Formulation and Development of Sertaconazole Ethosomal Gel for Antifungal Activity on Dermatophytosis

Dermatophytosis

Dermatophytosis, commonly referred to as tinea or ringworm, is a superficial fungal infection affecting keratinized tissues such as skin, hair, and nails. It typically presents as red, itchy, scaly, circular lesions, and may sometimes lead to localized hair loss. Symptoms generally appear within 4-14 days after exposure. The infection is classified based on the anatomical site involved, and multiple body areas can be affected simultaneously^[1-3].

Dermatophytes are a group of fungi that invade keratinized tissues and are categorized into genera such as Trichophyton, Microsporum, and Epidermophyton, among others. Based on their habitat, they are further classified as anthropophilic (human-associated), zoophilic (animal-associated), and geophilic (soil-associated) ^[3,5,7].

Globally, fungal skin infections are highly prevalent, affecting nearly one billion people, with dermatophytosis being the most common type. Risk factors include poor hygiene, excessive sweating, close contact with infected individuals or animals, and weakened immunity. Diagnosis is usually based on clinical features and can be confirmed through microscopic examination or fungal culture ^[4].

Ethosomes:

Ethosomes are ethanol-rich lipid vesicles designed to enhance transdermal drug delivery. They are soft, flexible carriers that facilitate the penetration of drugs into deeper skin layers and may also enable systemic absorption. Structurally, ethosomes consist of phospholipids dispersed in a hydroalcoholic or hydroalcoholic-glycolic medium containing a relatively high concentration of ethanol. The formulation typically includes phospholipids such as phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, along with ethanol or isopropyl alcohol, water, and glycols like propylene glycol. Cholesterol may be added in small amounts to improve vesicle stability, while surfactants and cationic lipids can be incorporated to modify permeability and charge. The ethanol content generally ranges from 20-50%, which plays a crucial role in disrupting the lipid structure of the stratum corneum, thereby enhancing drug permeation ^[8-14].

The high alcohol concentration imparts flexibility to the vesicular membrane, allowing better interaction with skin lipids and improved drug distribution. Ethosomes offer advantages such as enhanced skin penetration, non-invasive delivery, and suitability for a wide range of drugs, including large molecules. However, limitations include relatively high cost, lower yield, and dependence on drug solubility and molecular size for effective delivery ^[16-18].

2. MATERIAL AND METHODS

Materials:

The standard raw material active pharmaceutical ingredient (Drug) of Sertaconazole are procurement from Dhamtec Pharma and Consultants B-204, Silver springs, Opposite MIDC office, Taloja, Navi Mumbai-410208.

Methods:

• Preformulation Studies

Preformulation studies represent the initial stage in dosage form development, focusing on the evaluation of physicochemical properties of the drug substance alone and in combination with excipients. These studies provide essential information for designing a safe, stable, effective, and bioavailable formulation.

1. Organoleptic Characteristics^[25-27]

The organoleptic properties of the drug were assessed through visual and sensory examination. Parameters such as appearance, colour, odour, taste, and texture were evaluated. The physical nature of the drug (crystalline or amorphous) was observed under adequate lighting. Colour and odour were determined by direct inspection and gentle smelling, respectively. Taste was assessed cautiously when permissible. Texture was examined manually to identify smoothness or grittiness. Solubility was evaluated in different solvents such as water, ethanol, and methanol at room temperature, and results were recorded according to standard pharmacopeial classifications.

2. Melting Point ^[27]

Melting point of Sertaconazole Nitrate was determined by using Thelie tube. Sertaconazole drug sample are filled half in a sealed capillary tube, attached to a thermometer with thread, was immersed in the tube. Heating is commenced and the temperature ranges at which Sertaconazole drug sample was melts was observed.

3. Solubility Study ^{[27, 28]}

The solubility of Sertaconazole was evaluated in various surfactants (Labrasol, Tween 80, Tween 20, Span 80) and cosurfactants (PEG 400, PEG 200, and propylene glycol). An excess amount of drug was added to 3 ml of each selected vehicle in stoppered vials. The mixtures were stirred continuously for 48 hours at 40±0.5°C using a magnetic stirrer, followed by equilibration at room temperature for 24 hours. The samples were then centrifuged at 3000 rpm for 20 minutes. The supernatant was collected and filtered through a 0.45 μm membrane filter for further analysis.

4. Identification of Drug and Polymer by FTIR [

Sertaconazole and polymers were identified using Fourier Transform Infrared (FTIR) spectroscopy. The sample was prepared by mixing the drug with IR-grade potassium bromide (KBr) in a 1:100 ratio and triturating uniformly. The mixture was then placed in the sample holder and scanned over a range of 4000-400 cm^-¹. The spectra were recorded using an FTIR spectrophotometer, and the obtained peaks were compared with standard reference spectra to confirm the identity of the drug and polymers.

