gels

Article
Development and Characterization of Gel-Based Buccoadhesive
Bilayer Formulation of Nifedipine
M. Alagusundaram 1, * , Nem Kumar Jain 2 , M. Yasmin Begum 3, * , S. Angala Parameswari 4 ,
Vinod Kumar Nelson 5 , Mohammad F. Bayan 6 and Balakumar Chandrasekaran 6

1 Department of Pharmaceutics, School of Pharmacy, ITM University, Gwalior 474001, Madhya Pradesh, India
2 Department of Pharmacology, School of Pharmacy, ITM University, Gwalior 474001, Madhya Pradesh, India;
nemjain.pharma@itmuniversity.ac.in
3 Department of Pharmaceutics, College of Pharmacy, King Khalid University, Abha 61421, Saudi Arabia
4 Department of Pharmaceutical Analysis, Jagan’s Institute of Pharmaceutical Sciences,
Nellore 524346, Andhra Pradesh, India; eswarialagusundaram@gmail.com
5 Department of Pharmaceutical Chemistry, Raghavendra Institute of Pharmaceutical Education and Research,
Anantapuramu 515721, Andhra Pradesh, India; vinod.kumar457@gmail.com
6 Faculty of Pharmacy, Philadelphia University, P.O. Box 1, Amman 19392, Jordan;
mbayan@philadelphia.edu.jo (M.F.B.); balakumar@philadelphia.edu.jo (B.C.)
* Correspondence: alagusundaram.sop@itmuniversity.ac.in (M.A.); ybajen@kku.edu.sa (M.Y.B.)

Abstract: A promising controlled drug delivery system has been developed based on polymeric
buccoadhesive bilayered formulation that uses a drug-free backing layer and a polymeric hydrophilic
gel buccoadhesive core layer containing nifedipine. The DSC thermogravimetric analysis confirms
the drug’s entrapment in the gel layer and reveals no evidence of a potential interaction. Various
ratios of bioadhesive polymers, including HPMC K100, PVP K30, SCMC, and CP 934, were combined
with EC as an impermeable backing layer to ensure unidirectional drug release towards the buccal
mucosa. The polymeric compositions of hydrophilic gel-natured HPMC, SCMC, and CP formed a
matrix layer by surrounding the core nifedipine during compression. Preformulation studies were
performed for all of the ingredients in order to evaluate their physical and flow characteristics. Ex
vivo buccoadhesive strength, surface pH, swelling index, in vitro and in vivo drug release, and
Citation: Alagusundaram, M.; Jain, ex vivo permeation investigations were performed to evaluate the produced gel-based system.
N.K.; Begum, M.Y.; Parameswari, Rapid temperature variations had no appreciable impact on the substance’s physical properties,
S.A.; Nelson, V.K.; Bayan, M.F.; pharmacological content, or buccoadhesive strength during stability testing using actual human
Chandrasekaran, B. Development saliva. It was clear from a histological examination of the ex vivo mucosa that the developed system
and Characterization of Gel-Based
did not cause any irritation or inflammation at the site of administration. The formulation NT5 was
Buccoadhesive Bilayer Formulation
the best one, with a correlation coefficient of 0.9966. The in vitro and in vivo drug release profiles
of Nifedipine. Gels 2023, 9, 688.
were well correlated, and they mimic the in vitro drug release pattern via the biological membrane.
https://doi.org/10.3390/gels9090688
Thus, the developed gel-based formulation was found to be novel, stable, and useful for the targeted
Academic Editor: Gary E. Wnek delivery of nifedipine.
Received: 21 July 2023
Revised: 20 August 2023
Keywords: nifedipine; buccoadhesive; triggered delivery; ex vivo permeation; polymeric gel
Accepted: 23 August 2023
Published: 26 August 2023

1. Introduction
The main goal of any drug delivery system is to deliver the medication precisely and
Copyright: © 2023 by the authors.
efficiently to the body’s site of action. This system should be able to trigger and control
Licensee MDPI, Basel, Switzerland.
the drug release at the intended spot, increasing drug availability, increasing therapeutic
This article is an open access article
efficacy, minimizing potential side effects, and reducing dose frequency [1]. The buccal
distributed under the terms and
portion of the mouth cavity is a desirable target for medication delivery [2], and the
conditions of the Creative Commons
administration of the desired medication via the buccal mucosal membrane lining of the
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
mouth cavity is known as buccal delivery.
4.0/).

Gels 2023, 9, 688. https://doi.org/10.3390/gels9090688 https://www.mdpi.com/journal/gels

Gels 2023, 9, 688 2 of 17

The mucosal lining of buccal tissues provides a significantly more hospitable envi-
ronment for drug absorption than oral drug delivery, with the latter presenting a hostile
environment for medications, especially proteins and polypeptides, due to acid hydrol-
ysis and the hepatic first-pass effect [3,4]. By overcoming the limitations of traditional
administration routes, the buccal mucosa is a valuable channel for treating either local
or systemic medicines. The buccal (inside the cheeks), sublingual (under the tongue),
and gingival (on the gums) areas of the oral cavity are the places where medications are
administered [5,6]. The best areas for medication delivery are the buccal and the sublingual
sectors, which can be used to treat either systemic or local disorders. The buccal mucosa
is a well-vascularized, easily accessible tissue for both administering and removing a de-
livery device [7,8]. This involves the ability to construct poly or single directional release
systems for local or systemic activities, and to incorporate permeability enhancers, enzyme
inhibitors, or pH modifiers in the formulation [9,10]. The oral mucosa is well supplied
with blood. After being absorbed from the oral cavity by the oral mucosa, drugs reach the
systemic circulation via the deep lingual or the facial vein, the internal jugular vein, and
the brachiocephalic vein [11]. Following buccal delivery, the drug enters the bloodstream
directly, thereby skipping the first-pass effect.
The three-dimensional polymeric networks of hydrophilic polymeric gel can absorb
large to small amounts of active pharmaceutical ingredients, and, as a result, they are
widely used to deliver medicines that are more biocompatible and have less discharge [12].
The main reason that HPMC (Hydroxy Propyl Methyl Cellulose), SCMC (Sodium Car-
boxy Methyl Cellulose), and CP (Carbopol) are so good at absorbing water is because
their molecular chains have hydrophilic groups, such as amino, amide, hydroxyl, and
carboxyl groups [13]. Polymeric hydrophilic gel systems have been shown to have different
physicochemical parameters, such as porosity, structural softness, swelling capacity, and
elasticity, because they have these hydrophilic groups. Bioadhesion occurs when two
surfaces, one of which must be a mucous tissue or membrane, come into contact with a
polymeric hydrophilic gel-based matrix layer, and this is the ability of a material to adhere
to the surface of the buccal mucosal layer [14]. When glandular columnar epithelial cells
release mucosal layers, they primarily consist of mucin and water with trace amounts of
lipids, other proteins, and muco-polysaccharides. More specifically, when thinking about
epithelial tissue or the mucus coat on the surface of a tissue, the connection to a mucus
coat is formed by mucoadhesive gel in a hydrophilic environment. The mechanism of
the bioadhesion process occurs in three steps: the wetting and swelling of the polymer
to form a hydrophilic gel by absorbing the mucin (Step 1), the interpenetration of the gel
chains and the mucosal membrane (Step 2), and there chemical bond formation between the
entangled chains (Step 3) [15]. The schematic illustrations of the above steps are represented
in Figure 1.
Nifedipine is a calcium channel blocker of the dihydropyridine type, and it is mainly
used to treat hypertension and angina pectoris [16]. Due to its short elimination time of
2–4 h, nifedipine is a good choice for controlled release delivery. The primary objective
of this study was to formulate and characterize polymeric buccoadhesive gel-based bi-
layer formulations that would release nifedipine in a unidirectional pattern at the site of
administration for an extended period of time without being washed away by saliva.
Gels 2023,9,9,688
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Figure1.
Figure 1. Mechanism
Mechanism of
of bioadhesion
bioadhesionincludes
includeswetting
wetting and
and swelling,
swelling, interpenetration
interpenetrationand
andformation
formation
ofchemical
of chemicalbonds.
bonds.

