LIU ET AL.
’ NO. 11
October 09, 2012
C2012 American Chemical Society
Chemical Transformations of
Nanosilver in Biological Environments
Jingyu Liu,†Zhongying Wang,†Frances D. Liu,‡Agnes B. Kane,§,^and Robert H. Hurt‡,^,*
†Department of Chemistry,‡School of Engineering,§Department of Pathology and Laboratory Medicine, and^Institute for Molecular and Nanoscale Innovation,
Brown University, Providence, Rhode Island 02912, United States
human or environmental receptors are
may involve adsorption, chemical reaction,
dissolution, or aggregation and can affect
transport, bioavailability, bioaccumulation,
toxicity, and ultimately risk.
A number of recent studies have focused
on the environmental transformations of
silver nanoparticles (Ag-NPs).5?20These
studies show that Ag-NPs undergo slow
oxidative dissolution by molecular oxygen
and protons,5,18,19reactions with reduced
sulfur species or chloride,7,8,10?12,17adsorp-
tion of polymers,20natural organic matter
(NOM),15,16,20or proteins,21,22and aggre-
coatings.14,15,17,20,23The transformed pro-
ducts can show reduced biological activity
(e.g., silver sulfide)24,25or enhanced bio-
logical activity (e.g., the free ion or soluble
profound transformation between the
time of synthesis and the time at which
complexes)26,27relative to the initial particle.
In the case of silver-based nanomaterials, it
has become quite clear that chemical trans-
Less is known about Ag-NP transforma-
tions in biological systems. The chemical
environments in biological systems have
some features in common with those in
the natural environment (presence of Cl?,
some organic ligands, reduced sulfur species)
butalsosignificant differences. Somebiologi-
cal environments have very low pH (gastric
fluid), very high concentrations of organic
potential for photochemistry in the near-skin
region. Biological compartments also contain
silver ion by mechanisms evolved in nature
for transport of sodium30or copper ion.31
Many aspects of Ag-NP biochemistry are
unexplored, such as the interactions with
*Address correspondence to
Received for review July 31, 2012
and accepted October 9, 2012.
ABSTRACT The widespread use of silver nanoparticles (Ag-NPs) in consumer and
medical products provides strong motivation for a careful assessment of their environ-
mental and human health risks. Recent studies have shown that Ag-NPs released to the
natural environment undergo profound chemical transformations that can affect silver
bioavailability, toxicity, andrisk.Lessis knownaboutAg-NPchemicaltransformations in
biological systems, though the medical literature clearly reports that chronic silver
ingestion produces argyrial deposits consisting of silver-, sulfur-, and selenium-contain-
ing particulate phases. Here we show that Ag-NPs undergo a rich set of biochemical
transformations, including accelerated oxidative dissolution in gastric acid, thiol binding
and exchange, photoreduction of thiol- or protein-bound silver to secondary zerovalent Ag-NPs, and rapid reactions between silver surfaces and reduced
selenium species. Selenide is also observed to rapidly exchange with sulfide in preformed Ag2S solid phases. The combined results allow us to propose a
conceptual model for Ag-NP transformation pathways in the human body. In this model, argyrial silver deposits are not translocated engineered Ag-NPs,
but rather secondary particles formed by partial dissolution in the GI tract followed by ion uptake, systemic circulation as organo-Ag complexes, and
sulfides and further into selenides or Se/S mixed phases through exchange reactions. The formation of secondary particles in biological environments
implies that Ag-NPs are not only a product of industrial nanotechnology but also have long been present in the human body following exposure to more
traditional chemical forms of silver.
KEYWORDS: silver.selenium.nanomaterial transformation.fate.health risks.photochemistry
LIU ET AL.
’ NO. 11
selenium species, the photochemistry of Ag biocom-
plexes in the near-skin region, or are incompletely
understood, such as the extent of dissolution in the
The medical literature does not systematically ad-
dress the chemical mechanisms and pathways of
Ag-NP transformation, but it does provide useful in-
formation on the final fate and form of silver in the
human body. Overexposure to silver through inges-
tion, inhalation, and dermal contact can raise silver
concentrations in blood and, in some cases, cause
irreversible skin discoloration (argyria) especially in
sun-exposed areas.33?46Silver-rich granules, mostly
collocated with sulfur and selenium, have been de-
tected in the connective tissue of the dermis in argyria
patients.33,34,36,41,42,44?46A recent study reported the
deposition of Ag-, S-, and Se-containing nanoparticles
in small intestine, liver, and kidney tissue after oral
long-term fate of silver in the body is as particulate
deposits, and the deposition is favored by light ex-
posure, but the chemical pathways have not been
systematically investigated and remain unclear.
