Future Journal of Pharmaceutical Sciences xxx (2017) 1e11

H O S T E D BY Contents lists available at ScienceDirect

Future Journal of Pharmaceutical Sciences

journal homepage: http://www.journals.elsevier.com/future-journal-of-
pharmaceutical-sciences/

Enhanced transdermal permeability of Terbinafine through novel

nanoemulgel formulation; Development, in vitro and in vivo
characterization
Maha E. Elmataeeshy a, Magda S. Sokar b, Mohammed Bahey-El-Din c, Dalia S. Shaker d, *
a
Pharmaceutics Department, Faculty of Pharmacy, Al Asher University, Cairo, Egypt
b
Pharmaceutics Department, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
c
Microbiology and Immunology Department, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
d
Pharmaceutics and Pharmaceutical Technology Department, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt
(FUE), Cairo, Egypt

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

Article history: Terbinafine Hcl (TB) is a poorly water soluble antifungal drug. Topical nanoemulsion based gel containing
Received 21 February 2017 TB was prepared with a view to improve its solubility and antifungal activity. In preparation of the
Received in revised form nanoemulsion (NE), excipients were selected based on the solubility study. Peceol was optimized as the
20 June 2017
oil phase. Tween 80 and propanol were optimized as the surfactant and co-solvent respectively, and were
Accepted 10 July 2017
Available online xxx
mixed (Smix) in different weight ratios (1:1, 1:2, 1:3, 1:4, 4:1, 3:1 and 2:1, respectively). Pseudoternary
phase diagrams were developed and Pecol and Smix were mixed in different weight ratios ranging from
1:9 to 9:1. Based on the NE region of each diagram, the formulae were selected. The formulated nano-
Keywords:
Terbinafine nanoemulsion
emulsions were characterized and evaluated for in vitro drug release and thermodynamic stability. The
Pseudoternary phase diagrams optimum nanoemulsion formulae containing 10 or 15% w/w oil, 45% w/w Smix (1:2/1:3) and 45-40% w/
Permeation study w aqueous phase) were incorporated into Carbopol 940 gel bases forming three different TB nano-
emulsion based emulgel formulae (F1-F3) which were examined for ex vivo drug permeation and in vivo
antifungal activity compared to the marketed product; Lamisil® emulgel. The results showed that TB skin
permeation from all the prepared nanoemulsion based gel formulae was significantly (p < 0.05)
improved in relation to the commercial emulgel. F3 exhibited a superior in vivo antifungal activity over
the marketed emulgel for the treatment of Candida infection.
© 2017 Production and hosting by Elsevier B.V. on behalf of Future University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction materials of less than 20 nm and function creates a drug depot in

One of the many challenges facing formulation scientists is the
development of dermal formulations. For topical delivery, poor
permeability of drugs leads to high cost of therapy and decreased
patient compliance. One of the techniques to overcome such
problem is by lipid based colloidal sub-micron drug delivery. This
technology allows high concentration of drug to penetrate the skin
as the lipophilic intracellular pathway of skin allows penetration of
* Corresponding author. Future University in Egypt, Pharmaceutical Technology
Department, College of Pharmacy, P.O. Box: 11477, Cairo, Egypt.
E-mail addresses: maha_elmataeeshy@hotmail.com (M.E. Elmataeeshy),
magdasokar@yahoo.com (M.S. Sokar), m.bahey-el-din@alexu.edu.eg (M. Bahey-El-
Din), dalia.samuel@fue.edu.eg (D.S. Shaker).
Peer review under responsibility of Future University.
the stratum corneum and epidermis [1].
Nanoemulsion (NE) with uniform distribution of particle size,
offers several advantages for topical and transdermal delivery of
pharmaceutical agents. Rather than being self-administered and
lowering side-effects, nanoemulsion has the ability to efficiently
dissolve lipophilic drugs, enhance skin permeation and extend the
release of lipophilic and hydrophilic drugs. Moreover, it exerts
complete dispersion onto skin and good skin hydration in cosmetic
products [2].
Terbinafine Hcl (TB) is a synthetic allyl amine derivative with
antifungal activity. TB exerts its effect through inhibition of squa-
lene epoxidase, thereby blocking the biosynthesis of ergosterol, an
important component of fungal cell membranes. As a result, this
agent disrupts the fungal cell membrane synthesis and inhibits the
fungal growth. It is used topically for superficial skin infections such

http://dx.doi.org/10.1016/j.fjps.2017.07.003
2314-7245/© 2017 Production and hosting by Elsevier B.V. on behalf of Future University. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
2 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11

as jock itch (tinea cruris), athlete's foot (tinea pedis), and other
types of ringworm (tinea corporis) and Candida species [3]. The
objectives of the present study are improving solubility and anti-
fungal activity of TB through incorporation in nanoemulsion based
gel.
2. Material and methods
2.1. Materials
Terbinafine Hcl was obtained from Apex pharma Egypt. Peceol
oil was obtained as a gift from Gattefosse Co., Lyon, France. Pro-
pylene glycol, Tween 80 and Propanol were purchased from El-Nasr
pharmaceutical chemicals Co., Alexandria, Egypt. Cremophor
RH40® (Polyoxy 40 hydrogenated castor oil) was purchased from
BASF Co., Germany. Maisine 35-1® (Glycerol monolinoleate), Cap-
royl 90® (propylene glycol monocaprylate), Labrafil® M1944CS
(oleoyl macrogol 6-glycerides) and Transcutol were manufactured
by Gattefosse Co., Lyon, France. Carbopol 940 was purchased from
Goodrich Chemical Co (London, England). Lamisil® skin emulgel
(Terbinafine1% w/w, manufactured by Novartis Pharma, Cairo,
Egypt). Lamisil® Solution, 1% (Terbinafine1% w/w, manufactured by
Novartis Pharma, Cairo, Egypt). YPD broth (peptone 20 g/L, yeast
extract 10 g/L, and dextrose 20 g/L) and YPD agar plates were ob-
tained from Oxoid Limited (Basingstoke, Hampshire, UK). Fetal
bovine serum (FBS); was purchased from Biowest, France. Candida
albicans (ATCC® 2091); was obtained from the American Type
Culture Collection, USA. Methylprednisolone acetate injection
(Depo Medrol); was purchased from Pfizer Co., Egypt. All other
chemicals used were of analytical grade.
2.4. Preparation of nanoemulsion formulae
Based on the NE region of each phase diagram different
formulae were selected, the appropriate oils and Smix weight ratio
used in the nanoemulsion were chosen from the constructed phase
diagrams. The composition of selected non medicated nano-
emulsion systems was shown in Table 1 obtaining codes NE-A1, NE-
A2, NE-B1, NE-B2, NE-C1, NE-C2, NE-D1 and NE-D2. The selection of
these ratios was expected to give o/w nanoemulsion systems due to
the relatively low weight ratio of oil to water. For each 5% of oil
selected, the formula that used the minimum concentration of
Smix for its NE formulation was selected from phase diagram [7].
Visual observation was achieved for transparency and physical
state of NE formulae.
2.5. Preparation of TB-nanoemulsion formulae
Twenty mg of TB was weighed (Sensitive electronic balance,
model PB303-S, Mettler Toledo Co., Switzerland) and dissolved in
the Smix at their determined ratios and mixed continuously for
2 min using the vortex mixer. The oil phase was then finally
incorporated into the mixture containing drug and mixed using the
vortex mixer followed by dropwise addition of the specified weight
of water (Table 1) to obtain homogenous TB- nanoemulsion
formulae obtaining new codes (SM1, SM1\, SM2, SM2\, SM3, SM3\,
SM4 and SM4\).
Transparency and physical state of the drug loaded nano-
emulsions were checked for the effect of loading the drug on the
selected formulae.

