International Journal of Biological Macromolecules 162 (2020) 629–644

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Multi-layer dressing made of laminated electrospun nanowebs and

cellulose-based adhesive for comprehensive wound care
Ahmed A. Nada a,⁎, Eman A. Ali b, Ahmed A.F. Soliman c, Jialong Shen d, Nabil Y. Abou-Zeid a, Samuel M. Hudson d
a
Pre-treatment and Finishing of Cellulosic Textiles Dept., Textile Research Division, National Research Centre (Scopus Affiliation ID 60014618), Dokki, Giza, Egypt
b
Polymers & Pigments Dept., Chemical Industries Research Division, National Research Centre, Dokki, Giza, Egypt
c
Department of Pharmacognosy, Pharmaceutical and Drug Industries Division, National Research Centre, Dokki, Giza, Egypt
d
Department of Textile Engineering Chemistry & Science, North Carolina State University, Raleigh, NC 27695-8301, United States

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

Article history: In this work, multi-layer wound dressing was made of laminated layers of electrospun fibers supported by adhe-
Received 27 April 2020 sive sheet. Graft copolymerization of methyl methacrylate (MMA) and 2-Ethyl-1-hexyl acrylate (EHA) onto
Received in revised form 5 June 2020 carboxymethyl cellulose (CMC) was conducted to obtain an adhesive sheet with 1.52 (N/cm2) loop tack, 1.7
Accepted 19 June 2020
(N/cm) peel strength and 25 s shear strength. Diclofenac sodium, anti-inflammatory drug, was loaded to the ad-
Available online 20 June 2020
hesive sheet with encapsulation efficiency 73%.
Keywords:
The contact layer to wound was made of synthesized anti-bleeding agents, chitosan iodoacetamide (CI) loaded
Multi-layer wound dressing into electrospun polyvinyl alcohol (PVA) fibers. It was fabricated from fiber diameter 300 nm by electrospinning
Graft copolymerization of 5% wt/v of CI (D.S. 18.7%) mixed with 10% wt/v PVA, at 20 kV and 17 cm airgap. The second, pain-relief layer
Encapsulation was fabricated by encapsulating up to 50% wt/wt of capsaicin into gelatin nanofibers (197 nm) crosslinked by
Electrospinning glyoxal. The third, antimicrobial layer was fabricated from PVA electrospun fibers loaded with 2% wt/wt gentami-
cin. Biocompatibility test showed insignificant adverse effects of the fabricated layers on fibroblast cells. Animal
test on rat showed accelerated wound healing from 21 to 7 days for the multi-layer dressing. Histopathological
findings corroborated the intactness of the epidermis layer of the treated samples.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction As a result, the US Defense Department has authorized three hemo-

Shortcomings in trauma care, on-site, are considered as the major
cause of mortality and impaired quality of life. Successful techniques
for trauma care such as junctional tourniquets, and blood-transfusion
equipment need well-trained medics available within minutes immedi-
ately after an injury [1]. Troops in front lines, those injured in car wrecks
and others who are severely wounded may not have enough time to be
served quickly and effectively by those tools. According to a US-military
medical report [1], nearly a quarter of the American soldiers killed in ac-
tion over the past 10 years, lost their lives of wounds that can be treated.
Therefore, providing a suitable wound dressing, produced using afford-
able local resources and handleable to untrained caregivers for acceler-
ating wound healing, could save many lives.
An understanding of the basic physiology of wound healing process
reveals that the healing process comprises of separated and overlapping
four phases, namely hemostasis, inflammatory, proliferation, and re-
modeling, which take place successively [2].
⁎ Corresponding author at: Textile Research Division, National Research Centre, Dokki,
Giza, Egypt.
E-mail address: aanada@ncsu.edu (A.A. Nada).
static agents: zeolite dressing called “Quikclot”, chitosan dressing called
“Hemcon” and the fibrin American Red Cross dressing. However, each
product has disadvantages and drawbacks as zeolite, an effective hemo-
static agent, may cause major thermal injuries, remain as a foreign body
in open wounds and are toxic in the eye or lung. Hemcon bandages, a ly-
ophilized chitosan foam, are not large enough or sufficiently flexible to
fill large wounds and work best on limited flat surfaces. The fibrin
American Red Cross dressing is highly effective but also limited in avail-
ability and costs 100 times over Quikclot and 10 times over Hemcon [3].
Therefore, many other products have been developed and commer-
cialized in the market to control bleeding as hemostatic agents [4–6].
For instance, chitosan-based films [3,7–9], oxidized cellulose [10], zinc
paste [11], silver nitrate [12] and aluminum chloride [13] have been
used as hemostatic agents. However, many others have been employed
to provide antimicrobial protection such as quaternary ammonium salts
[14], honey [15], iodine complexes [16], and antibiotic agents [17]. How-
ever, each product has benefits and drawbacks and can only serve one
phase in the wound healing process. In some cases, chitosan for in-
stance, can be used as both a hemostatic agent and an antimicrobial
agent [18].
On the other hand, drug delivery systems have been recently
employed as a new pharmacological approach to improve the efficacy

