gels

Article
Synthesis and Characterization of Acrylamide/Acrylic Acid
Co-Polymers and Glutaraldehyde Crosslinked
pH-Sensitive Hydrogels
Munir Ahmad Khan 1,† , Abul Kalam Azad 2, *,† , Muhammad Safdar 1 , Asif Nawaz 1 , Muhammad Akhlaq 1 ,
Pijush Paul 3 , Md. Kamal Hossain 4 , Md. Habibur Rahman 5 , Roua S. Baty 6 , Attalla F. El-kott 7,8 ,
Mohamed Kamel 9 , Simona G. Bungau 10 and Mohamed M. Abdel-Daim 11,12, *

1 Department of Pharmaceutics, Faculty of Pharmacy, Gomal University, Dera Ismail Khan 29050, Pakistan;
[email protected] (M.A.K.); [email protected] (M.S.);
[email protected] (A.N.); [email protected] (M.A.)
2 Pharmaceutical Technology Unit, Faculty of Pharmacy, AIMST University, Bedong 08100, Kedah, Malaysia
3 Department of Pharmacy, Gono Bishwabidyalay, Mirzanagar, Savar, Dhaka 1344, Bangladesh;
[email protected]
4 Institute of Health and Sports, Victoria University, Melbourne 3011, Australia; [email protected]
5 Department of Global Medical Science, Wonju College of Medicine, Yonsei University,
Wonju 26426, Gangwon-do, Korea; [email protected]
6 Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
[email protected]
7 Biology Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61421, Saudi Arabia;





 [email protected]
8 Zoology Department, Faculty of Science, Damanhour Univesity, Damanhour 22511, Egypt
Citation: Khan, M.A.; Azad, A.K.; 9 Department of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University,
Safdar, M.; Nawaz, A.; Akhlaq, M.;
Giza 12211, Egypt; [email protected]
Paul, P.; Hossain, M.K.; Rahman, 10 Department of Pharmacy, Faculty of Medicine and Pharmacy, University of Oradea, 410087 Oradea, Romania;
M.H.; Baty, R.S.; El-kott, A.F.; et al. [email protected]
Synthesis and Characterization of 11 Department of Pharmaceutical Sciences, Batterjee Medical College, P.O. Box 6231, Jeddah 21442, Saudi Arabia
12 Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt
Acrylamide/Acrylic Acid
Co-Polymers and Glutaraldehyde * Correspondence: [email protected] (A.K.A.); [email protected] (M.M.A.-D.)
Crosslinked pH-Sensitive Hydrogels. † These authors contributed equally to this work.
Gels 2022, 8, 47. https://doi.org/
10.3390/gels8010047 Abstract: This project aims to synthesize and characterize the pH-sensitive controlled release of
5-fluorouracil (5-FU) loaded hydrogels (5-FULH) by polymerization of acrylamide (AM) and acrylic
Academic Editors: Francisco
acid (AA) in the presence of glutaraldehyde (GA) as a crosslinker with ammonium persulphate as
Fernández-Campos and
an initiator. The formulation’s code is named according to acrylamide (A1, A2, A3), acrylic acid
Mireia Mallandrich
(B1, B2, B3) and glutaraldehyde (C1, C2, C3). The optimized formulations were exposed to various
Received: 20 December 2021 physicochemical tests, namely swelling, diffusion, porosity, sol gel analysis, and attenuated total
Accepted: 5 January 2022 reflection-Fourier transform infrared (ATR-FTIR). These 5-FULH were subjected to kinetic models
Published: 9 January 2022 for drug release data. The 5-FU were shown to be soluble in distilled water and phosphate buffer
Publisher’s Note: MDPI stays neutral media at pH 7.4, and sparingly soluble in an acidic media at pH 1.2. The ATR-FTIR data confirmed
with regard to jurisdictional claims in that the 5-FU have no interaction with other ingredients. The lowest dynamic (0.98 ± 0.04% to
published maps and institutional affil- 1.90 ± 0.03%; 1.65 ± 0.01% to 6.88 ± 0.03%) and equilibrium swelling (1.85 ± 0.01% to 6.68 ± 0.03%;
iations. 10.12 ± 0.02% to 27.89 ± 0.03%) of formulations was observed at pH 1.2, whereas the higher dynamic
(4.33 ± 0.04% to 10.21 ± 0.01%) and equilibrium swelling (22.25 ± 0.03% to 55.48 ± 0.04%) was
recorded at pH 7.4. These findings clearly indicated that the synthesized 5-FULH have potential
swelling characteristics in pH 6.8 that will enhance the drug’s release in the same pH medium. The
Copyright: © 2022 by the authors.
porosity values of formulated 5-FULH range from 34% to 62% with different weight ratios of AM,
Licensee MDPI, Basel, Switzerland.
AA, and GA. The gel fractions data showed variations ranging from 74 ± 0.4% (A1) to 94 ± 0.2%
This article is an open access article
distributed under the terms and
(B3). However, formulation A1 reported the highest 24 ± 0.1% and B3 the lowest 09 ± 0.3% sol
conditions of the Creative Commons fractions rate among the formulations. Around 20% drug release from the 5-FULH was found at
Attribution (CC BY) license (https:// 1 h in an acidic media (pH1.2), whereas >65% of drug release (pH7.4) was observed at around 25 h.
creativecommons.org/licenses/by/ These findings concluded that GA crosslinked 5-FU loaded AM and AA based hydrogels would be a
4.0/). potential pH-sensitive oral controlled colon drug delivery carrier.

