Azerbaijan Pharmaceutical and Pharmacotherapy Journal

Received July 14, 2023 Accept December 22, 2023 Publish January 25, 2024.
DOI https://doi.org/10.61336/appj/22-3-1

Formulation and Evaluation of Minoxidil Loaded Submicron

Emulsion Based Topical Gel For Treatment of Alopecia
Jasjeet Kaur Narang1, Ramandeep Singh Narang2, Mehak1
1
Department of Pharmaceutics, Khalsa College of Pharmacy, Amritsar
2
Department of Oral and Maxillofacial Pathology, Sri Guru Ram Das Institute of Dental Sciences and Research, Amritsar, Punjab, India
Corresponding author: Jasjeet Kaur Narang (e-mail: jasjeet2975@yahoo.com).
©2023 the Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0

Abstract: Introduction: Alopecia is a benign hair condition that causes reduced hair growth on the scalp and has afflicted
population worldwide. Aim: The present study is designed to formulate and characterize submicron emulsion based topical
gel containing an anti-hypertensive drug like minoxidil to overcome the adverse effects of conventional oral dosage forms &
to provide an improved, targetted therapy for treatment of alopecia. Methods: The minoxidil loaded submicron emulsion was
prepared by aqueous titration method using a suitable combination of Clove oil (oil phase), Tween 20 (surfactant) and
Transcutol P (co-surfactant). Results: The maximum submicron emulsion area obtained after constructing pseudo-ternary
phase diagrams was found in S mix ratio 4:1. The optimized submicron emulsion formulation (M5) exhibited a pH of 5.4 ±
0.17, in vitro release of 95.08 ± 0.36 %, ex vivo permeation of 79.36 ± 0.18%, particle size of 181.3nm with an uniform
particle size distribution (<1) and optimum zeta potential (-8.80 mV). The optimized formulation (M5) was then converted
intogel formulation by adding different concentrations (1%, 1.5% and 2% w/v) of gelling agent like Carbopol 934. Among
them, M52%w/v was considered as the optimized gel formulation on the basis of different evaluation studies. The optimized
submicron emulsion based gel formulation also showed an inherited anti-oxidant potential and remain stable for 3 months at
4℃. Conclusion: The minoxidil loaded submicron emulsion based topical gel formulation could be considered as a
beneficial nano-approach in contrast to other traditional topical dosage forms for treating alopecia.

Key Words: Alopecia, Benign, Submicron emulsion, Pseudo-ternary, Antioxidant.