Here we study the chemical transformations of Ag-
NPs in biological media with emphasis on compart-
ments in the human body. We report accelerated
dissolution in gastric acid, photoreduction of Ag?thiol
and Ag?protein complexes to metallic Ag-NPs, and
surfaces and to exchange with sulfur on silver surfaces
that have been previously sulfidated. The combined
results suggest a pathway for argyrial deposits that
involves partial gastric digestion to soluble silver, ion
uptake, and systematic transport as thiol complexes,
photoreduction of Ag(I) to immobilize silver in the
form of Ag-NPs inthe near-skin region, and then in situ
transformation to sulfides and selenides. We thus
propose that argyrial deposits are secondary particles
ratherthan translocatedprimaryparticles and, assuch,
are not unique to Ag-NP exposure but occur upon
exposure to a variety of silver compounds and silver-
Because of the wide range of nanosilver applica-
tions, a variety of exposure routes are relevant includ-
ing ingestion, inhalation, dermal contact, wound
surface application, and insertion or implantation of
medical devices.48These exposure routes bring Ag-
NPs in contact with a range of different fluid environ-
ments, and we begin by examining oxidative dissolu-
tion at the initial point of entry.
Oxidative Dissolution in Biological Media. Nanosilver is
unstable to oxidation and releases ions through gra-
of pre-existing oxide films in fluid media. Oxidative
dissolution is a complex chemical reaction influenced
by pH, coatings, and ligands in the surrounding
acterized in environmental fluid phases,5,11,12,14,16,49
but less so in biological media,6,11,50though the im-
portant role of released silver ions in Ag-NP toxicity
is well-recognized.26,27,51,52Biological compartments
particle surface area (particle size and aggregation
state)6,18,19,27,52,53to be important factors influencing
biodissolution rates.Figure1explorestheeffects ofpH
and primary particle size in four media representing
the (1) stomach (pH 1.5),54(2) lysosome (pH 4.5),55
(3) inflammatory phase of acute wounds (pH 5.7),56
and (4) extracellular environment/blood/lung fluid
(pH 7.4),54using previously published kinetics for a
model citrate-capped Ag-NP material system:5
An area normalized form useful for estimating the size
dependence has (1/S)(?dm/dt) on the left-hand side
Figure 1. Effects of pH and particle size on Ag-NP oxidative
dissolution in simple media estimated using previously
published kinetics.5(A) pH effect for 5 nm diameter Ag-NPs.
(B) Primary particle size effect at pH 1.5. The calculations use
wt % of total input silver released as ions.
LIU ET AL.
’ NO. 11
and a pre-exponential factor A = 7.6 ? 1016μg release/
Ag-NPs in the stomach, but that dissolution will be
incomplete for most particles due to limited residence
time (10?240 min in stomach).57Ligand, coating, and
salt effects limit our ability to accurately predict dis-
solution rates in complex media at this time from
simple approaches such as the use of eq 1, so experi-
mental data on gastric fluid and wound fluid simulants
are needed to confirm this conclusion. Our previous
work used ultrafiltration/atomic adsorption (AA) for
release rate measurement, which is quantitative but
incompatible with the presence of proteins or other
macromolecules, whichcanbind Agþandbe removed
by the ultrafilter, leadingto incorrect assignment ofAg
to the condensed (particle) phase. At high silver doses
(above those relevant to the natural environment),
and chloride has been reported to cause matrix effects
that interfere with the atomic absorption method.58
For experiments in biological media, therefore, we
need an alternative technique and chose to adapt an
in situ plasmon resonance tracking method.11,59
Ag-NPs are well-known to exhibit strong localized
surface plasmon resonance (LSPR), and the corre-
sponding UV?vis absorption peak is sensitive to par-
ticle size, shape, aggregation state, and the external
dielectric environment60and can be used to monitor
total Ag concentration.59,61We adapted the technique
of Espinoza59and Zook11with modification by using 1
wt % polyvinylpyrrolidone (PVP) to protect particles
from aggregation and UV?vis absorbance to track ion
release. The technique was first validated by oxida-
tively dissolving Ag-NPs in 0.084 M HNO3, which gives
the same pH as synthetic gastric acid. Figure 2A shows
a rapid decrease of the LSPR signal at ∼397 nm,
corresponding to Ag0oxidation. Assuming that optical
absorption at the LSPR peak height is proportional to
be compared to direct measurement by ultrafiltration/
AA (Figure 2B). Figure 2B validates the LSPR technique,
which can now be used in complex media containing
Ag-binding macromolecules that cannot pass the
ultrafilter (MW cutoff 3K).
Figure 3 uses the LSPR technique to measure silver
ion release profiles in gastric fluid and pseudoextracel-
lular fluid (PECF) with BSA as a simulant for wound
agreement with the behavior in simple media (dashed
black curve) and is very slow in PECF. The elevated pH
in PECF is not sufficient to explain the slow dissolution
the PECF buffer. Interestingly, the presence of albumin
greatly accelerates ion release. It is possible that the
strong affinity of BSA for Agþ62facilitates the mobiliza-
tion of surface-bound Agþto the solution phase,
though more work is needed to understand this
Interaction of Agþwith Chloride and Thiol. The Agþ
produced by oxidative dissolution can further trans-
form in biological media, whose composition deter-
mines the speciation, mobility, and bioactivity of silver.
