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. 2024 Oct 18;14(11):270. doi: 10.1007/s13205-024-04116-1

Augmentation of antifungal activity of fluconazole using a clove oil nanoemulgel formulation optimized by factorial randomized D-optimal design

Shaimaa M Badr-Eldin 1,2, Hibah Mubarak Aldawsari 1,3, Sabna Kotta 1,3,✉, Mahmoud Abdelkhalek Elfaky 4
PMCID: PMC11489362  PMID: 39430772

Abstract

In the present study, fluconazole (FLU) showed the highest solubility in clove oil and was selected as the oil phase for the FLU-loaded nanoemulsion (FLU-NE). Among the studied cosurfactants, Labrafac was better than ethanol at providing globules with acceptable sizes and a lower polydispersity index (PDI) when Tween 80 was the surfactant. This optimized FLU-NE was thermodynamically stable. Furthermore, FLU-NE stored at 40 ± 2 °C and 75 ± 5% relative humidity for 6 months demonstrated good stability. The FLU-NE was converted to a FLU-loaded nanoemulsion gel (FLU-NEG) using 2% w/v sodium carboxymethyl cellulose. The FLU-NEG was acceptable in terms of visual appearance and spreadability. Rheological studies revealed pseudoplastic behavior for FLU-NEG. The viscosity of FLU-NEG decreased when the applied rpm was increased. FLU-NEG showed greater drug release than that from a FLU-GEL formulation. Furthermore, the FLU release from FLU-NEG followed the Higuchi model. The results from the in vitro antifungal evaluation of FLU-NEG on Candida albicans ATCC 76615 strain confirmed the increase in the antifungal activity of FLU by clove oil. Significant differences were observed in the zones of inhibition produced by FLU-NEG compared to those produced by the blank nanoemulsion gel (B-NEG), fluconazole suspension (FLU-SUS), and nystatin samples. Thus, the increase in the antifungal activity of FLU using clove oil as the oil phase in its nanoemulsion formulation was quite evident from our results. Therefore, the developed FLU-NEG could be considered a potential candidate for further preclinical and clinical studies.

Keywords: Antifungal, Clove oil, Experimental design, Fluconazole, Nanoemulsion

Introduction

Recently, nanotechnology and its applications in biomedicine have been explored for improved drug delivery and therapy. In line with these developments, nanoemulsion formulations have contributed significantly to enhance the bioavailability or drug-loading capacity of these materials (Wilson et al. 2022). Furthermore, nanoemulsions have been proven to be useful in targeted delivery, ophthalmic drug delivery, intranasal drug delivery, transdermal drug delivery, and brain-targeted drug delivery, and the list goes on (Karami et al. 2019; Choradiya and Patil 2021; Alaayedi and Maraie 2023; Gerber et al. 2023). Nanoemulsions are also useful in the intravenous delivery of drugs (Norouzi et al. 2020). A nanoemulsion is considered a stable system that is isotropic and consists of two immiscible liquids formed with the use of surfactants, often with globule sizes less than 200 nm (Kotta et al. 2012; Guttoff et al. 2015). The phase inversion composition technique (PICT), a low-energy technique, can be used to manufacture good-quality nanoemulsions (Kotta et al. 2015).

Fluconazole (FLU) is an FDA-approved antifungal drug and is a fluorine-substituted bis-triazole. The solubility and dissolution enhancement of FLU have been the focus of research on this drug. The low aqueous solubility of FLU has always been a concern for drug delivery, and solid dispersions have been suggested as a promising method to overcome this obstacle (Papageorgiou et al. 2008). Very recently, coprocessing of FLU with other excipients, such as menthol, has been carried out successfully (Shiekh Ali et al. 2023). The importance of nanoemulsions for solubility and bioavailability has been well established (Kotta et al. 2012). Interestingly, nanoemulsions have been reported to be better than salt synthesis at enhancing drug solubility (Groo et al. 2017). Therefore, a nanoemulsion was selected as the drug-delivery platform for FLU. In this study, we further aimed to increase the antifungal activity of FLU.

The selection of appropriate oil is always key to the success of nanoemulsion drug-delivery systems. The ability of the oil to solubilize the drug is a prerequisite for the selection of the oil phase. Moreover, oil, which has pharmacologic action similar to that of drugs, would be an added advantage. Oils with antimicrobial activities and antifungal activity, particularly in the case of FLU, would be beneficial for nanoemulsion treatment of microbial infections, and plant-derived volatile oils have been found suitable for this purpose (Li et al. 2024; Chouhan et al. 2017). According to a literature review, neem, almond, olive, and clove oils have been found to possess antifungal activity (Rodrigues et al. 2019; Geng et al. 2016; Goel et al. 2016; Chee and Lee 2007). Therefore, these volatile oils were chosen for the oil phase in the present study.

