Preparation and Characterization of Lidocaine-Loaded, Microemulsion-Based Topical Gels

Abstract

: Microemulsion-based gels (MBGs) were prepared for transdermal delivery of lidocaine and evaluated for their potential for local anesthesia. Lidocaine solubility was measured in various oils, and phase diagrams were constructed to map the concentration range of oil, surfactant, cosurfactant, and water for oil-in-water (o/w) microemulsion (ME) domains, employing the water titration method at different surfactant/cosurfactant weight ratios. Refractive index, electrical conductivity, droplet size, zeta potential, pH, viscosity, and stability of fluid o/w MEs were evaluated. Carbomer® 940 was incorporated into the fluid drug-loaded MEs as a gelling agent. Microemulsion-based gels were characterized for spreadability, pH, viscosity, and in-vitro drug release measurements, and based on the results obtained, the best MBGs were selected and subsequently subjected to ex-vivo rat skin permeation anesthetic effect and irritation studies. Data indicated the formation of nano-sized droplets of MEs ranging from 20 - 52 nm with a polydispersity of less than 0.5. In-vitro release and ex-vivo permeation studies on MBGs showed significantly higher drug release and permeation in comparison to the marketed topical gel. Developed MBG formulations demonstrated greater potential for transdermal delivery of lidocaine and advantage over the commercially available gel product, and therefore, they may be considered as potential vehicles for the topical delivery of lidocaine.

Full text

Iranian Journal of Pharmaceutical Research 21 (2022) e1: 1-21
DOI: 10.22037/ijpr.2021.115214.15256
Received: March 2021
Accepted: June 2021
Original Article
Preparation and Characterization of Lidocaine-loaded,
Microemulsion-Based Topical Gels
Mahshid Daryaba, Mehrdad Faizib, Arash Mahboubia, c* and Reza Aboofazelia, d*
aDepartment of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical
Sciences, Tehran, Iran. bDepartment of Pharmacology and Toxicology, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran. cFood Safety Research Center,
Shahid Beheshti University of Medical Sciences, Tehran, Iran. dProtein Technology Research
Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
* Corresponding author:
E-mail: a.mahboubi@sbmu.ac.ir; raboofazeli@sbmu.ac.irE-mail: a.mahboubi@sbmu.ac.ir; raboofazeli@sbmu.ac.ir
Abstract
Microemulsion-based gels (MBGs) were prepared for transdermal delivery of lidocaine and
evaluated for their potential for local anesthesia. Lidocaine solubility was measured in various
oils, and phase diagrams were constructed to map the concentration range of oil, surfactant,
cosurfactant, and water for oil-in-water (o/w) microemulsion (ME) domains, employing the
water titration method at dierent surfactant/cosurfactant weight ratios. Refractive index,
electrical conductivity, droplet size, zeta potential, pH, viscosity, and stability of uid o/w MEs
were evaluated. Carbomer 940 was incorporated into the uid drug-loaded MEs as a gelling
agent. Microemulsion-based gels were characterized for spreadability, pH, viscosity, and in-vitro
drug release measurements, and based on the results obtained, the best MBGs were selected and
subsequently subjected to ex-vivo rat skin permeation anesthetic eect and irritation studies. Data
indicated the formation of nano-sized droplets of MEs ranging from 20-52 nm with a polydispersity
of less than 0.5. In-vitro release and ex-vivo permeation studies on MBGs showed signicantly
higher drug release and permeation in comparison to the marketed topical gel. Developed MBG
formulations demonstrated greater potential for transdermal delivery of lidocaine and advantage
over the commercially available gel product, and therefore, they may be considered as potential
vehicles for the topical delivery of lidocaine.
Keywords: Lidocaine; Microemulsion; Microemulsion-based gel; Phase diagrams; Skin
permeation; Local anesthesia.
Introduction
Topical anesthetics, as valuable tools in
the eld of dermatology, are widely used
to control cutaneous pain associated with
medical procedures, prevent or treat chronic
conditions such as post-herpetic neuralgia,
complex regional pain syndrome, and cancer-
related pains. These compounds are expected
to cause painless, cutaneous analgesia with a
quick onset of action and sucient duration
(1-3). Based on the chemical structure of
their intermediate chain, these weak bases are
classied into aminoesters (e.g., benzocaine,
procaine, tetracaine, etc.) and aminoamides
(e.g., bupivacaine, lidocaine, prilocaine)
classes (1, 4).
Local anesthetic (LA) drugs are
commercially available in various
pharmaceutical dosage forms such as gels,

2
Daryab M et al. / IJPR 21 (2022) e1: 1-21
creams, ointments, solutions, and patches (5,
6), and many of them are available over-the-
counter without the need for a prescription.
The purpose of such transdermal formulations
is to increase skin permeability, reduce the
eective concentration, provide painless,
cutaneous analgesia and numbness with a
quick onset of action and sucient duration of
action and minimize side eects (6, 7).
Lidocaine is a low soluble and good
penetrating drug (Biopharmaceutical
Classication System, class II) with a
molecular weight of 234.3 g/mol and log p
equal to 2.84, which makes it a good candidate
for skin delivery (Scheme 1). This amide
derivative is the most commonly used as well
as an eective and reliable LA drug formulated
as topical products due to its desirable
properties such as the low risk of allergic
reactions, intermediate duration of action, and
low systemic toxicity (8-10). However, the
major problems encountered with the use of
commercial lidocaine products are late-onset
and insuciency of local anesthetic eect. It
has been reported that commercial creams or
gels are not capable of eectively delivering
lidocaine base (or its HCl salt) through the
intact skin. To overcome these problems, an
increase in the skin permeability to dermally
applied lidocaine is required (8).
Several strategies have been adopted to
enhance the skin permeability of LAs and
improve their onset and duration of action,
as well as prevent systemic absorption
and reduce side eects. Among the most
common physical techniques, one can
mention iontophoresis, sonophoresis,
magnetophoresis, electroporation,
microporation, and microneedle technologies
(9, 11 and 12). However, using these methods
is restricted because of the high cost, need for
special devices, and qualied sta (13). Other
delivery strategies include the incorporation
of LAs into innovative colloidal carrier
delivery systems such as liposomes, niosomes,
ethosomes, nanospheres, nanoparticles, and
microemulsions (5, 13).
Microemulsions (ME), rst introduced
by Hoar and Schulman (14), are transparent,
spontaneously formed, dispersed systems in
which the interfacial layer is stabilized by
a layer of surfactant molecules (usually in
combination with a co-surfactant) (15). These
transparent, low viscose, thermodynamically
stable (no tendency for occulation or
coalescence) colloidal dispersions with
droplets less than 120 nm in diameter oer
several advantages for ecient transdermal
delivery of drugs. MEs can be formulated
as water-in-oil (w/o), oil-in-water (o/w),
and bicontinuous systems (16-18). The ease
of preparation, relatively high solubilizing
capacity for a variety of hydrophilic and
lipophilic molecules because of the existence
of two microdomains in a single-phase
solution, long-term thermodynamic stability,
and good production feasibility have made
them promising drug delivery systems (19-
21). The greater amount of drug incorporated
in MEs, compared to conventional topical
formulations, could increase the ux of drug
through the skin. Moreover, enhancement of
drug solubility can increase the concentration
gradient and thermodynamic activity of
the drug, which could favor its partitioning
into the skin. The possibility of employing
ingredients for ME formulation with skin
penetration enhancing eect can also aect
the barrier function of stratum corneum (SC),
promoting permeation of drug (22-25).
Since MEs are less viscose in nature, their
low skin adherence has restricted their topical
application (26). To overcome this challenge
and retain the applied dose on the skin for
a sucient time, ME-based gel (MBG)
formulations have been, therefore, developed,
utilizing a suitable thickening agent to modify
the rheological behavior. MBGs, also known
Scheme 1. Structure of lidocaine

