Carbohydrate Polymers 238 (2020) 116175

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

Wound healing of nanofiber comprising Polygalacturonic/Hyaluronic acid T

embedded silver nanoparticles: In-vitro and in-vivo studies
M.R. El-Aassara,b,*,1, Omar M. Ibrahimb,c,1, Moustafa M.G. Foudad,**, Nagham G. El-Beherib,
Mona M. Agwae
a
Department of Chemistry, College of Science, Jouf University, Sakaka 2014, Saudi Arabia
b
Polymer Materials Research Department, Advanced Technology and New Material Institute, City of Scientific Research and Technological Applications (SRTA-City), New
Borg El-Arab City, Universities and Research Institutes District, Alexandria 21934, Egypt
c
Department of Medicine and Translational Research, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
d
Pre-Treatment and Finishing of Cellulosic-based Fibers Department, Textile Industries Research Division, National Research Center, 33 El- Behooth St, Dokki, Giza,
12311, Egypt
e
Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, 33 El- Behooth St, Dokki,
Giza, 12311, Egypt

ARTICLE INFO ABSTRACT

Keywords: The current study is pertaining to develop a novel wound dressing, comprising natural biologically absorbable
Polygalacturonic acid materials for wound healing In-vivo. Wound dressing is composed of Polygalacturonic acid, Hyaluronic acid
Silver nanoparticles embedded silver nanoparticles, which is further fabricated to form nanofibrous mat, using electrospinning. Silver
Nanofiber nanoparticles was prepared using PGA. AgNPs in this formula will serve as an antioxidant and anti-inflammatory
Hyaluronic acid
that protect cells from destructive effect of elevated ROS and accelerate wound healing. The physical perfor-
Antibacterial
Wound dressing
mance and water contact angle for nanofiber was evaluated. The produced nanofiber was characterized by
Fourier-transform infrared (FTIR), scanning electron microscopy and thermal analysis. Also, the embedded
AgNPs was also characterized by UV–vis spectroscopy and TEM. The nanofiber mates embedded AgNPs was
applied to the wounded site of albino rats in-vivo. Histopathological assessment for the wound was fully per-
formed. Also, the antimicrobial activity for the fabricated wound dressing was evaluated against gram+ve and
gram −ve bacteria.

1. Introduction
The skin of the human has a prominent protective role due to
stratum corneum layer that make skin acts like a permeation barrier
(Boer, Duchnik, Maleszka, & Marchlewicz, 2016). Cutaneous injury is
major problems for health care system all over the world as the skin
loses its protective function if any damage of this layer occurs, either by
wounds or burns (Ho, Walsh, Yue, Dardik, & Cheema, 2017). The
wound generally refers to a skin damage caused by surgery or trauma.
Wound infection is one of the fundamental causes that hampered and
delay the healing process (Sarhan, Azzazy, & El-Sherbiny, 2016). For
the antibiotics to be effective against and or prevent wound infection, a
high concentration at the specified wound area is guaranteed that leads
to higher local concentration and lower serum level instead of systemic
administration that lead to sever side effect due to higher serum level
(Musters, Burger, Buskens, Bemelman, & Tanis, 2015). So, a better
protection against microbial infection by fabrication of suitable anti-
microbial wound dressing is necessary for faster wound tissue re-
generation (Li et al., 2016).
The process of wound healing proceeds through a cascade of events
which include four different major phases; coagulation, inflammation,
proliferation and tissue remodeling (Shalaby et al., 2020). The in-
flammatory response is a substantial phase of wound healing and it
begins within minutes after the injury and characterized by the man-
ufacturing of cytokines (pro-inflammatory) and stimulation of cellular
immunity (Wilkinson, White, & Chipman, 2011). Not only cytokines are
vital in initiating, sustaining and regulating the cellular response after
injury but also, they have been involved in wound healing impairment.

Corresponding author at: Department of Chemistry, College of Science, Jouf University, Sakaka 2014, Saudi Arabia.
⁎

Corresponding author at: Pre-treatment and Finishing of Cellulosic-based Fibers Department, Textile Industries Research Division, National Research Centre,
⁎⁎

Dokki, Giza, 12622, Egypt.

E-mail addresses: mrelaassar@ju.edu.sa (M.R. El-Aassar), drmmfouda@gmail.com (M.M.G. Fouda).
1
M. R. El Aassar and Omar M. Ibrahim contributed equally to this work.

