Article

Volume 11, Issue 4, 2021, 12037 - 12054

https://doi.org/10.33263/BRIAC114.1203712054

Design, Formulation, In-Vitro and Ex-Vivo Evaluation of

Atazanavir Loaded Cubosomal Gel
1 2 2
Ananda Kumar Chettupalli , Madhubabu Ananthula , Padmanabha Rao Amarachinta ,
2 3, *
Vasudha Bakshi , Vinod Kumar Yata
1 Department of Pharmaceutics, Centre for Nano-medicine, School of Pharmacy, Anurag University, Venkatapur,
Ghatkesar, Medchal, Hyderabad, Telangana-500088, India
2 Department of Pharmaceutics, Centre for Nano-medicine, School of Pharmacy, Anurag University, Venkatapur,
Ghatkesar, Medchal, Hyderabad, Telangana-500088, India
3 Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal-132001, India
* Correspondence: vinodyata@gmail.com;
Scopus Author ID 36553530200
Received: 13.11.2020; Revised: 20.12.2020; Accepted: 21.12.2020; Published: 30.12.2020

Abstract: In this study, Atazanavir (ATZ) was designed into the Nano formulation called cubosomes
to improve its bioavailability and curtail the adverse effects by the transdermal route delivery of ATZ -
loaded cubosomes. Around twenty cubosomal formulations were formulated using a Central composite
factorial design. The effect of glyceryl monooleate (GMO), surfactant (Pluronic F 127), and
Cetyltrimethylammonium bromide (CTAB) were studied using processes of emulsification and
homogenization. Different concentrations of independent variables on particle size distribution, zeta
potential, and entrapment efficiency were determined. FTIR, DSC, X-ray, and SEM, TEM results
established that the drug was encapsulated in the cubosomes. The results suggested that the optimal
formula exhibited a particle size of 100±7.9 - 345±6.4 nm and entrapment efficiency ranging from
61±4.6 - 93±0.8, zeta potential values ranging from -24.51 to -32.45 mV, polydispersity index values
ranged from 0.35±0.01-0.54±0.02 of ATZ. The in vitro studies showed a controlled release pattern of
drug release up to 24h. The ATZ cubosomal gel application on the in vivo absorption studies of the drug
was studied in rats and compared with oral ATZ solution. The in vivo study results showed that the
transdermal application of ATZ cubosomal gel considerably improves the absorption of drug compared
to that of oral ATZ solution and found that the relative bioavailability is 4.6 times greater of oral ATZ
solution. Thus it can be concluded that the ATZ cubosomal gel application via transdermal delivery
route has the potential in increasing the bioavailability of the drug.

Keywords: ATZ-loaded cubosomes; central composite design; Pluronic F 127; bicontinuous structures;
homogenization processes.
© 2020 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction

Antiretroviral (ARV) treatment guidelines currently recommend ARV regimens

containing a Nucleos(t)ide Reverse Transcriptase Inhibitors (N(t)RTIs) based backbone with a
Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) or ritonavir-boosted Protease
Inhibitor (PI/r). However, significant toxicity has been associated with N(t)RTI(s) and PI/r
containing regimens. Recent data presented by Gupta et al. show that the combination of
raltegravir (RAL) plus unboosted atazanavir (ATV) may be an alternative effective ARV
regimen demonstrating good virologic and immunologic response [1]. Furthermore, the
combination is well tolerated and has a low incidence of adverse effects [2]. Moreover, side
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effects reported by Zhu et al. during a study in healthy subjects were generally “mild-to-
moderate” in intensity. Common side effects were seen when both drugs were taken, such as
jaundice and headache [3]. Ripamonti et al. evidenced that after five to seven months of therapy
based on RAL plus ATV no patients discontinued treatment due to drugs used in therapy,
adverse events. No one had grade 3 or 4 lab toxicity [4]. For these reasons, this combination of
antiretroviral therapy based on RAL plus ATV attracts the scientific community's attention
because both drugs have a good safety profile coupled with potent antiviral activity. Their
combined use would avert nucleoside- and ritonavir-related toxicities.
Transdermal drug delivery is a non-invasive delivery of medications across the skin's
surface through its layers to the circulatory system [5]. Because of its enormous advantages
over conventional delivery systems, the transdermal drug delivery system remains of great
interest for researchers [6]. This is because of its ability to deliver different drugs into blood,
eliminate hepatic first-pass metabolism, and enhance patient compliance [7]. Cubosomes were
selected as a nanocarrier because they possess many advantages. Cubosomes are formed by the
fragmentation of lipid base in the presence of surfactant using excess water [8]. Cubosomes
differ from emulsions. An emulsion is a biphasic system consisting of two immiscible liquids,
one of which is finely and uniformly dispersed as globules throughout the second phase [9].
Cubosomes can penetrate skin and mucosa because of the similarity between their inner
structure and epithelium cell; they can enhance drug bioavailability [10]. One more advantage
of cubosomes is their hydrophilic, hydrophobic, and amphiphilic nature of drugs ranging from
small molecules to large molecules.[11].
The lipophilic drug carrier has the ability to improve the therapeutic efficacy of the drug
[12-14]. Cubosomes are nanostructured aqueous dispersions having a characteristic feature of
dispersed particles. Their name, cubosomes, reflects their dispersion particle shape, consisting
of cubic liquid crystalline phase consisting of highly twisted bi-continuous structures, two
congruent non-intersecting water channels separated by continuous lipid bilayer [15-17].
Cubosomes can be prepared from biodegradable lipid materials like mono-glycerides
such as monoolein (MO). MO forms bilayers of continuous inverted cubic phase separating
two intercrossing water canals at NRT. Cubosomes are formed due to the emulsification of
lipid phases in water [18-19]. These cubosomes are potential drug nanocarriers since they are
tailored for solubilizing higher amounts of amphiphilic, hydrophobic, and hydrophilic drugs in
their structures [20]. Moreover, cubosomes are greatly biocompatible and bio-adhesive
nanoparticles. The presence of the same cubic phase structure between the cubosomes and
stratum corneum has a penetration enhancing effect on the skin due to the stratum corneum's
fluidization [21]. Furthermore, cubosomes are known to be skin-adhesive, flexible drug
nanocarriers administered by transdermal as potential carriers in the drug delivery system.
Response surface methodology (RSM) is considered n efficient statistical technique
used to reveal process parameters and their interactive effects [22-23]. The Central Composite
design (CDD) is the main tool of response surface methodology design and has been
successfully used to model and optimize the processes in many fields [24-26]. Atazanavir
(ATZ) based cubosomes provide potential effects in the form of biocompatible and bio-
adhesive transdermal dosage form with a decrease in ATZ side effects. Therefore, this study is
aimed to prepare ATZ -loaded cubosomes and look for the parameters meters influencing the
properties of MO-based cubosomes. Additionally, to assess the ability of cubosomes to act as
a transdermal carrier in drug delivery.