5. Selection of Solvent [27, 28]

The solubility of drug was determined in verity of polar and non-polar solvents as per IP specification. The common and stable solvent for Sertaconazole was found to be Methanol.

6. Preparation of Standard Stock Solution [25, 27]

10mg drug (Sertaconazole) was weighed accurately and dissolved with sufficient quantity of methanol solvent then solution was transfer in to a 100ml of volumetric flask and volume up to 100ml with methanol solvent in volumetric flask. Thus, the stock solution (100µg/ml) of Sertaconazole drug in methanol solvent was prepared.

7. Calibration Curve of Pure Drug [27]

The stock solution (100µg/ml) was prepared to get concentration, 5-30µg/ml. The of concentration v/s peak area absorbance was plotted and data was subjected to linear regression analysis on the maximum absorbance (λ max= 200).

8. Drug-Excipient Interaction Study by FTIR [27,53]

Drug-excipient compatibility was evaluated using FTIR spectroscopy. Spectra of the pure drug, excipients, and drug-loaded formulation were recorded over 4000-400 cm?¹ using the KBr pellet or ATR method. The characteristic peaks of the drug were compared with those of the formulation to identify any changes. No significant shifts or new peaks were observed, indicating absence of interaction and good compatibility between the drug and excipients.

• Formulation of Ethosomes (Cold Method) ^[56,59,64]

Sertaconazole-loaded ethosomes were prepared by the cold method to preserve drug stability. Phospholipids and lipophilic components were dissolved in ethanol with gentle stirring at room temperature. A cold aqueous phase (distilled water) was prepared separately and added dropwise to the ethanolic solution under continuous stirring (700 rpm) to form a vesicular suspension. The mixture was further stirred for 30-60 minutes and sonicated to reduce vesicle size and ensure uniformity.

The prepared ethosomal suspension was stored at 4°C and later incorporated into a gel base containing gelling agents such as HPMC and Carbomer 934. The gel was prepared separately and mixed gently with the ethosomal suspension to obtain a uniform ethosomal gel suitable for topical delivery.

• Characterization of Ethosomes ^[59,64]

1. Particle Size:

The particle size of ethosomal vesicles was determined using the dynamic light scattering (DLS) technique. The gel sample was diluted with distilled water to obtain a clear dispersion and analyzed using a particle size analyzer at controlled temperature. The average particle size and polydispersity index (PDI) were recorded to assess vesicle size distribution, uniformity, and stability of the formulation.

2. Zeta Potential Measurement

Zeta potential was measured to evaluate the surface charge and stability of ethosomal vesicles. It indicates the degree of electrostatic repulsion or attraction between particles, which influences aggregation and dispersion stability. Generally, values above ±30 mV indicate good stability, while lower values may lead to aggregation. The zeta potential of the formulation was determined using a zeta sizer at 25°C. Ethosomal systems typically show values in the range of -10 to -20 mV, indicating moderate stability.

3. SEM Analysis:

Scanning Electron Microscopy (SEM) was used to study the surface morphology of the formulation. A small quantity of the sample was mounted on an aluminum stub using double-sided adhesive tape and coated with a thin layer of gold under vacuum to enhance conductivity. The sample was then examined under an SEM at suitable magnifications to observe surface characteristics such as shape, texture, and uniformity.

• Formulation of Ethosomal Gel ^[28,33]

5. Preparation of Carbapol Gel Base

For prepare the Carbopol 934 gel base, 1 g of Carbopol 934 was dispersed in 90 mL of hot distilled water pre-mixed with 10 mL of glycerol to prevent clumping. After complete dispersion, accurately weighed quantities of methyl paraben, propyl paraben, HPMC, and Span 60 were added to enhance stability and emulsification. The entire mixture was stirred until uniform thickening occurred. Finally, the gel was neutralized with dropwise addition of 50% w/w triethanolamine to adjust the pH and promote gel formation. The resulting gel was smooth, homogenous, and transparent, suitable as a base for ethosomal incorporation.

5. Incorporation of Ethosome in the Gel Base ^[54,55]

The prepared ethosomes was slowly added in carbopol 934 gel base with gentle stirring. Finally, the ethosomal gel was mixed using a mechanical stirrer for 5 min. Total six formulations were prepared and evaluated.

• Evaluation of Ethosomal Gel ^[28,29]

1. Physical appearance

The formulated Sertaconazole ethosomal gel formulations were observed visually for their color, homogeneity and consistency, presence of any clog and sudden change in viscosity.

2. Clarity Test

The clarity of the prepared ethosomal gel formulation was evaluated by visual inspection. A small quantity of the gel was placed in a clean, transparent glass container and observed against both black and white backgrounds under adequate lighting conditions. The formulation was checked for the presence of any particulate matter, turbidity, or phase separation. The gel was considered clear if it appeared transparent and free from any visible impurities or suspended particles. The observations were recorded to assess the overall quality and homogeneity of the formulation.

