The indirect method was used for the determination of entrapment efficiency (Pandit et al., 2023). Transfersomal dispersion was placed in centrifugation tubes and centrifuged at 8,000 rpm (C-24 plus, Remi Elektrotechnik Ltd., India) at 4°C for 2 h, followed by decantation of a clear supernatant solution. A clear decanted solution (1 mL) was diluted with methanol up to 10 mL and analyzed at 260 nm using a UV-visible spectrophotometer (UV1800, Shimadzu Corporation, Japan). Entrapment efficiency was calculated as entrapment efficiency (% EE) = (total amount of drug added–the amount of free drug in the supernatant/total amount of drug added) X 100.

Diffusion of fluconazole from transfersomes was evaluated using a Franz diffusion cell apparatus (DBK Instruments, Mumbai, India). Transfersomal dispersion (corresponding to 10 mg of fluconazole) was accurately weighed and transferred on a cellophane membrane (150 LA 401, HiMedia, Mumbai, India). A membrane was introduced between the receptor and donor compartment. The receptor compartment was filled with phosphate buffer at pH 5.5 as the diffusion medium. The medium was stirred with a magnetic stirrer and maintained at a temperature of 37°C ± 0.5°C. A sample (1 mL) was removed periodically at 30 min intervals, and the same volume of the diffusion medium was introduced to the cell to maintain the sink condition. The sample was further diluted by pH 5.5 phosphate buffer to 10 mL volume, and the absorbance was measured at 260 nm.

Dispersion of transfersomes was sonicated for 5 min before testing. Dispersion (1 mL of) was diluted with the freshly prepared phosphate buffer pH 5.5. The drop of dispersion was applied over carbon taps on the aluminum stub and air-dried. The dried sample was subjected to SEM studies by the model (SEM Quanta 200–EDX system, ICON laboratories, Mumbai, India) (Sarwar et al., 2018).

Particle size, polydispersity index (PDI), and zeta potential were measured using Zetasizer (Horiba-SZ100, Japan) (Duangjit et al., 2013). Transfersomal dispersion was sonicated using a probe sonicator for 5 min before testing. Furthermore, 0.5 mL of transfersomal dispersion was diluted with phosphate buffer at pH 5.5 and exposed to dynamic light scattering. Each test was carried out in triplicate (Sohail et al., 2018).

For the DSC thermogram of the optimized formulation, we followed a method analogous to the one described for the drug–excipient compatibility study. In this case, the formulation was positioned in a sample pan, and an empty pan served as the reference. It is noteworthy that, unlike the drug–excipient compatibility study where pure excipients were used, the DSC study for the optimized formulation involved the use of the actual formulation in the sample pan. This nuanced approach was undertaken to provide a more accurate representation of the thermal properties of the formulated product. In response to concerns about the specific lipid and edge activator chosen for this study, we will incorporate a brief but comprehensive explanation in the article to justify our selection, ensuring that this aspect is adequately addressed.

pH of the gel was measured using a digital pH meter (LMPH-9, MAA Scientific Instrument Lab, India) (Gupta and Singhvi, 2012). In a 10 mL beaker, 5 gm of gel was placed, and the pH was measured at room temperature in triplicate (Yadav et al., 2019).

The transfersomal gel and marketed gel (M, gel, Flumet, Leeford Healthcare Ltd) were tested for the measurement of viscosity using a using a viscometer (LVDV, Brookfield, USA). In a beaker, 10 gm of gel was placed, and the viscosity of the gel was measured using spindle 64 at 30, 60, and 100 rpm (Gul et al., 2022).

In vitro drug diffusion from the transfersomal and the marketed gel was carried out using a Franz diffusion cell apparatus (Waqas et al., 2022). The transfersomal and marketed gel dose corresponding to 10 mg of fluconazole was weighed precisely and transferred to the cellophane membrane. The cellophane membrane was sandwiched between the receptor and donor compartment. The rest of the evaluation was performed similarly, as described under in vitro drug diffusion of transfersomal formulations.

An ex vivo skin permeation study was accomplished through the goat skin (hair removed from the dorsal side) using a Franz diffusion cell. pH 5.5 phosphate buffer was used as a receptor medium, maintained at 35°C ± 0.5°C. Transfersomal gel (T-gel), gel loaded with pure fluconazole (P gel), and marketed gel (M gel) were mounted on goat skin separately, further introduced between a receiver and donor compartment. At regular intervals, 1 mL of the sample was withdrawn and replaced with a fresh medium to maintain the sink for up to 7 h. Samples were analyzed using a UV-visible spectrophotometer at 260 nm. After completion of the test, the goat skin was cautiously detached, and the formulation attached to the skin was scraped off. The skin was cut into small pieces and immersed in a 10 mL mixture of methanol and phosphate buffer solution with pH 5.5 (Kumar, 2015). The mixture was stirred for 30 min, and the extract was analyzed using a UV-visible spectrophotometer at 260 nm to determine the drug retained in the skin.

