Carbohydrate Polymers 232 (2020) 115786

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Wound dressing from polyvinyl alcohol/chitosan electrospun fiber T

membrane loaded with OH-CATH30 nanoparticles
Pengfei Zoua, Wen-Hui Leeb,c, Zhiqin Gaoc, Di Qinc, Yuxia Wanga, Jiao Liua, Tongyi Sunc,**,
Yuanyuan Gaoa,*
a
School of Pharmacy, Weifang Medical University, Weifang, 261053, Shandong, China
b
Key Laboratory of Bioactive Peptide of Yunnan Province/Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences,
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China
c
Key Laboratory of Biological Medicine in Universities of Shandong Province, School of Bioscience and Technology, Weifang Medical University, Weifang, 261053,
Shandong, China

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

Keywords: Novel nanomaterials have been developed for antimicrobial and wound healing applications. Here, we report the
OH-CATH30 preparation of a polyvinyl alcohol/chitosan (PVA/CS) nanofiber with carboxymethyl chitosan nanoparticles
PVA (CMCS-OH30 NPs) encapsulating the antibacterial peptide OH-CATH30 (OH-30). The PVA/CS nanofibers con-
Wound healing taining OH-30 NPs (NP-30-NFs) obtained via electrospinning could achieve a secondary embedded OH-30. The
Nanofibers
effect of NP-30-NFs on the release of OH-30 was investigated through high-performance liquid chromatography.
Nanoparticles
The antibacterial activities of NP-30-NFs against Escherichia coli and Staphylococcus aureus were studied by
bacterial plate counting. NP-30-NFs containing different concentrations of NPs were applied to mouse skin
wounds to determine their effectiveness in promoting wound healing. Results showed that NP-30-NFs exhibited
antibacterial properties and promoted skin wound healing.

1. Introduction and cytokine production without eliciting harmful immune responses to

Surgery, burns, abrasions, and chronic ulcers are among the key
factors that cause skin trauma (Sun, Siprashvili, & Khavari, 2014). Skin
damage is often accompanied with self-repair of the skin. These repair
processes are essential for organismal homeostasis and survival (Rankin
& Artis, 2018). Skin tissue repair is a dynamic and complex process that
involves inflammation, angiogenesis, granulation tissue growth, foreign
body reaction, and re-epithelialization (Gao et al., 2018; Zhou et al.,
2016). Unfortunately, wound infections can easily occur during wound
healing, thereby delaying the healing process. Therefore, bacterial in-
fection is a major concern in the treatment of open wounds and burns
(Akia, Rodriguez, Materon, Gilkerson, & Lozano, 2019).
Antibacterial peptides (AMPs) of natural origin may be clinical
candidates because of their broad-spectrum antibacterial activity, dif-
ficulty in causing drug resistance, and regulatory effect on the body’s
immune system. As a possible candidate drug against clinical drug-re-
sistant isolates (Li, Lee, & Zhang, 2012, 2013), cathelicidin OH-CATH30
(OH-30) derived from king cobra selectively upregulates chemokine
induce a regulatory effect on inflammatory responses and to treat
various bacterial infections (Li et al., 2014; Zhang et al., 2010; Zhao
et al., 2018). Notably, the dynamic balance of inflammatory response
and reduced inflammation is an important guarantee for tissue repair
(Eming, Krieg, & Davidson, 2007; Rankin & Artis, 2018). AMPs, as
antibacterial agents, have great potential for wound dressing applica-
tions (Gomes, Mano, Queiroz, & Gouveia, 2015). However, limited
stability and bioavailability restrict their applications because AMPs are
easily hydrolyzed by proteases (Silva et al., 2015). Therefore, effec-
tively avoiding the enzyme degradation of AMPs and accurately con-
trolling their release are desired for active peptide nanomaterials (Silva
et al., 2015). A drug delivery carrier should be developed to protect
active peptides from proteases and release these peptides. We reported
that our prepared biodegradable drug nanoparticle (NP) CMCS-OH-30
delivery system can promote OH-30 sustained release. Our results also
demonstrated that the prepared CMCS-OH-30 NPs accelerate the
healing of skin lesions and reduce scars on mice (Sun et al., 2018).
The application of occlusive wound dressings is a routine external

⁎
Corresponding author at: School of Pharmacy, Weifang Medical University, Baotong Road, Weifang, 261053, Shangdong, China.
⁎⁎
Corresponding author at: Key Laboratory of Biological Medicine in Universities of Shandong Province, School of Bioscience and Technology, Weifang Medical
University, Baotong Road, Weifang, 261053, Shangdong, China.
E-mail addresses: sd_sty@126.com (T. Sun), yyg20062006@126.com (Y. Gao).

