Original Research

Topical delivery of aceclofenac as

nanoemulsion comprising
excipients having optimum
1. Introduction
2. Materials and methods
emulsification capabilities:
3. Results and discussion preparation, characterization and
in vivo evaluation
4. Conclusion
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Sandipan Dasgupta, Sanjay Dey, Supratik Choudhury &

Bhaskar Mazumder†
†
Dibrugarh University, Department of Pharmaceutical Sciences, Assam, India

Objective: The aim of the present study was to investigate the potential of a
nanoemulsion for topical delivery of aceclofenac using different excipients
having optimum emulsifying ability rather than their solubilizing capacity.
Methods: The oil-in-water nanoemulsions were prepared by screening the
excipients from the nanoemulsion region of pseudoternary phase diagram.
The prepared nanoemulsions were subjected to different thermodynamic
stability tests. The nanoemulsion formulations that passed thermodynamic
For personal use only.

stability tests were characterized for viscosity, droplet size, transmission

electron microscopy, refractive index and in vitro skin permeation. The
in vitro skin permeation profile of optimized nanoemulsion formulation
(NE31, containing 23.85% Polyoxy-35-castor oil, 7.95% PEG 400 and 13.6%
Triacetin) was compared with that of nanoemulsion gel (NG31) and marketed
gel formulation (HIFENAC GEL (HIG)). In vivo anti-inflammatory efficacy
studies were also carried out for NE31, NG31 and HIG.
Results: The significant (p < 0.001) increase in in vitro permeability and in vivo
anti-inflammatory efficacy of the NG31 formulation was observed as
compared with HIG formulation.
Conclusion: It can be concluded that the selection of surfactant and cosurfac-
tant on the basis of their emulsification capabilities other than the solubiliz-
ing capacity of drug is an important criterion for the formulation
of nanoemulsion.

Keywords: aceclofenac, emulsification capability, nanoemulsion, topical delivery

Expert Opin. Drug Deliv. (2013) 10(4):411-420

1. Introduction

In the last few years, the delivery of highly lipophilic drugs in the form of nanoe-
mulsion have gained a potential interest to most research scientists due to its certain
advantages like ease of formulation, non-toxicity and optimum permeable capacity
through skin.
Osteoarthritis (OA) is the most common disease after the age of 65 in about 60%
of men and 70% of women [1]. OA is primarily a non-inflammatory degenerative
joint disease characterized by progressive loss of articular cartilage, subchondrial
bone sclerosis, osteophyte formation, changes in the synovial membrane and an
increased volume of synovial fluid with reduced viscosity and hence, changed lubri-
cation properties. Current treatment options for OA are limited. They include

