fabrication of a novel scaffold of clotrimazole-microemulsion-containing nanofibers using an...

Accepted Manuscript

Title: Fabrication of a novel scaffold ofclotrimazole-microemulsion-containing nanofibers using anelectrospinning process for oral candidiasis applications

Author: Prasopchai Tonglairoum Tanasait NgawhirunpatTheerasak Rojanarata Ruchadaporn Kaomongkolgit PraneetOpanasopit

PII: S0927-7765(14)00685-7DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2014.12.009Reference: COLSUB 6784

To appear in: Colloids and Surfaces B: Biointerfaces

Received date: 14-9-2014Revised date: 3-11-2014Accepted date: 5-12-2014

Please cite this article as: P. Tonglairoum, T. Ngawhirunpat, T. Rojanarata, R.Kaomongkolgit, P. Opanasopit, Fabrication of a novel scaffold of clotrimazole-microemulsion-containing nanofibers using an electrospinning process fororal candidiasis applications, Colloids and Surfaces B: Biointerfaces (2014),http://dx.doi.org/10.1016/j.colsurfb.2014.12.009

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

http://dx.doi.org/doi:10.1016/j.colsurfb.2014.12.009

http://dx.doi.org/10.1016/j.colsurfb.2014.12.009

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Original Research Article1

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Fabrication of a novel scaffold of clotrimazole-microemulsion-containing nanofibers using 3

an electrospinning process for oral candidiasis applications4

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Prasopchai Tonglairouma, Tanasait Ngawhirunpata, Theerasak Rojanarataa, Ruchadaporn 6

Kaomongkolgitb and Praneet Opanasopita,*7

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a Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy, 9

Silpakorn University, Nakhon Pathom, Thailand10

b Department of Oral Diagnosis, Faculty of Dentistry, Naresuan University, Phitsanulok, 11

Thailand12

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* To whom correspondence should be sent:14

Praneet Opanasopit15

Faculty of Pharmacy, Silpakorn University, 16

Nakhon Pathom, 73000, Thailand17

Tel: 66-34-25580018

Fax: 66-34-25094119

E-mail address: [email protected]; [email protected]

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Running head: Clotrimazole-microemulsion incorporated nanofibers24

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Abstract 26

Clotrimazole (CZ)-loaded microemulsion-containing nanofiber mats were developed 27

as an alternative for oral candidiasis applications. The microemulsion was composed of oleic 28

acid (O), Tween 80 (T80), and a co-surfactant such as benzyl alcohol (BzOH), ethyl alcohol 29

(EtOH) or isopropyl alcohol (IPA). The nanofiber mats were obtained by electrospinning a 30

blended solution of a CZ-loaded microemulsion and a mixed polymer solution of 2% w/v 31

chitosan (CS) and 10% w/v polyvinyl alcohol (PVA) at a weight ratio of 30:70. The 32

nanofiber mats were characterized using various analytical techniques. The entrapment 33

efficiency, drug release, antifungal activity and cytotoxicity were investigated. The average 34

diameter of the nanofiber mats was in the range of 105.91-125.56 nm. The differential 35

scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD) results revealed the 36

amorphous state of the CZ-loaded microemulsions incorporated into the nanofiber mats. The 37

entrapment efficiency of CZ in the mats was approximately 72.58-98.10%, depended on the 38

microemulsion formulation. The release experiment demonstrated different CZ release 39

characteristics from nanofiber mats prepared using different CZ-loaded microemulsions. The 40

extent of drug release from the fiber mats at 4 h was approximately 64.81-74.15%. The 41

release kinetics appeared to follow Higuchi’s model. In comparison with CZ lozenges (10 42

mg), the nanofiber mats exhibited more rapid killing activity. Moreover, the nanofiber mats 43

demonstrated desirable mucoadhesive properties and were safe for 2 h. Therefore, the 44

nanofiber mats have the potential to be promising candidates for oral candidiasis applications. 45

Keywords: clotrimazole; microemulsion; chitosan; nanofibers; oral candidiasis46

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1. Introduction51

Candida is a normal commensal organism in the oral cavity of most healthy individuals 52

and becomes overgrown and causes opportunistic infections in human immunodeficiency 53

virus (HIV) and immunocompromised patients [1-2]. Oropharyngeal candidiasis (OPC) is a 54

common, treatable oral mucosal infection caused by the overgrowth of Candida spp. The 55

principal species associated with OPC is C. albicans [3]. Several groups of effective 56

antifungal drugs are currently available and can be used for OPC management. The most 57

conventional and efficient drugs are polyenes and azoles [4-5]. Topical dosage forms are 58

becoming increasingly popular in OPC therapy due to greater compliance and reduced side 59

effects [6-7].60

Clotrimazole (CZ), 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole, is an imidazole-61

type antimycotic drug that is highly lipophilic and is utilized for the treatment of fungal 62

infections of skin and mucus diseases [8-10]. However, the compound exhibits poor aqueous 63

solubility (0.49 µg/ml) [11] that might affect its antimycotic activity. Therefore, the 64

enhancement of the CZ solubility and release rate is necessary for rapid antimycotic activity. 65