https://doi.org/10.1016/j.carbpol.2020.116175
Received 31 December 2019; Received in revised form 11 March 2020; Accepted 13 March 2020
Available online 09 April 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
M.R. El-Aassar, et al.
Through the inflammatory stage of the wound healing macrophages
and neutrophils invade the wound and produce great quantities of su-
peroxide radical anions. This phenomenon is characterized as the “re-
spiratory burst’. Moreover, a proinflammatory cytokines can stimulate
other cells like fibroblast and chondrocytes to produce reactive oxygen
species (ROS) (Meier et al., 1989).
Reactive oxygen species (ROS) are considered chemically reactive
species comprising oxygen. Examples include peroxides, superoxide,
hydroxyl radical, and singlet (Hayyan, Hashim, & AlNashef, 2016). It
plays a fundamental role in the modulation of cell survival, cell death,
differentiation, cell signaling, and inflammation-related factor produc-
tion (Abdal Dayem et al., 2017). The production of these ROS in the
wound is needed for the defense against invading pathogenic bacteria
as its part of the innate immune system (auf dem Keller, Kumin, Braun,
& Werner, 2006). The substantial goal of the generation of higher ROS
levels during inflammatory stage of the wound healing is the destruc-
tion and elimination of the invading pathogenic microorganisms and/or
degradation of the damaged tissue structures (Bonferoni et al., 2018). In
addition to the beneficial role of ROS in killing pathogenic bacteria, it
also has a series of negative effects. At low concentration ROS are the
critical mediators of various intracellular signaling (D’Autreaux &
Toledano, 2007). It was reported that low ROS level that secreted in a
sustained manner at wound site participate in a variety of biological
processes, comprising angiogenesis, epithelialization and granuloma-
tous tissue formation (Nocchi, Björklund, Svensson, Engblom, &
Ruzgas, 2017). On the other hand, higher ROS level can lead to sever
cellular damage (Forman, Ursini, & Maiorino, 2014). So, the regulation
of ROS levels in wound site is essential and required for efficient repair.
Over production of ROS for an extended period is a substantial
cause for hindering wound healing process resulting in epithelial cell
deterioration, oxidative stress, and cytotoxicity that supports the ad-
vantage of the antioxidant agent for these symptoms (Bonferoni et al.,
2018; Pothireddy et al., 2016).
Silver nanocarriers induce its antioxidant activity through reduc-
tion, electron donating and radical scavenging abilities. So, silver na-
noparticles have been recognized as scavenger of ROS and can decrease
the amount of free radicals emerging through inflammatory stage of
wound healing (Gopukumar, Sana-Fathima, Alexander, Alex, &
Praseetha, 2016). They have the ability to cross the biological com-
partment with a greater rate than bulk silver ions since that it gained a
great interest in the reduction of wound bioburden (Wilkinson et al.,
2011). The anti-inflammatory activity of silver nanoparticles has been
reported by many researchers which helps in accelerating and adjust-
ment of wound healing process (Tian et al., 2007; Wong et al., 2009).
For the modulation of the healing process, diverse inflammatory med-
iators are produced within the wound area so that, the clarification of
both the pro- and anti-inflammatory cytokines is essential to protect the
newly regenerative tissue from damage caused by cytokines imbalance,
oxidants and antioxidants around the wound area (Eming, Krieg, &
Davidson, 2007). The successful tissue regeneration and wound repair
related with in vivo regulation of the inflammatory response as pro-
longed inflammatory response is among the causes of delaying wound
healing (Hafez, El-Aassar, Khalil, Al-Deyab, & Taha, 2011). Tian et al.
showed that the anti-inflammatory activity of silver nanoparticles is
mediated through cytokines modulation (Tian et al., 2007).
As previously mentioned in the above section that cytokines can
stimulate fibroblast and chondrocytes to produce ROS (Meier et al.,
1989). So, modulation of cytokines production by silver nanoparticles
can participate in lowering ROS levels to avoid sever cellular damage
and delay wound healing (Yu et al., 2016). In previous study the rate of
wound healing of rat group treated with collagen nanofiber im-
pregnated silver nanoparticles were faster than plain collagen nanofiber
with no silver nanoparticle. Histological examination showed an ac-
celerated collagen production, re-epithelization, and improved wound
contraction with silver nanoparticles composite collagen nanofibers.
These results were attributed to the anti-inflammatory and aseptic
2
Carbohydrate Polymers 238 (2020) 116175
conditions provided by silver nanoparticles that lead to reduced re-
active cell infiltration, which further supported the fibrous connective
tissue proliferation and sequential keratin layer regeneration (Rath,
Hussain, Chauhan, Garg, & Goyal, 2016).
Several methods in the literature, for the preparation of silver na-
noparticles have been disclosed extensively. One of the common and
cost-effective ways is the chemical reduction method (Abdel-Mohsen
et al., 2014; Abdelsalam et al., 2019; Abu-Saied, Abdel-Halim, Fouda, &
Al-Deyab, 2013; Dahlous, Abd-Elkader, Fouda, Al Othman, & El-Faham,
2019; El-Aassar, El Fawal, Kamoun, & Fouda, 2015; El-Aassar, Fouda, &
Kenawy, 2013; El-Aassar, Hafez, El-Deeb, & Fouda, 2014; El-Hamshary
et al., 2015; El-Naggar, Shaheen, Fouda, & Hebeish, 2016; El-Rafie
et al., 2011; Fouda et al., 2016; Fouda, El-Aassar, & Al-Deyab, 2013;
Fouda et al., 2015; Fouda, EL Naggar, Shaheen, & Al Deyab, 2014;
Hebeish, Abdel-Mohdy et al., 2011; Hebeish, El-Naggar et al., 2011; El-
Naggar et al., 2018; Přichystalová et al., 2014; Shaheen et al., 2016;
Textor, Fouda, & Mahltig, 2010). Silver nitrate is frequently being used
as a precursor for silver ions., in addition, natural and/or synthetic
polymer is being utilized this way in the reduction of silver ions, and in
the meantime, stabilize the formed silver nanoparticles as well.
Polygalacturonic acid (PGA) in this current study is being used as
natural candidate for reducing the silver ions into silver nanoparticles;
