Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Nanoparticles
Zong-ming Xiu,
Qing-bo Zhang,
Hema L. Puppala,
Vicki L. Colvin,
ASSOCIATED CONTENT
*S Supporting Information
Experimental methods for AgNPs characterization, E. coli
growth inhibition assay, anaerobic PEG-AgNPs synthesis, air-
exposed AgNPs preparation, AgNPs ltration, statistical
analysis, Figures S1,S2, Tables S1,S2, and corresponding
discussions are provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: alvarez@rice.edu. Phone: (713)348-5903.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
This research was supported by a Joint US-UK Research
Program (Grant RD-834557501-0 by U.S.-EPA and U.K.-
NERC-ESPRC). We thank Xiaoyu Chai (Biostatistician of
Clinical Statistics, Fred Hutchinson Cancer Research Center)
for his assistance with the statistical analysis.
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Nano Letters Letter
dx.doi.org/10.1021/nl301934w | Nano Lett. 2012, 12, 42714275 4275
1
SUPPORTING INFORMATION
Negligible Particle-Specific Antibacterial Activity
of Silver Nanoparticles
Zong-ming Xiu, Qing-bo Zhang, Hema L. Puppala, Vicki L. Colvin, Pedro J. J. Alvarez*
a
Dept. of Civil & Environmental Engineering, Rice University, Houston, TX 77005
b
Dept. of Chemical Engineering, Rice University, Houston, TX 77005
* Corresponding Author; Email: alvarez@rice.edu, PHONE: (713)348-5903
9 pages in total, with 3 figures and 2 tables
2
AgNPs and Chemicals
Glycol-thiol coated AgNPs (PEG-AgNPs: 3, 5 and 11 nm) were synthesized in the lab
1
and transferred to an anaerobic chamber for storage. Commercial polyvinylpyrrolidone-coated
AgNPs of three different sizes (PVP-AgNPs: 18, 51 and 72 nm), which have been widely used in
previous studies,
2-6
were obtained from NanoAmor (Houston, TX). The PVP-AgNPs powders
were suspended in DI water and homogenized by an ultrasonic cleaner (5510, Branson, CT). The
sizes and zeta-potential () of the six particles (Table S1) were characterized in the exposure
medium (2 mM sodium bicarbonate buffer), using JEM 2100F TEM (JEOL 2100 Field Emission
Gun Transmission Electron Microscope, Japan) and dynamic light scattering with a Malvern
Zetasizer (ZEN 3600, Malvern Instrument, UK) respectively. The morphologies of the PVP-
AgNPs are provided in Figure S1.
AgNO
3
and HNO
3
(~69.0%) was obtained from Sigma-Aldrich (St. Louis, MO); NaCl,
LB (Luria-Bertani) broth, NaHCO
3
and H
2
O
2
(30%) were all obtained from Fisher Scientific
(Fair Lawn, NJ). All chemicals used were reagent grade or better unless otherwise specified.
Table S1. Characterization of PEG- and PVP-AgNPs stock suspensions
PEG-AgNPs Sample 1 Sample 2 Sample 3
Particle size 2.8 0.5 nm 4.7 0.2 nm 10.5 0.6 nm
Zeta-potential (mV) -16.1 1.2 -13.5 0.5 -17.1 2.1
Stock concentration 44.5 mg/L 70.3 mg/L 82.0 mg/L
(Dissolved Ag
+
) (0.31 mg/L) (0.38 mg/L) (0.28 mg/L)
PVP-AgNPs Sample 1 Sample 2 Sample 3
Particle size 17.5 2.9 nm 51.4 18.7 nm 71.5 20.3 nm
Zeta-potential (mV) -27.5 1.4 -35.8 0.6 -37.1 0.6
Stock concentration 8,200 mg/L 5,700 mg/L 6,700 mg/L
(Dissolved Ag
+
) (2.93 mg/L) (7.90 mg/L) (7.15 mg/L)
3
Figure S1. TEM characterization of the commercial PVP-AgNPs (a) PVP-18nm (17.5 2.9 nm),
(b) PVP-51nm (51.4 18.7 nm) and (c) PVP-72nm (71.5 20.3 nm).
