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Research Article

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Therapeutic
Delivery

Investigation of ethyl cellulose


microsponge gel for topical delivery of
eberconazole nitrate for fungal therapy

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.

Topical agents are mainstays in both cosmetics


and the treatment of dermatological disorders.
Conventional dermatological products provide active ingredients in relatively high concentrations, but for a short duration. This may
lead to a cycle of short-term overmedication
followed by long-term undermedication [1] .
Rashes or other serious side effects can occur
when a more active ingredient penetrates into
the skin [2] . Various controlled drug-delivery
systems, such as microcapsules, microspheres,
nanoemulsion, liposomes and niosomes, have
been investigated in order to maximize the
duration of active ingredients being present
either on the epidermis or within skin layers,
while minimizing their transdermal penetration into the body [36] . However, the release
rate of active drugs from microcapsules cannot be controlled once the capsule wall is
ruptured. Similarly, liposomes are relatively
expensive, difficult to manufacture and have
a low holding capacity of the active drug [7] .
One of the novel techniques used to control the release of active ingredients from

10.4155/TDE.14.43 2014 Future Science Ltd

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

topical formulations is polymeric microsponge-based drug delivery [8] . This system


provides maximum efficacy, reduced irritancy, extended product stability, enhanced
formulation flexibility, increased elegance
and better esthetic properties. Microsponges
are polymeric, porous and tiny, sponge-like
spherical particles. This delivery system
contain a huge number of interconnecting
voids within a noncollapsible structure that
imparts a large porous surface. It can adsorb
or entrap a wide range of pharmaceutical
active ingredients and can be formulated into
gels, creams, liquids and powders [9] . Being
relatively large in size (5300 m), upon
topical application, microsponges do not pass
through stratum corneum (SC) and remain
on the skin surface. The porous nature of
microsponges favors controlled release of
the encapsulated drug, leading to minimal
accumulation of the drug in the epidermis
and dermis [10] . Recently, microsponges have
been developed for benzoyl peroxide, retinoid and 5-fluorouracil that provided maxi-

Ther. Deliv. (2014) 5(7), 781794

part of

ISSN 2041-5990

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Research Article Bothiraja, Gholap, Shaikh & Pawar


mal accumulation in the dermis, minimal permeation
across the skin and minimal irritation to the skin without sacrificing the efficacy of drug [9,11,12] . The success
of microsponge technology is reflected in the successful approval of Retin-A Micro (Janssen Ortho LLC,
Manati, Puerto Rico; 0.1 or 0.04% tretinoin) and
Carac (Dermik Laboratories, Canada) (0.5% 5-flurouracil) for the treatment of acne and actinic keratoses,
respectively, by the US FDA.
Eberconazole nitrate (EB) is an imidazole derivative that is widely administered topically for the treatment of fungal infections. It is known to inhibit the
synthesis of ergosterol, an essential component of the
fungal cytoplasmic membrane, leading to structural
and functional changes. It is favored in the management of inflamed dermatophytic infections as it exhibits potent anti-inflammatory effects [13,14] . It is also
effective against triazole- and fluconazole-resistant
fungal infections [15] . EB is available as a tablet and
3% ointment. Topical infections showed poor response
to the oral EB tablet due to poor oral bioavailability
(30%), which is attributable to its high lipophilicity
and high distribution (50%) into the cerebrospinal
fluid. The EB 3% ointment applied three-times daily
has demonstrable activity. However, topical ointment
preparations are less acceptable to patients due to their
high viscosity, leading to inappropriate application to
skin lesions. Patients may report garment soiling from
greasy residues. Furthermore, Phase II and III clinical studies revealed that multiple applications of 1%
cream to human skin resulted in pruritis, local cutaneous irritation, skin dryness, erythema and reddening of
skin. This might be due to excessive accumulation in
the epidermis and dermis of the lipophilic drug [16] . In
addition, the systemically absorbed fraction of the dose
is excreted in the urine [17] . Thus, for effective topical antifungal therapy, the cutaneous availability of EB
needs to be modulated; a need that can be met by a
suitable drug-delivery system. To date, the approach of
controlled topical delivery of EB has not been investigated by researchers to the best of authors knowledge.
Thus, the aim of the present investigation was to design
ethyl cellulose microsponges as a novel carrier for the
controlled topical delivery of EB. This novel carrier
is expected to prevent the excessive accumulation of
the drug in the skin, control its side effects, improve
its efficacy and decrease the frequency of application
and the systemic absorption. The work undertaken
included the preparation, optimization and evaluation of EB microsponges. A 32 factorial design assisted
in the statistical optimization. The optimized microsponges were dispersed into a hydrogel and evaluated
for their performance.

