1

Formation, Physicochemical

Characterization and Toxicity of

Fullerene Nanoparticles

Ji DAI and Guanghao CHENHKUST

11 Nov 2011

2

Nanomaterial (NM) is a material with one dimension under 100nm. (EPA, 2007)

nanometer1 10 100 1000450

NM aggregation in water and soil

NM in molecular form

NM in airPM0.1

Background---Definition

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Background---NanomaterialApplication

First Generation ~2001: Passive nanostructuresNano-structured coatings, nano-particles, nano-structured metals, polymers, ceramics, catalysts, composites, displays

Second Generation ~2006: Active nanostructuresTransistors, amplifiers, targeted drugs and chemicals, actuators, adaptive structures, sensors, diagnostic assays, fuel cells, solar cells, high performance nano-composites, ceramics, metals

Third Generation ~2010: 3-D nano-systemsVarious assembly techniques, networking at the nano-scale and new architectures, biomimetic materials, novel therapeutics/targeted drug delivery

Fourth Generation ~2015: Molecular Nano-systemsMolecular devices “by design”, atomic design, emerging functions(Maynard and Michelson, 2005)

4http://www.nanotechproject.org/inventories/consumer/analysis_draft/

5http://www.nanotechproject.org/inventories/consumer/analysis_draft/

2006

2009

2006

2009

2006

2009

2006

2009

6

ManufacturedNM

Point Source

Accidental Release

Non-point Source

Intentional Release

Natural NMSource

SingleNP

AggregatedNP

AdsorbedNP

Water/Soil

Groundwater

Human

Air

environment

Background---Pathways into Environment

(Nowack and Bucheli, 2007)

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NM

Disruption of membrane and cell wall

Oxidize or damage protein

Damage DNA

Possible Causes of Biological Toxicity

(Klaine et al., 2008)

ROS

photoexcitation

Reactive Oxygen Species

8http://www.nanotechproject.org/inventories/consumer/analysis_draft/

Silver is not biodegradable.

Carbon is biodegradable and easy effect on environment and human. Therefore, carbon based nanomaterials were most wildly studied.

Typical carbon nanomaterials

Fullerene (C60, C70, C76, C84, etc.)

Nanomaterial (NM)

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Toxicityof C60

Mechanisms Study

toxicity enhancement

C60 is the most representative NM, therefore, they were wildly studied. Although C60 is virtual insoluble, there are still some methods to disperse C60into water and produce C60 suspensions (nC60), which achieve nC60s with different physicochemical properties. It may affect the toxic study results.

Formation and Physical Properties

of nC60 Agglomerates in Water

Chemical Propertiesof nC60 Agglomerates

In water

Reactive oxygen species generation (Hotze et al., 2008)

pH, Ionic strength, Organic components (NOM, medium, protein) (Wiesner et al., 2008)

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Objectives

The major objective:

Toxicity of nC60

The major objective:

Toxicity of nC60

Different nC60 dispersal methods via solvent exchange (mainly used in toxicity studies)

Different nC60 dispersal methods via solvent exchange (mainly used in toxicity studies)

THF/nC60(hydrophilic)

Tol/nC60(hydrophobic)

The details will be

shown in next slide.

The details will be

shown in next slide.

Comparison physical properties

Bacteria are at the bottom of food chain.∵

∴ Choose E. coli as the targeted species for toxicity study.

Toxicity tests.Toxicity tests.

E. coli toxicity performance

Possible Mechanisms

growth

metabolism

live/dead double staining

surface modification

reactive oxygen generation

adsorption or absorption

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nC60 Preparation Methods

-

Dissolve C60 into THF

Stir over night and filtrated

Add ultra-pure water with 1:1 ratio

Remove THF with rotary evaporator by a stepwise approach

C60 in toluene

ultra-pure water

Tetrahydrofuran (THF) toluene

Tol/nC60

THF/nC60

several to 100 nm

100 to 500 nm

sonification

Number-weighted Diameter (nm)

0 50 100 150 200

Par

ticle

Num

ber

20

40

60

80

100

120

Tol/nC60

THF/nC60

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For E. coli Testsa

Bacteria

Escherichia coliATCC11229

nC60 particle sizesnC60 concentrations

a: 0 mg/lb: 0.01 mg/lc: 0.1 mg/ld: 1 mg/l

b c d

Transfer E. coli to the bottle and make it 106/ml then incubate at 37oC with 135 rpm.

The E. coli growth, acetate production were studied during the incubation. Live/dead double staining were conducted after 4 hours of incubation.