The free Agþconcentration in biological media is
extremely low due to complexation and possibly pre-
cipitation of Agþwith Cl?, typically on the order of
10?9M (Ksp= 1.77 ? 10?10for AgCl).65The maximum
concentration of soluble silver species (sum of Agþ,
AgCl(aq), AgCl2?, and AgCl32?) estimated using visual
MINTEQ (version 3.0)66is 0.51 and 0.58 mg/L in syn-
thetic gastric acid and albumin-free wound simulant,
respectively. Above this concentration, AgCl(s) will
appear and limit further increases in silver bioavail-
ability.67In many exposure scenarios, the total silver
Figure 2. Development and validation of plasmon reso-
nance tracking technique for Ag-NP (average diameter
5 nm) dissolution in complex media. (A) UV?vis spectra
recorded during incubation of Ag-NP in dilute HNO3, show-
ing rapid decrease of peak absorbance due to oxidative
dissolution. (B) Comparison of time-resolved Ag-NP disso-
lution measured by UV?vis and ultrafiltration/AA, demon-
strating UV?vis as an alternative tool for quantifying silver
ion release. The gray dashed curve gives calculated release
kinetics at pH 1.1 using eq 1. The release experiment was
conducted using Ag-NP suspension (initial concentration
5 mg/L) in 0.084 M HNO3supplemented with 1 wt % PVP at
room temperature in the dark.
LIU ET AL.
’ NO. 11
dose is less than these values (<0.5 ppm), so AgCl(s)is
not expected as a product, but it can occur at high
doses are used to produce acute effects.68,69
The Agþand its soluble complexes in the GI tract
can be taken up into systemic circulation by active
transport routes for Naþand Cuþ70and enter the
bloodstream, where it is expected to bind to proteins
and distributed to a variety of tissues and organs.71
Serum albumin, the most abundant plasma protein, is
involved in the transport of other ions including Cu2þ,
albumin (HSA) toward Agþand the abundance of HSA
in blood (50 g/L)76make serum albumin a likely trans-
reported with the formation constants of 105and 104,
and cysteine, methionine, and disulfide bridge resi-
dues are the major functional groups involved.77In
general, the soft acid Agþbinds strongly to thiol (?SH)
groups (log Kformation= 11.9 for Agþ-cysteine)78includ-
ing small molecule thiols such as reduced glutathione
a typical blood concentration of 1 mM.80Here we use
GSH as a model thiol compound to investigate Agþ
binding, exchange, and competition with chloride
species in biological fluids.
We first observed that free Agþand free GSH show
tion, Figure S1) consistent with the strong binding
reaction: Agþþ GSH T GS-Ag þ Hþ.81At high GSH/Ag
ratio, the stoichiometry of the complex is Ag-GSH,
while at low ratios, the stoichiometry approaches
Ag2-GSH. The removal of reaction products using
centrifugal ultrafiltration (MW cutoff 3K) indicates
the formation of high molecular weight silver?GSH
polymer complexes. Silver?thiol polymers were re-
ported to have a two-dimensional layered structure
with Ag?S bonds in a three-coordinate mode,82and
silver-rich complexes were also identified where ex-
cess silver binds to sulfur groups or forms Ag?Ag
bonds with mercaptide-bound silver atoms.83,84More
information on silver?thiol polymers is found in the
An important question for biological partitioning is
whether Agþcan exchange easily between high affi-
nity ligands such as Cl?and RSH and remain mobile.
To test chloride/thiol exchange, we first formed AgCl
precipitates and exposed them to GSH while tracking
particle size by dynamic light scattering (DLS).
Figure 4A shows that addition of AgNO3to PBS buffer
causes rapid precipitation of AgCl, and the particle
Figure 3. Time-resolved silver ion release from Ag-NPs
(average diameter 5 nm) in synthetic gastric fluid (pH
1.12) and wound fluid (pH 7.52). The black and red dashed
lines represent the calculated silver ion release in simple
media pH 1.5 and 7.4 buffer, respectively. The release
tion of 5 mg/L in simulant biological media at 37 ?C in the
dark. The PECF63and PECF supplemented with 20 mg/mL
BSA64were used to mimic wound exudate. At each time
point, a 300?800 nm UV?vis spectrum was recorded and
the absorbance at 397 nm was used to determine total
Figure 4. Demonstration of Agþinteractions with chloride
and preferred binding to thiol. (A) Time-resolved measure-
ment of hydrodynamic particle sizes during formation and
growth of AgCl particles upon addition of AgNO3into PBS
AgNO3was mixed with PBS buffer for 30 min, followed by
addition of 1 mM GSH. The DLS signal disappears after
45 min, indicating complete dissolution of AgCl(s).
