Saudi Pharmaceutical Journal 31 (2023) 462–471

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Original article

Potential benefits of using chitosan and silk fibroin topical hydrogel for
managing wound healing and coagulation
Soumya Narayana a,⇑, Arfa Nasrine a, Mohammed Gulzar Ahmed b, Rokeya Sultana c, B.H. Jaswanth Gowda a,
Suprith Surya d, Mansour Almuqbil e, Syed Mohammed Basheeruddin Asdaq f,⇑, Sultan Alshehri g,
Syed Arif Hussain h
a
Department of Pharmaceutics, Yenepoya Pharmacy College & Research Centre, Yenepoya (Deemed to be University), Mangalore 575018, India
b
Department of Pharmaceutics, Yenepoya Pharmacy College & Research Centre, Yenepoya (Deemed to be University), Mangalore 575018, India
c
Department of Pharmacognosy, Yenepoya Pharmacy College & Research Centre, Yenepoya (Deemed to be University), Mangalore 575018, India
d
Veterinary Surgeon and in charge Advanced Surgical Skill Enhancement Division (ASSEND), Yenepoya (Deemed to be University), Mangalore 575018, India
e
Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
f
Department of Pharmacy Practice, College of Pharmacy, AlMaarefa University, Dariyah, 13713, Riyadh, Saudi Arabia
g
Department of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Ad Diriyah 13713, Saudi Arabia
h
Respiratory Care Department, College of Applied Sciences, AlMaarefa University, Dariyah 13713, Riyadh, Saudi Arabia

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

Article history: Background & Objectives: The intricate process of wound healing involves replacing the cellular or tissue
Received 6 January 2023 structure that has been destroyed. In recent years various wound dressings were launched but reported
Accepted 27 January 2023 several limitations. The topical gel preparations are intended for certain skin wound conditions for local
Available online 1 February 2023
action. Chitosan-based hemostatic materials are the most effective in halting acute hemorrhage, and nat-
urally occurring silk fibroin is widely utilized for tissue regeneration. So, this study was conducted to
Keywords: evaluate the potential of chitosan hydrogel(CHI-HYD) and chitosan silk fibroin hydrogel (CHI-SF-HYD)
Topical hydrogel
on blood clotting and wound healing.
Blood clotting
Wound healing
Methods: Hydrogel was prepared using various concentrations of silk fibroin with guar gum as a gelling
Chitosan agent. The optimized formulations were evaluated for visual appearance, Fourier transforms infrared
Silk fibroin spectroscopy (FT-IR), pH, spreadability, viscosity, antimicrobial activity, HR-TEM analysis, ex vivo skin
permeation, skin irritation, stability studies, and in vivo studies by using adult male Wistar albino rats.
Results: Based on the outcome of FT-IR, no chemical interaction between the components was noticed.
The developed hydrogels exhibited a viscosity of 79.2 ± 4.2 Pa.s (CHI-HYD), 79.8 ± 3.8 Pa.s (CHI-SF-
HYD), and pH of 5.87 ± 0.2 (CHI-HYD), 5.96 ± 0.1 (CHI-SF-HYD). The prepared hydrogels were sterile
and non-irritant to the skin. The in vivo study outcomes show that the CHI-SF-HYD treated group has
significantly shortened the span of tissue reformation than other groups. This demonstrated that the
CHI-SF-HYD could consequently accelerate the regeneration of the damaged area.
Interpretation & Conclusion: Overall, the positive outcomes revealed improved blood coagulation and re-
epithelialization. This indicates that the CHI-SF-HYD could be used to develop novel wound-healing devices.
Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding authors at: Department of Pharmacy Practice, College of Pharmacy, AlMaarefa University, Dariyah, 13713, Riyadh, Saudi Arabia (S.M.B. Asdaq); Yenepoya
Pharmacy College and Research CentreYenepoya (Deemed to be University)Deralakatte-575018 Mangalore, Karnataka, India (S.Narayana).
E-mail addresses: kalikollur123@gmail.com (S. Narayana), arfanasrine14@gmail.com (A. Nasrine), mohammedgulzar1@gmail.com (M. Gulzar Ahmed), rokeya009ster@
gmail.com (R. Sultana), jashgowda20@gmail.com (B.H. Jaswanth Gowda), suprithsurya@gmail.com (S. Surya), mmetwazi@ksu.edu.sa (M. Almuqbil), sasdaq@gmail.com,
sasdag@mcst.edu.sa (S.M.B. Asdaq), sshehri.c@mcst.edu.sa (S. Alshehri), pulmoarif@gmail.com (S. Arif Hussain).
Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

https://doi.org/10.1016/j.jsps.2023.01.013
1319-0164/Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Narayana, A. Nasrine, M. Gulzar Ahmed et al. Saudi Pharmaceutical Journal 31 (2023) 462–471