Bacteria
E. coli strain K12 (ATCC 25404) was chosen as a model microorganism for inactivation
experiments. This facilitates comparison with numerous other related antimicrobial studies, and
(because E. coli is facultative) allows for testing under both aerobic and anaerobic conditions. E.
coli exhibits equal susceptibility to silver ions under aerobic and anaerobic conditions (Figure S2)
7
. The detailed bacteria stock suspension preparation method was provided in our previous paper
7
. Sodium bicarbonate (2 mM), which is commonly used as exposure medium
8
, was chosen to
avoid ligands present in complex growth media that could bind with Ag
+
/AgNPs and affect silver
bioavailability and/or promote precipitation or other confounding effects. All AgNPs/Ag
+
toxicity assays were below the Ag
2
CO
3
precipitation potential (K
sp
=0.8110
-12
).
(a) (b) (c)
4
Figure S2. E. coli strain K12 (ATCC 25404) exhibits equal susceptibility to the silver ion (which
is the definitive molecular toxicants) under aerobic and anaerobic conditions. Reproduced with
permission from reference 7. Copyright 2011 American Chemical Society.
Preparation of air-exposed AgNP stock suspensions
To prepare air-exposed AgNPs samples, PEG-AgNPs (3, 5, 11 nm) and PVP-AgNPs (18,
51, 72 nm) stock suspensions (~2g/L) were transferred out of the chamber and exposed to air for
5 days (with pH adjusted to 4.0 to accelerate the oxidation of AgNPs and the release of Ag
+
).
The released Ag
+
concentration of each sample was monitored by ICP-OES/MS (Perkin Elmer,
Waltham, MA) to avoid complete oxidation of AgNPs. The air-exposed AgNPs suspensions
were then transferred to anaerobic chamber and resuspended in NaHCO
3
buffer solution to
achieve a constant pH (8.1) for Ag
+
dissolution equilibration (5 days). The final Ag
+
concentration for PVP-AgNPs are 9.5 mg/L, 17 mg/L. 10.5 mg/L respectively.
Anaerobic synthesis of the PEG-AgNPs
The anaerobic PEG-AgNPs were synthesized by mixing the pre-synthesized PEG-AgNPs
suspension with sodium borohydride under vigorous agitation in an anaerobic chamber. All
5
precursors (including AgNPs suspensions and NaBH
4
solution) were transferred and equilibrated
inside the anaerobic chamber for 24h to allow for oxygen depletion. Excess amount of NaBH
4
was added to ensure a complete reduction of all Ag
+
.
The AgNPs were sealed in an Amicon ultra centrifugal filter unit (molecular weight
cutoff 10,000, 2~5nm in pore size, Millipore, MA) and moved out of the chamber for
centrifugation (J2-MC Centrifuge, Beckman Coulter, CA) to get rid of the impurities (e.g.,
NaBH
4
). The centrifuged samples were transferred back to the chamber to collect the filtrate and
refilled with 2mM NaHCO
3
buffer. This washing process was repeated continuously four times
and the filtrates were monitored for dissolved silver with ICP-Ms. The final PEG-AgNPs stock
suspensions were re-suspended in 2mM NaHCO
3
buffer solution, with no detectable Ag
+
(by
ICP-Ms, detection limit: 1 g/L) in supernatants.
Preparation of filtered AgNP stock suspensions
To purify the AgNPs, PEG- and PVP-AgNPs stock suspensions were washed by DI water
and filtered through the Amicon ultra centrifugal filter units by centrifugation. AgNPs stock
suspensions were filtered continuously for ~10 times to get rid of the residual Ag
+
as much as
possible. The filtrates were analyzed for total dissolved silver with an ICP-OES/MS to obtain the
concentrations of dissolved silver.
After filtration, the retentates were resuspended and transferred to 50-ml corning tubes
(Corning, Lowell, MA). The concentrations of the AgNPs stocks were determined by nitric
acid/hydrogen peroxide digestion as described earlier
7
. These stocks were stored and tested
inside the anaerobic chamber to avoid any confounding effects caused by oxidative Ag
+
release
during the dose-response assays.
6
The minimum lethal concentration (MLC) of the six filtered AgNPs to E. coli was
compared to Ag
+
to assess their relative toxicity contribution to AgNPs. The MLC is defined as
the minimum lethal concentration of AgNPs in the exposure medium (bicarbonate buffer) that
causes statistically significant E. coli mortality (p < 0.05) relative to the control set without
AgNPs, as we reported earlier
7
.
