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Real Time Cell Electronic Sensing (RT-CES)

for Nanotoxicity Evaluation

Reyes Sierra1, Lila Otero1, Scott Boitano2 & Jim Field1

1

1Dept Chemical & Environmental Engineering2Dept Cell Physiology1 Dept Cell Physiology

The University of Arizona

RSIERRA@EMAIL ARIZONA [email protected]

SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

NANOPARTICLES Nanoparticles (NPs) are particles sized in less

than 100 nmthan 100 nm.

100,000 smaller

2Nano = Dwarf (Greek) = 10-9

http://www.homecents.com

2


Nano = Dwarf (Greek) = 10

INTRODUCTIONINTRODUCTIONConventional

Filtration

Ultrafiltration

Microfiltration

Filtration

RO

33

Nanoparticles

3


3

Unique Properties of Nanoscale MaterialsUnique Properties of Nanoscale Materials

Hi h ifi f ( 100 2/ )

• Small size

• High specific surface area (> 100 m2/g)

• Quantum effects(dual behavior, wave- and particle-like) unique mechanical,electronic photonic and magnetic propertieselectronic, photonic, and magnetic properties

44

SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

Increase of Surface Area with Decreasing Particle Size

5Nel et al Science 2006 311:622-627 5Nel et al. Science, 2006, 311:622-627


NPs - Applications

I i i d t i l / i l li tiIncreasing industrial / commercial applications

MedicineCatalysis

Environmental technology

Cosmetics

S i d t

Microelectronics

Semiconductors

http://www slashgear comhttp://www.slashgear.com

Nanotechnology 1 trillion US $ market by 2015Nanotechnology 1 trillion US $ market by 2015.


NP i S i d t M f t iNPs in Semiconductor Manufacturing

CMP slurries

• SiO2SiO2

• Al2O3

• CeO2

NPs for immersion lithography

Carbon nanotubes


Colloidal silica (10-130 nm)(Source: www.bjgrish.com) 7

NPs in Chemo-Mechanical Planarization

Untreated CMP effluentUntreated CMP effluent (50-500 mg/L)


8

Nanomaterials ESH ConcernsNanomaterials – ESH Concerns

Concern about the adverse effects of NPs on biological systems- ENM: unusual properties due to their small sizeENM: unusual properties due to their small size

- Increasing evidence that some NP cause toxicity

Poor understanding of “nanotoxicity”- Uncertainty about the real-life hazards of engineered NPs

Need for improved bioassays to evaluate “nanotoxicity”


Problems Assessing NanotoxicityProblems Assessing Nanotoxicity

Reduction of MTT dye by NP surfaceQuenching of fluorescence Sequestration of insoluble C

Interference in classical methods dependent on

y yQ gMTT dye by CNT

Interference in classical methods dependent on colorimetric or fluorimetric measurements.

NP characterization most studies do not include characterization of NPs in biological medium.

1010


Real Time Cell Electronic Sensing (RT-CES)

E-Plate 96

The RT-CES system measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of culture plates.

SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing 11

E-plate

1212

Chem. Res. Toxicol. 2005, 18, 154-161

Cell Index

CI- Quantitative measure of the overall status of the cells:

Cell number Cell adhesion and spreadingp g Cell morphology

C ll h l b f (B) d ft 3h t t tIncrease in Cell Index with cell number Cell morphology before (B) and after 3h treatment with As(III) (C)

Increase in Cell Index with cell number

Chem. Res. Toxicol. 2005, 18, 154-161

Chem. Res. Toxicol. 2005, 18, 154-161

13

Impedance-based Real Time Cell Analysis

Advantages (Fluorescent) label free

Dynamic data of the biological status of the cells

Noninvasive

High throughput technique

Limitations Requires adherent cells

Correlation analysis between RT-CES and classical toxicity

14

endpoints performed on a limited number of compounds

Limited information of its applicability to NPs.14


OBJECTIVESOBJECTIVES

Assess the applicability of a real time cell electronic sensing (RT-CES) technique based on impedance measurements to

l t th t t i it f i d i i id evaluate the cytotoxicity of nanosized inorganic oxides used in semiconductor manufacturing

Validation of the RT-CES assay results: RT-CES vs. MTT

Characterizing the aggregation of nanomaterials in the g gg gbiological medium.

