Preparation and characterization of liposomes encapsulating Calophyllum inophyllum oil

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Preparation and characterization of liposomes encapsulating Calophyllum inophyllum oil. Callophyllum inophyllum oil, also known as Tamanu oil, is reported to treat a wide range of skin problems such as acne, eczema, psoriasis, herpes, hemorrhoids, and injuries caused due to wounds, among others. Liposomes, which are effective carriers for topical treatment of dermal diseases, could enhance the therapeutic efficiency of Tamanu oil. Therefore, the purpose of this study was to formulate and characterize liposomes loading Tamanu oil. Liposomes encapsulating Tamanu oil with different ratios of Phospholipon 90G and L-α-lecithin were prepared using the thin-film hydration technique. Liposomal formulations were characterized in terms of aspect, particle size, size distribution, zeta potential, and morphology by using light microscope and dynamic light scattering analysis (DLS).
Life ScienceS | Pharmacology
Preparation and characterization
of liposomes encapsulating Calophyllum inophyllum oil
Huu Trong Phan, Van Thanh Tran*
Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City
Received 18 July 2017; accepted 14 November 2017
Abstract:
oil
can
lead
to
skin
irritations;
just
Callophyllum inophyllum oil, also known as Tamanu oil, is reported to treat
a wide range of skin problems such as acne, eczema, psoriasis, herpes,
hemorrhoids, and injuries caused due to wounds, among others. Liposomes,
which are effective carriers for topical treatment of dermal diseases, could
enhance the therapeutic efficiency of Tamanu oil. Therefore, the purpose of
this study was to formulate and characterize liposomes loading Tamanu oil.
Liposomes encapsulating Tamanu oil with different ratios of Phospholipon
90G and L-α-lecithin were prepared using the thin-film hydration technique.
Liposomal formulations were characterized in terms of aspect, particle size,
size distribution, zeta potential, and morphology by using light microscope and
dynamic light scattering analysis (DLS). Furthermore, the best formulation
was tested with the storage stability after 30 days, and Tamanu oil loaded
in the liposomes was identified as dictated in Vietnamese pharmacopoeia.
The data demonstrated that an average liposome diameter of 53 nm with a
narrow polydispersity (0.289) was obtained at a Phospholipon 90G to L-α-
lecithin molar ratio of 4:6, and a Tamanu oil to phospholipid mass ratio of
1:3 approximately. In addition, according to the DLS results, the particle size
and the zeta potential were quite stable at 2-8oC during 30 days of storage.
The study achieved the promising results for developing a novel formulation
containing Tamanu oil, which may be valuable to treatment of skin diseases.
like other oils, it can clog skin pores,
often resulting in acnes and other skin
infections [1, 2].
Tamanu oil came to be used in
cosmetics about 40 years ago, and it
was approved for clinical uses about 20
years ago [3]. Liposomes are used as a
drug delivery system offering several
benefits including biocompatibility,
adjustable membrane to control their
pharmacokinetic properties, increasing
efficacy and therapeutic index of active
agents [4, 5]. In terms of liposomal
composition, liposomes are nanometric
or sub-micrometric vesicles consisting
of an internal aqueous core and one or
more external phospholipid bilayer.
This makes it possible for liposomes to
load both hydrophobic and hydrophilic
molecules. Hydrophobic compounds
are inserted into a lipid layer, while
Keywords: liposome, Tamanu oil, thin-film hydration method.
Classification number: 3.3
hydrophilic compound can be entrapped
in an aqueous center. This contributes
to protecting these compounds from
degradation
and
any
other
adverse
environment
al
factors,
thereby
Introduction
Calophyllum inophyllum L.,
Guttiferae, locally called Tamanu, is a
tropical tree that is widely distributed
throughout Africa, Asia, and Pacific
countries. Calophyllum inophyllum
oil extracted from the Tamanu nuts is
composed of a mixture of lipids and other
components including xanthone, flavon,
and terpene derivatives. Traditionally,
Tamanu oil has topically been used for
skin care and to relieve skin problems
the 20th century, a number of researches
have reported the pharmacological
properties of Tamanu oil. Those include
anti-inflammatory, antimicrobial,
wound-healing, tissue-regenerative, and
skin-protective properties. However, the
direct application of pure Tamanu oil on
the skin presents some disadvantages:
The permeable efficiency of Tamanu
oil across the skin is low due to the
hydrophobicity of its lipid compositions;
Tamanu oil is slightly rubefacient, so,
improving their stability. Moreover,
owing to possessing a similar lipid
bilayer with that of the skin, liposomes
are easily attracted to dermal cells in
different ways, for instance, adsorption,
endocytosis, lipid exchange, or fusion.
