nanotechnology: toxicological considerations. terry tetley · effects of ambient environmental...

Nanotechnology: Toxicological considerations.

Terry Tetley

Lung Cell Biology

Section of Pharmacology and Toxicology National Heart and Lung Institute

Imperial College

AIR POLLUTION

Friday 5 to Tuesday 9 December 1952

Estimated that up until 8 December 4,000 people had died prematurely

and 100,000 more were made ill.

Recent research suggests that the total number of fatalities was considerably

greater, at about 12,000

Effects of ambient environmental particulate air pollution on cardio vascular health

• Increases in PM2.5 from a few hours up to weeks causes increased ischemic heart disease events and hospitalisation

• Increased myocardial infarct, both acute and chronic exposure, and increased risk of death from myocardial infarct with chronic exposure

• Raised blood pressure

• Reduced heart rate variability

• Development (in previously healthy individuals) and acceleration of atherosclerosis

• Deaths form heart attacks

• Deaths from strokes

Effects of ambient environmental particulate air pollution on respiratory health.

• Increased coughing and wheezing, particularly children

• Increased use of medication for asthma

• Attacks of asthma in patients with pre-existing asthma, especially children

• Exacerbation of chronic obstructive pulmonary disease (COPD) and associated increased hospital admissions (doubling for each 10mg/m3 increase in PM2.5) and increased rate of decline in FEV1

• Reduced growth in lung function in children

• Increased risk of microbial infection

• Deaths from cancer and other respiratory causes

• Prenatal exposure relates to reduced lung function/function and increased airway hyperresponsiveness, airway inflammation, mucus secretion

LONDON, DECEMBER 1952

AIR POLLUTION: 1952 SMOGS – MIX OF SOOT AND FOG

Today: Deisel exhaust particles are <100nm in diameter

ENGINEERED NANOPARTICLES?

EFFECTS UNKNOWN

Nanoparticles/nanomaterials are structures which have at least one dimension of 100nm or less. Include nanofibres, nanotubes as well as spherical structures with a diameter of <100nm.

Nanomaterials

Why use nanoparticles?

• When materials enter the nano-range, their properties alter from that of their micron-sized equivalents.

• Nanoparticles have enhanced thermal, magnetic, electrical and optical properties

• As size decreases, the surface area:volume ratio increases

• The surface is more reactive, improving drug labelling

Nanoparticle: Less than 100nm in one dimension

1 1cm3 sugar cube,

1 x 1015 100nm3 cubes

6.4 x 1016 25nm3 cubes

Nanoparticles can be natural, anthropogenic or manufactured

Sugar cube analogy

Nanoparticle use, number of products listed as containing nanoparticulate material

Over 1300 products-

Medicine Engineering Construction industry Cosmetics Clothing Food Sports goods

Antibacterial sprays and medical products

Cosmetics, sun cream

Sports goods

Low calorie food products

Diagnosis, treatment

Clothing

Use of engineered nanoparticles

Nanoparticle use

2002 – Publication of Prey, a novel by Michael Crichton. A medic with a vivid imagination. The book describes how an artificial swarm of nanorobots develop intelligence and threaten their human inventors. The novel generated concern within the nanotechnology community that the public perception of nanotechnology might be affected, creating fear of a similar scenario in real life.

Nanotoxicology Following concerns raised by Prince Charles and others, the Royal Society and Royal Academy of Engineering published a report, Nanoscience and nanotechnologies: opportunities and uncertainties, July 2004. Nanotoxicology emerged as a subject in its own right. http://www.nanotec.org.uk/finalReport.htm.

Perceived risks: • Occupational exposure

• Exposure during use/application/disposal

• Accidental exposure

Concerns: • Safety: Potential adverse effects

via inhalation- lungs, dermal-skin, ingestion-gut, systemic delivery

(e.g. intravenous administration)

• Susceptible subpopulations

• Implications for society

Risks and concerns

Deposition and impact of inhaled nano-sized ambient air pollution particles

Structure of the lung

TT1 cells - Transformed human alveolar epithelial type-1-like cell (TT1) model. Kemp, S. J. et al Am. J. Respir. Cell Mol. Biol. 2008

AT2 cells - Primary alveolar epithelial type 2 cells, isolated from normal regions of human lung tissue.