1. Introduction 2. Methods

Cell proliferation occurs due to the stimulation of growth fac- 2.1. Materials
tors that will help tissue repair by involving various changes, such
as the multiplication of parenchymal cells and the production of an The supplier of chitosan was Yarrow Chem Products in Mumbai.
extracellular matrix (Shaw and Martin, 2009, Eming et al., 2007). We received a sample of silk fibroin as a gift from Sericare in Ban-
The interruption in the wound healing mechanism occurs due to galore. The supplier of guar gum was Loba Chemicals in Mumbai.
some modifications in the process of coagulation. The thrombin Finally, acetic acid was bought from Hyderabad’s Avra Synthesis
and fibrin formations are induced when the sub-endothelium of Pvt ltd. Without additional purification, analytical-grade chemicals
injured blood vessels and capillaries are accumulated by platelets, were utilized for all other substances.
leading to the wound’s healing by coagulation (Gowda et al.,
2023a; Hoffman et al., 2006). 2.2. Procurement and maintenance of animals
Further, the fibrin network establishes a framework for the
subsequent process, which also balances out the underlying The Institutional Animal Ethics Committee, Yenepoya Univer-
platelet plug (Drew et al., 2001). In this manner, the underlying sity, Deralakatte, Mangalore, approved the study protocol (YU/
fibrin is used as a framework by fibroblasts that come in IAEC/15/2019, dated 09/11/2019). Animals were procured from
contact with the injury bed. Angiogenesis will develop new Liveon Biolabs, Tumakuru, Karnataka (1610/ROBiBt/S/2012/
blood vessels (Hadjipanayiet al., 2015). Throughout this process, CPCSEA). Healthy male Wistar albino rats weighing 150–200 g
the keratinocytes will move and multiply over the injury site to were used in the study. They were housed in ventilated cages in
restore the skin. (Piipponen et al., 2020, McDonald et al., 2007). maintained conditions (25C, 12 h light and dark cycles), having
Many formulations are available for wound healing, such as access to regular rat pellets and water ad libitum. All the animal
ointments, creams, lotions, etc. However, the hydrogel formula- experiments were performed following the guidelines of the Com-
tion is gaining attention nowadays due to its advantages over mittee for Control and Supervision of Experiments on Animals
others, such as bioadhesion, biocompatibility, better spreadabil- (CPCSEA).
ity with sustained drug-releasing ability, etc. Hydrogels are a
three-dimensional, cross-linked network of polymers, and water 2.3. Formulation of chitosan hydrogels and chitosan silk-fibroin topical
penetrates these networks, causing swelling and giving the hydrogels
hydrogel a soft and rubbery consistency but will not dissolve
(Monica and Gautami, 2014). 2.3.1. Chitosan hydrogels (CHI-HYD)
Chitosan-based hemostatic materials are the most effective in Overnight soaked chitosan in 40 mL of water and 1 mL acetic
halting acute hemorrhages. It is a larger molecular weight copoly- acid was stirred well using a digital magnetic stirrer at 250 rpm
mer that bears glucosamine radicals and acetyl glucosamine in its for one hour. Then guar gum was added in small aliquots with con-
structure that forms viscous polyelectrolytes upon dissolution in tinuous stirring to avoid the formation of lumps (500–600 rpm).
inorganic and organic acids. Chitosan is widely used for its various Then 0.1 g of sodium benzoate was added and continued stirring
advantages, such as biodegradability and biocompatibility. for another 30 min.
Recently many studies have also shown its ability to act as an
anti-bacterial and bind to LDL to convert them into HDL, which fur- 2.3.2. Chitosan silk fibroin hydrogel (CHI-SF-HYD)
ther helps to reduce the risks associated with hyperlipidemia. It To prepare CHI-SF-HYD, the same steps were followed until the
was also found that it can be a good procoagulant (Hattori et al., addition of guar gum (Table 1). Then silk-fibroin (0.3 g) was added
2017). Chitosan adheres to the erythrocytes and encourages plate- slowly with continuous stirring. Then sodium benzoate 0.1 g was
let adhesion, activation, and aggregation at the injured site. It leads added and stirred for another 30 min (Misal et al., 2012).
to the activation of coagulation factors and achieves hemostasis
(Stricker-Krongrad et al., 2018). 2.4. Evaluation
Silk is a natural protein polymer extracted from mulberry silk
by removing the outer silk sericin. USFDA also approves it for wide 2.4.1. FT-IR spectroscopy
medical applications (Nasrine et al., 2022). The silk may produce FT-IR studies were performed using CHI, SF, guar gum (GG), and
health benefits associated with fibroin (Altman et al., 2003). Along- CHI-SF HYD formulation with an FT-IR spectrophotometer (Shi-
side this, silk-fibroin involves multiple advantages such as biocom- madzu, Japan). IR spectral analyses were used to examine the
patibility, biodegradability, low immunogenicity, etc. Thus, it is
widely utilized as a medicine for tissue engineering and regenera-
Table 1
tive medicine, for example, cartilage, cornea, and bone repair. Also,
Composition of hydrogels.
its hemostatic ability could be a potent biomaterial for skin-
repairing activity (Chen and Liu, 2016, Gil et al., 2013). The studies INGREDIENTS FORMULATION CODE
also indicated that SF is non-harmful under acute dermal toxicity CHI-HYD CHI-SF-HYD
(Padol et al., 2011). Chitosan 0.5 g 0.5 g
Here, we have developed chitosan hydrogels (CHI-HYD) and Acetic acid (1 %) 1 mL 1 mL
chitosan silk fibroin hydrogels (CHI-SF-HYD) as novel drug delivery Guar gum 0.5 g 0.5 g
Silk-fibroin – 0.3 g
systems for effective wound healing treatment. Further evaluated
Sodium benzoate 0.1 g 0.1 g
for coagulation and wound healing properties to determine the Deionized water q.s q.s
potential of combinational polymers in tissue regeneration.