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Ther. Deliv. (2014) 5(7)

Materials & methods


EB was purchased from Sigma-Aldrich Chemical
Private Ltd (India). Eberconazole commercial cream
(Ebernet 1% w/w; Dr. Reddys Laboratories Limited,
Hyderabad, India) was purchased from a pharmacy.
Ethyl cellulose (46 centipoise [cp]), polyvinyl alcohol
(molecular weight: 1:25,000) and dichloromethane
were procured from Merck Chemicals (India). Carbopol 934 NF and triethanolamine were purchased from
Loba Chemie Private Ltd (India). Sabouraud dextrose
agar was purchased from HiMedia Laboratories Private Ltd (India). All other chemicals and solvents were
of analytical reagent grade.
Preparation of EB microsponges

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

The effects of different weight ratios of drug to ethyl


cellulose (0.5:1, 1:1, 1.5:1, 3:1 and 5:1) on PY, particle size and entrapment efficiency of microsponges
were investigated. The effects of stirring rate (1000,
1500, 2000 and 2500 rpm) and stirring time (60, 90,
120 and 150 min) were also studied. Furthermore, a
32 factorial design was adopted in order to optimize
the amount of internal solvent volume (X1) and emulsifier concentration (X 2), which were identified as the
independent variables affecting the particle size and
entrapment efficiency (dependent variables).

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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

where Mact is the actual EB content in weighed


quantity of microsponges, Mms is the weighed quantity
of powder of microsponges and Mthe is the theoretical
amount of EB in the microsponges calculated from the
quantity added in the process.
Particle size

Particle size was determined by the laser diffraction


technique using a Malvern Mastersizer 2000 SM
(Malvern Instruments, UK). Analysis was carried out
at room temperature, keeping the angle of detection at
90. The average particle size was expressed in terms
of d(0.9) m.

Research Article

were heated in hermetically sealed aluminium pans at


a heating rate of 10C/min over a range from room
temperature to 100C under a nitrogen atmosphere
(flow rate of 50 ml/min).
Powder x-ray diffraction

The powder x-ray diffraction (PXRD) patterns were


recorded using an x-ray diffractometer (PW 1729;
Philips, The Netherlands) employing Cu K radiation (1.542 ) with a voltage of 30 kV and a current
of 30mA. Samples were scanned from 5 and 50 2.
Preparation of EB microsponge-loaded gel

The gel-forming polymer Carbopol 934 NF (1%) was


soaked in distilled water for 2 h and then dispersed
by agitation at approximately 500 rpm with a magnetic stirrer in order to obtain a smooth dispersion.
It was allowed to stand for 15 min in order to expel
the entrained air. The obtained viscous solution was
neutralized to pH 7 with triethanolamine with slow
agitation [18] . At this stage, either EB (1%) as ethanolic
solution or EB microsponges (equivalent to 1% w/w
of EB) were incorporated in order to obtain homogenous hydrogel-based topical delivery systems termed
eberconazole-loaded gels (EBGs) and eberconazole
microsponge-loaded gels (EBMGs), respectively. The
pH and viscosity of the formulation was measured
using a digital pH-meter (model: 355M/s; Systronic,
India) and a Brookfield digital viscometer (model:
DVIII+ rheometer; Brookfield Engineering Laboratories, Inc., MA, USA). The drug concentration was
determined by a process similar to that of microsponge
drug content determination.