2.
2. Results
Results and
and Discussions
Discussions
The
The purpose of
purpose of this
this research
research was
was to
to both
both produce
produce and
and evaluate
evaluate polymeric
polymeric buccoad-
buccoad-
hesive
hesive gel-based
gel-based bilayer
bilayer formulations
formulations that
that deliver
deliver the
the drug
drug in
in aaunidirectional
unidirectionalpattern
pattern at
at
the
the site
siteof
ofadministration
administration over
over time.
time. Using
Using HPMC-K100M,
HPMC-K100M, SCMC, SCMC, PVP-K30,
PVP-K30, and
and CP934,
CP934,
the
the bilayer
bilayer gel-based systems were
gel-based systems werecreated
createdthrough
throughdirect
directcompression
compression[17,18].
[17,18].Because
Becauseof
of its minimal water permeability and its flexibility in the buccal environment,
its minimal water permeability and its flexibility in the buccal environment, EC was cho- EC was
sen as the backing layer [19]. The HPMC, SCMC, and CP form a gel coating over the
Gels 2023, 9, 688 Gels 2023, 9, x FOR PEER REVIEW 4 of 17 4

chosen as the backing

nifedipinelayer [19]. The
nucleus. WhenHPMC, SCMC, and
these polymers areCP form a with
hydrated gel coating
water, over the viscou
they form
nifedipine nucleus. When these polymers are hydrated with water, they form viscous
with a long duration of retention on mucosal surfaces, resulting in the formation of a
gels with a long duration
sive contactsof[20].
retention on mucosal
In conjunction surfaces,
with resulting
an increase in concentration
in the the formation ofof the HPM
adhesive contacts [20]. In conjunction with an increase in the concentration of the HPMC,
change in the morphology of nifedipine from cubical to plate-like was observed, and
a change in theled
morphology of nifedipine
to an increase from cubical
in the dissolution rate to plate-like
during the inwas observed,
vitro and this
release experiments.
led to an increase in the dissolution rate during the in vitro release experiments.
2.1. Drug Polymer Interaction Studies through DSC
2.1. Drug Polymer Interaction Studies through DSC
DSC is used for investigation into any physicochemical interactions that may
DSC is used for investigation into any physicochemical interactions that may exist
between the drug and the matrix polymer. Thermal analysis of nifedipine alone and
between the drug and the matrix polymer. Thermal analysis of nifedipine alone and
in the presence of various polymers is performed in an incremental series of weight
also in the presence of various polymers is performed in an incremental series of weight
tions. Thermogram of the first run indicated that the melting point of pure nifedip
fractions. Thermogram of the first run indicated that the melting point of pure nifedipine
approximately 173 °C as an endothermic peak. Due to the dehydration process o
is approximately 173 ◦ C as an endothermic peak. Due to the dehydration process of the
HPMC, a large, broad endothermic effect was observed over a temperature range o
HPMC, a large, broad endothermic effect was observed over a temperature range of 100 to
to 120 °C, with a shift to 112.2 °C as a larger endothermic peak [21]. The difference i
120 ◦ C, with a shift to 112.2 ◦ C as a larger endothermic peak [21]. The difference in the
chemical composition of cellulose in the SCMC causes the observed difference in the
chemical composition of cellulose in the SCMC causes the observed difference in thermal
decomposition behavior and thermal stability. The thermogram of the SCMC show
decomposition behavior and thermal stability. The thermogram of the SCMC shows the
fraction
fraction of weight of weight
loss as loss as 80.4%,
80.4%, indicating thatindicating that the neat
the neat cellulose cellulose
and the SCMCand the SCMC con
contains
the fraction of non-volatile components. The thermal decomposition process of cellulose of cell
the fraction of non-volatile components. The thermal decomposition process
occurs approximately
occurs approximately 189 °C as anpeak.
189 ◦ C as an endothermic endothermic peak. Theofthermogram
The thermogram carbopol showsof carbopol s
a broad endothermic peak at 241 °C, which indicates the evaporation
a broad endothermic peak at 241 ◦ C, which indicates the evaporation of adsorbed water of adsorbed w
on CP 934, and, on after,
CP 934, theand, after, point
melting the melting point decomposition
decomposition begins. Thebegins. The DSCof
DSC diagram diagram o
physical mixture of nifedipine-polymer mixture shows a
the physical mixture of nifedipine-polymer mixture shows a similar sharp well-defined similar sharp well-define
endothermic peakdothermic peak ofwhich
of nifedipine, nifedipine, which
indicates thatindicates that no
no interaction interaction
persists betweenpersists
the betwee
drug and polymer. The DSC thermogram of nifedipine, HPMC K 100, SCMC, CP 934, and CP 934
drug and polymer. The DSC thermogram of nifedipine, HPMC K 100, SCMC,
nifedipine-polymer
nifedipine-polymer mixture is shownmixture is shown
in curves A, B,inC,curves
D, andA,EB,inC, D, and
Figure 2, Erespectively,
in Figure 2, respect
and is individually shown in Figures
and is individually shown in Figures S1–S5, respectively. S1–S5, respectively.