3. Phase Separation

The phase separation study of the ethosomal gel formulation was carried out to evaluate its physical stability. The prepared gel was stored in a closed container at different temperature conditions, including room temperature and accelerated conditions, for a specified period. The samples were periodically observed for any signs of phase separation, such as formation of layers, syneresis, or liquid separation. The gel was visually inspected for changes in consistency, homogeneity, and appearance. The formulation was considered stable if no phase separation or visible changes were observed throughout the study period. All observations were recorded systematically.

• Antifungal Activity of Sertaconazole Ethosomal Gel ^{[49, 50, 53]}

The antifungal activity of Sertaconazole-loaded ethosomal gel was evaluated using the agar well diffusion method. Dextrose agar medium was used, and fungal strains such as Candida albicans and Aspergillus niger were selected. Standardized fungal inoculum was spread uniformly on sterile agar plates, and wells (≈6 mm) were created aseptically. The test sample was added into the wells, while plain gel and marketed formulation served as negative and positive controls, respectively.The plates were incubated at 28±2°C for 48 hours. After incubation, zones of inhibition were observed and measured in millimeters. The results were compared with controls to assess antifungal efficacy, indicating improved activity of the ethosomal gel due to enhanced drug penetration and sustained release.

3. RESULTS AND DISCUSSION

Results

• Pre-formulation Studies:

1. Organoleptic Character:

Table 1: Organoleptic Character of Sertaconazole Drug

2. Melting point

The melting point of Sertaconazole was determined through three measurements. The recorded values were 161?°C, 162?°C, and 162?°C. Based on these results, the mean melting point was calculated to be approximately 162?°C.

Table 2: Melting Point of Sertaconazole

3. Solubility Studies

The solubility profile of Sertaconazole was determined in various solvents and surfactants to evaluate its potential for formulation into topical delivery systems like ethosomal gel. The results are presented in the table below:

Table 3: Solubility of Sertaconazole

4. FTIR Studies of Drug and Polymer

• FTIR of Drug

Figure 1: FTIR of Sertaconazole

Table 4: Peak table with principal peak of Sertaconazole

• FTIR of Polymer (Carbopol 934):

Figure 2: FTIR graph of Carbopol934

Table 5: Peak table with principal peak of polymer (Carbapol)

5. Calibration Curve of Pure Drug:

Standard calibration curve of Sertaconazole was carried out in methanol at 262 nm. The absorbance values obtained are shown in table. Using concentration and absorbance data, a beer lumbert’s plot was obtained. The plot in the given figure. The R² value of Sertaconazole was found to be 0.997, which is near to1, which signifies linearity.

Table 6: Standard calibration curve data of Sertaconazole

Figure 3: Calibration Curve of Sertaconazole Drug

6. Drug Excipient Interaction Studies by FTIR:

Figure 4: FTIR Graph of Drug- Exipient Interaction

Table 7: Peak table with principal peak of Drug and Gel Interaction

• Formulation of Ethosome Formulation:

Table 8: Formulatiom Table of Sertaconazole Ethosomes

• Characterization of Ethosome Formulation;

1. Particle Size

The particle size of ethosomal vesicles was determined using DLS and found to be 170.2 nm, indicating nanosized vesicles suitable for enhanced skin penetration. The PDI was below 0.3, showing uniform size distribution and good stability. These results confirm that the formulation is homogeneous and appropriate for effective transdermal drug delivery.

Figure 5: Particle Size of Ethosomes

2. Zeta Potential Measurement:

The zeta potential of formulation F2 was found to be -10.6 mV, indicating that the ethosomal vesicles possessed a negative surface charge. This negative charge helps reduce particle aggregation by providing electrostatic repulsion between vesicles. Although values above ±30 mV are generally considered highly stable, the obtained value suggests moderate stability of the formulation. The zeta potential distribution also showed slight variation in surface charge, which may be attributed to differences in vesicle composition, while phospholipids and surfactants contributed to maintaining dispersion stability.

Figure 6: Zeta potential Measurement of Ethosomes

3. SEM Analysis

The particle size observed in the micrograph is in the nanometer range (below 200 nm), which correlates well with the particle size analysis obtained by DLS. The clear and distinct vesicular structures confirm the successful formation of ethosomes.

Figure 7: SEM of Ethosomes

4. Drug Entrapment Efficiency

Table 9: Drug Entrapment Efficiency of ethosome formulation

• Formulation of Sertaconazole Ethosomal Gel:

Table 10: Composition of Ethosomal Gel formulation

• Evaluation of Sertaconazole Ethosomal Gel:

1. Physical Appearance:

Table 11: Physical Appearance of Ethosomal Formulations

2. Clarity Test

Table 12: Clarity Test for Ethosomal Gel Formulations

3. Phase Separation Study

Table 13: Phase Separation Study of Ethosomal Gel

• Anti-Fungal Activity of Sertaconazole Formulation:

Table 14: Anti-Fungal Activity of Sertaconazole Formulations

Figure 8: Anti-Fungal activity of ethosome and standard formulation