Acta Biomaterialia 30 (2016) 397–407

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Investigation of functional selenium nanoparticles as potent

antimicrobial agents against superbugs
Xiaoquan Huang 1,a, Xu Chen 1,a, Qingchang Chen a, Qianqian Yu a, Dongdong Sun a,b, Jie Liu a,⇑
a
Department of Chemistry, Jinan University, Guangzhou 510632, China
b
School of Life Sciences, Anhui Agricultural University, Hefei 230036, China

a r t i c l e i n f o a b s t r a c t

Article history: Developing highly effective antibacterial agents is important for a wide range of applications. However,
Received 1 June 2015 the emergence of multiple antibiotic-resistant bacteria poses a public health threat. Many developed
Received in revised form 8 October 2015 agents have limited practical application due to chemical instability, low biocompatibility, and
Accepted 26 October 2015
poor long-term antibacterial efficiency. In the following study, we synthesize a synergistic nanocompos-
Available online 27 October 2015
ite by conjugating quercetin (Qu) and acetylcholine (Ach) to the surface of Se nanoparticles
(Qu–Ach@SeNPs). Quercetin has been reported to exhibit a wide range of biological activities related
Keywords:
to their antibacterial activity and acetylcholine as a neurotransmitter, which can combine with the recep-
Selenium nanoparticles
Quercetin
tor on the bacterial cell. Arrows indicate NPs and arrowheads indicate compromised cell walls. The study
Acetylcholine demonstrated how Qu–Ach@SeNPs exhibit a synergistically enhanced antibacterial performance
Antimicrobial agents against the multidrug-resistant superbugs (MDRs) compared to Qu@SeNPs and Ach@SeNPs alone.
Superbugs Qu–Ach@SeNPs are effective against MDRs, such as Methicillin-resistant Staphylococcus aureus (MRSA),
at a low dose. The mechanistic studies showed that Qu–Ach@SeNPs attach to the bacterial cell wall, caus-
ing irreversible damage to the membrane, and thereby achieving a remarkable synergistic antibacterial
effect to inhibit MRSA. The findings suggested that the synergistic properties of quercetin and acetyl-
choline enhance the antibacterial activity of SeNPs. In this way, Qu–Ach@SeNPs comprise a new class
of inorganic nano-antibacterial agents that can be used as useful applications in biomedical devices.

Statement of significance

The Qu–Ach@SeNPs have low cytotoxicity when tested on normal human cells in vitro. Qu–Ach@SeNPs
are effective against MDRs, such as Methicillin-resistant S. aureus (MRSA), at a low dose. Importantly,
Qu–Ach@SeNPs showed no emergence of resistance. These results suggest that Qu–Ach@SeNPs have
excellent antibacterial activities. These agents can serve as good antibacterial agents against superbugs.
Our data suggest that these antibacterial agents may have widespread application in the field of medicine
for combating infectious diseases caused by MDRs, as well as other infectious diseases.
Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Infectious diseases caused by bacteria have attracted wide
spread attention during the past few decades, afflicting millions
of people worldwide each year [1,2]. Multidrug-resistant super-
bugs (MDRs) have emerged worldwide due to the abuse of antibi-
otics. MDRs, including Mycobacterium tuberculosis, Enterococcus
faecium, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus
aureus, Acinetobacter baumannii and Pseudomonas aeruginosa [3–5],
⇑ Corresponding author.
E-mail address: tliuliu@jnu.edu.cn (J. Liu).
1
Both authors contributed equally to this work.
are becoming a major public health problem [6]. In the few past
years, numerous non-antibiotic drugs have been developed, such
as cationic polymers [7–12], antimicrobial peptides [13–15],
photothermal/photodynamic agents [16,17]. While these non-
antibiotic drugs show promising antibacterial activity, there are
still shortcomings associated with these methods, For example,
cationic polymers showed a certain degree of hemolysis in vivo,
proving a barrier for utilization as an-antibacterial agent during
infection [18], In addition, antimicrobial peptides are natural
antimicrobial agents but are easily degraded under physiological
conditions. Due to these problems, finding safe and effective non-
antibiotic antibacterial agents has become a major goal in medical
research. Recently, there has been renewed interest in developing