in the meantime, PGA is used as good stabilizer for the formed AgNPs
too. Polygalacturonic acid (PGA), which is namely sometimes, as Pectin
acid, is considered as natural occurring polymer, and it looks as
transparent, gelatinous acid present in mature fruits and some other
vegetables (Peng et al., 2011). It is formed by the degradation of pectin,
and is produced through the interaction between pectinase and pectin.
Hyaluronic acid (HA) is being used together with PGA in this current
work also to improve the healing process (Fouda et al., 2016). As
known, HA considered one of the best biomaterials based Poly-
saccharide utilized for wound care management particularly, due to its
biocompatibility, biodegradability, and atypical physicochemical fea-
tures. HA is naturally occurring, nonimmunogenic, anionic, non-sul-
fated Glycosaminogly can distributed broadly thru connective tissues,
epithelia, neural tissues, and synovial fluids. HA is highly soluble water
and more hydrophilic than any other natural occurring polymers,
therefore, it has a strong role in getting rid of lots of wound exudates,
hence, enhance the chance for wound healing as well (El-Aassar et al.,
2015).
So, the main objective in this current study is to develop a wound
dressing nanofiber mat, comprising natural biologically absorbable
materials; Hyaluronic, Polygalacturonic acid embedded silver nano-
particles for wound healing application; in-vivo. Silver nanoparticles in
this formula, will serve as an antioxidant and anti-inflammatory that
protect the cells from the destructive effect of elevated ROS and in the
meantime, accelerate wound healing process (Scheme 1).
2. Materials and methods
2.1. Materials
Polygalacturonic acid (PGA) was purchased from TCI Chemicals
(Tokyo, Japan). Silver nitrate (AgNO3) (99.9%), Hyaluronic acid (HA)
and Poly (Vinyl) Alcohol (PVA) were obtained from Sigma-Aldrich
(Schnelldorf, Germany). Microorganisms were collected from Protein
Department, Genetic Engineering and Technological Application
Institute (City of Scientific Research and Technological Application).
They were completely purified on LB media (Luria Bertani medium (LB)
and incubated at 37 °C for 24 h.
2.2. Preparation of silver nanoparticles-Polygalacturonic acid/Hyaluronic
acid/ poly (vinyl) alcohol (Ag-PGA/HA)-PVA antibacterial nanofiber
2.2.1. Synthesis of silver nanoparticles (AgNPs)
Polygalacturonic acid was dissolved in bi-distilled water to produce
M.R. El-Aassar, et al.
Scheme 1. Diagram illustrating the formation of (Ag-PGA/HA)-PVA.
1% (w/v) solutions. Complete dissolution was obtained by addition of
1 M NaOH (pH = 11.7) and magnetic stirring at room temperature for
30 min. Then 400 μl of 0.45 mM AgNO3 was added under magnetic
stirring for 1 h until yellow solution was obtained.
2.2.2. Preparation of Hyaluronic- Polygalacturonic acid/silver
nanoparticles (Ag-PGA/HA) blend solution
Blend solution was obtained by adding 0.25% (w/v) of HA to Ag-
PGA solution under continuous magnetic stirring and raising the tem-
perature till 50 °C for 1 h, until homogenous solution was produced.
This was followed by 24 h freeze drying.
2.2.3. Preparation of (Ag-PGA/HA)-PVA spinnable solution
The spinnable solution was prepared by dissolving PVA 8% (w/v) at
30 °C, and then 1% of Ag-PGA/HA dry powder was added. This solution
was left under magnetic stirring overnight till the solution became
homogeneous.
2.2.4. Electrospinning process
The prepared spinnable solution was inserted into syringe made of
glass having metal capillary needle (with inner diameter = 0.5 mm).
“The electrospinning setup (nanoNC needle laboratory machine, Model:
ESR100D, Seoul, Korea)” contained syringe pump and collector of
grounded plate coated with aluminum foil, as well as high voltage
power source was applied to the polymer formulation; (Ag-PGA/HA)-
PVA blend solution in a syringe thru an alligator clip adhered to the
syringe needle. The electrospinning was accomplished at room tem-
perature, and the working solutions were inserted from the syringe
pump with a feed rate of 1.3 ml/h. The distance between the needle tip
and the grounded collector plate was 17 cm, and the high voltage was
applied at 11 kV. Dry nanofibers were gathered directly from the alu-
minum foil and kept at room temperature.
2.3. Characterization
2.3.1. UV–vis spectroscopy
Silver nanoparticles optical properties and synthesis was verified by
using UV- (LAMBDA 650) range 300–600 nm. 1 ml of pure PGA was
used as blank before sample measurement. Synthesis was optimized by
observing AgNPs formation under several conditions; AgNO3 con-
centration (range: 0.12-0.45 mM) and Polygalacturonic Acid
3
Carbohydrate Polymers 238 (2020) 116175
concentration (range: 0.12–1% w/v).
2.3.2. Scanning (SEM) and transmission (TEM) electron microscopy
Nanofibers and Silver nanoparticles morphology were captured by
scanning electron microscope “(JEOL-JSM-6360LA-Japan)”, while
powder by transmission electron microscope by a “JEM-1010 unit
(JEOL Ltd., Tokyo, Japan)”, provided with an AMT XR-80 digital
camera (Advanced Microscopy Techniques Corp., Woburn, MA, USA).
Furthermore, Image J software was used to measure AgNPs mean
particle size, and particle distribution was indicated by polydispersity
index (PDI) according to the following formula (Clayton, Salameh,
Wereley, & Kinzer-Ursem, 2016):
2
Standard Deviation
Poly Dispersity Index (PDI) =
Mean Diameter (1)
2.3.3. Fourier transform infrared (FTIR) and Raman spectroscopy
The main changes in chemical composition of polymer blend were
verified by FTIR (Shimadzu FT-IR8400 S, Japan), by measuring spectral
range of 4000–400 cm−1 of the fine powder. Raman spectra of the
copolymer fine powder were obtained by Bruker FT-Raman
Spectrometer Multi RAM (Bruker Optics Inc. Billerica, MA USA) with
FT-Raman microscope – Raman scope III was used.
2.3.4. Thermogravimetric analysis (TGA)
The thermal degeneration attribute of nanofibers was verified by
thermogravimetric analyzer “(Shimadzu Thermal Gravimetric Analysis
(TGA)—50, Japan)”, that was achieved under nitrogen at a heating rate
of 10 °C/min until 600 °C, with starting weight of6 mg sample.
2.3.5. Tensile strength
Nanofibers mechanical properties were assessed by tensile tester
(model AG-I, Shidmadzu, Japan). Nanofiber with 6 mm × 1 mm di-
