Electrospun PVP Nanofibrous Co-Loaded with Miconazole and Lidocaine for the Treatment of Oral Candidiasis

Abstract

Introduction

Oral candidiasis caused by Candida albicans is common among immunocompromised individuals. Conventional topical dosage forms have several limitations, including imprecise dosing, rapid salivary clearance, short residence time at the site of application, and inconsistent therapeutic efficacy.

Rationale

To overcome these challenges, this study developed an electrospun nanofibrous patch for localized co-delivery of miconazole (MZ) and lidocaine (LD) to oral mucosa. MZ and LD were co-encapsulated in electrospun polyvinylpyrrolidone (PVP) nanofibers and evaluated for morphology, drug loading, release profile, minimum inhibitory concentration (MIC), cytotoxicity, and in vivo antifungal efficacy.

Results

Scanning electron microscopy showed smooth fibers with an average diameter of 490 ± 122nm. Drug loading values were 41 µg/mg for LD and 46 µg/mg for MZ. Release studies demonstrated rapid LD release, with approximately 70% released within the first few minutes and complete release after 30 minutes. In contrast, MZ reached about 95% cumulative release within 120 minutes. The MZ/LD combination (1:1) showed potent antifungal activity, with an MIC of 0.004 μg/mL and an inhibition zone of 20 ± 1 mm. No in vitro cytotoxicity was observed at concentrations of < 16 µg/mL for MZ, < 1250 µg/mL for LD, and < 16 and 78 µg/mL for MZ and LD, respectively, for their combination. In a murine oral candidiasis model, MZ/LD-loaded nanofibers significantly (p< 0.0001) reduced fungal burden to 22 CFU/mL and improved tongue lesion scores compared with MZ-loaded nanofibers alone (1100 CFU/mL) and the commercial MZ formulation.

Conclusion

These findings demonstrate that the developed dual-drug nanofibrous system is a safe and promising localized delivery system for treating oral candidiasis.

Introduction

Oral candidiasis is a group of superficial fungal infections of the oral mucosa, including pseudomembranous, erythematous, hyperplastic, and angular forms.^1–5 Although Candida albicans remains the most frequent isolate species, recent clinical data indicate a growing number of non-albicans Candida species (NAC) such as C. glabrata, C. tropicalis, and C. krusei, particularly in immunocompromised individuals, including the elderly, infants, individuals with acquired immunodeficiency syndrome, and those receiving cancer treatment or immunosuppressant medications.^1,6–8 This changing epidemiology, together with persistent treatment failure and recurrence among susceptible individuals, highlights the need for more effective local therapeutic strategies for oral candidiasis.^3,4,8,9

Management of oral candidiasis includes both topical and systemic antifungal therapy, selected based on disease severity, extent of involvement, and host-related factors.^3,4,9–11 Topical azoles such as miconazole (MZ) are commonly used for mild to moderate disease because they provide high local drug exposure with limited systemic effect.^2,3,9 On the other hand, systemic azoles (eg, fluconazole) are generally reserved for extensive, refractory, or high-risk cases. Still, their use may be limited by adverse effects, drug interactions, and emerging azole resistance among Candida albicans and non-albicans isolates.^4,7–9,11 Conventional topical dosage forms, including gels, suspensions, and lozenges, often have a short residence time on the oral mucosa and are readily diluted or removed by saliva, leading to variable drug exposure at the site of infection and inconsistent clinical outcomes.^2,3,10,12,13

In addition to fungal burden, oral candidiasis may be accompanied by burning, soreness, and discomfort that impair oral function and quality of life in vulnerable patient groups.^1,4,5 Lidocaine (LD) is a widely used local anesthetic for temporary relief of oral and/or oropharyngeal pain; however, it is usually administered in rapidly cleared solutions, sprays, or lozenges that do not provide sustained contact with the affected mucosa.^14–17 Beyond its anesthetic effect, LD has demonstrated intrinsic antifungal activity against Candida species, suggesting that its combination with azoles may provide additive or synergistic therapeutic benefit.^17–20 Accordingly, co-loading MZ and LD into a single localized delivery system may provide simultaneous antifungal therapy and symptomatic relief while potentially enhancing antifungal efficacy.^17–20

Mucoadhesive systems have been explored to improve oral drug retention and maintain therapeutic concentrations at the mucosal surface.^10,12,13 Polyvinylpyrrolidone (PVP) is a suitable, water-soluble polymer widely used in pharmaceutical formulations and is suitable for processing into electrospun drug-loaded fibers.²¹ Electrospinning enables the production of nanofibrous matrices with high surface area, tunable architecture, and efficient drug incorporation, properties that can promote close contact with mucosal tissue and allow modulation of release behavior beyond what is achievable with conventional bulk formulations.^16,21–23 Although PVP has moderate mucoadhesive properties, its nanofibrous form can provide rapid hydration, extensive interfacial contact, and reproducible drug distribution, which may support effective local delivery to oral mucosa.^16,21