Residual solvent content determination

The amount of total residual solvent in EB microsponges


was determined by using a thermal gravimetric analyser
(TGA-60WS0; Shimadzu Corp., Japan). Thermal gravimetric analysis was performed by heating a weighed
amount of sample (1020 mg) in nitrogen atmosphere
from 2580C at the rate of 2C/min and the loss of
weight as a function of temperature was recorded.
Scanning electron microscope

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

Thermal properties were recorded using a DSC 821e


(Mettler-Toledo, Switzerland). Samples (5 mg each)

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In vitro release studies

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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783

Research Article Bothiraja, Gholap, Shaikh & Pawar


were compared with those of EBG and eberconazole
commercial cream (EBCC; 1% w/w). Analysis of the
data was performed using PCP Disso software, version
3 (Poona College of Pharmacy, India).

moderately high sensitivity [19] . The amounts of drug


detected in SC are indicative of drug deposition in the
skin. Measurements were performed in triplicate and
their means were reported.

Ex vivo diffusion studies

Primary skin irritation studies

The ex vivo diffusion study was performed on excised


Wistar rat skin according to the study protocol
approved by the Institutional Animal Ethics Committee constituted under the Committee for the Purpose
of Control and Supervision on Experimental Animals.
The abdominal skin of the rat was shaved, carefully
excised and defatted in order to remove the subcutaneous fat. The rat skin was placed on the Franz diffusion cell with the epidermal side facing the donor
compartment and the dermal side in contact with the
receptor solution. Further procedures were similar to
those mentioned in the In vitro release studies section.
Measurements were performed in triplicate and their
means were reported.

Primary skin irritation studies of the EBMG and


EBCC were performed using nine male Wistar rats in
accordance with the guidelines of the Consumer Product Safety Commission [20] . Hair present on the back
of each rat was removed using a hair removal cream
and an area of 4 cm 2 was marked. Nine rats were randomly assigned to three experimental groups of three
rats each. Group I served as a control, group II was
treated with EBMG while group III was treated with
EBCC. 10 mg of sample were applied to each rat (2.5
kg-1 body mass) to groups II and III every day for a
period of 7 days. The skin was cleaned before each
dose application and the resulting reactions, such as
erythema and edema, were scored after 1, 3, 5 and 7
days, as per the Draize patch test [10] . The selection
criteria for the Draize test and skin irritation score
scale is shown in Table 1. The primary irritation index
(PII) was calculated using Equations 46. The results
of triplicate measurements and their means were
reported.

In vivo skin deposition study

Fifteen male Wistar rats with undamaged skin, aged


23 months and with 180200 g body mass were used
for the skin deposition study. The rats were purchased
from Lacsmi Biofarms (India). The protocol of the
experiment was approved by the Institutional Animal
Ethics Committee of Sharadchandra Pawar College of
Pharmacy (Otur, Maharashtra, India). The rats were
housed in polypropylene cages with free access to a
standard laboratory diet and water. They were kept at
25 1C and 4555% relative humidity (RH) with a
12-h lightdark cycle.

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

Tape stripping method

The in vivo skin deposition study was performed


using the tape stripping technique [19] . On the day
of the experiment, rats were randomly assigned to
three experimental groups of five rats each. Hair was
removed from the back of each rat and an area of 2
cm2 was marked. EBG, EBMG and EBCC formulations containing 1 mg equivalent of EB were applied
on the skin and gently distributed on the marked area
of different groups. At 12 h post application, the skin
surface of each group of animal was carefully washed
with distilled water and wiped with a cotton swab in
order to remove excess formulation.
The SC was removed by tape stripping with 15
pieces of adhesive tape. Tapes containing the SC were
immersed in 5 ml methanol, vortex stirred for 2 min,
filtered using a 0.45-m membrane and analyzed for
EB content by UV spectrophotometry. UV spectroscopy was selected as the method for determination as it
is universal, cheap, rapid and easy to use and also has

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Ther. Deliv. (2014) 5(7)

In vitro antifungal testing

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

Description of irritant response

No reaction

Weakly positive reaction (usually


characterized by mild erythema across
most of the treatment site)

