contemporary issues in nanotoxicology: continuing to ......2012/05/31 · nanotoxicology &...

RTI International

ERC/SRC center at University of Arizona May 31, 2012

Contemporary Issues in Nanotoxicology: Continuing to

Relate Material Properties to Biological Response

Christie Sayes, Ph.D.

Program Manager Nanotoxicology & Nanopharmacology

Center for Aerosol and Nanomaterials Engineering

RTI International

[email protected]

Engineering and Technology

ACKNOWLEDGMENTS

Support

Sayes Lab Department of Vet. Physiology & Pharmacology and the Department of Biomedical Engineering

o Michael Berg

o Amy Romoser

o Aishwarya Sooresh

o Allen Sho

o Peter “Alex” Smith

o David Figuero Center for Aerosols and Nanomaterials Engineering

o Spencer West

o Phil Durham

o Laura Haines

Texas A&M University RTI International

2

http://www2.dupont.com/DuPont_Home/en_US/index.html


NP

Advantages to Nanotechnology

Enhanced Drug

Solubility

Increased Drug

Specificity

Imaging

Applications

Diagnostics

Altered Optical

Properties

Increased

Insulating Ability

Improved

Electronic Efficacy

Bactericidal

Consumer Products Medicine & Research

But what are the risks? 3


The most accepted toxicological

evaluation is an in vivo study. Because

tissues are composed of multiple cell

types, in vitro toxicology must use

multiple cell types in the study design.

Effects must be related to particle PCC

• Size

• Morphology

• Surface

Nanotoxicology is the study of the environmental and human health

effects of nanomaterials designed to improve our way of life.

3-step process to characterize nanotoxicity

1. Delivery

2. Chemical/biochemical reaction with target

3. Cellular dysfunction and resultant toxicities

What is Nanotoxicology?

4


Themes in Nanotoxicology that Influence Other Fields

Approach: The scale of health using the hierarchical oxidative stress model

DIS

EA

SE

-FR

EE

DIS

EA

SE

D

no observable adverse effects

immunological response

no extended adverse effects

This approach fits well within RTI International

“turning knowledge into practice”

Level of Oxidative Stress Increasing

Increasing Particle Concentration

5


0 5 10 15 20

0

1

2

3

4 media & UV

Time (min)

0

1

2

3

4 media & sonicate

0

1

2

3

4 media

RL

U (

x 1

0,0

00

)

0

1

2

3

4water

nano-TiO2 anatase

nano-TiO2 rutile

Structure Chemistry Toxicity

Toxicological evaluations require comprehensive material characterization including both

physical attributes and chemical surface reactivity.

Physical & chemical features of nanomaterials

6


Nanoparticle Properties Relevant

To Nanotoxicology

1) Chemical composition

2) Size & size distribution

3) Surface area

4) Surface chemistry,

stability, REDOX

5) Crystallinity & purity

6) pH & ISP

SO3H

7


Raw Materials Production

Product Manufacturing

Consumer Use

Product End of Life

A Life-Cycle Approach

Step 1: Material Characterization of Pristine Engineered Nanomaterial

Step 2: Formulate Nanocomposite or Other Nano-Enabled Bulk Material

Step 3: Simulate Wear-and-Tear or Weathering Conditions

Step 4: Measure Exposures

Step 5: Perform Focused Toxicity Testing

Step 6: Assess and Manage Risks

8


CASE STUDY:

Importance of Material

Characterization

9


Material Characterization

Berg & Sayes, Nanotoxicology, 2009

Effects of pH on a metal

oxide nanoparticle

10


0

25

50

75

100

1hr 24hr 48hr

0

500

1000

1500

2000

2500

-80

-60

-40

-20

0

20

40

60

80

1 3 5 7 9 11 13

Zeta

po

ten

tia

l (m

V)

pH

model NP

control cells

Ce

llu

lar

via

bil

ity (

%)