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Results & Discussion

1. Comparison Physical Properties2. E.coli Toxicity Performance3. Possible Mechanisms

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Particle Sizestored THF/nC60 Tol/nC60

number-weighted mean (nm)

fresh 111.2 39.8

7 d 122.5 86.3

30 d 140.6 107.7

intensity-weighted mean (nm)

fresh 140.5 98.8

7 d 131.8 173.9

30 d 146.0 168.9

Polydispersity index (PDI)

fresh 0.068 0.198

7 d 0.032 0.154

30 d 0.025 0.104

Number-weighted Diameter (nm)

0 50 100 150 200

Par

ticle

Num

ber

20

40

60

80

100

120

Tol/nC60

THF/nC60

The particle sizes of THF/nC60 and Tol/nC60 are almost in the same range after long-term of storage, which will be used to conduct following comparison experiments.

Number-weighted and intensity-weighted mean diameters indicated the major particle size of nC60. PDI represent the particle distribution status: smaller PDI means more uniform distribution.

1616

TEM Image

20 nm20 nm20 nm20 nm

tol/nC60THF/nC60

Fast Fourier transformations (FFT) of TEM Images show that nC60 in both cases are face-centered cubic crystals (fcc).

17

E. coli Growth

Time (hr)

0 1 2 3 4 5 6 7

OD

600

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

controlTHF/nC60=0.01 mg/l

THF/nC60=0.1 mg/l

THF/nC60=1 mg/l

Time (hr)

0 1 2 3 4 5 6 7O

D 6

000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

controlTol/nC60=0.01 mg/l

Tol/nC60=0.1 mg/l

Tol/nC60=1 mg/l

E. coli were incubated under 37oC overnight (12 hrs) prior to testing.

THF/nC60 is more toxic than Tol/nC60.The toxic affecting mainly respond at the lag growth phase.

Therefore, the incubation time changed from 12 hrs to 19 hrs to prolong the lag period.

When the concentration of THF/nC60 reached 1 mg/L, E. coli growth was inhibited greatly.

There is not any apparent growth inhibition on E. coli even when Tol/nC60concentration reached 1 mg/L.

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E. coli Growth (cont.)E. coli were incubated under 37oC for 19 hrs prior to testing.

Time (hr)

-2 0 2 4 6 8 10 12cu

ltura

ble

cell

amou

nt (

cells

/ml)

1e+4

1e+5

1e+6

1e+7

1e+8

1e+9

1e+10

1e+11

OD

600

0.0

0.5

1.0

1.5

2.0

controlTHF/nC60 = 0.01 mg/L

THF/nC60 = 0.1 mg/L

THF/nC60 = 1 mg/L

controlTHF/nC60 = 0.01 mg/L

THF/nC60 = 0.1 mg/L

THF/nC60 = 1 mg/L

Time (hr)

-2 0 2 4 6 8 10 12

OD

600

0.0

0.5

1.0

1.5

2.0

cultu

rabl

e ce

ll am

ount

(ce

lls/m

l)

1e+4

1e+5

1e+6

1e+7

1e+8

1e+9

1e+10

1e+11

THF/nC60 =1 mg/L

tol/nC60 =1 mg/L

controlTHF/nC60 =1 mg/L

tol/nC60 =1 mg/L

control

nC60 concentration (mg/L)

0.0 0.2 0.4 0.6 0.8 1.0

inhi

bitio

n ra

te (

%)

0

10

20

30

40

50

60

70

80

90

100

Y=0.9352X-0.0004R2=0.9997EC50 = 0.54 mg/L

For THF/nC60, median effect concentration EC50=0.54 mg/l, it is very toxic.

For Tol/nC60, it may inhibit the growth at lag growth phase, and enhance the growth at exponential phase.

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Time (hr)

1 2 3 4 5 6 7

Ace

tate

pro

duct

ion

(mg/

l)

-50

0

50

100

150

200

250

300

350

controlTHF/nC60=0.01 mg/l

THF/nC60=0.1 mg/l

THF/nC60=1 mg/l

Acetate Production

Time (hr)

1 2 3 4 5 6 7

Ace

tate

pro

duct

ion

(mg/

l)

-50

0

50

100

150

200

250

300

350

controlTol/nC60=0.01 mg/l

Tol/nC60=0.1 mg/l

Tol/nC60=1 mg/l

The acetate accumulation is the result of tricarboxylic acid cycle (TCA cycle). E. coli (aerobic organisms) use glucose as substrate producing acetate through TCA cycle, which indicates the growth condition of bacteria.

THF/nC60 is very toxic. And Tol/nC60 enhance bacteria growth at exponential phase.

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Live/Dead Double Staining

deathenhanced but see moredeath

enhancement

control Tol/nC60 = 1 mg/l

THF/nC60 = 0.1 mg/l THF/nC60 = 1 mg/l

live cells

dead cells

Chemical Property --- Modification

Pure C60 exhibits four sharp bands at 526, 575, 1182 and 1428

The strongest bands of toluene on IR (729, 696)

The strongest bands of THF on IR (2865, 2975) 21

Strain moves absorption to right from 1428 to 1410 shows the breaking down of C=C to C-C

Strain moves absorption to left from 1182 to 1183 shows small part of C-C changing to C-H

aromatic C=C stretching Methyl symmetrical C-H bending aromatic in-place C-H bending

aldehyde C-H stretching Aliphatic aldehyde C=O stretching or aromatic combination bands

Aromatic out-of plane C-H bending

Tol/nC60

THF/nC60

C60

The surface chemistry of Tol/nC60 was much more modified during the preparation than THF/nC60. It indicated that Tol/nC60 is less toxic than THF/nC60

possibly because of the surface modification.

