Biomaterials 162 (2018) 109e122

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Bio-inspired redox-cycling antimicrobial film for sustained generation

of reactive oxygen species
Huan Liu a, Xue Qu a, *, Eunkyoung Kim b, Miao Lei a, Kai Dai a, Xiaoli Tan d, Miao Xu e,
Jinyang Li b, Yangping Liu d, Xiaowen Shi c, Peng Li e, Gregory F. Payne b,
Changsheng Liu a, **
a
Key Laboratory for Ultrafine Materials of Ministry of Education, The State Key Laboratory of Bioreactor Engineering, East China University of Science and
Technology, Shanghai, 200237, China
b
Institute for Biosystems and Biotechnology Research and Fischell, Department of Bioengineering, 5115 Plant Sciences Building, College Park, MD, 20742,
USA
c
Department of Environmental Science, College of Resource and Environmental Science, Wuhan University, Wuhan, 430072, China
d
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University,
Tianjin, 300070, China
e
Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced
Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing, 210009, China

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

Article history: Open wounds and burns are prone to infection and there remains considerable interest in developing
Received 24 June 2017 safe and effective mechanisms to confer antimicrobial activities to wound dressings. We report a bio-
Received in revised form mimetic wound dressing for the in situ and sustained generation of reactive oxygen species (ROS).
20 December 2017
Specifically, we fabricate a catechol-modified chitosan film that mimics features of the melanin capsule
Accepted 31 December 2017
generated during an insect immune response to infection. We use an electrochemical reverse engi-
Available online 2 January 2018
neering approach to demonstrate that this catechol-chitosan film possesses redox-activities and can be
repeatedly oxidized and reduced. In vitro tests demonstrate that this film catalyzes the transfer of
Keywords:
Antimicrobial
electrons from physiological reductant ascorbate to O2 for sustained ROS generation, and confers
Bio-inspiration ascorbate-dependent antimicrobial activities. In vivo antimicrobial experiment with a rat subcutaneous
Catechol model indicates the catechol-chitosan film at reduced state inhibits the bacterial growth and alleviates
Chitosan the infection of the incisions. Open wound healing tests with a mouse model indicate that the catechol-
Reactive oxygen species chitosan film suppresses the bacterial population at the wound site, induces less inflammation and
Redox-activity promotes wound healing. We envision this biomimetic approach for the sustained, localized and in situ
generation of ROS could provide new opportunities for wound management by protecting against
pathogen infection and potentially even enlisting ROS-mediated wound healing mechanisms.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction recent approach is to create dressings that mimic the antimicrobial

Open wounds and burns are vulnerable to infection and there is
considerable interest in developing protective antimicrobial
dressings [1e4]. One conventional approach is to create dressings
that slowly release antimicrobial agents (e.g., silver or antibiotics)
with safety concerns such as inducing antibiotic resistance and also
depletion of active agent from such passive dressings [5e7]. A more
* Corresponding author.
** Corresponding author.
E-mail addresses: quxue@ecust.edu.cn (X. Qu), liucs@ecust.edu.cn (C. Liu).
defense mechanisms of the host immune system (e.g., dressings
containing antimicrobial peptides) [8e10]. One common immune
response to pathogen attack is the oxidative burst in which NADPH
oxidase enzymes catalyze the transfer of electrons from NADPH to
O2 to generate reactive oxygen species (ROS) [11]. While the active
in situ generation of ROS is an attractive antimicrobial mechanism
[12,13], it is difficult to incorporate labile biological components
(enzymes and cells) into engineered materials to recapitulate this
oxidative burst pathway. Here, we investigated an alternative bio-
inspired pathway for the sustained non-enzymatic generation of
ROS.

https://doi.org/10.1016/j.biomaterials.2017.12.027
0142-9612/© 2018 Elsevier Ltd. All rights reserved.
110 H. Liu et al. / Biomaterials 162 (2018) 109e122

Our approach is inspired by an incompletely understood innate
immune response in insects: the localized synthesis of a melanin
coating to encapsulate invading pathogens [14,15]. Melanin is
generated by the phenoloxidase-mediated conversion of tyrosine
[15] into reactive quinones that undergo subsequent non-
enzymatic reactions to generate the macromolecular melanin
capsule [16]. Traditionally, it is believed that this immune response
confers antimicrobial activity through (i) the active generation of
reactive species (e.g., quinones) during melanin synthesis [17], and
(ii) the passive barrier properties of the capsule that restricts
pathogen mobility, growth and reproduction [18]. Twenty years ago
it was suggested that the melanin capsule might be redox-active
and may offer an additional active antimicrobial mechanism by
catalyzing the transfer of electrons from endogenous extracellular
reducing equivalents (e.g., from ascorbate) to O2 for the sustained
generation of ROS [19]. This proposed mechanism is illustrated in
Scheme 1A but it has been difficult to test in vivo. Interestingly,
more recent clinical research suggests an analogous mechanism
may explain the pro-oxidant activities of human pheomelanin: this
melanin appears to accept electrons from biological reducing
equivalents (e.g., glutathione) [20] and/or transfer electrons to O2 to
generate ROS [21,22]. In addition, recent reports emphasize that
melanin's functions are context dependent making it even more
challenging to understand it's in vivo activities [23].
Here, we investigated a catechol-chitosan (Cat-Chit) film that
mimics the structure (catechol group) and redox activities of mel-
anins [24,25] and show this film possesses reversible redox-
activities capable of catalyzing the repeated transfer of electrons
from ascorbate to O2 for the sustained generation of ROS (H2O2) as
illustrated in Scheme 1A. Scheme 1B shows this film is prepared in
two steps. First, a film of the pH-responsive self-assembling ami-
nopolysaccharide chitosan (Chit) is formed by electrodeposition
through a cathodic neutralization mechanism [26]. Chitosan is
chosen as the matrix material because its hemostatic [4,27] and
antimicrobial properties [28,29] are assets for wound management,
and because its physicochemical properties are convenient for film
fabrication [30] and catechol modification [31e33]. Second, the
electrodeposited chitosan film is modified by the oxidative grafting
of catechol moieties. Catechols (including those derived from
tyrosine) can be readily oxidized into reactive o-quinones that can
undergo uncatalyzed conjugation reactions [34]. In particular,
catechol can be oxidized spontaneously (at high pH as in the case of
polydopamine) [35e38], enzymatically (by phenoloxidases) [39],
anodically (by electrochemistry) [40] and chemically (by periodate
as illustrated in Scheme 1B) [32,41]. Quinone conjugation reactions
can be quite complex and remain poorly understood but likely