Different properties of the gel formulation such as tackiness, firmness, stringiness, and work of adhesion were tested using a Brookfield texture analyzer CT3-100 (Brookfield Engineering Labs, Inc., United States). The test was fixed at the compression mode by means of probe TA3/100 and TA-BT-KIT fixture, and trigger load was kept at 5.0 g. T-gel (without any air pockets into the gel sample) was filled into the female probe. A tapering 35-mm male probe at an angle of 45°C was enforced down into individual specimens at a rate of 0.5 mm/s to a 10-mm depth. From the resulting time plot, cohesiveness (work essential to distort the hydrogel in the downward movement of the probe), hardness (strength obligatory to achieve a specified deformation), and adhesiveness (energy essential to surpass attractive forces among the sample and probe) were read carefully. Spreadability was calculated from the energy required to deform the sample (Pandit et al., 2023).

The antifungal activity of the transfersomal gel (T-gel) and marketed gel (M-gel) was checked against Candida albicans using the agar well diffusion method (Rajan and Vasudevan, 2012). Precisely, 2.1 gm of Mueller–Hinton agar (MHA) was incorporated into a 250-mL a conical flask composed of distilled water (100 mL). It was thoroughly heated to obtain a clear solution. The resultant solution was sterilized for 15 min at 15 lb pressure at 121°C using an autoclave. The sterile agar solution was added to the MHA solution. The resulting medium was poured into a sterilized petri plate and allowed to solidify. Strains of C. albicans were streaked on a plate. Wells were created in sterile media with a sterile borer approximately at a depth of 6 mm. Finally, samples were transferred into wells and incubated at 37°C for 48 h (Choudhary et al., 2021). The zone of inhibition around the well was measured.

Fluconazole is soluble in methanol, whereas soya lecithin found in the outer layer of transfersomes is insoluble. Soya lecithin was precipitated out, and fluconazole was dissolved after the addition of methanol, which ultimately correlated with the quantity of free drug and consequently drug entrapped into vesicles. The entrapment efficiency of transfersomes ranged between 95.03% ± 0.08% and 97.28% ± 0.42% (Figure 2). Fluconazole is a moderately lipophilic drug; therefore, it has a greater affinity toward the lipid matrix. The amount of both the lipid and surfactant had a major influence on entrapping drugs in the vesicular system (Durán-Lobato et al., 2013). Soya lecithin contributed to the formation of more rigid vesicles to avoid the leaking of drugs from vesicles. Conversely, the enhanced concentration of Span 80 reduced drug entrapment, owing to the solubilization of lecithin and the generation of a more porous membrane, causing an enhanced leakage of the drug. Initial trials were conducted to optimize the amount of soya lecithin and Span 80. Different formulations were selected to achieve maximum entrapment.

The diffusion of the drug from the transfersomes was performed over 7 h at a temperature of 37°C in a phosphate buffer with pH 5.5 (Figure 3). The diffusion of the drug from transfersomes ranged between 64% and 85%. The difference in the diffusion pattern from transfersomes is attributed to the difference in the concentration of Span 80 and soya lecithin. The concentration of the surfactant had a direct correlation, while phospholipids had an inverse correlation for drug diffusion. The reason for this might be enhanced solubilization of fluconazole by Span 80, and drug retardation was endorsed by the formation of a compact bilayer structure by soya lecithin. Span 80 contains lipophilic tails with long and unsaturated (C18) moieties, which leads to a permeable vesicle membrane (Balata et al., 2020). Formulations B9 and B10 showed a higher drug diffusion of 85.62% ± 0.5% and 75.66% ± 0.3%, respectively, within 7 h, as they were composed of a higher amount of the surfactant. Conversely, slower diffusion was noticed in formulations B2, B3, B5, and B6, with diffusion rates of 77.45% ± 0.52%, 82.44% ± 0.41%, 74.28% ± 0.3%, and 67.48% ± 0.51%, respectively, within 7 h, as they were composed of a higher amount of soya lecithin (El-Gizawy et al., 2020). The diffusion data are indicative of the impact of soya lecithin and Span 80; hence, these variables were designated as independent variables, and drug diffusion was chosen as the dependent variable. Drug diffusion from different formulations was analyzed using the ANOVA test, and graphs were obtained using Design Expert Software. The contour plot and 3D surface (Figures 4A, B) of drug diffusion revealed that the enhanced concentration of Span 80 was responsible for higher drug diffusion, while the increased amount of soya lecithin retarded the diffusion of fluconazole. The ANOVA equation also supports these results, where a negative value associated with soya lecithin showed drug retardation and a positive coefficient of Span 80, indicative of faster drug diffusion. The p-value 0.0096 (<0.05) revealed the significance of the test. Y = 77.45 ‐ 2.53 * A + 5.01 * B + 4.76 * AB ‐ 0.7150 * A 2 ‐ 2.99 * B 2 .

By taking into account soya lecithin and Span 80, the concentration overlay plot was obtained, as shown in Figure 4C. The overlay plot helped select an optimized formulation. The concentration of soya lecithin and Span 80 in the optimized formulation was chosen to achieve a prolonged release with maximum entrapment efficiency. The yellow region in the overlay plot showed a concentration of 337 mg of soya lecithin and 1.46 mL of Span 80 to achieve the desired drug diffusion of 80% within 7 h. The prepared formulation was evaluated further. The entrapment efficiency of optimized transfersome formulations was 95.0% ± 2.2%.