Gels 2022, 8, 47. https://doi.org/10.3390/gels8010047 https://www.mdpi.com/journal/gels

Gels 2022, 8, 47 2 of 18

Keywords: hydrogels; pH-sensitive; colon drug delivery; acrylamide; acrylic acid; glutaraldehyde

1. Introduction
The worldwide prevalence of colonic disease is increasing with over one hundred
thousand colorectal cancer patients newly diagnosed every year [1]. Traditional non-
targeted therapy has unwanted side-effects and offers low efficacy owing to the systemic
absorption of the drug before it reaches the disease site [2]. Therefore, colon-targeted
drug delivery systems for the local treatment of colonic diseases are urgently sought [3].
Depending on the colonic milieu, colon targeted drug delivery systems specifically release
the drug where it is needed, thus circumventing the untimely release of the drug in
the upper gastrointestinal (GI) tract [4]. It is important to take into consideration the
area neighboring the disease site(s) as well as the colon physiology, since the GI tract
experiences constant changes in motility, fluid composition, pH from the small to the
large intestine, and enzymatic activity [5]. Numerous formulation strategies have been
investigated to develop colonic drug delivery (i.e., enzyme-sensitive systems, magnetically
triggered systems, and pH-responsive systems) [6]. Hydrogels are a three-dimensional
network of crosslinked polymer that can absorb and hold a significant amount of water
inside the gap between the chains [7]. Hydrogels are extensively explored in a wide
range of applications including medical, biological, and pharmaceutical fields due to
their high-water content, swelling characteristics, biocompatibility, high permeability,
and nontoxicity [8]. Besides this, stimuli-sensitive hydrogels are gaining attention as
smart drug delivery systems as they can respond to the surrounding environment (e.g.,
temperature, pH, and enzymes). pH-sensitive hydrogels are preferable to other systems
due to their higher controllability [9,10]. Several types of pH-responsive hydrogels have
been investigated for site specific drug delivery owing to significant pH variations in the
GI tract, where the drug release characteristics change with the variations in the medium
pH because of relaxation of the chain [11].
AA is a pH-sensitive polyelectrolyte, which is superabsorbent as well as possessing
concentration-dependent mucoadhesive properties and has been exclusively explored in
colon drug delivery systems [12]. The advantage of AA is that variation in the composition
of the polymers results in different drug release characteristics depending on the surround-
ing pH [13]. Temperature-sensitive and pH-responsive behavior has also been displayed
by the copolymers AA and the interpenetrating networks (IPNs) [14]. Also, pH-sensitive
macromolecular structures designed by the poly(AA) (PAA) have been explored for use in
site-specific drug delivery systems [15]. Owing to the ionization of the carboxylic acid (CA)
functionalities, the PAA based hydrogels swell considerably at pH 7.4 since its pKa value
ranges from 4.5–5 [16].
AM has been employed in various biomedical applications including protein drug
delivery due to its biocompatibility, pH-responsiveness and mucoadhesivity [17]. Success
has been reported using an AM grafted N-succinyl chitosan-based hydrogel with a pH
dependent release of insulin when administered orally [18].
GA is used as a crosslinking agent for the preparation of hydrogels [19]. It is also
employed for cell and enzyme immobilization due to its higher aqueous solubility, ease
of accessibility, low cost and efficiency of biomaterials stabilization [20]. GA contains
two aldehyde groups which can form immediate covalent bonds with the functionalities
available (i.e., imidazoles, phenols, hydroxyl, and thiols) [21]. It has been reported that at a
pH of around 7, GA reacts more quickly with amine groups [22] as well as forming better
chemical and thermal crosslinks than those of other aldehydes [23].
5-FU is frequently used as one of the mainstays in potential antitumor drugs in clinical
chemotherapy, specifically for the treatment of colon cancer [24]. It has a short biological
half-life (10–20 min), causing partial absorption where? Upper intestine? since it is a
sparingly water-soluble pyrimidine antimetabolite. The drug is rapidly metabolized by
Gels 2022, 8, 47 3 of 18

dihydro pyrimidine dehydrogenase after oral delivery [25]. The clinical use of 5-FU affects
the normal cells, leading to the adverse effects on GI tracts, the central nervous system and
bone marrow, owing to the lack of tumor selectivity [26,27]. In recent studies, pH-sensitive
hydrogels have been investigated by researchers for controlled delivery of 5-FU to reduce
these side effects and to enhance the antitumor activity [28]. PAA and its derivatives-based
pH-responsive hydrogels can circumvent the effect of the highly acidic surroundings on
drugs [29]. The pKa of PAA is around 3.5, therefore at low pH, the hydrogels based on
PAA become contracted resulting in the premature release of the drug in the stomach.
Consequently, at higher pH, they remain swollen, leading to drug release in the intestine.
Nevertheless, avoiding premature drug release before entering the colon section is very
challenging due to the pH resemblance between the small intestine and the colon [30].
The goal of this study is to synthesize pH-responsive hydrogels using AM, AA and
GA. Hydrogels were prepared by a simple crosslinking method, employing 5-FU as a
model drug. The ATR-FTIR spectroscopy was used to investigate the interaction between
the drug and the excipients. Swelling characteristics, diffusion coefficient, drug loading,
in vitro release and release kinetics were also studied.

2. Results and Discussion

2.1. Synthesis Hydrogel
The mechanism of polymerization of acrylic acid and acrylamide in the presence of
glutaraldehyde (GA) cross-linker was adapted from Wang et al. and partially modified the
synthesis reaction mechanism shown in Scheme 1. Acrylic acid (1), acrylic acid sodium
(2) and acrylamide (3) can react to Poly(acrylic acid-co-acrylamide) (4) using KPS as a
radical initiator. The persulfate initiator was decomposed under heating to generate sulfate
anion radical. The cross-linker glutaraldehyde (5) was added after polymerization finished
in order to avoid the crosslinking reaction of Poly(acrylic acid-co-acrylamide) (4) during
polymerization. Through heat-treatment at 110–160 ◦ C the amide groups and hydroxyl
Gels 2022, 8, x FOR PEER REVIEWgroups can react with aldehyde groups from glutaraldehyde (5) to form poly(AA-co-AM)
4 of 19
(6) network structure. Grafted hydrogels exhibit pH-sensitive swelling properties and
pH-controlled drug release behavior.

Scheme1.1.General
Scheme Generalmechanism
mechanism for
for polymerization ofAA
polymerization of AAand
andAM
AMininthe
the presence
presence of of glutaraldehyde
glutaraldehyde
cross-linker[31].
cross-linker [31].

2.2. Solubility Study

The 5-FU is a nucleobase analogue having a pKa value of (8 ± 0.1). It has a water
solubility of 12.2 g/L at 20 °C. (repeated) It is freely soluble in a phosphate buffer of pH
Gels 2022, 8, 47 4 of 18

2.2. Solubility Study

The 5-FU is a nucleobase analogue having a pKa value of (8 ± 0.1). It has a water
solubility of 12.2 g/L at 20 ◦ C. (repeated) It is freely soluble in a phosphate buffer of
pH 7.4 as well as in distilled water compared to pH 1.2 (Table 1). The solubility study is an
important parameter which conforms to the dissolution experiments.
Table 1. Solubility study of 5-FU at 37 ◦ C.