It is for this reason that the use of
liposomes as a topical drug delivery
system in the treatment of skin diseases
facilitates the penetration of active
ingredients into the deeper layer of the
skin where their action should occur
[6, 7]. Finally, the entrapment of drugs
for centuries. Ever since the first half of
long-term dermal exposure to the pure
into liposomal vesicles overcomes some
*Corresponding author: Email: tranvanthanh@uphcm.edu.vn
56
Vietnam Journal of Science,
Technology and Engineering
December 2017 Vol.59 Number 4
Life ScienceS | Pharmacology
inconveniences that are present in free
Methods
Zetasizer.
drugs, such as irritation, unpleasant odor,
clogging pores. Due to these benefits,
liposomes are likely to be a promising
choice for loading Tamanu oil in order to
enhance clinical efficacy of the oil.
In addition, when it comes to
Preparation of liposomes loading
Tamanu oil:
Liposomes were prepared by the
thin-film hydration technique. In brief,
the lipid phase (consisting of accurately
weighed quantities of CIO, and a PL-LL
Light microscopy:
The liposomal vesicles were
monitored for their morphological
attributes with the help of a digital
optical microscope at 100X Objective
(Olympus, Moticam 1000, Japan).
physicochemical properties of liposomes
that influence skin permeation of active
ingredient entrapped in liposomes, a few
researches indicated that small-sized
liposomal vesicles appear to bring out a
higher degree of penetration, and some
surfactants act as a skin penetration
enhancer.
For these reasons, this study
was aimed at the preparation and
characterization of liposomes containing
Tamanu oil. In this way, liposomal
suspensions encapsulating Tamanu oil
with various organic solvents dissolving
the lipid phase, speed of rotor/stator
homogenization, lipid compositions,
and tween 80 concentration have been
studied based on their particle size,
size distribution, and stability in order
to optimize the liposomal formulation
loading Tamanu oil.
Then, the result would be useful
in the creation of effective liposomal
topical formulations loading Tamanu
oil for cosmetic and dermatological
applications.
Materials and methods
mixture in different molar ratios) was
dissolved in organic solvent (ethanol,
chloroform, or petroleum ether) in a
round bottom flask; the organic solvent
was then removed under reduced
pressure by using a rotary evaporator
(Buchi R-200/205) at 70°C, then a thin
lipid layer appeared in the flask. Thus,
the lipid film obtained was kept on
evaporating for three hours to eliminate
the trace of the organic solvent. Finally,
the hydration of the film with 50 ml of
distilled water was carried out on the
rotary evaporator under fast spin, at
50°C, for one hour to favor the vesicle
formation. Liposomes were stored at
temperatures between 2-8°C.
Homogenization of prepared
liposomes:
The prepared liposomal suspension
was homogenized by using Rotor/
Stator Homogenizer, heated at 50°C
(homogenization takes place at higher
temperatures than phase transition
temperatures of lipids) for 15 minutes.
The rotor speed was set up at 11,000
rpm, 15,000 rpm, and 19,000 rpm in
turns.
Measurement of liposome size and
Identification of Tamanu oil in
liposomes:
Thin-layer chromatography
identification test: 10 µl of standard
solution (0.1 g of CIO dissolved in 0.5
ml diethyl ether), test solution (0.1 g of
liposomes sediment dissolved in 0.5 ml
diethyl ether), and placebo solution (0.1
g of a mixture of PL, LL, and tween 80,
dissolved in 0.5 ml diethyl ether) were
applied on a parallel line of the thin-
layer chromatography plate coated with
silica gel-G. The plate was placed in a
pre-saturated chromatographic chamber
with a solvent system consisting of a
mixture of benzene and ethyl acetate
(8:2). The chromatogram was developed
with the developing solvent system until
the solvent front had moved about three-
fourths of the length of the plate. Thus,
the plate was removed from the chamber,
the solvent front was marked and dried
at a temperature from 100-105oC for 5
minutes. The location of the spots on
the plate was observed under UV light
(at the wavelengths of 254 nm and 365
nm). Later, the plate was sprayed with
a vanillin-sulfuric acid reagent, and
heated at 100-105oC for 5 minutes.