http://dx.doi.org/10.1016/j.actbio.2015.10.041
1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
398 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407
nanoparticle-based antibacterial agents, due to their antibacterial
efficiency performances and relatively low toxicity in human cells.
It has been reported that Ag nanoparticles exhibit excellent
antibacterial activity by binding the thiol groups in enzymes,
generating reactive oxygen species (ROS), and disrupting the con-
served bacterial respiratory chain in different kinds of bacteria.
However, further studies showed that AgNPs caused potential
adverse responses by the immune system, both in vitro and
in vivo [19,20].
Due to the antibacterial potential of metal nanoparticles, and
the potential toxic disadvantage of silver nanoparticles, we focused
our research on the widely studied selenium nanoparticle. Sele-
nium nanomaterials, including nanoparticles, nanorods, nano-
wires, and nanotubes, have been widely-explored and used in
many fields, owning to the excellent anticancer activities and
low toxicity [21–23]. At present, there are a few studies concerning
the antibacterial properties of selenium nanomaterials. For exam-
ple, in one study S. aureus growth was inhibited in the presence
of selenium nanoparticles in vitro [24]. In another study Yip et al.
studied the effects of fabric padded with highly stable selenium
nanoparticles (PSP-SeNPs) as antifungal and antibacterial materials
[25]. These prior studies used selenium nanoparticles that were
not targeted to bacteria. Using compounds with targeting ability
to bacteria on the surface of selenium nanoparticles will improve
the antibacterial ability of selenium nanoparticles. Our results
showed that, when compared with the selenium nanoparticles
without targeting ability to bacteria, the antibacterial activities of
the bacterial-targeting selenium nanoparticles were significantly
improved. This suggests that selenium nanoparticles may be useful
in biomedical devices.
We designed and synthesized Qu@SeNPs, Ach@SeNPs and
Qu–Ach@SeNPs to test the antimicrobial activity of these nanoma-
terials. Quercetin (Qu), a bactericidal compound found in plants,
has been shown to have antibacterial action. Its activity is limited
to certain bacterial species, such as S. aureus [26]. Acetylcholine, a
neurotransmitter, has the ability to interact with the acetylcholine-
receptor and increase the permeability of cell membranes. Acetyl-
choline is also used as a permeability-active unit in antibacterial
compounds [27,28]. Acetylcholine, as a drug, for treating vasospas-
tic disease, such as Raynaud’s disease, embolism angiitis and
treatment of tinnitus, deaf, ear vertigo et al. (0.1 g at a time)
Nederberg et al. have reported that acetylcholine did not induce
significant toxicity in mice [27]. Taking advantage of the antimicro-
bial properties of quercetin and the cell membrane targeting of
acetylcholine, we attempted to prepare a synergistic material of
acetylcholine chloride and quercetin on Selenium nanoparticles
for use against superbugs. The following experiments show
that Qu–Ach@SeNPs has efficient antibacterial and bactericidal
activities, proving they are good antimicrobial alternatives for the
development of antimicrobial agents. The minimal inhibition
concentration (MIC) of Qu–Ach@SeNPs showed high antibacterial
and bactericidal activities against superbugs. Zone of inhibition
tests, and LIVE/DEAD bacterial viability assays were used to char-
acterize the antibacterial activity of Qu–Ach@SeNPs. In addition,
the specific antimicrobial mechanism of Qu–Ach@SeNPs action
for has been clarified. Furthermore, a cytotoxicity assay showed
low toxicity to human embryonic kidney cells (HEK 293T) in vitro.
2. Materials and methods
2.1. Materials and reagents
Na2SeO3, quercetin, acetylcholine chloride (Ach) and sodium
borohydride (NaBH4, 98% min) were purchased from Sigma–
Aldrich Chemical Co. Escherichia coli ATCC 8739, S. aureus ATCC
6538, P. aeruginosa ATCC 27853, A. baumannii ATCC11038 were
from Guandong Microbiology Culture Center. MDR E. coli E76227,
MDR P. aeruginosa BJ915, MDR K. pneumonia R12K2637, MDR S.
aureus ATCC 25213, MDR A. baumannii GIM 1.650 were from
domestic hospitals in China. Ultrapure MilliQ water (18.2 MW)
was used in all experiments and all solutions were stored in the
refrigerator at 4 °C.
2.2. Synthesis and characterization of SeNPs
A mixture of quercetin (C15H10O7
2H2O) (1 mmol/L, dissolved in
methanol), Na2SeO3 (0.5 mmol/L, dissolved in water) and acetyl-
choline chloride (1 mmol/L, dissolved in water) was stirred in the
presence of acetic acid for 10 min in an ice-water bath, then an
aqueous solution of NaBH4 (1 mL 0.01 mol/L) was added dropwise
with vigorous stirring [29]. After half an hour of gentle stirring, the
solution turned red. Finally, NPs were centrifuged at 8000 rpm for
10 min and the red precipitate was collected. The particles were
washed three times with PBS. Ach@SeNPs were synthesized using
Na2SeO3 (0.5 mmol/L) and acetylcholine chloride (2 mmol/L) with-
out the addition of quercetin. Qu@SeNPs were synthesized using
Na2SeO3 (0.5 mmol/L) and quercetin (2 mmol/L). The morphologies
of SeNPs were observed with transmission electron microscopy
(TEM, Hitachi H-7650). The Zeta potential was tested by Nano-ZS
instrument (Malvern Instruments Limited).
2.3. Antibacterial activity test of SeNPs
A 96-well microtiter plate dilution was applied to determine
the minimum inhibitory concentration (MIC). Logarithmic-phase
bacteria (OD600nm 0.5) were diluted with nutrient broth 100 folds
and then a sterile pipette was used to add 190 lL of the
logarithmic-phase bacterial suspension (the final density of bacte-
ria was 2.5  107 CFU/mL) and 10 lL of both varied concentrations
of SeNPs. Then added 100 lL turn draw down, decreasing as double
dilution, and finally every hole filled 200 lL, the concentration of
each sample set of three parallel samples, incubated them at