++

Moderate positive reaction (usually


distinct erythema, possibly spreading
beyond the treatment site)

+++

Strongly positive reaction (strong, often


spreading erythema with edema)

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Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy

(1 mg/ml) were placed into cups of size 8 mm, then into


wells of a Sabouraud dextrose plate previously seeded
with the test organism (Aspergilus niger). After allowing diffusion of the solution for 2 h, the plates were
incubated at 27C for 48 h. The zone of inhibition
measured around each cup was compared with that of
the standard. The results of triplicate measurements
and their means were reported.
Results
In this study, ethyl cellulose microsponges have been
developed and investigated as topical carriers for the
controlled release and deposition of EB on the skin surface. The effects of the drug:polymer ratio on PY, drug
content, entrapment efficiency and mean particle size
were studied. The effects of rate and time of stirring,
internal solvent volume and emulsifier concentrations
were also studied during formulation optimization.
Effects of drug:polymer ratio on the EB
microsponges

The PY, entrapment efficiency and particle size of


microsponges were greatly affected by the drug:polymer
ratios (Table 2) . Microsponges could not be obtained
with the drug:polymer ratios of 6:1 and 7:1 and free
EB crystals were observed under the optical microscope, while the drug:polymer ratios 0.5:1 to 5:1 gave
spherical microsponges.
The PY was increased with increasing the
drug:polymer ratio up to 1.5:1, whereas further
increase to 5:1 did not significantly affect the PY. At all
drug:polymer ratios, the drug content was less than the
theoretical value, which also indicated that the entrapment efficiency was less than 100%. The free-drug
concentration decreased with increasing drug:polymer
ratios from 1.5:1 to 5:1 and was in the range of 8.56
0.9% to 20.16 1.2%. However, further increases from

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1.5: 1 to 5:1 caused higher free-drug concentrations,


probably due to polymerpolymer interactions overruling the polymerdrug interactions. The mean particle size of microsponges obtained from drug:polymer
ratios of 0.5:1 and 1.5:1 were 39.4 2.2 m and 24.5
2.0 m, respectively. Decreases in particle size was
observed with increases in the drug:polymer ratio. At
still higher ratios, much larger particles were observed.
Optimum entrapment efficiency and narrow particle
size distributions were shown for the drug:polymer
ratio of 1.5:1 (EBM3).
Effects of stirring time & stirring rate

The effects of stirring time and stirring rate on particle


sizes and entrapment efficiencies of the formulated
EB microsponges were examined for the formulation
EBM3. The EB microsponges were analyzed after 60,
90, 120 and 150 min of stirring and at 1000, 1500,
2000 and 2500 rpm stirring rates, respectively. Larger
particles were obtained at low stirring rates and times,
whereas smaller particle sizes were observed at high
stirring rates and times. At still higher stirring rates,
much larger particles were observed. Focusing on minimal particle size, the optimum stirring condition for
EB microsponges was found to be 2000 rpm for 120
min (Figure 1A) . The stirring conditions had a similar
effect on the entrapment of drug. Maximum entrapment of the drug was obtained at stirring conditions of
2000 rpm for 120 min (Figure 1B) .
Effects of solvent volume & emulsifier
concentration by 32 factorial design

As per the 32 factorial design, nine different batches


were prepared for EBM3 at 2000 rpm for 120 min.
The coded levels and actual values of the variables
along with the measured responses are shown in
Table 3. The data obtained were subjected to multiple

Table 2. Optimization of drug:polymer ratios for preparation of eberconazole nitratemicrosponges.


Formulation

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

Values are presented as means standard deviation (n = 3).


EB: Eberconazole nitrate; EBM: Eberconazole microsponges.