In the present study, the formulation and optimization of a nanoemulsion of FLU (FLU-NE) were performed via the phase inversion technique. The selection of the oil phase for the FLU-NE was based on the presence of antifungal activity and the extent of solubility of FLU. Furthermore, while Tween 80 was chosen as the surfactant, the use of Labrafac and ethanol was based on miscibility. The suitability of ethanol and Labrafac as cosurfactants was evaluated using an experimental design approach. We further planned to convert the optimized FLU-NE to a FLU-loaded nanoemulsion gel (FLU-NEG) for topical application. The effect of the FLU-loaded nanoemulsion with volatile oil on the increase in antifungal activity was subsequently studied.

Materials and methods

Materials

Fluconazole (Spimaco, Riyadh, KSA), almond oil (Delmon Product Ltd., Jeddah, Saudi Arabia), olive oil (Al Jouf, Jeddah, Saudi Arabia), clove oil B.P., neem oil, and Tween 80 (Sigma‒Aldrich, St. Louis, USA), and Labrafac PG (Gattefosse, France) were used in the study. All chemicals utilized were of analytical quality and readily available on the market.

Solubility studies

The solubility of FLU was determined in the different oil samples. Briefly, FLU was added in excess quantities to neem, almond, olive, and clove-oil samples. Furthermore, the samples were mixed by vortexing for 5 min and subsequently placed in a shaking water bath for a period of 72 h at a temperature of 37 °C. The undissolved FLU was removed by centrifuging the obtained samples at 5000 rpm for 30 min. The upper portion of the sample subjected to centrifugation contained the dissolved FLU, and a predetermined volume of this upper layer was taken and diluted with methanol to determine the solubility of FLU. The estimation of FLU in the samples was carried out at 260 nm using UV–Vis spectrophotometry (UV-2600 Shimadzu, Japan) (Göğer and Aboul-Enein 2001; Kotta et al. 2021).

Design, evaluation, and optimization of the formulation

A factorial randomized D-optimal design was used in this study. Tween 80 was chosen as the surfactant in all formulations. Ethanol and Labrafac were used as cosurfactants. Surfactant mix (Mixture of surfactant and cosurfactant; Smix) were prepared with Tween 80 as the surfactant and ethanol or Labrafac as the co-surfactant. The formulation design employed Oil:Smix ratio and cosurfactant type as the independent factors. Furthermore, the globule size and polydispersity index (PDI) were selected as the formulation-dependent factors.

Nanoemulsion formulations were prepared using PICT. Briefly, fluconazole was first dissolved in clove oil. Later, Smix was prepared by mixing Tween 80 and selected cosurfactants (ethanol or Labrafac) at a ratio of 2:1. Then, this mixture was added to a predetermined amount of clove oil as per the design and finally thoroughly mixed with a vortex shaker. After, water was added slowly, the solution was mixed continuously using a vortex mixer (Kotta et al. 2015, 2021; Khan et al. 2012). Design-Expert® software (Stat Ease, Inc., Minneapolis, USA) was used for designing the experiments and evaluating the results.

The evaluation of the globule size and PDI was carried out in triplicate (Zetasizer Nano, Malvern Instruments, Worcestershire, UK).

Optimization of formulation

Optimization of the FLU-NE was carried out numerically (Venugopal et al. 2016). The minimum values for the globule size and PDI were set as the targets during optimization. The independent variable levels suggested by the software were utilized to prepare the optimized FLU-NE.

Evaluation and characterization of the optimized nanoemulsion

Thermodynamic stability

The thermodynamic stability of the optimized FLU-NE was examined following a previously reported method (Kotta et al. 2015). In the centrifugation test, the FLU-NE sample was centrifuged at 5000 rpm for 30 min. In the heating–cooling cycle test, the FLU-NE sample was alternatively kept at 4 °C and 40 °C for 48 h. The heating–cooling cycle was repeated thrice. In the freeze‒thaw cycle test, the FLU-NE sample was incubated at − 20 0 °C and 25 °C for 48 h at each temperature for three cycles, after which the thermodynamic stability was evaluated, i.e., absence of cracking, creaming, and phase separation.

Globule size, PDI, and zeta potential

The evaluation of the globule size, PDI, and zeta potential of the optimized nanoemulsion was carried after a tenfold dilution using (Zetasizer Nano, Malvern Instruments Limited, Worcestershire, UK). The determinations were carried out in triplicate.