Previous studies have investigated mucoadhesive antifungal systems for oral candidiasis, including fluconazole-contacting mucoadhesive nanoparticles and MZ-based buccal films.^2,10,13,24 In addition, co-loaded MZ-LD films and nanoparticulate platforms have demonstrated the feasibility of combining antifungal and anesthetic agents in topical candidiasis treatment.^8,22,24 Also, electrospun antifungal nanofibers have shown promise as localized delivery platforms. Yet, the application of a mucoadhesive electrospun PVP nanofibrous patch co-loaded with MZ and LD for oral candidiasis has not been systematically established.^16,21–23

Therefore, this study hypothesizes that electrospun mucoadhesive fiber patches will (i) enhance mucosal residence and site-specific delivery of MZ, (ii) provide rapid and sustained local analgesia via LD, and (iii) augment antifungal efficacy through combined pharmacologic and physicochemical effects. To test this hypothesis, the study aims to develop an electrospun fiber-based delivery system to treat oral candidiasis, with a focus on controlling the direct release of MZ and LD onto the oral mucosa. By leveraging the precision and flexibility of electrospinning technology, this research aims to fabricate mucoadhesive electrospun patches that adhere to the buccal mucosa, thereby ensuring extended drug contact time and enhanced local drug concentrations. The objectives are to optimize the fiber formulation to maximize mucoadhesive properties and drug-release efficiency, evaluate the patches’ antifungal and analgesic properties, and assess their safety, biocompatibility, and efficacy in vitro and in vivo models. This approach aims to overcome the limitations of traditional treatments for oral candidiasis by providing a targeted, efficient, and patient-friendly therapeutic option with dual therapeutic benefits.

Materials and Methods

Materials

Polyvinylpyrrolidone (PVP, average molecular weight ~1,300,000), absolute ethanol (≥ 99.8%), and lidocaine hydrochloride monohydrate (LD) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Miconazole free base (MZ) was purchased from Biosynth Carbosynth (Compton, UK), and distilled water was generated using Milli-Q Water (Millipore, Billerica, MA, USA). Candida albicans (ATCC 66027) reference fungus was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Mueller–Hinton agar (MHA) and Mueller-Hinton broth (MHB) were purchased from Scharlau (Barcelona, Spain) and prepared according to the manufacturer’s instructions.

Methods

Preparation of Electrospun Fibers

PVP was dissolved in ethanol at a concentration of 10% (w/v) and stirred at room temperature for two hours to ensure complete dissolution and homogeneity. Following this, MZ and LD were incorporated into the solution at concentrations of 0.5% (w/v) each, and the mixture was stirred for an additional hour to ensure uniform homogenization. The electrospinning process was conducted using a modified method by Tawfik et al²⁵ using a Spraybase^® system (Dublin, Ireland), with the prepared mixture transferred into a 5 mL syringe. The electrospinning parameters included a 0.7 mm needle, a tip-to-collector distance of 15 cm, an applied voltage of 9 kV, and a flow rate of 0.8 mL/hour, all maintained at ambient temperature and humidity between 30% and 40%. Blank nanofibers were prepared under the same conditions, but without drug additives and at a reduced voltage of 6.2 kV.

For the preparation of single-drug nanofibers, a similar procedure to that for drug-loaded fibers was used. Briefly, PVP (10% w/v) was dissolved in ethanol and stirred for one hour, after which either MZ or LD was added individually at a concentration of 0.5% (w/v), followed by an additional hour of stirring. Electrospinning was carried out using a 0.7 mm needle, a tip-to-collector distance of 15 cm, and a flow rate of 0.8 mL/h. The applied voltage was 7 kV for the MZ-loaded nanofibers and 9 kV for the LD-loaded nanofibers.

Assessment of Surface Morphology via Scanning Electron Microscopy (SEM)

The morphological characteristics and size of the nanofibers were examined using a JEOL SEM (JSM-IT500HR, ASIA PTE. Ltd., Singapore). Samples were prepared for imaging by placing them on SEM stubs and coating them with a 2 nm platinum layer using the Auto Fine Coater (JEC-3000FC, JEOL, ASIA PTE. Ltd., Singapore). Fiber diameters were measured using ImageJ (National Institutes of Health, MD, USA) from 100 individual fibers per formulation, including blank and drug-loaded fibers, collected from two independent batches, with four images per batch. The data were then analyzed using OriginPro 2021 (OriginLab Corporation, Northampton, MA, USA).

