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Research Article
Pharmaceutical and Pharmacological Evaluation of the Effect
of Nano-Formulated Spironolactone and Progesterone on Inflammation
and Hormonal Levels for Managing Hirsutism Experimentally Induced in Rats
Reham I. Amer,
1,2
Ghada E. Yassin,
1,2
Reem A. Mohamed,
3
and Ahmed M. Fayez
3,4
Received 28 December 2020; accepted 27 March 2021
Abstract. Hirsutism is a dermatological condition that refers to the excessive growth of
hair in androgen-sensitive areas in women. Recently, the enhancement of the visible signs of
a hairy female has taken special concern that affected the quality of life. The present study
was developed to compare the follicular targeting effect of topical spironolactone (SP) or
progesterone (PG)-loaded nanostructured lipid carrier (NLC) on the management of
hirsutism. Four NLC formulations were prepared using cold homogenization techniques
and pharmaceutically evaluated. SP-NLC and PG-NLC topical hydrogels were prepared to
explore their pharmacological effect on letrozole induced polycystic ovarian syndrome
(PCOS) in rats. Inflammatory mediators, antioxidant, and hormonal parameters were
assayed. Additionally, histopathological examination was carried out to confirm the
successful induction of PCOS. Results confirmed that all NLC formulations have a spherical
shape with particle size ranged from 225.92 ± 0.41 to 447.80 ± 0.66 nm, entrapment efficiency
> 75%, and zeta potential (−31.4 to −36.5 mV). F1 and F3 NLCs were considered as
selected formulations for SP and PG, respectively. Female Wistar rats treated with F1
formulation for 3 weeks displayed better outcomes as manifested by the measured
parameters as compared to the other tested groups. A significant reduction in hair follicle
diameter and density was observed after topical application of SP or PG nano-gels. Finally,
the outcomes pose a strong argument that the development of topically administered SP-
NLC can be explored as a promising carrier over PG-NLC for more effectual improvement
in the visible sign of hirsutism.
KEY WORDS: Hirsutism; Hydrogel; Spironolactone; Polycystic ovary Syndrome; Nanostructure lipid
carriers.
INTRODUCTION
Hirsutism refers to the excessive growth of hair in
androgen-sensitive areas in women. Growing hair is usually
dark, thick, and coarse. The commonly affected areas are the
upper lips, chin, and central chest (1). This condition is one of
the most distressing and embarrassing conditions for a
woman. The perception of hirsutism is widely subjective,
and the quality of life of the hairy females can be adversely
affected by this phenomenon. Hence, the present study will
offer a new possible solution for this problem by the use of
topically applied nano-formulated drugs.
A hyper-androgenic condition such as polycystic ovary
syndrome (PCOS) is considered the most common cause of
hirsutism representing approximately 70% of hirsutism cases (2).
PCOS is usually accompanied by weight gain, infertility, and
insulin resistance (3). The latter leads to hyperinsulinemia which
will cause the ovaries to increase the production of androgen,
leading to anovulation (4). Additionally, the relative alteration in
LH/FSH levels, which are two major pituitary hormones, is
another pathophysiological perturbation in PCOS. Luteinizing
hormone (LH) stimulates the production of androgen substrates,
which are converted for the production of sex hormones such as
testosterone and estrogen. On the other hand, follicle-stimulating
hormone (FSH) stimulates the maturation of the ovarian follicle
before being released. LH and FSH need to be in a precise
balance for normal physiological function (5).
Spironolactone (SP) is a lipophilic drug which belongs to
a class of medications known as potassium-sparing diuretics
(6). Food and Drug Administration approved spironolactone
1
Department of Pharmaceutics and Industrial Pharmacy, Faculty of
Pharmacy, Al-Azhar University, Cairo, Egypt.
2
Department of Pharmaceutics, Faculty of Pharmacy, October
University for Modern Sciences and Arts (MSA), Giza, Egypt.
3
Department of Pharmacology, Faculty of Pharmacy, October
University for Modern Sciences and Arts (MSA), Giza, Egypt.
4
To whom correspondence should be addressed. (e–mail:
afayez@msa.eun.eg)
AAPS PharmSciTech (2021) 22:204
DOI: 10.1208/s12249-021-02003-z
1530-9932/21/0000-0001/0 #2021 American Association of Pharmaceutical Scientists
(SP) for clinical use as an antihypertensive agent (7). SP has
been reported in dermatological therapy due to its anti-
androgenic effect resulting from dual mechanisms, namely,
reduction of androgen production and competitive blocking
of androgen receptor in target tissues (8). Numerous clinical
studies have demonstrated the efficacy of oral SP by the doses
50–200 mg per day for 6 months on hirsute women. The
observed results in multiple studies showed improvement of
all factors measured. Yet, this oral regimen is commonly
associated with dose-dependent adverse effects (9).
Progesterone (PG) is a natural lipophilic steroid hor-
mone secreted primarily by the corpus luteum of the ovary
and the placenta. PG acts on a wide range of tissues, and it is
involved in milk production, aging, and hormonal disorder in
menopausal women, besides its role as an ovulation inhibitor
(contraceptive hormones) (10). The first line of treatment for
hirsutism is oral contraceptive (OCP) hormonal medication,
especially in those women desiring contraception. Estrogen/
progesterone combinations act by suppressing FSH and LH
levels. Also, they reduce gonadotropin secretion leading to a
decrease in ovarian androgen production (11). Many side
effects of PG such as breast tenderness, irregular vaginal
bleeding, mood changes, and mild fluid retention were
reported after the administration of OCP medication.