INTRODUCTION with 80% first pass metabolism [10]. Therefore, it

Alopecia is defined as a non-cancerous autoimmune hair
disorder in which the extensive loss of hair occurs from
various regions of body especially from scalp [1]. It is the
most common dermatologial complaint with a varying
prevalence rate throughout the globe [2]. It has an
immense psychosocial influence over the person’s mind
[3]. The pathophysiological mechanism of alopecia is an
enigma, but hormones, infections, medications and hair
styling practices are the various triggering factors which
are also responsible for the occurrence of alopecia [4].
Treatment options of alopecia include masks, wigs and
sunscreen, administration of plasma injection rich in
platelets and corticosteroids, administration of topical
prescription drugs like anti-androgens, vasodilators etc
[5,6,7,8]. Among the different drugs used for alopecia
treatment, minoxidil is the first USFDA approved poorly
water-soluble anti-hypertensive drug which is known to
prolong the growth phase (anagen phase) in hair follicle
with prominent anti hair loss results [9]. It does not bind to
plasma proteins and exhibits a shorter elimination half-life
(4.2 hours). The oral ingestion of this drug however, leads
to different adverse effects such as hypotension,
palpitations, weight gain, hypertrichosis and itching etc
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
necessitates the formulation of nanocarrier which would
overcome the drawbacks associated with oral traditional
formulation and improve the overall efficacy of drug.
Further, it would be advantageous if the formulated
nanocarrier provides controlled release of the drug as it
would also contribute in reducing the dose and the dose
related side effects. The controlled drug delivery systems
developed and reported in the literature for the treatment
of alopecia include nanolipid carriers[11-12] solid lipid
nanoparticles[13-14], polymeric nanoparticles [15-16],
Nanoemulsions [17-18], liposomes [19-20],
Transferosomes [21-22], Niosomes [23-24], Ethosomes
[25-26], cubosomes [27], metallic nanoparticles[28-29],
Liquid Crystalline nanoparticles [30]. However, since most
of the above mentioned dosage forms are in liquid form,
they limit the retention of the drug at the application site.
In order to overcome this drawback in the present study a
submicron emulsion based gel was formulated for delivery
of minoxidil.
Submicron emulsions are thermodynamically stable
isotropic mixtures of drug with suitable concentrations of
oil, surfactants, co-surfactants and water with a mean
droplet size of 10-500 nm. This nanocarrier has gained
attention of many scientists due to its maximum
solubilization capability, smaller droplet size with
maximum stability [31]. However, to overcome the
problem of its low viscosity, formulation of submicron
emulsion as a topical gel is a better option. The submicron
emulsion based gel is defined as the gelling system which
can be obtained after the addition of submicron emulsion
system into gel matrix. The presence of sebum in hair
follicles makes it a most promising approach for delivering
minoxidil [32]. This nanocarrier enhances the permeation
of active drug moeity into skin and provides sustained
release which leads to improved patient compliance and
higher therapeutic effect [33]. This study provides the
concept of preparation and characterization of submicron
emulsion based topical gel containing minoxidil for
treating alopecia. The gel would not only provide a
targeted delivery of the drug at the site of application but
due to its gel state would enhance the retention of the
drug at the application site whoich would inturn lead to an
overall improvement in the therapeutic efficacy of the drug
and inturn patient compliance.
MATERIAL AND METHODS
The study was conducted at Khalsa college of Pharmacy,
Amritsar and didnot utilise any living animals or humans
and therefore no permission was taken from the
Institutional Ethical Committee. Minoxidil was procured as
a gift sample from Kwality Pharmaceutical Ltd. (Amritsar,
India). Capryol 90 and Transcutol P were procured from
Saint-Priest, France (Gattefosse) as a gift sample.
Polysorbate 20and triethanolamine wereobtained from
S.D. Fine Chem Ltd. (Tamilnadu, India). Carbopol 934
and ferric chloride were obtained from Himedia
laboratories Pvt. Ltd. (Mumbai, India). All other analytical
reagents& chemicals were purchased from Merck
Specialities Pvt. Ltd. (Mumbai, India) and Qualikem Fine
Chemicals Pvt. Ltd. (New Delhi, India).
Formulation of submicron emulsion
Solubility and Miscibility Studies
The solubility determination is the prerequisite step to
select the appropriate components for the formulation
ofsubmicron emulsion. To commence with the solubility
studies, an excess amount of drug was added in
eppendorf tubes containing different oils (Clove oil, Oleic
acid, Isopropyl Myristate, Capryol 90, Tea tree oil and
Olive oil), surfactants (Kolliphor HS 15, Tween 40, Tween
20, Span 80, Span 20, Tween 60 and Tween 80) and co-
surfactants (Transcutol P, PEG 400 and PEG 600)