Scheme 1. (A) Left: innate immune response in insects: melanin capsule offers antimicrobial activity by catalyzing the transfer of electrons from endogenous reductants to O2 for
ROS generation; Right: a mimetic film that possesses reversible redox-activities serves as a catalyst accepting electrons from ascorbate to O2 for sustained ROS generation. (B)
Schematic illustration of the fabrication of Cat-Chit film by coupling chitosan electro-deposition with subsequent oxidative grafting of catechol.
 H. Liu et al. / Biomaterials 162 (2018) 109e122
include the Schiff base and Michael-type adducts shown in Scheme
1B [33,42e46].
The aims of this study were to explore the capabilities of this
redox-active biomimetic film to generate ROS, confer antimicrobial
activities and promote wound healing. We envision that such films
could be incorporated into dressings for advanced wound
management.
2. Materials and methods
2.1. Materials
The following materials were purchased from Sigma-Aldrich:
chitosan from crab shells (85% deacetylation and 200 kDa), cate-
chol (reagent grade
99%), ascorbate (reagent grade: 99%), catalase
(2 k-5k units/mg protein), 5,5-Dimethyl-1-pyrroline N-oxide
(DMPO), Hexaammineruthenium (III) chloride (Ru(NH3)6Cl3) (re-
agent grade: 98%), ferrocene dimethanol (reagent grade
98%), 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
(MTT). Hydrogen Peroxide assay kit was purchased from AAT Bio-
quest®. LIVE/DEAD BacLight Bacterial Viability Kit was purchased
from Invitrogen. Hydrochloric acid, sodium hydrate, glutaralde-
hyde, Ethylenediaminetetraacetic acid (EDTA), dimethyl sulphoxide
(DMSO) were purchased from Shanghai Lingfeng. Sodium iodate
was purchased from Beijing J&K. All cell-culture related reagents
were purchased from Gibco (Grand Island, NY). The three-electrode
system (PARSTAT2273) that was used for electrodeposition
employed gold-coated silicon wafer as the working electrode, Pt
wire as the counter electrode and Ag/AgCl as the reference
electrode.
2.2. Fabrication of the catechol-modified chitosan film
2.2.1. Electrodeposition of chitosan film
The chitosan film is fabricated using electrodeposition method.
Briefly, the chitosan solution was prepared by dissolving chitosan in
1% hydrochloric acid with overnight stirring. The resultant solution
was diluted to 10 mg/mL by adding deionized water, and the pH
was adjusted to 5e6. Electrodeposition of chitosan was performed
by immersing the gold-coated electrode (working electrode) into
the chitosan solution described above that was supplemented with
20 mM H2O2 (H2O2 is used during chitosan electrodeposition to
allow a pH gradient to be generated at lower reducing voltages). For
chitosan's cathodic electrodeposition a reducing voltage was
applied to achieve a constant current density (16 A/m2) for 5 min.
The resultant chitosan-coated electrode was rinsed extensively
with water and phosphate buffer (PB, pH 7.0). After exposure to
NaOH solution (100 mM) for 30 min, the free-standing chitosan
film was peeled off from the electrode.
2.2.2. Catechol grafting
The chitosan film was immersed into a catechol/NaIO3-con-
taining solution (catechol conc. 20 mM, NaIO3 conc. 10 mM) over-
night with gentle shaking for oxidative grafting of catechols. The
obtained film (catechol-chitosan, denoted as Cat-Chit) was
collected and rinsed with water extensively.
2.3. Characterization of catechol-modified chitosan film
SEM analysis was performed on S-4800 (Hitachi) scanning
electron microscope. Chemical analysis of films was performed on
an Attenuated total reflectance Fourier-transform infrared system
(ATR-FTIR, Nicolet 5700), UV/vis spectrometer (SpectraMax M2,
Molecular Probes) and X-ray photoelectron spectroscopy (XPS,
ESCALAB 250Xi).
111
2.4. Electrochemical reverse engineering
To confirm the reversible redox-activity of Cat-Chit film, chito-
san film was first deposited on a Pt electrode (1.5  1.5 cm2), and
then the chitosan-coated electrode was immersed into a catechol/
NaIO3-containing solution as described above. After fabrication, the
film-coated electrodes were rinsed with water and then tested by
obtaining cyclic voltammograms (CVs) in the presence of soluble
mediators (0.1 M PB, pH 7.0). Scan rate 5 mV/s.
2.5. ROS generation testing
2.5.1. Transfer of electrons from ascorbate to Cat-Chit film
Cat-Chit film acquires electrons from reducing equivalents and
converts to its reduced state. As discussed, the reduced Cat-Chit
film was obtained by incubation in ascorbate solution for 15 min
followed by extensive rinsing in water. FolineCiocalteu colori-
metric assay was used to check that ascorbate was completely
removed from the films during the rinsing step [47]. 300 mM
ascorbate was typically used for in vitro studies. This reduced Cat-
Chit film is designated Cat-Chit (red). The oxidized catechol-
chitosan film was obtained by oxidation via O2 bubbling for 2 h.
The oxidized Cat-Chit film is designated Cat-Chit (ox).
2.5.2. ROS detection
To demonstrate the ROS generation activity, we first converted
the Cat-Chit film to its reduced state by incubation with ascorbate
(300 mM; 15 min) followed by extensive water rinsing. Next, Chit,
Cat-Chit (ox), Cat-Chit (red) films and Cat-Chit (red) film with
additional catalase were first incubated with 1 mL of air-saturated
water for predetermined time respectively, after which an aliquot
of the solution was diluted and tested for ROS-generation using
electron paramagnetic resonance (EPR) and Chemiluminescent
measurement.
For EPR measurement, this aliquot (films incubated for 2 h) is
mixed with the Fenton Fe2þ reagent and radical trapper DMPO [48].
Briefly, 27.5 mL of sample aliquots were mixed with 27.5 mL of a
reaction solution containing (NH4)2Fe(SO4)2 (200 mM), EDTA
(200 mM) and DMPO (100 mM), then quickly mounted onto EPR
instrument. EDTA was used to chelate metal ions that could affect
ROS (OH) generation. Data collection started after mixing the
liquids above for 126 s. Catalase used is 100 mg/mL. Each group has
singular sample.
Chemiluminescent measurement was performed using horse-
radish peroxidase enzyme mediated hydrogen peroxide assay kit
(Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit, AAT Bio-
quest®). Different films were first incubated with 1 mL of water for
12 h respectively. Aliquots of the solutions were measured ac-
cording to the assay protocol. Catalase used is 100 mg/mL. Each
group has triplicate samples.
2.5.3. Dynamics of ROS-generation
Dynamics of ROS-generation was examined using EPR assay. The
films with 1  1 cm2 were separately immersed into 1 mL of
distilled water. At predetermined time interval, 27.5 mL of sample
aliquots were mixed with 27.5 mL of a reaction solution containing
