Development and evaluation of clotrimazole microemulsions for topical application: Effects of HLB value of surfactant mixture and cosurfactant type on formulation design

Abstract

Clotrimazole is a synthetic imidazole with broad-spectrum antimycotic effect that has been widely used for the topical treatment for athlete's foot (tinea pedis), oropharyngeal and vulvovaginal candidiasis. The objective of the study was to develop a nano-delivery system containing to improve the penetration capacity of Clotrimazole. Microemulsions were formulated and the chemophysical properties, permeability through rat skin and irritancy examined by HET-CAM test of drug-loaded formulations and stability were evaluated. The average droplet size and viscosity of all clotrimazole-loaded microemulsion formulations were respectively between 126.7–228.13 nm with PDI value less than 0.31 and 24.53–155.16 mPa·s. The penetration capacity of clotrimazole was markedly improved by using microemulsion formulations as delivery carriers: the 24-h cumulative permeated amount from the optimized microemulsion was approximately three-fold higher than that from the clotrimazole solution (drug dissolved in 30 % ethanol) and about 6.5-fold higher than that from the commercial product. The HET-CAM test showed the irritancy on skin of designed clotrimazole-loaded formulation was acceptable compared to the positive control of 0.8 % paraformaldehyde aqueous solution. The stability studies showed that the physicochemical characteristics and residual drug percentage (about 95.3 %) of F2 clotrimazole-loaded formulation were fairly stable after thermodynamic and storage tests, indicating designed microemulsions as a delivery carrier could be considered as a potential strategy for clotrimazole topical dosage form deployment.

Graphical abstract

1. Introduction

Clotrimazole (1-((2-Chlorophenyl)diphenylmethyl)-1H–imidazole, molecular formula: C₂₂H₁₇ClN₂, MW: 344.84 g/mol) is a synthetic imidazole with potential antimycotic effect discovered in the 1960s. It is a hydrophobically active pharmaceutical ingredient with aqueous solubility of 0.49 mg/L, Log P of 6.1 and pKa 6.7.(Bolla et al., 2019; Ravani et al., 2013b; Santos et al., 2013) Clotrimazole inhibits microsomal cytochrome P450-dependent 14-α-lanosterol demethylation and leads to a reduction in biosynthesis of ergosterol in fungal form. Consumption of ergosterol disrupts normal membrane permeability and fluidity. (Crowley and Gallagher, 2014; Hitchcock, 1991; Hitchcock et al., 1990) Clotrimazole is a potent antifungal agent with a broad spectrum and is widely used for treatment of tinea cruris, tinea corporis and tinea pedis (athlete's foot) as well as oropharyngeal candidiasis and vulvovaginal, etc.(Gelone and O'Donnell, 2006) Currently, it is only used as a topical agent due to its limited oral absorption and systemic toxicity. Clotrimazole is available in conventional topical dosage forms such as cream, lotion and ointment; (Bolla et al., 2019; Crowley and Gallagher, 2014) however, its topical bioavailability is quite low, about 0.5–10 %, owing to its low aqueous solubility and poor permeability. (Bolla et al., 2019).

