Apixaban Ultrafine O/W Nano Emulsion Transdermal Drug Delivery System: Formulation, In Vitro and Ex Vivo Characterization

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Sys Rev Pharm 2020; 11(2): 82 94
A multifaceted review journal in the field of pharmacy
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82 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
Apixaban Ultrafine O/W Nano Emulsion Transdermal Drug
Delivery System: Formulation,
In Vitro
and
Ex Vivo
Characterization
Mustafa R. Abdulbaqi 1,*, N A Rajab 2
1Department of Pharmaceutics, College of Pharmacy, Al-Bayan University, Iraq
2Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Iraq
E-mail: drmustafa1986@yahoo.com
Article History: Submitted: 10.11.2019 Revised: 05.12.2019 Accepted: 10.01.2020
ABSTRACT
Apixaban (APX) is a potent oral anticoagulant drug that directly inhibit
coagulation factor Xa for prevention of venous thromboembolism (VTE)
following total hip or knee replacement surgery. Orally APX has poor
water solubility (0.028 mg/mL) a nd relative low bioavailability (50%).
Transdermal APX delivery was utilized as a convenient alternative route
to control oral limitations. This study designed to formulate ultrafine APX
o/w nanoemulsion with self-permeation enhancing properties through
skin barrier utilizing the ultrafine (> 50 nm) nanosized droplets as well as
nanoemulsion components themselves to act as a permeation enhancer.
Solubility study result in s electing triacetin oil, triton-x-100 and carbitol as
oil phase, surfactant and cosurfactant respectively, while pseudoternary
phase diagram construct nanoemulsion area for choosing formulations.
Twenty-one o/w nanoemulsions prepared and characterized for droplet
size, pH values, percent transmittance, electroconductivity, APX content,
in vitro
APX release, and
ex vivo
permeation through Albino Wistar rat
abdominal ski n to simulate human skin. Among formulations, ten
preparations demonstrate ultrafine APX o/w nanoemulsions with high
percent transmittance and electroconductivity, pH values appropriate for
skin application, ultrafine droplet sizes (> 50 nm) and accepted APX
content.
In vitro
release studies reveal significant (p ≤ 0.05) increase in
APX dispersibility and diffusion through dialysis membrane.
Ex vivo
APX
permeation through rat abdominal skin was significantly (p ≤ 0.05)
increased in compa rison with pure drug as assured by significant ( p ≤
0.05) enhancement in permeation parameters Jss, KP and ER with
shorter Tlag, which could be attributed to permeation enhancing
properties of nanoemulsion formulation itself.
Key words: Apixaban (APX); Ultrafine o/w nanoemulsion; transdermal
drug delivery.
Correspondence:
Mustafa R. Abdulbaqi
Al-Bayan Univversity
Iraq
E-mail: drmustafa1986@yahoo.com
DOI: 10.5530/srp.2020.2.14
@Advanced Scientific Research. All rights reserved
INTRODUCTION
Apixaban (APX) is a potent oral anticoagulant drug that
selectively and directly inhibit coagulation factor Xa and
used as a prophylactic therapy for the prevention of venous
thromboembolism (VTE) following total hip or knee
replacement surgery(1). It was approved by FDA on
December 28, 2012, for the prevention of stroke and
systemic embolism in patients with non-valvular atrial
fibrillation (AF) and marketed by Bristol-Myers
Squibb/Pfizer with trade name Eliquis. Unfortunately, APX
has poor water solubility of 0.028 mg/mL at 24 °C and
relative low bioavailability of about 50% after oral
administration of a single 10 mg dose. This low
bioavailability could be attributed to the incomplete
absorption of APX in the gastrointestinal tract (GIT), and
from the effect of first-pass metabolism in gut and liver(2).
Additionally, tablet dosage form is the only available form
for APX, which consider somewhat expensive for most
patients(3), although APX treatment was the dominant
strategy in the prevention of stroke and systemic embolism
over warfarin therapy, as APX shown to be safer than
warfarin with superior therapeutic activity in the prevention
of stroke(4). Therefore, regarding the limitations of the
marketed APX tablet in bioavailability and economic cost,
the development of new APX formulation was of interest
and important to obtain desirable pharmacokinetic
properties, including increased bioavailability, and more
cost-effective dosage form. Transdermal drug delivery has
several advantages over the oral route, as transdermal route
can overcome the limited APX absorption through intestine
and evade first-pass metabolism problems in gut and liver,
and hence, improved absorption via the skin and enhanced
bioavailability(5). Consequently, transdermal APX
administration through the skin was the most attractive
dosage form. One of the most promising tools for
transdermal drug administration is the lipid-based
dispersion nanoemulsion, due to desired features of the
nanoemulsion system that demonstrate enhanced solubility
of lipophilic or poorly soluble drugs, good thermodynamic
stability and improved dermal and transdermal drug
delivery by permeation enhancing properties of its
components through biologic membranes, excellent drug
loading capacity and negligible or minimum skin irritation
tendency(6, 7). Nanoemulsion is a transparent heterogenous
system consisting of two immiscible liquids, water and oil,
stabilized by an interfacial layer of surfactant / cosurfactant
mixture, or namely Smix, forming isotropic system that
contain the drug molecules in solubilized form
within the oil phase droplets(8). Nanoemulsion dispersion
display uniform distribution with droplet size ranged from
20 - 200 nm, this permit high drug flux and penetration
through the intracellular lipophilic pathway of the skin that
allow the nano-sized droplets of less than 20 nm to permeate
easily and creates a drug depot within the stratum corneum
and epidermis(9). Recently, ultrafine nanoemulsion
formulation developed as an advanced approach of emulsion
system, which designate clear isotropic nanoemulsion with
droplet size of below 50 nm. The key step for such
nanoemulsion preparation is to find the appropriate blend of
oil and surfactant that able to dissolve the required dose of

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
83 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
drug, which is 5 mg of apixaban in our study, and eventually
form clear isotropic mixture of oil, water, surfactant and
cosurfactant to form nanoemulsion system within ultrafine
droplet size below 50 nm(10). Transdermal application of
such ultrafine formulation offer several advantages over
other traditional transdermal delivery systems due unique
properties of the ultrafine nanoemulsion, including better
spreading ability of ultrafine nano-sized drug particles over
larger surface of the skin caused by increased effective area
of available drug particles exposed to the skin; increased size
to volume ratio of ultrafine sized particles (below 50 nm)
and thereby decrease the amount of final formulation and
increase its capacity for drug loading; in addition to the
permeation enhancing properties of the components of
nanoemulsion, the formulation itself of ultrafine
nanoemulsion can act as a permeation enhancer without
using any chemical or physical permeation facilitating
technique(11, 12).
