Iranian Journal of Pharmaceutical Research (2016), 15 (1): 35-52 Copyright © 2016 by School of Pharmacy

Received: February 2014 Shaheed Beheshti University of Medical Sciences and Health Services
Accepted: Jun 2014

Original Article

Enhancement of Anti-Dermatitis Potential of Clobetasol Propionate by

DHA [Docosahexaenoic Acid] Rich Algal Oil Nanoemulsion Gel

Mohammad Sarfaraz Alama*, Mohammad Sajid Alia , Foziyah Zakira , Nawazish Alama, b,
Mohammad Intakhab Alama , Faruque Ahmadc , Masoom Raza Siddiquid , Mohammad Daud Alie ,
Mohammad Salahuddin Ansarif , Sarfaraz Ahmada and Maksood Alia

College of Pharmacy, Jazan University, Jazan, KSA. bSBS College of Pharmacy, Patti,
a

Amritsar, Punjab, India. cDepartment of Clinical Nutrition, College of Applied Medical

Sciences, Jazan University, Jazan, KSA. dChemistry Department, College of Science, King Saud
University, Riyadh, KSA. eDepartment of Pharmacy, Mohammad Al-Mana college for Health
Sciences,Al-Rakkah, Dammam, KSA. fCollege of Pharmacy, AlDawadmi, Shaqra University,
KSA.

Abstract

The aim of the present study was to investigate the potential of nanoemulsion formulation for
topical delivery of Clobetasol propionate (CP) using algal oil (containing omega-3 fatty acids)
as the oil phase. CP has anti-inflammatory, immunomodulatory and antiproliferative activities.
However, its clinical use is restricted to some extent due to its poor permeability across the skin.
Algal oil was used as the oil phase and was also exploited for its anti-inflammatory effect along
with CP in the treatment of inflammation associated with dermatitis. Nanoemulsion formulations
were prepared by aqueous phase titration method, using algal oil, tween 20, PEG 200 and water
as the oil phase, surfactant, co-surfactant and aqueous phase respectively. Furthermore, different
formulations were subjected to evaluate for ex-vivo permeation and in-vivo anti-inflammatory,
irritation and contact dermatitis studies. The optimized nanoemulsion was converted into
hydrogel-thickened nanoemulsion system (HTN) using carbopol 971 and had a viscosity of 97.57
± 0.04 PaS. The optimized formulation had small average diameter (120 nm) with zeta potential of
-37.01 mV which indicated good long-term stability. In-vivo anti-inflammatory activity indicated
84.55% and 41.04% inhibition of inflammation for drug loaded and placebo formulations
respectively. The assessment of skin permeation was done by DSC and histopathology studies
which indicated changes in the structure of epidermal membrane of skin. Contact dermatitis
reveals that the higher NTPDase activity in the treatment with the CP-loaded nanoemulsion could
be related to the higher anti-inflammatory effect in comparison with placebo nanoemulsion gel.

Keywords: Algal Oil; Anti-inflammatory study; Clobetasol Propionate; Contact Dermatitis;

Nanoemulsion.

Introduction condition of the skin resulting from exposure to

allergens or irritants. Only the superficial regions
Contact dermatitis is a frequent inflammatory of the skin i.e., epidermis and dermis are affected
(1-3). It can cause red, itchy and scaly skin and
* Corresponding author: sometimes the skin becomes blistered, dry and
E-mail: [email protected] cracked. It is more common in women than men.
Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

It is experienced by approximately 1 in 5 women diameters ranging from 50 to 500 nm (12).

on their hands at some point during their lives (4). Studies have proved that smaller particle
The symptomatic treatment of contact dermatitis size in nanoemulsions ensure close contact
involves the use of topical corticosteroids with stratum corneum thereby improving the
particularly because of their vasoconstrictive, absorption and therapeutic concentration of
anti-inflammatory, immunosuppressive and poorly water soluble drug in the target tissue.
antiproliferative effects (5). Among the various In addition it ensures low skin irritation, high
corticosteroids, Clobetasol propionate (CP) solubilization capacity for hydrophilic and
is the drug of choice for the reason that it lipophilic drugs, and high drug-loading capacity
is highly potent (6). It exerts its action by for topical delivery (13). Nanoemulsions act
inhibition of phospholipase A2 which leads to as a depot for slow and controlled release and
the inhibition of synthesis of arachidonic acid reduce the frequency of drug administration
and controls the biosynthesis of prostaglandins thus diminishing adverse effects. In our
and leukotrienes (7). However, there are few previous study, we have successfully prepared
drawbacks associated with the use of topical CP o/w nanoemulsion of CP using eucalyptus oil,
such as skin atrophy, steroid acne, telangiectasia, tween-20 and ethanol as oil, surfactant and co-
hypopigmentation and poor absorption through surfactant respectively (14). Fontana et al.,
skin (8). The main limitation lies in the barrier (2009) studied the physicochemical and in-vitro
function of the skin, which is considered as one release characteristics of CP-loaded polymeric
of the most impermeable epithelia of the human nanoparticles (nanocapsules and nanospheres)
body to exogenous substances. Therefore, the and CP-loaded  nanoemulsion (15). The
major challenges for a topical formulation are to controlled release characteristic of nanoparticles
provide a sufficient increase in drug penetration was found to be better than nanoemulsion.
into the skin, without inducing any significant Moreover the photostability of nanoemulsion
irreversible alteration to the skin barrier function was better than nanospheres but worse than
(9-11). There has been increased interest during nanocapsules. The influence of the polymeric
recent years in the use of topical vehicle systems wall of CP-loaded lipid-core nanocapsules or
that could modify drug permeation through nanoemulsion was evaluated in a model of
the skin. Many of the dermal vehicles contain contact dermatitis after topical administration in
chemical enhancers and solvents to achieve these rats. The nanocapsules exhibited better control
goals. But use of these chemical enhancers may of the drug release and provided better in-vivo
be harmful, especially in chronic application, dermatological efficacy (16). The physical and
as many of them are irritants. Therefore, it is chemical degradation of clobetasol propionate
desirable to develop a topical vehicle system that (CP) incorporated in nanoemulsion was
does not require the use of chemical enhancers to evaluated as per ICH guidelines. The changes
facilitate drug permeation through the skin. in different physicochemical characteristics
Although several attempts have been made parameters were found to be statistically
to alleviate the adverse effects and improve the insignificant (p ≥ 0.05) after three months of
therapeutic efficacy. Various colloidal carriers storage. CP exhibited enhanced stability in the
such as poly (d, l-lactic-co-glycolicacid) nanoemulsion formulation (17). The novelty
(PLGA) microspheres, solid lipid nanoparticles, in the present work lies on the use of algal oil
lipid nanospheres, nanostructured lipid carriers, which is rich in omega 3-fatty acid that gets
polymeric nanocapsules and lecithin/chitosan incorporated in the cell membrane and competes
nanoparticles containing CP have been developed with arachidonic acid to reduce the formation
to enhance the therapeutic efficacy. of inflammatory mediators. Moreover the drug
Recent novel nanotechnology based penetration mechanistic approaches in the dipper
delivery systems offer unique properties and layer of skin and anti-inflammatory activity on
substantially improves applications in topical animal model have been explained.
delivery. Nanoemulsions are submicron oil- Thus, the main aim of the present study was to
in-water (o/w) emulsions with mean droplet evaluate the potential of algal oil which contains

