Silver nanoparticles induce apoptotic cell death in

Candida albicans through the increase of hydroxyl radicals

In-sok Hwang1, Juneyoung Lee1, Ji Hong Hwang1, Keuk-Jun Kim2 and Dong Gun Lee1
1 School of Life Sciences and Biotechnology, Kyungpook National University, Buk-gu, Daegu, Korea
2 Department of Clinical Pathology, Tae Kyeung College, Gyeongsan-si, Korea

Keywords
antifungal activity; apoptosis;
Candida albicans; hydroxyl radicals; silver
nanoparticles
Correspondence
D. G. Lee, School of Life Sciences and
Biotechnology, College of Natural Sciences,
Kyungpook National University, Daehak-ro
80, Buk-gu, Daegu 702-701, Korea
Fax: +82 53 955 5522
Tel: +82 53 950 5373
E-mail: dglee222@knu.ac.kr
(Received 25 November 2011, revised 19
January 2012, accepted 8 February 2012)
doi:10.1111/j.1742-4658.2012.08527.x

Silver nanoparticles have been shown to be detrimental to fungal cells

although the mechanism(s) of action have not been clearly established. In
this study, we used Candida albicans cells to show that silver nanoparticles
exert their antifungal effect through apoptosis. Many studies have shown
that the accumulation of reactive oxygen species induces and regulates the
induction of apoptosis. Furthermore, hydroxyl radicals are considered an
important component of cell death. Therefore, we assumed that hydroxyl
radicals were related to apoptosis and the effect of thiourea as a hydroxyl
radical scavenger was investigated. We measured the production of reactive
oxygen species and investigated whether silver nanoparticles induced the
accumulation of hydroxyl radicals. A reduction in the mitochondrial membrane potential shown by ow cytometry analysis and the release of cytochrome c from mitochondria were also veried. In addition, the apoptotic
effects of silver nanoparticles were detected by uorescence microscopy
using other conrmed diagnostic markers of yeast apoptosis including
phosphatidylserine externalization, DNA and nuclear fragmentation, and
the activation of metacaspases. Cells exposed to silver nanoparticles
showed increased reactive oxygen species and hydroxyl radical production.
All other phenomena of mitochondrial dysfunction and apoptotic features also appeared. The results indicate that silver nanoparticles possess
antifungal effects with apoptotic features and we suggest that the hydroxyl
radicals generated by silver nanoparticles have a signicant role in mitochondrial dysfunctional apoptosis.

Introduction
It has been known since ancient times that silver and
its compounds are effective antimicrobial agents
[1,2]. In the 19th century, microbial infections were
treated with 0.5% AgNO3, which was also used for
the prevention of infections in burns. When the era
of antibiotics began with the discovery of penicillin,
the use of silver slowly declined [3]. Currently,
due to the appearance of micro-organisms insensitive

to conventional drugs, the use of silver for treating

infections has once again gained importance. However, the use of silver ions has one major drawback;
they are easily inactivated by complexation and precipitation and the use of silver ions has therefore
been limited [4].
Here, silver nanoparticles (nano-Ag), which are not
electrocharged, can be a valuable alternative to ionic

Abbreviations
DHR-123, dihydrorhodamine; DiOC6(3), 3,3-dihexyloxacarbocyanine iodide; FITC, fluorescein isothiocyanate; H2O2, hydrogen peroxide;
HPF, 2-[6-(4-hydroxy) phenoxy-3H-xanthen-3-on-9-yl]-benzoic acid; nano-Ag, silver nanoparticles; MIC, minimum inhibitory concentration;
PI, propidium iodide; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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I.-s. Hwang et al.

