Topically Applied Biopolymer-Based Tri-Layered Hierarchically Structured Nanofibrous Scaffold with a Self-Pumping Effect for Accelerated Full-Thickness Wound Healing in a Rat Model

Abstract

Wound healing has grown to be a significant problem at a global scale. The lack of multifunctionality in most wound dressing-based biopolymers prevents them from meeting all clinical requirements. Therefore, a multifunctional biopolymer-based tri-layered hierarchically nanofibrous scaffold in wound dressing can contribute to skin regeneration. In this study, a multifunctional antibacterial biopolymer-based tri-layered hierarchically nanofibrous scaffold comprising three layers was constructed. The bottom and the top layers contain hydrophilic silk fibroin (SF) and fish skin
collagen (COL), respectively, for accelerated healing, interspersed with a middle layer of hydrophobic poly-3-hydroxybutyrate (PHB) containing amoxicillin (AMX) as an antibacterial drug. The advantageous physicochemical properties of the nanofibrous scaffold were estimated by SEM, FTIR, fluid uptake, contact angle, porosity, and mechanical properties. Moreover, the in vitro cytotoxicity and
cell healing were assessed by MTT assay and the cell scratching method, respectively, and revealed excellent biocompatibility. The nanofibrous scaffold exhibited significant antimicrobial activity against multiple pathogenic bacteria. Furthermore, the in vivo wound healing and histological studies demonstrated complete wound healing in wounded rats on day 14, along with an increase in the
expression level of the transforming growth factor-β1 (TGF-β1) and a decrease in the expression level of interleukin-6 (IL-6). The results revealed that the fabricated nanofibrous scaffold is a potent wound dressing scaffold, and significantly accelerates full-thickness wound healing in a rat model.

Full text

Citation: Hamza, K.H.; El-Shanshory,
A.A.; Agwa, M.M.; Abo-Alkasem,
M.I.; El-Fakharany, E.M.; Abdelsattar,
A.S.; El-Bardan, A.A.; Kassem, T.S.;
Mo, X.; Soliman, H.M.A. Topically
Applied Biopolymer-Based Tri-
Layered Hierarchically Structured
Nanofibrous Scaffold with a Self-
Pumping Effect for Accelerated
Full-Thickness Wound Healing in a
Rat Model. Pharmaceutics 2023,15,
1518. https://doi.org/10.3390/
pharmaceutics15051518
Academic Editors: Ewa Kłodzi ´nska
and Marek Konop
Received: 20 December 2022
Revised: 17 April 2023
Accepted: 15 May 2023
Published: 17 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceutics
Article
Topically Applied Biopolymer-Based Tri-Layered Hierarchically
Structured Nanofibrous Scaffold with a Self-Pumping Effect
for Accelerated Full-Thickness Wound Healing in a Rat Model
Kholoud H. Hamza 1, Ahmed A. El-Shanshory 2, * , Mona M. Agwa 3, * , Mohamed I. Abo-Alkasem 3,
Esmail M. El-Fakharany 4, Abdallah S. Abdelsattar 5, 6, * , Ali A. El-Bardan 1, Taher S. Kassem 1, Xiumei Mo 7
and Hesham M. A. Soliman 2
1Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Alexandria 21321, Egypt;
kholoudhamza135@gmail.com (K.H.H.); alielbardan@yahoo.com (A.A.E.-B.); taherkasem@gmail.com (T.S.K.)
2
Composites and Nanostructured Materials Research Department, Advanced Technology and New Materials
Research Institute (ATNMRI), City of Scientific Research and Technological Applications (SRTA-City),
New Borg Al-Arab, Alexandria 21934, Egypt; h.soliman@srtacity.sci.eg
3Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries
Research Institute, National Research Center, Dokki, Giza 12622, Egypt; alkasem88@yahoo.com
4Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI),
City of Scientific Research and Technological Applications (SRTA-City), Alexandria 21934, Egypt;
esmailelfakharany@yahoo.co.uk
5Center for Microbiology and Phage Therapy, Zewail City of Science and Technology, October Gardens,
6th of October City, Giza 12578, Egypt
6Center for X-Ray and Determination of Structure of Matter, Zewail City of Science and Technology,
October Gardens, 6th of October City, Giza 12578, Egypt
7Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, College of Chemistry,
Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China; xmm@dhu.edu.cn
*
Correspondence: shansho.medo@gmail.com (A.A.E.-S.); mona.m.agwa@alexu.edu.eg or mm.agwa@nrc.sci.eg
or magwa79@gmail.com (M.M.A.); p-abdallah.abdelsattar@zewailcity.edu.eg (A.S.A.)
Abstract:
Wound healing has grown to be a significant problem at a global scale. The lack of multi-
functionality in most wound dressing-based biopolymers prevents them from meeting all clinical
requirements. Therefore, a multifunctional biopolymer-based tri-layered hierarchically nanofibrous
scaffold in wound dressing can contribute to skin regeneration. In this study, a multifunctional
antibacterial biopolymer-based tri-layered hierarchically nanofibrous scaffold comprising three layers
was constructed. The bottom and the top layers contain hydrophilic silk fibroin (SF) and fish skin
collagen (COL), respectively, for accelerated healing, interspersed with a middle layer of hydrophobic
poly-3-hydroxybutyrate (PHB) containing amoxicillin (AMX) as an antibacterial drug. The advanta-
geous physicochemical properties of the nanofibrous scaffold were estimated by SEM, FTIR, fluid
uptake, contact angle, porosity, and mechanical properties. Moreover, the
in vitro
cytotoxicity and
cell healing were assessed by MTT assay and the cell scratching method, respectively, and revealed ex-
cellent biocompatibility. The nanofibrous scaffold exhibited significant antimicrobial activity against
multiple pathogenic bacteria. Furthermore, the
in vivo
wound healing and histological studies
demonstrated complete wound healing in wounded rats on day 14, along with an increase in the
expression level of the transforming growth factor-
β
1 (TGF-
β
1) and a decrease in the expression level
of interleukin-6 (IL-6). The results revealed that the fabricated nanofibrous scaffold is a potent wound
dressing scaffold, and significantly accelerates full-thickness wound healing in a rat model.
Keywords:
biopolymer; wound healing; tri-layered; hierarchically structured; self-pumping effect;
unidirectional exudates discharge; nanofibrous scaffold
Pharmaceutics 2023,15, 1518. https://doi.org/10.3390/pharmaceutics15051518 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2023,15, 1518 2 of 25
1. Introduction
The skin tissue serves as a vital barrier against the external environment [
1
,
2
]. The
skin loses its protective function when exposed to injuries such as wounds or burns, which
allows bacteria to enter, causing severe systemic infections [
3
,
4
]. Normal wound healing is
a complex and dynamic process involving multiple steps, including hemostasis, inflam-
mation, proliferation, and remodeling [
5
]. The optimal wound dressing materials should
act as a barrier between the wound and the external environment to protect the wound
site, inhibit the growth of pathogenic micro-organisms, and provide a humid environment
around the wound in order to accelerate healing [
6
,
7
]. However, a wound site with ex-
cessive amounts of wound exudates creates a favorable microenvironment for bacterial
growth, which is detrimental to the wound healing process, and thus the removal of the
wound exudates is urgently required [
8
]. Furthermore, it should be fabricated from safe
and non-toxic bio-adhesive materials [
9
,
10
]. Consequently, developing a wound dressing
with unidirectional excessive wound site exudates, draining, and antimicrobial properties
is vital for accelerating the wound healing process. In recent years, various strategies in-
volving surface functionalization [
11
] and in situ polymerization [
12
] have been developed
to fabricate hydrophilic–hydrophobic dual-layer configurations for unidirectional fluid
transportation textiles. Nevertheless, the majority of these methods were not cost-effective,
lacked robustness, and were difficult to apply [13,14].
Nanotechnology is the study of nano-sized materials and has garnered significant interest
due to its numerous applications in the pharmaceutical and biomedical fields [
15
–
19
]. The vast
majority of skin preparations require frequent reapplication, necessitating the search for
the most effective and optimal alternative [20].
Electrospinning is a simple, inexpensive, and widely utilized method for nanofiber
manufacturing from a polymer solution with a size ranging from tens of nanometers to
micrometers [
21
]. Nanofibers demonstrate several advantages, such as a small diameter
with narrow distribution, a high surface area to volume ratio, excellent mechanical prop-
erties, a soft surface with no sharp corners, and increase patient comfort via decreasing
the frequency of dressing changes, [
17
,
22
–
24
]. In addition to these distinctive characteris-
tics, nanofibers permit skin regeneration, cell respiration, water retention, and effective
exudate absorption, making them ideal for wound healing applications [
25
]. Polymers
used in various wound dressing nanofibers can be categorized as either natural or syn-
thetic. The synthesis and modulation of synthetic polymers such as poly(ethylene oxide)
(PEO), poly(caprolactone) (PCL), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and
poly(lactic-co-glycolic acid) (PLGA) have considerable flexibility [
26
,
27
]. At the same time,
natural polymers such as collagen (COL), silk fibroin (SF), and hyaluronic acid (HA) dis-
played excellent biocompatibility, high safety, biodegradability, low toxicity, and lower
immune resistance [
28
,
29
]. Due to their poor mechanical strength, high surface tension,
and poor solubility in organic solvents, it is challenging to fabricate nanofibers from natural
polymers; however, they could be combined with synthetic polymers to produce nanofibers
with enhanced mechanical strength [30].
Among these natural polymers, collagen (COL) is a naturally existing animal protein
that is abundant in fibrous tissue such as tendons, skin, blood vessels, cartilage, bone,
cornea, ligament, and the gut. Fish skin is primarily composed of collagen, mostly type I,
characterized by its lower antigenicity and excellent biocompatibility [31].
SF is a natural polymer originating from silk worm cocoons. Its excellent biodegrad-
ability, biocompatibility, and convenient mechanical properties make it an excellent material
for biomedical areas, including tissue engineering [
32
,
33
]. PEO is a biodegradable, bio-
compatible, chemically stable, and water-soluble polymer that can be spun into nano and
microfibers, thus it could be combined with natural polymers to stabilize the electrospin-
ning process [34].
PHB is a hydrophobic, biocompatible, and biodegradable polymer produced by bac-
teria and has been extensively applied in wound healing and tissue engineering applica-
tions [
21
]. Bacterial infection is considered among the leading causes of delayed wound