36
 Algal oil nanoemulsion gel

DHA and EPA (18) along with CP in the treatment added to each 5-mL capacity stopper vial and
of inflammation observed in dermatitis, in the mixed using a vortex mixer (Nickel-Electro
form of nanoemulsion using algal oil as the oil Ltd., Oldmixon Crescent, UK). The mixture
phase and other non irritating pharmaceutical vial was then kept at 37 ± 1°C in an isothermal
acceptable ingredients without using penetration shaker (Nirmal International, New Delhi,
enhancers. Algal oil was used as an excipient as India) for 72 h to get to equilibrium. The
well as an active ingredient. The low viscosity equilibrated samples were removed from the
of nanoemulsion restrains its clinical application shaker and centrifuged at 3000 rpm for 15 min.
due to inconvenient use, therefore hydrogel- The supernatant was taken and filtered through
thickened nanoemulsion (HTN) system were a 0.45 µm membrane filter. The concentration
formulated with good stability, powerful of CP was determined in algal oil by UV
permeation ability and suitable viscosity for the spectrophotometer (Shimadzu, Kyoto, Japan)
topical delivery which provided longer contact at 241 nm. For selection of surfactant and co-
with skin. The hydrogel in the formulation will surfactant, miscibility of algal oil was done
enable close proximity of the formulation with with a number of surfactants like Tween-20,
the skin facilitating cutaneous absorption of the Tween-80, Labrasol and Tween-60 and co-
active moiety. This will lead to accumulation surfactants like ethanol, Transcutol-P, Plurol
of drug in the skin and prevent leaching of the oleique, PEG-200 and PEG-400, in 1: 1 ratio
drug into systemic circulation. The present (oil: surfactant/co-surfactant). Observations
investigation focused on the preparation and were done visually for miscibility. The mixtures
characterization of HTN system with CP, ex-vivo which were clear/ transparent in a ratio of 1: 1
permeation studies, irritation study, in-vivo anti- (v/v) were considered for further studies.
inflammatory activity and their nickel induced
dermatitis study of the specialized delivery Phase Studies
systems. The long-term goal of this work was to On the basis of solubility/miscibility studies,
develop safe topical CP formulations for clinical Tween-20 as a surfactant and Transcutol-P as a
use to increase the anti-dermatitis activity. co-surfactant were selected for the preparation of
nanoemulsion of algal oil. Double distilled water
Experimental was used as an aqueous phase to avoid surface
active impurities. Surfactant and co-surfactant
Materials were mixed (S ) in different weight ratios (1:,0
mix
Clobetasol propionate was obtained as a 1 :5 ,1 :4 ,1 :3 ,1 :2 ,1 :1) with increasing amount
gift sample from (Mumbai, India). Omega 3 of surfactant with respect to co-surfactant.
fatty acid enriched Microalgae DHA Oil was Sixteen different combinations of oil and S (1:
mix
obtained as a gift sample from. China. PEG-400, :1 ,7 :3 ,3 :1 ,3.5 :1 ,4 :1 5 :1 ,6 :1 ,7 :1 ,8 :1 ,9
Tween-80, Tween-20 and ethanol were purchased 2 :8 ,3 :7 ,4 :6 ,5 :5 ,6 :4 ,2 and 9:1 ) were made
from Merck (Merck, India). Caprylocaproyl so that maximum ratio could be covered for the
macrogol-6 glycerides (Labrasol), diethylene study to delineate the boundaries of the phases
glycol monoethyl ether (Transcutol-P) and Plurol formed precisely in the phase diagrams. For the
Oleique were obtained as a kind gift sample from determination of existing zone of nanoemulsion,
Gattefosse (Mumbai, India). All other chemicals pseudo ternary phase diagrams were constructed
were of analytical grade. using aqueous phase titration method. Slow
titration with the aqueous phase was done for
Screening of Excipients each weight ratio of oil and Smix, and visual
An important criterion for screening of observations were made for transparent and easily
components for high loading of drug and more flowable oil-in-water (o/w) nanoemulsions. The
stability in nanoemulsions is the solubility/ physical state of nanoemulsion was marked on
miscibility of drug in oil, surfactant and co- a pseudo three component phase diagram with
surfactant. For determination of solubility of one axis representing the aqueous phase, second
CP in algal oil, an excess amount of CP was representing oil and the third representing a

