Studies on Development and Evaluation of Topical Ethosomal Gel Embedded Antifungal Agent for Athlete's Foot

Introduction

Fungal infections affect the skin surface or mucous membranes of the body with approximately around 50 million people suffering with these infections all over the world, especially in developing and under developed nations.¹ Athlete's foot, also known as ‘tinea pedis’ is a fungal skin infection caused by different fungi including species of Trichophyton, Epidermophyton,and Microsporum which is associated with itching, scaling, cracking and redness.² There are numerous topical antifungal drugs available for the treatment of athlete's foot including miconazole nitrate, clotrimazole, terbinafine hydrochloride, butenafine hydrochloride. A novel drug, Econazole nitrate (ECN) (1- [2-(4-chlorophenyl) methoxy]-2-(2, 4-dichlorophenyl) ethyl)-1H imidazole mononitrate is a broad-spectrum imidazole antifungal drug used for topical application on the skin or mucous membrane for the treatment of fungal infection.³ ECN belongs to the BCS class (IV) drug with low aqueous solubility and low permeability which can have a negative impact on antifungal efficacy.⁴ The solubility of poorly water-soluble ECZ can be altered in many ways such as addition of co-solvents, addition of surfactants, modification of drug crystal forms by solid dispersion and inclusion complexation with cyclodextrin.⁵

Fungal infection can be treated with topical antifungal agents available in the form of lotions, aerosol foams and sprays, powders, creams, ointments, pastes, jellies or gels.⁶ These dosage forms are associated with potential bioavailability drawbacks such as less residual time, poor skin permeability and absorption, and also show poor patient compliance.⁷ In the new era of pharmaceutical dosage forms, transdermal drug delivery system (TDDS) established itself as an integral part of novel drug delivery systems.⁸ Ethosomes are novel vesicular carriers developed for transdermal delivery of several drugs to enhance skin permeation and therapeutic activity of drug.⁹

In the present study, ethosomal vesicular suspension formulation was prepared by solvent dispersion technique by mixing ECN with other excipients such as, soya lecithin (0.5-1.5% w/v) ethanol (5-20% v/v), propylene glycol (10% v/v) and water. Further, the vesicular suspension was converted into a gel formulation ES-3 by adding 0.5, 0.8, 1.0, 1.5% w/v of Carbopol for gelling into the vesicular suspension. This study aimed to evaluate the drug-excipients interactions, gel rheological characteristics, vesicular shape and surface morphology, particle size distribution, zeta potential, and entrapment efficiency, spreadability, viscosity and skin irritation. The drug embedded ethosomes in the form gel may produce significant effects to alter the drug related pharmacokinetic characteristics and improve the efficacy of drug to treat athlete’s foot disease.

Materials and Methods

Materials

Econazole nitrate (ECN) was obtained as a gift sample from Maharshi Laboratories Pvt. Ltd, Panoli, Gujarat, India. L-α-Lecithin, soya bean was gift sample from Sigma-Aldrich Chemical Pvt Limited, Bengaluru, India. Propylene glycol and Carbopol were purchased from S.B. Fine chemicals Ltd, Mumbai, India. All other reagents and solvents used were of analytical grade satisfying pharmacopoeia specification.

Development of calibration curve

A stock solution of ECN was prepared by dissolving 100 (mg) of pure drug with pH 7.4 phosphate buffer in a 100 (mL) volumetric flask to obtain the concentration 1000 µg/mL. Accurately measured 10 (mL) of the above stock solution was further diluted up to 100 (mL) with phosphate buffer to obtain a working standard solution containing 100 µg/mL. From the solution, different concentrations (5, 10, 15, 20, 25 µg/mL) of aliquots were prepared and were subjected to scanning between 200-400 nm in a UV-Visible spectrophotometer (Shimadzu 1201, Japan).¹⁰ The absorption maxima of ECN were obtained at λ max 271.5 nm. The procedure was performed in triplicates to validate the development of calibration curve.

Fourier-transform infrared (FT-IR) spectroscopic analysis

Drug polymer interactions were studied by FT-IR spectroscopy by using potassium bromide (KBr) press pellet technique. The pure drug and mixture of drug and excipients with KBr (1:5) were compressed under 10 tones pressure in a hydraulic press to form a transparent pellet. The IR- spectrum of the pellet from 450-4000 cm-1 was recorded taking air as the reference and compared to study any interference.