S.No Solvent Remarks

1 Distilled Water Soluble
2 Phosphate buffer pH 7.4 Soluble
3 Acidic media pH 1.2 Sparingly soluble

2.3. FTIR
FTIR spectral analysis of 5-FU and 5-FULH is shown in Figure 1. Pure 5-FU, drug loaded,
and unloaded hydrogel samples were assessed using FTIR in the array of 400–4000 cm−1 . FTIR
represents the distinct peak at 3177 cm−1 , 2927 cm−1 , 1658 cm−1 in pure 5-FU spectra and
3196 cm−1 , 2924 cm−1 and 1654 cm−1 in 5-FULH. In the case of pure 5-FU, the bands at
3412 cm−1 , 1165 cm−1 show evidence of N-H stretching (free) and –C-F band; after drug
loading some of the bands disappear and N-H stretching (free) appears at the same wave
number, while –C-F band appears at 1165 cm−1 . This indicates that the 5-FU is molecularly
dispersed into the prepared hydrogels [32]. In the case of unloaded hydrogel, see Figure 1c,
the AM peak appears at 1647 cm−1 for primary amide (C=O) stretching. The peak for the
alkene group (HC=CH) appears at 3065 cm−1 . In the band of GA, the peak assigned to
the C-OH stretching at 1243 cm−1 is very clear, suggesting that the desired material has
been successfully prepared [33]. Owing to the electrostatic interaction among the various
functional groups of AM, AA and GA, the characteristic peaks of AM, AA and GA are
shifted to developed 5-FULH. Figure 1b shows that the loading of 5-FU by the developed
hydrogel was successful and no interaction between the 5-FU and hydrogel ingredients
was detected [34].

2.4. Swelling Studies

The release of the drug from the 5-FULH occurs once the polymer network is dissolved,
followed by drug diffusion from the surface of the structure which is associated with the
swelling behavior of the 5-FULH [35]. The advantage of hydrogel is that it can swell in the
surrounding medium due to its chemical structure, which allows affinity with the water
molecules [36]. Swelling studies of 5-FULH were conducted using various pH mediums of
1.2, 6.8, and 7.4. Equilibrium and dynamic swelling proportions of different 5-FULH are
depicted in Table 2.
Table 2. % Dynamic and equilibrium swelling study of 5-FULH.

pH 1.2 pH 6.8 pH 7.4

F.
Code Dynamic Equilibrium Dynamic Equilibrium Dynamic Equilibrium
Swelling Swelling Swelling Swelling Swelling Swelling
A1 0.98 ± 0.04 1.65 ± 0.01 1.85 ± 0.01 10.12 ± 0.02 4.33 ± 0.04 29.67 ± 0.05
A2 0.99 ± 0.03 2.98 ± 0.03 3.89 ± 0.03 16.27 ± 0.03 5.99 ± 0.03 39.68 ± 0.03
A3 1.32 ± 0.01 4.11 ± 0.05 4.13 ± 0.05 28.15 ± 0.01 7.71 ± 0.03 47.98 ± 0.02
B1 0.99 ± 0.02 3.55 ± 0.02 3.35 ± 0.02 12.89 ± 0.04 5.77 ± 0.02 33.46 ± 0.07
B2 1.21 ± 0.04 5.02 ± 0.01 5.02 ± 0.01 17.65 ± 0.05 7.44 ± 0.03 47.96 ± 0.05
B3 1.43 ± 0.02 6.88 ± 0.03 6.68 ± 0.03 27.89 ± 0.03 10.21 ± 0.01 55.48 ± 0.04
C1 1.90 ± 0.03 3.99 ± 0.01 4.99 ± 0.01 18.67 ± 0.02 6.88 ± 0.02 42.78 ± 0.06
C2 1.33 ± 0.01 4.64 ± 0.02 3.14 ± 0.02 16.74 ± 0.01 6.10 ± 0.03 34.53 ± 0.05
C3 1.10 ± 0.01 3.45 ± 0.04 2.05 ± 0.04 11.87 ± 0.04 5.66 ± 0.02 22.25 ± 0.03
 Gels 2022, 8, x FOR PEER REVIEW 5 of 19

Gels 2022, 8, 47 5 of 18
developed hydrogel was successful and no interaction between the 5-FU and hydrogel
ingredients was detected [34].

Gels 2022, 8, x FOR PEER REVIEW 6 of 19

Figure 1. ATR-FTIR spectra of (a) Pure 5-FU, (b) 5-FULH, and (c) Unloaded hydrogels.
Figure 1. ATR-FTIR spectra of (a) Pure 5-FU, (b) 5-FULH, and (c) Unloaded hydrogels.
2.5. Effect of AM
2.4. Effect
2.5. SwellingAM Studies
Figures 2ofand 3 describe the swelling attitude of 5-FULH with various concentrations of
AM. The The
Figuresrelease
swelling 2 andof3 the
values ofdrug
describe from
thesethe the
were5-FULH
swelling
gels attitude
measured occurs 37once
ofat5-FULH the
°C andwithpolymer
various
medium network
pH is dis-
concentrations
of 1.2, 6.8,
solved, followed byincreased
drug diffusion ◦ C andwhich
andof AM.
7.4. The
The swelling
swelling values ofwith
theseanfrom
gels the surface
were
increase AM of
measured
in the structure
at 37
concentration andmediumis associated
a raise pH of 1.2,
in me-
with
6.8, the
and swelling
7.4. The behavior
swelling of the
increased 5-FULH
with an[35]. The
increase advantage
in AM of hydrogel
concentration
dium pH. This swelling behavior of the polymer was due to its hydrophilic nature. The is
and that
a it can
raise in
medium
swell in pH.
the This swelling
surrounding behavior
medium dueof the
to polymer
its chemical was due to
structure,
physical nature and hydrogen bond formation restricts the release of the drug. its hydrophilic
which allows nature.
affinity The
with
physical
the waternature and hydrogen
molecules bondstudies
[36]. Swelling formation restrictswere
of 5-FULH the release of theusing
conducted drug.various pH
mediums of 1.2, 6.8, and 7.4. Equilibrium and dynamic swelling proportions of different
10
5-FULH are depicted A1 in Table
A2 2. A3
9
Table 2. % Dynamic and equilibrium swelling study of 5-FULH.
8
pH 1.2 pH 6.8 pH 7.4
Dynamic swelling (%)

