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RESEARCH ARTICLE

Antifungal Activity of Eugenol Loaded Electrospun PAN Nanofiber Mats Against Candida Albicans kourosh Semnania, Masoomeh Shams-Ghahfarokhia*, Mehran Afrashi b, Aref Fakhralib and Dariush Semnani b* a b

Department of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-331, Iran; Department of Textile Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

ARTICLE HISTORY Received: May 27, 2017 Revised: February 09, 2018 Accepted: February 14, 2018 DOI: 10.2174/1567201815666180226120436

Abstract: Eugenol, as the major phenolic component of clove essential oil due to its desired properties in medical field, was loaded into polyacrylonitrile (PAN) nanofibers with various percentages. Our main purpose in this study was to determine the in vitro antifungal activity of eugenol loaded on PAN nanofibers against Candida albicans as the most common causative agent for candidiasis. Also, the surface morphology and the mechanical properties of nanofibers were studied by scanning electron microscope (SEM) and a tensile tester, respectively. The average diameters of nanofibers in pure PAN nanofibers were found to be 127 nm. The results showed that the average diameter of nanofibers after increasing the eugenol ratio (from 127 to 179-218 nm) was increased. Drug release profile of the samples was gradual and was completed after 150 hours. According to the results, these nanofiber mats loaded with eugenol can be used for treating cutaneous mucocutaneous candidiasis in high risk patients as a coating on a fabric substrate or temporary wound dressing.

Keywords: Candida albicans, eugenol, electrospinning, antifungal activity, PAN nanofiber. 1. INTRODUCTION Fungal infections pose a serious threat to human health especially to immunocompromised patients. Recently, the occurrence of systemic candidiasis has consistently increased [1]. C. albicans is one of dimorphic fungus that can grow in a yeast phase depending on environmental conditions [2]. Oral infections and vaginal candidiasis are two major types of infections in humans that are caused by this fungus [3]. Vulvovaginal candidiasis (VVC) is a common disease in women [4]. Antifungal therapy can be effective in controlling and treating this infection. It can be carried out with topical or systemic agents [5]. Eugenol is a naturally occurring phenolic component from clove oil, and is widely used in pharmaceutical, cosmetics, antimicrobial and antifungal properties [6-8]. Nevertheless, it has some disadvantages such as low sensitivity, poor water solubility, highly volatility, instability and sensitivity to oxygen. These disadvantages lead to difficulties in to formulating it into aqueous solution [9]. *Address correspondence to these authors at the Department of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115331, Iran; Tel: +98-21-82884505; Fax: +98-21-82884555; E-mail: [email protected] & [email protected] Department of Textile Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran; Tel: +98 31 3391 5006; Fax: +98 31 33912444; E-mail: [email protected] 1567-2018/18 $58.00+.00

In recent years, nanoencapsulation method has become a technique that has numerous benefits, such as ease of handling, enhanced stability, protection against oxidation, controlled release, consecutive delivery of multiple active ingredients, improved water solubility, reduced toxicity and side effects of hydrophobic ingredients, and also enhanced bioavailability and efficacy of drugs [10, 11]. The development of polymeric fibers for many applications has been studied in the past years [12]. For example, encapsulation of eugenol into polymeric nanoparticles has been studied to enhance its stability against light oxidation [13, 14]. Drug delivery system (DDS) for control drug release was developed by encapsulating drugs into a specific delivery system in order to provide a controllable release of certain ratio of drugs at a proper time or targeted site over the duration from wide length of period time [15, 16]. The use of fibrous mats in the biomedical field is now gaining accrued interest due to a number of advantages such as improved therapeutic index, possibility for localized delivery and reduced toxicity of drugs. Electrospun nanofibers method exhibits various unique features, which can be controlled by varying the processing and solution parameters [17, 18]. This method is capable of producing nano and micro fibers from most polymeric solutions [19]. In this process, a polymer liquid is first loaded into a container with a nozzle by a certain feed rate and then charged with a high © 2018 Bentham Science Publishers

