nanomaterials

Article
Functionalized ZnO Nanoparticles with Gallic Acid
for Antioxidant and Antibacterial Activity against
Methicillin-Resistant S. aureus
Joo Min Lee 1 , Kyong-Hoon Choi 2 , Jeeeun Min 3 , Ho-Joong Kim 4, *, Jun-Pil Jee 5, *
and Bong Joo Park 2,6, *
1 Department of Food and Nutrition, Chosun University, Gwangju 61452, Korea; joominlee@chosun.ac.kr
2 Institute of Biomaterials, Kwangwoon University, 20 Kwangwoongil, Nowon-gu, Seoul 01897, Korea;
solidchem@hanmail.net
3 Halla Energy and Environment, Seoul 05769, Korea; iseeiget@naver.com
4 Department of Chemistry, Chosun University, Gwangju 61452, Korea
5 College of Pharmacy, Chosun University, Gwangju 61452, Korea
6 Department of Electrical & Biological Physics, Kwangwoon University, 20 Kwangwoongil, Nowon-gu,
Seoul 01897, Korea
* Correspondence: hjkim@chosun.ac.kr (H.-J.K.); Jee@chosun.ac.kr (J.-P.J.); parkbj@kw.ac.kr (B.J.P.);
Tel.: +82-62-230-6643 (H.-J.K.); +82-2-940-8629 (B.J.P.)

Received: 30 September 2017; Accepted: 31 October 2017; Published: 2 November 2017

Abstract: In this study, we report a new multifunctional nanoparticle with antioxidative and
antibacterial activities in vitro. ZnO@GA nanoparticles were fabricated by coordinated covalent
bonding of the antioxidant gallic acid (GA) on the surface of ZnO nanoparticles. This addition imparts
both antioxidant activity and high affinity for the bacterial cell membrane. Antioxidative activities
at various concentrations were evaluated using a 2,20 -azino-bis(ethylbenzthiazoline-6-sulfonic acid)
(ABTS) radical scavenging method. Antibacterial activities were evaluated against Gram-positive
bacteria (Staphylococcus aureus: S. aureus), including several strains of methicillin-resistant S. aureus
(MRSA), and Gram-negative bacteria (Escherichia coli). The functionalized ZnO@GA nanoparticles
showed good antioxidative activity (69.71%), and the bactericidal activity of these nanoparticles
was also increased compared to that of non-functionalized ZnO nanoparticles, with particularly
effective inhibition and high selectivity for MRSA strains. The results indicate that multifunctional
ZnO nanoparticles conjugated to GA molecules via a simple surface modification process displaying
both antioxidant and antibacterial activity, suggesting a possibility to use it as an antibacterial agent
for removing MRSA.

Keywords: ZnO@GA; antioxidative activity; antibacterial activity; gallic acid; antibiotic resistance;
methicillin-resistant Staphylococcus aureus

1. Introduction
Infectious diseases caused by bacteria are a significant burden on public health and threaten the
economic stability of societies worldwide [1]. In particular, the widespread incorrect use of conventional
antibiotics has led to the adaptation of microorganisms to these therapies, and the appearance of
antibiotic-resistant bacteria is a serious problem [2,3]. Currently, MRSA is the most commonly found
antibiotic-resistant bacteria in many parts of the world [4]. Over the last decade, MRSA strains
have become one of the main causes of mortality among hospital-acquired infectious diseases [5].
However, the development of novel antibiotics to solve this problem had limited progress. Therefore,
the development of alternative methods to treat infections caused by antibiotic-resistant bacteria is an
urgent challenge in medical biotechnology.