Stability studies

The optimized FLU-NE samples were kept for six months at 40 ± 2 °C and 75 ± 5% relative humidity. After 6 months of storage, the mean globule size and PDI were determined and compared with the values observed initially (Khan et al. 2019).

Formulation and evaluation of fluconazole-loaded nanoemulgel (FLU-NEG)

Formulation of FLU-NEG

The optimized FLU-NE formulation was selected for the preparation of nanoemulogels. Sodium carboxymethyl cellulose (2% w/v) was added to the prepared O/W FLU-NE by stirring using a magnetic stirrer (Hussain et al. 2016).

Evaluation of FLU-NEG

Physical examination

The color, appearance, and homogeneity of the prepared FLU-NEG were visually inspected. The pH was measured using a pH meter (Jenway, Staffordshire, ST15 OSA, UK).

Spreadability test

The spreadability of the FLU-NEG was estimated as follows. Briefly, 0.5 g of FLU-NEG was placed within a circle (1-cm diameter) marked on a glass plate, and another glass plate was kept over the FLU-NEG sample. Thereafter, a weight of 500 g was kept on the second glass plate kept on the FLU-NEG sample for 5 min. The spreadability was determined in terms of the enhancement in diameter due to the spreading of FLU-NEG (Bachhav and Patravale 2009).

Rheological studies

With the use of a viscometer (Brookfield Metek DV2T, Middleboro, MA, United States), the viscosity of the FLU-NEG was measured. Before starting the measurements, samples from the gels were permitted to settle for 30 min at 25 ± 0.5 °C (Hussain et al. 2016). Five, 10, 15, 20, 25, and 30 rpm were applied to the gel. The matching dial reading was recorded for each speed. The recordings were performed thrice (Dantas et al. 2016).

In vitro drug-release study

A dissolution-dialysis method was used to perform a straightforward dialysis approach to measure the in vitro release of FLU-NEG, which was subsequently compared with that of a FLU-loaded plain gel (FLU-GEL). A hollow glass cylinder with an interior diameter of 2.9 cm and a length of 15 cm made up the dissolving cell. Rubber bands were used to secure the donor-gel reservoir backing membranes to the glass tubes at the end of the 2.9-cm internal diameter. The donor-gel reservoirs were then covered with semipermeable membranes. The dissolution device had tubes linked to it. A 250-mg weight of gel, or 0.5% FLU, was added to each tube. The tubes were then agitated at 50 rpm in 300 mL of pH 7.4 phosphate buffer that was kept at 37 ± 0.5 °C. For the drug, this volume was sufficiently high to solubilize all the dissolved molecules and thus provided the perfect sink conditions. To maintain a constant volume throughout the experiment, approximately 2 mL of sample was removed at predetermined intervals (0.5, 1, 2, 3, 4, 5, and 6 h), after which the mixture was replaced with an equivalent amount of new buffer solution. At 260 nm, the samples were examined using a spectrophotometric apparatus (UV-2600 Shimadzu, Japan) (Hussain et al. 2016). Kinetic analysis of the release data was performed according to the zero, first, and Higuchi models (Shah et al. 2019).

Antifungal evaluation

Three samples, blank nanoemulsion gel (B-NEG), FLU-NEG, and fluconazole suspension (FLU-SUS), were examined for their antifungal activity using standard strains of Candida albicans ATCC 76615 obtained from the King Abdulaziz University Hospital’s microbiology laboratory in Jeddah, Saudi Arabia. A B-NEG, FLU-NEG, and FLU-SUS were tested for comparison. Moreover, nystatin (20 µg) was used as the standard in the present study.

Using the previously mentioned agar-diffusion approach, a preliminary screening of the antifungal activity was carried out (Clinical and Laboratory Standards Institute 2006). In brief, 25 mL of Sabouraud dextrose agar (SDA) containing 1 mL of fungal culture (1 × 106 CFU/mL) was placed into 90-mm Petri dishes. On the seeded agar plates, four wells of equal diameter were created. Next, 50 μL of each sample was added to the wells; Nystatin (20 µg) was used as a positive control. The Petri dishes were then incubated at 30 °C for 24 h. The definition of inhibitory activity was the lack of fungal growth in the vicinity of the wells. A caliper was used to measure the inhibition zone in millimeters. The experiments were repeated three times.

Statistical analysis

The data for each experiment are presented as the means ± standard deviations of three determinations. The statistical significance of the data obtained for the selection of oil, stability studies, and antifungal evaluation was determined using InStat Software (GraphPad, CA, USA), and the Tukey–Kramer test was used for comparison.