Consequently, numerous studies have attempted to improve the bioavailability by using novel drug delivery systems such as ethanolsomes, liposomes, microemulsions, nanogels, solid lipid nanoparticles and ufosomes etc., (Alam et al., 2017; Bolla et al., 2019; Esposito et al., 2013; Maheshwari et al., 2012; Manca et al., 2019; Ravani et al., 2013a) to increase the aqueous solubility and membrane permeability of clotrimazole. Among these drug delivery systems, microemulsions containing both aqueous and oil phases, surfactant and cosurfactant with nanoscale particle size about 200 nm have been applied as promising vehicles for transdermal and topical drug delivery due to their composition (can be used as an enhancer) and small particle size. Microemulsions can be formed by spontaneous emulsification, are easy to manufacture, and are convenient for future industrial scale-up (Chen et al., 2018; Hung et al., 2021; Lawrence and Rees, 2012; Scomoroscenco et al., 2021; Souto et al., 2022; Yasir Siddique et al., 2021) Furthermore, permeation enhancers incorporated in microemulsion systems have been reported to enhance dermal delivery by improving wetting of the skin surface, reducing interfacial tension at the formulation–skin interface, and inducing a moderate and reversible perturbation of stratum corneum lipid domains (Szumała and Macierzanka, 2022; Williams and Barry, 2012). At the same time, earlier studies have shown that appropriately designed microemulsions do not cause severe or irreversible barrier disruption and may even reduce membrane irritation caused by active pharmaceutical ingredients (APIs), despite usually containing relatively high levels of oil and surfactants (El Maghraby, 2008; Lin et al., 2018a; Spernath et al., 2007).

The effects of composition and proportion of clomatrimazole-loaded microemulsions on average droplet size, viscosity and permeability through rat skin (permeation rate (flux), deposition amount in skin at end of experimental (D_24h) and lag (LT)) times were evaluated by permeation study through rat skin. The applied irritancy on rat skin caused by formulations was determined by HET-CAM test. Additionally, the stability of clotrimazole-loaded formulations was also evaluated to validate the utility of the experimental formulations.

2. Materials and Methods

2.1. Materials and animals

Clotrimazole, Cremophor EL, Diazepam and Paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Capryol 90, Labrafac cc and Labrasol, Transcutol HP were purchased from Battefosse (Nanterre, France). Isopropyl myristate was obtained from Tokyo Chemical Industry (Tokyo, Japan). Kolliphor HS15 was obtained from BASF (Ludwigshafen, Germany). 1,2-propanediol, 1,3-butanediol, and 1,5-pentanediol were purchased from Alfa Aesar (Massachusetts, USA). Commercial clotrimazole cream (Canesten® 1 % w/w cream) was purchased from Bayer AG (Leverkusen, Germany). All other chemicals and solvents were of analytical reagent grade.

2.2. Solubility determination

Clotrimazole is a hydrophobically active therapeutic compound. To determine the suitable components for preparation of clotrimazole-loaded microemulsions, drug solubility in oil (Capryol, Peceol, Labrafac and Isopropyl myrisate) and cosurfactant (1,2-propanediol, 1,5-pentanediol, ethanol and transcutol) was determined. An excess of drug was taken in an Eppendorf container with 1 g of oil or cosurfactant, then the samples were placed in a horizontal shaker (for homogeneous mixing) at 25 °C for 24 h. These samples were then centrifuged at 9520 G for 10 mins, with the supernatant being collected and diluted with methanol. The diluted sample was analyzed for clotrimazole content by a modified HPLC method.(Das et al., 2021; Garcia Ferreira et al., 2019; Yasir Siddique et al., 2021).

2.3. Pseudoternary phase diagram construction

Despite the many microemulsions reported in the literature, the challenge for drug pharmaceutical formulation designers is to predict which type, proportion of oil and surfactant should be chosen for a particular application, as these vary significantly from case to case. For example, different oils have different required HLB values of surfactants that might have particular effects on phase behavior, so a pseudoternary phase diagram construction is rather necessary and important for the preparation of microemulsion systems.(Chen et al., 2018; Shinde et al., 2018; Tsai et al., 2023).

The water titration method was used to build a pseudoternary phase diagram. Oil and surfactant phases were blended in a ratio of 1:9 to 9:1 respectively, and the mixtures of oil and surfactant at certain weight ratios were titrated by adding distilled water drop-wise with vortex shaking; then, the mixture was judged visually as to whether it was a multiphase microemulsion or a two-phase mixture. The component percentages of micoremulsion were then calculated and the pseudoternary phase diagrams were constructed; and finally, the transparent ME areas were determined.

