gels

Article
Numerical Optimization of Prednisolone–Tacrolimus Loaded
Ultraflexible Transethosomes for Transdermal Delivery
Enhancement; Box–Behnken Design, Evaluation, Optimization,
and Pharmacokinetic Study
Munerah M. Alfadhel 1 , Randa Mohammed Zaki 1,2, * , Basmah Nasser Aldosari 3 and Ossama M. Sayed 4

1 Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173,
Al-Kharj 11942, Saudi Arabia; [Link]@[Link]
2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni-Suef University,
Beni-Suef 62514, Egypt
3 Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; baldosari@[Link]
4 Department of Pharmaceutics, Faculty of Pharmacy, Sinai University-Kantara Branch, Ismailia 41612, Egypt;
[Link]@[Link]
* Correspondence: [Link]@[Link]; Tel.: +966-540617870

Abstract: The aim of the present study is to formulate highly permeable carriers (i.e., transetho-
somes) for enhancing the delivery of prednisolone combined with tacrolimus for both topical and
systemic pathological conditions. A Box–Behnken experimental design was implemented in this
research. Three independent variables: surfactant concentration (X1), ethanol concentration (X2), and
tacrolimus concentration (X3) were adopted in the design while three responses: entrapment effi-
ciency (Y1), vesicle size (Y2), and zeta potential (Y3) were investigated. By applying design analysis,
Citation: Alfadhel, M.M.; Zaki, R.M.; one optimum formulation was chosen to be incorporated into topical gel formulation. The optimized
Aldosari, B.N.; Sayed, O.M. transethosomal gel formula was characterized in terms of pH, drug content, and spreadability. The
Numerical Optimization of gel formula was challenged in terms of its anti-inflammatory effect and pharmacokinetics against oral
Prednisolone–Tacrolimus Loaded prednisolone suspension and topical prednisolone–tacrolimus gel. The optimized transethosomal gel
Ultraflexible Transethosomes for achieved the highest rate of rat hind paw edema reduction (98.34%) and highest pharmacokinetics
Transdermal Delivery Enhancement;
parameters (Cmax 133.266 ± 6.469 µg/mL; AUC0-∞ 538.922 ± 49.052 µg·h/mL), which indicated
Box–Behnken Design, Evaluation,
better performance of the formulated gel.
Optimization, and Pharmacokinetic
Study. Gels 2023, 9, 400. https://
Keywords: prednisolone; tacrolimus; Box–Behnken design; transdermal delivery; transethosomes
[Link]/10.3390/gels9050400

Academic Editors: Ying Huang,

Zhengwei Huang and
Xuanjuan Zhang 1. Introduction
Received: 26 March 2023 For many years, prednisolone (PRED) has been utilized extensively in the treatment of
Revised: 27 April 2023 inflammatory illnesses (both acute and chronic). For many rheumatic illnesses, a long-term
Accepted: 3 May 2023 remedy with these medications is frequently required to manage the symptoms. Long-
Published: 10 May 2023 term use of PRED in therapy has a number of negative consequences on the heart and
metabolism of bones. One of the most common adverse effects of long-term PRED use is
bone loss. The anti-inflammatory effect of PRED is underpinned by very intricate processes
that obstruct the operation of numerous systems. In addition to the risks connected to
Copyright: © 2023 by the authors.
the prolonged use of PRED at a super-physiological dose, there are issues connected to
Licensee MDPI, Basel, Switzerland.
stopping the steroid medication. Reduced unfavorable side effects would be extremely
This article is an open access article
beneficial for therapeutic usage of PRED, which is the greatest anti-inflammatory medicine
distributed under the terms and
conditions of the Creative Commons
currently on the market. For patients, worry regarding their safety has always been the
Attribution (CC BY) license (https://
main barrier to the use of oral PRED [1,2]. One of the means to reduce the side effects of
[Link]/licenses/by/
PRED is to shift the route of application to the transdermal route.
4.0/).

Gels 2023, 9, 400. [Link] [Link]

Gels 2023, 9, 400 2 of 19

Recently, the name “trans-ethosomes” was given to lipid-based nanovesicles that

include an edge activator and ethanol. The benefits of both ethosomes and transfersomes
are present in transethosomes (TETSMs). TETSMs provide a number of advantages over
other drug delivery methods because of the inclusion of these components. Ethanol
improves medication penetration through the minute holes created in the stratum corneum
as a result of fluidization by enhancing the lipid’s fluidity and decreasing the density
of the lipid bilayer [3]. These vesicles’ edge activator weakens the phospholipid bilayer,
making them ultradeformable and extremely elastic [4]. Therefore, it is anticipated that
these drug-loaded nanovesicles will have a positive effect on therapeutic activity.
Tacrolimus (TAC) alters the humoral and cell-mediated immune reactions linked to in-
flammation in a number of different ways. The key method of action involves reducing the
immunophilin FK506 binding protein 12’s ability to activate calcineurin as a phosphatase,
preventing the generation of interleukin (IL)-2, and inhibiting the signal transduction
pathway that activates T cells. Although cyclosporin also inhibits calcineurin by forming a
compound with a different immunophilin, in vitro and in vivo research shows that TAC
has 10–100 times greater immunosuppressive activity than cyclosporin. Additionally, nitric
oxide synthase activation and apoptosis may be inhibited by TAC, which may also enhance
the effects of corticosteroids in these processes [5].
Many sources in the literature confirmed that combining corticosteroids therapy with
TAC leads to lower required doses of corticosteroids in renal transplant operations [6]. This
could be beneficial in terms of fewer side effects from steroids.
The aim of this research is to examine the possibility of combining PRED and TAC
in TETSMs carriers and utilizing the transdermal route to increase the anti-inflammatory
effect and pharmacokinetics of PRED.

2. Results and Discussion

2.1. Effect of Formulation Variables on EE%, Vesicle Size, and Zeta Potential
The chief objective of this study was to assess the effectiveness of TETSMs in improving
the penetration of PRED through the skin for the management of inflammatory disorders.
TETSMs are capable of effectively penetrating the stratum corneum due to their high
alcohol content and surfactant properties [7]. Additionally, the study also examined the
impact of TAC on the delivery of PRED to the skin.
An analysis was conducted to examine the impact of various formulation variables,
including concentration of surfactant (X1), concentration of ethanol (X2), and concentration
of TAC (X3) on EE% (Y1), vesicular size (Y2), and zeta potential (Y3). The results of the
regression analysis, which were used to determine the best fitting model for each response,
are summarized in Table 1.

Table 1. Composition of PRED-TAC TETSMs Formulations and the results of different responses (mean ± SD).