(NH4)2Fe(SO4)2 (200 mM), EDTA (200 mM) and DMPO (100 mM),
then quickly mounted onto EPR instrument. Data collection started
after mixing the liquids above for 126 s, each group had a singular
sample. The incorporated H2O2 amount was quantified using a
standard H2O2 calibration curve which was made by measuring
gradient diluted standard H2O2 solutions using EPR assay respec-
tively. The EPR spectra for H2O2econtaining solutions has a char-
acteristic DMPO-OH· signal with 1:2:2:1 quartet, of which the
second peak intensity of the corresponded standard vs. its
112 H. Liu et al. / Biomaterials 162 (2018) 109e122
concentration was fitted to be the calibration curve.
Chemiluminescent measurement was also performed to
examine the correlation between ascorbate concentration used for
film reduction and the H2O2-generation activity of the reduced film.
Films were first incubated with different concentration of ascorbate
solutions for 15 min followed by extensive rinsing in water, and
then incubated with 1 mL of water for 12 h respectively. Aliquots of
the solutions were measured according to the assay protocol. Each
group has triplicate samples.
2.5.4. Regenerating ROS-generation
Two pieces of Cat-Chit films were converted to their reduced
form by incubating in 2 mL of ascorbate solution (300 mM; 15 min),
rinsed with water, and then separately incubated in 1 mL of air-
saturated water (12 h). After the 1st incubation the water solu-
tions were sampled for H2O2 measurement by enzyme assay. The
Cat-Chit films were then recovered and one of them was replen-
ished with electrons by re-incubation with ascorbate and then re-
used for a subsequent incubation in water (12 h). The other
(serving as a control) was re-incubated in water (12 h) in the sub-
sequent cycles without ascorbate treatment. Each group has trip-
licate samples.
2.6. In vitro antimicrobial experiments
2.6.1. Bacteriostatic activity assay
Bacteria of S. aureus (ATCC 25923), E. coli (ATCC 25922) and
methicillin resistant Staphylococcus aureus acquired from hospital
(MRSA, Second Affiliated Hospital of Zhejiang University) were
used in this study. The Cat-Chit (red) films used for these studies are
prepared by ascorbate (300 mM) incubation for 15 min. Four sam-
ple groups were tested: Chit, Cat-Chit (ox), Cat-Chit (red) and Cat-
Chit (red) films with additional catalase. For each group three
pieces of films (z1  1 cm2) were added into 1 mL of bacteria so-
lution (106 CFU/mL), and incubated at 37  C. The OD600 was moni-
tored every 3 h. Each group had 3 replicate samples. The bacteria
solution in the absence of films or with 10 mM of H2O2 was used as
control.
2.6.2. The inhibition zone assay
200 mL of bacteria solutions (1  105 CFU/mL) were firstly
introduced onto the agar plates, and then the films were placed
onto the plates and incubated for 12 h at 37  C.
2.6.3. Morphology study
The bacteria before or after Cat-Chit (red) films treatment for 4 h
was examined by SEM. Before measurement, the bacteria were
fixed by 2% glutaraldehyde for 2 h, and then experienced gradient
dehydration. The bacteria solution was dropped onto silicon wafer,
followed by freeze-drying.
2.6.4. Live/dead bacterial assay
Live/dead bacterial assay was performed to examine the
viability of bacteria before and after Cat-Chit (red) films treatment.
After incubation with or without films for 4 h, bacteria solution was
mixed with 6 mL of dye solution containing 1.67 mM of SYTO 9 dye
and 20 mM of propidium iodide for 20 min at room temperature.
The bacteria were then imaged using a confocal microscope (Nikon
A1R). SYTO 9 dye with green color labeled both live and dead
bacteria while propidium iodide dye with red color stained only
dead bacteria. The orange color in samples contact with film is
overlapped by green and red colors, indicating dead bacteria [49].
2.6.5. Ascorbate concentration dependent antimicrobial activity
testing
The antimicrobial activity of Cat-Chit films with different
ascorbate treatment were also examined using E. coli. Films
(z1  1 cm2) were first respectively incubated with different con-
centration of ascorbate solutions for 15 min followed by extensive
rinsing in water. For each group three pieces of films were added
into 1 mL of bacteria solution (105 CFU/mL), and incubated at 37  C
for 12 h. Cell media were sampled for measuring the survivors us-
ing plate counting. Each group has 3 replicate samples.
2.6.6. Regenerating antimicrobial activity evaluation
Three pieces of Cat-Chit films were converted to their reduced
form by incubating in 3 mL of ascorbate solution (300 mM; 15 min),
rinsed with water, and then incubated in 1 mL of bacterial cultures
(106 CFU/mL) for 12 h. After the 1st incubation, cell media were
sampled for measuring the remaining viable bacteria using dilution
plate counting, and also the films were recovered and replenished
electrons by contacting with ascorbate (300 mM, 15 min), and then
re-used for a subsequent incubation. In parallel, three other Cat-
Chit films (serving as a control) were used in the same manner as
described above, except for that after the 1st incubation the films
were reused without ascorbate treatment in subsequent incubation
cycles. Each group has a singular sample.
2.7. Biocompatibility evaluation
The HaCaT cell line is used to estimate the biocompatibility of
the Cat-Chit films reduced by varying ascorbate levels. For this
study, the films were first cut into 8 mm diameter disks and then
sterilized by UV light overnight. Following the incubation of HaCaT
cells (2.6  104 cells/cm2) in a culture medium for 24 h at 37  C
containing 5% CO2, each of the culture media was added with one
piece of film and then further cultured for an additional 24 h. The
films were then removed and the cells were washed with PBS. 1 mL
of culture media containing 1 mg/mL MTT were added to each well
and the plates were incubated for another 4 h. The yielded purple
formazan crystals inside cells were dissolved by DMSO and OD
values were measured at 492 nm using a Spectramax M2 plate
reader (Molecular Devices, USA). Cell viability was calculated by
assuming 100% viability in the control set (media without films).
2.8. In vivo antimicrobial activity of Cat-Chit film
All animal experiments adhered to the NIH guidelines for the
care and use of laboratory animals (NIH Publication no. 85-23 Rev.
1985) and were approved by the Research Center for Laboratory
Animals of Shanghai University of Traditional Chinese Medicine.
Male Sprague Dawley (SD) rat (180e200 g and 6e7 weeks of age)
were used for this study. Animals were divided into 3 groups: (i)
Chit; (ii) Cat-Chit (ox); (iii) Cat-Chit (red) (reduced with 300 mM