Results and discussion

Solubility studies

The results from the solubility studies of the different oils are shown in Fig. 1. A highly significant difference (p < 0.001) in the solubility of FLU in clove oil (200.49 ± 3.03 mg/ml) was observed compared to that in neem (7.25 ± 0.63 mg/ml), almond (4.83 ± 0.81 mg/ml), and olive oils (6.32 ± 0.81 mg/ml). The very high solubility of FLU in clove oil was in good agreement with its reported solubility (Vlaia et al. 2021). Based on the results of the solubility study, clove oil was selected as the oil phase for FLU-NE.

Fig. 1.

Fig. 1

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Solubility of fluconazole (FLU) in different oils [Statistical evaluation: @, p < 0.05, compared to neem oil; †, p < 0.05, compared to almond oil; #, p < 0.05, compared to olive oil; $, p < 0.001, compared to clove oil]

Design, evaluation, and optimization of FLU-loaded nanoemulsion formulation

Table 1 provides the responses obtained for various FLU-loaded nanoemulsion formulation trials.

Table 1.

Runs and responses for FLU nanoemulsions

Run Independent variables Responses
A: oil: smix ratio B: co-surfactant type Globule size PDI
1 1:4 Ethanol 63.22 0.249
2 1:3 Labrafac 112.8 0.128
3 1:5 Ethanol 59.78 0.240
4 1:6 Ethanol 22.70 0.190
5 1:5 Ethanol 60.05 0.242
6 1:2 Labrafac 373.87 0.352
7 1:3 Labrafac 113.40 0.132
8 1:3 Ethanol 151.83 0.487
9 1:5 Labrafac 81.23 0.126
10 1:3 Ethanol 150.29 0.481
11 1:4 Labrafac 99.34 0.135
12 1:4 Labrafac 99.06 0.137
13 1:6 Labrafac 34.55 0.118
14 1:4 Ethanol 64.11 0.253
15 1:2 Ethanol 421.5 0.517
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The analysis of variance data for globule size are listed in Table 2. The design model for globule size was found to be significant based on the F and p values. Furthermore, the Oil:Smix ratio, cosurfactant type, and interaction effect between independent factors were also found to be significant (p value < 0.05).

Table 2.

Analysis of variance data for globule size

Source Sum of squares Degrees of freedom Mean square F-value p value
Model 1.893 × 105 9 21,034.25 57,236.05  < 0.0001
A—oil: Smix ratio 1.850 × 105 4 46,238.33 1.258 × 105  < 0.0001
B—co-surfactant type 10.16 1 10.16 27.66 0.0033
AB 4200.94 4 1050.23 2857.78  < 0.0001
Pure error 1.84 5 0.3675 – –
Total (corr.) 1.893 × 105 14 – – –
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The predicted versus actual plot for globule size was found to be acceptable (Fig. 2a). A predicted versus actual plot can be used to assess the suitability of the model for predicting responses. The closeness of the observed and predicted values inferred the suitability of the design model for the prediction of the globule size. From the interaction (Fig. 2b) and 3D surface (Fig. 2c) plots, the effects of Oil:Smix ratio and cosurfactant type could be elucidated. The interaction and 3D surface plots showed that a decrease in the globule size occurred when the Oil:Smix ratio was changed from 1:2 to 1:6. The possibility of a decreased globule size resulting from an increased Smix content has been previously established (Kotta et al. 2015). This reduction in globule size was more evident when the Oil:Smix ratio was changed from 1:2 to 1:3 than when it was changed from 1:3 to 1:6.

Fig. 2.

Fig. 2

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Plots obtained for globule size of fluconazole -loaded nanoemulsion formulations (a) predicted versus actual plot (b) interaction plot (c) 3D surface plot