Solid-State Characterization Using X-Ray Diffraction (XRD)

A modified method described by Alzahrani et al²⁶ was followed with some modifications. In brief, the crystallography and amorphous structure of PVP, MZ, LD, their physical mixture (PM, which corresponds to the same ratio of the drug-loaded fibers but in powder form), blank, and drug-loaded fibers were evaluated using Rigaku Miniflex 300/600 X-ray Diffraction (Tokyo, Japan) with a voltage of 40 kV, a current of 15 mA, and Cu Kα radiation (1.54059 Å). PM was prepared at a 10:0.5:0.5 (PVP:MZ:LD) ratio. Then, each sample was placed in an aluminum holder to initiate the analysis over the range of 2° to 60° at a speed of 5°/minute.

Fourier-Transform Infrared Spectroscopy (FTIR)

The chemical bonds and characteristic functional groups of PVP, MZ, LD, their PM, blank, and drug-loaded fibers were analyzed as described by Alzahrani et al²⁶ using the Thermo Smart ATR IS20 Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) to identify any structural changes, interactions, or modifications that occurred during electrospinning. The spectra were analyzed over the wavenumber range 4000 to 500 cm⁻¹, with 32 scans per sample and a spectral resolution of 4 cm⁻¹.

Quantitative Analysis of Pure Drugs Using High-Performance Liquid Chromatography (HPLC)

MZ and LD were quantitatively analyzed using a Waters e2695 HPLC system comprising a Waters^® 717 plus autosampler, a Waters 600 binary pump, and a Waters 2489 UV detector (Waters Technologies Corporation, Milford, MA, USA). The separation was achieved by gradient elution using a mobile phase consisting of 1% triethanolamine (pH adjusted to 3.9 with formic acid) as solvent A and methanol as solvent B. A linear gradient was run at a flow rate of 1.2 mL/minute, with an initial condition of 90% solvent A and 10% solvent B. This ratio was then increased to 75% solvent B from t=0 to 5.79 minutes, then maintained at that ratio until t=10 minutes. The gradient was then returned to 90% solvent A and 10% solvent B from t=10 to 12 minutes, and held at that composition until 14 minutes. Chromatographic analysis was carried out using an XSelect HSS CYANO (5 µm, 4.6×250 mm) maintained at 20°C. The mobile phase flow rate was set at 1.2 mL/minute, with a 10 µL injection volume, and detection was performed at 210 nm and 270 nm for MZ and LD, respectively. Stock solutions of MZ and LD were prepared in the Mobile phase at a 1:1 ratio, and calibration curves were generated using serial dilutions ranging from 200 to 6.25 µg/mL.

Determination of Encapsulation Efficiency (EE%) and Drug Loading (DL)

Predetermined amounts of MZ/LD-loaded fibers were dissolved in a solution comprising 2.5% dimethyl sulfoxide (DMSO), 1.25% Tween 80, and 96.25% phosphate-buffered saline (PBS) at pH 6.8. The samples were maintained at room temperature for at least 4 hours to ensure complete dissolution. Subsequently, the concentrations of MZ and LD were quantified using a previously developed HPLC method detailed in the previous section. The EE% and DL were then calculated using the equations below based on the measured drug concentrations and the initial amounts of drugs and polymers used in the fiber preparation.

(1)

(2)

The results represent the mean ± standard deviation (SD) of at least three replicates.

Quantification of MZ and LD Drug Release

To assess the release profile of MZ and LD from nanofibers, at least three samples of the fibrous mat, each weighing 20 ± 0.1 mg, were dissolved in 15 mL of a pre-warmed solvent mixture. This mixture consisted of 96.25% (v/v) phosphate-buffered saline (PBS) at pH 6.8, 2.5% (v/v) dimethyl sulfoxide (DMSO), and 1.25% (v/v) Tween 80, contained in glass vials. The release profiles of MZ and LD from the fabricated nanofibers were monitored using a shaking incubator (Excella E24 Incubator Shaker Series, New Brunswick Scientific Co., Enfield, CT, USA) set to 100 RPM and 37°C.

At designated time intervals ranging from 1 to 120 minutes, 1 mL of the solution was withdrawn and replaced with an equal volume of the solvent mixture. The concentrations of MZ and LD released were quantified using the same HPLC method mentioned above. The cumulative release percentage was calculated as a function of time using the following equation:

(3)

Results are expressed as the mean ± SD from independent triplicates.