LIU ET AL.
’ NO. 11
size increases quickly from ∼300 nm to micrometers.
Addition of GSH (Figure 4B) dissolves the AgCl pre-
cipitates within 45 min to produce Ag-GSH complexes
that can be monomers or soluble oligomers undetect-
able by DLS. We note that silver?GSH complexes have
been shown to be bioavailable to aquatic organism85
and were reported to enhance silver transport from
AgCl across simulated biological membranes.86Silver
is also reported to readily exchange between different
thiol groups87despite the strong Ag?thiol bond. Well-
dispersed silver?GSH complexes are likely important
transporters that deliver silver to biological targets
through thiolate ligand exchange.87By this mechan-
ism, silver can be transported and distributed across
absorption.71Of special interest here are clinical cases
of argyria where silver is located in the basement
membrane of skin as particles,34,36,38,41,42,44with an
unknown formation mechanism.
Biological Photochemistry of Silver. Argyria, character-
ized by the irreversible bluish-gray discoloration of
skin, is most prominent in light-affected skin areas
and hasbeen characterized as aparticulate phasewith
Ag collocated with S and/or Se.34,36,38,41,42,44,45The
clinical pattern of sunlight dependence suggests the
with the silver?thiol and silver?protein complexes
described in the previous section and study their
behavior under UV?visible light.
DI water gradually darken over 6 h under 365 nm UV
irradiation but not in room light. Figure 5B shows full
spectra, which develop a broad 400?550 nm band
over time under UV irradiation but not in room light.
TEM (Figure 5C and Figure S2) and XRD (Figure 5D)
show that this process is accompanied by nanoparti-
cle formation in face-centered cubic (fcc) Ag0phase.
Similar phototransformations were also observed in
different GSH/Agþratios or using cysteine and oxi-
dized glutathione (GSSG) as thiol sources. In each case,
the product was zerovalent nanosilver (Supporting In-
formation, Figure S3). Note that prolonged incubation
of AgNO3/GSH for several days in room light or dark
causes a similar color change to that in Figure 5, which
must be uncatalyzed GSH reduction88such as may
occur in dark deep tissues, but more slowly.
It is clear that UV irradiation of Ag complexes with
small molecule thiols produces Ag-NPs. Much of the
thiol content in blood and tissue is found in protein,
and the main component of basement membrane
connective tissue, where argyrial deposits are often
Figure 5. UV irradiation induces Ag-NP formation in AgNO3/GSH mixtures at concentration ratio of 1/0.3 in DI water.
(A) Photographs after 1?6 h light exposure show nanoparticle formation only at UV wavelengths. (B) UV?vis spectra of
samples in (A). (C) TEM image of 3 h UV-exposed sample, confirming the generation of nanoparticles. (D) Identification of
product phase by XRD. Experiments were conducted by exposure of 5 mL of AgNO3(1 mM) and GSH (0.3 mM) mixture to UV
light (365 nm) or ambient lab light.
LIU ET AL.
’ NO. 11
found, is the protein collagen. Here we exposed type I
collagen solutions to AgNO3and UV and observed
irregularly shaped silver nanostructures (Figure 7A,B).
SAED pattern identifies the phase again as polycrystal-
showing that photoreduction progresses over time
and even occurs to some extent under room light.
Photoreduction of AgNO3in collagen solution is fast;
30 to 50% between 1 and 5 h exposures (Figure 7D).
Experiments using a gel form of collagen that more
closely mimics the physical form of tissue also provide
evidence of Ag-NP formation under UV?vis light with
Together, these data show that a range of thiol-
containing biomolecules and proteins produce zero-
valent Ag-NPs upon UV photodecomposition with
here is related to that in black-and-white photography
position products are zerovalent Ag rather than sulfide
phases, the photodecomposition alone is not a suffi-
deposits and an additional mechanism is needed (next
Sulfide and Selenide Reactions. We thought originally
that Ag/S/Se argyrial particles may come directly from
Ag?thiol/selenol photodecomposition, but the data
show that the photodecomposition products are not
sulfides but zerovalent Ag particles. It has been estab-
lished that Ag2S-NPs can be generated by direct
sulfidation of such Ag-NPs under conditions relevant
to the natural environment.7?10Sulfides are present in
the human body at concentrations reported from the
nanomolar range90up to 10?100 μM,91and the
corresponding sulfidation time scales range from
hours to days,7making Ag-NP sulfidation a plausible
transformation in biological systems as it is in the
natural environment. Because previous literature has
focused on Ag-NP sulfidation reactions, we focus here
the biological setting.