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All rights reserved: reproduction in whole or in part not permitted
 S. Choudhury et al.
intra-articular (IA) injection of glucocorticoids and hyalur-
onic acid (HA) preparations or symptomatic treatment with
simple non-steroidal anti-inflammatory drugs (NSAIDs) [2].
Due to the localized nature of the disease, the IA injection
of glucocorticoids and HA seems to be an attractive approach
for OA, but they provide only short-term pain relief and/
or often do not provide adequate pain relief and have no
patient compliance [2].
On the other hand, rheumatoid arthritis (RA) is a wide-
spread chronic systemic inflammatory disease that is primarily
characterized by inflammation of the synovium with destruc-
tion of cartilage and bone as the disease progresses. More
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severe disease may be associated with vasculitis, pericarditis,
pleural effusion, pulmonary interstitial fibrosis, peripheral
neuropathies, subcutaneous and pulmonary nodules and
scleritis [3,4]. Since there is no cure, symptomatic treatment
is the only choice to reduce the pain and inflammation.
Formulations of NSAIDs are the first-choice remedies which
would fulfill the need of providing significantly long
remission of pain with improved patient compliance.
Aceclofenac is an NSAID, which is used for the treatment
of OA and RA [5-7]. It comes under the category II drug
under Biopharmaceutical Classification System (BCS). It
has an intermediate half-life of 3 -- 4 h and it requires fre-
For personal use only.
quent oral dosing because of its short half-life. It undergoes
substantial first-pass metabolism. Oral administration of ace-
clofenac may lead to various side effects like gastrointestinal
ulcer and bleeding on chronic use which results in anemic
condition. Therefore, the topical delivery of aceclofenac
would be possible alternative offering distinct advantages
such as elimination of the absorption variable rate, first-
pass intestinal and hepatic metabolism inherent with oral
dosing and delivering the drug directly to the inflamed
site and thereby, producing high local concentrations and
avoiding the side effects [8].
The designing of a topical drug delivery system of aceclofe-
nac could not only increase payload of drug locally and
improve the release of drug for a prolonged period of time
but also reduce the risk of systemic toxicity. Nanoemulsions,
owing to their small size, provide a large interfacial area for
rapid drug release, thereby enhancing bioavailability, reducing
dose size, providing consistent temporal profiles of drug
absorption and protecting the drugs from the hostile environ-
ment of the body. In addition, nanoemulsions could be used
as a reservoir for sustained release of drug for prolonged
period of time, thus avoiding high concentration of drug in
the blood [9,10]. Though, nanoemulsion formulations of ace-
clofenac have been already reported and the selection of ingre-
dients for the formulation of nanoemulsion was made based
on their solubilizing capacity rather than their emulsification
ability [11-13]. The present research work has enlightened the
selection of ingredients for the formulation of nanoemulsion
based on their emulsification capacity rather than their
solubilizing properties and their potential on aceclofenac
permeability through topical route.
412 Expert Opin. Drug Deliv. (2013) 10(4)
The present research work was aimed to explore the feasibil-
ity of nanoemulsion as a novel carrier system for topical
application of aceclofenac for modulation of drug release.
The in vitro permeation of drug and anti-inflammatory
activity of nanoemulsion gel were also evaluated.
2. Materials and methods
2.1 Materials
Aceclofenac was obtained as gift sample from Cipla Ltd.
(Sikkim, India). Isopropyl myristate (IPM), oleic acid, castor
oil, ethyl oleate, Tween 80 and Cremophore EL were pur-
chased from HiMedia (Mumbai, India). Span 80 was pur-
chased from Loba Chemicals (Mumbai, India). Glycerol
triacetate (Triacetin) was purchased from E-Merck (Mumbai,
India). HIFENAC GEL (HIG) (30 g; batch no. M12 02/12;
manufactured by Intas Pharmaceutical Ltd., Ahmedabad,
India) was purchased from local pharmacy. All other chemicals
used in the study were of analytical grade.
2.2 Screening of oil
The solubility of aceclofenac in various oils was determined
by adding an excess amount of drug in 5 ml of selected oils
(castor oil, Triacetin, IPM, ethyl oleate, oleic acid) and kept
under magnetic stirring at temperature of 25 ± 1.0
C for
72 h. The equilibrated sample was centrifuged at 3000 rpm
for 15 min to separate the undissolved drug. The supernatant
was taken and filtered through 0.45 µm membrane filter. The
concentration of aceclofenac in oil was determined using
UV-Visible spectrophotometer (UV-1700, Shimadzu, Tokyo,
Japan) at 273 nm.
2.3 Screening of surfactants
The emulsification ability of the surfactants was evaluated by
adding 300 mg of surfactant into 300 mg of the selected oily
phase. The mixture was gently homogenized at 45 -- 60
C.
The isotropic mixture of 50 mg was accurately weighed and
diluted with distilled water to yield a fine emulsion volume
of 50 ml. The ease of formation of emulsion was monitored
by noting the number of volumetric flask inversions required
to give uniform emulsion. The emulsions were allowed to
stand for 2 h to note for any change in turbidity through
visual observation and their transmittance was assessed at
273 nm by colorimeter (6051 Jenway, UK) using distilled
water as blank.
2.4 Screening of cosurfactant
The cosurfactant was selected by mixing 100 mg of cosurfac-
tant in 200 mg of the previously selected surfactant and the