The release medium used was selected to maintain sink conditions throughout the release study. The solubility of both MZ and LD in the selected release medium was experimentally confirmed to be at least 1000 µg/mL for each drug. During the release study, 20 mg of drug-loaded nanofibers were immersed in 15 mL of release medium. Based on the experimentally determined drug loading (46 ± 1 µg/mg for MZ and 41 ± 2 µg/mg for LD), the maximum theoretical drug concentrations were approximately 61 µg/mL for MZ and 54.6 µg/mL for LD. Therefore, the release medium met the sink condition criterion (at least three times the saturation solubility), ensuring that drug release was not limited by solubility.

In addition, according to regulatory guidance, the In Vitro Release Testing (IVRT) receptor medium must be selected to ensure sink conditions, meaning it should have sufficient capacity to dissolve the actives so that the system does not reach saturation during testing.^27,28 For topical formulations, the US Food and Drug Administration (US FDA) recommends aqueous buffers for soluble drugs or hydroalcoholic or surfactant media for poorly soluble actives.²⁷ PBS is frequently modified with co-solvents or surfactants to enhance the solubility of the drug used.²⁸ The selected receptor medium in this study aligns with FDA/USP guidelines by being primarily aqueous (<5% organic additives) while achieving sufficient solubilization to maintain sink conditions.^27,28 PBS was chosen to establish a biological pH and ionic strength comparable to body fluids, with a pH of 6.8, representative of the physiological range of saliva (pH 6.2–7.6).²⁹

MZ free base faces solubility challenges, exhibiting a poor water solubility of less than 1 µg/mL and minimal solubility in hydrophilic media. These limitations are mainly due to its weak basicity, which impedes dissolution in aqueous environments.³⁰ In its salt form, MZ nitrate also shows higher but still very limited solubility (≈ 0.1–0.2 mg/mL).³¹ Therefore, PBS alone cannot maintain the sink conditions, and DMSO (2.5% v/v) was used as a cosolvent to improve solubility. Also, Tween-80 (1.25% v/v), a non-ionic surfactant, provided micellar solubilization above its critical micelle concentration (CMC). This dual system enhances the solubility of both drugs without exceeding levels known to interfere with diffusion or membrane integrity.²⁸ Overall, this medium provides sink conditions for LD and MZ, facilitating diffusion-controlled release, and is consistent with FDA/USP guidelines.

Several studies have provided insight into effective solubilization techniques for MZ and other hydrophobic drugs. For instance, Zhang et al utilized 5% DMSO to suspend MZ for in vivo treatment in mice.³² Additionally, Zhang et al explored various aqueous–organic solvent systems, including an ethanol–water (1:1) mixture with Tween 80, to facilitate the release of hydrophobic drugs such as curcumin from BSA nanofiber/protein scaffolds.³³ In another study, Maha et al investigated the impact of DMSO on the release of MZ nitrate gel and skin permeation using Franz-type diffusion cells with rabbit skin. Gels containing 3%, 5%, and 7% DMSO were tested, revealing a concentration-dependent increase in drug permeation, with 7% DMSO achieving a markedly higher release (52.76%) compared to the control (2.83%).³⁴

In Vitro Cytotoxicity Assessment of Pure Drugs

Cytotoxicity of MZ, LD, and their combination was measured and evaluated by detecting formazan product formation in the colorimetric MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], resulting from tetrazolium reduction. Human Foreskin Fibroblast (HFF-1) cell line with reference number (ATCC- SCRC-140) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin 100 U/mL, and streptomycin 100 μg/mL, all of which were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell viability was evaluated as described by Alamer et al³⁵ Cells at 80% confluence were harvested and seeded into 96-well plates at a density of 1.5×10⁴ cells per well, then incubated for 24 hours (plate 1) and 48 hours (plate 2) in 5% CO₂ at 37°C. After the incubation period, the cells were treated with LD, MZ, and an LD: MZ combination at a 5:1 ratio to ensure cell viability and biocompatibility.

The synergistic activity demonstrated in previous studies between MZ and LD against Candida albicans supports the rationale to evaluate the combination, where checkerboard assay evaluation of LD and MZ was reported with concentration ranges (15 to 2500 µg/mL for LD and 0.015 to 32 µg/mL for MZ.³⁶ However, in this study, the concentrations were selected at levels exceeding those investigated in the previous study, in a 5:1 ratio (LD: MZ), which was experimentally convenient to investigate the cytotoxicity of both drugs. For LD, serial dilutions from 5000 to 78 µg/mL were tested, while the range for MZ was 1000 to 16 µg/mL. For the combination, a 5:1 ratio was maintained within the same concentration range as the drugs alone (5000 to 78 µg/mL for LD and 1000 to 16 µg/mL for MZ).