This step was carried out to detect the
Materials
The Calophyllum inophyllum oil
(CIO) was provided by the Traditional
Medicine Institute in Ho Chi Minh city
(Vietnam). Phospholipon 90G (PL) from
soybean and L-α-Lecithin (LL) from egg
yolk were purchased from Lipoid® and
Calbiochem®, respectively. The solvents
zeta potential:
The mean particle size, size
distribution, and zeta potential analysis
of the liposomes were determined at
25°C by Dynamic Light Scattering
using a Malvern Zetasizer (Malvern
Instrument Limited, Malvern, UK).
Experiments were run in triplicate.
presence of other compositions that
weren’t visible under UV light. The
test solution chromatogram should be
mainly similar to that of the standard
solution and different from that of the
placebo solution.
Results and discussions
Homogenization of liposomes
for phospholipids including chloroform,
Storage stability studies:
loading Tamanu oil
ethanol 90 percent, and petroleum ether
(30-60) were purchased from Sigma
Aldrich (Germany). Tween 80 which
was used to enhance the solubility of
The liposomal suspension was stored
in darkness at 8°C and 25°C for 30 days.
The storage stability of the liposomes
was based on the change of both
The multi-lamellar vesicle (MLV)
liposomes prepared by thin-layer
hydration method are fairly large (several
micrometers) and heterogeneous. In this
phospholipids in ethanol was purchased
their
particle
size
and
polydispersity
way, it is compulsory to reduce and
from Sigma Aldrich (Germany).
index (PDI) measured by the Malvern
homogenize the liposomal particle size.
December 2017 Vol.59 Number 4
Vietnam Journal of Science,
Technology and Engineering
57
Life ScienceS | Pharmacology
The particle size and size distribution
of the liposomal vesicles obtained after
homogenization using rotor/stator
Table 1. Particle size and polydispersity index of formulations homogenized
at different speeds.
homogenizer at different rotor speeds
Formulation
Particle size (d.nm)
Polydispersity index
are presented in Table 1.
The
particle
size
of
the
non-
H0 (non-homogenized)
410.4
0.568
homogenized liposomes was about four
times larger than that of the homogenized
H11 (11,000 rpm)
119.26
0.292
liposomes. Furthermore, the rotor/stator
homogenization decreased the size
H15 (15,000 rpm)
106.26
0.298
distribution
of
the
liposomal
vesicle,
H19 (19,000 rpm)
106.06
0.279
which
related
to
the
stability
of
the
liposomes. These results showed that
the homogenization played an important
role in the preparation of liposomes.
Table 2. Particle size and polydispersity index of formulations prepared by
using three different organic solvents dissolving the lipid phase.
The
particle
size
and
size
Formulation
Particle size (d.nm)
Polydispersity index
distributions of liposomes obtained
through homogenization at 15,000 rpm
A1 (ethanol)
110.70
0.309
and at 19,000 rpm are mainly similar.
A2 (chloroform)
114.04
0.270
Moreover, the homogenization at high
speed may result in degradation of
A3 (ethanol:petroleum ether (8:2))
114.98
0.286
Tamanu
oil
and
also
lipid
materials.
Therefore,
the
speed
of
15,000
rpm
product can cause adverse side effects
for 30 minutes). It was seen that the
was chosen to homogenize liposomal
in cases of long-term application. As a
change of the lipid molar ratio could
vesicles.
result, ethanol was used as an organic
result in the change in the stability of
Organic solvent used to dissolve the
lipid phase
Solubility study proved that the lipid
phase consisting of CIO, PL, and LL
was dissolved well in chloroform or in
a mixture of ethanol and petroleum ether
(8:2). Yet it formed a suspension after
being dispersed in ethanol.
solvent for dissolving the lipid phase.
Encapsulation of Tamanu oil
Almost all the compositions in CIO
are lipophilic. So, the CIO was added to
the lipid phase (passive encapsulation
method) and would be integrated into
the phospholipid bilayer during the
formation of the thin-layer film. The
liposomal vesicles. To be more precise,
the formulations containing a mixture of
PL and LL with molar ratios from 8:2 to
4:6 presented a higher stability than the
others. Then, the particle size and the size
distribution of these formulations were
measured in order to find out the best
formulation. The results are displayed in
Table 3.