individually. The contents in eppendorf tubes were mixed
using vortex mixer and then kept on biological shaker for
3 days at 37℃±2℃. After this, the centrifugation of
samples was done at 3000 rpm for 15 minutes and the
supernatant layer was removed followed by filtration. It
was then analysed using U.V spectrophotometer at λ max
286 nm.The estimations performed in triplicate.
For the miscibility studies, one ml of each selected
surfactants and co-surfactants were taken in 1:1 ratio in
eppendorf tubes and mixed using vortex mixer at 25±1℃.
The obtained mixtures were kept overnight to determine
the miscibility of Smix [18].
Pseudo-ternary phase diagram construction and
formulation development
The method used for construction of pseudo-ternary
phase diagrams was aqueous titration method in which
the different Smix (Surfactant: Co-surfactant) ratios (1:0,
1:1, 1:2, 2:1, 3:1 and 4:1) were prepared followed by the
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
preparation of various oil:S mix ratios. The aqueous phase
was slowly added into these oil:Smix ratios to produce a
clear, transparent and less viscous submicron emulsion
system. The prepared emulsions were vigrously vortexed
and kept for 24 hours for attaining equilibirium [34]. After
this, the combinations for the preparation of placebo
submicron emulsions were selected from the constructed
phase diagrams to achieve a formulation with suitable oil
concentration for better solubilization of drug with
minimum Smix and high water content. The same
procedure was repeated for the preparation of submicron
emulsion containing minoxidil in a dose of 1%w/v [29].
Physical stability studies
The minoxidil containing submicron emulsions were
checked for stability by subjecting them to different
physical stability tests. The first test i.e. heating-cooling
cycle was performed by storing the emulsion formulations
at 4℃ and 40℃ each for 48 hrs. The second stability test
which was conducted was centrifugation test in which the
centrifugation of prepared submicron emulsion
wasperformed for 30 minutes at 3000 rpm to examine any
kind of physical instability. The third test i.e. freeze thaw
cycle or accelerated stability testing was done by storing
the formulations at -21℃ and +25℃ for atleast 2 days [35].
The stable formulations were further characterized on the
basis of different parameters.
Characterization of submicron emulsion
pH
The pH of submicron emulsions was examined using
digital pH meter at room temperature as too high or too
low pH could lead to unwanted side effects [35].
In vitro drug release study& determination of release
kinetics
For the conduct of drug release study, the dialysis
membrane was firstly treated with 0.3% w/v sodium
sulphide solution and other reagents. It was then mounted
on Franz diffusion cell between two half cell
compartments. The phosphate buffer pH 5.5 was added in
receptor compartment as release medium at 37℃ with
continuous stirring at 75 rpm. The donor compartment
consisted of one ml of 1% w/v drug loaded submicron
emulsions and the alliquots were taken at pre-defined
time periods upto 6 hours from the receptor compartent.
The assessment of collected samples was done using
U.V spectrophotometer at λmax 286 nm and the results
obtained were compared with those obtained using drug
solution (1%w/v) in ethanol. The experiment was repeated
in triplicate [36].
The data produced from in vitro release investigation was
plotted in different release models to study the release
kinetics of prepared submicron emulsions. The model for
which the coefficient correlation (R2) value was found to
be near unity was considered as release model [37].
Particle size, PDI and droplet charge assessment
The size of particles and polydispersity index of
submicron emulsion were examined by dynamic light
scattering method. For this, the dilution of the samples
was performed 100 times with distilled water and
assessed using zeta-sizer. After this, they were directly
placed into module [38]. The charge on the average
globules was measured by using Malvern zeta-sizer [39].
Ex vivo skin permeation study& data analysis
For the evaluation of drug permeation, the excised rat skin
sample was obtained from pharmacology department,
followed by complete removal of hair and underlying fat
from the body. After this, the excised skin was completely
stabilised and franz diffusion cell was used to conduct this
study. Phosphate buffer (pH 7.4) was put into the receptor
compartment, while the donor compartment consisted of 1
mL of the 1% w/v minoxidil-loaded submicron emulsion.
The samples were taken at pre-defined time intervals (6-
hrs study) andanalysed by U.V spectrophotometer at λ max
286 nm. The experiment was done in triplicate and the
comparison was done with 1%w/v minoxidil ethanolic
solution (Benson et al., 2018).The drug flux (Jss) was
obtained by plotting the amount of minoxidil submicron-
sized emulsion permeated in steady state conditions v/s
time. The permeability coefficient (kp) was measured by
dividing the flux obtained to the initial drug concentration
