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European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics


journal homepage: www.elsevier.com/locate/ejpb

Research paper

The clinical efcacy of cosmeceutical application of liquid crystalline


nanostructured dispersions of alpha lipoic acid as anti-wrinkle
Saly Sherif a, Ehab R. Bendas b,, Sabry Badawy a
a
b

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-

Corresponding author. Faculty of Pharmacy, Cairo University, Kasr El-Ainy


Street 11562, Egypt. Tel.: +20 225311260; fax: +20 223628426.
E-mail address: ihabrasmy@gmail.com (E.R. Bendas).
0939-6411/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejpb.2013.09.008

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

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S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

nature [6]. GMO, as the major structure lipid of cubosomes, has


been reported to be an effective transdermal enhancer [7], which
makes GMO dispersions more desirable to be used in the cosmeceutical eld. This effect might be due to the structural organization of cubosomes, which is similar to that found of
biomembranes [8,9].
Poloxamer 407 (P407) is a triblock copolymer with a central
hydrophobic chain of polyoxypropylene (PPO) and two identical
lateral hydrophilic chains of polyoxyethylene (PEO) [10]. The widespread application of P407 gel in topical delivery systems is due to
the reversible solgel property that allows cool solution to ow
onto the skin and permit good contact with skin on formation of
a non-occlusive gel at body temperature. The gel could also be easily removed by washing with cold water [11]. Increasing the P407
concentration increases the viscosity of the gel, which can change
the releasing process of additives (as ALA or ALA cubosomes) from
the gel. The presence of drugs can also change some rheological
characters of these gels [10].
ALA is a natural occurring fatty acid with potent antioxidant
activity which exists in the mitochondria of all kinds of prokaryotic
and eukaryotic cells [12,13]. Structure of ALA is illustrated in Fig. 2.
ALA is known as a network antioxidant due to its ability to regenerate/recycle itself, as well as other antioxidants such as vitamins C
and E, so that they can continue destroying free radicals [12]. Since
available data of formulations containing 5% ALA were successful
to produce a dramatic reduction in facial lines in cases associated
with photo-aging, this compound has gained the attention of cosmetologists and dermatologists.
The objective of the present work was to explore the potential
of P407 solution to be used as a vehicle for the novel lipid base
particulate system (cubosome dispersions), to prepare a cosmetically acceptable preparation that could stabilize and sustain the
delivery of ALA when used as anti-wrinkle agent.
2. Materials and methods
2.1. Materials
Myverol 1899 K (Myverol) was used as the source of GMO
and was kindly supplied as a gift from Kerry Bioscience (Norwich,
NY, USA). Poloxamer 407 (P407), absolute ethanol and palladium
(II) chloride were purchased from SigmaAldrich (St. Louis, MO,
USA). Alpha lipoic acid (ALA) was kindly supplied by EVA pharma
(Cairo, Egypt). Milli-Q puried water was used for all experiments.
Other reagents were of analytical grade.
2.2. Preparation of cubosome dispersions
Cubosome dispersions were fabricated using two different
methods. The rst conventional method involved emulsication

L2

Fig. 2. Structure of ALA.

of GMO/P407 mixtures in water followed by ultrasonication. Three


formulations (D1, D2 and D3) were prepared by weighing appropriate amounts of GMO and P407. The mixture was gently melted
in a water bath (Clifton, England) at 70 C. This was injected into
preheated water at 70 C and maintained under mechanical
stirring at 1500 rpm. Dispersions were cooled to room temperature
then ultrasonicated at maximum power of 130 kW (Elma, Transsonic, Germany) for 1 min according to the method published by
Esposito et al. [14]. Fifty milligrams of ALA was added to water
prior to the addition of GMO and P407. The second method
adopted was dilution of an isotropic liquid by a hydrotrope. An
isotropic liquid was prepared by combining GMO with ethanol.
This isotropic liquid was diluted with P407 solution as described
by Spicer and Hayden [15]. Fifty milligrams of ALA was added to
ethanol prior to the dilution (D4). The detailed compositions of
the dispersions are listed in Table 1.
2.3. Preparation of P407 gels
P407 gels were prepared using the cold method [16]. Concentrations of P407 and ALA were expressed by weight percent (%
w/w). Appropriate amounts of poloxamer 407 were slowly added
to cold distilled water at 5 C to yield 20% and 30% gels and constant stirring was maintained [17]. The poloxamer solution was
kept refrigerated until a clear solution was obtained (612 h). For
the gel to be formed, the solution should be kept at 30 C. The
appropriate amounts of ALA or ALA cubosome dispersions were
then added to the gels to yield 5%.
2.4. Assay of alpha lipoic acid
Stock solution of ALA having a concentration of 1  103 M was
freshly prepared in a 1:1 mixture of ethanol and water. To ensure
complete drug dissolution, the solution was sonicated at 60 kW for
2 min. Palladium (II) chloride standard solution (1  102 M) was
prepared by dissolving palladium (II) chloride in water (to which
0.1 ml of concentrated hydrochloric acid has been added). The mixture was then warmed in a water bath to ensure complete dissolution. The ionic strength (l) of the nal solution for determination
was kept constant at 0.2 M, by the addition of a 2 M potassium
chloride solution. Britton Robinson buffer was used to adjust the