7
F. Code Dynamic Equilibrium Dynamic Equilibrium Dynamic Equilibrium
6 Swelling Swelling Swelling Swelling Swelling Swelling
5
A1 0.98 ± 0.04 1.65 ± 0.01 1.85 ± 0.01 10.12 ± 0.02 4.33 ± 0.04 29.67 ± 0.05
A2 0.99 ± 0.03 2.98 ± 0.03 3.89 ± 0.03 16.27 ± 0.03 5.99 ± 0.03 39.68 ± 0.03
4
A3 1.32 ± 0.01 4.11 ± 0.05 4.13 ± 0.05 28.15 ± 0.01 7.71 ± 0.03 47.98 ± 0.02
3 0.99 ± 0.02 3.55 ± 0.02
B1 3.35 ± 0.02 12.89 ± 0.04 5.77 ± 0.02 33.46 ± 0.07
B2
2 1.21 ± 0.04 5.02 ± 0.01 5.02 ± 0.01 17.65 ± 0.05 7.44 ± 0.03 47.96 ± 0.05
B3 1.43 ± 0.02 6.88 ± 0.03 6.68 ± 0.03 27.89 ± 0.03 10.21 ± 0.01 55.48 ± 0.04
1 1.90 ± 0.03 3.99 ± 0.01
C1 4.99 ± 0.01 18.67 ± 0.02 6.88 ± 0.02 42.78 ± 0.06
0 1.33 ± 0.01 4.64 ± 0.02
C2 3.14 ± 0.02 16.74 ± 0.01 6.10 ± 0.03 34.53 ± 0.05
C3 1.10 ± 0.01 pH 1.2
3.45 ± 0.04 pH 6.811.87 ± 0.04 5.66 ±pH
2.05 ± 0.04 7.4 22.25 ± 0.03
0.02
Figure 2. Effect
Figure of AM
2. Effect on on
of AM dynamic swelling
dynamic of 5-FULH.
swelling of 5-FULH.

60
A1 A2 A3
50
g (%)

40
 2
1
0
pH 1.2 pH 6.8 pH 7.4
Gels 2022, 8, 47 6 of 18
Figure 2. Effect of AM on dynamic swelling of 5-FULH.

60
A1 A2 A3
50

Equilibrium swelling (%)

40

30

20

10

0
pH 1.2 pH 6.8 pH 7.4
Figure 3. Effect
Figure of AM
3. Effect on on
of AM equilibrium swelling
equilibrium of 5-FULH.
swelling of 5-FULH.

2.6.2.6. Effect
Effect of AA
of AA
Figures
Figures 4 and
4 and 5 show
5 show the the swelling
swelling attitude
attitude of 5-FULH
of 5-FULH withwith various
various concentrations
concentrations of
of AA. The swelling values of these gels were measured at 37 ◦ C and medium pH of 1.2,
AA. The swelling values of these gels were measured at 37 °C and medium pH of 1.2, 6.8,
and6.8,
7.4.and
The7.4. The swelling
swelling increased
increased with anwith an increase
increase in AA concentration
in AA concentration and in
and a raise a raise
me- in
medium pH. This swelling behavior of the polymer was again due to its hydrophilic
dium pH. This swelling behavior of the polymer was again due to its hydrophilic nature. nature.
TheThe physical
physical nature
nature andand hydrogen
hydrogen bond
bond formation
formation again
again restricts
restricts the release
the release of drug.
of the the drug.

2.7. Effect of GA
Introduction of a crosslinking agent such as GA affects the swelling behavior of 5-FULH.
Figures 6 and 7 show GA at different concentrations and the effect over the swelling of
hydrogel at 37 ◦ C in medium pH of 1.2, 6.8, and 7.4. Results show that when the concentration
of GA increases, the swelling ratio decreases. This might be due to increased cross linking of
GA. Generally, mobility of the polymer chain is affected by the crosslinking, ensuring the low
Gels 2022, 8, x FOR PEER REVIEW solubility of the polysaccharide [37]. GA promotes the degree of crosslinking which 7 of
in 19
turn
results in folding of the polymeric chains and the subsequent attainment of reticulation point,
thus affecting the aqueous absorption capacity [38].
12
B1 B2 B3
10
Dynamic swelling (%)

0
pH 1.2 pH 6.8 pH 7.4
Figure
Figure 4. Effect
4. Effect of AA
of AA on on dynamic
dynamic swelling
swelling of 5-FULH.
of 5-FULH.

70
B1 B2 B3
60
(%)
 2

0
pH 1.2 pH 6.8 pH 7.4
Gels 2022, 8, 47 7 of 18
Figure 4. Effect of AA on dynamic swelling of 5-FULH.

70
B1 B2 B3
60

Equilibrium swelling (%)

50

40

30

20

10

0
Gels 2022, 8, x FOR PEER REVIEW
Gels 2022, 8, x FOR PEER REVIEW 8 of 19 8 of 1
pH 1.2 pH 6.8 pH 7.4
Figure 5. Effect
Figure of AA
5. Effect on equilibrium
of AA swelling
on equilibrium of 5-FULH.
swelling of 5-FULH.

8 of GA
2.7. Effect 8
C1 C2 C1
C3 C2 C3
Introduction
7 of a crosslinking
7 agent such as GA affects the swelling behavior of 5-
FULH. Figures 6 and 7 show GA at different concentrations and the effect over the swell-
6 in medium pH of 1.2, 6.8, and 7.4. Results show that when the
ing of 6hydrogel at 37 °C
Dynamic swelling (%)
Dynamic swelling (%)

concentration of GA increases, the swelling ratio decreases. This might be due to increased
5
cross linking 5
of GA. Generally, mobility of the polymer chain is affected by the crosslink-
ing, ensuring the low solubility of the polysaccharide [37]. GA promotes the degree of
4 4
crosslinking which in turn results in folding of the polymeric chains and the subsequent
attainment of reticulation
3 point, thus affecting the aqueous absorption capacity [38].
3
2
2
1
1
0
0 pH 1.2 pH 6.8 pH 7.4
pH 1.2 pH 6.8 pH 7.4
Figure 6. Effect of GA over dynamic swelling of 5-FULH.
Figure 6. Effect
Figure of GA
6. Effect overover
of GA dynamic swelling
dynamic of 5-FULH.
swelling of 5-FULH.