Independent Variables Dependent Variables

Formula Code Surfactant Ethanol Tacrolimus

Vesicles Size Zeta Potential
Concentration Concentration Concentration EE% (Y1) PDI
(nm) (Y2) (mv) (Y3)
w/v % (X1) v/v % (X2) w/v % (X3)
1 1 20 0.065 87.6 ± 2.14 330.5 ± 10.12 −32.2 ± 0.92 0.132 ± 0.08
2 1 40 0.065 80.4 ± 1.87 307.8 ± 8.75 −36.1 ± 0.87 0.315 ± 0.09
3 1 30 0.1 84.4 ± 1.46 320.7 ± 7.35 −33.8 ± 1.01 0.326 ± 0.11
4 0.2 20 0.065 62.4 ± 2.01 253.6 ± 6.54 −19.3 ± 1.17 0.321 ± 0.12
5 0.6 30 0.065 72.3 ± 3.27 283.5 ± 5.43 −30.4 ± 1.28 0.276 ± 0.03
6 0.6 30 0.065 72.1 ± 2.98 283.2 ± 6.98 −30.1 ± 0.84 0.376 ± 0.11
7 0.2 30 0.1 58.4 ± 1.54 242.1 ± 4.33 −22.1 ± 0.58 0.123 ± 0.02
8 0.6 30 0.065 72.4 ± 1.63 282.9 ± 7.21 −30.3 ± 0.23 0.265 ± 0.06
9 0.2 30 0.03 56.7 ± 3.21 231.5 ± 3.75 −21.8 ± 0.67 0.225 ± 0.14
10 0.6 20 0.03 75.6 ± 2.19 290.4 ± 7.25 −27.3 ± 0.95 0.431 ± 0.10
11 0.6 30 0.065 72.6 ± 1.92 283.7 ± 3.98 −30.5 ± 0.26 0.259 ± 0.07
12 0.6 20 0.1 76.5 ± 1.64 302.6 ± 9.43 −28.1 ± 1.11 0.239 ± 0.13
13 0.6 40 0.1 68.7 ± 2.54 278.5 ± 5.38 −32.7 ± 0.83 0.185 ± 0.05
14 0.2 40 0.065 51.3 ± 2.73 230.4 ± 6.27 −24.8 ± 0.63 0.173 ± 0.16
15 0.6 30 0.065 71.9 ± 3.81 283.3 ± 4.79 −30.6 ± 0.27 0.265 ± 0.08
16 1 30 0.03 83.2 ± 1.26 307.6 ± 10.3 −33.1 ± 1.03 0.439 ± 0.14
17 0.6 40 0.03 66.5 ± 1.87 266.7 ± 7.83 −31.8 ± 0.68 0.286 ± 0.12
Gels 2023, 9, 400 3 of 19

2.1.1. Effect of Formulation Variables on EE%

EE% data of the formulated TETSMs are shown in Table 1. Model fitting showed
a fairly good fit with a quadratic interaction model, with a correlation coefficient R2 of
0.9994, adjusted R2 of 0.9986, and predicted R2 of 0.9932. The small difference (less than 0.2)
between the adjusted and predicted R2 confirms the validity of the model [8]. Additionally,
the model showed high adequate precision of 125.84 (greater than 4), indicating the ability
of the model to navigate the design space [9], as represented in Table 2. ANOVA in
Table 3 showed that EE% was significantly affected by all independent variables (p < 0.05)
with the following equation representing the combined effect of independent variables
on EE% of TETSMs:

EE% = +72.26 + 13.35X1 − 4.40X2 + 0.7500 X3 + 0.9750X1X2 − 0.1250X1X3

(1)
+0.3250X2X3 − 1.49X12 − 0.3425 X22 − 0.0925X32

where X1 is the concentration of surfactant, X2 is the concentration of ethanol, and

X3 is the concentration of TAC. The response surface curves of the EE% values from the
interaction of different independent variables are shown in Figures 1A and 2A.

Table 2. Output data of Box–Behnken design for optimization of PRED–TAC-loaded TETSMs.

Adequate
Dependent Variables R2 Adjusted R2 Predicted R2
Precision
Y1: % EE 0.9994 0.9986 0.9932 125.8424
Y2: Vesicle size (nm) 0.9999 0.9998 0.9990 325.6015
Y3: Zeta potential (mV) 0.9988 0.9974 0.9877 89.5319

Table 3. ANOVA for Box–Behnken design of PRED–TAC-loaded TETSMs.

Dependent
Source SS Df Mean Square F Value p Value
Variable
Model 1599.74 9 177.75 1313.87 <0.0001 significant
A-Surfactant concentration 1425.78 1 1425.78 10,539.03 <0.0001
B-Ethanol concentration 154.88 1 154.88 1144.84 <0.0001
C-Tacrolimus concentration 4.50 1 4.50 33.26 0.0007
AB 3.80 1 3.80 28.11 0.0011
Y1
AC 0.0625 1 0.0625 0.4620 0.5185
BC 0.4225 1 0.4225 3.12 0.1205
A2 9.38 1 9.38 69.33 <0.0001
B2 0.4939 1 0.4939 3.65 0.0977
C2 0.0360 1 0.0360 0.2663 0.6217
Model 13,537.78 9 1504.20 9232.25 <0.0001 significant
A-Surfactant concentration 11,942.85 1 11,942.85 73,301.15 <0.0001
B-Ethanol concentration 1106.85 1 1106.85 6793.48 <0.0001
C-Tacrolimus concentration 288.00 1 288.00 1767.65 <0.0001
AB 0.0625 1 0.0625 0.3836 0.5553
Y2
AC 1.69 1 1.69 10.37 0.0146
BC 0.0000 1 0.0000 0.0000 1.0000
A2 145.21 1 145.21 891.22 <0.0001
B2 41.18 1 41.18 252.77 <0.0001
C2 16.80 1 16.80 103.11 <0.0001
Gels 2023, 9, 400 4 of 19

Table 3. Cont.

Dependent
Source SS Df Mean Square F Value p Value
Variable
Model 345.94 9 38.44 671.83 <0.0001 significant
A-Surfactant concentration 278.48 1 278.48 4867.32 <0.0001
B-Ethanol concentration 42.78 1 42.78 747.74 <0.0001
C-Tacrolimus concentration 0.9112 1 0.9112 15.93 0.0053
AB 0.6400 1 0.6400 11.19 0.0123
Y3
AC 0.0400 1 0.0400 0.6991 0.4307
BC 0.0025 1 0.0025 0.0437 0.8404
A2 21.84 1 21.84 381.72 <0.0001
B2 0.0000 1 0.0000 0.0005 0.9835
C2Gels 2023, 9, x FOR PEER REVIEW
0.6821 1 0.6821 11.92 0.0106 5 of 21

Y1: % EE, Y2: Vesicle size (nm), Y3: Zeta potential (mV), SS: sum of squares, Df: degree of freedom.

