Original Article

In Vitro Skin Permeation and Antifungal Activity of Naftifine Microemulsions


  • Meryem Sedef ERDAL
  • Aslı GÜRBÜZ
  • Sevgi GÜNGÖR
  • Yıldız ÖZSOY

Received Date: 13.07.2018 Accepted Date: 18.10.2018 Turk J Pharm Sci 2020;17(1):43-48 PMID: 32454759


Microemulsions are fluid, isotropic, colloidal systems that have been widely studied as drug delivery systems. The percutaneous transport of active agents can be enhanced by their microemulsion formulation when compared to conventional formulations. The purpose of this study was to evaluate naftifine-loaded microemulsions with the objective of improving the skin permeation of the drug.

Materials and Methods:

Microemulsions comprising oleic acid (oil phase), Kolliphor EL or Kolliphor RH40 (surfactant), Transcutol (co-surfactant), and water were prepared and physicochemical characterization was performed. In vitro skin permeation of naftifine from microemulsions was investigated and compared with that of its conventional commercial formulation. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to evaluate the interaction between the microemulsions and the stratum corneum lipids. Candida albicans American Type Culture Collection (ATCC) 10231 and Candida parapsilosis were used to evaluate the antifungal susceptibility of the naftifine-loaded microemulsions.


The microemulsion formulation containing Kolliphor RH40 as co-surfactant increased naftifine permeation through pig skin significantly when compared with the commercial topical formulation (p<0.05). ATR-FTIR spectroscopy showed that microemulsions increased the fluidity of the stratum corneum lipid bilayers. Drug-loaded microemulsions possessed superior antifungal activity against Candida albicans ATCC 10231 and Candida parapsilosis.


This study demonstrated that microemulsions could be suggested as an alternative topical carrier with potential for enhanced skin delivery of naftifine.

Keywords: Naftifine, microemulsion, colloidal drug carrier system, topical antifungal


Naftifine hydrochloride (naftifine) is a synthetic, topical allylamine antifungal compound that is effective in the management of superficial dermatomycoses. Naftifine shows primarily fungicidal activity against Candida species including Candida albicans (C. albicans) and Candida parapsilosis (C. parapsilosis) and it has been proven to be especially effective in moderate to severe cutaneous candidiasis caused by these two species.1,2

The strongly lipophilic nature of naftifine (log p: 5.4) leads to its accumulation in high concentrations within the stratum corneum, which is the outermost barrier layer of the skin. However, the therapeutic efficacy of antifungal drugs applied topically depends on the ability of the formulation to overcome the stratum corneum barrier and improve the uptake in deeper skin layers.3 Therefore, a drug delivery system aiming to increase the topical penetration of naftifine is important to enhance its local antifungal efficacy. Microemulsions are colloidal nano-sized carriers with a dynamic microstructure that form spontaneously by combining appropriate amounts of oil, water, surfactant, and a co-surfactant.4,5 They are thermodynamically stable and isotropic systems and they have been extensively explored for a variety of pharmaceutical applications, including dermal and transdermal drug delivery.6,7,8,9 The high solubilization capacity of microemulsions and their ability to modify the diffusional barrier of the stratum corneum can facilitate drug penetration into deeper skin layers compared to conventional formulations.10,11 The aim of the present study was to formulate and evaluate the in vitro skin permeation and antifungal activity of naftifine-loaded microemulsion formulations comprising oleic acid (oil phase), Kolliphor EL or Kolliphor RH40 (surfactant), Transcutol (co-surfactant), and water. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to evaluate the interaction between the microemulsions and the stratum corneum lipids. The antifungal susceptibility of naftifine-loaded microemulsions was assessed against two Candida species, namely C. albicans American Type Culture Collection (ATCC) 10231 and C. parapsilosis.



Naftifine was kindly provided by Eczacıbaşı Drug Company (İstanbul, Turkey), and the polyoxyl castor oils (Kolliphor® EL and Kolliphor® RH40) were kind gifts from BASF (Limburgerhof, Germany). Oleic acid was purchased from Sigma (St. Louis, MO, USA). Diethylene glycol monoethyl ether (Transcutol P®) was kindly provided by Gattefossé (Lyon, France). RPMI-1640 medium and 4-morpholinepropanesulfonic acid (MOPS) were purchased from Sigma (St. Louis, MO, USA). All other chemicals used in the study were of analytical grade.

