International Journal of Biological Macromolecules 226 (2023) 383–396

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International Journal of Biological Macromolecules

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

Incorporation of Saqez essential oil into polyvinyl alcohol/chitosan bilayer

hydrogel as a potent wound dressing material
Ali Rezaei , Hamide Ehtesabi *, Somaye Ebrahimi
Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Nowadays, many studies are conducted on multilayer hydrogels for wound dressing. On the other hand,
Biopolymer considering the emergence of bacterial resistance to common antibiotics, studies on the use of natural essential
Antibacterial oils and their derivatives that have antibacterial and antioxidant activity can be useful. Herein, a novel bilayer
Antioxidant
hydrogel developed from polyvinyl alcohol and chitosan with the incorporation of Saqez essential oil (SEO) was
synthesized. The results showed a gel-type structure with specific compression and flexibility, while the
microscopic images confirmed the formation of a bilayer hydrogel. Further, the data showed that increasing the
concentration of SEO reduces the swelling and water vapor permeability and increases the water retention and
hydrophobicity of the hydrogel surface. The effects of the combination of SEO in the bilayer hydrogel led to a
strong antioxidant property and increased antimicrobial activity. Also, the in vitro results demonstrated that the
bilayer hydrogels are biocompatible, non-toxic, and blood compatible. Finally, the results of the in vivo tests
showed that these bilayer hydrogels had good homeostatic efficiency. Overall, the obtained results indicate that
these bilayer hydrogels are promising candidates for wound dressing.

1. Introduction
Wounding can be defined as disrupted epithelial integrity of the
tissues. The process of wound healing is a steady and inherent phe­
nomenon that leads to the restoration of tissue integrity in a great
number of cases. Nonetheless, wounds cannot be healed naturally in
some cases and become a medical challenge, requiring particular
treatments and care [1]. Human health is frequently endangered by
improper treatments, which result in diseases like tissue dehydration,
disorders of the immune system, bacterial infections, and even more
acute secondary traumas [2]. Through the provision of a favorable
medium, modern techniques of wound treatment have paved the way
for the regulation of the procedure and sped up healing [3]. Wound
dressings are capable of providing temporary protection in order to
replace the injured skin in the course of the treatment and healing
processes, which makes it possible to control or avoid wound infections
effectively and provide a favorable healing environment [4]. As one of
the modern wound care materials, hydrogels are 3D networks of hy­
drophilic polymers capable of holding large volumes of water within
their structures. The macromolecular structure of hydrogels resembles
that of biological tissues. Hydrogels are applicable as a replacement
material for skin in order to prevent the inflammation resulting from
wound infections [5]. Nonetheless, conventional hydrogels have certain
disadvantages, which are attributable to their weak antibacterial po­
tential and poor mechanical characteristics, making them unfavorable
materials for being used as wound dressings. Thus, developing an ideal
hydrogel dressing featuring multi-functional benefits, including
breathability, softness, transparency, flexibility, antibacterial charac­
teristics, free formability, easy application, and biocompatibility, is
crucial [6].
In the current research, new wound dressings designed as bilayer
composites have been shown to perform better than single-layer
hydrogels [7]. The top layer is usually designed to prevent bacteria
from penetrating, allowing oxygen to penetrate the wound and provide
higher mechanical strength, and the bottom layer is designed to better
regenerate the skin bed and help kill bacteria on the wound surface [8].
One of the hydrophilic, non-toxic, biocompatible, and biodegradable
materials commonly used in biomedical fields, including drug delivery,
tissue engineering, and wound healing, is polyvinyl alcohol (PVA). In
addition, manufactured hydrogel PVA has shown good mechanical
properties [9]. However, hydrogels that use PVA as the main component
usually lack insufficient swelling capacity and do not affect the wound

* Corresponding author.
E-mail address: h_ehtesabi@sbu.ac.ir (H. Ehtesabi).

https://doi.org/10.1016/j.ijbiomac.2022.12.036
Received 3 September 2022; Received in revised form 26 October 2022; Accepted 5 December 2022
Available online 7 December 2022
0141-8130/© 2022 Elsevier B.V. All rights reserved.
A. Rezaei et al.
healing process [10]. Chitosan (C) is the second-most common
biopolymer and a chitin-derived biopolymer. C is a cationic linear
polysaccharide consisting of β - (1, 4)-linked D-glucosamine and N-
acetyl-D-glucosamine. Many hydroxyls (-OH) and amino (-NH2) groups
are available along the C chain. As a result of the protonation of the
-NH2 groups on the polymer chains, C is soluble in water under acidic
media conditions [11]. Following that, the active and positively charged
surface of C can interact with PVA via electrostatic interaction, and so
on. Therefore, this interaction is useful for the formation of bilayer PVA
and C. Furthermore, C is widely used as an antimicrobial agent to pre­
vent infections. Also, biocompatibility is also biodegradable and inex­
pensive. As a result, it has been used in biomedical applications,
particularly wound dressings [12].
The use of essential oils as bioactive agents in numerous fields, such
as pharmaceuticals, food-related applications, cosmetics, etc., has
received increased interest in recent years [13]. Antioxidant and anti­
bacterial effects, as well as antiviral, insecticidal, analgesic, and anti-
inflammatory characteristics, are the primary benefits of essential oil
[14]. essential oil are composed primarily of terpenes and terpenoids,
sesquiterpenes, phenolic compounds, and aldehydes, which are complex
and unique substances. The hydrophobic character of essential oil is
determined by their chemical composition, which can be viewed as both
an advantage and a disadvantage depending on the intended application
area [15]. Through steam distillation, one can extract essential oils from
plants, which are volatile aromatic secondary compounds generally
made from phenylpropanoids and terpenoids [16]. Given their capa­
bility of modifying the cellular metabolic activity, permeability, and
toxicity of a variety of strains of microorganisms, essential oils can be
regarded as natural bioactive alternatives to antibiotics [17]. Pistacia
atlantica is a small evergreen shrub or tree belonging to the Anacardia­
ceae family, native to parts of Eurasia from the Iranian plateau to North
Africa, which has three subspecies: mutica, kurdica, and carbolic. The
subspecies motica is the main source of exudate gum (Saqez), which is
not well known in the world and is traditionally used as a therapeutic
agent for infections [18]. The gum of this plant and the compounds
derived from it have many uses in various industries [19] and medicine
[20], and a large number of studies have confirmed its significant
antimicrobial, anti-inflammatory, and antioxidant activity [21].
