Drug Development and Industrial Pharmacy

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Optimization of nutraceutical coenzyme Q10

nanoemulsion with improved skin permeability
and anti-wrinkle efficiency

Eman S. El-Leithy, Amna M. Makky, Abeer M. Khattab & Doaa G. Hussein

To cite this article: Eman S. El-Leithy, Amna M. Makky, Abeer M. Khattab & Doaa G. Hussein
(2017): Optimization of nutraceutical coenzyme Q10 nanoemulsion with improved skin
permeability and anti-wrinkle efficiency, Drug Development and Industrial Pharmacy, DOI:
10.1080/03639045.2017.1391836

To link to this article: http://dx.doi.org/10.1080/03639045.2017.1391836

Published online: 02 Nov 2017.

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 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, 2017
https://doi.org/10.1080/03639045.2017.1391836

RESEARCH ARTICLE

Optimization of nutraceutical coenzyme Q10 nanoemulsion with improved skin

permeability and anti-wrinkle efficiency
Eman S. El-Leithya,b, Amna M. Makkyc, Abeer M. Khattabd and Doaa G. Husseind
a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Cairo,
Egypt; bDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Ain Helwan, Cairo, Egypt;
c
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; dNational Organization for Drug
Control and Research, Cairo, Egypt

ABSTRACT ARTICLE HISTORY

Coenzyme Q10 (CoQ10) is an insoluble, poorly permeable antioxidant with great biological value which Received 30 May 2017
Downloaded by [Gothenburg University Library] at 06:48 03 November 2017

acts as anti-aging and anti-wrinkle agent. To improve its permeability through topical application, the cur- Revised 26 September 2017
rent study aimed at formulating oil/water (o/w) nanoemulsion (NE) as an efficient vehicle for delivering Accepted 1 October 2017
(CoQ10) through the skin barriers. The solubility of (CoQ10) was tested for various oils, surfactants (S), and
co-surfactants (CoS). The NE region was determined by constructing pseudoternary phase diagrams. NE KEYWORDS
formulae containing 1, 2, and 3% w/w drug have been subjected to thermodynamic stability test. The for- Coenzyme Q10;
mulae that passed thermodynamic stability tests were characterized by physical properties as pH, viscosity, nanoemulsion; phase
refractive index, droplet size, zeta-potential, TEM, electroconductivity, in vitro release, and ex vivo perme- diagram; permeability;
ation. The formula ‘F2’ containing 10% w/w isopropyl myristate (oil phase), 60% w/w of Tween 80: anti-wrinkle
Transcutol HP mixture (S/CoSmix) at ratio 2:1, 30% w/w water and 2% w/w drug was evaluated for its anti-
wrinkle efficiency using an animal model. The ‘F2’ formula showed 11.76 ± 1.1 nm droplet size,
1.4260 ± 0.0016 refractive index, 0.228 PDI,
14.7 ± 1.23 mv zeta potential, 7.06 ± 0.051 pH, 199.05 ± 0.35 cp
viscosity, and the highest percentage of drug release in the selected dissolution media. About 47.21% of
the drug was released in phosphate buffer 7.4 containing 5% w/v Labrasol and 5% w/v isopropyl alcohol
through 24 h. It also showed the highest drug flux (Jss ¼ 3.164 mg/cm2/h), enhancement ratio (Er ¼ 8.32),
and permeability coefficient (Kp ¼ 22.14  10
4 cm2/h). CoQ10 NE reduced the skin wrinkles and gave the
skin smooth appearance. Our investigation suggests the potential use of NE as a vehicle for enhancing
solubility and permeability of CoQ10 and thus improving its anti-wrinkle efficiency.

Introduction
Skin is the outermost tissue of the body that represents the larg-
est organ per body weight or surface (10% of the body weight
and nearly 2 m2 of the body surface area) [1]. It has robustness
barrier’s activity against environmental stress, including hydration,
dehydration, temperature, and UV radiation [2]. Stratum corneum
(SC) consists of horny cells that made up of peripheral nonviable
layers of epiderm is considering the control barrier rate of trans-
dermal absorption, which directly relates to the lipophilicity of
drugs [3].
Skin aging is a complex biological process that is directly influ-
enced by endogenous and exogenous (intrinsic and extrinsic) fac-
tors [4]. These factors lead to a progressive increase in wrinkles
due to cumulative changes in structural, physiological, and appear-
ance of skin layers, especially those exposed to sun [5].
The ultra violet (UV) radiation is an important factor that causes
an imbalance between pro-oxidants and antioxidants which in
turn leads to oxidative stress and photoaging of the skin [6]. The
constant action of UV rays on the skin diminishes the antioxidants
existing inside the skin over time. The antioxidants commonly
placed over stratum corneum are susceptible to ultra violet (UV)
exposure and a single dose can decrease their concentration to
almost half amount [7].
Rabe et al. [8] proposed three strategies for treatment and pre-
vention of skin aging. One of which is based on applying the
retinoic acid, alpha hydroxy acids, antioxidants, estrogens, and
growth factors to reduce signs and symptoms of skin aging.
Antioxidants include (CoQ10), alpha-lipoic acid (ALA), niacina-
mide (B3 vitamin), L-ascorbic acid, and a-tocopherol (vitamin E)
can quench free radicals, which can accelerate the process of
aging [9]. CoQ10 has a tail of 10 isoprene units made up of 50 car-
bon atoms attached to its benzoquinone head structure, that pro-
vides it with highly lipophilic characteristics (as a freely fat soluble)
and very low water solubility (<1 mg/L) [10]. CoQ10 exists in mito-
chondria of all body cells that have an essential role as an electron
and proton transporter [11]. CoQ10 concentration in the skin epi-
dermis layer is 10-folds higher than the dermis layer [12]. Recent
investigations proved the obvious anti-aging effect of CoQ10 on
the skin, which attributed to increase the production of basement
membrane components such as keratinocytes, laminin, and colla-
gen fibers, as well as increasing the fibroblast proliferation
together with protecting cells against oxidative stress [13].
Potential sensitivity and photo-degradation of most antioxi-
dants to light exposure are considered factors that reduce their
activities. Furthermore, inefficient percutaneous penetration is
deemed as another problem that limits their permeation through
the viable epidermis and dermis layers. Thus, the real challenge of
antioxidant is to increase the permeation through stratum cor-
neum and reach to the deep of cutaneous layers without signifi-
cant absorption to blood circulation [14].