in argyrial deposits were initially unclear. We found no
literature on Ag/Se biochemistry, although selenium
reactions with silver nanoclusters are the basis for the
“selenium toning” technique used for stylistic and sta-
bility enhancement in black-and-white photography.92
silver surfaces in a manner similar to sulfur.7Because
selenide oxidizes more readily than sulfide, we used
selenite (SeO32?) and NaBH4to generate reduced sele-
in a glovebox (with continuous measurement of O2at
<0.2 ppm). Addition of silver foil to this solution leads to
1 week shows the formation of crystalline silver selenide
trace O2in analogy to oxysulfidation.7More likely at this
low oxygen content is partial reduction of selenite by
sodium borohydride to diselenide94followed by dispro-
portionation to Ag2Se and Se0. We do observe the char-
acteristic red nanoparticles of Se0as a byproduct in some
experiments. Both selenide and diselenide are important
intermediates in human selenium metabolism.95
The much lower abundance of Se in the human
body relative to S (∼1/10000)96,97makes it unlikely
that selenium could compete kinetically with sulfur in
its reaction with silver surfaces. This kinetic advantage
suggests initial formation of silver sulfides, but this
would require another separate mechanism to explain
the incorporation of Se in argyrial deposits. Silver
sulfide is highly insoluble (Ksp = 5.92 ? 10?51for
Ag2S)28but silver selenide even more so (Ksp= 3.1 ?
10?65for Ag2Se),98which makes the replacement
reaction thermodynamically favorable:
rxn¼ ? 81:5 kJ=mol
One might expect that the very low solubility of Ag2S
would make this reaction slow unless it proceeds
through a solid-state mechanism. We were unable to
tion into Ag/S phases to evaluate the possibility of this
exchange reaction. We therefore conducted experi-
ments in which Ag2S surfaces were created and ex-
reaction with sulfidated silver foils was readily ob-
served: within 1 day, selenium replaces sulfur on
S-tarnished silver films as determined by EDS (Table
S1, Supporting Information). The exchange reaction
was studied in detail for Ag2S nanoparticles by pre-
sulfidating Ag-NPs and incubating them in reduced
selenium solutions at equal Se/S molar ratios. The XRD
spectra in Figure 9 show that the Se/S exchange
reaction is complete after 3 days and the final product
Figure 6. UV irradiation induces formation of small-
after exposed to UV for 3 and 12 h. (B) TEM image of 12 h
UV-exposed sample in (A). Experiments were conducted by
in PBS buffer under UV light (365 nm).
LIU ET AL.
’ NO. 11
for Se over S is both thermodynamically predicted and
to completion over times easily accessible in laboratory
SUMMARY AND CONCLUSIONS
The goal of this study was to investigate the chemi-
cal pathways for Ag-NP transformation in biological
media relevant to human exposures. Our results show
accelerated oxidative dissolution in the GI tract, pre-
ferential thiol binding and exchange reactions, photo-
decomposition of Ag biocomplexes to zerovalent Ag-
NPs, and reactions with sulfur and selenium. Particu-
larly interesting findings are the selenium tarnishing
of silver surfaces and the ability of selenide to rapidly
replace sulfide in Ag2S-NPs and Ag2S films through
Of particular interest in this study were the chemical
pathways leading to argyria. The medical literature
provides multiple case studies that document argyrial
deposits as Ag/S/Se particulate phases located in the
near-skin region, and that deposition occurs preferen-
tially in light-affected regions. The combined results of
the present study allow us to propose a conceptual
model for argyrial pathways (Figure 10). Ingestion or
inhalation followed by macrophage clearance can
bring Ag-NPs into the GI tract. The very low pH in the
stomach leads to ion release, but the short residence
time should cause the extent of dissolution to be
incomplete in many cases. Silver ion and its complexes
uptake channels and circulate systematically. Note that
NPs can also enter the body directly through wounds,
tract, but the ability of particles to cross the gut epithe-
lium is limited,99and in this case, ion uptake is likely the
main route to systemic circulation (vida infra).
The majority ofsilverincirculation ispredicted tobe
bound to thiol complexes, which have high binding
Figure 7. Photoreduction produces Ag-NPs in collagen solutions. (A) Photograph of AgNO3(1 mM) in collagen solution
(0.1 mg/mL) at pH 3.9 and 7.4 after UV irradiation for 3 h. (B) TEM image of 3 h UV-exposed sample at pH 7.4; the inset gives
SAED pattern corresponding to fcc Ag0. (C) UV?vis spectra of sample solutions under various exposure conditions.
(D)Dissolved silvermeasurement in samplesolutionsduring dark or UV-irradiatedincubation, showingthat photoreduction
onlyoccursunderUV exposure.Experiments wereconductedbyexposureof 5mLof AgNO3(1 mM)and 0.1mg/mLcollagen
of Ag-NPs by centrifugal ultrafiltration.
LIU ET AL.