Nanostructure lipid carriers (NLCs) have attracted growing
attention in pharmaceutical research for dermal and transdermal
delivery enhancement. This colloidal drug delivery system is
produced using blends of solid lipid (long chain) and liquid lipid
(short chain) (12). NLCs nowadays are widely used in topical
cosmetic or dermatological follicular targeting preparations as it
offers the benefits of reducing both the drug dose and the systemic
side effects accompanied by oral delivery of medication (13). The
increase in drug distribution to the target site within the hair
follicles may result from the small size of the lipid particles that
assures close connection to the stratum corneum, thus promoting
drug penetration into the hair follicle (14). On the other hand,
NLCs were developed as promising drug delivery carriers through
the skin due to its lipid nature and biocompatibility. Due to the
high ability of NLCs to entrap medications, they have great
contribution in resolving the insolubility problem of lipophilic
drugs upon reaching systemic circulation (15). Additionally, NLCs
are characterized by large surface area which enables longer
contact time of the drug with the skin for maximum penetration
(16). Generally speaking, the aim of the current study is to
formulate SP-loaded NLC and PG-loaded NLC as a topically
applied hirsute medication to achieve dual effects, locally on the
hair follicles and systemically on the hormonal level.
MATERIALS AND METHODS
Materials
SP was kindly supplied by Eipico (Cairo, Egypt).
Progesterone and Tween 80 were purchased from Sigma-
Aldrich Chemical Co. (ST Louis, MO, USA). Stearic acid and
oleic acid (OA) were donated by Gattefossé (Saint-Priest,
France). Poloxamer 188, tri-ethanolamine, and Carbopol 934
were obtained from El-Nile Pharmaceutical Co. (Cairo,
Egypt). Letrozole® was obtained from Novartis pharmaceu-
ticals (El Amiria, Cairo, Egypt); other solvents used were of
analytical reagent grades.
Preparation of SP-NLCs and PG-NLCs
Four different formulations of NLCs are prepared by the
cold homogenization technique Table I. Firstly, solid lipid (stearic
acid) was indirectly heated (up to 65 °C), and then, liquid lipid
(oleic acid) was added in two different ratios 8:2 and 6:4 w/w and
then mixed. Next, either SP or PG was dissolved in an aqueous
surfactant solution (Tween 80 and Poloxamer 188). After that, the
previous aqueous solution was added to the melted lipid phase
while stirring with subsequent rapid cooling in ice. Homogeniza-
tion was done using a high-speed homogenizer (AH-2010; ATS
Engineering Inc., Suzhous Branch, Jiangus, China) at 12000 rpm
for 20 min with subsequent probe sonication at 90 W for 5 min,
whereas they were still immersed in the ice-bath (17). The
resultant NLCs formulations were stored at room temperature
for further investigation.
Characterization of SP-NLCs and PG-NLCs
Physicochemical Characterization
NLCs of either SP or PG were characterized in terms of
the average particle size (PS), polydispersity index (PI), and
zeta potential (ZP). The previous parameters were estimated
using a Malvern particle size analyzer (Zeta seizer 4000S,
Japan) at 25 °C after proper dilution of the samples with
double distilled water. The recorded results in Table II are the
means ± standard deviations (SD) of three determinations.
Transmission Electron Microscopy (TEM)
Surface morphology of SP-NLC and PG-NLC were
observed using transmission electron microscopy JEOL 1010
(JEOL Ltd, Tokyo, Japan) at 200 kV. One drop of each NLC
was diluted to 50-fold with pure water before dropping onto a
Formvar/Carbon 230 Mesh copper grids (Zhongjingkeyi
Technology Co. Ltd., Beijing, China), air-dried at room
temperature of about 25 ± 2 °C for 24 h, and then negatively
stained with (1% w/v) phosphotungstic acid for approxi-
mately 20 min before observation (18).
Entrapment Efficiency (EE%)
To calculate the amount of either SP or PG entrapped
inside the prepared nano-carrier system, about 100 mg of
NLCs equivalent to a known amount of each drug was
subjected to centrifugation for 20 min at 25,000 rpm, filtered
through disk filter (pore size: 0.45 μm, Sigma-Aldrich, USA).
Finally, the amount of either SP or PG in each supernatant
(free drug) was determined spectrophotometrically at
ʎ
max
220 nm and 240 nm for SP and PG, respectively. Drug
entrapment efficiency (EE%) was finally calculated using the
following equation:
EE% ¼Wt:of added drug−Wt:of free drug
Wt:of added drug 100
where wt. of added drug is the initial weight of either SP or
PG added in the formulations, and Wt. of free drug is the
amount of each drug in the supernatant (19).
204 Page 2 of 11 AAPS PharmSciTech (2021) 22:204
Ex vivo Permeation Study
Ex vivo release studies of SP and PG from the selected
NLC formulations (F1 and F3), respectively, across hairless
rat skin, were carried out using Franz diffusion cells (20).
Firstly, the skin section was washed with water and kept for
about 30 min in phosphate buffer pH 7.4 to ensure complete
saturation (21). Then, the skin section was positioned
between receptor and donor compartments. About 2 ml of
each NLCs dispersion was located in the donor compartment.
The receptor medium (15 ml) consisted of a mixture of
phosphate-buffered (PBS) pH .4: ethanol (4:1) (v/v). The
addition of ethanol to the receptor fluid was done to enhance
the solubility of both SP and PG upon release in the aqueous
buffer (22,23). The temperature was maintained at 37 ± 1 °C.