CONTACT Abeer Mostafa Khattab abeer_khattab75@yahoo.com National Organization for Drug Control and Research, Cairo, Egypt
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 E. S. EL-LEITHY ET AL.
Although the conventional encapsulation techniques such as
the inclusion complex of CoQ10 with cyclodextrin succeed to
increase its solubility, oral bioavailability, and stability [15], it was
reported that the usage of the chemical permeation enhancers as
cyclodextrin is not considered as the best choice for transdermal
delivery. The capability of such permeation enhancer for enhanc-
ing transport across the skin does not accomplish the desired skin
disturbance. They demonstrate poor permeation across the stra-
tum corneum themselves and therefore their activity is limited to
the top layer of the stratum corneum. Furthermore, they should
be used in high concentration to be effective as skin permeation
enhancers with which it may increase the risk of skin irritation.
Consequently, cyclodextrin application as a chemical permeation
enhancer offers poor transdermal delivery of candidate drug mole-
cules [16].
Various novel strategies are currently being developed for effi-
cient delivery of poorly water-soluble drugs, such as nanoemulsion
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(NE) [17,18]. Nanoemulsion is a thermodynamically stable system
of two immiscible liquids (oil and water), which is stabilized by an
interfacial film of surfactant and co-surfactant molecules. It is iso-
tropically clear colloidal dispersion that has droplets size less than
100 nm [19]. Nanoemulsion is easily used in skin care due to its
good sensorial properties (merging textures, rapid penetration)
and its biophysical properties (hydrating power).The high solubility
potential for drugs of the NE system might increase the thermo-
dynamic activity towards the skin [20]. Ingredients of NE act as
permeation enhancers that increase the drug flux via the skin [21].
The permeation rate of drug increases as a result of increasing of
drug affinity of NE internal phase, which can be modified easily
partitioning into the stratum cornea [22].
Accordingly, the present research study is focused on develop-
ing a new successful nanoemulsion as a potential carrier for
CoQ10 to increase its solubility, skin permeability, and thus
enhancing its anti-wrinkle efficiency.
Materials and methods
Materials
CoQ10 was kindly donated as a gift from Mebaco Company, Cairo,
Egypt. Tween 80 with the hydrophilic/lipophilic balance
(HLB ¼ 15), Span 20 (HLB ¼ 8.6), olive oil, sodium lauryl sulfate
(SLS), Tween 20, oleic acid, ethanol, and isopropanol (HPLC grade)
were purchased from El-Nasr Pharmaceutical Chemical Company,
Cairo, Egypt. Avocado oil was obtained as a gift from (Jan Dekker
Nederland B.V, Wormerveer, Holland). Caproyl 90, Labrasol
(HLB ¼ 14), Labrafil M1944 and Transcutol HP (HLB ¼ 4.2) were
given as a gift from Gattefosse (Cedex, France). Cremophor EL
(HLB ¼ 13.5), isopropyl myristate (IPM) and propylene glycol
(HLB ¼ 4.45) were purchased from Sigma-Aldrich, Germany.
Solubility study
Successful development of NE is based on selecting oil, surfactant,
and co-surfactant that showed higher solubility capacity for drug.
Solubility of CoQ10 in various oils such as oleic acid, avocado oil,
Labrafil M1944, IPM, caproyl 90, olive oil, IPM:oleic acid at a ratio
of 3:1 w/w and IPM:oleic acid at a ratio of 2:1 w/w, surfactants
(e.g. Labrasol, Tween 80, and Cremophor EL) and co-surfactants
(e.g. Transcutol HP, propylene glycol, and Span 20). An excess
amount of CoQ10 was allowed to dissolve in 1 g of each vehicle in
capped vials. The mixtures were vortexed to facilitate drug solubil-
ity and kept at 37  C ± 1.0 in an isothermal shaker for 72 h to reach
equilibrium state. The equilibrated samples were centrifuged at
3000 rpm for 15 min. The supernatant was filtered through a
0.45 mm membrane filter and diluted with the mobile phase (etha-
nol:isopropanol) at a ratio of 75:25 v/v [23]. The concentration of
CoQ10 was determined by HPLC method reported by Joe and
Sunil [24]. Briefly, the chromatographic conditions used were rep-
resented as follows; Hypersil column ODS C18 250 mm  4.6 mm,
5 mm particle size, and the mobile phase consists of 75:25 (%v/v)
mixture of ethanol:isopropanol, the injection volume of 20 mL and
the flow rate of 2 mL/min. The effluent was monitored at 275 nm.
The sample was analyzed in triplicate.
Screening of surfactants/co-surfactants mixture
Selection of surfactant/co-surfactant (S/CoS) mixture that forms oil
in water NE is based on using those mixtures with (HLB) value
greater than 10 [25]. Theoretical calculation of HLB values for three
surfactants (Labrasol, Tween 80, and Cremophor EL) and three co-
surfactants (Span20, Transcutol HP, and Propylene glycol) at differ-
ent ratios (1:1, 1:2, 1:3, 1:4, 2:1, 3:1, and 4:1) w/w were carried out
using an internet program (HLB-surfactant-Tripod Iran-surfactant.-
tripod.com). The S/CoS mixtures with HLB values at the range of
10–13 were screened for o/w NE formation following the method
developed by Ljiljana and Marija [26]. The S/CoS mixture was
added dropwise to the weighed amounts of oils and water at
fixed ratio 1:1 w/w at 25  C. During titration, samples were mag-
netically stirred in order to reach equilibrium state. The amounts
of S/CoS mixture required to emulsify the oil with water and form
transparent emulsion were recorded [27].
Constructing of pseudo-ternary phase diagram
Based on the solubility and screening studies, IPM and IPM:Oleic
acid mixture at ratio of 3:1 w/w were selected as oil, Labrasol,
Cremophor EL, and Tween 80 were selected as surfactants; mean-
while Transcutol HP and propylene glycol were selected as co-sur-
factants. Various pseudoternary phase diagrams were constructed
using aqueous titration method. The weight ratios of S/CoS were
4:1, 3:1, and 2:1 w/w. Different combinations of different weight
ratios of oil and S/CoS mixture 1:9–9:1 w/w were made to con-
struct phase diagrams. Briefly, water was added dropwise under
magnetic stirring at 25  C to each weight ratio of oil and S/CoS
until it turned turbid [28]. The volume of water used was then
recorded in each system. The physical state of NE was marked on
pseudo-3-component phase diagrams. One axis of the pseudo-
three-component phase diagram represented the aqueous phase,
the other represented the oil phase, and the third represented a
mixture of surfactant and co-surfactant phase at a fixed weight
ratio [29]. The existence of the micro/nanoemulsion field was
monitored by clear and transparent areas in phase diagrams.
Optimization of CoQ10 loaded NE formulations
From the selected phase diagrams, different formulae were pre-
pared using 10% of oil, S/CoS mixture ranged 50–60%, and water
ranged 30–40% to optimize their composition to form NE and
ensuring emulsification stability in the presence of the drug. Drug-
loaded NE formulations were prepared by dissolving accurately
weighed amount of CoQ10 into the pre-weighed oil-S/CoS mix-
ture. The specified amount of water was added dropwise under
ultrasonication till a clear and transparent liquid was obtained.
To optimize the NE formulations, the prepared formulae were