In vitro alveolar models

AM – Alveolar macrophages lavaged from human lung tissue

Human lung cell monolayer

Alveolar epithelium, alveolar macrophages, fibroblasts,

Incubated with nanoparticles

Up to 24 hours (acute)

Medium Cells

Pro-inflammatory mediators Particle uptake, Viability, Cytokines, chemokines Reactive oxygen species

Nanoparticle exposure models

4hr

Carboxyl

24hr

Neutral

ATII cell uptake of 50nm polystyrene latex nanoparticles

4hr

Carboxyl

24hr

Neutral

ATI cell uptake of 50nm polystyrene latex nanoparticles

0 30 60 90 120 150 180 210 2400

1000

2000

3000

4000

5000

6000

7000

Time (mins)

MFI

0 30 60 90 120 150 180 210 2400

1000

2000

3000

4000

5000

6000

7000

Time (mins)

MFI

50nm 100nm

ATI cell uptake of 50nm and 100nm polystyrene latex nanoparticles

Amine-modified Carboxyl-modified Unmodified

Particle cytotoxicity

0 50 100 150 2000

20

40

60

80

100UnmodifiedAmine ModifiedCarboxyl Modified

Dose (g/ml)

% V

iabi

lity

0 50 100 150 2000

20

40

60

80

100UnmodifiedAmine ModifiedCarboxyl Modified

Dose (g/ml)

% V

iabi

lity

50nm Nanoparticles

0 50 100 150 2000

20

40

60

80

100

Dose (g/ml)

UnmodifiedAmine ModifiedCarboxyl Modified

% V

iabi

lity

100nm Nanoparticles

Carboxyl-modified NP

A

E

Amine-modified NP

Hopping probe ion conductance microscopy of AT1 epithelial cells exposed to carboxyl-modified and amine-modified 50nm

latex particles for 4 hours.

Carboxyl-modified NP

A

E

Amine-modified NP

Hopping probe ion conductance microscopy of AT1 epithelial cells exposed to carboxyl-modified and amine-modified 50nm

latex particles for 4 hours.

Ruenraroengsak et al. Respiratory epithelial cell cytotoxicity and membrane damage (holes) caused by amine-modified nanoparticles, Nanotoxicology 2012, 6:94-108

500nm

Cellular fate of nanoparticles

Engineered nanoparticles/nanomaterials • Carbon-based – fullerenes, carbon nanotubes, wires, graphene etc

• Metals – gold, silver

• Metal oxides – titanium dioxide (self cleaning surfaces), zinc oxide (both in

sunscreens), copper oxide, cerium oxide (in deisel to reduce particle numbers)

• Organic/soft – as synthesised for drug delivery

• Quantum dots <10nm diameter

ARGYRIA – was he born with a silver spoon in his mouth?

Medical applications of silver nanoparticles

Au 5 nm

Au 15 nm

1 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml

Differences in cellular uptake of gold and silver nanoparticles

Phagocytic alveolar macrophage cell exposure (4 hr)

Does this impair phagocytic activity? eg during bacterial infection

Au 5 nm

1 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml

Au15 nm

Alveolar epithelial Type 1 cell exposure (4 hr)

Differences in cellular uptake of gold and silver nanoparticles

Au 5 nm

Au 15 nm

Alveolar epithelial type 2 cell exposure (4 hr)

1 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml

Differences in cellular uptake of gold and silver nanoparticles

Effect of Ag on ATI cells – not cytotoxic, but stimulate pro-inflammatory mediators

Ag (g/ml)

% C

ell V

iabi

lity

0 10 20 30 40 500

20

40

60

80

100 10nm Ag Spheres50nm Ag SpheresAg Nanowires

Ag (g/ml)IL

-8 (p

g/m

l)

0 10 20 30 40 500

50100150200250300350400

10nm Ag Spheres50nm Ag SpheresAg NanowiresPristine

Ag (g/ml)