A few studies have attempted to improve the solubility of CZ by complexation with 66

cyclodextrin [10, 12]. However, this formulation exhibits low drug-loading and incomplete 67

drug dissolution. The solubility of CZ is improved at acidic pHs, but CZ is unstable at acidic 68

pHs. Nevertheless, the lipophilicity of CZ allows CZ to serve as a potential candidate for a 69

lipid-based system such as microemulsion and nanoemulsion. Additionally, for oral 70

candidiasis applications, CZ concentrations should not only achieve high levels rapidly but 71

also be maintained at levels above the minimum inhibitory concentration (MIC) for an 72

extended period of time [13-14]. Conventional formulations for local oral delivery include 73

lozenges, mouthwash, oral gels and suspensions. The drug release from these formulations 74

usually exhibits an initial burst release and rapidly declines to sub-therapeutic concentrations 75

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[15]. An ideal dosage form for the treatment of oral candidiasis is one that provides sustained 76

drug release, is retained in the oral cavity and produces antifungal effects for prolonged 77

periods of time. These characteristics are possible if the drug delivery system demonstrates 78

sustained release, as well as mucoadhesive properties [16-18].79

Recently, nanofiber-based scaffolds have become popular. These materials exhibit 80

outstanding characteristics such as low density, high porosity, large specific surface areas and 81

very small pore sizes. Nanofibers have been applied in many fields (tissue engineering, drug 82

delivery systems, wound dressing, filtration, etc.) [18-20]. Electrospinning is one of the most 83

promising techniques for the fabrication of nanofibrous scaffolds because of its efficiency, 84

simplicity in fabrication, versatility, low cost and potential to be scaled up to industrial levels85

[21-22]. 86

In recent years, electrospinning of emulsions has generated growing interest [23-24]. 87

The electrospinning of emulsions can produce composite nanofibers with nanoscale drug 88

particles surrounded by emulsifiers and distributed in a biocompatible nanofiber or 89

biodegradable polymer. The properties of the composite nanofiber can be adjusted by 90

selecting the appropriate polymer, drug, solvent, emulsifier and electrospinning process 91

conditions [25]. In this study, CZ-loaded microemulsion-containing chitosan-92

ethylenediaminetetraacetic acid/polyvinyl alcohol (CS-EDTA/PVA) nanofibers were prepared 93

using different microemulsion formulations. The microemulsion formulations were selected 94

from the literature. The microemulsions were subsequently incorporated into electrospun 95

nanofibers using CS-EDTA/PVA, which is mucoadhesive and electrospinnable, as the fiber-96

forming polymer to increase the contact time with the oral mucosa. The nanofibers were 97

investigated for entrapment efficiency, drug release, antifungal activity and cytotoxicity.98

2. Materials and Methods99

2.1 Materials100

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Clotrimazole (CZ), chitosan (degree of deacetylation 0.85, MW 110 kDa), 101

ethylenediaminetetraacetic acid and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium 102

bromide (MTT) were purchased from Sigma-Chemical Co. (St. Louis, MO, USA). Polyvinyl 103

alcohol (PVA) (degree of polymerization 1600, degree of hydrolysis 97.5-99.5 mol%) 104

was purchased from Fluka, Switzerland. Oleic acid was purchased from Fluka Chemie AG 105

(Seelze, Germany). Medium chain triglycerides (Estasan® 3580) were obtained from 106

Uniqema Asia Pacific (Kuala Lumpur, Malaysia). Sabouraud dextrose broth was purchased 107

from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Human gingival 108

fibroblasts (HGF) were obtained from the Faculty of Dentistry, Naresuan University, 109

Thailand. Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA, penicillin-110

streptomycin antibiotics and fetal bovine serum (FBS) were obtained from GIBCO-111

Invitrogen (Grand Island, NY, USA). All other reagents and solvents were of analytical grade 112

and were used without further purification. 113

2.2 Phase diagram construction114

Pseudo-ternary phase diagrams were produced to obtain the concentration range of 115

components for the existing range of microemulsions. The microemulsion systems consisted 116

of oil, surfactant and cosurfactant. Oleic acid (O) was used as the oil phase, Tween 80 (T80) 117

was used as the surfactant and the cosurfactant was varied. The benzyl alcohol (BzOH), ethyl 118

alcohol (EtOH) or isopropyl alcohol (IPA) was used as the cosurfactant in F1, F2 and F3, 119

respectively. The weight ratio of surfactant to cosurfactant (Smix) was 3:1. For each pseudo-120

ternary phase diagram at a specific Smix weight ratio, the mixtures of oil and Smix were 121

prepared at the weight ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 6:4, 7:3, 8:2, 9:1 and 0:10. These 122

mixtures were titrated dropwise with water under magnetic stirring. After being equilibrated, 123

the systems were visually characterized. Transparent fluid systems were characterized as 124

microemulsions. 125

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2.3 Preparation of CZ-loaded microemulsion systems126