Drug release from different nano-carrier formulations was
assessed for 24 h by intermittently sampling the receptor
compartment (5 ml) and fresh mixture of PBS: ethanol
solution within the same ratio as previously mentioned was
replaced. Samples were filtered, and the amount of either SP
or PG released was determined using UV spectrophotometer
(Shimadzu, UV-Vis 1601 PC spectrophotometer, wavelength
range of 200-1000 nm, Tokyo, Japan)
FT-IR Analysis
Fourier transform infrared (FTIR) analysis was per-
formed on the lyophilized form of the selected SP-NLC and
PG-NLC formulations along with pure SP and PG. The
samples were mixed with KBR (IR grade) at a ratio of 100:1
and scanned over a wave range of 4000–400 cm
−1(
Shimadzu
IR/FTIR spectrophotometer (435 U-O4), Japan. Each data
point was recorded in three replicates using absorbance mode
to facilitate quantitative analysis (24).
DSC Analysis
Differential scanning calorimetry (DSC) measurements
were done for pure SP and PG, and their nano-formulations
using (PerkinElmer DSC Calorimeter, Waltham, MA, USA),
both selected SP-NLC and PG-NLC formulations were
lyophilized before the investigation and accurately weighed
for 3 mg and then sealed in an aluminum pan.Finally, the
experimental parameters were programed to reach 400 °C
withaheatingrateof10°C/mininadrynitrogen
environment. An empty pan, sealed in the same way as the
sample, was used as a reference (25).
Preparation of SP-NLC and PG-NLC Hydrogels
The selected SP-NLC and PG-NLC formulations (F1 and
F3) were formulated as a topical hydrogel using 1% w/v
Carbopol 934 as a gelling agent. Carbopol 934 (1% w/v) was
added to the nano-carrier dispersion under overhead stirring
at 300 rpm. Stirring was continued for 1 h until the Carbopol
got dispersed. The gel dispersion was then neutralized using
tri-ethanolamine and then left for 24 h for complete swelling
and equilibration of Carbopol. The final concentrations of SP
and PG in the NLC gels were maintained at 2% and 8%,
respectively.
Table I. Formulation of spironolactone and progesterone nanostructure lipid carrier
Ingredients NLCs Formulations
F1 F2 F3 F4
Spironolactone (mg) 30 30 ——————— ——————
Progesterone (mg) —————— ——————— 100 100
Stearic acid (gm) 1.8 2.4 1.8 2.4
Oleic acid (gm) 1.2 0.6 1.2 0.6
Tween 80 (mg) 750 750 750 750
Poloxamer 188 (mg) 750 750 750 750
H
2
OQSQSQSQS
Four different formulations of NLCs containing SP and PG were prepared by the cold homogenization technique. Stearic acid used as a solid
lipid while liquid lipid (oleic acid) was added in two different ratios 8:2 and 6:4 w/w then mixed. Finally; either SP or PG was dissolved in an
aqueous surfactant solution (Tween 80 and Poloxamer 188). QS quantity sufficient, NLCs nanostructured lipid carriers
Table II. Physicochemical characterization of different spironolactone and progesterone nanostructure lipid carrier formulations
NLCs Formulations Particle size (nm) ± SD PDI (%) ± SD Zeta potential ( mv ) % Entrapment Efficiency ± SD
F1 225.92 ± 0.41 0.428 ± 0.01 −36.5 ± 0.6 89.99 ± 0.45
F2 318.75 ± 0.36 0.712 ± 0.09 −31.4 ± 0.8 70.35 ± 0.56
F3 270.10 ± 0.12 0.393 ± 0.11 −36.3 ± 0.9 90.91 ± 0.82
F4 447.80 ± 0.66 0.539 ± 0.12 −33.1 ± 0.6 73.70 ± 0.55
Results of particle size, PDI, zeta potential measurements using a Malvern particle size analyzer and entrapment efficiency of different SP-NLC
and PG-NLC formulations. Values were represented as mean of triplicate ± standard deviation (Mean ± SD, n= 3). PDI polydispersity index
204 Page 3 of 11AAPS PharmSciTech (2021) 22:204
Evaluation of SP-NLC and PG-NLC Hydrogels
SP- and PG-loaded NLC hydrogels were examined for their
physical appearance, pH, and rheological properties using
rotational Brookfield viscometer (HBDV-III, USA) (26).
Pharmacological Evaluation
Animals
Adult female Wistar albino rats weighing 120–150 g.
were purchased from the National Institute of Ophthalmol-
ogy, Giza, Egypt. The animals were kept in the animal house
of the faculty of pharmacy, MSA University. They were
housed under constant environmental conditions of 12/12 h
dark/light cycles and a temperature of 25 ± 2 °C. The animals
were fed commercially available rat normal pellet diet and
water ad libitum and were left 7 days for acclimatization.
Animal experiments were conducted in full compliance with
local, national, ethical, and regulatory principles and local
licensing regulations, per the spirit of Association for
Assessment and Accreditation of Laboratory Animal Care
(AAALAC) International’s expectations for animal care and
use/ethics committees. The study was approved by the ethics
committee of the Faculty of Pharmacy, MSA University
(PH2/Ec2/2018PD).
Experimental Design
Twenty-four Wistar rats were evenly divided into 4
groups; the first group received a topical drug-free formula-
tion to serve as a normal control group, while the second
group received letrozole 1 mg/kg P. O using an oral tube for
21 days and served as a PCOS positive control group. The
third and fourth groups received letrozole 1 mg/kg P. O using
an oral tube for 21 days and were simultaneously treated by
either topical SP-NLC or PG-NLC hydrogels, respectively,
for 21 days (27).
Pharmacological Study
At the end of the treatment period, rats were anesthe-
tized, and blood was collected from the jugular vein and
centrifuged (4000 rpm, 4 °C) for 10 min to separate sera.