surfactant--cosurfactant mixture (Smix) was added to the
selected oily phase. The mixture was gently heated at
45 -- 60
C for homogenizing the components. The 50 mg
of isotropic mixture was accurately weighed and diluted
with distilled water to 50 ml to yield fine emulsion. The
ease of formation of emulsions was monitored by noting the
 Topical delivery of aceclofenac as nanoemulsion comprising excipients having optimum emulsification capabilities
number of volumetric flask inversions required to give
uniform emulsion. The resulting emulsions were observed
visually for the relative turbidity. The emulsions were allowed
to stand for 2 h to note for any change in turbidity and their
transmittance was assessed at 273 nm by colorimeter using
distilled water as blank. As the ratio of cosurfactants to surfac-
tant/s is the same, the turbidity of resulting nanoemulsions
will help in assessing the relative efficacy of the cosurfactants
to improve the nanoemulsification ability of surfactant/s.
2.5 Pseudoternary phase diagram
On the basis of the solubility studies of drug, Triacetin,
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Polyoxy-35-castor oil and PEG 400 were selected as the oil
phase, surfactant and cosurfactant, respectively. Distilled
water was used as an aqueous phase. Surfactant and cosurfac-
tant (Smix) were mixed in different weight ratios (1:3, 1:2, 1:1,
2:1 and 3:1). These Smix ratios were chosen in increasing con-
centration of surfactant with respect to cosurfactant and
increasing concentration of cosurfactant with respect to sur-
factant for detailed study of the phase diagrams. For each
phase diagram, oil and Smix at specific ratio was mixed thor-
oughly in different weight ratios from 1:9 to 9:1 in different
glass vials. Sixteen different combinations of oil and Smix,
1:9, 1:8, 1:7, 1:6, 1:5, 2:8 (1:4), 1:3.5, 1:3, 3:7 (1:2.3), 1:2,
For personal use only.
4:6 (1:1.5), 5:5 (1:1), 6:4 (1:0.7), 7:3 (1:0.43), 8:2 (1:0.25),
9:1 (1:0.1), were made so that maximum ratios were covered
for the study to delineate the boundaries of phases precisely
formed in the phase diagrams. Pseudoternary phase diagrams
were developed using aqueous titration method. Slow titra-
tion with aqueous phase was performed for each weight ratio
of oil and Smix and visual observations were made for trans-
parent and easily flowable oil-in-water (o/w) nanoemulsions.
The physical state of the nanoemulsion was marked on a
pseudo-three-component phase diagram with one axis repre-
senting aqueous phase, the other representing oil and the third
representing a mixture of surfactant and cosurfactant at fixed
weight ratios.
2.6 Selection of nanoemulsion formulations
From each phase diagram constructed, different formulas
were selected from the nanoemulsion region so that the drug
could be incorporated into the oil phase; 1.5% (m/m) of
aceclofenac, which was kept constant in all the selected
formulations, was dissolved in the oil phase of nanoemulsion
formulation. Selected formulations were subjected to different
dispersion stability tests.
2.7Thermodynamic stability studies
To overcome the problem of metastable formulation, thermo-
dynamic stability tests were performed, which were as follows:
i) Centrifugation: Selected formulations were centrifuged
at 3500 rpm for 30 min.
ii) Heating and cooling cycle: Those formulations that did
not show any phase separation on centrifugation were
Expert Opin. Drug Deliv. (2013) 10(4)
taken for heating and cooling cycle. Six cycles between
refrigerator temperature of 4
C and 45
C with storage at
each temperature of not less than 48 h were studied. Those
formulations which were stable at these temperatures were
subjected to freeze-thaw cycle test.
iii) Freeze-thaw cycle: Three freeze-thaw cycles were done
between -21
C and +25
C with storage at each tempera-
ture for not less than 48 h for the formulations.
Those formulations which passed these thermodynamic
stability tests were selected for further studies.
2.8 Characterization of nanoemulsion
2.8.1 Droplet size analysis
The globule size of the nanoemulsion was determined by
photon correlation spectroscopy. The nanoemulsions (0.1 ml)
were diluted with 50 ml of water in a volumetric flask and gently
mixed by inverting the flask. Measurement was done using a
Zetasizer 1000 HS (Malvern Instruments, UK). Light scattering
was monitored at 25
C at a 90
angle. Droplet size and
polydispersity index were obtained as the average of
three measurements.
2.8.2Viscosity
The viscosity of the formulations (0.5 ml) was determined
without dilution using Brookfield DVE viscometer (Brook-
field Engineering Laboratories, Inc., Middleboro, MA,
USA) using spindle no. 63 at 25 ± 0.5
C. The software
used to calculate the viscosity was Rheocalc V2.6. Average
and standard deviation of three data at shear rate of 120.0 s-1
were reported.
2.8.3 Transmission electron microscopy
The surface topography of the nanoemulsions were studied
using transmission electron microscopy (TEM). A JEMCX
100II operating at 200 kV capable of point-to-point resolu-
tion was used. A combination of bright-field imaging at
increasing magnification and of diffraction modes was used
to reveal the form and size of the nanoemulsion. To perform
the TEM observations, the nanoemulsion formulations were
diluted hundred times with water. A drop of the diluted nano-
emulsion was directly deposited on the holey film grid and
observed after drying.
2.8.4Refractive index
The refractive index of placebo formulations and drug-loaded
formulations was determined using an Abbes-type refractometer
(Macro Scientific Works, Delhi, India).
2.8.5In vitro skin permeation studies
In vitro permeation studies were carried out on modified