Positive control cells were untreated, and negative control cells were treated with 0.2% Triton X-100 and then incubated for 24 hours (plate 1) and 48 hours (plate 2). After the incubation period, the growth medium was discarded, and 100 µL DMEM and 20 µL MTS Reagent (CellTiter 96^® Aqueous One Solution Cell Proliferation Assay) were added to each well under dark conditions. The wells were then incubated for 3 hours. The absorbance of the formazan products formed in the cell culture medium was measured at 490 nm using a microplate reader (BIOTEK Instruments Inc., Winooski, VT, USA). The cell viability percentage was calculated according to the equation described by Aburayan et al³⁷ as follows:

(4)

Where S is the absorbance of cells treated with MZ, LD, or their combination; T is the absorbance of cells treated with Triton X-100; and H is the absorbance of cells treated with culture medium only.

Determination of the Minimum Inhibitory Concentration (MIC)

Antifungal susceptibility was assessed using a modified broth dilution method previously reported for Candida albicans cultured in Mueller–Hinton broth (MHB) supplemented with 5% glucose.^38,39 The absolute MIC values reported here are not directly comparable to those generated using the CLSI reference method and should not be interpreted using clinical breakpoints; rather, they were used to confirm that antifungal activity was retained after encapsulation.

MZ and LD were serially diluted two-fold from 2 to 0.001 μg/mL. Then, 100 μL of Candida albicans (ATCC 66027, handled in a BSL-1 facility) inoculum (1.5 × 10⁵ CFU/mL) was added to each well. Positive-control wells contained Candida without drugs, whereas negative-control wells contained only MHB supplemented with 5% glucose. The 96-well microtiter plates were incubated overnight at 30 ± 2°C with continuous shaking at 200 RPM. The MIC was defined as the lowest drug concentration at which no visible growth was observed. Candida growth inhibition was measured at 600 nm using a PowerWave XS2 plate reader (bioMérieux, Marcy L’Etoile, France). Results are expressed as mean ± SD (n = 3).

Determination of the Antimicrobial Activity of MZ/LD–Loaded PVP Fibers

The CLSI M44-A2 guidelines for antifungal susceptibility testing were followed.⁴⁰,100 μL of Candida albicans (ATCC 66027) suspension at a cell density of 1.5×10⁵ CFU/mL was plated onto MHA. One mg of the MZ/LD—loaded PVP fibers and blank fibers was placed on each plate, along with a sterile microbiological disc (GE Healthcare, USA) supplemented with the same drug dose as the fibers. Then, agar plates were incubated at 25 ± 2°C for 24 hours. The diameters of the clear zones of fungal inhibition were measured in millimeters (mm), and the images of the plates were taken. The results are presented as mean ± SD (n=3).

Animal Infection Model

Candida and Culture Conditions

Candida albicans (ATCC 10231), obtained from ATCC (Manassas, VA, USA), was kept on Sabouraud’s dextrose agar (SDA) plates for 48 hours at 35°C. Colonies were suspended in 5 mL of sterile 0.85% PBS. The final concentration was adjusted to 1×10⁶ CFU/mL.

For the in vivo study, a more virulent strain (Candida albicans ATCC 10231) was used and maintained in a BSL-2 laboratory facility. In contrast, the in vitro study described in Determination of the Minimum Inhibitory Concentration (MIC) used Candida albicans ATCC 66027, which was handled in a BSL-1 facility.

Animal Handling Conditions

Male adult Swiss mice weighing 25–30 gm were obtained from the animal house of the King Fahad Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. The in vivo study protocol was approved by the Local Committee of Ethics of Research on Living Creatures in King Abdulaziz City for Science and Technology- Institutional Review Board (KACST-IRB#25007), in adherence with the Declaration of Helsinki, the Guiding Principle in Care and Use of Animals (DHEW production NIH 80–23) and the Standards of Laboratory Animal Care (NIH distribution #85-23, reconsidered in 1985). Mice were kept in cages housing 4 animals, adapted for at least 2 weeks in naturally controlled enclosures (20 ± 1°C and a 12/12-h dark/light cycle) and fed food pellets and tap water ad libitum. In this study, male mice were selected to reduce biological variability associated with hormonal fluctuation during the estrous cycle and improve experimental consistency.

