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Background: The aim of the study was to investigate ethyl cellulose microsponges
as topical carriers for the controlled release and cutaneous drug deposition of
eberconazole nitrate (EB). Materials & method: EB microsponges were prepared
using the quasiemulsion solvent diffusion method. The effect of formulation
variables (drug:polymer ratio, internal phase volume and amount of emulsifier) and
process variables (stirring time and stirring speed) on the physical characteristics of
microsponges were investigated. The optimized microsponges were dispersed into a
hydrogel and evaluated. Results & discussion: Spherical and porous EB microsponge
particles were obtained. The optimized microsponges possessed particle size, drug
content and entrapment efficiency of 24.5 m, 43.31% and 91.44%, respectively.
Microsponge-loaded gels demonstrated controlled release, nonirritancy to rat skin
and antifungal activity. An in vivo skin deposition study demonstrated fourfold
higher retention in the stratum corneum layer as compared with commercial cream.
Conclusion: Developed ethyl cellulose microsponges could be potential pharmaceutical
topical carriers of EB in antifungal therapy.
Chellampillai Bothiraja*,1,
Amol D Gholap2,
Karimunnisa S Shaikh3
&Atmaram P Pawar1
1
Department of Pharmaceutics, Bharati
Vidyapeeth University, Poona College
of Pharmacy, Erandwane, Pune 411038,
Maharashtra, India
2
Department of Pharmaceutics,
Sharadchandra Pawar College of
Pharmacy, University of Pune, Otur, Pune
412409, Maharashtra, India
3
Department of Pharmaceutics, Modern
College of Pharmacy, Nigdi, Pune
411044, Maharashtra, India
*Author for correspondence:
Tel.: +91 20 25437237
Fax: +91 20 25439383
pounbothi@yahoo.com
part of
ISSN 2041-5990
781
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EB microsponges were prepared by the quasiemulsion solvent diffusion method [9] . The organic internal
phase containing EB and ethyl cellulose (60 mg) in 5
ml dichloromethane was gradually added into 30 ml
distilled water (external phase), which contained polyvinyl alcohol (40 mg) as the emulsifying agent. The
mixture was stirred on a magnetic stirrer at 2000 rpm
for 120 min at 35C in order to remove dichloromethane. The formed microsponges were filtered through
Whatman filter paper no. 41 (Whatman, UK),
washed with distilled water, dried at 40C for 12 h and
weighed. The production yield (PY) was calculated
using Equation 1:
PY (%) = Practical mass (miscrosponges)/
Theoretical mass (polymer + drug) 100
The free EB was estimated by measuring the absorbance of the filtrate after suitable dilutions with phosphate buffer (pH 5.4) at 237 nm using an ultraviolet
(UV) spectrophotometer (V-630, Jasco, Japan). The
drug concentration was calculated using the calibration curve equation y = 0.013x + 0.019. Here, y is
the measured absorbance and x is the concentration
ing/ml.
Optimization of formulation parameters
&process variables
Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy
Characterization of EB microsponges
Drug content & entrapment efficiency
The weighed samples of EB microsponges were dissolved in methanol under ultrasonication for 1 h
at 30C, The samples were filtered using a 0.2-m
membrane filter and absorbance were read at 237 nm
using an UV double-beam spectrophotometer (V-630;
Jasco) after suitable dilutions with phosphate buffer (pH 5.4) were obtained. The data presented are
mean values of three independent samples produced
under identical production conditions. The drug content and entrapment efficiency were calculated using
Equations 2 & 3 :
Actual drug content (%) =
Mact/Mms 100
Entrapment efficiency (%) =
Mact/Mthe 100
Research Article
Surface topography was studied by using a scanning electron microscope (SEM). Microsponges were
mounted on double-faced adhesive tape and coated
with a thin goldpalladium layer with a sputter-coated
unit (VG-Microtech, UK) and analyzed by scanning
electron microscopy (VG-Microtech, Uckfield, UK)
operated at a 10-kV acceleration voltage.
Differential scanning calorimetry
In vitro release studies were performed using an artificial cellophane membrane (Membra-Cel MD 34-14;
cut-off: 12 kDa; Viskase Co, MS, USA). For this
experiment, a vertical Franz diffusion cell with a surface area of 2.54 cm2 and a reservoir capacity of 32
ml was used. The artificial membrane was securely
placed between the two halves of the diffusion cell.
The receptor compartment contained phosphate buffer (pH 5.4), and its temperature maintained at 37
0.5C and stirred continuously using a magnetic stirrer. A predetermined amount of EBMG (2.7 mg) containing 1 mg of EB was placed on the donor side. A
total of 2 ml of the sample was withdrawn from the
receptor compartment at definite time intervals and
replaced with an equal volume of fresh receptor fluid.
The aliquots were suitably diluted with the receptor
medium and analyzed by an UV spectrophotometer.
