Journal of Colloid and Interface Science 559 (2020) 313–323

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Journal of Colloid and Interface Science

journal homepage: www.elsevier.com/locate/jcis

Bienzymatic synergism of vanadium oxide nanodots to efficiently

eradicate drug-resistant bacteria during wound healing in vivo
Weishuai Ma a, Tingting Zhang a, Ronggui Li a,⇑, Yusheng Niu b, Xuecheng Yang c, Jing Liu c, Yuanhong Xu a,c,⇑,
Chang Ming Li a,⇑
a
Institute of Advanced Cross-field Science, College of Life Science, Qingdao University, Qingdao 266071, China
b
School of Tourism and Geography Science, Qingdao University, Qingdao 266071, China
c
Department of Urology, Key Laboratory of Urinary System Diseases, the Affiliated Hospital of Qingdao University, Qingdao 266003, China

g r a p h i c a l a b s t r a c t
Vanadium oxide nanodots (VOxNDs) with bienzymatic synergism were constructed and applied to wound healing in vivo with their highly efficiently
antibacterial properties.

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

Article history: Antibiotic resistance is a common phenomenon observed during treatment with antibacterials. Use of
Received 15 August 2019 nanozymes, especially those with synergistic enzyme-like activities, as antibacterials could overcome
Revised 11 September 2019 this problem, but their synthesis is limited by their high cost and/or complex production process.
Accepted 12 September 2019
Herein, vanadium oxide nanodots (VOxNDs) were prepared via a one-step bottom-up ethanol-thermal
Available online 12 September 2019
method using vanadium trichloride as the precursor. VOxNDs alone possess bienzyme mimics of perox-
idase and oxidase. Accordingly, highly efficient antibacterials against drug-resistant bacteria can be
Keywords:
obtained through synergistic catalysis; the oxidase-like activity decomposes O2 to generate superoxide
Vanadium oxide nanodot
Enzyme mimic
anion radical (O

2 ) and hydroxyl radicals ( OH), and the intrinsic peroxidase-like activity can further

Bienzymatic induce the production of OH from external H2O2. Consequently, H2O2 concentration could decrease up
Antibacterial to four magnitude orders with VOxNDs to achieve an antibacterial efficacy similar to that of H2O2 alone.
Wound healing Wound healing in vivo further confirms the high antibacterial efficiency, good biocompatibility, and

⇑ Corresponding authors.
E-mail addresses: lrg@qdu.edu.cn (R. Li), yhxu@qdu.edu.cn (Y. Xu), ecmli@qdu.edu.cn (C.M. Li).