2.2. Solubility study 2.6. Thermodynamic stability studies

The solubility of TB was determined in various excepients; oils The prepared nanoemulsion formulae were subjected to
(Maisine, Caproyl 90 and Peceol), surfactants (Labrasol, Cremophor different thermodynamic stability tests [8].
RH40 and Tween 80) and co-solvents (Transcutol, Propylene glycol
and Propanol). An excess quantity of TB (0.5 gm) was mixed with
2.6.1. Heating-cooling cycle
2 mL solvent (oils, surfactants and co-solvents) separately in
The nanoemulsion formulae were subjected to six cooling,
stoppered vials for 72 h at 37
C and 100 rpm in isothermal shaker
heating cycles between 4
C and 45
C by storing at each temper-
(Kottermann, type 3047, Hanigsen, Germany). The solvent-drug
ature for 48 h. Samples were then observed for precipitation or
mixture was then centrifuged at 3000 rpm (Fanem, 206-R Centri-
separation.
fuge, Brazil) for 15 min then left to reach equilibrium for 72 h. The
supernatant was filtered through 0.45 mm. Millipore membrane
filters and diluted with methanol. The amount of TB solubilized was 2.6.2. Centrifugation
quantified spectrophotometrically (UV-160A Double Beam Spec- Those formulae which were stable under heating-cooling cycle
trophotometer, Shimadzu, Japan) at lmax 283 nm using methanol as were suspected to centrifugation for 30 min at 3500 rpm. Formulae
blank [4]. that did not show any phase separation after centrifugation were
subjected to freezeethaw cycle.
2.3. Development of pseudoternary diagram

Table 1
On the basis of the solubility study pseudoternary phase dia-
Composition of selected non-medicated/medicated nanoemulsion formulae.
grams were developed in order to obtain the nanoemulsion zone
and determine the concentration ratios of the components of Smix ratio Matrix code Ingredients (% w/w)
nanoemulsion formulae. The Smix weight ratios (1:1, 1:2, 1:3, 1:4, Peceol oil Smix (Tween80/Propanol) Water
2:1, 3:1, 4:1) were screened for nanoemulsion formation. For each 1:1 NE-A1/(SM1) 10 40 50
phase diagram, Peceol and Tween 80/Propanol were combined in NE-A2/(SM1\) 15 45 40
different weight ratios ranging from 1:9 to 9:1 in separate glass 1:2 NE-B1/(SM2) 10 45 45
vials. Slow titration with an aqueous phase in a dropwise manner at NE-B2/(SM2\) 15 45 40
1:3 NE-C1/(SM3) 10 45 45
25
C with vortex (GEMMY, vortex mixer; VM-300, Germany), was
NE-C2/(SM3\) 15 45 40
performed at each weight ratio of 2 gm of Smix and oil phases until 1:4 NE-D1/(SM4) 10 40 50
first turbidity as previously described [5]. All mixtures that formed NE-D2/(SM4\) 15 45 40
transparent o/w systems were marked and plotted on the triangle The total concentration of oil, S/CoS and water in all formulae was 100% (w/w). NE
graph using CHEMIX 3.51 software tool (Arne Standnes, Norway) codes represent non medicated formulae while SM codes represent TB loaded
[6]. nanoemulsion.

Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11
2.6.3. Freeze thaw cycle
The selected formulae were exposed to three freeze-thaw cycles
in between 21
C and þ25
C for 48 h at each temperature which
were observed for phase separation.
2.7. Characterization of optimized TB nanoemulsion formulae
2.7.1. Measurement of droplet size
The average droplet size and polydispersity index (PDI) of the
nanoemulsion formulae were measured by Dynamic Light Scat-
tering technique (DLS) or sometimes called as photon correlation
spectroscopy (PCS) using a Malvern Zetasizer Nano ZS90, UK. The
measurements were performed at a fixed angle of 90
and at 25
C.
About 0.1 mL of drug eloaded nanoemulsion was dispersed in
10 mL of distilled water under gentle stirring in a glass beaker [9].
Then sample cell was filled with the dispersion for droplet size
measurement. Each size value was the mean of triplicate samples
±SD. The PDI was measured for the uniformity of particles diameter
[8].
2.7.2. Zeta potential measurement
Zeta potential of TB nanoemulsions was measured by an elec-
trophoretic light scattering technique using a Malvern Zetasizer
Nano ZS (Malvern Instruments, Ltd., UK) Series ZEN3600 with a
dynamic light-scattering particle-size analyzer at a wavelength of
633 nm by applying 1v electric field. Nanoemulsions were
dispersed in prefiltered, double distilled water at ratio 1:100 [9].
The average ± SD of three independent measurements were re-
ported [8].
2.7.3. Determination of pH
The pH values of TB nanoemulsion formulae were determined
using a digital pH meter (Schott Gerate digital pH meter, model pH-
meter-CG-820, Germany), the measurements of pH data was done
in triplicate [10].
2.7.4. Viscosity determination
Brookfield DV III ultra V6.0 RV Cone and Plate Rheometer
(Brookfield Engineering Laboratories, Inc., Middleboro, MA) using
spindle # CPE40 at 25 ± 0.5
C the spindle speed began at 200 rpm.
The software used for the calculations was Rheocalc V2.6 was used
to measure the viscosity of nanoemulsion formulae (0.5 gm) [10].
2.7.5. Refractive Index
Refractive Index of formulae was measured to evaluate the
nanoemulsion transparency at 25
C using Refractometer Abbe,
Baush and Lomb Optical Company, NY [10].
2.7.6. Transmission electron microscopy (TEM) analysis
Morphology and structure of the nanoemulsion were studied
using TEM (Jeol, JEM-100 CX electron microscope, Japan). Selected
samples are diluted with water (1:25), and is placed on a car-
bonecoated copper grid, stained with 2% uranyl acetate aqueous
solution for 30 s [11].
2.7.7. In vitro drug release study
Drug release from optimized nanoemulsion formulae was
assessed using the dialysis bag method through semipermeable
membrane [12,13]. The dialysis bag (Visking ® 36/32, 27 mm,
MWCO 12000e14000, Serva, USA) was fixed to a 500 mL stoppered
glass container containing 350 mL of the release medium pH 5.5
(methanol 10%/acetate buffer 1:9, respectively) [6]. Considering
sink condition, in a thermostatic shaking water bath at 37 ± 0.5
C
and 50 strokes per minute [14]. The optimized nanoemulsion
formulae (1 mL), equivalent to 10 mg TB, were separately added
Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
3
into the dialysis bag. Samples (3 mL) were withdrawn at fixed time
intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and a corresponding aliquot of the
fresh dissolution medium was replaced. The release of the drug
from the selected formulae was compared with that of aqueous
drug suspension (1 mL) and with the marketed topical Lamisil®
solution (1 mL). The samples were analyzed spectrophotometri-
cally at l max 283 nm. All experiments were performed in duplicate.
2.8. Preparation of Terbinafine nanoemulsion based gel
Nanoemulsion based gel was prepared by dispersing 1 gm car-
bopol 940 in a sufficient quantity of distilled water preheated at
70
C, using a magnetic stirrer (RH-basic, IKA, Germany). After the
complete dispersion, the formed dispersion was kept in the dark for
24 h to swell completely. Triethanolamine was added into the
swollen polymer to adjust its pH. Optimized TB loaded nano-
emulsion formulae (SM2, SM2\ and SM3) were incorporated with
the polymer gel matrix separately in a ratio 1:1 [15] forming
viscous and smooth nanoemulsion based emulgel (F1-F3, respec-
tively) using a magnetic stirrer for 15 min at 250 rpm, whose
composition was depicted in Table 2.
2.9. Characterization of TB nanoemulsion based emulgel
TB nanoemulsion based gel was inspected visually for their
color, homogeneity and consistency. pH value and viscosity were
measured [16].
2.9.1. Drug content
The drug content of the different preparations TB nanoemulsion
based gel (F1-F3) was determined separately by dissolving 0.1 g of
the gel, containing 0.5 mg of the drug in 25 mL of methanol. The
resulting solutions were filtered with 0.45 mL filter to obtain clear
solutions. The drug content was measured spectrophotometrically
at l max 283 nm against methanol as blank [17].
2.9.2. Ex vivo study (permeation study)
The protocol to carry out in vitro permeation studies was
approved by the Institutional Animal Ethics Committee, Faculty of
Pharmacy, Alexandria University, Egypt. In vitro skin permeation
studies were performed on a fabricated Franz diffusion cell with an
effective diffusional area of 3.14 cm2 and 9.5 mL of receiver
chamber capacity using a rat abdominal skin.
The full-thickness of fresh rat skin was excised from the
abdominal region of female albino rats, weighing 200 ± 10 gm and
abdominal skin hair was removed using an electric clipper. The
adipose tissue was removed then the skin was hydrated with
normal saline solution.
The skin sample was mounted between the donor and receiver
compartment of the Franz diffusion cell, where the stratum cor-
neum side faced the donor compartment and the dermal side faced
the receiver compartment. The receiving chamber was filled with
acetate buffer, pH 5.5/methanol 10%; 9:1, respectively [18]. Diffu-
sion cells were placed in a thermostatically controlled shaking
water bath (type 3047; Ko € ttermann, Ha €nigsen, Germany). Water
bath with fixed diffusion cells was shaken at 25 strokes per minute,
while the temperature was maintained at 37 ± 0.5
C as described
in literature [14,19]. One gm (containing 5 mg TB) of each TB
nanoemulgel formulae (F1-F3) was placed separately in the donor
compartment in direct contact with the skin and sealed with
paraffin film to provide occlusive conditions. Samples were with-
drawn at regular time intervals (1, 2, 3, 4, 5, 6, 7 and 12 h), filtered
through 0.45 mm membrane filter and analyzed spectrophotomet-
rically at lmax 283 nm. Withdrawn samples were replaced with an
equal volume of the fresh dissolution medium [20].
4 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11