Increasing water content


Fig. 1. Transformation of mesophases from L2 (reversed micellar) to La (lamellar) to C (bicontinuous cubic phase), by increasing the water content.

S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259


Table 1
Composition of dispersions of ALA cubosomes D1, D2, D3 and D4.
Dispersion

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

pH of the nal solution for determination at 2.2. This was prepared


by using a ratio of 1:1:1 of 0.04 M boric acid, 0.04 M phosphoric
acid and 0.04 M acetic acid, and the pH was adjusted by using
0.2 M sodium hydroxide solution. Serial dilutions were prepared
by adding known aliquots from the stock solution, followed by
5 ml of Britton Robinson buffer, then 1 ml of potassium chloride
solution and nally 0.2 ml of palladium (II) chloride solution. The
volume was then diluted to 10 ml by addition of distilled water.
The mixture was mixed well then left to stand for at least
10 min. The absorbance was then measured spectrophotometrically at the predetermined kmax 250 nm against a reagent blank.
All measurements were done at a temperature of 25 1 C. Base
line correction was carried out to delete any absorbance reading
of the blank. The assay method was validated.
2.5. Characterization of cubosomes
2.5.1. Particle size and zeta potential measurements
The particle size and zeta potential of cubosomes were determined by dynamic light scattering (DLS) (Zetasizer, Malvern
Instruments, UK). Each sample was diluted with deionized water,
adjusted to a suitable light scattering intensity (300 Hz) and measured at 25 C in triplicate. Following the particle size analysis of
the cubosomes, the mode was switched from Size to Zeta,
and three measurements of zeta potential were recorded.
2.5.2. Light microscopy
Samples of the prepared cubosome dispersions were suitably
diluted with deionized water and examined under a Leica microscope (Leica image analyzer, Model Q 550IW equipped with Leica
DMLB microscope Cambridge, England. Connected to Camera,
Model TK-C1380 JVC, Victor Company, Japan) calibrated with a
micrometer slide using polarized light or differential interference
contrast at magnications between 10 and 100.
2.5.3. Transmission electron microscopy (TEM)
The samples were prepared by placing 5 ll droplet of the
cubosome dispersion onto a 300 mesh carbon-coated copper grid,
and letting the cubosomes settle for 35 min. Excess uid was
removed by wicking it off with an absorbent paper. The samples
were then viewed on a JEOL Model (JEM 1400, USA) 120KV transmission electron microscope.
2.5.4. Entrapment efciency
A sample of each dispersions containing 50 mg of ALA was prepared as explained previously. In order to determine the amount of
drug that was successfully entrapped inside the cubosomes (EE%),
it was rst mandated to separate the cubosomes from the resulting
dispersion. Then the amount of free drug in the dispersion was
then analyzed spectrophotometrically at kmax 250 nm, which was
then subtracted from the total amount of drug initially added.
A volume of 1 ml from each of the dispersions was diluted with
4 ml of deionized water. A volume of 1 ml from this diluted dispersion was further diluted with another 4 ml of deionized water. The
resulting diluted dispersion was then passed through a syringe

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.