Keshary-Chien diffusion cell with an effective diffusional
area of 3.14 cm2 and 100 ml receiving chamber capacity,
using rat abdominal skin. The full thickness of rat skin was
413
 S. Choudhury et al.
Table 1. Solubility of aceclofenac in different oils*.
Oil Solubility (mg/ml)
Castor oil 45.13 ± 0.97
Ethyl oleate 22.19 ± 0.16
IPM 3.42 ± 0.10
Oleic acid 4.70 ± 0.05
Triacetin 49.89 ± 0.18
*Mean ± SD, n = 3.
IPM: Isopropyl myristate.
excised from the abdominal region and hair were removed with
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an electric clipper. The subcutaneous tissue was removed surgi-
cally and the dermis side was wiped with isopropyl alcohol to
remove adhering fat. The cleaned skin was washed with dis-
tilled water and stored at -21
C until further use. The skin
was brought to room temperature and mounted between the
donor and receiver compartment of Keshary-Chien diffusion
cell where the stratum corneum side was facing the donor com-
partment and the dermal side was facing the receiver compart-
ment. The receiver chamber was filled with methanolic
phosphate buffer saline pH 7.4 (30:70%, v/v). The receiver
fluid was stirred with magnetic stirrer at a speed of 100 rpm
and the temperature was maintained at 37 ± 1
C. One milliliter
For personal use only.
of nanoemulsion formulation (1.5% mg/ml aceclofenac) or 1 g
of aceclofenac gel (1.5% mg/g) was placed into the donor com-
partment and sealed with paraffin film to provide occlusive
conditions. Samples were withdrawn at regular intervals (0.5,
1, 2, 4, 8, 12, 18, 24 h), filtered through 0.45 µm membrane
filter and analyzed for drug content by UV-Visible
spectrophotometer (UV-1700) at 273 nm.
2.8.6 Preparation of nanoemulsion gel formulation
On the basis of permeation study, nanoemulsion formulation
of NE31 was formulated to gel formulation as it shows highest
permeability through rat skin. One gram of Carbopol 934P
was dispersed in sufficient quantity of distilled water. After
complete dispersion, the solution was kept in dark for 24 h
for complete swelling of Carbopol 934P. The aceclofenac-
loaded nanoemulsion (NF31) was slowly added to the viscous
solution of Carbopol 934P under magnetic stirring. The 1%
w/v of Carbopol 934P was used for the formulation of gel.
The pH was adjusted between 6 and 8 using 0.5 ml of trietha-
nolamine and 1 ml of PEG 400 was incorporated to prevent
the evaporation of moisture. The nanoemulsion gel formula-
tion was kept at ambient condition after which in vitro perme-
ation study was performed using similar procedure as
mentioned in Section 2.8.5. The in vitro permeation study
was also carried out for marketed gel formulation (HIG).
Permeation data analysis
2.8.7
The permeation profiles were constructed by plotting the
cumulative amount of aceclofenac permeated per unit dialysis
membrane area (µg/cm2) versus time. The steady-state flux
414 Expert Opin. Drug Deliv. (2013) 10(4)
(Jss, µg/cm2/h) [14] of aceclofenac was calculated from the
slope of the plot using linear regression analysis. The perme-
ability co-efficient (Kp) of the drug through the membrane
was calculated using the following equation [15]:
Jss
Kp =
C
Where, C is the initial concentration of the drug in the
donor compartment.
The penetration-enhancing effect was calculated in terms of
enhancement ratio (Er) by using the following equation:
Jss of nanoemulsion formulation
Er =
Jss of control (HIG)
2.9 Skin irritation study
Skin irritation study was performed on four Wistar albinos.
About 5 cm2 area of hair from the back side were depleted
with the help of depilatories and area was marked on both the
side. One side served as control while other as test. After 24 h,
nanoemulsion dispersion (NE31) and nanoemulsion gel for-
mulation (NG31) were applied (500 mg/rat). Once a day for
7 days observation were made for any sensitivity and the
reaction if any was graded as: A: no reaction; B: slight, patchy
erythema; C: moderate but patchy erythema; D: moderate
erythema; E: severe erythema with or without edema.
2.10 In vivo efficacy study
The anti-inflammatory activity was evaluated using the
carrageenan-induced hind paw edema method developed by
Winter [18]. Wistar albino rats (six rats each group) of either
sex weighing 120 -- 150 g were used for these studies. Prior
to the experiment, the approval was taken from the Institu-
tional Animal Ethical Committee, M.K.C.G. Medical College
and Hospital, Odisha, India. All the rats were divided into four
groups: Group I: served as control; Group II: rats were treated
with HIG; Group III: rats were treated with nanoemulsion
(NE31) and Group IV: rats were treated with nanoemulsion
gel (NG31). The animals were housed in institutional animal
house under standard conditions with free access to food and
water. Inflammation was induced by sub-plantar carrageenan
injection and after 1 h, formulations were applied topically
on the inflamed paw of rats by gently rubbing with index fin-
ger and the volume of the paw was measured. The thickness of
the paw was measured using a digital plethysmometer (Orchid
Scientifics, Nasik, India) before injection (0 h) and at regular
intervals (1, 2, 4, 8, 12, 24 h) after injection. The amount of
paw swelling was determined for 24 h and expressed as percent
edema relative to the initial hind paw volume. Percent inhibi-
tion of edema produced by each formulation-treated group
was calculated against the respective control group.
2.11 Statistical analysis
Statistical analysis of anti-inflammatory activity was done by
means of one-way ANOVA (analysis of variance) followed
 Topical delivery of aceclofenac as nanoemulsion comprising excipients having optimum emulsification capabilities