Scheme 1. Structure of lidocaine

3
Lidocaine-loaded, Microemulsion-based Topical Gels
as hydrogel-thickened MEs, are nanocarriers
derived from o/w MEs composed of dispersed
oil phase within a continuous aqueous phase,
which is thickened with a suitable hydrophilic
gelling agent (27, 28). By the addition of
gelling components, the application of
MEs to the skin becomes easier compared
to runny uid MEs. Various gelling agents
such as Carbopol, xanthan gum, chitosan,
poloxamer, hydroxypropyl methylcellulose,
and carrageenan have been utilized for the
preparation of MBGs (27, 28).
Based on the type of polymer used, dierent
procedures for the preparation of MBGs have
been employed. A mixture of oil, surfactant,
and cosurfactant with the dissolved drug is
added to the previously prepared hydrogel
matrix in a two-stage procedure. Alternatively,
o/w ME is prepared and then gelled by directly
dispersing a suitable thickening agent (28).
These gels have the advantages of both MEs
and hydrogels, including ease of preparation,
enhanced drug solubility and permeability,
optical clarity, longer shelf-life, water
solubility, and spreadability (29, 30). In recent
years, numerous studies have demonstrated
that MBGs are potential transdermal
delivery systems for a wide variety of drugs
commonly used in dierent skin disorders or
even systemic diseases (31, 32). Negi et al.
showed that phospholipid MBGs containing
lidocaine and prilocaine have enhanced skin
permeation and improved the analgesic eect
signicantly, compared to the commercial
cream (13), while a remarkable analgesic
activity has been observed with ropivacaine-
loaded MBGs, formulated by Transcutol
HP and Capryol 90 (33). In 2017, Okur et
al. investigated the permeation of Carbopol
940-based, benzocaine-loaded MBGs and
conrmed high permeability through the skin
with less systemic side eects with no sign of
inammation and irritation (34).
Since the dermal delivery of lidocaine is
still a concern, the suitability of MBGs for
its transdermal delivery was examined in this
investigation. Thus, this study was planned
to develop and characterize lidocaine-loaded
MBGs, formulated with pharmaceutically
acceptable components. It was hypothesized
that a rapid onset and longer duration of
anesthetic eect of lidocaine might be
produced when incorporated in MBGs.
Experimental
Materials
Lidocaine base and Cremophor RH40
(PEG-40 hydrogenated castor oil) were
supplied by Daroupakhsh Pharma Chem.
Co. (Tehran, Iran) and Osvah Pharmaceutical
Co. (Tehran, Iran), respectively. Kolliphor®
ELP (Cremophor EL, PEG-35 hydrogenated
castor oil) was purchased from BASF
(Germany). Labrasol (caprylocaproyl
macrogol-8-glycerides) and Transcutol P
(diethylene glycol monoethyl ether) were
gifted by Gattefossé (France). Olive & castor
oils were provided from Sigma Aldrich (USA)
and GPR Rectapur (France), respectively.
Triacetin (glycerol triacetate), isopropyl
myristate (IPM), polysorbate 80 (Tween 80),
polyethylene glycol 400 (PEG 400), propylene
glycol (PG), ethanol 96% (v/v), glacial acetic
acid (HPLC grade), acetonitrile (HPLC grade),
sodium hydroxide and potassium dihydrogen
phosphate were all obtained from Merck
Chemical Co. (Germany). Carbomer 934
and 940 were purchased from BF Goodrich
(USA). Metolose 90SH (hydroxypropyl
methylcellulose) was provided from Shin-
Etsu Chemical Co., Ltd. (Japan). Puried
water was prepared by a Millipore Milli-Q
water purication system (USA).
High-performance liquid chromatography
(HPLC) method
A quantitative assay of lidocaine base
was carried out by an HPLC method outlined
in the United States Pharmacopoeia (USP
41-NF 36). Chromatographic studies were
implemented on the Knauer HPLC system
(Germany), equipped with a UV detector
(Smartline 2500), pump (Smartline 1000), and
software (Chromgate V3.1.7). The separation
process was carried out on a reversed-phase
C18 column (5 μ, 250 × 4.6 mm), using a
freshly prepared and degassed mobile phase
consisting of acetonitrile (A) and water/glacial
acetic acid (930:50; pH 3.4) in the ratio of 1:4.
The ow rate was xed at 1.2 mL/min, and
all measurements were performed at room
temperature. The injection of samples was
performed on a Reodyne injector equipped

4
Daryab M et al. / IJPR 21 (2022) e1: 1-21
with a 20 μL loop. The UV detector was set at
254 nm. The calibration curve was found to be
linear in the concentration range of 10-100 μg/
mL (r2 = 0.9985).
Determination of lidocaine oil solubility
The solubility of lidocaine was evaluated in
triacetin, IPM, castor oil, and olive oil by the
shake-ask method (35). An excess amount of
lidocaine was added to 1 mL of the oil. The
mixture was then continuously stirred, using
a magnetic stirrer, at room temperature (25
℃) for 72 h in order to achieve equilibrium.
After removing the undissolved drug, samples
were centrifuged (Sigma 1-14, Osterode am
Harz, Germany) at 5000 rpm for 10 min;
the supernatant was separated and ltered
through a 0.22 μm membrane lter and then
diluted with a suitable solvent (chloroform or
ethanol 96% v/v). Finally, the amount of the
drug dissolved in each oil was assayed by a
UV spectrophotometer (UV-2601, Rayleigh,
China) at the wavelength of 263 nm, using oil
samples with known drug concentrations.
Construction of phase diagrams
Castor oil and triacetin (selected based
on the oil solubility studies), four non-ionic
surfactants (Tween 80, Labrasol, Cremophor®
EL and Cremophor RH40) and three co-
surfactants (PEG 400, Transcutol P and PG)
were chosen to construct the phase diagrams
and determine the o/w microemulsion domains.
Surfactant/co-surfactant weight ratios (Rsm)
were kept constant at dierent values of
1:1, 1:2, 2:1. Clear oil-surfactant mixtures
with various weight ratios of 1:9 to 9:1 were
prepared by weighing appropriate amounts
of each component into screw-capped vials
and mixing thoroughly at room temperature.
Samples were then titrated with small aliquots
of triple distilled water while stirring for
a sucient time to attain equilibrium. The
course of each titration was inspected visually
and through cross-polaroids for determining
the clarity and the possible formation of a
birefringent liquid crystalline phase. The
triangle diagrams were mapped with the top
apex representing a xed Rsm (1:1, 1:2, or
2:1), the right and left apices representing
the oil and water, respectively. All mixtures
which produced optically transparent, non-
birefringent solutions at relatively water-rich
parts of the phase diagrams were designated
as o/w MEs.
Preparation of lidocaine-loaded MEs
Following the determination of o/w ME
regions on the phase diagrams, those oil/
surfactant/oil systems which demonstrated
a relatively extended o/w ME area on the
phase diagrams, composed of a minimum
of 5% (w/w) oil and not more than 25%
(w/w) surfactant mixture, were selected for
drug loading. Lidocaine-loaded MEs were
prepared by the spontaneous emulsication
method. A given amount of lidocaine base was
dissolved gradually in the oil phase to which
the surfactant mixtures were then added, and
the required amount of distilled water was
nally added dropwise while stirring the
mixture gently until a transparent solution
was obtained. The formulations were stored
at room temperature and were evaluated
for clarity, drug precipitation, and phase
separation within 72 h.
Characterization of uid MEs
Refractive index (RI), pH, and conductivity
The Refractive index of drug-loaded MEs
was measured by Abbe Refractometer (2WAJ,
Bluewave Industry Co., Ltd., Shanghai,
China). The pH and electrical conductivity
of MEs were determined using a calibrated
pH-meter (744, Metrohm AG, Switzerland)
and a conductivity meter (712, Metrohm AG,
Switzerland), respectively.
Determination of particle size and zeta
potential
Mean droplet size (Z-ave), polydispersity
index (PDI), and zeta potential of ME
formulations were measured at 25 °C, using
a Malvern Zetasizer (Nano-ZS, Malvern
Instruments, Worcestershire, UK), equipped
with a Nano ZS Software for data acquisition
and analysis. Each sample was analyzed in
triplicate, and the results were reported as
mean ± SEM.
Determination of viscosity and rheological
behavior
A Brookeld DV2T cone and plate
viscometer (LV, Brookeld Engineering