37 °C for 24 h. The MIC was the concentration at which no signifi-
cant increased.
The zone of inhibition test was employed to observe the inhibi-
tory effects of quercetin (15.1 mg/mL), acetylcholine chloride
(9.1 mg/mL), mixture of quercetin and acetylcholine chloride
(15.1/9.1 mg/mL) and SeNPs (25 mg/mL) intuitive. Logarithmic-
phase bacteria (OD600nm 0.5) were diluted to approximately
1.0  107 CFU/mL with LB broth. Then, 20 lL of the bacterial sus-
pension was inoculated on LB agar plates evenly. Subsequently,
the sample disk containing the antimicrobial agent solution was
gently placed at the center of the LB agar plates and cultured over-
night at 37 °C (at least three times each group). The diameter of
the zone of inhibition around the disk is the standard of measure
of the antibacterial activity.
2.4. Fluorescence microscopic observation (live/dead)
Logarithmic-phase bacteria (1.5 mL OD600nm 0.5) were cen-
trifuged at 5000 rpm for 5 min and washed by phosphate buffer
solution (PBS, 0.01 mol/L, pH 7.4) three times. The supernatant
was discarded and the remaining precipitates, which were bacte-
ria, were resuspended in 1.5 mL of PBS. Bacteria were treated with
100 lL of 25 lg/mL SeNPs. Then 100 lL of fluorescent dye was
added and stained in the dark for 15 min. The fluorescent dyes
were mixed, by mixing 10 mg of acridine orange (AO, Fluk) and
10 mg of ethidium bromide (EB, Fluk) in 10 mL of PBS (0.01 mol/
L, pH 7.4). After rinsing with PBS three times, the bacterial cells
were imaged using laser scanning confocal microscope (LSM, Leica
 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407
Microsystems, Wetzlar, Germany). The experiment performed
without SeNPs treatment were used as control assay.
2.5. ROS assay
For total ROS, the bacterial (E. coli and S. aureus) cells were incu-
bated with SeNPs for 10, 30 and 60 min, respectively. Then accord-
ing to the total ROS assay kit, the samples were stained with 10 lM
20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min in
the dark at the room temperature and washed once with PBS.
Total ROS of the bacterial cells were examined under a laser
scanning confocal microscope and the Tecan infinite 200 micro-
plate reader with the excitation at 488 nm and emission at
525 nm, respectively.
2.6. Membrane permeability assay
We prepared membrane permeability assay samples using
reported methods [29]. The logarithmic-phase E. coli or S. aureus
(optical density, OD600nm 0.5) was incubated with 100 lL of
25 lg/mL SeNPs at 37 °C for 1 h and 2 h, respectively, and stained
using PI (3 lM). Incubated Tenfold-dilution of logarithmic-phase
bacteria (OD600nm 0.05) with 4 lM diSC3-5 for 1 h, 100 mM KCl
was used immediately on ion equilibration. Subsequently, they
were treated with SeNPs (25 lg/mL) for 1 h and 2 h, respectively.
The fluorescence was measured with excitation and emission
wavelengths of 622 nm and 670 nm, respectively. The control
assay is done with an equal volume of water.
The b-galactosidase assay test has been reported by Koepse
and Russell [30]. 100 lL of 100 mg/mL of o-Nitrophenyl-b-D-
Galactopyranoside (ONPG) was added to 1.5 mL of the
logarithmic-phase E. coli (optical density, OD600nm 0.5) suspension.
Then, the SeNPs were added to the above mentioned suspension.
To evaluated Ortho-nitrophenol (ONP), we measured the increase
in OD420nm using UV-vis spectroscopy. Suspensions without the
addition of SeNPs are used as a control.
2.7. Preparation of bacterial samples for SEM and TEM
Samples for SEM were prepared according to the previous
reports [31,32]. The bacteria in the logarithmic phase were treated
with SeNPs (25 lg/mL) for 2 h at 37 °C on a shaker bed at 200 rpm.
The bacteria were collected with centrifugation at 5000 rpm for
5 min and washed with PBS three times, then fixed with 2.5% glu-
taraldehyde overnight at 4 °C and dehydrated with a sequential
treatment of 50%, 70%, 85%, 90%, and 100% ethanol for 10 min,
respectively, gold sputter-coated, observed SEM.
After incubating with SeNPs (25 lg/mL) for 2 h at 37 °C on a
shaker bed at 200 rpm, the super-thin slice samples for TEM were
prepared using reported methods [33]. The bacterial cells were
collected by 5000 rpm for 5 min and washed with PBS three times.
Then cells were fixed with 2.5% glutaraldehyde for 4 h at 4 °C. The
treated samples were washed twice with PBS and fixed with 0.1%
osmic acid for 2 h. Samples were washed three times with PBS
and dehydrated with sequential treatment of 50%, 70%, 85%, 90%,
and 100% ethanol for 15 min, respectively, and further cut into
ultrathin slices and stained with 2% uranyl acetate and 0.2% lead
citrate before observation.
2.8. Cytotoxicity assay
HEK 293T cells were used to evaluate the cytotoxicity of SeNPs.
The cells were cultured in Dulbecco’s modified Eagles medium in a
humidified atmosphere (5% CO2, and 95% O2), then incubated with
1  104 cells per well of HEK 293T in 96-well plates with a function
of concentrations of SeNPs at 37 °C for 24 h and 48 h. Then 10 lL of
399
5% MTT solution was added into each well and the cells were incu-
bated for a further 4 h. The culture medium containing MTT was
removed and 100 lL of DMSO was added into each well to dissolve
the formazan crystals with low-speed shaking for 15 min. A micro-
plate reader was used to measure the OD545nm value. Using a cyto-
toxicity assay kit intracellular lactate dehydrogenase (LDH) leakage
was tested. The release of LDH into the cell culture supernatant
was quantified using a microplate reader at a wavelength of
450 nm.[34] The positive control assay was done with untreated
cells, their proliferation rate was set to 100%.
3. Results and discussion
3.1. Preparation and characterization of SeNPs
SeNPs were synthesized via reduction of Na2SeO3 by NaBH4 as
previously reported [29,35]. Selenium nanoparticles were capped
with quercetin, acetylcholine molecules, or both to form compact,
globular nanocomposites with high stability. The morphology