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2. Materials and Methods

Materials: Atazanavir (ATZ) were given by Hetero Labs, Hyderabad, as a source of

glyceryl-monooleate (GMO), Pluronic® F-127was purchased from Sigma-Aldrich Chemical
Company. Ethanol was purchased from S.D. Fine chemicals, Mumbai. Cetyl-trimethyl-
ammonium bromide (CTAB) was purchased from Loba Chemie, India. All other chemicals
were of HPLC grade.

2.1. Experimental design.

An (Central composite design) experimental design with two factors, three levels
(DESIGN EXPERT® software, version 12.0.12.0, Stat-Ease Inc. Minneapolis, MN, USA.) was
adopted optimization of atazanavir cubosomes. The effect of three independent variables,
namely GMO, Pluronic F127and CTAB concentrations, and dependent variables are on
cubosomal % Entrapment efficiency, % CDR, the particle size of ATZ-loaded cubosomes was
evaluated. The analysis of the experimental data was performed by an ANOVA. The Ex-vivo
and in-vitro drug permeation studies were performed with prior approval from the IAEC
committee wide No. No.006/09/AGI/CPCSEA/2019/16.
Desirability was calculated for the selection of optimized formula, which was subjected
to further evaluation. Table 1 shows the independent and dependent variables the actual and
coded levels of variables used in the CCD. GMO (X1), F127(X2), and CTAB (X3) are defined
as the independent variable. In contrast, entrapment efficiency (Y1), %Cumulative drug release
(Y2), and Particle size (Y3) are defined as the dependent variable in this study. The
experimental design included 20 experiments, and 5 center points are listed in Table 2. The
dependent variable (Y) 's relevance to independent variables (X) is evaluated by a second-order
polynomial equation. The model is described as follows:
Y = β0 +β1X1+ β2 X2 + β3X3+ β12X1X2+ β13X1X3 + β23X2X3 + β11X21+ β22X2 2 + β33X2 3
Where Y is the response; X1, X2, and X3 are the independent Factors; β0 is the intercept
coefficient; β1, β2, and β3 are the linear interaction coefficients; β13, β12, and β23 are the
squared effects terms and β11, β22, and β33 are the interaction coefficients. The ANOVA
analysis of data obtained from the experiments is carried out by Design-Expert software (trial
version 12.0.12.0, Stat-Ease, USA).

Table 1. Levels of three independent variables used in Central composite design.

Independent variable Coded symbol Levels
-1 0 +1
GMO (Glyceryl monooleate) (mg) X1 100 175 250
Pluronic F127(mg) X2 10 25 40
CTAB (Cetyltrimethylammonium bromide) (mg) X3 3 6.5 10
Dependent Variables Coded symbol Levels
Entrapment Efficiency (%) Y1 Maximize
Cumulative Drug Release (%) Y2 Maximize
Vesicle Size (nm) Y3 Minimum

2.2. Preparation of ATZ-loaded cubosomal dispersions.

ATZ-loaded cubosomal formulations were designed and developed as per the Nasr
method [22]. Atazanavir was weighed into a glass vial and heated at 40°C until free-flowing.
Aqueous solutions containing different GMO concentrations, Pluronic F-127, and CTAB were
dissolved in the obtained molten mixture by continuous stirring. Deionized water (0.5 mL) was
added to the mixture drop-wise while maintaining a high, stirring speed at room temperature
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to achieve a homogenous state(KLM-307, Elektrocraft Ind.pvt.LTD)at 40°C, amplitude 80%,

pulse cycle 1 for 30min until a milky dispersion was formed. The mixture was equilibrated for
48 h at room temperature. To prepare the Cubosomal dispersion, the obtained gel was dispersed
with the distilled water by vortex at high speed for 3 min. Then the cubosomal dispersions were
sonicated for 15 min using water bath sonicator (CD-4820, Citizen India). Twenty formulae of
different combinations were prepared, as shown in Table 2.