Based on the generated pseudo-ternary phase diagrams, an oil:Smix ratio of 1:2 was 127

selected for the CZ-loaded microemulsion formulations. The selected variables were the type 128

of cosurfactant (BzOH, EtOH and IPA for F1, F2 and F3, respectively). Oil and Smix were 129

mixed vigorously under magnetic stirring, and CZ (25% w/w to oily phase) was added to this 130

oily phase and mixed. Then, the mixture was heated at 70°C until the CZ was completely 131

dissolved (< 5 min). Both the physical and chemical stability of CZ in the formulation was 132

determined by visual observation and HPLC analysis [26]. Afterwards, water was133

incorporated for self-microemulsification. The Particle size was determined immediately 134

without dilution whereas Zeta potential were determined by diluted with various USP buffer 135

of pH 4, 7 and 9 using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). All 136

samples were measured in triplicate at room temperature.137

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2.4 Electrospinning process139

The 2% w/v CS solution was prepared by dissolving CS and EDTA in distilled water at 140

a weight ratio of 2:1. The 10% w/v PVA solution was prepared by dissolving PVA in 141

distilled water at 80C, and then the solution was stirred for 4 h. The CS-EDTA solution was 142

mixed with a PVA solution at a weight ratio of 30:70 [27]. The blended polymer solutions143

were mixed with CZ-loaded microemulsions at 40% w/w to polymer. The mixtures were 144

stirred for 12 h at room temperature. The viscosity and conductivity of the mixed solutions 145

were determined using a Brookfield viscometer (DV-III ultra, Brookfield Engineering 146

Laboratories, USA) and a conductivity meter (Eutech Instruments Pte Ltd, Singapore), 147

respectively. The spinning solution was placed in a 5 mL glass syringe connected to a 148

stainless steel needle with a 0.9 mm inner diameter. The needle was connected to the emitting 149

electrode of positive polarity from a Gamma High Voltage Research device. The 150

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electrospinning process was conducted at 25 °C, with a fixed applied voltage of 15 kV, a 15-151

cm distance between the tip and the collector, and a feeding rate of 0.3 ml/h. The electrospun 152

nanofibers were collected on aluminum foil covering the rotating collector.153

2.5 Characterization of the nanofibers154

2.5.1 Scanning electron microscope (SEM)155

The morphological appearances and diameters of the nanofiber mats were observed 156

using a scanning electron microscope (SEM, Camscan Mx2000, England). For this process, a 157

small section of the fiber was sputtered with a thin layer of gold before the SEM 158

observations. The average diameter of the fibers was measured using image analysis software159

(JMicroVision V.1.2.7, Switzerland).160

2.5.2 Fourier transform infrared spectrophotometry (FT-IR)161

The chemical structure of the fibers was characterized using a Fourier transform 162

infrared spectrophotometer (FT-IR, Nicolet 4700, USA) with a wave number range of 400–163

4000 cm−1. The fiber samples were ground and pressed into KBr dishes prior to the FT-IR 164

analysis.165

2.5.3 Differential scanning calorimetry (DSC) 166

A differential scanning calorimeter (Pyris Diamond DSC, PerkinElmer instrument, 167

USA) was used to determine the thermal behavior of the nanofiber mats and the physical 168

status of CZ in the nanofiber mats. The experiments were conducted using dry samples, under 169

nitrogen flow, weighing approximately 5 mg. The DSC traces were recorded from 50 to 170

250°C at 10°C/min.171

2.5.4 Powder X-ray diffractometry (PXRD)172

Powder X-ray diffraction analysis (PXRD, Miniflex II, Rigaku, Japan) of the samples 173

was performed to investigate the physical state of the CZ in the nanofiber mats using nickel-174

filtered Cu radiation generated in a sealed tube operated at 30 kv and 15 mA. The diffraction 175

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patterns of the nanofiber mats were recorded in the scan range of 5–45, with a scan speed of 176

5°Cmin-1.177

2.5.5 Mechanical characterizations178

Tensile testing of the nanofiber was performed using a texture analyzer by applying a 179

5 kg load cell equipped with tensile grip holder. All samples were cut into rectangle shapes 180

with dimensions of 25×5 mm2. The sample thicknesses of these samples ranged from 20-30 181

µm.182

2.5.6 Ex vivo mucoadhesion study183

The ex vivo mucoadhesion study was performed using a texture analyzer device 184

equipped with a 5 kg load cell. The porcine cheek pouch was used as the model surface for 185

bioadhesion testing. After the cheek pouch was excised and trimmed evenly, it was then 186

washed in simulated salivary fluid (2.38 g Na2HPO4, 0.19 g KH2PO4, and 8 g of NaCl per 187

liter of distilled water adjusted with the phosphoric acid to pH 6.80.05) [28] and then used 188

immediately. The nanofiber mats were cut into circular shapes with a diameter of 13.7 cm2189

and were fixed to a cylindrical perspex support (diameter 2 cm; length 4 cm; surface area 190