5
Lidocaine-loaded, Microemulsion-based Topical Gels
Laboratories, Middlesboro, USA), equipped
with a CP-42 spindle, was used to measure the
viscosity and examine the rheological behavior
of ME formulations. To evaluate thixotropic
behavior, measurements were carried out at a
rotation speed ranging from 2 to 70 rpm for
both up curves and down curves, at 25 ± 1 °C.
Results within the 10-100% range of torque
were considered acceptable and recorded. The
shear stress (Pa) was plotted vs. shear rate
(1/s), and the viscosity was calculated based
on the slope of the linear portion of the plots.
Preparation of lidocaine-loaded MBGs
MBGs containing 5 wt% lidocaine were
fabricated by dispersing dierent amounts
of various polymers, namely Carbomer 934
(1-3 wt%), Carbomer 940 (0.5-1.5 wt%)
or Metolose 90SH (3-6 wt%), in the drug-
loaded uid MEs under magnetic stirring.
Gel characterization
Spreadability
To measure the spreadability of MBGs, a
circle with 1 cm in diameter was marked on
a glass plate. Half a gram of the test gel was
placed on the circle, and a second glass plate
was placed on the gel. A 5 g weight was put
on the upper glass plate, and after 5 min, the
weight was removed, and the diameter of the
spread gel was measured and reported (36).
pH measurement
One gram of MBGs was mixed with 99 g
distilled water and stirred thoroughly until a
uniform mixture was obtained. The pH was
measured in triplicate, using a calibrated pH
meter (744, Metrohm AG, Switzerland).
Determination of viscosity and rheological
behavior
Viscosity and rheological properties of the
MBGs were determined at 25 ± 1 °C, using
a Brookeld DV-III Ultra Programmable
Rheometer (Brookeld Engineering
Laboratories, Middlesboro, USA), attached
with spindle no. 51. Measurements were
performed at a rotation speed ranging from 0.5
to 250 rpm for both upward and downward ow
curves. Flow curves (rheograms) were plotted,
and the viscosities were then calculated based
on the slope of the linear portion of the plots.
Stability tests
Fluid MEs were stored in sealed glass vials
at 25 °C for 15 months and observed for any
macroscopic changes, including turbidity,
phase separation, drug precipitation, and color
change (37). The stability of the selected
MBGs was also evaluated for 6 months at
ambient temperature, 2-8 °C and 40 ± 2 °C
(Relative humidity: 75 ± 5%), and checked for
their appearance and viscosity. In addition, the
optimum gel formulations were centrifuged
(5702, Eppendorf AG, Hamburg, Germany) at
5000 rpm for 30 min, subsequently subjected
to seven heating/cooling cycles (24 h at 4 °C
followed by 24 h at 40 °C) and three 24-hour
freeze-thaw (FT) cycles (-5 and 25 °C) (38-
40).
In-vitro drug release
Cellulose acetate membrane
In-vitro permeation study of lidocaine-
loaded MBGs was carried out using vertical
Franz diusion cell with 1.767 cm2 eective
diusion surface area. Synthetic cellulose
acetate membrane (MW cut-o 12,000 Da),
previously soaked in phosphate buer pH
7.4 for 24 h at 2-8 °C, was placed between
the donor and receptor compartments of the
diusion cell. The receptor chamber (25 mL)
was lled with phosphate buer solution
(0.1 M, pH 7.4) and thermostated at 37 ± 0.5
°C, while continuously stirring (400 rpm). A
quantity of 200 mg of the gel was applied to
the membrane, and the donor chamber was
covered with Paralm. At predetermined time
intervals (5, 7, 10, 15, 20, 30, 45, 60, 75, 90,
105, and 120 min), an aliquot of 2 mL sample
was taken from the release medium, and the
same volume of the fresh buer was added
to the receptor chamber to maintain the sink
condition. During the test, the diusion cells
were checked for the presence of a bubble on
both sides of the membrane. The cumulative
amount of drug released from MBGs at
each time was measured. As a control, a
commercially available 5 wt% lidocaine gel
was used.
Ex-vivo permeation study
The ex-vivo permeability study protocol
was approved by the local Animal Ethics
Committee of Shahid Beheshti University of

6
Daryab M et al. / IJPR 21 (2022) e1: 1-21
Medical Sciences, Tehran, Iran (approval No:
1399.002). Male Wistar albino rats (200-250
g) were sacriced by ether inhalation. The
hair of test animals was carefully trimmed
with electrical clippers, and the full-thickness
skin was excised carefully from the abdominal
region and wiped with acetone to remove
adhering fat and connecting tissues. The
prepared skins were wrapped in aluminum foil
and stored at -20 °C for further use. Prior to the
test, the skins were kept for 30 min at ambient
temperature, then mounted between the donor
and receiver compartments of a static Franz
diusion cell, while the dermis side was in
contact with the release medium for 12 h
(41). After skin hydration and replacement
of fresh phosphate buer (pH 7.4), the skin
permeation study was carried out with the
same procedure described for the in-vitro drug
release experiments, except that the samples
were taken from the receptor chamber after 7,
10, 15, 20, 30, 45, 60, 75, 90, 105, 120, 150,
180, 210, 240, 300, 360, 480 and 600 minutes
and ltered through a 0.45 μm membrane
lter. The cumulative percentage of lidocaine
in withdrawn samples was calculated, and the
results were plotted as a function of time (in
a minute) and compared with those obtained
from the commercial gel. The ex-vivo release
prole was tted into various mathematical
models, i.e., zero-order, rst-order, Higuchi
and Korsmeyer-Peppas, in order to elucidate
the kinetic release model. All the experiments
were performed in triplicate, and the results
were reported as mean ± SEM. Data were
statistically analyzed by one-way analysis
of variance (ANOVA), followed by Tukey’s
post hoc using GraphPad Prism version 8.0.1
(GraphPad Software, Inc., USA). A 0.05 level
of probability was considered as the level of
signicant dierence (*p < 0.05: signicant,
**p < 0.01: very signicant, and ***p < 0.001:
extremely signicant).
Evaluation of the local anesthetic eect
Male Wistar albino rats (200-250 g) and
New Zealand white male albino rabbits (2.0-
2.5 kg) were obtained from Pasteur Institute
(Tehran, Iran) and used for local anesthetic
studies and skin irritation tests, respectively.
The animals were housed in suitable cages
at a controlled temperature (20-24 °C), on a
12:12 h, day/night cycle with free access to
a pellet diet and water ad libitum. All animal
experiments were performed in accordance
with the National Institute of Health (NIH)
Guide for the Care and Use of Laboratory
Animals (8th edition), approved by the
Institutional Animal Care and Use Committee
and local Animal Ethics Committee of Shahid
Beheshti University of Medical Sciences (No.
IR.SBMU.PHARMACY.REC.1399.002). The
local anesthetic eect of formulations was
assessed by performing a manual von Frey
test. All experiments were carried out between
9:00 and 16:00. Before the experiments, the
adult male rats were placed in an individual
clear acrylic box with an elevated plastic wire
mesh oor which allowed acclimating for 30
min in the testing environment. Animals were
divided randomly into the following groups (n
= 8):
Placebo control group (control A, B, and C)
Treated group with selected lidocaine-
loaded MBGs (formulation A, B, and C)
Treated group with the commercial
lidocaine gel
In this behavioral study, 0.5 g of each gel
was topically applied to the rat hind paw.
Then, a series of 10 von Frey laments with
logarithmic incremental stiness (4, 6, 8, 10,
15, 26, 60, 100, 180, and 300 g) in ascending
order was used to determine the mechanical
allodynia threshold of the animals, 10-210
min following the application of the gel with
10-min intervals. Each nylon lament was
applied ve times through the mesh oor
on the plantar surface of the rat paw till it
bends (buckles). Brisk paw withdrawal,
licking, or shaking of the stimulated paw
was considered as a positive response (42).
The strongest lament inducing up to two
responses out of ve stimuli was recorded
as the mechanical threshold at each time
point. Results were reported as mean ± SEM.
Statistical dierences were evaluated using
two-way ANOVA with Bonferroni’s post-
test to compare the mechanical threshold at
each time, and one-way ANOVA followed by
Tukey’s post-test to evaluate the created area
under the time-course curve (AUC10-210 min) of
the mechanical threshold by each formulation
during the test. As stated earlier, p < 0.05 was
considered statistically signicant (*p < 0.05,