2.3. Determination of particle size, polydispersity index (PDI).

The mean particle size, PDI of ATZ-cubosomes were determined using Zetasizer Nano-
ZS (Malvern Instrument Ltd., Worcestershire, UK) at 25°C, based on photon correlation
spectroscopy and electrophoretic mobility principles. All samples were appropriately diluted
with deionized water before measurements were taken. The cubosomes formulations' zeta
potential were determined using a zeta sizer (Malvern instrument, UK). They were diluted in
the ratio of 1:2500 (v/v) with distilled water.

2.4. Entrapment efficiency.

In order to determine entrapment efficiency (EE %), the total amount of ATZ
incorporated in 1 mL cubosomal dispersion was determined after the addition of 9.0mL
ethanol. The resultant solution was assayed for the total ATZ content by UV spectrophotometer
(Lab India UV-320, Mumbai, India) at 249 nm using ethanol as blank. High-Speed
centrifugation was used to separate free ATZ from cubosomal dispersion [27]. One mL of
freshly prepared ATZ-loaded cubosomal dispersions was diluted to 10 mL with purified water,
and 3 mL of the diluted samples were placed in Amicon Ultra 3000 MWCO centrifuge tubes
(Millipore, USA) and centrifugation at 9000rpm for 45 min at 4◦C. Free ATZ contained in the
filtrate was measured spectrophotometrically at 249 nm [28]. The amount of entrapped ATZ
was calculated by subtracting the determined amount of ATZ in the filtrate from the total
amount of drug incorporated in 1mL cubosomal dispersion. The entrapment efficiency (EE %)
was calculated using the equation:
EP = [(Ct – Cr)/ Ct] * 100
Where,
Ct, concentration of total Atazanavir,
Cr, concentration of free Atazanavir.

2.5. Optimization and validation.

To attain an optimum cubosomal formulation that fulfills the previously decided

constraints on the % entrapment efficiency, % CDR, Particle size, Zeta potential, PDI (Table
2), simultaneous optimization technique via the desirability function was applied using
Design® expert software. The suggested optimized formulation was prepared, and the required
dependent variables were evaluated as previously. To objectively assess the validity of the final
selected models, the 95% two-sided prediction intervals (95% PIs) for the predicted values
were calculated. The mean of the measured values (actual value) for each dependent variable
of the optimized formulation was checked to ascertain if it lied within the 95% PIs or not [29].
Additionally, the actual values were also compared to the predicted values using % prediction
error.
% prediction error = 100 [Ym predicted – Ym actual] / Ym predicted (2).
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2.6. Preparation of gel containing ATZ-loaded cubosomes.

The gel formulation containing ATZ-loaded cubosomes was prepared. Briefly,

cubosomal dispersion of optimized formulation was mixed with carbopol 934 1% and left to
soak overnight. A mixture of absolute ethanol, glycerin, and deionized water was added
portion-wise with continuous stirring until the homogeneous gel is obtained. The final carbopol
934 and ATZ concentrations were 3% and 0.01% w/w, respectively, concerning the gel's final
weight.

Table 2. CCD Experimental design and response for the dependent variables.
GMO Pluronic F CTAB Particle PDI Zeta potential Desirability
Std Run %EE %CDR
(mg) 127 (mg) (mg) size (nm) (mV)
9 1 48.8655 25 6.5 82±1.1 84±4.6 300±4.6 0.54±0.01 -26.81 0.791
14 2 175 25 12.3863 72±2.6 68±6.1 275±8.6 0.52±0.02 -32.45 0.681
3 3 100 40 3 89±2.4 70±5.8 150±7.6 0.48±0.03 -29.43 0.846
15 4 175 25 6.5 93±0.9 90±1.3 168±5.6 0.35±0.01 -24.51 0.879
19 5 175 25 6.5 93±0.8 90±1.1 140±6.4 0.32±0.03 -24.53 0.978
2 6 250 10 3 66±3.4 92±1.2 310±8.4 0.43±0.01 -31.56 0.843
13 7 175 25 0.613725 69±4.6 78±2.6 264±8.2 0.42±0.01 -30.60 0.681
6 8 250 10 10 88±1.7 75±5.7 100±9.1 0.39±0.02 -24.91 0.789
7 9 100 40 10 65±3.6 70±4.9 345±6.4 0.42±0.02 -28.73 0.761
12 10 175 50.2269 6.5 73±1.4 65±6.4 160±5.8 0.41±0.01 -27.62 0.879
18 11 175 25 6.5 93±0.7 90±1.2 150±3.7 0.40±0.03 -25.40 0.941
1 12 100 10 3 67±3.6 70±5.6 430±7.9 0.58±0.02 -26.13 0.861
16 13 175 25 6.5 93±1.2 90±1.0 155±6.4 0.38±0.02 -24.50 0.923
10 14 301.134 25 6.5 79±2.7 80±3.5 100±7.9 0.39±0.01 -28.32 0.845
5 15 100 10 10 86±2.2 87±1.6 230±6.4 0.49±0.03 -28.11 0.645
4 16 250 40 3 72±2.9 85±2.3 120±9.2 0.51±0.02 -29.72 0.789
20 17 175 25 6.5 93±1.2 92±1.1 150±8.7 0.35±0.02 -24.53 0.856
8 18 250 40 10 61±4.6 60±5.4 253±5.6 0.43±0.02 -29.41 0.843
17 19 175 25 6.5 93±0.7 90±0.6 150±4.5 0.45±0.02 -24.50 0.910
11 20 175 -0.226892 6.5 78±1.3 80±2.3 215±8.8 0.48±0.01 -32.41 0.875

2.6.1. Evaluation of gel formulation.

2.6.1.1. pH evaluation.