https://doi.org/10.1016/j.ijbiomac.2020.06.184
0141-8130/© 2020 Elsevier B.V. All rights reserved.
630 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644
and the safety of drug administration. Vesicles, micelles, electrospun fi-
bers [19–23], emulsions [24], microspheres [25–27], hydrogels [28–32],
and biodegradable nanoparticles [33,34] have been extensively studied
[35] as carriers for biological substances such as drugs, genes, proteins,
and etc.
Few studies have been devoted to prepare laminated material com-
prising an absorbent substrate and a sheet-shaped carrier [36] loaded
with biological active substances. Such a new generation of medicated
dressings has been reported to overcome some of the disadvantages of
the topical application of pharmaceutical agents. Hydrogels, hydrocol-
loids, alginates, polyurethane foam/films and silicon gels have been
used to deliver active agents to the wound bed [37]. The latter materials
have been constructed to trap a single compound serving one part of the
targeting process and there is no single agent that can serve the entire
set of phases of the wound healing process, all at once. Meanwhile, it
is difficult to combine two or more wound healing agents together in
one substrate and to provide a dressing that can be placed on wounds
for a few days without the need for replacement or cleaning of the
wound bed. As a result, none has been reported on producing multi-
functional wound dressing for comprehensive wound care.
In this work, a laminated multifunctional electrospun dressing using
different electrospun mats was prepared based on different electrospun
fibers supported by an adhesive sheet. Cytotoxicity tests and pre-clinical
studies on a rat wound model were conducted to reveal the wound
dressing potentials.
2. Materials and methods
2.1. Materials
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC-HCl) and
chitosan high molecular weight (HMW), were purchased from Sigma-
Aldrich (Germany). 2-(N-morpholino)ethanesulfonic sodium salt
(MES), tranexamic acid (TA) and N-hydroxysulfosuccinmide sodium
salt (NHS) were purchased from Acros Organics (Belgium).
Methyl methacrylate (MMA) and 2-Ethyl-1-hexyl acrylate (EHA)
monomers were purchased from Acros Organics (Belgium) and used
as received. Carboxymethyl cellulose (CMC), (viscosity 700 cP at 1% in
water at 25 °C) was purchased from Fisher Scientific (USA) with degree
of substitution (D.S.) 0.7–0.85. Sodium persulfate (SPS), acetonitrile, hy-
droquinone, sodium thiosulfate (Na2S2O3), potassium bromide (KBr),
potassium bromate, potassium iodide (KI) and diclofenac sodium
were purchased from Acros Organics (Belgium) and used without fur-
ther purification.
Poly [vinyl alcohol] (PVA), Mw 89,000-134,000, 99 + % hydrolyzed,
were purchased from Sigma-Aldrich (Germany). Gelatin (Type A: from
Porcine Skin) and capsaicin were of analytical grade and were pur-
chased from Sigma Aldrich (USA). Acetic acid (AA), and hydrochloric
acid (HCl), dimethylformamide (DMF), sodium chloride and 40%
glyoxal solution were obtained from Fisher Scientific (USA) and used
without further purification.
2.2. Fabrication of comprehensive wound dressing
The proposed wound care composite is constructed of an adhesive
sheet that is supporting three different electrospun layers as shown in
Fig. 1. Each layer that contains or releases wound healing agents has
specific role in wound healing process. The preparation details of each
layer are described in the following subsections.
2.2.1. Preparation of pressure sensitive adhesive (PSA) sheet based on
carboxymethyl cellulose
Pressure-sensitive adhesive (PSA) represents an adhesive that re-
quires a finger pressure to stick to surfaces including human skin.
Graft copolymerization of methyl-methacrylate and ethyl-hexyl acry-
late on the CMC backbone is considered as one type of such PSA.
An aqueous solution of CMC (40 mL of 2.5%wt/v) was placed into a
250 mL three-necked flask equipped with a reflux condenser, thermom-
eter, and a nitrogen gas line. The required amount of initiator, sodium
persulfate (SPS) 0.9–3.6 mmol, was dissolved first in the CMC solutions
at room temperature. Next, the co-monomers of MMA (4.5–14 mmol)
and EHA (15–45 mmol) were added all together with 0.5 mL Tween
80 and vigorously stirred, for 1 h, at room temperature in order to obtain
homogenous white emulsions. The reaction solution was then purged
with a stream of nitrogen gas also at room temperature to remove the
dissolved oxygen from solutions. Finally, the solution was heated up
by a water-bath to the desired temperature and for a desired duration.
The reaction was quenched with 2 mL of 5% wt/v hydroquinone aque-
ous solution. The mixture was allowed to cool down, filtered and cast
onto Teflon plates. The cast films were dried at room temperature for
24 h, extracted by water for 24 h each to remove the unreacted mono-
mers, cut to strips and stored in vacuum dissector for further use.
2.2.1.1. Encapsulation and characterization of diclofenac sodium into PSA.
Diclofenac sodium (1 mg/mL) as anti-inflammatory drug was added
to the latex at 30oc and left for stirring for 1 h. Mixture was allowed to
cool down, filtered and cast onto Teflon plates. PSA sheet loaded with
the drug was rinsed with distilled water and acetonitrile to remove
the free, unentrapped drug and unreacted monomers.
The release rate of diclofenac sodium (drug) from PSA sheet was de-
termined by incubating 50 mg of PSA/drug samples in 10 mL PBS
(pH 7.4) at 37 °C in shaking water bath [38]. At certain time intervals,
1 mL of the releasing medium was taken, and 1 mL of fresh medium
was replaced. The 1 mL solution was filtered through a 0.2 μm mem-
brane and the absorbance readings of the supernatant were recorded
at 263 nm using UV–Vis spectrophotometer (SHIMADZU, UVmini-
1240). The released amount was calculated from standard calibration
curve [39].
The encapsulation efficiency (EE %) was defined as the measurement
of the remaining content of the drug encapsulated in PSA compared to
the starting theoretical amount (Eq. (1)):
EE% ¼ ½Practical loading drug=½Theoretical loading drug  100 ð1Þ
The practical loading drug was measured by incubating 50 mg of the
PSA/drug samples in 10 mL PBS (pH 7.4) at 37 °C in shaking water bath.
After 2 days, 1 mL of the released medium was taken and filtered, and
the absorbance readings of the supernatant were recorded.
2.2.1.2. PSA characterization. The percentage of the graft add-on is calcu-
lated as the weight of the grafted polymer (HEC-g-MMA/EHA) divided
by the weight of HEC used, multiplied by 100. The HEC-g-MMA/EHA
and the homopolymers of MMA and EHA were dried first at room
Fig. 1. Explanatory diagram of the wound dressing composite.
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644
temperature for 24 h. The HEC-g-MMA/EHA was washed from MMA
and EHA homopolymers by solvent extraction for 6 h with acetonitrile
and water using Soxhelt system [40]. The purified HEC-g-MMA/EHA
was dried to calculate graft add-on values [41].
The percentage of total conversion (TC %) is calculated by a quantita-
tive estimation of the free double bonds of MMA and EHA after and dur-
ing polymerization time according to a reported method [42,43] with
some modifications [32].
Typically, an accurate 2.5 g of the polymerized solution was trans-
ferred to a 100-ml flask and 50 mL of 2 wt%/v hydroquinone/ acetoni-
trile solution (acetonitrile has been used effectively as a common
solvent to MMA, EHA and hydroquinone), was used for quenching the
polymerization process at the desired conversion [44]. 10 mL of the lat-
ter solution was transferred to 250-mL flask and 10 mL of 0.2 N KBr/
KBrO3 mixture and 10 mL of 2 N sulfuric acid (H2SO4) were added. To
avoid losses of bromine, pressure in the flask was reduced slightly by
cooling, so that the reagents were sucked into the flask.
The solution was left to stand in the dark for 20 min and shaken fre-
quently. 10 mL of 20% KI was then added to the flask to be closed quickly
and stored in dark for 30 min. The iodine was then titrated with 0.5 N
Na2S2O3 solution. % TC is calculated using the following equation
(Eq. (2)):
%TC ¼ ½ðVt −V0 Þ=ðVb −V0 Þ  100
where, Vb = volume of 0.5 N Na2S2O3 solution by blank (10 mL of hy-
droquinone/acetonitrile solution). V0 = volume of 0.5 N Na2S2O3 solu-
tion at zero time. Vt = volume of 0.5 N Na2S2O3 solution at time t.
Tack, peel strength and shear strength were measured using Pres-