Figure 2. DSC thermogram: (A) Nifedipine (173.0 ◦ C); (B) HPMC K 100 (112.2 ◦ C); (C) SCMC
Figure 2. DSC thermogram: (A.) Nifedipine (173.0 °C); (B.) HPMC K 100 (112.2 °C); (C.) SCMC
(189.0 ◦ C); (D) CP 934 (241.0 ◦ C); (E) Nifedipine-polymer mixture.
°C); (D.) CP 934 (241.0 °C); (E.) Nifedipine-polymer mixture.
Gels 2023, 9, 688 5 of 17

2.2. Physicochemical Evaluation of Buccoadhesive Bilayered Formulations

Following the procedures that are outlined in the pharmacopoeia, the prepared gel-
based formulation was characterized for thickness, weight variation, hardness, and friability
in order to determine the stability of the product during transportation, packaging, and
storage. All of the formulations revealed an acceptable thickness, weight uniformity, rigid-
ity, friability, and drug content as per the accredited pharmacopoeia. In order to estimate
the amount of the drug to be used, the prepared formulations were evaluated for drug
content in triplicate, and the drug content was found to be in a range between 19.78 ± 0.22
to 20.32 ± 0.21 mg. Using polymers, the hardness of the formulations was maintained
within a range of 4.0 to 4.3 kg/cm2 . Concentrations of the HPMC and film-forming PVP
resulted in a marginal increase in hardness. Changes in hardness had no significant effect
on the release profile of the nifedipine. The observed results indicated reproducibility with
minimum intra-batch variability. The formulation NT5 comprised of HPMC K100, and
PVP K30 was discovered to have a hardness of 4.2 kg/cm2 . Due to its strong and viscous
gel layer, HPMC is frequently used for the preparation of hydrophilic matrix gels [22].
This layer shields the matrix from rapid disintegration, and it regulates the rate of drug
release without altering the release profile. Compared to all of the other formulations,
NT5 exhibited superior performance across all of the physicochemical parameters. The
post-compressional evaluation results are presented in Table 1 below.

Table 1. Physicochemical evaluation of buccoadhesive bilayer formulations of nifedipine.

Formulation Thickness Weight Variation Hardness Friability Drug Content Surface pH ±

Code (mm) ± SD mg ± SD (Kg/cm2 ) (%) In mg SD
NT1 2.38 ± 0.03 149 ± 0.52 4.1 ± 0.12 0.78 ± 0.03 19.91 ± 0.41 6.78 ± 0.06
NT2 2.39 ± 0.02 149 ± 0.76 4.0 ± 0.21 0.69 ± 0.03 19.85 ± 0.19 6.76 ± 0.03
NT3 2.39 ± 0.03 150 ± 0.69 4.1 ± 0.32 0.67 ± 0.04 20.32 ± 0.21 6.68 ± 0.02
NT4 2.38 ± 0.05 149 ± 0.74 4.0 ± 0.28 0.65 ± 0.04 20.26 ± 0.41 6.76 ± 0.04
NT5 2.42 ± 0.03 150 ± 0.22 4.2 ± 0.24 0.59 ± 0.01 20.02 ± 0.15 6.80 ± 0.02
NT6 2.38 ± 0.04 149 ± 0.89 4.1 ± 0.24 0.84 ± 0.02 20.19 ± 0.01 6.75 ± 0.06
NT7 2.39 ± 0.07 149 ± 0.98 4.1 ± 0.36 0.67 ± 0.04 19.78 ± 0.22 6.69 ± 0.06
NT8 2.40 ± 0.02 149 ± 0.76 4.3 ± 0.29 0.63 ± 0.03 19.79 ± 0.65 6.72 ± 0.06
NT9 2.38 ± 0.02 150 ± 0.87 4.2 ± 0.34 0.72 ± 0.01 20.21 ± 0.31 6.75 ± 0.04
NT10 2.41 ± 0.02 150 ± 0.26 4.1 ± 0.51 0.74 ± 0.03 20.15 ± 0.15 6.79 ± 0.04

Surface pH
The surface pH of the produced gel-based formulations was determined in light of the
fact that an acidic or alkaline pH can irritate the buccal mucosa and can affect the rate of
hydration of polymers [23]. The observed surface pH range for the samples was 6.68 to
6.80. These results demonstrate that there is no significant difference in the surface pH of
any of the formulations, and that the pH range falls within the range of salivary pH, i.e.,
6.5 to 6.8, which does not cause irritation, and which thereby assists patient compliance.
Interaction between the hydroxyl groups of HPMC and the carbonyl groups of PVP in
formulation NT5 occurred. This system either disperses or interacts with hydrophobic
nifedipine, whereas PVP is thought to inhibit nifedipine-HPMC by interacting with the
co-crystal surfaces, which results in a pH close to neutral or a salivary pH of 6.8.

2.3. Swelling Index

Based on reports, the swelling behavior of the gel is crucial to its bioadhesive properties
and its drug release profile [1]. The interaction between the hydroxyl groups of HPMC and
the carbonyl groups of PVP improves the mucoadhesive properties of the formulations in
comparison to those of the unaltered HPMC. The adhesion increases with the degree of
hydration until the point of disentanglement at the surface of the gel tissue, and this results
in a sudden decrease in adhesive strength due to overhydration. The adhesion is initially
strong, but it progressively weakens after swelling, and can last for up to six hours. The
 Gels 2023, 9, x FOR PEER REVIEW 6 of 18

Gels 2023, 9, 688 6 of 17

and this results in a sudden decrease in adhesive strength due to overhydration. The ad-
hesion is initially strong, but it progressively weakens after swelling, and can last for up
swelling indexThe
to six hours. increased as index
swelling the formulation
increased weight gain increased
as the formulation proportionally
weight with pro-
gain increased the
rate of HPMC hydration. The formulation NT5 with the highest concentration of HPMC
portionally with the rate of HPMC hydration. The formulation NT5 with the highest con-
and carbopol had the highest index of swelling after 6 h. The results are represented in
centration of HPMC and carbopol had the highest index of swelling after 6 h. The results
Figure 3 and Table S1.
are represented in Figure 3 and Table S1.

Figure3.3.Swelling
Figure Swellingindex
index%%of
ofNT1-NT10.
NT1-NT10.

