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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?