The pH of the gel was determined using a pH meter (SAB 5000 Lab India), which was
calibrated before every use with standard buffered solutions of pH 4, 7, and 10.

2.6.1.2. Viscosity measurement.

Gel viscosity was determined using the Brookfield viscometer (Brookfield Viscometer,
LVDV-11+Pro) using spindle S06 at a rotation speed of 60 rpm. The gel's measurement
samples were allowed to settle over 30 min at room temperature before the measurements were
taken.

2.6.1.3. Effect of storage.

The effect of storage on the optimized ATZ-loaded cubosomal gel formulation was
implemented by placing freshly prepared samples of the gel at room temperature
(25 °C ± 2 °C) for 3 months. The cubosomal gel was then evaluated for its pH, percent drug
content, and viscosity at different storage stages.

2.7. Physicochemical characterization of optimum cubosomal dispersion.

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The cubosomal formula that fulfilled the implemented Central composite design's
optimum criteria was subjected to further investigation and characterization.

2.7.1. Fourier transform infrared (FTIR).

An appropriate amount of KBr was dried under an infrared lamp, mixed with the
different samples (i.e., pure ATZ, Pluronic F 127, alcohol, Poloxamer 407). Finally, cubosomal
powder was identified using an FTIR spectrometer (Bruker, Alpha). It was made into a plate
at a pressure of 300 kg/cm2. The plate was scanned by an infrared spectrometer at 400–4000
wave-number with a resolution of 2 cm-1 [30].

2.7.2. Differential scanning calorimetry (DSC).

DSC was performed using a thermal analysis system (DSC- 60, Shimadzu, Japan) to
identify possible changes in the physical state of ATZ entrapped in cubosomal dispersion. DSC
was performed on pure ATZ powder, GMO, Pluronic F 127, CTAB, Ethanol, cubosomal
dispersion, and cubosomal gel. The cubosomal samples of about 5 mg were subjected to
heating at a rate of 10 °C/min in an aluminum pan under a nitrogen atmosphere condition. A
similar empty pan was used as the reference [30].

2.7.3. X-ray diffraction (XRD).

X-ray diffraction patterns of the prepared cubosomal dispersion as well as pure ATZ,
Cubosomal dispersion, and cubosomal gel samples were obtained using the X-ray
diffractometer (PHILIPS® X’pert multi-purpose diffractometer) with Cu as tube anode. The
diffractograms were recorded under the following conditions: the voltage 40 kV, the current
30 mA, the steps 0.02°, and the counting rate 1 s/step at room temperature. Data were collected
using a scattering angle (2θ) ranged 4–60°.

2.8. Surface morphology using SEM& TEM.

The study of cubosomal dispersion and cubosomal gel surface morphology was studied
by using scanning electron microscopy. The sample of formulations has first adhered to the
carbon-coated metallic stub. This was sputter-coated with a Platinum coating machine (JFC-
1600 Auto fine coater, JEOL, Tokyo, Japan) and mounted in SEM (JSM-6510LA, JEOL,
Tokyo, Japan) for surface analysis. Imaging was carried out in a high vacuum [23-25].
To determine the morphology of cubosomal dispersion, a transmission electron
microscope (JEOL, Japan), model JEM-2100 equipped with super twin lens, was used. A
droplet of cubosomes dispersion was placed on a carbon-coated copper grid and then stained
with 1% sodium phosphotungstate solution; after that, the excess fluid was removed by an
absorbent filter paper, and finally, the sample was subjected to dry for 15 min at room
temperature for studying the morphology of cubosomes.

2.9. In vitro drug release study.

In-vitro release studies were performed by unjacketed vertical Franz diffusion cells with
a diffusional surface area of 5.96 cm2 and 20 mL of receptor cell volume. Before initiation of
the study, the dialysis membrane was immersed in a buffered solution (pH 7.4). Formulation
equivalent to 5mg of Atazanavir was placed in the donor compartment. The receptor

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compartment consisting of PB pH 7.4 (containing 0.02% w/v of ethanol to retard microbial

growth) was maintained at 37±2°C under constant stirring up-to 24 hrs. The donor chamber
and the sampling port were covered with a lid to prevent evaporation during the study. Aliquots
of 5 mL were withdrawn periodically at different time intervals (0, 5, 10, 15, 30, 1, 2, 3, 4, 5,
6, 7, 8, 12, and 24hrs) replaced with equal volume to maintain constant receptor phase volume.
At the end of the study, the samples were suitably diluted, and the amount of the drug was
determined spectrophotometrically [31-33].

