Chemical Engineering Journal 385 (2020) 123464

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Chemical Engineering Journal

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

Anti-oxidant electroactive and antibacterial nanofibrous wound dressings T

based on poly(ε-caprolactone)/quaternized chitosan-graft-polyaniline for
full-thickness skin wound healing
⁎
Jiahui Hea, Yongping Lianga, Mengting Shia, Baolin Guoa,b,
a
Frontier Institute of Science and Technology, and State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710049, China
b
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an 710049, China

H I GH L IG H T S G R A P H I C A L A B S T R A C T

• Nanofibrous wound dressing with

comparable mechanical property to
soft tissue was designed.
• The dressing showed good electro-
activity and anti-oxidant ability.
• The dressing exhibited good bio-
compatibility and effective anti-
bacterial activity.
• the
The dressing significantly accelerated
healing process of full-thickness
skin wound.

A R T I C LE I N FO A B S T R A C T

Keywords: Developing a novel nanofibrous wound dressing with multi-functional properties, integrating suitable me-
Electroactive nanofiber chanical property, electroactivity, anti-oxidant and inherent antibacterial activity to promote wound healing
Electrospinning process is urgently desired, which could meet the increasing requirements of clinical needs. Herein, a series of
Anti-oxidant property antibacterial, anti-oxidant and electroactive nanofibrous membranes were fabricated by electrospinning poly(ε-
Antibacterial activity
caprolactone) (PCL) and quaternized chitosan-graft-polyaniline (QCSP) polymer solutions which combined the
Wound dressing
good mechanical property of PCL and multi-functionality of QCSP. The nanofibrous wound dressings exhibited
electroactivity, similar mechanical properties to soft tissue, free radical scavenging capacity, antibacterial
property, and biocompatibility. In particular, PCL/QCSP15 (15 wt% of QCSP in the sample) showed a good
balanced ability between antibacterial activity and cell proliferation, which significantly accelerated the healing
process of wound in a mouse full-thickness wounds defect model than commercial dressing (Tegaderm™ Film)
and pure PCL (PCL/QCSP0) nanofibrous membrane. Moreover, the histopathological examination and im-
munofluorescence staining showed that the wounds treated by PCL/QCSP15 nanofiber dressing exhibited higher
collagen deposition, granulation tissue thickness, and more angiogenesis. In one word, these antibacterial, anti-
oxidant, electroactivity nanofibrous membranes showed promising applications for full-thickness skin repair.

⁎
Corresponding author at: Frontier Institute of Science and Technology, and State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University,
Xi’an 710049, China.
E-mail address: baoling@mail.xjtu.edu.cn (B. Guo).