Nanomaterials 2017, 7, 365; doi:10.3390/nano7110365 www.mdpi.com/journal/nanomaterials

Nanomaterials 2017, 7, 365 2 of 10

Material science researchers have focused on designing multifunctional nanoparticles, which

combine various functionalities such as fluorescence, magnetism, and photoactivation to generate
reactive oxygen species (ROS), and biotargetability [6–9]. In this respect, metal and metal oxide
nanoparticles have been extensively researched for their photocatalytic, optoelectronic, bioengineering,
magnetic, antimicrobial, wound-healing, and anti-inflammatory properties [10–13] arising from
their unique physical and chemical characteristics, different from those of the bulk phase [14,15].
Among metal oxide nanoparticles, zinc oxide has extensive applications in various fields such as
optics, piezoelectricity, bioimaging, and biosensing [16–18]. In particular, ZnO presents sufficient
antimicrobial efficacy when the particle size is decreased to the nanometer range, as ZnO nanoparticles
can interact with the bacterial core or surface as soon as they contact the cell and then exhibit distinct
antibacterial mechanisms [19]. Therefore, ZnO nanoparticles are of great interest to biologists because
of their excellent antibiotic properties and good biocompatibility. This distinct activity has opened
new frontiers in the biological sciences. However, excessive ROS generated by ZnO nanoparticles
may cause cell membrane disintegration, membrane protein damage, and genomic instability, which
can initiate or enhance the development of many diseases [20,21]. Therefore, the development of
novel multifunctional ZnO nanoparticles with antioxidant activity could minimize damage caused by
excessive ROS.
In this study, we report a method for fabricating ZnO@GA nanoparticles by a simple surface
modification process. The nanoparticles display strong antioxidant and antibiotic effects and were
particularly effective against MRSA, suggesting that they can be a useful novel antibacterial agent for
removing MRSA.

2. Results and Discussion

Antioxidant-functionalized ZnO@GA nanoparticles were synthesized via a simple coating of
ZnO nanoparticles with gallic acid (GA). The ZnO nanoparticles were prepared by a sol-gel process.
Then, the carboxyl group of GA was covalently bonded to surface Zn2+ ions, forming multifunctional
ZnO@GA nanoparticles.
Pure ZnO and ZnO@GA nanoparticles were analyzed using various analytical tools.
Field emission scanning electron microscopy (FE-SEM) confirmed that the ZnO@GA nanoparticles
were nearly spherical in shape, with good size uniformity (Figure 1a). High-magnification scanning
electron microscopy (SEM) images of the ZnO@GA nanoparticles showed a variety of spherical shapes
(inset of Figure 1a), indicating that the nanoparticles aggregated spontaneously. Figure 1b indicates
the size distribution of pure ZnO nanoparticles, which was estimated by sampling 300 particles in
the Transmission electron microscopy (TEM) image. The average size of the ZnO nanoparticles was
11.5 ± 4.4 nm. TEM confirmed that ZnO and ZnO@GA nanoparticles both had globular shapes and
assembled in aggregates on the TEM grids (Figure 1c,d). It is notable that there was almost no change
in particle size or morphology after the surface modification. However, comparing the images in
Figure 1c,d, the ZnO@GA nanoparticles appeared blurrier than pure ZnO nanoparticles owing to the
conjugated antioxidant molecules.
Nanomaterials 2017, 7, 365 3 of 10
Nanomaterials 2017, 7, 365 3 of 10

Figure 1.
Figure 1. (a)
(a)Low-magnification
Low-magnificationFE-SEM
FE-SEMimage
imageofofthe
theZnO@gallic
ZnO@gallic acid
acid(GA)
(GA)nanoparticles. A
nanoparticles.
corresponding high-magnification FE-SEM image is shown in the inset; (b) Histogram
A corresponding high-magnification FE-SEM image is shown in the inset; (b) Histogram of the of the
ZnO@GA nanoparticle
ZnO@GA nanoparticle size
size distribution;
distribution; TEM
TEM images
images of
of (c)
(c) ZnO
ZnO nanoparticles
nanoparticles and
and (d)
(d) ZnO@GA
ZnO@GA
nanoparticles. Corresponding high-resolution TEM images are shown in the
nanoparticles. Corresponding high-resolution TEM images are shown in the insets. insets.