Exposure time

0

25

50

75

100

1hr 24hr 48hr

0

500

1000

1500

2000

2500

3000

-80

-60

-40

-20

0

20

40

60

2 4 6 8 10

pH

TiO2

TiO2

Ce

llu

lar

via

bil

ity (

%)

Exposure time

0

25

50

75

100

1hr 24hr 48hr

0

500

1000

1500

2000

2500

3000

3500

-50

-30

-10

10

30

50

2 4 6 8 10

Siz

e (

nm

)

pH

Al2O3

Ce

llu

lar

via

bil

ity (

%)

Al2O3

Exposure time 11


Gastric Acid Lysosomal

Fluid

Intestine &

Urine Blood

pH Level <2 4.5 5 7.4

Metal oxide

nanomaterial Zeta potential (mV) / Average agglomerate size (nm)

TiO2 +46/1573 +22/1860 +7/2390 -37/460

ZnO +50/360 +44/945 +16/1200 -3/1170

Al2O3 +45/561 +38/1750 +27/2400 -4/3050

CeO2 +32.6/1444 +26/2340 +20/2590 -6/2850

Fe2O3 +25.4/1800 -9/1740 -15/1700 -47/830

Can we predict how nanomaterials would behave in physiological compartments?

TiO2 and Fe2O3 nanoparticles demonstrate strongly charged agglomerates at pH=7.4

Material Characterization

12


CASE STUDY:

TOXICOLOGICAL EFFECTS

(AND MECHANISTIC ANALYSES)

OF SILICA NANOPARTICLES

13


In vitro cellular systems will need to be further developed, standardized,

and validated (relative to in vivo effects) in order to provide useful

screening data on the relative toxicity of inhaled particles

Each particle should be tested on a case-by-case basis

Cannot assume that nanomaterials are the same as

their bulk counterpart

but also cannot assume that they are more toxic

Risk = Hazard Exposure

Most nanotoxicology studies include hazard

identification

only some include exposure assessment

14


• Schematic diagram of the aerosol

nanoparticle reactor with

characterization instrumentation

• TEOS was pyrolyzed to generate SiO2

nanoparticles that are charged with an

aerosol neutralizer

• Characterized with a long or nano DMA,

and measured for particle concentration

with a condensation nucleus counter or

aerosol electrometer

Experimental Design

Exposure Groups

Group 1 (3 day exposure)

Sham (5 rats/group)

Particle-exposed (5 rats/group)

Targeted particle sizes = 35 nm

and 80 nm

Group 2 (1 day exposure)

Sham (5 rats/group)

Particle-exposed (5 rats/group)

Targeted particle sizes = 35 nm

and 80 nm

Inhalation

Post-exposure evaluation via

lung fluid and lung tissue

24 hr 1 wk 1 mo 2 mo

(lung fluid) (tissue)

Dose Metrics for Inhalation Studies

15


Particle Physicochemical Characterization

Aerosol Concentrations and Sizing

of 37 nm Amorphous Silica Particles (1x Day 1)

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

1 10 100 1000

Size (nm)

Co

nc

en

tra

tio

n (

pa

rtic

les

/mL

)

initial concentration 7:00 am

animal exposure 7:15 am


animal exposure 9:20



Aerosol Concentrations and Sizing

of 71 nm Amorphous Silica Particles (3x Day 3)

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

1.4E+07

1.6E+07

1.8E+07

1 10 100 1000

Size (nm)