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S. Narayana, A. Nasrine, M. Gulzar Ahmed et al.
interaction between the API and pharmacological excipients by
looking for any change in the peaks. The sample was put immedi-
ately beneath the probe, to which it was securely fastened. The
search was then scanned in the 4000–500 cm
1 wavenumber
range. Functional groups at each wavenumber were identified
from the collected IR spectra (cm
1) (KumirskaJ et al., 2010;
Negrea et al., 2015; Sanjana and Ahmeda, 2021a).
2.4.2. Thermal analysis
To clarify any interactions between chitosan and the other poly-
mers utilized in the study, thermal analysis using differential scan-
ning calorimetry (DSC) was conducted. Shimadzu’s DSC-60 Plus,
made in Japan, was used for DSC. The instrument was built with
components from the Japanese Shimadzu Corporation, including
a thermal analyzer (TA 60), a flow controller (FCL 60), a calorimeter
(DSC 60), and operational software (TA-60 WS). The samples were
placed in a sealed aluminum pan and heated from 30 to 300 °C at a
15 °C/min scanning rate while under nitrogen flow (30 mL/min).
DSC thermograms were then determined for test samples
(Narayana et al., 2022a, Mudgil et al., 2012).
2.4.3. Organoleptic properties
Tests were done on the hydrogel formulations for appearance,
texture, etc. Visual observation was used to evaluate the hydrogel’s
appearance. The texture was evaluated by pressing a small amount
of the prepared gels between the thumb and index finger. To assess
the surface, the formulation’s uniformity and the presence of large
particles were considered (Chen et al., 2016).
2.4.4. Spreadability test
Applying the gel on a flat surface and looking for grainy hydro-
gel properties can help determine spreadability. For example, on a
glass plate, the 0.5 g of produced gel was spread over a 2 cm diam-
eter circle that had been marked. Then, half the weight of the gel
was allowed to rest on the upper glass plate for five minutes. The
method was carried out three times, and the circle’s diameter after
the gels had been disseminated was measured (Sanjana et al.,
2021b, Al-Suwayehet al., 2014).
2.4.5. Determination of pH and viscosity
The formulation’s pH was measured using a digital pH meter
after the weighed amount of gel (1 g) was dissolved in 25 mL of
distilled water. The pH analysis was carried out in triplicates for
both formulations (Kumar et al., 2018). The viscosity of the gel for-
mulations was then measured using a Brookfield viscometer at a
temperature of 25 °C and spindle number 7 at 100 rpm (Sanjana
et al., 2022c, Monica et al., 2014, Misal et al., 2012).
2.5. Drug content analysis
Chitosan distribution in both gels was determined using a drug
content analysis. With deionized water, the gels were thinned out.
The gels and deionized water solutions were vortexed for five min-
utes to achieve uniform mixing. The mixes were then centrifuged
for 20 min at 1000 rpm. Each gel formulation’s drug content was
assessed using UV–vis spectrophotometry (SHIMADZU 1900,
Japan) (Fong et al., 2015).
2.6. Anti-bacterial activity
Using the cup plate method, an agar diffusion test was used to
measure the anti-bacterial activity. The substance permitted diffu-
sion across a dense agar media. Then, the nutritional agar medium
was made and sterilized for 15 min at a pressure of 15 lb/sq inch.
Then prepared agar with bacterial suspension was poured into
control and test Petri dishes and were incubated for 24 h. In the
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Saudi Pharmaceutical Journal 31 (2023) 462–471
test plate, when the inoculated agar was solidified, cups were
made using a sterile borer and then filled with prepared formula-
tion and incubated for 24 h. The zone of inhibition, a measure of
anti-bacterial action, was discovered after 24 h (Mandal et al.,
2012, Anbarasan et al., 2019).
2.7. Sterility testing
A different thioglycolate medium and a soybean casein digest
media were used to assess the hydrogel sterility. Both the positive
and negative control test was conducted. The microbes Bacillus
subtilis and Candida albicans were employed. In every instance,
incubation was done, and growth was observed (Singh et al.,
2010, Narayana and Ahmed, 2022b).
2.8. Blood clotting test
A lancet was used to puncture the rat tail vein, and the blood
was then allowed to run continuously. The timer was started con-
currently with the pricking. Blood drops were blotted every 30 s-
time intervals on one strip at a time. The same procedure was
repeated for the remaining two strips with tested compounds,
and blood clotting time was measured. All operations were carried
out with anesthesia (Parasuraman et al., 2010).
2.9. Morphological analysis using high resolution-transmission
electron microscopy (HR-TEM) evaluation
With the aid of an HR-TEM analyzer (JEOL, JEM-2100 electron
microscope; Tokyo, Japan), the shape of a particle and its size dis-
tribution was analyzed. The sample was placed over carbon coated