High-resolution TEM (HR-TEM) and X-ray diffraction (XRD) analysis were used to obtain a
High-resolution TEM (HR-TEM) and X-ray diffraction (XRD) analysis were used to obtain a
more detailed crystal structure of the ZnO@GA nanoparticles. The selected area electron diffraction
more detailed crystal structure of the ZnO@GA nanoparticles. The selected area electron diffraction
(SAED) pattern is presented in Figure 2a. The interlayer distances of the ZnO@GA nanoparticles were
(SAED) pattern is presented in Figure 2a. The interlayer distances of the ZnO@GA nanoparticles were
calculated to be 0.282 and 0.259 nm, which are comparable to the (100) and (002) planes of hexagonal
calculated to be 0.282 and 0.259 nm, which are comparable to the (100) and (002) planes of hexagonal
ZnO, respectively [22]. This result indicates that each of the ZnO@GA nanoparticles has a single
ZnO, respectively [22]. This result indicates that each of the ZnO@GA nanoparticles has a single
crystalline nature; thus, the ZnO@GA nanoparticles displayed high crystallization. Figure 2b shows
crystalline nature; thus, the ZnO@GA nanoparticles displayed high crystallization. Figure 2b shows
the powder XRD data of the ZnO@GA nanoparticles. The strong Bragg reflection peaks
the powder XRD data of the ZnO@GA nanoparticles. The strong Bragg reflection peaks (2θ = 31.7◦ ,
(2θ = 31.7°, 34.4°, 36.1°, 47.6°, 56.5°, 62.8°, 67.9° and 72.1°), matched by their Miller indices ((100),
34.4◦ , 36.1◦ , 47.6◦ , 56.5◦ , 62.8◦ , 67.9◦ and 72.1◦ ), matched by their Miller indices ((100), (002), (101),
(002), (101), (102), (110), (103), (112), and (004)), were obtained from a standard wurtzite ZnO structure
(102), (110), (103), (112), and (004)), were obtained from a standard wurtzite ZnO structure (JCPDS
(JCPDS Card No. 36-1451) [23]. Therefore, hexagonally structured ZnO was identified as a single
Card No. 36-1451) [23]. Therefore, hexagonally structured ZnO was identified as a single crystalline
crystalline phase in the ZnO@GA nanoparticles. The diffraction peak profile (2θ = 36.1°) was fairly
phase in the ZnO@GA nanoparticles. The diffraction peak profile (2θ = 36.1◦ ) was fairly well fitted by
well fitted by a convolution of Lorentzian functions (inset of Figure 2b). The mean crystalline size of
a convolution of Lorentzian functions (inset of Figure 2b). The mean crystalline size of the ZnO@GA
the ZnO@GA nanoparticles was 5.8 nm, calculated based on Scherrer’s equation.
nanoparticles was 5.8 nm, calculated based on Scherrer’s equation.
Nanomaterials 2017, 7, 365 4 of 10
Nanomaterials 2017, 7, 365 4 of 10