sure Sensitive Tape Council standards PSTC-6, PSTC-I and PSTC-7, re-
spectively. The quenched and cooled polymer solutions (latex) were
filtered using glass funnel and cast onto Teflon plates. The cast film
was dried at room temperature for 24 h. A Universal Instron tester (lo-
cated at the National Research Centre, Egypt) was used to evaluate loop
tack and peel strength.
Loop tack test was measured according to the Pressure Sensitive
Tape Council standards PSTC-6 [45,46]. Typically, a strip of
2.5 cm × 17.7 cm was cut from the film and used to form a loop. Approx-
imately 2.5 cm at both ends of the strip was masked with tape and
inserted into the upper grip. The instrument moved the upper grip
downward at a speed of 300 m/min until an area of 2.5 cm came into
contact with the stainless-steel substrate mounted into the lower grip.
After 1 min contact time, the tester moved the upper grip upwards at
the same speed, while recording the force needed to debond the loop
from the substrate. The maximum force per surface area necessary to re-
move the adhesive was reported as loop tack.
PSTC 1 test standard method evaluates peel strength at a peel angle
of 180° [46]. A specimen of 2.5 cm × 30.5 cm was cut. The strip was lam-
inated onto a stainless-steel substrate. The dwell time of the strip on the
stainless-steel surface do not exceed 1 min. The substrate and the strip
were inserted into the grips and the upper grip was set to move upward
at a speed of 300 mm/min. The average force required to peel the strip
from the substrate was recorded and reported as peel strength.
Cohesion test was measured as shear strength according to the Pres-
sure Sensitive Tape Council standards test PSTC-7 [46]. A specimen of
2.5 cm × 152.4 cm of the film was cut. The strip was laminated onto a
stainless-steel substrate and then placed in a home-built shear tester
using a C-clamp. After 1 min of contact, a 500 g weight was suspended
at the end of the strip. The time to failure was recorded.
2.2.2. Preparation of the hemostatic agent, chitosan iodoacetamide (CI)
The synthesis of chitosan iodoacetamide was carried out as reported
in our previous work [18]. Typically, in the first step, 0.58 g of iodoacetic
acid (IA), 0.36 g of NHS, 0.6 g of EDC, and 2 mL of DMF were stirred con-
tinuously for 24 h to form stable reactive ester intermediate. In the sec-
ond step, 0.5 g of chitosan was hydrolyzed by 4 mL of 1 M HCl first
631
before adding 16 mL of DI water and was continuously stirred until
fully dissolved. Then, the reagents mixture from first step was pipetted
into the chitosan solution with continuous stirring. The pH of the mix-
ture was adjusted by 1 M NaOH until pH value of 5 was reached.
This reaction was continued for 24 h and the reaction mixture was
then dialyzed against 3 L of DI water five times over 6 days. The purified
solution was then filtered and lyophilized. The lyophilized samples are
in the sponge form and were stored in desiccator until further use.
2.2.3. Preparation of PVA electrospun mat containing CI (first layer-contact
layer)
The contact layer to wound was made of synthesized anti-bleeding
agents, chitosan iodoacetamide (CI) loaded into electrospun polyvinyl
alcohol (PVA) fibers. Electrospun solution was prepared by mixing the
same volume of 5% wt/v of CI (D.S. 18.7%) in distilled water and 10%
wt/v PVA in distilled water. Electrospinning process was carried out
on house-made apparatus using 20 kV, 17 cm airgap and 0.2 mL/h
flow rate.
2.2.4. Preparation of gelatin electrospun mat containing capsaicin (second
layer)
The second, pain-relief, layer was fabricated using capsaicin as the
effective analgesic drug loaded into gelatin electrospun fibers. Gelatin
ð2Þ is a desirable candidate due to its biocompatibility and its melting
point that is close-to body temperature. An optimal condition was
predetermined for pure gelatin electrospun nanofiber from previous
work [28] as follows: 15 kV over 15 cm with a rate of 0.3 mL/h for 24%
gelatin in 70% Acetic Acid. For the purpose of repeatability, the viscosity
of the spinning solution was measured and reported over a range of
shear rates at 20 °C. Capsaicin dissolves well in 70% acetic acid and, be-
cause of its small molecule nature, it does not significantly affect the vis-
cosity and spinnability of the gelatin solution. Therefore, 24% gelatin in
70% acetic acid was used as stock solution and was mixed with different
amount of capsaicin ranging from 5% to 50%. In all cases, nanowebs were
obtained and their morphologies were observed under SEM.
2.2.4.1. Crosslinking of gelatin nanowebs. The electrospun nanowebs
were peeled off from aluminum foil substrate and were cut into pieces
with weight of 40 mg each. They were placed on a metal mesh that
was sitting on top of a crystallization dish filled with 40% Glyoxal solu-
tion. The whole unit was placed in a large desiccator and was sealed
for crosslinking for a predetermined amount of times. The crosslinked
nanowebs were then placed in another vacuum desiccator for 2 days
for removing excess unreacted glyoxal.
2.2.4.2. Controlled drug release profile for crosslinked capsaicin/gelatin
nanoweb. The drug release experiment was conducted for capsaicin gel-
atin nanowebs. 40 mg each of the crosslinked 20% capsaicin gelatin
nanowebs that were cross-linked for different amount of times were
placed into 20 mL vials with diameter of 28 mm. Co-solvent for both
component was determined to be 1:0.4 (v:v) of PBS and Ethanol and
was used as the release medium. 10 mL release medium was added
into each vial which is capped and placed into a dry aluminum temper-
ature shaker bath maintained at a constant temperature of 37 °C and a
rate of 180 RPM. At each time interval, 1 mL of the liquid aliquot was col-
lected into a micro-centrifugal tube for later analysis and another 1 mL
of fresh releasing medium was added back to the vial. All solid was dis-
solved after 5 h and the release experiment was concluded at that time.
200 μL of each collected aliquot was added into a well in the 96-well UV
plates and their absorbance were measured using a TECAN Spark spec-
trophotometer. A calibration curve was calculated using standard solu-
tion of capsaicin in the release medium with concentrations at 0.5, 0.25,
0.125, 0.0625, 0.05, 0.03125 mg/mL. The absorbance of the solution was
first scanned between 200 and 300 nm wavelength and a maximum ab-
sorbance for capsaicin was found at 279 nm and was used to calculate
its concentration.
632 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644
The encapsulation efficiency was defined as the measurement of the
remaining content of the drug encapsulated in matrix compared to the
starting theoretical amount as described in Eq. (1).
2.2.5. Preparation of PVA electrospun mat with antibiotic (3rd layer)
PVA solution (10% wt/v) was mixed with Gentamicin, water-soluble
antibiotic, and optimum condition of PVA electrospinning process was
taken in consideration to produce beads-free and smooth electrospun
fibers. Typically, 20 kV, 17 cm airgap and 0.2 mL/h flow rate and Genta-
micin 5% wt/wt was used to obtain antimicrobial electrospun mat.
2.3. Fourier transform infra-red spectroscopy (FT-IR)
FT-IR instrument model 460 plus Jasco, (Micro Analytical Center, Cairo
University, Egypt) was used to analyze the IR spectra of samples. A DTGS
(Globar) laser source provided different wavenumbers (ν) through the
interferometer to produce a (4000–400 cm−1) wavenumbers range. Soft-
ware was set up to scan the background and samples at certain number of
scans (64), and at certain resolution (4). A proper time should be taken
(ca. 15 min) to attain a clear spectrum without the influence of moisture
and carbon dioxide [47].
2.4. Scanning electron microscopy (SEM)
The electrospun fibers morphology was assessed using Quanta 250
FEG (Field emission Gun) scanning electron microscopy (SEM), located
at the national research center (NRC), and Verios 460 L field-emission
scanning electron microscopy (FE-SEM) located in the Analytic Instru-
mentation Facility (AIF) at North Carolina State University. Samples
(electrospun mats), deposited on aluminum sheet, were fixed on the
sample holder and coated with a layer of gold in vacuum using sputter
coater S150A Edwards-England to produce conductive surface. Gold-
coated mats were placed in the microscope chamber. Features of sample
morphology were obtained at 5–10 kV [48]. In case of Verios 460 L FE-