’ NO. 11
affinities but are easily exchangable, giving Ag(I) a
significant biomolecular mobility. (We note that sul-
fides and selenides have even higher binding affinities
than thiols but are orders of magnitude lower in
concentration in physiological fluids, so binding ki-
netics strongly favor complexation of silver to thiol as
a first step in the pathway.) The Ag(I) that reaches the
near-skin region in light-affected areas can be easily
photoreduced to metallic Ag-NPs, which effectively
immobilizes the silver. The immobilization is both
physical, due to low particle diffusivity, and chemical
since the thiol exchange reactions of Ag(I) are not
possible with Ag(0). Once formed, the Ag-NPs trans-
form to sulfide phases in analogy to the documented
biological systems is the incorporation of selenium.
The low concentrations of selenium relative to sulfur
make it difficult for selenium to compete kinetically
with sulfur, but over longer times, the higher affinity of
Se for Ag (lower Ksp, free energy) predicts selenides as
the equilibrium state. Our results demonstrate for the
first time the ability of selenide to exchange with
sulfide in silver phases, giving a plausible route for
the known formation of biological silver selenides in
argyrial deposits. Both the sulfide and selenide trans-
reactions due to the low bioavailability of silver in the
highly insoluble products.
One implication of these results is that argyrial
deposits are primarily secondary particles rather than
translocated primary particles. As such, they are not
uniquetoAg-NPexposure but occurupon exposureto
a variety of silver compounds and silver-containing
Figure 8. “Tarnishing” of metallic silver foil with reduced
selenium species generated in situ in aqueous media. (A)
Optical image of silver foil (99.9% Ag) incubated with DI
(B) XRD spectra of 7 day sampling, indicating formation
of crystalline Ag2Se. Experiments were performed in low-
oxygen atmospheres for stability with Sex?(x = 1 or 2)
species ([selenium] = 5 mM) generated in situ by NaBH4
reduction of Na2SeO3.
Figure 9. Demonstration of Se/S exchange reactions in Ag-
containing nanoparticles. (A) XRD pattern of pristine silver
nanopowder (20?40 nm, QuantumSphere), giving fcc me-
tallic silver. (B) XRD spectrum of sulfidation product, show-
ing conversion to acanthite Ag2S phase with some residual
Ag0(star labeled peaks). Experiment was conducted by
incubating silver nanopowder (10 mM) in Na2S (5 mM) for
3 days in air-saturated water. (C) XRD spectrum of sulfida-
tion product exposed to reduced selenium species, con-
firming the exchange reaction from Ag2S to Ag2Se. Sample
was prepared by incubating presulfidated silver nanopow-
der (5 mM) in reduced selenium generated in situ from
Na2SeO3 (5 mM) with NaBH4 (25 mM) in an anaerobic
atmosphere (O2∼0.1 ppm) for 3 days.
Figure 10. Summary of the biological transformations of
Ag-NPs and conceptual model for formation of argyrial
deposits. The Ag/S/Se argyrial particulate is proposed to
be the result of gastric dissolution, ion uptake, circulatory
thiol transport, and photoreduction to immobilesecondary
particles of zerovalent silver followed by sulfidation and
reduced in deep tissues at slower rates by nonphotochem-
LIU ET AL.
’ NO. 11
materials. This secondary particle idea and the pro-
sulfidation/selenation pathway in Figure 10 is consis-
tent with the observed fact that some argyrial cases
occur in patients who have ingested silver forms that
do not appear to contain particles.33,34,100?102Particu-
larly relevant support for our proposed pathway is
found in a recent article in this journal,102in which rats
were orally exposed to either soluble silver or two
different silver nanoparticles. The biodistribution pat-
terns were observed to be similar for soluble and NP
forms, and particles were observed in tissue even
when the rats were only fed soluble (nonparticulate)
must be formed in vivo, and that the combined
evidence suggests that ionic silver is the main bio-
The spontaneous formation of silver-containing na-
noparticles in this and other studies103?105remind us
that Ag-NPs not only are the product of industrial
nanotechnology but also may form spontaneously in
environmental or biological systems following expo-
sure to other more traditional chemical forms of silver.
MATERIALS AND METHODS
Materials. Citrate-stabilized Ag-NPs with average diameter
of 4?5 nm were synthesized by borohydride reduction.5Typi-
cally, a 59.2 mL solution containing trisodium citrate (0.6 mM)
and NaBH4(2 mM) was prepared in DI water (Millipore, 18.3
MΩ3cm). While being vigorously stirred in an ice bath, a 0.8 mL
solution of 15 mM AgClO4was quickly added into the mixture
followed by 3 h additional stirring. The resulting brownish
yellow Ag-NP suspension was purified with 2 cycle DI water
wash using centrifugal ultrafiltration (Amicon Ultra-15 3K, Milli-
pore, MA), concentrated to ∼40 mg/L, and stored at 4 ?C in
the dark for later use. Silver nanopowder (average diameter
20?40 nm) manufactured by a vapor condensation process
(QuantumSphrere, CA, USA) and silver foil (Strem Chemicals,
silver materials. Type I collagen (10.12 mg/mL in 0.02 M acetic
acid) from rat tail tendon was purchased from BD Biosciences
(NJ, USA), and 10? phosphate buffered saline (PBS) was ob-
used as fluorescent probe for measuring GSH concentration,
was purchased from Calbiochem, Inc. (CA, USA).