1515


RT-CES VS. MTT BIOASSAYS

H b hi l ith li l llHuman bronchial epithelial cells (16HBE14o-)

• Impedance based RT-CES assay:RT CES Impedance based RT CES assay:• (xCELLigence, Roche)

RT-CES

• O2 uptake

C ll b i iMTT

• Cell membrane integrity

1616


I d b d R l Ti C ll A l i (RTCA)IMPEDANCE-BASED RT-CES

Impedance-based Real Time Cell Analysis (RTCA)(RT-CES, xCELLigence, Roche) - Lung epithelial cells: 16HBE14o-

16 HBE culture E Plate 9616 HBE culture•MEM1 (10% FBS2)•37ºC

E-Plate 96•MEM (3.75% FBS)•100,000 cells/well•37ºC

RT-CES system•37ºC

37 C

1 Minimum Essential Medium2 Fetal Bovine Serum

17

o Cells are transferred to the E-Plate at 100,000 cells/well.o NPs are dosed after 16 h of incubation.o Cells are monitored for at least 48 h 17


o Cells are monitored for at least 48 h.

RT-CES Bioassay

Experiment stages9

F

6

7

8

ex

nano-Fe

ControlWashing

3

4

5

Cel

l Ind

e

10 mg/L

100 mg/L

1,000 mg/L

Washing

0

1

2

0 10 20 30 40 50 60 700 10 20 30 40 50 60 70

Time (hours)

18

Overnightgrowth

Exposure to Response to

nanoparticles 18pnanoparticles

nanoparticles


RT-CES Bioassay

CELL INDEX

R El t d i d ith ll t

19

Rcell = Electrode impedance with cells presentRb = Electrode impedance without cellsN= Number of points measured = 3

19


Example Output RT-CES with As(III)

8

p p ( )

ControlDosing of

As(III)

6

7

4

5

ell Ind

ex

0.5 mg/L

2

3

Ce

2 5 /L30 mg/L ->

0

1

0 10 20 30 40 50 60 70

2.5 mg/L10 mg/L100 mg/L ->

30 mg/L >

20

0 10 20 30 40 50 60 70

Time (hours)

Control As 500 ppb As 2,500 ppb As 10,000 ppb As 30,000 ppb As 100,000 ppb20


RT-CES: Al2O3 Nanoparticles

Al2O3 Nanoparticles (50 nm) - IC50 = 300 mg/L

control

250 ppm Al2O3

500 ppm Al2O3

1000 ppm Al2O3

2121


RT-CES: SiO2 nanoparticles

SiO2 Nanoparticles (10-20 nm) - IC50 = 225 mg/L

Control

2 p ( ) 50 g

100 mg/L

200 mg/L200 mg/L

300 mg/L600 mg/L

2222


RT-CES: CeO2 nanoparticles

CeO2 Nanoparticles (50 nm) - IC50 > 1,000 mg/L

2323


MTT BioassayMTT Bioassay

Assay relies on the reduction by live cells of the water-soluble tetrazolium MTT salt to a colored formazan dye.

Indicator of cell redox activity and viability

2424


MTT BIOASSAY

Cells• MEM (10% FBS)• 37°C, 5% CO2

24 wells plate• MEM (3 75% FBS)MEM (3.75% FBS)• 500,000 cells/well

Incubation• 37°C, 5% CO2

Staining• MTT reagent• 0.4 mg/mL

Measurement• Plate reader• 550 nm

25o Cells are transferred to a 24-well plate (5 105 cells/well)

NP d d ft 24 h f i b ti 25o NPs dosed after 24 h of incubationo After 48 h, cells are washed and stained with MTT reagent

MTT BIOASSAY: NANO- AL2O3

1.2

Al2O3 NP (IC50 = 980 mg/L)

0.8

1.02 3 ( 50 g )

0.6

bsorba

nce

0.2

0.4Ab

0.0

0 100 250 500 750 1,000 1,000 (no cells)

Blank (DMSO)

26

(no cells) (DMSO)

Concentration (mg/L)

NP did not interfere with the MTT analysis. Cell-free controls with the 26yhighest NP level caused a marginal increase of the absorbance relative to the DMSO control (2-3% of max. absorbance, depending on the NP used)

RT-CES & MTT BIOASSAY: NANO- SIO2

SiO2 NPs- IC50 = 225 mg/L (RT-CES), 172 mg/L (MTT)