50
50 C1 C2 C3
C1 45 C2 C3
Equilibrium swelling (%)

45
Equilibrium swelling (%)

40
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
0 pH 1.2 pH 6.8 pH 7.4
pH 1.2 pHGA
6.8 over the equilibrium swelling
pH 7.4 of 5-FULH.
Figure 7. Effect Figure 7. Effect
of GA over the of
equilibrium swelling of 5-FULH.
Figure 7. Effect of GA over the equilibrium swelling of 5-FULH.
2.8. Porosity Measurement
The porosity values of 5-FULH range from 34% to 62% with different weight ratios o
2.8. Porosity Measurement
AM, AA, and
The porosity values of GA. It was
5-FULH found
range that
from 34%thetohigher the concentration
62% with of polymer
different weight ratios of and monome
Gels 2022, 8, 47 8 of 18

2.8. Porosity Measurement

The porosity values of 5-FULH range from 34% to 62% with different weight ratios of
AM, AA, and GA. It was found that the higher the concentration of polymer and monomer,
the higher the porosity values. While enhancing the crosslinker concentration, the porosity
values decrease. Table 3 (B3) shows a higher % of gel fraction (94 ± 0.2%) with a higher %
of porosity (62 ± 0.06%); a similar type of relationship was found in previous studies. The
controlled release of the drug, around 65% at 25 h, could be attributed to the reduction of
water entry and subsequent diffusion of the drug from the hydrogel network [39].
Table 3. Diffusion coefficient, porosity %, gel fraction %, and sol fraction % of formulated 5-FULH.

F. Code DC (cm2 /s) Porosity % Gel Fraction % Sol Fraction %

A1 0.19 ± 0.02 34 ± 0.03 74 ± 0.4 24 ± 0.1
A2 0.09 ± 0.03 42 ± 0.04 86 ± 0.3 22 ± 0.2
A3 0.05 ± 0.01 55 ± 0.02 90 ± 0.4 15 ± 0.1
B1 0.08 ± 0.01 41 ± 0.03 85 ± 0.5 17 ± 0.3
B2 0.07 ± 0.03 43 ± 0.04 91 ± 0.3 11 ± 0.2
B3 0.03 ± 0.02 62 ± 0.06 94 ± 0.2 09 ± 0.3
C1 0.15 ± 0.01 39 ± 0.05 83 ± 0.4 16 ± 0.4
C2 0.09 ± 0.03 39 ± 0.04 89 ± 0.4 11 ± 0.4
C3 0.07 ± 0.02 47 ± 0.03 93 ± 0.3 09 ± 0.3

2.9. Gel Fraction

Table 3 shows the results of gel and sol fraction. In relation to the value of gel fraction,
it was observed that the gel fraction depended on the AM and AA. The gelling strength of
the prepared 5-FULH may increase with the higher content of AM, AA, and GA. As the
concentration of the AA is increased, the polymerization reaction is also enhanced due to
the accessibility of more binding sites. The higher gel fraction is attributed to the increased
bulk density of the hydrogel structure. A firm and robust hydrogel network is established
due to the higher concentration of polymer, resulting in a greater degree of crosslinking
which in turn leads to a lower porous structure of hydrogels [40]. However, sol fraction is
decreased since the concentration of AA and GA is increased due to the inverse relationship
with gel fraction [41].

2.10. Drug Loading

Drug loading was determined by using swelling, extraction, and weight values. The
amount of 5-FU was presented as g/g of dry gel and the data were presented in Table 4.
Table 4. Amount of 5-Flurouracil loaded in different formulations of acrylamide/acrylic acid hydrogels.

Amount of 5-FU Loaded (g/g of Dry Gel)

F. Code
By Swelling By Extraction By Weight
A1 0.0394 0.0373 0.0347
A2 0.0402 0.0392 0.0369
A3 0.0416 0.0423 0.0391
B1 0.0372 0.0393 0.0357
B2 0.0349 0.0385 0.0341
B3 0.0331 0.0377 0.0328

2.11. Cumulative Drug Release Measurement

From the polymeric 5-FULH, it can be seen that the release pattern of a formulation
depends on the swelling behavior of these hydrogels. This section discusses some of the
factors which influence the release of 5-FU from the prepared hydrogels.
Gels 2022, 8, 47 9 of 18

2.12. Effect of AM
These 5-FULH were designed by varying AM concentrations at 37 ◦ C and pH of 1.2
and 7.4. The release of 5-FU is very low in an acidic medium, increasing to 67% in a basic
medium. This might be due to greater swelling in a basic medium and less swelling in
an acidic medium. Mundargi et al. reported that the release of 5-FU increases when AM
content increases in the matrix [42]. Figure 8 shows that drug release was varied based on
the pH of the medium i.e., around 20% drug release was found at 1 h in an acidic media
(pH 1.2), whereas >65% of drug release (pH 7.4) was observed at around 25 h from or with?
the 5-FULH containing a higher amount of AM due to increased polymer chain flexibility,
Gels
Gels2022, 8, 8,
2022, x FOR PEER
x FOR REVIEW as shown in Figure 9. This release behavior of the drug was in agreement with the previous
REVIEW
PEER 1010ofof1919
study where single polymeric chains in pure AM allowed higher drug transportation
compared to the co-polymeric chain [43].

2020 A1 A2 A3
A1 A2 A3
1818
1616
Average Release (%)
Average Release (%)

1414
1212
1010
88
66
44
22
00
00 200
200 400
400 600
600 800
800 1000
1000 1200
1200 1400
1400
Time (min)
Time (min)
Figure AtAt
8. 8.
Figure pH 1.2,
pH effect
1.2, ofof
effect AM onon
AM 5-FU release.
5-FU release.

7070 A1
A1 A2
A2 A3
A3

6060
% drug release
% drug release

5050

4040

3030

2020

1010

00
00 300
300 600
600 900
900 1200
1200 1500
1500
Time
Time(min)
(min)
Figure 9. At pH 7.4, effect of AM on 5 FU release profile.
Figure 9. 9.
Figure AtAt
pH 7.4,
pH effect
7.4, ofof
effect AM onon
AM 5 FU release
5 FU profile.
release profile.