https://doi.org/10.1016/j.cej.2019.123464
Received 29 July 2019; Received in revised form 23 October 2019; Accepted 11 November 2019
Available online 12 November 2019
1385-8947/ © 2019 Elsevier B.V. All rights reserved.
J. He, et al.
1. Introduction
The largest organ of human body is skin tissue, which acts as the
first barrier to the external environment against dehydration, chemical/
radiological damages, and microorganisms invasion [1–3]. For those
damaged or destroyed skin tissue, the wound repair process appears
very complicated and needs highly orchestrated, which not only in-
volves the complex coordination between dermal and epidermal com-
partments, but also between local microenvironment, resident cells,
and immigrant cells [4–6]. Moreover, the lost tissue will be replaced by
scar tissue when the wounds affect the dermis because the dermis lacks
the regenerative ability which is similar to the epidermis [7,8]. For
serious skin defects, the wound will have a severe impact on people's
health and even threaten their life [9]. Thus, a large number of different
kinds of biomaterials have been fabricated to accelerate wound healing
process and reduce the formation of scar tissue, such as rubbers,
sponges, films, hydrogels, and nanofibrous membranes (NFMs), etc
[1,10–14]. Among these dressings for wound healing application, NFMs
developed by electrospinning have become one of the current research
hotspots which are attributed to NFMs with high surface-volume ratio
and microporous structure, and these are crucial properties for cell’s
migration, adhesion, growth, differentiation, and angiogenesis [15–17].
Specifically, NFMs with the morphology similar to the extracellular
matrix (ECM) could attract fibroblasts to the dermis, which can secrete
growth factor, collagen, and other ECM components to accelerate the
repair of damaged tissue [18–20].
Synthetic polymers including poly(ε-caprolactone) (PCL), poly-
urethane, poly(vinyl acetate), polyacrylonitrile, poly(L-lactic acid-co-
glycolic acid), and poly(vinyl alcohol) have been widely used to fab-
ricate wound dressings [21,22]. Particularly, PCL (a FDA approved
material) has been often selected as a main component to produce
nanofibers due to its biodegradability, biocompatibility, spinnability,
and high toughness [23,24]. Unfortunately, for the lack of functional
groups, PCL was often incorporated with other polymeric materials or
natural materials as wound dressing, such as antibacterial agents and so
on [25–28]. The wound sites are often in a moist, warm and nutrient-
rich environment which increases the risk of bacterial infection of the
wound and further results in the prolonged and excessive inflammatory
response [29,30]. Besides, the wound healing process can also be dis-
turbed by other several factors, such as free radicals, necrosis, hypoxia,
and so on [31]. To address this issue, developing a new type of NFM
wound dressing with multifunctional properties including inherent
antibacterial activity, antioxidant ability, and electroactivity is highly
desirable.
Quaternized chitosan-graft-polyaniline (QCSP) successfully synthe-
sized in our previous work exhibited superior cytocompatibility and
antibacterial activity than pure quaternized chitosan because of the
combined antibacterial property of QCS and polyaniline [32]. It also
showed good electroactivity and can reduce excessive free radicals in
the wound sites which can lead to tissue damage, infection, and pro-
longed wound healing. We have further demonstrated that hydrogels
containing QCSP greatly accelerated the healing process of wound in a
mouse full-thickness wounds defect model, indicating that QCSP is an
excellent component for wound dressing. However, designing of elec-
troactive nanofiber wound dressing based on quaternized chitosan-g-
polyaniline and PCL as a novel multifunctional wound dressing has not
been reported.
By combining the good mechanical property of PCL and multi-
functionality of QCSP, a series of antibacterial, electroactivity, anti-
oxidant nanofiber wound dressing were designed and produced in this
work, which showed biomimetic multifunctional features for wound
healing application. The healing efficiency of PCL/QCSP NFM wound
dressings was further evaluated by performing a mouse full-thickness
wounds defect model. The nanofiber wound dressings were fabricated
by electrospinning PCL/QCSP polymer solutions at a high DC voltage.
The morphology, chemical structure, mechanical properties,
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Chemical Engineering Journal 385 (2020) 123464
electroactivity, and antioxidant ability were systematically character-
ized. Moreover, the antimicrobial activity of these nanofiber wound
dressings was assessed by performing an in vitro surface antibacterial
experiment. The blood compatibility and cytocompatibility of PCL/
QCSP NFMs were evaluated by co-culturing with red blood cells (RBC)
and mouse fibroblasts (L929), respectively. Furthermore, quantitative
statistical analysis of wound contraction, histopathological examina-
tions, collagen content, and immunofluorescence staining was em-
ployed to investigate the therapeutic efficacy of PCL/QCSP NFM wound
dressings. All the results demonstrated that these electroactive anti-
bacterial NFM wound dressings with suitable mechanical properties
and good free radical scavenging capacity have enormous potential as a
novel biomaterial for wound healing application.
2. Materials and method
2.1. Materials
Chitosan (CS) with a molecular weight of 100–300 kDa was ob-
tained from J&K Scientific Ltd. Poly(ε-caprolactone) (Mn = 80 kDa),
acetic acid (AcOH), glycidyltrimethylammonium chloride (GTMAC),
hydrochloric acid (HCl), ammonium persulfate, sodium hydroxide
(NaOH), and trifluoroacetic acid (TFA) were obtained from Sigma. All
other materials or chemical reagents were used without further pur-
ification unless otherwise stated.
2.2. Synthesis of quaternized chitosan grafted polyaniline (QCSP)
QCSP was synthesized according to the references with modification
[32]. Materials and methods in Supporting Information (SI) shows
specific synthesis procedures of QCSP.
2.3. Preparation of PCL/QCSP polymer solution
PCL/QCSP NFMs were fabricated by electrospinning. Briefly, PCL
was dissolved in AcOH to form 15% (wt/vol) solution. QCSP was dis-
solved in TFA at the concentration of 1.5–7.5 wt% overnight at room
temperature. The electrospinning polymer solutions were obtained by
mixing PCL with QCSP polymer solution in a ratio of 2:1 (v/v) at 25 °C
under vigorous stirring for 15 min. The samples with the QCSP contents
(weight ratio) of 0%, 5%, 10%, 15% and 20% to PCL/QCSP NFMs were
named as PCL/QCSP0, PCL/QCSP5, PCL/QCSP10, PCL/QCSP15 and
PCL/QCSP20, respectively.
2.4. Electrospinning parameters
The specific parameters for electrospinning were set based on our
previous study with some modifications [33]. Briefly, the electrospin-
ning polymer solution was fed into a 5 mL glass syringe fitted with a
gauge 19 stainless steel needle as the nozzle. The positive voltage and
negative voltage were set to 17 kV and −4 kV, respectively. The tip-to-
collector distance and flow rate of feed solutions were fixed at 15 cm
and 0.04 mL h−1, respectively. Materials and methods in SI shows de-
