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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 μm, 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 [3–6] . 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 [email protected]

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 (5–300 μm), 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), 781–794

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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 author’s 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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Materials & methods EB was purchased from Sigma-Aldrich Chemical Private Ltd (India). Eberconazole commercial cream (Ebernet® 1% w/w; Dr. Reddy’s 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 35°C in order to remove dichloromethane. The formed microsponges were filtered through Whatman® filter paper no. 41 (Whatman, UK), washed with distilled water, dried at 40°C 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 in μg/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 30°C, The samples were filtered using a 0.2-μm 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) μm.

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were heated in hermetically sealed aluminium pans at a heating rate of 10°C/min over a range from room temperature to 100°C 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 Kα radiation (1.542 Å) with a voltage of 30 kV and a current of 30 mA. Samples were scanned from 5 and 50° 2θ. 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 (10–20 mg) in nitrogen atmosphere from 25–80°C at the rate of 2°C/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 gold–palladium 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 ± 0.5°C 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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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 4–6. The results of triplicate measurements and their means were reported.

In vivo skin deposition study

Fifteen male Wistar rats with undamaged skin, aged 2–3 months and with 180–200 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 ± 1°C and 45–55% relative humidity (RH) with a 12-h light–dark cycle.

Average scores = ΣErythema grade* + ΣEdema 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-μm 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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In vitro antifungal testing

In vitro antifungal testing was determined by the Sabouraud dextrose agar disk diffusion test employing the ‘cup–plate 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

0

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 27°C 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 polymer–polymer interactions overruling the polymer–drug 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 μm and 24.5 ± 2.0 μm, 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 (µm)

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

B

A

100 Entrapment efficiency (%)

45

Particle size (µm)

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 2500 rpm (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 = β0 + β1X1 + β2X2 + β11X12 + β22X22 + β12X1X2

where Y is the measured response, X is the level of the factors and β 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 9.7–44.4 μm and 80.62–96.01%, 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 B1–B3) yielded uniform spherical microsponge particles, yet these were less porous, due to poor diffusion of complete solvent from

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the surface (Figure 2A & 2B) . However, at high solvent volume (batches B7–B9), spherical shapes could not be obtained and a rupturced surface was observed (Figure 2C & 2D) . However, batches B4–B6 produced uniform spherical microsponges with numerous pores (Figure 2E & 2F) . EB crystals were also not observed. The solvent volume–emulsifier (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 193.82°C with an enthalpy

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

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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 (μm) efficiency (%)

B1

-1, -1

2

30

30.7 ± 1.2

93.04 ± 1.52

B2

-1, 0

2

60

37.2 ± 1.5

96.01 ± 1.86

B3

-1, 1

2

90

44.4 ± 2.1

98.34 ± 1.25

B4

0, -1

5

30

20.1 ± 1.0

88.17 ± 1.23

B5

0, 0

5

60

24.5 ± 1.3

91.44 ± 1.76

B6

0, 1

5

90

31.3 ± 1.5

92.32 ± 1.45

B7

1, -1

7

30

9.7 ± 0.9

80.62 ± 1.92

B8

1, 0

7

60

13.1 ± 0.7

83.62 ± 1.75

B9

1, 1

7

90

18.3 ± 1.3

85.31 ± 1.35

Values are presented as means ± standard deviation (n = 3). PVA: Polyvinyl alcohol.

(ΔH) of 224.55 J/g, 194.89°C with a ΔH of 18.20 J/g and 232.23°C with a ΔH 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 : 0.987–0.997) 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 μg/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

A

B

C

D

E

F

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 μg/ cm2 and 288.3 ± 3 μg/cm2 as compared with EBG and BBCC, respectively.

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In vivo skin deposition study

The extraction efficiency of the tape stripping method showed 204.8, 84.6 and 52.8 μg 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 μg/ cm2, which was 2.5- and 4-fold higher than that of EBG (42.3 ± 2.3 μg/cm2) and EBCC (26.4 ± 3.4 μg/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 

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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

B

0.678

 9.572

Entrapment efficiency

0.643

0.524

0.227

5.358

B

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

(1 mg/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

2

120 4

6

8

180

240

(ii)

280 °C

10 12 14 16 18 20 22 24 Time (min)

0

10

20

30

40

50

2ѳ

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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A

B

100

EBG EBMG

400 Q (µg/cm2)

80 Drug release (%)

500

60 40

EBCC

300 200

EBG 20

100

EBMG EBCC

0

0 0

2

4

8 6 Time (h)

10

0

12

2

4

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 liquid–liquid 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 (μg/cm2/h)

Intercept (μg/cm2)

r2 

Q12 h (μg/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

2

2

2

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

A

Research Article

B

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 volume–emulsifier 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 oil–water 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.

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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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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 matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript

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