and size of the resulting Qu@SeNPs, Ach@SeNPs, and Qu–Ach@
SeNPs were characterized by transmission electron microscopy
(TEANAI-10). TEM images suggest the SeNPs are spherical and with
a narrow size distribution (Fig. 1(A–C)). The sizes of the resulting
SeNPs are as follows: Qu@SeNPs were 80 ± 10 nm, Ach@SeNPs
were 53 ± 15 nm, and Qu–Ach@SeNPs were 120 ± 23 nm.
These results are consistent with hydrodynamic diameters of
90 ± 10 nm, 60 ± 15 nm, and 140 ± 21 nm, respectively, as observed
by dynamic light scattering (DLS, Zetasizer Nano ZS, Fig. S1(A–C),
Zeta-Potential Fig. S1(D–F)).
To confirm the formation of Qu@SeNPs, Ach@SeNPs, and Qu–
Ach@SeNPs, UV–vis Spectra and IR spectrum assays were per-
formed. Fig. 1D shows quercetin has two characteristic absorption
peaks at 254 nm and 374 nm. Similar results were observed for
Qu@SeNPs and Qu–Ach@SeNPs. Characteristic absorption peaks
could not be clearly observed for Ach and Ach@SeNPs. The IR spec-
trum assay showed peaks for Qu@SeNPs and Qu–Ach@SeNPs at
1613–1637 cm1 (deformation vibration of benzene) and 3388–
3460 cm1 (stretching vibration of –CH3) offset, compared to
quercetin. The IR spectrum assay showed peaks for Ach@SeNPs
and Qu–Ach@SeNPs at 1000–1500 cm1 (telescopic vibration of
C–N) decreased, compared to acetylcholine chloride (Fig. 1E).
3.2. Testing the antibacterial activity of SeNPs
We hypothesized that SeNPs have potent antibacterial activity
[25,36]. The activity of SeNPs was measured by a zone of inhibition
assay, where the compounds create a circular inhibition zone, with
diameter as a read-out. The Fig. 2 shows the inhibition zone results
for E. coli and S. aureus, respectively. Results suggest Ach@SeNPs
and Qu–Ach@SeNPs have significant antibacterial activities. Qu–
Ach@SeNPs showed the highest antibacterial activity (The inhibi-
tion zone close to 2 cm). Qu@SeNPs have different activities against
E. coli and S. aureus (The inhibition zones were 0.2 cm and 0.6 cm).
Qu@SeNPs had a higher antibacterial activity against S. aureus,
compared to E. coli. Perhaps the outer membrane permeability bar-
rier in Gram-negative E. coli is the likely reason for this observation
[26]. Quercetin and the mixture of quercetin and acetylcholine
chloride exhibited antibacterial activity against E. coli and S. aureus.
Acetylcholine chloride had no observable antibacterial activity,
compared to the negative control. This assay also proves the syner-
gistic effects of quercetin with acetylcholine on SeNPs on killing
bacteria.
The antibacterial activity of SeNPs as evaluated in vitro, by mon-
itoring the optical density at 600 nm (OD600nm) of a bacterial sus-
pension using a multifunctional microplate reader. SeNPs alone
400 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407
Fig. 1. Characterization of SeNPs. (A–C) TEM images of Qu@SeNPs (A), Ach@SeNPs (B), and Qu–Ach@SeNPs (C); (D) UV–vis spectra of Qu, Ach, Qu@SeNPs, Ach@SeNPs, and
Qu–Ach@SeNPs; (E) FTIR spectra of Ach (1), Ach@SeNPs (2), Qu (3), Qu@SeNPs (4), and Qu–Ach@SeNPs (5).
Fig. 2. Inhibition zones of without NPs (a), Ach (b), Qu (c), Ach/Qu (d), Qu@SeNPs
(e), Qu–Ach@SeNPs (f), Ach@SeNPs (g) against MDR E. coli (A and B) and MDR S.
aureus (C and D). The assay done without NPs is used as thecontrol.
are indicated by the minimum inhibitory concentration (MIC)
tested via a microbroth dilution method [29]. MIC values of
Qu@SeNPs, Ach@SeNPs, and Qu–Ach@SeNPs against E. coli,
P. aeruginosa, S. aureus, K. pneumoniae and A. baumannii were
tested. MIC values of Qu@SeNPs against these bacteria were 32.0,
24.0, 8.0, 24.0, and 8.0 lg/mL, respectively. MIC values of Ach@-
SeNPs were 6.0, 4.0, 4.0, 6.0 and 6.0 lg/mL, respectively, and those
of Qu–Ach@SeNPs were 4.0, 4.0, 2.0, 4.0, and 6.0 lg/mL, respec-
tively (Table S1). There is a little bit of difference for the SeNPs
against MDRs as can be observed from the Table S1. This could
be due to differences in the MDR cell walls, causing a difference
in sensitivity to nanocomposites. The MICs of Ach@SeNPs and
Qu–Ach@SeNPs were much lower than those of Qu@SeNPs. This
was because the Qu@SeNPs could not interact with the bacterial
cell membrane (which was mainly based on their high local
mass density). Acetylcholine, a neurotransmitter, has been used
as a permeability-active unit in antibacterial agents [27]. By pene-
trating the bacterial cell membrane, the acetylcholine-conjugated
nanoparticles had enhanced antibacterial activity. More SeNPs could
cross the membrane barriers of the bacteria, which increased the
antibacterial activity of SeNPs. Qu–Ach@SeNPs showed the highest
antibacterial activity against all the bacteria tested, especially
against the multi-drug resistant-superbugs. Synergistic effects
occurred only when both Qu and Ach were presented as co-
ligands on SeNPs during the reduction of Na2SeO3.
3.3. High antibacterial activities of SeNPs
We tested the antibacterial activity of three types of SeNPs
against Gram-positive and Gram-negative bacteria using MDR
E. coli and MDR S. aureus as model organisms. Actively dividing,
log phase bacterial cells, were used in all experiments. As shown
in Fig. 3(A–C), bacteria were incubated with nanoparticles at a
concentration of 25.0 lg/mL for 10–60 min. Bacterial survival
was determined by measuring the OD630nm using a UV–vis spec-
troscopy. The results show, all nanocomposites, except Qu@SeNPs,
significantly decrease the viability of E. coli and S. aureus cells over
time. When E. coli and S. aureus were treated with Ach@SeNPs and
Qu–Ach@SeNPs for 60 minutes, Ach@SeNPs reduced the viability of
both types of bacteria by 60%. Qu–Ach@SeNPs reduced the viability
of E. coli and S. aureus cells by 91.7% and 92.3%, respectively. These
results show the highly antibacterial activities of Qu–Ach@SeNPs,
and also proved that Qu–Ach@SeNPs exhibit a synergistically
enhanced antibacterial performance against the multidrug-
resistant superbugs (MDRs).
 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407 401