tailed procedures.
2.5. Characterizations
The physical and chemical characterizations of the NFMs including
Fourier transform infrared spectroscopy (FT-IR), scanning electron
microscope (SEM), mechanical properties, and swelling ratio of PCL/
QCSP NFMs was assessed according to our previous studies, and the
detailed procedures were shown in Materials and methods in SI
[34–37].
J. He, et al.
2.6. Electroactivity of PCL/QCSP NFMs
Cyclic voltammetry of PCL/QCSP was carried out on electro-
chemical Workstation (CHI 660D Instruments) to assess electroactivity
[38]. Briefly, the electrospinning polymer solution was dropped on the
surface of an indium tin oxide (ITO) glass and then placed at 45 °C for
4 h to obtain the electroactive PCL/QCSP film. The electrochemical
property of these PCL/QCSP NFMs was performed by using an Elec-
trochemical Workstation (CHI 660D Instruments) with a scan rate of
25 mV s−1. Materials and methods in SI shows detailed procedures.
2.7. Anti-oxidant ability of PCL/QCSP NFMs
The anti-oxidant abilities of PCL/QCSP samples were tested by
scavenging efficiency for the stable 1, 1-diphenyl-2-picrylhydrazyl
(DPPH) free radical according to our previous study [9]. Materials and
methods in SI shows specific procedures.
2.8. Surface antibacterial activity
The antibacterial activity of all PCL/QCSP NFMs was measured by a
surface antibacterial activity test by against both Staphylococcus aureus
(ATCC 29213) and Escherichia coli (ATCC 8379), which are re-
presentatives of gram-positive and gram-negative bacteria, respec-
tively. In brief, the sterilized PCL/QCSP NFMs were placed 24-well
culture plate. 10 μL of bacterial suspension in sterilized PBS was added
onto the surface of PCL/QCSP NFMs. The inoculated PCL/QCSP NFMs
were incubated at 37 °C in a relative humidified atmosphere (not less
than 90%) to evaluate the antibacterial property of these wound dres-
sings. The bacteria-killing ratios of PCL/QCSP NFMs were calculated by
the following equation:
bacteria count of control − survivor count on PCL/QCSPNFMs
kill% =
bacteria count of control
× 100%
The detail procedures for assessing the surface antibacterial activity
of PCL/QCSP NFMs are available in Materials and methods in SI.
2.9. Hemolytic activity assay of PCL/QCSP NFMs
The blood compatibility of PCL/QCSP NFMs was evaluated ac-
cording to hemolysis activity assay by using the mouse blood [39,40].
Materials and methods in SI shows specific procedures.
2.10. Cytocompatibility evaluation of PCL/QCSP NFM wound dressings
The cytotoxicity was evaluated to assess the cytocompatibility of all
PCL/QCSP NFMs by a direct contact test between the NFM groups and
the L929 cells (mouse fibroblast cells) [41–43]. Materials and methods
in SI shows specific procedures.
2.11. In vivo wound healing in a mouse full-thickness wounds defect model
A mouse full-thickness wounds defect model was conducted to as-
sess the in vivo wound healing capacity of PCL/QCSP NFMs according
to the publications [9,44]. Female Kunming mice (30–40 g, 5–6-week
age) were employed to conduct the in vivo experiments. All mice were
randomly divided into 3 groups including the group of Tegaderm™ film,
PCL/QCSP0, and PCL/QCSP15. Each group contained 15 mice. The
process of wound regeneration was studied by wound area monitoring.
Materials and methods in SI shows specific procedures about animal
surgery. All animal experiments were performed in accordance with
current guidelines for the care of laboratory animals and were approved
by the animal research committee of Xi'an Jiaotong University.
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Chemical Engineering Journal 385 (2020) 123464
2.12. Histological analysis
Histomorphological determination during different phases of wound
regeneration was carried out by Hematoxylin-Eosin (H&E) staining for
assessment of the inflammation and epidermal regeneration in wound
area. Briefly, the regenerated wound tissue collected on 3rd, 7th, and
14th day were fixed with 4% paraformaldehyde for 24 h. The sample
was then embedded in paraffin and cross sectioned to 40-μm thick
slices. The slices were stained with Hematoxylin-Eosin (Beyotime,
China) and analyzed and photo-captured by microscope (IX53,
Olympus, Japan). Materials and methods in SI shows specific proce-
dures about animal surgery.
2.13. Collagen deposition analysis
Commercial kits (Jiancheng bioengineering, China) was used to
estimate the content of hydroxyproline to evaluate the amount of col-
lagen in the wound. Briefly, the regenerated wound tissue collected on
the 3rd, 7th, and 14th day at the weight of 30 mg were made into disc-
shaped tissue (diameter = 1 cm), and collagen amount was evaluated
by estimating hydroxyproline content using a commercial kit. All op-
erations were carried out strictly in accordance with manufacturer's
instructions. Materials and methods in SI shows specific procedures.
2.14. Immunohistochemistry examination
The wound sections were immunohistochemistry stained with VEGF
and TNF-α to evaluate inflammation and blood vessels regeneration
under a standard protocol [12]. Briefly, for immunofluorescence
staining, the fixed and frozen regenerated wound tissue sections col-
lected on the 3rd, 7th, and 14th day were stained with TNFA antibody
(5 μg/mL) and Anti-VEGFA antibody (5 μg/mL). FITC-conjugated goat
anti-mouse IgG (cwbiotech) and FITC-conjugated goat anti-rabbit IgG
(cwbiotech) were used as the secondary antibody to reveal TNF-α and
VEGF expression. The nuclei were stained with DAPI containing
mounting solution. Slides were observed under an inverted fluorescence
microscope. Materials and methods in SI shows specific procedures.
2.15. Statistical analysis
All the results were analyzed statistically and expressed as a
mean ± standard deviation (SD). One-way ANOVA was used to de-
termine statistical differences and followed by a Bonferroni post hoc
test for multiple comparisons with SPSS, version 24 (IBM). Differences
were considered significant if P < 0.05.
3. Results and discussions
3.1. Fabrication and characterization of PCL/QCSP NFM wound dressings
A series of PCL/QCSP NFMs with suitable mechanical properties,
electroactivity, anti-oxidant ability, good biocompatibility, and ex-
cellent antibacterial activity were prepared for wound healing appli-
cation in this study (Fig. 1a). With PCL as the main component, con-
tinuous fibers with diameters ranging from 60 to 200 nm were prepared
with the increase of QCSP content in PCL/QCSP NFMs. Furthermore,
with the addition of QCSP, the NFM wound dressings were endowed
with anti-bacterial, anti-oxidant, and electroactive property. It had been
demonstrated that electroactive scaffolds are conducive to the elec-
trically excitable cell’s proliferation and differentiation, such as osteo-
blasts, fibroblasts, neurons, myoblasts, and cardiomyocytes
[9,34,45–47]. QCSP polymer was firstly synthesized and then mixed
with PCL under vigorous stirring for 15 min before electrospinning. The
detailed parameters of each PCL/QCSP NFMs are shown in Fig. 1b. All
the NFMs exhibited good stability according to testing the mass loss by
immersing each PCL/QCSP NFM in PBS at 37 °C (Fig. S1), which was
J. He, et al. Chemical Engineering Journal 385 (2020) 123464