In this study, a bilayer hydrogel was fabricated by a simple method as
a novel wound dressing. The top layer was fabricated and designed with
PVA polymer and glycerol. The lower layer was fabricated with hydro­
phobic SEO loaded in the matrix of C, polyethylene glycol, and glycerol
in order to make a layer in direct contact with the wound surface, and
this layer is simply connected to the upper layer. The obtained bilayer
hydrogels were characterized using Fourier transform infrared spec­
troscopy (FTIR), light microscopy, and scanning electron microscopy
(SEM) analysis. The effect of SEO on gel fraction, mechanical properties,
rheological properties, swelling behavior, and water retention capacity
was determined. Also, the antibacterial and Antioxidant activity of this
bilayer hydrogel was investigated. In the following, cytotoxicity, he­
molysis ratio, and blood clotting in vitro were investigated compre­
hensively. In addition, an in-vivo homeostasis test was performed to
check the effectiveness and homeostatic capability of the corresponding
bilayer hydrogels.
2. Materials and methods
2.1. Materials
PVA (Mw 89,000-98,000, 99 + % hydrolyzed), polyethylene glycol
for synthesis (PEG, MW 5000–7000), C (medium molecular weight and
75–85 % deacetylated), and glycerol (MW 92.09 (anhydrous basis))
were purchased from Sigma-Aldrich. All chemicals were of analytical
grade and were used without any further purification. For evaluating
antimicrobial activities, Pseudomonas aeruginosa (ATCC® number
27853) and Staphylococcus aureus (ATCC® number 25923) were used as
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International Journal of Biological Macromolecules 226 (2023) 383–396
bacterial contamination models.
2.2. Extraction of SEO
The oleoresin gums of the Pistacia Atlantica tree of the Motica sub­
species were collected from the city of Eghlid (Fars-Iran) in August,
dried in the air for 3 days, and then pulverized with a laboratory grinder
at 5000 rpm for 5 min. The average size of powdered gums was calcu­
lated from 0.35 to 1.5 mm by horizontal sieve test. The powdered gums
were distilled for 4 h using a Clevenger-type apparatus. The collected
pale yellow essential oils were dried on anhydrous sodium sulfate and
stored in sealed vials at 4 ◦ C until use. The purity of the resulting
essential oil was considered to be Pure SEO and this essential oil was
used without dilution in the synthesis and further measurements were
not done to check the concentration of essential oil.
2.3. Preparation of bilayer hydrogel
The first or lower layer (PL) was the PVA layer with glycerol. First, (5
% w/v) PVA granules were dissolved in 100 ml of warm distilled water at
90 ◦ C and 800 rpm speed for 4 h to obtain a clear and uniform solution.
Next, 2 % (w/v) glycerol was added to the PVA solution for better
softness and flexibility of the first layer under stirring for 45 min. After
cooling at room temperature, the PVA solution was poured into plastic
petri dishes and dried in an oven at 50 ◦ C for 24 h. The second or higher
layer (CL) (the layer associated with the wound), the C layer, and PEG
and glycerol were associated with different concentrations of SEO. To
begin with, C 2 % w/v was made by dissolving C in 1 % V/V acetic acid.
The C solution was then combined with 1 % V/V in PEG and glycerol and
stirred for 45 min. The impurities in the C solution were then removed
by centrifugation for 30 min at 25 ◦ C at 4000 rpm. After that, concen­
trations of 0, 0.5, 1, and 2 % V/V of pure SEO were added to the above C
solution and stirred for 30 min before being poured on the dried first
layer (PVA). Then, the two layers were dried in an oven with hot air at
45 ◦ C for 24 h. Finally, PVA/C/SEO (PCS) bilayer hydrogels loaded with
different concentrations of SEO were named PCS0, PCS1, PCS2, and
PCS3, respectively (Fig. 1).
2.4. Characterization of PCS bilayer hydrogel
2.4.1. Morphology study
The average thickness of each layer was determined by using mi­
crometers at six separate places on the film with 0.01 mm accuracy.
Moreover, the bilayer morphology of hydrogel samples was observed
under a light microscope (Zeiss Primo Star Binocular Microscope, Ger­
many). For this purpose, a thin cross-sectional smear of the sample was
prepared on a clean glass slide for preliminary observation. Also, the two
sides of the surface and cross-section morphologies of the PCS bilayer
hydrogels were observed on a field emission scanning electron micro­
scope (SEM; Tescan Mira3, Czech Republic) with an acceleration voltage
of 10 kV. Using a sputtering device, each sample was coated with a thin
layer of gold. The cross-section of each sample was prepared by cracking
with liquid nitrogen.
2.4.2. Attenuated total reflection-Fourier transform infrared (ATR-FTIR)
analysis
FTIR (Tensor 27, Bruker, Germany) was used to determine the
presence of specific chemical groups in the materials and hydrogels
produced. The FTIR spectrum was recorded in the range of 400–4000
cm− 1 using KBr pellets under controlled environmental conditions.