Co

ncen

trati

on

(p

art

icle

s/m

L)

initial concentration 7:15 am





animal exposure 12:15 pm

Aerosol nanoparticle size distributions for SiO2 exposure in the inhalation chamber

as a function of exposure time demonstrating aerosol stability

Typical aerosol exposure run on day 1

for the d50 = 37 nm particle generation

experiment

Typical aerosol exposure run on day 3

for the d50 = 83 nm particle exposure

experiment

37 nm 83 nm

16


Inflammatory Response Dead Cells

Pulmonary Effects

Lung lavage fluid percent polymorphed neutrophil

(%PMN) values

Lung lavage fluid lactate dehydrogenase (LDH) values

0

100

200

300

400

500

24 Hour

1 Week

1 Month

0

100

200

300

400

500

Sham 1X Exp 1X Sham 3X Exp 3X

37 nm SiO2

83 nm SiO2

0%

25%

50%

75%

100%

0%

25%

50%

75%

100%

Sham 1X Exp 1X Sham 3X Exp 3X

37 nm SiO2

83 nm SiO2

17


sham (unexposed) animals animals exposed to

83 nm aerosolized silica

nanoparticles

AD

TB

AD

TB

AD

Lung pathology after 2 month exposure

animals exposed to

37 nm aerosolized silica

nanoparticles

AD

TB

Exposed Sham

Catalase Activity

(mU • mL-1 • mg protein-1) 34.35 ± 0.2 31.63 ± 0.01

Total Glutathione

(mM/10k cells) 2.34 ± 0.19 1.69 ± 0.14

Pulmonary Effects

18


Lung Tissue of Rat Exposed to Positive Control Particle after 1 week

19


0

100

200

300

0.01 0.1 100 0.01:4 0.1:4 100:4 0.01:4 0.1:4 100:4 0.01:4 0.1:4 100:4

Control Fe2O3 Fe2O3:ECB Fe2O3:ECB + AA Fe2O3:ox-ECB

RF

U

Nanomaterial dose (mg/L)

*

*

* *

2011

Berg and Sayes, CRT (2010)

Oxidative Stress is a Theme in Nanotoxicology

20


Differential Cellular Uptake Mechanisms

21


Tier 1

Normal

Tier 2

Antioxidant Defense

Tier 3

Inflammation

The Role of In Vitro Toxicology in Nanotoxicology

Due to the enormous range of nanomaterial-types, coupled with the infinite variety of surface

coatings, in vitro toxicology will play a major role in hazard identification

Hierarchical Oxidative Stress Model

But, are we considering if the effects are reversible? 22


Characteristics of A549 and MeT-5A Cells

A549 Cell Line

• Isolated through explant culture of

lung carcinomatous tissue

• Considered a type II lung epithelial cell

epithelial (surfactant producing)

• Reported to have high levels of

antioxidant enzymes

MeT-5A Cell Line

• Isolated from pleural fluids obtained

from non-cancerous individuals

• Transfected with a plasmid containing

SV40

• Normal cell precursor to mesothelioma

Berg JM, Figueroa DE, Romoser AA, Sayes CM. (2012)

Toxicology In Vitro, submitted.

Cu/Zn SOD

Mn SOD

A549 MeT-5A

Catalase

GAPDH

23


Silica at the Nanoscale: How safe is it?

Micrometer crystalline SiO2 is a Class II IARC

probable carcinogen often associated with

respiratory diseases such as:

• Silicosis

• Fibrosis

Endpoints in toxicological studies involving

SiO2 are often dependent upon particle

surface characteristics

Micrometer amorphous SiO2 is under GRAS

classification and often used as a negative

control in toxicological studies

Since SiO2 particle toxicity has often been reported as a function of surface

parameters, and since nanoparticles exhibit high surface area to mass ratios, it

is necessary to reevaluate the safety of SiO2 particles at the nanoscale

SEM: Nanoscale Amorphous SiO2

24


250 nm

5 µm

Transmission Electron Micrograph

Scanning Electron Micrograph

Energy Dispersive X-ray Spectroscopy

Silica Nanoparticle Characterization

25


Concentration of H2O2 (µM)

Differential Response to Oxidative Stress

MTT Assay Live:Dead Cell Counts

0

20

40

60

80

100

120

50 100 250 500 800 1500 2000

0

20

40

60

80

100

120

140

5 50 100 250 500 800 1000 1500 2000

*

* * * *

*

*

*

* * * *

Cell

Via

bili

ty (

% o

f C

ontr

ol)