7
Lidocaine-loaded, Microemulsion-based Topical Gels
**p < 0.01 and ***p < 0.001).
Skin irritation test
The acute dermal irritation potential of the
nal formulation was evaluated in accordance
with the OECD guideline (43). The animals
were acclimatized for one week before the
beginning of the study and had access to a
standard diet and food. The hairs on the back of
rabbits were trimmed by an electrical clipper
24 h prior to administration of the formulation.
The animals were divided into three groups (n
= 3) as follows:
No application (control)
Blank MG4
Drug-loaded MG4
Half a gram of gel formulations was applied
uniformly to the test area (approximately 6
cm2). At the end of the 4-h exposing duration,
the residual gel was wiped o with water. All
rabbits were observed for any visible change
such as erythema or edema after 1, 24, 48, and
72 h of the gel application. If skin damage
cannot be recognized as irritation or corrosion
after 72 h, the observation should continue
until day 14 in an attempt to determine the
reversibility of the eects. Erythema and
edema were graded according to the following
criteria: 0, no visible reaction; 1, very slight
reaction; 2, well-dened erythema; 3, moderate
to severe reaction; 4, severe reaction. Finally,
the irritation scores of the test area were
calculated using the following equation and
interpreted according to Table 1.
Equation 1.
Results and Discussion
Drug solubility in the oil phase
The ability of the oil phase for drug
solubilization is considered as the most
important criterion for o/w microemulsion
formulations (44). Lidocaine solubility results
for dierent oils are given in Figure 1. As can
be seen, the highest solubility was obtained in
castor oil and triacetin (538.460 ± 7.457 mg/
mL and 530.727 ± 6.029 mg/mL, respectively).
This represents the potential of these oils to
Table 1. Interpretation of PII scores.*
PII score Irritation grade
PII = 0 no irritation
0 < PII ≤ 2 mild
2 < PII ≤ 5 moderate
5 <PII  8 severe
*Appraisal of the safety of chemicals in foods, drugs, and cosmetics: Association of Food
and Drug Officials of the United States, 1959.
Figure 1. Solubility study of lidocaine in various oils. Data expressed in mean  SEM (n = 3).
Figure 1. Solubility study of lidocaine in various oils. Data expressed in mean ± SEM (n = 3).
Table 1. Interpretation of PII scores.*
󰇛󰇜





8
Daryab M et al. / IJPR 21 (2022) e1: 1-21
solubilize lidocaine, and therefore, they were
selected for ME and MBG preparations.
Phase diagrams and o/w ME domains
Phase diagrams of four-component systems
were constructed to determine the appropriate
concentration ranges of the components to form
MEs. Unlike the systems containing castor oil,
the o/w ME region was observed on triacetin-
based phase diagrams. Figure 2 indicates that in
nearly all phase diagrams (except for triacetin/
Tween 80/PEG 400/water systems at Rsm of
1:2 and triacetin/Labrasol/PEG 400/water,
regardless of Rsm), a transparent, isotropic
o/w ME region was formed in the oil-poor
part of the phase diagrams. It should be noted
that because of the diculties in accurately
determining the boundaries between the ME
domains and surfactant-rich area on the top
of the phase diagrams, samples with up to 50
wt% surfactant mixture were considered as
MEs, above which the area was considered as
surfactant-rich area.
The following generalizations could be
Figure 2. Phase diagrams of systems consisting of triacetin as the oil phase (right apex), distilled
water (left apex), (A) Transcutol P, (B) PG, and (C) PEG 400 as co-surfactant and various
surfactants (top apex) namely Tween 80 (green), Labrasol (red), Cremophor EL (yellow), and
Cremophor RH40 (purple) at various Rsm of a) 1:1, b) 1:2, and c) 2:1. The colored area in the oil-
poor part of the phase diagram represents the o/w ME domain.
Figure 2. Phase diagrams of systems consisting of triacetin as the oil phase (right apex), distilled water (left apex), (A)
Transcutol P, (B) PG, and (C) PEG 400 as co-surfactant and various surfactants (top apex) namely Tween 80 (green),
Labrasol (red), Cremophor EL (yellow), and Cremophor RH40 (purple) at various Rsm of a) 1:1, b) 1:2, and c) 2:1.
The colored area in the oil-poor part of the phase diagram represents the o/w ME domain.

9
Lidocaine-loaded, Microemulsion-based Topical Gels
made about the investigated systems:
Tween-based systems showed higher water
solubilization capacity in comparison to the
other surfactants.
Irrespective of the type of surfactant and
Rsm, the largest and smallest ME areas were
seen in the presence of Transcutol P and PEG
400, respectively.
Regardless of the type of surfactant and
co-surfactant, Rsm did not have a signicant
inuence on the extent of the o/w ME region.
Various ME formulations were selected
from the relatively extended o/w ME area on
the phase diagrams, considering the minimum
possible concentration of surfactants. Those
with no drug precipitation and phase separation
at the time of preparation and after 72 h storage
were chosen for further characterization tests
(Table 2).
ME characteristics
The prepared formulations (Table 2) were
found to be macroscopically identical, i.e.,
homogeneous, single-phase, and transparent
by visual inspection. The colloidal nature
of these systems was also conrmed by
observing the Tyndall eect. Table 3 lists
the data of Z-average, PDI, zeta potential,
pH, conductivity, and viscosity of the drug-
loaded ME formulations. The isotropic nature
of the formulations was also conrmed as a
completely dark eld was observed under the
cross-polarized light microscope.
The refractive indices of all formulations
were ranged between 1.3750 and 1.3890 and
close to that of water as the external phase
(1.334). Electrical conductivity measurement
is a useful tool to dierentiate w/o droplets
from o/w-type droplets and bicontinuous
structures. Generally, low conductivity
exhibits the formation of w/o droplet MEs
(because water makes the internal phase),
while systems showing high conductivity are
dened as bicontinuous or o/w-type MEs as
the presence of water in the continuous phase
allows the measurement of conductivity. In
this study, data obtained from both RI and
conductivity measurements (162.592-198.340
Table 2. Composition of the selected microemulsion systems for drug solubilization.
Microemulsion Triacetin (%) (oil phase) Surfactant (%) Co-surfactant (%) Water
(aqueous phase)
X1 X
2 X
3 X
4 X
5 X
6
ME1 5.04 16.59 - - 8.29 - - 70.08
ME2 5.04 12.49 - - - 12.49 - 69.98
ME3 5.04 16.59 - - - 8.29 - 70.08
ME4 5.04 16.59 - - - - 8.29 70.08
ME5 5.04 - 12.49 - 12.49 - - 69.98
ME6 5.04 - 16.59 - 8.29 - - 70.08
ME7 5.04 - 9.97 - - 9.97 - 75.02
ME8 5.04 - 8.29 - - 16.59 - 70.08
ME9 5.04 - 13.23 - - 6.74 - 74.99
ME10 5.04 - 9.97 - - - 9.97 75.02
ME11 5.04 - 16.59 - - - 8.29 70.08
ME12 5.04 - - 16.59 8.29 - - 70.08
ME13 5.04 - - 12.49 - 12.49 - 69.98
ME14 5.04 - - 16.59 - 8.29 - 70.08
X1: Tween 80; X2: Cremopho
r
 EL; X3: Cremopho
r
 RH40; X4: PEG 400, X5: TranscutolP; X6: PG.
Table 2. Composition of the selected microemulsion systems for drug solubilization.
Table 3. Characterization of fluid MEs (mean  SEM).
ME Z-average (nm) PDI Zeta potential (mV) pH Conductivity (