Figure 2. (a) High-resolution transmission electron micrograph of the ZnO@GA nanoparticles; (b)
Figure 2. (a) High-resolution transmission electron micrograph of the ZnO@GA nanoparticles; (b) X-ray
X-ray diffraction (XRD) pattern of ZnO@CA nanoparticles. a.u., arbitrary units.
diffraction (XRD) pattern of ZnO@CA nanoparticles. a.u., arbitrary units.
To confirm the binding between the carboxyl group of the GA molecules and Zn2+ cations on the
2+
To confirm
surface of ZnO, theFourier-transform
binding between infrared (FT-IR) group
the carboxyl spectraofofthe pureGA GAmolecules
moleculesand and Zn cations on the
the ZnO@GA
surface of ZnO, Fourier-transform
nanoparticles were compared (Figure infrared
3a). The(FT-IR) spectra
main peaks of of
thepure GA molecules
ZnO@GA andand
nanoparticles thepure
ZnO@GA
nanoparticles
GA were very were compared
similar, (Figure
resembling 3a). The main
the characteristic peaks
peaks of the
of GA. ZnO@GA
This indicates nanoparticles
that GA moleculesand pure
GAremain
were veryon the surfaceresembling
similar, of ZnO nanoparticles even after
the characteristic washing
peaks of GA.with
Thisethanol. In particular,
indicates that GA both
molecules
samples
remain on theshowedsurface the presence of a carboxyl group
of ZnO nanoparticles even(2700
after towashing
3600 cm−1 ), hydroxyl
with ethanol.phenolic groups both
In particular,
(3284, 3382 cm −1 ), and an aromatic moiety (1541, 1618 cm −1 ), as
samples showed the presence of a carboxyl group (2700 to 3600 cm ), hydroxyl phenolic shown in Figure
− 1 3a [24]. However, thegroups
pure GA molecules had absorption peaks at 1613, 1427, and 1268 cm −1, according to the stretching
(3284, 3382 cm−1 ), and an aromatic moiety (1541, 1618 cm−1 ), as shown in Figure 3a [24]. However,
modes of the free carbonyl double bond (υC=O), the C–O single bond (υC–O), and the oxygen-hydrogen
the deformation
pure GA molecules had absorption peaks at 1613, 1427, and 1268 cm−1 , according to the stretching
(υC–OH) (top panel of Figure 3a). This result indicates that pure GA molecules have
modes of the free
protonated carbonyl
carboxyl double
groups bond (υC=O
(COOH), ), the C–OConversely,
as expected. single bond the (υC–O ), and thenanoparticles
ZnO@GA oxygen-hydrogen
deformation (υC–OHnovel
displayed strong ) (toppeaks
panelatof1560
Figure
and 3a).
1376 This
cm−1. result
These indicates
new bandsthat can pure GA molecules
be attributed to the have
protonated
asymmetric carboxyl
(υas = 1560 groups
cm−1(COOH), as expected.
) and symmetric (υs = 1376Conversely, the ZnO@GA
cm−1) stretching modes of the nanoparticles displayed
carboxyl group,
as shown
strong novel in Figure
peaks at3a (bottom
1560 panel).cm
and 1376 −1 . These
These results new
indicate that can
bands the carboxyl group to
be attributed bound to the
the asymmetric
(υassurface
= 1560ofcm the−1ZnO symmetric (υs = 1376 cm−1 ) stretching modes of the carboxyl group, as shown
) andnanoparticle.
Figure 3b shows
in Figure 3a (bottom panel). the These
photoluminescence
results indicate andthat
photoluminescence
the carboxyl group excitation
bound (PL and
to the PLE) of the
surface
spectra of pure GA molecules and ZnO@GA nanoparticles. The peak at 315 nm is an absorption
ZnO nanoparticle.
Nanomaterials 2017, 7, 365 5 of 10
band typical of pure GA molecules, which may be attributed to the aromatic ring. Also, pure GA
molecules had one strong emission peak, located at 368 nm (λex = 310 nm). After surface
modification, the PL and PLE spectra of the ZnO@GA nanoparticles exhibited characteristics very
similar to those of pure GA molecules. However, the emission spectrum of the ZnO@GA
nanoparticles also displayed emission at 450 to 650 nm. Broad, red-shifted emissions are typically
observed with ZnO nanomaterials and are attributed to a recombination process through electronic
states originating from oxygen vacancies or surface defects [25].

Figure
Figure 3.
3. (a)
(a) Fourier-transform
Fourier-transform infrared
infrared spectroscopy
spectroscopy spectra
spectra of
of pure
pure GA
GA molecules
molecules and
and ZnO@GA
ZnO@GA
nanoparticles;
nanoparticles; (b) PL and PLE spectra of pure GA molecules and ZnO@GA nanoparticlesininwater.
(b) PL and PLE spectra of pure GA molecules and ZnO@GA nanoparticles water.
The
The excitation
excitation and
and detection
detection wavelengths
wavelengths for
for both
both spectra
spectra were
were 310 and 380
310 and 380 nm.
nm. a.u.,
a.u., arbitrary
arbitrary units.
units.