ascorbate for 15 min), and each group contains 4 replicate samples.
Rats were anesthetized using a flow of pentobarbital solution (1.0%)
and kept warm on sterile gauze during surgery. The backs of the
rats were shaved and disinfected with iodophor. Two incisions with
1e2 cm in length were made on the back of each rat. All the sample
films each with 8 mm in diameter were individually pre-seeded
with 10 mL of inoculums containing 107 CFU/mL of MRSA, and
then air-dried for 10 min. After that, one sample film was implanted
subcutaneously into one incision at random. The incisions were
subsequently sutured with stitch. After 3 days implantation, the
animals were euthanized by inhalant anesthetic. The films were
removed and bacteria on it were quantified by dilution plate assay.
 H. Liu et al. / Biomaterials 162 (2018) 109e122
2.9. In vivo wound healing tests
Male BALB/c mice (20e25 g and 7e8 weeks of age) were used
for this study. Animals were divided into 3 groups: (i) Chit; (ii) Cat-
Chit (ox); (iii) Cat-Chit (red) (reduced with 10 mM ascorbate for
15 min), and each group contains 4 replicate samples. Mice were
anesthetized using a flow of pentobarbital solution (1.0%) and kept
warm on a sterile gauze during surgery. The standard splinted
wound model was used for each study [50e52]. Briefly, an aseptic
puncher was used to create two excisional wounds with 5 mm in
diameter on the backs of each mouse. Silicon rings were sutured in
place around the wounds to prevent healing by contraction. The
one wound was bare (not covered) and served as negative control;
the other wound was dressed by a single film with 8 mm in
diameter. Finally, the mice were wrapped with tagaderm trans-
parent dressing (3 M, 2e3/8 inches  2e3/4 inches) on the backs
and fixed with adhesive tape. Films were changed at day 3, 7 and 10
after surgery and wound healing pictures were recorded. On day 3
and day 7, the exudate around the wound bed was collected by a
cotton swab. The swab was then incubated with LB media for
bacterial amplification and bacteria counting. After 14 days of
healing, animals were euthanized by inhalant anesthetic. The skin
including the wound was removed and fixed in 10% formaldehyde
solution. The fixed tissue were sliced and analyzed by H&E staining
for histological testing, and also Masson's trichrome staining for
examining the formation of collagen.
2.10. Statistical analysis
All data were expressed with mean standard deviation (SD) and
analyzed using one-way ANOVA with post hoc tests. Significance
was set at p < .05 (***p < .001, **p < .01, *p < .05).
3. Results and discussion
3.1. Film fabrication and characterization
Films were prepared by electrodeposition (chitosan control)
followed by oxidative catechol grafting (catechol-chitosan film).
The morphology and the thickness of freeze-dried films were
characterized by SEM. Fig. 1A show a relatively smooth surface for
the chitosan film, whereas the surface of the catechol-chitosan film
appears to have considerably more roughness and porosity. The
thickness of films was about 100 mm (data not show).
Chemical evidence for the oxidative grafting of catechol to chi-
tosan is provided by three independent methods. First, the photo-
graphs and UVevis spectra in Fig. 1B show that catechol grafting
converts the initially-transparent chitosan film into a reddish
brown film with strong UV absorption consistent with a covalent
grafting of quinones to amines [53]. Fig. 1C shows ATR-FTIR spectra
of Chit and Cat-Chit films. As indicated by the black arrows, two IR
bands at 1643 and 1593 cm1 correspond to the stretching vibra-
tions of carbonyl (eNHeC]O) and amine (eNH2) moieties of chi-
tosan film [54]. The spectrum for the Cat-Chit film reveals that after
reaction distinct changes occur in the amine stretching vibration,
suggesting catechol oxidation products undergo covalent grafting
to the amino group on chitosan. We also observed a new peak at
1620 cm1, which could be partially assigned to C]N stretching
vibration from the Schiff base or tautomeric form of Michael's-type
adducts [55]. More rigorous evidence for covalent bond formation
between amines of chitosan and catechol is provided by XPS. Fig. 1D
compares the N1s spectra of the chitosan control and catechol-
chitosan films. Deconvolution of the spectra yielded a single peak
at 399.2 eV for the chitosan control film, while the catechol-
chitosan films has a new peak at 401.5 eV which can be assigned
113
to C]N structure on Schiff base or tautomer of Michael's-type
adducts [55].
Taken together, these results provide independent physical and
chemical evidence for the oxidative grafting of catechol to the
chitosan film. Due to the complexity of this reaction system, it is not
possible to definitively identify the specific linkages (Michael-type
adduct or Schiff base), or whether monomeric or oligomeric cate-
chols are grafted to the chitosan. Nevertheless the results in Fig. 1
are consistent with previous observations catechol modified chi-
tosan films [56].
3.2. Reversible redox-activity of Cat-Chit film
Because of the complexity of melanin and melanin-mimetic
materials, the characterization of structure, properties and func-
tion has been challenging [42,57e62]. Electrochemical reverse
engineering methodology was introduced to probe the redox-
activity of electro-fabricated catechol-chitosan and melanin-
chitosan films [63e65]. Here, we applied an electrochemical
reverse engineering methodology to probe the redox-activity of
catechol-chitosan film. As illustrated in Fig. 2A, the Cat-Chit film is
expected to have redox-capacitor properties [64,65] which can
accept and donate electrons through reduction and oxidation re-
actions, and it can store electrons when the film is maintained in a
reduced state [24]. Importantly, the Cat-Chit film is also expected to
be non-conducting with electron transfer requiring diffusible re-
ductants and oxidants. The reverse engineering method is based on
the use of mediators to engage the film in electrochemical redox-
cycling reactions that serve to transfer electrons between the un-
derlying electrode and Cat-Chit film. The upper reaction in Fig. 2A
shows that one mediator Ru(NH3)6Cl3 (Ru3þ; E ¼ 0.22 V vs Ag/
AgCl) engages in reductive redox-cycling by accepting electrons
from the electrode under reducing potentials and donating these
electrons to the film (the putative reaction shows the reduction of
film quinone moieties to catechols). The second mediator ferrocene
dimethanol (Fc; E ¼ þ0.25 V vs Ag/AgCl) engages in oxidative
redox-cycling by donating electrons to the electrode under
oxidizing potentials and then accepting electrons from the film (the