Pharmaceutics 2023,15, 1518 3 of 25
healing [
35
]. AMX is a semi-synthetic antibiotic with a broad-spectrum activity against
several Gram-positive and Gram-negative micro-organisms [
36
]. Nonetheless, several
reports revealed that systemic administration of antibiotics did not have a significant
outcome and was accompanied by several side effects. In addition, the local application
of antibiotic powder at the wound site is easily detached from the wound area, causing
severe inflammation [
36
]. The sustained release of antibiotics promoted wound healing by
enhancing the nanofiber’s ability to inhibit bacterial growth.
Due totheir varying hydrophilicity and hydrophobicity, unidirectional fluids discharging
from electrospun nanofibrous membranes have been extensively used in a variety of biomedical
applications [
37
–
39
]. Utilizing the hydrophilic–hydrophobic gradient structure to generate
an additional pressure difference between the hydrophobic region (middle layer) and the
hydrophilic region (top layers) could be exploited to achieve unidirectional fluid discharge and
self-pumping effects capable of absorbing the excessive wound exudates [40–42].
Nevertheless, the majority of electrospun nanofibrous membranes have a bi-layered
structure, which limits their applications in preventing reverse osmosis [
43
]. Therefore, it
is anticipated that tri-layered electrospun nanofibrous membranes accelerate the wound
healing process due to their improved fluid pumping and the ability of reverse osmosis
prevention [
44
]. Many researchers have developed a tri-layered nanofibrous composite
for a wide variety of biomedical applications such as, wound healing [
45
–
47
], bone tissue
regeneration [48], cardiac tissue engineering [49], and tendon rupture repair [50].
To our knowledge, this paper describes, for the first time, the fabrication and design of
a tri-layered hierarchically structured nanofibrous scaffold with potent properties based on
an electrospinning strategy and sequential layered stacking. The thin layer of electrospun
nanofibrous membrane rapidly degrades. The bottom layer consists of a permeable thin
layer of electrospun hydrophilic SF, which serves as a source of nutrition for the myo-
fibroblasts differentiation and proliferation in the skin extracellular matrix (ECM). The
middle layer consists of hydrophobic polymer PHB loaded with antimicrobial drug AMX,
respectively, to offer prolonged sustained drug release at the wound site. The hydrophilic
polymer COL on the top layer is a component that simulates the ECM to enhance cell growth
and absorb the excessive exudates discharged by the unidirectional fluid discharge effect.
The designed tri-layered hierarchically structured nanofibrous scaffold exhibited
prolonged drug release, slow
in vitro
degradation, and a potent antimicrobial effect. The
scaffold also exhibited good
in vitro
biocompatibility. Furthermore,
in vivo
wound closure,
histological and Q-RT-PCR studies on rat models treated with the tri-layered hierarchically
structured nanofibrous scaffold demonstrated a complete wound closure, accompanied by
increasing the expression level of TGF-
β
1 and alleviating the expression level of interleukin-
6 (IL-6). As a result, we believe that the designed tri-layered hierarchically structured
nanofibrous scaffold is a promising candidate for accelerated wound healing applications.
2. Materials and Methods
2.1. Materials
Raw Bombyx mori silk cocoons (B. mori) were purchased from the Agricultural Re-
search Institute (Cairo, Egypt). Dialysis tubing cellulose membranes were also pur-
chased (Mwt cut-off 14 Kda, SERVAPOR, HD, Germany). Poly(ethylene oxide) (PEO)
(Mwt = 900,000), calcium chloride anhydrous (CaCl
2
), and 2,2,2-trifluoroethanol (TFE)
were obtained from Acros-organics (NJ, USA). Poly-(3-hydroxy butyrate) (PHB), amox-
icillin sodium (AMX), butyl alcohol anhydrous (CH
3
(CH
2
)
3
OH, glutaraldehyde (GTA),
dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and ethanol (EtOH), at HPLC
grade, were purchased from Sigma-Aldrich (St Louis, MO, USA). Sodium carbonate an-
hydrous (Na
2
CO
3
) was procured from Oxford laboratory reagents. Sodium hydroxide
(NaOH) was obtained from El-Nasr pharmaceutical chemicals Co. Fetal bovine serum (FBS),
Dulbecco’s phosphate-buffered saline (PBS), and Dulbecco’s modified Eagle’s medium
(DMEM) were purchased from Gibco-BRL (Grand Island, NY, USA). Acetic acid glacial
(CH3COOH) was purchased from Oxford laboratory reagents.

Pharmaceutics 2023,15, 1518 4 of 25
2.2. Animal and Ethical Approval
Using 21 adult male Wistar rats, the
in vivo
wound healing rating for the prepared
nanofibers was determined (8 weeks old; 180–200 g). The
in vivo
experimentation complied
with the guidelines and protocols and was confirmed by the Research Ethical Committee of
Institutional Animal Care and Use Committee at Alexandria University (ALEXU-IACUC)
with approval number; AU14-210126-3-3.
2.3. Preparation of SF
The SF was prepared according to the previous strategy conducted by Chen, W. et al. [
46
].
Briefly, raw silk cocoons were degummed in triplicate for 30 min in boiling water containing
0.5% (w/w) Na
2
CO
3
, followed by washing in distilled water each time. The degummed silk
cocoons were dissolved in a CaCl
2
/H
2
O/EtOH solution (molar ratio 1:8:2) at 70
◦
C under
continuous stirring for 1 h. In cellulose dialysis tubes with a 14 Kda cut-off, the solution
was dialyzed against distilled water for 3 days at room temperature. Every 5 h, freshly
distilled water was changed. The preparation steps ended with filtering, centrifugation,
and freeze-drying the solution to obtain a regenerated sf sponge.
2.4. Preparation of COL from Tilapia Fish Skin
The fresh tilapia skin was purchased from a local fish market and stored at
−
20
◦
C
until usage. Fins, fat, and muscle fragments were scraped from the skins before being cut
into small sections (0.5 cm
2×
0.5 cm
2
) and mixed well. Following Treesin Potaros et al.’s
method, acid-solubilized collagen was extracted from the skin of Tilapia fish [
51
]. Briefly,
the fragments were dissolved in 10 volumes of 0.1 M NaOH, and the suspension was
agitated with a magnetic stirrer overnight. The skin fragments were re-suspended after
decanting in 20 volumes of 0.1 M NaOH solution. The alkaline-insoluble components
were filtered through a cloth and repeatedly rinsed with distilled water to achieve a
neutral pH. The insoluble parts of the collagen were removed using 10 volumes of 0.5 M
acetic acid over the course of three days. Furthermore, the resulting viscous solution
was centrifuged at 10,000
×
gfor 20 min at 4
◦
C. The residue was extracted again using
10 volumes of 0.5 M acetic acid for three days, and the extract was then centrifuged. The
two extracts’ supernatants were mixed and salted by adding NaCl at a final concentration
of 0.9 M. The precipitate was obtained by centrifuging at 10,000
×
gfor 20 min after
standing overnight. Afterward, it was dissolved in 10 volumes of 0.5 M acetic acid. The
solubilization and salting-out processes were carried out three times. The resulting solution
was dialyzed against 0.1 M acetic acid in a membrane with a 14,000 kDa cut-off, followed
by lyophilization to obtain an acid-solubilized collagen sponge.
2.5. Fabrication of a Tri-Layered Nanofibrous Scaffold
The electrospun nanofibrous scaffold was prepared using an electrospinning machine
(Nano NC laboratory machine, South Korea Republic, ESR 100) with a grounded aluminum
foil-covered drum collector. Briefly, the tri-layered hierarchically structured nanofibrous
scaffold was prepared by sequentially layering stacking electrospinning with three different
polymeric solutions of SF/PEO, AMX-loaded PHB, and COL/PEO in three different sol-
vents. The bottom layer was made according to the method of Chen et al. [
52
]; SF/PEO was
mixed at a mass ratio of 8:2 and then blended and dissolved in 5 mL distilled water with
constant stirring for 24 h at room temperature to obtain the total polymeric concentration of
14% (w/v). SF/PEO polymeric solution was filled into a 2.5 mL plastic syringe with a blunt-
ended needle 7G (ID = 3.81 mm) at a distance of 20 cm from the rotating drum collector at
speed = 460 rpm, dispensing rate of 1 mL/h and under applied high voltage of 18–20 Kv.
The middle layer was made one according to the method of El-shanshory et al. [
21
], with
some modifications.
In brief, 0.35 g of PHB was dissolved in 5 mL TFE with constant stirring for 4 h at 50
◦
C.
Subsequently, a PHB with a concentration of 7% (w/v) was mixed with two concentrations
of AMX 5% (w/w) and 10% (w/w), depending on the total polymeric solution concentration.