https://doi.org/10.1016/j.jcis.2019.09.040
0021-9797/Ó 2019 Published by Elsevier Inc.
314 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323
application potential of the synergistic antibacterial system due to the ‘‘nano” structure of VOxNDs. The
method of synthesis of nanodot antibacterials described in this paper is inexpensive, and the results of
this study reveal the multi-enzymatic synergism of nanozymes.
1. Introduction
Bacterial infections are a worldwide concern that claims mil-
lions of lives annually [1]. They have especially caused great panic
due to the development of antibiotic resistance [2]. To date, many
antibacterial materials such as metal ions [3], quaternary ammo-
nium compounds [4], biocides, carbon nanotubes [5], metal oxide
nanoparticles [6], and noble metal materials [7] have been devel-
oped to treat bacterial infections. However, the above-mentioned
antibacterial materials still have some limitations including
biotoxicity, high cost, complicated preparation processes, and/or
potential to cause environmental pollution [8]. H2O2 is also a
widely used antibacterial agent [9]; however, a high dose of
H2O2 is harmful to the human body and can delay wound healing
by hindering connective tissue functions. Therefore, there is a great
need to develop efficient low-toxic antibacterials in a simple and
cost-effective way.
Nanozymes, a class of nanomaterials such as metal oxide or
noble metal nanoparticles with intrinsic enzyme-like activities,
have been of great interest because of their remarkable advantages
including low cost and high stability. They are suggested to be
promising substitutes for the natural enzymes in various biological
and chemical applications [10]. Importantly, nanozymes such as
graphene quantum dots [11], vanadium pentoxide nanowires
[12], and magnetic iron oxide nanoparticles [13] show synergistic
antibacterial effects with H2O2 due to their intrinsic peroxidase-
mimic property, which can decompose H2O2 into hydroxyl radicals
(OH), thus showing significantly improved antibacterial effect of
H2O2 at a relatively lower concentration. Nevertheless, there is still
a large development gap in reducing the concentration of H2O2 in
antibacterial application. In addition, synergistic nanozymes,
which can synchronously actuate different catalytic reactions, pos-
sess multicatalytic functions and find applications in colorimetric
glucose sensing [14], tumour therapy [15], and intracellular antiox-
idant defence [16]. Further, Qu et al. confirmed the promising
antibacterial application potential of gold nanoparticles/silica
nanocomposite with both peroxidase and oxidase-mimic proper-
ties [17]. However, the application potential of synergistic nano-
zymes with antibacterial effects has been downplayed because
their synthesis is either costly or complex.
Among the nanomaterials explored, vanadium oxide (VOx) has
attracted special attention due to its potential nanozyme or
antibacterial activities [18]. Due to their different valency states
[18], many VOx-based nanomaterials such as nanorods [19], nano-
fibers [20], and nanowires [21] have been found to possess
peroxidase-like [22] or multiple-enzyme-mimetic properties [23].
When cut into smaller sizes (generally <10 nm), they show better
water dispersibility [24], more catalytically active sites [25,26],
and significantly lower toxicity than their bulk form [27]. Although
synthesis of the highly efficient enzyme-like VOx nanodots (VOx-
NDs) has been reported [23], their antibacterial potential has not
been reported.
We constructed a powerful bienzyme synergistic antibacterial
system in this study. VOxNDs were prepared using a one-step
ethanol-thermal strategy using vanadium (III) trichloride (VCl3)
as the precursor. The as-prepared VOxNDs exhibited bienzyme
mimic activities of peroxidase and oxidase. Systematic
experiments were conducted to study the possible enhanced
Ó 2019 Published by Elsevier Inc.
antibacterial activities based on bienzyme properties of VOxNDs
without and with H2O2. The possible mechanism was proposed
based on electron spin resonance (ESR) measurements, ultravio-
let–visible spectroscopy (UV–vis) spectra assays, fluorescence
method, and other assays. Assays to determine the antibacterial
efficacy on non-resistant and drug-resistant bacteria in vitro and
wound healing activity in vivo were conducted.
2. Experimental
2.1. Materials
VCl3 (97%) was purchased from Sinopharm Chemical Reagent
Co. Ltd., ChinaH2O2 solution (30% wt aqueous), glucose, 3- (4,5-d
imethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT),
dimethyl pyridine N-oxide (DMPO), and 5-tert-butoxycarbonyl-5-
methyl-1-pyrroline N-oxide (BMPO) were bought from Sigma-
Aldrich. Riboflavin (MW 376.36, 98%) was bought from Alfa Aesar
Co., Inc. 3,30 ,5,50 -Tetramethylbenzidine (TMB) and horseradish
peroxidase (HRP) were bought from the Shanghai Yuanye
Bio-Technology Co. Ltd. Luria-Bertani (LB) medium, 40 -6-diami
dino-2-phenylindole (DAPI), and propidium iodide (PI) were
bought from Thermo Fisher Scientific Inc. Non-resistant bacteria,
extended-spectrum b-lactamase (ESBL)-producing Escherichia
coli, methicillin-resistant Staphylococcus aureus (MRSA), and
kanamycin-resistant E. coli were obtained from the Experimental
Teaching Centre for Biology at Qingdao University. HeLa cells and
human umbilical vein endothelial cells (HUVECs) were obtained
from the Affiliated Hospital of Qingdao University. All the reagents
were of analytical grade and were used without further purifica-
tion. All the experiments involving animals were approved by the
Institutional Animal Care and Use Committee (IACUC) at Qingdao
University. Ultrapure water (18.2 MX; Millipore Co., USA) was
utilised in all experiments.
2.2. Apparatus
Transmission electron microscopy (TEM) and high-resolution
TEM (HRTEM) were carried out using a JEOL JEM-2100 microscope
at an acceleration voltage of 200 kV. Scanning electron microscopy
(SEM) image was obtained using an S-4800 scanning electron
microscope (Hitachi, Japan). X-ray diffraction (XRD) characterisa-
tions were performed using a Bruker D8 Advance diffractometer
with a copper Ka (k = 0.154056 nm) radiation source. X-ray photo-
electron spectroscopy (XPS) measurements were carried out using
a PHI5000 Versaprobe-II spectrometer with a monochromatic Al
Ka (1486.6 eV) source. The UV absorption spectra were obtained
with a Mapada UV-6300 spectrophotometer (Shanghai, China).
The photoluminescence (PL) spectra were obtained using an FS5
spectrophotometer (Edinburgh, UK). The fluorescence images were
obtained with a Leica TCS SP8 CARS fluorescence confocal
microscope.
2.3. Synthesis of VOxNDs
VOxNDs were prepared via a solvothermal method using VCl3
and ethanol as the vanadium precursor and the solvent, respec-
tively. In brief, 0.2 g vanadium trichloride was added into 20 mL
 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323
ethanol. The mixture was treated at 180 °C for 10 h. VOxNDs were