subjected to two tests, namely, dilution test followed by thermo-
dynamic stability test.
Dilution test and thermodynamic stability study
From each formula, 1 g of the sample was diluted with 100 ml dis-
tilled water. The formula is judged good in case of a transparent
and homogeneous NE is formed with no phase separation and
drug precipitation. The formulae that passed the dilution test were
subjected to thermostability test [30]. In this stability test, the for-
mulae were firstly centrifuged at 3500 rpm for 30 min. The formu-
lae that did not show any phase separations were subjected to
heating-cooling cycle test. Six cycles between 4 and 45  C with
storage at each temperature for not less than 48 h were under-
taken. The formulae that passed these circumstances and showed
good stability were subsequently subjected to three freeze-thaw
cycles test between
21  C and 25  C temperature [31]. Formulae
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that survived the thermodynamic stability tests were selected for
further characterization.
Characterization of CoQ10 NE
Percentage of drug content
Approximately 1 g of NE was dissolved in 50 mL (ethanol:isopropa-
nol at a ratio of 75:25 v/v) and drug content was measured using
HPLC method. The percentage of drug content was estimated fol-
lowing the given equation:
% Drug content ¼ Actual content=Theoretical content  100:
pH determination
The pH for each formula was determined in triplicate at 25  C with
a digital pH meter (HANNA Instrument, Woonsocket, RI) [32].
Viscosity determination
The viscosity of 0.5 g NE was determined at 50 rpm using spindle
(CPE40) at 25 ± 0.5  C (Brookfield Digital Viscometer DV III,
Middleboro, MA) [31].
Globule size and distribution analysis
The average of droplet size and polydispersity index (PDI) of NE
were determined using Malvern ZetaSizer (NanoZS90, Malvern
instrument Ltd., UK) with a 50 MV laser. The measurements were
performed at 25  C at a fixed angle of 90 . The measurement time
was 3 min. One gram of each formula was dispersed into 100 ml
of distilled water with gentle stirring in a glass beaker. An aliquot
of diluted NE was transferred into the cell sample holder for drop-
let size analysis. The data was represented triplicate samples ± SD
[33].
Zeta potential measurement
Zeta potential was measured using a zeta potential analyzer,
Malvern ZetaSizer (NanoZS90, Malvern Instrument Ltd., UK).
The diluted NE formulation was exposed to an electric field (1 V).
The data was represented triplicate samples ± SD.
Refractive index
The refractive index of NE has been determined at 25  C using an
Abbe refractometer (Digital Abbe refractometer, WAG-25,
Shanghai, China) [31].
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 3
Electroconductivity study
The electrical conductivity (r) of NE was determined using digital
conductometer (HANNA Instrument, H1255, Romania) at a dilution
ratio of 1:50 and 1:100 v/v [34].
Transmission electron microscopy examination
Morphology and structure of the NE globules were performed
using transmission electron microscope (TEM, Tecnai G20, Super
twin, double tilt, FEI, Netherlands) which is operated at 200 kV. A
drop of diluted nanoemulsion mixed with water at ratio 1:10 v/v
was directly deposited on the holey film grid, stained by 1%w/v
aqueous solution of phosphotungstic acid, and observed after dry-
ing it [35].
In vitro release studies
In vitro release of CoQ10 from NE formulae was investigated
through a semipermeable membrane (Sigma, molecular weight
cut off is 12,000 Da) using the modified dissolution apparatus I.
Different dissolution mediums were applied to determine the opti-
mal and most discriminating dissolution medium that allows effi-
cient dissolution and release the highly lipophilic drug (CoQ10).
Evaluated dissolution mediums were phosphate buffers with differ-
ent compositions: phosphate buffer at pH 5.5, phosphate buffer at
pH 7.4, phosphate buffer at pH 7.4 containing 0.25% w/v SLS,
0.25% w/v Tween 20, and 10% w/v isopropyl alcohol [36], phos-
phate buffer at pH 7.4 and 5%w/v Labrasol [37], and a newly
developed one (phosphate buffer at pH 7.4, 5% w/v Labrasol and
5% w/v isopropyl alcohol). A glass cylindrical tube of 2.5 cm diam-
eter and 6 cm length was tightly covered with semipermeable
membrane and hanged on the shaft of dissolution apparatus. One
gram of CoQ10 loaded NE was transferred to the cylindrical tube,
dipped into 100 ml of dissolution medium. Release study was car-
ried out for 24 h at the 37  C with stirring speed of 100 rpm [38].
At pre-determined time intervals: 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16,
and 24 h; an aliquot (1 mL) of dissolution medium was withdrawn,
filtered through a 0.45 mm filter, and replaced with an equal
volume of fresh medium to maintain sink condition. The
collected samples were analyzed for drug concentrations using
HPLC at k max 275 nm.
Kinetic analysis of CoQ10 nanoemulsion release data. To examine
the kinetics and mechanism of the drug release from different for-
mulae of NE, the data obtained from the drug release study was
fitted to models representing zero-order, first-order, and Higuchi's
square root of time. The results were also analyzed according to
the Korsmeyer–Peppas equation [39]:
Mt =M1 ¼ k  tn
where Mt/M1 represents the fraction of the drug released after
time t, k is the kinetic constant and n is the slope value of log mt/
m1 versus log time. The ‘n’ exponent is a characteristic value for
the release mechanism. The ‘n’  0.5 is for Fickian diffusion,
whereas n (0.5–1) indicates anomalous mechanism due to matrix
relaxation or erosion. (n ¼ 1) is expected for zero-order release.
Ex vivo skin permeation studies
The ex vivo permeation studies were carried out using Franz diffu-
sion cell, which is a reliable method for prediction of drug trans-
port across the skin [40]. These studies were conducted by
employing excised skin of Wistar rats and phosphate buffer at pH
7.4 containing 5% w/v Labrasol and 5%w/v isopropyl alcohol as
 4 E. S. EL-LEITHY ET AL.
dissolution medium. For preparing the skin for permeation study,
the hair on the dorsal side of the skin was firstly removed by a
hair removing cream, followed by removing the adhering fat layer
on the other skin side using a clipper and the whole skin was
washed with distilled water. The skin was wrapped in aluminum