putative reaction shows the oxidation of film catechol moieties to
quinones). The thermodynamic plot in Fig. 2A illustrates that the
reductive and oxidative redox-cycling reactions can be cycled by
oscillating the imposed potential (i.e., voltage).
In the first experiment, we probed for reversible redox-activity
by immersing the film-coated electrode in a mixed solution of
the two redox mediators (500 mM Fc, and 500 mM Ru3þ) and
applying a cyclic potential to the underlying electrode
(between 0.5 V and þ0.5 V; 10 cycles; scan rate 5 mV/s). Fig. 2B
shows the results from this experiment represented as both input-
output curves and cyclic voltammograms (CVs). The control in this
experiment is an unmodified chitosan film and the CV for this
control shows small peaks in the voltage regions for the Ru3þ and Fc
mediators. The CV for the Cat-Chit film shows considerable
amplification of both Ru3þ-reduction and Fc-oxidation. These am-
plifications in output currents are a signature for the redox-cycling
mechanisms of Fig. 2A. Fig. 2B also shows that there was no
noticeable change in the output currents over 10 repeated cycles.
This “steady” output for the Cat-Chit film suggests that the transfer
of electrons to the film during reductive redox-cycling is balanced
by the transfer of electrons from the film during oxidative redox-
cycling. These results indicate that the Cat-Chit film is redox-
active and can repeatedly donate and accept electrons.
Two additional reverse engineering experiments were pre-
formed to confirm the redox activity of the film. In these studies,
conditions were imposed to yield unbalanced redox-cycling. The
results in Fig. 2C show that for the initial cycles, a large amplified
114 H. Liu et al. / Biomaterials 162 (2018) 109e122
Fig. 1. Characterization of Cat-Chit film. (A) SEM, (B) UVevis, (C) ATR-FTIR, and (D) XPS provide evidence for film modification during catechol oxidative grafting.
output current is observed in the Ru3þ-reduction region for the Cat-
Chit film (compared to the control Chit film) and this amplification
becomes progressively less with each consecutive cycle. This un-
steady output response is consistent with the Cat-Chit film being
reduced by Ru3þ-mediated redox-cycling but in the absence of
oxidative redox-cycling the film becomes “saturated” with elec-
trons and unable to accept electrons. The results in Fig. 2D show
that a large amplification of output current is observed in the Fc-
oxidation region for the Cat-Chit film (compared to the control
Chit film) and this amplification progressively diminishes with each
consecutive cycle. This unsteady output response is consistent with
the Cat-Chit film being progressively depleted of electrons by the
Fc-mediated redox-cycling reactions.
In summary, the results in Fig. 2 indicate that the catechol-
chitosan film possess redox activity and it can repeatedly ex-
change electrons through oxidation and reduction reactions.
3.3. Transfer of electrons from ascorbate to O2 for ROS generation
Scheme 1A shows the underlying hypothesis of this research is
that the redox-activity of the grafted catechol moieties allows
endogenous reducing equivalents (e.g., ascorbate) to provide the
electrons needed to partially reduce O2 to generate ROS (e.g., H2O2)
[56]. To demonstrate ascorbate-mediated reduction, we incubated
a catechol-chitosan film (z1  1 cm2) with ascorbate (300 mM;
15 min) followed by extensive water rinsing, and then XPS analysis.
The photographs in Fig. 3A show an interesting color change when
Cat-Chit films are subjected to ascorbate reduction. Chemical evi-
dence for ascorbate-mediated reduction of Cat-Chit is presented in
Fig. 3A which shows the XPS spectra for Cat-Chit films before and
after ascorbate treatment. The ascorbate treated (i.e., reduced) Cat-
Chit film has an increased CeO peak at ~532.5 eV and a decreased
C]O peak at ~531.2 eV compared to the oxidized Cat-Chit film. This
observed difference is consistent with an ascorbate-mediated
reduction of oxidized quinone moieties to catechol moieties
(Fig. S1 shows additional XPS spectra for these films).
Next, we demonstrated the transfer of electrons from a reduced
catechol-chitosan film to O2 to generate ROS. For this experiment,
we first reduced a catechol-chitosan film (z1  1 cm2) by incuba-
tion with ascorbate (300 mM; 15 min). This reduced film was then
incubated in 1 mL of air-saturated distilled water for 2 h after which
an aliquot of the solution was diluted and tested for ROS-generation
using EPR. For EPR measurement, this aliquot is mixed with the
Fenton Fe2þ reagent and radical trapper DMPO [48]. The upper two
EPR spectra in Fig. 3B show results for incubation of this aliquot and
a positive control (an H2O2 solution). Both spectra show the char-
acteristic 1:2:2:1 quartet which indicates the reduced film had
generated ROS. We also analyzed two additional samples, (i) in-
cubation of a Cat-Chit (red) film in the presence of the H2O2 scav-
enging enzyme catalase, and (ii) incubation of a Cat-Chit (ox) film.
Fig. 3B shows the characteristic EPR signals was completely absent
from these two samples. The final spectrum in Fig. 3B is for incu-
bation of a chitosan film and this negative control also shows no
EPR signals. These results indicate that the reduced Cat-Chit film
can donate electrons to O2 for ROS (i.e., H2O2) generation.
Independent evidence for ROS-generation was obtained by the
horseradish peroxidase chemiluminescence method. Amplex Red is
used as the indicator, which is colorless but reacts with H2O2 to
form a pink product (resofurin) in the presence of HRP. Experi-
mentally, films were incubated in air-saturated water for 12 h and
then aliquots were tested by this H2O2-reporting assay. Fig. 3C
shows that only Cat-Chit (red) group with no catalase yield pink
color, indicating the presence of H2O2. The Cat-Chit (red) film
generated about 310 mM of H2O2. Negligible H2O2-generation was
observed for three controls with incubation of: (i) an unmodified
Chit film, (ii) a Cat-Chit (ox) film, and (iii) a Cat-Chit (red) film with
catalase. These results further confirm that Cat-Chit film can accept
electrons from ascorbate and transfer them to O2 to generate ROS
(Note: the reduced Cat-Chit film can be slowly oxidized in air over
the course of 7e10 days, and thus limit subsequent ROS generating
activities.).
The dynamics of ROS-generation using EPR was then examined.
Experimentally, reduced and oxidized Cat-Chit films (z1  1 cm2)
were separately incubated in air-saturated water and aliquots of the
incubation solutions were taken at various times for H2O2 deter-
mination. The H2O2 amount was quantified using a standard H2O2
 H. Liu et al. / Biomaterials 162 (2018) 109e122 115