Differential scanning calorimeter (DSC)

DSC was performed using DSC-60 (Shimadzu, Tokyo, Japan) calorimeter to study the thermal behaviors of drug alone and mixture of drug and excipients. The analysis was performed at temperature 40-400ºC at the rate of 10ºC/m. Nitrogen gas was introduced at a pressure of 2 bars and a flow rate of 20 mL/min and the data was analyzed by using TA-60 collector software.¹¹

Preparation of Econazole nitrate (ECN) ethosomal vesicular suspension Ethosomal vesicular suspension formulations (ES1- ES4) were prepared by solvent dispersion technique. ECN drug, soya lecithin (0.5-1.5% w/v), propylene glycol (10% v/v) were stirred on magnetic stirrer for 10 min at 200 rpm. Distilled water was slowly added drop wise with a syringe and then the whole system was stirred at 1000 rpm at 500 o C up to 45 min. The obtained shaken ethasomal vesicles were then sonicated by using ultrasonic bath sonicator (ICH sonicator, Mumbai) 10- min cycle for 30 min to form uniform vesicular ethosomal suspension.¹² Finally, all the prepared formulations were stored in a refrigerator.

Four batches of drug-loaded ethosomal vesicular suspension formulations were subjected to investigate the effect of certain formulation and process variables such as vesicular shape and surface morphology, particle size distribution, Polydispersity Index (PDI), zeta potential, drug entrapment efficiency (DEE), spreadability, viscosity, skin irritation and in vitro drug release. The detailed composition of the various formulations is stated in Table 1.

Preparation of Econazole nitrate (ECN) ethosomal gel

Based on the characterization results, from all the prepared ECN vesicular ethasomal suspensions, one formulation (ES-3) was further incorporated into a gel base by using Carbopol (0.5, 0.8, 1.0 1.5% w/v) as gelling agent. The gel base was prepared by dispersing the required quantity of carbopol in sufficient quantity of distilled water. Twenty milliliters of ECN vesicular ethasomal suspension was added to the gel base and stirred at 30ºC on a magnetic stirrer at 50 rpm for 30 min to form clear transparent gel.¹³ The composition and characteristics of drug loaded gel formulations are mentioned in Table 2.

Characterization of Econazole nitrate (ECN) ethosomal vesicular suspension

Scanning electron microscopy (SEM) analysis The vesicular shape and surface characteristics of ethosomal suspension were determined by scanning electron microscopy (model-JSM, 35CF, jeol, Japan) using gold sputter technique. Few drops of ethosomal suspension were mounted on a stub covered with clean glass. The sample was coated with polaron E5 100 sputter coat to 200 Ao thickness with gold palladium. The samples were examined by adjusting a working distance of 20 nm, a tilt of zero-degree and accelerating voltage of 20 kV. Photographs were taken within a range of 50–5000 magnifications.¹⁴

Particle size distribution, Polydispersity index (PDI), and Zeta potential

The mean particle vesicle size distribution of the sample was determined by Malvern Zetasizer (Nano ZS90) by dynamic light scattering (DLS) by monitoring at 25ºC at a scattering angle of 173º which measured size range between 6 nm and 0.6 µm with approximately 3 mW output at 632.8 nm digital correlator.¹⁵ The ethosomal vesicular suspension was diluted to 10 times with distilled water and vesicles size, PDI, zeta potential was determined. The nanometric size range of the particles were retained even after 100 times dilution with water which proves the compatibility of the system with excess of water. The zeta potential values were calculated by using the software Smoluchosky equation.

Entrapment efficiency (DEE)

Five milliliters of ethosomal vesicular suspension was transferred to 50 mL centrifuge tube and diluted with distilled water. ECN drug content of the ethosomal vesicular suspension was estimated by centrifuging a sample at 15000 rpm for 2(h) on an ultra-centrifuge (Hettich, Germany). The clear supernatant liquid and sediment were diluted with phosphate-buffer (PBS, pH = 7.4). The absorbance of the solution was measured by using spectrophotometry (Shimadzu 1201, Japan) at 271.5 nm. The drug concentration was calculated using a calibration curve (Y = 0.0136X - 0.004, R2 = 0.9988).