The antioxidant efficacies of pure GA molecules and ZnO@GA nanoparticles were evaluated
using an ABTS radical scavenging method. The ZnO@GA nanoparticles contained an average of 2.89
GA molecules per particle and were suggested to have excellent diffusion and stability in water. The
antioxidant activities of the ZnO@GA nanoparticles and pure GA are shown in Table 1. Pure GA
molecules scavenged ABTS radicals proportionally to the concentration. The hydroxyl groups of GA
Nanomaterials 2017, 7, 365 5 of 10

Figure 3b shows the photoluminescence and photoluminescence excitation (PL and PLE) spectra
of pure GA molecules and ZnO@GA nanoparticles. The peak at 315 nm is an absorption band typical
of pure GA molecules, which may be attributed to the aromatic ring. Also, pure GA molecules had
one strong emission peak, located at 368 nm (λex = 310 nm). After surface modification, the PL and
PLE spectra of the ZnO@GA nanoparticles exhibited characteristics very similar to those of pure GA
molecules. However, the emission spectrum of the ZnO@GA nanoparticles also displayed emission at
450 to 650 nm. Broad, red-shifted emissions are typically observed with ZnO nanomaterials and are
attributed to a recombination process through electronic states originating from oxygen vacancies or
surface defects [25].
The antioxidant efficacies of pure GA molecules and ZnO@GA nanoparticles were evaluated
using an ABTS radical scavenging method. The ZnO@GA nanoparticles contained an average of
2.89 GA molecules per particle and were suggested to have excellent diffusion and stability in water.
The antioxidant activities of the ZnO@GA nanoparticles and pure GA are shown in Table 1. Pure GA
molecules scavenged ABTS radicals proportionally to the concentration. The hydroxyl groups of GA
are important for its free radical scavenging efficiency. In particular, the OH at the para-position to
the carboxyl group appears to be essential for maintaining scavenging activity, as the scavenging
activity is diminished by its methylation [26]. The ZnO@GA nanoparticles also robustly scavenged
ABTS radicals. The decrease in the antioxidant activity of the ZnO@GA nanoparticles compared to
that of pure GA molecules may be attributable to steric repulsion between the nanoparticles and
ABTS radicals.
To confirm the antibacterial effects of GA, ZnO, and ZnO@GA nanoparticles, a static culture
method was used after mixing the bacteria and nanoparticles, as previously described [27].
The antibacterial effect of each sample was tested in five bacterial strains (a Gram-negative strain,
i.e., E. coli, and four Gram-positive strains, i.e., one S. aureus and three MRSA). The antibacterial
activities of each sample were evaluated by counting the colony-forming units (CFUs) of each strain as
a measure of the total number of viable bacteria (Figure 4). The ZnO@GA nanoparticles showed strong
antibacterial activity, two to four fold higher than that of ZnO nanoparticles, and displayed higher
antibacterial activity against S. aureus and MRSA than against E. coli. The ZnO@GA nanoparticles
at 50 and 100 µg/mL completely inhibited MRSA-1 and MRSA-2 strains, and they more effectively
killed the MRSA strains than the S. aureus strain at 50 µg/mL, suggesting that ZnO@GA nanoparticles
are specifically effective against MRSA strains. The selective inhibition effects were also confirmed
using confocal fluorescence microscopy, as shown in Figure 5. The confocal fluorescence microscopy
showed that the ZnO@GA nanoparticles have strong killing effects against Gram-positive bacteria,
with complete killing of the cells in the S. aureus and MRSA samples. The viability of Gram-negative
E. coli decreased as well, although many cells remained viable. This result also confirmed that
the ZnO@GA nanoparticles have selective inhibitory activity against Gram-positive bacteria and
particularly against MRSA.

Table 1. ABTS radical scavenging activity.