2.10. Ex vivo skin permeation study.

A freshly excised hairless abdominal rat skin was selected and placed between the
compartments of donor and receptor of Franz-type diffusion cells with an effective permeation
area of 2 cm2 and with the stratum corneum facing the donor compartment. The receptor
solution consisted of 200 mL of phosphate buffer pH 7.4 maintained at 37 ± 0.5 °C and
continuously stirred with a magnetic bar at 60 rpm. Then, 1 mL of the cubosomal dispersions
and ATZ aqueous solution was added to the donor compartment. Samples from the receptor
compartment (1 mL) were withdrawn periodically over 24 h and analyzed for drug content
spectrophotometrically at 249 nm. The 1 mL aliquots were substituted by an equal volume of
phosphate buffer pH 7.4 maintained at 37 ± 0.5 °C. At the end of the experiment and in order
to determine the amount of ATZ deposited in the skin, the rat skin was cleaned 5 times with a
cotton cloth soaked in ethanol. The skin was then finely divided and immersed for 6 h in 6 mL
of ethanol under constant stirring at room temperature. Extraction dispersions were centrifuged
at 4000 rpm for 15 min and filtered through a 0.22 μm filter. Diffusion cells free of formula
were also established. Samples collected from permeation of drug-free systems were used as a
blank, and filtrates were studied at 249nm using a spectrophotometer. The slope of the curve
plotted for the cumulative amount of ATZ infused per unit area as a function of time was used
to determine the drug steady-state flux (Jss) [34-36]. The permeability coefficient (kp) of ATZ
through the skin from the investigated cubosomes was calculated as follows:
Kp = Jss/C
Where Jss: steady-state drug flux
C: drug concentration in the donor compartment.

2.11. Statistical analysis.

Statistical analysis (SPSS program version 17 software) was of the in-vitro studies were
performed using one-way analysis of variance (ANOVA), followed by the least significant
difference (LSD) as a post hoc test., was applied using. The mean differences between the
samples were considered significant if P<0.05.

3. Results and Discussion

3.1. Preparation of ATZ-loaded cubosomes nanoparticles.

The ATZ cubosomal dispersion was prepared devoid of organic solvents, making a way
to green synthesis in drug delivery systems based on lipid carriers. Ethanol aqueous solution
was used as a steric barrier due to its absorption to the surface as an emulsion droplet,
preventing emulsion droplets' aggregation and stabilizing. Ethanol forms small particles with
uniform distribution, which can be easily dispersed in an aqueous medium. Stabilizers were

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used in cubosomal dispersion for modifying the surface properties and stability. The
stabilization of ATZ cubosomal dispersion by Pluronic F 127 formed due to the adsorption of
GMO, CTAB moieties into the outer surface of the cubosomal dispersion, which intern resulted
in the inverted-type self-assembled lipid nanostructure from the surrounding aqueous medium,
whereas the GMO copolymer’s hydrophilic moieties dangled in the water. Increasing the
concentration of Pluronic F 127 in the cubosomes formulations allowed smaller droplets to
form by increasing the interfacial stability of cubosomal nanoparticles.

3.2. Determination of particles size, PDI and zeta potential.

The particle size analysis for cubosomal dispersion loaded with ATZ exhibited particle
size values within the nano range (100±7.9–345±6.4 nm). As indicated in Table 1, the particle
size of cubosomes is indirectly proportional to the increase in Pluronic F 127 concentration.
Upon reducing Pluronic F 127 concentration, larger size cubosomes nanoparticles were formed
due to the condensed interfacial stability and an insufficient amount of the surfactant, leading
to aggregation of nanoparticles. The particle size distribution of the cubosomes nanoparticles
specified by the polydispersity index values ranged from 0.32 ± 0.03 to 0.58 ± 0.02, which was
an acceptable range. The higher values of zeta potential deliver sufficient electric repulsion,
which in turn prevents particle aggregation. The results of the potential zeta study show that
ATZ-loaded cubosomes nanoparticles carried a negative charge with mean values of –24.51 to
–32.45. This might be due to the presence of the GMO, Pluronic F 127. Moreover, the negative
surface charge may be due to the CTAB hydroxyl group. In general, Pluronic F 127 adding to
the cubosomal dispersion medium resulted in negative charge values of cubosomes due to the
interaction between Pluronic F 127 hydroxyl ions with the aqueous medium. Particle charge is
an important parameter suggested by Kohli and Alpar as the only negatively charged particles
are able to infuse through the skin due to the channels formed by the repulsive forces between
negatively charged skin lipids and particles.

3.3. Cubosomes nanoparticles encapsulation efficiency (EE %).

The formulated cubosomes nanoparticles were mainly composed of the lipophilic

material GMO and both Pluronic F 127and CTAB surrounding the nanoparticles. It is estimated
that cubosomes nanoparticles can carry and deliver lipophilic drugs that can dissolve in lipid
nanostructure. Different formulations were compared to assess the amount of drug incorporated
in the nanoparticles. It was found that due to the strong affinity between ATZ and the GMO in
the cubosomes nanoparticles, it was ‘grabbed’ in the liquid crystal structure. Thus the
encapsulation efficiency values were ranged from 61±4.6 to 93 ±0.8%. Such high drug
encapsulation efficiency is desirable to produce a therapeutic effect with less volume.