silver [5]. Nano-Ag are clusters of silver atoms that

range in diameter from 1 to 100 nm and are attracting
interest as antibacterial and antimicrobial agents. In
particular, because of recent advances in research on
metal nanoparticles, nano-Ag have received special
attention as a possible antimicrobial agent. Nano-Ag
are known to be a nontoxic and safe antibacterial
agents for the human body. In addition, nano-Ag have
also been reported to possess antifungal activity [6],
anti-inammatory properties [7], antiviral activity [8]
and anti-angiogenic activity [9]. Although the antimicrobial effects of nano-Ag are well known, their
mechanisms of action have been addressed only sporadically in the literature. Recent studies have shown
that nano-Ag interact with three main components
of micro-organisms to produce the antimicrobial
effect: the membrane or cell wall [6,10], DNA [11]
and microbial proteins [10]. In addition, there is
substantial evidence that nano-Ag produce reactive
oxygen species (ROS) [12]. The accumulation of
intracellular ROS is well known as an important regulator of apoptosis accumulating in the early apoptosis phase [13]. Subsequently, the level of intracellular
ROS accumulation increases, which initiates mitochondrial fragmentation [14]. Some other studies
have shown that hydroxyl radicals are linked to cell
death [15]. Because apoptosis is one of the mechanisms of cell death, we investigated whether there
are any connections between apoptosis and hydroxyl
radicals.
Candida albicans is probably one of the most successful opportunistic pathogens in humans. Under
conditions of a weakened immune system, colonizing
C. albicans can become opportunistic, causing recurrent mucosal infections and life-threatening contagious
infections with high mortality rates. Furthermore,
the number of known multidrug resistant bacteria
and fungi is increasing rapidly. Thus, the development
of more effective antifungal therapies is of great
importance. Understanding the mechanisms and decisions of cell death in fungi may provide new developments in the search for diverse novel antifungal
nanoparticles.
According to previously reported studies, nano-Ag
possess antifungal effects and cell-cycle analysis has
shown signicantly arrested cell cycles during the
G2 M phase [6]. There are many studies showing
G2 M-phase-mediated apoptosis [16]. For these reasons, we investigated whether nano-Ag could exert
apoptotic cell death in C. albicans and found a relationship between mitochondrial dysfunction and
hydroxyl radicals, which was induced by nano-Ag,
during apoptotic cell death.
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Results
Intracellular ROS accumulation
In a previous study, nano-Ag showed anticandidal
activity against C. albicans (Fig. 1). This substance
exhibited a minimum inhibitory concentration (MIC)
value of 2 lgmL)1, which was as efcient as that of
3 mm hydrogen peroxide (H2O2) on C. albicans (data
not shown). We used H2O2 as a positive control to
determine programmed cell death [16].
ROS are continuously formed because of cellular
oxygen metabolism. Recent studies have suggested that
the accumulation of ROS induces and regulates the
induction of apoptosis in metazoans and yeasts [17].
Therefore, to determine the production and accumulation of intracellular ROS induced by nano-Ag, we
chose to use the ROS-sensitive dye dihydrorhodamine
(DHR-123), which has been used previously as a general indicator of cellular ROS levels. Multiple ROS
directly oxidize DHR-123 to the highly stable, uorescent derivative rhodamine-123 in such a way that an
increase in the uorescent signal reects ROS production [18]. Cells treated with nano-Ag exhibited high
ROS levels compared with untreated cells. In the positive control, there was a signicant increase in the
amount of uorescence when the cells were treated
with H2O2 (Fig. 2).
First, we investigated the activity of nano-Ag
for chemically generated ROS. The iron-catalyzed

Fig. 1. Transmission electron micrograph of the nano-Ag used in

this work. The bar marker represents 20 nm.

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Silver nanoparticles induce apoptotic cell death

Measurement of mitochondrial membrane

potential (DWm)

Fig. 2. Flow cytometric analysis of ROS accumulation in nano-Ag

(blue) and H2O2 (red solid line) treated C. albicans cells stained with
DHR-123.

HaberWeiss process is known to be a promoter of

oxygen radicals under aerobic conditions. Ferritin, the
iron storage protein, is the principal reservoir for iron
within the cell [19]. For this reason, we used ferrous
perchlorate as a positive control in solely aqueous
solution. To detect hydroxyl radicals (OH) formed
in the Fenton reaction, we used the uorescent dye
2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]-benzoic
acid (HPF). The uorescence intensity did not increase
upon the addition of H2O2 alone, but did increase
substantially upon the addition of nano-Ag or ferrous
perchlorate in the presence of H2O2. The results clearly
show that nano-Ag could transmute H2O2 into OH
(Fig. 3A). We thought that nano-Ag induces apoptotic
cell death through the formation of highly ROS such
as OH.
We examined OH formation with HPF, which is
oxidized by OH with high specicity, because hydroxyl radicals have been suggested to be a crucial component of apoptosis in many studies [20]. Consistent
with the increase in intracellular ROS, the level of
intracellular OH was markedly increased in nano-Agtreated cells (Fig. 3B). These results indicate that ROS
induced by nano-Ag accumulated in the interior of
C. albicans cells, and most were converted into the
strong oxidant OH, considered to be a signicant factor in aging and apoptosis in yeast cells. To demonstrate that thiourea acts as an OH scavenger, we also
treated cells exposed to nano-Ag with thiourea. Thiourea signicantly reduced OH formation in nano-Ag
treated cells (Fig. 3B,c). We used thiourea in subsequent experiments to show the effect of decreased
hydroxyl radicals on mitochondria-mediated apoptotic
cell death.