As shown in Fig. 4(A–B), as the concentration of SeNPs
increased, the viability of both E. coli (A) and S. aureus (B) decreased
significantly. After bacteria were treated with 25.0 lg/mL SeNPs
for 6 h, Ach@SeNPs and Qu–Ach@SeNPs reduced the viability of
both E. coli and S. aureus cells by nearly 90%. These results suggest
SeNPs not only kill Gram-negative and Gram-positive bacterial
cells, but also inhibit bacterial cell division.
Bacterial viability was further measured by performing LIVE/
DEAD staining of E. coli and S. aureus using AO&EB fluorescent dyes
[33]. In this assay, the bacterial cells appear green or red when
viewed under a laser scanning confocal microscope (LSM), Green
or red cells are considered live/dead with intact/damaged mem-
branes. Fig. 5 shows the fluorescent micrographs of E. coli (Fig. 5A)
and S. aureus (Fig. 5B) treated with solutions of Qu@SeNPs, Ach@
SeNPs, and Qu–Ach@SeNPs for a predetermined time. The bacterial
cells of the control group fluoresced green (in web version),
indicating that the cells were alive. However, after Ach@SeNPs
and Qu–Ach@SeNPs treatment for 60 min, almost all the bacterial
cells fluoresced red and rarely fluoresced green (in web version),
indicating that the cells were killed. When bacteria were treated
with Qu@SeNPs for 60 min, only S. aureus cells fluoresced red.
These results show the antibacterial activity of quercetin against
S. aureus, compared to a weak antibacterial activity against
E. coli. These results agree with previous reports, wherein the
antibacterial activity of quercetin was limited to certain bacterial
species [26]. In summary, all these in vitro experiments indicate
our synthetic Qu–Ach@SeNPs have strong antibacterial properties
when tested against both Gram-positive and Gram-negative
bacteria. All the results show that Qu–Ach@SeNPs exhibit a syner-
gistically enhanced antibacterial performance against the
multidrug-resistant superbugs (MDRs).
3.4. Mechanisms of antibacterial action of Qu–Ach@SeNPs
Since SeNPs can induce the generation of ROS, we next exam-
ined the production of reactive oxygen species (ROS) in SeNPs-
treated E. coli and S. aureus. In these experiments, we tested
whether bacterial death resulted from oxidative damage [35]. Bac-
tericidal antibiotics induce the generation of ROS to kill bacteria
[37]. It has been reported that V2O5 NPs, as oxidase mimics inhibit
the bacterial biofilms via ROS [38]. Composition and catalysis-
related ROS effects on mammalian cells of Fe3O4-doped silica NPs
have been reported [39]. By measuring the total ROS concentra-
tions with 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) and
hydroxyl radical (a type of ROS) concentrations with hydrox-
yphenyl fluorescein (HPF), the results showed that Qu–Ach@SeNPs
significantly increase ROS production in of E. coli and S. aureus
(Fig. 6). It is likely that the antibacterial activity of Qu–Ach@SeNPs,
is linked to the generation of reactive oxygen species in bacteria.
Both Qu@SeNPs and Ach@SeNPs did not significantly increase
ROS production in E. coli. But in S. aureus, as shown in Fig. 6, it is
evident that levels of ROS produced by Qu@SeNPs after 60 min
are on par with the positive control for S. aureus. These results
show that one of the mechanisms by which Qu–Ach@SeNPs
kill bacteria is the generation of reactive oxygen species in
bacteria.