Hypothesis: The majority of CIO
compositions are lipophilic, which could
dissolve the particles of phospholipid
during the preparation of the thin-layer
film. This might lead to a homogeneous
layer film.
amount of CIO into the lipid phase was
established by evaluating the stability
(storage at a temperature between 2-8oC
for 30 days) of liposomal suspensions
with a varying amount of CIO.
At concentrations of up to 24.1%,
The potential zeta is a good tool to
investigate the stability of liposomal
products. In this study, the incorporation
of two kinds of phospholipids contributes
to the augmentation of potential zeta
(from -20 mV to -42 mV). This suggests
Therefore, ethanol, chloroform, and
petroleum ether were chosen to dissolve
the lipid phase. The particle size and size
distribution of the liposomal vesicles
were prepared by using these three
different solvents as shown in Table 2.
With regard to the particle size, there
is no significant change between the
formulations. Despite the increase in
the polydispersity index, this value is a
the liposomal suspensions obtained
were stable during the storage phase (no
surface phenomena were observed).
Optimization of lipid composition
The composition of the bilayer, in
particular phospholipids, influences
the fluidity as well as the stability of
liposomes considerably. First of all, the
stability of liposomal suspensions with
that the incorporation of PL and LL
improved liposome stability. The
formulation with a mixture of PL and LL
at the ratio of 6:4 gave the smallest mean
particle size. Therefore, this ratio was
chosen for the liposomal formulation
loading CIO.
Influence of tween 80 on physical
properties of liposomes
little small and is acceptable. Besides,
different molar ratios was investigated
The application of liposomes as a
the trace of the organic solvents such as
on the basis of the centrifuge stability
tropical and transdermal drug delivery
chloroform, petroleum ether in a topical
testing
(centrifugation
at
17,000
rpm
system
necessitates
some
specific
58
Vietnam Journal of Science,
Technology and Engineering
December 2017 Vol.59 Number 4
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Preparation and characterization of liposomes encapsulating Calophyllum inophyllum oil. Callophyllum inophyllum oil, also known as Tamanu oil, is reported to treat a wide range of skin problems such as acne, eczema, psoriasis, herpes, hemorrhoids, and injuries caused due to wounds, among others. Liposomes, which are effective carriers for topical treatment of dermal diseases, could enhance the therapeutic efficiency of Tamanu oil. Therefore, the purpose of this study was to formulate and characterize liposomes loading Tamanu oil. Liposomes encapsulating Tamanu oil with different ratios of Phospholipon 90G and L-α-lecithin were prepared using the thin-film hydration technique. Liposomal formulations were characterized in terms of aspect, particle size, size distribution, zeta potential, and morphology by using light microscope and dynamic light scattering analysis (DLS)..

Nội dung

Life ScienceS | Pharmacology Preparation and characterization of liposomes encapsulating Calophyllum inophyllum oil Huu Trong Phan, Van Thanh Tran* Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City Received 18 July 2017; accepted 14 November 2017 Abstract: Callophyllum inophyllum oil, also known as Tamanu oil, is reported to treat a wide range of skin problems such as acne, eczema, psoriasis, herpes, hemorrhoids, and injuries caused due to wounds, among others. Liposomes, which are effective carriers for topical treatment of dermal diseases, could enhance the therapeutic efficiency of Tamanu oil. Therefore, the purpose of this study was to formulate and characterize liposomes loading Tamanu oil. Liposomes encapsulating Tamanu oil with different ratios of Phospholipon 90G and L-α-lecithin were prepared using the thin-film hydration technique. Liposomal formulations were characterized in terms of aspect, particle size, size distribution, zeta potential, and morphology by using light microscope and dynamic light scattering analysis (DLS). Furthermore, the best formulation was tested with the storage stability after 30 days, and Tamanu oil loaded in the liposomes was identified as dictated in Vietnamese pharmacopoeia. The data demonstrated that an average liposome diameter of 53 nm with a narrow polydispersity (0.289) was obtained at a Phospholipon 90G to L-α-lecithin molar ratio of 4:6, and a Tamanu oil to phospholipid mass ratio of 1:3 approximately. In addition, according to the DLS results, the particle size and the zeta potential were quite stable at 2-8oC during 30 days of storage. The