S/cm) Refractive index Viscosity (mPa.s)
ME1 47.073  0.524 0.408 0.005 0.635 0.029 8.04 0.030 194.005  0.116 1.3850 0.0002 27.10 0.058
ME2 36.710  0.541 0.460 0.009 -0.711 0.075 8.28 0.015 167.327  0.122 1.3815 0.0002 11.90 0.033
ME3 45.290  0.593 0.389 0.004 0.0886 0.035 7.82 0.012 193.940  0.035 1.3830 0.0003 18.50 0.044
ME4 44.140  1.400 0.380 0.009 0.0255 0.040 7.76 0.010 194.721  0.124 1.3830 0.0002 19.70 0.015
ME5 52.196  0.248 0.404 0.002 0.523 0.046 7.80 0.010 190.037  0.043 1.3840 0.0002 20.90 0.010
ME6 30.950  0.163 0.363 0.003 -0.265 0.058 7.90 0.012 169.172  0.588 1.3865 0.0003 26.8 00.007
ME7 38.850  0.117 0.357 0.007 0.366 0.035 8.11 0.010 198.340  0.116 1.3770 0.0005 8.59 0.006
ME8 59.506  0.532 0.452 0.008 0.285 0.046 8.09 0.015 194.288  0.130 1.3805 0.0002 7.85 0.006
ME9 32.380  0.121 0.371 0.001 0.134 0.023 8.04 0.025 188.365  0.289 1.3765 0.0003 11.20 0.007
ME10 28.016  0.112 0.314 0.001 0.832 0.080 8.10 0.009 176.834  0.324 1.3750 0.0005 8.41 0.010
ME11 20.626  0.225 0.350 0.001 -0.447 0.052 7.98 0.015 162.591  0.176 1.3820 0.0002 19.80 0.030
ME12 45.400  5.506 0.428 0.050 0.0318 0.064 8.16 0.010 179.647  0.472 1.3890 0.0005 18.80 0.046
ME13 57.060  14.475 0.178 0.006 -0.310 0.035 8.20 0.015 173.398  1.041 1.3810 0.0005 9.10 0.006
ME14 26.910  0.736 0.232 0.006 0.201 0.098 8.22 0.012 173.012  1.882 1.3820 0.0003 14.00 0.055
Table 3. Characterization of uid MEs (mean ± SEM).

10
Daryab M et al. / IJPR 21 (2022) e1: 1-21
µS/cm) approved the o/w structure of the MEs
studied (45). The conductivity results also
depicted that the addition of lidocaine to the
internal phase of MEs did not aect the system
stability.
The average particle size and size
distribution of MEs were evaluated by the
dynamic light scattering technique. PDI was
also determined to provide information about
the deviation from the mean size. Table 3
represents the results of size and PDI analysis.
As can be seen, in all systems, the average
size of the ME droplets was less than 60 nm
(ranged from 20.626-59.506 nm) which lies
in the proposed range for ME systems (<120
nm). All formulations exhibited unimodal
droplet size distribution patterns (diagrams not
shown). In most cases, as a measure of droplet
size uniformity, PDI values were found to be
less than 0.4, suggesting that droplets in nearly
all MEs were relatively uniform-sized.
To analyze the charge of the droplets, zeta
potential is determined. Zeta potential values
indicated that the interface had a low surface
charge (-0.711 to +0.832 mV). The charge in
the interfacial area, in general, may originate
from many factors the composition of oil, the
presence of electrolytes in the water phase,
and the nature of surfactants. In this study,
very low zeta potential (nearly zero potential)
values obtained in this study could be ascribed
to the presence of non-ionic surfactants.
Zeta potential is not usually considered as an
important measure for the stability prediction
of MEs prepared with non-ionic surfactants
(46).
The pH values of all MEs were found to vary
between 7.76 and 8.28. A slight increase in pH
could be attributed to the presence of lidocaine
in the formulations. MEs possessed very low
viscosity (7.85 to 27.1 mPa.s), independent
of shear rate. For all formulations, a linear
section was observed on the ow curves,
constructed with shear stress vs. shear rate (r2
≥ 0.99). Figure 3 illustrates the rheogram of
the ME5 formulation.
MBGs
Three dierent gelling agents were used to
increase the viscosity of MEs. In the presence
of Carbomer 934, all gels were found
opaque. HPMC was also unable to yield clear,
homogenous MBGs with desired viscosity.
Turbidity and lack of homogeneity were
resolved by substituting HPMC and Carbomer
934 with Carbomer 940 (47). As described by
Chen et al., the reason might be associated with
the dissociation of Carbomer 934 and HPMC
matrices from the hydrated state by surfactant
and co-surfactant in the microemulsion (48).
The gels were also evaluated in terms of
stickiness, ease of spreading, and coarseness
Figure 3. Rheogram of ME5 formulation.
Figure 3. Rheogram of ME5 formulation.