Fig. 3. Survival rates of MDR E. coli (A) and MDR S. aureus (B) treated with Qu@SeNPs, Ach@SeNPs, Qu–Ach@SeNPs at different times of coincubation with or without SeNPs
(25 lg/mL). ($p < 0.05, $$p < 0.01 Qu@SeNPs-treated group vs. no Qu @SeNPs-treated group; #p < 0.05, ##p < 0.01 Ach@SeNPs-treated group vs. no Ach@SeNPs-treated
group;*p < 0.05, **p < 0.01 Qu–Ach@SeNPs-treated group vs. no Qu–Ach@SeNPs-treated group).

Fig. 4. Optical density at 600 nm (OD600nm) of bacterial (MDR E. coli (A) and MDR S. aureus (B)) suspension treated with SeNPs at different concentrations (from 0 lg/mL to
and 25 lg/mL) at 1 h.
402 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407

The bacterial cell membrane can be destroyed by nanocompos-
ites. The effect of SeNPs on the cell membrane was quantified using
fluorescent dyes. Propidium iodide (PI) can pass through a broken
cell membrane and bind to nucleic acids, causing an increase in flu-
orescence [40]. The cyanine dye diSC3-5 (3,30 -dipropylthiadicarbo
cyanine iodide) self-quenches inside the organized cytoplasmic
membrane. When the cell membrane is permeable, the dye is
released and fluorescence is recovered. [41–43]. PI and diSC3-5
were used to determine total cell membrane and cytoplasmic
membrane.
For both dyes, increased fluorescence indicates membrane dam-
age. Qu@SeNPs did not cause a significant increase in fluorescence
for either dye, compared to the negative control. When cells were
treated with Ach@SeNPs and Qu–Ach@SeNPs, total cell membrane
permeability significantly increased for both E. coli and S. aureus.
Ach@SeNPs and Qu–Ach@SeNPs also caused cytoplasmic mem-
brane permeability to increase for both Gram-negative E. coli and
Gram-positive S. aureus. Qu@SeNPs showed a weak effect on the
cell wall permeability of S. aureus. These results suggest that Ach@
SeNPs and Qu–Ach@SeNPs cause wide-spread damage to the bac-
terial cell membrane and cytoplasmic membrane (Fig. 7). These
results suggest that Qu–Ach@SeNPs show wide-spectrum activities
by the increase in the permeability of both cell wall and cytoplas-
mic membranes.
To further test the effects of SeNPs on bacterial cell integrity, we
performed another assay to measure the b-galactosidase enzyme,
which is present in the cytoplasm of the Enterobacteriaceae
(such as E. coli) [44]. When the cytoplasmic membrane is broken,