study achieved the promising results for developing a novel formulation containing Tamanu oil, which may be valuable to treatment of skin diseases. Keywords: liposome, Tamanu oil, thin-film hydration method. Classification number: 3.3 Introduction the 20th century, a number of researches have reported the pharmacological Guttiferae, locally called Tamanu, is a properties of Tamanu oil. Those include tropical tree that is widely distributed wound-healing, tissue-regenerative, and skin-protective properties. However, the oil extracted from the Tamanu nuts is direct application of pure Tamanu oil on composed of a mixture of lipids and other the skin presents some disadvantages: components including xanthone, flavon, The permeable efficiency of Tamanu and terpene derivatives. Traditionally, oil across the skin is low due to the Tamanu oil has topically been used for hydrophobicity of its lipid compositions; skin care and to relieve skin problems Tamanu oil is slightly rubefacient, so, for centuries. Ever since the first half of long-term dermal exposure to the pure oil can lead to skin irritations; just like other oils, it can clog skin pores, often resulting in acnes and other skin infections [1, 2]. Tamanu oil came to be used in cosmetics about 40 years ago, and it was approved for clinical uses about 20 years ago [3]. Liposomes are used as a drug delivery system offering several benefits including biocompatibility, adjustable membrane to control their pharmacokinetic properties, increasing efficacy and therapeutic index of active agents [4, 5]. In terms of liposomal composition, liposomes are nanometric or sub-micrometric vesicles consisting of an internal aqueous core and one or more external phospholipid bilayer. This makes it possible for liposomes to load both hydrophobic and hydrophilic molecules. Hydrophobic compounds are inserted into a lipid layer, while hydrophilic compound can be entrapped in an aqueous center. This contributes to protecting these compounds from degradation and any other adverse environment al factors, thereby improving their stability. Moreover, owing to possessing a similar lipid bilayer with that of the skin, liposomes are easily attracted to dermal cells in different ways, for instance, adsorption, endocytosis, lipid exchange, or fusion. It is for this reason that the use of liposomes as a topical drug delivery system in the treatment of skin diseases facilitates the penetration of active ingredients into the deeper layer of the skin where their action should occur [6, 7]. Finally, the entrapment of drugs into liposomal vesicles overcomes some *Corresponding author: Email: tranvanthanh@uphcm.edu.vn 56 Vietnam Journal of Science, Technology and Engineering December 2017 • Vol.59 Number 4 Life ScienceS | Pharmacology inconveniences that are present in free drugs, such as irritation, unpleasant odor, clogging pores. Due to these benefits, liposomes are likely to be a promising choice for loading Tamanu oil in order to enhance clinical efficacy of the oil. In addition, when it comes to physicochemical properties of liposomes that influence skin permeation of active ingredient entrapped in liposomes, a few researches indicated that small-sized liposomal vesicles appear to bring out a higher degree of penetration, and some surfactants act as a skin penetration enhancer. For these reasons, this study was aimed at the preparation and characterization of liposomes containing Tamanu oil. In this way, liposomal suspensions encapsulating Tamanu oil with various organic solvents dissolving the lipid phase, speed of rotor/stator homogenization, lipid compositions, and tween 80 concentration have been studied based on their particle size, size distribution, and stability in order to optimize the liposomal formulation loading Tamanu oil. Then, the result would be useful in the creation of effective liposomal topical formulations loading Tamanu oil for cosmetic and dermatological applications. Materials and methods Materials The Calophyllum inophyllum oil (CIO) was provided by the Traditional Medicine Institute in Ho Chi Minh city (Vietnam). Phospholipon 90G (PL) from soybean and L-α-Lecithin (LL) from egg yolk were purchased from Lipoid® and Calbiochem®, respectively. The solvents for phospholipids including chloroform, ethanol 90 percent, and petroleum ether (30-60) were purchased from Sigma Aldrich (Germany). Tween 80 which was used to enhance the solubility of phospholipids in ethanol was purchased from Sigma Aldrich (Germany). Methods Preparation of liposomes loading Tamanu oil: Liposomes were prepared by the thin-film hydration technique. In brief, the lipid phase (consisting of accurately weighed