2.13.
2.13.Effect
EffectofofAA
AA
Figures
Figures 10and
10 and1111present
presentthe
therelease
releaseprofile
profileofof5-FU
5-FUunder
underthe
theinfluence
influenceofofAA.
AA.The
The
higher
higher the concentration of AA, the higher the release of 5-FU. Enhancing the quantityofof
the concentration of AA, the higher the release of 5-FU. Enhancing the quantity
Gels 2022, 8, 47 10 of 18

2.13. Effect of AA
Figures 10 and 11 present the release profile of 5-FU under the influence of AA. The
higher the concentration of AA, the higher the release of 5-FU. Enhancing the quantity of
polymer AA increases the release rate of 5-FU due to the development of channels and
Gels 2022,
Gels 8, 8,
2022, x FOR PEER
x FOR REVIEW
PEER REVIEW pores in the hydrogel matrix resulting from relaxation of the polymer network. This 1111
of of1919
result
is in agreement with previous studies where drug release was enhanced with the higher
pH and the amount of AA [44].
3030
B1B1 B2B2 B3B3
2525
Average Release (%)
Average Release (%)

2020

1515

1010

55

00
00 200
200 400
400 600
600 800
800 1000
1000 1200
1200 1400
1400
Time
Time(min)
(min)
Figure
Figure 10.
Figure
10. Effect
10.
Effect of
Effect AA
ofof
AA on
AA the
onon release
the
the profile
release
release of
profile
profile 5-FU
ofof at
5-FU
5-FU pH
atat
pH 1.2.
pH 1.2.
1.2.

8080 B1B1 B2B2 B3B3

7070
% drug release
% drug release

6060

5050

4040

3030

2020

1010

00
00 300
300 600
600 900
900 1200
1200 1500
1500
Time
Time(min)
(min)
Figure 11. Effect of AA on the release profile of 5-FU at pH 7.4.

2.14.
FigureEffect
11.11.
Figure
of GA
Effect ofof
Effect AA onon
AA the release
the profile
release ofof
profile 5-FU atat
5-FU pH 7.4.
pH 7.4.
Figures 12 and 13 describe the release of 5-FU under different concentrations of GA.
The
2.14. release
2.14.Effect of
Effectofof
GA5-FU in an acidic medium was almost the same irrespective of different
GA
concentrations
Figures ofand
Figures1212and GA. The release
1313describe
describe the of
the 5-FU of
release
release was lower
of5-FU
5-FU with
under
under a higherconcentrations
different
different concentration
concentrationsofof GA
ofGA.
GA.
when
The it
Thereleasecomes to
releaseofof5-FU basic medium
5-FUininananacidic pH
acidicmedium7.4. This
mediumwas could
wasalmost be attributed
almostthe thesame to the higher
sameirrespective H-bonding
irrespectiveofofdifferent
different
and lower swelling
concentrations
concentrations of 5-FULH
ofofGA.
GA. The withofaof5-FU
Therelease
release high
5-FU concentration
waswaslower
lowerwithof a
with crosslinking
ahigher agent. It has
higherconcentration
concentration been
ofof
GAGA
reported
when that the gelation ratio is faster in GA crosslinked hydrogel, therefore higher GA
whenit itcomes
comestotobasic
basicmedium
mediumpH pH7.4.
7.4.This
Thiscould
couldbebeattributed
attributedtotothethehigher
higherH-bonding
H-bonding
and
andlower
lowerswelling
swellingofof5-FULH
5-FULHwith witha ahigh
highconcentration
concentrationofofcrosslinking
crosslinkingagent.
agent.It Ithas
hasbeen
been
reported
reportedthat thatthe
thegelation
gelationratio
ratioisisfaster
fasterininGA
GAcrosslinked
crosslinkedhydrogel,
hydrogel,therefore
thereforehigher
higherGA GA
concentration
concentrationincreases
increasesthethedrug
drugloading
loadingcapacity
capacity[45].[45].AsAsseen
seenininFigure
Figure13,
13,the
the5-FU
5-FUre-re-
lease
leasepercentages
percentagesfrom fromthethe5-FULH
5-FULHatatpH pH7.4
7.4were
weremoremorethan
than50%50%ofofthe
thepH
pH1.21.2atat18%
18%inin
 Gels 2022, 8, 47 11 of 18

concentration increases the drug loading capacity [45]. As seen in Figure 13, the 5-FU
release percentages from the 5-FULH at pH 7.4 were more than 50% of the pH 1.2 at
Gels 2022, 8, x FOR PEER REVIEW 18% in every formulation. The present findings are in line with the previous data which 12 of 19
investigated 5-FU loaded GA blended pH-responsive hydrogel and found 5-FU release of
Gels 2022, 8, x FOR PEER REVIEW around 64.0% to 85.99% at pH 7.4 but only 13.33% to 19.64% at pH 1.2 [46]. 12 of 19

30
C1 C2 C3
30
25 C1 C2 C3
25
Average release (%)

20
Average release (%)

20
15
15
10
10
5
5
00
00 200
200 400
400 600 800
600 800 1000
1000 1200
120014001400
Time(min)
Time (min)
Figure
Figure 12.Effect
12.
Figure12. Effectof
Effect ofGA
of GAon
onthe
on therelease
thereleaseprofile
releaseprofileofof5-FU
profile 5-FU
of atatpH
5-FU at1.2.
pH 1.2. 1.2.
pH

80 80 C1C1 C2 C2 C3 C3

70 70
60
60
% drug release

50
% drug release

50
40
40
30
30
20
20 10
10 0
0 200 400 600 800 1000 1200 1400
0
0 200 400 600 Time (min)
800 1000 1200 1400

Time profile
Figure 13. Effect of GA over 5-FUrelease (min)at pH 7.4.
Figure 13. Effect of GA over 5-FUrelease profile at pH 7.4.
The protonation of sulfonate ions occurs at acidic pH 1.2, which results in the gener-
ation of strong hydrogen bonds and physical
occurs interaction between the functional groups
Figure The protonation
13. Effect of sulfonate
of GA over ions
5-FUrelease profile at
at acidic
pH 7.4.pH 1.2, which results in the gener-
ofation
the hydrogel, causing a subsequent reduction in
of strong hydrogen bonds and physical interaction between swelling [47]. the
However, at pH
functional 7.4,
groups
deprotonation
of the of sulfonate ions occurs, owing to the enhanced electronic density on the
Thehydrogel,
polymeric protonationcausingsulfonate
structure andofless
a subsequent
physical ions
reduction at in
occursamong
interaction
swelling
acidic [47].
pH −1.2,
the –SO
However,
which
3 moieties,
at pH
results
thereby
7.4,gener-
in the
gener-
deprotonation of sulfonate ions occurs, owing to the enhanced electronic density on the
ation
ating aofgreater
strongdegree
hydrogen bonds
of swelling and5-FULH
of AA physical interaction
[48]. All of thesebetween the reduction
factors cause functional in groups
polymeric structure and less physical interaction among the –SO−3 moieties, thereby gen-
of the hydrogel,
intermolecular causingbonding,
hydrogen a subsequent
resultingreduction in swelling
in higher swelling [47]. However,
[49]. Likewise, the COOH at pH 7.4,
erating a greater degree of swelling of AA 5-FULH [48]. All of these factors cause reduc-
groups of AA are
deprotonation ofprotonated
sulfonate ations
pH 1.2 whichowing
occurs, leads toto a the
fall in swelling. electronic
enhanced However, atdensity
higher on the
tion in intermolecular hydrogen bonding, resulting in higher swelling [49]. Likewise, the
polymeric
COOH groups structure
of AAand less physical
are protonated interaction
at pH 1.2 whichamongleads tothe –SO
a fall
−3 moieties, thereby gen-
in swelling. However,
erating
at higher pH 7.4, carboxylic acid (CA) functionalities are deprotonated, resulting in cause
a greater degree of swelling of AA 5-FULH [48]. All of these factors higherreduc-
tion in intermolecular
swelling of the 5-FULHhydrogen
[50]. Also, bonding,
the osmotic resulting
pressurein of higher
the ionsswelling
is increased[49].
dueLikewise,
to the the
increase
COOH in protonation
groups of AA are of protonated
the COOH groups at pH[51]. The carboxylic
1.2 which leads toacid
a fall(CA) moieties were
in swelling. However,
Gels 2022, 8, 47 12 of 18