2.4.3. Swelling ratio test
Through a general gravimetric technique, the swelling ratio was
estimated for the hydrogels [4]. Of the dry samples, three sets of disc
specimens (with a diameter of 2 cm) were prepared, which were
weighed (md) and then submerged and soaked in phosphate-buffered
A. Rezaei et al.
Fig. 1. Schematic of PCS bilayer hydrogel synthesis method.
saline (PBS; pH 7.4) for 24 h at 37 ◦ C. After reaching the swelling
equilibrium, the specimens were removed, and their excess moisture
was discarded from the surface of the membrane using filter paper, and
then they were weighed (ms) again. The tests were carried out in trip­
licate, and the average values were recorded. In accordance with for­
mula (1) presented below, the swelling ratios (%) of the specimens were
determined [11]:
ms − md
Swelling ratio = × 100% (1)
md
2.4.4. Water retention capacity
The capacity of water retention was tested in accordance with earlier
reports [22]. In order to reach the swelling equilibrium, the specimens
were submerged in deionized water. The weights of specimens were
recorded after the removal of the surface water and shown by Weq. Then,
the wet hydrogels were kept in an oven at 37 ◦ C. At pre-determined
periods, they were weighed, and their weights were recorded. The
following formula describes the water retention ratio for hydrogels:
/ A direct immersion technique was used to determine the gel fraction
Water retention ratio = Wt Weq × 100% (2)
In which Weq and Wt stand for the initial (t = 0 min) and time-
dependent weights of hydrogels.
2.4.5. Water vapor transmission rate (WVTR)
The WVTR of hydrogels was determined according to ASTM E96/
E96M-10 (ASTM, 2010). The hydrogels were cut into round pieces
(10 mm in diameter), attached to the mouths of bottles containing 15 ml
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International Journal of Biological Macromolecules 226 (2023) 383–396
of deionized water using paraffin tape, and preserved for 24 h in an
incubator at 37 ◦ C and 70 % relative humidity. All bottles were weighed
at regular intervals (24, 48, and 72 h). Three bottles without caps were
considered controls, and each experiment was repeated three times. The
water vapor permeability of the films was determined by the following
equation:
Wt − W0
WVTR (g/m2h) = × 100 (3)
tA
2.4.6. Water contact angle
The water contact angle of specimens was determined at room
temperature via a contact angle analyzer device (manufactured by Data
physics Instruments, GmbH, Germany). An approximate amount of 3 μL
ultrapure water was utilized as the probe liquid dropped on the surface
of the hydrogels. To determine the average value, three replicates were
prepared for each sample.
2.4.7. Gel fraction
of the hydrogels. The PCS bilayer hydrogels were weighed after being
dried to a consistent weight, then put in separate beakers and soaked in
distilled water. Until the PCS bilayer hydrogels reached a consistent
weight, the water was replaced every 4 h. They were then weighed again
after being placed in a vacuum oven at 37 ◦ C until they reached a
consistent weight.
W₁
F(%) = × 100% (4)
Wₒ
A. Rezaei et al.
F (%) represents the gel fraction rate, W0 denotes the beginning weight
of the PCS bilayer hydrogels, and W1 represents the final weight of the
double-layer hydrogel films.
2.4.8. Mechanical test
The hydrogels' bending strain, elongation at break, and stress were
measured with a SANTAM Machine Model C/1010074 Universal Testing
Machine. All dried specimens were sliced into rectangular forms with
diameters of 20 mm and 40 mm. All samples were pulled at a rate of 10
mm/min with a 1 kN load cell until deformation occurred. Before
testing, the hydrogel samples were put in PBS for 15 min at 37 ◦ C to
enable wet conditions and reduce drying. Tensile strength and elonga­
tion at the break of the results were tested. The tensile strength
parameter was calculated using the following formula:
Fmax
Tensile Strength = (5)
A
2.4.9. Rheological analysis
The rheological properties of PCS bilayer hydrogels were determined
by testing them at 37 ◦ C on a rotary rheometer with a 25 mm diameter
parallel plate (MCR300 SN599139; FW2.04; Slot5).
2.4.10. In vitro antibacterial activity
The disk diffusion method was used to assess the antibacterial
properties of PCS bilayer hydrogels against Staphylococcus aureus (gram-
positive) (ATCC® number 25923) and Pseudomonas aeruginosa (gram-
negative) (ATCC® number 27853). Efficacy in vitro against critical
threat pathogens Staphylococcus aureus and Pseudomonas aeruginosa is
widely studied [23]. First, the PCS bilayer hydrogels were sterilized
under UV for 30 min. The bacteria were then suspended in sterilized
BCIindex = 100 × (abs of blood which had been in contact with sample at 542 nm)/abs of ACD whole blood in water at 542 nm
physiological saline, and the turbidity was adjusted to 1.5 × 108 CFU/
ml, the McFarland standard. The bacterial suspension was then cultured
on Müller-Hinton agar medium with a sterile swab (Merck Millipore,
Germany). The PCS bilayer hydrogels were then cut into disks about 5
mm in diameter, placed on Petri dishes, and incubated for 24 h at 37 ◦ C.
Ciprofloxacin antibiotic discs were also used as a control group. The
inhibition of bacterial growth was investigated after the specified time.
2.4.11. Antioxidant activity
The antioxidant activity of PCS bilayer hydrogel wound dressing
samples was evaluated using the Diphenyl-2-picrylhydrazyl radical
scavenging (DPPH) method. After dissolving 6 mg of DPPH in 50 ml of
methanol (0.3 mmol), 2.5 ml of each bilayer hydrogel extract and 2.5 ml
of the DPPH solution were thoroughly mixed. The tube was then incu­
bated in the dark for 30 min at room temperature. Their absorption was
then measured at 517 nm with a microplate reader (BioTek Instruments,
Epoch2, Winooski, VT, USA). The reactions were repeated three times,
and the percentage of radical scavenging was calculated by the
following equation:
DPPH scavenging (%) = [(Ac − As)/Ac )] × 100 (6)
where Ac represents the absorbance of the control DPPH solution and as
represents the absorbance of the PCS bilayer hydrogel extract following
reaction with the DPPH solution.