3.4. Selection of the optimum formula.

The analysis of formulations’ variables showed that there is a strong relationship

between GMO/Pluronic F 127 ratio and CTAB concentration and EE %, particle size
distribution, and %CDR. Three-dimensional response surface diagrams were plotted to
emphasize the effects of the interaction of the Pluronic F 127, CTAB, and GMO concentrations
on particle size, EE %, and % CDR, respectively.
The independent variables and their interactions on dependent responses were studied
using RSM and represented graphically by 3D surface plots. The effect of the amount of GMO,
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amount of Pluronic F127, and amount of CTAB on % EE, % CDR and vesicle size, are
represented in Figure 1. When % EE (Y1) was indicated as the response, a good correlation
was shown between observed and predicted values as revealed by R2 of 0.9092(Table 3 and 4).
Thus Y1 was significantly influenced by amounts of A, B, C and their interactive term (ABC)
and polynomial model of lipid concentration A2 with a p<0.0001. The magnitude of the positive
coefficient (328.21) of the term suggests that elevated levels of GMO, Pluronic F127, and
CTAB concentrations in the formulation could increase the %EE drastically. The size of
Cubosomes depends on GMO concentration can be explained in terms of the tendency of GMO
to coalesce at high GMO concentration. Increasing GMO content in the cubosomal formulation
leading to higher surface tension and thus increased the %EE. Figure 1(A) and (B) show the
response surface model for %EE in response to the investigated factors. The independent
variables have a positive influence on %EE. Figure 1(C) and (D) showed a linear relationship
between the amount of GMO and %CDR of Cubosomal dispersion. Quantitative estimation of
the significant models indicated that Pluronic F127 concentration had the prime influence on
the % CDR for its large positive coefficient (+7.59), suggesting that increasing the amount of
Pluronic F127 increased the CDR and the amount of CTAB has little effect on the % CDR in
the formulation (Equation (1-3)). A high concentration of Pluronic F127 results in increasing
the %CDR. Enhanced drug release may be due to micelles' formation at increased Pluronic
F127 concentration, resulting in sufficient spare space to accommodate more drugs. This
tendency could be attributed to the high lipophilicity and limited water solubility of ATZ. From
Figure 1(E) and (F), the effect of formulation type and process variables on Particle size can
be evaluated. When the amounts of GMO and Pluronic F127 were altered from lower to higher
levels, significant vesicle size changes were noticed, but at low levels of Pluronic F127 amount
alone, an increasing marginal trend in vesicle size was observed. This result implied that
Pluronic F127 concentration showed a promising effect on the vesicle size, confirmed by a
statistical ANOVA result. Point desirability suggested by Design-Expert® software actual and
predicted values for entrapment efficiency and percent cumulative drug release and particle
size (Figure 2).

Table 3. Analysis of variance in RSM and regression analysis predicted a second-order polynomial model for
entrapment efficiency.
Y1 (% EE) Y2 (Cumulative % DR) Y3 (Vesicle size)
Model R² Adjusted R² Predicted R² R² Adjusted R² Predicted R² R² Adjusted R² Predicted R²
Linear 0.0489 0.1294 0.4837 0.2132 0.0657 0.2207 0.2836 0.1493 0.2425
2FI 0.3985 0.1209 0.5600 0.4662 0.2198 0.2586 0.7463 0.6292 0.2228
Quadratic 0.9882 0.9775 0.9092 0.9729 0.9485 0.8130 0.9780 0.9583 0.8470
p value ≤0.0001 ≤0.0001 ≤0.0001

Table 4. ANOVA analysis of dependent parameters.

Parameter Data Degree of Sum of Mean of squares F Value p-Value
Source Freedom squares
% EE Model 9 2302.44 255.83 92.84 <0.0001
Residual 10 27.56 2.76
Lack of fit 5 24.72 4.94 0.0164
Pure error 5 2.83 0.5667
% Cumulative DR Model 9 1985.62 220.62 39.87 <0.0001
Residual 10 55.33 5.53
Lack of fit 5 9.30 5.26 0.0462
Pure error 5 1.77
Vesicle size Model 9 1496.05 16623.04 49.47 <0.0001
Residual 10 3360.40 336.04
Lack of fit 5 2939.57 587.91 6.99 0.0262
Pure error 5 420.83 84.17

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Figure 1. Contour plots and response surface plots showing the interactive effects Point desirability as
suggested by Design-Expert® software Effect of the interaction of the Pluronic F 127, GMO and CTAB
concentrations on A & B entrapment efficiency %, C & D %CDR and E & F particle size distribution.

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Figure 2. Point predictions (color points by the value of A. entrapment efficiency, B. % CDR, C. particle size
and D. total overly plot of design) as suggested by Design-Expert® software actual and predicted values

3.5. Fourier transform infrared (FT-IR) studies.

FTIR spectra of ATZ (Pure API), GMO, Pluronic F127, ethanol, cubosomal dispersion,
and cubosomal gel are shown in Figure 3.

Figure 3. FTIR Spectral Studies.

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3.6. DSC.

DSC was carried out to determine the crystalline properties of the loaded ATZ in
cubosomal nanoparticles (F5) compared with pure ATZ, CTAB, Pluronic F 127, and GMO.
The DSC thermogram of pure ATZ showed a characteristic peak at 163.48°C, correspondings
to its melting point Figure 4.

Figure 4. DSC thermogram of A. ATZ, B. GMO, C. Pluronic F127, D. CTAB, E. cubosomal dispersion and G.
cubosomal gel.