Fig. 5. Laser scanning confocal microscope of MDR E. coli (A) and MDR S. aureus (B) after being treated with Qu@SeNPs, Ach@SeNPs, Qu–Ach@SeNPs for 60 min, respectively.
The assay without NPs is used as the control. The bacterial cells that appeared green (AO)/red (EB) when visualized under fluorescence microscopy were considered live/dead
with intact/damaged membranes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407 403

b-galactosidase is released into the surrounding medium, where it
catalyzes the hydrolysis of o-Nitrophenyl-b-D-Galactopyranoside
(ONPG) to produce ONP, ONP production is determined by measur-
ing the OD420nm via UV–vis spectroscopy. Hence, in the present
study, we using ONPG analyzed the b-galactosidase. Fig. 8 shows,
the release of cytoplasmic b-galactosidase increased in E. coli sus-
pensions as nanoparticle concentration increased, over time. These
results indicate that treatment with Ach@SeNPs and Qu–Ach@
SeNPs causes compromised membrane integrity. This leads to
leakage of the cytoplasm into the surrounding medium. In the
present study, the ability of Ach@SeNPs and Qu–Ach@SeNPs to
get attached and penetrate into the bacterial cell was dramatically
improved with the introduction of acetylcholine.
Morphological changes to E. coli and S. aureus treated with
SeNPs were observed using scanning electron microscopy (SEM)
and transmission electron microscopy (TEM). In the absence of
SeNPs (Figs. 9A, E and 10A, E), the shapes of E. coli and S. aureus
were typically rod-shaped and rounded, respectively. Both bacteria
had smooth, intact cell walls. After incubation with SeNPs,
the results showed differences in bacterial morphology. After
treatment with Qu@SeNPs, the E. coli cells were typically rod-
shaped, smooth, with intact cell walls, same as the control
(Figs. 9B and 10B). As shown in Fig. 9(C–D), Ach@SeNPs and Qu–
Ach@SeNPs induced cell lysis, causing the quantity of E. coli cells
to decrease. The cell walls separate with membrane and are dam-
aged. The form and size of cells also changed significantly. In addi-
tion, for most E. coli cells, leakage of intracellular contents was
observed in Fig. 10(C and D). Changes in S. aureus also could be
observed morphologically as shown in Fig. 9(F–H). The antibacte-
rial activity of quercetin has been previously reported [45–47].
When cells were treated with Qu@SeNPs, significant morphologi-
cal changes to S. aureus could be observed (Fig. 9F and Fig. 10F),
compared to untreated-cells. Treated S. aureus cell walls were
sunken and damaged, similar to previous reports [26]. After incu-
bation with Ach@SeNPs and Qu–Ach@SeNPs, similar results were
observed. Cell walls of S. aureus were significantly damaged and
sunken. Damage was evident due to cytoplasmic release of cyto-
plasm and disorganization of the cell wall (Fig. 9(H and G) and
Fig. 10(H and G), arrows). These results suggest that acetylcholine
(Ach) is an effective antibacterial agent for delivering nanocompos-
ites to cell walls. Ach causes nanocomposites to combine with the
acetylcholine-receptor, damaging bacteria cell membranes and
killing bacteria (see Scheme 1).
All these results suggest the synergy of acetylcholine and quer-
cetin when combined on SeNPs causes enhanced antibacterial
activity. The nanocomposites work to kill bacteria by penetrating
the bacterial cell membranes and increasing the ROS production.
Our investigation into the antibacterial mechanisms of SeNPs
showed that the high antibacterial activity of Qu–Ach@SeNPs is
due to: (1) compromised bacteria cell membranes integrity, and
the (2) increased bacterial intracellular ROS level production, and
(3) entry into bacterial cells and disruption of DNA structure. All
these actions result in inhibition of bacterial growth and cause
bacterial cell death.
3.5. Bacterial resistance to SeNPs
To investigate the potential resistance of MDR superbugs to
SeNPs, we added Ach@SeNPs and Qu–Ach@SeNPs to cultures of
E. coli and S. aureus. After 30 passages, we found that the MIC of

Fig. 6. (A) MDR bacterial cells incubated with 10 mM DCF–DA in PBS for 30 min were treated with 25 lg/mL of Qu@SeNPs, Ach@SeNPs, Qu–Ach@SeNPs, respectively, for
60 min at 32 °C, and then imaged under a Laser scanning confocal microscope. The assay without NPs is used as the control. (B) Cellular total ROS probed with 20 ,70 -
dichlorofluorescein diacetate (DCFH-DA), MDR E. coli and MDR S. aureus without the addition of NPs were the control in all assays or the negative control. The positive control
was commercial Ros-up producing ROS in the kit. (*p < 0.05, **p < 0.01 SeNPs-treated group vs. no SeNPs-treated group) Each value represents the mean SD (n = 5).
404 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407

Fig. 7. Permeability of MDR bacterial cell membrane probed by PI (A) and diSC3-5 (B). The assay without NPs is used as the control. The increase of fluorescence corresponds
with the degree of the compromise of total cell membrane (A) (*p < 0.05, **p < 0.01 SeNPs-treated group vs. no SeNPs-treated group) and cytoplasmic membrane (B)
(&&p < 0.01 SeNPs-treated group vs. no SeNPs-treated group). Each value represents the mean SD (n = 5).