Figure 1. A 3D response
Figuresurface graphsurface
1. A 3D response for the effect
graph for theof independent
effect of independentfactors: surfactant
factors: surfactant concentration,
concentration,
Ethanol concentration, and Tacrolimus concentration on the dependent responses, EE% (A), vesicle
Ethanol concentration, and Tacrolimus concentration on the dependent responses,
size (B), and zeta potential (C) of PRED–TAC-loaded TETSMs. Red dots indicate the repli- EE% (A), vesicle
size (B), and zeta potential
cates in our(C) of PRED–TAC-loaded TETSMs. Red dots indicate the replicates
design.
in our design.
Gels 2023, 9, 400 5 of 19
Gels 2023, 9, x FOR PEER REVIEW 6 of 21

Figure 2. Contour graph for the effect of independent factors on different responses, EE% (A), vesi-
Figure 2. Contour graph for the effect of independent factors on different responses, EE% (A), vesicle
cle size (B), and zeta potential (C) of PRED–TAC-loaded TETSMs. Red dots indicate the repli-
size (B), and zeta potential (C) of PRED–TAC-loaded TETSMs. Red dots indicate the replicates in
cates in our design.
our design.
2.1.2. Effect of Formulation Variables on TETSMs Vesicle Size
Increasing the surfactant concentration led to an increase in EE% of PRED as confirmed
The vesicle
by the positive signsizes of different
of the formulated
correlation TETSMs
coefficient are shown(1).
in Equation in Table
This 1. Model
could befitting
explained
revealed a quadratic interaction between independent variables, with R2 of 0.9999, ad-
based on the low HLB value of span 60, which increases the lipophilic domain of the lipid
justed R2 of 0.9998, and predicted R2 of 0.9990. There was a small difference (less than 0.2)
bilayer and hence increases the entrapped PRED in this hydrophobic domain [10,11].
between the adjusted and predicted R2. Additionally, the model showed high adequate
Ethanol concentrations have a negative
precision of 325.6, as represented in Table 2. Theeffect
dataindividually
from ANOVAon inthe EE%
Table of PRED in
3 revealed
TETSMs,
that vesicle size of different formulations was significantly affected by all independent (1).
as represented by the negative sign of the correlation coefficient in Equation
The decrease in the EE% may be due to the increase in the fluidity and the leakage of the
vesicles [12–15]. This result is in contrast to the previous research, which reported that
enhancing the concentration of ethanol from 20 to 40% will have a positive impact on the
EE% [16]. In addition, TAC showed an improvement in EE% by increasing its concentration
from 0.03 to 0.1%, which aids the solubilization of drugs in the lipid mixture. Including
lipid soluble TAC could increase the solubility of PRED.
Gels 2023, 9, 400 6 of 19

2.1.2. Effect of Formulation Variables on TETSMs Vesicle Size

The vesicle sizes of different formulated TETSMs are shown in Table 1. Model fitting
revealed a quadratic interaction between independent variables, with R2 of 0.9999, adjusted
R2 of 0.9998, and predicted R2 of 0.9990. There was a small difference (less than 0.2) between
the adjusted and predicted R2 . Additionally, the model showed high adequate precision of
325.6, as represented in Table 2. The data from ANOVA in Table 3 revealed that vesicle size
of different formulations was significantly affected by all independent variables (p < 0.05)
with the following equation representing the combined effect of independent variables on
vesicle size of TETSMs:

Vesicles size = +283.32 + 38.64 X1 − 11.76X2 + 6.00X3 + 0.1250 X1X2 + 0.6500 X1X3
(2)
+0.0000X2X3 − 5.87X12 + 3.13X22 − 2.00X32
where X1 is the concentration of surfactant, X2 is the concentration of ethanol, and
X3 is the concentration of TAC. Figures 1B and 2B show the surface response curves of
the combined effects of prepared independent variables on vesicle size. Generally, the
surfactant concentration range in ethosomal formulations is 0.2–1% [17]. It was observed
that increasing the surfactant (span 60) ratio resulted in a slight or moderate increase in
vesicle size. The positive effect of surfactant on the vesicle size agrees with the earlier
outcomes and can be due to the reduction in the hydrophilic portion of the surfactant in
the presence of low HLB surfactant at high concentrations [10,11]. In addition, TAC had
a positive impact on the vesicle size values, meaning that increasing TAC concentration
from 0.03 to 0.1% resulted in a simultaneous increase in vesicle size. This condition was
observed parallel to the increase in span 60. In contrast, the concentration of ethanol was
found to have a negative impact on the vesicle size values and this is confirmed by the
negative sign of the correlation coefficient (X2) in Equation (2). This observation agrees
with the earlier literature [18].
Ethanol has been found to be a potent penetration enhancer. Ethanol concentration
has been reported in the literature to be in the range of 10–50% [7,19]. Enhancing the
concentration of ethanol resulted in a decrease in vesicle size, as stated in the previous
works [7,15,20–32].

2.1.3. Effect of Preparation Variables on Zeta Potential

Zeta potential is an indicator for the stability of nanosystems as it gives information
about the magnitude of attraction and repulsion between nanoparticles. As zeta potential
values increase, the repulsion between nanoparticles increases, and therefore there is
improvement in the system stability. As shown in Table 1, the zeta potential values of all
TETSMs formulations ranged from −19.3 ± 1.17 to −36.1 ± 0.87, indicating high stability
for the fabricated formulations.
The model fitting of the combined effects of TETSMs variables on zeta potential
proposed a quadratic model with R2 of 0.9988, adjusted R2 of 0.9974, and predicted R2 of
0.9877. There was a small difference (less than 0.2) between the adjusted and predicted R2 .
In addition, the model showed high adequate precision of 89.53 as represented in Table 2.
The data from ANOVA in Table 3 revealed that the zeta potential of different formula-
tions was considerably affected by all independent variables (p < 0.05) with the following
equation representing the effect of independent variables on zeta potential of TETSMs:

Zeta potential = + 30.38 + 5.90X1 + 2.31X2 + 0.3375X3 − 0.4000X1X2 + 0.1000X1 + 0.0250 X2X3
(3)
−2.28X12 − 0.0025X22 − 0.4025X32
where X1 is the concentration of surfactant, X2 is the concentration of ethanol, and X3 is
the concentration of TAC. As clear from the above equation, all independent variables
caused an increase in the zeta potential values. Increasing the zeta potential is so beneficial.
The increase in zeta potential values with the increase in span 60 concentration may be
attributed to the low HLB value (hydrophilic lipophilic balance) of span 60 resulting in
Gels 2023, 9, 400 7 of 19

high adsorption of OH ions from the hydration medium on the nanoparticles causing an
increase in zeta potential values [15].
The positive impact of ethanol concentration on zeta potential values may be due to
the negative charge imparted by ethanol on the particles’ surfaces. Ethanol’s effect on zeta
Gels 2023, 9, x FOR PEER REVIEW 8 of 21
potential is well documented in the literature [17,33–35].
The effect of different independent variables on zeta potential is shown in Figures 1C and 2C.