(Co) present in the donor compartment [40].
Formulation of submicron emulsion based topical gel
Submicron emulsion based topical gel was prepared by
dissolving different quantities of selected gelling agent
i.e.carbopol 934 into the optimized submicron emulsion
(M5) formulation with continuous stirring till the
equilibirium was attained. The pH adjustment to neutral
value was done using triethanolamine. The gel
formulations (M51%w/v, M51.5%w/v, M52%w/v) were then kept
overnight and further evaluated [41].
Characterization of submicron emulsion based topical
gels (M51%w/v, M51.5%w/v, M52%w/v)
Physical appearance, pH, homogeneity & grittiness
The prepared submicron gel formulations were firstly
checked under normal sunlight to examine their physical
appearance. The pH was assesed by using digital pH
meter while the homogeneity and grittiness was measured
by rubbing the gel formulations on the back of hand [42].
Drug content
To determine the drug content,minoxidil-loaded submicron
gels weremixed with methanol and shaken for 2 hours.
The obtained solutions were filtered followed by removal
of supernatant layer. The dilution of supernatant layer was
done using methanol and analysed by U.V
spectrophotometer at wavelength of286 nm [43].
Ex vivo skin permeation studies of topical submicron
gel formulations & data analysis
The ex vivo skin permeation study of topical submicron
emulsion based gels was done usingfranz diffusion cell.
The skin was properly excised, stabilized and placed
between the half-cell compartments. The different
concentrations (M51%w/v, M51.5%w/v, M52%w/v) of submicron
topical gel were separately placed in donor compartment
while the phosphate buffer pH 7.4 was used as release
medium. The study was continued for 6 hours with the
continous withdrawl of sample at predetermined time
intervals followed by replacement with fresh release
medium. The comparison of results were done with the
results obtained from plain minoxidil gel. The experiment
was performed in triplicate [44]. The flux and permeability
coefficient was also calculated.
Drug retention studies
After the permeation studies, the remaining formulation
was removed from the skin and cleaned with cotton
soaked in 0.05% SLS followed by subsequent washing
using purified water. Further, the skin was weighed and
chopped into small pieces. It was then dissolved in
suitable solvent and kept forsonication. The resulting
solution was centrifuged and filtered.It was then analysed
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
using U.V spectrophotometer at λmax 286nm [32].
Histopathological investigation
The skin samples utilized in ex vivo permeation studywere
subjected to histopathological study. For control, skin
samples wereexcised from rat body and placed in saline
solution. Both the treated and control samples were kept
for storage in 10% formaldehyde solution. Afterwards, the
samples were chopped vertically and stained with paraffin
wax. After staining, the treated and untreated samples
were examined using microscope [45].
Viscosity and in vitro bioadhesion Study
Viscosity of the submicron topical gels was determined by
Anton paar rheometer. The gel was put on the plate and
kept for equiliberation at 25 ± 0.1℃ for few minutes. The
measurement was performed in triplicate [46].
For in vitro bioadhesion study, an agar plate was firstly
prepared. The gel samples to be studied were placed in
the centre of plate and further attached to IP disintegration
assemby. The plates were coninuously moved up and
down in phosphate buffer media (pH 7.4) at room
temperature. The residence time of the test samples on
agar plate was measured visually. The experiment was
repeated in triplicate [47].
Antioxidant activity of submicron emulsion based
topical gel formulation
The antioxidant activity of minoxidil loaded submicron
emulsion based topical gel was determined by using two
methods:
DPPH method
Firstly, the stock solution (1mg/ml) of standard (ascorbic
acid) and test formulation was prepared separately in
methanol. Following this, the different serial dilutions (1-20
µg/ml) of standard and test formulations were also
prepared separately. From these dilutions, one ml was
taken and dissolved in methanolic solution of DPPH
(0.004%w/v). After 30 minutes, the prepared solutions
were analyzed at 515 nm against methanol as blank [48].
The formula used for measuring percent inhibition was as
follows:
% inhibition = [(ABlank-AFormulation)/ABlank]
Stability studies
The stability studies of optimized gel formulation was
performed by storing the submicron emulsion based gel
formulations at referigerator temperature (4℃) for the
period of 3 months [49].
Statistical analysis
All the results were represented as mean ± standard
deviation. The software used for analysis of
resultsproduced by various test groups was Graph pad
Instat 3 (two tailed unpaired t-test). The p-values ≤ 0.05
were found to be significant.
RESULTS
Solubility studies & miscibility assessment
The determination of minoxidil solubility was done in various components and is depicted in Table 1.