37
Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

mixture of surfactant and co-surfactant at fixed dielectric constant of the medium were set at
weight ratio (S ratio) (19). 4.55 m Pas and 79.4 respectively. Zeta potential
mix
was determined by using second generation
Selection of Formulations PALS (Phase Analysis Light Scattering), called
Among the pseudoternary phase diagrams M3PALS which measures the particle velocity.
showing maximum nanoemulsion area, a Refractive Index, pH and Viscosity
number of formulations were selected covering Viscosity of nanoemulsion was determined
the entire range of nanoemulsion occurrence in by using Brook-field DV III ultra V6.0 RV cone
the phase diagrams with minimum surfactant and and plate rheometer (Brookfield Engineering
maximum water concentration (20-21). Exactly Laboratories, Middleboro, MA). Refractive
0.05% w/w of CP, which was kept constant in all index was determined for different nanoemulsion
the selected formulations, was added to the oil formulations by using Abbe’s refractometer
phase during the formulation of nanoemulsions. (Nirmal International, Delhi, India) at 25°C
Selected formulations were subjected to various in triplicate. The pH was determined for the
physical stability tests. optimized nanoemulsions by using a calibrated
digital pH meter (Mettler Toledo MP 220,
Physical Stability Studies Greifensee, Switzerland) in triplicate at room
To overcome the problem of metastable temperature.
formulations, physical stability tests were
performed. The selected nanoemulsions were Ex-Vivo Skin Permeation Studies
subjected to centrifugation at 5000 rpm for Ex-vivo skin permeation studies were
30 min. The formulations that did not show performed on a fabricated Franz diffusion cell
any phase separations were taken for the with an effective diffusional area of 3.14 cm2
heating and cooling cycle. Six cycles between and 5 ml of receiver chamber capacity using
refrigerator temperature (4°C) and (45°C) with rat abdominal skin. The full-thickness rat skin
storage at each temperature of not less than 48 h was excised from the abdominal region, and
were done. The formulations which were found hairs were removed with an electric clipper. The
stable were subjected to a freeze-thaw cycle subcutaneous tissue was removed surgically,
test. Formulations were kept in deep freezer and the dermis side was wiped with isopropyl
(Vestfrost, Delhi, India) at 20°C for 24 h. After alcohol to remove adhering fat. The cleaned skin
24 h the nanoemulsions were removed and kept was washed with distilled water and stored in the
at room temperature. The physically stable deep freezer at -21°C until further use. The skin
nanoemulsions returned to their original form was brought to room temperature and mounted
within 2-3 min, 3 such cycles were repeated between the donor and receiver compartment
(22-23). of the Franz diffusion cell, where the stratum
corneum side faced the donor compartment and
Characterization of Nanoemulsions the dermal side faced the receiver compartment.
Particle Size and Zeta Potential Initially the donor compartment was empty and
The average size and polydispersity index of the receiver chamber was filled with acetate
the nanoemulsion droplets were determined by buffer pH 5. The receiver fluid was stirred with
photon correlation spectroscopy (Nano ZS90, a magnetic rotor at a speed of 100 rpm, and the
Malvern Instrument, U.K.) which is based on assembled apparatus was placed in the oven and
the principle of dynamic light scattering. The the temperature was maintained at 37 ± 1°C. All
measurements were performed using a He- the receiver fluid was replaced every 30 min to
Ne laser at 633 nm by using Avalanche photo stabilize the skin. It was found that the receiver
diode detector. Light scattering was monitored fluid showed negligible absorbance after 4.5 h
at 25°C at a 90° angle. Droplet size distribution and beyond, indicating complete stabilization of
studies were performed at refractive index the skin. After complete stabilization of the skin,
of 1.40 because the refractive index for all 1 ml of nanoemulsion formulation (0.5 mg/mL
formulation was in this range. The viscosity and CP) was placed into each donor compartment

38
 Algal oil nanoemulsion gel

and sealed with paraffin film to provide 1% w/v concentration of the polymers was
occlusive conditions. Samples were withdrawn slowly mixed with nanoemulsion under stirring
at regular intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, until equilibrium was attained. The formed
10, 12, 20, 22, and 24 h), filtered through a 0.45 hydrogel were kept for 24 h and checked for
µ membrane filter, and analyzed for drug content physical stability. The clear hydro-gel thickened
by UV spectrophotometer at 241 nm (24). nanoemulsion was evaluated for viscosity, pH,
content uniformity and homogeneity. In order to
Permeation and Distribution Data Analysis optimize polymer concentration, hydrogel was
The cumulative amount of CP permeated prepared with different polymer concentrations
through the albino rat skin (Q, g/cm2 was plotted (0.5%, 0.8%, and 1%) and evaluated for in-vitro
as a function of time (t, h) for each formulation. permeation study, viscosity and consistency.
The permeation rate (flux) at the steady state Content uniformity was carried out to ascertain
(Jss, g/cm2 /h) and lag time were calculated that concentration of drug in each portion
from the slope and intercept of the straight line was uniform. For that an accurately weighed
obtained by plotting the cumulative amount of quantity of gel (6 g) from 3 different portions
CP permeated per unit area of skin versus time at was taken and extracted with methanol which
steady state condition respectively. Permeability was analyzed by using UV spectrophotometer.
coefficient (Kp was calculated by dividing the HTN equivalent to 0.5 mg CP was applied gently
flux by initial drug concentration (Co) in the on stratum corneum and in-vitro permeation
donor portion of cell as given below: study was performed. At the end of the test, the
Kp = Jss/Co HTN that remained on the skin was removed,
Enhancement ration (Er) was calculated by cleaned with cotton soaked in a 0.05% sodium
dividing the Jss of the respective formulation by lauryl sulphate and washed with distilled water.
the Jss of the control formulation as given below: In order to determine the drug disposition in the
Er = Jss of formulation/Jss of control skin, it was weighed, cut into small pieces and
sonicated for 15 min with methanol in order to
Surface Morphology by Transmission extract the CP content. The resulting solution was
Electron Microscopy centrifuged and passed through 0.25 µm filter
Morphology and structure of the nanoemulsion and drug content (g/mg of skin) was determined
were studied using Morgagni 268D transmission spectrophotometerically (25).
electron microscopy (TEM) (FEI, Netherland)
operating at 70 KV and capable of point to point Assessment of Skin Permeation
resolution. Combination of bright field imaging The structural and chemical changes in
at increasing magnification and diffraction epidermal layer of skin due to the permeation
modes were used to reveal the form and size of drug were determined by DSC and
of nanoemulsion droplets. In order to perform histopathological study.
the TEM observations, a drop of nanoemulsion
was applied on carbon coated grid with 2% DSC Studies
phosphotungstic acid (PTA) and was left for 30 In order to assess mechanism of permeation,
sec. The dried coated grid was taken on a slide thermal transitions in desiccated stratum
and covered with a cover slip. The slide was corneum membranes of rats were investigated
observed under the electron microscope. using differential scanning calorimetry (DSC).
Both treated and untreated skin samples were
Hydrogel-Thickened Nanoemulsion previously hydrated over a 27% sodium bromide
The very low viscosity often exhibited by solution for 48 h to ensure 20% hydration. The
nanoemulsion is inappropriate for topical use. In skin samples were stored in a desiccator, over
order to increase the viscosity of nanoemulsion, silica gel, for at least 3 days before thermal
carbopol-971, carbopol-940, HPMC and sodium analysis. The sheets of skin were cut into small
alginate were selected as gelling agents. For pieces and 4 mg pieces were hermetically sealed
preparation of nanoemulsion hydrogel, initially in aluminium pans and kept in the DSC unit