Concentration of H2O2 (µM)

* * * *

* * *

*

0

20

40

60

80

100

120

0.01 0.1 1 10 25 50 75 100

Concentration of 30 nm SiO2 (µg/mL) Concentration of 30 nm SiO2 (µg/mL)

* * *

0

20

40

60

80

100

120

140

0.01 0.1 1 10 25 50 75 100

* * *

* * *

p<0.05 A549

MeT-5A

26


Ub

sMaf

Cytosol

Nucleus

ROS

Nrf-2 P

P P

ARE

↑CAT ↑other antioxidants

Ke

ap

1

Nrf-2 P

P

P

Kinase

Signaling

Cascade(s)

SiO2

SiO2

Nrf-2

Cul3

Rbx1 E2

Ub

Ub

HS

HS

Keap1

S S

NF-E2 Related Factor (Nrf2)

• Critical regulator of

intracellular

antioxidants and

phase II

detoxification

enzymes.

• May be induced by:

• Electophilic

Stress

• Kinase Signaling

Pathways

• Activation of Nrf2

can be cyto-

protective at low

levels of ROS

The Good The Bad • Elevated Nrf2 is a major

obstacle to the

successful treatment of

many cancers

The Ugly We hypothesize that SiO2

nanomaterials, which has

been shown to produce

ROS, may activate the

Nrf2 signaling pathway.

This induction might lead

to the induction of many

phase II genes which can

have implications in both

resistance against cell

death and carcinogenesis

27


Ub

sMaf

Cytosol

Nucleus

ROS

Nrf-2 P

P P

ARE

↑CAT ↑other antioxidants

Kea

p1

Nrf-2 P

P

P

Kinase

Signaling

Cascade(s)

SiO2

SiO2

Nrf-2

Cul3

Rbx1 E2

Ub

Ub

HS

HS

Keap1

S S

SiO2 exposed epithelial cells values given in hours

Cont. 0.5 2 4 8 12 24 48 1

NRF2

GAPDH

SiO2 exposed mesothelial cells Cont. 0.5 2 4 8 12 24 48 1

NRF2

GAPDH

Nrf2 Stabilization

28


Nrf2 Cytoplasmic Stabilization & Nuclear Translocation

Unexposed Control tBHQ (50 mM) SiO2 (75 µg/mL)

ep

ith

elia

l m

eso

the

lial

Scale bar = 10 mm

Cytoplasmic stabilization & nuclear translocation of NRF-2 (GREEN) was evident following

treatment with tBHQ or SiO2 nanoparticles for 24 hrs in both cell lines

29


CASE STUDY:

MIXTURES NANOTOXICOLOGY –

ITS HARDLY EVER JUST ONE

30


Exposure to Nanoparticle Mixtures

Reported Exposure to Nanoparticle Mixtures

Reference

Manganese from nearby welding during Li4Ti5O12 handling Peters, 2009

Iron and nickel as catalysts in carbon nanotube synthesis Maynard, 2004

Silicon & asbestos from insulation during CNTsynthesis Han, 2008

Combustion-derived particles from forklifts, heaters, & traffic Kuhlbusch, 2006 & 2010

Therefore, nanoparticle exposures are often mixtures of

combustion derived

carbonaceous particles and transition metals 31


Nanoparticles Representative of Mixtures

Carbonaceous Particle

• Engineered Carbon Black

• Used as a surrogate for elemental

carbon in particulate matter

• Extensively used in airborne

toxicological studies

Fe2O3

ECB

Transition Metal

• Iron oxide (Fe2O3)

• Represent transition metal oxides in

particulate matter

• Water insoluble particle, but soluble in

acidic pH

32


Guo and Sayes, P&FT, 2009

Aim

Determine if Co-exposures to

Fe2O3 and ECB results in additive

or synergistic cytotoxicological

effects

Conclusion

“Co-exposure to carbon black and

Fe2O3 particles can cause

oxidative stress that is

significantly greater than the

additive effects of exposures to

either particle type alone.”