Sample Concentration (µM) % Inhibition

ZnO@GA 20 33.29 ± 0.12
ZnO@GA 40 57.17 ± 0.96 a
ZnO@GA 100 69.71 ± 5.26 a
Gallic acid 20 43.38 ± 0.48 b
Gallic acid 40 72.76 ± 0.12 b
Gallic acid 100 93.25 ± 0.43 b
Data are expressed as the mean ± standard deviation (SD) of independent experiments (n = 3). Statistical significance
(p < 0.05) was analyzed using a one-way analysis of variance with a post-hoc Tukey’s test. a Activity at the lowest
concentration of the same sample. b Two different samples at the same concentration.
Nanomaterials 2017, 7, 365 6 of 10
Nanomaterials 2017, 7, 365 6 of 10

Figure 4. Antibacterial
Antibacterialeffects
effectsofofZnO@GA
ZnO@GAnanoparticles.
nanoparticles.(a) (a)
E. coli; (b) (b)
E. coli; S. aureus; (c) MRSA-1;
S. aureus; (c) MRSA-1;(d)
MRSA-2;
(d) MRSA-2;andand(e) (e)
MRSA-3.
MRSA-3. Data areare
Data shown
shownasasthe
themean
mean±±S.D S.D(n(n == 6).
6). Analysis
Analysis of
of statistical
significance (* p < 0.05, ** p << 0.005
0.005 versus
versus control)
control) was
was performed
performed using
using Student’s t-test.

As
As shown
shown in in Figure
Figure 4,
4, although
although thethe bactericidal
bactericidal effects
effects were
were relatively
relatively weak
weak compared
compared to to those
those
of
of the
the ZnO@GA
ZnO@GAnanoparticles,
nanoparticles,the theZnO
ZnO nanoparticles
nanoparticles also showed
also showedinhibitory effects
inhibitory against
effects the five
against the
strains at 100atand
five strains 100200 μg/mL.
and GA is known
200 µg/mL. GA is to have antibacterial
known activity, with
to have antibacterial a minimum
activity, inhibitory
with a minimum
concentration of 8 mg/mLof
inhibitory concentration for8S.mg/mL
aureus [28]. However,
for S. in this
aureus [28]. study, GA
However, in was
this used
study,at GA
0.6 towas
4.5 used
μg/mL. at
At these concentrations, GA alone did not display antibacterial activity against the
0.6 to 4.5 µg/mL. At these concentrations, GA alone did not display antibacterial activity against bacterial strains
the
(Figure
bacterial4).
strains (Figure 4).
Although
Although GA GA treatment
treatment alone
alone was
was not effective, the
not effective, ZnO nanoparticles
the ZnO nanoparticles conjugated
conjugated withwith low
low
concentrations
concentrations of GA, relatively lower concentrations than those in other published reports [28–31],
of GA, relatively lower concentrations than those in other published reports [28–31],
had enhanced antibacterial
had enhanced antibacterial properties
properties compared
compared to the ZnO
to the ZnO nanoparticles,
nanoparticles, dramatically
dramatically reducing
reducing
the
the cell viability of
cell viability Gram-positive bacteria,
of Gram-positive bacteria, particularly
particularly MRSA.
MRSA. TheThe strong antibacterial activity
strong antibacterial activity and
and
selectivity may be attributed to the high affinity of GA for the bacterial cell membrane
selectivity may be attributed to the high affinity of GA for the bacterial cell membrane and the increased and the
increased lipophilicity
lipophilicity upon theofaddition
upon the addition GA [31].of GA [31].
Overall, the results in this study suggest that ZnO nanoparticles functionalized with GA have
antioxidant activity, as well as selective antibacterial activity, against MRSA. However, future studies
will be required to evaluate their efficiency and bio-safety in vitro and in vivo.
 Although GA treatment alone was not effective, the ZnO nanoparticles conjugated with low
concentrations of GA, relatively lower concentrations than those in other published reports [28–31],
had enhanced antibacterial properties compared to the ZnO nanoparticles, dramatically reducing the
cell viability of Gram-positive bacteria, particularly MRSA. The strong antibacterial activity and
selectivity
Nanomaterials may
2017, 7,be
365attributed to the high affinity of GA for the bacterial cell membrane and the
7 of 10
increased lipophilicity upon the addition of GA [31].

Figure 5. Qualitative assay of antibacterial activity using live and dead cell staining of Gram-negative
Figure 5. Qualitative assay of antibacterial activity using live and dead cell staining of Gram-negative
and Gram-positive bacteria. Fluorescent images show live cells stained by SYTO-9 (green) and dead
and Gram-positive bacteria. Fluorescent images show live cells stained by SYTO-9 (green) and dead
cells stained by PI (red) after 24 h of incubation. Scale bars represent 50 μm.
cells stained by PI (red) after 24 h of incubation. Scale bars represent 50 µm.