pH 7.4, carboxylic acid (CA) functionalities are deprotonated, resulting in higher swelling
of the 5-FULH [50]. Also, the osmotic pressure of the ions is increased due to the increase
in protonation of the COOH groups [51]. The carboxylic acid (CA) moieties were converted
to the salt form which led to maximum swelling, as the pH of the medium was elevated
from a lower to a higher number [52].

2.15. Release Kinetics

Several kinetic models were employed to help understand the release mechanism
of the 5-FU from the hydrogels, and the regression coefficients (r) were selected for the
evaluation of the most suitable drug release [53]. The (r) values of most samples were
greater for zero-order kinetics compared to the first-order kinetics, which could be ascribed
to the variation in the amounts of AA and GA. From the results of the Higuchi model, it
was obvious that the release of the drug follows the diffusion-controlled release mechanism.
Among the models, the (r) values of 5-FULH for zero order (0.9632–0.9954) was compre-
hensively higher than the first order (0.9378–0.9932). The results indicated that the release
pattern of the drug corresponded to the zero-order kinetics.

3. Conclusions
The 5-FULH were processed for oral delivery using AM, AA and GA crosslinked.
These prepared 5-FULH were physio-chemically characterized. ATR-FTIR shows no such
interaction between the excipients and the model drug (i.e., 5-FU). Swelling studies show
that minimum swelling was achieved at acidic pH and maximum at alkaline Ph, which
depends on the monomer, polymer, and crosslinking agent. By increasing AM and AA
concentrations, swelling increased, whereas by increasing GA, it decreased. In vitro release
studies show that the most drug was released on alkaline pH, whereas the least drug was
released on acidic pH. The release of the drug also increases with increasing AM and AA
content but decreases with an increase in cross linker. The drug release kinetics models
followed a non-Fickian order of release. These findings concluded that AM and AA based
5-FULH would be appropriate for controlled drug delivery with pH reactive characteristics.

4. Materials and Methods

4.1. Materials
5-Fluorouracil (5-FU) was obtained from Biolabs Pharma Pvt. Ltd., (Biolabs Pharma,
Islamabad, Pakistan). Acrylamide (AM), acrylic acid (AA), glutaraldehyde (GA), disodium
hydrogen phosphate, sodium hydroxide, and monobasic potassium phosphate were pur-
chased from Sigma Aldrich (Sigma Aldrich, Darmstadt, Germany). Hydrochloric acid and
acetic acid were supplied by Icon Chemicals (Icon Chemicals, Ludhiana, Punjab, India).

4.2. Pre-Formulation Studies and Standard Curve

The stock solution was prepared using 10 mg of 5-FU dissolved in 100 mL of phosphate
buffer pH 7.4. A series of dilutions were made in descending order using phosphate buffer
pH 7.4. Samples from such mixtures were taken and spectrophotometrically analyzed at
256 nm (Shimadzu 601, Japan). The results were taken in triplicate and plotted.

Y = MC + B (1)

where:
Y = the absorbency of the solution, M = the angle of standard curve of identified
concentration, C = concentration that must be calculated and B = the curve’s cut off.

4.3. Solubility Study of Drug

The solubility study of 5-FU was conducted using different solvents of varying pH at 37 ◦ C.
Gels 2022, 8, 47 13 of 18

4.4. Preparation of 5-FULH

The 5-FULH were produced by a simple crosslinking method, with slight modifications
as presented in Table 5. In the first step a specific amount of distilled water was put into a
beaker and placed on a magnetic stirrer and heated. Thereafter the AM was poured into
the beaker and heated up till a clear solution was achieved. Similarly, by applying slight
modifications in temperature as well as in revolution time, an AA solution was obtained.
These solutions were then thoroughly mixed with continuous stirring on a magnetic stirrer
until a clear homogeneous liquid was produced. Finally, by adding distilled water, the
volume of the solution was made up to a hundred milliliters. Consequently, in a drop
wise method and with regular stirring, cross linker was added to the mixture, to produce a
uniform clear solution. This solution was then poured into the deoxygenized test tubes
and left to congeal in them for four days. The 5-FULH, once they were formed, were then
removed from the tubes. With the help of sharpened blades, these gels were cleaved as
well as properly scrubbed with Methanol. The discs were then put into petri dishes and
desiccated in an oven at 40 ◦ C for seventy-two hours [54,55].
Table 5. Composition of formulated 5-FULH.

F. Code AM AA GA TEMED APS D/Water

A1 4.08 g 0.48 g 23 µL 235 µL 0.20 g 13 mL
A2 5.88 g 0.48 g 23 µL 235 µL 0.20 g 13 mL
A3 7.5 g 0.48 g 23 µL 235 µL 0.20 g 13 mL
B1 4.08 g 0.96 g 23 µL 235 µL 0.20 g 13 mL
B2 4.08 g 1.5 g 23 µL 235 µL 0.20 g 13 mL
B3 4.08 g 2g 23 µL 235 µL 0.20 g 13 mL
C1 4.08 g 0.48 g 19 µL 235 µL 0.20 g 13 mL
C2 4.08 g 0.48 g 13 µL 235 µL 0.20 g 13 mL
C3 4.08 g 0.48 g 7 µL 235 µL 0.20 g 13 mL

4.5. Hydrogel’s Characterizations

4.5.1. ATR-FTIR
Physical interactions between drug and excipients were investigated using ATR-FTIR
(Perkin Elmer, Waltham, MA, USA). The samples were placed on ATR-FTIR and scanned
from 400–4000 cm−1 [56].

