Preparation and Characterization of Liposome Containing Minoxidil and Rosemary Essential Oil

Abstract

Minoxidil and Rosemary essential oil have been used for several years to stimulate hair growth. Therefore, co-administration of both Minoxidil and Rosemary essential oil could enhance hair growth. The chemical/biological characteristics of liposomes, which encapsulate both hydrophobic and hydrophilic drugs, can be utilized to encapsulate the herbal and chemical drug concoction concomitantly. A thin-film hydration method was used to prepare the liposomes. The entrapment efficacy of the liposomes was determined for Minoxidil and Rosemary essential oil using UV spectrophotometry and hydrodistillation; as dictated in the European pharmacopeia. Furthermore, a dynamic light scattering (DLS) analysis was conducted to determine the particle size and zeta potential of the prepared liposome. In addition, the storage stability of the liposome was checked after 60 days. The results showed that co-encapsulation of Minoxidil and Rosemary essential oil increased the encapsulation efficacy of Minoxidil while the entrapment efficacy of essential oil was not significantly influenced. In addition, according to the DLS results, the particle size and zeta potential of prepared liposomes didn't change significantly during 60 days of storage.

Full text

Preparaon and Characterizaon of Liposome Containing Minoxidil and
Rosemary Essenal Oil
Gita Kiaee1*, Hamid Akbari Javar1, Bita Kiaee2 and Shadi Kiaei3
1Tehran University of Medical Sciences, Tehran, Iran
2Islamic Azad University of Medical Science, Tehran, Iran
3Portland State University, USA
*Corresponding author: Gita Kiaee, Doctor of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, Tel: 98218889 6692; E-
mail: gkiaee@gmail.com
Received date: July 2, 2016; Accepted date: Aug 3, 2016; Published date: Aug 10, 2016
Copyright: © 2016 Kiaee G, et al. This is an open-access arcle distributed under the terms of the Creave Commons Aribuon License,
which permits unrestricted use, distribuon, and reproducon in any medium, provided the original author and source are credited.
Citaon: Kiaee G, Javar HA, Kiaee B, et al. Preparaon and Characterizaon of Liposome Containing Minoxidil and Rosemary Essenal Oil. J In
Silico In Vitro Pharmacol. 2016, 2:3.
Abstract
Minoxidil and Rosemary essenal oil have been used for
several years to smulate hair growth. Therefore, co-
administraon of both Minoxidil and Rosemary essenal
oil could enhance hair growth. The chemical/biological
characteriscs of liposomes, which encapsulate both
hydrophobic and hydrophilic drugs, can be ulized to
encapsulate the herbal and chemical drug concocon
concomitantly. A thin-lm hydraon method was used to
prepare the liposomes. The entrapment ecacy of the
liposomes was determined for Minoxidil and Rosemary
essenal oil using UV spectrophotometry and
hydrodisllaon; as dictated in the European
pharmacopeia. Furthermore, a dynamic light scaering
(DLS) analysis was conducted to determine the parcle
size and zeta potenal of the prepared liposome. In
addion, the storage stability of the liposome was
checked aer 60 days. The results showed that co-
encapsulaon of Minoxidil and Rosemary essenal oil
increased the encapsulaon ecacy of Minoxidil while
the entrapment ecacy of essenal oil was not
signicantly inuenced. In addion, according to the DLS
results, the parcle size and zeta potenal of prepared
liposomes didn’t change signicantly during 60 days of
storage.
Keywords:
Liposomes; Hydrodisllaon; Spectrophotometry; Co-
encapsulaon
Introducon
Minoxidil and Rosemary essenal oil have been known for
their hair growth smulatory properes for several years
(Arcle 1999) [1,2] and there are several explanaons that
aribute to the Minoxidil mechanism of acon [3]. One of the
biochemical acons of Minoxidil for hair growth is its
smulatory eect on prostaglandin and Vascular endothelial
growth factor (VEGF) synthesis [4]. However, a limitaon to the
applicaon of Minoxidil is its poor skin penetraon ability and
water solubility [5]. In order to enhance Minoxidil’s
penetraon and solubility it has to be formulated in an ethanol
based soluon [5] which has been known to cause dermas
irritaon, pruritus, erythema, scaling and dryness of skin [6]. In
addion, Terpene compounds such as cineol, limonene, and
Nerolidol have been observed to improve the penetraon of
Minoxidil in the skin [7]. These Terpene compounds, especially
cineol constute the major fracon of Rosemary essenal oil
[8-10]. Therefore, co-administraon of Minoxidil and
Rosemary essenal oil can lead to the high potency for a hair
growth formulaon.