% C

ell V

iabi

lity

0 10 20 30 40 500

20

40

60

80

100 10nm Ag Spheres50nm Ag SpheresAg Nanowires

Silver spray products NAME DESCRIPTION CODE

Colloidal Silver Spray Used as cosmetic spray, dermatologic applicatio S1CS

Schudheo Used to prevent unpleasant odors in shoes S3CS

Skin and Body Protectant Used for skin enhancement S5CS

General Hygiene Antiseptic Used to clean skin S6CS

Mesosilver Dermal application as antifungal spray. S7HS

Colloidal Silver Nasal Spray Used to improve breathing S8HS

Sovereign Silver Used as dietary supplement S9HS

Smelly Shoe Spray Used to prevent unpleasant odors in shoes S10OS

Silver Care Used as cosmetic for skin protection S11CS

Ultra Colloidal Silver Spray Used orally as a dietary supplement S12HS

Nanofix Used to clean surfaces S13OS

Nan Health Silver Used orally to prevent diseases (no built-in sprayer)

S2HO

Silver 22 Used orally as a dietary supplement (no built-in sprayer)

S4HO

Slide: courtesy Gedi Mainelis, Rutgers

Smelly Shoe Spray

• 3.86 µg/ml Colloidal Silver

• Isopropanol

• Water

• Peppermint essential oil

MesoSilver Nanofix Silver Nasal Spray

• 15.86µg/ml Colloidal silver

• Deionized water

• 1.21µg/ml silver

• 11.096 µg/ml silver

• Deionized water

SILVER CONTAINING PRODUCTS

S1 S2

• 505.4ug/ml Pyrithione Zinc

• SDA-40 Alcohol

• Isopropyl Myristate

• SLS needles • Undecy lenic acid

• 5115µg/ml Zinc

• Vegitable Glycerin

• Peppermint Oil

• Clovebud oil

• Echinacea extract

• Menthol

DermaZinc Therazinc

Zinc containing Products

Z1 Z2

Effect of silver products on type-1 epithelial cell viability 24 hr exposure

0.048 0.096 0.3877 0.198 0.397 1.5882

particle concentration micrograms/ml

6.32 12.65 50.6 63.93 127.85 511.5

Effect of zinc products on Type -1 epithelial cell viability 24 hour exposure

particle concentration micrograms/ml

Nanoparticles can induce reactive oxygen species in alveolar cells

Control

AT2

+NPs NPs +NAC +NPs NPs+NAC +NPs NPs+NAC

MAC

i j k l

m n o p

TT1 a b c d

e f g h

T=4h

q r s t

u v w x

Co-application of N-acetylcysteine prevents induction of ROS in epithelial cells, but has no effect on macrophages

DOSE

Silver-induced genotoxicity

nt 1 10 30 500

2

4

6

8

10 ***

**

C (g/ml)

H2A

.X /

GA

PD

HGAPDH

H2A.X

nt 1 10 30 50 Ag (μg/ml) Induction of double-stranded DNA breaks

Collaborative in vivo studies show that inhalation of silver causes acute: • Inflammation

• Reduction in elastance/elastic

recoil

• Reduced lung surfactant function

"

Qualities of graphine:

•A thin flake of carbon, one atom thick

•Thinnest, strongest material ever

•Conducts electricity as effectively as copper

•Conducts heat better than all other materials

•Almost completely transparent, yet so dense,

not even helium can pass through it.

Human hair 70m

Nanowire 1/10,000th

Carbon nanotubes

Multi-walled and single walled

Less than 100nm diameter.

Can reach many micrometres in length.

Use of carbon nanotubes, wires, fibres: Engineering Electronics Construction industry Cosmetics Clothing Sports goods Medicine

Bhirde,A.A. et al. ACS Nano 3, 307-316 (2009).

Bhirde,A.A. et al. ACS Nano 3, 307-316 (2009).

-EGF

+EGF

Carbon nanostructures may exist in many formats including tubes, wires and fibres

Crocidolyte asbestos Chrysotile asbestos

Inhalation of asbestos can cause lung disease

NORMAL LUNG

ASBESTOSIS

LUNG CANCER & MESOTHELIOMA

TT1 - 200nm 4VP CNTs, T=24h

Transmission EM of TT1 cell uptake of 200nm MWCNTs

TT1 - 700nm 4VP CNTs, T=24h

28.4 1

7.4

21.9

20.0

20.2

SHORT MWNTs LONG MWNTs

Effect of long and short multiwalled carbon nanotubes on human alveolar cells

AT2 cells

0 0.1 1 5 10 50 1000

102030405060708090

100CNT ACNT BCNT CCNT DCNT E

**

* ***

CNT sample (g/ml)