https://doi.org/10.1016/j.carbpol.2019.115786
Received 23 March 2019; Received in revised form 12 December 2019; Accepted 26 December 2019
Available online 28 December 2019
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
P. Zou, et al.
intervention that helps accelerate re-epithelialization and alters the
inflammatory milieu to favor healing (Sun et al., 2014). Traditional
wound dressings, such as gauze, bandages, and sponges, are used to
prevent bacterial invasion, but limited swelling capacity restricts their
applications (Fu et al., 2016). Modern wound dressings, such as hy-
drogels and nanofibers produced by electrospinning active components
derived from natural sources, have been widely explored to overcome
this issue in regenerative medicine and trauma (Zhou, Sui, Mo, & Sun,
2017). However, biological dressing that creates a moist environment
to promote wound healing is also an ideal place for the colonization and
reproduction of pathogenic microbes (Pei, Sun, Sun, Wang, & Zhao,
2015). Therefore, effectively reducing bacterial infection and creating a
relatively sterile microenvironment are important prerequisites for
developing an effective wound dressing. Stimulus-responsive CS and
poly (N-vinyl-2-pyrrolidone) (PVP) hydrogels for wound healing ap-
plication exhibit antibacterial activity (Rasool, Ata, & Islam, 2019), and
injectable hydrogels (Xan-CHO and NOCC) accelerate the reconstruc-
tion of the abdominal wall in rats (Huang et al., 2018). In addition,
wound dressings containing antibiotics (Garcia et al., 2017), non-
steroidal anti-inflammatory drugs, such as ibuprofen (Morgado, Miguel,
Correia, & Aguiar-Ricardo, 2017), titanium dioxide (El-Aassar, El
Fawal, El-Deeb, Hassan, & Mo, 2016), and silver nanoparticles (Ag-NPs)
(Das, Kumar, Patil, Viswanathan, & Ghosh, 2015; Kohsari, Shariatinia,
& Pourmortazavi, 2016; Lee et al., 2014; Mokhena & Luyt, 2017), have
shown good results against bacteria and are beneficial to wound
healing.
Electrospun nanofibers have been considered as applicable mate-
rials for wound dressing applications because of their intrinsic prop-
erties, such as high surface-area-to-volume ratio and swelling (Miguel
et al., 2019). Nanofibers in these membranes can act as drug delivery
systems (Gao et al., 2016), which prompt the incorporation of biomo-
lecules within the fiber structure to prevent skin infections and improve
healing (El-Aassar et al., 2016; Miguel et al., 2019). CS is a biode-
gradable natural polysaccharide that promotes blood clotting and pain
relief (Charensriwilaiwat et al., 2012). PVA is a water-soluble polymer
widely used in wound dressings because of its excellent film-forming
and mechanical properties (Fu et al., 2016; Zhao, Chen, Yao, & Li,
2017). Besides, blended fiber membranes composed of PVA and CS are
extensively used in various fields, such as tissue engineering scaffolds,
drug delivery (Miguel et al., 2019; Zarandi et al., 2015), new wound
dressings (Zhao et al., 2017; Zhou et al., 2013), heavy metal adsorption
(Habiba, Afifi, Salleh, & Ang, 2017), and air filtration sterilization
(Wang et al., 2018). Therefore, a PVA/CS system is expected to be a
good carrier of OH-30 and beneficial to the sustained release of re-
agents.
In this study, NP-30-NFs were used as dressing for mouse skin
wound to test the applicability of PVA/CS nanofibers as a drug delivery
carrier of OH-30 and wound dressing material via FTIR, SEM and LSCM.
We envisaged that NP-30-NFs could further enhance the bioavailability
and stability of OH-30. The release behaviour of OH-30 in NP-30-NFs
was evaluated compared with CMCS−OH-30 NPs. In addition, anti-
bacterial tests and mouse wound healing experiments were also eval-
uated. These characteristics showed the advantage of using the nano-
fibers as wound dressing.
2. Materials and methods
2.1. Materials
OH-CATH30 (amino acid sequence, KFFKKLKNSVKKRAKKFFKKP-
RVIGVSIPF; purity > 96 %) was synthesized by GL Biochem (Shanghai,
China). PVA (average molecular weight of 5.9―7.1 kDa) with a degree
of polymerization of 1750 ± 50 was bought from Sinopharm Chemical
Reagent Corporation (Shanghai, China). CS (average molecular weight
of 60―120 kDa) with a degree of deacethylation of > 95 % was pur-
chased from Aladdin Industrial Corporation (Shanghai, China).
2
Carbohydrate Polymers 232 (2020) 115786
2.2. Preparation of CMCS
CMCS preparation was slightly modified in accordance with a pre-
viously described method (Jiang, Han, Li, Yang, & Liu, 2015). In brief,
2.5 g of CS was accurately weighed and dissolved in 45 mL of 40 % (w/
w) NaOH solution and stirred overnight. CS was filtered and dissolved
in 80 % (v/v) isopropanol solution in a three-necked flask and a con-
densing device. Within 30 min, 15 mL of 50 % chloroacetic acid in
isopropanol solution was added dropwise, and the reaction was carried
out in a water bath at 50 °C for 4 h. A total of 40 % NaOH solution was
added to adjust pH to approximately 8.0. At the end of the reaction, the
sample was suction filtered, washed with 95 % ethanol solution six
times, and dried prior to futher use.