11
Lidocaine-loaded, Microemulsion-based Topical Gels
by rubbing a sucient amount of gels between
index and thumb ngers. In general, results
showed that all Carbomer 940-based gels
were homogeneous, transparent, and smooth
without any particulate matter, grittiness,
or lumps and, therefore, MBGs with 1 wt%
of Carbomer 940 were nally prepared for
further investigations.
MBGs properties
The data obtained from the characterization
of MBGs in terms of spreadability, pH, and
viscosity are given in Table 4. The spreadability
of gel formulations, that is, the ability of gels
to spread uniformly on the skin surface, is a
property upon which the therapeutic eciency
of a gel depends and helps in the uniform
gel application. Values in Table 4 refer to
the extent to which the formulations readily
spread on the glass plates by applying a small
amount of shear.
Results indicated that the highest
spreading diameter (4.2 cm) was obtained
for the formulation MG8, which possessed
the lowest viscosity, whereas the lowest
spreading diameter (3.1 cm) was found for the
system MG11 with the highest viscosity. The
appropriate spreadability of MBGs may be
related to the loose gel matrix nature of MBGs
due to the presence of oil globules (49).
As depicted in Table 4, it was observed
that pH values of MBGs were within the
physiological range varying from 6.87 to 7.42.
This pH range suggests that the gels could
result in less irritation to the skin. A decrease
in pH of MBGs in comparison with MEs
may be attributed to the acidic properties of
Carbomer 940 (17).
The use of MEs on the skin is very
dicult because of their uidity. For a dermal
pharmaceutical or cosmetic product, an
appropriate viscosity with sucient retention
time on the skin is required. Hence, MBGs
were developed by using Carbomer 940 in
an attempt to modify rheological behavior.
Viscosity values for lidocaine-loaded gels are
also shown in Table 4. As expected, following
the incorporation of the gelling agent into
MEs, the viscosity of systems increased
signicantly (from 224.83 to 871.62 mPa.s),
and pseudoplastic behavior was observed.
The latter could facilitate and improve the
spreading features of the formulation. The
ow indices (n) were found to be less than
1 (0.2788-0.4479), indicating that all MBGs
were shear-thinning in nature according to
the power law equation (13). Rheograms also
revealed the absence of thixotropy in the gels
investigated (Figure 4).
Stability studies of uid MEs and MBGs
The stability of MEs was evaluated after 15
months of storage at room temperature. MGBs
were also kept at dierent storage conditions
(5 ± 3 °C, 25 ± 2 °C and 40 ± 2 °C) for 9
months, and their transparency and consistency
were monitored. As shown in Figure 5, all
ME formulations (except ME13) were clear
without any turbidity or sedimentation. The
gels also remained clear with homogenous
structures and displayed no macroscopic
physical changes following storage at ambient
temperature and in a refrigerator (Figure 6).
However, loss of their viscosity was observed
Table 4. Characterization of the MBGs (mean ± SEM).
Formulations Spread diameter (cm) pH Viscosity (mPa.s) Flow index Nature/type of flow
MG1 3.3 0.058 7.23 0.012 696.65 0.870 0.3397 Shear-thinning / pseudoplastic
MG2 3.6 0.033 7.42 0.015 428.85 0.731 0.3438 Shear-thinning / pseudoplastic
MG3 3.7 0.058 6.98 0.010 705.75 0.463 0.3247 Shear-thinning / pseudoplastic
MG4 3.8 0.033 6.88 0.009 796.08 0.511 0.3245 Shear-thinning / pseudoplastic
MG5 3.6 0.058 6.97 0.012 712.89 0.932 0.3156 Shear-thinning / pseudoplastic
MG6 3.7 0.067 7.10 0.030 773.89 1.261 0.3404 Shear-thinning / pseudoplastic
MG7 4.1 0.058 7.21 0.010 235.51 0.334 0.3467 Shear-thinning / pseudoplastic
MG8 4.2 0.033 7.16 0.015 224.83 0.327 0.4479 Shear-thinning / pseudoplastic
MG9 3.9 0.100 7.18 0.009 379.50 0.464 0.3140 Shear-thinning / pseudoplastic
MG10 3.2 0.058 7.22 0.030 660.12 1.359 0.2788 Shear-thinning / pseudoplastic
MG11 3.1 0.067 7.14 0.003 871.62 1.458 0.2988 Shear-thinning / pseudoplastic
MG12 3.3 0.033 7.28 0.010 677.93 1.034 0.3265 Shear-thinning / pseudoplastic
MG13 3.8 0.100 7.31 0.009 397.85 1.172 0.3489 Shear-thinning / pseudoplastic
MG14 3.5 0.153 7.33 0.015 516.76 1.023 0.3502 Shear-thinning / pseudoplastic
Marketed gel 3.9 0.100 6.87 0.025 328.18 0.572 0.4776 Shear-thinning / pseudoplastic
Table 4. Characterization of the MBGs (mean ± SEM).

12
Daryab M et al. / IJPR 21 (2022) e1: 1-21
after 60 days of storage at 40 °C.
The stability of MBGs was evaluated
under stressed conditions by visual inspection.
When subjected to centrifugation at 5000
rpm for 30 min, it was found that this stress-
induced no damage, and the formulations
remained homogeneous and exhibited no sign
of phase separation or breakdown. The eect
Figure 4. Rheogram of MG6 formulation.
Figure 4. Rheogram of MG6 formulation.
Figure 5. Stability of MEs after 15 months of storage at room temperature. As seen, all
formulations except ME13 were clear without any turbidity or sedimentation.
Figure 5. Stability of MEs after 15 months of storage at room temperature. As seen, all formulations except ME13 were
clear without any turbidity or sedimentation.

13
Lidocaine-loaded, Microemulsion-based Topical Gels
of heating-cooling cycles on the stability of
MBGs was also veried. In each heating-
cooling cycle, the sample was rst heated to
40 °C for 24 h and subsequently cooled to 4 °C
for 24 h. Seven heating-cooling cycles were
run to record the gel responses to temperature
uctuations. Finally, the inuence of the
repeatedly freeze-thawed treatment (-5 and 25
°C for 24 h) on the stability of the gels was
investigated. The results obtained from these
stability tests foresee MBGs to have good
physical stability since no phase separation
was observed and the textural properties were
not inuenced by temperature variation.
Permeation study
Drug release and permeation studies through
cellulose acetate membrane and rat skin,
respectively, from MBGs, were carried out
using vertical Franz diusion cells. Although
human skin is considered the gold standard
in permeation study of topical formulations,
however, limited availability, variability, and
ethical reasons have led to employ the animal
skin models (50). Some structural similarities
between rat skin and human skin (e.g., thickness,
lipid content, and water uptake) can propose
rat skin as a surrogate for permeation studies
(51, 52). For preliminary drug permeation
screening, the lipophilic articial membrane
was employed, and subsequently, an ex-vivo
permeation study on rat skin was conducted
for the formulations with the highest ux value
through an articial membrane.
In-vitro drug release through an articial
membrane
This part of the investigation was aimed
to select the best MBG formulations for ex-
vivo skin permeation and animal tests. The
cumulative percentage of released lidocaine
was plotted as a function of time (Figure 7).
In general, it was observed that in all MBGs,
the drug release percentage at all sampling
points was signicantly greater than that of
the commercial gel, suggesting that MBGs
could improve the release pattern of the drug
in comparison with the marketed product. In-
vitro drug release proles also revealed that
the formulations MG3 (triacetin/Tween 80/
Transcutol P at Rsm of 2:1), MG5 (triacetin/
Cremophor EL/PEG 400 at Rsm of 1:1), and
MG4 (triacetin/Tween 80/PG at Rsm of 2:1)
released the maximum amount of lidocaine
(61.65 ± 1.62%, 61.24 ± 0.70% and 61.04 ±
0.76%, respectively) after 2 hours (p < 0.01)
(Figure 7), while the system MG11 (triacetin/
Cremophor EL/PG at Rsm of 2:1) displayed
the lowest amount of released drug (50.42
± 0.76%) with no statistically signicant
dierence compared to the commercial gel
(p > 0.05). Therefore, MG3, MG4, and MG5
were considered as the optimum gel systems
and chosen for ex-vivo drug permeation
investigations.
Ex-vivo drug permeation through the skin
The ex-vivo drug permeation through
the skin was carried out in an attempt to
formulate a vehicle with suitable skin uptake
Figure 6. Stability of MBGs after 9 months of storage at room temperature. No textural change or
breakdown was observed in the formulations investigated.
Figure 6. Stability of MBGs after 9 months of storage at room temperature. No textural change or breakdown was
observed in the formulations investigated.