2.4.
2.4.Ex
ExVivo
VivoBuccoadhesive
BuccoadhesiveStrength
Strength
With
Withaamodified
modifiedphysical
physicalbalance
balancemethod
method and andfresh
freshbuccal
buccalmucosa
mucosa from fromaanearby
nearby
slaughterhouse,
slaughterhouse, the vivo buccoadhesive
the ex vivo buccoadhesivestrengthstrengthwas wasmeasured
measured [24].
[24]. TheThe carboxyl
carboxyl and
and hydroxyl
hydroxyl functional
functional groups
groups of HPMC
of HPMC make make it for
it easier easier for hydrogen
hydrogen bonds to bonds
form to form
between
between the gel
the gel and the and the mucous
mucous membrane.membrane. The presence
The presence of moreofhydrophilic
more hydrophilic
functionalfunctional
groups
groups facilitates the formation of more hydrogen bonds. A gel’s
facilitates the formation of more hydrogen bonds. A gel’s molecular structure determinesmolecular structure
determines its level of Hydration
its level of hydration. hydration. isHydration
necessary is fornecessary
the gel tofor the gelontothe
enlarge enlarge
mucusonlayer,
the
mucus layer, maximizing exposure for the gel and mucin interpenetration,
maximizing exposure for the gel and mucin interpenetration, and providing entangle- and providing
entanglement
ment between between themThe
them [25]. [25].carbonyl
The carbonyl
groups groups
of PVPof PVP reduce
reduce thethe excess
excess hydration
hydration of
of HPMC,and
HPMC, andthey
theyincrease
increaseits
itsmucoadhesive
mucoadhesive properties.
properties. In In addition
addition toto its
its mucoadhesive
mucoadhesive
properties,
properties,HPMC
HPMCisisalso alsowidely
widelyused
usedfor forits
itscontrolled
controlledrelease
releasemechanism.
mechanism.The Theobserved
observed
buccoadhesive strength may be satisfactory in all formulations,
buccoadhesive strength may be satisfactory in all formulations, and this ensuresand this ensures thatthat
all of
all
the samples are successfully maintained in the buccal cavity. The results
of the samples are successfully maintained in the buccal cavity. The results are illustrated are illustrated in
Figure 4 and
in Figure 4 andTable S2.S2.
Table Due to to
Due thethe
miscibility
miscibility ofofHPMC
HPMCand andPVP,
PVP,thetheformulation
formulationofofNT5 NT5
possesses substantial buccoadhesive
possesses substantial buccoadhesive strength. strength.
Gels 2023,9,9,688
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Figure4.4.Ex
Figure Exvivo
vivobuccoadhesive
buccoadhesivestrength
strengthof
ofNT1-NT10.
NT1-NT10.

2.5.
2.5.In
InVitro
VitroDrug
DrugRelease
ReleaseStudy
Study
In
In all
all of the
theformulations,
formulations,the therelease
releaseof of nifedipine
nifedipine differed
differed significantly,
significantly, andand this
this may
may be attributable
be attributable to thetovariable
the variable proportions
proportions of polymeric
of polymeric substances.
substances. The The formulations
formulations pro-
produce a reasonable
duce a reasonable nifedipinedischarge
nifedipine dischargeatatthe theend
endof of12
12h.
h. Formulations
Formulations NT1, NT2, NT3, NT3,
NT4,
NT4,NT5,
NT5,andandNT6NT6containing
containingcombinations
combinationsofofHPMC, HPMC,PVP, PVP,SCMC,
SCMC, andandcarbopol
carbopol gave
gavea
reasonable Nifedipine release of up to 12 h. The constituents of NT1, NT2,
a reasonable Nifedipine release of up to 12 h. The constituents of NT1, NT2, NT3, NT4, NT3, NT4, NT5,
and
NT5, NT6
anddemonstrated
NT6 demonstratedrespective rates ofrates
respective release of 98.3%,
of release 97.1%, 97.1%,
of 98.3%, 97.4%, 97.4%,
98.6%, and 97.6%.
98.6%, and
The regression coefficient (R), Higuchi’s plot, and the in vitro drug
97.6%. The regression coefficient (R), Higuchi’s plot, and the in vitro drug release all release all showed
that the drug
showed that release
the drug followed
release zero-order kinetics. Peppa’s
followed zero-order kinetics.plot showed
Peppa’s plotthat the diffusion
showed that the
exponents (n) were 0.93067,
diffusion exponents (n) were0.85066,
0.93067,0.89323,
0.85066,0.85197,
0.89323,0.91961,
0.85197,and 0.89929,
0.91961, andrespectively.
0.89929, re-
This shows This
spectively. that the
showsdrug release
that the drugmechanism was non-Fickian
release mechanism release for release
was non-Fickian NT2 and forNT4,
NT2
and Super Case II transport type for NT1, NT3, NT5, and NT6. The
and NT4, and Super Case II transport type for NT1, NT3, NT5, and NT6. The release of release of Nifedipine
from NT7-NT10
Nifedipine fromwith HPMC,
NT7-NT10 SCMC
with HPMC,alone, or a combination
SCMC of HPMC, of
alone, or a combination SCMC,
HPMC, PVP, and
SCMC,
Carbopol, was good for up to 12 h. The release rates of NT7-NT10
PVP, and Carbopol, was good for up to 12 h. The release rates of NT7-NT10 were 97.4%, were 97.4%, 98.4%,
98.2%,
98.4%,and 98.1%,
98.2%, andrespectively. The release
98.1%, respectively. Therate of nifedipine
release was dependent
rate of nifedipine on the swelling
was dependent on the
index and buccoadhesive strength, which can vary depending on the
swelling index and buccoadhesive strength, which can vary depending on the character- characteristics and
composition of matrix-forming
istics and composition gels. In general,
of matrix-forming gels. the rate of drug
In general, the release
rate of increased as the
drug release in-
proportion of hydrophilic polymer increased. Due to increases in swelling index and
creased as the proportion of hydrophilic polymer increased. Due to increases in swelling
buccoadhesive strength, it was possible to attribute the maximal cumulative percentage
index and buccoadhesive strength, it was possible to attribute the maximal cumulative
release of nifedipine from formulation NT5 to proportions of HPMC, PVP, and CP.
percentage release of nifedipine from formulation NT5 to proportions of HPMC, PVP, and
The in vitro drug release (Figure 5) demonstrated that the drug release followed
CP.
zero-order kinetics, which was supported by the regression coefficient (R). Higuchi’s plot
The in vitro drug release (Figure 5) demonstrated that the drug release followed zero-
(Figure 6) as cumulative % drug released versus square root of time, and the obtained
order kinetics, which was supported by the regression coefficient (R). Higuchi’s plot (Fig-
values were presented in the Table S3. Peppa’s plot (Figure 7 and Table S4) was drawn,
ure 6) as cumulative % drug released versus square root of time, and the obtained values
revealing slope values of 0.8421, 0.94699, 0.83522, and 0.85655, respectively, for NT7 to
were presented in the Table S3. Peppa’s plot (Figure 7 and Table S4) was drawn, revealing
NT10, confirming that the diffusion mechanism involved in the drug release was non-
slope values of 0.8421, 0.94699, 0.83522, and 0.85655, respectively, for NT7 to NT10, con-
Fickian release for formulations NT7, NT9, and NT10, and Super Case II transport type for
firming that the diffusion mechanism involved in the drug release was non-Fickian release
NT8. With the addition of HPMC, SCMC, PVP, and Carbopol bilayer buccal formulations,
for formulations
maximal NT7,was
drug release NT9, and NT10,
observed andend
at the Super
of 12Case
h. II transport type for NT8. With the
addition of HPMC, SCMC, PVP, and Carbopol bilayer buccal formulations, maximal drug
release was observed at the end of 12 h.
 Gels2023,
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Figure 5. In vitro drug release data of NT1 to NT10.