2.4. Clotrimazole-loaded formulations preparations

The composition of clotrimazole-loaded formulations is shown in Table 1. Accurately weighed quantities of oil phase (capryol, peceol), surfactants (Kolliphor, Labrasol, and Kolliphor/Labrasol mixture) and cosurfactant (1,2-propanediol, 1,3-butandiol, 1,5-pentanediol, Transcutol) were mixed thoroughly by vortex for 1 min at ambient temperature. Then, distilled water was added into the mixture by vortex for a further 1 min to obtain a homogeneous and transparent microemulsion. The 1 % clotrimazole was dissolved into the blank microemulsions by shaking for 24 h, then the clotrimazole-loaded microemulsion formulations were recorded for any change in turbidity or phase separation; finally, they were stored in brown glass bottles at ambient temperature until use.

Table 1.

The compositions of designed clotrimazole-loaded formulations.

Code	Surfactants (%)		HLB	Cosurfactants (%)
F1	Labrasol	25	12	Transcutol	28
F2	Kolliphor HS15/Labrasol	20	13	Transcutol	28
F3	Kolliphor HS15/Labrasol	20	14	Transcutol	28
F4	Kolliphor HS15	25	15	Transcutol	27
F5	Kolliphor HS15/Labrasol	20	13	Transcutol 1,2-propanediol	20 8
F6	Kolliphor HS15/Labrasol	20	13	Transcutol 1,3-butanediol	20 8
F7	Kolliphor HS15/Labrasol	20	13	Transcutol 1,5-pentanediol	20 8

Microemulsions containing 10 % oil, distilled water, surfactant, and cosurfactant.

2.5. Physicochemical properties determination

The viscosities of clotrimazole-loaded microemulsions were measured using a cone-plate of viscometer (Brookfield, Model LVDV-II, USA). Samples of 0.5 mL were placed in the cone-plate then heated, and the cone-plate temperature was maintained at 37 °C for 3 min. The viscosity measurement was recorded after 30 s at rotational speed of 100 rpm.

The average droplet size and polydispersity index (PI) of clotrimazole-loaded formulations were measured using a zetasizer analyzer (Malvern, 3000HSA, UK). The determination conditions included a scan angle of 90°, a wavelength of 658 nm, and a temperature of 25 °C. Samples were placed in a standard quartz cuvette to measure the mean droplet size and PI, with each sample conducted in triplicate and the average value was obtained.

2.6. In vitro skin permeation and drug deposition study

The transdermal permeation studies were used to determine the permeation capacity of clotrimazole-loaded microemulsions compared to clotrimazole ethanol solution (control group). The experiments were carried out by utilizing Franz diffusion compartments with an effective diffusion area of 3.46 cm², and the processes were as follows.(Hsieh et al., 2021).

The study protocol was reviewed and approved by the Kaohsiung Medical University Committee on Animal Use (Approval No. 110028). To prepare the belly skin of Sprague Dawley (SD) rat as barriers for permeability study, the animals were sacrificed and the hair of their abdominal region was shaved without causing any damage to the skin. The full-thickness skin was excised then the subcutaneous fat was carefully removed by surgical scissors. The barrier was set on top of the receptor chamber. Each receptor compartment was loaded with 20 mL mixture medium of ethanol/distilled water/transcutol at a ratio of 5/4/1 to maintain sink condition and stirred at 600 rpm with a temperature set at 37 ± 0.5 °C during the entire experiment. One mL each of the test formulation and the control group formulation (1 % clotrimazole dissolved in 30 % ethanol) was loaded evenly on the skin surface. At the predetermined time, 0.5 mL of the receiving fluid was collected from the receptor chamber, and then the same volume of fresh receptor medium was added back into the receptor chamber. The amount of clotrimazole was determined by a modified HPLC condition.(Das et al., 2021; Garcia Ferreira et al., 2019; Yasir Siddique et al., 2021).