ELISA technique was carried out using the corresponding
ELISA kit for the assessment of serum LH, FSH, estrogen,
PG, and testosterone in addition to levels of TNF-αand IL-6.
All ELISA tests were based on sandwich method, which
measures the amount of antigen (analyte) between 2 layers of
antibody (i.e., capture and detection antibodies).
Histopathological Examination
Rats’ovaries and skin were removed and preserved in
10% paraformaldehyde. After fixation in formalin, the ovary
specimens were dehydrated in alcohol, cleared in xylene, and
embedded in paraffin wax. Paraffin blocks were sectioned and
stained with Hematoxylin and Eosin (H&E) for histopatho-
logical examination by a light microscope (Olympus BX50,
Tokyo, Japan) under a magnification of × 40 for histopatho-
logical examination. Additionally, hair follicle mean diameter
was measured under the microscope, and density was
determined using Born-Viewer Image Analyzer.
Statistical Analysis
Values are expressed as mean ± SE of 6 rats, and the
difference between groups was tested for significance using
analysis of variance (ANOVA), followed by Tukey’s multiple
comparison test as the post-hoc test. The level of statistical
significance was accepted at P< 0.05.
RESULTS AND DISCUSSION
Preparation of Nanostructured Lipid Carrier
In the present study, four different formulations of NLCs
loaded with SP and PG were successfully prepared using cold
homogenization technique. This technique is selected during
this study to avoid accelerated degradation of PG (thermo-
labile hormone) due to the elevated temperature of the lipid
mixture throughout the preparation (28), as shown in Table I.
Stearic acid was selected as a solid lipid due to its ability to
solubilize lipophilic drugs (29) as the higher solubility of the
drug in the solid lipid is a very critical issue for the NLCs
formulation. Oleic acid was chosen as liquid lipid due to its
ability to uniform the monondisperse systems; in addition, it
was reported in some publications that oleic acid vesicle-
loaded medications enhanced the epidermal accumulation of
drug (30) with the subsequent deep localized effect of our
tested medication on the hair follicle. Tween 80 was selected
among various surfactants due to its approved regulatory
status and success in preparing various NLCs (31). Addition-
ally, poloxamer 188 was chosen as a second non-ionic
surfactant for its ability to increase the mechanical stability
of the formed NLC (32). Furthermore, Iti Chauhan et al.(33)
stated the ability of hydrophilic polymers like poloxamines or
poloxamers in forming a coating layer around the lipid
particles; thus, they have a great contribution in increasing
the residence time of drug molecules in the systemic
circulation.
Physicochemical Characterization
The small size of nanoparticles makes the dispersion
kinetically stable against sedimentation. In addition, particle
size can be used as an indicator of instability (34). The
particle size of all NLCs formulations (F1–F4) is presented by
the z-average diameter was between 225.92 ± 0.41 and 447.80
± 0.66 nm, as shown in Table II. It was observed that the
particle size of SP-NLC and PG-NLC formulations was
mainly affected by the liquid lipid concentrations. As on
increasing the oleic acid concentration up to 40% of the total
lipid content, the mean particle size of NLCs was consecu-
tively decreased. The decrease in size of the particles with a
higher amount of OA might be due to the incompatible
mixing between OA and stearic acid; as a result, the free OA
might form nano-emulsion with an additional surfactant
which results in the formation of smaller particles. Further-
more, the presence of non-ionic surfactants in all formulations
may result in stabilization of the lipid-based vesicles (35).
204 Page 4 of 11 AAPS PharmSciTech (2021) 22:204
The dispersity index of either SP-NLC or PG-NLC is in the
range between 0.393 ± 0.11 and 0.712 ± 0.12, as shown in Table II,
which indicates the uniformity of the prepared samples with
respect to the particle size as well as the homogeneity and stability
of the prepared nanoparticles. Hu et al.(36)andAgrawalet al.’s
(31) studies conducted that the PI of NLCs formulations was
decreased by increasing the oleic acid content. Their previous
conduction was significantly observed in F1 and F3 formulations.
ZP values of all NLCs are in the range of −31.4 ± 0.8 to −36.5 ±
0.9 mV Table II. The higher electrostatic repulsions between the
particles reflect higher stability. All NLCs formulations showed
negatively charged values which were favorable since it indicates
long-term physical stability and particle adhesion properties (37).
Additionally, it was observed that increasing the liquid lipid
concentration in F1 and F3 formulations was associated with an
increase in ZP, probably due to the increase in the number of
ionized carboxylic groups of oleic acid present at a higher liquid
lipid concentration.
Transmission Electron Microscopy
Transmission electron micrograph (TEM) of either SP-
NLC or PG-NLC formulations portrays that the particles
were spherical in shape with smooth morphology and no
aggregated particles were observed. Regardless of the
concentration of solid lipid: liquid lipid used, NLCs particles
were nanometer-sized with a proper size distribution (181.99–
307.48 nm). The round shapes observed assure the mallea-
bility of the formed colloidal vesicles. The stabilized spherical
particles were formed during homogenization after the
addition of Tween 80, which plays a significant role in
reducing interfacial tension and thus reduced particle aggre-
gation. Likewise, the incorporation of poloxamer as a
hydrophilic surfactant in both formulations initiated a signif-
icant reduction in the particle size as a result of its steric
stabilization effect (32). The average particle size observed in
the TEM images is in good agreement with the size obtained
from the PS analyzer, as shown in Fig. 1.