mensions were analyzed at a crosshead speed of 30 mm/min. Digital
micrometer (Mituto, Tokyo, Japan), with a precision of 0.001 mm was
used to measure nanofiber thickness. The average of five times was
obtained.
2.3.6. Water contact angle
Water contact angle of each nanofiber was assessed by measuring
their static contact angle (θ) (Ramé-Hart Instrument Goniometer, model
M.R. El-Aassar, et al.
Fig. 1. UV–vis absorption spectra of A. PGA concentration B. AgNO3 concentration effect on the preparation of Ag-PGA.
500-F1, France) by assigning a drop of ultra-pure water on it at three
different locations.
2.4. In-vitro antibacterial study
Nanofibers were cut into equal-sized pieces and sterilized by UV.
(Ag-PGA/HA)-PVA and (PGA/HA)-PVA were incubated with 100 μl of
Bacillus Subtilis, Staphylococcus Aureus and Escherichia Coli on agar
plates at 37 °C for 24 h (Fouda, Knittel, Hipler, Elsner, & Schollmeyer,
2006; Selvaraj, Thangam, & Fathima, 2018).
2.5. In-vivo study
All carried experiments on rodents were following guidelines and
protocols, and approved by the Research Ethical Committee of
Institutional Animal Care and Use Committee at Alexandria University
(ALEXU-IACUC) with approval number; AU14-190806-3-1.
2.5.1. Preparation of full thickness wound model
Twenty male rats 60 days old, with a mean weight of approximately
180 g were used. The animals were accommodated in a separate
birdcage at an orderly temperature (25 ± 3 °C; 35–60% humidity)
below a 12 h light/dark cycle and received a standard diet including
pelleted food “(Labina; Purina Nutrients Ltda, Sao Paulo-SP, Brazil)”
and mineral water ad libitum. This study was accepted by Local Ethical
Committee of Animal Research. The animals were counted, weighed
and then divided into four groups with five animals in each as follows;
group I: applied PGA/HA/PVA nanofiber, group II: Ag-PGA/HA/PVA
nanofiber, group III: vehicle control and applied commercial ointment
(Garamycin®) and group IV: vehicle control and applied saline.
As for surgical proceedings, the animals were subjecting to weight
calculation, followed by anesthetized using intramuscular administra-
tion of 10% Ketamine Hydrochloride “(Dopalen®, 0.1 ml/100 g body
weight)” and 2%xylazine hydrochloride “(Calmium®, 0.1 ml/100 g
body weight)”. After shaving, Antisepsis of the area was achieved with
4% alcohol-based iodine. In the center of the shaved area and with a
fine scissor, a surgical skin lesion (Ø = 2.5 cm), 1 cm below the bone
prominence on the back of the animals, was formed using a metallic
circular marker to delimit the area, and with the aid of a surgical
scalpel, the dorsal muscle fascia was exposed. The wound was cleaned
daily with alcohol, followed by topical application of treatments.
Finally, wounds were covered with sterile gauze and fixed with circular
adhesive bands and the wound areas were calculated (Kodati, Burra, &
Kumar, 2011; Rath, Johal, & Goyal, 2011).
4
Carbohydrate Polymers 238 (2020) 116175
2.5.2. Photo documentation and histological analysis
Starting immediately after surgery, images of the skin wounds at 3,
5, 8,10 and 14 days after surgery were captured with an optical digital
camera at a fixed focal distance (Abercrombie, Flint, & James, 1956).
Dressings were soaked with sterile saline until they could be removed
with minimal disturbance of the surface of the wound. From the healed
wound, a specimen sample of tissue is isolated from each group of rats
for histopathological examination at day 10 and day 14 (Biswas, Maity,
& Mukherjee, 2004). The animals were anesthetized using the anes-
thetic procedure described above, followed by intracardiac adminis-
tration of 0.5 ml of potassium chloride. Specimens of the wound area
were removed, fixed in 10% formalin, and sent for histopathological
examination and slide preparation according to routine procedures.
Slices of 5 μm from the skin pass through the center of the wound were
stained with hematoxylin-eosin (H&E) and picrosirius, and analyzed
under a polarized light microscope using the ACT-@U program for
histological examination and Masson’s trichrome staining (MTS) for
estimation of collagen deposition (Sarhan et al., 2016). The reduction
percentage in wound area was calculated from the following Eq. (2):
Healed Area
Wound Contraction %=
Total Area (2)
3. Results and discussion
3.1. UV–vis spectroscopy and TEM analysis of produced silver nanoparticles
PGA was used as a green synthesis platform of AgNPs. Because it is
very rich in carboxylic and hydroxylic groups that transfer electrons to
Ag+, resulting in Ag° nanoparticles. Therefore, PGA is acting as a re-
ducing agent for AgNPs synthesis. UV–vis spectroscopy was used to
confirm synthesis and optical response of colloidal AgNPs. PGA con-
centration is a limiting factor in the synthesis process of AgNPs. In
Fig. 1A, maximum UV–vis spectra were observed upon using 0.5–1%
PGA concentrations at wavelength of 415 nm. While lower spectra were
observed at 0.12-0.25% PGA concentrations at wavelength of 418 nm.
In Fig. 1B, 0.45 mM of AgNO3showed the highest spectra at wavelength
of 415 nm. While 0.25 mM and 0.12 mM AgNO3 concentrations showed
intermediate and lowest spectra at wavelengths of 412 nm and 427 nm,
respectively. The most predominant absorption band was 410–415 nm,
which confirmed successful production of AgNPs and suggested that the
produced particle size is ranging between 5–10 nm (Zahran, Ahmed, &
El-Rafie, 2014).
TEM was used for further characterization of synthesized Ag-PGA
powder. It displayed spherical silver particulates which were
M.R. El-Aassar, et al.
Fig. 2. (1) TEM image of A. Ag-PGA powder B. (Ag-PGA/HA)-PVA nanofiber, (2) SEM images of a. (Ag-PGA)-PVA b. (PGA/HA)-PVA c. (Ag-PGA/HA)-PVA na-
nofibers.
encapsulated within PGA with an average particle size of 8.6 nm con-
firming what has been shown by UV–vis (Fig. 2.1.A). Polydispersity
index was used to estimate stability and distribution of produced
AgNPs. Synthesized AgNPs had PDI = 0.18 which indicated successful
encapsulation and homogenous distribution of produced nanoparticles.
Therefore, UV–vis and TEM confirmed that PGA is an efficient reducing
and stabilizing agent for the synthesis of AgNPs.
3.2. Morphological analysis of nanofibers structure
Surface morphology and size of (Ag-PGA)-PVA, (PGA/HA)-PVA and