3.7. X-ray diffraction studies.

In order to further confirm the physical state of ATZ, X-ray diffraction patterns of the
prepared ATZ-loaded cubosomes, as well as the pure drug powder samples, were obtained
(Figure 5). The diffractogram of the pure ATZ clearly showed strong characteristic peaks
within the range of 2Θ 10–30°; meanwhile, those characteristic peaks disappeared in ATZ
loaded cubosomal nanoparticles (F5), indicating that the drug is either molecularly dispersed
in cubosomes or possibly transformed into an amorphous form.

Figure 5. XRD diffractograms of A. Pure ATZ, B. cubosomal dispersion, and C. cubosomal gel.

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3.8. Cubosomes nanoparticles morphology.

The cubosomal dispersion was studied for surface morphology at 30.0 kV

magnification using SEM. The morphology of nanoparticles was found to be nearly spherical
in shape, exhibited polydispersity, and possessed a smooth surface shown in Figures 6A and
B.
Figure 6 C and D shows TEM images taken for optimized cubosomal dispersion (F5)
and Cubosomal gel formulations. Cubic shapes of particles with zero mean curvature.
However, a small population of hexagonal vesicles was observed. Particle sizes were in the
nano-range and were well disconnected from each other. Cubosomes nanoparticles F5 showed
smaller particles size. These findings are verified before by particle size and can be elucidated
by the presence of a larger amount of Pluronic F 127 adsorbed on the cubosomes surface, which
acts as a coating layer for stabilizing the surface area of nanoparticles.

Figure 6. SEM micrograph of A. cubosomal dispersion b. cubosomal gel& TEM micrograph of C& D.
cubosomal dispersion cubosomal gel.

3.9. Ex vivo permeation study.

Animal skin models have been successfully utilized as alternatives for human skin.
Accordingly, the rat skin is considered a successful ex vivo model for studying the different
drug carrier permeation systems. Figure 7 showed the cumulative amount of ATZ permeated
through a unit area of abdominal rat skin from formulae F5 and cubosomal gel compared with
aqueous ATZ solution. The calculated permeation parameters are illustrated. Evidently, no lag
phase was identified. ATZ was detected in the receptor compartment after the first hour,
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indicating the rapid drug release and its permeation across the skin. Similar results were
previously reported in the literature.
All tested preparations showed relatively low amounts of ATZ released within the first
1.5 h, but the amount of ATZ released was significantly (P < 0.05) higher for F2 (1409.29 ±
150 μg/cm2) after 24 h compared with those of cubosomal dispersion (1100 ± 68.17 μg/cm2)
and ATZ aqueous solution (890.45 ± 324.61 μg/cm2). It was obvious that the amount of ATZ
deposited in the skin of F5 and cubosomal dispersion was 1.54 and 1.06 times greater than that
of ATZ aqueous solution, respectively. This is favored when ATZ deposition in the skin is
needed in certain dermatological conditions such as atopic dermatitis, actinic keratosis, and
psoriasis.
The amount of ATZ deposited in the skin of cubosomal dispersion is relatively lower
than that of F5. This may be attributed to the high zeta potential value of F5 because of the
higher amount of CTAB used in F5 than that in remaining formulations. It was previously
reported that the positively charged nano-emulsions containing phytosphingosine were found
to be more effective in terms of skin diffusion of fludrocortisone acetate and flumethasone
pivalate through porcine skin than the negatively charged ones. The permeation parameters are
illustrated. The transdermal flux (Jss) was determined from the slope of a Cartesian plot of the
collective amount of drug present in receptor compartment versus time; meanwhile, ER2
enhancement ratio is the ratio of the amount of drug deposited from formulation to drug
solution. ER3 enhancement ratio is the ratio of transdermal flux from formulation to drug
solution. The steady-state drug flux (Jss) values for ATZ aqueous solution, cubosomal
dispersion, and F5, were nearly 28.64±5.1, 42.37±2.1, and 56.51±4.3 μg/cm2.hr, respectively;
meanwhile, the permeability coefficient (kp) values for ATZ aqueous solution, cubosomal
dispersion, and F5, were 0.005±0.002, 0.009±0.001, and 0.013±0.002 cm/h. The high values
of Jss and kp for F5 could be due to the higher amounts of GMO and Pluronic F127 in F5
compared with cubosomal dispersion and ATZ solution as it was previously reported that both
GMO and Pluronic F127 are penetration enhancers.

Figure 7. Ex vivo permeation studies of ATZ-loaded cubosomal Suspension F5, Cubosomal Gel compared with
ATZ aqueous solution through excised rat skin.

3.9.1. Characterization of cubosomal ATZ gel.

The ATZ cubosomal gel drug content was estimated by dissolving 1 gram of gel in 8
mL ethyl alcohol. The volume was made up to 10 mL. Later 1 mL of solution was diluted with
ethyl alcohol and measured spectrophotometrically at λmax 249 nm. The drug content was
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99.18 ± 0.63% (Figure 8), and the measured pH of the gel was 6.02 ± 0.05, which lies within
the acceptable pH range of the skin. The cubosomal ATZ gel viscosity was 14,635 ± 296 (cP),
which complies with the optimum gel viscosity range 13,000–16,000 (cP) Table 5.