Entrapment Efficiency (EE%)
The effect of oleic acid (OA) on drug entrapment
efficiency in NLCs is investigated in Table II. It has been
observed that the drug entrapment efficiency of NLCs had
increased to (89.99% and 90.91%) by increasing the percent-
age of oleic acid from 20 to 40% w/w in SP-NLCs (F1) and
PG-NLCs (F3), respectively. This observation might be due
to the incorporation of liquid lipid OA into solid lipids
(stearic acid), which have led to massive crystal order
disturbance. Greater imperfections in the crystal lattice leave
enough space to accommodate drug molecules, which subse-
quently improved drug-loading capacity and drug entrapment
efficiency (38). Higher entrapment in all NLCs formulations
containing 40% w/w oleic acid indicates higher solubility of
either SP or PG in oleic acid, compared to stearic acid.
To sum up the results of the previous pharmaceutical
experiments, it was observed that F1 and F3 shown minimum
particle size, maximum ZP value, and highest entrapment
efficiency %. Accordingly, they had been chosen as a selected
NLC formulation to carry out the further investigations.
Ex vivo Permeation Study
The ex vivo release profiles of SP-NLC (F1) and PG-
NLC (F3) formulations prepared by binary lipid phase (10%)
in comparison with their drug suspensions containing the
same drug concentration are evaluated using Franz diffusion
cell as shown in Fig. 2. The prepared nanoparticles showed
initial burst release within 30–40 min of 30.05% for SP and
36.20% for PG, followed by slow diffusion of both medica-
tions out of the core. The initial burst of drugs might be due
to the presence of oleic acid on the outer shell of nanopar-
ticles, while the slow diffusion pattern of both medications
after the initial burst might be due to the penetration of the
diffusion medium into the hydrophobic lipid core. Addition-
ally, there was an observed reduction in the drug release at
the end of diffusion that reflecting the depletion of the SP and
PG from the core lipid matrix, resulting in a reduced
concentration gradient. Drug suspension (used as references)
release about 45% for SP compared with 85% from SP-NLC
formulation and about 33% PG in comparison with 76% from
PG-NLC formulation at the end of 24 hours release study
using the same experimental conditions.
FT-IR Analysis
FT-IR spectra of SP (pure), PG (pure), and their selected
NLCs formulations are illustrated in Fig. 3. The spectrum of
Fig. 1. Transmission electron micrographs of aSP-NLCs and bPG-NLCs
204 Page 5 of 11AAPS PharmSciTech (2021) 22:204
SP (pure) indicated the presence of C-H aliphatic bands at
2926 and 2857 cm
−1
. The sharp absorption band of lactone
carbonyl group C=O appeared around 1769 cm
−1
, while the
retinoic c=o groups appeared at 1686 cm
−1
. The spectrum of
SP-NLC revealed the presence of absorption bands at 2949,
1770, and 1682 cm
−1
corresponding to C-H aliphatic and C=O
groups, respectively. Shifting of carbonyl groups confirms the
possibility of interaction between SP and nano-lipid vehicles
via intermolecular hydrogen bonding (39). The FT-IR spec-
trum of PG (pure) indicated the presence of C-H aliphatic
bands at 2938 and 2853 cm
−1
along with C=O absorption
band at 1697and 1662 cm
-1
, while the spectrum of PG-NLC
showed no change in the wavenumbers related to carbonyl or
aliphatic C-H, enlighteningthattherearenosignsof
interaction between the PG and the NLC components.
DSC Analysis
Generally, DSC is a widely used tool to characterize
raw materials used in lipid-based drug delivery systems
(40). In this study, DSC has been carried out to investigate
the melting and recrystallization behavior of material that
has crystalline structures and allows us to evaluate the
compatibility of drugs with the lipid excipients used (41).
The DSC thermogram of SP (pure), PG (pure), and both
their NLCs lyophilized dried form is shown in Fig. 4.The
thermal curves of SP (pure) showed a characteristic sharp
endothermic peak at 205.39 °C; this characteristic peak
became less intense and shifted to 217.72 °C in SP-NLCs,
indicating the entrapment of SP in the lipid matrix. A
similar event has been detected in the thermogram of PG-
NLC as the characteristic sharp endothermic peak of PG
(pure) was clearly visualized at 125 °C; this peak was
shiftedintheDSCapparatusinPG-NLCsto119.65°C.
The most probable reason for the shifting in the charac-
teristic peaks of both SP and PG in their formulations
could be due to the higher hydrophobic nature of both
which enhances their dissolution in the molten stearic acid
(42). On the other hand, the presence of the characteristic
peak of both SP and PG in their lipid nano-formulations
proved the absence of any interaction between both drugs
and the chosen lipids.
Fig. 2. Ex vivo permeation study of SP-suspension, PG-suspension, SP-NLC, and PG-NLC
optimized formulation in PBS of pH 7.4
Fig. 3. FT-IR spectra of SP (pure), SP-NLCs, PG (pure), and PG-NLCs
204 Page 6 of 11 AAPS PharmSciTech (2021) 22:204
Evaluation of SP-NLC and PG-NLC Topical Hydrogels
To investigate the dermatological effect of both SP-NLC
and PG-NLC formulations on the treatment of hirsutism,
topical hydrogels were prepared using 1% w/v Carbopol 934
as a gelling agent. Visual inspection of the prepared hydrogels
indicates a suitable homogeneity and consistency with the
absence of lumps. pH values of the developed hydrogels were
Fig. 4. DSC thermograms of SP (pure), SP-NLCs, PG (pure), and PG-NLCs
Fig. 5. Effect of Letrozole and 21 days administration of SP-NLC and PG-NLC topical hydrogels
on serum aFSH, bLH, cPG, and dtestosterone. Statistical analysis was performed using one-way
ANOVA followed by Tukey’s post-hoc test (P< 0.05). Values are mean ± SE of (6 animals) as
compared with normal control (a), letrozole (b), SP-NLC hydrogel (c), and PG-NLC hydrogel (d)
treated groups
204 Page 7 of 11AAPS PharmSciTech (2021) 22:204
ranged from 5.50 ± 0.14 to 6.4 ± 0.60 for SP-NLC and from
5.95 ± 0.20 to 6.25 ± 0.15 for PG-NLC, which are considered
an acceptable value to avoid the risk of irritation after skin
application. The rheological parameter of both NLC gel
formulations exhibited shear thinning flow pattern with a
viscosity value (1570 ± 33.67 and 1743.75 ± 13.15 cps) for SP-
NLC and PG-NLC gels, respectively, thus, indicating the non-
Newtonian pseudoplastic behavior of the examined gels.