EE %of cubosomes Ct  Cf=Ct  100

2.5.5. In vitro drug release from cubosomes


A modied stainless steel disk assembly (USP Apparatus 5, paddle over disk assembly), was used for the assessment of the release
of the drug from the dispersions. A sample containing 50 mg of ALA
was placed in a disk and covered by a membrane then placed at the
bottom of the dissolution vessel. The membrane was used to retain
the formula inside the disk. The dissolution medium used was
700 ml of hydro-alcoholic solution (1:1) to ensure sink condition.
The apparatus was equilibrated to 32 0.5 C and the stirrer paddle
speed was set at 50 rpm. Aliquots were withdrawn at appropriate
time intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and ltered through a syringe lter having a pore size of 0.1 lm then analyzed spectrophotometrically at wavelength of 250 nm (according to the method of
drug assay). The amount of drug released was calculated from the
standard curve. This procedure was performed in triplicates for
each formulation. Cumulative % drug released were calculated
out and plotted against time. ALA release from cubosomes in gels
has been shown to be primarily controlled by diffusion through
the matrix [18] and consequently can be described by the Higuchi
diffusion equation given by:

Q Dm C d 2A  C d t 1=2

where Q is the mass of ALA released at time t, and is proportional to


the apparent diffusion coefcient of the drug in the matrix, Dm, the
initial amount of ALA in the matrix, A and the solubility of the drug
in the matrix Cd [19]. The slope of the linear t to the data from this
plot is proportional to the apparent diffusion coefcient for the drug
in the matrix, and permits rstly, assessment of diffusion as the primary means of drug release from the correlation coefcient for the
linear t, and second, a means to compare the diffusion of a drug
from the different matrices into the release medium.
2.6. Characterization of P407 gels
2.6.1. Gelation temperature
The gelation temperatures of the examined formulations of
P407 solutions were determined using the Visual Tube Inversion
Method [20]. Briey, two glass vials, one containing 1 g of the
P407 solution and the other 1 g of water, were placed in a water
bath. The temperature was slowly increased and the temperature
at which the solution stopped owing upon tilting was noted as
the gelation temperature (t1). Similarly, the temperature of the
water bath was lowered and the temperature, at which the gel
started owing, was noted (t2). The thermo-couple of a digital thermometer (Fluke, USA) was placed in the water tube. The mean SD
of t1 and t2 is reported as the critical gelation temperature (CGT).
Each measurement was repeated three times.
2.6.2. Rheological measurements
P407 gels were assayed by a continuous shear method using a
Rheotest 2.1 viscometer with concentric cylinders, designed for
use with preparations having viscosities between 20,000 and

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S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

380,000 cP. Samples were subjected to shear rates between 0 and


165 s1. The full scale torque was 5500 dyne cm2.

Table 2
Classication of volunteers according to the Glogau scale.

2.6.3. In vitro drug release from the gels


The same procedure that was used for the evaluation of ALA
release from cubosomes was adopted for evaluating release from
P407 gels loaded with ALA or ALA cubosomes Aliquots were withdrawn at appropriate time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h).
Percentage ALA released from each of the two concentrations of
P407 gel loaded with 50 mg ALA or ALA cubosomes (D3) has been
plotted against the time.
2.7. Statistical analysis
a

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.

Clinical efcacy was assessed subjectively using a 5-grade


Global Aesthetic Improvement Scale (GAIS). The volunteers were
graded the overall esthetic change (worse 1, no change 0,
somewhat improved 1, moderately improved 2 or very much
improved 3) by comparing the patients visual appearance at
follow-up against an archival photograph taken prior to treatment.

3.1. Study design


The study was designed as a double blinded placebo controlled
study. Ethical approval was granted by Research Ethics Committee
of the Faculty of Pharmacy, Cairo University and the protocol complies with the declarations of Helsinki and Tokyo for humans. The
nature and purpose of the study were fully explained to volunteers
and written informed consent was obtained from all of them.
Twelve healthy female volunteers within the age of 3864 years
were chosen to participate in our study. They are divided randomly
into two groups; Group I: consisting of eight volunteers, received
P407 gels loaded with ALA cubosomes (Treatment). Group II:
consisting of four volunteers, received P407 gels loaded with
cubosomes free from the drug (Placebo). The volunteers could
not be pregnant, lactating or smokers nor received any cosmetic
procedures in the face including botulinum toxin treatment of
any serotype, dermal llers, injection lipolysis, laser treatment,
dermabrasion or anti-aging treatments (creams and tablets) during
the preceding 12 months. Exclusion criteria also comprehended for
patients with active inammation, infection, cancerous or precancerous lesions and unhealed wounds of the face region. Patients
with collagen related diseases, alteration of blood clotting (e.g.
hemophilia or on anticoagulant treatment), diabetes as well as
those treated with systemic retinoids within 2 years were excluded. Volunteers eligibility for study participation was determined on the rst visit through a thorough history taking and a
detailed clinical examination. Pregnancy test was done only when
appropriate. At the start of the study volunteers were subjected to
an assessment of wrinkles using Glogau Photo-aging Classication
dened as follows; type I no wrinkles, type II winkles in motion, type III wrinkles at rest and type IV only wrinkles as described in Table 2. Volunteers were instructed to apply the
prepared formulation on the right side of their faces twice daily,
for a duration treatment of 3 months. Volunteers were followed
up on a weekly basis till the end of the treatment plan.
3.2. Clinical assessment of response to treatment
3.2.1. Clinical photography
High resolution digital photographs were obtained for both
sides of the face, at each visit, by a xed digital camera, set at a
xed distance and constant settings for standardization.