Fig. 8. Absorption of ONP at different times and concentrations treat with MDR E. coli. Qu@SeNPs (A), Ach@SeNPs (B), and Qu–Ach@SeNPs (C).
 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407 405

Fig. 9. Morphological changes of MDR E. coli (A–D) and MDR S. aureus (E–H) without (A and E) and treated with SeNPs (25 lg/mL, 2 h). Qu@SeNPs (B and F), Ach@SeNPs (C
and G) and Qu–Ach@SeNPs (D and H) visualized with SEM. Arrowheads indicate compromised cell walls. The assay without NPs is used as the control.

Fig. 10. TEM images of MDR E. coli and MDR S. aureus treated with 25 lg/mL of Qu@SeNPs (B and F), Ach@SeNPs (C and G), Qu–Ach@SeNPs (D and H), and untreated (A and E).
Arrows indicate NPs and arrowheads indicate compromised cell walls in several types: deformed (B and F), broken (C, D, G, H). The assay without NPs is used as the control.

Scheme 1. Schematic illustration of the synthesis of Qu–Ach@SeNPs, and for the targeted bacteria.
406 X. Huang et al. / Acta Biomaterialia 30 (2016) 397–407

Fig. 11. (A) Cell viability after incubation as a function of Qu@SeNPs, Ach@SeNPs and Qu–Ach@SeNPs concentrations determined by MTT assays for 24 and 48 h. (B) Cell
cytotoxicity after incubation as a function of Qu@SeNPs, Ach@SeNPs and Qu–Ach@SeNPs concentrations determined by lactate dehydrogenase (LDH) release assays for 24 and
48 h. (*p < 0.05, **p < 0.01, SeNPs-treated group vs. no SeNPs-treated group). Each value represents the mean SD (n = 5). The assay without NPs is used as as the control.

E. coli and S. aureus to SeNPs remained constant compared to the
starting MIC. These results suggest that resistance to the Ach@-
SeNPs and Qu–Ach@SeNPs did not emerge. We suspect that this
is due to the multiple antibacterial mechanisms of bacterial mem-
brane disruption, inhibition of enzymatic activity, and DNA or RNA
interaction of synthesized Qu–Ach@SeNPs. Previous studies have
shown SeNPs may disrupt DNA structure in vitro [48].
3.6. Cytotoxicity of SeNPs
For antibacterial drugs, the cytotoxicity to human cells is
unsolved. In order to determine cytotoxicity of SeNPs, the cytotox-
icity of SeNPs to human embryonic kidney cells (HEK 293T) was
evaluated using the MTT and LDH release assays. As shown in
Fig. 11A, the cell viability of Qu@SeNPs, Ach@SeNPs and Qu–Ach@
SeNPs was measured as a function of concentration (from 5 to
100 lg/mL) and incubation time. The cell viability of nanoparticles
was over 80% at concentrations P100 lg/mL, the concentration of
Qu–Ach@SeNPs exceeded the MIC of these nanoparticles against
E. coli and S. aureus by 20-fold. Results of Fig. 11B showed no
significant effects on human embryonic kidney cells. Therefore,
Qu–Ach@SeNPs are effective antibacterial agents with low cytotox-
icity and efficient antibacterial activity.
4. Conclusions
the permeability of cell membranes. This causes membrane disrup-
tion and leads to the leakage of the cytoplasm, allowing nanocom-
posites to subsequently invade bacterial cells and disrupt DNA
structure. Qu–Ach@SeNPs have low cytotoxicity when tested on
normal human cells in vitro. Importantly, Qu–Ach@SeNPs showed
no emergence of resistance. These results suggest that Qu–Ach@
SeNPs have excellent antibacterial activities. These agents can
serve as good antibacterial agents against superbugs. These data
suggest that these antibacterial agents may have potential applica-
tion in the field of medicine for combating infectious diseases
caused by MDRs, as well as other infectious diseases.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21171070 and 21371075), the Natural
Science Foundation of Guangdong Province (2014A030311025)
and Chinese Postdoctoral Science Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.actbio.2015.10.
041.

In this study, we have successfully designed three highly stable References

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SeNPs. Qu–Ach@SeNPs showed efficient antibacterial and bacteri-
cidal activities against superbugs. Due to the presence of acetyl-
choline, Qu–Ach@SeNPs shows the ability to combine with the
acetylcholine receptor on the bacteria cell membrane and increase
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