3.14 cm2) using double-side adhesive tape. The perspex support was then screwed onto the 191

upper probe of the instrument. During the measurement, 500 µL of simulated salivary fluid 192

was distributed onto the surface of the tissue. The probe was lowered at a speed of 2 mm/s to 193

come in contact with the tissue at a force of 0.03 N for a contact time of 15 min. 194

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2.6 Determination of CZ content196

The total amounts of CZ incorporated in the CZ-loaded microemulsion-containing 197

nanofiber mats were quantified in triplicate using HPLC (Agilent Technologies, USA) with a 198

detector operating at 215 mm, a Phenomenex® C18 column (150 x 4.60 mm, 5 µm particle 199

size) and a C18 guard column. The elution process was performed using a solvent system 200

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composed of methanol and ammonium carbonate buffer solution (75:25) at 2 ml/min.201

Accurately weighed samples (1 mg) of the fiber mats were dissolved in 1 mL of methanol 202

and were continuously shaken in an incubator (Orbital Shaking Incubator Model: SI4) at 150 203

rpm for 24 h. The experiments were performed in triplicate. The entrapment efficiency (%) 204

and loading capacity (%) were calculated according to equations (1) and (2), respectively:205

% Entrapment efficiency = (Pt/ Lt) X 100 (1)206

where Pt is the amount of CZ embedded in the nanofiber mats and Lt is the theoretical 207

amount of CZ (from the feeding solution) incorporated into the nanofiber mats.208

% Loading capacity = (Pt/ Mt) X 100 (2) 209

where Pt is the amount of CZ embedded in the nanofiber mats and Mt is the weight of 210

nanofiber mats.211

2.7 In vitro release212

The in vitro release studies were adapted from those of Singh et al. [29]. Briefly, 10 mg 213

of the CZ-loaded microemulsion-containing nanofiber mats was placed in a 100 mL bottle 214

containing 50 mL of artificial saliva (pH 6.8) with 20% PEG-400 that was incubated at 37°C 215

and shaken at 150 rpm. In contrast, the CZ lozenges (10 mg CZ, Candinas troche; Thailand 216

Jan Laboratories) were placed in a 1000 mL bottle containing 500 mL of artificial saliva (pH 217

6.8) with 20% PEG-400 that was incubated at 37°C and shaken at 150 rpm. To determine the 218

amount of CZ released from the fiber mats and CZ lozenges after a given interval, an aliquot219

(3.0 ml) of the release medium solution was withdrawn and replaced with the same volume of 220

fresh medium to maintain a constant volume. The amounts of CZ in the sample solutions 221

were analyzed by HPLC (Agilent Technologies, USA). The experiments were conducted in 222

triplicate.223

2.8 Antifungal activities of the nanofiber mats224

2.8.1 Candida strains and inoculum preparation225

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The ATCC 90028 strain of oral candida, Candida albicans, was used in this study. 226

Sabouraud dextrose broth (SDB) was used to revive the culture. An active culture was 227

prepared by inoculating fresh nutrient broth medium with a loop full of cells from stock 228

cultures at 37 ºC overnight. The candida suspensions were diluted in SDB and 229

spectrophotometrically standardized to 1×106 CFU/mL (Perkin Elmer Lambda 2, Germany).230

2.8.2 Time-kill curve studies231

The time kill analyses were adapted from a previously described protocol [30] using 232

SDB. Ten microliters of the adjusted inoculum suspension (approximately 2×106 CFU/ml) 233

was dispensed into a plastic Eppendorf tube containing 1 mL of SDB, providing the starting 234

inoculum of approximately 2×104 CFU/ml. The samples of the different CZ-loaded 235

microemulsion-containing nanofiber mats or CZ lozenges (10 mg, Candinas troche; Thailand 236

Jan Laboratories) with an equivalent CZ content (final concentration of 1.0 mg/mL) were 237

incubated with Candida suspensions under agitation at 37°C. After the predetermined contact 238

times (5, 15, 30, 60 and 120 min), small aliquots of the samples were removed, and the 239

microorganisms were counted by spreading each sample onto an SDA agar plate. The plates 240

were incubated for 24 h, and the viable colonies were assessed. The kill curves were 241

constructed by plotting the CFU/mL surviving at each time point in the presence and absence 242

of the nanofiber mats or lozenges.243

2.9 Evaluation of cytotoxicity244

The cytotoxicity of the CZ-loaded microemulsion-containing nanofiber mats was 245

evaluated to assess their biological compatibility. The human gingival fibroblast (HGF) cells 246

used in this experiment were primary cultures obtained from healthy marginal gingival tissue 247

obtained during the surgical extraction of third molars in three healthy donors. Ethical 248

approval for the study was obtained from Naresuan University. The HGF cells were cultured 249

in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum 250

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(FBS), 1% antibiotic and antimycotic solutions. The cells were seeded in 96-well plates at a 251

density of 1×104 cells/mL and were cultured under a humidified atmosphere of 5% CO2, 99% 252

relative humidity (RH) and 37 °C. When the culture reached confluence, the cells were 253

treated with the CZ-loaded microemulsion-containing nanofiber mats. The cytotoxicity tests 254

were performed based on a procedure adapted from the ISO10993-5 standard testing method 255