Ag-NP Oxidative Dissolution in Simulated Biological Media. Ag-NP
oxidative dissolution experiments were conducted in synthetic
media are listed in Table 1. Pseudoextracellular fluid (PECF) and
BSA-supplemented PECF were used to simulate wound exu-
dates.63,64A solution containing 10 mg/L Ag-NPs and 2 wt %
PVP was first prepared by adding fresh PVP solution (10 wt %)
into citrate-stabilized Ag-NP stock suspension; after incubation
at 4 ?C for 5 min, this solution was mixed with equal volume of
2? concentrated synthetic biological fluids to produce testing
solution with a Ag-NP concentration of 5 mg/L and PVP con-
centration of 1 wt %. The Ag-NP suspensions were then in-
cubated at 37 ?C in the dark for up to 24 h, during which period
silver ion release was monitored by measuring the localized
surface plasmon resonance (LSPR) absorbance using UV?vis
spectrometry. Typically, an aliquot of Ag-NP suspension was
MD) using particle-free media as background. The peak shift
was negligible (<5 nm) over the course of the experiment,
indicating that PVP successfully protects particles from aggre-
gation. The Ag-NP suspension gives a LSPR peak at ∼397 nm,
absorptivity, listhepathlength, andcisAg0concentration. The
mass percentage of Ag-NP dissolution can be derived by
[Agdis]=[Ag-NP]input ¼ (A0? At)=A0? 100%
Control experiments using Ag-NP dissolution in HNO3were
performed to validate this method. A test solution containing
5 mg/L Ag-NP and 1 wt % PVP was prepared in 0.084 M HNO3
(the same proton concentration as in synthetic gastric acid).
During incubation in the dark at room temperature, silver ion
dissolution was quantified with both LSPR absorbance-based
method as described above or graphite furnace atomic absorp-
of Ag-NPs using centrifugal ultrafiltration (Amicon Ultra-15, 3K).
Silver?Glutathione Complex Formation. The stoichiometry of
silver?GSH complex formation was measured by tracking the
performed by mixing AgNO3and GSH both at final concentra-
tionof1mMinDIwater,followed withquantification ofsoluble
silver using AA after collection of aqueous phase by centrifugal
ultrafiltration. On the basis of therapid reaction kinetics, 30 min
was used for the dose-dependent experiments. Typically, the
final AgNO3concentration was fixed at 1 mM, and GSH was
added into AgNO3at a concentration of 0.1 to 4 mM; after
incubation in the dark at room temperature for 30 min, the
aqueous phase was isolated with ultrafiltration filter. An aliquot
of the filtrate was analyzed by AA for soluble silver concentra-
tion, and another aliquot was used to measure the GSH con-
centration using ThioGlo-1 fluorescence reagent.107Details on
the fluorescence assay are provided in Supporting Information.
The decomposition of AgCl precipitate by GSH was studied
in PBS buffer. AgNO3was first added into PBS buffer at 1 mM,
and the immediate formation and growth of AgCl precipitates
were monitored using dynamic light scattering (DLS) on a
Zetasizer Nano ZS system (Malvern Instruments). After 30 min,
GSH was added into the AgCl precipitate at 1 mM, the white
precipitate gradually disappeared, and the hydrodynamic size
ofthedecomposed particles was tracked withDLS forup to1 h.
Photodecomposition of Silver Complexes. The further transforma-
tion of silver after dissolution and complex formation was
investigated by exposure of various test solutions containing
1 mM AgNO3to UV light. The thiol (?SH) or disulfide (?S?S?)
compounds tested included glutathione (0.1, 0.3, and 0.5 mM),
TABLE 1. Synthetic Biological Fluids
simulant fluids componentspH
BSA-supplemented pseudoextracellular fluid64
2 g/L NaCl, 7 mL/L 37% HCl
6.8 g/L NaCl, 2.2 g/L KCl, 25 g/L NaHCO3, 3.5 g/L KH2PO4
6.8 g/L NaCl, 2.2 g/L KCl, 25 g/L NaHCO3, 3.5 g/L KH2PO4, 20 g/L BSA
LIU ET AL.
’ NO. 11
cysteine (0.3 mM), and oxidized glutathione (0.3 mM). Typically,
5 mL of test solution in a 10 mL glass beaker was prepared by
mixing the thiol or disulfide with AgNO3in DI water and then
subjecting to UV irradiation for 0?24 h. Selected experiments
with GSH (1 mM) were conducted in PBS buffer, and a control
experiment was carried out using 1 mM AgNO3in DI water.