Table 2
Composition of different TB nanoemulsion based emulgel formulae.

Ingredients (%w/w)

Formulae Code TB-NE Code Terbinafine Hcl Carbopol 940 gel (1%) Peceol oil Smix Smix ratio Water Triethanolamine

F1 SM2 0.5 50 5 22.5 1:2 22.5 q.s

F2 SM2\ 0.5 50 7.5 22.5 1:2 20 q.s
F3 SM3 0.5 50 5 22.5 1:3 22.5 q.s

q.s means quantity sufficient.

Results of permeation study were compared to that from the
commercial product (0.5 gm containing 5 mg TB) under the same
experimental conditions.
2.9.2.1. Permeation and distribution data analysis. The cumulative
amount of TB permeated the albino rat skin (Q, mg/cm2) was plotted
as a function of time (t, h) for the prepared nanoemulsion emulgel
of TB and marketed product. The permeation rate (flux) at the
steady state (Jss, mg/cm2/h) and lag time were calculated from the
slope and intercept of the straight line obtained by plotting the
cumulative amount of TB permeated per unit area of skin versus
time at steady-state condition, respectively. Permeability coeffi-
cient (Kp) was calculated by dividing the flux by initial drug con-
centration (C0) in the donor portion of cell as given below [21].
Kp ¼ Jss /C 0
Enhancement ratio (Er) was calculated by dividing the Jss of the
respective formulation by the Jss of control formulation as given
below:
Er ¼ Jss of formulation/ Jss of control
culture containing 7.6  10 6 colony forming units (cfu) in the
middle of the shaved area of individual rat [22]. Animals were
divided into six groups, each containing six rats. The first group
served as a control receiving no formula, the second was treated
with the placebo vehicle, while the third group was treated with
the commercial TB emulgel (Lamisil®). The fourth, fifth and sixth
groups were treated with the prepared test TB loaded nano-
emulsion Carbopol based emulgel formulae (F1, F2 and F3,
respectively).
Treatments (equivalent to 5 mg drug) were topically applied to
the rats' skin separately once daily for three consecutive days
starting 24 h after infection [24], and results in terms of reduction
in skin fungal burden were compared. All animals were euthanized
24 h after the last treatment dose, where 3.0 cm2 of infected skin
was excised. The infected skin samples were collected, homoge-
nized in sterile saline and plated onto YPD agar plates containing
10 mg/mL chloramphenicol. Plates were incubated for 24 h at
37 ± 1
C, and then cfu values were recorded. Statistical analysis
was carried out by ANOVA test followed by student-Newman-Keuls
multiple comparison post hoc test. Graphpad Instat® version 3.10
was used to perform the statistical analysis.
2.11. Statistical analysis

The results were depicted as mean ± SD. All statistical com-

2.10. In vivo studies parisons were performed using Microsoft Excel 2010. The obtained
data of repeated measurements were subjected to Student's t-test
2.10.1. Preparation of the microorganisms and a p-value  0.05 was considered as significant.
Candida albicans (ATCC® 2091) was grown overnight in YPD
broth (peptone 20 g/L, yeast extract 10 g/L, and dextrose 20 g/L)
3. Results and discussion
containing 10% fetal bovine serum (FBS). After 14e18 h of vigorous
shaking at 30
C, the overnight culture was subcultured into fresh
3.1. Solubility study
YPD broth containing 10% FBS and was further shaken at 37
C for
2 h before skin infection of rats. This procedure was necessary to
Solubility of TB was determined in various oils, surfactants and
convert Candida to the virulent pseudohyphae form which has the
co-solvents mixtures (Fig. 1). Solubilizing capacity of an oily phase
ability to establish significant skin infection [22].
is critical for development of nanoemulsion as it determines drug
loading efficacy [25]. Oily phase screened include; peceol oil,
2.10.2. Preparation of the animal
Female Spargue Dawley rats weighing 190e210 g were used in
the experiment. Animals were housed under standard laboratory
conditions and received pathogen free diet and water ad libitum.
An area of approximately 3.0 cm2 was shaved in the back of each rat
two days before getting infected. To induce a heavy cutaneous
infection, animals were immunosuppressed by intraperitoneal in-
jection (IP) of three doses of methylprednisolone acetate (100 mg/
kg) for three successive days starting 48 h before infection (on
days 1, 0 and þ 1 of C. albicans infection) [23]. All animal exper-
imentation procedures were approved by the Animal Care and Use
Committee (ACUC) of the Faculty of Pharmacy, Alexandria
University.

2.10.3. Induction of C. albicans cutaneous infection and treatment

regime
Rats were intradermally infected with 200 ml of C. albicans Fig. 1. Solubility of TB in different oils, surfactants and co-solvents.

Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11
caproyl 90 and maisine (Fig. 1). Among all the screened oils high
solubilization capacity was exhibited in peceol (62.4 mg/mL ± 3.20)
followed by caproyl 90 with the lowest solubility obtained in
maisine. Therefore, peceol was selected for further investigation.
This difference may be due to the relative hydrophobicity of the oils
as peceol® has the lowest HLB value; 3 compared to high HLB values
of caproyl 90 & maisine; 6 and 4 [26] respectively in which the
hydrophobic TB exhibited the fewest solubility.
Non-ionic surfactants are usually utilized for fabrication of
nanoemulsion as they are less toxic compared to the ionic coun-
terparts [27]. Another important criteria in surfactants' selection is
bioactivity aspect. Some surfactants are recognized as safe (GRAS)
bioactive excipients due to hydrophilic character as Tween 80 or
inhibitory effect on P-glycoprotein as cremophor RH40 [28]. In this
study 3 different non-ionic surfactants; Tween 80, cremophor
RH40 and labrasol were studied. Tween 80 has the highest solu-
bility capacity of TB (27.4 mg/mL±1.88) and it is an HLB value of
14.5, which was suitable for the preparation of oil in water NE.
Different co-solvents namely; Transcutol P, Propylene glycol,
labrafil and propylene glycol were assessed in this study for the
solubilization ability of TB. The results were depicted in Fig. (1) that
demonstrated that the highest solubility for TB was in propanol;
185.2 mg/mL ± 6.1. Consequently propanol was chosen for further
investigation.
3.2. Development of pseudoternary diagram
Pseudoternary phase diagrams were constructed in the absence
of TB to recognize the zone of nanoemulsion and to optimize the
concentration of oil, surfactant and co-solvent in the nanoemulsion
formulae. The ratio of surfactant to co-solvent was very effective for
stable and efficient nanoemulsion formation. The phase diagrams
were constructed at the ratio of surfactant/co-solvent 1:1, 1:2, 1:3,
1:4, 2:1, 3:1 and 4:1 (w/w). The shaded area on each diagram is the
nanoemulsion region. The diagrams showed the biggest nano-
emulsion areas are; 1:1, 1:2, 1:3, 1:4; 50.18%, 50.09%, 49.56% and
52.61%, respectively compared to areas in case of surfactant/co-
solvent 2:1, 3:1, 4:1; 44.25%, 39.59% and 38.35%, respectively as
shown in Fig. 2.
The larger the area of the nanoemulsion field, the greater the
nanoemulsification efficiency of system [9,14]. It was noticed that
the increase of the co-solvent concentration with respect to sur-
factant caused corresponding increase in the nanoemulsion area
Fig. 2 (AeD). It was reported [29] that the relative length of hy-
drophobic chains of co-solvent had influence on nano-
emulsification and therefore the nanoemulsion area. Moreover,
since the drug is more soluble in co-solvent (Fig. 1), therefore
concentration is an important factor to solubilize required amount
of drug dose.
The decrease in the nanoemulsion area as shown in Fig. 2 (EeG)
is possibly due to the presence of low concentration of propanol
(co-solvent) which reduces micelles formation and consequently
decreases the solubilization capacity of nanoemulsion [30].
3.3. Selection of formulae
Hundreds of formulae can be prepared from the nanoemulsion
region of the pseudo-ternary phase diagrams. While going through
the diagrams, oil could be solubilized up to 40% w/w (Fig. 2).
Therefore, from each phase diagram below different concentrations
of oil that formed a nanoemulsion was selected at 5% intervals
(i.e.10% and15%) so that the largest number of formulations could
be selected covering the nanoemulsion area of the phase diagram.
Based on diagrams, the oil ratio could be increased up to 40% w/w
without changing nanoemulsion limits of particle size, as
Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
5
increasing oil conc may increase particle droplets of emulsion [31].
For drug loading study and further characterization, 5%, 10% and
15% oil ratio concentrations were selected.
As reported in literature, large amounts of surfactants, particu-
larly ionic surfactants, cause irritation, so for drug delivery,
nonionic surfactants are preferred in a low concentration as
possible [32]. Consequently, selection of formulae was based on the
criterion of being containing a minimum concentration of Smix. So
for each percentage of oil selected, the formulae which taken from
the phase diagram were only those having the minimum concen-
tration of Smix [33].
Another criterion was used for the selection of the different
formulae from the phase diagrams is the oil concentration which
should be such that it solubilizes the single drug dose completely
depending on the solubility of the drug in the oil [33]. Twenty mg of
TB [16] was selected as the dose for incorporation into the oil phase
and for convenience, 2 mL was selected as the nanoemulsion
formulation volume. Knowing that the solubility of TB in peceol;
62.4 mg/mL (Fig. 1), the 20 mg dose of TB will dissolve easily in 0.2
and 0.3 mL of the selected oil concentrations; 10 and 15%,
respectively.
Four pseudoeternary phase diagrams (Fig. 2, A-D) were chosen
which have the largest nanoemulsion areas; Smix (1:1, 1:2, 1:3 and
1:4; 50.18%, 50.09%, 49.56% and 52.61%, respectively). Two points
from each pseudoternary phase diagram were chosen for further
study, as shown in Table 1.
3.4. Evaluation of TB-loaded nanoemulsions
3.4.1. Visual characterization
All investigated TB-loaded nanoemulsions (SM1, SM1\, SM2,
SM2\, SM3, SM3\) were physically stable with no obvious change in
visual appearance i.e. no precipitation, phase separation or color
change except for SM4 & SM4\, they were turbid.
3.4.2. Thermodynamic stability tests
To overcome the problem of metastable formation, thermody-
namic stability tests were performed. The selected formulae were
subjected to different stress tests, such as centrifugation, heating-
cooling cycle, and freeze-thaw cycle tests. The formulae that sur-
vived thermodynamic stability tests were subjected to further
characterization, such as droplet size, viscosity determination, and
TEM [33].
All the tested formulae passed the tests, i.e. there was no sign of
phase separation, turbidity or drug precipitation observed.
3.4.3. Measurement of droplet size and polydispersity index
The droplet size of optimized formulae is given in Table 3. The
average droplet size of the investigated formulae ranged from 52.87
to 93.85 nm, which indicated that emulsion droplets are in nano-
metric range (<100 nm). In contrast, SM4 & SM4\ systems con-
taining Smix 1:4, produced emulsion with larger particle size of
138.5 nm ± 2.6 (data not shown) than the other TB-loaded systems
containing lower Smix ratios. Generally, it was observed that
incorporation of 1% TB into SM4 & SM4\ systems increased particle
size of the formed emulsions decreasing their stability.
It was observed that systems containing 15% peceol produced
nanoemulsion with a larger particle size than systems containing
10% peceol i.e. the larger the oil percentage, the larger was the
mean droplet size. These findings were consistent with a previous
report showing that the mean droplet size increased significantly
when more oil is incorporated owing to the expansion of oil
droplets of the nanoemulsion by the further oil addition [31] and
due to the simultaneous decrease in the Smix proportion [34].
The surfactant used in the preparation of this nanoemulsion
6 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11

Fig. 2. Pseudoternary phase diagrams of Peceol, Tween 80 and propanol at different Smix ratios; A (1:1), B (1; 2), C (1:3), D (1:4), E (4:1), F (3:1) and G (2:1).* Shaded regions
represent the clear nanoemulsion regions.

Table 3
Characterization parameters of prepared TB- nanoemulsion formulae.

TB-nanoemulsion formulae Particle size (nm)a Polydispersity index (PDI)a ZP (mV) RIa pHa Viscosity (cp)a

SM 1 56.52 ± 31.59 0.652 ± 0.20 þ22.924 1.39 ± 0.004 3.37 ± 0.4 26.685 ± 7.9
SM1\ 85.67 ± 26.25 0.572 ± 0.05 þ0.216 1.40 ± 0.003 3.455 ± 0.3 26.485 ± 11.1
SM2 52.87 ± 10.52 0.314 ± 0.18 þ12.302 1.39 ± 0.003 3.44 ± 0.1 8.785 ± 4.2
SM2\ 74.37 ± 3.45 0.284 ± 0.09 þ8.225 1.39 ± 0 3.33 ± 0.1 11.22 ± 6.0
SM3 68.63 ± 0.93 0.336 ± 0.07 þ22.34 1.39 ± 0.001 3.525 ± 0.4 5.7 ± 1.7
SM3\ 93.85 ± 5.64 0.262 ± 0.06 þ23 1.39 ± 0.0007 3.35 ± 0.3 8.505 ± 0.9
a
Values were expressed in mean ± SD (n ¼ 3).

system is Tween 80 achieving small mean droplet size based on
being esters of long chain fatty acids [35].
An inverse correlation was observed between the surfactant
concentration and mean droplet size, i.e.SM2 < SM3 and
SM2\ < SM3\ (Table 3). Similar findings were reported by Gao et al.
[36] who concluded that the droplet size of the microemulsion
decreases with increasing of the Smix ratio. The higher the sur-
factant ratios facilitated the interfacial film to condense and sta-
bilize, whereas increasing the concentration of the co-solvent
causes this film to expand [37e39]. Small mean droplet size of most

Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11
systems could be related to the penetration of co-solvent molecules
into the surfactant film. This would decrease the surface viscosity of
the interfacial film (as will be discussed later under section 3.4.6),
lowering the radius of curvature of the droplets and forming
transparent systems [40]. Thus, the relative proportion of surfac-
tant to co-solvent has varied effects on the droplet size [41];
nonsignificant difference was observed in the droplet size of SM1
and SM2 or SM1\ and SM2\ (p > 0.05).
The polydispersity (PDI) is a measure of homogeneity of parti-
cles and it varies from 0.0 to 1.0. The higher the PDI, the lower the
homogeneity of the particles in the formulation [42]. Polydispersity
index (PDI) of all formulae was less than 0.5 (Table 3), indicating
uniform and narrow globule size distribution [43,44].
Based on these results, the developed NEs with mean droplet
size less than 90 nm with PDI values < 0.5 (SM2, SM2\ and SM3)
were chosen for further studies.
3.4.4. Zeta potential measurement
Zeta potential (zp) is a function of the surface charge whose
value can be related to the physical stability of colloidal dispersions.
High absolute zeta potential values (±30 mV) should preferably be
achieved in most of the emulsions prepared in order to ensure the
creation of a high-energy barrier against coalescence of the
dispersed droplets [45]. However, this suggested zeta potential cut-
off point is only an experience-based value and cannot be reliably
used to predict the stability of nanoemulsion because a wide range
of absolute zeta potential values (i.e., 1.5, 12.5, 45.5 mV) have been
reported for nanoemulsion in previous studies [45e48].
The results of the zeta potential of the selected promising TB-
nanoemulsion formulae were shown in Table 3 showing that the
prepared formulae were positively charged and majority of values
are not low values, indicating stable system and well separated
emulsion globules [49]. The electrostatic repulsion forces between
the positively charged droplets get an avoidance of the coalescence
of the nanoemulsion [50].
3.4.5. Refractive index and pH measurements
Refractive index (RI) is taken as a measure for nanoemulsion
transparency [8]. The values of RI of selected TB-loaded nano-
emulsion formulae were similar to that of water (1.334) as depicted
in Table 3 indicating clear and transparent formulae and the pre-
pared nanoemulsions were of o/w type [51].
The pH values of the TB-loaded nanoemulsion formulae were
found to be 3.4125 ± 0.26 (Table 3) indicating the acidic nature of
the drug.
3.4.6. Viscosity determination
It was obvious from the viscosity data (Table 3) that the devel-
oped nanoemulsions had a low viscosity. Low viscosity is one of the
characteristic features of the nanoemulsions, which exhibit New-
tonian flow behavior [52,53].
The study revealed that the viscosity has a tendency to increase
with an increase in the oil content. As the oil content was increased
from 5% w/w to 10% w/w, an increase in the viscosity of the
formulae was observed which might be due to higher oil content. A
similar observation was published by Shafiq-un-Nabi et al. [33].
When the formulae with different Smix ratios, containing the
same peceol percentage, were compared, the maximum viscosity
values were obtained for SM1 and SM1\ formulae (26.685 ± 7.9 cp,
26.485 ± 11.1 cp, respectively) due to their minimum content of
propanol and the highest amount of Tween 80 (a fatty acid poly-
hydric alcohol ester having a high intrinsic viscosity) in the Smix
[20].
Moreover, the viscosity determination showed that as the con-
centration of co-solvent increased, the viscosity of the TB-loaded
Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
7
nanoemulsion formulae also get decreased. This could be related
to increased penetration of co-solvent molecules in the surfactant
film decreasing the surface viscosity of the interfacial film, and
forming transparent systems [35,54].
3.4.7. Transmission electron microscopy (TEM)
Representative micrographs are shown in Fig. 3. The developed
particles are spherical, discrete and have uniform droplet size. The
droplets in the nanoemulsion appeared dark, and the surroundings
were bright, and a “positive” image was seen. It is clear that the
droplet size of all the tested formulae appears to be smaller
compared to that calculated by photon correlation spectroscopy;
the transit aggregation that might occur during the Brownian
motion could be a possible explanation for the higher values
observed with the latter technique. A similar observation was
noticed in literature [35].
3.4.8. In vitro drug release study
In the process of biological permeation, the drug should first
release from the vehicles, and then it can be partitioned into or
absorbed by the skin or gastrointestinal tract [55]. As a conse-
quence, assessment of in vitro drug release is a crucial step in paving
the way for effective permeation.
The in vitro release of TB from the selected nanoemulsion
formulae (SM2, SM2\ & SM3) was assessed using the dialysis bag
method. As demonstrated in Fig. 4, the release from the selected
optimized nanoemulsion formulae was significantly (p < 0.05)
higher than that from the drug suspension and the marketed
product under the same experimental conditions. This was attrib-
uted to the fact that prepared nanoemulsion formulae are having a
smaller particle size of nano-size range (Table 3) and hence larger
surface area and higher interfacial area required for dissolution
[13,34].
SM2\ revealed the least amount of TB released; 10.32 mcg/mL
compared to the highest amount released of SM3 14.91 mcg/ml, in
6 h (Fig. 4) which may be due to higher oil conc. in SM2\ (15%)
compared to 10% in SM2 & SM3. However, they all have the same
conc. of Smix which was not sufficient to emulsify the increased
conc. of peceol in SM2\ [13].
On the other hand, SM3 exhibited the highest amount of TB
release; 14.91 mcg/mL in 6 h, which may be due to its high content
of the COS; propanol. COS in the nanoemulsion systems reduce the
interfacial tension and increase the fluidity of the interface. They
also increase the mobility of the hydrocarbon tail and allow greater
penetration of the oil in this region. Nanoemulsion area was also
used as an assessment criteria for the evaluation of co-solvents/co-
surfactants [53]. SM3 pseudoternary phase diagram showed the
largest area of the nanoemulsion region (Fig. 2 D); 52.61%, proving
the greatest nanoemulsification efficiency [9,14], hence maximum
penetration of the COS into the surfactant monolayer interface
which further enhance the nanoemulsification performance of the
system increasing cumulative % drug release. These results agreed
with what was reported in literature [56].
3.5. Preparation of nanoemulsion based gel
In the present study, TB-loaded nanoemulsion type is of o/w
type, so we have selected Carbopol 940 as the gel forming polymer.
A small amount of triethanolamine as neutralizing agent was added
to carbopol, which is a polycarboxylic acid, to start a partial ioni-
zation that in turn promotes the gel formation [57]. It has been
previously reported that when the polymer concentration is close
to 1%, the viscoelastic and flow properties of the nanoemulsions are
suitable to ensure an adequate performance during topical and
transdermal administration [58]. Consequently, Carbopol 940, the
8 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11

Fig. 3. Transmission electron micrographs of selected TB-loaded nanoemulsion formulae; SM2 (a), SM2\ (b) and SM3 (c).

Fig. 4. In vitro release profiles of TB from optimized nanoemulsion formulae; SM2, SM2\ and SM3 compared to that from Lamisil® and aqueous drug suspension in pH 5.5 methanol/
10%/acetate buffer 1:9, respectively at 37 ± 0.5
C and 50 strokes per minute.

Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11
gel forming polymer, was prepared as 1% w/w. At such a polymer
concentration, a gel behavior was exhibited by the aqueous Car-
bopol system and the corresponding nanoemulsion having good
consistency and a clear appearance.
3.6. Characterization of Terbinafine nanoemulsion based emulgel
3.6.1. Physicochemical properties and drug content
The pH values of all prepared emulgel formulae were
5.715 ± 0.26 which is within the physiological range and considered
acceptable to avoid the risk of irritation upon application to the skin
adult skin PH is 5.5 [59].
The gel system has an increased viscosity compared to that of
the corresponding nanoemulsion being more suitable to be applied
topically [15]. In summary, different formulae can be ordered ac-
cording to their viscosity values as follows; F2 (SM2\) > F1
(SM2) > F3 (SM3); 16800 ± 1.22, 10200 ± 2.65, 6650 ± 3.88 cp,
respectively.
Drug contents of the optimized nanoemulsion based gel
formulae (F1, F2 and F3) were found to be 97.95 ± 0.4, 96.02 ± 0.37
and 97.40 ± 0.41, respectively.
3.6.2. Ex vivo study (permeation study)
A steady increase of TB in the receptor chamber with time was
observed. There were no significant difference in the amount of TB
that permeated through the skin by the end of 12 h after application
from F1, F2 and F3 formulae, but was higher than that compared to
the 55 mcg from the marketed product (Fig. 5).
The results demonstrated that the permeation rate and
permeation coefficient of all the NE emulgel formulae (F1-F3)
through rat skin are significantly higher (P < 0.05) in comparison to
the marketed product (Table 4). The values of transdermal flux
(5.429 mcg/cm2/h) for the different NE formulae are approximately
threefold greater than that of the marketed emulgel, (2.034 mcg/
cm2/h) indicating that the permeation parameters of TB from
nanoemulsions were markedly influenced by the composition of
the formulae. Since all the nanoemulgel formulae and marketed
product carried equal drug load, it could, therefore, be concluded
that the concentration gradient is not the factor governing the
permeation process.
This high permeability may also be due to the small nano
droplets which settle down to close contact with the skin,
Fig. 5. Permeation Profiles of TB through the excised rat skin from different TB-nanoemulsion emulgel formulae compared to that from the marketed product.
Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
9
providing a larger surface area for permeation of drug and releasing
high drug concentration on the affected area [6,20]. Moreover,
hydration of the stratum corneum, due to the external water phase
of the NE, causes the corneum cells to swell, thus making the
channels for drug passage wider, resulting in high diffusivity of the
lipophilic drug as the droplet size approaches to molecular
dispersion [20,31]. As some lipid chains in the stratum corneum are
covalently attached to the corneocytes, hydration of these proteins
will also lead to the disorder of the lipid bilayers [31]. From liter-
ature and our previous work, the incorporation of penetration
enhancers facilitates the permeation of lipophilic drugs by altering
the barrier property of the stratum corneum through fluidization of
lipids. Therefore, the most probable possible mechanism is that
nanoemulsion enhances transdermal permeation of lipophilic
drugs-despite increasing water solubility-through alteration of SC
barrier, as many common components of nanoemulsion are
chemical penetration enhancers [60e63].
A Comparison of the permeation of the NE formulae with the
corresponding droplet size and viscosity indicated that F3 with the
smallest droplet size and the lowest viscosity had the highest
transdermal flux. In addition, F3 contains the highest water content
(45%) which improved the cumulative permeation from the system,
this finding was consistent with previous studies [53,64].
Another possible reason that could have an effect on the
permeation is the Smix concentration. All the tested formulae
contain the same Smix conc.; 45% (Table 2) but F1 & F2 contain
more surfactant conc. than in F3 and they exhibited lower rates of
drug permeation (Fig. 5). This might be due to a decreased ther-
modynamic activity of the drug in the nanoemulsion at the higher
content of surfactant [65,66]. The thermodynamic activity of a drug
in the formulation is a significant driving force for the release and
penetration of the drug into the skin. With increased surfactant
concentration, affinity to the vehicle became greater, and there is a
slow release of the drug and/or a poor transfer from the vehicle to
the skin.
Therefore, the high content of the surfactant lowers the
permeability rate due to decreased thermodynamic activity of the
drug in the nanoemulsion [65,66] which explains the permeation
difference between the three formulae despite being insignificant
(p > 0.05).
No lag time was obtained for all the prepared nanoemulgel
formulae (F1-F3). It is assumed that by incorporation into the
10 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11
Table 4
Permeability parameters of TB-NE emulgel formulae.
Formula The permeation rate at Permeability Enhancement
Code steady-state (steady-state Coefficient Ratio ER
flux, Jss), mcg/cm2/h Kp (cm h1)
F1 5.414 1.0828 2.6617
F2 5.4098 1.08196 2.659
F3 5.4631 1.09262 2.685
Lamisil® 2.034 0.4068 e
colloidal carrier, better contact with the skin is achieved, which
could be one of the reasons why the effect appeared sooner,
resulting in no lag times. Similar observations were reported in
literature [51,59]. As a result of this study, which matches many
other studies nanoemulsion has always been a promising vehicle
for transdermal delivery of lipophilic drugs [67].
3.6.3. In vivo study
Based on enhanced drug permeation, lowest droplet size, lowest
viscosity, and lowest polydispersity index formulae F1, F2 and F3
were selected for in vivo antifungal effects. The in vivo antifungal
activity of F3 showed significant reduction in C. albicans burden in
the rat skin when compared with the untreated group or the pla-
cebo vehicle treated group (p < 0.05). Described under the exper-
imental conditions, no significant fungal burden reduction was
observed with F1, F2 and Lamisil® (Fig. 6). Log CFU after F3 appli-
cation was found to be 6.13 ± 0.09 compared to 6.98 ± 0.23,
6.88 ± 0.11 and 6.73 ± 0.13 in case of F1, F2 and placebo treatment
respectively while log CFU in the untreated group was 6.83 ± 0.07
(Fig. 6).
It is important to point out that treatment was carried out for
only 3 successive days followed by animal euthanization on the
fourth day. During this short period of the experiment, only F3
revealed promising results indicating its relatively powerful effi-
cacy and penetration. F3 efficiency may be attributed to slightly
higher conc of propanol, it may act as a transdermal enhancer
through fluidization of SC lipids. This mechanism may confirm
that the enhancing effect of nanoemulsion is attributed to its ex-
cipients as discussed in permeation section. Furthermore, nano-
emulsion delivery systems are reported to prolong residence time
and they could reach greater surface areas, resulting in a
Fig. 6. In vivo antifungal efficacy of different TB-nanoemulsion emulgel formulae
compared to the marketed product and untreated group (control) after three succes-
sive days of a single application per day. Data were expressed as mean ± SEM. Ab-
breviations: C. albicans, Candida albicans; CFU, colony-forming unit; SEM, standard
error of mean.
Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
comparatively higher drug uptake. In addition, would show the