3.9.2. Effect of storage.

After 3 months of storage of the freshly prepared ATZ-loaded cubosomal gel

formulation at room temperature (25 °C ± 2 °C), the drug content, pH, and gel viscosity were
99.61 ± 0.28%, 5.95 ± 0.03, and 14,620 ± 125 cP, respectively. These results were found to be
statistically insignificant (P > 0.05, paired t-test) compared with the same results obtained
before storage, indicating the stability of ATZ-loaded cubosomal gel when stored at
25 °C ± 2 °C.

3.10. In vivo absorption study.

The parameters of LC-MS/MS method were validated according to ICH guidelines.

The method showed high accuracy and precision with linear regression in the range of analysis.
The mean (±SD, n = 6) plasma ATZ concentration-time profiles following administration of
single doses (0.03 mg/kg) of oral ATZ solution and topical ATZ-loaded cubosomal gel to fasted
rats were shown in Figure 7. It was obvious from ATZ plasma concentration-time profile that
ATZ shows two absorption peaks for both oral solution and transdermal cubosomal gel. The
second absorption peak is attributed to entero-hepatic circulation. This finding is in good
agreement with previously reported results. The bioavailability parameters (Cmax, Tmax, and
AUC0–48) were calculated from the individual ATZ plasma concentration-time curves. The
mean values ± SD are presented.
The obtained Cmax after oral administration was 0.31 ±0.17 ng/mL at Tmax 3.15 ±
1.82 h, which indicated that ATZ is rapidly absorbed when given orally. However, a
significantly higher value of Cmax (0.86 ± 0.23 ng/mL at 13.80 ± 4.60 h) was observed after
transdermal application of ATZ cubosomal gel (Table 6). The significantly delayed higher
Cmax values of ATZ cubosomal gel indicate a slow release of ATZ from cubosomal dispersion.
AUC0–48 of oral COL solution was 2.45 ± 1.2 ng.hr/mL, while AUC0–48 of ATZ cubosomal
gel was 11.85 ± 5.92 ng.hr/ mL, and the calculated relative bioavailability of ATZ cubosomal
gel, based on AUC0–48, is 4.8367 times compared with ATZ oral solution.

Table 5. Permeation parameters of optimized formulation compared with a solution.

Formula Jss μg/cm2.hr Permeability coefficient (cm/h) ER2 ER3
ATZ Solution 28.64±5.1 0.005±0.002 0.27 1.90
Cubosomal Dispersion 42.37±2.1 0.009±0.001 1.55 1.25
F5 46.51±4.3 0.013±0.002

Table 6. Bioavailability parameters of optimized formulation compared with a solution.

Formula ATZ oral Solution Cubosomal Dispersion Significance
Cmax (ng/ml) 0.31±0.17 0.86±0.23 0.031
Tmax (hr) 3.15±1.82 13.80±4060 0.042
AUC0-48 (ng.hr/mL) 2.45±1.2 11.85±5.92 0.0245

The exact mechanism for the enhanced skin penetration from cubosomal nanoparticles
is still under investigation. However, the statistically significant increase (P < 0.05) in the
relative bioavailability of the transdermal cubosomal dispersions may be due to its structure
and its similarity to the skin, which provides high flexibility in transdermal drug delivery for
both hydrophilic and lipophilic drugs. Another factor is the penetration enhancer effect of
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GMO, which acts by modifying the intercellular ordered structure of lipid bilayer in the stratum
corneum and increasing its fluidity.

Figure 8. ATZ plasma concentration-time curves in rats after administering a single dose (0.02 mg/kg) of oral
ATZ solution and topical ATZ cubosomal gel. Mean (±SD, n = 6).

Moreover, the presence of Pluronic F127 in cubosomes allows the drug to penetrate
deeply into the stratum corneum, change in the lipid arrangement, improving fluidity, and
finally enhancing transdermal drug permeation. Also, the higher skin permeability of
cubosomes may be attributed to the bio-adhesive characteristic and permeation enhancement
of their building units. The previous results agreed with several literature works that have
reported the potential of cubosomes in increasing the transdermal absorption of different drugs
such as etodolac, indomethacin, vitamin K, triclosan, and vaccine derived from peptide.
Contrary to the earlier reports, cubosomes may improvise the penetration of drugs, and GMO-
based cubosomes loaded with capsaicin decreased the skin absorption compared to that of
conventional creams.

4. Conclusions

Transdermal cubosomal gel was developed for enhancing the oral bioavailability of
ATZ drug. It was apparent that cubosomes containing ATZ are potential in delivering the ATZ
through the transdermal route for overcoming the side effects of oral administration. Moreover,
cubosomes containing ATZ duplicated the percent of ATZ deposited in the skin, which is
favored when deposition of ATZ in the skin is needed. But, because of the large variations in
its bioavailability, it is recommended to use many volunteers to obtain more accurate
pharmacokinetic parameters.

Funding

This research received no external funding.

Acknowledgments

The authors would like to thank Hetero Drugs. Pvt Ltd. Hyderabad for their enormous support
and providing the API as a gift sample. The authors would like to thank Anurag University
Chairman and Management for encouraging Research and Development. Vinod Kumar Yata
would like to thank the Department of Biotechnology, Government of India, for providing
financial support from “DBT-RA Program in Biotechnology & Life Sciences”.

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Conflicts of Interest

The authors declare no conflict of interest.

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