Drug encapsulation efficiency was calculated using the following equation:

Percentage DEE = ((T-C))/C

Where T is the total amount of drug in the supernatant and sediment, and C is the amount of drug in the supernatant. Three different formulations were tested in triplicates and the average ± SD was calculated.

Characterization of Econazole nitrate (ECN) ethosomal gel

Determination of drug content

Weighed 10 (g) of gel formulation was transferred to 250 (mL) volumetric flask containing 20 mL of alcohol and was stirred for 30 min. The volume was made up to 100 (mL) and filtered through vacuum filter. One (mL) of the filtrates was further diluted with 10 (mL) alcohol, and the absorbance was measured by using spectrophotometry¹⁶ (Shimadzu 1201, Japan) at 271.5 nm.

Determination of pH

The pH meter (Sartorius, Switzerland) was used to evaluate the pH values of formulations at room temperature. This pH meter was three-point calibrated (pH 4, pH 7, and pH 10) with standard buffer solutions to ensure instrument validity. Weighed 50 gm of gel formulation was transferred to 10 (mL) beaker and estimation of pH of the formulations in the range 3 to 9 was carried out in triplicates and the results were declared as average value ± standard deviation.

Estimation of viscosity

Brookfield viscometer DVII was used to determine the rheological properties of the samples at room temperature with a speed rate of 50 to 200 rpm. Spindle T 95 was used for the measurement of viscosity and it moved up and down indicating the viscosity of the sample at number of points along the path. The torque reading was always greater than 10% of all the gels. All measurements were carried out in triplicates, and the results were represented as average value ± standard deviation. 

In vitro drug release studies

The drug release from formulated ECN-loaded ethosomal gel was performed by using Franz diffusion cell (Labindia instruments, Bangalore). The donor compartment with 17 mm orifice diameter displayed a permeation area of 2.52 cm² . A suitable thickness of pre-treated cellophane membrane was fixed between donor and receptor compartment of the diffusion cell. The required quantity of gel (equivalent to 150 (mg) of ECN) was kept in donor compartment. The entire surface of membrane was in contact with the receptor compartment containing 85 (mL) of phosphate-buffer (PBS, pH = 7.4), with a rotation speed of 50 rpm for 3h and the temperature was maintained at 37±1°C. At the defined times (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 h), the sample (5 mL) was withdrawn and replaced with same volume of fresh medium. The withdrawn samples were filtered through a 0.45 µm membrane filter and after appropriate dilution, the concentration of drug in releasing medium at each of these times were measured at 271.5 nm spectrophotometrically (Shimadzu 1201, Japan).¹⁷ The percentage drug release vs time of each formulation was represented graphically.

Kinetics of Drug Release

In order to understand the mechanism and kinetics of drug release and the best fit model for the formulations was used by PCP-Disso-V2 software. The drug release data of the in vitro dissolution study was analyzed with various kinetic equations like zero-order (% Release v/s Time), first-order (Log % retained v/s time), and Korsmeyer and Peppas equations (Mt/M∞ = Ktn). Where Mt is the amount of drug released at time t, M∞ is the amount of drug released at infinite time, K is the kinetic constant incorporating the structural and geometric characteristics of the gel, and n is the diffusional exponent indicative of the release mechanism. Where n= 0.5 represents Fickian diffusion, <1.0 represents non-Fickian diffusion, < = 1.0 case-II transport, and n > 1.0 super case-II transport. Coefficient of correlation (r) values were calculated for the linear curves obtained by regression analysis of the above plots.

Results

Development of calibration curve The standard calibration curve of ECN in 7.4 PBS was generated by UV-visible spectrophotometric technique at absorption maxima 271.5 nm. The calibration curve was developed by a plot absorbance vs. concentration, which showed good linearity within a particular range (Figure 1) obeying beer’s law in concentration range of 2 – 20 µg/mL. The linear regression equation was generated and amount of drug present in the dosage form was calculated using a calibration curve (Y = 0.0136X - 0.004, R2 = 0.9988).