2.4.12. Hemolysis test
An approach from the American Society for Testing and Materials
was used to perform the hemolytic tests [24]. First, 7 mL of PBS was
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International Journal of Biological Macromolecules 226 (2023) 383–396
added to the hydrogels (2 cm × 2 cm), and they were then maintained at
37 ◦ C for 24 h. After that, 1 mL of diluted acid-citrate-dextrose (ACD)
blood was added, and it was incubated at 37 ◦ C for another 3 h. To make
the negative and positive controls, blood was mixed with 7 mL of PBS
and incubated at 37 ◦ C for 3 h each. The fluids were collected after in­
cubation and centrifuged at 8000 rpm for 15 min. The optical density
(OD) of the supernatants was measured at 540 nm. The following
calculation was used to determine the percentage of hemolysis:
Hemolysis (%) = (ODs − OD (− ) )/(OD (+ ) − OD (− ) ) × 100 (7)
where ODs, OD (− ), and OD (+), respectively, represent the sample's
OD, negative control, and positive control.
2.4.13. Whole blood clotting
In the plates, the prepared PCS bilayer hydrogels were inserted. For
5 min, these plates were thermostated in a 37 ◦ C water bath. The surface
was then slowly covered with 0.25 mL of blood sample (0.3 mL of ACD-
whole blood with the addition of 0.024 mL of CaCl2 (0.2 mol/l)). Plates
containing blood samples were then incubated in a thermostated incu­
bator at 37 ◦ C. After 10 min, 10 ml of deionized water were added
without disturbing the coagulated blood. 10 ml of solution was then
removed from the plates and centrifuged at 1000 rpm for 30 s. The su­
pernatant was decanted into a tube with 40 ml of extra deionized water
and kept at 37 ◦ C for 60 min. The blood clotting test was performed by
spectrophotometry at 542 nm, which measured the relative absorbance
of blood samples diluted to 50 ml. The absorbance of 0.25 ml of ACD-
whole blood in 50 ml of deionized water at 542 nm was considered to
be 100 as a reference value. The formula below can be used to calculate
biomaterials' blood clotting index (BCI):
(8)
2.4.14. In vitro cytotoxicity
According to ISO Specification 10,993, the MTT technique was used
to determine the test's cytotoxicity on L929 cells [25]. At a density of 1
× 104 cells per well, L929 cells in the logarithmic growth phase were
sown on 96-well plates and incubated at 37 ◦ C with 5 % CO2. After 12 h
of incubation, the culture media was taken out and 100 μL of the
hydrogel extract was added to each well. The blank culture medium was
used as a negative control. The plates underwent 24, 48, and 72 h of
incubation at 37 ◦ C, respectively. Each well received 10 μL of MTT (5
mg/mL) at the conclusion of incubation. The media were sucked out of
the plates after they had been continuously incubated for 4 h. Then, each
well received 150 μL of DMSO, and cultures were continued for an
additional 10 min at 37 ◦ C. A microplate reader was used to measure the
absorbance value at 490 nm (Tecan Austria GmbH, Untersbergstrasse 1A
A-5082, Austria). Finally, using the below formula, the cell viability was
determined.
Cell viability = OD490e /OD490c × 100% (9)
where the absorbance values for the experimental and adverse control
groups, respectively, are OD490e and OD490c.
2.4.15. In vivo hemostasis evaluation
Six rats (200 ± 20 g, male) were randomly assigned into two groups.
A 500 mg/1000 g intraperitoneal dose of 5 % chloral hydrate was used
to anesthetize the rat for 2 min before surgery, after which it was fixed
on surgical board. After that, the model was created by amputating the
A. Rezaei et al.
tail with surgical scissors three centimeters from the end and suspending
it in the air for 15 s to guarantee appropriate blood loss. The PCS bilayer
hydrogel sheet was then placed over the rat tail wound into one group,
who also used tweezers to provide some pressure. Used as a control was
the other group that wasn't given any treatment. Following the cessation
of bleeding, the hemostatic times of the two groups were calculated, and
the amount of blood lost was measured [26]. All animal experiments
were approved by the Research Ethics Committees of Shahid Beheshti
University (ethical code number # IR.SBU.REC.1400.196).
2.4.16. Statistical analysis
All quantitative information was presented as a mean ± standard
deviation (SD). The statistical analysis was performed using ANOVA
test, and p < 0.05 was determined statistically significant.
3. Results and discussion
3.1. Morphology study
The average thickness of the PL was 0.30 mm (±0.01), the CL was
0.33 mm (±0.01) and the bilayer was 0.63 mm (±0.01). The CL and the
PL indicated an approximate pH of 5.22 and 5.42, respectively, which is
favorable for wound dressing purposes because skin features a slightly
acidic pH (ranging from 4.7 to 5.75), and following the administration of
membranes, no irritation would be reported. It is noteworthy that
chronic wounds have higher alkaline pH (ranging from 7.15 to 8.9), and
their complete healing takes longer. In these conditions, contact with a
membrane featuring a lower pH will speed up the healing procedure. As
a result, applying a dressing with a slightly acidic and/or neutral pH (as
shown for membranes) is critical to improvement of the restoration
process of skin tissues (Fig. 2.A). As well, Fig. 2B represents two-layer
hydrogels whose color becomes darker with the addition of essential
oil, which indicates an increase in the concentration of essential oil in
layer C.
Light microscopy was performed to observe PCS bilayer hydrogels
prepared for general morphology, to confirm the formation of PCS
bilayer hydrogel s (Figs. 2.C1–3). Observations were performed with
×40, ×100, and ×400 magnification respectively. As shown in the
Figures, the PCS bilayer hydrogel is fully formed and the two layers of
PVA and CS with SEO are completely joined together. Figs. 2.D1–3
represent SEM cross-sectional micrographs of PCS bilayer hydrogels and
Figs. 2.D4–6 demonstrated pictures of the surface PCS bilayer hydrogels.