This study designed to prepare ultrafine oil in water (o/w)
nanoemulsion formulations for transdermal delivery of
apixaban (APX) as a novel technology with improved
pharmaceutical physical properties of the drug, including
increased solubility and decreased crystallinity, and to
evaluate the permeation enhancing properties of the
prepared ultrafine o/w nanoemulsion formulations for their
impact as being themselves permeation enhancers using
abdominal skin of Wistar Albino rat for the ex vivo
permeation study.
MATERIALS AND METHODS
Instruments and Materials
Instruments used in this study include, Water Bath Shaker
(Kottermann, type 3047, Hanigsen, Germany) for solubility
study, Centrifuge (Fanem, 206-R Centrifuge, Brazil), UV
VIS Spectrophotometer (Spectrumlab 752Pro, China),
Vortex Mixer (Labinco L46, Netherland), Electrical
Conductivity Meter (DDS-11A, China), Brookhaven
instrument (Zeta Plus, Serial NO: 21521, USA), Intelligent
Transdermal Diffusion Instrument (TP-6, China), Digital
pH meter (BP 3001, Singapore), OriginLab 2018 software
program was used to plot pseudo-ternary phase diagram,
Dialysis Membrane (M.W 8000 - 14000 Da, USA).
Materials used include, Pure Apixaban obtained from
ZHEJIANG CP CHEMICAL CO., LTD; Methanol and
Ethanol Lab grade solvents (Sigma Aldrich, USA); Oils
include oleic acid and triacetin (Hangzhou Hyper Chemicals
Limited), castor, sesame, black seed, jojoba, argan, olive,
coconut, avocado, anise, almond, funnel and wheat oils
(NOW® CO., USA); Surfactants include cremophor EL,
cremophor RH 40, tween 20, tween 80, span 20, span 80 and
triton-X100 (Sigma Aldrich, USA); Cosurfactants include
carbitol, methyl carbitol, glycerin, PEG 200, PEG 400 and
propylene glycol (Sigma Aldrich, USA).
Preparation of Apixaban Loaded O/W Nanoemulsion
Formulations
Apixaban Saturated Solubility Study
An excess amount of APX was mixed with 5 mL of oils,
surfactants and cosurfactant each separately in screw
stoppered 5 mL vials and vortex for 1 min, then place each
sample in water bath shaker for 72 h at 32 +
rpm followed by equilibrium for 24 hours. After reaching
equilibrium, each mixture was centrifuged at 3500 rpm for
20 min to separate the excess of insoluble drug and then the
supernatant was filtered through 0.45 µm membrane filters
and diluted with methanol. The solubilized amount of APX
was quantified spectrophotometrically using UV-Visible
spectrophotometer at 278 nm (𝛌max of APX in methanol)
using methanol as blank(13, 14).
Development of Pseudoternary Phase Diagram
Pseudoternary phase of oil, surfactant/cosurfactant (Smix),
and water was developed using aqueous titration method.
Surfactant/cosurfactant weight ratios of (1:1, 1:2, 1:3, 1:4, 2:1,
3:1, 4:1) were screened for nanoemulsion formation, these
ratios used for detailed study of phase diagram which reflect
increasing concentrations of cosurfactant with respect to
surfactant and increasing concentrations of surfactant with
respect to cosurfactant(15). For each phase diagram,
different weight ratios of oil and Smix were combined in a
range from 1:9 to 9:1 in separated vials, where a
homogenous mixture of oil and Smix was formed using
vortex for 5 minutes, then aqueous phase of deionized water
continuous stirring and visual observation until first
turbidity and clear transparent oil in water (o/w)
nanoemulsion was obtained(16).
Criteria for Selection Nano emulsion Formulations
Subjected for Thermodynamic Stability Testing
To make a selection of optimum formulae, different o/w
nano emulsion formulations were prepared from each phase
diagram within nano emulsion region for APX loading and
to subject thermodynamic stability studies depending on the
following criteria:
1. The selected dose of APX for incorporation into
the oil phase was 5 mg for the preparation of 2 g
oil in water (o/w) nano emulsion.
2. The oil concentration in the selected formulations
should able to solubilize the used dose (single
dose) of APX easily, which is 5 mg.