Fig. 1. (a) Schematic diagram of production and characteristics of PCL/QCSP NFMs; (b) Parameters of each PCL/QCSP NFM samples; (c) FT-IR spectra of CS, QCSP,
PCL, and PCL/QCSP20 NFM; (d) SEM images of pure PCL and PCL/QCSP nanofibers at low magnification (5000×); (e) SEM images of pure PCL and PCL/QCSP
nanofibers at high magnification (20000×); (f) Diameter distribution of pure PCL and PCL/QCSP nanofibers.

mainly because PCL chains in PCL/QCSP polymer solution can make
strong entanglement with QCSP chains. The chemical structure of PCL/
QCSP NFMs was confirmed by FT-IR as shown in Fig. 1c. The char-
acteristic peak of PCL at about 1722 cm−1 is attributed to carbonyl
stretching, and –COC– is seen at 1240 cm−1 and 1290 cm−1. The
characteristic peak of chitosan (CS) and QCSP at about 1640 cm−1 is
attributed to carbonyl stretching vibration of acetylated amino group.
The peak corresponding to the methyl band of GTMAC at 1475 cm−1
was present in the spectra of QCSP, indicating that GTMAC was suc-
cessfully conjugated to the amine group on chitosan backbone. A pair of
characteristic peaks at about 1572 cm−1 and 1490 cm−1 are attributed
to quinine diimine ring and benzonoid diamine ring stretching of
polyaniline in QCSP. The FT-IR spectrum of PCL/QCSP20 shows all
these characteristic peaks. The morphologies of PCL/QCSP NFMs at
different magnification were observed by SEM (Fig. 1d, e). A series of
nanofiber wound dressings with a smooth surface and random or-
ientation were fabricated successfully. The nanofiber diameters of pure
PCL and PCL/QCSP nanofibers were 91 ± 24 nm (PCL/QCSP0),
135 ± 33 nm (PCL/QCSP5), 169 ± 38 nm (PCL/QCSP10),
174 ± 38 nm (PCL/QCSP15), and 175 ± 35 nm (PCL/QCSP20), re-
spectively (Fig. 1f).