Table 1: Drug solubility in various emulsion components

S.No Solubility of Minoxidil
Oil Phase Surfactant Phase Co-surfactant Phase
Oil Solubility Surfactant Solubility Co-surfactant Solubility
(mg/ml) (mg/ml) (mg/ml)
1. Clove oil 51.83 ± 0.52 Kolliphor HS 15 1.8 ± 0.35 Transcutol P 11 ± 0.3
2. Oleic acid 48.6 ± 0.54 Tween 40 1.0 ± 0.25 PEG 400 1.0 ± 0.3
3. Capryol 90 7.0 ± 0.51 Tween 20 5.82 ± 0.4 PEG 600 0.3 ± 0.3
4. Tea-tree oil 0.56 ± 0,058 Span 80 1.3 ± 0.45 - -
5. Olive oil 0.74 ± 0.001 Tween 60 0.9 ± 0.3 - -
6. Isopropyl 0.4 ± 0.041 Tween 80 1.4 ± 0.45 - -
Myristate

Construction of Pseudoternary phase diagrams for formulation selection

Pseudoternary phase diagrams were constructed for each Smix ratios (1:0, 1:1, 1:2, 2:1, 3:1 & 4:1) by aqueous titration method
(Figure 1) to determine the extent of submicron emulsion region.

(A) (B)

(C) (D)
Sub-
micron
sized
Emul-
sion

VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
 (E) (F)
Figure 1: Pseudoternary Phase diagrams constructed for different S mix ratios for preparation of submicron emulsion.
(A) represents 1:0 Smix ratio, (B) – 1:1, (C) – 1:2, (D) – 2:1, (E) – 3:1, (F) – 4:1 S mix ratio.