Figure 5. In vitro drug release data of NT1 to NT10.
Figure 5. In vitro drug release data of NT1 to NT10.

Figure 6. Higuchi’s plot of NT1 to NT10.

Figure6.6.Higuchi’s
Figure Higuchi’splot
plotof
ofNT1
NT1totoNT10.
NT10.
Gels 2023,9,9,688
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Figure7.7.Peppa’s
Figure Peppa’splot
plotofofNT1
NT1totoNT10.
NT10.

Todetermine
To determinethe themechanism
mechanismof ofdrug
drugrelease
releasefrom
fromhydrophilic
hydrophilicmatrices,
matrices,ininvitro
vitrodis-
dis-
solution data of each formulation were analysed in conjunction with
solution data of each formulation were analysed in conjunction with various kinetic drugvarious kinetic drug
releaseequations
release equations[26,27].
[26,27].ToTobebeprecise,
precise,these
theseare
arezero
zeroorder:
order:QQ==K0t;K0t;Higuchi’s
Higuchi’ssquare
square
rateatattime:
rate time:QQ==KHt1/2
KHt1/2 and
and Peppa’s: Kmtn
Peppa’s: FF == Km tn, where Q Q is
is the
the amount
amountof ofdrug
drugrelease
releaseatat
timet;t;FFisisthe
time thefraction
fractionofofdrug
drugrelease
releaseatattime
timet;t;K0
K0isisthe
thezero-order
zero-orderkinetic
kineticdrug
drugrelease
release
constant;
constant;KH KHisisHiguchi’s
Higuchi’s square
squareroot of time
root kinetic
of time drug
kinetic release
drug constant;
release Km is
constant; Kma constant
is a con-
incorporating
stant incorporatingthe geometric and structural
the geometric characteristics
and structural of theofpolymeric
characteristics gel; and
the polymeric gel;nand
is
the diffusion exponent indicative of the release mechanism. The values of
n is the diffusion exponent indicative of the release mechanism. The values of the correla- the correlation
coefficient (r2 ) indicate
tion coefficient that the
(r2) indicate kinetics
that of drug
the kinetics of release was zero.
drug release wasThe mechanism
zero. of drugof
The mechanism
release was determined
drug release using Peppa’s
was determined model,model,
using Peppa’s which indicates super case
which indicates II transport
super based
case II transport
on diffusion
based exponent
on diffusion values (n).
exponent values (n).

2.6.
2.6.Ex
ExVivo
VivoPermeation
PermeationStudies
Studies
Oral
Oral mucosa constitutesaabarrier
mucosa constitutes barrierto
todrug
drugpermeation,
permeation,and
andits
itspermeability
permeabilitycharacter-
character-
istics
istics are intermediate between those of the skin epidermis and the gastrointestinaltract.
are intermediate between those of the skin epidermis and the gastrointestinal tract.
Ex
Exvivo
vivopermeation
permeationstudies
studiescan
candetermine
determinethe theefficacy
efficacyofofthe
thebuccal
buccalbarrier
barrierand
andwhether
whether
buccal absorption could be used to administer NT5. The formulation, NT5, was subjected
buccal absorption could be used to administer NT5. The formulation, NT5, was subjected
to permeation tests and the results presented in Figure 8 and Table S5. The results of drug
to permeation tests and the results presented in Figure 8 and Table S5. The results of drug
permeation of nifedipine through the sheep buccal mucosa showed that the drug was
permeation of nifedipine through the sheep buccal mucosa showed that the drug was re-
released from the formulation and permeated through the buccal membrane. This suggests
leased from the formulation and permeated through the buccal membrane. This suggests
that the drug could possibly permeate through the human buccal membrane. Ex vivo
that the drug could possibly permeate through the human buccal membrane. Ex vivo per-
permeation studies can determine the effectiveness of the buccal barrier and whether buccal
meation studies can determine the effectiveness of the buccal barrier and whether buccal
absorption can serve as a route of administration for nifedipine. Overall, the permeability
absorption can serve as a route of administration for nifedipine. Overall, the permeability
studies have helped figure out how well the formulation NT5 absorbs nifedipine, how it
studies have helped figure out how well the formulation NT5 absorbs nifedipine, how it
moves across the human buccal mucosa, and how buccoadhesive bilayered formulations
moves across the human buccal mucosa, and how buccoadhesive bilayered formulations
make permeability higher.
make permeability higher.
Gels 2023, 9, x FOR PEER REVIEW 10 of 18
Gels 2023,9,9,688
Gels2023, x FOR PEER REVIEW 10 of
10 of 17
18

Figure 8. Ex vivo permeation studies of NT5 through sheep buccal mucosa.

Figure8.8.Ex
Figure Exvivo
vivopermeation
permeationstudies
studiesof
ofNT5
NT5through
throughsheep
sheepbuccal
buccalmucosa.
mucosa.
2.7. Ex Vivo Muco Irritation by Histological Examination
2.7.
2.7.Ex
Ex Vivo
VivoMuco
During Muco Irritation
Irritationby
the administration byHistological
Histological Examination
Examination
of buccoadhesive samples, pathological alterations in cell
During
During the
the administration
administration of
ofbuccoadhesive
morphology and tissue organisation were evaluated buccoadhesive samples,
samples, pathological
pathological
via histological alterations
alterations
analysis. in
incell
Observa-cell
morphology
morphology
tions and tissue
under a microscope organisation
and tissue organisation were
indicate that NT5 evaluated
werehas
evaluated via histological
via histological
not significantly analysis.
damaged Observations
analysis. Observa-
the structure of
under
tions
the a microscope
under
buccal mucosa. indicate
a microscope
Following that
indicate NT5
that has
permeation NT5 not
hassignificantly damaged
not significantly
examinations, neither damaged
cell the structure
necrosis the of the
structure
nor complete of
buccal
removal mucosa.
the buccalof mucosa. Following
Following
the epithelium permeation
from permeation examinations,
the buccal mucosa wasneither
examinations, neither cell
observed. cellnecrosis
necrosis
There nor
werenorcomplete
nocomplete
indica-
removal
removal
tions ofofthe
theepithelium
of irritation, epithelium fromfrom
such as vascular thethe
buccal mucosa
buccal
congestion andwas
mucosa observed.
was There
observed.
subepithelial were
There
oedema. no indications
were no indica-
Furthermore, no
of irritation,
tions of such
irritation,
severe indicators, as vascular
such as congestion
vascular and
congestionsubepithelial
and oedema.
subepithelial Furthermore,
oedema. no severe
Furthermore,
epithelial necrosis, epithelial cell shedding, or bleeding, were no
indicators,
detected such
severe indicators, as
on any surfaceepithelial
such ofasthenecrosis,
epithelial epithelial cell
necrosis,Neither
buccal mucosa. shedding,
epithelialthecell or bleeding,
shedding,
basal membrane were detected
or bleeding, on
were
nor the super-
any
ficial surface
detected onofany
portions thethe
of buccal
surface mucosa.
of
submucosa Neither
the buccal
were the basal
mucosa.
altered membrane
inNeither tonor
the basal
comparison the superficial
membrane
untreated portions
nor the
mucosa. super-
Figure
9of the portions
ficial submucosa
depicts the resulting were
of the alteredfor
submucosa
images inwere
comparison
altered
untreated and totreated
in untreated
comparison mucosa.
buccal tomucosa Figure
untreated 9 depicts
mucosa.
as well the
Figure
as Figures
resulting
S69 depicts images for untreated and treated buccal mucosa as well as
and S7.the resulting images for untreated and treated buccal mucosa as well as Figures Figures S6 and S7.
S6 and S7.