best effect as it has the least viscosity so best spreadability &
therefore expected to exhibit faster release of active ingredients
[30]. Therefore, the results obtained from the in vivo study
confirmed our ex vivo observation that F3 exhibited the best
permeation outcomes.
4. Conclusion
The present investigation demonstrated that optimum nano-
emulsion drug delivery system of TB was prepared from peceol
(oil), tween80 (surfactant) and propanol (co-solvent). Nano-
emulsion formulae passing the thermodynamic stability tests were
characterized for morphology, pH, viscosity, RI, droplet size, PDI
and zeta potential. Optimized nanoemulsion formulae were
incorporated in carbopol based gel. Ex vivo study (permeation
study) results revealed that Sm3 nanoemulsion-based Carbopol gel
formula which consisted of 1% TB, 50% carbopol gel, 22.5% water, 5%
peceol oil and 22.5% Smix exhibited the highest permeability of
nanoemulsion based gel formulae. Small globule size of the SM3
and its low viscosity were the main factors of enhanced drug
permeation. In vivo antifungal activity emphasized the powerful
efficacy and penetration of Sm3 formula as it showed significant
reduction in C. albicans burden in the skin when compared to all
other groups (p < 0.05) during short treatment period of the
experiment (3 days).
Acknowledgments
Authors are highly thankful to Dr. Abdelwahab Kassem, Alex-
andria University (Egypt) and Dr. Mariana Fedorovska, Ivano
Frankvisk National Medical University (Ukaine) for providing
facilities.
References
[1] K.H. Reza, Nanoemulsion as a novel transdermal drug delivery system, Int. J.
Pharm. Sci. 2 (8) (2011) 1938e1946. http://dx.doi.org/10.13040/IJPSR.0975-
8232.2(8).1938-46.
[2] S.S. Abolmaali, A.M. Tamaddon, F.S. Farvadi, S. Daneshamuza, H. Moghimi,
Pharmaceutical nanoemulsions and their potential topical and transdermal
applications, Iran. J. Pharm. Res. 7 (3) (2011) 139e150.
[3] N.S. Ryder, S. Wagner, I. Leitner, In vitro activities of terbinafine against
cutaneous isolates of Candida albicans and other pathogenic yeasts, Anti-
microb. Agents Chemother. 42 (5) (1998) 1057e1061.
[4] Chauhan, F. Muzaffar, S. Lohia, Design, development and evaluation of topical
microemulsion, Int. J. Pharm. Pharm. Sci. 5 (2) (2013) 605e610.
[5] R.B.D. Reddy, C.T.L. Kumari, G.N. Sowjanya, S.L. Sindhuri, P. Bandhavi, Nano-
emulsions an emerging trend, IJPRD 4 (06) (2012) 137e152.
[6] U. Syamala, Development &optimization of Ally Amine antifungal nano-
emulgel using factorial design: for treatment of tinea pedis, Eur. Sci. J. 4 (2013)
597e605.
[7] G.H. Elosaily, Formulation and in-vitro evaluation of nystatin nanoemulsion-
based gel for topical delivery, J. Am. Sci. 8 (12) (2012) 541e548.
[8] H.A. Makadia, A.Y. Bhatt, R.B. Parmar, J.S. Paun, H.M. Tank, Self-nano emulsi-
fying drug delivery system (SNEDDS), Asian J. Pharm. 3 (1) (2013) 21e27.
[9] M.A. Elsheikh, Y.S. Elnaggar, E.Y. Gohar, O.Y. Abdallah, Nanoemulsion liquid
preconcentrates for raloxifene hydrochloride: optimization and in vivo
appraisal, Int. J. Nanomed. 7 (2012) 3787e3802, http://dx.doi.org/10.2147/
IJN.S33186.
[10] G.C. Soni, S.K. Prajapati, N. Chaudhri, Self nanoemulsion: advance form of drug
delivery system, WJPPS 3 (10) (2014) 410e436.
[11] M. Wais, A. Samad, A. Khale, M. Aqil, M. Khan, Investigation of nanoemulsion
system for transdermal delivery of glibenclamide, Int. J. Pharm. Pharm. Sci. 4
(2012) 482e487.
[12] K. Harika, S. Debnath, M.N. Babu, Formulation and evaluation of nanoemulsion
of amphotericin B, IJNTPS 5 (2015) 114e122.
[13] A.D. Mantena, B.R. Dontamsetti, A. Nerella, Formulation, optimization and
in vitro evaluation of rifampicin nanoemulsions, Int. J. Pharm. Sci. Drug Res. 7
(6) (2015) 451e455.
[14] Y.S. Elnaggar, M.A. El-Massik, O.Y. Abdallah, Self-nanoemulsifying drug de-
livery systems of tamoxifen citrate: design and optimization, Int. J. Pharm. 380
(2009) 133e141.
[15] S. Rao, T. Barot, K.S. Rajesh, L.L. Jha, Formulation, optimization and evaluation
 M.E. Elmataeeshy et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e11
of microemulsion based gel of Butenafine Hydrochloride for topical delivery
by using simplex lattice mixture design, Int. J. Pharm. Investig. 46 (2016)
1e12.
[16] V.V.S.N.R. Karri, S.K. Raman, G. Kuppusamy, S. Mulukutla, S. Ramaswamy,
R. Malayandi, Terbinafine hydrochloride loaded nanoemulsion based gel for
topical application, Int. J. Pharm. Investig. 45 (2015) 79e89.
[17] D. Kaur, A. Raina, N. Singh, Formulation and evaluation of carbopol 940 based
glibenclamide transdermal gel, Int. J. Pharm. Pharm. Sci. 6 (2015) 434e440.
[18] B.S. Barot, P.B. Parejiya, H.K. Patel, M.C. Gohel, P.K. Shelat, Microemulsion-
based gel of terbinafine for the treatment of onychomycosis optimization of
formulation using D-optimal design, AAPS PharmSciTech 13 (2012) 184e192.
[19] S.M. Talaat, Y.S.R. EL Nagaar, M. Bahey-El-Din, O.Y. Abdallah, Novel lecithin-
integrated liquid crystalline nanogels for enhanced cutaneous targeting of
terconazole: development, in vitro and in vivo studies, Int. J. Nanomed. 11
(2016) 5531e5547.
[20] S. Akhter, G.K. Jain, F.J. Ahmad, R.K. Khar, N. Jain, Z.I. Khan, S. Talegaonkar,
Investigation of nanoemulsion system for transdermal delivery of Domper-
idone: ex-vivo and in vivo studies, Curr. Nanosci. 4 (2008) 381e390.
[21] W.W. Zheng, L. Zhao, Y.M. Wei, Y. Ye, S.H. Xiao, Preparation and the in vitro
evaluation of nanoemulsion system for the transdermal delivery of granise-
tron hydrochloride, Chem. Pharm. Bull. 58 (2010) 1015e1019.
[22] H.R. Conti, A.R. Huppler, N. Whibley, S.L. Gaffen, Animal models for candidi-
asis, Curr. Protoc. Immunol. 105 (2014) 1e17.
[23] A.A. Kassem, A.M. Mohsen, R.S. Ahmed, T.M. Essam, Self-nanoemulsifying
drug delivery system (SNEDDS) with enhanced solubilization of nystatin for
treatment of oral candidiasis: design, optimization, in vitro and in vivo
evaluation, J. Mol. Liq. 218 (2016) 219e232. http://dx.doi.org/10.1016/j.
molliq.2016.02.081.
[24] U. Gupta, S.K.D. Dwivedi, H.K. Bid, R. Konwar, N.K. Jain, Ligand anchored
dendrimers based nanoconstructs for effective targeting to cancer cells, Int. J.
Pharm. 393 (2010) 186e197.
[25] J. Patel, A. Patel, M. Raval, N. Sheth, Formulation and development of a self-
nanoemulsifying drug delivery system of irbesartan, J. Adv. Pharm. Tech. 2
(2011) 9e16.
[26] H. Mahmoud, S. Al-Suwayeh, S. Elkadi, Design and optimization of self-
nanoemulsifying drug delivery systems of simvastatin aiming dissolution
enhancement, Afr. J. Pharm. Pharmacol. 7 (2013) 1482e1500.
[27] C. Chen, D. Han, C. Cai, X. Tang, An overview of liposome lyophilization and its
future potential, J. Control. Release 142 (2010) 299e311.
[28] F.S. Nielsen, K.B. Petersen, A. Müllertz, Bioavailability of probucol from lipid
and surfactant based formulations in minipigs: influence of droplet size and
dietary state, Eur. J. Pharm. Biopharm. 69 (2008) 553e562.
[29] D. Sakloetsakun, S. Dunnhaupt, J. Barthelmes, G. Perera, A. Bernkop-Schnurch,
Combining two technologies: multifunctional polymers and self-
nanoemulsifying drug delivery system (SNEDDS) for oral insulin administra-
tion, Int. J. Biol. Macromolec. 61 (2013) 363e372.
[30] S.A. Chime, F.C. Kenechukwu, A.A. Attama, in: Ali Demir Sezer (Ed.), Nano-
emulsions d Advances in Formulation, Characterization and Applications in
Drug Delivery, Application of Nanotechnology in Drug Delivery, InTech, 2014,
http://dx.doi.org/10.5772/58673 (chapter 3). Available from: http://www.
intechopen.com/books/application-of-nanotechnology-in-drug-delivery/
nanoemulsions-advances-in-formulation-characterization-and-applications-
in-drug-delivery.