(Ag-PGA/HA)-PVA nanofibers were observed by using SEM. Fig. 2.2 is
showing that (Ag-PGA)-PVA nanofibers have an approximate diameter
of 244 nm, while (PGA/HA)-PVA and (Ag-PGA/HA)-PVA nanofibers
diameters are 320 nm and 326 nm, respectively. Upon comparing
average diameter of (Ag-PGA)-PVA nanofibers with (PGA/HA)-PVA and
(Ag-PGA/HA)-PVA nanofibers, 30% increase in diameter was observed.
This increase could be due to the addition hyaluronan content in PGA
solution, which led to the increased electrical charges on the surface of
liquid droplet. No prominent changes were observed in nanofibers
morphology. While TEM was used to observe AgNPs component in-
corporated within (Ag-PGA/HA)-PVA nanofibers. Fig. 2.1.B is showing
that AgNPs are homogenously distributed within (Ag-PGA/HA)-PVA
nanofiber. Therefore, SEM and TEM confirmed successful
5
Carbohydrate Polymers 238 (2020) 116175
electrospinning process of (PGA/HA)-PVA loaded with AgNPs.
3.3. FTIR and Raman powder analysis
FTIR and Raman spectra were used to identify characteristic peaks
and major changes occurred within polymers blend and AgNPs loading.
In Fig. 3A, infrared absorption spectra of PGA/HA were compared
without and with AgNPs by observing shifting in O-H and NeH
stretching peaks from 3446 cm−1 to 3566 cm-1, C–H stretching from
2935 cm-1 to 2941 cm-1, CeOeC from 1620 cm-1 to 1604 cm-1 and
CeOH bending from 1417 cm−1 to 1425 cm-1. Both PGA and HA have a
similar chemical structure and infrared spectra. However, PGA/HA and
Ag-PGA/HA were showing a more prominent transmittance % ranging
between 3136 cm-1 and 3639 cm-1 due to NeH stretching of HA com-
ponent. The Raman spectra of Ag-PGA, PGA/HA and Ag-PGA/HA
powders were also observed. In Fig. 3B, Ag-PGA and PGA/HA exhibited
characteristic peaks at 1452 cm-1 and 1119 cm-1due to the CH2 group of
PGA, and glycosidic bonds CeOeC present between the repeating units
from HA, respectively. The intensity ratio of the peaks at 1458 cm-1
varied with the mixing combination of PGA, HA and AgNPs. The Ag-
PGA/HA exhibited vibrational bands at 1458 cm-1 and 2912 cm-1which
can be assigned to the NH2 group and CH group, respectively. There-
fore, FTIR and Raman spectra confirmed PGA and HA successful
blending and AgNPs presence.
M.R. El-Aassar, et al.
Fig. 3. A. FTIR spectra B. Raman spectra of PGA/HA, Ag-PGA and Ag-PGA/HA powder.
3.4. Wettability, thermal and mechanical analysis of nanofibers
TGA was used to characterize thermal stability property of nanofi-
bers (Fig. 4A). AgNPs component did not alter thermal weight loss
pattern of (PGA/HA)-PVA. Both (PGA/HA)-PVA and (Ag-PGA/HA)-
PVA nanofibers underwent through three major steps of weight loss.
They underwent 16% weight loss at 25–215 °C stage, 31% loss at
216–310 °C stage and 14% final loss at 311–600 °C stage. On the other
hand, (Ag-PGA)-PVA underwent 6% weight loss at 25–215 °C 1st stage,
10% loss at 216–310 °C 2nd stage and 59% loss at 311–600 °C 3rd stage.
These variations in weight loss could be accounted to rapid rupture of
HA glycosidic bond content in both (PGA/HA)-PVA and (Ag-PGA/HA)-
PVA compared to (Ag-PGA)-PVA nanofiber.
Fig. 4B illustrates the mechanical properties of the (Ag-PGA)-PVA,
(PGA/HA)-PVA, and (Ag-PGA/HA)-PVA nanofibers. The tensile stress
of the (Ag-PGA)-PVA nanofibers (∼4.1 MPa) was almost two time
stronger than the (PGA/HA)-PVA (∼ 1.9 MPa), and (Ag-PGA/HA)-PVA
(∼1.6 MPa). However, the tensile strength of the (Ag-PGA/HA)-PVA
nanofibers (∼30%) was increased to 22.5, and 10% for (PGA/HA)-PVA
and (PGA/HA)-PVA, respectively. The (PGA/HA)-PVA nanofibers
showed a significant improvement in stress-strain curve, indicating that
their ability to resist network deformation was decreased with AgNPs
content increase. This improvement in stress-strain value of the nano-
fiber due to the HA structural that acts as additional crosslinking effect
from the physical interaction between hyaluronan chains, PGA, and
PVA chains.
The wettability of produced nanofibers was observed by measuring
contact angle (Fig. 4C). (Ag-PGA)-PVA, (PGA/HA)-PVA and (Ag-PGA
HA)-PVA nanofibers were exhibiting water contact angle of 67.9°, 59.2°
and 64.2°, respectively. (Ag-PGA HA)-PVA nanofibers are showing the
lowest contact angle and best hydrophilicity. This may be associated
Fig. 4. A. TGA results of the weight loss B. tensile strength and C. Wettability test by using a contact angle tester of (Ag-PGA)-PVA, (PGA/HA)-PVA, and (Ag-PGA/
HA)-PVA nanofibers.
6
Carbohydrate Polymers 238 (2020) 116175
with the fact that biopolymers with a higher content of amine-func-
tionalized hyaluronic acid induces higher values of contact angles were
observed.
3.5. Antibacterial effect
Fig. 5 is showing the antibacterial effect of (PGA/HA)-PVA and (Ag-
PGA/HA)-PVA nanofibers. It was obvious that (PGA/HA)-PVA nano-
fiber did not exhibit any zone inhibition/antibacterial effect. While
upon adding AgNPs, (Ag-PGA/HA)-PVA nanofibers became very ef-
fective against gram-positive bacterial strains; Bacillus Subtilis and Sta-
phylococcus Aureus, as well as gram-negative bacterial strain; Escherichia
Coli. The antibacterial effect of AgNPs might be due to disruption of
bacterial cell membrane proteins and interaction with DNA.
3.6. Wound healing effect and histopathological assessment
Fig. 6 displays the macroscopic appearance of the wounds during
the time course of the experiment. No sign of abscess formation or
hypertrophic scars both in the early phase; 5th day or in the final phase;
14th day, respectively, were observed. The digital images of the wounds
allowed the estimation of the wound area progression in the groups
during the time course of the experiment.
The trial shows that the contraction force is exerted by many factors
within the wounded area. The scab is responsible for a rapid and early
contraction in the initial stages which is probably due to simple dehy-
dration. After five days there was a further abrupt onset of contraction,
predominant in the prepared (Ag-PGA/HA)-PVA nanofiber and blank
(PGA/HA)-PVA nanofiber over others. This can be related to the de-
creased capillary permeability and dehydration of the wound bed. With
the prepared nanofiber the contraction proceeds faster until epithelial
M.R. El-Aassar, et al. Carbohydrate Polymers 238 (2020) 116175