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electrical potential across a finite distance between the nozzle and the conductive collection device [20]. As the jet travels to the collector, the solvent evaporates and ultrafine fibers are obtained on the collector. The morphology of the electrospun fibrous mat is influenced by a number of factors, such as electrospun solution properties and processing conditions [21]. These fibrous mats could be used as scaffolding materials [22] and carriers for the delivery of drugs and active agents [23]. Efficiency of electrospun fibrous mats as drug delivery vehicles, relates to their ability to incorporate high drug loading capacity, high surface area, interconnecting porous structure and the simplicity of fabricating the required form of the delivery vehicles [24]. Drug release from electrospun nanofibrous mats can occur via various mechanisms namely diffusion, desorption and degradation [25]. Due to the side effects of oral administration of drugs in the treatment of C. albicans infections, the aim of this study was to investigate the possibility of using eugenol in a drug delivery system made of electrospun PAN nanofibers which can be produced cost-effectively with good applicability as wound dressings for local usage. Also, this polymer was selected to local antifungal therapy due to high drug loading capacity, high mechanical properties and long-term release characteristics [26]. Various tests were carried out such as characterization of the obtained nanofiber mats by Fouriertransform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), strength test and spectrophotometry to investigate the performance of nanofibrous drug loaded mats. 2. EXPERIMENTAL 2.1. Materials Polyacrylonitrile powder (PAN, Mw=100000 g/mol) was received from Polyacryl Co. (Isfahan, Iran) as polymer. Dimethylsulfoxide (DMSO) from Merck Co. (Germany) was used as the solvent. Eugenol was purchased from Sigma Aldrich. C. albicans ATCC 10231 from the Pathogenic Fungi Culture Collection of Pasteur Institute of Iran was used in this study.

Semnani et al.

2.3. Characterization For measuring the diameters of nanofibers, scanning electron microscope (SEM, XL30, Philips, Voltage: 15 kV, Secondary Electron Mode) and Sputter coater SCD 005 by BAL-TEC were used. Then, the average diameter of nanofibers was obtained by measuring the diameters of 100 randomly selected nanofibers from SEM images by using Image J software (National Institute of Health, USA). The drug loaded in PAN nanofibrous mat were analyzed by FTIR (BOMEM FTIR MB-series, MB-100 (Hartmann & Braun, Canada)) spectral studies method. The scan no. was selected to be 8 and wavenumber resolution was 8 cm1 for spectral range of 4000 to 400 cm1. The electrospun nanofibers were cut into small pieces and mixed with KBr powder and then compressed into the disc by applying pressure. For data collection, Omnic software provided by Nicolet was used. Tensile properties of produced nanofibers were examined using a tensile tester (Zwick 1446-60) in a constant rate of elongation mode (CRE). Each sample was tested 5 times, and the results are reported. The selected gauge length and crosshead speed were 20 mm and 10 mm/min, respectively. The dimension of the samples were: 2 cm long and 0.5 cm wide (at a relative humidity of 65±5% and temperature of 25±2 °C) [27, 28]. The amount of drug in the release aliquots was measured by UV–visible spectrophotometer (UVmini-1240, Shimadzu, Japan). First, the standard solutions were prepared to determine the calibration curve. For this purpose, different concentrations of drug solutions were prepared by diluting a stock solution. The absorbance values of these solutions were measured at a max of (283 nm) and the calibration curve was obtained and shown in Fig. (1). For drug release assay, a certain amount of drug loaded samples were immersed in PBS at 37 °C. By drug releasing from the fibrous mat, the concentration of the drug was increased gradually into the buffer solution.

2.2. Nanofiber Production First, PAN polymer solution was prepared using 15 wt% of polymer concentration in DMSO and stirred for about 5 hours at room temperature. Then, eugenol with initial polymer weight percentages of 0%, 10%, 20%, 30% and 40% were added into the polymer solution 30 min before electrospinning. Briefly, the electrospinning setup used in this study consisted of a high-voltage power supply and positive displacement syringes (TOP-5300, Japan) with a needle tip gauge of 22. Also, a grounded drum covered with aluminum foil was used as the collector. The distance between the collector and needle tip was 18 cm, electrospinning voltage applied to the needle was 18 kV, and the feeding rate of the polymer solutions was 0.35 ml/h. The collector rotation speed was about 60 RPM to collect uniform nanofiber mats. Needle was fixed and the width of each tape of mat was 5 cm. Electrospinnig was carried out at a temperature of 25°C and constant relative humidity of 35%.

Fig. (1). Eugenol calibration curve.