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Research Article Bothiraja, Gholap, Shaikh & Pawar

100
Entrapment efficiency (%)

45

Particle size (m)

40
35
30
25
20

90
80
70
60
50
40

60

90

120

150

Stirring time (min)

60

90

120

150

Stirring time (min)

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.

regression analysis using PCP Disso version 3 software


and fitted with the flowing Equation 7.
Y = 0 + 1X1 + 2X2 + 11X12 + 22X22 + 12X1X2

where Y is the measured response, X is the level of


the factors and is the coefficient computed from the
responses of the formulations.
The results of the multiple regression analysis for
particle size and percentage entrapment efficiency are
as follows:
Yps = 25.60 - 11.66X1 + 5.58X2 - 1.18X1X2
YEE = 90.64 - 6.30X1 + 2.35X2 - 1.15X1X2

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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Ther. Deliv. (2014) 5(7)

the surface (Figure 2A & 2B) . However, at high solvent


volume (batches B7B9), spherical shapes could not
be obtained and a rupturced surface was observed
(Figure 2C & 2D) . However, batches B4B6 produced
uniform spherical microsponges with numerous pores
(Figure 2E & 2F) . EB crystals were also not observed.
The solvent volumeemulsifier (X1X 2 ) interaction
also played a significant role, although to a lesser
extent than the other variable terms. The optimized
microsponge (B5) composition was selected based on
average particle size and entrapment efficiency of the
microsponges. During validation, adequate measurement of the signal-to-noise ratio was determined in
order to ensure that the model could be used to navigate the design space. The predicted r2 values were in
reasonable agreement with the adjusted r2 values in all
responses; adequate precision and significance factors
are shown in Table 4. The optimized EB microsponge
formulation (B5) was composed of a drug:polymer
ratio of 1.5:1, 60 mg polyvinyl alcohol (PVA) and 5
ml dichloromethane. The results showed that the
observed values of the optimized formulation were
highly similar to the predicted values. This optimized
microsponge formulation was selected for further
investigation.
Characterization of EB microsponges

The total residual solvent in the EB microsponges was


below 0.22% (w/w). The thermal behavior of EB,
ethyl cellulose, polyvinyl alcohol and the optimized
EB microsponges formulation is shown in Figure 3A .
Endothermic peaks (Tmax) at 193.82C with an enthalpy

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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)

Mean particle Entrapment


size (m)
efficiency (%)

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

Values are presented as means standard deviation (n = 3).


PVA: Polyvinyl alcohol.

(H) of 224.55 J/g, 194.89C with a H of 18.20 J/g


and 232.23C with a H of 32.45 J/g were detected,
indicating the melting points of crystalline pure EB,
ethyl cellulose and PVA, respectively. No drug peak
was observed in the thermogram of the optimized EB
microsponge, suggesting that most of the drug present remained in an amorphous sate and was dispersed
homogenously throughout the microsponge. The
PXRD study corroborates the results of differential
calorimetry (DSC). Distinct peaks of crystalline drug
were not observed in the PXRD of EB micropsonges,
indicating the existence of the drug in its amorphous
form (Figure 3B) .
Characterization of EB microsponge gels

Batch B5 EB microsponges were incorporated into a


Carbopol 934 NF hydrogel that would hold the active
ingredient without aggregation until it was applied to
the surface of the skin due to its viscous nature and
form a thin transparent film intended for controlled
release when applied on the surface of the skin. It was
designated as EBMG.
The developed EBMG formulation was a white,
viscous preparation with a smooth and homogeneous
appearance. It was thixotropic, easily spreadable and
had good esthetic qualities. The pH and viscosity were
5.5 0.4 and 3990.66 114.33 cps, respectively. The
drug content was 99.27 0.16%, indicating that drugloaded microsponges was uniformly dispersed in the
gel formulation [3] .
In vitro release study

The influence of the composition and vehicle on the


release profiles of the different formulations was investigated using an artificial cellophane membrane with

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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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Research Article Bothiraja, Gholap, Shaikh & Pawar

Figure 2. Scanning electron microscopy of microsponges. (A & B) batch B3, (C & D) batch B9 and (E & F) batch B5.

with EBG and EBCC, respectively. The cumulative


amount of drug permeated per unit surface area after
12 h was also found to be lower for EBMG as compared with EBG and EBCC. From these results, it was
observed that drug permeation became slower when
the drug was added to the formulation in its entrapped
form rather than in an unentrapped form. At the end
of 12 h, the EBMG flux was reduced to 158.1 5.0 g/
cm2 and 288.3 3 g/cm2 as compared with EBG and
BBCC, respectively.