Figure 9. Controlled untreated (A) and nifedipine buccoadhesive bilayer gel treated buccal mucosa
Figure 9. Controlled untreated (A) and nifedipine buccoadhesive bilayer gel treated buccal mu-
(B).
Figure 9. Controlled untreated (A) and nifedipine buccoadhesive bilayer gel treated buccal mucosa
cosa (B).
(B).
2.8.
2.8. In
In Vivo
Vivo Drug
Drug Release
Release Study
Study
2.8. In
In Vivo Drug
rabbits, inRelease
vivo Study diffusion investigations for NT5 revealed a zero-order re-
buccal
In rabbits, in vivo buccal diffusion investigations for NT5 revealed a zero-order release
lease In
pattern. rabbits,
pattern.
WhenWhen in vivo buccal
nifedipine
nifedipine diffusion investigations
buccoadhesive
buccoadhesive for
formulations
formulations NT5
werewererevealed
givengiven toa rabbits
to rabbitszero-order re-
in real
in real life,
lease pattern. When nifedipine buccoadhesive formulations were given to rabbits in real
 Gels2023,
Gels
Gels 2023,9,9,
2023, 9,688
xx FOR
FOR PEER
PEER REVIEW
REVIEW 11
11 of 17
11 of
of 18
18

there
life, was no
life, there
there evidence
was
was no of inflammation,
no evidence
evidence of irritation,
of inflammation,
inflammation, or other
irritation,
irritation, orsensitization
or at the site
other sensitization
other sensitization where
at the
at the site
site
the medicine
where the was given.
medicine was The representations
given. The are shown
representations are in Figure
shown in 10 and
Figure Table
10 and
where the medicine was given. The representations are shown in Figure 10 and Table S6.S6.
Table S6.

Figure 10.
Figure 10. In
In vivo
vivo drug
drug release
release of
of NT5.
NT5.
Figure 10. In vivo drug release of NT5.

2.9.In
2.9.
2.9. InVitro—In
In Vitro—InVivo
Vitro—In VivoCorrelation
Vivo Correlation
Correlation
Correlationsbetween
Correlations
Correlations betweenin
between invitro
in vitroand
vitro andin
and invivo
in vivodrug
vivo drugproperties
drug propertieswere
properties wereused
were usedto
used toshow
to showthat
show thatthe
that the
the
therapeutic
therapeutic effectiveness
effectiveness of of buccoadhesive
buccoadhesive nifedipine
nifedipine formulations
formulations is is
based
therapeutic effectiveness of buccoadhesive nifedipine formulations is based on both in based
on on
both inboth
vitroin
and
vitroin vivo
and drug
in vivoproperties.
drug Figure
properties. 11 shows
Figure 11 a graph
shows awith cumulative%
graph with in
cumulative%
vitro and in vivo drug properties. Figure 11 shows a graph with cumulative% in vitro vitro release
in vitro
on the x-axis
release
release on the
on and
the cumulative%
x-axis
x-axis and in vivo drug
and cumulative%
cumulative% release
in vivo
in vivo drug
drug onrelease
the y-axis
release for the
on the
on the same
y-axis
y-axis forperiod
for the same
the sameof
time
periodand ofthe
timevalues
and represented
the values in Table
represented S7.
in The
Tablerelease
S7. rate
The followed
release rate zero order,
followed
period of time and the values represented in Table S7. The release rate followed zero order, zeroand the
order,
correlation
and the
and coefficient
the correlation
correlation value was
coefficient
coefficient 0.9966.
value
value was 0.9966.
was 0.9966.

Figure11.
Figure
Figure 11.IVIVC
11. IVIVCplot
IVIVC plotStability
plot Stabilitystudy
Stability studyin
study inhuman
in humansaliva.
human saliva.
saliva.

2.10.
2.10.Stability
StabilityStudy
Study
2.10. Stability Study
Stability studies
studiesshould
Stability studies shouldevaluate
evaluate product
product properties
properties that
thatare
aresusceptible
susceptibletotochange
change
Stability should evaluate product properties that are susceptible to change
during
during storage
storage and that
and are
that anticipated
are to influence
anticipated to quality,
influence safety,
quality, and and
safety, efficacy. The stability
efficacy. The sta-
during storage and that are anticipated to influence quality, safety,◦ and efficacy. The sta-
studies
bility of NT5of
studies were
NT5carried
were out at accelerated
carried out at conditions
accelerated of 40 ± 2of C
conditions 4075± ±°C
2 5%75RH± overRH
5% a
bility studies of NT5 were carried out at accelerated conditions of 40 ± 2 °C 75 ± 5% RH
Gels 2023, 9, 688 12 of 17

three-month period, and periodically checked for appearance, buccoadhesive strength, and
in vitro drug release. The result was analysed by one-way ANOVA followed by Tukey’s
test, which indicates that the sample was stable and that the p value is non-significant. The
results are shown in Table 2. It was demonstrated that neither the physical appearance nor
the buccoadhesive strength nor the in vitro drug release changed.

Table 2. Stability studies of NT5.

Parameters 1st Month 2nd Month 3rd Month p Value

Physical appearance No Change No Change No Change -
Buccoadhesive strength 34.88 ± 1.09 ns 35.3 ± 1.09 ns 36 ± 0.34 ns 0.1539
In vitro drug release 98.06 ± 0.55 ns 98.13 ± 0.32 ns 98.26 ± 0.5 ns 0.8709
All values are expressed as Mean ± SD of triplicate measurements; ns = non-significant.