SEM, extremely low voltage of 500 V was used which does not require
samples to be coated. Fiber diameters were calculated from the SEM mi-
crographs using ImageJ software.
2.5. Viscosity measurement of the spinning solution
The viscosity of the spinning solution was measured using Anton
Paar MCR 302 rheometer attached with a concentric cylinder measuring
system. Ten data points were collected for every decade of the shear
rates between 0.1 and 1000 1/s with a duration of 5 s for each data point.
2.6. Cytotoxicity assessment
The cytotoxicity test was conducted with an adaptation from ISO
10993-5 standard test method in which human skin fibroblasts
(HFB4) were used for all studies. Cell line was obtained from the
Egyptian company for production of vaccines, sera & drugs (Vacsera),
Giza, Egypt. The choice of fibroblast cell line is based on their role on
producing proteins associated with extracellular matrix (ECM) synthe-
sis and their crucial role in wound healing process [5,6,31,32,47]. In typ-
ical procedure, cells were maintained in Dulbecco's modified eagle
medium (DMEM):F12 Medium (nutrient mixture)/10% (w/v) fetal bo-
vine serum (FBS) and were incubated at 37 °C in 5% CO2 and 95% humid-
ity. Cells were seeded into wells in 96-well plate at a density of 30,000
cells per well. After 48 h of incubation, the culture mediums were re-
placed by extracted media of different concentrations (12.5, 25, 50
and 100 ppm [μg/mL]) of the test compounds (each sample was tested
in three different wells). Samples were sterilized under ultraviolet (UV)
light for 20 min in a laminar flow before extraction. Cell culture me-
dium, without additional reagents, was used as control. The plate was
incubated again for 24 h. The number of living cells was determined
by the MTT assay. The culture medium was aspirated and replaced by
40 μL of MTT solution (2.5 mg/mL). Then, the solution was incubated
for another 4 h at 37 °C. The solution was aspirated and 200 μL 10% so-
dium dodecyl sulphate (SDS) in deionized water was added to each well
to dissolve the formazan crystals and incubated overnight at 37 °C. The
absorbance was measured using a ChroMate microplate (Awareness
Technology, U.S.A.) reader at 595 nm and a reference wavelength of
690 nm [49] and recorded as optical density (OD). The reported value
for each sample is the average of 8 measurements in the same column.
The percent of cell viability of the samples were calculated relative to
the control with fresh complete cell culture medium according to the
following equation (Eq. (3)).
½avðxÞ=avðNCÞ  100 ð3Þ
where Av: average, X: absorbance of sample, NC: absorbance of negative
control.
In this test, doxorubicin was used as positive control to show the ad-
verse effect of toxic material on cell viability and thus distinguish be-
tween safe and toxic materials.
2.7. In vivo test
The tested compounds that showed no significant toxicity were used
with animal models, in controlled laboratory environment to simulate
the clinical environment [4,50]. Experiments perform on animal models
have to provide justifiable comparisons to the human beings [51]. All
rats were handled in accordance with the NIH Guidelines for the Care
and Use of Laboratory Animals and with the recommendations of the In-
stitutional ethical committee in the National Research Center, Cairo,
Egypt (Reg. No. 17-055).
2.8. Experimental animals
Male rats weighing 110–120 g were used for in vivo studies and were
obtained from the animal house, National Research Center (NRC), Dokki,
Giza. Rats were housed in separate cages in standard environmental con-
ditions and were fed commercial rat feed and water ad libitum.
2.9. Wound creation
Excision wounds were used for the study of wound contraction.
Assigned area of surgery was swiped with betadine prior to excision.
All wounds were of full-thickness type extending up to the adipose tis-
sue. The backs of rats were shaved with electric clippers, hair removed
and then anaesthetized by open mask method with anesthetic ether.
Excision wounds of size (about) 1 cm2 were made by cutting 1 × 1 cm
piece of the skin from the shaven area in one side using punch biopsy.
2.10. Histopathological test
Rats were sacrificed after 21 days in different groups and autopsy
samples were taken from the skin of rats to include the entire wound
area. Specimens of all animals were dissected immediately after death
and fixed in 10% neutral-buffered formal saline for 72 h at least. All
the specimens were washed in tap water for half an hour and then
dehydrated in ascending grades of alcohol (70%–80%–90% and finally
absolute alcohol), cleared in xylene, impregnated in soft paraffin wax
at 56 °C and embedded in hard paraffin. Serial sections of 4 μm thickness
were cut by sledge microtome and stained with Haematoxylin and
eosin [52] for histopathological investigation. Images were captured
and processed using Adobe Photoshop version 8.0.
2.11. Statistical analysis
Results were expressed as a mean value with its standard deviation
(mean ± S.D.) of each sample that is repeated three times (n = 3).
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644
Statistical analysis was performed with Student's t-test and differences
were considered as significant at p-values below 0.05.
3. Results and discussion
3.1. Pressure sensitive adhesive (PSA) sheet based on grafted
carboxymethyl cellulose (CMC)
Water-soluble cellulose derivatives have been reported for graft co/
polymerization with different types of co/monomers via chemical initi-
ators, UV light, gamma rays and plasma excitation [39]. Fig. 2 shows the
proposed mechanism of the graft co-polymerization of CMC with MMA
and HEA in the presence of SPS as a thermal initiator. The reaction initi-
ated when SPS decomposed at 75 °C (A) to create active intermediates
species with free radicals (B). These free radicals transfer to the oxygen
atoms of CMC (C) to form covalent bonds with the double bonds of
MMA and/or EHA and CMC backbone (D). The final product was ex-
tracted by water and acetonitrile for 24 h each.
The proposed mechanism was confirmed using IR analysis by compar-
ing the CMC and PSA spectrum. Fig. 3 shows the IR spectra of CMC and
CMC-g-Poly(MMA-co-EHA). A sharp peak was observed at 1729 cm−1
corresponding to the carbonyl groups of polyacrylates as a certain evi-
dence of grafting. Peaks located at 2857 and 2960 cm−1 were observed
in the grafted substrate that is attributed to the CH stretching of the ali-
phatic part of EHA.
According to the proposed mechanism, such copolymerization reac-
tion relies on specific parameters to enhance the total conversion of the
acrylate monomers and the graft add-on. In the light of that, the initiator
Fig. 2. Proposed mechanism of graft co-polymerization of MMA (n) and EHA (m) onto carboxyethyl cellulose sodium salt.
633
concentration, the polymerization duration, and the reaction tempera-
ture were optimized to obtain the best condition.
3.1.1. Effect of polymerization duration on total conversion and graft add-
on
Fig. 4 shows the effect of the duration of the grafting copolymeriza-
tion of MMA and EHA onto CMC backbone on the total conversion (%)
and the graft add-on (%). Data shows that the TC % and graft add-on %
increase by increasing the reaction duration to reach 99% TC and 540%
in add-on by 3 h. It is understandable that time is one of the major fac-
tors affecting the grafting performance especially during the first 3 h
and by increasing the polymerization time the CMC-g-MMA/EHA
weight increases significantly. Thus, 3 h was taken as optimum condi-
tion for the further experiments.
3.1.2. Effect of initiator concentration on total conversion and graft add-on
The effect of SPS concentration in mmol on the total conversion (TC
%) and the graft add-on % is shown in Fig. 5. Data shows that TC % in-
creased as SPS increased up to 1.4 mmol and by increasing concentra-
tion any further, graft add-on decreased. Data reveals that lower SPS
concentration leads to less graft add-on which adversely affects the co-
hesion property. Therefore, the concentration 1.4 mmol of SPS was
taken as optimum condition for the further experiments.
3.1.3. Effect of polymerization temperature on total conversion and graft
add-on
The effect of the polymerization temperature (25, 60, 75 and 90 °C) on
the total conversion (%) of the MMA and EHA monomers and on the graft
634 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644