2.3. Preparation and evaluation of CMCS-OH-30 NPs
CMCS-OH-30 NPs were prepared via the electrostatic droplet
method in accordance with previously described methods (Sun et al.,
2018) with slight modifications. In brief, OH-30 was dissolved in
deionized water, and the previously prepared CMCS was dissolved in an
appropriate amount of deionized water (OH-30:CMCS = 1:2). OH-30
solution was aspirated into a sterile syringe added with 0.65 kV positive
voltage clamped on an electrospinning instrument (Beijing, China). OH-
30 solution was pushed into the CMCS solution at a bolus rate of
0.5 mm/min with magnetic stirring (250 rpm) until completion. After
the solution was stirred for 10 min, the sample was placed in an ul-
trasonic ice bath for 5 min at approximately 20 W for 3 s and stopped
for 2 s. The mixture was centrifuged at 12,000 rpm at 4 °C. After the
supernatant was removed, the precipitate was resuspended in deionized
water, and ultrasonication was repeated. The prepared NPs were pre-
cooled for 24 h at −80 °C before they were freeze dried.
The particle size distribution of CMCS-OH-30 NPs was determined
using a Malvern laser scattering particle size analyzer (Nano-ZS90,
Malvern, UK), and their morphological characteristics were observed
using a transmission electron microscope (JEM-2100; JEOL, Akishima-
shi, Japan). The encapsulation efficiency (EE) of CMCS-OH30 NPs was
identified. In brief, the free Cy5-labeled OH-30 released from CMCS-
OH30 NP in the supernatants was evaluated using a fluorescence ana-
lyzer (F-4600, Japan Corporation, Japan). EE was calculated with the
following Eq. (1):
EE = (Wt − Wn) / Wt × 100, (1)
where Wt and Wn refer to the weights of the total OH-30 used and the
nonencapsulated OH-30, respectively (n = 3).
2.4. Preparation of NP-30-NFs
PVA/CS nanofibers with a CS concentration of 20 % or higher have
a bactericidal effect (Abdelgawad, Hudson, & Rojas, 2014). Nanofibers
containing 10 % CS were prepared to eliminate the bactericidal action
of the nanofiber itself and highlight the antibacterial effect of AMPs.
The preparation method of the electrospun fiber membrane was slightly
modified on the basis of previously studied equations. Subsequently, 1
% (w/v) CS was dissolved in 15 % (v/v) acetic acid solution, and 9 %
(w/v) PVA was dissolved in deionized water and stirred at 95 °C for
1.5 h at a constant temperature. After the PVA solution was cooled to
25 °C, different final concentrations (i.e., 0.08 %, 0.12 %, and 0.27 %
w/v) of NPs were slowly added to CS, continuously stirred for 0.5 h and
transferred to the PVA solution. The bubbles were then allowed to
stand. The hydrogel solution was aspirated into a 5 mL sterile syringe
for electrospinning. The nanofiber filaments formed by the spinning
solution jet were received by a foil wrapped receiver at a positive
voltage for 24 h. The distance between the nozzle (1 mm) and the re-
ceiver was 23 cm, the height of the nozzle was 39 ± 5 cm, the speed of
the spinning solution was 0.03 ± 0.01 mm/min, the receiving voltage
P. Zou, et al.
was −1.44 ± 0.5 kV, the experimental temperature was room tem-
perature, and the relative humidity was 40 % ± 5 %. Pure nanofiber
(Pure) without NPs served as a control. The nanofibers were heated at
40 °C for 12 h and then stored in a vacuum-drying oven.
2.5. Scanning electron microscopy (SEM) analysis
The samples were gold coated using a sputter coater. The structure
inside the electrospun fiber membrane was observed through SEM
(TESCAN, VEGA3, CZ).
2.6. Fourier transform infrared spectroscopy (FTIR) analysis
The solid sample was prepared using a potassium bromide tableting
method. An appropriate amount of the sample was placed in an agate
mortar with potassium bromide and dried overnight in an oven at a
constant temperature of 60 °C. The mixture was added to a tableting
mold, pressed to a transparent pressure under the pressure of a ta-
bleting machine, and detected by FTIR (Thermo, IS10, Shanghai,
China). All of the operations were performed in an infrared oven to
prevent moisture absorption. The background of potassium bromide
was deducted as a blank. Each sample was tested three times.
2.7. Laser scanning confocal microscopy (LSCM) analysis
The preparation method was the same as Sections 2.2 and 2.3, but
the antimicrobial peptide was rhodamine B-labeled OH-30. A certain
area of the cut nanofiber membrane was placed on a slide and observed
under LSCM (Leica TCS SP8, Wetzlar, DE, USA). The fluorescence on
the 50 μm × 30 μm × 20 μm nanofiber membrane with 0.01 % OH-30
NPs was observed to determine the drug distribution on the fiber
membrane.
2.8. Degree of swelling
The swelling of the sample was quantified in accordance with pre-
viously described methods (Arthanari et al., 2016; Charernsriwilaiwat,
Rojanarata, Ngawhirunpat, & Opanasopit, 2012) with slight modifica-
tions. The samples were swelled into PBS (pH 7.4) at 37 °C for 2, 4, 6, 8,
10, and 12 h. The swelling ratio was determined with Eq. (2) as follows:
Degree of swelling (%) = (W − Wd) / Wd × 100, (2)
where W is the wet weight of the sample after it was immersed in PBS,