2.4. Antifungal Assay Tests The eugenol loaded electrospun PAN nanofiber mats and fluconazole (FCZ) antifungal activity were determined by the Clinical Laboratory Standards Institute (CLSI) via disk diffusion method (M44-A4 document guidelines). C. albicans ATCC 10231 was cultured on Sabouraud dextrose agar containing chloramphenicol (0.005%) and incubated at 37 °C for 48 h. Cells were washed three times with distilled water and yeast cells were adjusted to the turbidity of a 0.5

Antifungal Activity of Eugenol Loaded Electrospun PAN Nanofiber Mats

McFarland standard (approximately 105 cells ml-1). 10 l of a suspension of yeast cells were swab on Mueller Hinton agar medium (Merck Co., Germany) supplemented with 2% glucose and 0.5 μg/mL methylene blue. PAN/eugenol nanofiber mats including 10-40% eugenol concentration in comparison with fluconazole disk (FCZ; 25 μg) were placed on the plates. The plates were incubated at 30 °C and checked daily for clear zones of inhibition around fungal colonies up to 48 hours [29]. 3. RESULTS AND DISCUSSION 3.1. Morphology of the Nanofibers Table 1 shows SEM images of PAN nanofibers and PAN/eugenol nanofibers at different percentages of eugenol. It can be observed that PAN nanofibers are uniform and without beads. The average diameter of PAN nanofibers was found to be 127±21 nm. By adding 10 % of eugenol into PAN nanofibers the diameter of nanofibers increased from 127±21 to 179±25 nm. The results show that by increasing eugenol from 10 to 20 and then from 20 to 30, the diameter of nanofiber was increased to 187±32 and 218±30, respectively. Also, the diameter of nanofiber in the nanofiber mats composed of 40 % eugenol was 212±29 nm. This phenomenon may be due to increasing of the viscosity of the polymer solution as a result of eugenol addition. Also, it can be seen that these nanofibers are without beads. Nanofiber diameter distribution of these mats is demonstrated in Table 1 confirming the uniformity of the nanofibers. The results are in good agreement with the results reported in previous works [30, 31, 32]. 3.2. FTIR Study The FTIR spectrometer was used to analyze the chemical reaction between PAN and eugenol. The spectrum of pure PAN nanofibers is shown in Fig. (2-b). Sharp peaks demon-

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strated in this spectrum are attributed to (-CN) nitrile group (2246 cm-1) and (C=O) carbonyl (1733 cm-1) groups. The absorbance peak at 2934 cm-1 demonstrates the stretching of CH in the structure, as well. In FTIR spectrum of eugenol (Fig. 2-a), the peaks at 1034 and 1272 cm-1 are due to the stretching vibration of symmetric and asymmetric C-O-C, confirming presence of the methoxy group of eugenol. Also, the absorbance peaks at 2840 and 2938 cm-1 are attributed to vibration of alkyl side chain of this drug. Other characteristic band in eugenol is at 1451 cm-1 (due to presence of the – CH). Moreover, (Fig. 2-c) shows the spectra of PAN loaded with eugenol. It can be observed that all functional groups of PAN and eugenol are present in the spectrum. This test also demonstrates the compatibility between eugenol and electrospinning PAN nanofibers. Moreover, there are no new absorption bands in the PAN/eugenol spectra and as a result, no chemical interaction between the polymer and the additive was found. Good agreement was found between the results of the present work and the experimental results in previous works [33]. 3.3. Tensile Properties Tensile and elongation at break of nanofibers mats were examined and results were plotted in Fig. (3). Tensile and elongation at break of pure PAN nanofibers mats were found to be 3.07 MPa and 29.09%, respectively. Results show that by adding 10% eugenol into PAN nanofibers, tensile properties decreased a little compared to pure PAN nanofibers mats, from 3.07 MPa and 29.09 % to 2.95 MPa and 27.56%, respectively. Also, the tensile strength and elongation at break of nanofibers mat containing 20% eugenol decreased to 2.08 MPa and 23.07 %, respectively. It can be observed that tensile properties for other samples were decreased, too. This trend might be attributed to length reduction of the polymer chain which is caused by eugenol molecules penetration and agglomeration in the PAN polymer chains.

Fig. (2). Spectra of pure PAN nanofibers, eugenol drug and PAN/eugenol nanofibers.

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Table 1.

Semnani et al.

SEM images analysis of PAN and PAN/eugenol Nanofiber Mats.

Sample

Typical SEM Image

Nanofiber Diameter Distribution

PAN nanofiber without eugenol

127±21

PAN mat containing 10% eugenol

179±25

PAN mat containing 20% eugenol

187±32

PAN mat containing 30% eugenol

218±30

PAN mat containing 40% eugenol

212±29

Antifungal Activity of Eugenol Loaded Electrospun PAN Nanofiber Mats

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Fig. (3). Relation between eugenol concentrations with stress and strain of the PAN/eugenol nanofiber mats.