100

120

80

100

control)

RT-CES40

60

ity (%

of

MTT20

40

Activ

i

27

0

0 200 400 600 800 100027Conc. (mg/L)

xCELLigence MTTSRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing

COMPARISON RT-CES vs MTT RESULTS

1400IC50 > 1,000 mg/L

1000

1200

mg/

L)

600

800

ry C

onc

(m

200

400

% In

hibi

to

0ZnO Mn2O3 Ag0 SiO2 Al2O3 CeO2 Fe0 Fe2O3 TiO2 ZrO2

50

Nanomaterial

SiO2 Al2O3 CeO2 ZrO2

28

Nanomaterial

RTCA MTTRT-CES

28Conclusions: Good correlation between RT-CES and MTT results

Al2O3 and SiO2 moderate toxicity, CeO2 not toxic

Aggregation of NPs in Biological MediumAggregation of NPs in Biological Medium

Particle size distribution (PSD) & chemical analysisSame conditions as in toxicity assays

PSD & Z t PSD & Zeta Potential (DLS)

Concentration (ICP-OES)

29

Sampling(ICP OES)

29


AGGREGATION OF NPS IN BIOLOGICAL MEDIUMAGGREGATION OF NPS IN BIOLOGICAL MEDIUM

Dispersion Aggregation Sedimentation

supernatant

total

3030


AGGREGATION OF NPS IN BIOLOGICAL MEDIUM

100CeO

80

rnat

ant)

CeO2

40

60

2 (in

sup

er

20% C

eO2

0H2O pH4.5 RT-CES PBS YEDP Microtox

Medium

31In bioassay media CeO2 NP agglomerated and the concentration that is 31In bioassay media, CeO2 NP agglomerated and the concentration that is effectively dispersed decreased many-fold

PROTEIN ADDITION (FBS)-Eff t St bilit f Al O NPEffect on Stability of Al2O3 NPs

36 39

50Al2O3 in the supernatant

4000

Particle size at different %FBS

36 36 39

30

40

over

y

3,0863000

size

(nm

)

10

20

% R

ec

1000

2000

Par

ticl

e s

2

00% FBS 2% FBS 5% FBS 10% FBS

219417

1940

0% FBS 2% FBS 5% FBS 10% FBS0 1.8 3.8 7.5 0 1.8 3.8 7.5

32FBS in the growth medium (MEM) stabilizes the Al2O3 NP dispersions

FBS (%) FBS (%)

32


CONCLUSIONSCONCLUSIONS

RT-CES is a useful, high throughput technique for dynamic monitoring ofNP cytotoxicity. The test relies on impedance measurements, avoidinginterference problems often associated with colorimetric/fluorimetric tests.

The inhibitory concentrations determined for NPs using the RT-CEStechnique correlated well with those generated by a commonly used

t t i it (MTT)cytotoxicity assay (MTT).

Al2O3 and SiO2 NPs showed moderate toxicity in the MTT and RT-CESassays CeO was not toxic at very high concentrations (1 000 mg/L)assays, CeO2 was not toxic at very high concentrations (1,000 mg/L)

Most of the nanoscale inorganic oxides tested showed a high tendency toaggregate in the RT-CES & MTT medium resulting in micron-sizegg g gaggregates that settled out of the dispersion.

3333


ONGOING RESEARCH:CYTOXICITY MECHANISMS OF INORGANIC OXIDE NPS

Release of toxic products

Disruption of cell membrane (flow cytometry)

ROS formation (intra- and extracellular generation)

Protein damage (ELISA bioassay)

3434

SRC/SEMATECH Engineering Research Center for Environmentally Benign Semiconductor Manufacturing34

ONGOING RESEARCH:REDUCING AGGREGATION OF NPS IN BIOLOGICAL MEDIUM

4045

CeO2+Dispex pH7

253035

%)

CeO2 pH7

101520

ensi

ty (%

05

10

0 1 10 1000

Inte

35

0.1 10 1000Particle Size Distribution (d.nm)

35


ACKNOWLEDGEMENTS

SEMATECH / SRCSEMATECH / SRC

UA Nanotoxicity group

SRC/Sematech Engineering Research Center for Environmentally Benign Semiconductor Manufacturing36

3737


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