3. Different concentrations of oil were selected from
each phase diagram in the nano emulsion region
with a difference of 5%.
4. Minimum concentrations of surfactant and co-
surfactant, hence Smix, and large percent of water
were selected from each phase diagram for
preparation of o/w nano emulsion
formulations(17, 18).
Formulation of Apixaban Loaded O/W Nano emulsions
A series of nano emulsion formulations were selected to be
prepared (Table 1.) using aqueous titration method, in
which, the assigned dose of APX of 5 mg was added to the
specified amount of oil phase and vortex until dissolving the
drug, then surfactant and co-surfactant (S mix) were added

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
84 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
to the oil loaded drug with the aid of vortex mixing. Then,
aqueous phase of deionized water was titrated gradually,
drop by drop, with gentle mixing until isotropic clear nano
emulsion was obtained(19). The preparation experiment was
performed in triplicate.
Table 1. Composition (w/w %) of apixaban nanoemulsion formulations
F-code
Smix
ratio
Triacetin
oil %
Water %
F-code
Smix
ratio
Triacetin
oil %
F-1
1:1
5
55
F-12
4:1
15
F-2
1:1
10
50
F-13
1:2
5
F-3
1:1
15
45
F-14
1:2
10
F-4
2:1
5
55
F-15
1:2
15
F-5
2:1
10
50
F-16
1:3
5
F-6
2:1
15
45
F-17
1:3
10
F-7
3:1
5
50
F-18
1:3
15
F-8
3:1
10
45
F-19
1:4
5
F-9
3:1
15
40
F-20
1:4
10
F-10
4:1
5
50
F-21
1:4
15
F-11
4:1
10
45
Thermodynamic Stability Study of the Prepared APX O/W
Nano emulsion Formulations
Three thermodynamic stability tests used to assess physical
stability of the prepared o/w nano emulsion formulations
and include centrifugation test, where nano emulsions
centrifuged at 3500 rpm for 30 min, followed by six heating-
cooling cycles utilized by storing each formulation between
refrigerator (4 °C) and heating (45 °C) temperatures for 48 h
in each temperature. Finally, accelerated stability assessment
by exposing the formulations to three freeze-thaw cycles
between freezing (
temperatures for 24 h at each temperature. After each test,
samples discarded if demonstrate phase separation,
precipitation or cracking by visual check(20, 21).
Characterization Techniques of the Prepared APX O/W
Nano emulsion Formulations
Droplet Size and Poly dispersity Index (PDI)
Measurement
The average droplet size of the prepared o/w nano emulsions
was measured by dynamic light scattering (DLS), which
analyze fluctuations in light scattering at 25 °C and
scattering angle of 90 ° caused by the Brownian motion of
the particles using photon correlation spectrophotometer
(PCS). Poly dispersity index (PDI) is a measure of
homogeneity in droplet size which ranges from 0 to 1 and
measured by electrophoretic light scattering technique(22).
pH Measurement
Digital pH meter used to measure pH values of APX o/w
nano emulsions, the pH of final transdermal APX
formulations is important for their compatibility with the
pH of skin to avoid possible irritation(23). The experiment
was performed in triplicate.
Transmittance Percent and Electrical Conductivity
Measurement
To confirm the type of the prepared nano emulsion,
transmittance percent of the prepared APX nano emulsions
was measured for optical transparency using UV-Visible
spectrophotometer at 650 nm, samples were not diluted
while keeping distilled water as blank(24), while electro-
conductivity
consisting of digital meter and two Pt / platinized electrodes.
The metal electrodes immersed in 5 mL of each sample at 25
reading was recorded (25). The experiment was
performed in triplicate.
Apixaban Content Measurement
Two grams of each APX nanoemulsion (supposed to contain
5 mg of APX) diluted with methanol and sonicated for 15
min for complete mixing, then filtered with 0.45 µm filter
syringe and analyzed spectrophotometrically at 278 nm
using methanol as a blank(26). The experiment was
performed in triplicate.
In Vitro Apixaban Release Study of Ultrafine APX O/W
Nano emulsions
In vitro APX release from ultrafine o/w nano emulsions was
achieved using vertical Franz cell diffusional system with
receptor part volume of 15 mL and donor part of 3 mL, and
dialysis membrane (M.W 8000 - 14000 Da, USA), as
diffusional barrier, mounted between donor and receptor
parts of Franz cell with diffusional area of 1.77 cm2. The
dialysis membrane was first soaked in phosphate buffer
saline (PBS) pH 7.4 for 24 h prior to use, in which PBS at 32
+ 0.5
Sodium Lauryl Sulphate (SLS) (to keep APX in solubilized
form) under continuous stirring of 600 rpm (to simulate in
vivo conditions) was used as releasing medium(27, 28).
Two grams of the prepared ultrafine o/w nano emulsions
loaded with single dose APX of 5 mg, as well as 2 g of PBS
pH 7.4 containing 5 mg of pure APX suspended in it as a
control, were placed separately in donor compartments of

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
85 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
Franz cell instrument under experimental parameters
mentioned above. Samples of 0.1 mL were withdrawn every
5 mins and replaced with equivalent volume of fresh PBS
dissolution medium after each withdrawal. APX content of
the samples was quantified spectrophotometrically at 280
nm using PBS pH 7.4 buffer as a blank and until complete
release of APX was achieved, then the cumulative amount of
APX released was calculated and plotted as a function of
time(29, 30). The experiment was performed in triplicate.