4
J. He, et al.
Fig. 2. Mechanical properties and swelling ratio of PCL/QCSP NFMs. (a) Tensile strain; (b) Tensile stress; (c) Young's modulus; (d) Stress-strain profile of PCL/QCSP
NFMs; (e) Swelling ratio of PCL/QCSP NFMs in PBS at 37 °C.
3.2. Mechanical properties and swelling behavior of PCL/QCSP NFMs
The essential requirements of the suitable multifunctional wound
dressing materials should include the desirable mechanical properties
and water uptake ability to provide protection on the wound site and
absorb the effluence to promote the healing process of wound [12,48].
For those joints that move and bend frequently like knee and ankle,
wound dressing should match the extensibility of the native skin to
maintain integrity of the dressing and prolong their lifespan during
wound healing process. Fig. 2a and b shows the tensile strain, tensile
stress and Young's modulus of PCL/QCSP NFMs, respectively. These
wound dressings have suitable stretchability between 81.7% and 48.1%
(Fig. 2d), which is close to human skin (75–60%) except for PCL/QCSP0
[49]. PCL/QCSP5 with the lowest QCSP content exhibited the highest
elongation-at-break (81.7%). Moreover, with the QCSP content in-
creasing, the elongation-at-break of PCL/QCSP NFMs were decreased
gradually. However, compared with PCL/QCSP0 NFM, the Young’s
modulus of PCL/QCSP samples obviously decreased with the addition
of QCSP in PCL/QCSP nanofibrous matrix, indicating that QCSP de-
creased the PCL/QCSP nanofibrous membrane’s modulus. The Young’s
modulus of PCL/QCSP NFMs ranged from 2.4 MPa to 4.9 MPa, which
are comparable to human soft tissue [50].
Swelling behavior of PCL/QCSP NFMs was determined in PBS at
37 °C (Fig. 2e). All the groups reached a swelling equilibrium within
3 h. The lowest water absorption about 70% was observed for the group
of PCL/QCSP20 after 3 h, and 130%, 120% and 100% for PCL/QCSP5,
PCL/QCSP10, and PCL/QCSP15, respectively, suggesting that the
swelling ratio decreased with increasing the content of QCSP in PCL/
QCSP NFMs. The water uptake ability of PCL/QCSP NFMs indicates that
these wound dressings can absorb the exudate from the wound, sug-
gesting that these dressings have great potential as wound dressings.
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Chemical Engineering Journal 385 (2020) 123464
3.3. Electrochemical property and antioxidant ability of PCL/QCSP NFMs
Electroactive polymers such as polyaniline, polypyrrole, and poly-
thiophene with the ability to regulate cellular activities of electrically
excitable cells bring a novel focus on designing biomaterials for skin
wound repair [51–53]. For example, fibroblast and myoblast cells,
which are one of the key players for orchestrating physiological tissue
repair, could adhere and proliferate on the electroactive polymers
based wound dressings and then to accelerate the wound healing pro-
cess [54–56]. It is known that electroactive materials can become
electrical conductors by reversible ion exchange between reduction
state and oxidation state [57]. Cyclic voltammetry measurement was
conducted to assess the electroactivity of PCL/QCSP NFMs. As shown in
Fig. 3a, two pairs of the oxidation/reduction peaks appeared at about
0.20 V, and 0.58 V, respectively. The first peak at 0.20 V was due to the
leucoemeraldine to emeraldine state transition, while the second peak
at 0.58 V was attributed to the emeraldine oxidation state to the per-
nigraniline state. The cyclic voltammograms of PCL/QCSP NFMs are
similar to each other differing only by the broadness of the oxidation/
reduction peaks, which shows QCSP dependence, and there is no oxi-
dation/reduction peak in PCL/QCSP0 NFM (Fig. S2). All the results
above demonstrated that these QCSP contained PCL/QCSP NFMs pos-
sess good electroactivity, indicating that they have great potential for
the treatment of skin wounds [9,36,58].
In the inflammatory phase of wound healing, neutrophils, leuco-
cytes, and monocytes will be attracted to the wound sites by biologi-
cally active mediators and then attack the microorganisms and foreign
debris via phagocytosis. This process will lead to the overproduction of
free radicals including superoxide anion, hydrogen peroxide, and hy-
droxyl anion [59]. The excessive level of these free radicals will induce
the disruption of the cellular oxidant/antioxidant balance and cause the
inactivation of enzyme, DNA breakage, and lipid peroxidation, which
further result in the extensive collateral damage to the surrounding skin
J. He, et al.
Fig. 3. (a) Electrochemical property of PCL/QCSP20 NFM; (b) Antioxidant ability of PCL/QCSP15 NFM dispersion liquid. *p < 0.05, **p < 0.01.
tissue over the wound sites and causing a prolonged process of wound
[60,61]. Therefore, an important strategy to enhance the healing pro-
cess of wound is to reduce/scavenge the free radicals around the wound
sites by the introduction of antioxidant agents, thereby to protect the
cells or tissues from damage and then to accelerate the wound healing
process [62,63]. Herein, we tested the scavenging efficiency for DPPH%
to evaluate the antioxidant abilities of these PCL/QCSP NFMs and PCL/
QCSP15 was taken as an example (Fig. 3b). The results suggested that
the antioxidant ability of PCL/QCSP15 NFM increased with the in-
creasing concentration of QCSP and almost 70% of free radicals can be
cleared by 6 mg mL−1 of PCL/QCSP15 dispersion liquid. Besides, the
scavenging efficiency for DPPH% more than 80% was achieved when
the content of PCL/QCSP15 dispersion liquid reached to 8 mg mL−1.
These data demonstrated the good antioxidant ability of these PCL/
QCSP NFMs. It has been confirmed that the dressings with antioxidant
ability could significantly accelerate the in vivo wound healing process
[64–66]. Thus, these polyaniline-contained PCL/QCSP NFMs, as an
anti-oxidant wound dressing, have a great potential in wound healing
application.
3.4. Antibacterial activity of PCL/QCSP NFMs
It is well known that bacterial infections may result in increased
exudate at the wound site and delay the healing process of wound
[67,68]. Hence, an optimum wound dressing material should possess
inherent antimicrobial properties to increase the wound healing rate by
casting down the number of pathogens and reducing the inflammatory
response of wounds [9,60]. A surface antibacterial activity test was
used to evaluate antibacterial activity of all PCL/QCSP NFMs by against
both S. aureus and E. coli in this study. Firstly, the antibacterial test was
conducted with 105 CFU mL−1 of the bacterial suspensions and all PCL/
QCSP NFM groups showed excellent killing ratio (> 90%) for both S.
aureus and E. coli expect for PCL/QCSP0 NFM group (Fig. 4a, c). Thus,
to further detect the difference of antibacterial properties among these
NFM wound dressings, 106 CFU mL -1 of the bacterial suspension was
used in the following test and the results were shown in Fig. 4b and d.
The lowest bacteria-killing ratio for S. aureus about 63% and about
71% for E. coli was observed for the group of PCL/QCSP5. Furthermore,