obtained by collecting the supernatant upon centrifugation at
12000 rpm for 10 min.
2.4. Preparation of VOxNDs for antibacterial and wound healing
experiments
Before the application experiment, evaporation was conducted
to remove the influence of the solvent, ethanol. In brief, an appro-
priate volume of as-obtained VOxNDs contained in a beaker was
placed in an electric thermostatic drying oven and heated at
50 °C until the ethanol was completely evaporated. Then distilled
water was added twice to reach VOxNDs aqueous suspension at
the initial volume.
2.5. Investigation of the peroxidase and oxidase mimetic activity of
VOxNDs
The peroxidase-mimic catalytic activity of VOxNDs was mea-
sured based on the oxidation of TMB with H2O2 [28]. However,
the oxidase-mimic behaviour of VOxNDs was measured based on
the oxidation of TMB with oxygen at different concentrations and
without H2O2 [29]. ESR was also carried out to confirm radical gen-
eration [15]. The reaction mechanism and kinetic study of the
peroxidase-like activity of VOxNDs were measured based on
Michaelis equation [28]. Details are indicated in Supporting Infor-
mation S1.1 and S1.2.
2.6. Bacterial culture and antibacterial tests
Single colonies of non-resistant and drug-resistant bacteria
were inoculated on solid LB medium were inoculated into 50 mL
of sterile liquid LB medium (containing bacto-tryptone [0.5 g],
bacto-yeast extract [0.25 g], and NaCl [0.5 g]). Then the suspen-
sions of both non-resistant and drug-resistant bacteria were cul-
tured at 37 °C overnight in a rotary shaker at 180 rev min
1.
Subsequently, the bacteria were diluted to 1  106 colony forming
units (CFU) mL
1 with sterile phosphate buffer saline (PBS). The as-
obtained bacterial solution (200 lL) was thoroughly mixed with
200 lg mL
1 VOxNDs and 50 lM H2O2 at 37 °C for 30 min. After
the treatment, the solution was cultured on a LB medium at
37 °C for 24 h, and the number of bacterial colonies was counted
using the CFU method. Control experiments were carried out in
parallel with H2O2 or VOxNDs alone using PBS as the blank control.
Characterisation of the bacteria by the fluorescence assay and char-
acterisation by SEM are shown in Supporting Information S1.3 and
S1.4, respectively.
2.7. Mouse wound model
Sprague-Dawley rats (6 weeks old; 200 g) were used to evaluate
the wound healing activity of H2O2 and VOxNDs. All the rats were
randomly distributed into four groups, and a wound (1 cm2) was
created on the dorsal portion. After that, the wound area was inoc-
ulated with 1  107 Staphylococcus aureus, which was then treated
with PBS, H2O2 (500 lM), VOxNDs (200 lg mL
1), or H2O2/VOxNDs
after 24 h. To harvest the wound tissue, the mice were sacrificed on
the sixth day of the experiment, and the skin, including the entire
wound with adjacent normal skin, was excised. The wound tissues
were then kept in PBS at 37 °C overnight, and the number of the
bacteria was assayed using the CFU method on the LB medium.
For histological examination, the wound tissues were fixed in
4% paraformaldehyde solution. Then, the tissue samples were
embedded in paraffin, sectioned into about 4-lm slices, and
stained with haematoxylin & eosin (H&E) [30].
315
2.8. Cytotoxicity and in vivo biocompatibility assay
The cytotoxicity of VOxNDs on HeLa cells and HUVECs was stud-
ied using the MTT assay [31]. Details are shown in Supporting
Information S1.5. To investigate the biosafety effect of VOxNDs
in vivo, VOxNDs at concentrations of 100 lg mL
1 and 200 lg mL
1
were injected intravenously into the rats. PBS was injected in con-
trol rats. After the six-day treatment, the main organs (spleen,
heart, lung, liver, and kidney) were collected for histological anal-
ysis. Blood samples from rats intravenously injected with
200 lg mL
1 VOxNDs were obtained after the sixth day of the
injection for physiological index examination [31].
3. Results and discussion
3.1. Characterisations of the as-prepared VOxNDs
As indicated, homogeneously distributed nanodots were
observed in the typical TEM images of the as-prepared super-
natant. This was indicative of the production of VOxNDs with good
uniformity (Fig. 1A). The mean lateral size VOxNDs was estimated
to be 3.36 ± 0.23 nm after analysing 100 particles in the TEM
images (Figure S1A). The corresponding HRTEM indicated the crys-
talline nature of VOxNDs (Fig. 1B). Two different lattice spaces of
0.228 nm and 0.233 nm were observed for VOxNDs (Figure S1B),
which was in accordance with the (1 1 3) diffraction planes of
V2O3 (JCPDS No. 34-0187) and (0 0 5) those of V2O5 (JCPDS No.
52-0794), respectively [32,33]. The AFM image and the corre-
sponding 3D form (Figure S2) with the corresponding height data
(Figure S1C) showed that the as-obtained VOxNDs were uniform
with a mean height of 3.16 ± 0.14 nm, indicating the few-layered
and near-spherical structure of as-prepared VOxNDs.
XRD analysis was carried out to confirm the crystalline struc-
ture of as-synthesised VOxNDs (Fig. 1D and S3). The main peaks
of 2h = 32.886° and 41.215° should correspond to (1 0 4) and
(1 1 3) planes of V2O3 (JCPDS No. 34–0187) [32]. The main peak
of 2h = 42.184° was ascribed to (0 0 5) planes of V2O5 (JCPDS No.
52-0794) [33]. These XRD results confirmed the crystalline nature
of VOxNDs, corroborating the results regarding the lattice analysis
obtained by HRTEM (Figure S1A). Furthermore, the intensity of
(1 1 3) reflection was broadened and decreased significantly
(Fig. 1D), also confirming the relatively low-crystalline structure
of these VOxNDs [34,35].
XPS analysis was further applied to confirm the elemental com-
ponents and the surface states of as-prepared VOxNDs. As indicated
in Fig. 1E, four major peaks at 285, 400, 517, and 532 eV were
observed, which resulted from the elements of C, N, V, and O,
respectively [36]. As shown in Fig. 1F, the XPS spectrum of V-2p
showed a small peak of V3+ (V1/22p at 515.9 eV) and a large peak of
V5+ (V3/2
2p at 516.9) [37,38]. These results further confirmed the
presence of V3+ and V5+ forms in the as-prepared VOxNDs. In addi-
tion, Fourier transform infrared (FTIR) spectrum for the as-
synthesised VOxNDs was used to analyse the surface functional
groups. The peaks in the regions of 3200–3600 cm
1, 2917–
3000 cm
1, and 518–930 cm
1 were typical vibrations of AOH
[39], ACAH [18], and VOx [40], respectively (Figure S4). The peak
at 1014 cm
1 was ascribed to the signals for V@O [41], and the
stretching frequency at 818 cm
1 and 530 cm
1 resulted from
VAOAV vibration in VOxNDs [42,43].
Due to the ‘‘nano” size of VOxNDs, novel optical properties
resulting from the edge and quantum effects were observed. Thus,
the optical properties of VOxNDs were investigated using UV–vis
absorbance and PL emission spectra. As can be seen from Fig. 1G,
VOxNDs showed maximum absorption at 310 nm. Meanwhile,
maximum excitation and emission wavelengths were found at
316 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323