Pharmacological Evaluation
Hormonal Plasma Assays
In the current study, PCOS was induced by daily oral
administration of letrozole (1 mg/kg), a non-steroidal aromatase
inhibitor, to female Wistar rats for 21 consecutive days.
Deficiency in aromatase activity plays an important role in
PCOS, affecting steroidogenesis and triggering ovarian failure
(43). Aromatase enzyme catalyzes the synthesis of estrogen from
androgens. Its deficiency results in hormonal imbalance with
decreased estrogen and increased androgen level. The circulat-
ing androgens, together with excess intra ovarian androgens,
cause polycystic ovaries (44). This condition has also been
associated with abnormal follicular development (45).
In the present study, 21 days of daily oral administration
of letrozole (1 mg/kg) brought about a significant increase in
serum FSH (263.7%), LH (3853.5%), and testosterone
(357%). Meanwhile, it reduces serum PG levels (53%) as
presented in Fig. 5. These results are in agreement with
previous reports where letrozole in a dose-dependent manner
decreased serum PG level, increased LH and testosterone,
and markedly elevated FSH level in the higher doses of
letrozole (0.5 and 1 mg/kg) (27). Furthermore, Fig. 6shows
that letrozole was able to increase both serum levels of TNF-
Fig. 6. Effect of Letrozole and 21 days administration of SP-NLC and PG-NLC topical hydrogels
on serum aTNF-αand bIL-6. Statistical analysis was performed using one-way ANOVA followed
by Tukey’spost-hoc test (P< 0.05). Values are mean ± SE of (6 animals) as compared with normal
control (a) and letrozole (b) treated group
Fig. 7. Effect of Letrozole and 21-day administration of SP-NLC and PG-NLC topical hydrogels on
hair follicle adiameter and bdensity. Statistical analysis was performed using one-way ANOVA
followed by Tukey’spost-hoc test (P< 0.05). Values are mean ± SE of (6 animals) as compared
with normal control (a) and letrozole (b) treated group
204 Page 8 of 11 AAPS PharmSciTech (2021) 22:204
α(66.2%) and IL-6 (126.8%) as indicators for inflammation.
These inflammatory-inducing effects of letrozole were also
previously observed in other models of PCOS (46,47). In the
current study, all biochemical parameters were assessed in
plasma using the ELISA technique.
SP is an aldosterone antagonist that is used in PCOS
because it blocks testosterone receptors, thus terminating its
actions (48). Daily application of SP-NLC topical hydrogel
reversed FSH level but had no effect on LH, PG, or
testosterone levels. A similar result was obtained by a
previous study documenting no change in testosterone or
LH levels in women with PCOS (49); this might be attributed
to the fact that SP acts on the receptor level and does not
affect hormonal production. In contrast, the application of
PG-NLC hydrogel restored the levels of all hormones,
perhaps due to a negative feedback mechanism. Meanwhile,
serum TNF-αand IL-6 levels are significantly reduced in rats
treated by either SP-NLC or PG-NLC hydrogels Fig. 6.
Hair Follicles Parameters
All of the rats in the treated groups showed a consider-
able moderated rate of hair growth on the areas in which both
topical nano-gel applied which was reflected on the duration
of time needed for shaving since the hair follicles became
looser and more comfortable to pluck, the hair follicles’
diameter and density (Fig. 7). Additionally, it was noticed that
no allergic reaction to the topical medication or skin eruption
in the treated areas in which the hydrogels were applied in all
tested groups.
Histopathological Examination
The induction of PCOS was also confirmed by histopath-
ological examination of the rats’ovaries, which revealed the
presence of numerous follicular cysts. Figure (8) shows (A)
ovary of rats in normal control group showing a normal
histological structure in the proestrus with different stages of
growing follicular, (B) ovary of letrozole-treated rats showing
numerous follicular cysts (★), (C) ovary of SP-NLC-treated
rat showing few outer growing follicles (→)andmany
corpora lutea with intramedullary congested BVs and extrav-
asation of blood (red arrow), and on the contrary, (D) ovary
of PG-NLC-treated rats showing normal histological struc-
tures with many growing follicles (→) and intact stromal
tissue.
CONCLUSION
In the present study, SP and PG nanostructured lipid
carriers were successfully prepared by cold homogenization
technique using various solid lipid: liquid lipid ratios. All
NLC formulations exhibited nanometer size, stable polydis-
persity index, and also acceptable zeta potential negative
charge. Regarding the pharmacological effect of the NLC
formulations, the null hypothesis was rejected; the pharma-
cological evaluation indicated that the increase in the hair
follicle diameter and density of rat-induced PCOS model was
significantly decreased after topical application of both SP-
NLCs and PG-NLCs nano-gels for successively 21 days. The
effect of the topically administered NLCs on hirsutism might
be attributed to their local action on the hair follicle resulted
from interaction of the lipid-based nano-carrier with the
lipophilic sebum present inside the follicular ducts and
systemic action on the hormonal level.
