Pedro J.J. Alvarez

Microbial Interactions with Fullerenesand Other Engineered Nanoparticles

2007 NSF Grantees ConfArlington, VA 12/6/07

CBEN

Acknowledgements: NSF to CBEN, EPA

> 475 Products Use of Nanomaterials

Nanomaterials are used for sensors, electronics, drug delivery, tissue engineering, imaging, catalysis,

cosmetics, sunscreens, etc.

Environmental Concerns & Opportunities

2. Applications: Enhanced or new capabilities to address existing and future environmental problems.

1. Implications: Create the information needed to use nanomaterials in an environmentally responsible and sustainable manner

CBEN Systems Goal: To develop effective water treatment systems

that exploit engineered nanoparticles

CBEN Societal Driver: To enable effective risk management for emerging nanotechnologies.

C60 (buckminsterfullerene)

Photocatalystand Antioxidant(sp2 hybridized)

R. Buckminster Fuller (Bucky)

Solid or solution nC60 (20-200 nm)

_ _ _

_

__

_

_

_ _ _ _

__

__

43-66Sodium azide2.0Benzene1.6nC60

EC50(mg/L)

Compound

E. coli respiration ceases after exposure to nC60

Vibrio fischeri (luminescent bacteria) with increasing concentrations of nC60

Standardized Microtox Assay

T im e (h o u rs )1 0 2 0 3 0 4 0 5 0

Rat

e of

E. c

oli C

O2

prod

uctio

n (μ

M/h

our)

0

1

2

3

C o n tro l0 .0 4 m g /L n C 6 00 .4 m g /L n C 6 04 .0 m g /L n C 6 05 m g /L C 6 0 O H

A m e n d m e n t ( 1 2 h o u rs )

nC60 is antibacterial

Developmental toxicity of nC60 (Zebrafish)

Zebrafish larva with pericardial edema due to nC60 exposure (1 mg/L)

0

20

40

60

80

100

48 60 72 84 96 108 120Hours Post-Fertilization

Per

icar

dial

Ede

ma

(%) nC60/THF

nC60/THF+GSH

Mitigation by GSH suggest that toxicity is related to oxidative stress

X. Zhu, Lin Zhu, Y. Li, Z. Duan, W. Chen and P.J. Alvarez (2006). Environ. Toxicol. Chem. 26(5):976-979.

Possible Antibacterial Mechanisms

1. nC60 punctures cells

2. nC60 exerts oxidative by generating photocatalytic reactive oxygen species

3. nC60 is a direct oxidant, harming membrane proteins and/or serving as “e- sponge” that interrupts electron transport phosphorylation

1. Does nC1. Does nC6060 puncture cells?puncture cells?

• Propidium iodide enters permeablized cells and stains nucleic acids

• Flow cytometry used to count stained cellsPe

rcen

t of P

erm

eabl

ized

Cel

ls

0

10

20

30

40

50E. coliB. subtilis

control nC60 toluene

nC60 does not compromise membrane integrity

DNA

cannot enter intact cellsPropidium iodide

membrane integrity, binds DNA to fluoresce

DNA

cannot enter intact cellsPropidium iodide

membrane integrity, binds DNA to fluoresce

DNA

cannot enter intact cellsPropidium iodide

membrane integrity, binds DNA to fluoresce

2. Oxidative Stress Due to ROS?

Do fullerenes produce enough ROS

to inactivate bacteria or virus

in conjunction with UV disinfection?

Light:

C60 is photosensitive.

C60 - high electron affinity

Photosensitization

MS2 virus inactivation by UV and fullerol

Fullerol + UVy = -0.0967xR2 = 0.48

-5

-4

-3

-2

-1

0

Contact Time (min)

Log

(N/N

0)

UV aloney = -0.034xR2 = 0.42

Fullerol in darky = -0.0071xR2 = 0.48

0 20 40 60 80 100 120 140

3 x faster

Hotze M., A.R. Badireiddy, S. Chelam, P.J.J.Alvarez and M. Wiesner (2007). Environ. Sci. Technol. 41: 6627-6632

Effect of illumination on antibacterial activity of photosensitive inorganic nanomaterials

B. subtilus E. coli

Toxicity: ZnO > SiO2 > TiO2

ROS production: TiO2 > ZnO > SiO2

(No correlation)

Cell death also occurs in the dark and in the absence of O2.Thus, an additional mechanism besides photocatalytic ROSproduction is involved.

OS by direct contact with cell?ROS produced by the (eukaryotic)cell’s immune response system?

Adams L.K. D. Y. Lyon, and P.J.J. Alvarez (2005). Wat. Res. 40(19):3527-3532.

ROSnC60

dye

Does nC60 produce ROS in bacteria?