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Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

along with a similar empty pan as a reference. to the initial hind paw volume (26). Percent
The sample was heated at the rate of 10°C/ inhibition of edema was calculated for placebo
min from the temperature range of 30-400°C. and drug loaded group with respect to control
Nitrogen was used as a purge gas and flow was group using the following formula:
adjusted to 20 mL/min.
% Edema (Control)-
Histopathology Studies % Edema (Formulation)
Abdominal skin of wistar rats was treated % Inhibition=
with the optimized CP nanoemulsion gel. After % Edema (Control)
24 h, the rats were sacrificed and skin samples Equation 1
were taken from untreated (control) and treated
areas. Each specimen was stored in 10% formalin In-vivo Skin irritation test
solution in phosphate buffer saline (pH 7.4). The All the materials used for preparation of
specimens were cut into sections vertically. Each nanoemulsion fall under generally regarded
section was dehydrated using ethanol embedded as safe (GRAS) category. Concentration of all
in paraffin wax for fixing and stained with materials is very critical issue for this formulation.
hematoxylin and eosin. These samples were then Large amount of surfactants is usually irritant
observed under light microscope (Motic, Japan) to the skin. Therefore skin irritation test was
and compared with control samples. performed to confirm concentration of materials
used for nanoemulsion preparation is safe. Van-
In-vivo Anti-Inflammatory Study Abbe et al. mentioned that a value between 0
The protocol to carry out in-vitro permeation and 9 indicates that the applied formulation is
studies was approved by the Institutional Animal generally non irritant to human skin (1). Skin
Ethics Committee S.B.S College of Pharmacy, irritation test was performed using either sex
Patti, Amritsar, Punjab, India. of wistar rats weighing 180–200 g. Wistar
The committee’s guidelines were followed rats were divided into 2 groups (n = 6) and
for the studies. The anti-inflammatory and applied the following formulations: optimized
sustaining action of the optimized formulation nanoemulsion and placebo nanoemulsion. The
was evaluated by the carrageenan induced animals were kept under standard laboratory
hind paw edema method by using digital conditions, temperature at 25 ± 1°C and relative
plethysmometer (Ugo Basile, Italy) in wistar humidity (55 ± 5%). The animals were housed
rats of either sex weighing 180 to 200 g. A left in polypropylene cages, six per cage, with free
hind paw of each rat was marked, just below access to standard laboratory diet and water
tibiotarsal junction, so that every time the paw as mention above. A single dose of 10 μL of
was dipped up to the fixed mark to ensure optimized nanoemulsion, Placebo nanoemulsion
constant paw volume. Animals were randomly and marketed cream were applied to the left
divided into 3 groups (control, placebo and ear of the rat and the right ear as a control. The
formulation treated) each containing 6 rats. The development of erythema was monitored for 14
placebo formulation contained only algal oil days using the reported method (27).
whereas the formulation treated were applied
HTN formulations containing algal oil and CP Dermatitis induction by 5% nickel sulfate
on the dorsal area of 9 cm2 gently with the help Contact dermatitis was induced by 5%
of micropore adhesive, 0.5 h prior to carrageenan nickel sulfate in solid Vaseline similar to the
injection. Acute inflammation was produced procedure adopted by Brum et al. (28-30).
by injecting 0.1 mL of 1% (w/v) carrageenan Animals (Wister rats) were divided into four
suspension in the sub plantar region of the left sets (n = 8). After tricotomization, all groups
hind paw 0.5 h after treatment with drug. The received sensitization with nickel sulfate in the
paw volume was measured at 0, 1, 2, 3, 6 and 12 abdomen, except the first group which received
h. The amount of paw swelling was determined only solid Vaseline and continued under the
for 12 h and expressed as percent edema relative same environmental and feeding conditions as