3.4. Drug Release Drug release profile of the eugenol from the PAN nanofibers was investigated by immersing of PAN/eugenol nanofiber mats into PBS at 37° C. The curves of drug release are demonstrated in Fig. (4). It can be observed that the release of eugenol from nanofibers was gradual. At first, the release rate was fast. This rapid release is due to diffusion of eugenol from surface of the nanofibers. According to the curves, 50% of eugenol was released from nanofibers after approximately 24 h. Then, the release of eugenol continued slowly until 150 h. By increasing the concentration of eugenol in the mats, the rapid release of drug was increased initially. The gradual release of eugenol-loaded PAN nanofibers is attributed to non-degradability of PAN polymer into PBS. Similar results were obtained when tamoxifen citrate, an anticancer drug, was loaded into PAN fibers for local cancer treatment [25].

Fig. (4). Accumulative release profiles of PAN/eugenol nanofibers mats.

Fig. (5). Representative images of agar plates containing pure PAN and PAN/eugenol impregnated disks: a,f: non drug-loaded nanofiber and control sample (fuloconazole), b-e : drug loaded nanofibers containing 10-40% eugenol, respectively.

3.5. Antifungal Activity Diameter of inhibition zone (DIZ) in disk diffusion method was used to analyze antifungal activity of PAN loaded with eugenol in constant mass/dimension. It can be observed in Fig. (5) that pure PAN nanofiber as a control sample have no antifungal activity whereas by loading the eugenol into nanofibers, these samples demonstrated antifungal activity. A few dimension deviations in the Fig. (5) is due to shrinkage of some of the nanofibers mats in condition

of disk diffusion method. Diameter of inhibition zone of PAN/eugenol nanofibers was brought together in Table 2. Diameter of inhibition zone of PAN nanofibers containing of 10% and 20% eugenol was 9 and 10 mm. Inhibition zone diameter of the drug containing PAN nanofibers is due to the diffusion of eugenol from PAN nanofibers structure. By increasing eugenol ratio to 30% and 40% into PAN nanofibers, the diameter of inhibition zone increased to 14 and 18 mm.

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Table 2.

Semnani et al.

Antifungal activity of PAN/eugenol Nanofiber Mats.

ACKNOWLEDGEMENTS This study has been financially supported by the Research Deputy of Tarbiat Modares University and Isfahan University of Technology.

Sample

DIZ (mm)

PAN

0

PAN/eugenol (10%)

9

PAN/eugenol (20%)

10

PAN/eugenol (30%)

14

REFERENCES

PAN/eugenol (40%)

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[1]

fuloconazole

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PATIENT CONSENT Declared none.

[2]

In the given electrospinning conditions, producing bead free and uniform PAN/eugenol nanofibers containing up to 40% eugenol was not possible. We were only able to produce uniform and smooth nanofibres by using eugenol concentrations until 40%. So, PAN nanofibrous mat containing 40% eugenol could be an optimal choice of localized drug delivery of eugenol due to its uniform morphology, acceptable tensile properties, suitable drug release and antifungal activity.

[3] [4]

[5]

CONCLUSION C. albicans is a member of fungi family that can cause various diseases and whose treatment usually has unsolicited side effects. Recently, drug release systems were used for treatment of various diseases. Therefore, eugenol was used as an antifungal drug in this study and various ratios of it were added to PAN nanofibers. The SEM images showed that both, the pure PAN and PAN loaded with eugenol were uniform and free of beads. Presence of eugenol inside the PAN nanofibers was confirmed by FTIR analysis and results of this test demonstrated that the inner-structure of the polymers was not changed by adding eugenol. On the other hand, eugenol was in polymers chains, physically. The tensile testing of the samples showed a decrease in tensile properties by adding of eugenol to the PAN nanofibers. Results of drug release demonstrated that the profile of eugenol release was slow and completed after 150 hours. Consequently, suitable antifungal activity of samples indicates that the drug loaded nanofibers mats can be used as promising drug carriers for fungi treatment. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Not applicable.

[6]

[7]

[8]

[9]

[10] [11]

[12] [13]

[14]

HUMAN AND ANIMAL RIGHTS No Animals/Humans were used for studies that are base of this research.

[15]

CONSENT FOR PUBLICATION

[16]

Not applicable. [17]

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

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