REFERENCES
1. Hunter MH, Carek PJ. Evaluation and treatment of women
with hirsutism. Am Fam Physician. 2003;67(12):2565–72.
2. Archer JS, Chang RJ. Hirsutism and acne in polycystic ovary
syndrome. Best Pract Res Clin Obstet Gynaecol.
2004;18(5):737–54.
Fig. 8. Effect of Letrozole and 21 days administration of SP-NLC and PG-NLC topical hydrogels on histopathological
examination in the ovary. ANormal control group. BLetrozole-positive control group. Cand DTreated by topical SP-NLC
and PG-NLC hydrogels, respectively
204 Page 9 of 11AAPS PharmSciTech (2021) 22:204
3. Chazenbalk G, Chen Y-H, Heneidi S, Lee J-M, Pall M, Chen Y-
DI, et al. Abnormal expression of genes involved in inflamma-
tion, lipid metabolism, and Wnt signaling in the adipose tissue
of polycystic ovary syndrome. J Clin Endocrinol Metab.
2012;97(5):E765–E70.
4. Diamanti-Kandarakis E, Christakou CD. Insulin resistance in
PCOS. Diagnosis and management of polycystic ovary syn-
drome: Springer; 2009. p. 35–61.
5. Rebar R, Judd H, Yen S, Rakoff J, Vandenberg G, Naftolin F.
Characterization of the inappropriate gonadotropin secretion in
polycystic ovary syndrome. J Clin Invest. 1976;57(5):1320–9.
6. Endou H, Hosoyamada M. Potassium-retaining diuretics: aldo-
sterone antagonists. Diuretics Handb Exp Pharmacol.
1995;117:335–61.
7. Rathnayake D, Sinclair R. Innovative use of spironolactone as
an antiandrogen in the treatment of female pattern hair loss.
Dermatol Clin. 2010;28(3):611–708.
8. Chen W-C, Zouboulis CC. Hormones and the pilosebaceous
unit. Dermatoendocrinol. 2009;1(2):81–6.
9. Cumming DC, Yang JC, Rebar RW, Yen SS. Treatment of
hirsutism with spironolactone. Jama. 1982;247(9):1295–8.
10. Macias H, Hinck L. Mammary gland development. Wiley
Interdiscip Rev Dev Biol. 2012;1(4):533–57.
11. Burkman RT Jr. The role of oral contraceptives in the treatment
of hyperandrogenic disorders. Am J Med. 1995;98(1):S130–S6.
12. Müller R, Radtke M, Wissing S. Nanostructured lipid matrices
for improved microencapsulation of drugs. Int J Pharm.
2002;242(1-2):121–8.
13. Knorr F, Lademann J, Patzelt A, Sterry W, Blume-Peytavi U,
Vogt A. Follicular transport route–research progress and future
perspectives. Eur J Pharm Biopharm. 2009;71(2):173–80.
14. Kaur S, et al. Nanostructure lipid carrier (NLC): the new
generation of lipid nanoparticles. Asian Pac J Health Sci.
2015;2:76–93.
15. Nandvikar NY, Lala RR, Shinde AS. Nanostructured lipid
carrier: the advanced lipid carriers. Int J Pharm Sci.
2019;10(12):5252–65.
16. Kurakula M, Ahmed OA, Fahmy UA, Ahmed TA. Solid lipid
nanoparticles for transdermal delivery of avanafil: optimization,
formulation, in-vitro and ex-vivo studies. J Liposome Res.
2016;26:288–96.
17. Upreti T, Senthil V. Nanostructured lipid carrier system for the
treatment for skin disease—a review. JSM Nanotechnol
Nanomed. 2017;5(3):1059–64.
18. Shekhawat PB. Preparation and evaluation of clotrimazole
nanostructured lipid carrier for topical delivery. Int J Pharm
Bio Sci. 2013;4:407–16.
19. Dong Y, Ng WK, Shen S, Kim S, Tan RB. Preparation and
characterization of spironolactone nanoparticles by antisolvent
precipitation. Int J Pharm. 2009;375(1-2):84–8.
20. Wissing S, Müller R. Solid lipid nanoparticles as carrier for
sunscreens: in vitro release and in vivo skin penetration. J
Control Release. 2002;81:225–33.
21. Csóka I, Csányi E, Zapantis G, Nagy E, Fehér-Kiss A, Horváth
G, et al. In vitro and in vivo percutaneous absorption of topical
dosage forms: case studies. Int J Pharm. 2005;291(1-2):11–9.
22. Yin-Ku L, Zih-Rou H, Rou-Zi Z, Jia-You F. Combination of
calcipotriol and methotrexate in nanostructured lipid carriers
for topical delivery. Int J Nanomedicine. 2010;5:117–28.
23. Friuli V, Bruni G, Musitelli G, Conte U, Maggi L. Influence of
dissolution media and presence of alcohol on the in vitro
performance of pharmaceutical products containing an insoluble
drug. J Pharm Sci. 2018;107(1):507–11.
24. Rohman A, Nugroho A, Lukitaningsih E, Sudjadi. Application
of vibrational spectroscopy in combination with chemometrics
techniques for authentication of herbal medicine. Appl
Spectrosc Rev. 2014;49:603–13.
25. Mura, et al. Characterization and evaluation of different
mesoporous silica kinds as carriers for the development of
effective oral dosage forms of glibenclamide. Int J Pharm.
2019;563:43–52.
26. Joshi B, Singh G, Rana A, Saini S, Singla V. Emulgel: a
comprehensive review on the recent advances in topical drug
delivery. Int Res J Pharm. 2011;2(11):66–70.