Fig. 1. Characterisation of the as-prepared VOxNDs: (A) TEM and (B) HRTEM images of VOxNDs, (C) AFM image, (D) XRD and (E) the survey XPS spectra, (F) narrow-scan XPS
spectra of V-2p. (G) UV–vis absorption (Abs), optimal emission (EM), and excitation (EX) in PL spectra. (H) 3D fluorescence spectra at gradually incremental excitation
wavelengths ranging from 280 nm to 405 nm, and (I) CIE 1931 chromaticity chart at excitation of 355 nm.

355 nm and 430 nm, respectively [44,45]. We further collected the
typical PL emissions of VOxNDs at a variety of excitation wave-
lengths between 280 nm and 405 nm (Fig. 1H). The maximum PL
emission at 430 nm showed little change with the varied wave-
lengths. The excitation-independent PL behaviour was due to the
uniform particle distribution of the as-prepared VOxNDs. The
1931 CIE chromaticity diagram of VOxNDs was further obtained.
This showed the location at (0.18, 0.13) under excitation at
355 nm (Fig. 1I), further confirming the distinct blue emission of
VOxNDs. Moreover, the PL lifetime of VOxNDs was studied. As
shown in Figure S5, the fluorescence lifetime of VOxNDs was
4.11 ns. This value changed to 5.54 ns upon the addition of 1 mM
H2O2. The insignificant variation in the fluorescence time indicated
the tiny change in the microenvironment of VOxNDs upon H2O2
addition [46].
3.2. The bienzyme mimics behaviour of VOxNDs
We studied whether VOxNDs have enzyme-mimic properties
utilising the substrates for the assays of their natural enzymes
under the corresponding circumstances. To verify the oxidase-
like activities of VOxNDs, the catalysis of O2 was carried out using
TMB as the chromogenic substrate. As indicated in Fig. 2A, VOxNDs
alone changed the colour of TMB solution to light blue with max-
imum absorbance at 652 nm (curve in Fig. 2A and inset c in Fig-
ure S6A) in the absence of H2O2. In addition, the absorbance at
652 nm for oxidised TMB increased significantly with an increase
in oxygen concentration (Fig. 2B). The results indicated that
VOxNDs possessed intrinsic oxidase-like activity using oxygen as
the substrate, which might lead to the generation of O

2 or OH.
Next, fluorescence experiments were carried out to explore the
possible reactive intermediate in the VOxNDs-based oxidation
reaction. Firstly, hydroethidine was used as the highly selective flu-
orescent probe to confirm the generation of superoxide anion rad-
ical (O

2 ). As shown, a significant increase in fluorescence intensity
was observed at 610 nm upon the addition of VOxNDs, indicating
that VOxNDs have the capability to generate O
2 (Figure S7A)
[47]. Moreover, the generated O
2 was further confirmed by ESR
using BMPO as the spin trap. As indicated in Fig. 2C, four obvious
typical peaks with a strength ratio of 1:1:1:1 were observed for
BMPO in the presence of VOxNDs compared to the control group
of BMPO alone. This indicates that a large number of O
2 was gen-
erated by decomposing oxygen in the presence of VOxNDs [48]. The
generation of OH could be further confirmed by terephthalic acid
(TA) based fluorescence experiments since OH can be captured
by non-fluorescent TA to generate 2-hydroxy terephthalic acid
(TAOH) showing distinct fluorescence at 435 nm [31]. As shown
(Figure S7B), fluorescence enhancement was observed upon the
addition of VOxNDs, indicating the production of OH in VOxNDs
alone [31]. These results were consistent with the ESR measure-
ment results using DMPO as the spin trap (Fig. 2D), which showed
four characteristic peaks with a strength ratio of 1:2:2:1.
 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323 317