fine mesh grid (2 0 0), dried at room temperature, and analyzed
(Alencastre et al., 2006).
2.10. Skin irritation study
The adult rats were placed in the cage. Water and regular feed
for rats were given ad libitum. The room temperature was main-
tained at 22 ± 3 0C with 30 – 70 % RH. The artificial lighting was
set up to last for 12 h every day, from 8 am to 8 pm. The rat’s hair
on the back (3 cm2) was cleanly shaved 24 h before the test. The
final gel formulations (gel base, CHI-HYD, and CHI-SF-HYD) were
evenly applied to the shaved area (2 cm2). Skin reactivity at the
application site was observed and graded once daily at 1, 24, 48,
72 h, 7, and 10 days, as appropriate (Alencastre et al., 2006).
2.11. Ex vivo permeation in rat skin
Franz diffusion cell was used to study the ex vivo diffusion of
hydrogels using 7.4 pH phosphate buffer as the receptor media. A
membrane for dialysis was made from rat abdomen skin. The stra-
tum corneum side of the skin was in close touch with the formula-
tion’s release interface within the donor cell. The rat’s skin was
covered with a weight of 1 mL of gel, which was then gently dipped
into 100 mL fresh receptor fluid media while still being repeatedly
stirred. At 37 °C, the temperature was maintained. After each sam-
ple withdrawal, the same quantity of media was replaced in the
receptor compartment to keep the sink condition. The collected
sample read spectrophotometrically at 247 nm (Alencastre et al.,
2006).
2.12. In vivo studies
Healthy adult Wistar albino male rats weighing 150–200 g were
chosen for the investigation. The institutional animal ethics com-
mittee of Yenepoya (Deemed to be University) approved the use
S. Narayana, A. Nasrine, M. Gulzar Ahmed et al.
Table 2
Organoleptic properties of CHI-HYD and CHI-SF-HYD.
Formulation code Visual appearance
CHI-HYD Thick, opaque
CHI-SF-HYD Thick, opaque
*Results are shown as mean ± SD (n = 3).
of animals in research. Intravenous ketamine was used to anes-
thetize the rats before and during the making of the wounds. Using
a razor, the hairs around the dorsal midline region were removed.
After that, a 2 cm diameter was determined and marked. Around
the circle that has been drawn, an excision is conducted using a
scalpel (1 cm). Three groups of six rats each were typically formed.
Group I: Topical application of base hydrogel (base hydrogel con-
tains other than chitosan and silk fibroin) negative control. Group
II: Topical application of CHI-HYD. Group III: Topical application of
CHI-SF-HYD. Then animals were observed to recover from wounds
(Aiyalu et al., 2016; Zhang, n.d., Nayeem et al., 2021). The wound
area was recorded by using transparent paper (tracing paper) on
the wound. Then the percentage of wound contraction was calcu-
lated using the below-mentioned formula. The data collected were
statistically analyzed using GraphPad Prism version 6.01 (Graph-
Pad Software, La Jolla, California, USA), and data were expressed
as mean ± SD. Statistical significance was determined by applying
variance analysis (one-way ANOVA) followed by Tukey’s multiple
comparisons tests. Statistical significance was accepted at the
95 % confidence level (p < 0.05) (Rajoo et al., 2021).
Wound area on 0 day
Wound area on Nth day
Nth day ¼  100
Wound area on 0 day
2.13. Stability studies
The primary goal of stability testing is to prove that the pre-
pared formulation’s efficacy is maintained over its shelf life.
Fig. 1. FT-IR spectrum of(a) CHI, (b) SF, (c) GG, (d) Formulation.
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Saudi Pharmaceutical Journal 31 (2023) 462–471
Texture pH Viscosity (Pa.s)
Smooth 5.87 ± 0.2 79.2 ± 4.2
Smooth 5.96 ± 0.1 79.8 ± 3.8
According to ICH recommendations, stability investigation for the
topical hydrogel formulations (25 ± 2 °C, and 60 ± 5 %, RH) was car-
ried out in a humidity chamber (LABON INSTRUMENTS). Samples
were collected at the beginning, 1st, 2nd, and 3rd respective
months to assess the visual appearance, spreadability, pH, and vis-
cosity. Any modifications to the evaluation parameters that were
reported. Tests were run in triplicate, and the mean and standard
deviation of the observed data were recorded (Ahamed et al.,
2011).
Ethical approval
The animal study protocol was approved by the Institutional
Ethics Committee of the Institutional Review Board (or Ethics Com-
mittee) of Yenepoya (Deemed to be University) with approval
number YU-IAEC (YU/IAEC/15/2019) dated 09/11/2019.
3. Results
3.1. Organoleptic properties
The prepared CHI-HYD and CHI-SF-HYDs were thick and opa-
que. The texture was smooth, and no phase separation was
observed. The pH of prepared hydrogels was found to be within
the acceptable range. The developed topical hydrogel’s viscosity
was 79.2 ± 4.2 Pa.s for CHI-HYD and 79.8 ± 3.8 Pa.s for CHI-SF
HYD. All results are displayed in Table 2.
S. Narayana, A. Nasrine, M. Gulzar Ahmed et al. Saudi Pharmaceutical Journal 31 (2023) 462–471