Fig. 3. FT-IR spectra of CMC and CMC-g-poly(MMA-co-EHA).

Fig. 4. Effect of the duration of the grafting copolymerization of MMA and EHA onto CMC backbone on the total conversion (%) and the graft add-on (%). PSA latex were prepared by using
40 mL of CMC (2.5 w/v), 0.5 mL of Tween 80, 1.4 mmol of SPS, 4.5 mmol of MMA and 30 mmol of EHA. All additives were added at room temperature and then polymerization temperature
raised to 75 °C.

add-on (%) is shown in Fig. 6. It was observed that TC % increased dramat-
ically by increasing the temperature until 75 °C and went down at higher
temperature. Data reveals that increasing polymerization temperature up
to 90 °C results in to more homopolymer rather than copolymerization.
3.1.4. Effect of molar ratios of MMA and EHA on PSA properties
Table 1 shows the PSA properties (tack, peel strength and shear
strength) as a function of different molar ratios of the monomers used
in this study (MMA and EHA). It was found that the CMC sheet was
very brittle and could not stand for testing. Meanwhile, CMC grafted
by 15 mmol of EHA did not have a sufficient adhesion for testing, and
CMC grafted by 45 mmol of EHA was too sticky to peel it off the Teflon
plate. Data revealed that CGME-1 sample in which CMC was grafted
by comonomers MMA (4.5 mmol) and EHA (30 mmol) showed good
tack and peel (adhesion properties) 2.1 and 2.1 respectively. However,
this CGME-1 latex showed the low shear strength (cohesion) about

Fig. 5. Effect of initiator (sodium persulphate) concentration (mmol) on the total conversion (%) and graft add-on (%). PSA latex were prepared by using 40 mL of CMC (2.5 wt/v) 0.5 mL of
Tween 80, 4.5 mmol of MMA and 30 mmol of EHA. All additives were added at room temperature and then polymerization temperature raised to 75 °C for 3 h.
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644 635

Fig. 6. Effect of polymerization temperature on the total conversion (%) and the graft add-on (%). PSA latex were prepared by using 40 mL of CMC (2.5 wt/v), 0.5 mL of Tween 80, 1.4 mmol
of SPS, 4.5 mmol of MMA and 30 mmol of EHA. All additives were added at room temperature and then polymerization temperature raised for 3 h.