At the end of the 24 h permeation test, the applied skin was carefully removed from the Franz cell and washed with deionized water 20 times to remove the residual drug on the surface of the smeared skin. The skin was then cut into small pieces and immersed in 4 mL receptor buffer, followed by extraction for 24 h with horizontal shaker at room temperature. After extraction, 1 mL of the extracted solution was collected and centrifuged at 9520 G for 10 min, then the supernatant of clotrimazole was quantified by modified HPLC method, using the Hitachi HPLC system (Hitachi, Tokyo, Japan) consisting of model L-7100 pump, L-5210 autosampler, model L-4000H detector, and Merck Lichrocart® RP-18e column (250 × 4 mm, 5 μm particle size).

The mobile phase consisted of 0.02 M phosphoric acid aqueous solution and methanol at ratios of 85/15 v/v. The flow rate was kept at 1.0 mL/min and the detection wavelength was set at 223 nm. The internal standard was 50 μg/mL diazepam dissolved in methanol. The analytical method successfully demonstrated linearity (1–100 μg/mL) from 1 to 200 μg/mL with a determination coefficient (R²) of 0.999, coefficient of variation of 5.66 %, and relative error of 4.76 %.

2.7. Irritancy

Hen's Egg Test – Chorioallantoic Membrane (HET-CAM) Test method has been widely used to evaluate the anti-irritation, anti-inflammatory potential and toxicity/ocular toxicity of materials or formulations;(Budai et al., 2021; Gonzalez-Pizarro et al., 2019; McKenzie et al., 2015; Ozturk et al., 2020). Accordingly, the HET-CAM test was performed herein to measure the irritation caused by the clotrimazole-loaded formulation, so 300 μL samples of test formulations were applied on the chorioallantoic membrane of fertilized chicken eggs at regular intervals of time. Various parameters like coagulation, hemorrhage, and vasoconstriction were detected during the experiment. Normal saline and 0.8 % paraformaldehyde aqueous solution were used as negative and positive (acceptable for topical administration) controls respectively. Irritation index was calculated by taking the sum of the scores of each injury as per the following equation:

$$\text{Irritation index}=\frac{\left(301-\mathrm{H}\right)\times 5}{300}+\frac{\left(301-\mathrm{V}\right)\times 7}{300}+\frac{\left(301-\mathrm{C}\right)\times 9}{300.}$$

where H, V and C indicate the times (s) when hemorrhaging, vasoconstriction and coagulation were observed respectively.

2.8. Stability study

The thermodynamic stability of clotrimazole-loaded formulation was evaluated by centrifugation at 1166 G rpm for 5 min and three cycles of freeze-thaw cycle test (samples were stored in amber glass bottles at temperatures of −21 °C and 25 °C, with storage time at each temperature not less than 48 h.(Baboota et al., 2007; Hung et al., 2021) The appearance was visually observed, then average droplet size and viscosity of the tested formulations were measured.

The stability of each clotrimazole-loaded formulation was evaluated by storage-tested formulation in an amber glass bottle at 25 ± 2 °C and 60 ± 5 % RH for 60 days. Samples were taken at scheduled time intervals for appearance and residual drug content analysis.

2.9. Data analysis

The cumulative permeated amount at 24 h of clotrimazole was plotted as a function of time (h) with the permeation rate (flux, μg/(cm² h) of the clotrimazole calculated by linear regression analysis and lag time (LT, h) defined as the first detected time. The flux and clotrimazole deposition in the skin over time (D_24h, μg/cm²) were used to assess the enhancement effect of microemulsion formulations. All experiments were performed in triplicate (n = 3) for each study to ensure reproducibility. Data was expressed as mean ± standard deviation of three determinations. The differences between the formulations were analyzed using One-way ANOVA and Tukey's multiple comparisons test by GraphPad Prism version 7 (GraphPad Software, Inc., California, USA), with p value <0.05 considered significant.