40
 Algal oil nanoemulsion gel

the other groups, this being the control group Results and Discussion
(C). The induction of dermatitis was done 6
days after sensitization by nickel sulfate in Criteria for Excipient Selection
solid Vaseline (5 applications with an interval The excipients were selected to be
of 72 h) in each ear after tricotomization. The nonirritating, nonsensitizing to the skin and
first group which received only solid Vaseline pharmaceutically acceptable, and in several
was euthanized 72 h after the last application of cases it should fall into the GRAS (generally
the sensitization agent. The second group was regarded as safe) category. Higher solubility of
induced to allergic contact dermatitis, which the drug in the oil phase was another important
was not managed, and the rats were euthanized criterion, as it would help the nanoemulsion to
72 h after the last application of nickel sulfate, maintain the drug in solubilized form. Safety
being the positive control (D). The third group is a major determining factor in choosing a
(E) received the topical administration in each surfactant, as large amounts of surfactant may
ear of the placebo nanoemulsion and the fourth cause skin irritation. Non-ionic surfactants are
(F) received topical administration in each considered to be less toxic than ionic surfactants.
ear of the CP-loaded nanoemulsion. Dose of Another important aspect to be taken into
application for nanoemulsion and marketed consideration is the selection of surfactants;
formulation was equal to 1 microgram of ideally the hydrophilic lipophilic balance (HLB)
CP. Group F and G were treated daily with value to form the o/w nanoemulsion should be
0.5ml of the drug loaded nanoemulsion and greater than 10. The right blend of low and high
marketed cream for 5 days on days 1, 3 and HLB surfactants leads to the formation of a
5. All formulations were applied uniformly stable nanoemulsion formulation. The presence
throughout the ear tissue with massage in order of co-surfactant decreases the bending stress of
to obtain a better drug penetration (29). After interface and allows the interfacial film sufficient
the completion of each treatment, the animals flexibility to take up different curvatures required
were euthanized and the blood was collected to form nanoemulsion over a wide range of
by cardiac puncture to determine the NTPDase composition.
activity. For NTPDase activity of lymphocytes,
mononuclear leukocytes were isolated from rat Screening of Excipients
blood collected with EDTA and separated using Drug loading per formulation is a very
Ficoll-Hypaque density gradients as described critical design factor in the development of
by Boyum. After the isolation of mononuclear nanoemulsion systems for poorly soluble drugs,
cells, NTPDase activity was determined by which is dependent on the drug solubility in oil
colorimetric assay in compliance with Leal et al. phase. Solubility of CP in algal oil was found
All samples were run in duplicate or triplicate, to be 19.59 mg/mL which is very good for
and specific activity is reported as nmol Pi topical delivery since the dose of CP is very
released/min/mg protein. Protein was measured less. But due to the presence of other fatty
by the Coomassie Blue method using bovine acid in algal oil, emulsification of algal oil is
serum albumin as the standard as described by very difficult. For getting good nanoemulsion
Bradford (31-32). region in ternary phase diagram, the miscibility
of oil with surfactant and co-surfactant is
Statistical Analysis important. Therefore the miscibility of algal
Each experiment was conducted in triplicate oil was performed with different surfactants
and data were analyzed using Excel 2007 and co-surfactants (Table 1). Another important
(Microsoft Office, Microsoft Inc., US) and criterion is the selection of surfactant with proper
expressed as a mean ± standard deviation (S.D.). HLB value. Hydrophilic surfactant and co-
Comparison between the differences of means surfactant are considered to prefer the interface
was performed by using paired t-test for paired and to lower the necessary energy to form the
comparisons where p-values of 0.05 or less were nanoemulsions, consequently improving the
considered significant. stability. For example, the required HLB value

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Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

Table 1. Miscibility of algal oil with surfactants and co-surfactants.

Miscibility of algal oil
S. No. With surfactant (1:1) Observation with surfactant With co-surfactant Observation with co-surfactant
1 Tween 20 Clear Ethanol Turbid
2 Tween 80 Turbid Transcutol P Turbid
3 Lecithin Turbid PEG 200 Clear
4 Unitop 100 Turbid Pleurol oleique Turbid

to form o/w nanoemulsion is greater than 10. So to sufficiently low level and thus was not able
selection of surfactant and co-surfactant with to reduce the free energy of the system to ultra
appropriate HLB value is necessary (8). low level desired to produce nanoemulsions. As
The miscibility of algal oil was found to be the surfactant concentration was increased in
highest with Tween-20 in case of surfactant and S ratio 2: 1 (Figure 1B) a higher nanoemulsion
mix
PEG 200 in case of co-surfactant in 1:1 ratio. region was observed, perhaps because of further
Apart from this, Tween-20 has high HLB value reduction of interfacial tension, increasing the
(13.2) which can provide good emulsification to fluidity of interface, thereby increasing entropy
the algal oil. PEG-200 is very good solublizing of system. As the surfactant concentration was
agent which can provide better penetration to further increased to 3: 1 and 4: 1 (Figure 1C
the lipophilic drug such as CP by increasing the and D), nanoemulsion region further increased
solubility of the drug in the lipophilic domain as compared to S ratio 1: 1 and 2: 1 and was
mix
of the stratum corneum. So for the development found to be maximum in case of S ratio 4: 1.
mix
of pseudoternary phase diagram algal oil was When S ratio 5:1 was studied, nanoemulsion
mix
selected as an oil phase, Tween-20 as surfactant region decreased slightly as compared to S
mix
and PEG-200 as a co-surfactant. 4: 1 which might be due to insufficient amount
resulting in the formation of large droplets by
Phase Studies diffusion processes driven by the gain in surface
The relationship between the phase behaviour free energy. When co-surfactant increases with
of a mixture and its composition can be captured respect to surfactant as observed in S (1: 2, 1:
mix
with the aid of a phase diagram (10). The aim 3, 1: 4), a decrease in nanoemulsion region is
of the construction of pseudo-ternary phase detected due to insufficient decrease in surface
diagram was to find out the existence range of tension. Another rational explanation may be
nanoemulsion. Care must be taken to ensure that that when temperature quench occurs during
observations are not made on metastable system. stress stability study, instability of nanoemulsion
Pseudoternary phase diagrams were constructed occurs due to separation of oil phase and droplet
separately for each S ratio for getting o/w distribution of smaller size is favoured by the
mix
nanoemulsion regions (Figure 1). The area of change in curvature free energy (33). Only
nanoemulsion isotropic region changed slightly those formulations, which showed no phase
as the ratio of surfactant in S was increased. separation, creaming, cracking, coalescence and
mix
In the phase diagrams, the existence of large phase inversion during stress stability tests, were
or small nanoemulsion region depends on the selected for further studies.
capability of the particular S to solubilize the
mix
oil phase. The extent of solubilization results in Selection of Formulation from Phase
a greater area with the formation of more clear Diagram
and homogenous solution. In Figure 1, the S It is reported that large amount of surfactant
mix
ratio 1:1 (Figure 1A) had a low nanoemulsion causes skin irritation and toxicity related issues
area. Thus, oil phase was solubilised to a lesser therefore it is important to use minimum amount
extent implying that the S was not able to of surfactant and co-surfactant in the formulation.
mix
reduce the interfacial tension of the oil droplets However, for topical delivery, where enhanced