Pharmaceutics 2023,15, 1518 5 of 25
PHB and AMX-containing PHB polymeric solution was filled into a 5 mL plastic syringe
with a blunt-ended needle 23G (ID = 0.34 mm) at a distance of 20 cm from the rotating drum
collector at a speed = 460 rpm, dispensing rate of 1 mL/h and under applied high voltage
of 18 Kv. Furthermore, for the top layer, COL/PEO at a weight ratio of 8:2 was blended
and dissolved in a 5 mL mixed solvent of glacial acetic acid/DMSO (4.65:0.35) (v/v) under
gentle stirring for 24 h at ambient temperature to obtain a total polymeric concentration
of 12% (w/v). COL/PEO polymeric solution was loaded into a 5 mL plastic syringe with
a blunt-ended needle 7G (ID = 3.81 mm) at a distance of 20 cm from the rotating drum
collector at speed = 460 rpm, dispensing rate of 1 mL/h and under applied high voltage of
18–20 Kv. For the stabilization and crosslinking of the tri-layered hierarchically structured
nanofibrous scaffold against dissolution in fluids, the bottom layer SF/PEO and the top
layer COL/PEO were sequentially placed in a sealed desiccator containing 75% EtOH
vapor for two days and another sealed desiccator containing 10% (w/w) GTA vapor for
two days, respectively. The stabilized tri-layered hierarchically structured nanofibrous
scaffold was dried in a vacuum oven at 40
◦
C for two days to remove any solvents or
crosslinker residuals.
2.6. Physicochemical Characterizations
The morphology and tri-layered hierarchically structured nanofibrous scaffold were
examined utilizing SEM (JEOL –JSM-6360LA, Tokyo, Japan). Before observation, nanofi-
brous scaffolds were sputtered with gold. Then, the average diameters of nanofibers were
measured using Image analysis software (Image J, National Institute of Health, Bethesda,
MD, USA) by randomly selecting 100 nanofibers from the SEM micrographs. FTIR (FT-IR,
Shimadzu FTIR-8400 S, Kyoto, Japan) with a wavelength range of 4000–400 cm
−1
was used
to evaluate the composition and chemical structure of the samples.
2.7. Swelling, Porosity, and Surface Wettability
The swelling capacity of the samples was determined according to the method con-
ducted by El-shanshory et al. [
21
]. The initial weight (Wd) of the synthesized nanofibrous
sample was recorded, and the sample was placed in phosphate buffer at room temperature.
After 24 h, samples were collected, and any excess surface water was gently wiped with
filter paper. At this point, the sample’s weight was recorded as (Ww). The following
formula was used to calculate the swelling (%).
Swelling(%)=(Ww −Wd/Wd)×100 (1)
The porosity of the samples was measured according to the liquid displacement
method [
53
]. A divided cylinder containing a given volume (V1) of absolute ethanol was
submerged in a known weight (W) of the sample. When no bubbles were discovered, the
resulting volume was then reported (V2). Finally, the absolute ethanol volume remaining
after the sample was removed from the absolute ethanol was determined (V3). The porosity
of the sample was determined according to the following equation:
Porosity(%)=((V1 −V3)/(V2 −V3))×100 (2)
The surface wettability of the nanofibrous scaffold samples was evaluated using a
contact angle meter, model VCA 2500 XE, with a CCD camera and software (AST Products,
Billerica, MA, USA). After 0.03 mL of deionized water was dropped onto the surface of the
nanofibrous scaffolds for 1 s, images were captured using the connected camera.
2.8. Mechanical Properties Evaluation
The tensile strength of nanofibrous scaffolds at room temperature was measured using
a universal testing machine (Shimadzu UTM, Kyoto, Japan). Crossheads were moved at
a constant rate of 5 mm/min at room temperature until samples rupture (n = 5), while

Pharmaceutics 2023,15, 1518 6 of 25
tensile strength was measured automatically. The elongation at rupture and tensile strength
were calculated.
2.9. In Vitro Antimicrobial Activity
Antimicrobial agents can kill or inhibit bacterial growth. Several techniques, including
agar dilution, disc -diffusion, and well diffusion, have been successfully applied to evaluate
and screen antimicrobial activity [
54
]. In this study, the antimicrobial activities of the
fabricated nanofibers (SF/PEO/PHB/COL/PEO, SF/PEO/AMX/PHB/COL/PEO) were
evaluated against Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212, according to
previously reported methods [
55
]. Briefly, the previously refreshed bacteria suspensions
were diluted with sterile 1% LB broth medium up to 100 fold, then 100
µ
L of the diluted
bacterial suspension was incubated with 10 mL of sterile 1% LB medium containing 0.030 g
of tested sample (nanofibers) while shaking for 24 h at 37
◦
C. Evaluation of the absorbance
of the culture medium at 600 nm with visible spectroscopy revealed the percentage of
bacterial growth inhibition. The percentage of bacterial inhibition was calculated according
to the following equation.
% inhibition A−B
A×100 (3)
where A and B are the absorbances of bacterial culture in the absence and the presence of
tested nanofiber, respectively.
2.10. Drug Release Assessment
To evaluate the
in vitro
drug release of nanofibrous scaffolds containing AMX, a
calibration curve was generated by measuring the absorbance values of progressively
diluted AMX concentrations at 273 nm. In brief, 3 mL of PBS buffer was added to two
cassettes containing 10 mg of two concentrations of AMX-loaded nanofibrous scaffold.
The cassettes were then placed in a shaker incubator set to 100 rpm at 37
◦
C. The buffer
was removed, and the same amount of new PBS buffer was added at a predetermined
time. The UV spectrophotometer-double beam (T80+, PG Instruments Ltd., England, UK)
was utilized for absorbance detection. Calculations were performed based on the AMX
concentration in the buffer, the AMX release percentage, and the cumulative release curve.
2.11. Cytotoxicity Assay of Nanofibrous Scaffolds
Following treatment, the effect of nanofibrous scaffolds on normal human cells of the
HSF (Primary skin fibroblasts) and HFB-4 (melanocytes) cell lines was evaluated using the
MTT cell viability assay. In 24-well sterile flat-bottom tissue culture plates, HSF and HFB-4
cells (1.0
×
10
3
each) were cultivated for 24 h in a CO
2
incubator. HSF and HFB-4 cells
were maintained in full DMEM media supplemented with 10% FBS. In triplicates, the discs
from each manufactured NF were cultured in the monolayer cells for two and four days at
weights of 0.5, 1.0, 2.0, and 4.0 mg/mL. After three rounds of washing with a new medium,
cells were treated with a 0.5 mg/mL MTT solution to remove dead cells and debris before
being cultured for approximately 2–3 h in 5% CO
2
. The formed formazan crystals were
dissolved in DMSO, and the optical density of each well was measured at 590 nm using
an ELISA reader and a microplate. Without including the prepared NFs, the relative cell
viability (%) in comparison to reference cells was calculated using the formula (X) test/(Y)
reference 100%.
2.12. Cell Scratching Assay of Nanofibrous Scaffolds
The effect of cell healing on the manufactured nanofibrous scaffold was determined
using the cell scratching method. Briefly, sterile 24-well cell culture plate (HFB-4, 1.0
×
10
5
)
cells were cultivated and then incubated in 5% CO
2
until the cell monolayers reached about
90% confluence. The cells were washed with fresh medium after being scratched by a sterile
micro-tip on the monolayers. After adding different discs of the nanofibrous scaffolds to