and Wd is the initial dry weight of the sample.
2.9. Drug release experiments
In our controlled drug release study, the highest concentration of
the nanofiber membrane (0.27 %) was selected as the representative. A
drug-loaded nanofiber mat (70 mg) was soaked in a centrifuge tube
containing 10 mL of PBS (pH 7.4) (Chen et al., 2015). The tube was
incubated in a thermostat shaker at 37 °C and a rock speed of 100 rpm.
At an appropriate time, 1 mL of the release medium was removed from
the centrifuge tube and replaced with an equal volume of fresh deio-
nized water. The sample was analyzed through a high-performance li-
quid chromatograph (HPLC) to determine OH-30 by using an injection
amount of 20 μL in a HPLC series system (Agilent 1260II, USA) with a
C18 column (120-EC, 4.6 mm × 15 cm, 4 μm, InfinityLab Poroshell).
The released OH-30 was detected at 280 nm. The mobile phase was
acetonitrile (ACN): water (H2O). The gradient was first maintained for
5 min at 80 % H2O/0.1 % trifluoroacetic acid (TFA) and 20 % ACN/0.1
% TFA followed by 20 min at 60 % H2O/0.1 % TFA and 40 % ACN/0.1
% TFA, and the flow rate was set to 1 mL/min. The average was ob-
tained from three parallel samples. The amount of NPs released was
used as the control.
3
Carbohydrate Polymers 232 (2020) 115786
2.10. Antimicrobial assay
Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC
25923) were selected as test strains for the antibacterial activity test of
electrospun fibrin membranes (Pei et al., 2015). The two kinds of
bacteria were pre-activated for two generations, inoculated into Lur-
ia―Bertani (LB) liquid medium, and cultured at 37 °C for 4–6 h until
the bacterial concentration reached 5 × 104 colony-forming units/mL.
After centrifugation (4000 rpm, 4 °C) was conducted for 10 min, the
bacterial suspension was resuspended with a fresh medium. Different
weights of nanofiber membranes (1–10 mg) were transferred in an
Eppendorf tube containing 1 mL of bacterial suspension. The optical
density (OD) at a wavelength of 570 nm was measured using a micro-
plate reader (Bio Tek, ELX800, USA) after 24 h of incubation at 37 °C.
The assay was performed in triplicate.
2.11. Cytotoxicity of the electrospun fiber membrane
The cytotoxicity of nanofiber mats was evaluated following a pre-
viously described method (Moura et al., 2014). The nanofiber mat was
sterilized in a UV environment for 30 min. The nanofiber membrane
was then immersed in a serum-free medium (MEM, 1 % v/v penicillin
and streptomycin, 1 % v/v non-essential amino acids and 1 % v/v so-
dium pyruvate) and cultured at 25 °C for 24 h to produce different
concentrations of extraction media (0.25, 0.5, 1, 3, 5, 7, and 9 mg/mL).
Afterward, 100 μL of HaCaT cells was seeded in 96-well plates (Corning
Incorporated, Corning, NY, USA) at a density of 5 × 104 cells/mL. After
48 h of culture, MEM supplemented with 10 % fetal bovine serum (FBS)
was aspirated, and the cells were incubated with different concentra-
tions of the extraction medium for 12 h. After the treatment was ad-
ministered, the cells were incubated with 10 μL of MTT (5 mg/mL) for
4 h. Subsequently, 100 μL of dimethyl sulfoxide was added to each well.
After 20 min of incubation, absorbance was determined at a wavelength
of 490 nm by using a microplate reader to calculate the relative cell
viability (%). The viability of untreated control cells was defined as 100
%.
2.12. Wound healing activity of electrospun fiber membranes
Five-week-old female specific pathogen-free (SPF)-grade KM mice
(average body weight of 20–25 g, 12 per group) were obtained from
Jinan Pengyue Animal Breeding Co., Ltd. The mice were first anesthe-
tized by intraperitoneally injecting chloral hydrate (0.1 mL/10 g).
Depilatory cream was used to clean the back skin of the mice. A Miltex®
Biopsy Punch (ID = 7 mm) with a plunger was placed perpendicular to
the back skin and cut by rotation. Surgical scissors were used to remove
the intermediate skin to create a round wound with full thickness. The
mice were randomized into six groups: untreated group, un-
encapsulated drug group, 0.08 % NPs group, 0.12 % NPs group, 0.27 %
NPs group and positive control group (JINCHUANG® chitosan wound
care film). A 1 cm × 1 cm dressing was applied to the wound, replaced
every 24 h, and continuously changed for 5 days. Each mouse was in-
dividually placed in a squirrel cage of an SPF animal house to prevent
mutual biting and the dressing from falling off. Each wound was ob-
served and photographed using a Nikon camera to record the wound
healing process. Three mice were then sacrificed on days 7 and 21
postoperatively, and the samples were obtained for subsequent hema-
toxylin―eosin (HE) staining experiments. The wound healing rate was
calculated using Eq. (3):
Wound contraction (%) =(A0-At) / A0 × 100 %, (3)
where Ao is the original wound wound area, and At is the wound area of
tissue examination on day t. The excised skin tissue was fixed with 4 %
paraformaldehyde tissue fixative for 24 h, rinsed, dehydrated, and
embedded in paraffin. Sections were stained with HE stain and
P. Zou, et al. Carbohydrate Polymers 232 (2020) 115786