Overall, the results in this study suggest that ZnO nanoparticles functionalized with GA have
3. Materials and Methods
antioxidant activity, as well as selective antibacterial activity, against MRSA. However, future studies
will be requiredoftoZnO@GA
3.1. Preparation evaluate Nanoparticles
their efficiency and bio-safety in vitro and in vivo.

ZnO nanoparticles
3. Materials and Methods were prepared by a process similar to that in a previous report [23]. A stock
solution of Zn(CH3 COO)2 ·2H2 O (0.1 M) was dissolved in 50 mL of methanol under vigorous stirring.
ThenPreparation
3.1. 25 mL of NaOH (0.2 M)
of ZnO@GA in methanol was added to this mixture, and the pH was maintained at 8.
Nanoparticles
This mixture was transferred to a Teflon-lined sealed stainless steel autoclave and maintained at 80 ◦ C
ZnO nanoparticles were prepared by a process similar to that in a previous report [23]. A stock
for 10 h. The resultant white solid products were washed with methanol several times, filtered, and
solution of Zn(CH3COO)2·2H2O (0.1◦ M) was dissolved in 50 mL of methanol under vigorous stirring.
then dried in a vacuum oven at 60 C for 6 h.
Then 25 mL of NaOH (0.2 M) in methanol was added to this mixture, and the pH was maintained at
A wet chemical process with GA was used to provide ZnO nanoparticles with antioxidant
8. This mixture was transferred to a Teflon-lined sealed stainless steel autoclave and maintained at
functionality, as follows. First, 20 mg of ZnO nanoparticles was diffused in EtOH (1 mL). This solution
80 °C for 10 h. The resultant white solid products were washed with methanol several times, filtered,
was added to a 2.08 × 10−5 M solution of GA/EtOH, and vigorous stirring was applied for 24 h.
and then dried in a vacuum oven at 60 °C for 6 h.
The resulting product was washed several times in EtOH and dried at 60 ◦ C.
A wet chemical process with GA was used to provide ZnO nanoparticles with antioxidant
functionality, as follows. First,
3.2. Physical Characterization of 20 mg of ZnO
ZnO@GA nanoparticles was diffused in EtOH (1 mL). This solution
Nanoparticles
was added to a 2.08 × 10 M solution of GA/EtOH, and vigorous stirring was applied for
−5

24 h. FE-SEM was performed

The resulting on awashed
product was SU-70 Analytical UltraHighResolution
several times in EtOH and driedSEM
at 60(Hitachi,
°C. Tokyo, Japan).
TEM SAED and high-resolution (HR) TEM were performed with a JEM-3100F TEM (JEOL, Tokyo,
Japan) operating at 200 kV. TEM samples were prepared by placing a drop of sample suspension onto
a standard carbon-coated copper grid. This grid was dried before the micrographs were recorded.
The phase structures of the sample were identified by XRD using a X’Pert Pro MPD (PANalytical,
Almelo, The Netherlands) with CuKα radiation (wavelength of the radiation, k = 1.54 Å). IR spectra
were obtained using a FT-IR spectrometer (Perkin-Elmer PE 100, Waltham, MA, USA). For IR
measurements, the samples were crushed on mortar and then prepared as pressed wafers (1% sample
Nanomaterials 2017, 7, 365 8 of 10

in KBr). The PL and PLE of the samples were measured on a F-4500 spectrofluorimeter (Hitachi, Tokyo,
Japan), using a Xe arc lamp (150W, Abet Technologies, Milford, CT, USA) as the excitation source.