Pharmaceutics 2023,15, 1518 7 of 25
each well individually, they were incubated in 5% CO
2
for 24 and 48 h to allow for cell
migration in the medium. Scratching healing and cell migration were then visualized and
recorded using a phase contrast microscope. Each experiment was carried out three times,
and the results were compared to untreated scratched cells.
2.13. In Vivo Wound Healing
The
in vivo
wound healing rating for the nanofibrous scaffolds was executed using
21 adult male Wistar rats (8 weeks old; 180–200 g). The
in vivo
experimentation followed
the guidelines and protocols and was approved by the Research Ethical Committee of the
Institutional Animal Care and Use Committee at Alexandria University (ALEXU-IACUC)
with approval number; AU14-210126-3-3. The animals were divided into three groups,
with seven animals in each one. Group one received sterile gauze, group two received
SF/PEO/PHB/COL/PEO (blank), and group three received SF/PEO/AMX/PHB/COL/PEO.
All groups were covered with plaster to secure the wound dressing at the wound place. The
rats were housed in separate stainless-steel cages, supplied with a standard laboratory diet
and mineral water ad libitum, and kept under planned environmental conditions (50–60%
humidity and 12 h light/dark cycle at 25
±
2
◦
C) for 7 days prior to the experiment to allow
for adaptation. The surgical operation was performed as previously reported [
2
,
4
,
56
,
57
].
The rats were anesthetized with intramuscular injection of 10% Ketamine Hydrochloride
“(Dopalen
®
-Sespo Indústria e Comércio Ltda, Vetbrands Saúde Animal Division, Paulínia,
Brazil, 0.1 mL/100 g body weight) and 2%xylazine hydrochloride (Calmium
®
-Agener
União, União Química, Embu-Guaçu, SP, Brazil, 0.1 mL/100 g body weight)”, the back
hair was shaved using an electric animal shaver, followed by sterilization of the skin using
ethanol solution (70%) and chlorhexidine. Then, using sterile surgical scissors, a surgical
scalpel, and forceps, a 1.5 cm diameter, a full-thickness circular excisional wound was
created in the center of the hairless skin. The dressed nanofibrous scaffolds and sterile
gauze were substituted for new ones every 3 days for 14 days. The wound areas were
calculated with the help of a digital caliper. The macroscopic photos of the wounds were
captured to evaluate the % contraction of the wound area using a digital camera on days
0, 3, 7,10, and 14 immediately after the surgical operation. The percentage of wound area
reduction was estimated according to the following equation.
Wound area reduction (%) = [1 −(Wound area at the given day/Wound area at the day 0)] ×100. (4)
For histological examination, the skins from the wound sites were removed before
scarification on the 7 and 14 days and then fixed in 10% formalin prior to slide preparation.
Subsequently, the skin slides were stained with both hematoxylin and eosin (H&E) and
Masson’s trichrome (MTS) and then examined under an optical microscope for epithelial-
ization, keratinization, and collagen deposition [
17
,
53
]. Finally, a section of the skin tissue
was preserved at −80 ◦C for further molecular analysis.
2.14. Quantitative Real-Time Polymerase Chain Reaction (RT PCR) Gene Expression Assay for
Interleukin-6 and TGF-β1
The total tissue RNA was isolated from the wounded skin samples collected on
day 14 using the Easy-spinTM Total RNA extraction kit (cat. No. 17221, South Korea,
iNtRON Biotechnology) following the manufacturer’s instructions. The purity and the
concentration of the extracted total RNA were evaluated utilizing a NanoDrop
™
UV–vis
spectrophotometer. Using the cDNA synthesis kit TOPscriptTM (cat. No. EZ005S, Dae-
jeon, South Korea, Enzynomics, Inc.), the reverse-transcription step for converting RNA to
complementary DNA (cDNA) was performed. The RT-PCR amplification reactions were
performed using SYBR green qPCR Master Mix (Thermo Fisher Scientific, Inc., Waltham,
MA, USA, cat. No. K0251) utilizing real-time PCR system, Applied Biosystems with
2.5
µ
L of cDNA and 1.5
µ
L)of each primer in a 25
µ
L reaction mixture final volume. The
expression level of the house-keeping gene Beta-actin (
β
-actin) was applied as the internal
control for the amplified samples. The sequences for the primers used for the amplifi-

Pharmaceutics 2023,15, 1518 8 of 25
cation of (cDNA) were as follows: 5-TTTCTCTCCGCAAGAGACTTCC-3 (forward) and
5-TGTGGGTGGTATCCTCTGTGA-3 (reverse) for IL-6; 5-TGACATGAACCGACCCTTCC-3
(forward) and 5-TGTGGAGCTGAAGCAGTAGT-3 (reverse) for TGF-
β
1 and 5-AGATCAAG
ATCATTGCTCCTCCT-3 (forward) and 5-ACGCAGCTCAGTAACAGTCC-3 (reverse) for
β
-actin. The alterations in the level of gene expression were estimated using a delta delta
comparative Ct (2-
∆∆
Ct) analysis technique. For the examined genes, the amplification tech-
nique consisted of one cycle of initial denaturation at 95
◦
C for 10 min, followed by 40 cycles
of 15 s at 95
◦
C, 30 s at 57
◦
C and 30 s at 72
◦
C. Melting curve analyses were performed for all
amplifications to ensure a single product was generated from each reaction [58].
2.15. Statistical Analysis
The collected data were statistically analyzed using costate software. One-way
ANOVA was performed, followed by the LSD test for multiple comparisons. Data were
expressed using the mean and standard deviation M
±
SD. p
≥
0.05 was regarded as
statistically significant across all analyses.
3. Results and Discussion
3.1. Preparation and Physicochemical Characterization
The morphological appearance and tri-layered hierarchical structures of the as-prepared
nanofibrous scaffolds (Figure 1) were evaluated under magnification, and the distribution
of their average diameters is depicted in supplementary data (Figures S1–S3). Smooth
surfaces, beadless, and no spindle on a string behavior were detected. Moreover, no AMX
accumulations were detected on the surface of nanofibrous scaffolds. According to these
results, the size distribution and average diameters are 250
±
82 nm, 261
±
49 nm, as well
as 266 ±55 nm for SF/PEO, PHB, and COL/PEO, respectively.
FT-IR was evaluated to detect the characteristic peaks and functional groups for the
ingredients of the as-prepared nanofibrous scaffolds SF, PHB, and COL. The basic charac-
teristic peaks of SF/PEO are explained by the appearance of amide I, C=O stretching bands
and were assisted by the presence of amide II and III at 1535 and 1238 cm
−1,
respectively.
Furthermore, the random coil of amide I appeared at 1654 cm
−1
, corresponding to the vibra-
tion band. PHB exhibited infrared absorption peaks at 1262 and 1725 cm
−1
, corresponding to
–CH and C=O, respectively, present in the ester group in the molecular chain. Additionally,
sharp peaks at 1034 and 1097 cm
−1
represent C–O stretching. Furthermore, absorption peaks
at 2926 and 2963 are attributed to C–H stretching vibrations of the methyl and methylene
groups [
59
]. AMX relevant major peaks are present at 1490, 1509–1520, 1685–1692, 2050,
3000, 3175, 3366, and 3448–3458 cm
−1
corresponding to N–H, C=C benzene ring stretching,
C=O stretching, amide I, C–C and C–N stretching, C–H benzene ring stretching and amide
N–H and phenol O–H stretching, respectively [
60
,
61
]. Collagen-derived tilapia fish skin
showed characteristic peaks at 3292–3315 cm
−1
corresponding to peptide N–H groups.
There are COL amide I band at 1656 cm
−1
, amide II at 1538–1548 cm
−1
, and amide III at
1232–1238 cm
−1
. Therefore, these results confirmed that the extracted COL is collagen
type I which are in concordance with the previously reported results by Elbaily et al. [
62
].
The successful loading of AMX within the nanofibrous composite can be confirmed by the
appearance of the peaks in AMX powder at 1509 cm
−1
and 1685 cm
−1
, which are assigned
to amide I and Amide II bond of AMX, respectively [
63
]. Moreover, the peaks at 1618 cm
−1
,
1774 cm
−1
and 3448 cm
−1
are present due to the absorption band of benzene ring, the vibra-
tion of carboxylic group and the stretching vibration of hydroxyl and amino group in the
AMX structure [
64
,
65
]. Additionally, the appearance of the AMX peak at 1509 cm
−1
in both
SF/PEO/5%AMX/PHB/COL/PEO and SF/PEO/10%AMX/PHB/COL/PEO confirms
the presence of AMX in both composites due to some weak van der Waals interactions
between AMX and nanofibrous composite. However, the detection of other AMX signals
was difficult perhaps due to some overlapping between vibration bands of AMX and the
nanofibrous composites [
66
]. The results obtained indicated the successful incorporation of
AMX into the nanofibrous scaffold. FT-IR graphs are demonstrated and plotted in Figure 2.

Pharmaceutics 2023,15, 1518 9 of 25
Pharmaceutics 2023, 15, x FOR PEER REVIEW 9 of 26
(a) (b)
(c) (d)
Figure 1. Nanofibrous scaffold morphology. SEM images of SF/PEO (a), PHB (b), and COL/PEO (c),
and a cross-sectional micrograph of a tri-layered hierarchically structured nanofibrous scaffold (d).
FT-IR was evaluated to detect the characteristic peaks and functional groups for the
ingredients of the as-prepared nanofibrous scaffolds SF, PHB, and COL. The basic
characteristic peaks of SF/PEO are explained by the appearance of amide I, C=O stretching
bands and were assisted by the presence of amide II and III at 1535 and 1238 cm
−1,
respectively. Furthermore, the random coil of amide I appeared at 1654 cm
−1
,
corresponding to the vibration band. PHB exhibited infrared absorption peaks at 1262 and
1725 cm
−1
, corresponding to –CH and C=O, respectively, present in the ester group in the
molecular chain. Additionally, sharp peaks at 1034 and 1097 cm
−1
represent C–O
stretching. Furthermore, absorption peaks at 2926 and 2963 are aributed to C–H
stretching vibrations of the methyl and methylene groups [59]. AMX relevant major peaks
are present at 1490, 1509–1520, 1685–1692, 2050, 3000, 3175, 3366, and 3448–3458 cm
−1
corresponding to N–H, C=C benzene ring stretching, C=O stretching, amide I, C–C and
C–N stretching, C–H benzene ring stretching and amide N–H and phenol O–H stretching,
respectively [60,61]. Collagen-derived tilapia fish skin showed characteristic peaks at
3292–3315 cm
−1
corresponding to peptide N–H groups. There are COL amide I band at
1656 cm
−1
, amide II at 1538–1548 cm
−1
, and amide III at 1232–1238 cm
−1
. Therefore, these
results confirmed that the extracted COL is collagen type I which are in concordance with
the previously reported results by Elbaily et al. [62]. The successful loading of AMX within
the nanofibrous composite can be confirmed by the appearance of the peaks in AMX
powder at 1509 cm
−1
and 1685 cm
−1
, which are assigned to amide I and Amide II bond of
AMX, respectively [63]. Moreover, the peaks at 1618 cm
−1
, 1774 cm
−1
and 3448 cm
−1
are
present due to the absorption band of benzene ring , the vibration of carboxylic group and
Figure 1.
Nanofibrous scaffold morphology. SEM images of SF/PEO (
a
), PHB (
b
), and COL/PEO (
c
),
and a cross-sectional micrograph of a tri-layered hierarchically structured nanofibrous scaffold (d).
The nanofibrous scaffold’s ability to manage wound exudates and drug delivery de-
pends on its ability to absorb fluids. Fluids uptake is affected by the hydrophilic nature
of the applied materials and their porosity. The swelling ability of the material affects
its weight, which decreases due to erosion during prolonged exposure to fluids, whereas
weight gain is due to fluid uptake during short exposure to fluids. The weight change
obtained after 72 h of immersion in PBS pH 7.4 at room temperature with partial weight
loss in the first 24 h was 25%, 5%, and 65% for SF/PEO, PHB, and COL/PEO, respectively.
Furthermore, All nanofibrous scaffolds maintained their structural stability for at least 48 h.
These results demonstrate the potency of the nanofibrous scaffold for various biomedical ap-
plications. Additionally, the porosity % of the nanofibrous scaffolds is a key factor affecting
their biomedical applications. The porosity % for SF/PEO, PHB, SF/PEO/PHB/COL/PEO
(blank), and COL/PEO are 75%, 80%, 85.71%, and 82.2%, respectively. These results demon-
strate the suitability of the materials for use in biomedical applications and the capacity of
the nanofibrous scaffold to facilitate the self-pumping effect and fluid movement from the
interior to the exterior surface of the matrix.
The hydrophilic behavior of nanofibrous scaffold significantly enhances cell differentiation
and adhesion. In general, the water contact angle of the SF/PEO is 79.2
◦
, while that for PHB
is 102.8
◦
. Moreover, the water contact angles for the COL/PEO, SF/PEO/COL/PEO, 5%