The SEM results indicate that the hydrogel membrane has a thick shape
and no pores, even at high magnifications. The dense surface of the
hydrogel membrane prevents microorganisms from entering and
approaching the wound. Due to the hydrophobic and immiscible prop­
erties of essential oil, their particles are visible on the surface of SEM
images. In general, the absence of pores on the surface shows that the
essential oil particles are trapped in the 3D structure of the hydrogel. At
the same time, the formation of macro pores indicates the rapid evap­
oration of essential oil. A cross-sectional examination of the bilayer
hydrogel confirmed that two layers of hydrogel were formed on top of
each other, as seen in the light microscope images, which clearly showed
a bilayer interface. These two layers of polymer matrix are almost
smooth and without any pores. Also, in these images, the particles of
essential oil can be seen from the surface.
3.2. ATR-FTIR analysis
As part of our research, we used FT-IR spectroscopy to identify
functional groups in SEO, CL, and PCS3 (Fig. 3.A). The SEO peaks at
3368 cm− 1, corresponding to the –OH alcohol. Furthermore, the peak at
2916 cm− 1 was associated with asymmetric C–H stretching, which is
probably assigned to alkyl stretch groups in SEO. Then, the peak at 1676
cm− 1 was associated with the C– – O groups. The peak at 1444 cm− 1 was
assigned to CHOH, and the observed peaks at 1265 and 786 cm− 1 are
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International Journal of Biological Macromolecules 226 (2023) 383–396
related to N–H (Amide III) and aromatic structure, respectively. In
addition, the C–H stretching vibration of aromatics can be seen at near
953 cm− 1 to 885 cm− 1. The CL solution bands appeared at 3330, 1629,
and 1215 cm− 1, which represent primary amide (N–H), carbonyl
groups (C=O), and amide ІІІ (C–– N stretch plus N–
– H in phase bending),
respectively. Peaks at 1367 cm− 1 correspond to the aromatic ring. The
FTIR spectra of PCS3 hydrogel demonstrate peaks at 3320 cm− 1, which
indicate the presence of primary amide (N–H). Also, two peaks at 1736
and 1629 cm− 1 indicate the presence of C– – O stretching vibration
probably related to aldehyde C– – O stretch groups and C– – C alkene
stretching. Next, peaks at 1367, 1215, and 950 cm− 1 indicate the pres­
ence of alkane bending vibration (C–H), C–O stretching vibration
groups, and C–– C bending vibration, respectively. The absence of a peak
at 2916 cm− 1 is probably due to the bonds between this group and the
active groups in chitosan and PEG. Generally, as shown in Fig. 3.A, the
functional groups present on the surface of SEO and CL hydrogel are
seen in the FTIR spectrum of the PCS3 [27,28].
3.3. Swelling ratio test
For the PCS bilayer hydrogels developed in this study, the swelling
performance was studied subject to a neutral medium (pH = 7.4) from
50 to 600 min, and Figs. 3.B and C present the obtained results. Over
time, the swelling ratio of sample PCS0 showed a significant difference
(p < 0.05) compared to other samples containing SEO. The maximum
swelling occurred in the sample PCS0, which is apparently attributable
to the hydrophilic nature of PVA and C. Generally, the addition of SEO to
the PCS bilayer hydrogel reduced the swelling significantly, which was
potentially ascribable to the hydrophobic nature of SEO. The swelling of
PCS hydrogels depends on the ionization of carboxyl or amino groups,
water diffusion, polymer relaxation, and dissociation of ionic and
hydrogen bonds. Furthermore, adding SEO to the hydrogel can result in
the restricted water uptake of the PCS network, which is potentially
ascribable to the increased interactions between hydrophobic SEO and
PVA and C chains [29]. In general, PCS hydrogels have a high-water
absorption capacity of 280 to 112 %. Amalraj et al., 2020, reported
that increasing the proportion of black pepper essential oil led to a
significant decrease in the swelling ratio, which they attributed to the
decrease in the interaction between water and the film matrix due to the
hydrophobic effect of the added essential oil [30].
3.4. Water retention capacity
In hydrogel wound dressings, the favorable potential of water
retention at various temperatures is a key issue for the provision of a
moist medium for dissolution of necrotic tissues, prevention of the for­
mation of scabs, and thus promotion of wound healing while providing
comfort without causing the pain resulting from dryness [31]. Fig. 3.D
illustrates the water retention capacity of PCS bilayer hydrogels. For
PCS0, after a rapid decrease during the first hour, the retention ratio
experienced a gradual decline within the next 5 h, and after 6 h, 32.8 %
of the initial weight was retained. An obviously different trend was
experienced as PCS1 was introduced. In the first 3 h, the rate of weight
loss declined. In addition, a higher retention ratio (35.3 %) was achieved
after 6 h. Given the weight loss of the entire procedure, PCS3 showed
better performance in improving the capacity of water retention (41.9
%) [10]. It was found that water retention capacity decreased more in
the second part of the study (6–12 h). This could be related to the
increased concentration of SEO, which allows less water to leave the CL
containing SEO, or related to the enhanced formation of the network or
changed interaction between the polymers and water [32].
3.5. Water vapor transmission rate (WVTR)
Besides maintaining a high humidity level between the dressing and
the wounded areas, effective wound dressings that employ hydrogels
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A. Rezaei et al.