[Link]. FormulationOptimization
Formulation Optimization and
and Validation
Validation
AA numeric optimization
numeric optimization was
wasperformed
performed by Design Expert Expert
by Design softwaresoftware
to select the
to opti-
select the
mum formula based on the highest desirability value. The optimum formula was found
optimum formula based on the highest desirability value. The optimum formula was
to consist of 0.999 (w/v %) span 60, 39.99 (v/v %) ethanol, and 0.03 (w/v %) TAC. The pre-
found to consist of 0.999 (w/v %) span 60, 39.99 (v/v %) ethanol, and 0.03 (w/v %) TAC.
dicted responses of EE%, vesicle size and zeta potential were 79.308%, 298.929 nm, and
The−35.047
predicted responses ofwith
mV, respectively, EE%, vesicle size
a desirability ofand
0.704zeta potential were
as represented 79.308%,
in Table 298.929 nm,
4 and Figure
and3. −
Validation of the optimum formula resulted in a % relative error less than 5% for all 4 and
35.047 mV, respectively, with a desirability of 0.704 as represented in Table
Figure 3. Validation
predicted responses,ofconfirming
the optimum formula
the fitness resulted
of the modelin a % relative error less than 5% for
[36].
all predicted responses, confirming the fitness of the model [36].
Table 4. The composition and validation of the optimized formula with its expected responses.
Table 4. The composition and validation of the optimized formula with its expected responses.
Independent Variables Predicted Responses Desirability
Surfactant
Independent
EthanolVariables
Con- Tacrolimus Predicted Responses Desirability
The Optimized Concentra- Vesicle Size Zeta Potential
The Surfactant centration Concentration
Ethanol Tacrolimus EE%
tion w/v %Concentration
FormulaConcentration (nm)
Vesicle Size (mv) Zeta Potential 0.704
Optimized v/v % (X2) Concentration
w/v % (X3) EE%
(nm) (mv) 0.704
Formula (X1)
w/v % (X1) v/v % (X2) w/v % (X3)
0.99990.9999 39.998
39.998 0.03000
0.03000 79.3083
79.3083 298.929 298.929 −35.0471 −35.0471
Validation of the optimum formula
Validation of the Optimum Formula
Responses Predicted value Experimental value % Relative error
Responses Predicted value Experimental value % Relative error
EE% EE% 79.3083
79.3083 81.892
81.892 3.258 3.258
Vesicle
Vesicle size size
298.929
298.929 305.325
305.325 2.139 2.139
(nm) (nm)
Zeta potential
Zeta potential −35.0471 −34.46 1.675
(mv) −35.0471 −34.46 1.675
(mv)

Figure 3. Cont.
Gels 2023, 9, x FOR PEER REVIEW 9 of 21

Gels 2023,
Gels9,2023,
400 9, x FOR PEER REVIEW 9 of 21 8 of 19

Figure
Figure 3. Desirability
3.
Figure 3. Desirabilityplot
Desirability plotof
plot of the
of the numericaloptimization
numerical
the numerical optimization
optimization of PRED–TAC-loaded
of PRED–TAC-loaded
of PRED–TAC-loaded TETSMs.
TETSMs.
TETSMs.

2.3. In In
[Link].
In Vitro
Vitro Release
Release
Vitro ofofPrednisolone
Releaseof Prednisolone fromOptimized
from
Prednisolone from Optimized
Optimized TETSMs
TETSMs
TETSMs Containing
Containing
Containing Gel
Gel Gel
The
The release
The releaseprofile
release profileof
profile of PRED
of PRED is
PRED is illustrated
isillustrated
illustrated inin Figure
Figure
in Figure 4.
4. The The release
release
4. The profile
profile
release showed
profile showed
that that
showed that
the
the PRED–TAC-loaded TETSMs gel achieved higher released PRED (82.93% ± 2.75) than
PRED–TAC-loaded
the PRED–TAC-loaded TETSMs
TETSMs gel
gel achieved
achieved higher
higher released
released PREDPRED (82.93%
(82.93% ± ±
2.75) 2.75)
than than
thethe
the PRED-loadedgel
PRED-loaded
PRED-loaded gel(42.56%
gel (42.56% ±
(42.56% ±± 3.11)
3.11)and
3.11) andPRED
and PRED
PRED suspension
suspension
suspension (50.45% ± 2.12).
(50.45%
(50.45% ± The higher
2.12).
± 2.12). The higher
The higher
release
release achieved could be attributed to the increased thermodynamic activity of PRED
release achieved could be
achieved could be attributed
attributed to to the
the increased
increased thermodynamic
thermodynamic activityactivity of of PRED
PRED
solubilizedininthe
solubilized theTETSMs
TETSMs lipidlipid bilayers,
bilayers,andand to to
thethe
nanosize
nanosizeof the
of vesicles
the that led
vesicles thatto led to
solubilized
higher
in the
release[37].
TETSMs
[37].All
Allgel
lipid bilayers,
gel formulations
formulations exhibited
and to the
Higuchi
nanosize
diffusion
of the
model
vesicles
release.
that led to
higher release exhibited Higuchi diffusion
higher release [37]. All gel formulations exhibited Higuchi diffusion model release. model release.

90
90
% prednisolone released

80
% prednisolone released

80 70
70 60
60 50
PRED loaded gel
50 40
30 PRED
PRED loaded gel
suspension
40
20 PRED-
PREDTAC loaded TETSMs gel
suspension
30
10
20 0 PRED- TAC loaded TETSMs gel
10 0 1 2 3 4 5 6 7
0 Time (hr)
0 1 2 3 4 5 6 7
Figure 4. InTime
vitro(hr)
release profile of PRED from the optimum PRED–TAC-loaded TETSMs gel com-
pared to PRED suspension and the PRED-loaded gel.

Figure 4. In
2.4. PRED
Figure 4. vitro release from
Permeation
In vitro release profile of PRED
PRED
Optimized
profile of from the
Formula
from the optimum
optimum PRED–TAC-loaded
PRED–TAC-loaded TETSMs
TETSMs gel
gel com-
com-
pared to PRED suspension and the PRED-loaded gel.
pared toThePRED suspension and the PRED-loaded gel.
permeation profile of PRED from the optimized-formula-loaded gel compared
to PRED-loaded gel and PRED suspension is illustrated in Figure 5, while permeation
PRED Permeation
[Link] from Optimized Preparations of PRED–TAC-loaded TETSMs gels
are presented in Table [Link]
from the
The permeation profile of PRED from the optimized-formula-loaded
optimized-formula-loaded gel compared
to PRED-loaded gel and PRED suspension is illustrated in Figure 5, while permeation
presented in
parameters are presented in Table
Table 5.
5. Preparations of PRED–TAC-loaded TETSMs gels
showed the highest percent of PRED (62.65% ± 2.87) permeated through the skin, compared
to PRED-loaded gels and PRED suspension (26.5% ± 2.21 and 36.76% ± 1.98, respectively),
 Gels 2023, 9, x FOR PEER REVIEW 10 of 21
Gels 2023, 9, 400 9 of 19

showed the highest percent of PRED (62.65% ± 2.87) permeated through the skin, com-
andpared to due
this is PRED-loaded gels rigidity
to the lower and PRED of suspension (26.5%
the vesicles, ± 2.21
which and 36.76%
permits ± 1.98, respec-
their squashing between
tively), and this is due to the lower rigidity of the vesicles, which permits their squashing
cells. The combined effect of the presence of both ethanol and edge activator (i.e., surfactant)
between cells. The combined effect of the presence of both ethanol and edge activator (i.e.,
enhanced the transepidermal flux of the gel formulation (45.27± 2.87 × 104 µg/cm42 ·h−1 )
surfactant) enhanced the transepidermal flux of the gel formulation (45.27± 2.87 × 10
compared
µg/cm2·hto−1) the PRED-loaded
compared gels and PRED
to the PRED-loaded suspension.
gels and PRED suspension.