Pharmaceutics 2023,15, 1518 10 of 25
AMX-loaded nanofibrous scaffold, and 10% AMX-loaded nanofibrous scaffold were 47
◦
, 43
◦
,
62
◦
, and 92
◦
, respectively. The photographs of water contact angles of the different nanofibrous
scaffolds are shown in Figure 3. The obtained results indicate the hydrophobic nature of PHB,
the moderate hydrophilicity of SF, COL, SF/PEO/COL/PEO, and the change in behavior of the
10% AMX-incorporated nanofibrous scaffold towards hydrophobicity.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 10 of 26
the stretching vibration of hydroxyl and amino group in the AMX structure [64,65].
Additionally, the appearance of the AMX peak at 1509 cm−1 in both
SF/PEO/5%AMX/PHB/COL/PEO and SF/PEO/10%AMX/PHB/COL/PEO confirms the
presence of AMX in both composites due to some weak van der Waals interactions
between AMX and nanofibrous composite. However, the detection of other AMX signals
was difficult perhaps due to some overlapping between vibration bands of AMX and the
nanofibrous composites [66]. The results obtained indicated the successful incorporation
of AMX into the nanofibrous scaffold. FT-IR graphs are demonstrated and ploed in
Figure 2.
(a)
Figure 2. Cont.

Pharmaceutics 2023,15, 1518 11 of 25
Pharmaceutics 2023, 15, x FOR PEER REVIEW 11 of 26
(b)
Figure 2. (a) FT-IR spectra of SF/PEO, PHB, COL/PEO, SF/PEO/5%AMX/PHB/COL/PEO,
SF/PEO/5%AMX/PHB/COL/PEO and AMX. (b) FT-IR spectra of SF/PEO/5%AMX/PHB/COL/PEO,
SF/PEO/5%AMX/PHB/COL/PEO, and AMX.
The nanofibrous scaffold’s ability to manage wound exudates and drug delivery
depends on its ability to absorb fluids. Fluids uptake is affected by the hydrophilic nature
of the applied materials and their porosity. The swelling ability of the material affects its
weight, which decreases due to erosion during prolonged exposure to fluids, whereas
weight gain is due to fluid uptake during short exposure to fluids. The weight change
obtained after 72 h of immersion in PBS pH 7.4 at room temperature with partial weight
loss in the first 24 h was 25%, 5%, and 65% for SF/PEO, PHB, and COL/PEO, respectively.
Furthermore, All nanofibrous scaffolds maintained their structural stability for at least 48
h. These results demonstrate the potency of the nanofibrous scaffold for various
biomedical applications. Additionally, the porosity % of the nanofibrous scaffolds is a key
factor affecting their biomedical applications. The porosity % for SF/PEO, PHB,
SF/PEO/PHB/COL/PEO (blank), and COL/PEO are 75%, 80%, 85.71%, and 82.2%,
respectively. These results demonstrate the suitability of the materials for use in
biomedical applications and the capacity of the nanofibrous scaffold to facilitate the self-
pumping effect and fluid movement from the interior to the exterior surface of the matrix.
The hydrophilic behavior of nanofibrous scaffold significantly enhances cell
differentiation and adhesion. In general, the water contact angle of the SF/PEO is 79.2°,
while that for PHB is 102.8°. Moreover, the water contact angles for the COL/PEO,
SF/PEO/COL/PEO, 5% AMX-loaded nanofibrous scaffold, and 10% AMX-loaded
nanofibrous scaffold were 47°, 43°, 62°, and 92°, respectively. The photographs of water
contact angles of the different nanofibrous scaffolds are shown in Figure 3. The obtained
Figure 2.
(
a
) FT-IR spectra of SF/PEO, PHB, COL/PEO, SF/PEO/5%AMX/PHB/COL/PEO,
SF/PEO/5%AMX/PHB/COL/PEO and AMX. (
b
) FT-IR spectra of SF/PEO/5%AMX/PHB/
COL/PEO, SF/PEO/5%AMX/PHB/COL/PEO, and AMX.
Materials used in biomedical applications should have suitable mechanical properties
to serve well as a scaffold. The results summarized in Table 1indicated that the SF/PEO
has a tensile strength of 9.1911 N/mm
2
and the value of elongation at break was 2.70500%,
whereas the tensile strength of COL/PEO is 27.777 N/mm
2
, and the value of elongation
at break was 1.1100%, Moreover, adding PHB and COL/PEO to SF/PEO layers exhibited
a tensile strength value for SF/PEO/PHB/COL/PEO scaffold to be 4.1666 N/mm
2
and
the value of elongation at break decreased to be 0.6000%. The incorporation of AMX (5%)
in SF/PEO/PHB/COL/PEO increased the values of tensile strength and elongation at
break, respectively, from 4.1666 N/mm
2
to 35.1732 N/mm
2
and 0.6000% to 1.84500%. The
addition of AMX(10%) in SF/PEO/PHB/COL/PEO increased the tensile strength and
elongation at break values, respectively, from 4.1666 N/mm
2
to 86.137 N/mm
2
and 0.6000%
to 2.50250% [67–69].

Pharmaceutics 2023,15, 1518 12 of 25
Pharmaceutics 2023, 15, x FOR PEER REVIEW 12 of 26
results indicate the hydrophobic nature of PHB, the moderate hydrophilicity of SF, COL,
SF/PEO/COL/PEO, and the change in behavior of the 10% AMX-incorporated nanofibrous
scaffold towards hydrophobicity.
Figure 3. Photographic demonstration of contact angle values for SF/PEO, PHB, COL/PEO,
SF/PEO/COL/PEO, 5%, and 10% AMX incorporated nanofibrous scaffolds.
Materials used in biomedical applications should have suitable mechanical
properties to serve well as a scaffold. The results summarized in Table 1 indicated that the
SF/PEO has a tensile strength of 9.1911 N/mm2 and the value of elongation at break was
2.70500%, whereas the tensile strength of COL/PEO is 27.777 N/mm2, and the value of
elongation at break was 1.1100%, Moreover, adding PHB and COL/PEO to SF/PEO layers
exhibited a tensile strength value for SF/PEO/PHB/COL/PEO scaffold to be 4.1666 N/mm2
and the value of elongation at break decreased to be 0.6000%. The incorporation of AMX
(5%) in SF/PEO/PHB/COL/PEO increased the values of tensile strength and elongation at
break, respectively, from 4.1666 N/mm2 to 35.1732 N/mm2 and 0.6000% to 1.84500%. The
addition of AMX(10%) in SF/PEO/PHB/COL/PEO increased the tensile strength and
elongation at break values, respectively, from 4.1666 N/mm2 to 86.137 N/mm2 and 0.6000%
to 2.50250% [67–69].
Table 1. Mean tensile strength and mean elongation at break values of SF/PEO, COL/PEO,
SF/PEO/PHB/COL/PEO, 5%, and 10% AMX incorporated nanofibrous scaffolds.
Sample Name Mean Tensile Strength
(N/mm2)
Mean Elongation at
Break (%)
SF/PEO 9.19 ± 0.05 2.70 ± 0.3
COL/PEO 27.77 ± 0.03 1.11 ± 0.2
SF/PEO/PHB/COL/PEO 4.16 ± 0.08 0.60 ± 0.5
SF/PEO/5%AMX/PHB/COL/PEO 35.17 ± 0.06 1.84 ± 1.1
SF/PEO/10%AMX/PHB/COL/PEO 86.13 ± 0.03 2.50 ± 1.2
Figure 3.
Photographic demonstration of contact angle values for SF/PEO, PHB, COL/PEO,
SF/PEO/COL/PEO, 5%, and 10% AMX incorporated nanofibrous scaffolds.
Table 1.
Mean tensile strength and mean elongation at break values of SF/PEO, COL/PEO,
SF/PEO/PHB/COL/PEO, 5%, and 10% AMX incorporated nanofibrous scaffolds.
Sample Name Mean Tensile Strength
(N/mm2)
Mean Elongation at
Break (%)
SF/PEO 9.19 ±0.05 2.70 ±0.3
COL/PEO 27.77 ±0.03 1.11 ±0.2
SF/PEO/PHB/COL/PEO 4.16 ±0.08 0.60 ±0.5
SF/PEO/5%AMX/PHB/COL/PEO 35.17 ±0.06 1.84 ±1.1
SF/PEO/10%AMX/PHB/COL/PEO 86.13 ±0.03 2.50 ±1.2
3.2. In Vitro Antimicrobial Activity
In several cases, wound infection may lead to severe problems and complications
associated with delayed healing as well as mortality. Consequently, it is necessary for the
newly developed wound dressing to estimate its ability to inhibit the growth of pathogenic
bacteria. The antibacterial activity was assessed regarding the % growth inhibition of
Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Staphylococcus aureus
ATCC 29213, and Enterococcus faecalis ATCC 29212bacteria after one day of contact with the
nanofiber. These bacteria are prevalent in wound discharges, especially in post-operative pa-
tients. SF/PEO/PHB/COL/PEO (blank) did not inhibit bacterial growth because it lacked
antimicrobial ingredients. Conversely, SF/PEO/5%AMX/PHB/COL/PEO demonstrated
highly significant antimicrobial activity against Staphylococcus epidermidis,Escherichia coli,
Staphylococcus aureus, and Enterococcus faecalis, with the calculated reduction in the number
of CFU of these bacterial cells reaching maximum of 83, 76.5, 83, and 89.5%, respectively.
3.3. In Vitro Drug Release and In Vitro Weight Loss
Figure 4summarizes the
in vitro
cumulative release profiles of nanofibrous scaffolds
incorporating 5% and 10% AMX. In addition, the AMX concentration in the release media