Kinetics of APX In Vitro Release
Various mathematical models were applied for the plotting
data obtained from in vitro release experiment to determine
the kinetics and mechanism of APX release from each
prepared nano emulsion, including zero order kinetic, first
order kinetic, higuchi model and korsmeyer - peppas model
using equations in Table 2.(31). The accuracy and
predictability of the applied models were compared on the
basis of plot linearity and calculated squared regression
coefficient (R2) constructed from graphs made for each
model, in which the plot that is linear with a highest value of
R2 would consider the selected kinetic model for APX release
from its formulation(32, 33).
Table 2. Kinetics Models for Apixaban In Vitro Release Study 1
Model
Equation
Zero order
Qt Q0 = K0t
First order
Ln (Qt /Q0) = K1t
Higuchi Model
Qt = KH
Hixson-Crowell Model
(Q0) (Qt) = KHCt
1 K0, K1, KH, KHC: rate constant for respective model; Qt: amount of APX released at time
(t); Q0: initial amount of APX in formulation
Ex Vivo Apixaban Permeation Study of Ultrafine APX O/W
Nanoemulsions
Permeation Skin Preparation of Wister Rat Abdomen
Abdominal skin of Wister Albino rat was used as diffusional
membrane for ultrafine APX o/w nanoemulsion permeation,
as it demonstrates comparable stratum corneum thickness
and water permeability to human skin(34). Albino male rats
of 2-3 months age and each weighing 200 ± 10 g were
scarified by ether inhalation and then the abdominal hair
was removed with care to avoid accidental skin damage
using electrical clipper. A fresh rat abdominal skin, with
rectangular shape of few centimeters in each dimension, was
excised from the animal using sharp surgical blade(35). To
remove the adipose tissue from the skin, diethyl ether
solvent was wiped on dermal side using cotton wool to
solubilize the adipose tissue and easily get rid the muscles
and blood vessels from the skin, followed by scrapping using
a scalpel carefully and washed with normal saline solution
for sterility and hygienic. The skin was then stabilized to
ensure complete removal of UV-visible absorbing materials
for 2-3 h with replacing the medium every 30 min until no
UV-visible absorbance was observed (36, 37). The prepared
sections of skin were then wrapped into aluminum foil and
the samples of the skin were thawed at room temperature for
at least 30 min and wiped carefully with PBS of pH 7.4 using
cotton wool balls.
Franz Cell - Ex Vivo Permeation Study through Rat
Abdominal Skin
According to the approval of animal ethical committee of
Baghdad University / College of Pharmacy, ex vivo skin
permeation study was performed utilizing the abdominal
skin of adult Wister Albino male rats.
Franz cells water bath system device used for the ex vivo
skin permeation study consisting of six Franz diffusion cells
and each diffusion cell consist lower receptor and upper
donor compartments. Skin cuts mounted onto receptor
compartment of Franz cells with effective diffusional area of
1.77 cm2 of skin and 15 mL capacity of receiver chamber.
The dermal side of the skin was exposed to the receptor
medium consisting of PBS pH 7.4 containing 1 % Sodium
Lauryl Sulphate (SLS), while the epidermal side was faced to
donor part of Franz cell under ambient conditions and
fastened with an O-ring(38). Prior to experiment, the skin
was equilibrated with the experimental temperature of 32 ±
0.5 oC to mimic skin surface temperature by filling both
compartments with receptor medium with magnetic stirring
at 600 rpm for 30 min to get rid of any air bubbles(39). Then
2 g of APX loaded ultrafine nanoemulsions each containing
5 mg of APX, was weighed and applied to the surface of skin
in the donor compartment. The receptor medium of PBS pH
7.4 / 1 % Sodium Lauryl Sulphate (SLS) was agitated using
magnetic stirrer within the device at 600 rpm and the
temperature was kept at 32 ± 0.5 oC throughout the
experiment to mimic in vivo conditions(29, 40). Samples of
0.1 mL were then withdrawn periodically from the sampling
port in the side of receptor cell part at predetermined time
intervals, which replaced with equal volume of fresh PBS pH
7.4 media to maintain sink condition, until complete
permeation of APX achieved using filtered syringe with pore
yzed using UV-Visible
spectrophotometer at 280 nm and the cumulative amount of
drug permeated was calculated and plotted against time(41).
The experiment was made in triplicate.
Ex Vivo Apixaban Permeation Data Analysis
Permeation profile obtained by plotting the cumulative
2) of APX permeated across the rat skin

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
86 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
on Y-axis as a function of time (t, min) on X-axis. This
profile used to calculate permeation parameters, including
permeation rate or transdermal APX flux at the steady state
(Jss, mg/cm2/h), which obtained from the slope of straight
linear portion of the regression line, Lag time (Tlag), which
was determined from the intercept of regression line.
Permeability coefficient (KP, cm/h), calculated from the
ratio of APX flux (Jss) divided by the initial concentration
(C0) of APX placed in donor compartment, while
enhancement ratio (ER), calculated by dividing flux (Jss) of
APX from tested formulation by control (pure APX) flux(42,
43).