(direct contact) [31]. The specimens were sterilized under an ultraviolet lamp for 1 h before 256

the cytotoxicity tests. Then, the CZ-loaded microemulsion-containing nanofiber mats 257

(equivalent to a final CZ concentration of 1 mg/ml) were added into each well of 48-well 258

plates containing 250 µL of medium. After 2 or 24 h, the nanofiber mats and the media were 259

removed. Finally, the cells were incubated with 100 µl of MTT-containing cell media (1 260

mg/ml) for 4 h. The cell medium was removed, the cells were rinsed with a phosphate buffer 261

(pH 7.4), and the formazan crystals that formed in the living cells were dissolved in 100 µl of 262

DMSO per well. Cell viability (%) was calculated based on the absorbance at 550 nm 263

determined using a microplate reader (Universal Microplate Analyzer, Model AOPUS01 and 264

AI53601, Packard BioScience, CT, USA). The viability of non-treated control cells was 265

arbitrarily defined as 100%.266

2.10 Statistical analysis 267

All experimental measurements were collected in triplicate. The values are expressed as 268

the mean standard deviation (SD). The statistical significance of the differences in each 269

experiment was examined using one-way analysis of variance (ANOVA), followed by a least 270

significant difference (LSD) post hoc test. The differences were significant at p < 0.05.271

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3. Results and Discussion273

3.1 Phase diagram construction274

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Pseudo-ternary phase diagrams were produced to obtain the concentration range of 275

components for the existing range of microemulsions. After the microemulsion regions in the 276

phase diagram were identified (transparent fluid systems), the appropriate oil, surfactant, 277

cosurfactant and water weight ratios used in the microemulsions were chosen from the 278

constructed phase diagrams (data not shown). The selected percent composition for all the 279

microemulsion systems was 30.32% w/w oleic acid oil (O), 9.06% w/w water, 60.62% w/w 280

Smix at a surfactant (T80) to cosurfactant weight ratio of 3:1. The F1, F2 and F3 281

microemulsion formulations were generated using benzyl alcohol (BzOH), ethanol (EtOH) 282

and isopropranol (IPA), respectively, as the cosurfactant.283

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3.2 Microemulsion evaluation285

Both the physical and chemical stability of CZ in the microemulsion formulation was 286

determined. The color of the CZ-loaded oily phase was not altered, and the recrystallization 287

or precipitation of CZ when stored at room temperature was not found in all formulations by 288

visual observation. Chemical stability due to thermal degradation (heated at 70°C for < 5 289

min) was another concern. No significant changes in drug content or degradation peaks were 290

observed by the HPLC method (data not shown). These results were in agreement with a 291

previous study, which indicated that CZ was resistant to thermal degradation (heated at 80°C 292

for 8 h) [26]. Three formulations (F1, F2 and F3) of microemulsions with and without CZ 293

were evaluated by measuring the particle size and Zeta potential, as listed in Table 1. The 294

average particle size and Zeta potential of the microemulsions were in the range of 57.80-295

88.11 nm and -58.73-2.45 mV, respectively. The results revealed that the type of alcohol 296

added to the solution as a cosurfactant did not significantly affect the particle size or Zeta 297

potential of the microemulsions. Moreover, the addition of CZ (25% w/w to oil phase) did 298

not significantly affect the particle size the particle size of the microemulsions but did alter 299

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the Zeta potential. The sharp increase in Zeta potential was observed at low pH (pH 4). This 300

outcome confirmed that the increase in Zeta potential resulted from the positive charge of CZ 301

molecules that were ionized.302

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3.3 Fabrication of nanofiber mats304

The CZ-loaded microemulsion-containing nanofiber mats were electrospun from a 305

mixture of the CZ-loaded microemulsion (40% wt to polymer) and CS-EDTA/PVA (weight 306

ratio of 30:70) solution. The conductivity of the CS-EDTA/PVA solution was 769.33±6.03 307

µS/mL and that of the CZ-loaded microemulsion (F1, F2 and F3)-containing CS-EDTA/PVA 308

solutions was 770.67±9.02, 839.33±8.08 and 892.67±11.59, respectively. The viscosity of the 309

CS-EDTA/PVA solution was 358.25±5.29 cP and that of the CZ-loaded microemulsion (F1, 310

F2 and F3)-containing CS-EDTA/PVA solution was 357.72±5.17, 355.24±3.80 and 311

356.40±2.10, respectively. The conductivity of the spinning solutions was increased after the 312

CZ-microemulsion was added due to an excess of CZ loaded in the oily phase, which could 313

be released and ionized in the polymer solution; however, the viscosity of the solution was 314

not altered. After the electrospinning process, the SEM images (Fig.1) indicated that bead-315

free and smooth fibers were generated with CS-EDTA/PVA at a weight ratio of 30/70. The 316

diameter of the CS-EDTA/PVA nanofibers was 141.75±42.14 nm. However, the nanofibers 317

obtained from the mixture of polymer solution and CZ-loaded microemulsions (F1, F2 and 318