Fig. 1. TEM micrographs showing the morphological features of CMCS-OH-30 NPs (A).
SEM images of nanofibers with different NP concentrations. Pure PVA/CS nanifibers (B), 0.08 % (w/v) NP-30-NFs (C), 0.12 % (w/v) NP-30-NFs (D), 0.27 % (w/v)
NP-30-NFs (E), and average diameter comparison (F) (n = 100).

horseshoe stain and photographed at 100× magnification. The animal p > 0.05 (ns) was considered not statistically significant.
protocol for this study was approved by the animal care and ethical
committee of the medical sector in Weifang Medical University. 3. Results and discussion

2.13. Statistical analysis 3.1. NP characterization

Data were collected and presented as the mean and standard error The cationic antibacterial peptide OH-30 could be ionically cross-
of the mean. The experimental data in different groups were compared linked with CMCS containing many carboxyl groups because of its
using one-way ANOVA in SPSS 15.0 (SPSS Inc., Chicago, IL, USA). chargeability. As such, it could self-assemble into NPs. The TEM pho-
*p < 0.05 and **p < 0.01 were considered statistically significant. tographs (Fig. 1A) and particle size distribution (Table 1) of CMCS-OH-

4
P. Zou, et al.
Table 1
Quality evaluation of CMCS-OH-30 NPs (n = 3).
Size (nm) PDI Zeta potential EE (%)
(mV)
−37.6 ± 1.5
Mean ± SD 164.6 ± 5.0 0.14 ± 0.02 92.14 ± 1.05
30 NPs are shown. In the TEM images, NPs were relatively round, and
their particle size was 164.6 ± 5.0 nm, thereby facilitating re-coating
for the subsequent electrospinning. The polydispersity index indicated
that the particle size distribution was uniform. The Z potential de-
monstrated that the particles did not easily settle, coagulate, or floc-
culate. The entire system was relatively stable and could meet the
stability requirement (> 15 mV) possibly because of the following
factors. A decrease in the surface tension of the droplets was due to the
application of a certain unit of positive charges on the OH-30 solution,
thereby causing the solution to drip into the low CMCS solution in a
uniform stable state. The application of a positive charge might have
distributed the originally disordered OH-30 molecules around the so-
lution under the action of an electric field, thereby increasing the
contact area between OH-30 and CMCS molecules and ion crosslinking.
Thus, the EE of the NPs prepared by the electrostatic droplet method
was improved compared with that of the extrusion method (Sun et al.,
2018).
3.2. Nanofiber characterization
3.2.1. SEM analysis
The SEM image shows electrospun nanofibers, suggesting that the
surface of the nanofiber filaments for each sample was smooth
(Fig. 1B–E). Fig. 1F illustrates the average diameter of the electrospun
fiber membranes with different NPs. A total of 100 fibers were analyzed
with Image Pro 8.0, and histograms were plotted. The average fiber
diameter was primarily determined in terms of solution viscosity and
conductivity (Haider, Haider, & Kang, 2018). The mean diameter of the
control group (mean ± SD) was 357.34 ± 110.45 nm, which de-
creased from 371.80 ± 110.31 nm to 327.48 ± 114.28 nm after the
drug was loaded. Given that the concentration of NPs in the solution
was high, the uniformity of the fiber diameter was less maintained
possibly because the viscosity and conductivity of the original spinning
solution changed as the CMCS-OH30 NPs increased (Table S1). As the
concentration of the NPs increased, the viscosity of the PVA/CS system
also increased, and the NPs had a certain unit of negative charge that
increased their conductivity. A high concentration of the fiber mem-
brane exhibited a small amount of bead structures possibly because of
an increase in surface tension resulting from uneven jetting and diffi-
culty in forming a Taylor cone. In terms of diameter, the particle size of
NPs was much smaller than the diameter of the electrospun fiber
membrane. Thus, the NPs could be encapsulated by the fiber mem-
brane. Charernsriwilaiwat et al. (2012) also confirmed that viscosity
and conductivity are important factors affecting the diameter of nano-
fibers.
3.2.2. LSCM of NP-30-NFs
After a full-layer scan of the prepared NP-30-NFs, the results were
intercepted (Fig. 2). Red fluorescence showed the NP distribution in any
direction of the fiber membrane, indicating that NPs were successfully
entrapped in the fiber membrane. The fiber membrane surface was
smooth as observed under the optical microscope. The NPs were en-
capsulated in the fiber membrane rather than irregularly adsorbed onto
the nanofiber surface. Nylon 6 nanofibers immersed in citrus juice so-
lution for 5 min can eradicate bacteria (Akia et al., 2019).
3.2.3. FTIR analysis
Fig. 3 illustrates the FTIR spectra of CS, CMCS, OH-30 NPs, and
5
Carbohydrate Polymers 232 (2020) 115786
nanofibers. As shown in the CS spectrum, the main characteristic peaks
were located at 3441.91 (OeH stretching vibration and NeH stretching
vibration), 2873.41 (C–H stretching vibration), 1597.11 (amide CeO
stretching), and 1077.56 cm−1 (primary hydroxyl, CeO tensile vibra-
tion). By contrast, the CMCS spectrum showed the characteristic peak
(1595.34 cm−1) of relevant carboxylate (−COO- not symmetrical
stretching) and the characteristic peak of carboxymethyl group
(1411.89 cm−1) (Mishra et al., 2011). The symmetrical stretching of the
carboxylate CeO indicated the presence of carboxymethyl groups in
CMCS. The main characteristic peak of OH-30 at 3284.98 cm−1 (OeH
stretching vibration and NeH stretching vibration) became a broad and
blunt peak at 3296.38 cm−1 after OH-30 was synthesized into NPs and
found in the fingerprint region. The multiple (−CH2)-in-plane bending
vibration at 721.71 cm−1 was successfully masked. These findings in-
dicated that CMCS and OH-30 were successfully crosslinked by inter-
molecular interactions. After the NPs were recoated, the peak of
3421.89 cm−1 was masked by the COO- of the PVA, showing a sharp
peak (Habiba, Siddique et al., 2017; Zhou et al., 2017).
The 1H NMR spectra of CMCS are presented in Fig. S1. The spectra
of CMCS illustrate that the chemical shifts at 4.4 ppm were due to the
protons of −CH2COO− at the O-position at C6 of CMCS. The results
showed that the carboxylated carboxymethyl chitosan at the C6 posi-
tion was successfully synthesized (Mi, Su, Fan, Zhu, & Zhang, 2013).
3.2.4. Degree of swelling
Swelling is one of the important properties of nanofiber membranes
for wound dressing applications (Arthanari et al., 2016). A wound
dressing with a good degree of swelling contributes to the absorption of
permeate from wound tissues, creating a moist microenvironment that
promotes wound healing. The degree of swelling of nanofibers plays an
important role in the loading and release behavior of the drug. Fig. 4A
shows the degree of swelling of various fiber membranes of different
NPs after they were soaked in PBS at 37 °C. The hydroxyl group on the
PVA molecule can interact with the amino group and the hydroxyl
group on the CS molecule through intermolecular hydrogen bonding; at
the same time, CMCS can form a hydrogen bond with CS. In this study,
the other components exhibited excellent swelling properties compared
with the control group, and the degree of swelling exceeded 100 %. The
degree of swelling of the nanofibers was related to the NP concentra-
tion. This result might be attributed to the increased amount of CMCS in
the NPs, promoting the degree of crosslinking in the PVA/CS solution.
However, in all cases, the maximum swelling was attained at 4–6 h
beyond which the degree of swelling was reduced. This behavior might
be due to the release of OH-30 and the degradation or leaching of
polymeric composites. The maximum swelling of gatifloxacin-loaded
alginate/PVA electrospun nanofibers was attained at 8 h (Arthanari
et al., 2016). Thus, the faster the swelling, the more conducive the
creation of a relatively clean wound environment. This phenomenon
accelerated wound healing.
3.3. In vitro drug release studies
Fig. 4B shows the in vitro release of OH-30 from NPs and nanofibers.
The prepared nanofibers exhibited good sustained release properties in
vitro. The average cumulative release of OH-30 released from the na-
nofibers was 14 % in the first 2 h and sustained release was observed for
at least 24 h. The final cumulative release was 66 % ± 7.5 % in 24 h.
The release mechanism of the prepared nanofibers might be caused by
the following scenarios. 1. The swelling degree of the nanofibers in-
creased and the intermolecular gap increased with time, providing a
spatial basis for NP release. 2. The degree of ionic crosslinking between
OH-30 and nanocarriers weakened, and OH-30 was released.
3.4. Antimicrobial assay
The results of antibacterial experiments showed that the electrospun
P. Zou, et al. Carbohydrate Polymers 232 (2020) 115786

Fig. 2. LSCM image of 0.27 % NP-30-NFs under different microscopes. Fluorescence microscope (A), o.ptical microscope (B), and combination of the two (C).

Fig. 4. Degree of swelling (%) of PVA/CS electrospun fiber membranes (A). OH-
30 conc. (0, 0.08 %, 0.12 %, 0.27 %) (n = 3).
In vitro release profile of OH-30 (plotted as a function of percentage cumulative
release vs. time) from nanofibers and CMCS-OH30 NPs at different time (B)
(n = 3).

Fig. 3. FTIR spectra of CMCS, CS, OH-30, NPs, PVA, and 0.27 % NP-30-NFs. Thus, an electrospun fiber membrane loaded with NPs could better
provide a relatively sterile microenvironment compared with a pure
electrospun fiber membrane. This phenomenon was beneficial to
fiber membrane loaded with NPs had superior antibacterial properties
wound healing.
compared with the simple electrospun fiber membranes (Fig. 5). This
finding was consistent with our previous research results (Sun et al.,
2018). When the concentration of the electrospun fiber membrane
carrying NPs reached 3 mg/mL, the inhibition rate was 80 % or more.