3.2.2. Patient satisfaction level


The volunteers were allowed to independently grade their
satisfaction level (PS) at each assessment time according to the criteria in Table 3. Meticulous reporting of any adverse skin reactions
such as erythema, itching or swelling, if any, was also recorded.
3.2.3. Ultrasound biomicroscopy (UBM)
Ultrasound measurements were performed using paradigm
ultrasound biomicroscope plus Model P45 (UBM plus), which is a
microprocessor-based digital instrument that uses very high frequency ultrasound (50 MHz) to produce a two dimensional section
view of the examined tissue in B-scan (brightness) mode for diagnostic evaluation, and A-scan mode that measures the axial length
with depth of penetration up to 5 mm. The ultrasound scan images
acquired by the UBM transducer are viewed on a high resolution
color monitor in real-time. The reected ultrasound waves are controlled and assembled by the computer and magnied to provide a
high resolution B-scan image. The cross-sectional picture can be
further enhanced by digital lters to improve image contrast or
brightness to identify interesting ndings without altering the
original scanned le image. Skin thickness measurements were
made above the zygomatic bone. The following parameters were
measured: epidermis and dermis thickness and dermis density.
Ultrasonic images were taken three times at each time for each
subject.
3.3. Data analysis
The differences between the percent increase in the thickness of
the epidermis and dermis layers, for both groups (Treatment and

Table 3
Patient satisfaction scoring criteria.
Score

Grade

Description

Dissatisfaction

1
2

Slightly satised
Moderately
satised
Satised

Patient feels worse than before/the same as


before
Patient feels slightly better but still not worth it
Patient feels good with need for slight
improvement
Patient feels optimal cosmetic result

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S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

Placebo) were assessed by a students t-test using the statistical


package SPSS version 16.0. Differences were considered statistically signicant at P-values less than 0.05.

Table 4 illustrates the EE % of each of the 3 dispersions. This may


be attributed to the high solubility of the drug in the GMO.

4.6. Gelation temperature and rheological measurements

4. Results and discussion


4.1. Preparation of ALA cubosomes
Formulations, D1, D2 and D3 produced uniform dispersions of
cubosomes. D4 was found to be non-uniform. Dispersions D1, D2
and D3 which were prepared by emulsication of GMO and P407
in water followed by ultrasonication were chosen for further
characterization whereas D4 was rejected, as there were many
aggregates produced and less cubic structures were found.

The gelation of poloxamer solution is known to be a reversible


process, i.e. gels revert to free-owing solutions when the temperature drops below the critical gel temperature (CGT). If the gelation
temperature is higher than 36 C, the formulation will remain liquid at body temperature, and thus does not control the release
of ALA. It would be convenient to have a gelation temperature between 30 and 36 C, to be liquid at room temperature, and thus
could be spread efciently on the skin, and form a gel instantly
upon contact with the skin [24]. The CGTs of 20 and 30% w/w of

4.2. Particle size and zeta potential

Formulation

0.0
D1

Zeta Potential (mV)

The particle size distribution for dispersions D1, D2 and D3 is


tabulated in Table 4. It can be observed that by increasing the concentration of GMO in the formulation the particle size becomes
smaller.
Zeta potential is an important parameter for the stability and
bio-distribution of colloidal dispersions [21]. Generally, high surface charges lead to electrical repulsion between particles and thus
prevent their aggregation [22]. As demonstrated in Fig. 3, results
showed that by increasing the GMO concentration used in the formulation the negative charge increases. This could be attributed to
the presence of free oleic acid with the GMO (as the purity of the
commercially available GMO is 95%). As it is lipophilic, free oleic
acid should be absorbed onto the surface of the cubosomes, some
of which might ionize to carry a negative charge. Therefore, the
negative charge on the surface of cubosomes was mainly due to
the ionization of the carboxylic end group of oleic acid. Although
the zeta potential in this system was not high enough to provide
effective electric repulsion to avoid the aggregation of particles,
however, P407 acting as a steric stabilizer, would not only stabilize
the cubic phase dispersions efciently but also preserve the inner
cubic structure of the particles [23].