quantities of CIO, and a PL-LL mixture in different molar ratios) was dissolved in organic solvent (ethanol, chloroform, or petroleum ether) in a round bottom flask; the organic solvent was then removed under reduced pressure by using a rotary evaporator (Buchi R-200/205) at 70°C, then a thin lipid layer appeared in the flask. Thus, the lipid film obtained was kept on evaporating for three hours to eliminate the trace of the organic solvent. Finally, the hydration of the film with 50 ml of distilled water was carried out on the rotary evaporator under fast spin, at 50°C, for one hour to favor the vesicle formation. Liposomes were stored at temperatures between 2-8°C. Homogenization of prepared liposomes: The prepared liposomal suspension was homogenized by using Rotor/ Stator Homogenizer, heated at 50°C (homogenization takes place at higher temperatures than phase transition temperatures of lipids) for 15 minutes. The rotor speed was set up at 11,000 rpm, 15,000 rpm, and 19,000 rpm in turns. Measurement of liposome size and zeta potential: The mean particle size, size distribution, and zeta potential analysis of the liposomes were determined at 25°C by Dynamic Light Scattering using a Malvern Zetasizer (Malvern Instrument Limited, Malvern, UK). Experiments were run in triplicate. Storage stability studies: The liposomal suspension was stored in darkness at 8°C and 25°C for 30 days. The storage stability of the liposomes was based on the change of both their particle size and polydispersity index (PDI) measured by the Malvern Zetasizer. Light microscopy: The liposomal vesicles were monitored for their morphological attributes with the help of a digital optical microscope at 100X Objective (Olympus, Moticam 1000, Japan). Identification of Tamanu oil in liposomes: Thin-layer chromatography identification test: 10 µl of standard solution (0.1 g of CIO dissolved in 0.5 ml diethyl ether), test solution (0.1 g of liposomes sediment dissolved in 0.5 ml diethyl ether), and placebo solution (0.1 g of a mixture of PL, LL, and tween 80, dissolved in 0.5 ml diethyl ether) were applied on a parallel line of the thin-layer chromatography plate coated with silica gel-G. The plate was placed in a pre-saturated chromatographic chamber with a solvent system consisting of a mixture of benzene and ethyl acetate (8:2). The chromatogram was developed with the developing solvent system until the solvent front had moved about three-fourths of the length of the plate. Thus, the plate was removed from the chamber, the solvent front was marked and dried at a temperature from 100-105oC for 5 minutes. The location of the spots on the plate was observed under UV light (at the wavelengths of 254 nm and 365 nm). Later, the plate was sprayed with a vanillin-sulfuric acid reagent, and heated at 100-105oC for 5 minutes. This step was carried out to detect the presence of other compositions that weren’t visible under UV light. The test solution chromatogram should be mainly similar to that of the standard solution and different from that of the placebo solution. Results and discussions Homogenization of liposomes loading Tamanu oil The multi-lamellar vesicle (MLV) liposomes prepared by thin-layer hydration method are fairly large (several micrometers) and heterogeneous. In this way, it is compulsory to reduce and homogenize the liposomal particle size. December 2017 • Vol.59 Number 4 Vietnam Journal of Science, Technology and Engineering 57 Life ScienceS | Pharmacology The particle size and size distribution of the liposomal vesicles obtained after homogenization using rotor/stator homogenizer at different rotor speeds are presented in Table 1. The particle size of the non-homogenized liposomes was about four times larger than that of the homogenized liposomes. Furthermore, the rotor/stator homogenization decreased the size distribution of the liposomal vesicle, which related to the stability of the liposomes. These results showed that the homogenization played an important role in the preparation of liposomes. The particle size and size distributions of liposomes obtained through homogenization at 15,000 rpm and at 19,000 rpm are mainly similar. Moreover, the homogenization at high speed may result in degradation of Tamanu oil and also lipid materials. Therefore, the speed of 15,000 rpm was chosen to homogenize liposomal vesicles. Table 1. Particle size and polydispersity index of formulations homogenized at different speeds. Formulation Particle size (d.nm) Polydispersity index H0 (non-homogenized) 410.4 0.568 H11 (11,000 rpm) 119.26 0.292 H15 (15,000 rpm) 106.26 0.298 H19 (19,000 rpm) 106.06 0.279 Table 2. Particle size and polydispersity index of