Table 2. Emulsification capability of surfactants and cosurfactants.

Surfactants Maximum number % Cosurfactants + Maximum number %

of inversions* Transmittance* Cremophor EL of inversions* Transmittance*

Cremophor EL 21 ± 2 90 ± 4 PEG 400 11 ± 1 98 ± 2

Span 80 42 ± 3 15 ± 2 Span 80 36 ± 4 42 ± 3
Tween 80 37 ± 2 47 ± 2 Tween 80 29 ± 2 64 ± 2
PEG 400 30 ± 1 62 ± 3

*Mean ± SD, n = 3.
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S:CoS = 1:1 S:CoS = 1:2 S:CoS = 1:3

Smix Smix
A. B. Smix C.
10 10
90 10 90
90
20 20
80 20 80
80
30 30 30
70 70 70
40 40 40
60 60 60
50 50 50
50 50 50
60 60 60
40 40 40
70 70 70
30 30 30
80 80 80
20 20 20
90 90 90
10 10 10
Aqueous Oil Aqueous Oil Aqueous Oil
10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90
For personal use only.

S:CoS = 2:1 S:CoS = 3:1

Smix Smix
D. E. 10
10 90
90
20
20 80
80
30
30 70
70
40
40 60
60
50 50
50
50
60 60
40
40
70 70
30 30
80 80
20 20
90 90
10 10

Aqueous Oil Aqueous Oil

10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90

Figure 1. Pseudoternary phase diagrams showing the o/w nanoemulsion (shaded area) regions of Triacetin (oil), Cremophore
EL (surfactant), PEG 400 (cosurfactant) at different Smix ratios A) Smix 1:1, B) Smix 2:1, C) Smix 3:1, D) Smix 2:1 and E) Smix 3:1.

by Dunnett’s t-test; p value < 0.05 was considered oil [16,19]. The solubility of aceclofenac in different oils is
statistically significant. presented in Table 1. Among the various oils, the solubility of
aceclofenac was found to be highest in Triacetin (49.894 ±
3. Results and discussion 0.184 mg/ml). Therefore, Triacetin was selected as the oil phase
for the development of nanoemulsion formulation.
3.1 Selection of excipients The selection of surfactant or cosurfactant in the further
The important criteria for the selection of excipients for the study was governed by their ability to reduce interfacial ten-
formulation of nanoemulsion should be pharmaceutically sion to a small value to aid the dispersion process during the
acceptable, non-sensitizing and non-irritating to the skin preparation of the nanoemulsion. The emulsification capabil-
and should fall into the category of generally regard as safe ity of different surfactant and cosurfactant is presented
(GRAS) [17]. The selection of suitable oil having maximum in Table 2. These studies indicated that Polyoxy-35-castor
solubilizing capacity is very important, as the drug in the nano- oil had very good ability to emulsify aceclofenac followed by
emulsion present in a solubilized form and the drug loading in PEG 400, Tween 80 and Span 80. The hydrophile--lipophile
the nanoemulsion is greatly influenced by the solubility in balance (HLB) values of the surfactants used in the