Pharmaceutics 2023,15, 1518 13 of 25
was plotted versus time to determine the amount of drug released
in vitro
from nanofibrous
scaffolds. The release of the drug was detected using a UV spectrophotometer and lasted
up to 120 h.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 13 of 26
3.2. In Vitro Antimicrobial Activity
In several cases, wound infection may lead to severe problems and complications
associated with delayed healing as well as mortality. Consequently, it is necessary for the
newly developed wound dressing to estimate its ability to inhibit the growth of
pathogenic bacteria. The antibacterial activity was assessed regarding the % growth
inhibition of Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212bacteria after one
day of contact with the nanofiber. These bacteria are prevalent in wound discharges,
especially in post-operative patients. SF/PEO/PHB/COL/PEO (blank) did not inhibit
bacterial growth because it lacked antimicrobial ingredients. Conversely,
SF/PEO/5%AMX/PHB/COL/PEO demonstrated highly significant antimicrobial activity
against Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, and Enterococcus
faecalis, with the calculated reduction in the number of CFU of these bacterial cells
reaching maximum of 83, 76.5, 83, and 89.5%, respectively.
3.3. In Vitro Drug Release and In Vitro Weight Loss
Figure 4 summarizes the in vitro cumulative release profiles of nanofibrous scaffolds
incorporating 5% and 10% AMX. In addition, the AMX concentration in the release media
was ploed versus time to determine the amount of drug released in vitro from
nanofibrous scaffolds. The release of the drug was detected using a UV spectrophotometer
and lasted up to 120 h.
Additionally, the degradation rate of the nanofibrous scaffolds incubated in PBS
solution at 37 °C was conducted for up to 6 weeks, as shown in Figure 4b. For the
nanofibrous scaffold containing 5% AMX, the weight loss percentage at week 1 was 22.4%,
whereas at week 6, it was 43.9%. In contrast, the percentage of weight loss at week 1 and
week 6 for the nanofibrous scaffold containing 10% AMX was 38.8% and 50%,
respectively.
Previous studies reported that AMX is more soluble in acidic media resulting in a
decrease in the swelling of the polymeric matrix with increasing AMX concentration [70].
Moreover, electrospinning process allows hydrophobic components of AMX to face the
surface of polymers due to the rapid solvent evaporation rate and this results in polymer
degradation retardment in the presence of AMX. These results are in agreement with the
results reported previously by Mollo et al. [71]. Based on the obtained results, we
hypothesize that a nanofibrous scaffold containing 5% AMX can serve as an effective
scaffold against bacterial invasion for wound healing applications.
(a)
Pharmaceutics 2023, 15, x FOR PEER REVIEW 14 of 26
(b)
Figure 4. Photographic illustration of (a) in vitro release profiles of AMX from
SF/PEO/5%AMX/PHB/COL/PEO and SF/PEO/10%AMX/PHB/COL/PEO and (b) in vitro weigh loss
of SF/PEO/PHB/COL/PEO, SF/PEO/5%AMX/PHB/COL/PEO, and
SF/PEO/10%AMX/PHB/COL/PEO.
3.4. In Vitro Cytotoxicity and Cell Scratching Assay
Cell viability and the effect of cell healing by the cell scratching method, as depicted
in Figures 5 and 6, are binding assays for scaffolds used in wound healing and other
biomedical applications. According to Figure 5, the cell viability of all tested nanofibrous
scaffolds after two and four days of incubation ranged between 50 and 80%, indicating
that these nanofibrous scaffolds have some cytotoxic effects on cells exposed to them for
four consecutive days. Consequently, based on the obtained results, it can be concluded
that blank nanofibrous scaffolds under study are cytocompatible, allowing more than 80%
cell viability, while the nanofibrous scaffold containing 5% and 10% AMX were found to
be almost 35–50% cell viability.
Moreover, from the results obtained from the cell scratching assay, the blank and
nanofibrous scaffold incorporated with 5%AMX at different cell concentrations
performed well for wound healing. In contrast, the nanofibrous scaffold incorporated
with 10%AMX showed relatively lower wound healing and some toxic effect on the cells.
Based on these findings, the tri-layered hierarchically structured nanofibrous scaffold is
recommended for wound healing applications. Furthermore, The viability of PIEC
cultured on pure SF, SF/P(LLA-CL) and SF/HBC nanofibrous scaffolds were good and
beneficial to cell growth in comparison with coverslips as reported by Zhang et al. [72,73].
Further, the previous studies conducted by Tian Zhou et al. have demonstrated that the
fish collagen/BG nanofibers induced proliferation on HaCaTs, indicating that fish collagen
nanofibers could effectively promote wound healing [74]. Moreover, the cell viability of
HPDLCs cultured was enhanced upon seeding on the Col/BG/CS membrane for
periodontal tissue regeneration as reported by Zhou et al. [75]. Additionally, it has been
reported that the biocompatibility results of the PHB/gelatin nanofibers against NIH-3T3
fibroblast cell lines were found to be non-toxic and aid in greater cell viability [76].
Additionally, it has been reported that the combination of PHB with SF supported the cell
aachment and proliferation of L929 and HaCaT cell lines [77].
Figure 4.
Photographic illustration of (
a
)
in vitro
release profiles of AMX from SF/PEO/
5%AMX/PHB/COL/PEO and SF/PEO/10%AMX/PHB/COL/PEO and (
b
)
in vitro
weigh loss of SF/
PEO/PHB/COL/PEO, SF/PEO/5%AMX/PHB/COL/PEO, and SF/PEO/10%AMX/PHB/COL/PEO.
Additionally, the degradation rate of the nanofibrous scaffolds incubated in PBS
solution at 37
◦
C was conducted for up to 6 weeks, as shown in Figure 4b. For the
nanofibrous scaffold containing 5% AMX, the weight loss percentage at week 1 was 22.4%,
whereas at week 6, it was 43.9%. In contrast, the percentage of weight loss at week 1 and
week 6 for the nanofibrous scaffold containing 10% AMX was 38.8% and 50%, respectively.
Previous studies reported that AMX is more soluble in acidic media resulting in a
decrease in the swelling of the polymeric matrix with increasing AMX concentration [
70
].
Moreover, electrospinning process allows hydrophobic components of AMX to face the