6
P. Zou, et al.
Fig. 5. The antibacterial activity of the nanofiber mats against (A) E. coli and (B) S. aureus after 24 h of incubation at 5 × 104 CFU/mL bacteria, 37℃ (n = 3).
Fig. 6. Cytotoxicity assay of HaCaT cells. Cells and different concentrations of
nanofiber membranes extracts were incubated for 12 h at 37℃ (n = 3).
3.5. Cytotoxicity evaluation
The indirect cytotoxicity of various concentrations of medium ex-
tracts from nanofiber mats with different NPs is shown in Fig. 6. At
1 mg/mL concentration, the cell viability of HaCaT decreased slightly
possibly because of the excessive concentration of the released OH-30,
which exerted a certain degree of killing effect on cells. When the
concentration reached 0.5 mg/mL, the cell survival rate increased sig-
nificantly, and the cells proliferated. At 0.25 mg/mL, proliferation was
observed, but its degree was not obvious possibly because of the slightly
low OH-30 concentration. These data indicated that nanofiber mats
appeared to have a certain proliferative effect on cells and may be
developed as wound dressings. The findings of this study provided a
basis for conducting subsequent animal wound experiments. The
HaCaT cell morphology was studied by calcein-AM fluorescence
staining (Fig. S2). The results indicated that the nanofibers might have
affected cell adhesion. This phenomenon might be advantageous for
skin wound healing. Secondary damage to skin wounds was avoided
during the change in dressing because the nanofibers acted as a drug
carrier for OH-30 rather than a tissue scaffold.
3.6. Evaluation of membrane performance during wound healing
Wound healing was observed on days 0, 3, 6, 9, and 12 as shown in
Fig. 7A. The wound healing rate throughout the process is shown in
Fig. 7B. On the first 6 days after surgery, the wound healing rate of the
medicated group was clearer than that of the blank group, the control
group and the positive control group possibly of the continued slow
release of OH-30 (Table S2). After dosing was terminated on day 5, the
wound healed slowly because the wound was not conducive to drug
penetration after scabbing. Six days after the operation, 0.27 % NP-30-
NFs showed a faster wound closure rate of 83.74 ± 1.41 %, whereas
the CMCS-OH30 NP group maintained a wound closure rate of
69.33 ± 2.41 % (Sun et al., 2018). OH-30 contained in NF-30-NP
7
Carbohydrate Polymers 232 (2020) 115786
could be released slowly from the nanofibers than CMCS-OH-30 NPs
during the healing process, which could against bacterias and accel-
erate wound healing. There is one advantage in using this nanofibers as
the wound dressing.
At day 12, 98.51 ± 0.37 % wound closure was observed in 0.27 %
NP-30-NFs while the chitosan/PVA/ZnO nanofibrous membranes
showed amuch lower percentage of contraction namely 90.5 ± 1.7 %
wound closure only (Ahmed et al., 2018). Consistent with our previous
findings, the present results indicated that the constructed OH-30 de-
livery system played an important role in the early wound healing
process.
Fig. 8A shows the histopathological analysis of the healing effects of
the samples stained with HE. The 7-day histopathological results
showed significant changes.
On day 21, the medicated group had a more uniform epithelial
surface thickness, a smoother surface layer, and a more consistent
connective tissue orientation compared with those of the control group.
Fig. 8B shows the histopathological analysis of healing by using Mas-
son’s trichrome-stained samples. On day 7, the extent of collagen de-
position after wound induction significantly increased in all the wound-
inducing groups compared with that in the control group. The degree of
inflammatory response in the control group was significantly higher
than that in the dosing group possibly because of the im-
munomodulatory effects of the previous OH-30. Epithelial regeneration
is necessary to promote wound healing. At 24 h after the wound is in-
flicted, basal cells isolated from the dermal layer of the wound interface
begin to divide and migrate to the defective area of the skin until the
thickness of the epidermis becomes normal (Jung et al., 2018). In this
experiment, the epithelial regeneration ability of the medicated group
was significantly better than that of the control group after 7 days.
Histopathological analysis confirmed that the acceleration of re-epi-
thelialization and collagen deposition was a wound healing effect in the
dosing group, and these effects promoted wound healing.
4. Conclusions
CMCS-OH-30 NPs were successfully encapsulated in nanofibers.
Nanofibers loaded with different concentrations of NPs showed a strong
antibacterial activity. HE and Masson’s staining demonstrated that the
wound healing ability of NP-loaded nanofibers was better than that of
the blank control group. These results revealed that NP-loaded nano-
fibers had dual antibacterial and wound healing functions.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (NSFC81673576 and 31600775), Shandong
Provincial Natural Science Foundation of China (ZR2016HM76),
Science and Technology Department of Yunnan Province (2019ZF003)
P. Zou, et al. Carbohydrate Polymers 232 (2020) 115786

Fig. 7. Wound healing effects on mice models for different treated groups (A); Wound closure rate of all groups (B) (n = 3).