A high-intensity UV lamp (B-100AP, UVP LLC, CA) was used
to provide 365 nm long-wave UV with an average intensity of
tion, and the UV intensity was measured each time before
experiment using a UVX radiometer (UVP LLC, CA) to guarantee
the consistency of the irradiation intensity.
Ag-NP formation was also examined in collagen solution
and in a collagen gel matrix. Collagen working solutions were
and the resulting solution was acidic with pH of 3.9 (Orion
at 1 mM, followed by UV irradiation (365 nm, ∼10 mW/cm2).
Selected experiments were carried out in the dark or ambient
lab light. The progress of photodecomposition was monitored
after removal of particulate phase with centrifugal ultrafiltra-
tion. Selected experiments were conducted in collagen gel
matrix to better reflect the physical state of tissue. The collagen
gel was prepared following vendor's protocol with modifica-
to 4 mg/mL, and AgNO3was added to concentration of 1 mM,
then 1 M NaOH was used to adjust pH to 7.4. The viscous
mixture was transferred into a 24-well plate with incubation at
37 ?C for 1 h, during which period solidified collagen gel was
formed. The collagen gel was then incubated in the dark, under
the ambient room light, sunlight, or UV lamp for up to 24 h.
Selenium Reactions with Ag0and Ag2S. Reduced selenium spe-
cies (Sex?, x = 1, 2) were generated in situ by reacting Na2SeO3
with NaBH493under anaerobic atmosphere in a glovebox
(O2∼0.1 ppm, Vacuum Atmospheres, CA). Typically, NaBH4
was mixed with Na2SeO3at 5/1 molar ratio in deoxygenated
DI water for 30 min. The formation of Ag2Se on Ag0surfaces
was first investigated using silver foil (99.9% Ag) of 0.127 mm
thickness cut into 4 mm ? 4 mm pieces. To remove possible
oxide layers, the small foil sections were dipped into 1% HNO3
for 5 min before use. A piece of silver foil was incubated in 5 mL
of in situ reduced selenium species ([Se] = 5 mM) for 7 days in
the glovebox. Silver foil incubated in DI water was used as
control. Se/S exchange reaction was conducted by treating
presulfidated silver nanopowder or silver foil with reduced
selenium species. A piece of silver foil or silver nanopowder
(10 mM) were first sulfidated with 5 mL of 5 mM Na2S solution
for up to 3 days, followed with purification of sulfidation
product with DI water wash using centrifugation. The Se/S
exchange was achieved by incubating the obtained sulfidated
products in reduced selenium ([Se] = 2.5 or 5 mM) for up to
3 days, followed by sample purification.
Sample Characterization. The UV?vis spectra of aqueous sam-
ples were recorded on a V-630 spectrophotometer (Jasco, MD)
between 300 and 800 nm. The morphology and size of UV-
induced Ag-NPs were determined with transmission electron
microscopy (TEM) on a Philips EM420 at 120 kV. TEM samples
were prepared by placing a drop of purified sample solution on
solvent evaporation at room temperature overnight. The com-
position and phase of UV irradiation products were identified
D8 Avance diffractometer with Cu KR radiation (λ = 1.5418 Å).
AgNO3and 0.3 mM GSH was exposed under a UV lamp for 3 h;
after samplewashwithDIwaterusingcentrifugal ultrafiltration,
the obtained concentrated nanoparticle suspension was added
onto a small glass slide and dried overnight in the dark. The Se-
tarnished silver foil surface and the products of Se/S exchange
water washing and by energy-dispersive X-ray spectroscopy
(EDS) on a LEO 1530 field-emission scanning electron micro-
scope (FE-SEM) at 20 kV accelerating voltage and 8.5 mm
working distance (see Supporting Information). Samples for
SEM and EDS were prepared on silicon substrates, and silver
sulfide films before/after selenation were placed directly on the
conductive tape, both of which meet the requirement of flat
surfaces in the ZAF correction method.
Conflict of Interest: The authors declare no competing
Acknowledgment. Financial support was provided by NSF
Grant ECCS-1057547, and the Superfund Research Program of
the National Institute of Environmental Health Sciences,
P42ES013660. Although sponsored in part by NIEHS, this work
does not necessarily reflect the views of the agency.
Supporting Information Available: ThioGlo-1 fluorescence
assay used for quantification of GSH concentration, experimen-
tal phenomena of silver?GSH polymer formation over a wide
range of GSH concentrations, reaction stoichiometry of sil-
ver?GSH polymer formation, TEM image of Ag-NPs produced
by UV irradiation of AgNO3/GSH mixture, Ag-NPs produced by
collagen gel matrix after incubated in the dark, exposure under
ambient lablight, sunlight, and UVlight, and elemental analysis
of sulfidation and Se/S exchange reaction products. This ma-
terial is available free of charge via the Internet at http://pubs.
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