In many systems, apoptosis is associated with loss of

the mitochondrial inner membrane potential (DWm),
which may be regarded as a limiting factor in the
apoptotic pathway. Reduction of DWm is among the
changes encountered during the early reversible stages
of apoptosis and is preceded by cytochrome c release
in several cell types [21,22].
To investigate whether nano-Ag decreased DWm, we
used the mitochondria-specic voltage-dependent dye
3,3-dihexyloxacarbocyanine iodide, DiOC6(3), which
aggregates inside healthy mitochondria and uoresces
green. When the mitochondrial membrane depolarizes,
the dye no longer accumulates and is distributed
throughout the cell, resulting in a decrease in green uorescence. The results show that nano-Ag-treated cells
had a decreased DWm, which was in agreement with
the pattern induced by H2O2 treatments as the positive
control (Fig. 4A). However, cells that were treated
with nano-Ag and thiourea did not undergo substantial changes (Fig. 4A,a).
We performed the mitochondrial DWm assay with
JC-1 to verify our results. JC-1 has advantages over
other cationic dyes in that it can selectively enter the
mitochondria and reversibly change color from red to
green as the membrane potential decreases. In healthy
cells with high mitochondrial DWm, JC-1 spontaneously
forms complexes known as J-aggregates with intense
red uorescence. However, in apoptotic or unhealthy
cells with low DWm, JC-1 remains in the monomeric
form, which shows only green uorescence [23]. The
ratio of green to red uorescence is dependent only on
the membrane potential and not on other factors such
as mitochondrial size, shape and density, which may
inuence single-component uorescence signals. Flow
cytometric analysis of JC-1 uorescence is best performed using 2D green versus red uorescence plots.
As shown in Fig. 4B, both nano-Ag and H2O2 treatments induced a signicant decrease in DWm, whereas
the combined treatment with nano-Ag and thiourea
appeared to have only a slight effect. Therefore, the
results suggest that nano-Ag induced the breakdown of
DWm, which is a critical step in cells undergoing apoptosis, and the loss of mitochondrial permeability. This
result suggests that restriction of OH formation helps
maintain the balance of the mitochondrial membrane.
Cytochrome c release
Translocation of cytochrome c from the mitochondria
to the cytosol is a pivotal event in apoptotic cell death.

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Fig. 3. (A) Detection of hydroxyl radicals in

the Fenton reaction using HPF (final 5 lM;
0.1% dimethylformamide as a cosolvent).
The fluorescence intensity was determined
at 515 nm with excitation at 490 nm. NanoAg (lower solid line) and ferrous perchlorate
(upper dotted line) were added at 40 s. (B)
Flow cytometric analysis of the formation of
hydroxyl radicals in C. albicans using the
dye HPF. (a) Control, (b) cells exposed to
nano-Ag, (c) cells exposed nano-Ag with
thiourea, (d) cells exposed to H2O2.

Cytochrome c, a soluble protein electrostatically bound

to the outer face of the inner mitochondrial membrane, is an essential component of the respiratory
chain acting as an electron carrier between the cytochrome bc1 and cytochrome c oxidase complex [24].
We assumed that cytochrome c would be detected
in the cytosol because of the results of the previous mitochondrial membrane potential assay. In this
regard, we investigated whether nano-Ag-treated
cells could induce cytochrome c release from the mitochondria. A large amount of cytochrome c was
detected in the cytosolic buffer medium following the
nano-Ag-treated cells, although cytochrome c rarely
appeared in supernatants that were additionally treated
with thiourea (Fig. 4C). These results show that
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nano-Ag induced the release of cytochrome c from the

mitochondria and suggest that the mitochondria of
nano-Ag-treated cells, which suppressed the formation
of OH by thiourea, are not directly affected by the OH.
Annexin Vpropidium iodide double staining
The early stages of apoptotic phenomenon can be
detected with uorescein isothiocyanate (FITC)
Annexin V staining, which binds to phosphatidylserine
with high afnity in the presence of Ca2 + [25], combined with the membrane-impermeable dye propidium
iodide (PI). Phosphatidylserine is only distributed in
the inner leaet of the lipid bilayer of the plasma
membrane, which is maintained by the ATP-binding