foil and stored at
20  C until used. Before the experiment, the
skin was taken from the freezer and allowed to be unraveling
from ice freezer then cut into 4.5 cm 4.5 cm pieces and soaked
in phosphate buffer overnight at 4  C to allow full skin hydration
[41]. The skin pieces were clamped between the donor and the
receptor compartment of Franz diffusion cells with an effective dif-
fusion area of 1.77 cm2 whereas; the stratum corneum layer was
facing the donor compartment. Aliquots of 0.5 g of NE formulae
(F2, F8, and F14) and CoQ10 suspension equivalent to 10 mg
CoQ10 were injected to the donor compartments. The CoQ10 sus-
pension was prepared according to Wen and Tung [10] (1% w/v
CoQ10 in 1% w/v sodium lauryl sulfate aqueous solution). The
receptor compartment with volume 7.5 ml was filled with the
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above mentioned the phosphate buffer and maintained at
37  C ± 1 with stirring at 500 rpm. At pre-determined time intervals:
0.5, 1, 2, 4, 6, 8, 10, 12, 24 h; 1 ml aliquot of the receptor medium
was withdrawn and replaced with the same volume of fresh
medium. All the collected samples were filtered using 0.45 mm and
then CoQ10 content was determined using HPLC method.
The cumulative amounts of permeated CoQ10 through the skin
per unit surface area were calculated using the given equation
and subsequently used for plotting the profiles of the drug perme-
ation per unit time (t).
Cumulative amount of drug permeated ¼
Concentration ð lg=mlÞ  dilution factor
Surface area of skin ðcm2 Þ
The rate of drug permeation at steady state (drug flux) (Jss) was
presented by the slope of the linear portion of the plotted pro-
files. The permeability coefficient (Kp) was calculated by dividing
the Jss with an initial concentration of the drug in the donor cell
(Co) mg/ml by using the following equation [42].
Kp ¼ Jss =Co
Enhancement ratio (Er) was calculated by dividing the Jss of the
respective formula by that of control using the following equation:
Er ¼ Jss of formulation=Jss of control:
The skin pieces were collected, rinsed with distilled water, gen-
tly dried with a cotton swab, cut into small pieces, and soaked in
a 10 mL mixture of ethanol:isopropyl alcohol at a ratio of 75:25 v/v
for 12 h in closed tubes. The tubes were sonicated for three cycles
each 15 min to avoid the rise of the temperature during the
extraction process. The supernatant was filtered using 0.45 mm and
the CoQ10 content was estimated using HPLC [43].
Statistical analysis
The experimental in vitro release and ex vivo permeation data
were further subjected to statistical analysis using one way
ANOVA test ‘SPSS version 17.0 for Windows’ (SPSS Inc., Chicago,
IL) followed by post-HOC LSD alpha (0.05) (95% confidence
intervals).
In vivo anti-wrinkle evaluation of CoQ10 NE
The anti-wrinkling activity of the optimized CoQ10 NE formula (F2)
was evaluated through an animal model. Twelve- to eighteen-
month-old female rats weighing 200–240 g were obtained from
the animal house of National Organization for Drug Control and
Research. This work was carried out according to the international
guidelines for caring and usage of laboratory animals. The experi-
mental protocol had been ethically approved by the Animal Care
Committee, Faculty of Pharmacy, Helwan University (No (3) for 14/
6/2016). In the current study, two methods were adopted for eval-
uating the anti-wrinkle activity of the optimum nanoemulsion
formula.
Morphological examination
Laboratory rats had free access to food and water and were
adapted to the air-conditioned room (23 ± 2  C and 50 ± 10%
humidity with 12 h light/12 h dark cycle) for 1 week before the
study. First, the rat dorsal hair was removed using hair removal
cream then divided into two groups, each group contains three
rats. The first group was marked as a control (untreated) and the
second group was treated with 1 g of the optimum NE formula
(F2) twice a day for 1 month. The formula was applied topically
on the skin on the square area of 10  5 cm of the rat dorsal part.
The morphological examination of the animal groups was carried
out after 0, 7, 14, 21, and 30 days of treatment by taking photo-
graphs for animal skin [44].
Histopathological evaluation
At the end of 30 days, the rats were sacrificed and the skin was
taken for histopathological examination. The skin of rats was cut
and fixed in 10% v/v formalin solution for 24 h. Before the examin-
ation, the skin was washed with tap water and followed by a
diluted alcohol for dehydration. The specimens were cleared in
xylene and embedded in paraffin at 56 in hot air oven for 24 h.
Paraffin beeswax tissue blocks were prepared for sectioning at 4 m
thickness by sledgemicrotome. The obtained tissue sections were
collected on glass slides, deparaffinized, and stained with hema-
toxylin & eosin stain for examination under the light electric
microscope [45].
Results and discussion
Solubility studies
The ability of NE to maintain the drug in a soluble form is greatly
influenced by its solubility in the oil phase. However, drug solubil-
ity due to the presence of surfactant or co-surfactant may lead to
a risk of drug precipitation [46]. Solubility limit of CoQ10 in differ-
ent oils (Figure 1) was arranged in the following descending order;
IPM: oleic acid at a ratio of 3:1 w/w > IPM > IPM: oleic acid at a
ratio of 2:1 w/w > oleic acid > avocado oil > caproyl 90 > Labrafil
M 1944 > olive oil. The solubility of IPM: oleic acid mixture at
mass ratio 3:1 w/w and IPM were 169.98 ±17.43 and
118.89 ± 0.084 mg/g, respectively, accordingly both were chosen as
optimum oil phases for subsequent study. The solubility of CoQ10
in surfactants was found to be 36.90 ± 0.04, 28.29 ± 1.76, and
13.22 ± 0.51 mg/g for Labrasol, Tween 80, and Cremophor EL,
respectively. For co-surfactants, the solubility has been as follows
24.29 ± 5.81, 0.55 ± 0.01, and 0.52 ± 0.03 mg/g for Span 20,
Transcutol HP, and propylene glycol, respectively (Figure 1).
Screening of surfactants/co-surfactants mixtures
All S/CoS mixtures with an HLB value greater than 10 were
selected and dropped into a mixture of water and oil (IPM or IPM
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 5
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Figure 1. Solubility of coenzyme Q10 in different oils, surfactants, and co-surfactants.