Animal Preparation and Oral Infection

To develop oral candidiasis in mice using Candida albicans, the method mentioned by Takakura et al was followed.⁴¹ The animals were immunosuppressed with two subcutaneous injections of prednisolone (Pfizer, NY, USA) at a dose of 100 mg/kg body weight, one day before and three days after infection with Candida albicans. Tetracycline hydrochloride (Novartis, Basel, Switzerland) was added to drinking water at a concentration of 0.83 mg/mL and administered to mice beginning one day before infection. Mice were anesthetized (according to the protocol described in Animal Anesthesia and Euthanasia), followed by soaking a small cotton pad in a Candida albicans suspension (2.5 × 10⁷ CFU/mL) and swabbing the entire oral cavity of the mice (inoculation) to form oral infections.⁴¹

Treatment of the Infection

The animal experiment was carefully designed to achieve the project’s goal. Accordingly, 28 mice total have been distributed to seven groups (n=4/group), control group or group 1 (not infected, not treatment), group 2 (infected, not treatment), group 3 (infected, treated with blank nanofibers), group 4 (infected, treated with MZ/LD nanofibers), group 5 (infected, treated with MZ nanofibers), group 6 (infected, treated with LD nanofibers), and group 7 (infected, treated with MZ market drug).

Evaluation of Infection Development

After mice were euthanized (according to the protocol described in Animal Anesthesia and Euthanasia), the severity of Candida albicans infection of the tongue was assessed macroscopically. The severity of oral candidiasis was scored according to the criteria described by Takakura et al⁴¹ 0 = normal; 1 = white patches covering ≤20% of the tongue surface; 2 = white patches covering 21–90% of the tongue surface; 3 = white patches covering >90% of the tongue surface; and 4 = thick pseudomembranous white patches covering >90% of the tongue surface.⁴¹ The present study was not performed under blinded conditions; therefore, observer bias cannot be excluded, particularly for subjective lesion scoring.

Microbiological Evaluation of Infection Progression

A cotton pad was used to swab the whole oral cavity, including the buccal mucosa, tongue, soft palate, and other oral mucosal surfaces. The cotton pad’s end was then sliced and inserted into a tube containing 5 mL of sterile saline, after which the tube was vortexed to release Candida albicans. Serial 100-fold dilutions of the cell suspension were cultured on Candida GS plates at 35°C for 20 hours. Finally, the Candida colonies were counted.⁴¹

Nanofibers Treatment

Half (0.5) mg of MZ, LD, and MZ/LD nanofibers was given to the mice orally at a dose of approximately 23 μg for MZ and 20.5 μg for LD, and was carefully placed directly onto the dorsal surface of the tongue using sterile forceps to ensure localized contact with the oral mucosa. The mice were gently manually restrained for a short period to facilitate adhesion of the nanofiber to the moist oral mucosa before being returned to their cages. The commercially available oral gel of MD (Jamjoom Pharma, Jeddah, Saudi Arabia) was also applied at a dose equivalent to 23 μg during the experiment. Treatment was initiated once daily on day 3 post-inoculation, when oral candidiasis was established according to the protocol of Takakura et al, and continued through day 7 to evaluate the therapeutic efficacy of the formulations during the active phase of infection.⁴¹

Animal Anesthesia and Euthanasia

Mice were anesthetized by an intramuscular injection into each femur with 50 μL of 2 mg/mL chlorpromazine chloride (TagiPharma, South Beloit, IL, USA). At the same time, they were euthanized by inhalation of isoflurane (Abbott, Chicago, IL, USA) overdose followed by cervical dislocation as a secondary physical method to ensure death. This protocol was in accordance with applicable ethical and veterinary guidelines, including recommendations outlined by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals.⁴¹ Moreover, the use of chlorpromazine in the present study was not introduced as a novel anesthetic approach. Still, it was adopted from the established murine oral candidiasis model described by Takakura et al, which served as the basis for our infection protocol. To ensure methodological consistency and facilitate comparison with this validated model, the same anesthetic regimen was employed during oral inoculation. All experimental groups were subjected to the identical anesthesia protocol; therefore, any potential effects of chlorpromazine on oral mucosal physiology or immune responses would have been consistent across groups and are unlikely to have influenced the comparative evaluation of treatment efficacy.⁴¹

Statistical Analysis

Experiments were performed in at least three independent replicates, and the results were analyzed, plotted, and expressed as the mean ± SD. The mean and SD of EE, DL, drug release, and fiber diameters, as well as the regression equation from the HPLC analysis, were determined using Microsoft Excel. OriginPro was used to plot the size distribution of nanofibers, the drug release profile, and the XRD and FTIR Figures. In vivo statistical analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test, in GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Statistical significance was considered at p < 0.05. Significance levels are indicated in the figures as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).