Formulation development and thermodynamic stability studies

The placebo submicron emulsions which were found to be stable after physical stability testing were further chosen for loading
the drug in a suitable dose (1%w/v). The prepared minoxidil loaded submicron emulsion were again checked for stability and
the composition of formulations which passed the stability testing is given in Table 2.

Table 2: Composition of Minoxidil containing submicron emulsions

Formulation Clove oil Tween20: Distilled Drug Volume of
Transcutol P (Smix) water (mg) Submicron
Code (ml) (ml) (ml) emulsion (ml)
M1 0.8 3.2 8 100 10

M5 0.4 3.6 6 100 10

N2 0.597 2.388 7.015 100 10

N6 0.750 1.750 7.5 100 10

H1 0.222 1.778 8 100 10

Characterization and optimization of submicron emulsion

pH
The pH of all submicron emulsion formulations were found to be in the range of 4.5 to 6.6.

In vitro drug release study & determination of release kinetics

The comparision of in vitro release study of different submicron emulsion formulations with minoxidil solution is illustrated in
Figure 2.
Percentage Cmulative drug

M1
released

M5
N6
H1
N2
Drug solution

Time (Hrs)

Figure 2: Comparison of in vitro release profiles of minoxidil loaded submicron emulsion formulations (M1, M5, N2, N6
& H1) with Minoxidil solution (1%w/v) over a period of 6 hours

Ex vivo skin permeation study and data analysis

The submicron emulsion (M5) was further subjected to ex vivo skin permeation study using excised rat skin obtained from the
pharmaclogy department. The results obtained were compared with those obtained by using minoxidil solution (1%w/v) and are
depicted in Figure 3.
Cumulative amount
of drug Permeated
(μg/cm2)

Submicron emulsion (M5)

Drug solution

Time (Hrs.)
Figure 3: Comparison of ex vivo permeation profile of submicron emulsion formulation (M5) and minoxidil solution
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
(1%w/v)
The data obtained from ex vivo skin permeation study is summarized in Table 2.

Table 2: Percent cumulative drug permeated, flux values & Permeability coefficient values of optimized submicron
emulsion and minoxidil solution
Formulation % Cumulative drug permeated [µg Flux Jss ± S.D Permeability
cm-2/ Co *100]± S.D (n=3) coefficient (Kp) ±
(n=3) S.D (n=3)

Submicron 79.36 ± 0.18% 904.96 ± 0.15

9.04 10-3± 1.24
emulsion (M5)
Minoxidil 49.79 ± 0.15% 522.4 ± 0.19
5.22 10-3 ± 1.15
Solution (1%w/v)

Particle size, PDI and Zeta potential

The graphs obtained from particle size, PDI and zeta potential determination are given in Figure 4.

(A) (B)
Figure 4: Fig. 4(A) shows the particle size and PDI value of optimized submicron emulsion formulation (M5) and Fig
4(B) represents the zeta potential of optimized submicron emulsion formulation (M5)

Formulation of Minoxidil loaded submicron emulsion based topical gel

The optimized minoxidil loaded submicron emulsion formulation (M5) was converted into gel formulation by using carbopol 934
as gelling agent.
Characterization and optimization of submicron emulsion based gel formulations(M51%w/v, M51.5%w/v, M52%w/v)
Physical evaluation, pH, Drug content, Homogeneity and grittiness
The gel formulations were visually evaluated to examine their physical appearance. The results are shown in Table 3.

Table 3: Physical appearance, pH, drug content and homogeneity profile of different submicron emulsion based gel
formulations
Property M5(1% w/v) containing 1% M5(1.5% w/v) containing 1.5% M5(2% w/v) containing
carbopol 934 carbopol 934 2% carbopol 934
Colour Slightly yellowish Slightly yellowish Slightly yellowish
Appearance Transparent Transparent Transparent
Odour Characteristic Characteristic Characteristic
Washability Washable Washable Washable
Phase separation No No No
Consistency + ++ +++
pH± S.D 5.6 ± 0.14 6.1 ± 0.23 6.3 ± 0.11
Homogeneity Homogeneous & no grittiness Homogeneous & no grittiness Homogeneous & no
grittiness
Percent drug content 90.74 ± 0.21 88.88 ± 0.15 98.14 ± 0.19
± S.D
+ = low, ++ = medium, +++ = high

Ex vivo skin permeation studyusing excised rat skin & data analysis
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
The cumulative amount of minoxidil permeated from different submicron emulsion-based gel formulations (M5 1%w/v, M51.5%w/v,
M52%w/v) was found to be 64.28±0.15%, 56.29±0.18% and 51.86±0.18% respectively, which was higher in contrast to the plain
gel (33.23 ± 0.18). Figure 5 depicts the outcome of ex vivo permeation study.