4.5.2. Dynamic Swelling Study

Dried slices of these preparations were properly weighed and immersed into buffers of
various pH (i.e., pH 7.4, pH 6.8, and pH 1.2). By removing discs from the medium, readings
were noted over regular time periods [57,58]. The surface water was removed from the
5-FULH using tissue paper. The discs were dipped again in the pH 6.8 and 7.4 buffer, after
having been weighed at definite intervals [59]. The following formula was used to obtain
the dynamic S:
Ws
S= (2)
Wd
where:
Ws = weight of swollen gel at specific time, Wd = weight of dry hydrogel, and
S = swelling ratio.

4.5.3. Equilibrium Swelling Study

ES studies were also performed on the 5-FULH, which were prepared for frequent
swelling. The % ES was determined using the following equation [60,61].

Ws − Wd
ES (%) = × 100 (3)
Wd
where:
Gels 2022, 8, 47 14 of 18

ES = equilibrium swelling, Ws = weight of swollen gel at specific time, and Wd = weight

of dry hydrogel.

4.5.4. Diffusion Coefficient (DC)

DC is the quantity of solvent that was absorbed/diffused across the unit area in unit
time through a concentration gradient [62,63]. The diffusion coefficient was calculated by
Formula (4):
2
h·θ

D=π (4)
4· Qeq
where:
D = DC of the 5-FULH, Qeq = the hydrogel’s swelling at equilibrium, θ = the angle of
straight portion of swelling curves and h = early sample thickness.

4.5.5. Sol Gel Fraction Analysis

Sol-gel analysis technique was performed for predisposing reactant quantity used
during the preparation of the 5-FULH. The prepared 5-FULH were dried without washing
and placed in deionized water at ambient temperature until constant weight was attained.
After that, extracted hydrogel was taken out and dried in an oven at 60 ◦ C [64,65].

M2 − M1
Sol fraction (SF) (%) = × 100 (5)
M1

where:
M2 = Final/extracted gel wt, M1 = Initial wt of dry gel and

Gel f raction = 100 − SF (6)

4.5.6. Porosity Measurement

Porosity is an important consideration mainly affecting the swelling attributes of the
5-FULH. The % porosity was calculated by solvent replacement technique. Hydrogel discs
were dried and soaked in ethanol (100%) overnight. Extra ethanol was removed using
blotting paper and then the 5-FULH were weighed [66]. The porosity was calculated and
attained by using the following equation:

Mt − Mo
Porosity (%) = × 100 (7)
ρv

where:
Mt = weight before immersion, Mo = weight after immersion, ρ = density of absolute
ethanol and v = volume of hydrogel.

4.5.7. Drug Loading

Samples which showed maximum swelling were used for drug loading and release
studies. The drug loading into the discs of hydrogel was achieved by soaking them for
one week in a solution of the drug. A 1% w/v 5-FU in pH 7.4 solution was used for drug
loading. After achieving the equilibrium value, the swelled 5-FULH were removed from
the drug solution, blotted with filter paper, first dried at room temperature, and then placed
in an oven at 40–45 ◦ C for one week to remove the absorbed solvent. To determine the
percentage of drug-loading, weighed drug loaded samples were extracted repeatedly using
a phosphate buffer solution of pH 7.4 up to exhaustion, and the concentration of the drugs
in pooled extract was determined spectrophotometrically at λ max 256 nm. The quantity of
drug loaded into the 5-FULH was also determined by the swelling method [67].
Gels 2022, 8, 47 15 of 18

4.5.8. In Vitro Release Study

To assess the amount of released drug from formulated 5-FULH, in vitro release studies
were performed, using dissolution apparatus (pharma test; Pt-Dt 7). These hydrogel discs
were positioned in a dissolution medium of 500 mL at 37 ◦ C temp in a pH of 1.2 and 7.4 and
shaken at a hundred revolutions per minute. Samples were selected at specific time periods
and replaced. These samples were then evaluated at 256 nm using a UV-spectrophotometer
(Shimadzu) [68].

4.5.9. Release Kinetics

To investigate the discharge of medicament from the gels and its mechanism, various
kinetic models were applied [58], which are given below.
First order kinetics ln (1 − F) = K1t
Zero order kinetics Mo − Mt = Ko t
Higuchi model Q = K2 t1/2
Korsmyer Peppas Model Mt/Mœ = Ktn

4.6. Statistical Analysis

Findings from all of the experiments were made in triplicate and presented as the
means ± S.D. These samples were analyzed statistically with the help of one-way ANOVA
and t-test. At p < 0.05, differences were considered as significant.

Author Contributions: Conceptualization, A.K.A., and M.S.; methodology, M.A.K., A.K.A., M.S.,
A.N., and M.A.; software, M.A., A.K.A., and M.S.; validation, A.K.A.; formal analysis, M.A., and
A.K.A.; investigation, M.A.K, M.A., and M.S.; resources, M.S., A.N., and M.A.; data curation, A.K.A.;
writing—original draft preparation, M.A.K, A.K.A., M.A., and M.S.; review and editing, A.K.A., P.P.,
M.K.H., R.S.B., A.F.E.-k., M.K., S.G.B., M.M.A.-D., and M.H.R.; visualization, A.K.A.; supervision,
M.S., A.N., and M.A.; project administration, A.K.A., and M.S.; Funding, A.K.A., and M.M.A.-D. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Taif University Researchers Supporting Program (Project
number: TURSP-2020/269), Taif University, Saudi Arabia. The authors would like to thank the
Deanship of Scientific Research at King Khalid University, Abha, KSA, for funding this work under
grant number (R.G.P.1/62/42).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data will be available upon request.
Acknowledgments: This work was supported by the Taif University Researchers Supporting Pro-
gram (Project number: TURSP-2020/269), Taif University, Saudi Arabia. The authors would like to
thank the Deanship of Scientific Research at King Khalid University, Abha, KSA, for funding this
work under grant number (R.G.P.1/62/42).
Conflicts of Interest: Authors have declared no conflict of interest with other forms of study.

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