Preparation and characterization of microemulsions

We previously reported the preparation and physicochemical characterization of microemulsions loaded with naftifine.12 Briefly, the concentration range of components necessary for the formation of microemulsions was determined by construction of pseudoternary phase diagrams based on water titration at ambient temperature (25±0.5°C). The oil phase to surfactant/cosurfactant mixture ratio varied from 1:9 to 9:1 (w/w) and the ratio of surfactant/co-surfactant (km) was fixed as 1:2. The microemulsions were formed spontaneously at room temperature as a clear monophasic liquid (Table 1). Naftifine was solubilized in the oil phase at 1% concentration before preparation of the microemulsions. In order to verify the isotropic nature of the optimized microemulsions ME1 and ME2, cross-polarized light microscopy (Olympus BX51 U-AN 360, Tokyo, Japan) imaging was performed. The droplet size and the polydispersity index of the microemulsions were determined using dynamic light scattering (Malvern Zetasizer Nano ZS, Malvern Instruments, Malvern, Worcestershire, UK) after pre-filtering (0.45 mm, Millex) of the microemulsions. The electric conductivity and pH of the microemulsions were measured using a combined device (EuTech PC 700; Eutech Instruments, Landsmeer, the Netherlands) at room temperature. The refractive index of the microemulsions was determined by a digital Abbe Refractometer (Atogo Co. Ltd., Tokyo, Japan). In order to measure the viscosity and rheological behavior of the microemulsions a Brookfield Rheometer (Brookfield DV3THACJ0, Middleboro, MA, USA) with a cone-plate measuring device was used. All measurements were conducted in triplicate in a temperature-controlled environment at 25°C.

In vitro permeation study

Preparation of skin

Pig skin (obtained from a local slaughterhouse) was carefully cleaned of fat and muscle and then dermatomed to a thickness of 750 µm (Zimmer Electric Dermatome, Warsaw, IN, USA). The integrity of the skin was confirmed by transepidermal water loss measurement with an open chamber device (Tewameter TM 300; Courage + Khazaka Electronic, Cologne, Germany) as stated in the literature.4

Permeation experiments

In vitro permeation experiments were performed using Franz type diffusion cells (diffusion area of 1.76 cm2, Permegear V6A Stirrer, Hellertown, PA, USA). Pig skin was placed on the receiver chambers with the stratum corneum facing upwards and then the donor chambers were clamped in place. The receptor chamber was filled with 12 mL of phosphate buffer pH 5.0 containing 30% ethanol to ensure sink conditions. The receptor phase maintained at 37°C under constant stirring (300 rpm) with a magnetic bar. Microemulsion formulation (500 µL) was placed in the donor compartments of diffusion cells. The compartments were sealed by Parafilm M® (Bemis, Oshkosh, WI, USA) immediately after the addition of the formulation in order to prevent evaporation. The permeation of naftifine from microemulsions was followed for 8 h. The commercial topical cream formulation naftifine (Exoderil) was used as the control formulation. Samples of 500 µL were removed at appropriate time intervals for analysis and replaced immediately with fresh receptor medium. The samples were analyzed for their drug content by HPLC. Permeation profiles of naftifine were constructed by plotting time (h) against the cumulative amount of the drug (µg/cm2) as measured in the receptor medium. Steady state flux (Jss, mg/cm2.h) was calculated from the steady state part of the curve. The effect of microemulsions as carrier on dermal administration [enhancement ratio, (ER)] is determined by the following equation:

ER=Flux from microemulsion formulation/Flux from commercial formulation

Data were determined from the average of at least six experiments.

HPLC analysis

For the HPLC (Shimadzu Model LC 20AT, Kyoto, Japan) analysis of naftifine we used a reversed phase C18 column (4.6 mm×150 mm, 5 µm, Merck, Darmstadt, Germany) preceded by a guard column (4×4 mm, 5 µm, Merck, Darmstadt, Germany). The mobile phase consisted of acetonitrile:tetrahydrofuran:tetramethyl-ammonium hydroxide buffer (pH 7.8) (62:10:28) and the flow rate was fixed at 1.2 mL/min. The wavelength of detection was set at 280 nm. The limit of quantification was found to be 0.025 µg/mL. The method was validated for selectivity, linearity, accuracy, and precision. It was found to be linear between the concentration range of 0.025-100 µg/mL with a high correlation coefficient (r2=0.999) and was precise (intra- and interday variation 2%) and accurate (mean recovery 99%).