Expert Opin. Drug Deliv. (2013) 10(4) 415

 S. Choudhury et al.
Table 3. Composition of selected nanoemulsion formulations.
Formulation Oil:Smix ratio Smix ratio
NE11 1:3.03 1:1
NE12 1:3.5 1:2
NE13 1:2.34 1:3
NE21 1:4.95 2:1
NE31 1:2.33 3:1
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For personal use only.
Figure 2. Transmission electron microscopic image of
aceclofenac nanoemulsion.
investigation were above 10 except for Span 80. An important
criterion for selection of the surfactants is that the HLB value
of the surfactant should be greater than 10 to form
o/w nanoemulsion. In the present study, the authors have
selected Polyoxy-35-castor oil as surfactant. The addition of
cosurfactant is necessary to achieve transient negative interfa-
cial tension and fluid interfacial film. The cosurfactant
decreases the bending stress of interface and allows the interfa-
cial film sufficient flexibility to take up different curvatures
required to form nanoemulsion over a wide range of composi-
tion. Among the different cosurfactants tested to evaluate the
emulsification capacity with Cremophore EL, the PEG
400 was found to have more emulsification capacity than
Span 80 and Tween 80 (Table 2). Therefore, PEG 400 was
selected as cosurfactant for the preparation of nanoemulsion.
3.2 Construction of pseudoternary phase diagram
The construction of pseudoternary phase diagram makes it
easy to determine the range of concentrations of components
at which nanoemulsion form. Phase diagrams were con-
structed to determine the individual component ratio for the
formulation of an o/w emulsion consisting of Triacetin,
Cremophore EL and PEG 400 as oil phase, surfactant and
cosurfactant, respectively.
Polyoxy-35-castor oil and PEG 400 were mixed in different
volume ratios (3:1, 2:1, 1:1, 1:2, 1:3) to form the Smix. These
416 Expert Opin. Drug Deliv. (2013) 10(4)
Components (% m/m)
Oil Surfactant Cosurfactant Water
7.7 11.7 11.7 68.9
7.4 8.6 17.3 66.7
6.6 3.9 11.6 77.9
11.11 36.7 18.3 33.89
13.6 23.9 7.9 54.6
Smix ratios were chosen to reflect increasing concentrations of
cosurfactant with respect to surfactant and increasing concen-
trations of surfactant with respect to cosurfactant for detailed
study of the phase diagrams in the nanoemulsion formation.
The shaded portion of the phase diagram indicated the
nanoemulsion region and it was increased with an increase
in the concentration of surfactant (Figure 1). The maximum
and minimum nanoemulsion region was obtained with the
Smix ratio of 3:1 and 1:1, respectively. As the surfactant
concentration was increased in the Smix ratio from 2:1 to
3:1, a higher nanoemulsion region was observed, perhaps
because of further reduction of the interfacial tension, increas-
ing the fluidity of the interface, thereby increasing the entropy
of the system. There may be greater penetration of the oil
phase in the hydrophobic region of the surfactant mono-
mers [20,21]. The nanoemulsion region formed by Smix ratio
of 4:1 was very small which may be due to absence of
sufficient quantity of cosurfactant. Hence, the Smix ratio of
4:1 was avoided.
3.3 Selection of nanoemulsion formulations
Aceclofenac is a highly lipophilic drug, and its physicochemi-
cal properties suggest that it has good potential for transdermal
drug delivery. Therefore, the composition of nanoemulsion
formulation should be such that it must be free from skin irri-
tation. It is well known that large amounts of surfactants cause
skin irritation [22,23]; therefore, it is very important to deter-
mine the surfactant concentration properly and use the
optimum concentration of surfactant in the formulation.
The pseudoternary phase diagrams were carefully observed
and from them the formulations in which the amount of oil
phase completely solubilized the drug and which could accom-
modate the optimum quantity of Smix and distilled water
were selected for the study. The composition of selected
formulations is shown in Table 3.
3.4 Thermodynamic stability tests
Nanoemulsions are thermodynamically stable systems and are
formed at a particular concentration of oil, surfactant and
water, which make them stable to prevent phase separation,
creaming or cracking (Table 4). Therefore, the different
formulations were subjected to stability studies by using
centrifugation, heating-cooling cycle and freeze-thaw cycle.
 Topical delivery of aceclofenac as nanoemulsion comprising excipients having optimum emulsification capabilities

Table 4. Thermodynamic stability studies (centrifugation, heating-cooling cycle and freeze-thaw cycle), droplet
size, polydispersity index, viscosity, refractive index of fresh and placebo different nanoemulsion formulations.

Code Centrifugation Heating-cooling Freeze-thaw Droplet PDI‡ Viscosity Refractive index*

cycle cycle size (nm)* (mPa s)*
Fresh Placebo

NE11 ü ü ü 58.17 0.228 130.61 ± 0.31 1.359 ± 0.002 1.353 ± 0.002

NE12 ü ü ü 64.75 0.431 112.80 ± 0.21 1.372 ± 0.009 1.374 ± 0.004
NE13 ü ü - Not performed
NE21 ü ü ü 41.45 0.224 221.42 ± 0.22 1.389 ± 0.010 1.384 ± 0.001
NE31 ü ü ü 39.48 0.230 339.51 ± 0.31 1.375 ± 0.003 1.372 ± 0.002

*Mean ± SD, n = 3.
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z
Polydispersity index.
ü : Test passed; -: Test failed.

causes the interfacial film to condense and stabilize, while

Table 5. Permeability parameters of different the cosurfactant causes the film to expand. The polydispersity
nanoemulsion formulations. values of all the formulations were found to be within the
range of 0.22 -- 0.43. The polydispersity value less than
Formulation Jss (mg/cm2/h)‡ Kp  10-2 (cm/h)‡ Er
0.30 indicated the uniformity of droplet size.
HIG* 43.67 ± 2.11 0.29 ± 0.01 -
NE31 254.90 ± 1.25 1.70 ± 0.01 5.84 3.5.3 Viscosity determination
NE21 204.03 ± 4.65 1.36 ± 0.03 4.67
The viscosity of formulation NE31 was found to be the high-
For personal use only.