FT-IR analysis

FTIR of ECN pure drug, soya lecithin, carbopol and corresponding physical mixture of drug with excipients are given in Figure 2. The FTIR spectra of the pure ECN showed characteristic absorption peaks of various groups NH- stretching at 3671.5cm^-1, C=H stretching at 3018.1cm⁻¹, –NO2 at 1547 cm⁻¹, C=C stretching at 1407.6cm⁻¹, C–N stretching at 1330 cm⁻¹, C–O stretching 1005.21cm⁻¹ and C–Cl stretching 824.1cm^-1 (Figure 2a). The characteristic absorption peaks of soya lecithin were observed in the region of 3010.4, 2292.4, 1482.6, 1051.7 and 826.8 cm^-1 (Figure 2b). FTIR spectra of Carbopol showed the characteristic peaks at wave numbers 3516.2, 2853.9, 1412.3, 1051.6 and 794.6 cm^-1 corresponding to C-H stretching for aromatic C=C stretching in the aromatic ring and peaks at 1485.32, 1385.36 cm^-1 which can be assigned to the C-H deformation (Figure 2c). The physical mixture of ECN and carbopol characteristic peaks were noted at wave numbers 3338.7, 2926.4, 1635.2, 1079.5 and 881.2 cm-1 corresponding to NH-stretching, C=H stretching of –COO and –COOH groups respectively (Figure 2d). FTIR spectrum of physical mixtures of ECN and soya lecithin showed absorption peaks at wave numbers 3326.2, 2944.7, 1637.1, 1080.3 and 883.4 cm-1 corresponding to C-H stretching and C-H deformation (Figure 2e).

DSC analysis

The thermal behaviour of the pure ECN and drug loaded ethosomal gel was characterized using DSC, as shown in Figure 3. The thermogram of pure ECN showed a sharp endothermic peak at 119.2˚C, followed by 165˚C corresponding to its meting point (Figure 3a). However, the drug-loaded ethosomal gel showed a broad endothermic peak at 166.6o C and a sharp exothermic peak at 205˚C (Figure 3b).

Vesicle size distribution, Polydispersity Index and zeta potential

Results of average particle size distribution, polydispersity index, zeta potential is mentioned in Table 1.

Drug entrapment efficiency

The entrapment efficiency of ethosomal preparations was determined by ultracentrifugation method. The percentage of ECN content in the drug loaded ethosomal gel of batches EG-5 to EG-8 was found to be in the range of 79.43±0.6 to 50.56±0.6, respectively (Table 1).

Drug content, pH and viscosity

The percentage of ECN content in the formulated ethosomal gel of batches EG-5 to EG-8 was observed to be in the range of 55.35±0.28 to 70.25±0.24, respectively. The pH and viscosity of the gel formulations are mentioned in Table 2. 

In vitro drug release studies

The drug release from formulated ECN-loaded ethosomal gel was performed by using Franz diffusion cell (Labindia instruments, Bangalore). The percentage of drug release was obtained in the range 05, 09, 14, 21, 26 and 38% w/w in the time periods 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs, respectively (Table 3).

Kinetics of drug release

The in vitro dissolution data were analyzed by different kinetic models in order to estimate the n-value, which describes the drug release mechanism. Cumulative % drug release was analyzed using PCP Disso-v2 08 software. The values of correlation (r) were calculated and were found to be more linear for first order release as compared to zero-order release.

Discussion

ECN belongs to the BCS class (IV) drug and having a low aqueous solubility and permeability can have a negative impact on antifungal efficacy. ECN is available in the form of powder aerosols, creams, foams for the effective treatment of various fungal infections. After topical application, these products show extremely low absorption, although most of the applied drug remains on the skin surface, followed by minimum inhibitory concentration for dermatophytes.¹⁸ Therefore, we prepared a novel formulation, ECN ethosomes in the form of gel and attempted to evaluate its physical characteristics and drug release potential.

IR spectra of the physical mixture of the drug and excipients were compared with the spectra of the pure drug and individual polymer spectra. No considerable changes in the IR peaks of ECN were observed in the physical mixture indicating the absence of any interaction (Figure 2).

DSC is a well-established technique, often used for qualitative measurement of physical and chemical changes in either enthalpy or heat capacity of a crystalline drug in the polymer gel matrix during the manufacturing process. In DSC curve of the gel, no significant shift of endothermic peak was found as compared to pure drug. It may indicate that there were no changes in thermal behavior and the drug was molecularly dispersed in hydrogel matrix.