42
 Algal oil nanoemulsion gel

Figure
Figure 1. Pseudo-ternary
1. Pseudo-ternary phase
phase diagram for diagram
optimizing for optimizing
nanoemulsion formulation. nanoemulsion formulation.

skin permeation is the aim, it is not purposeful phase diagram, oil could be solubilised up to
to select the lowest surfactant concentration. The the extent of 40% w/w but in such cases the Smix
surfactant concentration should be chosen so that concentration was very high. For the preparation
it gives the maximum flux, which is an important of drug-loaded nanoemulsions, 0.05% CP was
criterion but its level should not be toxic to dissolved in oil phase. Therefore, from each
cause any irritation to the skin. This is usually phase diagram nanoemulsion formulations
not obtained with formulations that contain the containing different concentration of oil were
highest amount of surfactant since high surfactant selected which contained minimum to maximum
concentration decreases the thermodynamic Smix concentration (Table 2).
activity of the drug in the vehicle, and the affinity
of the drug to the vehicle becomes greater (5, Physical Stability Studies
34, 35). While going through pseudoternary Nanoemulsions are considered to be

Table 2. Composition of various CP loaded nanoemulsions.

Formulation Code Algal oil (% w/w) S (% w/w) Distilled water (% w/w) CP (% w/w)
mix

A1 7 50 43 0.05
A2 8 46 46 0.05
A3 10 55 35 0.05
B1 5 45 45 0.05
B2 5 40 55 0.05
B3 7 57 36 0.05
B4 9 50 41 0.05
C1 10 51 39 0.05
C2 15 57 28 0.05
C3 20 61 19 0.05

43
Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

Table 3. Physical stability studies of drug loaded formulations.

Formulation Code Heating Cooling Cycles Freeze thaw cycles Centrifugation Studies
A1 Passed Failed Failed
A2 Passed Passed Passed
A3 Failed Failed Passed
B1 Passed Passed Passed
B2 Passed Passed Passed
B3 Failed Passed Failed
B4 Passed Passed Passed
C1 Passed Passed Passed
C2 Passed Passed Passed
C3 Failed Failed Failed

kinetically stable systems which are formed Particle Size and Zeta Potential
at a particular concentration of oil, surfactant The average droplets size of different
and water, with no phase separation, creaming nanoemulsions was in the range of 100 to 200
or cracking. Selected formulation from phase nm. The polydispersity index is a ratio that
diagram were subjected to different stress stability gives information about the homogeneity of the
testing like heating cooling cycle, centrifugation particle size distribution in a given system. The
and freeze thaw cycle. During physical stability polydispersity index (Table 4) showed that all
testing some formulations became turbid and the nanoemulsions had narrow size distribution.
some showed phase separation. Additionally, When the concentration of oil phase was kept
when temperature quench occurs during stress constant, it was observed that the decrease in
stability study, instability of nanoemulsion particle size was inversely proportional to the
occurs due to separation of oil phase and droplet concentration of S . However, the droplet size
mix
distribution of smaller size is favoured by the of all the formulations was in the nano-range.
change in curvature free energy. Formulations The average particle size and polydispersity
with negligible phase separation, creaming, index of the formulation B1 was found to be 120
cracking, coalescence and phase inversion nm (Figure 2) and 0.325 respectively indicating
during stress stability tests, were selected for nano-range of droplets with minimum variation
further studies (Table 3). in particle size. The zeta potential depends on
both the particle surface and the dispersant.
Characterization of Nanoemulsions Particles interact according to the magnitude of
The formulations which passed physical zeta potential and not their surface charge and
stability test were evaluated for droplet size, therefore zeta potential can be used to predict
polydispersity index, zeta potential, viscosity, dispersion stability of the system. Zeta potential
pH, conductivity and refractive index. for formulation B1 was found to be -37.01 mV,

Table 4. Evaluation of various drug loaded formulations.

Formulation Average droplet size Polydispersity Zeta potential Viscosity (PaS) pH ± S.D. Refractive index ±
code (nm) ± S.D. (n = 3) indexc ± S.D. (n = 3) (mV) ± S.D. (n = 3) ± S.D. (n = 3) (n =3) S.D. (n = 3)
A2 172 ± 2.12 0.671 ± 0.03 -24.34 ± 1.05 4.55 ±0.03 5.30 ± 0.05 1.402 ± 0.01
B1 120 ± 1.95 0.325 ± 0.02 -37.01 ± 0.95 4.42 ±0.02 5.40 ± 0.04 1.412 ± 0.02
B2 135 ± 3.13 0.512 ± 0.05 -12.39 ± 0.61 3.91 ±0.03 5.34 ± 0.03 1.415 ± 0.04
C1 258 ± 4.01 0.413 ± 0.06 -25.13 ± 1.04 4.67 ±0.05 5.27 ± 0.04 1.424 ± 0.05

44
 Algal oil nanoemulsion gel

Figure 2. Average droplet size of nanoemulsion formulation B1.

which indicated good dispersion stability as it between excipient and drug. Acceptable pH of
represents significant distance between charged the formulation is another important aspect taken
particles in dispersion system. into consideration. Very high or low pH can lead
to skin irritation. pH of the formulations were
Refractive Index, pH and Viscosity of found to be in the range of 5.20-5.44 which are
Nanoemulsion very close to skin pH (pH 4.5–5.5). It was found
Refractive index of a formulation indicates that viscosity of the formulations depends upon
isotropic nature of formulation. Refractive amount of surfactant mixture. As the amount of
index (Table 4) of nanoemulsion was measured Smix decreased in a formulation, viscosity also
which basically signify the chemical interaction decreased.
between drug and excipients. There was no
significant difference in the refractive index Ex-Vivo Skin Permeation Studies
value of placebo and drug loaded nanoemulsions The permeation ability of the various
so it was concluded that the nanoemulsion nanoemulsions loaded CP and control was
formulations were chemically stable and evaluated using the ex-vivo permeation
remained isotropic thus showing no interaction experiments. The ex-vivo permeation profiles of

Figure 3. Ex-vivo permeation nanoemulsion study of formulations.

