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