Fig. 2. Bienzyme-like activity of VOxNDs: (A) the study of POD-like activity: UV–vis spectra of different systems. The inset shows the corresponding photographs of (a)
VOxNDs + H2O2; (b) TMB + H2O2; (c) VOxNDs + TMB; and (d) VOxNDs + TMB + H2O2. (B) The study of OXD-like activity: the effect of O2 concentration on TMB oxidation by
VOxNDs. Generation of (C) O

2 as well as (D) OH during POD-like catalysis. (E) UV–Vis absorption spectrum of the VOxNDs-TMB-H2O2 system with different concentrations of
H2O2. (F) Fluorescence spectra of PBS solution including TA alone; VOxNDs; TA and H2O2; VOxNDs and H2O2; TA and VOxNDs; TA, VOxNDs, and H2O2 after 12 h reaction. The
concentrations of TA, H2O2, and VOxNDs were 0.5 mM, 1 mM, and 200 lg/mL, respectively. kex: 315 nm, kem: 435 nm. (G) Histograms of DFL intensity showed the catalytic
effect of VOxNDs. The optimisation of (H) pH and (I) temperature for the peroxidase-like activity of VOxNDs and HRP. The error bars represent the standard deviation for three
measurements (n = 3).

Accordingly, the formed O


2 and OH resulted from the oxidation of
TMB within the oxidase-like VOxNDs alone. Furthermore, the
oxidase-like behaviours of VOxNDs were evaluated under different
pH and temperature conditions. As shown in Figure S8, VOxNDs
exhibited excellent enzymatic behaviour over a broad range of
pH and temperature conditions, showing good stability of the
oxidase-like catalytic ability of VOxNDs under various reaction
conditions.
Additionally, to verify the peroxidase-like activity of VOxNDs,
the catalysis of H2O2 was studied using 3,30 ,5,50 -tetramethylbenzi
dine (TMB) as the substrate. As indicated in Figure 2A, H2O2 in
the presence of either VOxNDs (curve a) or TMB (curve b) alone
did not produce obvious colour change or show UV–vis absorbance.
However, H2O2 with both TMB and VOxNDs (VOxNDs + TMB
+ H2O2) resulted in dark blue colour as well as strong absorbance
at 652 nm, which was much darker than that of TMB in VOxNDs
alone. This phenomenon demonstrated that VOxNDs could acceler-
ate the oxidation of TMB in the presence of H2O2. Furthermore, the
absorbance at 652 nm increased with increment in H2O2 concentra-
tion (Fig. 2E). The results proved that VOxNDs possessed
peroxidase-like activity, which should generally produce OH dur-
ing the decomposition of H2O2. Based on fluorescence measure-
ment, more significant fluorescence enhancement was observed
for TAin the presence of VOxNDs and H2O2 than other control
groups, confirming the large production of OH in the
corresponding system (Fig. 2F). Meanwhile, ESR characterisations
using DMPO as a spin trap were also performed to verify the forma-
tion of OH. Compared to the control group of pure H2O2, the four
characteristic peaks for OH can be obviously observed for H2O2 in
the presence of VOxNDs (Fig. 2D). Meanwhile, the intensity of OH
signal for VOxNDs with H2O2 measured by both the TA-based fluo-
rescence and ESR was much stronger than that without H2O2, indi-
cating the presence of both OXD- and POD- like enzyme properties
for VOxNDs. Accordingly, the strong oxidising OH could result in
the oxidation of TMB with much darker blue colour and absorption
peak at 652 nm in the presence of H2O2 (Fig. 2A).
In addition, we optimised the reaction conditions of peroxidase-
like VOxNDs and we compared them with that of natural HRP. It
was observed that the optimal pH and temperature of VOxNDs
were 3.5 and 40 °C, respectively, which were similar to those of
natural HRP (pH 4.0 and optimised temperature 37 °C). However,
as shown in Fig. 2H and I, VOxNDs showed more tolerance to harsh
reaction conditions.
3.3. Kinetic studies of the enzyme-mimic activities of the as-prepared
VOxNDs
To study the catalytic mechanism of the peroxidase-like activi-
ties of VOxNDs, the steady-state kinetic factors of the reaction were
studied under optimal pH (3.5) and temperature (40 °C). The initial
318 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323