D2

D3

-2.0
-4.0
-6.0
-8.0
-10.0
-12.0

Fig. 3. Chart illustrating zeta potential measurements of ALA cubosome dispersions


D1, D2 and D3 (n = 3). (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.).

4.3. Optical microscopy


The outer cubic structure of the cubosomes was clearly obvious
in each of the three dispersions as viewed in Fig. 4.
4.4. Transmission electron microscope
When the samples were viewed under the electron microscope,
discrete particles with an outer cubic structure could be viewed as
presented in Fig. 5.
4.5. Encapsulation efciency
D3 showed an entrapment efciency of 86.48% which is considered as the highest encapsulation efciency amongst the 3 formulations tested. The EE % increased as the GMO content increased.

Table 4
Particle size distribution and encapsulation efciency (EE) of ALA cubosomes D1, D2
and D3 (n = 3).
Dispersion

Particle size (nm) SD

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.).

S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

Gelation temperature (oC)

256

30

P407 placebo solution

25

P407 solution loaded with


ALA
P407 solution loaded with
ALA cubosomes

20
15
10
5
0
20

30

P407 gel concentration (% w/w)

P407 solution loaded with ALA or ALA cubosomes compared with


P407 placebo solution are shown in Fig. 6. Results indicate that
by increasing the concentration of P407 in aqueous solutions, the
gelation temperature is lowered [24]. The inuence of loading
P407 gel with ALA or ALA cubosomes on the gelation temperature
is a further lowering the CGT. The cubosomes utilized in this study
are dispersions of GMO bulk cubic phase. The cubosomes formed
are stabilized by the addition of P407, where the hydrophobic
PPO portion is presumed to adsorb to the surface of cubosomes
while the hydrophilic PEO chains extend out into the aqueous environment to provide steric shielding [25].
The presence of P407 on the surface of the cubosomes increases
the apparent concentration of polymer in the solutions and thereby
increasing the viscosity of the gel as demonstrated in Fig. 7 and
lowering the CGT [17]. Similar effects were previously reported
when introducing inorganic salts to P407 systems, which have
been described as salting-out effects. This phenomenon has been
ascribed to the ability of the salts to reduce the water activity, by
hydrogen bonding to the water and thereby increasing the effective aqueous concentration of the polymer [19,26]. Similarly, the
addition of cubosomes might also result in decreased water activity leading to an increased effective concentration of P407 and
thereby lowering of the CGT of the system [17].

P407 placebo gel


P407 gel loaded with ALA
P4O7 gel loaded with ALA
cubosomes

600

Apparent viscosity (Poise)

Fig. 5. Transmission electron micrograph of ALA cubosomes dispersion D3.

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

P407 gel concentration (% w/w)


Fig. 7. Apparent viscosities of 20 and 30% w/w poloxamer gels as function of
loading gel with ALA or ALA cubosomes, in comparison with P407 placebo gel, at
25 C (n = 3). (For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.).

4.7. In vitro drug release from cubosomes


100
90
80

% ALA released

In vitro release prole of ALA from cubosomes was performed in


hydro-alcoholic solution using the paddle over disk method. For
cubosomes of D1, 97.54% of the drug was released within 6 h. For
cubosomes of D2, 94.18% of the drug was released within 6 h.
For cubosomes of D3, 90.04% of the drug was released within 6 h
(Fig. 8). The results indicated a trend toward a decrease in release
rate with increasing GMO concentration, although this was not statistically signicant (p > 0.05). This may be attributed to the high
solubility of ALA in GMO. The release rate constants and correlation coefcients were calculated after tting the release data to
square root Higuchi model and are summarized in Table 5. The correlation coefcients (R) calculated for each formulation were above
0.99, which indicated that in vitro drug release proles of ALA
cubosomes t well with the square root Higuchi model. Hence formulation D3 was selected as the optimized formulation by virtue
of slowest drug release and maximum entrapment efciency.
The release proles of ALA from 20 and 30% w/w P407 gel as
illustrated in Fig. 9, display a trend of a decrease in the release rate
as P407 concentration increases [10], in which 98.9% and 98.02% of

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).

