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International Journal of Pharmaceutics

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

Pharmaceutical nanotechnology

Microemulsion and poloxamer microemulsion-based gel for

sustained transdermal delivery of diclofenac epolamine using in-skin
drug depot: In vitro/in vivo evaluation
Shahinaze A. Fouad a , Emad B. Basalious b, , Mohamed A. El-Nabarawi b , Saadia A. Tayel b
a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ahram Canadian University, Cairo, Egypt
b
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-aini Street, Cairo 11562, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Microemulsion (ME) and poloxamer microemulsion-based gel (PMBG) were developed and optimized
Received 17 April 2013 to enhance transport of diclofenac epolamine (DE) into the skin forming in-skin drug depot for sustained
Accepted 1 June 2013 transdermal delivery of drug. D-optimal mixture experimental design was applied to optimize ME that
Available online xxx
contains maximum amount of oil, minimum globule size and optimum drug solubility. Three formulation
variables; the oil phase X1 (Capryol ), Smix X2 (a mixture of Labrasol /Transcutol , 1:2 w/w) and water
Keywords:
X3 were included in the design. The systems were assessed for drug solubility, globule size and light
Diclofenac epolamine
absorbance. Following optimization, the values of formulation components (X1 , X2 , and X3 ) were 30%,
In-skin depot
Sustained transdermal delivery
50% and 20%, respectively. The optimized ME and PMBG were assessed for pH, drug content, skin irritation,
D-optimal design stability studies and ex vivo transport in rat skin. Contrary to PMBG and Flector gel, the optimized ME
Microemulsion showed the highest cumulative amount of DE permeated after 8 h and the in vivo anti-inammatory
efcacy in rat paw edema was sustained to 12 h after removal of ME applied to the skin conrming the
formation of in-skin drug depot. Our results proposed that topical ME formulation, containing higher
fraction of oil solubilized drug, could be promising for sustained transdermal delivery of drug.
2013 Elsevier B.V. All rights reserved.

1. Introduction non-woven polyester felt backing and covered with a polypropyl-

Musculoskeletal pain is a common problem often treated
with topical NSAIDs. Topical NSAIDs have a reduced risk of
upper GI complications such as gastric and peptic ulcers, dys-
pepsia as well as a lack of drugdrug interactions (McCarberg
and Argoff, 2010). Diclofenac epolamine is a NSAID, known as
diclofenac-N-(2-hydroxyethyl)-pyrrolidine) (DHEP) (Conte et al.,
2002). The diclofenac molecule, in its acidic form, is hydropho-
bic with very low solubility in water. The epolamine salt of
diclofenac has greater solubility in water and non-polar solvents
(n-octanol) than other diclofenac salts. High concentrations of
aqueous diclofenac epolamine solutions exhibit surfactant behav-
ior (McCarberg and Argoff, 2010). The solubility and the surfactant
properties of diclofenac epolamine enhance its membrane perme-
ability (OConnor and Corrigan, 2001).
DE is currently available as topical gel and patch marketed under
the brand name of Flector. Flector patch (10 cm 14 cm) con-
tains an adhesive material containing 1.3% DE which is applied to a
Corresponding author. Tel.: +20 1200010002.
E-mail addresses: dremadbasalious@cu.edu.eg,
dremadbasalious@gmail.com (E.B. Basalious).
ene lm release liner under patch (Petersen and Rovati, 2009). The
use of external drug reservoir (topical patch) is the common tech-
nique used to sustain the transdermal delivery of water soluble
drugs. The major disadvantages of transdermal patches are their
sophisticated method of manufacture and the possibility that a
local irritation will develop at the site of application. Erythema and
itching can be caused by the drug and the adhesive in the patch
formulation. Topical NSAID gels or creams are applied up to four
times daily. Moreover, patches and gels are inconvenient to patients
regarding discrepancy with cleaning and washing of skin. The com-
bination of all advantages of gels (simple method of manufacture
and ease of application by patients) with that of patches (sustained
delivery) is the goal of this study. Our hypothesis was to increase
the penetration of DE through epithelial tissue for loading of the
drug into the skin forming in-skin drug depot where skin acts as
in situ skin patch.
Effective penetration of drugs through the stratum corneum is
a major challenge in transdermal drug delivery. The presence of
lipid of the stratum corneum represents a lipophilic barrier that
restricts the permeation of molecules. Several approaches have
been proposed to increase skin permeation. Microemulsions (MEs)
which are clear, thermodynamically stable mixtures of oil, water
and surfactant, have been shown to be able to deliver drugs through