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Ther. Deliv. (2014) 5(7)

In vivo skin deposition study

The extraction efficiency of the tape stripping method


showed 204.8, 84.6 and 52.8 g of EB for EGMG, EBG
and EBCC formulations, respectively, from which their
depositions were calculated. The amount of EB deposited in the skin (SC) from EGMG was 102.4 4.2 g/
cm2, which was 2.5- and 4-fold higher than that of EBG
(42.3 2.3 g/cm2) and EBCC (26.4 3.4 g/cm2),
respectively, at the end of 12 h. This indicated that the
microsponges improved the drug residence of the skin.

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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

Adjusted R2 Predicted R2 Adequate


precision

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.

Primary skin irritation studies

The scores for erythema and edema were totaled for


all of the rats at the first, third, fifth and seventh days.
The PII was calculated based on the sum of the scored
reactions divided by 48 (four scoring intervals multiplied by two test parameters multiplied by three rats).
EBMG did not produce erythema and was a nonirritant (PII: 0.00), whereas EBCC showed some erythema and a PII of 0.06, indicating barely perceptible
irritation to the rat skin (Figure 5A) . The preceding
sections demonstrated controlled release of EB for
antifungal action, while the current section demonstrates the safety or nonirritant nature of the EBMG
formulation.
In vitro antifungal testing

No significant differences in antifungal activity were


observed between EBMG (1 mg/ml) and EBCC
A

(1mg/ml), as they produced zones of inhibition of 14.2


1.6 mm and 17.4 1.1 mm, respectively. The standard EB (1 mg/ml) formulation showed comparatively
smaller zones of inhibition (22.3 1.8 mm; Figure 5B) .
The results suggest that EBMG exhibited controlled
antifungal activity due to the controlled release of EB.
Discussion
EB is used in the treatment of topical fungal infections
and produces common side effects in the skin due to
excessive penetration and accumulation in the skin. It
was hypothesized that controlled release of the drug to
the skin could reduce the side effects by reducing percutaneous absorption. Microsponges are an attractive
approach for the topical delivery of drugs. This investigation aimed at utilizing ethyl cellulose microsponges
as carriers for the controlled release and cutaneous
drug deposition of EB.
B

(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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Research Article Bothiraja, Gholap, Shaikh & Pawar

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.

Microsponge preparation methods are limited


in terms of complexity and cost. The commercially
available microsponges are prepared by suspension
polymerization in a liquidliquid system. However,
most of the active pharmaceutical compounds would
decompose at the polymerization temperature [23] . The
quasiemulsion solvent diffusion method seemed to be
promising for the preparation of EB microsponges as
it is easy, reproducible, rapid and has the advantage
of avoiding solvent toxicity [24] . In the present study,
dichloromethane, which is capable of dissolving both
the drug and the polymer, was selected as the internal
solvent. The rapid diffusion of dichloromethane into
the aqueous medium could reduce the solubility of the
water-insoluble polymer ethyl cellulose in the droplets.
The instant mixing of the dichloromethane and water
at the interface of the droplets induced precipitation of
the polymer, thus forming a shell enclosing the dichloromethane and the dissolved drug. Counter diffusions
of dichloromethane and water through the shell pro-

moted further precipitation of the drug in the droplets


from the surface inwards. The finely dispersed droplets
of the polymer solution of the drug were solidified in
the aqueous medium via diffusion of the solvent [1] .
The PY, entrapment efficiency and particle
size of microsponges were greatly affected by the
drug:polymer ratios. The 0.5:1 to 5:1 ratios gave
spherical microsponges. It was previously reported that
mupirocin microsponges in the drug:polymer ratios of
0.25:1 and 2:1 [2] and benzyl peroxide microsponges
in the drug:polymer ratios of 7:1 and 13:1 [9] could be
prepared with ethyl cellulose. As shown in Table 2, the
increased PY at high drug:polymer ratios could be due
to the reduced diffusion rate of dichloromethane from
concentrated solutions into the aqueous phase, which
provides more time for droplet formation and may
improve the yield of microsponges [25] . The entrapment
efficiency was of less than 100% at all drug:polymer
ratios. This may be due to dissolution of the drug in
the solvent or aqueous phase. The higher drug entrap-

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

Zero order (r ) First order (r ) Higuchi (r )

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

0.990 0.003 209.9 6.1

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

All data are means standard deviation (n = 3).