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S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259


Table 5
Release rate constants and correlation coefcients of ALA cubosome dispersions D1,
D2 and D3, calculated in accordance with the release proles obtained using square
root Higuchi model.
Dispersion

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

20%w/w P407 gel with ALA


cubosomes

20% W/W P407 gel with ALA

10

30% W/W P407 gel with ALA

30%w/w P407 gel with ALA


cubosomes

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).

ALA were released within 8 h respectively and the difference was


not statistically signicant (p > 0.05). The release rate constants
and correlation coefcients were calculated after tting the release
data to square root Higuchi model and are summarized in Table 6.
The correlation coefcients (R) calculated for each gel concentration were above 0.99, which indicated that in vitro drug release
proles of ALA from P407 gel t well with the square root Higuchi
model.
The release prole of ALA from 20 and 30% w/w P407 gel loaded
with ALA cubosomes, showed that 65.34% and 55.52% of the drug
were released after 8 h respectively, as illustrated in Fig. 10. The release rate constants and correlation coefcients were calculated
after tting the release data to square root Higuchi model and
are summarized in Table 7. The correlation coefcients (R) calculated for each gel concentration were above 0.99, which indicated
that in vitro drug release proles of ALA from P407 gel t well with
the square root Higuchi model. From these results it can be
deduced that by increasing the P407 concentration of the gel, the
release rate of ALA was prolonged. This could be explained that
by increasing the concentration of P407, the viscosity of the gel increases as well, making it harder for the cubosomes to diffuse out
of the gel matrix.
4.8. In vivo evaluation of skin rejuvenation effect in volunteers
Clinical outcome was evaluated based on the GAIS level of
improvement. It was observed that no marked improvement was
seen after 1.5 months of treatment for group II (Placebo) but 75%
of volunteers showed some sort of improvement for group I (Treatment). However, after 3 months of treatment moderate improvement was seen in about 58.33% of the volunteers treated by
(Treatment) and 25% of volunteers of group I (Treatment) showed
very much improvement and satisfaction, while none of the volunteers of group II showed such improvement, as presented in Fig. 11.
After 3 months of treatment, it was noted that there was
impressive changes in the ne lines and also the pore sizes on

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.

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S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

1.5 months (Treatment)


1.5 months (Placebo)
3 months (Treatment)

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.).

The results indicate an increase in the thickness of the epidermis


and dermis layers after 3 months of treatment for the Treatment
group. The average % increase in the thickness of the epidermis
dermis layer was 8.84 4.93% for the Treatment and was
2.95 2.14% for the Placebo. Statistical analysis revealed a significant difference in the % increase in the thickness of epidermis
dermis layers between the Treatment and Placebo groups after
3 months of treatment as (p-value = 0.048).
It is worthy to note that application of ALA cubosomes twice
daily for 3 months leads to satisfactory progress in treatment of
wrinkles and scars in the studied volunteers. The results conrm
that cubosomes are useful in topical drug delivery, particularly
due to their bioadhesive properties, their characteristics as

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.).

S. Sherif et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 251259

259

References

Fig. 15. High resolution ultrasonic images of pre- and post-treatment (A and B) for
two representative volunteers.

sustained-release delivery systems, and their ability to protect the


encapsulated drug from degradation. It is also important to mention that Cubosomes show a structural organization identical to
that found in biomembranes which make them biocompatible
and of fewer side effects.
5. Conclusions
From the results, it could be concluded that ALA cubosomes
could be formulated using the conventional emulsication/ultrasonication technique, whereas the dilution method was not
successful in producing uniform dispersions. By increasing the
GMO content in the dispersion, the cubosomes size decreases,
the encapsulation efciency increases, zeta potential becomes
more negative. From the in vitro study, it was demonstrated that
cubosomes can incorporate and deliver the potent antioxidant
ALA, with a release prole that ts with the square root Higuchi
model. It was also apparent that 90.04% of the drug was released
from formula D3 within 6 h. When ALA cubosome dispersions
were loaded into 30% P407 gels, formulation signicantly
decreased the drug release rate. In summary, preliminary clinical
evidence indicates that 30% w/w P407 gel loaded with ALA cubosomes is safe and effective in creating esthetic correction of the facial region when used for at least 3 months in the great majority of
subjects. Further clinical studies will be conducted on large
number of volunteers to conrm the clinical efciency of ALA
cubosomes as anti-wrinkle.
Acknowledgment
The authors would like to express their deep gratitude to
Mohamed Hussein Medhat El-Komy, Suzan Shalaby and Rehab A.
Hegazy, Department of Dermatology, Faculty of Medicine, Cairo
University, who had conducted and supervised the clinical part
of this project.

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