Cumulative amount of drug

permeated (μg/cm2)

M5(1%w/v)
M5(1.5%w/v)
M5(2%w/v)
Plain gel

Time (Hrs)

Figure 5: Comparison of ex vivo skin permeation profile of different minoxidil loaded submicron emulsion based gels
and plain minoxidil gel

The flux and permeability coefficient values of different minoxidil loaded submicron emulsion based gels and plain minoxidil gel
were also measured and reported in Table 4.

Table 4: Percent cumulative drug permeated, flux values & Permeability coefficient values of different submicron
emulsion based gelsand minoxidil plain gel
Formulation % Cumulative drug permeated [µg Flux Jss ± S.D Permeability
cm-2/ Co *100]± S.D (n=3) coefficient (Kp) ±
(n=3) S.D (n=3)

M51% w/v 64.28±0.15 759.42 ± 0.29

7.59 10-3± 1.07
M51.5% w/v 56.29±0.18 609.5 ± 0.13
6.09 10-3± 1.15
M52% w/v 51.86±0.18 555.2 ± 0.27
5.55 10-3± 1.02
Plain minoxidil 33.23 ± 0.18 527.92 ± 0.18
Gel 5.27 10-3± 1.24

Drug retention study

The amount of drug retained (mg) and percent drug retained in skin layers were also measured and results are given in Figures
6(a) and Figure 6(b). The drug retention from submicron emulsion based gel formulation M5 2%w/v (16.5 ± 0.28%) was also
significantly higher (p≤ 0.05) as compared to the drug retention from plain gel (5 ± 0.2%).
Percentage of drug retained in
Amount of drug retained in
skin layers (mg)

skin layers

Formulation Formulations

Figure 6(a) Figure 6(b)

VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
Figure 6 (a) & 6(b) represented the comparision of amount of drug retained in skin layers (mg) and percentage of drug
retained in skin layers from differentminoxidil loaded gels.

Histopathological study
The histopathological study of treated and saline treated skin samples to demonstrate the safety of the developed formulation
was done as per the given procedure. The data collected from this study is shown in Figure 7 and 8.

Figure 7: Saline treated excised rat skin Figure 8: Excised rat skin sample treated (magnification 10X)
with submicron emulsion based gel

Viscosity determination & in vitro bioadhesion study

The optimized submicron emulsion based gel formulation M52%w/v exhibited viscosity of 3854 ± 0.027 mpas and bioadhesion of
104 minutes ± 11.24. The bioadhesion time possesed by minoxidil loaded submicron emulsion based gel was significantly
higher (p≤ 0.05) in contrast to plain minoxidil gel (23 minutes ± 6.73).

Antioxidant activity of optimized submicron emulsion based gel

DPPH method
The results of DPPH assay are given in Figure 9.

Ascorbic acid M5
% inhibition

% inhibition

Ascorbic acid
M5

Concentration Log concentration

Fig. 9 (a) Fig. 9(b)

Figure 9(a): Antioxidant activity activity of Ascorbic acid and drug loaded submicron emulsion, Figure 9(b): Log dose
response of Ascorbic acid and drug loaded submicron emulsion based gel

Stability Study
The results of stability study are given in Table 5. The gel formulation (M5 2%w/v) after 3 months storage is given in Figure 11.

Table 5: Stability study of optimized submicron emulsion based gel (M5 2%w/v) stored at 4 ºC
Time (days) Viscosity (mPas) ± S.D pH ± S.D Drug Content
(n=3) (n=3) (mg) ± S.D (n=3)