velocity was estimated by the slopes of the linear portion of the

absorbance–time curve for TMB-derived oxidation products [49].
The Michaelis–Menten curves were calculated with H2O2 and
TMB as the substrates, whereby one substrate had a fixed concen-
tration and the other had varying concentrations. As indicated in
Figure S9A and 9B, the initial reaction rates showed obvious depen-
dence on the respective substrate concentration. In addition, the
Lineweaver–Burk plot was used to study the enzymatic activity
of VOxNDs. As can be seen in Figure S9C and 9D, the reciprocal of
the initial rate showed an obvious linear relationship against the
reciprocal of the substrate concentration. The catalytic parameters
(Km and Vmax) were calculated based on the double reciprocal of
the Michaelis–Menten equation. The comparison between the
parameters of VOxNDs and those of other peroxidase mimics are
presented in Table S1 [28,50–53]. As shown, the apparent Km val-
ues of VOxNDs with H2O2 substrate were significantly lower than
those of other nanozymes including TiO2@CeOx(1.39 mM) [50],
VO2 (B) (1.69 mM) [51], and GO-COOH (3.99 mM) [53]. In addition,
the Km value obtained using H2O2, as the substrate for VOxNDs was
0.077 mM, which was 48 times lower than that of HRP. The lower
Km value indicated that VOxNDs had a strong affinity to H2O2 [28]
compared to HRP and most of the other metal-based nanozymes
[50–52]. In addition, the Vmax value of VOxNDs using TMB as a sub-
strate was 26 times larger than that of HRP. This suggested that
VOxNDs possessed stronger peroxidase-like activity towards the
catalytic oxidation of TMB than HRP [53], which resulted from
the more active catalytic sites and larger specific surface area of
the nanodots nanostructures of VOxNDs [54].
Further study on the enzymatic mechanism of VOxNDs was con-
ducted by measuring Lineweaver–Burk curves by varying the three
concentrations of H2O2 (0.04 mM, 0.08 mM, and 0.12 mM) with
fixed TMB concentrations (0.08, 0.2 and 0.6 mM) or vice versa. As
shown, lines parallel to each other were obtained (Figure S9E
and 9F). This phenomenon was indicative of the ping-pong mech-
anism for the enzymatic activities of VOxNDs, which was similar to
that of natural HRP. In other words, VOxNDs first reacted with one
of the substrates (H2O2) catalytically and liberated the first product
before reacting with the other substrate (TMB). The first product
was OH radical, which was produced by the decomposition of
H2O2 during the catalytic reaction of VOxNDs [28,53].
We also evaluated the steady-state kinetic factors for the
oxidase-like catalytic reaction of TMB. Figure S10 shows the
Michaelis–Menten curves of VOxNDs, which were plotted by vary-
ing TMB concentrations. The as-prepared VOxNDs exhibited lower
Km values of 0.3022 mM compared to other oxidase mimics [55,56]
such as CeO2 nanoceria (Table S2) [55], indicating that maximal
activity of VOxNDs could be achieved at a lower TMB concentra-
tion. The above results proved that the as-prepared VOxNDs had
excellent oxidase-like activity.
3.4. Antibacterial activity and wound healing applications of VOxNDs
H2O2 is popularly utilised in treating bacterial infections due to
its strong oxidising properties [9]. However, compared to H2O2, the

OH or O

2 was much stronger oxidative and thus showed much
higher antibacterial capacity. As characterised, the as-prepared
VOxNDs in our study possessed two enzyme mimics including
peroxidase- and oxidase-activities, which could generate OH and
O
2 both by itself and with H2O2. As illustrated in Fig. 3A, synergis-
tic enzymatic actions between the bienzymes of VOxNDs occurred
as follows: In the absence of H2O2, because of their oxidase-like
activity, VOxNDs transformed O2 to produce O

2 and OH, which
was toxic to bacteria [17]. When H2O2 was externally added to
the above reaction system, with the intrinsic peroxidase-like activ-
ity of VOxNDs, it caused more OH to be produced, which was more
toxic to bacteria [57]. Accordingly, the synergistic catalytic
Fig. 3. (A) The antibacterial mechanism of bienzymatic synergism of as-prepared
VOxNDs. (B) Representative SEM images of E. coli and S. aureus cells after different
treatments. Cultured bacterial cells were treated with (a) PBS as control, (b) H2O2
(50 lM), (c) VOxNDs (200 lg mL
1), or (d) H2O2/VOxNDs.
activities of VOxNDs nanozyme are expected to boost the antibac-
terial efficiency of H2O2. Accordingly, the effective concentration of
H2O2 can be significantly reduced in antibacterial application.
Accordingly, the antibacterial behaviours of as-prepared VOx-
NDs were studied in the absence and presence of H2O2. S. aureus
and E. coli were selected as the representatives of gram-positive
and gram-negative bacteria, respectively. Based on the CFU
method (Figure S11A-11E), VOxNDs (200 lg/mL) or H2O2 (50 lM)
alone exhibited good antibacterial activities compared to the con-
trol groups of PBS. The antibacterial activity of VOxNDs resulted
from their multi-enzyme activities that could generate high oxidis-
ing OH and O
2 . Furthermore, the growth of both bacterial strains
was almost completely depressed by the synergistic antibacterial
system of VOxNDs and H2O2. Thus, in the presence of VOxNDs,
H2O2 could suppress the viabilities of both E. coli and S. aureus
much more efficiently. Figure S11E showed the CFU amount for
bacteria on LB agar plate. Compared to control groups, bacterial
colonies were hardly observed in the groups treated with the
H2O2/VOxNDs synergetic system. We further studied the dose-
dependent antibacterial properties of H2O2 in the absence and
presence of VOxNDs. As shown in Figure S11C and 11D, with the
assistance of VOxNDs, the concentration of H2O2 decreased by
about 2–4 orders of magnitude to achieve similar antibacterial
effects of H2O2 alone [17].
SEM was applied to characterise the morphology variation of
E. coli and S. aureus before and after treatment with VOxNDs and/
or H2O2 (Fig. 3B). As shown, the untreated E. coli and S. aureus cells
were rods and spheres, respectively. In addition, they both have
intact and smooth cell walls. When treated with H2O2 or VOxNDs
alone, the surface of the bacteria became slightly rough and wrin-
kled. Notably, in the presence of both VOxNDs and H2O2, the bacte-
ria cells became very rough and seriously damaged. These results
were consistent with that of the CFU method, which suggest that
H2O2 combined with VOxNDs have the highest antibacterial effi-
ciency against both E. coli and S. aureus.
To evaluate further the boosted antibacterial activities of H2O2
and VOxNDs, the DAPI and PI fluorescence-based live/dead assays
were carried out using E. coli as an example. In the control groups,
 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323 319