Fig. 2. Electrochemical reverse engineering demonstrates repeatable redox-cycling of the film. (A) Schematic illustration on the mediated reductive and oxidative redox-cycling
reactions with the Cat-Chit film. (B) Amplified and steady outputs are observed under conditions that allow both reductive and oxidative redox-cycling (500 mM Fc and 500 mM
Ru3þ). (C) Amplified but unsteady output is observed under conditions that allow only reductive redox-cycling (500 mM Ru3þ; scan rate 5 mV/s). (D) Amplified but unsteady output
is observed under conditions that allow only oxidative redox-cycling (500 mM Fcþ; scan rate 5 mV/s).

calibration curve that is obtained by the second peak intensity of that when a Cat-Chit (ox) film was incubated with air-saturated
the corresponded DMPO-OH· spin adduct. The EPR results in Fig. 3D water, no ROS evolution was observed over the course of the in-
show the generation of H2O2 from the Cat-Chit (red) film increased cubation. (Fig. S2 shows analgous experiments with enzymatic
progressively over the 6 h experiment. In contrast, Fig. 3D shows detection of H2O2). In summary, the EPR and enzyme methods for
116 H. Liu et al. / Biomaterials 162 (2018) 109e122
Fig. 3. Cat-Chit film can accept electrons from ascorbate and donate electrons to O2 for ROS generation. (A) XPS evidence for film reduction by ascorbate (300 mM for 15 min). (B)
EPR spectra and (C) enzyme assays show reduced Cat-Chit films can generate H2O2. (Inserted photograph shows the chemiluminescence reaction). (D) Kinetics of H2O2-generation
measured by EPR. (E) Cat-Chit films can be repeatedly replenished by ascorbate for the sustained generation of ROS.
measuring H2O2 are in qualitative agreement and indicate that
reduced films can generate ROS while the oxidized films have
limited ROS-generating abilities.
The ability of the Cat-Chit films to repeatedly generate H2O2 was
then examined. In this experiment, the Cat-Chit films were (i)
converted to their reduced state by ascorbate treatment (300 mM;
15 min), (ii) rinsed with water, and (iii) incubated in air-saturated
water (12 h) after which the water solutions were sampled for
H2O2 measurement. Fig. 3E shows that approximately 350 mM of
H2O2 was generated in this 1st cycle. The Cat-Chit films were then
recovered and replenished with electrons by re-incubation with
ascorbate before re-incubation with air-saturated water (12 h). The
results from these “ascorbate replenished” films show H2O2-levels
again reached approximately 350 mM for this second cycle and for
subsequent cycles. The control Cat-Chit films in Fig. 3E were only
incubated with ascorbate once (in the beginning of the experiment)
and their ability to generate H2O2 was observed to decay progres-
sively with each subsequent cycle. These results indicate that the
ascorbate can repeatedly replenish the Cat-Chit film with electrons
for subsequent H2O2-generation (Fig. S3 shows H2O2 generation
increases with increasing ascorbate concentration.).
3.4. In vitro antimicrobial activity of Cat-Chit film
We next evaluated the antimicrobial properties of the Cat-Chit
film. In these experiments, we treated the Cat-Chit film with
300 mM ascorbate to test our proposed mechanism that the film
can access electrons from ascorbate and generate ROS to confer
antimicrobial activities. Initially, we tested in vitro antimicrobial
activities using E. coli, S. aureus and MRSA as model Gram-negative,
Gram-positive and drug resistant pathogens. In the initial test,
1  106 CFU/mL of bacteria were inoculated into growth medium
(50% LuriaeBertani broth medium) at 37  C and growth was
monitored by intermittent measurements of optical density (OD).
The growth curves in Fig. 4A show three general responses. One
response is uninhibited growth and this response was observed
when bacteria were cultivated in the (i) absence of films, (ii) the
presence of unmodified Chit control film (note: chitosan is often
reported to offer antimicrobial activities although these activities
appear to be pH dependent and related to whether there are pos-
itive charges on the polymer backbone) [66,67], or (iii) the presence
of a Cat-Chit (red) film (reduction with 300 mM ascorbate for
15 min) plus the H2O2-scavenging enzyme catalase. The second
response is complete growth inhibition and this was observed
when bacteria were cultivated with (i) 10 mM H2O2, or (ii) Cat-Chit
(red) film (reduction with 300 mM ascorbate for 15 min). The third
response is intermediate and shows partial growth inhibition and
this was observed when bacteria were cultured with Cat-Chit (ox)
film. In summary, the results in Fig. 4A show the Cat-Chit film
confers antimicrobial activities. Several reports indicate that phe-
nolics possess antimicrobial activities [68e70] and our results
suggest that ROS-generation may provide one mechanism for such
activities.
Further evidence for the film's antimicrobial activity is provided
by inhibition zone assays for the three types of bacteria (Fig. 4B). In
these experiments, film samples were placed onto the surface of an
agar plate after the plate was inoculated with bacteria. After incu-
bation for 12 h, Fig. 4B shows a growth inhibition-zone for the Cat-
Chit (red) film, which is consistent with the release of a diffusible
biocide (i.e., H2O2). No inhibition-zone is observed for the two
control films (unmodified Chit and the Cat-Chit (ox)). The genera-
tion of a diffusible antimicrobial species is also consistent with the
ROS generating mechanism of Scheme 1A.
Next, we examined if incubation with the reduced Cat-Chits film
 H. Liu et al. / Biomaterials 162 (2018) 109e122 117