Table 1 function of MMA and EHA molar ratios. The highest Tg was recorded
Effect of molar ratios of MMA and EHA on PSA properties. for CMC pure sample, 115 °C.
Code Molar ratios Loop tack Peel strength Shear strength
MMA: EHA (N/cm2) (N/cm) (sec) 3.1.6. Release study of diclofenac sodium
CMC 0:0 – – – In this part, PSA (CGME-2) was explored for its capability to encap-
CGE 0:30 – – – sulate anti-inflammatory drug, diclofenac sodium, and its release profile
CGME-1 4.5:30 2.1 2.1 20 in PBS, pH 7, at 37 °C was investigated. In Fig. 7, the total amount of the
CGME-2 9:30 1.52 1.716 25
drug released over 48 h was recorded to show a gradual release in the
CGME-3 14:30 1.07 0.981 25
first 18 h to reach 70% of the total drug. However, the released amount
PSA latex were prepared by using 40 mL of CMC (2.5 wt/v), 0.5 mL of Tween 80, and of drug stayed constant at 71–73% over the next 24 h. Data reveals that
1.8 mmol of SPS. All additives were added at room temperature and then polymerization
temperature was raised to 75 °C for 3 h.
no burst release was observed, and PSA matrix showed a systematic
control of the release rate over the first 18 h. Also, encapsulation effi-
ciency was found to be 73.3% after 48 h. This decline is attributed to
the affinity between the PSA and the drug so that the reminder drug
20 s. Nevertheless, CGME-3 samples in which MMA molar ratio in-
molecules did not diffuse out of the PSA matrix.
creased by threefold (14 mmol), the cohesion strength has increased
to 25 s and loop tack and peel strength decreased by 50%. Data agrees
3.2. Hemostatic layer: PVA electrospun mat containing chitosan
with the PSA principles in which EHA as an alkyl acrylate that possesses
iodoacetamide (CI) (first layer)
lower Tg is suitable monomer to impart flexibility and tack of the adhe-
sive. However, modifying monomers such as MMA impart better cohe-
This layer is considered as the first layer to be in contact with the
sion strength to the adhesive sheet [53]. CGME-2 sample gained a
wound bed. This layer was designed to contain CI for its hemostatic ef-
moderate and acceptable adhesive and cohesion values and was chosen
fect. Typically, PVA solution 10%wt/v was dissolved in distilled water
for further use.
and electrospun under reported condition in previous work [21]. Typi-
cally, PVA solution gives beads-free and smooth electrospun fibers at
3.1.5. Differential scanning calorimetry (DSC) analysis 20 kV, 17 cm air gap and 0.2 mL/h flow rate of the solution. PVA 10%
Glass transition temperature (Tg) is one of the most important fac- wt/v solution was mixed with 5% wt/v CI solution on volume ratio 1:1
tors affecting the performance of PSA. Tg affects the major adhesive and electrospun under the same condition of PVA solutions.
properties as it affects the segmental motions necessary for the adhesive SEM images and the distribution curve of PVA and PVA/CI
flow and thus, bonding to other surfaces. It also affects the flexibility of electrospun fibers are shown in Fig. 8. Data shows the average of fiber
adhesive sheets over a range of temperatures. Typically, the lower the Tg
the more segmental motion is present at room temperature, the
smoother surface of final film and the higher tackiness. Consequently,
adhesive sheet will be much easier to reshape to the patient skin.
In this work, EHA was used to lower the Tg final value of final latex to
enhance the adhesive properties. Table 2 shows the Tg values as a

Table 2
Glass transition temperature of sheets made of CMC and PSA sheet with different MMA
and EHA molar ratios.

No. Latex Tg (°C)

1 CMC 115
2 CGE −55
3 CGME-1 −49
4 CGME-2 −35
5 CGME-3 −22

CGME-1, CGME-2 and CGME-3 composition are mentioned in Table 1. Fig. 7. The release profile of diclofenac sodium encapsulated into PSA sheet (CGME-2).
636 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644

Fig. 8. SEM images and distribution curves of PVA (a and b) and PVA/CI electrospun fibers (c and d).

Table 3
Fiber diameters of electrospun nanowebs. 3.3. Pain-relief layer: gelatin loaded capsaicin (second layer)
Materials Fiber diameter (nm) Standard deviation
This second, pain-relief layer was constructed using gelatin
Neat gelatin 136.2 9.97
electrospun mat as the carrier and capsaicin as the effective analgesic
5%Capsaicin+gelatin 166.3 34.73
10%Capsaicin+gelatin 164.2 25.48 drug. The spinning solvent was 70% acetic acid/water solution and
15%Capsaicin+gelatin 191.3 50.49 therefore was able to dissolve both the hydrophilic gelatin and the hy-
20%Capsaicin+gelatin 177.7 29.53 drophobic drug (capsaicin).
50%Capsaicin+gelatin 197.6 32.69 The viscosity of gelatin solution (24% wt/v) manifested a plateau of
0.348 Pa·s at shear rates lower than 100 1/s. At shear rates beyond
diameter of PVA electrospun fibers is 695 nm. In addition of CI to PVA, 100 1/s, gelatin solution showed an obvious shear thinning behavior.
fiber diameters decreased to 300 nm. This is attributed to the increase For electrospinning, whose spinning solution was extruded at very
of the electro-conductivity of the mixed solutions [54]. slow rate, the plateau value for the Newtonian behavior region best

Fig. 9. SEM of Gelatin nanowebs and Gelatin nanowebs loaded with increasing amount of Capsaicin.
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644 637

Fig. 10. SEM of Gelatin nanowebs loaded with 50% capsaicin.

approximates the spinning viscosity of the gelatin solution. It was ob-
served that the viscosity and spinnability were not been affected by
adding the drug as it recorded 0.351 Pa.s at 50% wt/wt of capsaicin.
Data shows that the average gelatin fiber dimeter is 136 nm and
197 nm when 50% wt/wt capsaicin was loaded (Table 3). SEM images
in Fig. 9 showed that the fiber surfaces manifest a transition from
smooth to wrinkled at about 15% loading. This feature was augmented
at a much higher loading of 50% as shown in Fig. 10. This structural fea-
ture can be caused by a microphase separation due to the dissimilar hy-
drophobicity between the two components (gelatin and capsaicin). At
the same time, it proves that an excess amount of drug has not included
into the fiber and crystallized by itself forming a nice hexagonal shaped
capsaicin crystal (Fig. 10). It is noticeable that electrospun nanowebs
eliminated the pungency of neat capsaicin remarkably well. Handling
nanowebs with 50% loading does not release pungent scent that
would cause uncomfortable allergic reactions while a trace amount of
the pure substance will normally do. The encapsulation method used
here therefore eliminated one of the drawbacks of capsaicin.
The release profile and the encapsulation efficiency carried out by
conducting an absorbance scans between 200 and 300 nm of pure re-
lease medium, the dissolved uncross-linked gelatin capsaicin nanoweb,
and the dissolved uncross-linked neat gelatin nanoweb. It was found
that the release medium selected does not absorb between these wave-
lengths and the absorbance of neat gelatin solution is negligible. On the
other hand, gelatin/ capsaicin solution possesses a large peak with its
lambda maximum at 279 nm as shown in Fig. 12. Due to its water solu-
bility at physiological temperature, gelatin, and its drug-loaded nanofi-
bers instantly dissolved in warm water. The dissolution can be slowed
down or delayed by chemical crosslinking of the electrospun fibers. In
Fig. 13, it was demonstrated that the release of capsaicin can be slowed
down by briefly expose the gelatin/drug nanoweb to glyoxal vapor.
Based on the consistent cumulative capsaicin amount, it is concluded
that capsaicin was encapsulated uniformly in the nanoweb with low
sample-to-sample variations. The release curve indicates that capsaicin
release completed for all samples at about 2 h mark with encapsulation
efficiency of up to 100%.