Interestingly, when we examined the effect of the type of cosurfactant on the globule size, a definite change in pattern was observed. At Oil:Smix ratios ranging from 1:2 to 1:3, ethanol produced nanoemulsions with larger globule sizes than those produced by Labrafac. However, with Oil:Smix ratios of 1:4, 1:5, and 1:6, ethanol produced nanoemulsions with slightly smaller globule sizes than those produced by Labrafac. Notably, these results were new to the current knowledge, and they were obtained owing to the unique design of the experiment employed in the present study. In practice, a surfactant system with a higher HLB would favor o/w emulsions. Therefore, ethanol, which has a higher hydrophilic–lipophilic balance (HLB) than Labrafac PG, could be expected to result in a smaller globule size. However, the observations at Oil:Smix ratios of 1:2 to 1:3 were not as expected. A possible explanation for these observations could be derived from the results of a recently reported study in which the effect of alcohols was studied during the preparation of surfactant-free microemulsions (Abrar and Bhaskarwar 2022). The study demonstrated that alcohols with high HLB values could migrate from the interface to bulk water. Thus, the hydrophilic ethanol, with an HLB of 7.95, potentially migrated to the bulk water of the nanoemulsion system. This could reduce the cosurfactant activity available to the globules, thus resulting in a greater globule size (Abrar and Bhaskarwar 2022). However, in the case of Labrafac PG with an HLB value of 2, this migration from the interface to bulk water was not favorable. Therefore, when used as a cosurfactant, more Labrafac PG was observed at the interface of the globules while the opposite is true when ethanol was used as the cosurfactant. The presence of a cosurfactant at the surfactant interface is necessary to enhance the fluidity of the surfactant film, thereby enhancing the stability and decreasing the globule size (Shaker et al. 2019). All the above mentioned mechanisms can be expected to be more pronounced at lower Smix concentrations. At low Smix concentrations, the percentage of ethanol that migrated from the interface to the bulk water might be very high. However, at high Smix concentrations, the percentage of ethanol that migrated from the interface to the bulk water would be lower; therefore, the influence of this loss of cosurfactant on the globule size would also be lower. However, detailed studies are required for further elucidation of the underlying mechanisms involved.

The analysis of variance data for the PDI are provided in Table 3. In the case of the PDI, the design model was significant according to the F and p values. Furthermore, the effects of the Oil:Smix ratio, cosurfactant type, and the interaction effect between independent factors on the PDI were found to be highly significant (p value < 0.001).

Table 3.

Analysis of variance data for polydispersity index (PDI)

Source Sum of squares Degrees of freedom Mean Square F-value p value
Model 0.2825 9 0.0314 4129.67  < 0.0001
A—Oil:Smix ratio 0.1297 4 0.0324 4268.07  < 0.0001
B- Co-surfactant type 0.1203 1 0.1203 15,829.57  < 0.0001
AB 0.0433 4 0.0108 1422.94  < 0.0001
Pure Error 0.0000 5 7.600E − 06 – –
Total (corr.) 0.2825 14 – – –
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Similar to the observations for globule size, the appropriateness of the selected design model was indicated by the predicted versus actual plot (Fig. 3a), wherein the correlation between the observed and predicted values was high. Regarding the interaction (Fig. 3b) and 3D surface (Fig. 3c) plots for PDI, a significant difference in pattern was observed for ethanol and Labrafac cosurfactants. In general, a higher Smix content and therefore cosurfactant content reduced the globule size. This effect of surfactant and cosurfactant has been well established in previous studies (Algahtani et al. 2022).

Fig. 3.

Fig. 3

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Plots obtained for polydispersity index (PDI) of fluconazole-loaded nanoemulsion formulations (a) predicted versus actual plot (b) interaction plot (c) 3D surface plot

With respect to the PDI, ethanol, as a cosurfactant, produced higher values than Labrafac at all studied Oil:Smix ratios. A further detailed examination of the results showed that a drastic decrease in the PDI was caused by ethanol when the Oil:Smix ratio was changed from 1:3 to 1:4. Moreover, a drastic change caused by Labrafac was observed by changing the Oil:Smix ratio from 1:2 to 1:3. Surprisingly, there was no significant effect of Labrafac on the PDI when the Oil:Smix ratio was changed from 1:3 to 1:6. This potentially occurred because the PDI reached the lowest possible level of approximately 0.1. However, in the case of ethanol, a decrease in the PDI was noted even after the Oil:Smix ratio was changed from 1:4 to 1:6. As mentioned for globule size, this detailed effect of the cosurfactants, ethanol, and Labrafac was only possible due to the specific design employed in the present study. To the best of our knowledge, no such reports on this relationship have been published. Thus, our study design could be extended to many aspects of nanoemulsion formulations to further tailor the system to achieve optimum characteristics. As discussed for globule size, the migration of ethanol from the interface to bulk water could be expected to influence the PDI. While the globule sizes were similar for ethanol and the Labrafac nanoemulsion, the PDI values were distinctly greater for ethanol. Here, we could further presume that the migration of ethanol did not follow a regular pattern and therefore resulted in different globule sizes or more toward a nonuniform globule size distribution. Several factors, such as mixing time, temperature, surfactant type/concentration, and oil type/concentration, could affect ethanol migration. A detailed study of the effect of ethanol migration on the PDI could reveal further mechanisms for these observations.