Measurements were performed in triplicate and their
means were reported. The release kinetics of EBMG
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Average scores =
Erythema grade* + Edema grade*/Number of animals
*at 1,3,5 and 7 day
Variable factor = types of skin time of reading
PII = Average scores variable factors
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In vitro antifungal testing was determined by the Sabouraud dextrose agar disk diffusion test employing
the cupplate technique using a previously sterilized
Petri dish [21] . EBMG (1 mg/ml) and EBCC (1 mg/
ml) formulations and pure eberconazole as a standard
Table 1. Skin irritation score scale.
Grading
No reaction
++
+++
Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy
Research Article
Drug:polymer
ratio (by weight)
Production
yield (%)
Theoretical drug
content (%)
Actual drug
content (%)
Entrapment
efficiency (%)
Mean
particle size
(m)
EBM1
0.5:1
68.62 1.34
23.07
18.42 1.75
79.84 1.02
39.4 2.2
EBM2
1:1
75.31 1.52
37.49
31.38 1.35
83.70 1.61
31.6 2.8
EBM3
1.5:1
84.26 0.17
47.36
43.31 1.42
91.44 1.25
24.5 2.0
EBM4
3:1
85.31 0.85
64.28
55.72 1.46
86.68 1.73
36.3 3.2
EBM5
5:1
85.89 1.01
75.00
66.43 1.86
88.90 1.28
43.8 3.7
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100
Entrapment efficiency (%)
45
40
35
30
25
20
90
80
70
60
50
40
60
90
120
150
60
90
120
150
Figure 1. Effect of stirring time and stirring speed on microsponge particle size and encapsulation efficiency.
(A)Effect of stirring time on particle size with respect to stirring rate: EB microsponges 1000 rpm (diamonds),
1500 rpm (triangles), 2000 rpm (crosses) and 2500 rpm (squares). (B) Effect of stirring speed on encapsulation
efficiency with respect to stirring speed: 1000 rpm (diamonds), 1500 rpm (squares), 2000 rpm (triangles) and
2500rpm (crosses). All data are means standard deviation (n = 3).
EB: Eberconazole nitrate.
The mean particle size (Yps) and entrapment efficiency (Y EE ) were in the ranges of 9.744.4 m and
80.6296.01%, respectively. The multiple regression
analysis of the mean particle size and entrapment efficiency of the factorial batches revealed a good fit (r2 =
0.9985 for particle size and r2 = 0.9940 for entrapment
efficiency), suggesting strong influences of the selected
variables.
Comparison of the coefficients of the terms of
Equation 8 & 9 suggested a predominant negative
influence of solvent volume and positive influence
of emulsifier concentration on the particle size and
entrapment efficiency. The effect of solvent volume on
the microsponges morphology was also investigated.
Low solvent volume (batches B1B3) yielded uniform
spherical microsponge particles, yet these were less
porous, due to poor diffusion of complete solvent from
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Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy
Research Article
Table 3. Optimization of solvent volume and emulsifier concentration by 32 factorial design for the
preparation of eberconazole nitrate microsponges.
Batch
number
Coded levels
(X1, X 2)
Solvent
(ml) (X 2)
PVA
(mg) (X 2)
B1
-1, -1
30
30.7 1.2
93.04 1.52
B2
-1, 0
60
37.2 1.5
96.01 1.86
B3
-1, 1
90
44.4 2.1
98.34 1.25
B4
0, -1
30
20.1 1.0
88.17 1.23
B5
0, 0
60
24.5 1.3
91.44 1.76
B6
0, 1
90
31.3 1.5
92.32 1.45
B7
1, -1
30
9.7 0.9
80.62 1.92
B8
1, 0
60
13.1 0.7
83.62 1.75
B9
1, 1
90
18.3 1.3
85.31 1.35
phosphate buffer (pH 5.4). Being a topical formulation, the release medium was adjusted to the pH of the
skin (i.e., pH 5.4). The in vitro release profiles of EB
from different formulations are shown in Figure 4A,
indicating that EBCC (1% w/w) and conventional
EBG released the drug within 3 and 8 h, respectively.
However, EB release from EBMG showed a biphasic
pattern, with an initial burst release (24%) within 1
h followed by a sustained release at up to 12 h with
74% release of drug. In order to determine the mechanism of drug release from these formulations, different
kinetics models (zero-order release, first-order release
and the Higuchi equation) were employed. The results
showed that the release kinetics from gel formulations
best fitted (r2 : 0.9870.997) the Higuchi kinetic model
(Table 5) .