The surface chemistry of Tol/nC60 was much more modified during the preparation than THF/nC60. It indicated that Tol/nC60 is less toxic than THF/nC60

possibly because of the surface modification.

nC60 Photoreactivity

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0C60

1C60

3C60

C60·-

Rel

ativ

e E

nerg

y S

tate

1 2

3

4

54

6

7λ

O2 1O2

O2

1O2

donor

O2

O2 ·-

1: electrons excited; 2: fluorescence; 3: intersystem crossing; 4:reaction with O2; 5: self- quenching; 6: oxidation and reduction reaction; 7: electron transportation(Hotze et al., 2008)

0

2

4

6

8

10

12

14

16

THF/nC60

Tol/nC60

waterE. coli11229

E. coliJM109

E. coli 11229+

NB

E. coli JM109+

LB

XT

T fo

rmaz

an p

rodu

ctio

n ra

te (

10-3

A.U

./min

)

23

0

20

40

60

80

THF/nC60

Tol/nC60

Flu

ores

cenc

e in

tens

ity in

crea

se r

ate

(A.U

./min

)

waterE. coli11229

E. coliJM109

E. coli 11229+

NB

E. coli JM109+

LB

−•2OSuperoxide generation ( )

Reactive Oxygen Species Generation

Singlet Oxygen generation ( )21O

THF/nC60 enhance the total production of singlet oxygen, while Tol/nC60 generate more superoxide than THF/nC60.

Superoxide generated by THF/nC60 is one possible reason for the toxicity performance. While singlet oxygen would not be a problem for E. coli growth.

Superoxide generated by THF/nC60 is one possible reason for the toxicity performance. While singlet oxygen would not be a problem for E. coli growth.

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When E. coli concentration is lower than 108 cfu/ml, the nC60 adsorption/absorption mainly happens after 2 hours mixing. However they adsorb quickly when E. coli concentration is 109 cfu/ml.

Time (hour)

0 1 2 3 4 5-0.1

0.0

0.1

0.2

0.3

0.4

0.5

E. coli = 109 cfu/mlE. coli = 108 cfu/mlE. coli = 107 cfu/mlE. coli = 106 cfu/mlcontrol

adso

rptio

n/ab

sorp

tion

perc

enta

ge (

%)

Adsorption/Absorption

E. coli (cfu/ml)

0

10

20

30

40

50

THF/nC60 @ 37 oC

THF/nC60 @ 25 oC

THF/nC60 @ 4 oC

Tol/nC60 @ 37 oC

Tol/nC60 @ 25 oC

Tol/nC60 @ 4 oC

0 106 107 108 109

adso

rptio

n/ab

sorp

tion

perc

enta

ge (

%)

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Adsorption/Absorption (cont.)

The toxic effect happens after the nC60agglomerates attached onto the surface of E. coli cell wall. nC60 then affects the metabolism of E. coli, thereby inhibiting the growth or kill the cells .

The toxic effect happens after the nC60agglomerates attached onto the surface of E. coli cell wall. nC60 then affects the metabolism of E. coli, thereby inhibiting the growth or kill the cells .

THF/nC60 is more easy to adsorb/absorb onto/into the bacteria.The increasing of adsorption/absorption percentage with rising of temperature indicates that the metabolism enhances the adsorption/absorption.

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ConclusionsSame size distribution of THF/nC60 and Tol/nC60

THF/nC60 is more toxic. Its EC50=0.54 mg/lTHF/nC60 is more toxic. Its EC50=0.54 mg/l

The surface of THF/nC60 is less chemically modified during the disperse process.

The surface of THF/nC60 is less chemically modified during the disperse process.

THF/nC60

produce more superoxideoxygen which is toxic to the bacteria.

THF/nC60

produce more superoxideoxygen which is toxic to the bacteria.

THF/nC60 is easily adsorbed or absorbed onto/into bacteria.

THF/nC60 is easily adsorbed or absorbed onto/into bacteria.

ROS generation is not the only reason of the toxicity effects. The chemically modification and adsorption or absorption are also important.ROS generation is not the only reason of the toxicity effects. The chemically modification and adsorption or absorption are also important.

Ongoing study: Conduct RT-PCR (reverse transcriptase PCR) to further investigate the toxic mechanisms

Ongoing study: Conduct RT-PCR (reverse transcriptase PCR) to further investigate the toxic mechanisms

27

Thanks!

Q&A