Increases in ROS-detection dye fluorescence

Perc

ent i

ncre

ase

as

com

pare

d to

neg

ativ

e co

ntro

l

0

100

200

300

400HydroethidineH2DCFDA

Dye + E. coli + nC60 Dye + E. coli + H2O2 Dye + nC60

nC60 w/o cells oxidizes H2DCFDA

H2DCFDAActivated by

esterases, Oxidized by ROS

to fluoresce

esterases

H2DCF

HydroethidineOxidized by

superoxide to fluoresce

ROS

H2DCFDAActivated by

esterases, Oxidized by ROS

to fluoresce

esterases

H2DCF

HydroethidineOxidized by

superoxide to fluoresce

ROS

Looking for lipid peroxidation as evidence of ROS damage

• Hallmark of lipid peroxidation is malonedialdehyde (MDA)

• MDA forms colored adducts with thiobarbituric acid (TBA)

Assessing MDA-TBA adducts in cells exposed to nC60

Abs

532

0.00

0.02

0.04

0.06

0.08w/ TBAdye-free control

E. coli E. coli + nC60nC60

nC60 w/o cells forms colored product with TBA.∴Need to need to re-evaluate previous studies that reported ROS-mediated oxidative stress

nC60 is more toxic to bacteria thanmany other common nanomaterials

Incr

easi

ng to

xici

ty

THF/nC60son/nC60 aq/nC60 PVP/nC60Ag+ nano-Ag NaOCl nZnO nZVI nTiO2 nSiO2

0.01

0.1

1

10

100

1000

10000

MIC

(ppm

)

20

nC60 is a broad-spectrum antibacterial agent

Ct (mg/l * min) for 99% kill:• 0.03-0.05 for free chlorine• ∼ 100 for nC60• 95-180 for chloramines

Bacteria MIC (mg/L) Bacillus subtilis 0.01-0.05 Burkholderia cepacia 0.0125 - 0.025 Desulfovibrio desulfuricans 0.1-0.2 Escherichia coli 0.01 - 0.05 Pseudomonas aeruginosa 0.05 - 0.066 Ralstonia pickettii 0.025 - 0.0375 Streptomyces albus <0.05

• High activity, as indicated by MIC’s (stronger than azide!)

• Kills Gram +, Gram -, facultative aerobes, anaerobes

100

Time (min)

20

40

60

80

0 10 20 30 40 50 60

% V

iabl

e E

. col

i(cf

u/m

L)

nC60 = 2 mg/L

Potential Leapfrogging Opportunities

What factors affect nC60’s toxicity to bacteria?

• Motivation– Elucidate factors that attenuate or amplify toxicity,

to prevent unintentional ecosystem damage and/or exploit its antibacterial properties In engineered systems.

• Factors considered– C60 Derivatization– Particle size– Salt (ionic strength)– Sorption

Dose Response Curve for Fullerenes Colvin, West & co-workers, Rice University

OHOH

OH

OH

HO

HO

OH

OHHO

HO

HOOH

OHHO

OHOH

COOHHOOC

HOOC

HOOC

HOOC

COOH

10-4 10-2 100 102 104 106 108

0

20

40

60

80

100

% D

ead

Fullerene Species Concentration (ppb)

HO

HO

OHHO

OH

O

OH

O

O

Na

Na

Sayes et al,. NanoLetters 2004, 4, 1881-1887

Less toxic butmore mobile

Centrifuge 25,400 x g for 20 minutes

Filter supernatant through 0.1 μm filter

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Hydrodynamic Diameter (nm)

Perc

ent I

nten

sity

PelletSupernatantoriginal

Effect of nC60 Particle Size?

Dynamic Light Scattering (DLS) Data

Large nC60 (pellet)crystalline

Small nC60 (filtered supernatant)amorphous

0.2 μm 100 nm

TEM of Size-separated nC60

Lyon* D. Y., L. K. Adams, J. C. Falkner, and P.J.J. Alvarez (2006). Environ. Sci. Technol. 40 (14):4360-4366

nC60 Particle Size vs Toxicity

0.12500.01-0.1<100 nm particles

0.0551107.5 - 10>100 nm particles

0.06100 0.75 - 1.0nC60

SurfaceArea:Volume

Average Diameter (nm)

B. subtilis MIC (mg/L)

x100 x 2

Lyon* D. Y., L. K. Adams, J. C. Falkner, and P.J.J. Alvarez (2006). Environ. Sci. Technol. 40 (14):4360-4366

Salts promote coagulation & precipitation = less toxicity

Io n ic S tre n g th (M , N a C l)0 .0 0 0 .0 2 0 .0 4 0 .0 6 0 .0 8 0 .1 0 0 .1 2 0 .1 4 0 .1 6 0 .1 8

Mea

n Pa

rtic

le D

iam

eter

(nm

)

0

2 08 0

1 0 0

1 2 0

1 4 0

1 6 0

Ionic Strength of Fresh WaterSeawater 0.7M

PAC

Adsorbed nC60

Sorption of nC60 onto PAC reduced its bioavailability and toxicity when added concurrently at the time of exposure (more PAC added = more attenuation).

PAC + nC60

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40Time (h)

nC60-free ControlnC60 only10mg PAC+nC6015mg PAC+nC6020mg PAC+nC60

CO

2pr

oduc

tion

by E

. col

i(μm

oles

/min

)

29

NOM reduces bioavailability & toxicity of nC60

200 nm

nC60

nC60 trapped by humic colloids

0

200

400

600

800

1000

1200

0 10 20 30 40 50Time (h)

Cum

ulat

ive

CO

2pr

oduc

tion

(μm

ole) 1400

0.27 mg humic acid+nC60

100 mg soil+nC6092 mg sand+nC60

nC60-free controlnC60 only

Humic acid concentrations as low as 0.1 mg/L eliminated toxicity

Conclusions• nC60 can be bactericidal

(oxidative stress)

• Implications: Ecotoxicology- Biodiversity and food webs? Biogeochemical cycling? Mitigated by NOM, salts

• Applications: DBP-free disinfection, antifouling or anticorrosion coatings? Membranes?

Any Questions?Any Questions?