RESULTS AND DISCUSSION
Preparation of Apixaban Loaded O/W Nanoemulsion
Formulations
Solubility study
Among tested oils, triacetin exhibit higher solubilizing
capacity (31.870 mg/mL), followed by oleic acid and sesame
oils with saturated solubility of 21.672 and 15.301 mg/mL
respectively, while lowest solubility obtained in wheat oil
(1.533 mg/mL). Therefore, triacetin oil was selected as oil
phase in preparation and further investigation of
nanoemulsion formulations. Triacetin has beneficial
properties for construction in transdermal preparations,
since it act as skin penetration enhancer through stratum
corneum, for example in Oxytrol® patch indicated for
overactive bladder which is available commercially and
contain triacetin as penetration enhancer(44). Triacetin also
display good miscibility with other components during
nanoemulsion formulation that enable facile preparation of
homogenous one phase system and good loading properties.
In surfactants, highest solubility of APX was achieved in
triton-X-100 (9.256 mg/mL) and cremophor EL (8.725
mg/mL), while span 20 displayed lowest saturated solubility
of APX (3.264 mg/mL). Therefore, triton-X-100 was selected
as a surfactant for nanoemulsion formulation, it is non-ionic
hydrophilic surfactant with hydrophilic lipophilic balance
(HLB) value of 13.4, which is appropriate for facile o/w
nanoemulsion formation with low toxicity(45). Triton-X-
100 also has membrane permeabilization properties for
living cells bio-membranes which rely on lipid composition,
triton-X-100 concentration and the ease to intercalate
between lipids(46). Carbitol demonstrate highest
solubilizing capability to APX (159.959 mg/mL), while
glycerol demonstrates lowest APX solubility (6.772 mg/mL),
therefore carbitol selected to mix with the surfactant triton-
X-100 to prepare Smix. It is frequently applied in the
formulation of transdermal nanoemulsion drug delivery
system, as carbitol spreads easily over the skin without
streaking, high skin biocompatibility and safety, its ability to
solubilize large number of drugs and most importantly, it
has skin penetration enhancing properties(47).
Pseudoternary Phase Diagram
Seven pseudoternary phase diagrams (Figure 1.) were
constructed by aqueous titration method using triacetin as
oil phase, triton-X-100 and carbitol as surfactant and
cosurfactant respectively at different ratios and deionized
water as aqueous phase. The results showed nearly same
nanoemulsion regions (shaded area) for all Smix ratios with
biggest nanoemulsion region at surfactant / cosurfactant
ratio 1:1, this may be caused by the greater penetration of the
oil phase into the hydrophobic tail region of the surfactant
triggered by the presence of hydrophobic cosurfactant
carbitol(48). Additionally, the increased entropy of
nanosystem would expect to force oil molecules with
enhanced penetration to interfacial surfactant layer due to
their smaller size molecule compared to surfactant molecule
used(49). Concerning the effect of surfactant/cosurfactant
ratio, there was slight increase in nanoemulsion area with
the increase in surfactant concentration with respect to
cosurfactant, and therefore diagrams with Smix ratios 2:1,
3:1 and 4:1 have wider nanoemulsion area than 1:2, 1:3 and
1:4 Smix diagrams, this increase in nanoemulsion area with
Smix ratio could be attributed to the increase in HLB value
of nanoemulsion system caused by increment of hydrophilic
surfactant triton-X-100 (HLB 13.4) and hence, increased
hydrophilicity of system with improved micelle formation,
enhanced solubilizing capacity of nanoemulsion and
eventually optimized aqueous miscibility(50).
Cosurfactant carbitol increase nanoemulsion solubility by
their insertion into void spaces between the surfactant
molecules and hence aid in the reduction of interfacial
tension and increase fluidity(51), it also improves APX
dispersibility in the system due to hydrophobic nature of
carbitol with HLB value of 4, therefore triton-x-100
surfactant and carbitol cosurfactant were able to form a
stable oil in water (o/w) nanoemulsion system with HLB
value above10 indicating hydrophilic surfactant(52).
(a) (b) (c)

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
87 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
(d) (e) (f)
(g)
Figure 1. Pseudo-ternary phase diagrams of triacetin, triton-X-100 and carbitol at different Smix ratios of (a) 1:2, (b) 1:3, (c) 1:4,
(d) 2:1, (e) 3:1, (f) 4:1 and (g) 1:1, Shaded area represent (o/w) clear nano emulsion regions.
Formulation of Apixaban Loaded O/W Nano emulsions
Pseudo ternary phase diagram can be used for
demonstration of lower and greater weight percent of oil and
Smix for the preparation of o/w nano emulsion
formulations(53). Twenty-one o/w nano emulsion
formulations (Table 1.) prepared using aqueous titration
method, constructed by selecting three formulations from
each phase diagram of different Smix ratio. Two criteria
were dependent for the preparation of the selected
formulations; first, the amount of oil was selected at 5 %
weight interval selecting 5, 10 and 15 % of triacetin oil ratio
concentration so that largest number of formulations could
be selected to cover the nano emulsion area for each phase
diagram(54). The second criterion based on selecting
formulations with minimum concentrations of Smix, as
mentioned previously, to avoid possible skin irritation by
surfactant application(55). Hence, nano emulsion
formulations taken from each phase diagram for each
selected percent of triacetin (5%, 10% and 15 %) were only
those having the minimum concentrations of Smix, which
were detected as 30 % and 40 % for Smix ratios 1:1, 1:2, 1:3,
1:4 and 2:1; while for Smix ratios 3:1 and 4:1, minimum
concentrations were detected at 35 % and 45 % for
emulsification. Additionally, the selected percentages of
triacetin oil (5, 10 and 15%) were able to completely
solubilize single dose of APX (5 mg) depending on the
results of solubility study. There was no obvious change in
visual appearance during formulation, i.e. phase separation,
turbidity or color change, as well as no drug precipitation of
APX was observed during deionized water addition.