0378-5173/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
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2 S.A. Fouad et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

the skin better than conventional systems such as gel, creams and (Spectrophotometer (UV 1601, PC UVVisible, Shimadzu, Japan)
ointment (Kreilgaard, 2002). ME and ME based gels were pre- after appropriate dilution with methanol at max about 276 nm.
pared in an attempt to increase the transdermal drug delivery of Appropriate diluted solutions of oils, surfactants and co-solvents
both hydrophilic and lipophilic drugs (Barot et al., 2012; Kreilgaard in methanol were taken as blank. Components which showed the
et al., 2000; Trotta et al., 1997). Moreover, NSAIDs are one of highest solubility of DE were used for further studies (Barot et al.,
the most important drug classes that have been formulated as 2012).
microemulsion-based hydrogels for both topical and transdermal
use such as ibuprofen (Chen et al., 2006), ketoprofen (Rhee et al.,
2.3. Construction of pseudo-ternary phase diagrams
2001) and diclofenac (Kweon et al., 2004).
MEs and ME based gels were found to have favorable solvent
The pseudo-ternary phase diagrams were constructed using
properties due to the potential incorporation of large fraction of
titration method to determine the ME region and to obtain the con-
lipophilic and/or hydrophilic phases (Malcolmson and Lawrence,
centration range of components for the existing range of MEs with
1993; Malcolmson et al., 1998). Only the dissolved fraction of a
different possible compositions of oil, surfactant/co-solvents, and
drug in a vehicle can enter the skin. The small globule size of MEs
water (Barot et al., 2012).
makes them a suitable vehicle to penetrate epithelial tissue and use
The ratio of surfactant to co-solvent (Smix) was altered at 1:1,
skin as a depot for sustained drug delivery (Yuan and Acosta, 2009).
1:2 and 2:1 and such mixtures were prepared. These mixtures
Development of a pharmaceutical formulation consumes a lot
(Smix) were mixed with the oil phase to give the weight ratios of
of time and is considered as a complex process. Thus, D-optimal
90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90.
mixture design is applied to develop pharmaceutical formulation
Applying the aqueous titration method, distilled water was titrated
because it was demonstrated to be an efcient method for opti-
drop by drop to the oil and Smix mixture under magnetic stirring at
mization of the formulation and to understand the relationship
ambient temperature. After each addition, the mixture was exam-
between independent variables and dependent variables in a for-
ined for the appearance. The end point of the titration was the point
mulation (Basalious et al., 2010; Gao et al., 2004).
where the solution becomes cloudy or turbid. The quantity of the
Literature lacks any data about the use of ME for loading of drug
aqueous phase required to make the mixture turbid was noted.
into skin to form in-skin depot for sustained transdermal delivery
The percentages of the different incorporated components were
of DE (a water soluble drug). Thus, the aim of this study was the for-
then calculated and the same procedure was followed for the other
mulation and optimization of ME and PMBG. D-optimal design was
Smix ratios to plot the pseudo-ternary phase diagram. Pseudo-
applied to optimize formulation that contains a maximum amount
ternary phase diagrams were constructed with Tri-plot software
of lipid, small globule size (<100 nm) and possess enhanced skin
Version 4.1.2. (Todd Thompson software). The clear ME zones were
transport of the drug forming in-skin drug depot. In vivo study of
identied and marked.
the anti-inammatory efcacy and sustained delivery of in-skin
depot of DE was carried out by carrageenan induced rat paw edema
method. 2.4. Formulation optimization of MEs

D-optimal mixture experimental study was designed based on a

2. Materials and methods
2.1. Materials
Diclofenac epolamine was kindly supplied by Marcyrl for
Pharmaceutical industries, El-Obour (Egypt), Tween 80 and Iso-
propyl Myristate were purchased from Sigma Aldrich Chemical
Co. (St. Louis, MO, USA.), Miglyol 812 and Miglyol 840 (was
obtained from Sasol (Witten, Germany). Polyethylene glycol 400
was from El-Nasr Pharmaceutical Chemicals Co. (Egypt), Labrasol ,
Dipalmitoylphosphatidylglycerol (DPPG ), Capryol 90, Cocoate
cc, Labrafac cc, and Gatuline Intense and Transcutol HP were
supplied by Gattefoss (France.), Poloxamer 407 was obtained
from BASF Corporation, Chemical Division (USA). Distilled water
was used throughout the study. All other chemical reagents and
solvents were of analytical grade and used as received.
2.2. Screening of oils, surfactants and co-solvents for ME
formation
For selecting solvents with good solubilizing capacity for DE,
the saturated solubility of DE in various oils such as Capryol 90,
Cocoate cc, isopropyl myristate , Gatuline in-Tense , Miglyol
812 and Miglyol 840, surfactants (Labrasol , Labrafac cc, Tween
80 and DPPG ) and co-solvents (Transcutol HP and polyethylene
glycol 400) was determined.
Excess amount of DE was added in 5 g of oils, surfactants and
co-solvents in 10-ml-capacity stoppered glass vials and shaken
on a shaker for 48 hours at ambient temperature. Suspension was
centrifuged at 4000 rpm for 10 min and the concentration of DE
in the supernatant was determined by UV spectrophotometer
three component system: the oil phase X1 (Capryol 90), Smix X2 (a
mixture of Labrasol /Transcutol , 1:2 w/w) and aqueous phase X3
(water). The total concentration of the three components summed
to 100%. Based on the previous results obtained from phase dia-
gram, the range of each component was selected as follows: The
amount of oil was chosen in the range 1030%. The Smix ranged
from 50% to 80%. Since hydration of the stratum corneum signif-
icantly affects penetration of drug into the skin, water range was
selected to be 1030%.
The solubility of drug in ME, mg/ml (Y1 ), mean globule size (Y2 )
and absorbance of ME (Y3 ) were used as the responses (dependent
variables). The responses of all model formulations were treated by
Design-Expert software (version 7; Stat-Ease, Inc., Minneapolis,
MN). Suitable models for mixture designs consisting of three com-
ponents include linear, quadratic and special cubic models. The best
tting mathematical model was selected based on the comparisons
of several statistical parameters including the multiple correlation
coefcient (R2 ), adjusted multiple correlation coefcient (adjusted
R2 ) and the predicted residual sum of square (PRESS), proved by
Design-Expert software. Among them, PRESS indicates how well
the model ts the data, and for the chosen model it should be
small relative to the other models under consideration (Huang et al.,
2004).
D-optimal design was selected since it minimizes the variance
associated with the estimates of the coefcients in the model (Holm
et al., 2006). The software selected a set of candidate points as a base
design. These included factorial points (high and low level from
the constraints on each factor, centers of edges, constraint plane
centroids, axial check point, and an overall center point). The base
design consisted of 16 runs (Table 1). The optimum formulation
was selected, which had the highest oil content, minimum content

