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Carbohydrate Polymers
Volume 324, 15 January 2024, 121526
Wound healing
potential of silver
nanoparticles
embedded in
optimized bio-inspired
hybridized chitosan
soft and dry hydrogel
Roxane Abdel-Gawad , Rihab Osman,
Gehanne A.S. Awad, Nahed Mortada
Department of Pharmaceutics and
Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, P.O. Box 11566, Cairo,
Egypt
Received 30 August 2023, Revised 15 October 2023,
Accepted 23 October 2023, Available online 24
October 2023, Version of Record 8 November 2023.
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Abstract
Interactive wound dressings combining
healing and antimicrobial potentials, besides
ensuring patient compliance with a
recognized wound care service gained
considerable interest recently. Both hydrogel
spray dried microparticles (HMP) and soft
hydrogel (G) were prepared. The bio-inspired
combinatory platform included natural bio-
macromolecules namely: chitosan (CS) and
collagen (COL) with wound healing
enhancement and connective tissue building
capabilities cross linked with the natural
genipin (GN) to build a three dimensional
structured matrix. The optimized plain
hydrogel obtained by a box behnken design
(BBD) program (G) scored maximum swelling
and porosity. The network was hosted with
green synthesized cefotaxime sodium (cef.Na)
AgNPs reduced by the anabolic folic acid (FA).
Both hydrogels exhibited good antimicrobial
activity against gram +ve and –ve bacteria. The
wound healing activity, evaluated in injured
rats, showed >98 % and complete wound
closure after two and three weeks
respectively. Oxidative stress minimization
was proved by the estimation of biochemical
markers malondialdehyde (MDA) and
superoxide dismutase (SOD) levels at the
wound site.
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Introduction
Developing novel interactive wound care
products that can achieve clean healing with
complete restoration of skin function with
minimal scaring in a short time frame had
been recently the focus of many research
works. Building such products with natural
bio-inspired macromolecules and cross-
linkers will improve their safety and
tolerability while guaranteeing sustainability.
Chitosan (CS) is a poly-cationic carbohydrate
bio-macromolecule composed of randomly
distributed β-(1–4)-D-glucosamine and N-
acetyl-D-glucosamine. It is a biocompatible,
biodegradable, non-immunogenic and non-
carcinogenic biopolymer. The monomeric unit
of CS, N-acetyl glucosamine, promotes wound
healing by stimulating hemostasis and
accelerating tissue regeneration. Also its
cationic nature offers an antimicrobial activity,
interacting with bacterial cell wall leading to
leakage of cytoplasmic contents and
microorganism death (Üstündağ Okur et al.,
2019). CS is thus a suitable candidate for
biomedical applications, such as tissue
engineering and drug delivery vehicle, for
sutures and wound healing dressings, skin
culture, vascular grafting and vaccine delivery
(Mi et al., 2003; Lewandowska et al., 2017;
Feng et al., 2021).
CS based wound dressing suffer from several
disadvantages, including low CS water
solubility, moderate antibacterial activity and
poor mechanical properties calling for the
urgency of cross-linkers, other polymers and
antibiotics incorporation (Wang et al., 2020;
Feng et al., 2021; Elangwe et al., 2022). Recent
researches have been combining CS with other
polymers producing combinatory platforms,
as with collagen, hyaluronic acid, xanthan
gum, grapheme oxide, … (Anisha et al., 2013;
Malik et al., 2020; Mahmoodi et al., 2021;
Yang et al., 2021).
Hydrogels made of hydrophilic polymers have
been regarded as a good contestant in tackling
the demanding challenge of providing moist
but bacteria-free environment with high
adhesiveness, elasticity, and durability
(Üstündağ Okur et al., 2019). They can also
achieve rehydration of non-viable tissue
facilitating the process of natural autolysis
(Jones et al., 2006). They present low
interfacial tension, high molecular and oxygen
permeability, good mechanical properties that
resemble physiological soft tissue (Üstündağ
Okur et al., 2019). Hydrogels are generally
transparent, offering the possibility for easy
wound monitoring and assessment, besides
protecting the wound bed from impact and
abrasion. They can be also be designed to be
multifunctional by introducing variable
functional aspects including antimicrobials,
cells, anti-inflammatory agents, antioxidants,
pro-angiogenics etc.…..(Niculescu &
Grumezescu, 2022). This study focuses on a
hydrogel system offering synergized acting
components, promoting a fast wound healing
especially on burns.
Collagen (COL), group of naturally occurring
proteins, is the most preferred biomaterial in
tissue engineering due to its biocompatibility,
biodegradability, low antigenicity and good
homoeostatic properties. It is the main
component of the connective tissue,
maintaining their structural integrity and
providing the hardness, stiffness and elasticity
of tissues (Shah et al., 2019). It has a vital role
in the natural wound healing process; starting
from the initial induction of clotting to the
formation and appearance of the final scar. It
stimulates formation of fibroblasts and
accelerates the migration of endothelial cells.
Recently, freeze-dried COL bio-matrices have
been utilized because of their ability to pick
up fluid, debris and inflammatory cells
containing phagocytosed bacteria. The major
disadvantages of COL-based biomaterials are
the rapid degradation and poor mechanical
properties which might hinder their use in
several tissue engineering applications.