14
Daryab M et al. / IJPR 21 (2022) e1: 1-21
and penetration. Results are depicted in Figure
8. As can be seen, the drug permeation from
formulations MG3, MG4, and MG5 started
immediately without any lag phase, followed
by a continuous increase over time. By
comparing the skin permeation proles, it is
observed that MG4 (triacetin/Tween 80/PG at
Rsm of 2:1) exhibited the highest cumulative
Figure 7. In-vitro drug release profiles of formulation MG3, MG4 and MG5 through an artificial
membrane. Data are shown as means ± SEM (n = 3). One-way ANOVA followed by Tukey’s post-
test multiple comparisons were conducted (*p < 0.05: significant, **p < 0.01: very significant, and
***p < 0.001: extremely significant, in comparison with the marketed product).
Figure 8. Drug permeation from MG3, MG4, and MG5 systems through abdominal rat skin. Data
are shown as means ± SEM (n = 3). The difference between the release percentage of the
formulations was statistically analyzed by one-way ANOVA followed by Tukey's post-test (*p <
0.05: significant, **p < 0.01: very significant, and ***p < 0.001: extremely significant, in
comparison with the marketed product).
Figure 7. In-vitro drug release proles of formulation MG3, MG4 and MG5 through an articial membrane. Data are
shown as means ± SEM (n = 3). One-way ANOVA followed by Tukey’s post-test multiple comparisons were conduct-
ed (*p < 0.05: signicant, **p < 0.01: very signicant, and ***p < 0.001: extremely signicant, in comparison with the
marketed product).
Figure 8. Drug permeation from MG3, MG4, and MG5 systems through abdominal rat skin. Data are shown as means ±
SEM (n = 3). The dierence between the release percentage of the formulations was statistically analyzed by one-way
ANOVA followed by Tukey’s post-test (*p < 0.05: signicant, **p < 0.01: very signicant, and ***p < 0.001: extremely
signicant, in comparison with the marketed product).

15
Lidocaine-loaded, Microemulsion-based Topical Gels
amount of lidocaine permeation versus time
after 10 h (5300.705 mg/cm2) and signicantly
enhanced lidocaine permeation compared
to MG5 and the control gel. No statistically
signicant dierence in permeation was
found between MG3 and MG4 until 15 min,
suggesting that their onset of action could be
almost the same. However, higher drug release
was observed from MG4, which supports a
longer duration of action.
Rapid onset of action for LAs is very
important, and therefore, a high initial
permeation is immediately required.
Cumulative drug release per unit area of
skin surface from all formulations after 10,
20, and 60 min demonstrated a signicant
enhancement of ux in comparison with the
commercial gel (p < 0.001), so that for MG4
as the optimized system, 4.09, 3.54, and
1.91-fold increase in the ux were observed,
respectively. It is crucial to determine the
minimum amount of drug permeation that
induces local anesthesia (i.e., the anesthetic
threshold). Lidocaine anesthetic threshold was
calculated to be 500 μg/cm2, based on the data
obtained from in-vitro drug release and in-vivo
anesthetic examination (tail-ick test) (8).
Considering this value, it could be expected
that formulation MG4 causes local anesthesia
faster than the other MGBs within 7 minutes
after applying the gel. Formulations MG3 and
MG5 also induced their eects after 10-15
min; however, the amount of drug needed to
initiate the anesthetic eect of the commercial
gel was released in 30 to 45 min. In general,
it is concluded that the faster local anesthesia
was achieved by the use of MBGs, compared
to the commercially available gel.
An increase in the permeation rate
within the rst two hours could be explained
as follows. Due to the presence of both
hydrophilic and lipophilic components and
the resulting combined eects, MEs possess
a favorable solubilizing behavior. This
increases the thermodynamic activity of the
drug, which is a driving force for drug release
and its penetration (49). Besides, it has been
previously reported that topically applied MEs
are expected to penetrate the skin and exist
intact in the stratum corneum (SC). Kweon
et al. have suggested that MEs, once entered
into the SC, could alter both the polar and lipid
pathways, and the subsequent interaction of the
lipid portion of the MEs with the SC makes the
dissolved drug partition into the existing lipids.
On the other hand, the bilayer structure of the
SC could be destabilized by the intercalation of
ME droplets between its lipid chains (53). The
hydration eect on the drug uptake of the SC
by the hydrophilic domain of MEs should also
be considered. It is thought that the aqueous
phase of MEs would increase the interlamellar
volume and disrupt the lipid bilayers due to the
swelling of the intercellular proteins, causing
a more easily penetration of the drug through
the lipid pathway of the SC (53). In conclusion,
the greater penetration enhancing the activity of
MEs may be attributed to the combined eects
of both the lipophilic and hydrophilic domains
of microemulsions.
As can be seen in Figure 8, the release of
lidocaine molecules from the investigated
MBGs (MG3-MG5) was sustained for 10
h. This phenomenon may be explained by
considering the release of the loaded lidocaine
from the internal phase, which might act as
a drug reservoir, to the external phase and
then from the continuous phase to the skin
through passive diusion. Lidocaine can also
be partially solubilized in the external phase
and interfacial lm of ME that can supply fast
release at the initial time of study leading to
the fast onset of action without any lag time.
It has been suggested that the gel formation in
ME limits the diusion of the drug dissolved
in the droplets and therefore slows down its
release. Thus, one can conclude that MBGs
are potentially able to sustain the release of
drugs as compared with their uid systems.
Therefore, the high permeation rate of MG4
could be related to its ability to create a high-
saturated vehicle which can result in high
thermodynamic activity (54).
The particle size of ME droplets plays
an important role in percutaneous drug
absorption. It has been reported in the
literature that by decreasing the droplet size,
the number of particles that can interact with
the skin surface is probably increased (49, 53
and 55). In this investigation, the particle size
of all ME formulations was in the range of
20-52 nm. This suggests that a large surface
area for the transfer of lidocaine to the skin is
available.

16
Daryab M et al. / IJPR 21 (2022) e1: 1-21
The higher lidocaine ux from formulated
MBGs compared to the commercial gel
originates from the penetration-enhancing
eect of applied components. Cao et al.
prepared celecoxib-loaded MBG using Tween
80 and Transcutol P and evaluated the ex-
vivo permeation of the drug into the mouse
skin. The results revealed that the interested
formulation could have a 4-fold greater
permeability than the conventional gel (56).
These ndings have also been previously
obtained by Shakeel et al., using penetration
enhancers such as Labral, triacetin, Tween
80, and Transcutol P for aceclofenac (57).
Similarly, other researchers have developed
MBGs containing a mixture of Cremophor
EL and PEG 400 as the surfactant phase to
co-delivery of evodiamine and rutaecarpine.
By application of this nano-based gel
formulation, it was shown possible to achieve
approximately 2.6-fold higher transdermal
ux compared with control hydrogel (58).
In general, numerous studies on ME gels
prepared with Tween 80 and PG have shown
that this surfactant mixture has an important
impact on increasing the skin permeability as
well as the stability of systems, and it has also
been stated that PG can exhibit an additive
eect on drug permeation in combination with
other penetration enhancers (59).
Drug release kinetics
To determine the kinetics of permeation
from these vehicles, the data obtained from ex-
vivo permeation experiments were kinetically
analyzed according to zero order, rst order,
Higuchi and Korsmeyer-Peppas models, and
the results of data tting into these models
were evaluated by the highest correlation
coecient (R2). Based on the best goodness
of t (see Table 5), it was found that MG3,
MG4, and the marketed product were followed
Higuchi kinetic model (MG3: R2 = 0.9942,
MG4: R2 = 0.9862, marketed gel: R2 = 0.9832).
Higuchi model-based permeation, previously
reported for indomethacin (chitosan-based),
terbinane (chitosan-based), itraconazole
(Lutrol F127-based) and ibuprofen
(Carbopol 940-based) MBGs and for topical
ketoprofen and pentoxifylline MEs (24, 60-
64), suggests that the release process could be
mainly controlled by the Fickian diusion of
dissolved lidocaine through the gel network of
Carbomer 940. However, the analysis of the
release plot for MG5 revealed that lidocaine
followed the rst-order model for controlled
permeation, suggesting that the release rate is
concentration-dependent (23, 65).
If diusion is the main drug release
mechanism regarding the Higuchi equation,
then a plot of the drug amount released versus
the square root of time should result in a straight
line. However, a deviation from the Fickian
equation may be observed, and the mechanism
of diusion from polymeric dosage forms may
follow a non-Fickian behavior. Korsmeyer-
Peppas equation (Equation 2) is a more
general relationship that describes a mixed
mechanism of drug release (polymer swelling
and/or diusion) from a polymeric system:
Equation 2.
where k is a constant incorporating the
geometrics and structural characteristics
of dosage form, n is the release exponent
indicative of the release mechanism, and
Mt/M∞ is the fractional release of the drug.
This equation relates the drug release to the
elapsed time (t). In this study, to elucidate
the drug release mechanism, the rst 60%
of drug release data was used to calculate
Table 5. Models used to assess the release kinetics from the best MBGs and the corresponding Korsmeyer-Peppas parameters.
Formulation R2 values Korsmeyer-Peppas parameters
Zero ordera First orderb Higuchic Korsmeyer-Peppasd n k
MG3 0.9409 0.9913 0.9942 0.9919 0.5062 2.9798
MG4 0.9613 0.9628 0.9862 0.9890 0.4840 3.7818
MG5 0.9528 0.9964 0.9936 0.9915 0.5609 2.2289
Commercial gel 0.9775 0.9585 0.9832 0.9940 0.8543 0.4476
aCumulative amount of drug permeated (g) versus time.
bLog of the amount of remaining drug (g) versus time.
cCumulative amount of drug permeated (g) versus square root of time.
dSee equation 2 in the text.
Table 5. Models used to assess the release kinetics from the best MBGs and the corresponding Korsmeyer-Peppas
parameters.