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
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Table 1
The formulations of mixture design and their characterization results.

Formulation A: Capryol B: Smix C: Water Solubility (mg/ml) Globule size (nm) Absorbance

1 20 70 10 292.27 62.93 0.075

2 10 70 20 268.61 49.15 0.023
3 30 50 20 374.70 77.2 0.045
4 30 50 20 277.04 28.19 0.01
5 25 60 15 188.29 48.9 0.032
6 15 65 20 248.00 37.55 0.012
7 10 60 30 304.44 54.8 0.034
8 30 60 10 262.29 50.4 0.036
9 10 80 10 171.19 151.6 0.089
10 30 60 10 260.42 60.5 0.027
11 20 50 30 358.31 43.45 0.059
12 22.5 55 22.5 273.77 54.6 0.022
13 15 55 30 480.09 51.6 0.023
14 10 60 30 359.09 59.6 0.024
15 20 50 30 370.02 54.68 0.025
16 10 80 10 248.71 140.6 0.091

of Smix, intermediate drug solubility (Y1 ) and the globule size (Y2 )
less than 100 nm.
2.5. Preparation of MEs
From the pseudo-ternary phase diagrams, Smix ratio with maxi-
mum ME region was selected. Different proportions of oil and Smix
were mixed based on the ratios presented in Table 1. The mixture
of oil and Smix was mixed using vortex (VSM-3 model, PRO Scien-
tic Inc., Oxford, England) at ambient temperature. The measured
amount of distilled water was added drop wise to the oily mixture
until clear and transparent liquid was obtained. All MEs were then
stored at ambient temperature.
2.6. Evaluation of the prepared MEs
2.6.1. Determination of drug solubility in the prepared MEs
The solubility of DE in MEs (in mg/ml: Y1 ) was determined.
Excess amount of DE was added in 5 g of each of the previously
prepared ME in 10-ml-capacity stoppered vials. The resultant mix-
ture was mixed initially by vortex mixer then, all the vials were
shaken in the shaker for 24 h at 25 C.
Afterwards, centrifugation was done at 4000 rpm for 10 min and
the concentration of DE in the supernatant was determined by UV
spectrophotometer after appropriate dilution with methanol at its
respective max . The plain ME without drug with the same compo-
sition was taken as blank after appropriate dilution with methanol.
2.6.2. Determination of globule size by photon correlation
spectroscopy
The globule size (in nm: Y2 ), was determined using photon
correlation spectroscopy that analyzes the uctuations in light
scattering due to the Brownian motion of particles using Malvern
Zetasizer Nano-ZS (Ver.6.20, Malvern Instruments Ltd., Worcester-
shire, England). All measurements were done at room temperature
(25 C) and at 90 C to the incident beam.
2.6.3. Measurement of spectroscopic absorbance at 400 nm
The optical clarity of aqueous dispersions of SNEDD formu-
lations was measured spectroscopically. The absorbance of each
formulation was measured at 400 nm, using distilled water as a
blank.
2.6.4. Transmission electron microscopy (TEM) of the optimized
DE loaded MEs
The morphology of the optimized ME systems was observed
using transmission electron microscopy. A drop of each ME was
placed on a copper grid and the excess was removed with a l-
ter paper. One drop of 2% aqueous solution of phosphotungistic
acid (PTA) was added onto the grid and left for 3060 s to allow
staining. The excess was removed with a lter paper. The grid was
nally examined under the transmission electron microscope (JEOL
(JEM-1400), Tokyo, Japan).
2.7. Formulation of DE-loaded ME and PMBG
As MEs have low viscosity, their retention at the affected parts
is quiet less. Therefore, their viscosity was required to be increased
by the addition of a suitable gelling agent. Poloxamer was used
as a gelling agent for the optimized ME formulation to formulate
thermosensitive microemulsion-based gel of DE.
Plain poloxamer gel (25%) was rstly prepared according to the
cold technique (Chang et al., 2002; Shin et al., 1999). ME contain-
ing the drug was added portion-wise onto the plain gel in a ratio
of gel:ME (2:1) with continuous stirring. The nal microemulsion-
based gel formulation contained 1.3% w/w DE. DE was dissolved
directly in the optimized ME to prepare drug loaded ME containing
1.3% w/w DE.
2.8. Evaluation of DE microemulsion and PMBG
2.8.1. pH measurements and drug content
The apparent pH of the formulations was measured by a pH
meter in triplicate at 25 C. For determination of drug content, one
gram of ME formulations was diluted with appropriate amount of
methanol. The concentration of DE was determined by UV spec-
trophotometer at its respective max . The plain ME formulations
without drug with the same composition was taken as blank after
appropriate dilution with methanol.
2.8.2. Stability study
The optimized DE loaded ME and PMBGl were stored at
40 C/75% RH for three months. Optical clarity and drug content
were performed for the stored drug loaded ME and microemulsion-
based gel using the same procedures adopted for the fresh samples.
Morphology of the stored drug-loaded ME was determined using
transmission electron microscopy.
2.8.3. Skin irritation test
Three male albino Wistar rats (130150 g) were kept under
standard laboratory conditions and housed in cages with free access
to a standard laboratory diet and water ad libitum. A single dose of
100 L of the optimized drug-loaded ME, optimized drug-loaded
PMBG and the market formulation (Flector gel) was applied to the

