic assay using cytochrome C to demonstrate the eletron transfer
ZnO, TiO2
0
5
10
15
20
25
30
35
40
Me
tal d
isso
luti
on
rat
e (%
)
Metal oxide nanoparticles
In water
In BEGM
In DMEM
ICP-MS analysis for metal dissolution in water and culture
medium
CuO
ZnO
Al2O3
SiO2
Y2O3
La2O3
Gd2O3
HfO2Yb2O3
ZrO2
In2O3NiO
Sb2O3CeO2
SnO2
TiO2
Ni2O3
Cr2O3
Mn2O3
CoO
Co3O4
CuO
ZnO
Fe2O3Fe3O4
WO3
-4
-3
-2
-5
Ec (
eV
)
20100 30 40
Metal Dissolution (%)
Dissolution in BEGM < 13.05
Al2O3, CeO2,
Gd2O3, HfO2, In2O3, La2O3,NiO, Sb2O3,
SiO2, SnO2, TiO2, Yb2O3,
Y2O3, ZrO2
Fe2O3
Fe3O4
WO3
CoO
Co3O4
Cr2O3
Mn2O3
Ni2O3
ZnO
CuO
Ec < -4.80
Ec < -4.22
Regression Tree
Metal dissolution in BEGM < 13.05 Area under LDH curve
ToxicSafe
7.00.5
In silico regression tree analysis for the toxicological impact of metal dissolution versus conduction band energy.
*
*
**
*
*
e-
Ec
Ev
Ec
Ev Ec
Ev
-4.12
-4.84
En
ergy t
o v
acu
um
(eV
)
Conduction band
Valence band
Biological redox potential
In vivo toxicity confirmation
0.0
1.5
1.0
0.5
Pu
lmon
ary
in
flam
mati
on
Oxidized redox couples
(Cytochrome c, NADPH, etc)
Cytochrome c
In vitro multi-parametric
toxicological responses
PI Fluo-4 MitoSox DCF JC-1
Co3O4
Cr2O3
WO3
Fe2O3
Ni2O3
CeO2
Al2O3
ZnO
24 Metal Oxide Nanoparticle Library
Summary
Acknowledgements
Andre Nel
Jeff Zink
Shuo Lin
Ivy Ji
Huan Meng
Saji George
Xiang Wang
Haiyuan Zhang
Yan Zhao
Vincent Castranova
Tina Sager
Funding support:
Lutz Madler, Bremen
Suman Pokhrel, Bremen