Fig. 5. Antibacterial effect of (PGA/HA)-PVA and (Ag-PGA/HA)-PVA nanofibers on Bacillus Subtilis, Staphylococcus Aureus and Escherichia Coli.

closure is nearly complete. The wound contraction is depicted in Fig. 6,
and is showing that the wound contracting ability of (Ag-PGA/HA)-PVA
nanofiber and blank (PGA/HA)-PVA nanofiber treated groups showed
significant wound healing from day 8 onwards and the epithelization
period was on day 14. Whereas in the Garamycin® cream and control
group (applied saline) it took more than 14 days (Fig. 6).
The results of excision wound model and % of wound contraction
are illustrated in Table 1. Both the (Ag-PGA/HA)-PVA nanofiber and
(PGA/HA)-PVA nanofiber treated groups exhibited significant wound
healing activity as compared to other groups that treated with Gar-
amycin® cream and control group (applied saline) in excision wound
model (Fig. 6).
The inflammation is the self defense mechanism of the body to re-
move critical stimuli, including irritants, pathogens or damaged cells
during the healing process. Many cells are involved directly or in-
directly through production of inflammatory cytokine in the pathology
of chronic inflammation including macrophages, eosinophils and neu-
trophils (Khansari, Shakiba, & Mahmoudi, 2009). The histopathological