Biopolymer based hydrogels with enhanced
mechanical strength and retarded dissolution
are prepared by cross linking either ionically
or covalently. The cross-linkers can be either
natural or synthetic, with the preference of
the former due to lesser or negligible toxicity
(Nayak et al., 2019). Genipin (GN) is an iridoid
glucoside extracted from Gardenia jasminoides
Ellis fruits (Chung et al., 2018). It is commonly
manufactured either by direct chemical
procedures using β-glucosidase enzyme or by
a microbiological process. Penicillium nigricans
produces ß-glucosidase, which in turn
hydrolyzes geniposide into aglycone genipin
active form (Dimida et al., 2015). In recent
years, GN has drawn considerable research
interests as a cross-linker in biomedical
applications (Chung et al., 2018). It provides
beneficial activity for wound products by
stimulating the peripheral nerve regeneration
allowing the perception of pain to prevent
chronic wounds incidence (Mekhail et al.,
2014).
This work suggests a combinatory platform
based on natural polymer components CS and
COL cross-linked with GN and loaded with a
novel greenly synthesized AgNPs loaded with
cef.Na, broad spectrum antibiotic. AgNPs were
prepared with the anabolic folic acid (FA) as
reducing agent to promote connective tissue
regeneration, fibroblast migration, and
keratinocyte proliferation (Rembe et al., 2018).
Synergistically, AgNPs, cef.Na, and CS will
enhance the microbial antibacterial activity
and prevent the development of antibiotic
resistance by the bacterial strains (Afridi et al.,
2021; Haji et al., 2022).
In spite of their unlimited advantages,
hydrogels are obsolete in highly exuding
wounds (severe burns) and gangrenous
wounds (diabetic foot) which call for dry
hydrogels, that absorb exudate and swell to
gain a gel structure with higher water
absorbing capabilities. They also offer the ease
of application even in inaccessible wounds
and long shelf life stability. Freeze dried
hydrogels are known as “Cryogel”, drying at
ambient air and oven produces “Xerogel” and
the supercritical assisted atomization forms
“Aerogel” (Auriemma et al., 2020). Recently,
spray drying was used to produce hydrogel
microparticles (HMP) or hydrogel
microspheres (Wei et al., 2020).
In this work, we aim at developing a bio-
inspired sustainable medicated hydrogel and
HMP based on cross-linked CS/COL
combinatory bio-macromolecules. The
mechanical strength and antimicrobial activity
of the gels will be modulated using GN natural
cross linker and cef.Na-AgNPs, respectively, to
provide a sustainable wound healing gel and
gel precursor. The dry and wet hydrogels were
evaluated for their physical properties and
pharmacodynamic activity to ensure effective
clean wound healing with complete
restoration of skin function and minimal
scaring.
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Section snippets
Materials
Chitosan (CS) 40,000 kDa, DD 75 %, Sigma-
Aldrich, UK. Dialysis tubing cellulose
membrane molecular weight cut-off: 14,000
and Hematoxylin & Eosin stain (H&E): Sigma-
Aldrich, UK. Collagen (COL): hydrolyzed
powder, Allied Chemical Group, Egypt. Ethyl
alcohol 96 %: International Company for
Supportive Medical Industries (ALAMIA), 10th
of Ramadan city, Egypt. Genipin (GN): Bio-
Tech Co., Ltd. China. Glacial acetic acid: El Nasr
Pharmaceutical Chemicals (ADWIC) Abou
Zaabal, Cairo, Egypt. Ultra-pure …
Box Behnken design
The complex effect of mixing different
concentrations of the bio-macromolecules
building the hydrogel network on its
properties was studied according to a 33
design using BBD. The optimum quality
attributes for a wound product is to absorb
excess exudate while keeping the
environment humid enough to preserve the
cell viability (Hasan et al., 2021). Accordingly,
S% will reflect the hydrophilicity of the
hydrogel's network and P% related to the
degree of crosslinking will affect the oxygen …
Conclusions
Cef.Na loaded green synthesized AgNPs were
embedded into the 3-dimentional structure of
a bio-inspired natural hydrogel. This hydrogel
components: CS and COL, contributed in
harmony with each other to prevent any
microbial infiltration at the wound site (CS
antimicrobial activity), and enhanced
connective tissue build up (COL). These
natural polymers were cross-linked with a
natural safe cross-linker (GN) forming a
hydrogel able to release its antimicrobial
payload in a constant manner, with …
Future prospective
The medicated G-cef.Na-AgNPs bio inspired
hydrogel and HMP in situ forming hydrogel
loaded with greenly synthesized AgNPs are
good candidate for human trial experiments.
After the approval of regulatory authorities,
excellent clinical results are expected in case
of severe burns and exuding wounds. …
CRediT authorship contribution
statement
1. Roxane Abdel-Gawad (Teaching
Assistant of Pharmaceutics and
Industrial Pharmacy, Faculty of
Pharmacy, Ain Shams University):
contributed to the design and
implementation of the research;
collected the data; contributed data or
analysis tools; performed the analysis;
wrote the paper. …
2. Rihab Osman (Professor of
Pharmaceutics and Industrial
Pharmacy, Faculty of Pharmacy, Ain
Shams University): contributed to the
design and implementation of the
research; wrote the paper; provided
critical feedback …
Declaration of competing interest
The authors have no known competing
financial interests or personal relationships
that could have appeared to influence the
work reported in this paper. …
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