Fig. 4. In vitro antimicrobial properties of Cat-Chit film. (A) The reduced Cat-Chit completely inhibits E. coli, S. aureus and MRSA growth. (B) Reduced Cat-Chit film generates a zone
of bacteria inhibition for in an agar plate assay consistent with the generation of diffusible ROS (zoom-in insets for highlighting the inhibition zone without bacteria). (C) Fluorescent
staining and SEM images of bacteria after contact with reduced Cat-Chit films indicate that the released ROS kills bacteria by inducing cellular membrane injury. (green color
indicates live and dead bacteria; red color indicates dead bacteria) (D) Cat-Chit films with ascorbate replenishment (300 mM; 15 min) retain high bactericidal activity against E. coli.
[Data represent mean values ± SD (n ¼ 3)]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
118 H. Liu et al. / Biomaterials 162 (2018) 109e122
induced significant morphological changes to E. coli and S. aureus.
The SEM images in the left panel in Fig. 4C demonstrates that
treatment with the Cat-Chit (red) films induced substantial
disruption of cellular structures which is consistent with ROS
induced damage [65,71]. The right panel in Fig. 4C shows a live
(green)/dead (red) staining assay for E. coli and S. aureus cells after a
4 h incubation. These images further demonstrate the bactericidal
activity of the Cat-Chit film.
Finally, the regenerability of antibacterial activity of Cat-Chit
films was examined. Specifically, we examined bactericidal activ-
ity by incubating E. coli (106 CFU/mL) in culture medium in the
presence of films for 12 h, and then measuring survivors using
dilution plate counting (Fig. S4) [66]. Killing efficiency was calcu-
lated by comparing the bacterial colonies formed when E. coli was
cultivated in the presence of films and when they were cultured in
growth media lacking films. The repeatability of the film's bacte-
ricidal activity was assessed by recovering films after an incubation,
replenishing electrons by contacting the films with ascorbate
(300 mM; 15 min), and then re-using the films for a subsequent
incubation. Fig. 4D shows that when the Cat-Chit films were
repeatedly treated with ascorbate, high bactericidal activity was
observed over multiple cycles. The control in Fig. 4D is E. coli that
was cultured in the presence of Cat-Chit films that were only
contacted with ascorbate prior to the first incubation: the bacteri-
cidal activity of these films was observed to decrease progressively
with each subsequent incubation consistent with a depletion of
electrons from the films. These results further support the mech-
anism of Scheme 1A which proposes that the Cat-Chit film cata-
lyzes the transfer of electrons from ascorbate to O2 for sustained
antibacterial activity.
The antimicrobial activities of Cat-Chit films treated with
different concentrations of ascorbate were also examined by
incubating E. coli for 12 h and then measuring survivors using plate
counting. The results shown in Fig. S5 indicates the Cat-Chit films
show significant bacteriostatic activity when the concentration of
ascorbate exceeded about 10 mM.
3.5. Biocompatibility of Cat-Chit film
The biocompatibility assay for Cat-Chit films were performed
using HaCaT cells which are model keratinocytes derived from
human skin. We incubated these cells with films for 24 h and then
measured the surviving cells using a MTT viability assay. The results
in Fig. 5 show the unmodified control Chit and the oxidized Cat-
Chit films have no cytotoxicities. The Cat-Chit films treated with
moderate levels of ascorbate (1 or 10 mM) also show no cytotox-
icities while films treated with high ascorbate levels (300 mM)
slightly induced cell growth inhibition as the cell viability was
decreased by 15%. Presumably the toxicity observed after treatment
of the films with high ascorbate levels is due to a high level of ROS-
generation (Fig. S3). These results suggest Cat-Chit films have good
biocompatibility only if we control the concentration of ascorbate
used for film reduction. Further these results suggest that endog-
enous levels of ascorbate (<0.1 mM) [72] should be too low to result
in the generation of high concentrations of ROS and associated
cytotoxicities for the Cat-Chit films.
3.6. In vivo antimicrobial activity of Cat-Chit film
We next evaluated the in vivo antimicrobial activities of Cat-Chit
films by using a SD rat subcutaneous implantation model. MRSA
was employed as a model bacterium for this study since it is
responsible for several difficult-to-treat infections in humans. After
pre-seeded with MRSA, the films (i) Chit; (ii) Cat-Chit (ox); (iii) Cat-
Chit (red) (reduced with 300 mM ascorbate) were respectively
Fig. 5. The biocompatibility estimation of chat-chit films using a MTT viability assay.
HaCaT cells were cultured in the presence of different films for 1 day. HaCaT cell
viability assays provide evidence that the Chit, Cat-Chit (ox), and Cat-Chit (red) films
are biocompatible. [Data represent mean values ± SD (n ¼ 4)]. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of
this article.)
implanted subcutaneously for 3 days. Fig. 6A shows two 1e2 cm
length incisions were created in the dorsum of each rat and sutured
after film implantation. After 3 days culturing, the wounds were re-
opened and the infection state was photographed as shown in
Fig. 6B. The wound implanted with the Cat-Chit (red) film pre-
sented a normal tissue, while the control wound embedded with
the Chit or Cat-Chit (ox) were found to have obvious festering with
serious inflammation and suppuration. The implanted films were
taken out and the attached bacterial were collected for quantifi-
cation by dilution plate counting. We observed an average 2-Log
reduction of bacteria number on the surface of Cat-Chit (red) film
in comparison with the controls of chitosan and oxidized Cat-Chit
surfaces (Fig. 6C and D). This result verified that an inhibition of
microbial growth was achieved in vivo for the Cat-Chit (red) film.
3.7. In vivo wound healing evaluation
Finally, we evaluated the catechol grafted chitosan films in vivo
using a mouse excisional wound splinting model [50e52]. Fig. 7A
shows two 5 mm diameter full-thickness wounds were created in
the dorsum of each mouse. One wound was uncovered and the
other was dressed by a single film of 8 mm diameter. The test Cat-
Chit film in this experiment was treated with 10 mM ascorbate in
order to balance the desired antimicrobial activities against the
undesired cytotoxicities. Fig. 7B shows photographs of post-
operative wound healing in mice treated with different films. These
images were analyzed (Image J) and Fig. 7C shows the percentage of
wound closure (relative to wound area at day 0). All wounds were
closed, while wounds dressed with films closed faster (compared to
bare wounds). On day 14, the wound treated with a reduced Cat-
Chit film was observed to have a statistically higher closure
compared to the other groups.
To test for bacterial contamination in the wound bed, exudates
were collected on day 3 and day 7 by removing the film and
swabbing the wound area. The swab was then placed in saline so-
lution with LB broth and incubated for 24 h after which the bacteria
were counted by dilution plate counting [66]. Fig. 7D shows a
considerably lower bacterial population generated from the wound
 H. Liu et al. / Biomaterials 162 (2018) 109e122 119