3. Conclusions
The HPMC K 100, SCMC, PVP K 30, and carbopol 934 polymers, along with an imper-
meable backing layer of ethyl cellulose, were directly compressed to produce nifedipine
buccoadhesive bilayer formulations with a polymeric hydrophilic gel-based matrix. The
DSC thermogravimetric analysis confirms the drug’s incorporation into a polymeric struc-
ture with only a physical process and reveals no evidence of a potential interaction. The
physicochemical characteristics of the produced samples, such as surface pH, swelling
percentage, thickness, weight fluctuation, hardness, friability, and drug content, all adhere
to pharmacopoeial standards for all samples. Reproducible findings were obtained in
rabbits for ex vivo buccoadhesive strength, in vitro drug release, ex vivo permeability, and
in vivo drug release. Histological analysis of ex vivo mucosa reveals that the sample did
not produce any irritation or inflammation at the delivery site. The best formulation overall
was NT5, which comprises 25 mg of HPMC, 10 mg of CP, and 12.5 mg of PVP. With a
correlation value of 0.996, the in vitro and in vivo drug release profiles were well correlated,
demonstrating the capacity of the system to mimic the in vitro release pattern via the
biological membrane. The reduced dose frequency suggests that the proposed gel-based
formulation could be used as a viable controlled drug delivery strategy for nifedipine.

4. Materials and Methods

Nifedipine was bought from Drugs India in Hyderabad, India, along with HPMC K
100, PVP K 30, SCMC, CP 934, and EC. Newly collected buccal mucosa of sheep for testing
the strength of the buccoadhesive and ex vivo permeation studies was obtained from a
local slaughterhouse in Gwalior, India. All other supplies obtained and utilized were of
an analytical standard. By using direct compression, the buccoadhesive bilayer gel-based
formulations were produced.

4.1. Drug Polymer Interaction Studies through Differential Scanning Calorimetry (DSC)
DSC is utilized to investigate any physicochemical interaction between the Nifedipine
and the polymer matrix [28]. The DSC thermograms of pure drug, polymer, and the
composition of drug polymers were recorded in the DSC analyzer model Universal V4.5A
at a heating rate of 20 ◦ C per minute from 0 to 350 ◦ C in a nitrogen environment. The
nonexistence of the drug melting peak in the DSC thermogram is typically indicative of the
substance’s porous or crystalline state in the polymer. Any sharp or significant alteration in
the thermal behavior of both the drug and the polymer also manifested as movements of
exothermic and endothermic peaks, and these alterations are typically attributed to drug
and polymer interactions.

4.2. Preparation of Buccoadhesive Bilayer Formulations of Nifedipine

Nifedipine containing bilayer buccoadhesive formulations were created using the
direct compression technique [29]. Different sets were made to find the best formulation
by adjusting the HPMC, SCMC, and PVP K 30 ratio. The medication was combined with
Gels 2023, 9, 688 13 of 17

the mucoadhesive polymers HPMC, SCMC, PVP K-30, CP, mannitol, and lactose in a glass
mortar, and mortared for fifteen minutes. The powder was completely mixed and screened
through a 60 m sieve before direct compression. Magnesium stearate was used to lubricate
the mixture for three to five minutes. The combination (100 mg) was then squeezed in a
nine-station rotary punching machine (Ahmadabad, India) using an 8 mm diameter die.
The impermeable EC backing layer was placed on the compact above, and the upper punch
was elevated. The two layers were then crushed into a buccoadhesive bilayer gel. The
components of the Nifedipine bilayer buccal formulations are listed in Table 3.

Table 3. The buccoadhesive bilayer formulations of Nifedipine.

Formulation Code NT1 NT2 NT3 NT4 NT5 NT6 NT7 NT8 NT9 NT10
Nifedipine 20 20 20 20 20 20 20 20 20 20
HPMC K 100 25 - 12.5 12.5 25 - 6.25 25 6.25 37.5
SCMC 12.5 25 - 25 - 12.5 6.25 6.25 25 -
PVP K 30 - 12.5 25 - 12.5 25 25 6.25 6.25 -
Ingredients (mg) CP 934 10 10 10 10 10 10 10 10 10 10
Mg. stearate 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Lactose 15 15 15 15 15 15 15 15 15 15
Mannitol 15 15 15 15 15 15 15 15 15 15
EC 50 50 50 50 50 50 50 50 50 50
Total weight in mg 150 150 150 150 150 150 150 150 150 150

4.3. Physicochemical Evaluation of Buccoadhesive Bilayered Formulations

Following the technique outlined for conventional oral formulations in the certified
pharmacopeia, all of the manufactured formulations were assessed for post-compressional
evaluations, such as thickness, weight variation, hardness, friability, and drug content [30].

Surface pH
To check for any potential side effects in the buccal environment, the surface pH of the
buccal formulations was measured. It was decided to maintain the surface pH as adjacent
to neutral as feasible because an acidic or basic pH may bother the buccal mucosa. The
gel-based sample was kept in contact with 5 mL of phosphate buffer containing 2% w/v
agar medium (pH 6.8 ± 0.01) for 2 h at room temperature in order to cause it to swell.
The electrode was placed in contact with the surface of the formulations, and the pH was
then determined after one minute of equilibration [29,31]. The average of three readings
was noted.

4.4. Swelling Index (SI)

The gravimetric method was used to determine the gel swelling index [32]. A 1% agar
gel plate was used to measure the gel’s rate of swelling. The gel’s typical weight (W1)
was computed. The formulations were put in a petri dish with a gel surface and kept at
37 ± 1 ◦ C in an incubator. At one-hour intervals of time up to six hours, formulations were
taken out, cleaned with regular filter paper, and reweighed (W2). The following formula
was used to determine the index of swelling:

Index of swelling (SI) = [(W2 − W1)/W1] × 100

4.5. Ex Vivo Buccoadhesive Strength

The ex vivo buccoadhesive strength was assessed using a modified physical balance
method [33,34]. Within two hours of the slaughter, fresh sheep buccal mucosa was obtained
from a nearby slaughterhouse and employed. The underlying fat and loose tissues were
cut out in order to separate the mucosal membrane. After being rinsed with distilled water,
the membrane was then treated with phosphate buffer, pH 6.8. By maintaining a 5-g saliva
solution at 37 ◦ C prior to the investigation, the two sides of the balance were made equal.
Gels 2023, 9, 688 14 of 17