the killing ratio for S. aureus about 70%, 86%, and 92% and for E. coli
about 82%, 94%, and 95% were observed for PCL/QCSP10, PCL/
QCSP15, and PCL/QCSP20, respectively, indicating that the anti-
bacterial activity of the NFMs gradually increased with increasing the
content of QCSP in PCL/QCSP NFMs.
PCL/QCSP NFMs exhibited good antibacterial activity from QCSP.
QCS possessed positive-charged amino groups and quaternary
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Chemical Engineering Journal 385 (2020) 123464
ammonium groups which is effective for killing bacteria by electrostatic
adherence [32,69]. What’s more, for those gram-negative bacteria, the
expression of genes that are vital to bacterial survival can be down-
regulated with the existence of polyaniline, which will further affect the
formation of cell wall and the metabolism of energy, and eventually
lead to the death of those gram-negative bacteria [70–72]. Hence, the
synergetic antibacterial activity of PCL/QCSP NFMs was assigned to the
complementary effect of positive-charged amino groups, quaternary
ammonium groups, and acidified polyaniline. Beyond this, PCL/
QCSP15 and PCL/QCSP20 groups with good antibacterial activity
suggested that they have great potential to effectively prevent bacterial
infection on the wound.
3.5. Hemocompatibility and cytocompatibility of PCL/QCSP NFM
Biomedical materials possessing good biocompatibility is the pre-
requisite for novel wound dressing [73]. Thus, we tested hemo-
compatibility and cytocompatibility of the NFMs to estimate bio-
compatibility of these NFM wound dressings. The in vitro hemolysis test
was first carried out. The macroscopical color of all the five NFM
groups, negative control group (PBS), and positive control group
(Triton X-100) were shown in Fig. 5a. All the NFM groups presented
light yellow that was similar to the negative control group, while the
positive control group was bright red. Furthermore, the quantitative
data of hemolysis test was shown in Fig. 5b. Hemolysis ratio of all the
NFM wound dressings decreased with the QCSP content decreasing
(1.09%–0.44%). compared with other NFM groups, PCL/QCSP20 ex-
hibited the highest hemolysis ratio (1.45%). All these data indicated
that all the NFM groups showed excellent hemocompatibility for wound
healing application.
PCL/QCSP NFMs have great potential to promote the proliferation
of electrically excitable cells due to the good electroactivity of QCSP
itself. Thus, the proliferation of L929 cells was assessed by a direct
contact test, PCL/QCSP0 NFM was taken as control by seeding the same
initial density. The results of Alamar-Blue assay exhibited a significant
growth trend from the 1st to 5th day (Fig. 5c), indicating that the L929
cells had good growth throughout the whole experiment. There was no
obvious difference in cell viability between the QCSP contained groups
and PCL/QCSP0 group on the first day. After 3 days of incubation, the
cell number with two or three times increase except for PCL/QCSP20,
indicating that PCL/QCSP20 group has negative effect on L929 cells.
Besides, compared with PCL/QCSP0 group (P < 0.01) and other QCSP
contained groups, PCL/QCSP15 group showed the highest cell pro-
liferation on day 3. On the fifth day, PCL/QCSP5, PCL/QCSP10, and
PCL/QCSP15 groups exhibited higher cell number than PCL/QCSP0
J. He, et al.
Fig. 4. Antibacterial activity for S. aureus of PCL/QCSP NFMs with the bacterial suspension at the concentration of 105 CFU mL−1 (a) and 106 CFU mL−1 (b);
Antibacterial activity for E. coli of PCL/QCSP NFMs with the bacterial suspension at the concentration of 105 CFU mL−1 (c) and 106 CFU mL−1 (d), *p < 0.05,
**p < 0.01.
and PCL/QCSP20 (P < 0.01), and there was no difference among the
group of PCL/QCSP5, PCL/QCSP10, and PCL/QCSP15. All these results
confirmed that the PCL/QCSP5, PCL/QCSP10, and PCL/QCSP15 had
good biocompatibility and the ability to enhance the proliferation of
L929 cells.
A series of PCL and PCL/QCSP15 NFM wound dressings extracts
with concentrations ranging from 0 to 20 mg/mL were obtained after
soaking each NFMs with cell culture medium for 72 h to further eval-
uate the biocompatibility of NFM wound dressings (Fig. 5). There was
no difference in cell number between all the five different concentra-
tions of extracts, which confirmed that the extracts from PCL/QCSP15
had no cytotoxic leaching content. Besides, most of L929 cells were
green in all the NFM’s extract groups after 24 h incubation and showed
a spindle-like morphology, which was consistent with the quantitative
data of alamarBlue® assay for wound dressing extracts of PCL/QCSP15
NFM. Overall, all these experiment results demonstrated that all these
NFM wound dressings had good biocompatibility except for PCL/
QCSP20. PCL/QCSP15 group with balanced property of antibacterial
activity and cell proliferation was chosen for further evaluation of
wound healing performance in vivo.
3.6. In vivo wound healing
All the above results indicated that the physical properties will be
significantly affected by the introduction of QCSP. The surface anti-
bacterial activities of PCL/QCSP NFMs also exhibited a QCSP depen-
dence and a more efficient antibacterial effect presented in the group
7
Chemical Engineering Journal 385 (2020) 123464
containing a higher content of QCSP. However, the cytotoxicity of PCL/
QCSP NFMs will also increase with QCSP content increasing.
Fortunately, PCL/QCSP15 NFM with a good balance between anti-
bacterial properties and cytocompatibility have great potential in
treatment of skin wounds. Thus, the wound healing performance of the
PCL/QCSP NFMs was further evaluated in a mouse full-thickness
wounds defect model. PCL/QCSP15 with a suitable effect for cell pro-
liferation and prominent antibacterial activity was selected as a re-
presentative. PCL/QCSP0 and Tegaderm™ film were taken as control
groups. On the 3rd, 7th, and 14th day, PCL/QCSP15 group showed the
advantageous wound contraction over Tegaderm™ film group and PCL/
QCSP0 group (Fig. 6a-c). In detail, on the 3rd day, the wound areas of
all the groups reduced to some extent after the surgery, the largest
wound contraction area (43%, P < 0.01) was presented in PCL/
QCSP15 group, suggested that it had a higher effect on promoting
wound healing. On the 7th day, PCL/QCSP15 group and PCL/QCSP0
group showed significant difference (P < 0.01) with Tegaderm™ film
group, while the wound contraction of PCL/QCSP15 group was higher
than the group of PCL/QCSP0 (P < 0.05), indicating that both two
PCL/QCSP NFM groups had better therapeutic efficacy than Tegaderm™
film group. On the 14th day, the wounds treated with PCL/QCSP15
NFM or PCL/QCSP0 NFM were observed to have closure and had about
9% lead than Tegaderm™ film group in wound contraction (P < 0.05).
Besides, even the regenerated skin of some mice had presented hair
after treated with PCL/QCSP15 for 14 days. These quantitative data
about the wound area demonstrate that PCL/QCSP15 NFM had better
wound healing effect than PCL/QCSP0 group and Tegaderm™ film
J. He, et al. Chemical Engineering Journal 385 (2020) 123464