NE11 187.27 ± 3.26 1.25 ± 0.02 4.29

NE12 162.03 ± 3.67 1.08 ± 0.02
NG31 199.60 ± 6.93 1.33 ± 0.05
*HIG: HIFENAC GEL was used as control.
z
Mean ± SD, n = 3.
Er: Enhancement ratio; Jss: Steady-state flux; Kp: Permeability coefficient.
Only those formulations that survived thermodynamic
stability tests were selected for further study.
3.5 Characterization of nanoemulsions
3.5.1 Transmission electron microscopy
The nanoemulsion appeared dark and with bright surroundings
(Figure 2). The droplet size ranged between 29 and 71 nm and
was in agreement with the droplet size distribution measured
using photon correlation spectroscopy (Table 4).
3.5.2 Droplet size analysis
The droplet size decreased with the increase in oil concentra-
tion in the formulation (Table 4). The droplet size of formu-
lation NE31, containing 13.6% of oil was 39.48 ± 5.06 nm,
which was lower as compared with other formulations. There
was only a slight difference in the mean droplet size of formu-
lations NE31 and NE21, which may be due to the increase in
the concentration of Smix in NE31. The droplet size in the
formulation of NE12 was more as compared with formula-
tion NE11, which may be due to the increased concentration
of cosurfactant. This result is in accordance with the report
that the addition of surfactant to nanoemulsion systems
3.71 est (339.50 ± 0.31 mPa s) compared with other formulations
4.57 (Table 4). This may be due to decreased concentration of
cosurfactant. This deferred from one characteristic of nanoe-
mulsion which is that they are usually of low viscosity.
Instead, a hypothesis can be put forward that due to the
higher viscosity of the nanoemulsions the droplets were not
able to coalesce and thus retained their nanosize. The viscosity
of the formulation NE12 was lower as compared with formu-
lation NE11. This may be due to the lower oil concentration.
3.5.4Refractive index
The values of the refractive index of drug-loaded formulations
and placebo formulations are given in Table 4. It was found
that there were no significant differences of refractive index
when drug-loaded formulations were compared with placebo
formulations. Therefore, it can be concluded that the nanoe-
mulsion formulations were not only thermodynamically sta-
ble but also chemically stable and remained isotropic; thus,
there were no interactions between nanoemulsion excipients
and drug.
3.5.5 In vitro skin permeation studies
The permeation ability of the various nanoemulsion formula-
tions (NE11, NE12, NE21 and NE31), nanoemulsion gel
(NG31) and aceclofenac gel (HIG) were evaluated using
in vitro permeation experiments. The permeation parameters
of nanoemulsion formulations, nanoemulsion gel and
aceclofenac gel are presented in Table 5. The permeation profiles
of NE11, NE12, NE21, NE31, NG31 and aceclofenac gel
through rat skins are shown in Figure 3. In vitro skin permeation

Expert Opin. Drug Deliv. (2013) 10(4) 417

 S. Choudhury et al.

30

Amount of aceclofenac permeated (mg cm-2)

25

20

15

10
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by University Library Utrecht on 03/18/13

0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

NE31 NE21 NE11 HIG NG31

Figure 3. In vitro skin permeation profile of aceclofenac (mean ± SD) from nanoemulsion (NE11, NE21 and NE31),
nanoemulsion gel (NG31) and aceclofenac gel (HIFENAC GEL, HIG) (mean ± SD; n = 3).
For personal use only.

Table 6. Anti-inflammatory effects of nanoemulsion (NE31), nanoemulsion gel (NG31) and aceclofenac gel (HIG).

Time (h) In vivo performance Group

I II III IV

1 Edema* (%) 36.50 ± 0.02 34.09 ± 0.02z 29.90 ± 0.01z 33.16 ± 0.02z
Inhibition (%) - 6.61 18.09 9.15
2 Edema* (%) 44.47 ± 0.02 38.89 ± 0.02z 28.39 ± 0.02z 30.89 ± 0.01z
Inhibition (%) - 12.56 36.16 30.55
4 Edema* (%) 46.79 ± 0.02 36.62 ± 0.03z 23.12 ± 0.02z 27.59 ± 0.02z
Inhibition (%) - 21.74 50.59 41.02
8 Edema* (%) 47.81 ± 0.01 36.87 ± 0.02z 18.09 ± 0.02z 22.28 ± 0.02z
Inhibition (%) - 22.89 62.17 53.41
12 Edema* (%) 43.70 ± 0.02 28.28 ± 0.02z 14.82 ± 0.02z 18.73 ± 0.02z
Inhibition (%) - 35.28 66.08 57.13
24 Edema* (%) 37.28 ± 0.02 21.72 ± 0.02z 10.05 ± 0.02z 12.91 ± 0.03z
Inhibition (%) - 41.74 73.04 65.36