8
P. Zou, et al.
Fig. 8. Histopathology of the healing effect of samples on wound-induced skin damage with hematoxylin and eosin (HE) (A) stain ×20 (7, 21 days) or Masson’s
trichrome stain (B) ×20 (7, 21 days).
and Youth Innovative Team Development Plan of Universities in
Shandong Province (2019KJM003).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115786.
References
Abdelgawad, A. M., Hudson, S. M., & Rojas, O. J. (2014). Antimicrobial wound dressing
nanofiber mats from multicomponent (chitosan/silver-NPs/polyvinyl alcohol) sys-
tems. Carbohydrate Polymers, 100, 166–178.
Ahmed, R., Tariq, M., Ali, I., Asghar, R., Noorunnisa Khanam, P., Augustine, R., et al.
(2018). Novel electrospun chitosan/polyvinyl alcohol/zinc oxide nanofibrous mats
with antibacterial and antioxidant properties for diabetic wound healing.
International Journal of Biological Macromolecules, 120(Pt A), 385–393.
Akia, M., Rodriguez, C., Materon, L., Gilkerson, R., & Lozano, K. (2019). Antibacterial
activity of polymeric nanofiber membranes impregnated with Texas sour orange
juice. European Polymer Journal, 115, 1–5.
Arthanari, S., Mani, G., Jang, J. H., Choi, J. O., Cho, Y. H., Lee, J. H., et al. (2016).
Preparation and characterization of gatifloxacin-loaded alginate/poly (vinyl alcohol)
electrospun nanofibers. Artificial Cells, Nanomedicine, and Biotechnology, 44(3),
847–852.
Charernsriwilaiwat, N., Rojanarata, T., Ngawhirunpat, T., & Opanasopit, P. (2012).
Electrospun chitosan/polyvinyl alcohol nanofibre mats for wound healing.
International Wound Journal, 11(2), 215–222.
Chen, W., Li, D., Ei-Shanshory, A., El-Newehy, M., Ei-Hamshary, H. A., Al-Deyab, S. S.,
et al. (2015). Dexamethasone loaded core-shell SF/PEO nanofibers via green elec-
trospinning reduced endothelial cells inflammatory damage. Colloids and Surfaces B
Biointerfaces, 126, 561–568.
Das, A., Kumar, A., Patil, N. B., Viswanathan, C., & Ghosh, D. (2015). Preparation and
characterization of silver nanoparticle loaded amorphous hydrogel of carbox-
ymethylcellulose for infected wounds. Carbohydrate Polymers, 130, 254–261.
El-Aassar, M. R., El Fawal, G. F., El-Deeb, N. M., Hassan, H. S., & Mo, X. (2016).
Electrospun polyvinyl alcohol/ pluronic F127 blended nanofibers containing tita-
nium dioxide for antibacterial wound dressing. Applied Biochemistry and
Biotechnology, 178(8), 1488–1502.
Eming, S. A., Krieg, T., & Davidson, J. M. (2007). Inflammation in wound repair:
Molecular and cellular mechanisms. The Journal of Investigative Dermatology, 127(3),
514–525.
Fu, R., Li, C., Yu, C., Xie, H., Shi, S., Li, Z., et al. (2016). A novel electrospun membrane
based on moxifloxacin hydrochloride/poly(vinyl alcohol)/sodium alginate for anti-
bacterial wound dressings in practical application. Drug Delivery, 23(3), 828–839.
Gao, Q., Huang, C., Sun, B., Aqeel, B. M., Wang, J., Chen, W., et al. (2016). Fabrication
and characterization of metal stent coating with drug-loaded nanofiber film for
gallstone dissolution. Journal of Biomaterials Applications, 31(5), 784–796.
9
Carbohydrate Polymers 232 (2020) 115786
Gao, Z. H., Deng, C. J., Xie, Y. Y., Guo, X. L., Wang, Q. Q., Liu, L. Z., et al. (2018). Pore-
forming toxin-like protein complex expressed by frog promotes tissue repair. FASEB
Journal, 33(1), 782–795.
Garcia, M. C., Aldana, A. A., Tartara, L. I., Alovero, F., Strumia, M. C., Manzo, R. H., et al.
(2017). Bioadhesive and biocompatible films as wound dressing materials based on a
novel dendronized chitosan loaded with ciprofloxacin. Carbohydrate Polymers, 175,
75–86.
Gomes, A. P., Mano, J. F., Queiroz, J. A., & Gouveia, I. C. (2015). Incorporation of an-
timicrobial peptides on functionalized cotton gauzes for medical applications.
Carbohydrate Polymers, 127, 451–461.
Habiba, U., Afifi, A. M., Salleh, A., & Ang, B. C. (2017). Chitosan/(polyvinyl alcohol)/
zeolite electrospun composite nanofibrous membrane for adsorption of Cr(6+), Fe
(3+) and Ni(2). Journal of Hazardous Materials, 322(Pt A), 182–194.
Habiba, U., Siddique, T. A., Talebian, S., Lee, J. J. L., Salleh, A., Ang, B. C., et al. (2017).
Effect of deacetylation on property of electrospun chitosan/PVA nanofibrous mem-
brane and removal of methyl orange, Fe(III) and Cr(VI) ions. Carbohydrate Polymers,
177, 32–39.
Haider, A., Haider, S., & Kang, I.-K. (2018). A comprehensive review summarizing the
effect of electrospinning parameters and potential applications of nanofibers in bio-
medical and biotechnology. Arabian Journal of Chemistry, 11(8), 1165–1188.
Huang, J., Deng, Y., Ren, J., Chen, G., Wang, G., Wang, F., et al. (2018). Novel in situ
forming hydrogel based on xanthan and chitosan re-gelifying in liquids for local drug
delivery. Carbohydrate Polymers, 186, 54–63.
Jiang, Z., Han, B., Li, H., Yang, Y., & Liu, W. (2015). Carboxymethyl chitosan represses
tumor angiogenesis in vitro and in vivo. Carbohydrate Polymers, 129, 1–8.
Jung, H. S., Kim, M. H., Shin, J. Y., Park, S. R., Jung, J. Y., & Park, W. H. (2018).
Electrospinning and wound healing activity of beta-chitin extracted from cuttlefish
bone. Carbohydrate Polymers, 193, 205–211.
Kohsari, I., Shariatinia, Z., & Pourmortazavi, S. M. (2016). Antibacterial electrospun
chitosan-polyethylene oxide nanocomposite mats containing bioactive silver nano-
particles. Carbohydrate Polymers, 140, 287–298.
Lee, S. J., Heo, D. N., Moon, J. H., Ko, W. K., Lee, J. B., Bae, M. S., et al. (2014).
Electrospun chitosan nanofibers with controlled levels of silver nanoparticles.