EBCC: Eberconazole commercial cream; EBG: Eberconazole-loaded gel; EBMG: Eberconazole microsponge-loaded gel; Q12 h: Cumulative amount of drug permeated
per unit surface area after 12 h.

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Ther. Deliv. (2014) 5(7)

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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.

ment efficiency obtained at high drug:polymer ratios


can be explained due to the fact of there being larger
amounts of drug present per unit of polymer at higher
drug:polymer ratios [26] . As indicated by the thermal gravimetric analysis, the total residual solvent in
microsponges was approximately 0.22% (w/w). The
higher yield and lower residual solvent content justifies
use of the quasiemulsion solvent diffusion method in
order to obtain microsponges.
A decrease in particle size was observed with
increases in the drug:polymer ratio, which could be
correlated with the kinetics of microsponge formation in the presence of comparatively lower concentrations of the polymer. As dichloromethane diffuses out,
nearly all of the dispersed phase containing the polymer and drug is converted into solid microsponges and
can be seen as separate particles. At high drug:polymer
ratios, this dichloromethane diffusion caused the
minor polymer concentration to only surround the
drug and eventually decrease the particle size. By
contrast, for the highly viscous dispersed phase containing comparatively higher polymer concentrations
formed larger droplets when poured into the dispersion
medium. The drug:polymer ratio of 1.5:1 (EBM3)
demonstrated the optimal entrapment efficiency and
narrow particle size distribution.
Researchers have reported that the particle size
increases with increasing stirring rates and stirring
times due to the higher kinetic energy of the system
with higher agitation forces for prolonged periods.
Because of this energy, smaller particles and even larger
particles have a tendency to bind together with other

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surrounding particles by overcoming the interfacial


energy barrier [27] . In this study, the effect of stirring
times and stirring rates on particle sizes and encapsulation efficiencies was also investigated. Larger particles
were obtained at low stirring rates and times, which
may be due to there being less time given for polymer
precipitation. Here, the dissolved polymers remain
free in the aqueous phase either alone or in agglomerate form with the emulsifier. At high stirring rates and
times, the particle size notably decreased, suggesting
that most of the polymer had undergone precipitation,
thereby forming particles. At still higher stirring rates,
much larger particles were observed due to interparticle aggregation. The stirring conditions had a similar
effect on the entrapment of the drug. Focusing on minimal particle size and maximum entrapment efficiency,
the optimal stirring conditions for EB microsponges
were found to be 2000 rpm for 120 min.
Furthermore, the effects of solvent volume and
amount of emulsifier on microsponges were investigated using a 32 factorial design. The solvent volume
showed a predominantly negative influence on the
particle size and entrapment efficiency. High solvent
volumes created a low-viscosity dispersed phase, which
formed emulsion globules that were easily divisible into
smaller droplets [28] . These smaller-sized particles consequently entrapped smaller amounts of the drug. By
contrast, the highly viscous dispersed phase containing
a low solvent volume hindered the easy diffusion of the
solvent, giving rise to larger droplets entrapping larger
amounts of the drug. The positive influence of emulsifier concentration on particle size and entrapment