Table 1. Selected system for subsequent phase diagram construction.

Oil Composition of S/CoS Ratio of S/CoS
IPM Tween 80:Transcutol HP 2:1, 3:1, 4:1
Cremophor EL:Transcutol HP 2:1, 3:1, 4:1
IPM:oleic acid Labrasol:PG 2:1, 3:1, 4:1
3:1 Tween80:Transcutol HP 2:1
Cremophor EL:Transcutol HP 2:1, 3:1, 4:1
Labrasol:Transcutol HP 2:1 ,3:1, 4:1
Concentrations of oil (10–30%), S/CoS (40–60%), and water (35–50%).

Figure 2. Pseudoternary phase diagrams of IPM:oleic acid (3:1; oil), Labrasol: Transcutol HP (S/COS) at ratio (A) 2:1, (B) 3:1, and (C) 4:1 and water as an example of a
system with a poor nanoemulsion region.
 6 E. S. EL-LEITHY ET AL.
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Figure 3. Pseudoternary phase diagrams of IPM (oil), Tween80:Transcutol HP(S/COS) at ratio (A) 2:1, (B) 3:1, and (C) 4:1 and water as an example of a system with a
large nanoemulsion region.

Table 2. Composition and thermodynamic stability evaluation for CoQ10 nanoemulsion.

Composition of
Nanoemulsion %w/wa Thermodynamic stability test
Formula Drug %w/w Dilution
Code S/CoSratio Oil S/CoS Water of drug Test Centrifugation Heating-cooling Freeze-thaw Inference
F1 2:1 10 60 30 1 Clear 冑 冑 冑 Pass
F2 10 60 30 2 Clear 冑 冑 冑 Passb
F3 10 60 30 3 Clear 冑 冑  Failed
F4 10 50 40 1 Clear    Failed
F5 10 50 40 2 Turbid _ _ _ Failed
F6 10 50 40 3 Turbid _ _ _ Failed
F7 3:1 10 60 30 1 Clear 冑 冑 冑 Pass
F8 10 60 30 2 Clear 冑 冑 冑 Passb
F9 10 60 30 3 Clear 冑 冑  Failed
F10 10 50 40 1 Clear    Failed
F11 10 50 40 2 Clear    Failed
F12 10 50 40 3 Clear    Failed
F13 4:1 10 60 30 1 Clear 冑 冑 冑 Pass
F14 10 60 30 2 Clear 冑 冑 冑 Passb
F15 10 60 30 3 Clear 冑 冑  Failed
F16 10 50 40 1 Clear    Failed
F17 10 50 40 2 Clear    Failed
F18 10 50 40 3 Clear    Failed
a
Oil ¼ IPM, S/CoS ¼ Tween 80: Transcutol HP.
b
Refer to the stable formulations that containing the optimum amount of the drug and subjected to further characterization.

and oleic acid mixture) at a ratio of 1:1 w/w until obtaining trans-
parent NE. The systems that gave transparent NE were judged
‘good’, while the systems that gave turbidly or no NE formation
were judged ‘bad’. The S/CoS mixture that succeeded to emulsify
the water/oil mixture with minimum concentration ranged
30–60% w/w were selected and subsequently used for
constructing pseudo-ternary phase diagrams to identify and opti-
mize the o/w NE regions as shown in Table 1. The results showed
that although Co-surfactant Span 20 exhibited the maximum solu-
bility for CoQ10, all of its NE formulae with both oil types were
judged bad and in turn, it was excluded from our study. Also, all
S/CoS mixtures (e.g. Labrasol–propylene glycol mixture) with
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 7

Table 3. Characterization of Q10 nanoemulsion. 60 F2

Characterization F2 F8 F14 F8
50 F14
pH 7.06 ± 0.051 7.16 ± 0.045 7.15 ± 0.180
Viscosity (cp) at 50 rpm 199.05 ± 0.35 236.75 ± 18.87 8035.5 ± 375.47 40

% Q10 release
Droplet size (nm) 11.76 ± 1.1 11.17 ± 0.95 11.73 ± 1.17
Polydispersity index 0.228 0.278 0.262 30
Zeta potential (mv)
14.7 ± 1.23
17.2 ± 1.70
11.3 ± 0.99
Drug content 101.58 ± 0.142 102.85 ± 0.500 101.08 ± 0.800 20
Refractive index 1.4260 ± 0.001 1.4262 ± 0.00077 1.4250 ± 0.001
Electroconductivitya 48.03 ± 0.55 52.36 ± 0.77 58.53 ± 0.5 10
Electroconductivityb 42.5 ± 2.29 50.06 ± 0.2 53.83 ± 0.76
a
At dilution ratio 1:100. 0
b
At dilution ratio 1:50 (mean value ± SD) (n ¼ 3). 0 5 10 15 20 25 30
Time hr

Figure 5. In vitro release studies of CoQ10 nanoemulsions in phosphate buffer

pH 7.4 containing 5% w/v Labrasol and 5% w/v isopropyl alcohol.
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Figure 4. Typical TEM image of CoQ10 nanoemulsion (a) and its size distribution (b) measured by zetasizer at room temperature.