3. Results and discussion

3.1. Solubility

The solubility of clotrimazole in different oil and surfactant samples is given in Table 2. Clotrimazole is a hydrophobically active therapeutic compound with a poor aqueous solubility of 0.49 μg/mL.(Saadatfar et al., 2018) The drug loading of each formulation will affect the total weight of the formulation used to deliver a therapeutic dose of drug. Choosing a suitable oil phase is very important as it affects the choice of other ingredients and microemulsion formulations, mainly in the case of o/w microemulsions. In general, the oil with the greatest solubilizing capacity for the selected drug is selected as the oil phase of the microemulsion formulation. As shown in Table 2, peceol had the highest solubility. Peceol containing long-chain fatty acids (C18:2) is an oily solvent used as a solubilizer for lipophilic-active pharmaceutical ingredients (APIs) in oral and topical formulations,(Grove et al., 2006; Mouri et al., 2014) is an approved pharmaceutical excipient, hence was selected as oil phase in this study.

Table 2.

Solubility of clotrimazole in different oil and cosurfactant formulations.

Solvent/Surfactant	Solubility(mg/mL)
Capryol 90	39.87 ± 0.36
Peceol	42.33 ± 2.03
Labrafac cc	5.49 ± 0.12
Isopropyl myrisate	1.86 ± 0.09
Ethanol	64.39 ± 1.58
1,2-Propanediol	33.51 ± 0.52
1,5-Pentanediol	21.93 ± 1.25
Transcutol HP	83.21 ± 2.52

Among these cosurfactants, Transcutol had the highest solubility. Transcutol is a kind of diethylene glycol monoethyl ether that can be used as a powerful solubilizer, penetrative and bioavailable enhancer in oral and parenteral routes, and is an excellent cosolvent in self-emulsifying formulations. Furthermore, it is available in approved drug products for oral and topical administration due to its safety profile, so it was chosen as the co-surfactant in this study. (Guo et al., 2019; Souto et al., 2022)

3.2. Pseudoternary phase diagram

Mixing of oil/water/surfactant/cosurfactant quantities in a suitable ratio is the key factor for microemulsion formations.(Trotta et al., 1995) Construction of pseudoternary phase diagrams is a useful method to determine the appropriate composition of microemulsion formulations,(Ali et al., 2017) using Kolliphor HS15/Labrasol, Trancutol, and different oils (capyrol and peceol). As shown in

Fig. 1, the blue-shaded area corresponds to compositions that yielded clear, transparent and single-phase systems, indicating a high likelihood of microemulsion formation. As indicated in

Fig. 1.

The pseudoternary phase diagrams of the different compositions of oil /surfactant /cosurfactant mixture.

Fig. 1 a and b, the construction microemulsion area by using peceol was larger than that of using Capryol. In using peceol, kolliphor HS15/labrasol, and different cosurfactants (Trancutol, 1,2 propanediol, 1,5-pentanediol and 1,3-butanediol) to construct pseudoternary phase diagrams (

Fig. 1 c, d and e), it was found that the type of cosurfactant also affected the construction microemulsion area. The 1,2 propanediol formulation showed the construction microemulsion area slightly decreased via increase in the C chain length (C3 ∼ C5) of cosurfactant. Among these cosurfactants, using transcutol as cosurfactant could obtain a larger area; hence, peceol and transcutol were selected for clotrimazole-loaded microemulsion preparations.

3.3. Physicochemical characteristics

As shown in Table 3, the average droplet size of all drug-loaded microemulsions ranged from 156.9 to 228.13 nm with PDI less than 0.31. Additionally, it was found that the average droplet size increased when part of the transcutol was replaced by other short-chain alcohols (F2 vs F5, F6, F7). This phenomenon might be due to the better emulsification effect of transcutol although the nanoscale microemulsion formulation could help advance drug delivery,(Kohli and Alpar, 2004; Lin et al., 2018b; Sabeti et al., 2014) where PDI value less than 0.31 indicated homogeneity of the clotrimazole-loaded microemulsion formulations. The viscosity of formulations containing single surfactant (F1 and F4) was higher than that of microemulsions prepared by mixed surfactants (F2 and F3); additionally, the viscosity did not show significantly change when part of the transcutol was replaced by other short-chain alcohols (F2 vs F5,F6,F7).