Fig. 4. Typical fluorescence images of live and dead E. coli cells after various treatments. (A) bacteria alone; (B) bacteria co-incubated with H2O2; (C) bacteria co-incubated
with VOxNDs; and (D) bacteria co-incubated with H2O2 and VOxNDs. Bacterial quasi-nuclear staining with DAPI exhibited blue fluorescence, while dead bacteria staining with
PI exhibited red fluorescence. Bar scale: 20 mm.

most bacteria were alive, as revealed by the little red fluorescence
(Fig. 4). In VOxNDs- or H2O2-treated groups, little more red fluores-
cent cells were observed. However, for bacteria treated with both
H2O2 and VOxNDs, a large amount of red fluorescence signal was
observed, indicating maximum bacterial cell death by the H2O2/
VOxNDs system. All the results were in accordance with those
obtained from CFU assays, further confirming the enhanced
antibacterial activity of H2O2 in the presence of VOxNDs.
To investigate the antibacterial activity of VOxNDs on drug-
resistant bacteria, ESBL-producing E. coli, MRSA, and kanamycin-
resistant E. coli were selected as representative bacteria, which
exhibit high-level tolerance against penicillin, kanamycin, and
methicillin, thus leading to serious chronic and recurrent infections
[2]. As shown in Fig. 5A-B, only a small number of drug-resistant
bacteria were inhibited in the presence of H2O2 or VOxNDs alone,
while almost all of the drug-resistant bacteria were suppressed
by the H2O2/VOxNDs system. The results indicated that the as-
prepared VOxNDs exhibited excellent antibacterial abilities in the
presence of a low concentration of H2O2 (50 lM) not only for
non-resistant bacteria but also for various drug-resistant bacteria.
In addition, the comparison of H2O2 concentration required for
VOxNDs and other antibacterial materials [2,11,13,17,58–65] are
listed in Table S3, demonstrating that the as-prepared VOxNDs
require a lower concentration of H2O2 to reach the comparative
antibacterial effects.
3.5. Wound infection treated by VOxNDs and/or H2O2
To assess further the feasibility of antibacterial efficacy of
H2O2/VOxNDs in wound healing, Sprague-Dawley male rats with
a wound on their dorsal portion were used for in vivo tests. S. aur-
eus cells of 107 CFU/mL were injected on the wound to induce
bacterial infection to the rats. Since high concentrations of
H2O2, usually 0.1 to 1.0 M, will be detrimental to normal tissue
and can lead to delayed wound healing, low concentration of
H2O2 was used for the treatment of the wound infection. Herein,
320 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323

Fig. 5. Survival rates of (A) kanamycin-resistant E. coli, (B) ESBL-producing E. coli, and (C) MRSA after treated with VOxNDs (200 lg/mL) and/or H2O2 (50 lM) as determined
by CFU method. (D) Photographs of kanamycin-resistant E. coli, ESBL-producing E. coli, and MRSA colonies after various treatments. Cultured bacterial cells were treated with
(a) PBS as control, (b) H2O2 (50 lM), (c) VOxNDs (200 lg/mL), and d) H2O2/VOxNDs. The error bars represent the standard deviation for three measurements (n = 3). Asterisks
indicate statistically significant differences (**p < 0.01).