Fig. 6. Macroscopic examination of the wounds treated with (PGA/HA)-PVA nanofiber (Group I), (Ag-PGA/HA)-PVA nanofibers (Group II), Garamycin® cream;
positive control (Group III) and saline; negative control (Group IV) for 14 days.

7
M.R. El-Aassar, et al. Carbohydrate Polymers 238 (2020) 116175

Table 1
Evaluation of the wound area cm2 and wound contraction % of different groups over a period of 14 days.
Observation Days Wound Area (cm2) (Mean ± SEM) and Percentage of Wound Contraction

Group I Group II Group III Group IV

Area (cm2) % contraction Area (cm2) % contraction Area (cm2) % contraction Area (cm2) % contraction

0 5 ± 0.46 Nil 4.8 ± 0.5 Nil 5.15 ± 0.4 Nil 4.8 ± 0.3 Nil
3 2.9 ± 0.8 41.85 1.6 ± 0.2 66.95 3.40 ± 0.59 33.99 3.8 ± 0.3 19.47
5 2.2 ± 0.67 55.85 0.91 ± 0.16 81.14 2.35 ± 0.45 54.33 3.1 ± 0.2 34.94
8 1.26 ± 0.52 75.08 0.36 ± 0.10 92.49 1.89 ± 0.31 63.27 2.3 ± 0.25 51.55
10 0.59 ± 0.27 88.31 0.18 ± 0.07 96.23 0.84 ± 0.32 83.50 1.54 ± 0.17 68.08
14 0.13 ± 0.062 97.42 0.02 ± 0.01 99.51 0.38 ± 92.43 0.79 ± 0.15 83.65

Fig. 7. A. Histological analysis of treated wounds on day 10 and 14 using H and E stain. Epi: Epidermis; K: keratin layer; BV: blood vessels; HF: hair follicles; SG:
sebaceous glands; U: ulcer; M: muscle B. Histopathological evaluation of treated wounds using Masson’s trichome Stain (MTS) on day 10 and 14 (original magni-
fication = 100).