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B
a

Silver nanoparticles induce apoptotic cell death

Fig. 4. (A) Loss of the mitochondrial inner membrane potential in C. albicans induced by treatment with nano-Ag (a), and H2O2 (b) for 1 h. In
each panel, the untreated control is the black background peak and the red solid lines represent individual treatment with nano-Ag or H2O2
only. Nano-Ag treatment with thiourea is shown by the blue solid lines (a). Cells were stained with DiOC6 and the fluorescence was measured by flow cytometry. A decrease in fluorescent signal (shift to the left) corresponds with a loss in the mitochondrial membrane potential.
(B) Quantitative mitochondrial membrane potential of C. albicans stained by JC-1 and measured by FACS. The area under the horizontal line
displays cells with decreased membrane potential. (a) Control, (b) cells exposed to nano-Ag, (c) cells exposed to nano-Ag with thiourea, (d)
cells exposed to H2O2. (C) Detection of cytochrome c released from C. albicans mitochondria following the incubation with nano-Ag. Cytosol
was ultracentrifuged and the supernatants were subjected to SDS PAGE and western blotting for released cytochrome c. The untreated
control (lane a) or cells cultured in nano-Ag (lane b), nano-Ag treated with thiourea (lane c), and H2O2 (lane d).

cassette transporters in C. albicans. To determine whether

nano-Ag could induce apoptotic features, the FITC
Annexin V and PI double-staining method was used.
As shown in Fig. 5, the cell population in the lower
right (LR) quadrant, which corresponds to the percentage of early apoptotic cells (Annexin V-positive and
PI-negative), increased to 35.87% and 45.37% after
treating the cells with nano-Ag and H2O2, respectively,
for 1 h. Curiously, the percentage of nano-Ag treated
with thiourea did not increase signicantly as when
treated solely with nano-Ag. To show the distinct difference, we drew a bar graph showing the percentage
of apoptotic cells at the bottom. These results demonstrate that it is possible for nano-Ag to induce apoptotic cell death in C. albicans cells. Hence, it was
conrmed that the generation and accumulation of
intracellular ROS, specically hydroxyl radicals,
induced by nano-Ag was related to an apoptotic mechanism in C. albicans cells.

Measurement of DNA damage

To further conrm the apoptotic features induced in
nano-Ag-treated C. albicans cells, a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) assay was conducted to detect apoptotic
DNA fragmentation by labeling 3-OH termini
with modied nucleotides catalyzed by terminal
deoxynucleotidyltransferase. The labeling of breaks in
the DNA by TUNEL, a reliable method for the
identication of apoptotic cells, is utilized to visualize
the apoptotic phenotype of cells [26].
A strong blue uorescence indicated a greater degree
of typical apoptotic DNA condensation and fragmentation in the nuclei of C. albicans cells exposed to
nano-Ag than in the intact nuclei of normal control
cells. 4-6-Diamidino-2-phenylindole staining of the
nano-Ag-treated cells showed the distributed nuclear
fragments (Fig. 6A). Similar results were obtained by

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Fig. 5. Effect of nano-Ag on the exposition of phosphatidylserine at the cytoplasmic membrane. C. albicans cells. Protoplasts were harvested, stained with FITCAnnexin V and PI, and observed with a FACS. The bottom bar graph shows the percentage of apoptotic cells. (A)
Control, (B) cells exposed to nano-Ag, (C) cells exposed nano-Ag with thiourea, (D) cells exposed to H2O2.