Via

bilit

y (%

of

Con

trol

)

AT2 cells

0 0.1 1 5 10 50 1000

102030405060708090

100CNT ACNT BCNT CCNT DCNT E

**

* ***

CNT sample (g/ml)

Viab

ility

(% o

f Con

trol) 15-20m

Long) 3-5m (medium) 0.6-2m (short)

TT1 cells

0 0.1 1 5 10 25 50 100 5000

102030405060708090

100110

CNT ACNT BCNT CCNT DCNT E

***

*

******

***

******

********

CNT sample (g/ml)

Viab

ility

(% o

f Con

trol

)

Effect of long and short MWCNT on cell viability (MTT) With and without Curosurf (pig surfactant)

AM cells

0 0.1 1 5 10 50 1000

102030405060708090

100CNT ACNT BCNT CCNT DCNT E

***

******

* *

CNT sample (g/ml)

Via

bilit

y (%

of

Con

trol

)

long

short

IL-8 release from TT1, AT2 and AMs exposed to short and long MWCNTs

TT1 cells

0

500

1000

1500

200000.111050

***

***

** * *

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL-8

(pg/

ml)

TT1 cells

0

500

1000

1500

200000.111050

***

***

** * *

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL-8

(pg/

ml)

AT2 cells

0

2000

4000

6000

8000

10000

1200000.111050

******

*

******

*****

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL -

8 (p

g/m

l)AT2 cells

0

2000

4000

6000

8000

10000

1200000.111050

******

*

******

*****

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL -

8 (p

g/m

l)

TT1 cells AT2 cells TT1 cells

0

500

1000

1500

200000.111050

***

***

** * *

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL-8

(pg/

ml)

AMs

0

2000

4000

6000

8000

1000000.11

5010

***

***

* ****

Purity

Length 20m3m0.6m

97%

CNT sample (g/ml)

IL -

8 (p

g/m

l)

AMs

0

2000

4000

6000

8000

1000000.11

5010

***

***

* ****

Purity

Length 20m3m0.6m

97%

CNT sample (g/ml)

IL -

8 (

pg

/ml)

AMs

TT1 cells

0

500

1000

1500

200000.111050

***

***

** * *

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL-8

(pg/

ml)

AT2 cells

0

2000

4000

6000

8000

10000

1200000.111050

******

*

******

*****

Purity

Length 20m 3m0.6m 20m

48% 74% 97% 97%

CNT sample (g/ml)

IL -

8 (p

g/m

l)

AMs

0

2000

4000

6000

8000

1000000.11

5010

***

***

* ****

Purity

Length 20m3m0.6m

97%

CNT sample (g/ml)

IL -

8 (pg

/ml)

g/ml

• Shorter CNTs are more reactive with epithelial cells. • Longer CNTs are more reactive with macrophages.

Same pattern of response for IL-6 and MCP1 release

Frustrated phagocytosis is a common feature of mesothelioma

Frustrated phagocytosis of carbon nanotubes by human alveolar macrophages

Effect of MWCNT of different lengths on human lung fibroblast viability (n=4 subjects)

Effect of MWCNTs of different lengths on soluble collagen release by human lung fibroblasts

N=4

Development of granuloma and mesothelioma following injection of MWCNT into peritoneal cavity

Can Carbon Nanotubes cause fibrosis?

Ma-Hock,L. et al. Toxicol. Sci. 112, 468-481 (2009) Shvedova, A.A et al. Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L552-65

Ag PVP

20nm 110nm

Curosurf Curosurf

g/ml Ag

IL-6 pro-inflammatory cytokine release by AT1 cells exposed to silver nanoparticles

67

SP-A

SP-D

Binding of surfactant proteins A and D to MWCNTs

36 72 108 KDa

KDa 43

SPA binding

SPD binding

MWCNTs Long (>15m) Short (<3m)

NT 1 5 10 25 50

AR MWCNT (µg/ml)

β-tubulin

SpD

SpA

Intracellular AT2 SP-A and SP-D following exposure to pristine Arkema MWNTs

Mercer et al, Particle and Fibre Toxicology, 2013 epub ahead of print

Verdict Some nanosized materials may have adverse health effects They need to be tested, safety procedures need to be put

into place

Imperial College London

MRC-HPA Centre for Environment and Health

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