Table 3 thermograms of CHI, SF, and the physical mixture of these were
Interpretation of FT-IR spectra. depicted in Fig. 2. The thermogram of CHI showed a strong sharp
Sample Absorption Classified by group Class of compound endothermic at 295.91 °C, close to the CHI melting point. At
cm
1 231.06 °C, SF displayed an exothermic peak. The thermogram of
CHI 3300–2500 Strong, broad OAH Carboxylic acid the physical mixture showed the same peaks, and it was confirmed
stretching that the formulation was thermally stable.
3000–2800 Strong, broad NAH Amine salt
stretching
1620–1610 Strong C@C stretching a, b unsaturated ketone 3.4. Spreadability test
1550–1500 Strong NAO stretching Nitro compound
1500–1300 Medium CAH bending Alkane methylene,
methyl group For the good therapeutic efficacy of topical gel formulation,
1075–1020 Strong S@O stretching Sulfoxide group spreadability plays an important role. Fig. 3 displays the spread-
900–800 C@C bending Alkene ability values, i.e., detected diameters within one minute. Results
750 ± 20 Strong CAH bending Monosubstituted indicated no gritty surface, and it quickly spread over the glass
SF 3300–2500 Strong broad OAH Carboxylic acid
stretching
plate.
1618 NAH bending Amine
1550–1500 Strong NAO stretching Nitro compound
1250–1020 Medium CAN Amine 3.5. Blood clotting test
stretching
840–650 C@C bending Alkene The obtained results revealed that CHI has hemostatic activity.
GG 3300–2500 Strong broad OAH Carboxylic acid
In addition, the time required for blood clotting was found to be
stretching
1670–1600 Weak C@C stretching Alkene comparatively less in CHI-HYD and CHI-SF-HYD than natural clot-
1390–1310 Medium OAH bending Phenol ting process. The results are shown in Table 4 and Fig. 4.
1000–650 Strong C@C bending Alkene