Optimization of formulation

The software provided results from the numerical optimization with an Oil:Smix ratio of 1:3 and Labrafac as a cosurfactant; this was considered the optimized formula for the FLU-loaded clove oil nanoemulsion (FLU-NE). When the Oil:Smix ratio was changed from 1:2 to 1:3, a drastic decrease in the globule size was observed. However, after changing the Oil:Smix ratio from 1:3 to 1:4, 1:5, or 1:6, no such distinct decrease in the globule size was observed. These results potentially influenced the selection of Oil:Smix ratio of 1:3 by the software. The PDI values were distinctly greater when ethanol was used as the cosurfactant at all studied Oil:Smix ratios. As discussed earlier, the migration of ethanol from the interface to the bulk water of the nanoemulsion potentially decreased its efficacy in acting as a better cosurfactant. Thus, the suggestion of Labrafac as a cosurfactant by the software was logical. Furthermore, the software predicted a globule size of 113.1 nm and a PDI of 0.130 for the optimized FLU-NE. The concentration of FLU in the optimized FLU-NE was 0.5% w/v.

Evaluation and characterization of the optimized nanoemulsion

Thermodynamic stability

The optimized FLU-NE formulation passed all conducted thermodynamic stability tests. The centrifugation, heating–cooling cycle, and freeze‒thaw cycle tests confirmed the thermodynamic stability of the optimized FLU-NE. Cracking, creaming, or phase separation were not observed in the tested optimized FLU-NE formulation. The thermodynamic stability of nanoemulsions has been established in previous studies (Kotta et al. 2015; Pramod et al. 2012a). Interestingly, the thermodynamic stability of the FLU-NE was comparable to that of reported nanoemulsions prepared via the PICT method using Capryol 90 (oil phase), Gelucire44/14 (surfactant), and Transcutol HP (cosurfactant); these are novel agents with better performance in nanoemulsions (Kotta et al. 2015; Pramod et al. 2012a). Furthermore, the thermodynamic stability of FLU-NE is comparable to that of eugenol (a major chemical constituent of clove oil), which is a nanoemulsion prepared with Tween 80 (a surfactant) and ethanol (a cosurfactant) (Kotta et al. 2015; Pramod et al. 2012a). Thus, these thermodynamic stability studies revealed that the formulation excipients selected for FLU-NE were appropriate for further studies.

Globule size, PDI, and zeta potential

The mean globule size of the optimized FLU-NE was 115.17 ± 1.04 nm, with a PDI of 0.137 ± 0.008. These results agreed well with the software-predicted values of 113.1 nm and 0.130 for globule size and PDI, respectively, indicating the suitability of the selected design model. A diameter of 300 nm or less can deliver the drug into skin layers. While the mean globule size is important, consideration should also be given to the PDI value. A value of 1 for the PDI indicates a highly polydisperse system. PDI values less than 0.1 indicate monodisperse samples, and values up to 0.2–0.3 indicate low or moderate polydispersion and are acceptable. Thus, a PDI of less than 0.3 is generally considered acceptable. Therefore, the observed values for the globule size and PDI were considered acceptable (Grijalvo and Rodriguez-Abreu 2023; Danaei et al. 2018). These observed mean globule sizes and Fig. 4a provide a representative size-distribution pattern of the optimized FLU-NE formulation. Moreover, the optimized FLU-NE had a mean zeta potential of 5.91 ± 0.197 mV. Even though the zeta potential of the optimized FLU-NE was lower than the desirable value of ± 30 mV, the fact that FLU-NE was converted to a nanoemulsion gel (FLU-NEG) validates its acceptability. Furthermore, the possibility of a stable nanoformulation with a low surface charge has also been reported. This could be explained by other factors, such as steric stabilization, which can also contribute to the stability of the dispersion (Gupta et al. 2016). Figure 4b shows a representative zeta potential plot of the optimized FLU-NE formulation.

Fig. 4.