8
M.R. El-Aassar, et al.
Fig. 8. Histological analysis of normal skin using hematoxylin-eosin (H&E) staining (A) and Masson’s trichome (MTS) Stain (B).
examination to the injured skin area showed in Fig. 7 was used to il-
lustrate the possible mechanism of wound contraction in Fig. 6 com-
pared to normal skin described in Fig. 8.
Fig. 7 A is showing all images of the histopathological examination
using H&E staining of the wounded skin sections from different groups
at day 10 and 14 respectively compared to the normal skin section
Fig. 8A. The normal skin section showed the complete structural ele-
ments of the healthy skin including intact epidermis (external epithe-
lium formed of 2–3 cell layers), dermis (layer of connective tissue),
sebaceous glands and hair follicles as illustrated in Fig. 8A.
These groups showed a varying response to the treatment at which
the best wound healing ability and complete epithelization was re-
corded for group II; (Ag-PGA/HA)-PVA nanofiber followed by group I;
applied blank (PGA/HA)-PVA nanofiber. The wound area showed no
ulcer with almost normal skin that filled with dense connective tissues
and was surrounded by a new dermal layer on both day 10 and 14
(Fig. 7A). Well-formed epidermis (external epithelium formed of 2–3
cell layers) and dermis (layer of connective tissue) were seen in the
tissues. The skin sections also showed the most basic structures of the
rat's skin like hair follicle (HF), sebaceous gland (SG) and blood vessels
(BV). Previous researches demonstrated the fundamental roles played
by the angiogenesis in the regeneration of both epidermis (EPI) and
dermis containing hair follicles (Gharibi, Yeganeh, Rezapour-Lactoee, &
Hassan, 2015; Jhong et al., 2014). The faster healing and epithelization
could be attributed to the promising wound healing action of (HA)
(Sabra, Ragab, Agwa, & Rohani, 2020).
On the other hand, the rat's skin section given group III; Garamycin®
cream showed a reduced size ulcer area on day 10 with early healing as
thin squamous epithelial layer and marked infiltration of both epi-
dermis and dermis with polymorph neutrophils (PMNLs), inflammatory
cells, more fibroblasts and blood vessels with absence of sebaceous
glands and hair follicles (Fig. 7A). On day 14 the skin showed no ulcer
characterized by re-epithelialization with highly distinct epidermis
(Epi) (external epithelium formed of 2–3 cell layers), and dermis (layer
of connective tissue represented by red arrow). The complete skin re-
generation could be clarified by the presence of many blood vessels and
the necessary cells like hair follicle and sebaceous gland. On the con-
trary the skin rat group that applied saline (group IV) showed a greater
ulcer width along with many invasive inflammatory cells, numerous
polymorph neutrophils (PMNLs), granulation tissues and absence of
both sebaceous glands and intact hair follicles at both day 10 and 14.
From the previous results it is clear that the histopathological ex-
aminations for the rat skin are compatible with the results of photo
documentary analysis for wound healing assessment. Accumulation of
9
Carbohydrate Polymers 238 (2020) 116175
inflammatory cells/neutrophils was high in control when compared
with treated group as inflammatory cells were reduced due to the
strong antimicrobial activity of silver nanoparticles in the open wound
site.
For estimation and analysis of the intensity of the deposition of
collagen fibers Masson’s trichrome (MTS) stain was used as collagen
synthesis is an essential step to ensure complete wound healing
(Fig. 7B). Collagen was observed as blue violet color. The stain was
successfully applied to estimate the collagen deposition progress during
the formation of granulation tissue and matrix remolding. The intensity
of the blue stain equivalent to the relative amount of total collagen fiber
deposited. The normal skin showed uniform, mature, compact and
normal collagen deposition as illustrated in Fig. 8B. Histopathological
evaluation of the skin sections using Masson’s trichome stained tissues
of wound treated groups are illustrated in Fig. 7B. On both 10th and 14th
day, the skin group treated with saline (group IV) showed very much
less collagen deposition compared to other treated groups and normal
skin. The skin section from group treated with Garamycin® cream
(group III) showed a very less collagen deposition on day 10 whereas on
day 14 the deposited collagen was moderate in the granulation tissue.
Skin tissue section treated with blank (PGA/HA)-PVA nanofiber (group
I) showed a moderate collagen deposition in the granulation tissue on
day 10 which increased to become more compact and mature on day
14. While the skin tissue section treated with (Ag-PGA/HA)-PVA na-
nofiber (group II) showed more compact, dense and matured collagen
fiber deposit parallel to the epidermis which mimic the normal collagen
deposition on normal skin on both day 10 and 14. These results in-
dicated that among the all treated groups (Ag-PGA/HA)-PVA nanofiber
(group II) exhibited a higher degree of collagen deposition followed by
blank (PGA/HA)-PVA nanofiber (group I).
4. Conclusion and perspectives
This research is elucidating the biomedical activity of a novel
electrospun Polygalacturonic/Hyaluronic acid nanofiber loaded with
AgNPs. Successful AgNPs synthesis by PGA was verified by UV–vis
spectra ranging between 410–415 nm and TEM image of 8.6 nm
average particulate diameter. SEM confirmed the electrospinning of
(Ag-PGA/HA)-PVA in nanoscale with an average diameter of 326 nm.
AgNPs component of (Ag-PGA/HA)-PVA nanofiber exhibited a robust
zone inhibition antibacterial activity against gram + ve and gram -ve
bacteria. While HA component was responsible for the enhanced hy-
drophilicity and strain activities. Additionally, the in-vivo study in al-
bino rat had shown maximum wound epithelization and collagen
M.R. El-Aassar, et al.
deposition after 14 days of nanofiber administration. Therefore, we are
offering an efficient nanofiber for a quick healing of wound infections.
Both the cellular and molecular mechanisms perform a major role in
the healing process. The healing quality is greatly governed by reg-
ulation of gene expression through healing. During different healing
stages, a marked change in the level of the gene expression takes place.
The most important genes involved in wound healing process includes
vascular endothelial growth factor (VEGF), transforming growth factor
beta (TGF-β) family, epidermal growth factor (EGF) family, interleukin
(IL) family, and tumor necrosis factor-α family (TNF-α). At the late
stage of healing the EGF family, TGF-β and VEGF are over expressed or
upregulated on contrary to TNF-α and IL family which is down regu-
lated. In the future its expected to study the molecular mechanism of
the healing process by estimating the level gene expression of the above
mentioned biomarkers quantitively by real time PCR in the skin of the
rat at the wound area at different healing stages during the healing
process after treatment with our developed nanofiber.
CRediT authorship contribution statement
M.R. El-Aassar: Conceptualization, Methodology, Funding acqui-
sition, Writing - original draft. Omar M. Ibrahim: Data curation,
Formal analysis, Investigation, Writing - review & editing. Moustafa
M.G. Fouda: Conceptualization, Methodology, Funding acquisition,
Writing - original draft. Nagham G. El-Beheri: Data curation, Formal
analysis, Investigation, Writing - review & editing. Mona M. Agwa:
Data curation, Formal analysis, Investigation, Writing - review &
editing.
Declaration of Competing Interest
Authors declare that no conflict of interest.
Acknowledgements
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at Jouf University for funding the work
through the research group project No. 40/18.
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