[31] Y. Yuan, S.M. Li, F.K. Mo, D.F. Zhong, Investigation of microemulsion system for
transdermal delivery of meloxicam, Int. J. Pharm. 321 (2006) 117e123. http://
dx.doi.org/10.1016/j.ijpharm.2006.06.021.
[32] M.J. Lawrence, G.D. Rees, Microemulsion-based media as novel drug delivery
systems, Adv. Drug Deliv. Rev. 45 (2000) 89e121.
[33] S. Shafiq-un-Nabi, F. Shakeel, S. Talegaonkar, J. Ali, S. Baboota, A. Ahuja,
Rk Khar, M. Ali, Formulation development and optimization using nano-
emulsion technique, AAPS PharmSciTech 8 (2007) E12eE17.
[34] J.B. Jeevana, K. Sreelakshmi, Design and evaluation of self-nanoemulsifying
drug delivery system of flutamide, J. Young Pharm. 3 (1) (2011) 4e8.
[35] S.A. Tayel, M.A. El-Nabarawi, M.I. Tadros, W.H. Abd Elsalam, Promising ion-
sensitive in situ ocular nanoemulsion gels of terbinafine hydrochloride:
design, in vitro characterization and in vivo estimation of the ocular irritation
and drug pharmacokinetics in the aqueous humor of rabbits, Int. J. Pharm. 443
(2013) 293e305.
[36] Z.G. Gao, H.G. Choi, H.J. Shin, K.M. Park, S.J. Lim, K.J. Hwang, C.K. Kim, Physi-
cochemical characterization and evaluation of a microemulsion system for
oral delivery of cyclosporin a, Int. J. Pharm. 161 (1998) 75e86.
[37] U.A. Fahmy, O.A.A. Ahmed, K.M. Hosny, Development and evaluation of ava-
nafil self-nanoemulsifying drug delivery system with rapid onset of action
and enhanced bioavailability, AAPS PharmSciTech 16 (2015) 53e58.
[38] K.M. Hosny, Z.M. Banjar, The formulation of a nasal nanoemulsion zaleplon in
situ gel for the treatment of insomnia,, Expert Opin. Drug Deliv. 10 (2013)
1033e1041.
[39] N.J. Kale, L.V. Allen, Studies on microemulsions using brij 96 as surfactant and
glycerin, ethylene glycol and propylene glycol as cosurfactants, Int. J. Pharm.
57 (1989) 87e93.
[40] S. Terjarla, Microemulsion systems: an overview and pharmaceutical appli-
cations, Crit. Rev. Ther. Drug Carr. Syst. 16 (1999) 461e521.
[41] R.C. Baker, A.T. Florence, T.F. Tadros, R.M. Wood, Investigations into the
Please cite this article in press as: M.E. Elmataeeshy, et al., Enhanced transdermal permeability of Terbinafine through novel nanoemulgel
formulation; Development, in vitro and in vivo characterization, Future Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/
j.fjps.2017.07.003
11
formation and characterization of microemulsions. I. Phase diagrams of the
ternary system waterdsodium alkyl benzene sulfonatedhexanol and the
quaternary system waterdxylenedsodium alkyl benzene sulfona-
tedhexanol, J. Colloid Interface Sci. 100 (1984) 311e331.
[42] A.M. Avachat, V.G. Patel, Self nanoemulsifying drug delivery system of stabi-
lized ellagic acidephospholipid complex with improved dissolution and
permeability, Saudi Pharm. J. 23 (3) (2015) 276e289.
[43] K. Balakumar, C.V. Raghavan, N.T. Selvan, R.H. Prasad, S. Abdu, Self nano-
emulsifying drug delivery system (SNEDD) of rosuvastatin calcium: design,
formulation, bioavailability and pharmacokinetic evaluation, Colloids Surf. B
Biointerfaces 112 (2013) 337e343.
[44] F. Shakeel, N. Haq, F. Alanazi, I.A. Alsarra, Polymeric solid self-nanoemulsifying
drug delivery system of glibenclamide using coffee husk as a low cost bio-
sorbent, Powder Technol. 256 (2014) 352e360, http://dx.doi.org/10.1016/
j.powtec.2014.02.028.
[45] A.H. Negussie, J.L. Miller, G. Reddy, S.K. Drake, B.J. Wood, M.R. Dreher, Syn-
thesis and in vitro evaluation of cyclic NGR peptide targeted thermally sen-
sitive liposome, J. Control. Release 143 (2) (2010) 265e273.
[46] Y. Wang, S. Tu, R. Li, X. Yang, L. Liu, Q. Zhang, Cholesterol succinyl chitosan
anchored liposomes: preparation, characterization, physical stability, and
drug release behavior, Nanomedicine 6 (3) (2010) 471e477. http://dx.doi.org/
10.1016/j.nano.2009.09.005.
[47] A. Sharma, U.S. Sharma, Liposomes in drug delivery: progress and limitations,
Int. J. Pharm. 154 (1997) 123e140.
[48] S.A. Wissinga, O. Kayserb, R.H. Müller, Solid lipid nanoparticles for parenteral
drug delivery, Adv. Drug Deliv. Rev. 56 (2004) 1257e1272.
[49] A.G. Agrawal, A. Kumar, P.S. Gide, Formulation of solid self-nanoemulsifying
drug delivery systems using N-methyl pyrrolidone as cosolvent, Drug Dev.
Ind. Pharm. 41 (4) (2015) 594e604.
[50] M. Badran, E.I. Taha, M. Tayel, S. Al-Suwayeh, Ultra-fine selfnanoemulsifying
drug delivery system for transdermal delivery of meloxicam: dependency on
the type of surfactants, J. Mol. Liq. 190 (2014) 16e22.
[51] M.S. Ali, M.S. Alam, N. Alam, M.R. Siddiqui, Preparation, characterization and
stability study of dutasteride loaded nanoemulsion for treatment of benign
prostatic hypertrophy, Iran. J. Pharm. Res. 13 (4) (2014) 1125e1140.
[52] M.J. Lawrence, G.D. Rees, Microemulsion-based media as novel drug delivery
systems, Adv. Drug Deliv. Rev. 45 (1) (2000) 89e121.
[53] A. Azeem, F.J. Ahmad, R.K. Khar, S. Talegaonkar, Nanocarrier for the trans-
dermal delivery of an antiparkinsonian drug, AAPS PharmSciTech 10 (4)
(2009) 1093e1103.
[54] S. Terjarla, Microemulsion systems: an overview and pharmaceutical appli-
cations, Crit. Rev. Ther. Drug Carr. Syst. 16 (5) (1999) 461e521.
[55] S. Küchler, W. Herrmann, G.P. Minkin, T. Blaschke, C. Zoschke, K.D. Kramer,
R. Bittl, M.S. Korting, SLN for topical application in skin dis-
easesdcharacterization of drugecarrier and carrieretarget interactions, Int. J.
Pharm. 390 (2) (2010) 225e233.
[56] N. Marasini, Y.D. Yan, B.K. Poudel, H.G. Choi, C.S. Yong, J.O. Kim, Development
and optimization of self-nanoemulsifying drug delivery system with
enhanced bioavailability by boxebehnken design and Desirability function,
J. Pharm. Sci. 101 (2012) 4584e4596.
[57] G. Bonacucina, S. Martelli, G.F. Palmieri, Rheological, mucoadhesive and
release properties of carbopol gels in hydrophilic cosolvents, Int. J. Pharm. 282
(2004) 115e130.
[58] R. Lapasin, M. Grassi, N. Coceani, Effect of polymer addition on the rheology of
o/w microemulsions, Rheol. Acta 40 (2001) 185e192, http://dx.doi.org/
10.1007/s003970000151.
[59] K.R. Sabu, G.D. Basarkar, Formulation, development and in-vitro evaluation of
terbinafine hydrochloride emulgel for topical fungal infection, Int. J. Pharm.
Sci. 21 (2) (2013) 168e173.
[60] E. Abd, S. Namjoshi, Y.H. Mohammed, M.S. Roberts, J.E. Grice, Synergistic skin
penetration enhancer and nanoemulsion formulations promote the human
epidermal permeation of caffeine and naproxen, J. Pharm. Sci. 105 (1) (2016)
212e220.
[61] I. Som, K. Bhatia, M. Yasir, Status of surfactants as penetration enhancers in
transdermal drug delivery, J. Pharm. Bioall Sci. 4 (2012) 2e9.
[62] A. Nanda, S. Nanda, N.M. Khan Ghilzai, Current Developments using emerging
Transdermal Technologies in physical enhancement methods, Curr. Drug
Deliv. 3 (2006) 233e242.
[63] D. Bhavna, G. Aggarwal, S.L. Harikumar, Enhanced transdermal permeability
of piroxicam through novel nanoemulgel formulation, Int. J. Pharm. Investig. 4
(2) (2014) 65e76.
[64] M.J. Alvarez-Figueroa, J. Blanco-Me ndez, Transdermal delivery of metho-
trexate: iontophoretic delivery from hydrogels and passive delivery from
microemulsions, Int. J. Pharm. 215 (2001) 57e65.
[65] V.P. Shah, Skin penetration enhancer: scientific prospective, in: D.S. Hsieh
(Ed.), Drug Permeation Enhancement: Theory and Applications, Marcel Dek-
ker Inc, New York, 1994, pp. 19e24.
[66] S.A. Yadav, S.S. Poddar, Formulation, in-vitro and in-vivo evaluation of
nanoemulsion gel for transdermal drug delivery of nimodipine, Asian J.
Pharm. Clin. Res. 8 (2) (2015) 119e124.
[67] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, M. Aqil, S. Shafiq, Nanoemulsions as
vehicles for transdermal delivery of aceclofenac, AAPS PharmSciTech 8 (4)
(2007) 124e135.