Table 3.

The droplet size, polydispersity index (PDI) and viscosity of designed clotrimazole-loaded formulations.

Formulation code	Particle size (nm)	PDI	Viscosity (mPa·s)
F1	123.70 ± 1.65	0.17 ± 0.00	19.60 ± 2.61
F2	126.70 ± 1.56	0.22 ± 0.01	24.53 ± 0.47
F3	227.37 ± 0.21	0.21 ± 0.02	34.57 ± 1.55
F4	174.90 ± 1.90	0.27 ± 0.01	79.17 ± 0.59
F5	228.13 ± 2.69	0.19 ± 0.01	26.83 ± 0.06
F6	203.20 ± 2.88	0.20 ± 0.02	26.70 ± 0.62
F7	182.13 ± 2.19	0.21 ± 0.02	24.53 ± 0.59

3.4. In vitro permeation study

Surfactants are key ingredients in the formation of microemulsions. Their type, HLB value and added amount significantly affect the characteristics and performance of the microemulsion.(Constantinides and Scalart, 1997; Gullapalli and Sheth, 1999; Vu et al., 2021; Wu et al., 2001) Kolliphor® HS 15 consists of 12-hydroxystearic acid and polyethylene glycol (HLB = 15), is a non-ionic, potent emulsifier and solubilizer with approved use in parenteral and ophthalmic drugs.(Liu et al., 2016; Rowe, 2020) Labrasol® consists of a small fraction of mono-, di- and tri-glycerides and mainly PEG-8 (MW 400) mono- and diesters of caprylic (C8) and capric (C10) acids. It is a nonionic surfactant with HLB value of 12, used as solubilizer in topical formulations, including foam, gel, microemulsion, solutions, and suspensions.(Rowe, 2020) Combinations of surfactants can exhibit several advantages such as lower CMC, improved thermodynamic and kinetic stabilities, specific HLB value, enhanced drug-loading capacity, improved capacity for modifications, accurate size control and reduced requirement for surface-active agents;(Cagel et al., 2017; Li and Chiang, 2012; Zhang et al., 2018) therefore, Labrasol® and Kolliphor® HS 15 and their combinations with different HLB values were used to prepare drug-loaded microemulsions. A permeation study was conducted to evaluate the enhancement effect by using microemulsion systems as a carrier vis-à-vis the control group (clotrimazole dissolved in 30 % ethanol) and commercial product (Canesten 1 % cream).

As indicated in Fig. 2, the permeation capacity of most microemulsion formulations were higher than that of the control group, indicating microemulsions possess topical enhancer effect. Similarly, numerous studies have validated that microemulsion systems can provide excellent solubilization and transdermal transport effect as the combined effect of the components of the microemulsion increase the thermodynamic activity of the whole system. Additionally, microemulsion systems with nanoscale size can reduce the interface tension between skin lipid and vehicle, leading to faster permeation.(Azeem et al., 2009; Hung et al., 2021; Jakab et al., 2018; Teichmann et al., 2007; Yuan et al., 2006)

Fig. 2.

The permeation profiles of clotrimazole-loaded formulations.(a. Formulations F1–F4 with different HLB values compared with the control formulation. b. Comparison of formulations F3, F5–F7 with different cosurfactants.)