3.4. Preparation of PVA electrospun mat with gentamicin (third layer)

3.3.1. Capsaicin controlled release
In the first place, encapsulation of capsaicin was confirmed by the This layer was made to protect the wound bed from bacterial inva-
FTIR (Fig. 11) based on the increase in the peak intensity in the C\\H sion by loading water-soluble antibiotic, gentamicin (Gen), to PVA
stretching region at 2930 cm−1. electrospun fibers. Typically, PVA solution (10% wt/v) was used

Fig. 11. FTIR spectra of Gelatin nanowebs and Gelatin nanowebs loaded with Capsaicin.
638 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644

Table 4
Cytotoxicity test of the wound healing composite substituents (100 μg/mL) on hu-
man normal cell line ((HFB4).

Samples code Cytotoxicity % at 100 μg/mL

CGME-2 2.1 ± 0.41

PVA/CI 5.3 ± 0.8
Gelatin/Capsaicin 3.7 ± 0.3
PVA/Gen 7.9 ± 0.9
Doxorubicin 88.93 ± 0.15

CGME-2; adhesive sheet was prepared based on carboxymethyl cellulose with

methyl methacrylate (MMA) and 2-Ethyl-1-hexyl acrylate (EHA) monomers in
molar ratios (9:30).

containing Gen (2% wt/wt). PVA/Gen electrospun fibers were produced

by applying the same electrospinning condition of PVA in which 20 kV,
17 cm and 0.2 mL rate flow were used. Fig. 14 shows SEM image and the
distribution curve of the PVA/Gen electrospun fibers. Data shows that
the average of the fiber diameter is 270 nm which decreased by two
folds than PVA electrospun fiber 695 nm, shown in Fig. 8a and b. This
is also due to the increase of the electro-conductivity of the mixture
solution.
Fig. 12. UV absorbance spectra of fresh releasing medium, Gelatin Capsaicin nanoweb, and
Gelatin nanoweb between 200 and 300 nm.
3.5. Cytotoxicity test

The biological compatibility of the substituents of the wound healing

multi-layer was demonstrated via MTT test using fibroblast cells. Such
cell type was chosen because it produces proteins associated with the
extracellular matrix (ECM) synthesis and accelerates wound healing
process. Table 4 shows the percentage of cell cytotoxicity (%) of the ad-
hesive sheet (CGME-2) loaded with diclofenac sodium (1 mg/mL); PVA
electrospun fibers loaded with IC (5% wt/v and D.S. = 18.7%); gelatin
electrospun fibers loaded with 50% (wt/wt) capsaicin; and PVA
electrospun fibers loaded with 2% wt/wt gentamicin. The biocompatibil-
ity of each component of the composites showed insignificant toxicity
with values less than 8% compared to doxorubicin (positive control)
89%. PVA/CI sample showed 5.3% cytotoxicity due to the presence of chi-
tosan iodoacetamides. Iodoacetamide derivatives react with SH func-
tional groups of cells that increases the cytotoxicity accordingly
[55,56]. This result is encouraging to use the wound dressing composite
in the pre-clinical studies on rat in the next subsection.

3.6. Evaluation of wound contraction rate on rat skin

The contraction rate of wound closure was investigated for different

combinations of the prepared layers mentioned above. Fig. 15 shows ex-
Fig. 13. Cumulative Capsaicin drug release over time for 20% Capsaicin Gelatin nanowebs planatory diagrams of different combinations for wound laminates used
cross-linked for 30 mins (Sample A), 1 h (Sample B), and 2 h (Sample C). in the wound healing test on rats. Sample number 1 was excluded for
the untreated rats. Sample number 2 represents PSA sheet (CGME-2)

Fig. 14. SEM image and distribution curve of PVA/Gen electrospun fibers.
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644
Fig. 15. Explanatory diagram of different combinations of wound laminates used in the
wound healing test on rats. 2: PSA sheet (CGME-2) loaded with 1 mg/mL diclofenac
sodium; 3: PSA sheet and PVA electrospun fibers loaded 2% wt/wt gentamicin; 4:
PSA + PVA/Gen + gelatin electrospun fibers loaded with 50% (wt/wt) capsaicin; 5:
PSA + PVA/Gen + Gelatein/Cap+ PVA electrospun fibers loaded by IC of (5% wt/v and
D.S. = 18.7%).
loaded with 1 mg/mL diclofenac sodium and sample number 3 repre-
sents PSA sheet holding PVA electrospun fibers loaded with gentamicin
(2% wt/wt). While, sample 4 represents PSA holding both PVA/Gen and
gelatin electrospun fibers loaded with 50% (wt/wt) capsaicin and sam-
ple number 5 represents PSA holding both PVA/Gen, gelatin/Cap and
PVA electrospun fibers loaded by IC of (5% wt/v and D.S. = 18.7%).
Fig. 16 shows photo images of sampling wound healing combina-
tions involving PSA sheets and electrospun laminates.
The wound areas of the untreated and treated wounds are recorded in
Table 6 in which readings were taken in days 0, 3, 7, 14 and 21. The visual
observation (Table 5) of the topical application of all combinations was
very benign to the hosts and enhances the healing process except sample
number 2 of the adhesive sheet. This is can be explained that when the
Fig. 16. Samples of different wound healing combinations including PSA sheet and electrospun laminates.
639
adhesive sheet directly sticks to the wound bed, it ruptures the new
built skin tissues as it is removed. Unlike sample number 2, all samples
showed lack of redness at all time points of administration which indi-
cates that the formulations of samples 3–5 did not induce extensive
acute inflammatory responses. Adhesive sheet that sticks to the back
skin and provide strong stabilization of the dressing on the rat back, sticks
to the wound cap too and causes pain and tissue damage. However, this
has not been observed with PSA covered by electrospun layers.
Data shows that the wounds of the untreated rats (group 1) stayed
unhealed even after 21 days. However, group 2 in which the adhesive
sheet used in direct contact with wound, showed adverse effect on
wounds. Contradictorily, group 3 which treated with the antimicrobial
layer showed a crest (black cap) after 3 days and showed complete
healing after 21 days. Group 4 showed an accelerated healing rate better
than group 3. In group 5, wounds are fully recovered in 7 days.
3.7. Histology assessments
The wound contraction rate is a quick indication of the treatment ef-
fectiveness. However, histopathological assay will reveal whether the
prepared formula play a role in the process of wound healing in normal
fashion.
In group 1, there was no histopathological alteration and the normal
histological structure of the epidermis, dermis, subcutaneous tissue and
musculature were recorded in Fig. 17 a, b and c.
In group 2, focal acanthosis was observed in the epidermal layer as-
sociated with focal haemorrhage, necrosis, and hyalinization with gran-
ulation tissue formation in the underlying dermal layer (Fig. 18a, b and
c). The deep dermal layer showed inflammatory cells infiltration with
granulation tissue formation (Fig. 18d).
In group 3, the epidermis showed focal acanthosis while the under-
lying dermis had focal haemorrhage and granulation tissue formation
(Fig. 19a and b). Focal haemorrhage was detected also in the deep
layer of the dermis with granulation tissue (Fig. 19c). The subcutaneous
tissue showed focal haemorrhage and oedema (Fig. 19d).
In group 4, mils acanthosis was noticed in the epidermis associated
with granulation tissue formation in the underlying dermis (Fig. 20a
and b). Few oedema was observed in the subcutaneous tissue (Fig. 20c).
In group 5, the epidermis was intact while the underlying dermis
showed granulation tissue formation with focal haemorrhages
(Fig. 21a, b and c).
Histopathological findings reveal that the healed skin of rats treated
with group 5 which has the three laminated electrospun layers, shows a
normal skin structure similar to that on unwounded skin.
4. Conclusions
A comprehensive wound dressing was fabricated based on lam-
inated electrospun fibers loaded with the necessary wound healing
640 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644