3.2. Ft-IR studies

The interactions between the components were studied by per-

forming FT-IR spectroscopy. The characteristic absorption bands in
the CHI, SF, and GG were listed. The same peaks were observed in
the formulation spectra, confirming that no interaction between
the components and formulation was compatible. The range is
shown in Fig. 1, and the interpretation of functional groups is dis-
played in Table 3.

3.3. Thermal analysis

To detect thermal stability or formulation incompatibility Fig. 3. Spreadability values for the CHI-HYD and CHI-SF-HYD *Results are shown
as mean ± SD (n = 3).
between the excipients DSC study has been carried out. DSC

Fig. 2. Shows the DSC thermogram of (a) CHI, (b) SF, and (c) physical mixture.

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Table 4
Blood clotting test for control, CHI-HYD, and CHI-SF-HYD.

Number of blood drops Blood coagulation (sec)

Trials Mean ± SD
1 2 3
Control 8 8 7 7.66 ± 0.5 229.8
CHI-HYD 5 6 5 5.33 ± 0.5 159.9
CHI-SF-HYD 4 4 5 4.33 ± 0.5 129.9

*Results are shown as mean ± SD (n = 3).

Fig. 4. Blood clotting test for Control, CHI-HYD& CHI-SF-HYD.

3.6. Drug content analysis zones of inhibition. The anti-bacterial activity outcomes are dis-
played in Table 6 and Fig. 5. The zone of inhibition was found to
The percentage of CHI in CHI-HYD was found to be 81 ± 3.5 %, be between 25 and 28.5 mm.
and in CHI-SF-HYD was found to be 83 ± 1.4 %. The obtained out-
comes indicated that CHI was distributed uniformly in the gels, 3.8. HR-TEM analysis
and the CHI loss during the formulation of gels was negligible
(Table 5). The CHI-SF-HYD were found to be spherical, and the porous
interconnected well-distributed particles with a smooth and
3.7. Anti-bacterial activity homogenous surface were observed. As depicted in Fig. 6, the par-
ticles’ size in the nano range with uniform distribution.
The antibacterial activity of the prepared formulations was
tested against the most common microorganisms like staphylococ- 3.9. Test for sterility
cus aureus and Escherichia coli. The gel formulation showed good
The test showed that the medium used was sterile. The results
of the test for sterility are shown in Table 7. The results indicated
Table 5
Drug content data for hydrogels.
no bacterial growth. Hence, the tested formulation passes the test
for sterility.
Formulation code Drug Content (%)
CHI-HYD 81 ± 3.5
3.10. Skin irritation study
CHI-SF-HYD 83 ± 1.4

*Results are shown as mean ± SD (n = 3). The gel base, CHI-HYD, and CHI-SF-HYDs were identically
applied to each rat’s shaved area (2 cm2). Any skin reaction at
the applied site was individually evaluated and recorded once daily
at 1, 24, 48, 72 h, 7, and 10 days. No allergy or swelling was spotted
Table 6
Anti-bacterial activity. for developed CHI-HYD, CHI-SF-HYDs, and gel base formulations.

Microorganism Zone of inhibition(mm)

3.11. Ex vivo permeation in rat skin
Marketed formulation CHI-HYD CHI-SF-HYD
Staphylococcus aureus& 28.5 ± 1.1 25 ± 0.6 25 ± 0.4 Fig. 7 shows the ex vivo permeation of gel in rat skin. The cumu-
Escherichia coli
lative amount of CHI permeated through CHI solution, CHI-HYD
*Results are shown as mean ± SD (n = 3). and CHI-SF-HYD were 2.1, 8.4, and 19.5 lg/ cm2, respectively. It
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Fig. 5. Antimicrobial activity (a). control, (b). marketed formulation, (c). CHI-HYD, (d). CHI-SF-HYD.