The advents of novel drug delivery system provide the
appropriate means to encapsulate both hydrophilic and
hydrophobic compounds [11]. Liposomes have capability of
increasing the concentraon of topically applied drugs in the
dermis while reducing the unfavourable risk by restricng the
absorbance of systemic drug [12]. Moreover, the similarity of
lipid composion of liposomes and membranes of intercellular
lamellae and keranocytes render the improvement in drug
release properes and skin compability [13]. Therefore,
liposomes appear to be an appropriate vehicle to co-
encapsulate Minoxidil and the Rosemary essenal oil [14].
Furthermore, previous studies of liposomal encapsulaon of
Minoxidil has led to the higher concentraon of drugs in the
pilosebace units in comparison to convenonal Minoxidil
formulaons [15]. In addion, the organic solvent deleon of
convenonal formulaons reduces the adverse side eects of
long term applicaon. Moreover, liposomal encapsulaon of
Rosemary essenal oil could protect the essenal oil against
degradaon factors such as pH and light, and increase its
stability [16].
Research Article
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Therefore, the aim of this study is to prepare liposomes
containing Minoxidil and rosemary essenal oil that presented
acceptable physicochemical properes.
Materials and Methods
Materials
The Rosemary essenal oil was purchased from Barij
essence company and the other consumed substances were
purchased from Merck Company.
Liposome preparaon
Liposomes were prepared by a thin-lm hydraon method
as reported in the literature accurately weighed quanes of
the Egg Phosphadyl choline (EPHC) and Cholesterol (CHOL)
with the molar rao of 7:3 were dissolved in methanol:
Chloroform (1:3) mixture. The soluon was placed in a rotary
evaporator (Rotavapor R 200/205, Buchi) at 55°C unl a thin
lipid lm on the wall of a round-boomed ask was obtained.
The resulng lipid lm was kept under a vacuum overnight in
order to eliminate traces of organic solvents. The lipid lm was
then hydrated with 10 mL of the aqueous soluon described in
(Table 1) for one hour. Aerwards the homogenous
suspension of the liposome was spun in the centrifuge for 45
minute at 15000 rpm. Following the separaon of the
supernatant, the sediment was rehydrated with dislled water
and it was sonicated for 10 min by using an ultrasound bath
(Transonic 460 H, Singen), and nally the liposome mixture
was extruded with a 400 nm lter.
Table 1: Content of aqueous soluon for hydrang a thin-lm
layer.
Formulatio
n 1
Formulatio
n 2
Formulatio
n 3
Formulatio
n 4
Minoxidil
e
- 1 mg/ml - 1 mg/ml
Essential
oil
- - 300 µl 300 µl
Determinaon of Minoxidil-entrapment
ecacy
The supernatant of centrifuged suspension containing
liposome were analyzed at 285 nm spectrophotometrically.
The percent drug entrapment for the prepared liposome was
calculated by using equaon (1).
(Total drug added – non entrapped drug/Total drug added) ×
100 (1)
Determinaon of Rosemary essenal oil-
entrapment ecacy
The quanty of entrapped essenal oil in the liposome was
determined via a method introduced in the European
pharmacopeia [17]. The method was carried out by means of
steam disllaon by placing the prepared liposomal soluon in
the ask described below and heat it for 30 min (Figure 1).
Aer 30 min, heang was stopped, and following 10 min of
cooling a room temperature the volume of the essenal oil
was read o. The drug entrapment percentage was calculated
using equaon (2).
(The volume of essenal oil in the tubed aer 30 minutes of
heang/Total volume of added essenal oil) × 100 (2)
Figure 1: Hydro disllaon method: Clevenger type
apparatus.
Size and zeta potenal
The vesicle size and zeta potenal analysis of the liposomes
were carried out by using a Malvern Zetasizer 2000 HS
(Malvern instrument limited, Malvern, UK, NIPER, SAS Nagar,
Punjab).
Storage stability studies
In order to determine the physical stability of the liposomes,
size of the parcle and polydispersity index (PDI) were
measured by Malvern Zetasizer. The vesicles were stored at
4°C for up to 2 months under light protecon [18]. In
predetermined me intervals, vesicles were characterized for
their vesicle size and PDI.