33


Chemically Active Groups on

ECB Surface Gives Rise to

Surface Redox Capabilities

H2Q(s) + 2Fe3+

(aq) ------> Q(s) + 2Fe2+(aq) + 2H+

Fenton Reaction:

Fe2+ + H2O2 → Fe3+ + OH· + OH-

Hypothesis: Oxidative stress formed in a co-exposure of

Fe2O3 and ECB can be eliminated by surface oxidation of

ECB (termed: ox-ECB).

Oxidation Decreases Reductive Capacity

34


XPS gives quantitative data on

elemental composition and chemistry on the particle surface

ECB (%) Ox-ECB (%)

%C [C/ C+O] 89.88 88.53

%O [O/ C+O] 10.12 11.47

O:C 0.1126 0.1295

Ratio of Q(s):H2Q(s) was found to be circa 5000 times greater in ox-ECB than in ECB.

The O:C ratio in ox-ECB is 15% greater than in ECB. This may not seem like a large

increase, but edge carbons comprise about 20% of the total carbon content for 50 nm

amorphous carbon particles.

H2Q(s) + 2Fe3+(aq) ------> Q(s) + 2Fe2+

(aq) + 2H+

Surface Chemistry Analysis with XPS

35


N

VV

Fe2O3

ECB

Fe2O3

ECB

V

V

V

ox-ECB

N

Fe2O3

Fe2O3 + ECB

2 mm 1 mm 0.5 mm

Fe2O3 + ECB Fe2O3 + ox-ECB

Intracellular Nanoparticle Incorporation

36


1. A majority of the Fe2O3 is internalized into the cells after 24 hours

2. Uptake of Fe2O3 is not altered following ECB co-exposure

Intracellular Nanoparticle Incorporation

37


Nanoparticle Agglomerates in Cellular Vesicles

STEM-HAADF micrograph

of internalized Fe2O3

nanoparticle agglomerates

Lysosomal Trafficking

• Nanoparticles accumulate in

lysosomes

• pH ≤ 5.2 (pKa Lysotracker Red)

• May alter dynamic nanoparticle

properties

• May solubilize water insoluble

particles

Fe2O3 Exposed

2μm

Control ECB Ox-ECB

Fe2O3

1 hr

3 hr

24 hr

Intracellular Trafficking of Nanoparticles

38


Oxidant Production

• Significant increases seen

in Fe2O3 and ECB co-

exposure groups

• Oxidant production

eliminated by addition of

L-ascorbic acid

• ox-ECB and Fe2O3 did not

differ from control groups

• Effects greater at 25

hours than at 2.5 hours

Statistically significant to control population; P<0.05

39


Nanomaterial physicochemical

features Induced toxicological effects Mathematical techniques

Cross-validation More

fundamental

More

complicated Immediate Systemic

Regression

Analysis Classification

Primary particle

size

Zeta potential (as

a measure of

surface charge)

Cell viability Metabolism Linear

Linear

Discriminant

Analysis

Inter-lab

comparisons

Shape Agglomerate size Tissue

damage

Distribution &

accumulation Non-linear

k-Nearest

Neighbor Beta-testers

Chemical

composition

Adsorption of

surrounding

matrix

Cytokine

production Inflammation

Machine

learning

Support Vector

Machines

Additional

physicochemical

data

Specific surface

area

Reductive

capacity

Membrane

damage

Immune

response

Causal

relationships

Decision Trees

or Neural

Networks

Additional

toxicology data

THREE KEY AREAS OF GROWTH:

Mathematical/Computational-Based Predictive Models

Alternative Toxicity Testing

Characterization of Real-World Exposure Scenarios

Where Do We Go From Here?

40

contemporary issues in nanotoxicology: continuing to ......2012/05/31 · nanotoxicology &...

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