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Research paper
Department of Pharmaceutical Technology, Faculty of Pharmacy, Misr International University, Cairo, Egypt
Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt
a r t i c l e
i n f o
Article history:
Received 8 May 2013
Accepted in revised form 9 September 2013
Available online 18 September 2013
Keywords:
Liquid crystals
Drug release
Antioxidant
Alpha lipoic acid
Glyceryl monooleate
Cubosomes
Poloxamer gel
Cosmeceutical application
Clinical study
a b s t r a c t
Topical 5% alpha lipoic acid (ALA) has shown efcacy in treatment of photo-damaged skin. The aim of this
work was to evaluate the potential of poloxamer (P407) gel as a vehicle for the novel lipid base particulate system (cubosome dispersions) of ALA. Cubosome dispersions were formulated by two different
approaches, emulsication of glyceryl monoolein (GMO) and poloxamer (P407) in water followed by
ultrasonication, and the dilution method using a hydrotrope. Three different concentrations of GMO were
used to formulate the cubosome dispersions using the rst method, 5% (D1), 10% (D2) and 15% w/w (D3).
In the second technique an isotropic liquid was produced by combining GMO with ethanol, and this isotropic liquid was then diluted with a P407 solution (D4). The dispersions were characterized by zeta
potential, light scattering techniques, optical and transmission electron microscopy, encapsulation efciency and in vitro drug release. Results showed that D4 was not a uniform dispersion and that D1, D2
and D3 were uniform dispersions, in which by increasing the GMO content in the dispersion, the size
of the cubosomes decreased, zeta potential became more negative, encapsulation efciency increased
up to 86.48% and the drug release rate was slower. P407 gels were prepared using the cold method.
Two concentrations of P407 gel were fabricated, 20 and 30% w/w. P407 gels were loaded with either
ALA or dispersions containing ALA cubosomes. P407 gels were characterized by critical gelation temperature, rheological measurements and in vitro drug release studies. Results suggested that by increasing
P407 concentration, the gelation temperature decreases and viscosity increases. Drug release in both
cases was found to follow the Higuchi square root model. Gel loaded with ALA cubosomes provided a signicantly lower release rate than the gel loaded with the un-encapsulated ALA. A double blinded placebo
controlled clinical study was conducted, aiming to evaluate the efcacy as an anti-wrinkle agent and
volunteers satisfaction upon application of topical 30% P407 gel loaded with ALA cubosomes. Results
indicated reduction in facial lines, almost complete resolution of ne lines in the periorbital region
and upper lip area and overall improvement in skin color and texture in most volunteers. There were
no instances of irritation, peeling or other apparent adverse side effects.
2013 Elsevier B.V. All rights reserved.
1. Introduction
In the recent years, self-assembled lyotropic liquid crystalline
phases of lipids and water have gained increasing interest. That
is due to their potential in different application elds, as encapsulation and administration of drugs, and the formulation of novel
delivery systems [1].
Glyceryl monoolein (GMO) is a long chain unsaturated monoglyceride that is able to form lyotropic liquid crystalline cubic
phases in water [2]. This polar lipid is essentially insoluble in
water, but self-associates, and may depending on the water con-
tent form a reversed micellar (L2), a lamellar (La), or a bicontinuous cubic phase (C) as visualized in Fig. 1. The cubic phases are
often referred to as reversed or inverse cubic phases, indicating
the curvature of the consistent bilayer toward the polar medium
[3]. The interesting properties of the cubic phase formed by this
monoglyceride, temperature stability, high internal surface area
and the low-cost raw material, which makes them desirable to
be used as consumer products and in the pharmaceutical industry
applications [4]. Cubosomes are discrete, submicron, nanostructured particles of bicontinuous cubic liquid crystalline phase,
which are able to incorporate large amounts of drugs, and can be
localized in body cavities, on the skin or different mucosal surfaces
[5]. This structure offers two separate lipid and water domains.
This compartmentalization can be used to introduce guest molecules that are either hydrophilic, lipophilic or amphiphilic in
252
L2
D1
D2
D3
D4
Content (% w/w)
GMO
P407
Ethanol
Water
5.0
10.0
15.0
68.0
1.0
1.0
1.0
0.3
5.0
94.0
89.0
84.0
26.7
253
lter having a pore size of 0.1 lm. The ltrate was analyzed
spectrophotometrically at kmax 250 nm. Concentration obtained
was multiplied by the total volume of the dispersion produced,
considering the dilution factor. This represented the concentration
of free drug (Cf, namely that not entrapped in cubosomes). This
was then subtracted from the total drug concentration (Ct) in the
formulation to give the amount of drug that was successfully entrapped inside the cubosomes. Each experiment was repeated
three times.
Q Dm C d 2A C d t 1=2
254
Table 2
Classication of volunteers according to the Glogau scale.
All experiments were performed in replicate for validity of statistical analysis. Results were expressed as mean SD. One-way
analysis of variance (ANOVA) was used to assess statistical significance where required. Differences were considered signicant for
P-values < 0.05.
3. In vivo evaluation of skin rejuvenation effect in volunteers
Volunteer number
Age (yr)
Glogau scale
1
2
3
4
5
6
7
8
9a
10a
11a
12a
38
45
41
42
48
42
51
64
39
47
43
42
I
II
III
II
III
III
III
III
II
III
III
I
Placebo group.
Table 3
Patient satisfaction scoring criteria.