Table 4. Percentage of Q10 released from nanoemulsion formulas in the different dissolution media at specified periods of time.
Percentage drug released from different formulas
In phosphate buffer at pH 7.4 containing
0.25% w/v SLS, 5% w/v
Tween 80, 10% w/v 5% w/v Labrasol,
Time (h) isopropyl alcohol 5%w/v Labrasol 5% w/v isopropyl alcohol
10 F2 1.61 ± 0.23 9.41 ± 0.50 7.31 ± 0.04
16 5.88 ± 0.06 10.86 ± 0.16 39.44 ± 1.02
24 6.79 ± 0.43 10.92 ± 0.48 47.21 ± 4.41
10 F8 0.59 ± 0.11 2.13 ± 0.22 0.94 ± 0.1
16 4.23 ± 0.29 3.53 ± 0.33 4.48 ± 0.2
24 5.08 ± 0.1 3.54 ± 0.33 5.49 ± 0.17
10 F14 0.57 ± 0.08 0.82 ± 0.116 2.41 ± 0.31
16 2.42 ± 0.12 1.98 ± 0.021 4.01 ± 0.19
24 2.66 ± 0.38 2.06 ± 0.097 4.08 ± 0.08
 8 E. S. EL-LEITHY ET AL.
Table 5. Kinetic analysis of the drug release data.
Zero-order
(m0
m) ¼ kt
Formulation R2 K R2
F2 0.948 5.89
F8 0.998 0.637
F14 0.921 0.262
120 Control
Cumulative amount of Q10
F2
100
permeated µg/cm2
F8
80 F14
60
40
20
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0
0 5 10 15
Figure 6. Ex vivo permeation studies of CoQ10 nanoemulsions in comparison to
control (CoQ10 suspension in 1%w/v SDS) in Phosphate buffer pH 7.4 containing
5%w/v Labrasol and 5%w/v isopropyl alcohol.
concentration more than 60%, even showed good NE formation,
was excluded to avoid the possibility of skin irritation [47]. Co-sur-
factants improve drug permeation, decrease bending stress of
interface, give sufficient interfacial film flexibility, and take up dif-
ferent curvatures required to form NE [48].
Construction of pseudoternary phase diagram
Various pseudo-ternary phase systems (68 systems) were prepared
from IPM or IPM:oleic acid as oil (10–30%), S/CoS (40–60%) at dif-
ferent ratios and water (35–50%). Table 1 illustrated 16 systems
selected based on their clarity, miscibility, and flowability. The rest
was turbid, immiscible, and viscous. Figure 2 shows three dia-
grams of IPM:oleic acid mixture 3:1 w/w as oil, Labrasol, and
Transcutol as S/CoS mixture at three ratios 4:1, 3:1, and 2:1 w/w
and water as an example for system with poor NE region. While
Figure 3 shows three diagrams of IPM as oil, Tween 80,
and Transcutol HP as S/CoS mixture at three ratios 4:1, 3:1, and
2:1 w/w and water as an example to a system with the large
nanoemulsion region. The black areas represent the o/w NE
regions and the rest regions represent the turbid or conventional
emulsions [49]. The results showed that all diagrams constructed
with IPM:oleic acid mixture had poor NE regions in comparison to
those constructed with IPM.
The figures clearly revealed that nanoemulsion formation was
highly dependent on the amount of oil, surfactant, and co-surfac-
tant and their mass ratio. Largest black zones for NE were shown
at higher S/CoS ratio, which was arranged descendingly as
4:1 > 3:1 > 2:1 w/w. Increasing the S/CoS ratio improves the
micelle formation and increases the solubilizing capacity of the
NE. Co-surfactant was inserted into the cavities between the sur-
factant molecules and consequently, it increases the solubility of
formed NE to maximum value [50].
Optimization of CoQ10 loaded NE
From the above results, 18 formulae from the three diagrams of
the IPM system were selected and prepared for discriminating
Higuchi Krosemeyer–Peppas
First order (lnm ¼ kt) (mt/m1 ¼ kt1/2) (mt/m1 ¼ ktn)
K R2 K R2 N Order of release
0.88 0.156 0.958 40.86 0.964 0.17 Higuchi
0.979 0.091 0.999 4.75 0.913 0.43 Higuchi
0.902 0.039 0.934 1.824 0.989 0.44 Higuchi
the efficient NE formulae with a high capacity for drug loading
and acceptable thermodynamic stability. These 18 formulae
were subjected to dilution test, the formula that showed a
transparent and clear solution was judged clear and the formula
showed precipitation or phase separation was judged turbid.
The results showed that all formulae passed the test except for-
mula (F5) and (F6) which showed drug precipitation upon dilu-
tion with water at ratio 1:100 v/v. This could be due to the
presence of a low concentration of the surfactant than other
20 25 30 formulae (Table 2).
Time hr The formulae that passed the dilution test were subjected to
thermodynamic stability test to differentiate NE from the emulsion
formulae [32]. Results presented in Table 2 revealed that the for-
mulae F3, F9, F12, F15, and F18 containing 3%w/w drug and those
containing 50% w/w S/CoS mixture (F4, F10, F11, F12, F16, F17,
and F18) were unstable and showed precipitation of the drug dur-
ing the thermodynamic stability tests. This might be attributed to
the internal phase coagulation at low temperature and the
unusual pressures exerted by the ice crystals on the dispersed
globules and the absorbing layers of S/CoS [51]. The formulae F1,
F2, F7, F8, F13, and F14 containing 1 and 2% w/w of the drug and
60% w/w S/CoS mixture was clear, transparent, and showed no
phase separation, cracking, or drug precipitation. The stability of
these formulae was due to low drug percentage (1 and 2% w/w)
and the higher percentage of S/CoS that increased the solubility
of the drug and prevented its precipitation [25]. As the drug per-
meation through the skin is highly dependent on the drug con-
centration, the stable formulae containing 2% w/w CoQ10 (F2, F8,
and F14) were selected for further characterization.
Characterization of CoQ10 NE
The pH, drug content, and viscosity determination
The topically delivered formulations should have a pH range of
6–8 to minimize the skin irritation [32]. The pH of the three
selected formulae F2, F8, and F14 was around 7 (Table 3). The per-
centage drugs content of the selected NEs F2, F8, and F14 were
101.58 ± 0.142, 102.85 ± 0.5, and 101.08 ± 0.8% w/w, respectively.
These results revealed the efficacy of the method of preparation
and the best capability of these formulas to be loaded with
CoQ10 up to 2% w/w. The viscosity values were found as
follows199.05 ± 0.35, 236.75 ± 18.87, and 8035.5 ± 375.47 cp for F2,
F8, and F14, respectively (Table 2). The high viscosity recorded
with the formula F14 was attributed to the high percentage of
Tween 80 (48% w/w) [52].
The TEM, droplet size, and particle size distribution
The TEM image of CoQ10 NE revealed spherical shaped droplets
with a nearly uniform particle size (<100 nm) (Figure 4(a)). These
results coincided with the results of particle size analysis that
recorded 11.76 ± 1.1, 11.17 ± 0.95, and 11.73 ± 1.17 nm as an aver-
age droplets size and polydispersity index (PDI) 0.228, 0.278, and
0.262 for F2, F8, and F14, respectively, refer to Figure 4(b) and