80
% prednisolone permeated

60

40 PRED loaded gel

PRED suspension
20 PRED- TAC loaded TETSMs gel

0
0 2 4 6 8
Time (hr)

Figure 5. Ex vivo permeation of prednisolone from the optimum-formula-loaded gel compared to

Figure 5. Ex vivo permeation of prednisolone from the optimum-formula-loaded gel compared to
PRED suspension and the PRED-loaded gel.
PRED suspension and the PRED-loaded gel.
Table 5. Ex vivo permeation parameters of PRED from the optimum-formula-loaded gel compared
Table 5. Ex suspension
to PRED vivo permeation parameters
and PRED-loaded gelof(nPRED from± SD).
= 3, mean the optimum-formula-loaded gel compared
to PRED suspension and PRED-loaded gel (n = 3, mean ± SD).
Formulation Flux (Jss) Permeability Coeffi- Partition Coefficient
Code
Formulation (µg/cm 2 h−1) × 104 cient (P) (cm/h) × 10−6
Flux (Jss ) Permeability Coefficient (K P) ×104
Partition Coefficient
PRED–TAC-loaded
Code 2
(µg/cm h−1 ) × 10 4 (P) (cm/h) × 10−6 (KP ) × 104
45.27± 2.87 9.21± 2.87 242± 2.87
TETSMs gel
PRED–TAC-loaded
45.27 ± 2.87 9.21 ± 2.87 242 ± 2.87
TETSMs
PRED gel
suspension 27.87 ±2.43 5.11 ±2.43 95.41 ± 2.43
PRED-loaded
PRED suspension gel 19.32±±2.43
27.87 3.12 3.425.11
± 3.12
± 2.43 38.54 ±95.41
3.12 ± 2.43
PRED-loaded gel 19.32 ± 3.12 3.42 ± 3.12 38.54 ± 3.12
2.5. Morphological Examination of PRED–TAC-loaded TETSMs
The transmission
2.5. Morphological electronofimage
Examination of the optimum TETSMs
PRED–TAC-Loaded formula is represented in Figure 6.
The image shows spherical vesicles with large aqueous cores. The vesicles are smooth,
The
well transmission
defined, electron image
and non-aggregated, of the optimum
confirming formula
their stability. is represented
The image in Figure 6.
is also in agree-
Thement
image shows spherical vesicles
with the size analysis data. with large aqueous cores. The vesicles are smooth, well
Gels 2023, 9, x FOR PEER REVIEW
defined, and non-aggregated, confirming their stability. The image is also in agreement 11 of 21
with the size analysis data.

[Link]
Figure 6. TEM image TEM image of formula.
optimum the optimum formula.

2.6. Characterization of Gel Formulations

Gel formulations of PRED were characterized regarding pH, % drug content, and
spreadability. The data of these characterizations are shown in Table 6. Regarding pH, all
formulations showed a pH around 6.5 which is dermatologically acceptable according to
Gels 2023, 9, 400 10 of 19

2.6. Characterization of Gel Formulations

Gel formulations of PRED were characterized regarding pH, % drug content, and
spreadability. The data of these characterizations are shown in Table 6. Regarding pH, all
formulations showed a pH around 6.5 which is dermatologically acceptable according to
the literature [38]. All formulations kept a good % drug content meaning good compatibility
of the mixed ingredients.

Table 6. Characterization of PRED containing gel formulations.

Formula pH %Drug Content Spreadability

PRED–TAC-loaded
6.92 ± 0.014 96.53 ± 1.34 4.23 ± 0.12
TETSMs gel
PRED-loaded gel 6.54 ± 0.015 98.23 ± 0.42 3.87 ± 0.23
PRED–TAC-loaded gel 6.73 ± 0.009 97.48 ± 1.51 3.92 ± 0.15

The spreadability has a substantial role in patient agreement and aids in uniform gel
administration to the skin. An acceptable gel needs less time to spread over the skin and
will have great spreadability. The values of spreadability (4.23–3.87 (g cm)/s) confirm that
the gels spread without difficulty by application of a sheer minimum and have satisfactory
bioadhesion [39].

2.7. In Vivo Evaluation of Anti-Inflammatory Effect of PRED Containing TETSMs Gels

The rat hind paw edema model is one of the most used anti-inflammatory models to
study the effect of topically applied anti-inflammatory agents. The percent edema inhibition
of PRED-loaded gel, PRED–TAC-loaded gel, and PRED–TAC-loaded TETSMs gel are shown
in Table 7 and Figure 7. It was noted that PRED–TAC-loaded TETSMs gel achieved the
highest
Gels 2023, 9, x FOR rate of percent inhibition (p < 0.05) among the other gel formulations (98.34% in
PEER REVIEW 12 of 21
180 min), followed by PRED–TAC-loaded gel (63.26%), and PRED-loaded gel (41.25%).

Table 7. % EdemaTable
inhibition of PRED
7. % Edema gel formulations.
inhibition of PRED gel formulations.

% Edema% Edema Inhibition

Inhibition
Time (min) Time (min) PRED–TAC-Loaded PRED–TAC-Loaded
PRED–TAC-Loaded
PRED-Loaded
PRED-Loaded Gel Gel
PRED–TAC-Loaded Gel
Gel TETSMsTETSMs
Gel Gel
30 30 5.34 5.34 10.12 10.12 21.56 21.56
60 60 9.34 9.34 16.24 16.24 33.25 33.25
90 90 14.67 14.67 31.27 31.27 62.87 62.87
120 22.11 43.24 78.43
150 120 34.32 22.11 55.32 43.24 91.2 78.43
180 150 41.25 34.32 63.26 55.32 98.34 91.2
180 41.25 63.26 98.34

120

100
% edema inhibition

80

60

40

20

0
30 60 90 120 150 180
Time (min)
PRED loaded gel PRED-TAC loaded gel PRED- TAC loaded TETSMs gel

Figure
Figure 7. % Edema 7. % Edema
inhibition inhibition
of PRED of PRED gel formulations.
gel formulations.

2.8. Comparative Pharmacokinetic Study of Prednisolone Gel Formulations against Oral PRED
Suspension
Pharmacokinetic parameters of PRED-containing gel formulations and oral PRED
suspension are shown in Table 8. Plasma PRED profiles are illustrated in Figure 8. It is
Gels 2023, 9, 400 11 of 19

PRED, as one of the corticosteroids, has the ability to suppress every step of the
inflammatory cascade including synthesis of inflammatory mediators and cell-mediated
immunity [40]. The TETSMs gel formulation enhanced the permeation of PRED and
its localization in skin layers due to both ethanol and edge activator (surfactant) effects.
Additionally, TAC has a synergistic effect on the inflammatory process due to its effect on
inhibiting the production of cytokines involved in the inflammatory cascade [41,42].