Score
Grade
Description
Dissatisfaction
1
2
Slightly satised
Moderately
satised
Satised
255
Formulation
0.0
D1
D2
D3
-2.0
-4.0
-6.0
-8.0
-10.0
-12.0
Table 4
Particle size distribution and encapsulation efciency (EE) of ALA cubosomes D1, D2
and D3 (n = 3).
Dispersion
EE (%) SD
D1
D2
D3
148 0.9
123 0.8
101 0.7
76.60 0.62
83.85 1.39
86.48 0.68
Fig. 4. Optical micrograph of ALA cubosome dispersions D1, D2 and D3. (For
interpretation of the references to color in this gure legend, the reader is referred
to the web version of this article.).
256
30
25
20
15
10
5
0
20
30
600
Fig. 6. Chart illustrating changes in the critical gelation temperature using the
Visual Tube Inversion Method as function of loading 20 and 30% w/w P407
solution with ALA or ALA cubosomes, in comparison with P407 placebo solution
(n = 3). (For interpretation of the references to color in this gure legend, the reader
is referred to the web version of this article.).
500
400
300
200
100
0
20
30
% ALA released
70
60
50
40
30
D1
20
D2
10
D3
0
0
Time (hrs)
Fig. 8. In vitro ALA release from dispersions of cubosomes with different concentrations of GMO, 5% w/w (D1), 10% w/w (D2) and 15% w/w (D3) using Paddle over
Disk Assembly method in (1:1) hydro-alcoholic solution at 32 0.5 C (n = 3).
257
Rate constant
Equation
D1
D2
D3
20.933
20.086
18.195
0.9902
0.9923
0.9941
y = 20.933t1/2 0.277
y = 20.086t1/2 0.2414
y = 18.195t1/2 0.0149
Table 6
Release rate constants and correlation coefcients of 20 and 30% w/w P407 gel loaded
with ALA calculated in accordance with the release proles obtained using square
root Higuchi model.
P407 gel loaded
with ALA
Rate constant
Equation
20% w/w
30% w/w
21.058
18.804
0.9964
0.9985
y = 21.058t1/2 5.2664
y = 18.804t1/2 4.9405
100
70
90
60
% ALA released
% ALA released
80
70
60
50
40
10
40
30
20
10
30
20
50
0
0
Time (h)
Time, hr
Fig. 9. Time dependent effect of P407 concentration on ALA release from P407 gel
loaded with 50 mg ALA, using Paddle over Disk Assembly method in (1:1) hydroalcoholic solution at 32 0.5 C (n = 3).
Fig. 10. Time dependent effect of P407 concentration on ALA release from P407 gel
loaded with ALA cubosomes using Paddle over Disk Assembly method in (1:1)
hydro-alcoholic solution at 32 0.5 C (n = 3).
Table 7
Release rate constants and correlation coefcients of 20 and 30% w/w P407 gel loaded
with ALA cubosomes calculated in accordance with the release proles obtained using
square root Higuchi model.
P407 gel loaded with
ALA cubosomes
Rate constant
Equation
20% w/w
30% w/w
14.401
12.809
0.9950
0.9924
y = 14.401t1/2 8.3812
y = 12.809t1/2 9.9124
the face were diminished (Fig. 12). A visible reduction in the depth
of ne periorbital lines and ne vertical lines on the upper lip was
also observed (Fig. 13), deeper periorbital lines appeared shallow
after completing the treatment regimen (Fig. 14). This assessment
was conrmed by comparison with photographs taken at the
beginning of the study. It is believed, however, that this effect
was due to increased collagen production by saturation of broblasts with ALA. Cellular membrane repair has been attributed to
antioxidant administration to tissues [27]. In addition to the above
ndings, the female volunteers also reported a noticeable difference in skin tone of the face, which was described as a healthy
glow (Fig. 14) [28]. Subjects reported no complaints regarding
irritation from daily use of the gel. Two of the subjects reported
improvement of facial scars, which was conrmed by comparison
with photographs taken at the beginning of the study.
Fig. 15 shows the visible change in the dermis density by following the 3 month treatment course which was conrmed by
ultrasound. The increase in dermal density implies an increase in
the amount of collagen. After the 3 month treatment course solar
scars were treated and are not apparent in the ultrasound image.
The activation of transcription factor AP-1, through production of
collagenases, could remodel the damaged collagen, resulting in
the clinical improvement of solar scars [28].
Fig. 15, visualizes the progressive replenishment of low
echogenic, dark areas seen over treatment course of 3 months.
258
80
3 months (Placebo)
70
% of Volunteers
60
50
40
30
20
10
0
No change
Somewhat
improved
Moderately
improved
Very much
improved
GAIS Category
Fig. 11. Categorical outcomes of the Global Improvement Scale (GAIS) score at 1.5
and 3 months post-treatment for both groups of volunteers (Treatment and
Placebo). (For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.).
Fig. 13. Photographic images depicting the facial area of a 48-years old volunteer
(A) before treatment and (B) 3 months after treatment with 30% P407 gel containing
ALA cubosomes D3. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.).
Fig. 12. Photographic images depicting the facial area of a 45-years old volunteer
(A) before treatment and (B) 3 months after treatment with 30% P407 gel containing
ALA cubosomes D3. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.).
Fig. 14. Photographic images depicting the facial area of a 51 years old volunteer
(A) before treatment and (B) 3 months after treatment with 30% P407 gel containing
ALA cubosomes D3. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.).
259
References
Fig. 15. High resolution ultrasonic images of pre- and post-treatment (A and B) for
two representative volunteers.