ATR-FTIR spectroscopy

The effect of microemulsions ME1 and ME2 and marketed formulation (Exoderil) on pig skin was investigated by ATR-FTIR spectroscopy. For this purpose, the surface of 4 cm2 pieces of dermatomed pig skin was treated with 300 µL of ME1, ME2, or commercial cream for 8 h. At the end of the treatment period ATR-FTIR spectra were recorded as the average of 40 scans with a spectral resolution of 4 cm-1 in the 4000-650 cm-1 range using a Perkin Elmer Spectrum 100 FTIR spectrometer (UK) equipped with a ZnSe ATR crystal. Attention was focused on characterizing the occurrence of peaks near 2850 and 2920 cm-1, which were due to the symmetric and asymmetric CH2 stretching vibrations, respectively. In order to minimize intersample variation, the same piece of skin before treatment was used for normalization.13

In vitro antifungal activity


C. albicans ATCC 10231 and C. parapsilosis were used to evaluate the antifungal susceptibility of naftifine-loaded microemulsions. The yeasts were cultured and maintained on Sabouraud dextrose agar for 24 h at 30°C prior to testing.


The minimum inhibitory concentration (MIC) of microemulsion formulations was determined by the microdilution technique using 96-well microplates. Serial two-fold dilutions ranging from 5000 to 5 mg/mL were done following the Clinical Laboratory Standards Institute (CLSI) recommendations, rule CLSI M27-A3 for yeasts.14,15 RPMI-1640 medium (Sigma, St. Louis, MO, USA) buffered to pH 7.0 with MOPS (Sigma, St. Louis, MO, USA) was used for the dilutions. The inoculum was prepared with RPMI-1640 medium, using 24 h cultures adjusted to a turbidity equivalent to a 0.5 McFarland standard, to give a final concentration of 0.5×103 to 2.5×103 cfu/mL in the test tray. The trays were covered and placed in plastic bags to prevent evaporation and incubated at 35°C for 46-50 h. The MICs were defined as the lowest concentration of compound giving complete inhibition of visible growth.16 Naftifine was used as reference antifungal for yeast. The MIC values of the naftifine were within the accuracy range in CLSI throughout the study.14 All tests were performed in triplicate.

Statistical analysis

All results are the mean ± standard deviation of at least three experiments. The statistical analysis was performed using Tukey’s multiple comparison test, with p<0.05 as level of significance (GraphPad Prism 5 Software, La Jolla, CA, USA).


Physicochemical characterization of microemulsions

The small droplet size (7.34±0.03-11.17±0.25 nm) together with its narrow distribution (0.15±0.01-0.19±0.01) indicated the homogeneous nature of the microemulsions and explained the clarity and isotropicity of the systems. The conductivity values revealed that the microemulsions were in oil in water (o/w) form and naftifine in salt form led to increased conductivity. The pH values of the microemulsions were reasonable for their cutaneous application (4.26±0.01-5.71±0.03). The refractive indices of the microemulsions confirmed the transparency of the formulations. The apparent viscosity values of the microemulsions were 70.76±0.12-83.58±0.46 Cp and they showed Newtonian flow behavior.12