formulations prepared by using three different organic solvents dissolving the lipid phase. Formulation Particle size (d.nm) Polydispersity index A1 (ethanol) 110.70 0.309 A2 (chloroform) 114.04 0.270 A3 (ethanol:petroleum ether (8:2)) 114.98 0.286 product can cause adverse side effects for 30 minutes). It was seen that the in cases of long-term application. As a change of the lipid molar ratio could result, ethanol was used as an organic result in the change in the stability of Organic solvent used to dissolve the lipid phase Solubility study proved that the lipid phase consisting of CIO, PL, and LL was dissolved well in chloroform or in a mixture of ethanol and petroleum ether (8:2). Yet it formed a suspension after being dispersed in ethanol. Hypothesis: The majority of CIO compositions are lipophilic, which could dissolve the particles of phospholipid during the preparation of the thin-layer film. This might lead to a homogeneous layer film. Therefore, ethanol, chloroform, and petroleum ether were chosen to dissolve the lipid phase. The particle size and size distribution of the liposomal vesicles were prepared by using these three different solvents as shown in Table 2. With regard to the particle size, there is no significant change between the formulations. Despite the increase in the polydispersity index, this value is a little small and is acceptable. Besides, the trace of the organic solvents such as chloroform, petroleum ether in a topical solvent for dissolving the lipid phase. Encapsulation of Tamanu oil Almost all the compositions in CIO are lipophilic. So, the CIO was added to the lipid phase (passive encapsulation method) and would be integrated into the phospholipid bilayer during the formation of the thin-layer film. The amount of CIO into the lipid phase was established by evaluating the stability (storage at a temperature between 2-8oC for 30 days) of liposomal suspensions with a varying amount of CIO. At concentrations of up to 24.1%, the liposomal suspensions obtained were stable during the storage phase (no surface phenomena were observed). Optimization of lipid composition The composition of the bilayer, in particular phospholipids, influences the fluidity as well as the stability of liposomes considerably. First of all, the stability of liposomal suspensions with different molar ratios was investigated on the basis of the centrifuge stability testing (centrifugation at 17,000 rpm liposomal vesicles. To be more precise, the formulations containing a mixture of PL and LL with molar ratios from 8:2 to 4:6 presented a higher stability than the others. Then, the particle size and the size distribution of these formulations were measured in order to find out the best formulation. The results are displayed in Table 3. The potential zeta is a good tool to investigate the stability of liposomal products. In this study, the incorporation of two kinds of phospholipids contributes to the augmentation of potential zeta (from -20 mV to -42 mV). This suggests that the incorporation of PL and LL improved liposome stability. The formulation with a mixture of PL and LL at the ratio of 6:4 gave the smallest mean particle size. Therefore, this ratio was chosen for the liposomal formulation loading CIO. Influence of tween 80 on physical properties of liposomes The application of liposomes as a tropical and transdermal drug delivery system necessitates some specific 58 Vietnam Journal of Science, Technology and Engineering December 2017 • Vol.59 Number 4 Life ScienceS | Pharmacology Table 3. Particle size distribution and potential zeta of different formulations with various molar ratios of PL and LL. Formulation Particle size (d.nm) Polydispersity index Potential zeta (mV) properties such as elasticity of liposomes. It was reported that the surfactant acted as an “edge activator” which enhanced the flexibility of liposomes. This helps B0 (10 PL: 0 LL) 143.08 0.333 -20.3 B2 (8 PL: 2 LL) 118.18 0.302 -42.0 B3 (7 PL: 3 LL) 114.06 0.309 -48.7 B4 (6 PL: 4 LL) 107.88 0.301 -53.7 B5 (5 PL: 5 LL) 110.70 0.309 -56.7 B6 (4 PL: 6 LL) 110.22 0.298 -56.8 the encapsulated agent to penetrate to the deeper layer of the skin. In the present paper, the effect of tween 80 is evaluated as well as that of its concentration on the particle size, and the size distribution of liposomal vesicles is also evaluated, which is summarized in Table 4.The film of the tween 80-free formulation (C0) did not completely detach during the hydration because of the hydrophobicity Table 4. Particle size and polydispersity index of formulations with and without different concentrations of tween 80. of the film compositions. The use of the surfactant