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
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left ear of the rat, with the right ear as a control. The development
of erythema was monitored for 24 h then the gel was removed,
and the application sites were graded according to a visual scoring
scale from no erythma, mild, moderate, high and severe erythema
(Azeem et al., 2009; Shakeel et al., 2007).
2.8.4. Study of ex vivo transport of DE from optimized
formulations into rat skin and ability to form in situ drug depot
Ex vivo skin transport studies were performed using newly born
rat skin (Azeem et al., 2009; Sarigullu Ozguney et al., 2006). Newly
born albino Wistar rats were sacriced and skin samples obtained
was inspected for the presence of any holes or irregularities. Fresh
skin used in the study was preserved in 10% glycerin solution
at 20 C. The study performed in this section was approved by
Research Ethics Committee, Faculty of Pharmacy, Cairo University.
Skin was slowly thawed and was cut into small circular pieces. The
lower surface of the skin was allowed to hydrate for 1 h at 37 C
prior to experimentation.
The Ex vivo skin transport studies of DE from the optimized ME,
PMBG and the market product (Flector gel) were performed in a
USP dissolution apparatus tester (USP apparatus II) at 37 0.1 C.
One gram of drug loaded ME, PMBG and the market formulation,
all containing 1.3% drug w/w were placed in double open-sided
glass cylindrical tubes (2.5 cm in diameter and 5 cm in length, with
area = 4.9 cm2 ) tightly covered from one side with rat skin. The
loaded tubes were attached from the second side to the shafts of
the USP dissolution tester apparatus. This assembly represents the
donor compartment. The shafts rotated at a speed of 50 rpm in
phosphate buffer pH 7.4. The dissolution vessels (receptor com-
partment) were lled with 300 ml of phosphate buffer pH 7.4.
Four milliliter samples were withdrawn periodically at pre-
determined time intervals of 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7
and 8 h and replaced instantly by equal amount of fresh phosphate
buffer pH 7.4 in order to maintain the same volume.
The drug concentration was determined by UV spectropho-
tometer at 276 nm. The skin transport studies were done in
duplicates and the average percentage drug permeated was plotted
versus time. Cumulative amount of drug in receptor chamber for
the three formulations was plotted as a function of time.
To study the ability of the three formulations to form in situ
depot in the skin, transport of DE in the skin was observed after
removal of the formulations from the donor compartment. Rat skin
was removed after 3 h. The formulations were wiped off with wet-
ted cotton pieces then the rat skin was mounted again on glass
cylinder to continue the skin transport study.
2.8.5. In vivo study of the anti-inammatory efcacy and
sustained delivery of in-skin depot of DE
The sustained anti-inammatory efcacy and the ability of the
optimized ME and PMBG to form in-skin depot were compared
in vivo using carrageenan induced rat paw edema test. Flector gel
is the market formulation and it is used as a reference product. Also,
plain PMBG is prepared to be used as a control. Each formulation
except the prepared plain gel contains 1.3% w/w DE.
Thirty two adult male albino Wistar rats, weighing 130150 g
were used in this study. They were purchased from Helwans Farm
of experimental animals (Cairo, Egypt). The animals were accli-
matized to environment for one week, they were housed under
controlled environment at 25 1 C with a 12 hur light/dark cycle.
All animals had free access to standard rodent pellet food consist-
ing of vitamin mixture (1%), mineral mixture (4%), corn oil (10%),
sucrose (20%), cellulose (0.2%), casein 95% (10.5%), starch (54.3%)
and water.
Animals were divided into four groups of eight rats each. The
plain PMBG was assigned to the rst group, the optimized ME was
assigned to the second group, PMBG was assigned to the third group
and the Flector gel was assigned to the fourth group.
In order to induce inammation, animals were rst injected with
0.1 ml of 1% carrageenan solution in saline in the plantar region of
the right hind paw. The initial paw thickness (Ti ) was measured
using a Micrometer Caliper, one hour after carrageenan injection.
Then, 1 g of each formulation was applied to the right hind paw
of the rats. After 3 h of formulation application (sufcient time for
skin loading and formation of in-skin drug depot), formulations
remaining on the surface of the paw were wiped off with cotton
then, the paw thickness (Tf ) was measured again using a Microm-
eter Caliper at different time intervals (3, 4, 5, 6, 7, 8 and 12 h). The
edema % was calculated from the mean effect in treated animals
according to the following equation:
Tf Ti
% edema = 100
Ti
where Tf is the thickness measured following administration of the
formulae at different time intervals. Ti is the thickness measured
1 h after carrageenan sodium injection. Data were analyzed statis-
tically by Students t-test at 5% signicance level using GraphPad
Prism 5 program (GraphPad Inc., USA).
3. Results and discussion
3.1. Screening of components for ME
The saturated solubility of DE in various oils, surfactants and co-
solvents was estimated as shown in Fig. 1. Amongst the various oily
phases that were screened, Capryol 90 provided the highest solu-
bility of DE so was chosen for further investigations. Solubility of DE
in Labrasol was the highest among the surfactants. Labrasol was
selected for further studies due to its solubility prole and its low
toxicity level as a non-ionic surfactant (Shaq-un-Nabi et al., 2007).
Transcutol HP, which is a solubilizer and absorption enhancer
(Basalious et al., 2010), was found to be a very efcient solubilizer
for DE, and so was chosen as a co-solvent in the development of
DE loaded ME formulations aiming to improve the drug loading
capabilities.
3.2. Construction of pseudo-ternary phase diagrams
To obtain the appropriate components and their concentration
ranges for MEs, pseudo-ternary phase diagrams were constructed
for different Smix ratios 1:1, 1:2 and 2:1, so that o/w ME regions
could be identied and ME formulations could be optimized.
The three ratios gave stable and clear MEs but the ratio which
gave the largest ME region was found to be 1:2 and therefore it
was selected for further studies. This is clearly shown in Fig. 2. The
phase study clearly reveals that with a decrease in the weight ratio
of Labrasol from 1 to 0.5, the ME region is expanded. This obser-
vation conforms to the results obtained from the study of Barot
et al. (2012). It is obvious also that an increase of the weight ratio of
Labrasol from 1 to 2 resulted also in expansion of the ME region.
This observation is in agreement with Shakeel et al. stating that
as the surfactant concentration was increased in the Smix ratio, a
higher ME region was observed, perhaps because of further reduc-
tion of the interfacial tension, increasing the uidity of the interface,
thereby increasing the entropy of the system (Shakeel et al., 2007).
Thus, the effect of Labrasol on ME area depends the other com-
ponents of ME especially co-solvent. This is because the reduction
of o/w interface is not achieved by single-chain surfactants alone.
The combination of short to medium chain length alcohols (such as
Transcutol HP) with single chain surfactants could result in lower-
ing the interfacial tension due to increased uidity at the interface
(Binks et al., 1989). Miscibility of aqueous and oily phases could