F3) contained globule particles, which might have been the microemulsion particles or 319

polymer beads, that were regularly distributed within the nanofiber mats, and the fiber 320

diameters decreased. The diameters of the fibers were 120.58±25.32, 125.56±41.93 and 321

105.91±18.13 nm for the F1, F2 and F3 formulations, respectively. These differences may 322

have been due to the increase in the conductivity of the spinning solution after the addition of 323

the CZ-microemulsion. The conductivity of the spinning solution affects the tensile force on 324

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the fiber in the presence of an electric field and affects fiber diameter. Solutions with high 325

conductivity will possess a greater charge carrying capacity and will be subjected to greater 326

tensile force in the presence of an electric field, resulting in a smaller diameter [32]. This 327

result is in agreement with previous studies that report that the average fiber diameter 328

decreases with the increasing conductivity of the solution [33]. Solution conductivity is one 329

of the key parameters in the electrospinning process. 330

3.4 Characterization of electrospun nanofiber mats331

3.4.1 FT-IR analysis 332

The FTIR spectra of the CZ powder, the blank CS-EDTA/PVA nanofiber mats and 333

different formulations of CZ-loaded microemulsion-containing nanofiber mats are presented 334

in Fig.2a. The spectrum of the blank nanofiber mats exhibited absorption peaks at 3414, 335

2937, 1629, 1432 and 1095 cm-1, which were attributed to the (O-H), s (CH2), (C=O), 336

(CH-O-H) and (C-O), respectively. The pure CZ powder exhibited dominant absorption 337

peaks at 1578, 1483, and 1314 cm-1 that correspond to benzene ring stretches. The bands at 338

905, 823, and 756 cm-1 were assigned to C-H stretching. The bands at 1083 cm-1 and 1208 339

cm-1 correspond to chlorobenzene and C-N stretching, respectively. The peaks observed in 340

the CZ powder spectrum were also observed in spectra of the CZ-loaded microemulsion-341

containing nanofiber mats with increased intensity. Therefore, CZ-loaded microemulsions342

were incorporated into the nanofiber mats.343

344

3.4.2 Differential scanning calorimetry (DSC)345

DSC studies were undertaken to evaluate the physical state of CZ in the electrospun 346

nanofiber mats, and the thermograms are displayed in Fig.2b. The thermogram of the CZ 347

powder exhibited an endothermic sharp peak at 144.13°C due to its melting temperature. The 348

blank CS-EDTA/PVA nanofiber mat exhibited a endothermic curve at 217.83°C. After the 349

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incorporation of various formulations of CZ-loaded microemulsions into the nanofiber mats, 350

the absence of a detectable crystalline domain was observed, indicating that CZ was 351

incorporated into the CS-EDTA/PVA nanofiber mats in an amorphous state. Moreover, the 352

melting point of CZ was lower than the melting point of the CS-EDTA/PVA nanofiber mats, 353

indicating that loading of the CZ into the nanofiber mats did not affect their thermal behavior. 354

355

3.4.3 X-ray diffractometry (XPRD) 356

The diffractogram of the CZ powder displayed strong crystalline peaks, indicating its high 357

degree of crystallinity. However, no such peak was found in the diffractograms of the blank 358

or CZ-loaded microemulsion (F1-F3)-containing nanofiber mats (data not shown); these 359

results are in accordance with the DSC-thermogram results, indicating that CZ was 360

incorporated into the nanofiber mats in an amorphous state. 361

362

3.4.4 Mechanical properties and ex vivo mucoadhesion studies363

The mechanical properties in terms of the tensile strength, strain at the maximum and 364

Young’s modulus of the CS-EDTA/PVA nanofiber mats with and without different 365

formulations of CZ-loaded microemulsions were evaluated using a texture analyzer, and the 366

results are presented in Table 2. The Young’s modulus of all tested nanofiber mats was in the 367

range of 2.8-6.1 MPa. The results demonstrated that the addition of the CZ-loaded 368

microemulsions into the fiber mats exerted little effect on the mechanical properties of the 369

nanofiber mats. The Young’s modulus of the mats decreased after the incorporation of the 370

CZ-loaded microemulsions. Moreover, the mucoadhesive strength of the mats also 371

moderately decreased after incorporation of the CZ-loaded microemulsions. The 372

mucoadhesive strength of the mats was in the range of 2.55-6.31 g. 373

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3.5 Drug content and loading capacity375

The total CZ content in the CZ-loaded microemulsion-containing nanofiber mats was 376

determined. The entrapment efficiency (%) and loading capacity (%) of CZ in the nanofiber 377

mats are listed in Table 2. The F3 formulation exhibited the highest entrapment efficiency 378

and loading capacity.379

380

3.6 In vitro release381

The in vitro release studies were performed in artificial saliva (pH 6.8) containing 382

20% PEG-400. The release behavior of CZ from different formulations of the CZ-loaded 383

microemulsion-containing nanofiber mats in comparison with CZ lozenges is presented in 384