Fig. 2. (A) Weight, thickness, and surface pH of PL, CL, and PCS bilayer hydrogels. (B) The images of PCS bilayer hydrogels. Optical microscopic image at (C1) ×40,
(C2) 100× and (C3) ×400. The SEM cross-sectional morphology of PCS bilayer hydrogels at (D1) 1.0 kx, and (D2) 500× (D3) 100×, and the SEM surface morphology
(D4) 1.0 kx, (D5) 500×, (D6) 100×.
must feature controllable transmission and absorption rates. Higher
WVTRs cause the wound area to dry much faster, which may lead to
scarring. However, lower WVTRs in wound dressings may make the
wound area susceptible to excessive exudates that may lead to an
increased chance of infection and delayed healing. It is recommended
that the WVTR value be in the range of 2000–2500 g/m2/day in order to
maintain an optimal amount of moisture in the wound environment
[33]. As shown in Fig. 3.E, The WVTRs of hydrogels were 2257 ± 356,
2127 ± 298, 352 ± 352, and 1854 ± 410 g/m2 per day for PCS0, PCS1,
PCS2 and PCS3 respectively. However, the WVTR for the control
(without covering) was found to be 4232 ± 598 g/m2 per day, which is
extremely high. As the concentration of SEO increases, the percentage of
WVTR decreases, but this decrease percentage was not significant (p <
0.05), which is in agreement with the previous report [34]. This
decrease in WVTR value after adding SEO can be due to the presence of
strong physical interaction between the polymer chains of layer C,
which leads to the unavailability of available free sites for the in­
teractions of water molecules in the PCS bilayer hydrogel. In addition, it
can be due to the hydrophobic nature of the essential oil, which reduces
the rate of water vapor passing through the matrix in the polymer ma­
trix, which is consistent with the results of the contact angle, water
retention, and swelling behavior tests. In general, the WVTR of the PCS
hydrogels was near to the range judged suitable for wound dressings that
might provide an adequate level of moisture to prevent severe dehy­
dration or excessive development of exudates in the wound region [34].
3.6. Water contact angle
For materials contacting the human skin, a balance between hydro­
phobic and hydrophilic properties is important. Also, chemical compo­
sition and surface topology determine a surface's wettability. The
increasing contact angle of the PCS bilayer hydrogels is shown in Fig. 4.
A. Due to its hydrophilic nature, PCS0 revealed a low water contact
angle of 33◦ . With the addition of SEO, the water contact angle of PCS1,
PCS2, and PCS3, which include mostly polyphenols, i.e., aromatic
compounds with a -OH group, on the surface of the films rose. As a
result, the contact angle increased from 33◦ to 78◦ for PCS3, which is
confirmed by previous results [35].
3.7. Gel fraction
A gel fraction test is conducted to determine the effectiveness of the
cross-linker. Gel fraction levels are determined by the crystallinity of the
network and the degree of crosslinking [36]. Fig. 4.B shows the findings
of evaluating the gel fraction of the PCS bilayer hydrogels. The values of
the gel fraction for the samples of PCS0, PCS1, PCS2, and PCS3 were 20
%, 48.78 %, 54.05 %, and 78.65 %. Generally, gel fraction results
showed significant differences (p < 0.05) correlated with the increase of
SEO. This increase in gel fraction can be due to the presence of O–H and
N–H functional groups in the SEO and the increase in crosslinks in layer
C due to the addition of SEO [37].
3.8. Mechanical test
Mechanical qualities are required to offer appropriate physical sup­
port for cell proliferation and their application to homeostasis in order to
be used as a wound dressing. Therefore, it is crucial that an ideal wound
dressing possesses the appropriate mechanical properties [38]. The
stress, bending strain, and elongation at break of PCS bilayer hydrogels
were examined. According to Fig. 5.A, the stress has increased signifi­
cantly (p < 0.05). That is due to the changed thickness of the bilayer
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International Journal of Biological Macromolecules 226 (2023) 383–396
hydrogel compared to the single-layer. In the following, bending prop­
erties show an increase with double layering, and we guess that the
reason is the addition of essential oil in the PCS bilayer hydrogel to the C
layer, that the oily state of the essential oil has played a significant role
in improving the bending, and that the placement of the essential oil
between the joints of the two polymers and C has helped in this situation
(Fig. 5.B). Elongation at break is an indication of the flexibility and
extensibility of film prior to breakage. Figs. 5.C and D show the addition
of essential oil also increases the elongation at break in the PCS bilayer
hydrogels, which is confirmed by previous results [39]. Also, this issue
can be due to the addition of essential oil to the PCS bilayer hydrogel,
that the oily state of the essential oil plays a significant role in increasing
the elasticity at the elongation at break. Also, according to Fig. 5.C, this
increase can be related to the characteristics of PVA polymer.
3.9. Dynamic rheology analysis
To obtain a greater understanding of the properties of PCS bilayer
hydrogels, their rheological properties are investigated in various
ambient atmosphere settings. The loss modulus is the viscous response,
while the storage modulus is the elastic solid-like behavior. As Figs. 6.A
and B show, the G′ of each of the two samples exceeds G′′ which
confirmed the results of previous studies [40]. As a result, the acquired
PCS bilayer hydrogels can be considered predominantly elastic. Adding
SEO leads to increased hydrogel elasticity. When the frequency becomes
equal to the reciprocal relaxation time, which is determinable as the
gelation time, G′′ and G′ will crossover. The results show the gelation
times of PCS3 and PCS0 begin at the start of the process. It shows that the
cross-linked structure of the hydrogels is formed rapidly, whereas the
effect of SEO on the formation of the network structure is insignificant.