3.3. Evaluation of ZnO@GA Nanoparticle Antioxidant Activity

The radical cation decolorization assay determines the capacity of substances to scavenge
ABTS [31]. The ABTS radical cation (ABTS + ) was prepared by mixing 2.45 mM potassium persulfate
and 7 mM ABTS stock solution (1/1) and was placed in a dark place until the absorption peak was
stabilized. After 20 h, this solution was diluted with MeOH, and the absorbance was maintained
at 1 (λmax = 734 nm). For photo-detection, 0.9 mL of ABTS·+ solution was combined with 0.1 mL
of different concentrations of GA and ZnO@GA nanoparticles, and mixed for 45 s. Measurements
were taken at 734 nm after 15 min. The antioxidative activities of GA and ZnO@GA nanoparticles
were estimated by detecting the decrease in absorbance at different concentrations using the
following equation:
E = (Ac − As /Ac ) × 100 (1)

where As and Ac are the respective absorbance of the samples and ABTS·+ , expressed in µmol.

3.4. Evaluation of ZnO@GA Nanoparticle Antibacterial Activity

To evaluate the antibacterial effects of the ZnO@GA nanoparticles against a Gram-negative and
four Gram-positive bacterial strains, we used previously described methods and bacterial strains [27].
Briefly, five bacterial strains were used: a Gram-negative bacteria, E. coli (ATCC 11775), and four
Gram-positive bacterial strains, which were clinically isolated strains, namely, S. aureus (ATCC 14458),
MRSA-1 (KCCM 40510), and MRSA-2 and MRSA-3, [32,33].
For the quantitative antibacterial test, E. coli and S. aureus were suspended at 106 to 107 CFU/mL
in nutrient broth, and the MRSA strains were suspended in brain heart infusion (BHI) broth. After the
10-fold dilution of each bacterial suspension, the bacterial cells (approximately 105 to 106 CFU/mL)
were inoculated in 24-well plates and incubated with various concentrations (0, 25, 50, 100, and
200 µg/mL) of GA, ZnO, and ZnO@GA samples at 35 ◦ C. After 24 h of incubation, the bacterial
cells in 24 well plates were diluted by a serial 10-fold dilution method and inoculated onto a plate
count agar (PCA) plate for E. coli and S. aureus and a BHI agar plate for the MRSA strains. The PCA
and BHI plates were incubated for a further 24 h, and the CFUs in each plate were counted. For the
antibacterial activity of each sample, each bacterial cell viability was expressed as CFU/mL versus
the concentrations of each sample, and an inhibition of >3 log in the cell viability of each strain was
defined as a positive antibacterial effect.
For fluorescence microscopy to confirm the antibacterial effects of the samples, each bacterial cell
type at 106 to 107 CFU/mL was plated onto a cover glass coated with poly-L-lysine and incubated for
1 h. Next, the cover glass was washed with 0.9% saline solution three times to remove the detached
cells from the glass, before incubation with samples for 24 h. Finally, live and dead bacterial cells were
stained with a LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA)
according to the manufacturer’s instructions. Each bacterial cell was analyzed using a confocal
fluorescence microscope (FV-1200, Olympus, Tokyo, Japan) with a 20× objective lens, excitation filters
at 485 nm for both SYTO 9 and propidium iodide (PI), and emission filters at 530 and 630 nm for
SYTO 9 and PI, respectively. Each fluorescence image was analyzed using imaging software (Imaris,
Bitplane, Concord, MA, USA).

3.5. Statistical Analysis

The experiments were repeated three times (n = 6), and the quantitative data are shown as the
mean ± standard deviation (S.D.). The statistical significance (p < 0.05) was analyzed by Student’s t-test.
Nanomaterials 2017, 7, 365 9 of 10

4. Conclusions
In this study, we successfully fabricated multifunctional ZnO nanoparticles conjugated with
GA via a simple surface modification process. These multifunctional ZnO@GA nanoparticles show
high antioxidant and antibacterial activity, and the functionality and potentiality on antioxidant and
antibacterial activity suggest that they can be useful as a novel antibacterial agent for MRSA.

Acknowledgments: This study was supported by research funds provided by Chosun University in 2014.
Author Contributions: J.L., K.-H.C. and J.M. designed the study and wrote the manuscript. These authors
contributed equally to this work. H.-J.K., J.-P.J. and B.J.P. gave many suggestions during the project. All authors
reviewed the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

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