2.8. Comparative Pharmacokinetic Study of Prednisolone Gel Formulations against Oral PRED Suspension
Pharmacokinetic parameters of PRED-containing gel formulations and oral PRED
suspension are shown in Table 8. Plasma PRED profiles are illustrated in Figure 8. It is
noticed that PRED–TAC-loaded TETSMs gel achieved higher pharmacokinetic parameters
Gels 2023, 9, x FOR PEER REVIEW (p < 0.05) compared to PRED-loaded gel (topical) and PRED oral suspension in terms 13 of of
21
Cmax and AUC 0-∞ (133.266 ± 6.469 µg/mL, 538.922 ± 49.052 µg·h/mL), (1.3 and 2.2-fold
increase in Cmax and 1.47 and 1.88 fold increase in AUC 0-∞ compared to oral suspension
and PRED gel, respectively), with significance (p < 0.05) according to ANOVA results in
Table 8. Pharmacokinetic parameters of PRED formulations (mean ± SD).
Table 9. The higher systemic concentration of PRED after application of PRED–TAC-loaded
TETSMs gel than after oral administration could be attributed to the presence PRED–TAC- of edge
Pharmacokinetic
activators PRED Suspension
in the TETSMs composition which increasePRED-Loaded
skin penetrationLoaded
by enhancing the
TETSMs
fluidity ofParameters (Oral) making them
the TETSMs phospholipid bilayer, Gel (Topical)
ultradeformable andGel extremely
elastic, and therefore
Cmax facilitating their squeezing
103.333 ± 5.686 into the skin
61.7 pores [43,44].
± 6.564 Additionally,
133.266 ± 6.469
the presence of ethanol in TETSMs improves drug penetration through the minute holes
Tmax 1.00 ± 0.000 2.00 ± 0.000 1.96 ± 0.057
created in the stratum corneum as a result of fluidization by enhancing the lipids fluidity
t1/2 3.449 ± 0.413 4.926 ± 0.344 3.665 ± 0.428
and decreasing the density of the lipid bilayer [11]. Moreover, the addition of TAC to
AUC 0-t 341.080 ± 33.666 240.056 ± 36.342 490.233 ± 32.855
TETSMs gel formulations resulted in higher pharmacokinetic parameters of PRED, as
AUC 0-inf_obs 365.769 ± 42.054
TAC is previously reported to increase corticosteroids 285.776 ± 45.123 [45].
accumulation 538.922
These±findings
49.052
MRT 0-inf_obs 4.120 ± 0.480 6.293 ± 0.348 4.852 ± 0.466
support the fact that transdermal delivery bypasses first-pass metabolism for many active
ingredients including corticosteroids.
Table 9. One way ANOVA of pharmacokinetic parameters of PRED formulations.
Table 8. Pharmacokinetic parameters of PRED formulations (mean ± SD).
Pharmacokinetic
SS Df MS F p-Value F Crit
Parameters
Pharmacokinetic PRED Suspension PRED-Loaded Gel PRED–TAC-Loaded
Parameters
Cmax 7751.127 (Oral)2 3875.563(Topical)
99.13899 TETSMs5.143253
0.017047 Gel
Cmax
Tmax 1.935556103.333 ± 25.686 0.96777861.7 ± 6.564
871 133.266 ±5.143253
0.001355 6.469
Tmax 1.00 ± 0.000 2.00 ± 0.000 1.96 ± 0.057
t1/2
t1/2 3.8160983.449 ± 0.413
2 1.908049 12.09148
4.926 ± 0.344 0.007855
3.665 ± 5.143253
0.428
AUCAUC0-t0-t 95040.8341.080 ± 2
33.666 47520.4
240.056 40.34386
± 36.342 0.000332 ±5.143253
490.233 32.855
AUC 0-inf_obs 365.769 ± 42.054 285.776 ± 45.123 538.922 ± 49.052
AUC 0-inf_obs
MRT 0-inf_obs
100463.8 2
4.120 ± 0.480
50231.89 24.26347
6.293 ± 0.348
0.001332 5.143253
4.852 ± 0.466
MRT 0-inf_obs 7.33255 2 3.666275 19.28791 0.002439 5.143253

160
140
prednisolone plasma

120
concentration

100 PRED suspension (oral)

(µg/ml)

80
PRED loaded gel (topical)
60
40
PRED- TAC loaded TETSMs gel
20 (topical)
0
0 2 4 6 8 10 12 14
Time (hr)

Figure 8.
Figure 8. Plasma concentration time
Plasma concentration time profiles
profiles of
of PRED
PRED formulations.
formulations.

3. Conclusions
The present research proved that incorporating TAC with PRED in TETSMs and
transdermal application increased the efficacy and optimized the pharmacokinetics of
Gels 2023, 9, 400 12 of 19

Table 9. One way ANOVA of pharmacokinetic parameters of PRED formulations.

Pharmacokinetic
SS Df MS F p-Value F Crit
Parameters
Cmax 7751.127 2 3875.563 99.13899 0.017047 5.143253
Tmax 1.935556 2 0.967778 871 0.001355 5.143253
t1/2 3.816098 2 1.908049 12.09148 0.007855 5.143253
AUC 0-t 95040.8 2 47520.4 40.34386 0.000332 5.143253
AUC 0-inf_obs 100463.8 2 50231.89 24.26347 0.001332 5.143253
MRT 0-inf_obs 7.33255 2 3.666275 19.28791 0.002439 5.143253

3. Conclusions
The present research proved that incorporating TAC with PRED in TETSMs and
transdermal application increased the efficacy and optimized the pharmacokinetics of
PRED, thus improving the efficacy of the therapy for both topical and systemic pathological
conditions. These findings focus the spotlight on a way to decrease the proposed doses of
corticosteroids, utilizing the benefits of both changing the route of administration and the
incorporation of TAC. This will lead to better management of inflammatory conditions and
fewer side effects linked to the use of corticosteroids.

4. Material and Methods

4.1. Materials
Prednisolone (PRED) and tacrolimus (TAC) were kindly gifted by Aljazeera Phar-
maceuticals Company. Phosphatidylcholine from soy lecithin (Phospholipon 90G, pure
phosphatidylcholine stabilized with 0.1% ascorbyl palmitate and max 0.3% tocopherol)
was a gift from LIPOID GmbH, Germany. Ethanol, span 60, and hydroxypropyl methyl
cellulose were purchased from Sigma-Aldrich.

4.2. Experimental Design Setup

A Box–Behnken design was utilized to optimize the formulation variables for creating
PRED-loaded TETSMs with the goal of achieving high encapsulation efficiency, small
vesicle size, and high zeta potential. Experiments were conducted using Design Expert
software, with 17 total experiments conducted, including 13 at the midpoint of each edge
of a multidimensional cube and 4 replicates of the cube’s center point. Three independent
variables were estimated, including surfactant concentration, ethanol concentration, and
tacrolimus concentration. The dependent variables were encapsulation efficiency, vesicle
size, and zeta potential. The study design and composition of the prepared PRED-loaded
TETSMs are presented in Tables 1 and 10. The optimized formula was selected by Design
Expert software based on the highest value of desirability for achieving our goal of high
encapsulation efficiency, high zeta potential, and small particle size. The selected optimized
formula was fabricated and assessed again for different responses to detect the accuracy of
the model.

Table 10. Dependent and independent variables in Box–Behnken design.