Fig. 4

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Representative images of (a) size-distribution pattern and (b) zeta potential of the optimized fluconazole-loaded nanoemulsion (FLU-NE) formulation

Stability studies

Accelerated stability studies serve the purpose of predicting the long-term stability of a product by subjecting it to exaggerated storage conditions over a shorter period of time. The stability studies were conducted according to the Q1A(R2) guideline of International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) wherein the storage conditions for the accelerated stability testing are mentioned as 40 ± 2 °C temperature and 75 ± 5% relative humidity. The results obtained for the stability studies of FLU-NE stored at 40 ± 2 °C and 75 ± 5% relative humidity for 6 months are shown in Fig. 5. The results revealed that for both the mean globule size and the PDI, there were statistically significant changes (p < 0.05) between the initial sample values and the sample values after 6 months of storage. Nevertheless, the globule size observed for globules was less than 200 nm, and the PDI was less than 0.3, even after storage for 6 months under accelerated temperature and humidity conditions. Nanoemulsions less than 200 nm in size are considered acceptable (Patel et al. 2017), and nanostructures with a PDI less than 0.3 are acceptable because they indicate homogeneity of globule size (Danaei et al. 2018). Therefore, the optimized FLU-NE is sufficiently stable. A stable nanoemulsion could lead to improved stability for the NEG prepared with it, and thus, it's reasonable to anticipate that FLU-NE could enhance the stability of FLU-NEG.

Fig. 5.

Fig. 5

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Results of stability studies of fluconazole-loaded nanoemulsion (FLU-NE) stored at 40 ± 2 °C and 75 ± 5% relative humidity for 6 months in comparison to initial samples

Formulation and evaluation of the fluconazole-loaded nanoemulgel (FLU-NEG)

Physical examination

The color, appearance, and homogeneity of the prepared FLU-NEG were found to be satisfactory upon visual inspection. FLU-NEG showed good homogeneity. The pH was 5.61 ± 0.34. Because the pH was close to that of skin, FLU-NEG would be compatible with skin and unlikely to irritate it after application. However, the absence of any skin irritation can be confirmed only by in vivo skin-irritation studies. Furthermore, the nanoemulsion had an opaque white appearance.

Spreadability test

The spreadability of topical formulations is a critical component of patient compliance. Furthermore, it is believed to be an essential part of patient adherence to therapy. Because fluconazole emulgel spreads quickly, it is believed to have a high spreadability. The therapeutic efficacy of gels can also be dependent on their spreading capacity. Good spreadability was shown by the spreading diameter of 7.5 ± 1.6 cm. Thus, FLU-NEG possesses sufficient spreadability to facilitate the application of the gel to the skin (Dantas et al. 2016; Shinde et al. 2012).

Rheological studies

Since the developed FLU-NEG formulation is applied to thin layers of skin, consistency is one of its most crucial components. The viscosity test results for FLU-NEG at six different rotational rates per minute (1, 2, 3, 4, 5, 10, 15, 20, 25, and 30 rpm) are shown in Fig. 6. Initially, at an rpm of 1, the viscosity of FLU-NEG was 2760 cP. This viscosity sharply decreased to 2076 cP when the rpm was changed to 5. However, at higher rpm, this significant decrease in viscosity was not observed. For instance, when the rpm was changed from 5 to 10, the viscosity decreased from 2076 to 1784 cP. Furthermore, when the rpm was further changed to 30 rpm, the viscosity decreased to a value of 1164 cP. Therefore, the reduction in viscosity was not linear with the change in rpm.

Fig. 6.

Fig. 6

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Results of rheological studies of fluconazole-loaded nanoemulgel (FLU-NEG)

In general, the gel formulation viscosity indicates consistency (Shinde et al. 2012). When applied under high shear conditions, the viscosity of FLU-NEG decreases with increasing shear rate, demonstrating a non-Newtonian flow, more precisely, pseudoplastic or shear-thinning behavior; this characteristic is desirable because of its low resistance to flow over high shear rates (Bousmina 1999). This shear-thinning behavior could be justified for FLU-NEG from the results of previous studies with hydrogels (Ramachandran et al. 1999; Pramod et al. 2012b). The reduction in viscosity of the FLU-NEG formulation, owing to its pseudoplastic or shear-thinning behavior, further confirmed its characteristic high spreadability upon application but without any draining of the formulation (Carvalho et al. 2010).

In vitro drug-release study

Unlike the usual biphasic release pattern observed for drug release from nanoemulsion gels, a steady drug-release pattern was observed from FLU-NEG. The oil barrier is likely the reason for the biphasic release of nanoemulsions or nanoemulsion gels (Prajapati et al. 2021). Thus, presumably, clove oil in the FLU-NEG does not hinder the ability of the FLU to reach the release medium. Nevertheless, a greater release of the drug from FLU-NEG than from its suspension form was observed; this result was in good agreement with previously reported observations (Gaber et al. 2023). At every sample point, the percentage of FLU released from FLU-NEG was substantially greater than that from FLU-GEL. Finally, 79.31 ± 3.1% of FLU was released from FLU-NEG after 6 h, while only 60.53 ± 3.57% of the FLU was released from FLU-GEL (Fig. 7).

Fig. 7.