Pharmaceutics 2023,15, 1518 14 of 25
surface of polymers due to the rapid solvent evaporation rate and this results in polymer
degradation retardment in the presence of AMX. These results are in agreement with
the results reported previously by Mollo et al. [
71
]. Based on the obtained results, we
hypothesize that a nanofibrous scaffold containing 5% AMX can serve as an effective
scaffold against bacterial invasion for wound healing applications.
3.4. In Vitro Cytotoxicity and Cell Scratching Assay
Cell viability and the effect of cell healing by the cell scratching method, as depicted
in Figures 5and 6, are binding assays for scaffolds used in wound healing and other
biomedical applications. According to Figure 5, the cell viability of all tested nanofibrous
scaffolds after two and four days of incubation ranged between 50 and 80%, indicating that
these nanofibrous scaffolds have some cytotoxic effects on cells exposed to them for four
consecutive days. Consequently, based on the obtained results, it can be concluded that
blank nanofibrous scaffolds under study are cytocompatible, allowing more than 80% cell
viability, while the nanofibrous scaffold containing 5% and 10% AMX were found to be
almost 35–50% cell viability.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 15 of 26
Figure 5. In vitro cell viability assessment: cytotoxic effect test of the prepared NFs by MTT assay
on both HFB-4 (A) and HSF; (B) human normal skin cell lines after 2 and 4 days of incubation.
Figure 5. In vitro
cell viability assessment: cytotoxic effect test of the prepared NFs by MTT assay on
both HFB-4 (A) and HSF; (B) human normal skin cell lines after 2 and 4 days of incubation.
Moreover, from the results obtained from the cell scratching assay, the blank and
nanofibrous scaffold incorporated with 5%AMX at different cell concentrations performed
well for wound healing. In contrast, the nanofibrous scaffold incorporated with 10%AMX
showed relatively lower wound healing and some toxic effect on the cells. Based on these
findings, the tri-layered hierarchically structured nanofibrous scaffold is recommended
for wound healing applications. Furthermore, The viability of PIEC cultured on pure SF,
SF/P(LLA-CL) and SF/HBC nanofibrous scaffolds were good and beneficial to cell growth
in comparison with coverslips as reported by Zhang et al. [
72
,
73
]. Further, the previous stud-
ies conducted by Tian Zhou et al. have demonstrated that the fish collagen/BG nanofibers
induced proliferation on HaCaTs, indicating that fish collagen nanofibers could effectively
promote wound healing [
74
]. Moreover, the cell viability of HPDLCs cultured was en-

Pharmaceutics 2023,15, 1518 15 of 25
hanced upon seeding on the Col/BG/CS membrane for periodontal tissue regeneration as
reported by Zhou et al. [
75
]. Additionally, it has been reported that the biocompatibility
results of the PHB/gelatin nanofibers against NIH-3T3 fibroblast cell lines were found to
be non-toxic and aid in greater cell viability [
76
]. Additionally, it has been reported that the
combination of PHB with SF supported the cell attachment and proliferation of L929 and
HaCaT cell lines [77].
Pharmaceutics 2023, 15, x FOR PEER REVIEW 15 of 26
Figure 5. In vitro cell viability assessment: cytotoxic effect test of the prepared NFs by MTT assay
on both HFB-4 (A) and HSF; (B) human normal skin cell lines after 2 and 4 days of incubation.
Figure 6.
Typical photomicrographs of cell scratching of HFB-4 melanocytes at 48 h (scale bar is
500
µ
m). The black bars indicate the distances of the wound closure. All experiments were performed
three times.
3.5. In Vivo Wound Healing
The effect of different treatment groups on
in vivo
wound healing efficiency is illus-
trated in Figure 7a and Table 1. Topical delivery of the antibiotics leads to bulk collection
of higher drug doses at the target site, thus reducing side effects such as systemic toxicity
associated with high drug doses and bacterial resistance. Therefore, the optimal wound
dressing material should contain antimicrobial agents with a sustained release behavior
in order to accelerate wound healing [
78
]. Additionally, in contrast to free antibiotics
that cannot arrive and spread evenly over infected areas, antibiotic-loaded nano-carriers
are characterized by their higher penetration efficiency and equal distribution around
the infected spots [
79
]. In this regard, AMX, a broad-spectrum antimicrobial compound,
was incorporated into the SF/PEO/PHB/COL/PEO nanofibrous scaffold to boost wound
repair. In comparison to the sterile gauze group, the SF/PEO/PHB/COL/PEO (blank)- and
5% AMX incorporated SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated
groups displayed significant progress in wound healing associated with normal tissue,
with no exudates at all healing stages.

Pharmaceutics 2023,15, 1518 16 of 25
Pharmaceutics 2023, 15, x FOR PEER REVIEW 18 of 26
(a)
(b)
Figure 7. (a): Photographic images of in vivo full-thickness excision wounds after treatment with
sterile gauze, collagen/silk fibroin NFs, and collagen/AMX/silk fibroin NFs for 14 days.
(b) The
graph illustrates (%) wound contraction during 14 days.
Figure 7.
(
a
): Photographic images of
in vivo
full-thickness excision wounds after treatment with
sterile gauze, collagen/silk fibroin NFs, and collagen/AMX/silk fibroin NFs for 14 days. (
b
) The
graph illustrates (%) wound contraction during 14 days.

Pharmaceutics 2023,15, 1518 17 of 25
In addition, the incorporation of AMX into the SF/PEO/PHB/COL/PEO nanofi-
brous scaffold accelerated the wound healing process. The wounds treated with the
SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold were partially healed after 7 days.
The % contraction of the wound area was studied as shown in Table 2and Figure 7b. It
was found that after 3 days from treatment, the percentage of wound closure was 9.09%,
24.18%, and 53.73% for the sterile gauze group, the SF/PEO/PHB/COL/PEO nanofibrous
scaffold, and the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold, respectively.
Interestingly, at day 14 after treatment, both the SF/PEO/5%AMX/PHB/COL/PEO nanofi-
brous scaffold and the SF/PEO/PHB/COL/PEO nanofibrous scaffold showed 99.63% and
98.40% wound closure, respectively. Conversely, the sterile gauze group showed a wound
closure of 86.54%. The previous results showed that the wound closure was slower in the
sterile gauze group than in the nanofiber-treated groups.
Table 2. Wound closure rates of nanofiber-treated groups and untreated positive control.
Wound Diameter (mm) (Mean ±SDM) and % of
Wound Closure (W.C.)
Observation Day
ZERO-DAY Day 3 Day 7 Day 10 Day 14
Positive
Control (sterile gauze)
Diameter
187.28
±
1.98 170.26
±
4.87 131.87
±
3.17
62.94 ±0.90 25.21 ±6.32
% W.C 0 9.09 29.59 66.39 86.54
SF/PEO/PHB/COL/PEO Diameter
187.42
±
1.40 142.10
±
2.79
73.46 ±4.40 22.72 ±2.81 2.96 ±2.75
% W.C 0 24.18 60.80 87.88 98.42
SF/PEO/5%AMX/PHB/COL/PEO Diameter
185.08
±
3.30
85.64 ±6.44 54.05 ±4.60 7.26 ±0.67 0.68 ±0.64
% W.C 0 53.73 70.80 96.08 99.63
The histopathological examination of the skin sections from different treated groups
was performed to estimate the degree of vascularization, inflammation, and skin regen-
eration. During the early stage of wound healing, the lower inflammation level can
promote factors responsible for wound healing. On the other hand, severe inflamma-
tion in wound sites hindered tissue repair. Figure 8a,b showed the histopathological
features of H&E and MTS stained skin sections of NF-treated wounds compared with
untreated control and normal skin on days 7 and 14 post-wounding, respectively. As
depicted in Figure 8a, the normal skin image presented the typical structural elements
of healthy skin, such as intact epidermis, dermis (connective tissue layer), muscles, se-
baceous glands (SG), blood vessels, and hair follicles. It is evident that the healing of
the skin section treated with the SF/PEO/PHB/COL/PEO nanofibrous scaffold and the
SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold was higher compared to the ster-
ile gauze group. Furthermore, the nanofibers loaded with AMX showed considerable heal-
ing over the early stages of post-operation. On day 7, the skin section from groups treated
with sterile gauze showed ulcers, inflammatory cells, no SGs, and intact hair follicles. On
the contrary, the SF/PEO/PHB/COL/PEO and SF/PEO/5%AMX/PHB/COL/PEO nanofi-
brous scaffold-treated group begins forming a thin coat of neo-epithelium associated with
mild inflammatory cells, mild new vessel, and fibroblasts. On day 14, the wound section
from the sterile-gauze-treated group showed a small ulcer, scab formation with a smaller
number of fibroblasts and vasculature, and ahigh number of inflammatory cells with mini-
mal re-epithelialization and the absence of both regenerated SGs, hair follicles, and other
adnexa in the renovated dermis. The wound site also showed loosely arranged connective
tissue bundles in the dermis. Conversely, the wound sections of SF/PEO/PHB/COL/PEO
and SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated group showed rela-
tively normal skin appearance loaded with connective tissues (CT) and enveloped by a new
dermal layer. The tissues contained a fully developed epidermis (2–3 layers) and dermis
(completely organized connective tissue layer). The wound section also displayed the most
basic skin structures, such as hair follicles, SG, and blood vessels. The degree of collagen