3.10. Antibacterial test
Wound infection is one of the most difficult problems to deal with the
wound. A good wound dressing should not only act as a barrier between
the wound site and the external pathogenic environment, but it should
also have antibacterial properties. S. aureus and P. aeruginosa are two of
the most pathogens for infecting human wounds. They are frequently
the source of coinfection of the same wounds, leading to a lengthy delay
in wound healing, a steady worsening of health conditions, and
extraordinary resistance to the most potent antimicrobial medicines
[41]. The antibacterial properties of hydrogels against S. aureus (gram-
positive) and P. aeruginosa (gram-negative) were evaluated by the disk
diffusion method. According to Fig. 7.A, PCS1-PCS3 bilayer hydrogels
show an inhibitory effect against S. aureus as a gram-positive bacterium,
which confirmed the results of previous studies [42]. The most
remarkable inhibition of the growth of the above bacterium occurred in
PCS3 (inhibitory zone = 11 mm), while the minimum effect was asso­
ciated with the basic group PCS0 (7 mm). According to Fig. 7.B, the
extent of bacterial growth inhibition by hydrogel groups was scrutinized
for P. aeruginosa as a gram-negative bacterium which is comparable to
the findings of previous research [28]. According to the results, PCS3
(14 mm) had the maximum inhibitory effect, whereas the minimum
effect was that of the basic group PCS1 (relatively resistant with a
diameter of 10 mm) (Fig. 7.C). Antimicrobial activity in this essential oil
can be attributed to compounds such as camphene and limonene,
linalool and alpha-terpineol, cineole, borneol, carvacrol, and alpha-
pinene [43]. When discussing the mechanism of action of essential
oils, it is essential to mention that treating microorganisms with essen­
tial oils destroys the integrity and function of the membrane. This can
eventually lead to a loss of cell self-sustainability, intracellular
A. Rezaei et al. International Journal of Biological Macromolecules 226 (2023) 383–396

Fig. 3. (A) FTIR of SEO, C layer and PCS, (B) Swelling ratio results as a function of time for PCS0, PCS1, PCS2, and PCS3 bilayer hydrogels, (C) Image of swelling
behavior for PCS bilayer hydrogels, (D) PCS water retention, (E) WVTR of hydrogels after 24 h.

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Fig. 4. (A) Contact angle measurements and Contact angle images of PCS bilayer hydrogels, (B) PCS bilayer hydrogels gel fraction.

Fig. 5. (A) Stress of the PCS bilayer hydrogels, (B) Bending strain of the PCS bilayer hydrogels, (C) Elongation at break of the PCS bilayer hydrogels, (D) Elongation
images of CL, PL and BL hydrogels.

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Fig. 6. (A) Rheological properties of PCS0 bilayer hydrogels, (B) Rheological properties of PCS3 bilayer hydrogels.
compound leakage, and cell death. In general, the bilayer hydrogel with
essential oil demonstrated the ability to kill S. aureus and P. aeruginosa,
two common pathogenic bacterial strains that cause inflammation and
delay wound healing [44].
3.11. Antioxidant activity
Massive amounts of free radicals would be present in the wound site
when the skin is injured, leading to oxidative stress. The DPPH assay was
looked into to determine the antioxidant capability of PCS bilayer
hydrogels. The antioxidant activity of the PCS0 bilayer hydrogel was
shown in Fig. 7.D to be approximately 19.35 %. However, the bilayer
hydrogel scavenging capacity increased to 61.32 % (PCS3), which shows
that it has improved significantly (p < 0.05) and confirms the findings of
previous research [45]. Because SEO free amino groups can combine
with unpaired electrons to generate ammonium groups, which are able
to scavenge free radicals, they can be credited with this great oxidative
ability. Due to the presence of amino groups, C was also said to possess
antioxidant effects [46].
3.12. Whole blood clotting
The in vitro blood coagulation assay is a common way to show how
well materials are at controlling bleeding. The findings of the in vitro
whole blood clotting test used to evaluate the hemostatic effects of PCS0,
PCS1, PCS2, and PCS3 hydrogels are displayed in Fig. 8.A and B. BCI is a
relative metric to quantitatively quantify hemostatic performance. A
lower BCI value indicates a better hemostatic property of materials.
When the concentration of SEO in the bilayer hydrogels increases, the
BCI values of the PCS hydrogels decrease (p < 0.05). The indices for the
bilayer hydrogels PCS0, PCS1, PCS2, and PCS3 were therefore 23.36,
17.55, 15.33, and 14.55, respectively. Overall, we believe that the
interaction of cationic C and positively charged amide functional groups
in SEO with negatively charged blood cells can result in the formation of
a blood-like viscous gel that promotes blood coagulation. Also, due to
the superabsorbent nature of PVA, the hydrogels exhibit some degree of
hemostatic properties, which can rapidly adsorb fibrinogen and plasma
proteins and enhance platelet aggregation and promote blood clot for­
mation in the hydrogels [47].
3.13. In vitro hemolysis test
Also, using an in vitro hemolysis assay, the hemocompatibility of PCS
bilayer hydrogels was estimated. In vitro blood compatibility, also
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International Journal of Biological Macromolecules 226 (2023) 383–396
known as hemolysis, is a study that assesses the adaptive behavior of red
blood cells (RBC) when they come into contact with foreign substances.
Wound dressings in direct contact with blood must also be compatible
and have a sufficient range of hemolysis in the field of tissue engineer­
ing. According to the ASTMF756–00 (2000) standard, biomaterials with
up to 5 % hemolysis are considered safe [48]. According to the statistical
analysis (p < 0.05), the hemolysis ratios of PCS hydrogels were lower
than 4 % compared to the positive control group of water (Fig. 8.C). The
supernatants of various groups after centrifugation have been shown in
Fig. 8.D. While the color of PCS3, PCS2, PCS1, and PCS0 was bright
yellow, the positive group (water) showed a bright red color as a result
of the broken red blood cells, which indicates negligible hemolytic ef­
fects. In general, the percentage of hemolysis decreased in bilayer
hydrogels containing continuous SEO, which was consistent with pre­
vious studies using plant-derived gums [49].