Thermodynamic Stability Study of the Prepared APX O/W
Nano emulsion Formulations
Thermodynamic stability tests were applied for all twenty-
one formulations and results demonstrate stability of all
formulations and therefore flocculation, aggregation, phase
separation, creaming, cracking or coalescence not take place
when prepared at particular proportions of surfactant, co-
surfactant, oil and water. The inherited high stability of the
prepared nano emulsions could be attributed to the stearic
stabilization of nonionic surfactant triton-X-100(56). This
thermodynamic stability confers long shelf life to nano
emulsions as compared to ordinary emulsions(57).
Characterization Techniques of the Prepared APX O/W
Nano emulsion Formulations
Droplet Size and Poly dispersity Index (PDI)
Average droplet size was measured for the entire prepared
o/w nano emulsions as all pass thermodynamic stability tests
successfully. Results presented in Table 3. and demonstrate
o/w nano emulsions formation with droplet sizes ranged
from 11.47 nm to 691.32 nm. The wide variation in droplet
size caused by using different ratios of o/w nano emulsion
components of Smix, oil and water, as well as by changing
Smix ratios of surfactant and co-surfactant (58), which affect
the surface curvature of the film by changing its flexibility
and consequently influences droplet size.
Among tested formulations, ten APX o/w nano emulsions
demonstrate ultrafine droplet size of less than 50 nm(10, 59)
including all three prepared formulations with Smix ratio of
3:1 (F-7, F-8 and F-9) with smallest size of 11.47 nm by F-7.
Although higher triton-x-100 surfactant concentration, only
one formulation with Smix ratio of 4:1 (F-10) display
ultrafine droplets with average size of 15.42 nm, the same
was observed at Smix ratios of 1:1, 1:2, 1:3 and 1:4 each
demonstrating one formulation within ultrafine nano-sized
droplets including F-1, F-13, F-16 and F-21 with average
sizes of 26.41, 13.12, 27.53 and 38.42 nm respectively, while
Smix ratio 2:1 showed two ultrafine sized formulations, F-5
and F-6 with average droplet size of 27.67 nm and 26.08 nm
respectively. According to the results, a decrease in droplet
size was demonstrated with the increase in surfactant
concentration, and consequently in Smix ratio, as smaller
sizes obtained at Smix ratios of 4:1 and 3:1, the reason for

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
88 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
the surfactant impact on the particle size could be attributed
to the increased HLB value and hence, hydrophilicity of
surfactant mixture which facilitate reduction in the
curvature of the triacetin oil interface which present with
fairly high solubility and therefore leading to droplet size
reduction(60). Additionally, stabilization effect of the
triacetin oil droplets by localization of triton-x-100
surfactant molecules at the oil/water interface resulting in
higher stability and smaller droplet size(61).
PDI was measured for all formulations and values ranged
between (0.206 0.431) as displayed in Table 3. The
majority of the prepared o/w nano emulsion formulations
presented with PDI of 0.3 or below, which considered to be
acceptable, indicating the formation of homogenous
monodispersed nano emulsions with good stability and very
narrow poly dispersity with good uniformity in droplet size
distribution upon dilution. Other formulations presented
with higher PDI of larger than 0.3 values, indicating the
formation of poly disperse emulsion systems with reduced
stability (62, 63). It is obvious from the results that, larger
droplet size distribution demonstrates higher PDI and
therefore reduced stability of droplets within their o/w nano
emulsion formulation and lesser uniformity of droplets
distribution or poly disperse system.
Table 3. Results of mean particle size distribution (PSD); poly dispersity index (PDI); pH values; electrical conductivity;
transmittance percent; percent of APX content; for the prepared APX o/w nano emulsion formulations F-1 to F-21, (mean ± SD,
n = 3).
F-
code
PSD
PDI
pH
Electrical conductivity
Transmittance %
% APX
content
F-1
26.41
0.296
5.68 + 0.07
174.52 + 0.93
98.38 + 0.02
99.72 + 0.15 %
F-2
343.31
0.263
5.53 + 0.01
162.65 + 1.24
99.62 + 0.02
97.06 + 0.22 %
F-3
127.49
0.412
5.47 + 0.04
144.38 + 0.88
99.98 + 0.03
97.55 + 0.1 %
F-4
482.62
0.312
5.64 + 0.03
173.06 + 0.67
98.15 + 0.04
96.91 + 0.12 %
F-5
27.67
0.328
5.52 + 0.07
165.23 + 0.38
99.87 + 0.03
99.03 + 0.11 %
F-6
26.08
0.259
5.44 + 0.04
141.86 + 0.46
97.88 + 0.06
99.78 + 0.23 %
F-7
11.47
0.206
5.73 + 0.02
161.56 + 0.58
98.89 + 0.04
99.95 + 0.25 %
F-8
15.89
0.251
5.62 + 0.07
149.66 + 1.03
97.80 + 0.03
99.04 + 0.19 %
F-9
46.06
0.352
5.48 + 0.03
132.55 + 0.76
98.89 + 0.01
99.71 + 0.28 %
F-10
15.42
0.268
5.76 + 0.04
159.83 + 0.96
97.04 + 0.01
98.99 + 0.22 %
F-11
92.46
0.423
5.65 + 0.06
136.22 + 1.17
97.92 + 0.01
97.38 + 0.17 %
F-12
355.58
0.405
5.59 + 0.02
128.45 + 0.73
99.46 + 0.04
96.92 + 0.16 %
F-13
13.12
0.276
5.57 + 0.02
182.26 + 1.42
97.07 + 0.02
99.78 + 0.12 %
F-14
184.77
0.421
5.51 + 0.04
169.06 + 1.01
99.99 + 0.01