Fig.3. The results revealed the CZ was released from the different formulations of the CZ-385

loaded microemulsion-containing nanofiber mats in a burst release manner. In the first 30 386

min, 46.7%, 33.3%, and 39.2% of the CZ contained in the nanofiber mats of the F1, F2 and 387

F3 formulations, respectively, was released. The percentage of drug release from the fiber 388

mats at 4 h was approximately 74.15, 71.75 and 64.81, respectively. However, the CZ was 389

released in a slower manner after the initial burst release and was sustained for 24 h. 390

Disparate CZ release profiles for the nanofiber mats prepared from different CZ-loaded 391

microemulsions were observed. The nanofiber mats of the F1 formulation exhibited a faster 392

initial release than the F2, F3 and CZ lozenges did. The drug release from the nanofiber 393

mats of the F1, F2 and F3 formulations was 90.5%, 98.8% and 75.6%, respectively, at 24 h. 394

The release kinetics appeared to follow the Higuchi model; the drug release versus the 395

square root of release time profile yielded a straight line over 60% of the total release 396

process. Higuchi used a pseudo-steady-state approach, which is valid for systems initially 397

containing a large excess of drug (drug loading >> drug solubility). In this model, the solid 398

drug is assumed to dissolve from the surface layer of the device first; when this layer 399

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becomes exhausted of drug, the next layer begins to be depleted by dissolution through the 400

matrix into the external solution [34].401

3.7 Antifungal studies402

The antifungal activity of the CZ-loaded microemulsion-containing nanofibers was 403

investigated by time-kill analysis to determine the exposure time required to kill standardized 404

microbial inoculums. The time-kill plots of different CZ-loaded microemulsion-containing 405

nanofibers in comparison with CZ lozenges and the control sample are presented in Fig.4. 406

The CZ-loaded microemulsion-containing nanofibers and CZ lozenges inhibited the Candida 407

cells within 5 min after contact and killed the Candida cells within 1 h. In addition, all 408

formulations of the CZ-loaded microemulsion-containing nanofibers exhibited rapid 409

antifungal activity and displayed significantly faster antifungal activity, compared to the CZ 410

lozenges. These results may be due to the initial rapid release of CZ from the CZ-loaded 411

microemulsion-containing nanofibers in comparison to the CZ lozenges, which require time 412

to disintegrate before CZ dissolution. Our previous studies demonstrated the very rapid 413

antimicrobial activity of chitosan-based nanofiber mats loaded with Garcinia mangostana414

extracts [26] and the antifungal activity of CZ composited PVP/HPβCD nannofibers [35]. 415

Moreover, electrospun nanofibers exhibit useful properties, including high porosity, very 416

small pore sizes, large specific surface areas and high surface-to-volume ratios [20-22], 417

resulting in rapid drug released from the mats and rapid antifungal activity.418

3.8 Cytotoxicity evaluation419

Cell culture tests were performed to assess the biological compatibility of the 420

nanofibers. The cytotoxicity of the CZ-loaded microemulsion-containing nanofiber mats was 421

investigated after a 2 or 24 h incubation using an MTT assay to determine the % viability, as 422

illustrated in Fig.5. A significant decrease in the cell viability (approximately 60% cell 423

viability) was observed when the HGF cells were incubated with CZ-loaded microemulsion-424

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containing nanofibers (F1, F2 and F3) for 24 h, compared to the control (untreated cells) 425

(p<0.05), whereas, after 2 h of incubation with culture media containing nanofiber mats, the 426

cell viability remained similar to that of the control cells for all formulations. These results 427

from the shorter time period (2 h) were in agreement with the time kill studies. Cytotoxicity 428

was observed only for the 24-h incubation with the culture media containing the nanofiber 429

mats. This result may have occurred because a large amount of CZ was loaded into the mats 430

and was toxic to the fibroblast cells. When the incubation time increased, a higher dose of CZ 431

was released, which resulted in a greater amount of CZ coming in contact with the cells. Our 432

previous studies demonstrated that CZ exhibits concentration-dependent cytotoxicity with 433

HGF cells at pH 7.4 when incubated for 2 and 24 h, with IC50 values of 24.9 and 6.1 µg/ml, 434

respectively. Moreover, the cytotoxicity of CZ-loaded PVP/HPβCD nanofiber mats was 435

significant increased when the CZ content in the mats increased [35]. In contrast, the blank 436

CS-EDTA/PVA nanofiber mats were also tested, and no significantly difference was 437

observed in the cytotoxicity between the blank nanofiber mats and control [36]. Thus, the 438

cytoxicity may have resulted from the presence of CZ. The results indicated that all 439

formulations were safe and less cytotoxic for a 2 h incubation. In addition, the cytotoxicity 440

did not differ among the nanofiber mats prepared from the different formulations.441