Table 5
Photo images of wounds contraction progress over 21 days of untreated and treated wounds by different combinations 2, 3, 4 and 5.

Time Animal groups

points

Untreated 2 3 4 5
Day 0

Day 3

Day 7

Day 14

Day 21

Samples codes are described in Fig. 15.

drugs supported by an adhesive sheet. The adhesive sheet was pre-
pared based on carboxymethyl cellulose grafted by methyl meth-
acrylate (MMA) and 2-ethyl hexyl acrylate (EHA). The graft-
copolymerization reaction was optimized at 75 °C, for 3 h, using so-
dium persulphate (thermal initiator) and different molar ratios of
MMA and EHA. First, a contact layer was made of chitosan
iodoacetamide as a synthetic hemostatic reagent loaded into PVA
electrospun fibers. The second layer was made to relieve pain in
the injured part and was made of gelatin electrospun fibers loaded
with capsaicin (2% wt/wt). The third layer, the protective shell was
made of electrospun fibers of PVA loaded by antibiotic. The wound
dressing substituents were examined for biocompatibility and
showed very insignificant adverse effects on fibroblast cells. Differ-
ent combinations of these three layers were considered for pre-
clinical study on rats. The closure of wound was recorded for each
combination and revealed that wounds of group 5 in which all
layers superimposed on the adhesive sheet were fully recovered
in 7 days with intact epidermis structure.
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644 641

Table 6 affiliation ID: 60014618) for facilities provided for analysis and to the fi-
wound area (cm2) of the treated rats by different formulas. nancial support from Science and Technology Development Fund
Time points (day) Wound area (cm2) (STDF) through US-Egypt project, cycle 17 and I.D. 114, entitled” A Med-
Group 1 Group 2 Group 3 Group 4 Group 5
ical Textile for Comprehensive Wound Care: A laminated Multifunc-
tional Electrospun Fabric that is Hemostatic, Anti-inflammatory and
0 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1
Anti-microbial”.
3 0.96 0.91 0.87 0.77 0.75
± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.2
7 0.91 0.81 0.66 0.41 0.1 ± 0.1 Author statement
± 0.2 ± 0.1 ± 0.2 ± 0.2
14 0.86 0.75 0.51 0.33 Healed Ahmed A. Nada: Developed the original idea of the research, con-
± 0.2 ± 0.2 ± 0.1 ± 0.1
21 0.71 0.52 Healed Healed Healed
ducted the adhesive sheet experiments including encapsulation and
± 0.1 ± 0.1 drawing mechanism, conducted the first layer of PVA and iodoacetamide
chitosan, conducted the third layer of PVA and gentamicin. Dr. Nada
Samples codes are described in Fig. 15.
wrote the original and the revised draft of the manuscript.
Eman A. Ali: Conducted PSA analysis, DSC analysis and animal test.
Acknowledgement Ahmed A.F. Soliman: Conducted the cytotoxicity test, collected, and
interpreted data.
The authors are grateful for the funding provided by U.S.-Egypt Sci- Jialong Shen: Conducted the iodoacetamide chitosan synthesis, anal-
ence and Technology Joint Fund, administered by the National Academy ysis and discussing data. Also, conducted the second layer of gelatin
of Sciences (US: CFDA # 98.000-AID, Subaward 2000007149). The au- nanofibers with capsaicin and its crosslinking. Dr. Shen conducted the
thors are grateful to National Research Centre (Egypt) (Scopus release profile of the second layer and helped writing the manuscript.

Fig. 17. Skin of rat in group 1 (unwounded skin) with two magnifications b and c.

Fig. 18. Skin of rat in Group 2 (PSA sheet (CGME-2) loaded with 1 mg/mL diclofenac sodium), with different magnifications b, c and d.
642 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644

Fig. 19. Skin of rat in Group 3 PSA/ diclofenac sodium sheet and PVA electrospun fibers loaded 2% wt/wt gentamicin.

Nabil Y. Abou-Zeid: Senior author and the Egyptian principle inves- Samuel M. Hudson: Senior author and US-principle investigator of
tigator of the research project funded this work. Supervisor and the research project funded this work. Supervisor and
conceptualization. conceptualization.

Fig. 20. Skin of rat in Group 4 PSA + PVA/Gen + gelatin electrospun fibers loaded with 50% (wt/wt) capsaicin, with two magnifications b and c.
 A.A. Nada et al. / International Journal of Biological Macromolecules 162 (2020) 629–644
Fig. 21. Skin of rat in Group 4; PSA + PVA/Gen + Gelatein/Cap+ PVA electrospun fibers loaded by IC of (5% wt/v and D.S. = 18.7%), with two magnifications b and c.
Declaration of competing interest
Authors have declared no conflicts of interest.
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