The morphology of optimized transfersome vesicles was studied by SEM. SEM images were confirmative of the good distribution of transfersomes with a size range of 350–403 nm (Figure 5). Spherical morphology of transfersomes confirmed the vesicular drug delivery system.

The average particle size of the optimized formulation of transfersomes was 397 ± 2 nm (Figure 6A), which is the optimum size of a vesicle for a topical drug delivery system. The PDI of the colloidal system indicates the distribution of particles in the system. Well-distributed particles in the system are indicative of a stable colloidal system. The PDI value below 0.7 denotes the monodispersity of dispersion (Massadeh et al., 2020). The PDI value for optimized transfersome formulation was 0.43, which indicated the system was moderately monodispersed. To study surface charges of transfersomes, zeta potential was determined as it is of crucial importance to stabilize the dispersion. Zeta potential values ranged between +30 and −30 mV, indicative of a high probability of flocculation of vesicles. The value for zeta potential was −38 ± 5 mV (Figure 6B, which was supportive of a better stability of transfersomes. The type of surfactant with their concentration affects the value of zeta potential (Tawfeek et al., 2020). The negative charge indicated more stability and enhanced penetration potential of formulation through the skin.

Transfersome formulation exhibited a peak at 102.89°C (Figure 7), and pure fluconazole exhibited a sharp peak at 139.6°C. The reduction in the peak was confirmative of the solubilization of the drug into the lipid phase.

The drug diffusion from the optimized formulation exhibited a remarkable result, with a diffusion rate of 81.05% ± 0.23% within 7 h (Figure 8). The sustained drug diffusion, as discussed earlier, can be attributed to the presence of soya lecithin.

The pH of the transfersomal gel and the plain gel was recorded thrice using a digital pH meter. The results ranged between 5.5 ± 0.23 and 5.6 ± 0.54 for the transfersomal gel and 5.1 ± 0.24 to 5.2 ± 0.41 for plain gel formulation. This pH range falls within the acceptable range of 4–7 for transdermal application (Pathan et al., 2018), indicating the suitability of the gel formulation for application on the infected skin.

The viscosity of gel formulations is an important parameter for ease of removal from the container and application on the skin, reflecting the consistency of the formulation (Shinde et al., 2012). The viscosity of transfersomal and marketed gel formulation was measured at different rpm values such as 30, 60, and 100 at room temperature with a Brookfield viscometer using spindle no. 64. The viscosity decreased with an increase in the rate of shear, displaying non-Newtonian flow characteristics (shear-thinning). This behavior is advantageous due to its low flow resistance when applied under high-shear conditions (Bousmina, 1999). Determinations were done in triplicate (Table 2).

The diffusion rates of the drug from the T-gel, M-gel, and P-gel were 78.85% ± 0.9%, 70.64% ± 1.5%, and 28.10% ± 1.2%, respectively, within 7 h (Figure 9A). The highest drug diffusion was from the T-gel, followed by the M-gel and P-gel. The reason for this might be attributed to the faster diffusion of transfersomes into skin layers. Transfersomes can change their membrane flexibility, contributing to the easy passage of drugs through the skin layer spontaneously. This ability is known as the self-optimizing deformability (Barani et al., 2021). As transfersomes are extremely deformable, these can easily cross the skin layer with very small pores (Yang and Merlin, 2020). A gel containing pure fluconazole showed minimum diffusion, owing to its highly lipophilic nature.

T-gel formulation significantly enhanced the diffusion of fluconazole across the goat membrane, as compared to the M-gel and P-gel. Drug diffusion was 77.32% ± 0.17%, 67.76% ± 0.38%, and 26.77% ± 0.23%, respectively, within 7 h (Figure 9B). The quantity of the drug reserved in the skin for these formulations was 21.86% ± 0.31%, 17.70% ± 0.45%, and 9.18% ± 0.61%, respectively.

The texture analysis of the transfersomal gel focused on firmness, tackiness, and work of adhesion, which collectively contribute to cohesion, stickiness, and spreadability of semisolids. The results, presented as firmness (gm) and tackiness (gm) in comparison to the marketed gel (Figures 9C–F), indicate superior spreadability of the transfersomal gel over the marketed gel (Kolimi et al., 2022). These values were suggestive of the better spreadability of the transfersomal gel as compared to the marketed gel.

The antifungal activity of the transfersomal gel was evaluated against a commercial gel using the agar well diffusion method on C. albicans. The antifungal activity was measured in terms of the zone of inhibition (Elmoslemany et al., 2012). Due to its widespread impact on humans, C. albicans was chosen as an infecting fungus. The zone of inhibition of the transfersomal gel was 30 ± 0.9 mm, and for the marketed gel, it was 20 ± 1.2 mm (Figures 9E, F), which demonstrates the good antifungal activity of the transfersomal gel against C. albicans.