442

4. Conclusion443

In this study, novel CZ-loaded microemulsion-containing nanofiber mats were 444

successfully electrospun from a mixture of different CZ-microemulsion formulations and 445

polymer solutions. The CZ was released from the nanofiber mats in an initial burst release, 446

followed by sustained release. The nanofiber mats exhibited excellent antifungal activity, 447

with low toxicity. Further in vivo studies are needed to evaluate this material for applications 448

in oral candidiasis.449

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450

451

Acknowledgments452

The authors would like to thank the Commission of Higher Education (Thailand), the 453

Thailand Research Funds through the Royal Golden Jubilee Ph.D. Program (Grant 454

No.PHD/0092/2554), the Thailand Research Funds and Faculty of Pharmacy, the Silpakorn 455

University, and the Silpakorn University Research and development institute for their 456

financial support.457

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devices – ISO 10993, Part 5: Tests for in vitro cytotoxicity. Geneva, Switzerland; 2009.509

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[33]. C.J. Angammana, S.H. Jayaram, IEEE. Trans. Ind. Appl. 47 (2011) 1109-1117.511

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Figure legends518

Figure 1 SEM images of a) a pure CS-EDTA/PVA nanofiber, b) CZ-loaded microemulsion 519

(F1)-containing nanofiber, c) CZ-loaded microemulsion (F2)-containing nanofiber and d) 520

CZ-loaded microemulsion (F3)-containing nanofiber.521

522

Figure 2 a) FT-IR spectra and b) DSC thermograms of a pure CS-EDTA/PVA nanofiber and 523

different formulations (F1-F3) of CZ-loaded microemulsion-containing nanofibers.524

525

Figure 3 Release profiles of CZ from (♦) CZ lozenges, and CZ-loaded microemulsion-526

containing nanofibers prepared from (■) F1, (▲) F2 and (●) F3 microemulsions. The data are 527

expressed as mean ± standard deviation from three independent experiments.528

529

Figure 4 Time kill curves for C. albicans treated with the (■) F1, (▲) F2 and (●) F3 530

formulations of CZ-loaded microemulsion-containing nanofibers; (♦) CZ lozenges and (x) 531

control. The data are expressed as mean ± standard deviation from three independent 532

experiments. *statistically significant (P<0.05) vs. control; **statistically significant 533

difference between nanofiber mats and CZ lozenges.534

535

Figure 5 The percentage of viable HGF cells after treatment for (■) 2 and (□) 24 h with 536

different formulations (F1-F3) of CZ-loaded microemulsion-containing nanofibers. Each 537

value represents the mean ± standard deviation of five wells. *Statistically significant 538

(P<0.05) vs. control group. 539

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Figure 1543

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Figure 2550

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Figure 3561

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Figure 4566

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Figure 5583

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Table 1 Particle size and Zeta potential of F1, F2 and F3 microemulsions with and without 591

25% w/w CZ. The data are expressed as mean ± standard deviation from three independent 592

experiments.593

594

Zeta potential (mV)Microemulsions Particle size (nm)

pH 4 pH 7 pH 9

F1(O:T80: BzOH) 70.97±8.09 -25.77±0.47 -38.13±2.20 -41.50±0.70

F2(O:T80: EtOH) 63.38±13.90 -25.60±0.36 -40.23±0.81 -58.73±0.93

F3(O:T80: IPA) 65.01±7.44 -26.47±0.72 -38.57±0.91 -41.83±0.32

F1+ 25 % w/w CZ 88.11±17.48 2.45±0.14 -17.57±0.68 -39.47±0.55

F2+25 % w/w CZ 57.80±8.23 -3.54±0.38 -16.87±0.15 -37.90±2.02

F3+25 % w/w CZ 64.44±9.24 3.46±0.36 -16.73±0.95 -39.47±3.04

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Table 2 The mechanical properties, ex vivo mucoadhesive properties, entrapment efficiency 610

(%) and loading capacity (%) of pure CS-EDTA/PVA nanofibers and different formulations 611

(F1-F3) of CZ-loaded microemulsion-containing nanofibers. Each value represents the mean 612

± standard deviation from three independent experiments.613

614

615

Nanofiber matsYoung’s modulus

(MPa)

Ex vivomucoadhesive

strength (g)

Entrapment efficiency

(%)

Loading capacity

(%)

CS-EDTA/PVA nanofibers

6.1 ± 1.2 6.31 ± 0.21 - -

CS-EDTA/PVA nanofibers+F1

4.8 ± 0.9 4.29 ± 0.25 72.86 ± 2.15 7.56 ± 0.22


4.9 ± 0.3 2.55 ± 0.18 72.58 ± 4.38 7.53 ± 0.45


2.8 ± 0.6 3.10 ± 0.14 98.10 ± 6.95 10.17 ± 0.72

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Nanofiber mats containing clotrimazole (CZ)-loaded microemulsion were developed 628as an alternative for oral candidiasis applications.629

Microemulsion particles were observed to be regularly distributed within the mats.630

The DSC and PXRD results revealed the amorphous state of the CZ-loaded 631microemulsions incorporated in the nanofiber mats.632

The nanofiber mats exhibited more rapid killing activity than commercial CZ 633lozenges did.634

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fabrication of a novel scaffold of clotrimazole-microemulsion-containing nanofibers using an...

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