500 lM H2O2, which was much lower than the usual concentra-
tion was tested for the synergistic antibacterial treatment with
VOxNDs. Fig. 6A shows the four groups of rats treated with PBS,
H2O2, VOxNDs, and H2O2/VOxNDs. Compared to othones, rats trea-
ted with H2O2/VOxNDs showed the fastest wound healing, which
was almost completely healed after the six-day therapy. Quanti-
tative analysis of the wound area was carried out on different
days (2, 4, and 6) using the size of the fresh wound on the first
day as control (100%). The results demonstrated that the H2O2/
VOxNDs group showed the greatest wound healing capacity
among all the tested groups (Fig. 6B). To investigate further the
antimicrobial efficacy of the different tested groups, S. aureus cells
were collected from the wound tissue of each group and the
abundance of S. aureus was measured by the CFU method. As
shown in Fig. 6C, much fewer bacterial colonies were observed
in H2O2/VOxNDs group, while no significant differences were
observed in the other three tested groups. This result suggested
that enhanced wound healing was proportionally related to the
antibacterial activity of the H2O2/VOxNDs system. Furthermore,
the wound healing process was also investigated via H&E staining
strategy. In Fig. 6D, it is shown that the skin sections treated with
H2O2/VOxNDs exhibited intact epidermis, while other control
groups possessed fragmentary epidermal layers at different levels
[55]. Meanwhile, the blood vessels and the hair follicles under the
wound tissue also recovered rapidly in the H2O2/VOxNDs group
compared to that in the other three groups.
3.6. In vitro and in vivo cytotoxicity study of VOxNDs
The above-mentioned results confirmed the practical applica-
bility of the as-prepared VOxNDs/H2O2 system in in vivo wound
healing. Therefore, it is necessary to study the biosafety of VOxNDs.
Thus, we first examined the effects of VOxNDs on the viability of
HUVECs and HeLa cells. Figure S12 shows the cell viabilities
depending on different concentrations of VOxNDs ranging from 0
to 200 lg mL
1 after 4 h and 24 h treatment. The results indicated
that even at VOxNDs concentration of up to 200 lg mL
1, the via-
bilities of HeLa cells and HUVECs remained 80% after 24 h treat-
ment, indicating that VOxNDs possessed good biocompatibility.
To investigate further whether VOxNDs causes toxic side effects
in vivo, VOxNDs were injected intravenously into healthy rats. As
shown in Fig. 7A, compared to the untreated mice, there were no
significant differences in the blood physiological index of
VOxNDs-treated mice. The major organs, including the liver, heart,
lung, spleen, and kidney were sliced and stained with H&E. No
obvious histological abnormalities or adverse effects were
observed for all the organs (Fig. 7B), and their histology was similar
to that of untreated rats, suggesting that VOxNDs have good biosaf-
ety in vivo. The results demonstrated that VOxNDs possessed low
cytotoxicity. This is consistent with the fact that although VOx is
suggested to cytotoxic, converting it into the ‘‘nano” size could sig-
nificantly reduce the cytotoxicity [27]. Thus, VOxNDs of only
3.36 ± 0.23 nm in lateral size and 3.16 ± 0.3 nm in thickness have
 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323 321

Fig. 6. Healing effect on the wound infected with S. aureus. The wounds were treated with (1) PBS, (2) H2O2 (500 lM), (3) VOxNDs (200 lg/mL), and (4) H2O2/VOxNDs. (A)
Photographs of wounds on the dorsal portion of the rats after different treatments (scale bar = 0.5 cm). (B) The size of the wound area of rats at different times. (C) The number
of bacteria obtained from wound tissues after different treatments. (D) Histological data of corresponding skin wounds after different treatments. Error bars represent the
standard deviation (n = 3). Asterisks indicated statistically significant differences (*p < 0.05, **p < 0.01).

Fig. 7. (A) Blood physiological index including alkaline phosphatase (ALP), alanine transaminase (ALT), albumin (ALB), globulin (GLOB), aspartate transaminase (AST), total
protein (TP), urea nitrogen (UREA) and the ratio of albumin and globulin (A/G) of rats treated with VOxNDs or the PBS control. Error bars represented the standard deviation
(n = 3). (B) Histological data of the major organs of rats treated with different concentration of VOxNDs or the PBS control.
322 W. Ma et al. / Journal of Colloid and Interface Science 559 (2020) 313–323
relatively low cytotoxicity as well as good biocompatibility. There-
fore, VOxNDs have excellent antibacterial activity and can promote
wound healing in the presence of H2O2 without causing any dam-
age to the main organs.
4. Conclusion
In this study, VOxNDs, synthesised by adopting a facile ethanol-
thermal strategy using VCl3 powder as the precursor, exhibited
bienzyme mimetic behaviours of peroxidase and oxidase. Based
on the bienzymatic synergism of VOxNDs, an efficient enzymatic
antibacterial system was constructed by utilising only 50 lM
H2O2, which was much lower than most previous enzyme
mimics-based antibacterial systems. As evidenced by TEM and
AFM, the average lateral size and thickness of VOxNDs were found
to be 3.36 ± 0.23 nm and 3.16 ± 0.14 nm, respectively. This antibac-
terial system exhibited striking antibacterial activities against not
only non-resistant bacteria (gram-positive S. aureus and gram-
negative E. coli) but also drug-resistant bacteria (ESBL-producing
E. coli, MRSA, and kanamycin-resistant E. coli). Moreover, this
antibacterial system showed great potential in accelerating wound
healing in vivo with low concentrations of H2O2. Due to their
‘‘nano” size, the as-prepared VOxNDs have satisfying biosafety as
confirmed via MTT assays, physiological index examination, and
H&E staining. The findings can provide new ideas for synthesising
multi-enzyme synergistic antibacterials and promote the develop-
ment of nanodot-based nanozymes for wound healing and treating
other diseases.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgment
This work was supported by the National Natural Science Foun-
dation of China (Nos. 21874079 and 21575071), Natural Science
Foundation for Outstanding Young Scientists of Shandong Province
(ZR2018JL011), Key R&D Project of Shandong Province
(GG201809230180), Qingdao Science & Technology Planning Pro-
ject (17-6-3-15-gx), and Science & Technology Fund Planning Pro-
ject of Shandong Colleges and Universities (J16LA13 & J18KA112).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jcis.2019.09.040.
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