allowed the film to become more fluid and to form vesicles easily. At concentrations up to 10% (w/w), Formulation C0 (0% w/w) C1 (5% w/w) C2 (10% w/w) Particle size (d.nm) - 109.8 102.74 Polydispersity index - 0.301 0.276 Potential Zeta (mV) - -53.7 -54.5 tween 80 was not only beneficial to the hydration, but it also contributed to the reduction in the particle size and the size distribution of liposomal vesicles. However, at the range from 15-20% (w/w) of tween 80 in the liposomal formulation, liposomal particles became C3 (15% w/w) 97.38 0.418 -53.9 more heterogeneous. Therefore, the content of 10% (w/w) of tween 80 was C4 (20% w/w) 106.32 0.455 -54.2 chosen to be added to the formulation for preparation of liposomes encapsulating the CIO. Characterization of the final liposomal formulation Physical appearance of liposomal suspension: The liposome suspension is homogeneous, and light green in color. The morphological attributes of liposomes before homogenization, as observed through an optical microscope, is shown in Fig. 1. Although the liposomes viewed by using the optical microscope were giant liposomes, the optical microscope image provided the morphology of liposomes loading CIO. The image showed that Tamanu oil (green color) was encapsulated into the liposomal bilayer. Fig. 1. Optical microscope image at 100X objective of the liposomal formulation. Storage stability: The stability results which displayed minimal changes of particle size and size distribution of the final liposomal formulation are summarised in Table 5. December 2017 • Vol.59 Number 4 Vietnam Journal of Science, Technology and Engineering 59 Life ScienceS | Pharmacology Table 5. Particle size and polydispersity index of the final liposomal formulation before and after storage for 30 days at the different temperatures. inophyllum oil was optimized with the lipid phase comprising 65.9% (w/w) of the Phospholipon 90G: L-α-lecithin Formulation S0 (day 0) S01 (30 days stored at 2-8oC) S02 (30 days stored at 25-30oC) Particle size (d.nm) 106.26 100.9 108.2 Polydispersity index 0.289 0.300 0.301 Potential zeta (mV) -49.8 -43.0 -51.4 combination in the 6:4 molar ratio, 10% (w/w) of tween 80, 24.1% (w/w) of CIO. The average size of the prepared liposomes was small (mean diameter of 102.74 d.nm) and homogeneous (PDI of 0.276). The high negative charge of liposomal vesicles (-54.5 mV), and the minimal modification of particle size as well as the polydispersity index of liposomal vesicles after the storage stability studies indicated a good stability of suspension of liposomes uV at 254nm uV at 365nm Vanilin-Sulfuric Fig. 2. Chromatograms of the standard solution (S), the test solution (T), and the placebo solution (P), stained under a UV light at 254 nm, 365 nm, and with Vanilin-Sulfuric respectively. encapsulating Tamanu oil. Interestingly, ethanol was used as a solvent dissolving the lipid phase in order to avoid the toxicity of the trace of organic solvent. REFERENCES [1] D.D. Verma, S. Verma, G. blume, A. Fahr (2003), “Particle size of liposomes influences dermal delivery of substances into skine”, Int. J. Pharm., 258(1-2), pp.141-151. [2] J.l. Ansel, e. lupo, l. mijouin, et al. (2016), “biological activity of polynesian Calophyllum inophyllum oil extract on human skin cells”, Planta. Med., 82(11-12), pp.961-966. [3] Y. rahimpour, H. Hamishehkar (2012), “liposomes in cosmeceutics”, Expert Opin. Drug Deliv., 9(4), pp.443-455. [4] A. Ahad, A.A. Al-Saleh, A.m. Al-mohizea, et al. (2017), “Formulation and characterization of Phospholipon 90 G and tween 80 based transfersomes for transdermal delivery of eprosartan mesylate”, Pharm. Dev. Technol., pp.1-7. The particle size and the size distribution of liposomal suspension had slightly modified after 30 days of storage. The liposomal vesicles entrapping Tamanu oil were fairly stable during the storage even at temperatures between 25-30oC. Identification of Tamanu oil into liposomes: The chromatograms (Fig. 2) showed that each separated spot obtained from the test solution corresponds to that of the standard solution. As a result, all components of CIO were encapsulated into the liposomal vesicles. Concluding remarks In the present study, liposomal formulation trapping Calophyllum [5] A.c. Dweck, T. meadows (2002), “Tamanu (Calophyllum inophyllum) - the African, Asian, Polynesian and Pacific Panacea”, Int. J. Cosmet. Sci., 24(6), pp.341-348. [6] K. egbaria, N. Weiner (1990), “liposomes as topical drug delivery system”, Adv. Drug Deliv. Rev., 5(3), pp.287-300. [7] r. banerijee (2001), “liposomes: Applications in medicine”, J. Biomater Appl., 16(1), pp.3-21.

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