Table 6. Permeability parameters and the extracted amount of Q10 from the skin.
Formulation code Jss (mg/cm2/h)
Control 0.38
F2 3.164
F8 2.666
F14 0.744
Jss: drug flux; Kp: permeability coefficient; Er: enhancement ratio.
1600
Extracted amount of Q10 from
1400
1200
skin µg/cm2
1000
800
600
400
200
Downloaded by [Gothenburg University Library] at 06:48 03 November 2017
0
F2 F8 F14
Buffer 7.4+5%L abrasol+5%isopropanol
Figure 7. Extracted amount of CoQ10 from the rat skin at the end of ex vivo per-
meation studies of CoQ10 nanoemulsions in comparison to control (CoQ10 sus-
pension in 1%w/v SLS).
Table 3. These results reflect the homogeneity and nanometric
droplet size for all NE formulae.
Zeta potential measurement
The zeta potential values of F2, F8, and F14 were negative values
of
14.7 ± 1.23,
17.2 ± 1.7, and
11.3 ± 0.99 mv, respectively
(Table 3). The negative charge on the emulsion droplets was
attributed to the free fatty acids and/or the surfactants and co-sur-
factants used since these two materials are mostly derivatives of
fatty acids [33].
Refractive index and electro-conductivity study
The mean values of the refractive index of the NE formulae were
in the range of 1.425–1.426 (Table 3). These values were much
closer to the refractive index of water (1.33) indicating o/w nanoe-
mulsions formation that had percentage transmittance more than
99% [53]. Meanwhile, the electrical conductivity was used to meas-
ure the electroconductive nature of the system. The conductivity
of the nanoemulsion formulae diluted at ratios 1:50 v/v and 1:100
v/v were high and ranged from 42.5 to 58.5 mS/cm (Table 3).
These high values were attributed to the presence of water as an
external phase which allows free mobility of ions [54,55].
In vitro release studies
Different dissolution mediums were evaluated for the drug release
from NE formulae (F2, F8, and F14) through a semipermeable
membrane. Our results revealed the undetectable concentration of
CoQ10 in phosphate buffers at pH 5.5 and 7.4. Both media were
recognized as unsuitable dissolution media for the highly lipo-
philic drug CoQ10. The percentage of CoQ10 released from the NE
formulae in the different dissolution media at specified periods of
time was presented in Table 4. It could be seen that the dissol-
ution media composed of phosphate buffer at pH 7.4, 5% w/v
Labrasol and 5%w/v isopropyl alcohol showed the highest per-
centage of drug release. The presence of 5% w/v Labrasol estab-
lished sink conditions and sustained better solubility of CoQ10 in
the dissolution media [56]. When a drug is practically insoluble in
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 9
Kp (cm2/h)  10
4 Er Amount of the drug extracted per area (mg/cm2) ± S.D
2.66 – 199.22 ± 18.3
22.14 8.32 1389.04 ± 15.66
18.66 7.015 882.41 ± 41.79
5.2 1.957 761.65 ± 14.05
the normal buffered aqueous media (with or without surfactants),
the use of a non-aqueous media is deemed as an alternative
approach. Isopropyl alcohol as ‘co-solvent’ reduces water–water
interactions and increases the solubility of lipophilic drugs [57].
The solubility of CoQ10 had been previously recorded as
36.9 mg/ml in (Labrasol) and was reported as 38.8 mg/ml in (iso-
propyl alcohol) [58].
Release profiles of nanoemulsion formulae F2, F8, and F14 in
the selected dissolution media were presented in Figure 5.
Control
Formula F2 exhibited the highest significant percentage of drug
release after 24 h (p < .001), that reached 8.5 folds higher than F8
and 11.57 folds higher than F14, while there is no significant dif-
ference between those amounts released from F8 to F14 (p > .05).
These results were attributed to lower viscosity of F2
(199.05 ± 0.35 cp), smaller droplet size (11.76 ± 1.1 nm) of its glob-
ules and low surfactant concentration. At higher surfactant con-
centration, the affinity of drug would be higher to the vehicle and
the thermodynamic activity would be lower leading to slow
release of drug and/or a poor transfer of drug from the vehicle to
the receptor medium [59].
Kinetic analysis data. Correlation coefficients (r2) and release con-
stants derived from kinetics analysis of the CoQ10 NE release data
are presented in Table 5. The mechanism of drug release is actu-
ally best fitted Higuchi model, whereas Korsmeyer–Peppas plots
showed high linearity (R2 values are between 0.913 and 0.989) and
slope values ‘n’ are less than 0.5 [60]. These results indicated that
the drug diffusion was the rate limiting step.
Ex vivo skin permeation studies
Permeation profiles of the cumulative amounts of CoQ10 released
in the selected phosphate buffer from F2, F8, F14 NE formulae,
and CoQ10 suspension (as control) per unit time are presented in
Figure 6. The three NE formulae showed a highly significant effect
on the amounts of CoQ10 permeated through rat skin compared
to CoQ10 suspension in 1% w/v SLS (p < .001) that enhances the
skin penetration by perturbing skin barrier and stratum corneum
skin layers [10].
The formula F2 showed a highly prominent effect on improv-
ing the drug permeation compared to the other formulae that
ordered descendingly as F8 > F14 > control. Our results revealed
the efficacy of the NE on improving the skin permeation of the
highly lipophilic drug CoQ10). NE penetrates the stratum cor-
neum skin layers and alters both lipid and polar pathways. The
drug dissolved in the lipid domain of the NE can directly pene-
trate the lipid of stratum corneum skin layer, thereby destabiliz-
ing its bilayer structure. These interactions will increase the lipid
pathway permeability to drugs. On the other hand, the hydro-
philic domain of NE can hydrate the stratum corneum skin
layers to a greater extent and enhance percutaneous uptake of
drugs [61].
NE presents drugs in nanosized and solubilized globules. The
presence of surfactants in NE formulae enhances skin permeation
due to their solubility or extracting effect on the lipid barrier of
 10 E. S. EL-LEITHY ET AL.

the stratum corneum skin layers, thus increasing the membrane
fluidity and leading to alterations in the tight junction properties
of stratum corneum skin layers [62].
Transcutol HP, which is used as co-surfactant in all NE formulae,
has been reported as an efficient permeation enhancer [63]. Also,
the IPM oil used in all NE formulae is an aliphatic ester having the
ability to penetrate the skin lipid bilayers of the stratum corneum
skin layers by disrupting their arrangement and orders. The IPM
has lower values of Hildebrand solubility parameter (d ¼ 8.02),
which is near to that of human skin (d ¼ 10.5). Thus, IPM was con-
sidered as an excellent selection for formulating the typical NE for-
mulae [64].
Permeability parameters including drug flux (Jss), permeation
coefficient (Kp), and enhancement ratio (Er) were calculated and
presented in Table 6. All parameters were significantly increased
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with NE formulae in comparison to control (p < .001). The values
of 3.164 mg/cm2/h, 8.32 and 22.14  10
4 cm2/h were recorded for
F2 as the highest flux (Jss), enhancement ratio (Er), and permeation
coefficient (Kp), respectively.
At the end of permeation experiment, the efficacy of the NE
formulae on penetrating into skin layer was evaluated by deter-
mining the amount of CoQ10 retained in rat skin [65]. The accu-
mulated amounts in rat skin after exposing to NE formulae and
CoQ10 suspension as control was determined and presented in
Figure 7. It was found that CoQ10 NE formulae allowed for high
drug retention in rat skin. The amounts of CoQ10 extracted from
the skin after 24 h were 1389.04 ± 15.66, 882.41 ± 41.79, and
761.65 ± 14.05 mg/cm2 for F2, F8, and F14, respectively. These val-
ues present about 6.97, 4.42, and 3.82 folds of CoQ10 accumulated
in skin treated with coenzyme (CoQ10) from suspension (control).