TUNEL assay staining of the breaks in the DNA

nuclear strands during the late stages of apoptosis.
TUNEL-positive cells, which showed a strong green
uorescence or intense green uorescent spots, were
observed in the population treated with nano-Ag
(Fig. 6B). In untreated cultures, the nucleus appeared
as a single round spot in the cells (Fig. 6A,a) or did
not show up well against the backgrounds (Fig. 6B,a).
Candida albicans is known to activate programmed
cell death with features reminiscent of apoptosis in
response to a variety of environmental stimuli such as
H2O2 [2628]. For this reason, we used cells treated
with H2O2 as a positive control. Supporting our observations, exposure of C. albicans cells to nano-Ag
resulted in apoptotic DNA damage. Furthermore, we
ascertained the oxidative stress-protecting effects of
thiourea.
Measurement of metacaspase activation
Caspases are typically activated in the early stages of
apoptosis and they play a central role in the apoptotic

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signaling network. Although caspases are not present

in fungi, orthologs of caspases in animals, termed
metacaspases, have been identied in fungi and plants,
and their activity can be assessed using the same detection marker [29,30]. In order to conrm metacaspase
activation, cells were incubated with the CaspACE
FITCVADFMK in situ marker that binds to
the active site of metacaspases, and detected using a
uorescence microscope. Cells with intracellular active
metacaspases stained uorescent green, whereas nonapoptotic cells appeared unstained. Fluorescence analysis
of the cells treated with nano-Ag showed a signicant
green uorescence in the FITCVADFMK-loaded
cells that was consistent with the positive control treated with H2O2 (Fig. 6C). In addition, the number of
activated metacaspases decreased, which also reduced

OH formation in thiourea-treated cells (Fig. 6C,c), as

expected. These results suggest that nano-Ag treatment
did initially lead to signicant generation of strong
oxidant hydroxyl radicals, which are well-known to be
important regulators of yeast apoptosis, and then the
hydroxyl radicals activated the metacaspases.

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Silver nanoparticles induce apoptotic cell death

Fig. 6. DNA and nuclear fragmentation were shown by 4-6-diamidino-2-phenylindole (A) and TUNEL (B) staining. Effect of nano-Ag on the
activity of metacaspase in C. albicans (C). Nano-Ag-treated cells were collected, stained and observed under a fluorescent microscope. (a)
Control, (b) cells exposed to nano-Ag, (c) cells exposed nano-Ag with thiourea, (d) cells exposed to H2O2.

Discussion
Apoptosis is a highly regulated cellular suicide program crucial for development and homeostasis in
metazoan organisms, resulting in the removal of
unwanted, mutated, damaged or simply dispensable
cells without an inammatory reaction occurring
[31,32]. Apoptosis has been accepted as a process that
is not exclusive to multicellular organisms, but rather
is a universal mechanism of cell elimination operating
according to a basic program, including in simpler and
more ancient forms of single-celled eukaryotes. The
full apoptotic program comprises two phases, one of
which has necrotic features [33]. Therefore, we analyzed the more denitive signs of the apoptosis process
in this study.
ROS, such as O2 , H2O2 and OH, are considered to
be crucial regulators of aging, and their accumulation
has been proven to play a key role in apoptosis [17].
We used DHR-123 to determine ROS accumulation
during exposure to nano-Ag in C. albicans. Nano-Agtreated cells displayed increased intracellular ROS levels compared with untreated cells (Fig. 2). In addition,

ROS damaged ironsulfur clusters, making ferrous

iron available for oxidation by the Fenton reaction
and these events appear to be mediated through the
Tricarboxylic acid cycle and the transient depletion of
NADH [34]. The Fenton reaction leads to OH formation, and OH damages DNA, proteins and lipids,
resulting in cell death. OH is the neutral form of the
hydroxide ion. OH is highly reactive and consequently
causes damage to oxidative cells. The HaberWeiss
reaction generates OH from H2O2 and superoxide
( O2 ) [19]. This reaction can occur in cells and is
therefore a possible source of oxidative stress. The
reaction is very slow, but is catalyzed by iron. For this
reason, we thought it possible that nano-Ag induces

OH formation as an iron catalyst. As expected, the

uorescence intensity increased substantially upon
the addition of nano-Ag in the presence of H2O2
(Fig. 3A). After that, we examined the intracellular
levels of hydroxyl radicals treated with nano-Ag and
tried to learn how the thiourea impacts OH accumulation in C. albicans cells treated with nano-Ag. We used
thiourea as a scavenger of OH. Thiourea is a potent