Levels
Independent Variables
Low High
Surfactant concentration w/v % (X1) 0.2 1
Ethanol concentration v/v % (X2) 20 40
Tacrolimus (TAC) concentration (X3) 0.03 0.1
Dependent values (Responses) Desirability
EE% (Y1) maximize
Vesicle size (Y2) minimize
Zeta potential (Y3) maximize
Gels 2023, 9, 400 13 of 19

4.3. Preparation of PRED-Loaded TETSMs

Fabrication of PRED-loaded TETSMs was performed according to the thin film hydra-
tion method [46]. Briefly, the accurate weight of phosphatidylcholine, PRED (100 mg), TAC,
and span 60 were dissolved in 10 mL organic solvent, which consisted of 2:1 chloroform:
methanol in a rounded flask. Then the organic solvent was evaporated under vacuum
using a rotary evaporator (Buchi Rotavapor R-200, Switzerland) at 60 ◦ C and 100 rpm. The
formed film was then hydrated with 10 mL of a certain ethanol concentration as specified
in Table 2. The hydration step was carried out at 60 ◦ C to be higher than the lipid transi-
tion temperature [11]. Finally, the formulated dispersions were kept in a refrigerator for
further analysis.

4.4. Characterization of Formulated PRED-Loaded TETSMs

4.4.1. Entrapment Efficiency Estimation
The entrapment efficiency (EE%) of PRED in the fabricated preparations was calculated
by determining the percentage of PRED found in the fabricated preparations. To separate
unentrapped drug, cooling centrifugation was performed at 16,000 rpm and 4 ◦ C using a
Sigma cooling centrifuge from Sigma Laborzentrifugen GmbH. The clear supernatant was
then analyzed for PRED content at λ max 254 nm using a Jasco UV–Vis spectrophotometer
from Jasco, Japan. The EE% can be computed by subtraction of the quantity of PRED
present in the supernatant from the initial quantity of PRED added [47].

4.4.2. Vesicle Size and Zeta Potential Analysis

The zeta potential, polydispersity index (PDI), and average vesicle size of all fabri-
cated preparations were measured using photon correlation spectroscopy with a Zetasizer
2000 (Malvern Instruments Ltd., Malvern, UK) [48]. The nanodispersions were exposed
to dilution before analysis and experiments were conducted in triplicate at a 90-degree
scattering angle and a temperature of 25 degrees Celsius prior to taking measurements.

4.4.3. Formulation Optimization

The optimized formulation for PRED was selected using Design Expert® software
(Ver. 12, Stat-Ease, Minneapolis, MN, USA), with the goal of achieving the highest EE%
and zeta potential, while minimizing vesicle size. The selected optimized formulation was
then fabricated and tested in triplicate to validate the accuracy of the model by comparing
the predicted responses from the software with actual measurements, using the equation
for relative error [49,50].

% Relative error = ((predicted value − actual value)/predicted value) × 100 (4)

4.5. Preparation of Gels Containing Optimized TETSMs:

The optimized formula was amalgamated into a gel base using hydroxypropyl methyl-
cellulose (HPMC, K4M) as a gelling agent at 2.5% (w/v). Briefly, the accurate weight of
HPMC (0.25 g) was dispersed in 10 mL distilled water while stirring at 1000 rpm to obtain
homogenous dispersion. Then the optimum TETSMs formula was dispersed into the
gel base with continuous stirring to obtain a final gel formulation with 5 mg PRED per
1 g gel [36].

4.6. Evaluation of Gel Formulations

4.6.1. pH Evaluation
A 100 mg sample of the TETSMs-loaded gel formulation was weighed and added
to a 50 mL volumetric flask. The volume was then filled to 50 mL with double-distilled
water. The pH of the HPMC gel loaded with TETSMs was noted down using a glass
microelectrode (Mettler Instruments, Giessen, Germany) by measuring the pH after 1 min
of equilibration. The experiments were repeated three times to confirm the neutralization
Gels 2023, 9, 400 14 of 19

of the gel from different batches. The pH was measured on the first, 15th, and 30th day
after preparation to check for any changes in pH over time.

4.6.2. Spreadability
A modified instrument called the spreadability apparatus was used to test spreadabil-
ity. It consisted of two glass slides with gel in the middle, with the upper slide attached to a
balance by a hook and the lower slide fixed to a wooden plate. On the basis of the gel’s slip
and drag characteristics, spreadability was measured [51]. Spreadability was computed
using the following equation:
s = m × l/t (5)
where s represents the spreadability, m is the weight in pan (g), l is the fixed distance moved
by the slide and t is the time.

4.6.3. Drug Content Determination

The created gels were given 48 h to rest before 1 g of each was taken in a 10 mL
volumetric flask, dissolved in methanol, and the remaining 10 mL was filled with water.
For PRED, maximum absorbance values were determined spectrophotometrically using
the Jasco V530, Tokyo, Japan at 254 nm. From a standard calibration curve in methanol,
concentrations of PRED were determined [52].

4.6.4. In Vitro Drug Release Studies

The release of PRED from the PRED–TAC-loaded TETSMs gel, PRED-loaded gel, and
PRED suspension was tested in vitro using a dialysis bag method. An amount of 3 mg of
PRED was placed in a dialysis bag with a molecular weight cut-off of 12,000 Da and released
into 50 mL of Sorensen’s phosphate buffer at pH 7.4. Samples were taken from the medium
at various intervals and replaced with fresh medium over a period of 6 h [53]. The samples
were analyzed using spectrophotometry at a wavelength of 254 nm (using a Jasco V530
from Tokyo, Japan). The release kinetics were determined by applying Korsmeyer–Peppas,
Zero-order, First-order, Higuchi, and Hixson models to the data obtained.

4.6.5. In Vitro Permeation Studies

The barrier membrane samples of hairless rat skin were used to conduct the study after
approval by the Institutional Animal Ethical Committee (IAEC) number SCBR-055-2022,
Prince Sattam Bin Abdulaziz University. Firstly, the animals were anesthetized by ether
and sacrificed; then the abdominal hair was cautiously shaved off. Secondly, skin samples
were excised, and the subcutaneous tissues were detached carefully and finally washed
with saline and stored at −20 ◦ C until use. The membrane samples were placed in vertical
Franz diffusion cells, consisting of a receptor compartment with a capacity of 5 mL and
a diffusion surface area of 1 cm2 . Isotonic phosphate buffer saline (1.55 M, pH 7.4) was
placed in the receptor compartments and stirred at 600 rpm in a temperature-controlled
water bath. Before starting the experiment, phosphate buffer saline was added to the donor
compartment at 37 ◦ C to equilibrate the biological membranes for thirty minutes. After this
time, the phosphate buffer saline was cautiously detached from the donor compartment.
Afterward, a suitable quantity of TETSMs gel equivalent to 3 mg PRED was added to the
donor compartment and spread over the membrane. To maintain occlusive conditions
in the skin, glass slips were put on the donor compartments during the experiment. The
in vitro permeation studies were conducted for 6 h and, at different time intervals, 1 mL
samples were withdrawn from the receptor compartment and substituted with the same
quantity of buffer. All experiments were repeated five times [53,54].
The cumulative amount permeated was computed using Equation (3) by taking into
account the volume of the receptor phase (VR ), the volume of the sample at each time point
(Vs ), and the quantified concentration of the sample taken at the nth time point (Cn ). This
Gels 2023, 9, 400 15 of 19

cumulative amount calculated at each time point was then divided by the diffusion area of
the Franz cells (measured in cm2 ) to obtain the final result.