98.38 + 0.27 %
F-15
316.59
0.394
5.43 + 0.05
155.71 + 0.07
97.28 + 0.05
97.27 + 0.22 %
F-16
27.53
0.317
5.46 + 0.07
175.48 + 0.97
99.62 + 0.02
99.48 + 0.26 %
F-17
142.90
0.431
5.34 + 0.01
162.88 + 0.69
98.18 + 0.06
97.08 + 0.13 %
F-18
260.72
0.356
5.22 + 0.03
144.83 + 0.83
97.34 + 0.01
97.16 + 0.2 %
F-19
691.32
0.399
5.46 + 0.05
183.45 + 0.48
98.86 + 0.02
96.84 + 0.24 %
F-20
150.62
0.431
5.33 + 0.06
168.84 + 1.04
98.78 + 0.06
97.38 + 0.28 %
F-21
38.42
0.334
5.21 + 0.03
143.98 + 0.98
99.08 + 0.04
99.82 + 0.16 %
PH Measurement
The pH measurements summarized in Table 3. and reveal
pH range for APX o/w nano emulsions of (5.21 5.76) for F-
21 and F-10 respectively, these values were suitable for
topical application due to comparable values with skin pH
which ranges from 4.5 to 6.5 and therefore evade skin
irritation and/or sensitivity(64). pH values obtained slightly
reduced with the increase in triacetin oil concentration
within APX nano emulsions, this could be attributed to the
increased acetic acid release caused by partial triacetin
hydrolysis into acetic acid and glycerol in the aqueous phase
of nano emulsion (65). Apixaban is a neutral compound that
does not ionize at physiologic pH range (1.2 6.8), therefore
it does not ionize at pH range (4.67 5.76) of the prepared
o/w nano emulsions (66).
Transmittance Percent and Electrical Conductivity
Measurement
All prepared formulations tested for their %T (Table 3.) and
results ranged from 97.04 % 99.99 % for F-10 and F-14
respectively, indicating transport light easily, optically clear,
transparent and nanosized droplets(51). Conductometer was
employed for all prepared APX o/w nano emulsions and
readings ranged from 128.45 183.45 for F-12 and F-
16 respectively as presented in Table 3. which revealed the
formulation of o/w nano emulsions with high degree of
electrical conductivity as water represents the external phase
and can conduct electrical current(67).
Apixaban Content Measurement
APX content within o/w nano emulsions was measured for
all formulations and range from 96.84 % to 99.95 % for F-19

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
89 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
and F-7 respectively, and hence, set within the official range
(85 % - 115 %) accepted according to the united states
pharmacopeia (USP) as presented in Table 3. APX was
therefore loaded successfully within all the prepared o/w
nano emulsions without any precipitation or degradation of
the drug.
In Vitro Apixaban Release Study
Ten formulations of ultrafine apixaban o/w nano emulsions
(droplet size less than 50 nm) selected for APX in vitro
release estimation as displayed in Figures 2 and 3, and
demonstrate variable time durations for complete APX
release, in which F-1, F-5, F-6 and F-16 ultrafine
formulations release APX completely after 45 min, while F-
7, F-10 and F-13 formulations take 35 min for complete
APX release, and eventually F-8, F-9 and F-21 demonstrate
nearly 100% release after 40, 55 and 50 min. Pure APX
suspended in phosphate buffer saline (PBS) of pH 7.4 was
also included for in vitro study as a control and demonstrate
significantly (p 0.05) slower release profile than other
tested samples of APX after formulation as ultrafine o/w
nano emulsion, in which only 16.12 % and 25.88 % of APX
released after 35 and 55 min respectively. APX release from
each ultrafine o/w nano emulsion was highly dependent on
droplet size, as faster APX release of 35 min was reported by
o/w nano emulsion F-7, F-10 and F-13 formulations with
droplet sizes below 20 nm, while longer release duration of
55 min was displayed by o/w nano emulsion F- 9
formulation with droplet size 46.06 nm. The cumulative
amount of APX released from F-7, F-10 and F-13 after 35
min, and F-9 after 55 min, was significantly ( ) higher
than plain APX suspended in PBS with about 6.203 and
3.863 folds respectively. This influence of droplet size on
APX release could be attributed to the pronounced increase
in the effective interfacial area of APX particles exposed to
PBS dissolution media and hence, higher dissolution rate
and faster drug release(68, 69). Although no significant
(p>0.05) difference observed in the in vitro release rate
profile between ultrafine APX loaded formulations with
nearly same droplet size, they differ in the proportion of
each component of Smix, triacetin oil and water, as well as in
the surfactant / co-surfactant ratio for Smix used in each
ultrafine formulation. Regarding the effect of Smix ratio
within each ultrafine o/w nano emulsion, it was observed
that formulations with higher Smix ratios (higher triton-x-
100 surfactant concentration) demonstrate faster APX
release profile, the reason for this effect caused by
solubilizing efficacy and hydrophilicity enhancement of APX
induced by using triton-x-100 surfactant in high
concentration and hence, present APX in dissolved form
that is the only form can cross the membrane(70, 71).
Additionally, smaller droplet size of ultrafine nano
emulsions produced by the increase in Smix ratio was
accompanied with the increase in APX release rate(72).