OH scavenger that has an established means of

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mitigating the effects of OH damage in both eukaryotes and prokaryotes [3537]. The results showed that
C. albicans cells treated with nano-Ag produced hydroxyl radicals, and thiourea was accompanied by a
reduction in OH formation (Fig. 3B).
Several other studies have linked cytochrome c
release, ROS formation and changes in the mitochondrial membrane potential to yeast apoptosis [38,39].
During apoptosis, the decrease in DWm is caused by
the opening of membrane pores that are located in the
mitochondrial membrane. Consequently, the decrease
in DWm leads to the translocation and activation of
various proapoptotic factors. Reduction of the mitochondrial inner membrane potential (DWm) is among
the changes encountered during the early reversible
stages of apoptosis and is related to cytochrome c
release [21,22]. Thus, we determined DWm. The results
showed that mitochondrial permeability in nano-Agtreated cells was damaged by the breakdown of DWm
(Fig. 4A,B). By contrast, cells with hydroxyl radical
accumulation inhibited by thiourea did not show substantial changes. The contents of cytochrome c
released into the cytosol and mitochondrial membrane
depolarization were measured to understand the inuence of substances on the intrinsic pathway. Cytochrome c, which is located in the mitochondrial
membrane, is released into the cytosol during the early
phases of apoptosis and a caspase-cascade is then activated as a representative of the other apoptotic protease [40]. As a result of defects in the mitochondrial
electron transport system, cytochrome c is reduced
when it is released into the cytosol because of the loss
of the cytochrome c oxidase activity. Upon the release
of cytochrome c into the cytoplasm, the protein binds
to apoptotic protease-activating factor [38]. The release
of cytochrome c requires an increase in the permiability of the mitochondrial outer membrane. The increase
in the mitochondrial transmembrane potential, which
has been predicted to promote osmotic matrix swelling, is associated with one model for cytochrome c
release from the mitochondria during apoptosis.
Because the mitochondrial inner membrane, with its
numerous cristae, has a considerably larger surface
area than that of the outer membrane, expansion of
the inner membrane upon matrix swelling can break
the outer membrane, which would be expected to trigger the release of cytochrome c to the cytosol [41].
Treatment with nano-Ag enhanced the content of cytosolic cytochrome c in C. albicans cells (Fig. 4C), suggesting that nano-Ag may trigger cytochrome
c-mediated intrinsic apoptosis. As expected, the addition of thiourea to nano-Ag-treated cells, which do not
produce hydroxyl radicals ordinarily, exhibited reduced
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cytochrome c release compared with those treated with

only nano-Ag. Thus, we believe that nano-Ag induces
apoptosis through the formation OH and that OH is
important to the apoptotic process.
Furthermore, we investigated a series of normally
apoptotic properties including the exposition of phosphatidylserine, DNA and nuclear fragmentation, and
the activity of metacaspases nally.
To discriminate between apoptotic and necrotic
cells, FITCAnnexin V and PI double staining were
used [25]. Candida albicans cells exposed to nano-Ag
stained Annexin V-positive and PI-negative, which was
similar to the response to H2O2, an inducer of apoptosis in yeast cells (Fig. 5). However, cells exposed
nano-Ag with thiourea showed decreasing apoptotic
features, which seemed to be protected by the thiourea.
In addition, we treated cells with nano-Ag and
monitored the proportion of cells positively stained for
4-6-diamidino-2-phenylindole and TUNEL staining to
study the development of the apoptotic phenotype,
including DNA and nuclei change (Fig. 6A,B). Finally,
cells exposed to nano-Ag exhibited metacaspase
activity, but cells treated with nano-Ag and thiourea
did not show any activity (Fig. 6C). These phenomena
indicate that nano-Ag induces apoptosis in C. albicans
and that highly reactive hydroxyl radicals are important to apoptosis triggered by nano-Ag.
In conclusion, this study demonstrated for the rst
time that nano-Ag promotes apoptosis in C. albicans
through phosphatidylserine exposure, DNA damage
and the activation of metacaspases. Ultimately, nanoAg disrupts the mitochondrial integrity and induces
cytochrome c release. Although the mechanisms of
nano-Ag in mitochondria-dependent apoptosis in
C. albicans have not been fully elucidated, this report
supports that nano-Ag induces programmed cell death
through ROS accumulation, especially OH. As shown
in Fig. 3, nano-Ag had the ability to generate OH
and cells treated with thiourea decreased OH production. Consequently, the reduction in OH accumulation
contributed to diminished mitochondrial dysfunctionmediated apoptosis. We conclude that nano-Ag induce
apoptotic cell death in C. albicans through OH generation, which deserves further study to provide elaboration on the apoptosis mechanisms of nano-Ag.