Results and Discussions

Surface Morphology of MZ/LD-Loaded Nanofibers

The morphological characteristics and size of the nanofibers were examined using SEM. Both blank and drug-loaded nanofibers exhibited a beadless, uniform structure (Figure 1A and B). The diameter assessment of the prepared fibers resulted in average values of 1102 ± 182 nm for the blank nanofibers and 490 ± 122 nm for the drug-loaded nanofibers. The use of high voltage led to a decrease in the diameter of the drug-loaded nanofibers, which was attributed to adjustments in the electrospinning parameters. Specifically, the voltage was increased during fabrication to stabilize the Taylor cone jet, resulting in nanofibers with smaller diameters (Figure 1B). This has also been observed in other studies, including Alamer et al and Tawfik et al^25,35 Nevertheless, the blank fibers are used as a control in the biological assessment of this study.

Figure 1.

Surface morphology of (A) blank and (B) drug-loaded nanofibers observed by SEM, with the corresponding fiber size distribution charts. The size distribution of (C) blank and (D) drug-loaded nanofibers was determined by measuring 100 individual fibers from four SEM images per formulation. The mean fiber diameter was 1102 ± 182 nm for the blank nanofibers and 490 ± 122 nm for the drug-loaded nanofibers.

Solid-State Characterization Using X-Ray Diffraction (XRD)

Figure 2 illustrates the XRD patterns of pure PVP, MZ, LD, PM, blank, and drug-loaded fibers. The PVP, blank fibers, and drug-loaded fibers exhibited a broad peak, indicating their amorphous nature. These findings align with the XRD results of PVP nanofibers reported by Hu et al, confirming its amorphous nature, as indicated by a broad halo peak.⁴² The absence of sharp diffraction peaks in the MZ/LD-drug-loaded fibers indicated that the drugs had been successfully incorporated into the fiber matrix without phase separation after electrospinning, suggesting their molecular dispersion within the nanofiber matrix. Numerous studies have shown that this amorphous structure enhances the drug’s solubility and the bioavailability of the loaded fibers.^26,43,44 In contrast, the XRD patterns of MZ and LD exhibited several sharp, distinct Bragg reflections, confirming their crystalline nature. MZ displayed sharp diffraction peaks at 2θ = 12.5°, 13.78°, 16.65°, 18.35°, 20.3°, 21.6°, 23.8°, 25.2°, 25.6°, 27.7°, and 30.08°, while LD exhibited peaks at 2θ = 13,5°, 14.4°, 16.8°, 19.53°, 20.4°, 25.2°, 26.14°, 30.47°, 32.13°, 33.18°, and 34.2°, consistent with the findings of Arshad et al, and Bakhtiari et al, respectively.^45,46 These diffraction peaks of the drugs were observed in the PM sample with very low intensities at 2θ = 12.08° and 21.11°, which might be attributed to the low drug-to-polymer ratio (10:0.5:0.5) for PVP:MZ:LD.

Figure 2.

XRD patterns of the raw materials (PVP, MZ, LD), their physical mixture (PM), and the blank and drug-loaded (DL) fibers.

Fourier-Transform Infrared Spectroscopy (FTIR)

FTIR was used to monitor changes in chemical composition during electrospinning. As shown in Figure 3, pure PVP, blank and drug-loaded fibers, and the PM exhibited a characteristic and broad band in the region between 4000 and 3500 cm⁻¹. This band was attributed to the stretching vibrations of the OH group, arising from the hygroscopic nature of the PVP polymer. This study aligns with Abdelghany et al, who reported an OH peak at 3451 cm⁻¹, attributed to PVP’s hygroscopicity.⁴⁷ Moreover, the pure PVP sample and blank PVP fibers exhibited identical signals, as the blank fibers are composed solely of PVP. These spectra displayed characteristic absorption bands of PVP polymer, including stretching vibrations of the C=O (carbonyl) group near 1650 cm⁻¹, stretching vibrations of the C-N group at around 1260 cm⁻¹, and CH-related bands (methylene) in the range of approximately 2930–3000 cm⁻¹.^48,49

Figure 3.

FTIR spectra of raw materials (PVP, MZ, LD), their physical mixture (PM), and the blank and drug-loaded (DL) fibers.