Fig. 7. Ex vivo permeation of Chitosan solution, CHI-HYD, and CHI-SF-HYD in rat

skin.

a significant difference in wound closure. Fig. 9 shows the % of

wound contraction for various groups on the 5th,10th,15th, and
20th days. For days 5, 10, and 15, the highest % wound contraction
was observed for CHI-SF-HYD treated group (33.5, 48.5, and 93.5 %,
respectively). The CHI-HYD-treated group showed % wound con-
traction of 20.66, 35, and 49.25 %, respectively, comparatively les-
ser than that of the CHI-SF-HYD treated. Interestingly on the 17th
day, the % of wound contraction for the CHI-SF-HYD-treated rats
Fig. 6. HR-TEM analysis of CHI-SF-HYD.
was 99.50 %. A highly significant difference in the % of wound con-
traction was observed for CHI-SF-HYD (p < 0.0001) throughout the
experiment period when compared with CHI-HYD and gel base
was confirmed that the permeation capacity of CHI has signifi- treated group.
cantly enhanced SF-containing hydrogel.

3.12. Invivo studies 3.13. Stability studies

Fig. 8 depicts images of the wound for all groups: base HYD
treated, CHI-HYD treated, and CHI-SF-HYD treated animals from
days 1 to 20. The CHI-SF-HYD-treated group exhibited a fast tissue
regeneration process in all the animals tested with wound cavities.
Compared to other groups, the CHI-SF-HYD treated groups showed
According to ICH criteria for developed gel formulations, a sta-
bility study was conducted to ensure safety and efficacy during
storage. The formulations have good quality characteristics. The
outcomes of the stability studies disclosed that the developed
hydrogels are stable.

Table 7
Test for sterility.

Days 1 2 3 4 5 6 7
F S F S F S F S F S F S F S
CHI-HYD – – – – – – – – – – – – – –
CHI-SF-HYD – – – – – – – – – – – – – –
Positive control + + + + + + + + + + + + + +
Negative control – – – – – – – – – – – – – –

(+) = growth of microorganism (-) = no growth F = fluid thioglycollate medium.

S = soyabean casein digest medium.

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Fig. 8. Representative images of the wound areas of the various rat groups (Base-HYD, CHI-HYD, and CHI-SF-HYD).
4. Discussion
The present study was planned to develop CHI-HYD and CHI-SF-
HYD and to determine the potential of combinational polymers in
the tissue regeneration process. The studies showed that SF is
widely reported for its ability in tissue regeneration and engineer-
ing (Chen and Liu, 2016, Gil et al., 2013). The developed CHI-HYD
and CHI-SF-HYD were smooth in texture with good spreadability,
as shown in Fig. 3. Chen et al. reported that the spreadability values
of gels with this range evidenced that easy spreading on the sur-
face by applying a low shear rate (Chen et al., 2006). The IR spectral
analyses exhibited that excipients used in the formulation were
compatible with each other (Fig. 1), and the DSC analysis curves
shown in Fig. 3 proved the thermal stability of hydrogels. Our
study found that blood clotting time was lesser when treated with
CHI-HYD and CHI-SF-HYD than natural blood clotting (Table 3 and
469
Saudi Pharmaceutical Journal 31 (2023) 462–471
Fig. 4). Radwan et al. reported that the interaction of blood with
CHI formulation increases platelet volume, and the distribution
width leads to platelet adhesion activation and aggregation.
Platelet activation stimulates coagulation factors and blood clots
(Radwan-Pragłowska et al., 2019). A good zone of inhibition con-
firmed the anti-bacterial activity of the developed formulation,
but the addition of SF does not significantly improve its anti-
bacterial action (Table 6, Fig. 5). Other researchers obtained similar
results where it was found that SF alone does not offer any anti-
bacterial activity. Still, antibiotics can be released sustainably from
SF biomaterials, killing the bacteria growth (El-KasedReham et al.,
2016). The HR-TEM results in Fig. 6 showed that the porous inter-
connected well-distributed particles with a smooth and homoge-
nous surface of particles in CHI-SF-HYD improved the adhesion
properties of the gel (Zhang et al., 2009). The skin irritation study
showed no allergy or swelling even after ten days of study,
S. Narayana, A. Nasrine, M. Gulzar Ahmed et al. Saudi Pharmaceutical Journal 31 (2023) 462–471

Fig. 9. Percentage of wound contraction Percentage of wound contraction for CHI-SF-HYD treated, CHI-HYD treated, and base-HYD treated rats for days 5, 10, 15, and 20.
* Indicates significant differences in wound contraction. ****=p < 0.0001, ***=p < 0.001.