45
Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

Figure 4. Cryo-electron microscopic images of formulations; A = Placebo; B = Drug loaded nanoemulsion.

CP through excised abdominal skins of rat are came in close contact with the skin and a large
shown in (Figure 3). A steady increase of CP in amount of algal oil in nanoemulsion might have
the receptor chambers with time was observed. penetrated into the skin. DHA present in algal
The permeation profiles of nanoemulsion were oil had strong permeation enhancing effect (37).
in accordance with the Fick’s diffusion equation. In addition, due to the small droplet diameters of
Statistical comparison of the flux throughout 24 nanoemulsion, the likely mechanism may also be
h showed that the nanoemulsion preparations the permeation of CP directly from the droplets
of CP provided flux (Table 5) higher than that into the stratum corneum without nanoemulsion
of the control (CP suspension) which had low fusion to the stratum corneum and subsequent
cumulative amount of CP at 24 h after application. permeation enhancement.
Cumulative amount of CP permeated from
nanoemulsions was 2.55– 5.22 times higher Surface Morphology of Particle
that of the control, 24 h post application. The The TEM studies were carried out to get
high permeation rate of nanoemulsions might more insight about the morphology of the
be attributed to several factors. Firstly, the high nanoemulsion systems. When TEM was
concentration of CP released in nanoemulsion performed for optimized formulation it was
resulted in high concentration gradient, which finally concluded that the particles were spherical
might be the main permeation mechanism of CP in shape and finely distributed within nanometer
into the skin from the formulation. Nanoemulsion range. Particle size for placebo and drug loaded
could act as drug reservoir where drug is released formulation was found between 85–110 nm
from the inner phase to outer phase and then and 96–141 nm (Figure 4) respectively. For CP
further into the skin (36). Secondly, due to the loaded formulation particle size had increased
small droplet size, droplets settled down and significantly due to drug entrapment inside the

Table 5. Ex-vivo permeation parameter of CP loaded nanoemulsion formulation and control.

−1
Nanoemulsion Flux Ratio (µg/cm2 h ) Permeability coefficient (Kp ) Enhancement Rate
A2 7.27 0.014 3.69
B1 10.27 0.020 5.22
B2 6.56 0.013 3.33
C1 5.03 0.010 2.55
Control 1.96 0.003 1

46
 Algal oil nanoemulsion gel

polymer was increased its viscosity increased

simultaneously. A small quantity of gel was
pressed between the thumb and index finger
and the consistency and homogeneity of the
gel were observed. The HTN showed absence
of any coarse particles. Carbopol-971 in HTN
resulted in a high viscosity and oily droplets
might be distributed in gel network, which might
contribute to the enhancement of the stability
of droplets in nanoemulsion. The pH value
for all three gel formulations was found in the
range 5.15–5.55 (Table 6) which is favourable
5. DSC study of rat skin
Figure 5. (A)
DSC andstudyCP-loaded
of rat skin nanoemulsion
(A) and CP-loaded treated skin (B).
for topical application. The gels prepared with
nanoemulsion treated skin (B).
0.5 and 0.8% w/v carbopol were not suitable
for topical delivery because the consistency
thology Studies of gels was not good. Hydrogel containing 1%
oil droplets.
ence of CP loaded nanoemulsionThese on results were in agreements
the anatomical structure ofcarbopol-971
the rat skin iswas found to have good viscosity
discussed
with the droplet size obtained from photon and maximum amount of drug was retained
aid of light microscopic
correlationfindings.
spectroscopy. The photomicrographs of untreated rat skin
in the skin during(control)
permeation study (Table 6).
Therefore it was further evaluated for in-vivo
ormal skin (Figure 6) with well defined epidermal and dermal layers. Keratin layer was
Hydrogel Thickened Nanoemulsion anti-inflammatory study, in-vivo irritation study
med and lied just adjacent
Previously,to the
the top
gel most
matrixlayer of the epidermis.
of nanoemulsion andDermis
contactwas devoid of
dermetitis.
has been prepared with carbomer-940, xanthan
mmatory cells.gum When andthe skin was
sodium alginatetreated with nanoemulsion
for improving the formulation,
Assessment definite
of Skin Permeation Mechanisms
were observed rheological
in the skinbehaviour
morphology. of nanoemulsion (38-40).
The disruption DSC Studies
and extraction of lipid bilayers
In this work, carbopol-971, carbopol-940, HPMC DSC thermogram showed one prominent
rly evident as (15
distinct
Cps)voids and empty
and sodium spaces
alginate werevisible in thepeak
selected epidermal
at 108°Cregion. The5) in untreated skin
(Figure
at 1% w/v concentration for the preparation which was attributed to intracellular keratin
n of epidermal layer indicated permeation of CP through stratum corneum (42). The
of HTN. However, when carbopol-940 and denaturation. The treated skin specimen
n of subcutaneousHPMC lipidswere
might causeindehydration
added nanoemulsion of SC
withleading to significant
shifted endothermsloss of lower melting point
towards
stirring, only white hydrogel was obtained 74.55°C. It was observed that nanoemulsion
and the nanoemulsion structure was disturbed. formulations decreased the protein endotherm
Likewise, when sodium alginate was added to lower melting points, suggesting keratin
in nanoemulsion system it formed hydrogel denaturation and possible intracellular
but after 24 h phase separation occurred. So permeation mechanism in addition to the
it was concluded that carbopol-940, sodium extraction of lipid bilayers. One small peak
alginate and HPMC were not good gel forming at 32°C was completely removed due to lipid
polymers for CP loaded nanoemulsion. When extracting solvent and it completely disappeared
carbopol-971 was added in nanoemulsion in formulation treated skin which indicated that
system a transparent stable hydrogel formed components of nanoemulsions enhanced skin
which also maintained nanoemulsion structure permeation of drugs through extraction of SC
of the formulation. As the concentration of lipids (41).
6. Histological findings of skin biopsies from wistar rats; control (A), treated with
lsion (B). Table 6. Evaluation of hydrogel thickened system.
Carbopol Viscosity (PaS) ± pH ± S.D. Flux Drug retained in
S. No. -1
concentration (%) S.D. (n = 3) (n = 3) (µg/cm2 h ) skin (µg/mg)
1 0.4 64.79 ± 0.03 5.15 ± 0.05 8.69 0.25
2 0.8 73.44 ± 0.01 5.43 ± 0.03 5.12 0.34
3 1 97.57 ± 0.04 5.55 ± 0.01 3.93 0.57

47
Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

Figure 6. Histological findings of skin biopsies from wistar rats; control (A), treated with nanoemulsion (B).