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
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S.A. Fouad et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx 5

Fig. 1. Solubility (mg/ml) of diclofenac epolamine in various microemulsion components.

Fig. 2. Pseudo-ternary phase diagrams of microemulsions composed of oil (Capryol 90), Smix (surfactant: Labrasol , co-solvent: Transcutol ) and water at various oil/Smix
ratios 1:2 (a), 1:1 (b) and 2:1 (c).

also be increased by medium chain length alcohols due to their
partitioning behavior between the two phases (Lawrence and Rees,
2000; Shaq-un-Nabi et al., 2007).
3.3. Formulation optimization of ME using D-optimal design
In order to rapidly obtain the optimal ME, D-optimal mixture
experimental design was applied in this study. The oil phase X1
(Capryol 90), Smix X2 (a mixture of Labrasol /Transcutol , 1:2
w/w) and aqueous phase X3 (water) were chosen as formulation
variables and the solubility of drug in ME, mg/ml (Y1 ), mean glob-
ule size (Y2 ) and absorbance of ME (Y3 ) were used as the responses
(dependent variables). The responses of these formulations are
summarized in Table 1. The independent and response variables
were related using polynomial equation with statistical analysis
through Design-Expert software. As shown in Table 2, the approx-
imation of response values of Y1 based on linear model was the
most suitable because its PRESS was smallest. The values of the
coefcients X1 , X2 and X3 are related to the effect of these variables
on the response. A positive sign of coefcient indicates a synergistic
effect while a negative term indicates an antagonistic effect upon
the response (Huang et al., 2005). The larger coefcient means the
independent variable has more potent inuence on the response.
As shown in Table 1, solubility of DE in the different ME formu-
lation varied between 171.1 and 480 mg/ml. It can be inferred that
the three independent factors have a profound effect on drug sol-
ubility. As illustrated in Table 3, a p-value of 0.05 for any factor
in analysis of variance (ANOVA) indicates a signicant effect of the
corresponding factors on the solubility of drug in ME (Y1 ). It can
be inferred that the terms X1 , X2 , and X3 have a signicant effect
on the drug solubility (p < 0.05). This result could be conrmed by
the positive value of these coefcients (Table 2). Fig. 3 shows the
contour diagrams illustrating the effect of varying ratios of (X1 ),
(X2 ) and (X3 ) on the solubility of drug in ME (Y1 ). It is obvious that
the water content in ME formulation has the highest positive effect
on the solubility of DE in ME. This means that increasing the water

Table 2
Reduced Regression results of the measured responses.

Response Model R2 Adjusted R2 Predicted R2 PRESS Regression equation for the responses

Y1 Linear 0.547 0.4774 0.342 58,715.09 Y1 = +307.68X1 + 117.74X2 + 832.50X3

Y2 Quadratic 0.8193 0.7537 0.5469 7792.269 Y2 = +526.53X1 + 384.48X2 + 830.97X3 1631.31X1 X2 2172.43X2 X3
Y3 Quadratic 0.7951 0.7439 0.6327 0.0036 Y3 = 0.29X1 + 0.26X2 + 1.25X3 2.70X2 X3

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
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Table 3 negative effect of combination of Smix and water content on the

ANOVA of the solubility of drug in microemulsion formulations (Y1 ).
globule size of the MEs and thus, their absorbance. Fig. 4a and b
Source Sum of squares dF Mean square F p-Value shows the contour diagrams illustrating the effect of varying ratios