In considering the effect of HLB value of surfactant on the drug permeability

Table 4 F1 ∼ F4), it was observed that this value significantly affected the permeability of clotrimazole. Better performance on skin permeability (highest permeation rate, highest D24h and shortened LT) was observed when the microemulsion was prepared by surfactant with HLB 13 (F2). Likewise, earlier studies have reported that the permeability of active pharmaceutical ingredients on rat and mouse skin was highly dependent on the HLB of the surfactant in the formulation.(Chen et al., 2012; Vu et al., 2021; Wu et al., 2001; Wu et al., 2010)

Table 4.

The permeation parameters of clotrimazole-loaded formulations.

	Flux (μg/(cm2h))	D24h(μg/cm2)	Lag time (h)
Control (1 %)	12.41 ± 9.90	44.12 ± 33.76	3.33 ± 2.31
Commercial product (1 %)	5.85 ± 2.85	10.19 ± 8.04	4.33 ± 1.53
F1	19.00 ± 1.90	33.13 ± 6.73	2.33 ± 0.58
F2	111.17 ± 99.53	61.00 ± 23.68	0.67 ± 0.29
F3	80.59 ± 42.20	37.10 ± 18.84	1.33 ± 0.58
F4	13.10 ± 3.47	15.51 ± 2.47	3.33 ± 0.58
F5	115.11 ± 2.03	19.37 ± 4.92	2.33 ± 0.58
F6	7.85 ± 0.15	14.94 ± 5.18	2.33 ± 0.58
F7	17.62 ± 6.65	14.38 ± 4.83	2.33 ± 0.58

To reduce the added amount of transcutol, various alkanols (including 1,2-propanediol (C3), 1,3-butanediol(C5), and 1,5-pentanediol (C5)) instead of partial transcutol of 8 % to prepare drug-loaded microemulsions were used. As shown in

Table 4 F2, F5 ∼ F7, the drug permeability decreased slightly with the increase of C chain length (C3 ∼ C5) of cosurfactant, with the transcutol showing the highest permeability. This result might be due to transcutol, diethylene glycol monoethyl ether (C6), being a powerful skin penetration enhancer;(Osborne and Musakhanian, 2018; Ryu et al., 2020; Ustundag Okur et al., 2020). Nevertheless, the clotrimazole-loaded microemulsion (F2) showed the best performance on permeability, hence it was selected to conduct the following irritancy test and stability studies.

3.5. Irritancy evaluation

The irritancy evaluation of experimental formulations is indicated in Table 5. and Fig. 3. Normal saline was used as negative control. No sign of hemorrhaging, vasoconstriction and coagulation phenomena were observed and the irritation index was 0 (Fig. 3A). The 0.8 % paraformaldehyde aqueous solution was used as a positive control to demonstrate acceptable irritation of transdermal formulations.(Azeem et al., 2012; Lin et al., 2018b) As shown in Fig. 3B; some hemorrhaging, vasoconstriction and coagulation was observed after 30 s application and the irritation index was 12.65 ± 0.50. As shown in Fig. 3C and D, in chorioallantoic membranes treated with clotrimazole-loaded microemulsion and blank microemulsion, slight coagulation phenomena were observed at about 3.2 min and 4.1 min with irritation indices of 9.28 ± 0.8 and 5.94 ± 0.61 respectively, indicating that the designed microemulsion was considered compatible for topical application.

Table 5.

Irritation indices of the negative control (0.9 % NaCl), positive control (0.8 % formaldehyde), clotrimazole-loaded microemulsion (F2), and blank microemulsion without clotrimazole (F2-Blank). Data are presented as mean ± SD (n = 3).

Formulation	Irritation index	Image after 5 min
0.9 % NaCl (Negative control)	0
0.8 % Formaldehyde (Positive control)	12.65 ± 0.50
F2-Blank	5.94 ± 0.61
F2	9.28 ± 0.80

Fig. 3.

Irritancy evaluation of experimental formulations on the chorioallantoic membrane of fertilized chicken eggs. (A: Negative control-normal saline; B: positive control-0.8 % formaldehyde; C: blank microemulsion without clotrimazole (F2-blank); D: clotrimazole-loaded micoremulsion (F2).