The vesicular shape and surface characteristics of ethosomal suspension were determined by scanning electron microscopy (model-JSM, 35CF, jeol, Japan) using gold sputter technique. The SEM photomicrographs are shown in Figure 4a, 4b. Morphology of the drug loaded ethosomal suspension containing 0.5% w/v of soya lecithin (ES-1) shows a separate cluster bridge type micron size vesicles (Figure 4a) and the discrete nanosized drug vesicles in entire formulation can also be observed. SEM images suggest that the drug is not evenly dispersed in low concentration soya lecithin. SEM microphotographs of the formulation (ES-3) containing 1.5% soya lecithin shows that the drug and soya lecithin are molecularly entrapped to form uniform nano sized lipid bridges (Figure 4b). Therefore, the concentration of soya lecithin was significantly influencing the morphology of vesicular formulation.

Particle size, zeta potential and polydispersity index (PDI) are essential parameters for vesicular characterization due to their safety, stability, efficacy and in vivo performance as drug delivery systems.¹⁹ While increasing the concentration of soya lecithin, ethanol significantly influenced the particle size distribution (Figure 5a, 5b), Zeta potential (Figure 6a, 6b). Polydispersity Index (PDI) values obtained were in the acceptable range (Table 1).

The concentration of drug entrapment efficiency was observed and the results revealed that the amount of soya lecithin used in the formulation was directly proportional to the entrapment efficiency. The high drug encapsulation efficiency observed for all formulations (ES-1 to ES-4) by increasing the concentration of lecithin might be attributed to the hydrophobic nature of ECN (water solubility ≤1 mg/mL), which facilitates its incorporation in the phospholipid matrix. The concentration of ethanol used in the formulation was inversely proportional to the percentage of drug entrapment efficiency. By increasing the ethanol concentration up to 15% (w/v), the entrapment efficiency also increased and on further increasing the ethanol concentration (>20% w/v), the vesicle membrane became more compact which hindered further entry of the drug. The unbound drug may be dissolved in ethanol that may have led to decrease in the entrapment efficiency.

Based on the characterization results from all the prepared ECN vesicular ethosomal suspension, select optimum formulation (ES-3) was further incorporated into a gel base by using Carbopol (0.5, 0.8, 1.0 1.5% w/v) as gelling agent. Several characteristics of ECN ethosomal gel such as percentage of drug content, pH and viscosity were investigated. The increase in the concentration (0.5, 0.8 and 1.0) of carbopol significantly improved the drug content in the drug embedded gel. Further by increasing carbopol up to 1.5% w/v, drug content slowly decreased due the formation of thick gel network. The percentage of carbopol also influenced the pH and viscosity of the gel.

In vitro drug release studies explained that the concentration of carbopol (0.5 to 1.0) increased the release of drug from the gel slowly for a prolonged period of time due to longer residual time of gel on the skin surface. Further when the concentration of carbopol increased above 1% w/w, the drug release was very slow at 3 hrs due to increased viscosity of gel forming an occlusive thick layer which hindered the diffusion of drug into dermal layer of the skin.

The kinetic data was best fitted to Peppa’s model and good regression coefficient was observed. The drug release from ECN ethosomal gel containing 1.0% w/v of Carbopol EG-7 showed Higuchi model, followed by first order kinetics (Figure 7). The values of diffusion coefficient ranged between n = 0.72480 and 0.8788 indicating the drug release from the gel followed case-II transport

Conclusion

It can be concluded from the above investigation that the proper selection of optimized formulation conditions is very important to achieve high drug entrapment efficiency, drug content and to release of ECN from the gel. Among the four sets of formulations, ES-3 was considered as optimized ethosomal formulation and it was used for the development of ethosomal gel. As the investigated results suggest, the ECN embedded ethosomal gel containing 1% w/v of Carbopol as gelling agent (EG-7) is a promising carrier to improve the potential bioavailability and can be used effectively for the treatment of Athlete's foot.

Conflict of Interest

None

Acknowledgement

The authors thank Maharishi Laboratories Pvt. Ltd, Panoli, Gujarat and the principal, chairman of Togari Veeramallapa Memorial College of Pharmacy, Ballari. We are very thanking to Rajiv Gandhi University Health Sciences Bengaluru for proving UG research grant for this research work.