In vitro permeation study

The permeability of naftifine from microemulsions was measured in vitro using excised pig skin. The cumulative percutaneous penetrating amounts of naftifine from the microemulsions and from the commercial cream are illustrated in Figure 1 and the permeation parameters are summarized in Table 2. Flux of naftifine from the microemulsion ME1 (109.99±1.58 µg.cm-2.h-1) was significantly higher than that of ME2 (45.59±2.10 µg.cm-2.h-1) and commercial cream (21.32±1.56 µg.cm-2.h-1) (p<0.05). It has been reported that percutaneous permeation is an essential requirement for satisfactory topically applied antifungals in order to achieve therapeutic concentration for deep fungal infections.17 The ability of a microemulsion to improve the transport of drugs to and across the skin is largely influenced by the composition and concentration of the microemulsion components, as well as the internal structure and type of the microemulsion used (o/w, w/o, or bicontinuous systems).10,18 Various oils have been used as components of the oil phase in microemulsions. Oleic acid is one of the most frequently selected ones and it has been demonstrated that oleic acid containing microemulsions increased the penetration of lipophilic drugs through the skin.19,20 Transcutol P is a well-known penetration enhancer in skin delivery and by its amphiphilic nature it has been known to reduce interfacial tension, resulting in balanced microemulsion systems.21 Although both of the microemulsions contained oleic acid and Transcutol P, the highest permeation was obtained by ME1, demonstrating a possible synergistic effect of the added surfactant Kolliphor® RH40. The content of the surfactant mixture in ME1 influenced the permeation rate of naftifine. In addition, the lower viscosity of ME1 (75.62±0.24 cP) than ME2 (83.58±0.46 cP) might provide better mobility for the drug molecules and consequently a faster release with a diffusion-controlled mechanism.12,22 The permeability coefficient (Kp) of naftifine in ME1 was fivefold higher than the value obtained for the commercial formulation. The microemulsion formulation ME1 had the highest ERFlux (5.16) with a ~5-fold enhancement rate compared to the commercial formulation. It was also found that the lag times were shortened when microemulsions were used (Table 2). This result was consistent with previous studies reporting that microemulsions remarkably shortened the lag time of formulations.23

ATR-FTIR spectroscopy

In Figure 2, the spectra of untreated pig skin (control) and pig skin treated with either microemulsion formulations (ME1 and ME2) or commercial cream (Exoderil) are shown. The most characteristic bands are the asymmetric and symmetric CH2 stretching absorbances at 2920 cm-1 and 2850 cm-1 arising from endogenous skin lipids. We observed that both the CH2 stretching vibration bands shifted towards higher wave numbers after microemulsion application, which could be attributed to a decrease in the conformational order of the stratum corneum lipids. Thus, disorder of the barrier might further have enhanced permeation of naftifine across the stratum corneum.17,24

In vitro antifungal activity of naftifine microemulsions

Naftifine is a topical antifungal agent having shown a specific inhibitory effect in sterol biosynthesis of C. albicans and C. parapsilosis.25 The in vitro antifungal activity of naftifine-loaded microemulsions ME1 and ME2 against C. albicans ATTC 10231 and C. parapsilosis (port blood) was higher than that of the commercial topical cream (Exoderil) as can be seen in Figure 3. These results proved that microemulsions had superior fungicidal efficacies due to considerable penetration of the drug into fungal cell membranes.26 Jadhav et al.27 observed greater activity of fluconazole-loaded microemulsions than of conventional gel formulations and this was attributed to the better diffusion of the drug provided by the presence of surfactants and oil phase. Similarly, El-Hadidy et al.28 (2012) observed considerably higher antifungal activity with voriconazole microemulsions against C. albicans when compared to the supersaturated solution of the same drug. Microemulsion formulation M1, which contains Kolliphor EL as surfactant, showed higher antifungal activity compared to that of the microemulsion M2 and Exoderil. Kolliphor EL is a nonionic surfactant and it is a well-known formulation vehicle for various hydrophobic drugs, including anesthetics, photosensitizers, sedatives, immunosuppressive agents, and anticancer drugs.29 Kolliphor EL-containing microemulsions have been considered effective topical carriers to deliver the antifungal agents oxiconazole and terconazole.30,31 The composition of microemulsion M1 in our study might alter the permeation of fungal cells more than the formulation M2 and commercial cream, which consequently resulted in higher penetration of microemulsion globules containing naftifine through fungal cell walls.


Our results showed significant naftifine permeation enhancement by a microemulsion formulation comprising oleic acid, Kolliphor RH40, Transcutol, and water when compared with its conventional topical formulation. ATR-FTIR spectra revealed that microemulsions increased the fluidity of the stratum corneum lipid bilayers. Drug-loaded microemulsions showed superior antifungal activity against two Candida species. In conclusion, this study demonstrated that a microemulsion formulation of naftifine could be a potential alternative to the conventional formulation of the drug.


This work was supported by the Scientific Research Projects Coordination Unit of İstanbul University (Project number NP-512 and BYP-2017-26514).

Conflict of Interest: No conflict of interest was declared by the authors.

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