Histopathology Studies epidermal region. The disruption of epidermal

The influence of CP loaded nanoemulsion layer indicated permeation of CP through stratum
on the anatomical structure of the rat skin is corneum (42). The extraction of subcutaneous
discussed with the aid of light microscopic lipids might cause dehydration of SC leading to
findings. The photomicrographs of untreated rat significant loss of moisture.
skin (control) showed normal skin (Figure 6)
with well defined epidermal and dermal layers. Anti-Inflammatory Studies
Keratin layer was well formed and lied just The anti-inflammatory effects of CP loaded
adjacent to the top most layer of the epidermis. in an optimized HTN were compared with the
Dermis was devoid of any inflammatory cells. placebo (algal oil) HTN formulations. The
When the skin was treated with nanoemulsion rat’s left footpad became edematous soon after
formulation, definite changes were observed injection of carrageenan and reached its peak at
in the skin morphology. The disruption and 3 h (76.25%). Significant (p < 0.05) amount of
extraction of lipid bilayers was clearly evident % inhibition (84.55%) (Table 7) was achieved
as distinct voids and empty spaces visible in the in case of CP loaded HTN formulation which

Table 7. Anti-inflammatory effects of drug loaded and placebo nanoemulsion gel in carrageenan-induced rat paw edema.
Group Formulation N Mean weight ± SD (g) Time (h) % Mean Edema ± SD % Inhibition
Control 6 180.0 ± 12.3 1 29.2 ± 2.3
(carrageenan only) 2 43.01 ± 4.2
I 3 76.25 ± 3.3
6 59.11 ± 3.7
12 39.3 ± 3.01
II Drug loaded 6 190 ± 10.01 1 25.5 ± 2.4 12.67
nanoemulsion hydrogel 2 33.15 ± 2.8 22.92
3 46.20 ± 4.1 39.40
6 14.10 ± 1.8 76.16
12 6.09 ± 1.2 84.55
III Placebo 6 195.0 ± 12.3 1 27.3 ± 3.1 6.50
nanoemulsion hydrogel 2 36.6 ± 3.7 14.90
3 53.19 ± 3.4 30.37
6 36.12 ± 3.1 38.89
12 23.17± 2.5 41.04
N = Number of rats in each group; SD = Standard deviation

48
 Algal oil nanoemulsion gel

Figure 7. ATP (7a) and ADP (7b) hydrolysis in lymphocytes obtained from the control group (C), contact dermatitis group (D), groups
with dermatitis treated with placebo nanoemulsion, and drug loaded nanoemulsion. Data were analyzed statistically by one-way ANOVA
followed by the Tukey-Kramer test (a) and Kruskal-Wallis Test (b), (p < 0.05).

indicated good anti-inflammatory activity at Contact dermatitis

the end of 12 h. Besides, 41.04% inhibition The in-vivo NTPDase activity of lymphocytes
was also achieved in placebo (algal oil) HTN after each treatment is shown in (Figure 7). A
formulation at the end of 12 h indicating some significant increase in NTPDase activity was
anti-inflammatory activity of algal oil which observed in lymphocytes of the group treated
might be due to presence of omega-3 fatty acids. with CP-loaded nanoemulsion, in relation to
Percentage edema was highest in case of control ATP and ADP (Figure 7a and 7b, respectively),
group and least in case of drug loaded group. compared to all other groups (p < 0.05). The
This finding revealed synergistic inhibitory ADP and the ATP hydrolysis values for the
effect on inflammation thus suggesting that the group treated with CP loaded nanoemulsion
optimized formulation could be a promising and the control groups were not statistically
delivery system for dermatitis treatment. different. The higher NTPDase activity may be
associated with the high levels of extracellular
Skin irritation test  ATP resulting from the inflammatory process,
The mean values of skin irritation score which occurs in cases of allergic contact
for drug loaded nanoemulsion gel and placebo dermatitis. During the dermatitis, these high
nanoemulsion gel were found to be 1.66 ± 0.81 levels of ATP would have an affinity for P2X7
and 0.83 ± 0.75 respectively (Table 8). From purinergic receptors, leading to a Th1 pattern
these results which were based on 14 days test, of immune response with the production of
it can be concluded that optimized nanoemulsion inflammatory cytokines. The NTPDase would
was safe to be used as topical drug delivery act by decreasing the levels of ATP, which in low
system. It clearly indicated that nanoemulsion concentration would bind to the P2Y receptors,
has more skin irritation potential due to high reversing the pattern of immune response to Th2
amount of surfactant in comparison to placebo with the release of anti-inflammatory cytokines.
nanoemulsion because drug itself may has Thus, it is possible that the increased hydrolysis
irritation potential. Overall all the formulation of adenine nucleotides also leads to an increase in
have low irritation score hence it is safe for the extracellular adenosine concentration, which
human use. has immunosuppressive and anti-inflammatory

Table 8. Skin irritation score of the placebo nanoemulsion, drug loaded nanoemulsion and marketed cream.
Score after Score after Score after Score after Score after Score after Mean
S. No. Group
(day) 1 (day) 2 (day) 3 (day) 4 (day) 7 (day) 14 score± SD
1 Drug loaded nanoemulsion hydrogel 1 1 3 2 1 2 1.66 ± 0.81
2 Placebo nanoemulsion hydrogel 2 0 2 0 1 2 0.83 ± 0.75

49
Sarfaraz A et al. / IJPR (2016), 15 (1): 35-52

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