Kinetics of Apixaban In Vitro Release
The in vitro release kinetics reveal pure APX in PBS pH 7.4
and all studied ten formulations of ultrafine APX o/w nano
emulsions with highest correlation coefficient (R2) fit with
zero order kinetic model, indicating concentration
independent APX release kinetic. This fitting of APX release
with zero order kinetics could be attributed to the APX flow
retardation effect produced by dialysis membrane, that
regulate and control APX release rate rather than slowing
it(73). While for pure APX, as drug demonstrate low
solubility in PBS pH 7.4, then APX solubility control its
release as reported by Noyes Whitney equation for solid
dissolution(74).
Figure 2. Comparative in vitro release study of ultrafine apixaban o/w nano emulsion formulation F-1, F-5, F-6, F-7 and F-8 with
pure APX in phosphate buffer saline pH 7.4.
0
20
40
60
80
100
020 40 60
cumulative % APX
released
Time (min)
F-1
F-5
F-6
F-7
F-8
Pure APX

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
90 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
Figure 3. Comparative in vitro release study of ultrafine apixaban o/w nano emulsion formulation F-9, F-10, F-13, F-16 and F-21
with pure APX (control) in phosphate buffer saline pH 7.4.
Ex Vivo Apixaban Permeation Study
Rat abdominal skin used as permeation barrier for ex vivo
evaluation of ultrafine APX o/w nano emulsions using Franz
cell diffusional system. Among ten ultrafine APX nano
emulsions, five formulations (F-1, F-6, F-7, F-13 and F-21)
were selected for ex vivo studies evaluation. The results, as
shown in Figure 4., indicate significantly
enhanced permeability of ultrafine APX nano emulsions in
comparison with that of pure APX, in which complete
permeation was observed after 7, 6, 5, 6 and 8 hours for F-1,
F-6, F-7, F-13 and F-21 respectively, while pure APX
demonstrate permeation percent of 13.32, 17.94, 24.93 and
28.07 after 5, 6, 7 and 8 hours respectively. This significant
increase in permeation of APX form ultrafine nano
emulsions could be attributed to several factors, first, the
ultrafine droplets (< 50 nm) of nano emulsion significantly
increase the rate of permeation, as the nanosized droplets
enable drug transfer through the skin barrier and therefore
easily reach stratum corneum (75, 76). Secondly, APX
present or hidden within triacetin droplets in solubilized
form, which was aided by the presence of co-surfactant
carbitol in Smix and hence, can permeate through lipophilic
skin layers more efficiently(77). Third, as this study was
aimed, the formulation itself of ultrafine nano emulsion act
as permeation enhancer due to its components of triacetin
oil and triton-x-100 surfactant which already demonstrate
permeation enhancing properties according to previous
literatures(78, 79).
Figure 4. Comparative En vitro permeation study of ultrafine apixaban o/w nano emulsion formulation F-1, F-6, F-7, F-13 and F-
21 with pure APX (control) through rat abdominal skin.
0
20
40
60
80
100
020 40 60
cumulative % APX released
Time (min)
F-9
F-10
F-13
F-16
F-21
Pure APX
0
20
40
60
80
100
0 5 10
Cummulative amount
permeated (ug/cm2)
Time (h)
F-1
F-6
F-7
F-13
F-21
Pure APX

Mustafa R. Abdulbaqi et al / Apixaban Ultrafine O / W Nano Emulsion Transdermal Drug Delivery System: Formulation, In vitro
and Ex Vitro Characterization
91 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020
Ex Vivo Apixaban Permeation Data Analysis
Permeation parameters Jss, KP and ER were calculated and
their values increased
with pure APX control as shown in Table 4. The lag time for
starting permeation (Tlag) also assure faster permeation of
APX nano emulsions than pure drug, which were 0.5 1.33
h, while it was found 2.5 h for pure APX. These outcomes
encourage APX formulation for transdermal delivery of drug
with more efficient and safer use than marketed tablet
form(80).
Table 4. Ex vivo permeability parameters of ultrafine APX o/w nano emulsion formulations and pure APX (control)
Parameter
F-code
Flux
2 .h)
KP (cm/h) ×
10 -3
Tlag (h)
ER
F 1
16.091
6.436
1
3.413
F 6
18.874
7.550
0.83
4.003
F 7
21.743
8.697
0.5
4.612
F 13
19.045
7.618
0.75
4.040
F 21
13.444
5.378
1.33
2.852
Pure APX
4.7561
1.886
2.5
1
CONCLUSION
As a conclusion from this work, apixaban was formulated
successfully into o/w nano emulsion with ultrafine sized
droplets (< 50 nm) with preferential properties for
transdermal application. Apixaban oral administration
drawbacks of limited bioavailability and poor solubility were
evaded by the optimized ultrafine APX o/w nano emulsion
as a novel delivery system for transdermal application of the
drug. Additionally, characterization techniques, in vitro
release studies and ex vivo permeation testing of the
prepared APX formulations approve their relevance for
effective, safe and practical transdermal application over the
skin without using chemical or physical permeation
enhancing techniques.
CONFLICT OF INTEREST
Authors declare no conflict of interest was reported.
ACKNOWLEDGEMENT
Authors were grateful to College of Pharmacy / Baghdad
University for their laboratory, experimental devices and
materials supplying and facilitation. Special thank was
afforded to Central Service Laboratory / College of
Education Ibn Al Haitham / Baghdad University for
performing characterization tests during research.
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