Dose-response assay of AgNPs
Six fresh and six air-exposed AgNPs stock suspensions were tested for dose-response on
E. coli mortality inside the anaerobic chamber following the procedure as showed in Xiu et al
7
.
Specially, antimicrobial assay of the air-exposed AgNPs was conducted after 5 days of
equilibration in anaerobic chamber to ensure a complete Ag
+
dissolution. Free Ag
+
concentrations were measured in the same medium and toxicity data of each AgNPs suspension
was plotted against the free Ag
+
concentration in it. E. coli mortality in different treatments was
then determined by viable plate counts
9
and was calculated as 1-N/N
0
100%, where N and N
0
are the remaining and initial concentrations of viable bacteria (CFU/mL), respectively. The dose-
response curves of E. coli mortality versus Ag
+
concentrations was fitted using a sigmoidal
model (Eq. S1)
10
(Sigma-Plot v10.0):
(Eq. S1)
Where y is the E. coli mortality rate, y
0
is the baseline mortality rate without silver addition,
x is silver concentration (expressed as released Ag
+
), and x
0
is the Ag
+
concentration that results
in 50% mortality (EC
50
). All tests were conducted in triplicate and repeated at least three times to
ensure reproducibility.
Statistical Analyses
7
Whether differences between EC
50
values for different treatments were statistically
significant was determined using Students t-test at the 95% confidence level. T-tests were also
used to determine MLC values (i.e., the lowest concentrations that resulted in a significant (p <
0.05) decrease in cell viability, assessed by plate counts) as well as the significance of hormesis
effect relative to unexposed controls. For the dose-response assays, E. coli mortality data for
each type of air-exposed AgNPs (expressed as their corresponding released Ag
+
concentrations)
and for the AgNO
3
treatment were fitted with the Sigmoidal model (Eq. S1) using non-linear
regression. The SAS PROC NLIN procedure and R software, version 2.9.2 (R Foundation for
Statistical Computing, Vienna, Austria) was used for this purpose.
11
Whether differences in
dose-response trends were statistically significant were determined by the F-test at the 95%
significance level using the SAS/STAT version 9.2 software (SAS Institute Inc., Cary, NC).
This analysis (Table S2) inferred that the sigmoidal curves were statistically indiscernible and
thus, that the antibacterial activity of AgNPs could be fully explained by the toxicity of the
released Ag
+
.
Table S2. Dose-response assay statistical analysis results
F-test
Ag
+
control
PEG-
3 nm
PEG-
5 nm
PEG-
11 nm
PVP-
18 nm
PVP-
51 nm
PVP-
72 nm
p value 0.80 0.71 0.56 0.79 0.65 0.22
Ag
+
release at low pH found near a cell membrane under the influence of the proton motive
force
The air-exposed PVP-AgNPs (three types in total) were transferred into the anaerobic
chamber and each type of AgNPs was added to different media with different pH values. The
lowest pH used (3.0, diluted HNO
3
solution) was mimicking the local pH of a cell membrane
8
under the influence of the proton motive force. An intermediate pH (5.5, DI water) was used to
represent the inside of a lysosome in eukaryotic cells. The highest pH (8.1, 2mM NaHCO
3
solution) is representative of bicarbonate-buffered natural waters and was the same as in the
toxicity assays. The released Ag
+
concentrations in the supernatants were measured by ICP-Ms
after 72-h equilibration. As shown in Figure S2, Ag
+
release was affected by the solution pH,
with higher Ag
+
release at lower pH, suggesting increased release as AgNPs become in contact
with these organelles.
0
5
10
15
20
25
30
35
86 nm 36 nm
A
g
+
r
e
l
e
a
s
e
d
(
m
g
/
L
)
25 nm
pH 3.0
pH 5.5
pH 8.1
Figure S3. Dissolution of air-exposed PVP-AgNPs at different pH, as encountered near bacterial
cell membranes influenced by the proton motive force (pH 3), or inside lysosomes of eukaryotic
cells (pH 5.5) or in bicarbonate-buffered natural water (pH 8.1). Ag
+
release increases with
decreasing pH.
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