Preparation, characterization and antibacterial activity. Carbohydrate Polymers, 111,
530–537.
Li, S. A., Lee, W. H., & Zhang, Y. (2012). Efficacy of OH-CATH30 and its analogs against
drug-resistant bacteria in vitro and in mouse models. Antimicrobial Agents and
Chemotherapy, 56(6), 3309–3317.
Li, S. A., Liu, J., Xiang, Y., Wang, Y. J., Lee, W. H., & Zhang, Y. (2014). Therapeutic
potential of the antimicrobial peptide OH-CATH30 for antibiotic-resistant
Pseudomonas aeruginosa keratitis. Antimicrobial Agents and Chemotherapy, 58(6),
3144–3150.
Li, S. A., Xiang, Y., Wang, Y. J., Liu, J., Lee, W. H., & Zhang, Y. (2013). Naturally oc-
curring antimicrobial peptide OH-CATH30 selectively regulates the innate immune
response to protect against sepsis. Journal of Medicinal Chemistry, 56(22), 9136–9145.
Mi, Y., Su, R., Fan, D. D., Zhu, X. L., & Zhang, W. N. (2013). Preparation of N,O-car-
boxymethyl chitosan coated alginate microcapsules and their application to
Bifidobacterium longum BIOMA 5920. Materials Science & Engineering C, Materials for
Biological Applications, 33(5), 3047–3053.
Miguel, S. P., Sequeira, R. S., Moreira, A. F., Cabral, C. S. D., Mendonca, A. G., Ferreira, P.,
et al. (2019). An overview of electrospun membranes loaded with bioactive
P. Zou, et al.
molecules for improving the wound healing process. European Journal of
Pharmaceutics and Biopharmaceutics, 139, 1–22.
Mishra, D., Bhunia, B., Banerjee, I., Datta, P., Dhara, S., & Maiti, T. K. (2011).
Enzymatically crosslinked carboxymethyl–chitosan/gelatin/nano-hydroxyapatite in-
jectable gels for in situ bone tissue engineering application. Materials Science and
Engineering C, 31(7), 1295–1304.
Mokhena, T. C., & Luyt, A. S. (2017). Electrospun alginate nanofibres impregnated with
silver nanoparticles: Preparation, morphology and antibacterial properties.
Carbohydrate Polymers, 165, 304–312.
Morgado, P. I., Miguel, S. P., Correia, I. J., & Aguiar-Ricardo, A. (2017). Ibuprofen loaded
PVA/chitosan membranes: A highly efficient strategy towards an improved skin
wound healing. Carbohydrate Polymers, 159, 136–145.
Moura, L. I., Dias, A. M., Leal, E. C., Carvalho, L., de Sousa, H. C., & Carvalho, E. (2014).
Chitosan-based dressings loaded with neurotensin–an efficient strategy to improve
early diabetic wound healing. Acta Biomaterialia, 10(2), 843–857.
Pei, Z., Sun, Q., Sun, X., Wang, Y., & Zhao, P. (2015). Preparation and characterization of
silver nanoparticles on silk fibroin/carboxymethylchitosan composite sponge as anti-
bacterial wound dressing. Bio-medical Materials and Engineering, 26(Suppl 1),
S111–118.
Rankin, L. C., & Artis, D. (2018). Beyond host defense: Emerging functions of the immune
system in regulating complex tissue physiology. Cell, 173(3), 554–567.
Rasool, A., Ata, S., & Islam, A. (2019). Stimuli responsive biopolymer (chitosan) based
blend hydrogels for wound healing application. Carbohydrate Polymers, 203,
423–429.
Silva, J. P., Dhall, S., Garcia, M., Chan, A., Costa, C., Gama, M., et al. (2015). Improved
burn wound healing by the antimicrobial peptide LLKKK18 released from conjugates
with dextrin embedded in a carbopol gel. Acta Biomaterialia, 26, 249–262.
Sun, B. K., Siprashvili, Z., & Khavari, P. A. (2014). Advances in skin grafting and
10
Carbohydrate Polymers 232 (2020) 115786
treatment of cutaneous wounds. Science, 346(6212), 941–945.
Sun, T., Zhan, B., Zhang, W., Qin, D., Xia, G., Zhang, H., et al. (2018). Carboxymethyl
chitosan nanoparticles loaded with bioactive peptide OH-CATH30 benefit nonscar
wound healing. International Journal of Nanomedicine, 13, 5771–5786.
Wang, Z., Yan, F., Pei, H., Li, J., Cui, Z., & He, B. (2018). Antibacterial and en-
vironmentally friendly chitosan/polyvinyl alcohol blend membranes for air filtration.
Carbohydrate Polymers, 198, 241–248.
Zarandi, M. A., Zahedi, P., Rezaeian, I., Salehpour, A., Gholami, M., & Motealleh, B.
(2015). Drug release, cell adhesion and wound healing evaluations of electrospun
carboxymethyl chitosan/polyethylene oxide nanofibres containing phenytoin sodium
and vitamin C. IET Nanobiotechnology, 9(4), 191–200.
Zhang, Y., Zhao, H., Yu, G. Y., Liu, X. D., Shen, J. H., Lee, W. H., et al. (2010). Structure-
function relationship of king cobra cathelicidin. Peptides, 31(8), 1488–1493.
Zhao, F., Lan, X. Q., Du, Y., Chen, P. Y., Zhao, J., Zhao, F., et al. (2018). King cobra
peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant
isolates. Zoological Research, 39(2), 87–96.
Zhao, L., Chen, D., Yao, Q., & Li, M. (2017). Studies on the use of recombinant spider silk
protein/polyvinyl alcohol electrospinning membrane as wound dressing. International
Journal of Nanomedicine, 12, 8103–8114.
Zhou, T., Sui, B., Mo, X., & Sun, J. (2017). Multifunctional and biomimetic fish collagen/
bioactive glass nanofibers: Fabrication, antibacterial activity and inducing skin re-
generation in vitro and in vivo. International Journal of Nanomedicine, 12, 3495–3507.
Zhou, T., Wang, N., Xue, Y., Ding, T., Liu, X., Mo, X., et al. (2016). Electrospun tilapia
collagen nanofibers accelerating wound healing via inducing keratinocytes pro-
liferation and differentiation. Colloids and Surfaces B Biointerfaces, 143, 415–422.
Zhou, Y., Yang, H., Liu, X., Mao, J., Gu, S., & Xu, W. (2013). Electrospinning of carbox-
yethyl chitosan/poly(vinyl alcohol)/silk fibroin nanoparticles for wound dressings.
International Journal of Biological Macromolecules, 53, 88–92.