Table 8
Physical parameters after accelerated stability study of the optimized formulation.

Physical Parameters 0th day 30th day 60th day 90th day
CH CSH CH CSH CH CSH CH CSH
Visual appearance Thick, opaque Thick, opaque Thick, opaque Thick, opaque Thick, opaque Thick, opaque Thick, opaque Thick, opaque
Spreadability 26 ± 1.2 27 ± 1.4 26 ± 2.1 27 ± 1.7 25 ± 1.5 26.5 ± 2.6 25 ± 1.8 26 ± 2.1
pH 5.87 ± 0.2 5.96 ± 0.1 5.91 ± 0.1 5.92 ± 0.3 5.89 ± 0.2 5.91 ± 0.1 5.88 ± 0.2 5.90 ± 0.4
Viscosity (cps) 79.20 ± 4.2 79.80 ± 3.8 7923 ± 3.5 7975 ± 2.9 7921 ± 1.8 7982 ± 4.5 7922 ± 2.9 7981 ± 3.9

*Results are shown as mean ± SD (n = 3).

signifying that the developed gel base, CHI-HYD, and CHI-SF-HYD
formulations were safe to apply for blood clotting and wound heal-
ing activity. Due to CHI’s excellent biodegradability and biocom-
patibility, skin irritation or local adverse effects are uncommon.
As a result, using CHI reduces the likelihood of developing contact
dermatitis compared to conventional anti-bacterial treatments
(Zheng et al., 2020, Aiyalu et al., 2016). The test for sterility result
is shown in Table 7. The results indicated no bacterial growth.
Hence, the tested hydrogel formulations are considered to pass
the test for sterility. Also, the permeation capacity of CHI was
significantly increased in CHI-SF-HYD than in CHI-HYD and simple
CHI solution, which may be due to the porous interconnected
structure of CHI-SF-HYD (Dubey and Prabhu, 2014, Yang et a.,
2004, Huang et al., 2003). In vivo animal studies evidenced
faster-wound healing with CHI-SF-HYD-treated animals, which
can be identified in Fig. 8. This may be due to the high amount
of b-sheet patterns present in SF intensifies cell adhesion, prolifer-
ation, and migration of cells, hence it boosts the tissue reformation
around the wounded area (Wang et al., 2016, Aljady et al., 2000).
Also, literature reported that SF’s porous and interconnected struc-
ture might lead to tissue regeneration and faster wound healing
(He et al., 2019). Complete wound recovery (99.50 %) was observed
for CHI-SF-HYD on day 17. A highly significant difference in the %
of wound contraction was observed for CHI-SF-HYD (p < 0.0001)
throughout the experiment period when compared with CHI-
HYD and gel base treated group.
The shelf life of the HYDs, shown in Table 8, indicates no change
in color, odor, spreadability, pH, and viscosity. The outcomes
470
revealed that the developed HYDs are stable (Massensini et al.,
2015, Meng et al., 2014).,
The present research has a robust design and positive results,
which pave the path for future wound healing treatment. However,
limitations of the study could be the small in vivo sample size and
lack of microscopic evaluation data for wound tissues.
5. Conclusion
In this study, CHI-HYD formulations alone and together with SF
were designed and evaluated for various characteristics. The CHI-
HYD and CHI-SF-HYD demonstrated excellent biocompatibility,
desired stability, and acceptable physical characteristics. In addi-
tion, CHI-SF-HYD remarkably shortened the span of tissue refor-
mation in an in vivo animal model compared with CHI-HYD
alone, and the gel base, which demonstrated these developed
hydrogels, could consequently accelerate the regeneration of the
damaged area. Overall, the positive findings indicate an improved
knowledge of the events contributing to blood coagulation and
re-epithelialization. This suggests that in the future, the CHI-SF-
HYD could be employed to develop novel wound-healing devices
to provide compliance for patients and the healthcare sector.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
S. Narayana, A. Nasrine, M. Gulzar Ahmed et al.
Acknowledgment
The authors would like to acknowledge the Researchers
Supporting Project number (RSP2023R115), King Saud University,
Riyadh, Saudi Arabia, for extending financial support to do this
research project. Furthermore, researchers thank Sericare,
Bangalore, for providing the silk fibroin gift sample used in the
study. Finally, the authors would like to thank Yenepoya Pharmacy
College and Research Centre for providing laboratory facilities to
perform the experimental works.
Funding
The authors are thankful to AlMaarefa University for providing
support to do this research.
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