Figure 8. Photographs showed thick and deep wrinkles on the skin of control (un-treated group) of rats (A), smooth skin surfaces with the absence of wrinkles after
30 days of treatment with NEF2 (B), multiple numbers of wrinkles folds associated with hyperkeratosis, parakeratosis and squamous thick horny layer in untreated group
(C) and mild wrinkles associated with thick prickle cell layer, lower keratosis, and parakeratosis and cells retaining their structure with obvious nuclei in NEF2 group (D),
declined sebaceous glands in control (E) while hypertrophy and hyperplasia of gland in NEF2 group (F), the sweat glands and hair follicles with narrow lumen and
pyknotic nuclei (G) in control and hyperplasia and hypertrophy in the sweat gland with wide lumen of the hair follicles in NEF2 group (H), hyalinization in the collagen
and elastin fibers in control group (I) while a define, well differentiated fibrillar structure of the collagen and elastin fibers in NEF2 group (J), hylonization in muscle
fiber (K) in control group while a defined, well differentiated fibrillar in muscle structure in NEF2 group (L).

Skin histopathological evaluation
Skin aging is characterized by changing the skin thickness, elasti-
city, color, vascular dilatation, and wrinkles. The formation of wrin-
kles and fine lines on the skin is attributed to epidermal atrophy,
degeneration of dermal collagen, and elastic fiber deterioration
[44]. Morphological examination of the skin of both old rat
groups (untreated and treated) from 0 to 30 days are shown in
Figure 8(A,B). It could be seen that the control group had no
changes in the skin that showed multiple numbers of wrinkles
folds in epidermis (Figure 8(A)), the rat group treated with NE F2
showed a complete decrease in the number of wrinkles and
appearance of smooth skin after 30 days of treatment (Figure 8(B)).
Further evaluation of the effect of CoQ10 NE on the skin wrin-
kles was carried out through the histopathological evaluation of
skin layers.
Figure 8(C,D) shows the histopathological examination of upper
epidermal layers of untreated and treated rat skin. The untreated
Downloaded by [Gothenburg University Library] at 06:48 03 November 2017
rat skin (Figure 8(C)) showed hyperkeratosis and parakeratosis in
the epidermal layer and flatten thick horny layer. Hyperkeratosis is
a thickening of stratum corneum layers caused by the accumula-
tion of excess numbers of stratum corneum cells and disorders of
keratinization because the process of desquamation is disturbed.
Parakeratosis is a nucleated keratinocyte in the stratum corneum
layers, which may occur due to accelerated keratinocyte turnover.
Parakeratosis can be used as morphological examination character
in differentiating and classifying certain inflammatory skin diseases
in the epidermal layer [66]. The treated group with CoQ10 NE (F2)
showed less hyperkeratosis and parakeratosis with the appearance
of the thick prickle cell layer and well retained healthy cells with
obvious nuclei.
The dermal layer contains sweat glands, hair follicles, and seba-
ceous glands which provide the subcutaneous fat and the plumpy
appearance of the skin and hair. The control untreated group
(Figure 8(E)) shows a decline in sebaceous gland size in compari-
son to the treated group with CoQ10 NE) (F2) that shows hyper-
trophy and hyperplasia (increase in the size and number of
sebaceous glands cells) (Figure 8(F)). The sweat glands and hair
follicles of the untreated group had a narrow lumen and pyknotic
nuclei (Figure 8(G)). While the treated group with CoQ10 NE (F2)
revealed hyperplasia and hypertrophy in the sweat gland with a
wide lumen of hair follicles (Figure 8(H)).
Figure 8(I,J) shows the extended effect of CoQ10 NE on the
hyalinization of collagen and elastin fibers of dermis and hypoder-
mis layers. The untreated group (Figure 8(I)) shows a well-defined
degenerated neoplastic smooth muscle cells, collagen fibers lost
their details, and appeared fused with a homogeneous, cellular
glassy eosinophilic appearance. The collagen elastin fibers of the
dermis and hypodermis also showed poor-defined histological
structure. On the other hand, treated group with the CoQ10 NE
F2 showed a well-differentiated fibrillar histological structure of
collagen and elastic fiber with less appearance of hyalinization
(Figure 8(J)).
Figure 8(K) showed that hyalinization was also noticed in the
skeletal muscle underneath the dermis and hypodermis layers for
the untreated group. While, the treated group with CoQ10 NE (F2)
showed defined, a well differentiated fibrillar histological structure
in the skeletal muscle with detectable nuclei in the underneath
layer of dermis and hypodermis (Figure 8(L)).
Conclusion
The extract of our investigation suggested a sufficient manipula-
tion of the stratum corneum barrier induced by the preparation
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 11
and characterization of o/w NE containing CoQ10 (2% w/v) with
minimum surfactant concentration. NE ‘F2’ showed the highest
release through 24 h (47.21%) and the highest drug flux (Jss),
enhancement ratio (Er), and permeability coefficient (Kp), their val-
ues were 3.164 mg/cm2/h, 8.32, and 22.14  10
4cm2/h, respect-
ively. Application of coenzyme CoQ10 NE on wrinkled skin showed
a decrease in the skin wrinkles with the appearance of the smooth
surface and less parakeratosis and hyperkeratosis in the epidermal
layer. Well-define fibrillar histological structure without hyaliniza-
tion appeared in the elastic and collagen fibers of the dermis and
skeletal muscles beneath dermis and hypodermis. It can be con-
cluded that the developed NE can be used as a potential vehicle
for enhancement of solubility and permeability of CoQ10 through
topical applications as well as improvement of its anti-wrinkle
efficacy.
Acknowledgements
The authors are thankful to Gattefosse (Cedex, France) for provid-
ing the gift samples. Authors would like also to acknowledge
National Organization for Drug Control and Research for providing
all necessary facilities during experimental work. The authors are
thankful to Prof. Dr. Adel Bakeer Kholoussy, Professor of
Pathology, Cairo University, Egypt.
Disclosure statement
No potential conflict of interest was reported by the authors.
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