Model 48,819.5 2 24,409.7 7.85143 0.0058 of (X1 ), (X2 ) and (X3 ) on the globule size (nm) of ME (Y2 ) and spec-
Linear Mixture 48,819.5 2 24,409.7 7.85143 0.0058 troscopic absorbance of ME (Y3 ), respectively. It is obvious that
Residual 40,416.4 13 3108.95 there is an optimum ratio of all the mixture components for ME
Lack of Fit 31,080 8 3885 2.08057 0.2179 formulation having small globule size and absorbance. Sufcient
Pure Error 9336.38 5 1867.28
concentration of water is needed for maximal effect of Smix on
Cor Total 89,235.8 15
emulsication of Capryol .
The aim of the optimization of pharmaceutical formulations
is generally to determine the levels of the variable from which a
robust product with high quality characteristics may be produced
(Basalious et al., 2010). Some of the measured responses have to
be minimized. In this case, these responses comprise the globule
size (<100 nm) and the absorbance (<0.09). The small globule size
of oil phase of ME with high fraction of drug entrapped allows bet-
ter penetration of these oil globules into the skin to act as in situ
depot releasing the drug in controlled rate. Some responses, such as
the solubility of drug in ME should have an optimal intermediate
range (200350 mg/ml) to increase the fraction of drug incorpo-
rated into the oil globules allowing the controlled release of drug
from ME globules. The very high solubility of drug in ME systems
(about 480 mg/ml in case of formulation 13) is not required for for-
mulation of in-skin depot MEs as most of the drug is entrapped in
the aqueous phase of ME systems showing poor absorption into
the lipid of stratum corneum. Under these conditions, these three
responses were then combined to determine an all over optimum
region. Fig. 5 shows an acceptable region met the requirement of
these responses. According to the selection criteria, those ME com-
positions with high oil content, minimum content of Smix, optimal
DE solubility and smallest globule size were chosen. A ME for-
mulation satisfying these criteria was prepared and evaluated. An
optimum response was found with Y1 , Y2 , and Y3 of 317.6 mg/ml,
Fig. 3. Contour plot of the effect of variables on the solubility of drug in microemul- 54.4 nm and 0.024 at X1 , X2 and X3 values of 30%, 50% and 20%,
sion formulations (Y1 ). respectively.
DE was dissolved directly in the optimized ME to prepare drug
loaded ME containing 1.3% w/w DE. PMBG (containing 1.3% w/w DE)
content in ME formulation increases the fraction of DE that is sol- was prepared by mixing plain poloxamer gel with the optimized
uble in the aqueous phase of ME. ME containing the drug in a ratio of gel:ME (2:1) with continu-
As shown in Table 1, the globule size of the different ME for- ous stirring. Our goal was to prepare thermosensitive PMBG that
mulation varied between 28.19 and 151.6 nm. As shown in Table 2, undergoes gelling at skin temperature. The low viscosity is required
the approximation of response values of Y2 and Y3 based on the at the application site to allow better penetration of oil globules
quadratic model was the most suitable. of the optimized ME into skin. However, Poloxamer gel had lost
ANOVA of the effect of variables on globule size (nm) of ME (Y2 ) its thermosensitive properties upon mixing with ME. PMBG and
and spectroscopic absorbance of ME (Y3 ) shows that the terms X1 , ME were subjected to further studies to investigate their abil-
X2 , and X3 have a signicant effect on both responses (p < 0.05). ity to enhance DE transport into skin and formation of in-skin
The coefcient of X2 X3 for both responses was largest, showing the depot.