27. Kafali H, Iriadam M, OzardalıI, Demir N. Letrozole-induced
polycystic ovaries in the rat: a new model for cystic ovarian
disease. Arch Med Res. 2004;35(2):103–8.
28. Li Q, Cai T, Huang Y, Xia X, Cole S, Cai Y. A review of the
structure, preparation, and application of NLCs, PNPs, and
PLNs. Nanomaterials. 2017;7(6):122.
29. Manjunath K, Reddy JS, Venkateswarlu V. Solid lipid nanopar-
ticles as drug delivery systems. Methods Find Exp Clin
Pharmacol. 2005;27(2):127–44.
30. Zakir F, Vaidya B, Goyal AK, Malik B, Vyas SP. Development
and characterization of oleic acid vesicles for the topical delivery
of fluconazole. Drug Deliv. 2010;17(4):238–48.
31. Agrawal Y, Petkar KC, Sawant KK. Development, evaluation
and clinical studies of acitretin loaded nanostructured lipid
carriers for topical treatment of psoriasis. Int J Pharm.
2010;401(1-2):93–102.
32. Paruvathanahalli SR, Jestin C. Development and evaluation of
nanostructured lipid carrier-based hydrogel for topical delivery
of 5-fluorouracil. Int J Nanomedicine. 2016;11:5067–77.
33. Chauhan I, et al. Nanostructured lipid carrier: the groundbreak-
ing approach for transdermal drug delivery. Adv Pharm Bull.
2020;10(2):150–65.
34. Gaba B, Fazil M, Khan S, Ali A, Baboota S, Ali J.
Nanostructured lipid carrier system for topical delivery of
terbinafine hydrochloride. Bull Fac Pharm Cairo Univ.
2015;53(2):147–59.
35. Jiao J. Polyoxyethylated nonionic surfactants and their applica-
tions in topical ocular drug delivery. Adv Drug Deliv Rev.
2008;60:1663–73.
36. Hu F-Q, Jiang S-P, Du Y-Z, Yuan H, Ye Y-Q, Zeng S.
Preparation and characterization of stearic acid nanostructured
lipid carriers by solvent diffusion method in an aqueous system.
Colloids Surf B: Biointerfaces. 2005;45(3-4):167–73.
37. Lu GW, Gao P. Emulsions and microemulsions for topical and
transdermal drug delivery. In: Kulkarni VS, editor. Handbook
of Non-Invasive Drug Delivery Systems; 2010. p. 59–94.
38. Xia D, Cui F, Gan Y, Mu H, Yang M. Design of lipid matrix
particles for fenofibrate: effect of polymorphism of glycerol
monostearate on drug incorporation and release. J Pharm Sci.
2014;103(2):697–705.
39. Shah S, Joshi S, Lin S, Madan P. Preparation and characteriza-
tion of spironolactone solid dispersions using hydrophilic
carriers. Asian J Pharm Sci. 2012;7(1):40–9.
40. Kumar N, Goindi S, Saini B, Bansal G. Thermal characteriza-
tion and compatibility studies of itraconazole and excipients for
development of solid lipid nanoparticles. J Therm Anal
Calorim. 2014;115:2375–83.
41. Seyfoddin A, Shaw J, Al-Kassas R. Solid lipid nanoparticles for
ocular drug delivery. Drug Deliv. 2010;17:467–89.
42. Revathy S, Vrinda S. Kumar and Sabitha M. Omega-3 fatty acid
based nanolipid formulation of atorvastatin for treating hyper-
lipidaemia. Adv Pharm Bull. 2019;9(2):271–80.
43. Yang H, Lee SY, Lee SR, Pyun B-J, Kim HJ, Lee YH, et al.
Therapeutic effect of Ecklonia Cava Extract In Letrozole-
induced polycystic ovary syndrome rats. Front Pharmacol.
2018;9:1325.
44. Caldwell A, Middleton L, Jimenez M, Desai R, McMahon A,
Allan C, et al. Characterization of reproductive, metabolic, and
endocrine features of polycystic ovary syndrome in female
hyperandrogenic mouse models. Endocrinology.
2014;155(8):3146–59.
45. Choi S-H, Shapiro H, Robinson GE, Irvine J, Neuman J, Rosen
B, et al. Psychological side-effects of clomiphene citrate and
human menopausal gonadotrophin. J Psychosom Obstet
Gynaecol. 2005;26(2):93–100.
46. Rezvanfar M, Rezvanfar M, Ahmadi A, Saadi HS, Baeeri M,
Abdollahi M. Mechanistic links between oxidative/nitrosative
stress and tumor necrosis factor alpha in letrozole-induced
204 Page 10 of 11 AAPS PharmSciTech (2021) 22:204
murine polycystic ovary: biochemical and pathological evi-
dences for beneficial effect of pioglitazone. Hum Exp Toxicol.
2012;31(9):887–97.
47. Pandey V, Singh A, Singh A, Krishna A, Pandey U, Tripathi
YB. Role of oxidative stress and low-grade inflammation in
letrozole-induced polycystic ovary syndrome in the rat. Reprod
Biol. 2016;16(1):70–7.
48. Christy NA, Franks AS, Cross LB. Spironolactone for hirsutism
in polycystic ovary syndrome. Ann Pharmacother.
2005;39(9):1517–21.
49. Spritzer PM, Lisboa KO, Mattiello S, Lhullier F. Spironolactone
as a single agent for long-term therapy of hirsute patients. Clin
Endocrinol. 2000;52(5):587–94.
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204 Page 11 of 11AAPS PharmSciTech (2021) 22:204