Materials and methods

Reagents and culture conditions
The H2O2 and thiourea used in this study were purchased
from the Sigma Chemical Co. (St. Louis, MO, USA).

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Nano-Ag were stored at 4 C. Candida albicans (ATCC

90028) cells were cultured in YPD broth (Difco, Franklin
Lakes, NJ, USA) containing yeast extract, peptone and
dextrose (50 gL)1) with aeration at 28 C.

Preparation of nano-Ag
One hundred grams of solid silver were dissolved in
100 mL of 100% nitric acid at 90 C, and 1 L of distilled
water was added. By adding sodium chloride to the silver
solution, the Ag ions were precipitated and then clustered
together to form monodispersed nanoparticles in an aqueous medium. The sizes and morphology of the nano-Ag
were examined by TEM (H-7600; Hitachi Ltd, Tokyo,
Japan). The results showed that nano-Ag was spherical in
form and its average size was 3 nm (Fig. 1). Because the
nal concentration of colloidal silver was 60 000 p.p.m.,
this solution was diluted, and then used to investigate the
apoptotic antifungal effects.

Intracellular ROS accumulation

Intracellular ROS production and the accumulation of
hydroxyl radicals (OH) were measured using the uorescent dye DHR-123 and HPF. In a previous study, nanoAg showed signicant antifungal activity at low concentrations, which was similar to the level of amphotericin B [6].
Since then, we have determined the most efcient
concentration of H2O2 for the induction of apoptosis [16].
Cells (2 108mL)1) were treated with 2 lgmL)1 nano-Ag
and 3 mm H2O2 for 1 h at 28 C, based on the MIC value
as a criterion (data not shown). After incubation, the cells
were washed with NaCl Pi before being stained with
5 lgmL)1 DHR-123 and analyzed using a FACSCalibur
ow cytometer (Becton Dickinson, San Jose, CA, USA).
The reactivity of nano-Ag for ROS was compared with
ferrous perchlorate [Fe(ClO4)2], which was used as the
Fenton reaction. We tried to detect OH formed in the
Fenton reaction, using HPF. Five micromoles of HPF was
added to sodium phosphate buffer (0.1 m, pH 7.4) containing
3 mm H2O2 and then 2 lgmL)1 nano-Ag or 100 lm
ferrous perchlorate was added. The OH formation was
detected as an increase in HPF uorescence by a Spectrouorometer (Shimadzu RF-5301PC; Shimadzu, Japan) at
490 nm excitation and 515 nm emission wavelength.
The intracellular OH accumulation was measured by
incubating the cells with 2 lgmL)1 nano-Ag and 3 mm
H2O2 in NaCl Pi containing 5 lm using the dye HPF for
1 h at 28 C. Subsequently, the cells were washed twice in
NaCl Pi and analyzed by ow cytometry [42]. For the OH
quenching experiments, 150 mm of thiourea was added
simultaneously with nano-Ag. Thiourea has been used
at mm levels in vitro as a OH scavenger [43]. Thiourea was
used for all subsequent tests.

Silver nanoparticles induce apoptotic cell death

Measurement of mitochondrial membrane

potential (DWm)
Fungal mitochondrial membrane depolarization was analyzed by DiOC6(3) staining. Cells (2 108mL)1) were harvested and incubated with 2 lgmL)1 nano-Ag and 3 mm
H2O2 for 1 h at 28 C. Subsequently, the cells were washed
with NaCl Pi and incubated with 2 ngmL)1 of DiOC6(3)
for 30 min. Cells were analyzed by ow cytometer.
JC-1 (Molecular Probes, Carlsbad, CA, USA) is a mitochondrial dye (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-benzimidazolylcarbocyanine chloride) that stains mitochondria
in living cells in a membrane potential-dependent fashion.
JC-1 was also used to conrm the decrease in membrane
potential and the number of mitochondria specically. Cells
(2 108mL)1) were treated with 2 lgmL)1 nano-Ag and
3 mm H2O2 for 1 h at 28 C. Treated cells were washed in
NaCl Pi, suspended in 200 lL staining solution containing
2 lgmL)1 of JC-1 for 20 min at 37 C. The cells were
centrifuged at 500 g for 5 min and then the pellet was
resuspended with 1 mL NaCl Pi. Cells were then analyzed
by ow cytometer.