Fig. 4. Contour plots of the effect of variables on the globule size (Y2 ) (a) and the spectroscopic absorbance (Y3 ) (b) of the microemulsion formulations.

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
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Fig. 5. Overlay plot for the effect of different variables on the three responses.
The solubility of drug in microemulsion, mg/ml (Y1 ), mean globule size (Y2 ) and
absorbance of microemulsion (Y3 ).
3.4. Stability studies of the optimized formulations
The optimized formulations were stable when stored at
40 C/75% RH for three months where there was no obvious change
in visual appearance. The drug content of the fresh and stored for-
mulations of the optimized drug-loaded ME were 100.62% 2.12
and 98.54% 2.82, respectively. The drug content of the fresh and
stored formulations of the optimized PMBG were 104.89% 3.22
and 101.98% 3.6, respectively. The pH values of the fresh and
stored optimized formulations ranged from 6.5 to 7. The morphol-
ogy of the optimized drug-loaded ME examined via TEM was not
changed before and after storage. Transmission electron micro-
graphs of the optimized formulation, Fig. 6 revealed that the
globules of the developed MEs are spherical, discrete and have
uniform droplet size distribution. Globules appear to have compa-
rable size to the calculated values obtained by photon correlation
spectroscopy (Table 1).
Fig. 6. Transmission electron micrographs of the fresh (a) and stored (b) optimized drug-loaded microemulsion taken at 30,000 magnication.
Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
7
3.5. Skin irritation test
Various formulations, when applied topically, might cause skin
irritation. Thus, rat skin irritation experiments were conducted in
order to assess the potential irritant effects of the optimized drug-
loaded ME, PMBG and the market formulation (Flector gel). All the
ME formulations and the market product show no erythema. Thus,
the optimized ME formulations were safe to be used for transder-
mal drug delivery.
3.6. Ex vivo transport of DE from optimized formulations into rat
skin and ability to form in situ drug depot
Ex vivo skin transport from the optimized drug-loaded ME,
PMBG and the market formulation (Flector gel) through newly
born rat skin are illustrated in Fig. 7.
One gram of each formulation was placed on skin of newly
born rat attached to a cylindrical tube having surface area 4.9 cm2 .
Amongst the formulations tested, the optimized drug-loaded ME
showed the highest cumulative amount of DE permeated after
8 h (345.45 g/cm2 29.8) followed by PMBG (57.45 g/cm2 9.8)
and nally the market formulation (9.45 g/cm2 2.9). The con-
tent of the surfactants mixture in MEs signicantly enhanced the
transport of drug through skin. Moreover, the small globule size
of the ME droplets also affects the percutaneous absorption of the
drug. When the droplet size is very small, there is a chance that the
number of vesicles that can interact with a xed area of stratum
corneum to increase, thereby increasing the efciency in percuta-
neous uptake (Shah et al., 2010). Thus, the high skin transport of DE
from ME is mainly due to the amount of drug solubilized in small oil
globules that easily transport through the lipid of stratum corneum
of the skin. Although containing the same surfactant mixture and
globule size as ME, PMBG showed signicant reduction in drug
transport in skin. An explanation for this observation may be due
to the high water content of PMBG (about 75%). The major amount
of DE is located in the aqueous phase interacting with Poloxamer
micelles and consequently lower transport rate through the skin
was observed (Xuan, 2011). The explanation is useful also for the
poor skin transport of DE from Flector gel compared with ME for-
mulations especially when we know that composition of Flector
gel contains about 75% water with nonionic surfactant such as PEG
400 monostearate. The lack of oil globules in the market product
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Fig. 7. Permeation proles of DE from ME, PMBG and Flector gel through rat skin.

explains the signicant lowering of DE skin transport compared to

PMBG.
To conrm that DE containing oil globules transport through
stratum corneum and act as in-skin drug depot for sustained deliv-
ery of drug, release of DE from the skin was observed after removal
of formulations after 3 h of permeation from the donor compart-
ment. As shown in Fig. 7, the cumulative amount of DE permeated
from ME (removed 3 h after permeation from donor compartment)
was continuously increased with lower rate compared with that
of normal ME. This observation conrms that skin acts as drug
reservoir (in-skin depot) after removal of ME. In case of PMBG
and Flector gel, there was no remarkable increase of the cumula-
tive amount permeated of DE after removal of these formulations
from donor compartment. Thus, the gel matrix rather than skin
acts as drug reservoir for these formulations. Contrary to ME, the
availability of the gel on skin surface is very important to main-
tain anti-inammatory efcacy. To conrm the previous results,
the three formulations were subjected to in vivo study for treating
inamed rat skin.