Fig. 5. Hemocompatibility and cytocompatibility analysis of PCL/QCSP NFM wound dressings. (a) Images of RBC; (b) Hemolysis ratio (%) of the NFMs by direct
contact with the nanofiber wound dressings for 1 h; (c) Quantitative analysis of L929 cell proliferation by a direct contact with PCL/QCSP wound dressings and PCL/
QCSP0 was taken as a control, *p < 0.05,**p < 0.01; (d) Quantitative analysis of L929 cell viability in the extracts of PCL and PCL/QCSP15 for 3 days at different
concentrations, *p < 0.05,**p < 0.01; (e) LIVE/DEAD staining of L929 cells after incubated with the extracts for 24 h.

group. Our previous work had confirmed that all the healing stages can
be accelerated by the wound dressing based on QCSP, which was due to
the synergistic effect of QCS and polyaniline [9]. Beyond that, wound
healing process was further accelerated in the midanaphase due to
antioxidative wound dressing [74]. Compared with Tegaderm™ film
group, PCL/QCSP0 group showed better therapeutic effect throughout
the whole healing stages because the NFMs with appropriate porosity
have the permeable ability to moisture and air, and maintain a partially
immobilized moist environment by absorbing the effluence from the
wound area [75]. Thus, PCL/QCSP15 NFM with suitable physical
structure and chemical properties could promote the wound healing
and showed better therapeutic efficacy than Tegaderm™ film and PCL/
QCSP0 NFM on wound healing.
3.7. Histological analysis of wound regeneration
Histological analysis was performed to further distinguish the
quality of the regenerated tissue in the wounds among commercial
Tegaderm™ film, PCL/QCSP0, and PCL/QCSP15 groups by H&E
staining, the indicators mainly included inflammatory cells infiltration,
fibroblast immigration, connective tissue synthesis, and re-epitheliza-
tion [76]. The mild acute inflammatory responses were shown on the
3rd day in Fig. 7a, fibroblasts and inflammatory cells migrated to the
wound site in these three groups. What’s more, compared with Tega-
derm™ and PCL/QCSP0 group, PCL/QCSP15 group exhibited less in-
flammatory cells and more fibroblast cells around the impaired region,
which may be attributed to the antioxidant and electroactivity of PCL/
QCSP15 NFM [9]. These results indicated that PCL/QCSP15 group with
the ability to partially suppress inflammatory cell infiltration and ac-
celerate wound healing process among these three groups. With the
further healing of the wounds, all three groups presented less in-
flammatory cells on the 7th day, and a layer of epithelium was formed
in the group of PCL/QCSP15 and PCL/QCSP0. Compared with Tega-
derm™ film and PCL/QCSP0 group, PCL/QCSP15 group exhibited a
higher regularity of both connective tissue and epithelium. On the 14th
day, Tegaderm™ film group showed the basic structure of regenerated
wound tissue including epithelium and dermis, while PCL/QCSP0
group showed a few hair follicles formation. Importantly, PCL/QCSP15
group showed the maximum amount of well-proliferated fibroblast,
thickened epidermis, hair follicles, and mature blood vessels, suggested
that PCL/QCSP15 had the best wound healing performance among
these thress groups. In summary, PCL/QCSP15 group showed a higher
density of fibroblasts (on the 3rd day), completely formated epithelium
structure (on the 7th day), and high differentiated epithelial tissue
which was closer to normal skin (on the 14th day), exhibiting the
prominent wound healing performance. PCL/QCSP0 group showed a