Ex vivo diffusion study
The ex vivo diffusion study was performed for conventional EBG, EBMG and EBCC formulations in order
to provide better comparisons between permeation
profiles through rat skin. The cumulative amount of
drug permeated per unit skin surface area was plotted against time (Figure 4B) . The EBMG exhibited a
burst effect within 2 h (98.3 7.0 g/cm2) due to the
presence of nonencapsulated drug closer to the surface
or on the surface of the microsponges, which does
not produce irritation to the skin. After that, a linear
relationship existed for up to 12 h due to the porous
nature of the microsponges providing the channel for
drug release [22] . The slopes (flux) of the linear portion
of the permeation profiles were calculated. The flux
represented the rate of permeation or flux of EB from
different formulations (Table 5) . Two- and three-fold
decreases in flux were observed for EBMG as compared
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Figure 2. Scanning electron microscopy of microsponges. (A & B) batch B3, (C & D) batch B9 and (E & F) batch B5.
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Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy
Research Article
Table 4. Validated output of the 32 factorial analysis of all eberconazole nitrate microsponge
formulation.
Responses
R2
Significance factors
Particle size
0.873
0.816
0.678
9.572
Entrapment efficiency
0.643
0.524
0.227
5.358
B: Solvent volume.
(i)
Intensity
exo
mW
(i)
(ii)
(iii)
(iv)
60
0
120
4
180
240
(ii)
280 C
10 12 14 16 18 20 22 24
Time (min)
10
20
30
40
50
Figure 3. Differential scanning calorimetry and powder x-ray diffraction of microsponges. (A) DSC thermograms
of (i) eberconazole nitrate, (ii) ethyl cellulose, (iii) polyvinyl alcohol and (iv) eberconazole microsponge batch B5.
(B) Powder x-ray diffraction patterns of (i) eberconazole nitrate and (ii) eberconazole microsponge batch B5.
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100
EBG
EBMG
400
Q (g/cm2)
80
Drug release (%)
500
60
40
EBCC
300
200
EBG
20
100
EBMG
EBCC
0
0
8
6
Time (h)
10
12
6
8
Time (h)
10
12
Figure 4. In vitro release and ex vivo diffusion profiles of microsponge. (A) In vitro release and (B) ex vivo
diffusion profiles of eberconazole from EBG, EBMG and EBCC. All data are means standard deviation (n = 3).
EBCC: Eberconazole commercial cream; EBG: Eberconazole-loaded gel; EBMG: Eberconazole microsponge-loaded
gel; Q: Cumulative amount of drug permeated per unit surface area.
Table 5. In vitro release kinetics in cellophane membrane and permeation characterization in rat skin of eberconazole
nitrate from different formulations.
Formulation
code
Cellophane membrane
Rat skin
Flux
(g/cm2/h)
Intercept
(g/cm2)
r2
Q12 h
(g/cm2)
EBG
0.901 0.013
0.476 0.025
0.987 0.001
21.3 2.3
102.9 2.9
0.971 0.013
340.1 8.3
EBMG
0.932 0.018
0.459 0.032
0.997 0.007
11.5 1.7
72.2 2.1
EBCC
0.744 0.042
0.528 0.033
0.815 0.014
33.6 2.6
133.7 3.4
0.862 0.011
498.2 7.4
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Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy
Research Article
Control
EBMG
EBCC
Figure 5. Skin irritation and in vitro antifungal study. (A) Skin irritation study of eberconazole from EBMG and EBCC. (B) In vitro
antifungal activity of G1 (eberconazole nitrate), G2 (EBMG) and G3 (EBCC).
EBCC: Eberconazole commercial cream; EBMG: Eberconazole microsponge-loaded gel.
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Executive summary
Preparation & evaluation of eberconazole nitrate-loaded microsponge gels
Eberconazole nitrate-loaded ethyl cellulose microsponges were prepared using the quasiemulsion solvent
diffusion method.
The effects of formulation (drug:polymer ratio, internal phase volume and amount of emulsifier) and process
variables (stirring time and stirring speed) on the physical characteristics of microsponges were investigated.
Optimized microsponges were dispersed into a hydrogel and evaluated for their performance.
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Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy
Research Article
carrier for EB in topical fungal therapy. Further clinical studies are planned to establish the efficacy and
safety of these formulation on human skin.
Future perspective
The progress in polymer technology has led to an
increased curiosity in the use of sensitive drug molecules
for therapy. The sensitive drug molecules that are primarily used to treat fungal infections have poor physiochemical properties. Delivery of these molecules to the
active site and the maintainence of structural integrity
are challenging due to the tough conditions provided
by the skin (e.g., rashes or other serious side effects can
occur when more active ingredients penetrate into the
skin). However, recent advances in polymeric microsponge-based drug-delivery systems can be explored in
order to provide maximum efficacy, reduced irritancy,
extended product stability, enhanced formulation flexibility, increased elegance and improved esthetic properties. Furthermore, our studies indicate that microsponge systems have great potential for the topical
delivery of antifungal drugs. In order to fully explore
References
Papers of special note have been highlighted as:
of interest; of considerable interest.
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