Pharmaceutics 2023,15, 1518 18 of 25
formation and deposition from the wound section were estimated among various groups by
using Masson’s trichrome staining (MTS) Figure 8b. Sterile-gauze-treated groups showed
loose collagenous fiber deposition at the lesion site on both day 7 and day 14. In contrast,
the highest collagen fiber deposition was observed in both SF/PEO/PHB/COL/PEO and
SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated groups characterized
by mild collagen deposition on day 7, which became denser on day 14 and resembled
the pattern of collagen deposition in the normal skin (basketweave), which confers both
flexibility and pliability [
22
]. The photographic images and histopathological examination
results suggested that the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffolds accel-
erate wound healing by promoting complete re-epithelialization, collagen deposition, and
arrangement. The efficient
in vivo
wound healing performance was previously reported
for collagen and zein nanofibrous membranes loaded with berberine [
80
]. Another study
observed an accelerated
in vivo
wound healing, re-epithelization, and collagen deposition
for collagen nanofibers loaded with AgNPs due to their intrinsic antibacterial activity [
81
].
The hybrid between collagen and silk fibroin might improve both the physical and biologi-
cal characteristics of the scaffold for both tissue engineering and biomedical applications.
This result may be due to the hybrid’s resemblance to the ECM skin, which promotes cell
adhesion and proliferation [82].
3.6. Gene Expression of IL-6 and TGF-β1
The wound repair process comprises successive steps that initiate immediately after
an injury and develop into a complete skin reconstruction. The process involves the coordi-
nated action of multiple cell types (resident and circulating cells homing to the wound site),
the ECM, and soluble mediators named cytokines [
83
]. The wound repair process begins
with coagulation and hemostasis, which halt bleeding and initiate the cellular response.
In addition, the activated platelets within the clot release significant growth factors and
cytokines that stimulate the resident cells to initiate angiogenesis, re-epithelialization, and
connective tissue restoration. Any perturbation to these steps leads to chronic wounds [
84
].
IL-6 is a potent immunologic mediator that plays a fundamental role in inflammation,
representing a substantial physiological phase in normal wound healing. The study of
the expression of IL-6 in wound healing revealed that its expression was upregulated
following injury in human, animal, and
in vitro
models. It was reported that overex-
pression of IL-6 following injury stimulates the production of various pro-inflammatory
cytokines from existing cells, including stromal cells, keratinocytes, endothelial cells,
and macrophages. In combination with the expression of anti-inflammatory mediators,
macrophages are reconfigured from M1 pro-inflammatory to M2 tissue repairing [85].The
delay in this conversion step results in delayed healing, an increased risk of infection,
and scar formation [
86
]. The results revealed that both blank SF/PEO/PHB/COL/PEO
and SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated wounds significantly
inhibited (p< 0.05) the expression level of IL-6 gene compared to sterile-gauze-treated
wounds (Figure 9a). The faster a wound heals, the lower the expression of inflammatory
factors, and vice versa. This significant decrease in IL-6 gene expression can be attributed
to the potent anti-inflammatory and immune-enhancing properties of the SF/COL nanofi-
brous scaffold. A hybrid SF/COL nanofibrous scaffold has been reported to hasten wound
closure and tissue restoration
in vivo
[
82
,
87
]. Moreover, the level of IL-6 gene expres-
sion was lower in wounds treated with SF/PEO/5%AMX/PHB/COL/PEO nanofibrous
scaffolds compared to wounds treated with blank SF/PEO/PHB/COL/PEO nanofibrous
scaffolds. Through incorporation into the SF/PEO/PHB/COL/PEO nanofibrous scaffold,
AMX is slowly released at the wound site, preventing bacterial infections and accelerating
the healing process, contributing to the superior anti-inflammatory activity of AMX-loaded
nanofibers. Furthermore, the released AMX is readily engulfed at the inflammation site by
immune cells such as macrophages and produces localized effects on the wound [78,88].
Growth factors are crucial for proper wound repair. TGF-
β
is a type of pluripotent
cytokine that is produced primarily by macrophages and plays a significant role in cell

Pharmaceutics 2023,15, 1518 19 of 25
proliferation and migration, immune regulation, apoptosis, and inflammatory response. It
is also involved in all healing processes, such as the stimulation offibroblast generation,
differentiation of fibroblasts into myofibroblasts, and synthesis of collagen I and II [89,90].
In addition, TGF-
β
1 can stimulate the healing process via activation of angiogenesis and
augment the ECM production via both fibroblast and differentiated myofibroblast [
62
]. As
shown in (Figure 9b), the expression level of the TGF-
β
1 gene in the group topically treated
with SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold was significantly enhanced
(p< 0.05) in comparison to SF/PEO/PHB/COL/PEO nanofibrous and sterile-gauze-treated
group. This enhanced expression level in the case of treatment with AMX incorporated
SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold might be due to induction in the
formation of contractile bundles of normal fibroblasts [91].
Pharmaceutics 2023, 15, x FOR PEER REVIEW 19 of 26
(a)
(b)
Figure 8. (a) Photographic illustration of histological analysis of treated wounds on days 7 and 14
using H&E stain. (b) Histopathological evaluation of treated wounds using MTS on days 7 and 14
(original magnification = 100).
3.6. Gene Expression of IL-6 and TGF-β1
The wound repair process comprises successive steps that initiate immediately after
an injury and develop into a complete skin reconstruction. The process involves the
coordinated action of multiple cell types (resident and circulating cells homing to the
wound site), the ECM, and soluble mediators named cytokines [83]. The wound repair
process begins with coagulation and hemostasis, which halt bleeding and initiate the
cellular response. In addition, the activated platelets within the clot release significant
growth factors and cytokines that stimulate the resident cells to initiate angiogenesis, re-
epithelialization, and connective tissue restoration. Any perturbation to these steps leads
to chronic wounds [84]. IL-6 is a potent immunologic mediator that plays a fundamental
Figure 8.
(
a
) Photographic illustration of histological analysis of treated wounds on days 7 and 14
using H&E stain. (
b
) Histopathological evaluation of treated wounds using MTS on days 7 and 14
(original magnification = 100).

Pharmaceutics 2023,15, 1518 20 of 25
Pharmaceutics 2023, 15, x FOR PEER REVIEW 21 of 26
(a)
(b)
Figure 9. Gene expression levels related to wound healing on day 14 post-wounding. (a) IL-6 and
(b) TGF-β. Different leers a, b, and c express the statistically significant difference for a p-value ≤
0.05.
Figure 9.
Gene expression levels related to wound healing on day 14 post-wounding. (
a
) IL-6
and (
b
) TGF-
β
. Different letters a, b, and c express the statistically significant difference for a
p-value ≤0.05.

Pharmaceutics 2023,15, 1518 21 of 25
A significant increase in both wound closure size and wound healing percentage was
previously reported by Abo El-Ela, F.I, and his colleagues after topical application of AMX
loaded into Layered Double Hydroxide (LDH) nanocomposite (AMOX/LDH) compared
to the non-treated and control group [
78
]. They attributed this result to the excellent
penetration capacity of the LDH nanocomposites carrying the antimicrobial agent that
accelerate the healing process via preventing infections. Moreover, a higher expression level
for the TGF-
β
1 gene was recorded for groups treated with blank nanofiber (collagen/silk
fibroin nanofibers) compared to the sterile-gauze-treated group. Due to the activation of
macrophages to produce chemotactic growth factors (GF), angiogenesis, and fibroblast
proliferation via upregulation of TGF-
β
1, bFGF (primary fibroblast growth factor), and
α
-SMA (
α
-small muscle actin) genes, topical application of collagen isolated from tilapia
skin resulted in optimal and standard cutaneous wound healing in the rat model [
62
].
It was previously reported that diabetic wounds in mice treated with silk fibroin/poly-
(L-lactide-co-caprolactone) nanofiber scaffolds had a significantly higher TGF-
β
1 gene
expression level than wounds in untreated mice.
4. Conclusions
Wound dressing nanofibrous scaffolds fabricated from natural components have gar-
nered significant interest as promising wound dressings. In the current research, three
layers of a biocompatible, multifunctional, antibacterial, biopolymer-based, hierarchically
structured nanofibrous scaffold were fabricated. The bottom and top layers contain hy-
drophilic silk fibroin from natural SF and fish skin COL for accelerated wound healing,
interspersed with a middle layer of hydrophobic PHB containing AMX as an antibacterial
drug. The fabricated hierarchically structured nanofibrous scaffold elucidated sustained
in vitro
AMX release, good mechanical properties, high cytocompatibility, and enhanced
antimicrobial effect. This hierarchically structured nanofibrous scaffold improves
in vivo
wound healing in rats, alleviating inflammation and increasing tissue epithelization. There-
fore, it can be concluded that this nanofibrous scaffold can be utilized as an effective
wound dressing.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pharmaceutics15051518/s1, Figure S1: Average diameter distri-
bution of SF/PEO. Figure S2: Average diameter distribution of PHB. Figure S3: Average diameter
distribution of Col/PEO. Figure S4: SEM cross-sectional photograph of SF/PEO/PHB/Col/PEO.
Author Contributions:
Conceptualization, K.H.H., A.A.E.-S., M.M.A., M.I.A.-A., E.M.E.-F., A.A.E.-B.,
T.S.K., X.M. and H.M.A.S.; methodology, K.H.H., A.A.E.-S., M.M.A., M.I.A.-A., E.M.E.-F., A.A.E.-B.,
T.S.K., X.M. and H.M.A.S.; writing—original draft preparation, K.H.H., A.A.E.-S., M.M.A., M.I.A.-A.,
E.M.E.-F., A.A.E.-B., T.S.K., X.M. and H.M.A.S.; writing—review and editing, K.H.H., A.A.E.-S.,
M.M.A., M.I.A.-A., E.M.E.-F., A.S.A., A.A.E.-B., T.S.K., X.M. and H.M.A.S.; supervision, K.H.H.,
A.A.E.-S., M.M.A., M.I.A.-A., E.M.E.-F., A.A.E.-B., T.S.K., X.M. and H.M.A.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
This study was conducted in accordance with the Declaration
of Helsinki and approved by the Research Ethical Committee of the Institutional Animal Care and Use
Committee at Alexandria University (ALEXU-IACUC), with the approval number AU14-210126-3-3
the date 26 January 2021.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All data are available in the manuscript and supplementary materials.
Conflicts of Interest: The authors declare no conflict of interest.

Pharmaceutics 2023,15, 1518 22 of 25
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