0 3854 ± 0.023 6.29 ± 0.018 9.814± 0.026

30 3850 ± 0.030 6.33 ± 0.020 9.812± 0.024

60 3847 ± 0.035 6.35 ± 0.037 9.809± 0.021

90 3843 ± 0.016 6.28 ± 0.029 9.807 ± 0.023

VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
Figure 11: Optimized minoxidil loaded gel formulation after storage period of 3 months at 4℃
DISCUSSION
Solubility studies & miscibility assessment
The solubility determination of drug in the oil, surfactant
and cosurfactant is considered as the most important
criterion in the formulation of submicron emulsion. Among
all the oils, the maximum solubility of minoxidil was found
in clove oil (51.83 ± 0.52 mg/ml) (Table 1). Thus, clove oil
was chosen for the formulation of submicron emulsion as
it would reduce the incidence of alopecia occurred from
oxidative stress at dermal papillae cells due to its own
antioxidant property [50].
On screening surfactants and cosurfactants (Table 1), it
was observed that the drug exhibited maximum solubility
in Tween 20 (5.82 ± 0.4 mg/ml) and Transcutol P (11 ±
0.3 mg/ml), which were chosen as surfactant and co-
surfactant phase repectively. The right blend of
surfactants leads to the formation of a stable submicron
emulsion upon dilution with water [51].
Construction of Pseudoternary phase diagrams for
formulation selection
Pseudoternary phase diagrams were constructed for each
Smix ratios (1:0, 1:1, 1:2, 2:1, 3:1 & 4:1) by aqueous
titration method to determine the extent of submicron
emulsion region (Figure 1). In Smix ratio 1:0, the submicron
emulsion region was found to be very less in contrast to
Smix ratio 1:1 indicating that the surfactant alone was not
able to solubilize the oil completely. When co-surfactant
(Transcutol P) was increased from 1:1 to 1:2, the
submicron emulsion area was found to be decreased.
This is due to the fact that further enhancement in
concentration of cosurfactant did not result in lower
interfacial tension. In case of 2:1 surfactant-cosurfactant
ratio, the area was comparitively higher than previous
ratios but lower than that found in case of 3:1. The largest
submicron emulsion area was found in Smix ratio 4:1,
which might be due to complete solubilization of oil phase,
reduced interfacial tension and formation of flexible film
surrounding the oil droplets of dispersed phase.
Therefore, combinations for development of placebo
formulations were selected from S mix ratio 4:1 and
subjected to physical stability testing. The drug was
loaded into the stable placebo submicron emulsion
formulations.
Formulation development and thermodynamic
stability studies
The placebo submicron emulsions which were found to be
stable after physical stability testing were further chosen
for loading the drug in a suitable dose (1%w/v). The
prepared minoxidil loaded submicron emulsion were again
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
checked for stability.
Characterization and optimization of submicron
emulsion
pH
The pH of all submicron emulsion formulations were found
to be in the range of 4.5 to 6.6, similar to the pH of skin
and hair [52]. This pH value indicated that the prepared
formulations were safe for topical application.
In vitro drug release study & determination of release
kinetics
This study measured the drug release from prepared
minoxidil containing submicron emulsions and minoxidil
solution (1%w/v) over a priod of 6 hours ( Figure 2). From
the results, it was found that minoxidil solution (1% w/v)
showed higher release (98.01±0.19%) as compared to the
prepared submicron emulsion formulations. It was due to
the presence of drug in its free form [53]. However, a
sudden decrease in drug release from minoxidil solution
was observed after 2 hours.
On the other hand, submicron emulsions showed
sustained and controlled release of drug. Among the
submicron emulsion formulations, M5 exhibited an in vitro
release of 95.08± 0.36%, which was found to be
comparitively higher than M1 (88.08± 0.36%), N2 (79.91±
0.26%), N6 (68.83 ±0.29%) and H1 (67.66 ± 0.22)
respectively. Therefore, M5 was considered as optimized
formulation and selected for further evaluation study.
Kinetic analysis of in vitro release profile of optimized
submicron emulsion (M5) was done by fitting the data
obtained in various kinetic models. The correlation
coefficient values obtained for zero order, first order,
higuchi model and Peppas-Korsemeyer were found to be
0.8659, 0.9648, 0.683 and 0.6415 respectively.Since the
correlation coefficient (R2) for first order model was near to
1, it was concluded that the release of minoxidil from
submicron emulsion formulation (M5) followed first order
kinetics.
Ex vivo skin permeation study and data analysis
The data obtained from this study revealed that minoxidil
loaded submicron emulsion (M5) exhibited maximum drug
permeation (79.36 ± 0.18%) in comparison with minoxidil
solution (49.79 ± 0.15%). The high drug permeation from
submicron emulsion was due to smaller globule size,
which resulted in direct and higher drug permeation into
skin. Another reason of higher drug permeation from
submicron emulsion was due to the presence of
Transcutol P as co-surfactant, which is reported to be a
potent permeation enhancer [54].
The flux and the permeability coefficient values were
found to be remarkably higher for submicron emulsion in
comparison with drug solution.
Particle size, PDI and Zeta potential
The average droplet size of submicron emulsion (M5) was
observed to be 181.3 nm with a PDI value of 0.239
(Figure 4), which is less than 1 respectively. The
polydispersity value less than 1 indicates monodisperse
and homogeneous nature of the formulation. These
results confirmed the ability of formulation to achieve the
desired transfollicular and transcellular penetration in skin
[49,12]. The formulation M5 exhibited the zeta potential of
-8.80 mV.
Formulation of Minoxidil loaded submicron emulsion
based topical gel
The optimized minoxidil loaded submicron emulsion
formulation (M5) was converted into gel formulation by
using different concentrations of carbopol 934 as gelling
agent.
Characterization and optimization of submicron
emulsion based gel formulations(M51%w/v, M51.5%w/v,
M52%w/v)
Physical evaluation, pH, Drug content, Homogeneity
and grittiness
The gel formulations were visually evaluated to examine
their physical appearance (Table 3) and found to be
stable and homogeneous.
Ex vivo skin permeation studyusing excised rat skin
& data analysis
The results of this study revealed that high drug
permeation was observed in the case of submicron
emulsion based gel formulations in contrast to the plain
gel , which might be due to the presence of transcutol P,
which is having a permeation enhancing activity [53].
As the quantity of gelling agent enhanced from 1% to 2%,
the drug permeation was found to be reduced. For
effective topical delivery, drug permeated from the
submicron emulsion-based gel must not come in contact
with blood circulation. Due to this, the formulation with
least permeation (M52%w/v)was selected as the optimized
submicron emulsion-based gel formulation.
The flux and permeability coefficient values of different
minoxidil loaded submicron emulsion gels and plain
minoxidil gel were found to be significantly higher than the
plain drug loaded gels due to the presence of suractants.
Drug retention study
The drug retention from submicron emulsion based gel
formulation M52%w/v was also significantly higher (p≤ 0.05)
as compared to the drug retention from plain gel which
was due to the submicron size and also due to the
presence of transcutol P, which is an efficient penetration
enhancer. The presence of sebum in the folliclular
compartment also results in accumulation of drug in lipid
environments or skin appendages, thus enhancing the
retention of hydrophobic drugs in the skin appendages
and follicles [55].
Histopathological study
From the observations of te histopathological studies
conducted, it was concluded that both the skin samples
(Treated or untreated) showed no evidence of
inflammation, indicating that the prepared formulation was
not irritating to the rat skin. Thus, the minoxidil loaded
submicron emulsion based gel was within the limit of skin
tolerance and safe to use for topical applications[56].
VOLUME 23, ISSUE Pharmacotherapy and Medical Science Advancements: A Cross-Continental Dialogue with East European and Asian Academics, Pages 92-102
Viscosity determination & in vitro bioadhesion study
The bioadhesion time possesed by minoxidil loaded
submicron emulsion based gel was significantly higher (p≤
0.05) in contrast to plain minoxidil gel, leading to higher
residence time and better therapeutic activity.
Antioxidant activity of optimized submicron emulsion
based gel
DPPH method
The significant antioxidant activity showed by minoxidil
loaded gel was due to the presence of clove oil, which is a
potent anti-oxidant [57].
Stability Study
The results of stability study revealed that there was no
significant change in the value of pH, viscosity and drug
content of optimized minoxidil loaded submicron emulsion
based gel formulation after the storage period of 3
months.
LIMITATIONS
The efficacy of the dosage form would be dependent on
the storage conditions. In this regards the patients have to
be counselled.
CONCLUSION
The minoxidil loaded submicron emulsion based topical
gel was formulated and showed a controlled drug release,
optimum particle size with PDI value less than 1,
significantly higher permeation and drug retention with
prolonged stability. It also showed a potent anti-oxidant
activity, which would inturn result in decreased incidence
of alopecia. Thus, the stabilized submicron emulsion
based topical gel of minoxidil would be a beneficial topical
nanostrategy in contrast to other topical dosage
formulations introduced for alopecia therapy.
ACKNOWLEDGEMENTS
The authors acknowledge the facilities provided by Khalsa
College of Pharmacy, Amritsar, Punjab.
CONFLICT OF INTEREST
All the authors of this research declared no conflict of
interest.
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