8
J. He, et al.
Fig. 6. (a) Photographs of the wound closure on the 3rd, 7th and 14th day for commercial Tegaderm™ film, PCL/QCSP0, and PCL/QCSP15 NFM; (b) The schematic
diagram of wounds closure on the 3rd, 7th and 14th day; (c) Quantitative statistical analysis of wounds closure for commercial Tegaderm™ film, PCL/QCSP0, and
PCL/QCSP15 NFM treatment. *P < 0.05, **P < 0.01.
better therapeutic effect than Tegaderm™ film group.
Granulation tissue consists of some growth factors, fibroblasts, ac-
cumulated ECM, and other cells [77], which can also work with par-
enchymal cells in the repair process of some inflammatory injury [78].
Thus, an important indicator in wound healing process is the thickness
of granulation tissue, which can evaluate the therapeutic effect of the
wound dressing. After 14 days of surgery, the granulation tissue in PCL/
QCSP15 group exhibited approximately 225 mm thicker than that in
the group of PCL/QCSP0 (P < 0.05), while the granulation tissue of
the Tegaderm™ film group was thinner than all the NFM groups (Fig. 7b
and c). In one word, according to in vivo experimental studies, PCL/
QCSP15 showed the best wound healing performance among these
three groups.
3.8. Collagen deposition analysis
One of the important markers of wound healing is the total collagen
level. Due to the metabolism of collagen, the therapeutic effects were
evaluated by analyzing the hydroxyproline content, because the hy-
droxyproline is a unique component in collagen fibers and possesses a
constant mass of about 13.4% [79]. Fig. 7d shows that the concentra-
tion of hydroxyproline in all groups increased continuously in the first
7 days and maintained at a stable level on the 14th day over the whole
healing stage. Compared with Tegaderm™ film group, PCL/QCSP NFM
groups exhibited higher collagen level during the 14 days (P < 0.01),
indicating that PCL/QCSP NFM groups had a better therapeutic effect
than Tegaderm™ films, and PCL/QCSP15 group showed the best wound
healing performance through the whole experiment. In conclusion, by
combining the results of H&E staining, the thickness of granulation
tissue, and the deposition of collagen, PCL/QCSP15 group exhibited the
superior therapeutic effects than Tegaderm™ film and PCL/QCSP0
group, which illustrated that the PCL/QCSP15 wound dressing greatly
increased the healing process of wound.
3.9. TNF-α and VEGF expression in regenerated wound tissue
During the wound healing process, many required cellular activities
were adjusted by cytokines, which mainly regulated the initiation and
9
Chemical Engineering Journal 385 (2020) 123464
cessation of the cell around wound bed [80]. Thus, we selected tumor
necrosis factor-α (TNF-α) as an indicator to verify the treatment effi-
cacy of PCL/QCSP NFMs in preventing infection by immuno-
fluorescence staining. Compared with the Tegaderm™ film and PCL/
QCSP0 NFM group, PCL/QCSP15 group showed the lowest expression
of TNF-α on day 7 and 14, PCL/QCSP0 group showed lower TNF-α
expression than Tegaderm™ group during the whole wound healing
stages (Fig. 8a), which was probably due to the introduction of QCS and
polyaniline that can endow the antibacterial ability to PCL/QCSP NFM
wound dressings to reduce inflammation. In addition to preventing
infection, angiogenesis of the wound was further estimated. Vascular
endothelial growth factor (VEGF) was selected as an indicator to
evaluate wound repair by immunohistochemical staining of regenera-
tion wound sections on day 7 and 14. The vascular angiogenesis in PCL/
QCSP15 group appeared grossly more VEGF expression than Tega-
derm™ film and PCL/QCSP0 NFM group on day 7 (P < 0.05) (Fig. 8).
Besides, VEGF expression was lowest in the Tegaderm™ film group and
there was no difference in the group of PCL/QCSP0 and PCL/QCSP15
on the 14th day. In conclusion, by synchronously reducing the pro-
duction of TNF-α and upregulating expression of VEGF, PCL/QCSP15
NFM group significantly accelerated the wound healing process than
Tegaderm™ films.
4. Conclusion
A series of antibacterial anti-oxidant and electroactive nanofiber
membranes were developed by electrospinning of PCL/QCSP polymer
solutions, and in vivo experiments demonstrated that the wound
healing process can be significantly accelerated by these multi-func-
tional NFM wound dressings. PCL/QCSP NFMs also exhibited tunable
water absorption and modulus by tuning the concentration of QCSP to
PCL/QCSP NFMs. Hemocompatibility and cytocompatibility were
confirmed by a co-culture with erythrocytes or L929 cells, respectively.
For the in vivo wound healing test, PCL/QCSP15 NFM exhibited a faster
wound healing rate than Tegaderm™ Film and PCL/QCSP0 NFM.
Moreover, the wound treated with PCL/QCSP15 NFM showed fewer
inflammatory infiltration, higher density of fibroblasts, thicker granu-
lation tissue thickness and collagen deposition. The
J. He, et al. Chemical Engineering Journal 385 (2020) 123464

Fig. 7. (a) Histological analysis (H&E staining) of wound regeneration with different treatment of Tegaderm™ film dressing, PCL/QCSP0, and PCL/QCSP15 NFM
wound dressing on the 3rd, 7th, and 14th day, green lines show the boundary of epidermis and dermis; (b) Images of granulation tissue thickness on the 14th day; (c)
Quantitative statistical analysis of granulation tissue thickness for each treatment of Tegaderm™ film dressing, PCL/QCSP0 NFM, and PCL/QCSP15 NFM on the 14th,
*P < 0.05, **P < 0.01; (d) Collagen content in Tegaderm™ film, PCL/QCSP0 group, and PCL/QCSP15 group by testing hydroxyproline concentration. *P < 0.05,
**P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

immunofluorescence staining of TGF-α and VEGF indicated the lowest are potential candidates for treatment of cutaneous skin wound healing.
production of TGF-α and the highest expression of VEGF after treated
with PCL/QCSP15 NFM for 7 days, which meant that PCL/QCSP15
Declaration of Competing Interest
wound dressing promoted the regeneration of blood vessel and reduced
the production of pro-inflammatory factor around the wound site. In
The authors declare that they have no known competing financial
summary, these multifunctional PCL/QCSP nanofiber wound dressings
interests or personal relationships that could have appeared to

10
J. He, et al. Chemical Engineering Journal 385 (2020) 123464

Fig. 8. Pictures of the regenerated wound tissue on 7th, and 14th day by immunofluorescence staining labeling with (a) TNF-α (green) and (b) VEGF (green), yellow
arrows show the expression of TNF-α and red arrows present VEGF; Quantitative statistical analysis of TNF-α-actinin (c) and VEGF (d) relative area percentage; For
all quantitative analysis, Tegaderm™ Film group on 7th day was taken as 100%, *P < 0.05, **P < 0.01. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

influence the work reported in this paper.
Acknowledgment
This work was jointly supported by the National Natural Science
Foundation of China (grant numbers: 51673155 and 51973172), State
Key Laboratory for Mechanical Behavior of Materials, the Fundamental
Research Funds for the Central Universities, and the World-Class
Universities (Disciplines) and the Characteristic Development Guidance
Funds for the Central Universities, Natural Science Foundation of
Shaanxi Province (No. 2018JM5026, and 2019TD-020), and Opening
Project of Key Laboratory of Shaanxi Province for Craniofacial Precision
Medicine Research, College of Stomatology, Xi’an Jiaotong University
(No. 2019LHM-KFKT008).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.cej.2019.123464.
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