3.6. Stability

Thermodynamic stability of clotrimazole microemulsion was quickly confirmed by centrifugation at 1166 G for 5 min and examination of three freeze-thawing cycles (−21 °C and 25 °C). After these tests, the appearance of the formulation did not change and no phase separation or precipitation was observed. As shown in Table 6, the viscosity and droplet size revealed almost no change after the centrifugation test; meanwhile, droplet size and viscosity showed slight change after freeze-thawing cycle tests, with results indicating the clotrimazole-loaded microemulsion was quite stable. Similarly, previous studies have reported that microemulsion systems are quite stable because their nanoscale droplet size and low interfacial tension make them thermodynamically stable.(Azeem et al., 2009; Lawrence and Rees, 2012; Tsai et al., 2016)

Table 6.

The average droplet size and viscosity of clotrimazole-loaded formulation before and after stability tests. (mean ± SD, n = 3).

	Droplet size nm	Viscosity mPa·s
Before test	126.70 ± 1.55	24.53 ± 0.47
Centrifugation test	124.67 ± 1.47	23.47 ± 0.32
Freeze-thawing cycle test	110.27 ± 0.50	38.37 ± 3.36

After storage, the appearance of clotrimazole-loaded microemulsion showed no significant changes, no drug crystallization was observed and no change in viscosity, but a slight increase in average droplet size from 126.70 nm to 177.50 nm was found (Table 7). The residual amount percentages of clotrimazole were 95.3 ± 5.3, indicating that the experimental preparations were quite stable.

Table 7.

Physicochemical properties of the F2 clotrimazole-loaded microemulsion following 2 months of storage (mean ± SD, n = 3).

Day	Particle size (nm)	PDI	Clotrimazole amount (%)	Viscosity (mPa·s)
0	126.70 ± 1.56	0.22 ± 0.01	100.0	24.53 ± 0.47
8	135.13 ± 1.27	0.23 ± 0.01	99.6	26.00 ± 4.19
30	154.93 ± 1.51	0.21 ± 0.02	100.1	23.53 ± 0.85
60	177.50 ± 2.84	0.19 ± 0.02	95.3	20.83 ± 0.29

4. Conclusions

In this study, peceol and transcutol had greatest solubility of clotrimazole, hence both were selected to prepare drug-loaded microemulsion formulations. The average droplet size and viscosity of experimental clotrimazole-loaded microemulsions ranged from 126.70 to 228.13 nm with less PDI indicating that all formulations were nanoscaled and homogeneously colloidal systems, so the permeability of clotrimazole was improved by using microemulsions as carriers. The type, HLB value of used surfactants and cosurfactants significantly affected the characterization and permeability of drug-loaded micormeulsions. The F2 formulation containing kolliphor/labrasol mixture with HLB 13 showed the highest permeability. In the HET-CAN test, the irritation index of F2 formulation (9.28 ± 0.8) was lower than that of the positive control (12.65 ± 0.50), indicating that the designed microemulsion was considered compatible for topical application. The stability studies demonstrated that the average droplet size, viscosity and

residual drug percentage (about 95.3 %) of F2 clotrimazole-loaded formulation were all fairly stable after thermodynamic and storage tests. Overall, the designed clotrimazole-loaded microemulsion demonstrated enhanced topical performance compared with both the clotrimazole solution and the commercial product, with acceptable irritation and preliminary physical stability, indicating potential safety and stability for short-term, localized topical application; however, its safety profile and long-term stability still need to be confirmed by further in vivo and extended-duration studies.

CRediT authorship contribution statement

Chih-Wun Fang: Funding acquisition, Formal analysis, Data curation. Yu-Wen Lin: Writing – original draft, Formal analysis, Data curation. I-Hui Chiu: Investigation, Data curation. Pao-Chu Wu: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All data generated or analyzed during this study are included in this published article.