Cytochrome c release
To investigate cytochrome c release from the mitochondria,
isolations of mitochondria were prepared [44]. Candida albicans cells were cultured in 500 mL of YPD medium for 24 h
at 30 C, collected by centrifugation at 500 g, and washed
twice with NaCl Pi and once with 1 m sorbitol. These cells
were treated with 2 lgmL)1 nano-Ag and 3 mm H2O2 for
2 h at 28 C. The treated cells were lysed with lysis buffer
(150 mm sodium chloride, 1% Triton X-100, 1 mm EDTA,
1 mm EGTA, 50 mm Tris, pH 8) and then centrifuged at
2000 g for 10 min to remove the cell debris and unbroken
cells. The supernatants were collected and centrifuged at
40 000 g for 1 h. The supernatants were collected to assay
for cytochrome c released from the mitochondira to the
cytoplasm. The protein content of these supernatants was
estimated using a NanoVue Plus Spectrophotometer (GE
Healthcare, Little Chalfont, Buckinghamshire, UK). Each
sample equivalent to 50 lg of protein was resolved on 12%
SDS PAGE. Separated proteins were transferred to a nitrocellulose membrane and analyzed by western blotting with
rabbit polyclonal anti-(yeast cytochrome c) [45]. Horseradish
peroxidase-linked goat anti-(rabbit IgG) was used as the
secondary antibody, and enhanced-chemiluminescence substrate was used for the detection of cytochrome c.

Annexin VPI double staining

Protoplasts of C. albicans were stained with FITC-labeled
Annexin V and PI using the FITCAnnexin V apoptosis
detection kit. Cells (2 108mL)1) were digested for 1 h at

FEBS Journal 279 (2012) 13271338 2012 The Authors Journal compilation 2012 FEBS

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Silver nanoparticles induce apoptotic cell death

I.-s. Hwang et al.

28 C in a potassium phosphate buffer (pH 6.0) containing

20 mgmL)1 lysing enzyme and 1 m sorbitol. Protoplasts
were incubated with 2 lgmL)1 nano-Ag and 3 mm H2O2
for 1 h at 28 C, based on the MIC value as a criterion,
and incubated for 20 min in an Annexin-binding buffer
containing 5 lL FITCAnnexin VmL)1 and PI. Protoplasts were then examined by a FACSCalibur ow cytometer
(Becton Dickinson).

Measurement of DNA damages

DNA strand breaks in C. albicans cells were analyzed by
TUNEL [46]. Cells (2 108mL)1) treated for 2 h with
2 lgmL)1 nano-Ag and 3 mm H2O2, were washed in
NaCl Pi, permeabilized for 2 min on ice and washed again
with a NaCl Pi. DNA ends were labeled with an in situ cell
death detection kit for 1 h at 37 C. The stained cells were
observed with a uorescence microscope.
Nuclear condensation and fragmentation were analyzed
by 4-6-diamidino-2-phenylindole staining [47]. Cells were
treated with 2 lgmL)1 nano-Ag and 3 mm H2O2 for 2 h
and then collected. For nuclear staining, cells were washed
twice with NaCl Pi, permeabilized in a permeabilization
solution (0.1% Triton X-100 and 0.1% sodium citrate) and
incubated with 1 lgmL)1 of 4-6-diamidino-2-phenylindole
in the dark for 20 min. Cells were then examined by a uorescence microscope.

Measurement of metacaspase activation

Activated metacaspases in C. albicans were measured using
the CaspACE FITCVADFMK in situ marker (Promega). Briey, each substance treated cell was washed in
NaCl Pi, suspended in 200 lL staining solution containing
10 lm of CaspACE FITCVADFMK in situ marker
and incubated for 30 min at room temperature in the dark.
Cells were then washed once and suspended in NaCl Pi.
Sample analysis was performed with a uorescence microscope, the Axio Imager A1, and Axio Cam MR5 (Carl Zeiss,
Thornwood, NY, USA).

Acknowledgements
This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MEST) (No. 2011-0000915) and by the
Next-Generation BioGreen 21 Program (No. PJ008158),
Rural Development Administration, Republic of Korea.

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