Results and Discussion
The inuence of rosemary essenal oil on
Minoxidil encapsulaon ecacy
The Minoxidil encapsulaon ecacy (EE) with and without
the essenal oil was 73% and 64% respecvely. Based on our
results, the liposome formulaon containing essenal oil
represented larger EE% of Minoxidil which was accompanied
by increase in the parcle size and zeta potenal of the
vesicles. The high zeta potenal frequently led to an increase
in the repulsion forces of the bilayer structure of the vesicles
Journal of In Silico & In Vitro Pharmacology
Vol.2 No.3:10
2016
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which consequently increased the size of the liposomes.
Minoxidil, being parally hydrophobic, was expected to be
localized in the membrane compartment of lipid vesicles [13].
In Baranl et al. [10] study it was shown that Terpen compounds
such as cineol increased the entrapment ecacy of
hydrophobic drugs [10], thus the increase in EE could be
related to Terpen compound of essenal oil.
The inuence of Minoxidil on Rosemary
essenal oil encapsulaon ecacy
The essenal oil encapsulaon ecacy with and without
Minoxidil was 50% and 55% respecvely. There was no
signicant change to the encapsulaon ecacy of Rosemary
essenal oil accompanied with Minoxidil.
Size and Zeta potenal of liposomes
Zeta potenal and parcle size of the formulated vesicles
aer probe sonicaon is presented in Table 2. The results
showed that the average size of liposomes containing
Minoxidil was 169 nm with a PDI of 0.261, while the average
size of the liposomes containing Minoxidil and essenal oil was
183 nm with a PDI of 0.174. In cases of vesicles containing
solely essenal oil, the vesicle size was 187 nm with a PDI of
0.105. The parcle size of free-drug liposomes was 118 nm.
The increasing of parcle size of liposomes containing Terpene
was observed. These ndings are in agreement with previous
study [10]. The PDI of the invesgated formulaons was below
0.3, which indicates the homogeneity of the prepared
liposomes [19]. Regarding the zeta potenal measurements,
all liposomal dispersions had a negave surface charge
indicang that the formulaons were more stable and
homogeneous in distribuon. Moreover, liposomes containing
Terpene are more negave than convenonal liposomes.
These negave charge values of the obtained liposomes are
aributed to the presence of ethanol [20].
Table 2: Size and Zeta potenal of Formulaon (1): Liposome
without drugs; Formulaon (2): Minoxidil loaded liposome;
Formulaon (3): Rosemary essenal oil loaded liposome;
Formulaon (4): Minoxidil and Rosemary essenal oil loaded
liposome formulaon.
Particle size Zeta potential
Formulation 1 118 -18.8
Formulation 2 169 -34
Formulation 3 187 -37.5
Formulation 4 183 -37
Stability studies
The physical stability of the four liposome formulaons
which were stored at 4°C for 60 days presented in Figures 2
and 3. The stability results showed minimal changes of parcle
size and PDI of the invesgated liposomes. The parcle size
and the PDI of liposomal dispersions had slightly increased
aer 60 days of storage. These results showed that the co-
existence of the Minoxidil and Rosemary essenal oil in the
vesicular formulaons did not aect the vesicle’s stability
during me.
Figure 2: Size of liposome parcle in 60 days of storage for
Formulaon (1): Liposome without drugs; Formulaon (2):
Minoxidil loaded liposome; Formulaon (3): Rosemary
essenal oil loaded liposome; Formulaon (4): Minoxidil
and Rosemary essenal oil loaded liposome formulaon.
Figure 3: Zeta potenal of liposome parcle in 60 days of
storage for Formulaon (1): Liposome without drugs;
Formulaon (2): Minoxidil loaded liposome; Formulaon
(3): Rosemary essenal oil loaded liposome; Formulaon
(4): Minoxidil and Rosemary essenal oil loaded liposome
formulaon.
Conclusion
Minoxidil and Rosemary essenal oil successfully entrapped
in the liposome with appropriate size and entrapment ecacy
which possible its consumpon as the future hair growth
smulator formulaon. The stability of formulaon in terms of
size and zeta potenal remained appropriate during 60 days of
storage.
Journal of In Silico & In Vitro Pharmacology
Vol.2 No.3:10
2016
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