Fig. 7

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Release profiles of fluconazole (FLU) from FLU-loaded nanoemulgel (FLU-NEG) and FLU-loaded plain gel (FLU-GEL)

The correlation coefficients (R2) for the zero-order models were 0.9658 and 0.9328 for FLU-NEG and FLU-GEL, respectively. Moreover, for the first-order models, the R2 values of the FLU-NEG and FLU-GEL samples were 0.9689 and 0.9660, respectively. However, the Higuchi model showed the highest R2 values for both FLU-NEG (0.9886) and FLU-GEL (0.9824). These results demonstrated that the Higuchi diffusion model fits the release kinetics of FLU from the FLU-NEG and FLU-GEL formulations. Thus, diffusion is the main method by which FLU is released from these gels. Similar to our observations, a recently reported study on a carbomer-based nanoemulsion gel also supported Higuchi diffusion as the most suitable drug-release model (Ma et al. 2021).

Antifungal evaluation

In the in vitro antifungal assay, B-NEG, FLU-NEG, and FLU-SUS samples were evaluated by an agar-diffusion assay with nystatin (20 µg) as a standard drug against Candida albicans ATCC 76615; the observed results are provided in Fig. 8. The results showed that there was a significant difference (p < 0.001) in the zones of inhibition produced by the samples. FLU-NEG had a significantly greater (p < 0.001) zone of inhibition than the other samples. This finding was in agreement with the reported results that nanoemulsions enhanced the in vitro antifungal activity of amphotericin B against Candida species (Marena et al. 2023). The enhanced permeation effects of nanoemulsions are major contributors to the enhancement of antifungal activity. Similarly, the ability of an essential oil component in nanoemulsions to enhance antifungal activity against Candida species has also been reported (Krishnamoorthy et al. 2021). These results indicated the synergistic effect of the essential oil component and the nanoglobules. This effect was also demonstrated in our study. The significantly greater antifungal effect of FLU-GEL was potentially due to the antifungal effect of clove oil causing an increase in the effect of FLU (Chee and Lee 2007; Pramod and Ansari 2010). This effect was further evident from the zone of inhibition of 16 ± 0.0 mm produced by the B-NEG. Therefore, the increase in the antifungal activity of fluconazole by clove oil nanoemulgel was evident from the results of the antifungal evaluation.

Fig. 8.

Fig. 8

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Antifungal activities of blank nanoemulsion gel (B-NEG), FLU-loaded nanoemulgel (FLU-NEG), and fluconazole suspension (FLU-SUS) samples against Candida albicans ATCC 76615 (a) bar diagram of zone of inhibition [Statistical evaluation: @, p < 0.05, compared to B-NEG; †, p < 0.05, compared to FLU-SUS; #, p < 0.05, compared to FLU-NEG; $, p < 0.001, compared to Nystatin] (b) photographs of microbiological plates

Conclusions

In the present study, the feasibility of increasing the antifungal activity of FLU was studied. Based on its significantly high solubility in clove oil, FLU was selected as the oil phase. Furthermore, Tween 80 was used as the surfactant, and the suitability of ethanol and Labrafac as cosurfactants was evaluated. The software provided results of numerical optimization with an Oil: Smix ratio of 1:3 and Labrafac as a cosurfactant; this was considered the optimized formula for FLU-NE. Furthermore, the software predicted a globule size of 113.1 nm and a PDI of 0.130 for the optimized FLU-NE, and these were in agreement with the observed values. The FLU-NE was stable during storage under accelerated temperature and humidity conditions. The optimized and evaluated FLU-NE was converted to FLU-NEG. The FLU-NEG had an acceptable appearance and spreadability. Rheological studies revealed pseudoplastic or shear-thinning behavior of the FLU-NEG. This behavior was advantageous for ensuring proper spreadability and application of the gel across the skin. A steady drug-release pattern was shown by the FLU-NEG model, and the pattern followed the Higuchi model. An in vitro antifungal assay on Candida albicans ATCC 76615 strain confirmed the increase in the antifungal activity of FLU by clove oil. Thus, in this study, we successfully showed an increase in the antifungal activity of FLU using clove oil as the oil phase in its nanoemulsion formulation. Therefore, our developed FLU-NEG could be considered for further preclinical and clinical studies toward its journey to a successful marketed antifungal nanoemulsion formulation.

Funding

The project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under Grant No. (G: 388-249-1442). The authors, therefore, acknowledge with thanks DSR for technical and financial support.

Declarations

Conflicts of interest

On behalf of all the authors, the corresponding author states that there are no conflicts of interest.

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