Fig. 8. Anti-inammatory efcacy of drug-loaded ME and PMBG compared to the

3.7. In vivo study of the anti-inammatory efcacy and sustained market product in rat paw edema.
delivery of in-skin depot of DE

The anti-inammatory efcacy of DE was taken as a measure

of in-skin depot formation and the extent of transport of drug
through the skin from the medicated formulations (ME, PMBG and
Flector gel). After 3 h of formulation application (sufcient time
for skin loading and formation of in-skin drug depot), formulations
remaining on the surface of the paw were wiped off with cotton.
Rat hind paw edema was used as a model for inammation
in this study (Winter et al., 1963). Results revealed that injection
of carrageenan (selected as inammagen) produced a pronounced
edema. Formulations applied to inamed area were removed after
3 h of application. Thus, the higher the amount of DE loaded into
skin (in-skin drug depot), the extended transdermal drug delivery
and the higher is the anti-inammatory efcacy as the skin itself
acts as drug reservoir.
The anti-inammatory efcacy of single dose application of
DE-loaded ME and PMBG was tested compared to Flector gel con-
taining the same concentration on the carrageenan induced rat hind
paw edema at different time intervals up to 12 h using plain base
as a control. As shown in Fig. 8, the inhibition of edema started 5 h
after formulation application (2 h after formulation removal). The
highest inhibition of edema was observed in case of drug-loaded ME
where the effect was sustained to 12 h and was signicantly differ-
ent from that of plain base 6 h (p < 0.05). Fig. 9 shows photoimages
of right hind rat paw showing edema before and six hours after
application of drug-loaded microemulsion. These results correlate
well with results previously obtained by ex vivo skin transport
study conrming that oil globules of ME containing solubilized DE
penetrate through stratum corneum and act as in-skin drug depot
for the sustained delivery of drug in the skin. This is highly useful
in dermal and transdermal deliver of drugs where the delivery sys-
tem is applied onto skin for few hours (at night before sleeping)
to load skin with drug then the in-skin depot continue the trans-
dermal delivery of the drug. This is advantageous in case of skin
where topical application of conventional systems for treatment
of soft tissue injuries is highly frequent reaching up to four times
daily. The inhibitory effect of PMBG and Flector gel was remark-
ably lower than that of ME conrming that these gels act as the

Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009
 ARTICLE IN PRESS
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IJP-13397; No. of Pages 10
S.A. Fouad et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx
Fig. 9. Photoimages of right hind rat paw showing edema before and 6 h after application of drug-loaded microemulsion.
drug reservoir and its availability on the skin surface is a must to
sustain the delivery of the drug into the skin.
4. Conclusion
In this study, ME and PMBG of DE were prepared and in vitro
evaluated. D-optimal mixture experimental design was applied in
order to rapidly obtain the optimal DE-loaded ME formulations
containing maximum amount of oil having minimum globule size
which allow transport of drug into skin forming in-skin depot
for sustained transdermal delivery of the drug. The optimized ME
formulation composed of 30% Capryol , 50% Smix (a mixture of
Labrasol /Transcutol , 1:2 w/w) and 20% water. The stability of the
optimized formulation was retained after storage at 40 C/75% RH
for three months. The ME formulations showed no skin irritation
and are safe to be used for transdermal drug delivery. Contrary
to PMBG and Flector gel, the optimized ME showed the highest
cumulative amount of DE permeated after 8 h and the release of DE
from the skin was observed even after removal of ME applied to the
skin. The high skin transport of DE from ME is mainly due to the
amount of drug solubilized in small oil globules that easily trans-
port through the lipid of stratum corneum of the skin. The in vivo
anti-inammatory efcacy in rat paw edema was sustained after
removal of ME applied to the skin conrming the formation of in-
skin drug depot. The signicant increase in DE transport through
the skin and the formation of in-skin drug depot by the developed
ME propose that the prepared system could be promising to sustain
the transdermal delivery of DE for treatment of soft tissue injuries.
The extended transdermal delivery of the optimized ME and clini-
cal evaluation on human patients with musculoskeletal pain needs
to be investigated.
Acknowledgements
We are very grateful for Marcyrl for Pharmaceutical Industries
and Gattefosse for providing the required chemicals for research
work. We are also grateful to Dr. Ayman El-Sahar (Department of
Pharmacology and Toxicology, Faculty of Pharmacy, Cairo Univer-
sity), for his kind help in the in vivo study in this paper.
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Please cite this article in press as: Fouad, S.A., et al., Microemulsion and poloxamer microemulsion-based gel for sustained
transdermal delivery of diclofenac epolamine using in-skin drug depot: In vitro/in vivo evaluation. Int J Pharmaceut (2013),
http://dx.doi.org/10.1016/j.ijpharm.2013.06.009