3.14. Cell viability test
The wound dressing cell compatibility test is required to determine
the extent to which the manufactured PCS bilayer hydrogels exhibit
favorable biocompatibility in response to common skin cells such as fi­
broblasts, keratinocytes, and epithelial cells during wound healing. Fi­
broblasts play an important role in the formation of connective tissues,
causing granulation of skin tissues and encouraging skin remodeling. We
used the MTT assay to investigate the cytotoxicity of fibroblast cells in
addition to cell interactions with wound dressings. As shown in Fig. 8.E,
during these three days of incubation, the cell viabilities on the PCS1,
PCS2, and PCS3 films are close to the PCS0, showing that SEO is suited
for cell incubation. Specifically, on the first day of the test, the viability
values of PCS0, PCS1, PCS2, and PCS3 were respectively 98 %, 95 %, 91
%, and 89 %, and PCS0 had the highest cell viability compared to other
groups, but there was no significant difference (p < 0.05). Further, on
the second day of incubation, there was a significant difference between
the control sample and PCS2, and PCS3, and on the third day, the con­
ditions were the same (p < 0.05). These results show that the cytotox­
icity of bilayer PCS hydrogels increases slightly with increasing SEO
concentration, but the cell viability of all samples was above 80 %,
which is considered non-toxic and was in agreement with the results of
previous studies [42]. These findings suggest that PCS bilayer hydrogels
have high biocompatibility and are good for cell survival and prolifer­
ation without causing cytotoxicity.
A. Rezaei et al.
Fig. 7. Antibacterial inhibitory effects of PCS bilayer hydrogels against (A) S. aureus and (B) P. aeruginosa (Disks' diameter = 6 mm), (C) Zone of inhibition diagram of
PCS bilayer hydrogels, (D) Antioxidant activity of PCS bilayer hydrogels.
3.15. In vivo hemostasis evaluation
At the moment, the most commonly utilized hemostasis technique
for deep traumas or penetrating wounds is to insert hemostatic gauze
into the wound and apply external pressure to reduce the body's rapid
blood loss. Nonetheless, such a procedure has drawbacks and a high
fatality rate among injured people [50]. The hemostatic effectiveness
and capability of PCS bilayer hydrogel were estimated using a mouse tail
amputation model and its associated results. Each group's hemostasis
time and blood loss were recorded. The mean blood loss and hemostasis
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International Journal of Biological Macromolecules 226 (2023) 383–396
time were 111 ± 42 mg and 145 ± 49 s for PCS3 hydrogel, 138 ± 48 mg
and 185 ± 54 s for gas, and 289 ± 58 mg and 457 ± 90 s for blank,
respectively (Figs. 9.A and B). Fig. 9.C also shows the hemostatic ability
and effectiveness of the PCS bilayer hydrogel using the mouse tail
amputation model. In general, the PCS bilayer hydrogel had a significant
difference with the control sample, which shows that the bilayer
hydrogel wound dressing reduces the time of hemostasis and the amount
of blood loss (p < 0.05). The reason for this increase in homeostatic
properties can be due to the functional groups with a positive load in C
and SEO. Through electrostatic interaction, it can increase the
A. Rezaei et al. International Journal of Biological Macromolecules 226 (2023) 383–396

Fig. 8. (A) The results of the blood clotting test, (B) Images of whole blood clotting of various PCS bilayer hydrogel, (C) Hemolysis rates for the PCS samples and
water as a positive control (D), Images of hemolytic activity assay of the PCS bilayer hydrogel (E) PCS0, PCS1, PCS2, and PCS3 cell viability tests.

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Fig. 9. (A) The results of the blood loss in bilayer hydrogel with SEO by using the mouse tail amputation model (bleeding blood volume in milligram), (B) The results
of the hemostasis time in bilayer hydrogel with SEO by using the mouse tail amputation model (bleeding blood time in seconds), (C) Images of the hemostatic ability
and effectiveness of bilayer hydrogel) the right-side image is BLEO sample and the left-side image is control (with SEO by using the mouse tail amputation model.
aggregation of negatively charged red blood cells [51]. All of these ob­
servations confirm that PCS bilayer hydrogels have potential as hemo­
static agents in biomedical fields.
4. Conclusions
In summary, a novel PCS bilayer hydrogel composed of C, SEO as
lower, and PVA as upper layers was successfully designed in an easy and
simple way for wound dressing. The effect of different concentrations of
SEO in the CL layer on the properties of the bilayer hydrogels was
investigated. The results demonstrated that the designed PCS bilayer
hydrogel achieved suitable physical and mechanical properties,
adequate WVTR, and good water swelling. It is also shown that the
incorporation of proper content of SEO could enhance the water reten­
tion capacity of the PCS0 hydrogel. The antioxidant test represented
excellent performance in eliminating free radicals as antioxidant
hydrogels. As well, antibacterial tests demonstrated high antibacterial
activity against pathogenic bacteria like S. aureus and P. aeruginosa. In
addition to this, cell viability, blood compatibility tests, and in vitro
hemostasis tests were performed on PCS bilayer hydrogel. It was found
that these bilayer hydrogels have good cell compatibility, suitable blood
compatibility, and hemostatic potential and are a good candidate for
wound dressing. Besides, it was indicated that compared to the un­
treated group, gas and PCS3 have a very good ability to reduce blood
loss and promote timely blood coagulation. As a result, and according to
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International Journal of Biological Macromolecules 226 (2023) 383–396
these findings, the designed bilayer wound dressing clearly shows that it
is a promising alternative to traditional products, and in the future, it
could be widely used as an antibacterial and antioxidant wound
dressing.
CRediT authorship contribution statement
Ali Rezaei: Investigation, Visualization, Writing – original draft,
Writing – review & editing. Hamide Ehtesabi: Conceptualization,
Methodology, Supervision, Writing – original draft, Writing – review &
editing. Somaye Ebrahimi: Investigation, Writing – original draft.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
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