Fig. 6. In vivo antimicrobial studies. (A) Photographs illustrate the rat subcutaneous implantation model. The film was pre-seeded with MRSA and implanted into the incision. (B)
Photographs of re-opened incisions after 3 days film implantation. (C) The bacteria were retrieved from the explanted films and cultured on agarose gel media. (D) Total bacteria
number of CFU (log) detected on the films. [The red line indicates the initial MRSA count; ***p < .001; Data represent mean values ± SD (n ¼ 4)]. (For interpretation of the references
to color in this figure legend, the reader is referred to the Web version of this article.)

treated with the reduced Cat-Chit film consistent with the film
possessing in vitro antimicrobial activity.
Fig. 8A shows histological analysis of wounds by hematoxylin
and eosin (H&E) staining. Low magnification of H&E staining of
these wounds is provided in Fig. S6, in which the areas closed by red
squares indicate the original wound sites. The surface of the bare
wound group, and wounds dressed with unmodified control Chit
films or oxidized Cat-Chit films did not achieve an enclosed
epithelial tissue (cyan arrows). In contrast, the wound dressed with
the reduced Cat-Chit film shows a complete formation of new
epithelium [73,74]. Additionally, more blood vessels (red arrows)
and hair follicles (green arrows) developed at wounds treated with
the reduced Cat-Chit film. In addition, wounds dressed with Chit or
oxidized Cat-Chit induced an immune response (black arrows
indicate significant inflammatory cell infiltration) [75e79], while
this response was not obvious for the wounds treated with the
reduced Cat-Chit film [80]. The extent of collagen deposition in the
wound site was estimated by Masson's trichrome staining (Fig. 8B).
In this method, the collagen fibers are stained blue. The wounds
dressed with the Cat-Chit film were observed to regenerate more
uniformly distributed collagen compared with other groups.
In summary, these in vivo results indicate that better healing
was achieved when the wound bed was dressed by a reduced Cat-
Chit film. The unique feature of this film is its ability to actively
generate ROS. ROS-generation is an important mechanism for im-
mune defense against bacterial infection and the evidence pre-
sented suggest the Cat-Chit film can perform this oxidative
bacterial killing. Previous studies have demonstrated that a low
level of ROS(H2O2) has no burden to the wound site, but on the
contrary can provide multiple positive effect on wound healing
[81]. We envision that the higher healing effect of red Cat-film may
be due to such beneficial effects of ROS. Therefore, it is possible that
the film's ROS generating ability may enlist additional mechanisms
that promote wound healing since emerging research indicates ROS
may promote wound healing through mechanism that involve
cytokine release (e.g. vascular endothelial growth factor, trans-
forming growth factor-b1) [82,83], cell proliferation (e.g. endothe-
lial cells, fibroblast) [84], collagen synthesis [85] and angiogenesis
[86]. Further work will be required to evaluate the diverse range of
mechanisms that enable this film to promote wound healing.
4. Conclusions
In summary, we fabricated a catechol-modified chitosan film to
mimic a mechanism proposed to explain the antimicrobial activity
of insect melanin. This mechanism involves a redox-based transfer
of electrons from reductants to O2 for the active in situ generation
of ROS. We provide in vitro evidence that (i) the catechol-chitosan
film is redox-active and can repeatedly undergo oxidation and
reduction reactions; (ii) it can catalyze the transfer of electrons
from the physiological reductant ascorbate to O2 for sustained ROS
generation, and (iii) this film possesses ascorbate-dependent anti-
microbial activities against both Gram-positive and Gram-negative
bacteria, and drug resistant bacteria. In vivo antimicrobial tests with
a rat subcutaneous model indicate the catechol-chitosan film at
reduced state inhibits the bacterial growth and alleviates the
infection of the incisions. In vivo wound healing tests with a mouse
model indicate that the catechol-chitosan film induces less in-
flammatory response and suppresses bacterial growth (compared
to untreated wounds or wounds treated with an unmodified chi-
tosan film). These in vivo studies further demonstrate that the
catechol-chitosan film promotes wound healing compared to un-
treated wounds. We envision this biomimetic approach for the
sustained, localized and in situ generation of ROS could provide
120 H. Liu et al. / Biomaterials 162 (2018) 109e122

Fig. 7. In vivo wound healing and antimicrobial studies. (A) Photographs illustrate the mouse wound healing model. (B) Photographs and (C) summary plots of wound closure. (D)
Assessing bacterial activity at wound site by swabbing, incubation and bacterial counting. (*p < .05, **p < .01, ***p < .001; Data represent mean values ± SD (n ¼ 4)).

Fig. 8. Histological analysis of wounds by (A) hematoxylin and eosin (H&E) staining and (B) Masson's trichrome staining (for collagen fibers). (cyan arrows indicate epithelial tissue,
red arrows indicate blood vessels, green arrows indicate hair follicle, black arrows indicate significant inflammatory cell infiltration). (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)
 H. Liu et al. / Biomaterials 162 (2018) 109e122
new opportunities for wound management.
Acknowledgements
The support from the National Natural Science Foundation of
China (51621002, 51573047), the 111 project (B14018), the Funda-
mental Research Funds for the Central Universities (222201718002,
222201717002, 222201717015), and the United States National
Science Foundation (CBET-1435957) and Defense Threat Reduction
Agency (HDTRA1-13-0037).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.biomaterials.2017.12.027.
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