After being divided into pieces, the sheep buccal mucosa was cleaned with phosphate
buffer, pH 6.8. The glass vial containing the phosphate buffer was connected to a piece
of buccal mucosa. A glass vial was fitted tightly with a glass beaker that held phosphate
buffer at pH 6.8 at 37 ◦ C that barely touched the mucosal surface. The buccal gel weighs
down the right-hand pan because it was attached to the bottom of a rubber stopper with
cyanoacrylate adhesive. The right pan has 5 g of weight removed from it. This brought
the pan and gel down over the mucosa. The balance was held in this posture for the entire
five-minute contact period. Using an infusion device, water was gradually infused at a rate
of 100 drops per minute (weight-equivalent) until the gel came away from the mucosal
layer to the right-hand pan. The mucoadhesive strength of the buccal gel is expressed in
grams by this detachment force:

Force of adhesion (N) = (Bioadhesive strength (g) × 9.8)/1000

Bond strength (N m−2 ) = Force of adhesion/surface area

4.6. In Vitro Drug Release Study

The drug release from the bilayer gel was investigated using the USP Type II rotating
paddle method [35,36]. The dissolution medium was 900 mL of pH 6.8 phosphate buffer.
At a rotational speed of 50 rpm and a temperature of 37 ± 1 ◦ C, the release research was
conducted. Cyanoacrylate adhesive was used to secure the buccal gel’s backing layer to the
glass slide. The disc was put in the dissolution vessel’s bottom. To maintain sink conditions,
aliquots (5 mL each) were taken out at regular intervals and refilled with fresh medium.
Filtered samples were then subjected to appropriate phosphate buffer pH 6.8 dilutions and
spectrophotometric analysis at 236 nm.

4.7. Ex Vivo Permeation Studies

Fresh sheep buccal mucosa was used in an ex vivo diffusion investigation of Nifedipine
formulations in a modified diffusion cell [37]. It was mounted between the donor and the
receptor compartments with the fresh sheep buccal mucosa. The donor compartment was
an open-ended cylinder to which sheep buccal mucosa was connected. The gel needed to
be positioned so that it adhered to the mucosal membrane. Isotonic phosphate buffer with
a pH of 6.8 was put inside the receptor compartment. The assembly was kept at 37 ◦ C while
being magnetically agitated. At regular intervals, samples were taken out in triplicate, and
they were subjected to UV spectrophotometer analysis at 236 nm.

4.8. Ex Vivo Muco Irritation by Histological Examination

Using fresh sheep buccal mucosa obtained from a nearby abattoir shortly after slaugh-
ter (sheep buccal mucosa was used for the histological evaluation within 2 h), ex vivo muco
irritations of Nifedipine buccal formulations (NT5) were carried out [38]. During ex vivo
permeation testing, the best formulation was applied over the sheep buccal mucosa and
put into contact with the polymeric gel-based matrix layer followed by an impermeable
ethyl cellulose backing layer, and the used mucosa was then histologically examined. In
order to assess the pathological alterations in tissue structure and cell morphology that
were brought on by the administration of buccoadhesive formulations, histological analysis
was conducted. The mucosa’s epithelial tissues were dehydrated with graded ethanol
(60 to 100%), were rinsed with distilled water for up to an hour, and were then fixed in 10%
neutral buffered formalin for two hours. After that, the mucosa received the usual xylene
permeation treatment and liquid paraffin embedding. Paraffin-embedded samples were
cut into 4 µm thick sections on a microtome using a disposable blade after 8 h of formalin
fixation, and they were then quickly stained with eosin.

4.9. In Vivo Drug Release Study

We chose six male New Zealand white rabbits 10–12 weeks old and weighing 2.5–3 kg.
A healthy rabbit weighing between 2.5 and 3 kg was selected, and its health was examined
Gels 2023, 9, 688 15 of 17

for any ailment [39,40]. The rabbit was not in a dorsal position, since the forelimbs and
the hind limbs were locked into the iron rod of the miniature operating table. With the
aid of the forceps, the prepared buccoadhesive bilayer gel was inserted into the buccal
membrane (cheek pouch). Dextrose solutions were regularly administered during the
research period. To stop blood clotting, 1 mL of blood was periodically drawn using
a syringe that also contained 1 mL of heparin solution. For around 30 min, these blood
samples were centrifuged at 2500 rpm. After appropriate dilution, one ml of the supernatant
was collected for spectrophotometric analysis at 236 nm. The resultant number represents
the quantity of the drug that was released from the rabbit buccal mucosa.

4.10. In Vitro—In Vivo Correlation

Nifedipine release was compared using in vitro and in vivo correlations. This release
is influenced by variables connected to the drug’s in vivo and in vitro properties [41,42].
Plots were made depicting the cumulative percentage of drug release both in vitro and
in vivo. Saliva samples were collected and filtered from ten people aged between 18 and
40. The formulations from the finest batch were placed in separate Petri dishes containing
5 mL of human saliva, and they were stored in a 37 ± 0.2 ◦ C oven for six hours [43,44]. At
regular intervals, the appearance of the buccoadhesive formulations, such as the colour
and the shape, and the Nifedipine concentration were both evaluated in order to determine
their stability.

4.11. Stability Study

The formulation NT5 was chosen, and stability tests were conducted in desicca-
tors under accelerated conditions of 40 ◦ C and 75◦ RH. The samples were stored in the
aforementioned settings for three months after being placed in amber-coloured screw-cap
containers [45]. The physical characteristics, buccoadhesive strength, and in vitro drug
release were evaluated on a regular basis. A one-way ANOVA test was used to analyse the
results, followed by Tukey’s test. At p 0.05, the differences were deemed to be statistically
significant.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/gels9090688/s1, Figure S1: DSC curve of Nifedipine; Figure S2:
DSC curve of HPMC K 100; Figure S3: DSC curve of SCMC; Figure S4: DSC curve of Carbopol 934;
Figure S5: DSC curve of Nifedipine and polymer mixture; Figure S6: Controlled buccal mucosa;
Figure S7: Test buccal mucosa; Table S1: Swelling index data for all formulations; Table S2: Ex-vivo
buccoadhesive strength; Table S3: In-vitro and Higuchi’s release data; Table S4: Peppa’s data; Table S5:
Ex-vivo permeation data of NT5; Table S6: In-vivo drug release of NT5; Table S7: In-vitro and In-vivo
correlation data of NT5.
Author Contributions: Conceptualization, M.A.; methodology, N.K.J.; software, N.K.J.; valida-
tion, S.A.P. and V.K.N.; formal analysis, S.A.P.; investigation, N.K.J., M.Y.B., V.K.N. and M.F.B.;
resources, V.K.N.; M.F.B. and B.C.; data curation, M.Y.B., S.A.P. and B.C.; writing—original draft,
M.A.; writing—review and editing, M.F.B. and B.C. All authors have read and agreed to the published
version of the manuscript.
Funding: The authors extend their appreciation to the Deanship of Scientific Research at King Khalid
University for funding this work through Research Group (Large) [Project Number RGP2/54/44].
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Gels 2023, 9, 688 16 of 17

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