Cumulative amount = VR × Cn + Vs ∑ C1 + · · · + Cn−1 c



(6)

4.6.6. Data Analysis for Permeation Studies

The different parameters of the skin permeation test were studied and assessed. The
steady state flux (Jss ) of the drug in µg·cm−2 ·h−1 at time t was computed from the slope
of the linear portion of the plot representing the cumulative amount of PRED permeated
per unit area over time. The cumulative amount of PRED in the receptor compartment
after 6 h was known as Qcum (µg·cm−2 ). The permeability coefficient (kp ) of PRED in each
formulation was computed by dividing the steady state flux (Jss ) by the initial concentration
of PRED in the donor compartment (which is considered the saturated solubility of PRED
in each formulation) (Co ) as presented in Equation (4). The results were represented as
mean ± S.D. [55].
Jss
kp = (7)
Co
ANOVA was used to analyze the data of flux and to compare the data of flux with
the cumulative amount of the drug in the receptor compartment of different formulations,
followed by a Tukey’s honestly significant difference test using IBM SPSS® version 23 for
Windows® . A probability of less than 5% (p < 0.05) was considered significant.

4.7. In Vivo Estimation of Anti-Inflammatory Effect of PRED-Loaded TETSMs Gels

Using adult male albino rats, the anti-inflammatory effectiveness of the best formu-
lation of PRED–TAC-loaded TETSMs gel was compared to PRED–TAC-loaded gel and
PRED-loaded gel. The study was conducted according to the guidelines of the Declaration
of Helsinki and approved by the Institutional Animal Ethical Committee (IAEC) number
SCBR-055-2022 of CPCSEA (Committee for Control and Supervision of Experiments on
Animals), Prince Sattam Bin Abdulaziz University. Rats ranged in weight from 100 to 185 g.
Before beginning the tests, they were housed for a week at the animal house at 25 ± 2 ◦ C
and a light: dark cycle of 12:12 h. Rats were fed a conventional rat pellet diet, which was
removed 12 h before the experiment, although water was still available. All studies were
conducted in accordance with the ethical standards for research using experimental animals
as well as the guidelines for the care of laboratory animals. In the present investigation,
24 rats were separated into four groups of six rats each (n = 6), and the following study
protocols were used.
First group: served as a positive control and received PRED-loaded gel.
Second group: received PRED–TAC-loaded gel.
Third group: received PRED–TAC-loaded TETSMs gel.
Fourth group: served as a negative control group.

4.7.1. Carrageenan-Induced Acute Inflammation

By using carrageenan-induced paw edema, the anti-inflammatory effect of the best
formulation of PRED–TAC-loaded TETSMs gel was compared to PRED–TAC-loaded gel
and PRED-loaded gel. The right paw of each animal was massaged 50 times with 0.5 g
of each treatment’s optimal formulation of PRED–TAC-loaded TETSMs gel, PRED–TAC-
loaded gel, and PRED-loaded gel an hour before carrageenan administration to allow
formulation penetration through the skin. Acute inflammation was generated in all groups
an hour after treatment by injecting 0.1 mL of 1% w/v carrageenan saline solution into each
rat’s right hind paw’s sub-plantar tissue. Following carrageenan administration, the rats
were monitored for three hours [56,57].
Gels 2023, 9, 400 16 of 19

4.7.2. Comparative Pharmacokinetics

Blood samples of 0.5 mL were collected at 1, 2, 4, 8, and 12 h from the abdominal aorta
after application of oral PRED suspension, topical PRED-loaded gel, and topical PRED–
TAC-loaded TETSMs gel at a dose of 50 mg/kg. To separate the plasma from different
blood samples, all blood samples were centrifuged at 2000× g for 15 min. Then plasma
was instantaneously conveyed into clean tubes and stored at –20 ◦ C for further analysis.

4.8. HPLC Conditions

HPLC assay of Prednisolone in Plasma
To prepare the plasma samples for the HPLC assay, all samples were vortexed; then
aliquots of 300 µL of plasma were put into falcon tubes. After that, 40 µL of 1 µg/mL
dexamethasone (internal standard) was added to the plasma samples. For Prednisolone
extraction, 1 mL of ethyl acetate was added and vortexed for 10 min then centrifuged at
2500× g for 10 min (4 ◦ C) to allow phase separation. Finally, the upper layer was transferred
to a glass tube and evaporated at 45 ◦ C to dryness then reconstituted with 300 µL of mobile
phase and injected into the HPLC system.
For quantitative determination of prednisolone in plasma samples, 100 µL aliquots
were injected in a Shimadzu HPLC system (SHIMADZU 1200 series HPLC system (Kyoto,
Japan) equipped with a quaternary pump, an online degasser, and an autosampler (SHI-
MADZU1200, Kyoto, Japan). a Ther-mosil® C-18 column (250 mm × 4.6 mm i.d., 5 µm
particle size) was used and operated at 30 ◦ C. The system was equipped with UV–Vis
detectors set at 254 nm. Isocratic elution was performed using acetonitrile and water (36:64)
as the mobile phase (1) with a flow rate of 1.2 mL/min and a total time of 12 min. The liquid
chromatography instrument was interfaced with a computer software using Microsoft
Windows 7 [58].

4.9. Pharmacokinetic Analysis

Pharmacokinetic parameters were calculated using WinNonlin software (version 1.5,
Scientific Consulting, Inc., Rockville, MD, USA). Pharmacokinetic parameters include C
max (maximum plasma concentration), Tmax (time required for peak concentration), AUC
(area under the curve), T1\2 (half-life), and MRT (mean residence time). The obtained
parameters were subjected to analysis by ANOVA and Fisher’s PSLD test for multiple
evaluations among groups. Results were considered significant if the p value was less than
0.05. All results were reported as the mean ± SD.

Author Contributions: Conceptualization, M.M.A. and O.M.S.; Methodology, R.M.Z. and B.N.A.;
Software, R.M.Z.; Validation, M.M.A. and R.M.Z.; Formal analysis, M.M.A. and O.M.S.; Investigation,
B.N.A. and O.M.S.; Resources, M.M.A., R.M.Z., B.N.A. and O.M.S.; Data curation, B.N.A. and O.M.S.;
Writing—original draft, O.M.S. and R.M.Z.; Writing—review & editing, R.M.Z.; Visualization, M.M.A.
and B.N.A.; Project administration, B.N.A. and M.M.A.; Funding acquisition, R.M.Z. All authors have
read and agreed to the published version of the manuscript.
Funding: The authors extend their appreciation to the Deputyship for Research and Innovation,
Ministry of Education in Saudi Arabia, for funding this research work through the project number
(IF-PSAU-2021/03/18810).
Institutional Review Board Statement: The study was conducted according with the Declaration of
Helsinki, and approved by the Institutional Animal Ethical Committee (IAEC) number SCBR-055-
2022 of CPCSEA (Committee for Control and Supervision of Experiments on Animals), Prince Sattam
Bin Abdulaziz University.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data is contained in the manuscript.
Acknowledgments: The authors extend their appreciation to the Deputyship for Research and
Innovation, and Prince Sattam Bin Abdulaziz University, Ministry of Education, Saudi Arabia.
Gels 2023, 9, 400 17 of 19

Conflicts of Interest: The authors declare no conflict of interest.

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