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791

Research Article Bothiraja, Gholap, Shaikh & Pawar


efficiency may be due to an increase in the apparent
viscosity of the continuous phase. Such high viscosity
might have resulted in larger emulsion droplets that
encapsulated larger amounts of the drug.
The solvent volumeemulsifier interaction had less of
an impact than the other variables in determining particle size and entrapment efficiency. The type and concentration of emulsifier have key roles in the preparation
of microsponges [29] . The emulsifier used in the preparation of microsponges was polyvinyl alcohol, which has a
nonionic nature. At some concentrations, this nonionic
emulsifier can associate in between the oilwater phase,
which can dissolve some portion of the drug, resulting
in a reduction in particle size and entrapment efficiency.
The in vitro release kinetic of the optimized EB
microsponges incorporated into Carbopol 934 NF
hydrogel best fitted the Higuchi kinetic model, indicating the presence of the diffusion controlled release
mechanism from the porous microsponge [30,31] . Similar release kinetics were observed for diclofenac sodium
from the microsponge-based gel system [1] . This controlled release of EB into the skin can alter its percutaneous absorption. The microsponge particles are too
large to pass through the SC and hence they would
remain on the skin surface, gradually releasing their
content over time, resulting in improved safety of the
applied drugs.
Two- and three-fold decreases in flux were observed
for EBMG as compared with EBG and EBCC, respectively, which may be due to the controlled release
mechanism of the drug from the porous microsponge
polymeric gel system, leading to low drug availability
for percutaneous absorption and resulting in improved
safety of the applied drugs. This result was consistent
with the report of Jelvehgari et al., who suggested that

the formation of a thicker wall in microsponges leads


to a longer diffusion path and consequently slower
drug release rate [32] .
Effective topical drug therapy requires sufficient
drug uptake into the skin over a particular period
of time for maximal pharmacological activity. The
higher amount of EB deposited in the skin (SC) from
the EBMG indicates that microsponges improved the
drug residence in the skin. This result is in agreement
with previous studies reporting that the use of particulate drug carriers, such as microparticles and nanoparticles, improved the drug residence in the skin without increasing transdermal transport [33] . The high
concentration of the drug in the skin after application
of EBMG could be explained by the occlusive effect,
since microsponge gels produce a film on the skin surface that reduces transepidermal water loss and favors
drug penetration into the skin. Moreover, the microsponge gel demonstrated nonirritancy to the rat skin
while retaining antifungal activity.
Conclusion
Ethyl cellulose microsponges were developed using the
quasiemulsion solvent diffusion method and characterized as an effective carrier for the topical delivery
of EB, a lipophilic antifungal drug. The properties
of the developed system were greatly affected by the
drug:polymer ratio, the volume of dichloromethane,
the amount of emulsifier, the stirring time and the stirring speed. The developed microsponge gel formulation demonstrated controlled release of EB. Primary
rat skin irritation tests revealed that the microsponge
gel formulation was nonirritant. Moreover, EB retained
antifungal activity on encapsulation in microsponges.
Ethyl cellulose microsponges proved to be a potential

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.

Effect of formulation & process variables


Optimal entrapment efficiencies and narrow particle size distributions were demonstrated for drug:polymer
ratios of 1.5:1.
Maximal entrapment of the drug was obtained at stirring conditions of 2000 rpm for 120 min.
Low solvent volumes yielded uniform spherical microsponge particles. By contrast, high solvent volumes
yielded a ruptured microsponge surface.

Ex vivo diffusion, in vivo skin deposition & in vitro antifungal studies


Microsponge gels demonstrated a controlled release profile.
The in vivo skin accumulation study demonstrated fourfold higher retention in the stratum corneum layer as
compared with the commercial cream.
Microsponges gels retained antifungal activity.

792

Ther. Deliv. (2014) 5(7)

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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.

this potential, Phase I/II clinical trials are needed for


the evaluation of the efficacy and safety of microsponge
systems on human skin for treating fungal infections.

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

Financial & competing interests disclosure

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Papers of special note have been highlighted as:
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The authors have received a Graduate Pharmacy Aptitude Test


Fellowship from the All India Council for Technical Education,
New Delhi, India. The authors have no other relevant affiliations or financial involvement with any organization or entity
with a financial interest in or financial conflict with the subject
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Ethical conduct of research


The authors state that they have obtained appropriate institutional review board approval or have followed the principles
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