Group I: Control group; Group II: Animals treated with aceclofenac gel (HIG); Group III: Animals treated with nanoemulsion (NE31); Group IV: Animals treated
with nanoemulsion gel (NG31).
*Mean ± SD, n = 6.
z
Edema rate (%) values were statistically significant from the saline control using one-way ANOVA followed by Dunnett’s t-test at p < 0.001.
ANOVA: Analysis of variance; HIG: HIFENAC GEL.

rate had the highest rate in the formulation NE31 as compared permeation profiles. The skin permeation profile of
with the aceclofenac gel. The flux (Jss) and permeability coeffi- NE31 was significantly (p < 0.05) different from the skin per-
cient (Kp) of NE31 and NG31 were found to be 254.90 ± meation profile of NE21 but the skin permeation profiles of
1.25 µg/cm/h, 1.70  10-2 ± 0.01 cm/h and 199.60 ± NE21 and NG31 were not significantly different. The signif-
6.93 µg/cm/h, 1.33  10-2 ± 0.05 cm/h. The enhancement of icant (p < 0.05) difference in the aceclofenac permeation
the flux of aceclofenac was increased 5.84 and 4.57 times between nanoemulsion formulation and aceclofenac gel might
for NE31 and NG31, respectively as compared with commer- be due to the smallest size of internal phase droplets [24]. The
cial gel formulation of aceclofenac. Formulations NE11, required flux for aceclofenac was approximately 197.54 µg/
NE12, NE21 and NG31 showed an intermediate skin cm/h and was obtained by formulation NG31 (199.60 ±

418 Expert Opin. Drug Deliv. (2013) 10(4)

 Topical delivery of aceclofenac as nanoemulsion comprising excipients having optimum emulsification capabilities

6.93 µg/cm/h). In order to reach the required flux, the area NE31. The enhanced anti-inflammatory effects of formula-
has to be decreased up to 3.11 cm2. The results of aceclofenac tion NE31 could be due to the enhanced permeation of
permeation from NG31 through the rat abdominal skin con- aceclofenac through the skin.
firmed that aceclofenac was permeated through the rat skin
and hence could possibly permeate through the human skin. 4. Conclusion

3.6 Skin irritation study
The skin irritation study of nanoemulsion dispersion (NE31)
and gel formulation (NG31) were performed to evaluate the
safety of the optimized nanoemulsion formulation. The
mean values of skin irritancy score following 7 days application
for formulation NE31 and NG31 were found to be 1.89 ±
Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by University Library Utrecht on 03/18/13
On the basis of lowest droplet size, lowest polydispersity,
optimum viscosity, optimum surfactant and cosurfactant
concentration highest skin permeation and highest in vivo
anti-inflammatory potential, the authors have selected
NE31 as optimized formulation, which contained Triacetin
(13.6%), Cremophore EL (23.9%), PEG 400 (7.9%) and dis-

0.45 and 1.9 ± 0.5, respectively. Van-Abbe et al. reported
that a value between 0 and 9 indicates that the applied formu-
lation is generally not an irritant to human skin [25]. This value
concluded that the optimized nanoemulsion formulations
were safe for topical delivery of aceclofenac.
3.7 In vivo efficacy study
Based on optimum skin permeation and lowest droplet size
formulation, NE31 was chosen for the study of in vivo anti-
inflammatory effects. The percentage inhibition after 12 h
administration was found to be high for NE31, that is,
73.04% as compared with HIG (41.74%); this difference
For personal use only.
tilled water (54.6%). The surfactant (Cremophore EL) and
cosurfactant (PEG 400) were selected on the basis of their
highest emulsification capacity. Therefore, it can be concluded
that the selection of surfactant and cosurfactant on the basis of
their emulsification capabilities other than solubilizing capac-
ity of drug is an important criterion for the formulation of
nanoemulsion. From the in vitro and in vivo data it can also
be concluded that nanoemulsion formulation NE31 has great
potential for the transdermal delivery of aceclofenac.
Declaration of interest

was significant (p < 0.01). The percent inhibition value for We are thankful to University Grants Commission (UGC),
formulations NG31 was found to be 65.36% (Table 6); the file no. 37-44/2009(SR)(ASM) for their financial support
difference is significant when compared with formulation for this work.

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Affiliation
Sandipan Dasgupta2, Sanjay Dey1,
Supratik Choudhury1 & Bhaskar Mazumder†1
†
Author for correspondence
1
Assistant Professor,
Department of Pharmaceutical Sciences,
Dibrugarh University, Dibrugarh-786004,
Assam, India
Tel: +919435256182;
E-mail: bhmaz@yahoo.co.in
2
Research Scholar,
Dibrugarh University, Department of
Pharmaceutical Sciences, Dibrugarh-786004,
Assam, India