 

17
Lidocaine-loaded, Microemulsion-based Topical Gels
values of n, k, and correlation coecient
(R2) (Table 5). Values of the release exponent
for MG3, MG4, and MG5 formulations and
the marketed product were calculated to be
between 0.484 and 0.854. Therefore, it was
concluded that the mechanism of transport for
all these formulations followed an anomalous
(non-Fickian) behavior, as described in Table
6, possibly including both diusion and/or
polymer erosion phenomena. These results
are in accordance with those reported for
zaltoprofen and griseofulvin MBGs (47, 66
and 67) and contraceptive vagino-adhesive
propranolol HCl gel (68).
Anesthetic eect
Paw withdrawal threshold (PWT) values
of the lidocaine-treated rats were found to
be signicantly higher than their respective
controls, conrming the induction of the
anesthetic eect of lidocaine (p < 0.001).
Repeated-measure, two-way ANOVA
(followed by Bonferroni’s post-test) revealed
that MG4 formulation showed a markedly
greater anesthetic eect in comparison with
the marketed gel. This nding supports the
results of the ex-vivo permeation test (Figure
9). Also, it was observed that MG3 showed
no statistically signicant dierence in PWT
value approximately during the rst two hours
of the study, and for MG5, the induction of local
anesthesia was similar to the marketed gel. In
order to compare the average pain threshold
during the complete period of observation
following the application of the formulations,
the area under the time-course curve was
calculated. As can be clearly seen in Figure
10, MG4 and MG3 induced a statistically
signicant high pain threshold in comparison
to the commercial product (p < 0.001, one-
way ANOVA followed by Tukey’s post-test),
although the dierence was not signicant
between that of MG5 and the marketed gel.
Skin irritation test
The irritation potential of any transdermal
formulation is a critical factor that could limit
its use and patient acceptability. In the present
Table 6. Interpretation of diffusion release mechanism.
Release exponent (n) Release mechanism
n ≤ 0.5 Fickian diffusion
0.45 < n < 0.89 non-Fickian (anomalous) transpor
t
n = 0.89 case II transpor
t
n > 0.89 super case II transpor
t
Table 6. Interpretation of diusion release mechanism.
Figure 9. Paw withdrawal threshold of the selected formulations (MG3, MG4, & MG5) to
mechanical stimulation (von Frey filaments). Data are shown as means ± SEM, n = 8 rats per group
(n = 8). Two-way ANOVA followed by Bonferroni post-test.
Figure 9. Paw withdrawal threshold of the selected formulations (MG3, MG4, and MG5) to mechanical stimulation
(von Frey laments). Data are shown as means ± SEM, n = 8 rats per group (n = 8). Two-way ANOVA followed by
Bonferroni post-test.

18
Daryab M et al. / IJPR 21 (2022) e1: 1-21
study, special consideration was given to the
selection of components used in the formulations
on the basis of solubility and the minimal skin
irritation tendency. Draize primary skin irritation
test was performed on the albino rabbit skin to
study the irritability of the optimum formulation.
The results obtained from skin irritation studies
after 1, 24, 48, and 72 h of the gel application
are listed in Table 7. The prepared gels were not
found to be skin irritants.
Conclusion
In the present study, various formulations
of lidocaine-loaded MBGs were prepared and
characterized. It was concluded that MBGs
could be considered as a more promising
approach for the transdermal delivery of
lidocaine due to their appropriate viscosity and
rheological behavior, spreadability, pH, high
penetration ability, and skin tolerability with
no irritation, high stability, and improvement
in PWT and anesthetic eect. However,
further research and clinical investigations
need to be conducted to elucidate the possible
mechanism(s) of lidocaine delivery to the skin
and conrm the therapeutic ecacy.
Acknowledgments
This research was nancially supported
by the Vice-Chancellor of Research, Shahid
Beheshti University of Medical Sciences. The
authors are grateful to Dr. Mona Khorram
Joy and Dr. Bahareh Alizadeh for their great
eorts and assistance in conducting the animal
studies and HPLC analysis, respectively.
Conict of interests
The authors declared that there is no
conict of interest.
Figure 10. The area under the curve (AUC10-210 min) of withdrawal threshold time-course of the
selected formulations (MG3, MG4, & MG5) to mechanical stimulation (von Frey filaments). Data
are shown as means ± SEM, n = 8 rats per group (n = 8). One-way ANOVA followed by Tukey's
post-test multiple comparisons were conducted.

Figure 10. The area under the curve (AUC10-210 min) of withdrawal threshold time-course of the selected formulations
(MG3, MG4, and MG5) to mechanical stimulation (von Frey laments). Data are shown as means ± SEM, n = 8 rats per
group (n = 8). One-way ANOVA followed by Tukey’s post-test multiple comparisons were conducted.
Table 7. Average response scores of skin irritation.
Group Primary Irritation Index (Mean 

SEM; n = 3)
1 h 24 h 48 h 72 h
No application (control) 0  0 0 0 0 0 0 0
Blank MG4 0  0 0 0 0 0 0 0
lidocaine-loaded MG4 0.44  0.11 0.22 0.11 0 0 0 0
Table 7. Average response scores of skin irritation.

19
Lidocaine-loaded, Microemulsion-based Topical Gels
Author contributions
All authors have made substantial
contributions to the experimental design of
this project. Experimental procedures and data
collection were carried out by M. Daryab.
Analysis and interpretation of data were
performed by all authors. This manuscript
was initially drafted by M. Daryab and
revised by A. Mahboubi and R. Aboofazeli
before submission. All authors approved the
nal manuscript for submission. This study
was the subject of the Pharm.D. thesis of M.
Daryab, proposed and approved by the School
of Pharmacy, Shahid Beheshti University of
Medical Sciences, Iran.
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