MZ, on the other hand, displayed characteristic peaks corresponding to its functional groups. Aliphatic methyl and methylene C- H stretching vibrations appeared at 2873 cm⁻¹, while aromatic C- H stretching was observed at 3107 cm⁻¹. The imidazole ring showed a characteristic C=N stretching vibration at 1690 cm⁻¹. N-H stretching and bending were detected at 3360 cm⁻¹ and 1588 cm⁻¹, respectively. The benzene ring displayed C=C stretching vibrations in the range of 1506–1396 cm⁻¹. Asymmetric and symmetric stretching of the C-O-C group appeared at 1230 cm⁻¹ and 1038 cm⁻¹, respectively. Additionally, C-H bending vibrations were observed in the fingerprint region, ranging from 915 cm⁻¹ to 564 cm⁻¹.^45,50,51 Furthermore, the LD spectrum exhibited characteristic peaks corresponding to its functional groups. Bands were observed at 3445 cm⁻¹ and 3383 cm⁻¹, corresponding to the symmetric and asymmetric N-H stretching vibrations of the secondary amine group—a peak at 3008 cm⁻¹ related to C-H stretching vibrations of methyl and methylene. The C=O stretching vibrations of the amide group appeared at 1653 cm⁻¹, while the C-N stretching vibration of the amide appeared at 1370 cm⁻¹. The C=C stretching vibrations of benzene were exhibited in the range of 1540–1472 cm⁻¹. Furthermore, N-H bending vibrations in the amide were observed at 1560 cm⁻¹, and C-H bending within the benzene ring was detected in the region of 950–628 cm⁻¹.^46,52,53

The IR spectrum of the PM sample exhibited low intensity and clear peaks in the region of 950–600 cm⁻¹, corresponding to the benzene ring in MZ and LD. In contrast, these peaks disappeared in the spectrum of the drug-loaded fiber, supporting the XRD finding and confirming that the drugs were successfully incorporated into the fiber matrix.

In this study, hydrogen bonding between PVP and the drug molecules cannot be completely excluded, and the FTIR spectra showed no notable shifts or broadening of the characteristic absorption bands. Consequently, this FTIR analysis did not provide clear evidence for hydrogen-bond formation in the drug-loaded electrospun nanofibers. This absence of clear evidence might be explained by the possible overlap between the characteristic absorption bands of the dominant PVP matrix and those of the incorporated drugs, as well as by the relatively low drug concentration (0.5% w/v) within the PVP matrix. Therefore, further investigation would be required to confirm or characterize any potential intermolecular interactions between PVP and the incorporated drugs.

Quantitative Analysis of MZ and LD Using HPLC to Assess Encapsulation Efficiency (EE%) and Drug Loading (DL)

Developing an analytical method to detect MZ and LD is an essential step in quantifying DL and EE% and evaluating the release of both drugs from the nanofibrous formulation. HPLC analysis confirmed the successful separation of MZ and LD, with retention times (Rt) of 11.8 minutes for MZ and 6.3 minutes for LD. The calibration curves for both drugs are presented in the Supplementary Materials Section (Figure 1). The electrospun PVP nanofibers demonstrated efficient drug encapsulation for both MZ and LD. LD demonstrated an EE% of approximately 89% ± 6%, with a DL of 41 ± 2 µg/mg. In comparison, MZ showed a higher encapsulation efficiency of 100% ± 2% and a DL of 46 ± 1 µg/mg. These results confirm that the PVP nanofibers effectively encapsulated both drugs, with LD achieving near-complete incorporation and MZ approaching full incorporation within the experimental variability.

Drug Release of MZ/LD-Loaded Nanofibers

As shown in Figure 4, MZ and LD from the monoaxial nanofibrous system exhibited distinct release kinetics. Approximately 70% of LD was released within the first few minutes, and complete release (100%) was observed after 30 minutes. In comparison, nearly 55% of MZ was released initially, reaching almost complete release (95%) after 120 minutes. These findings highlight the rapid and efficient release of both drugs, but a slightly quicker release profile for LD owing to its hydrophilic nature. MZ is classified as a Biopharmaceutics Classification System (BCS) Class II compound, characterized by low solubility and high permeability.⁵⁴ However, it exhibited a rapid release rate when incorporated into this polymeric nanofibrous system. This enhanced release can be attributed to the molecular dispersion of MZ within the nanofiber matrix achieved during electrospinning, as indicated by the XRD results.

Figure 4.

In vitro release of MZ/LD PVP electrospun fibers shows complete release of LD and MZ after 1 and 2 hours, respectively. Results are expressed as the mean ± SD from independent triplicates.

Previous studies have shown that hydrophobic drugs with poor solubility and low bioavailability can be rapidly released when incorporated into amorphous hydrophilic polymeric nanofibers for buccal or sublingual use. For example, Sipos et al delivered aceclofenac (a BCS Class II drug) using trolamine/PVP nanofibers, thereby achieving supersaturation upon dissolution due to the drug’s amorphous form. The polymer matrix dissolved quickly and completely, and the aceclofenac-loaded nanofibers exhibited rapid, complete dissolution in the release medium within minutes.⁵⁵ Li et al have also delivered a hydrophobic drug (carvedilol) on a hydrophilic nanofiber system (PVP/PEG-400) and shown rapid release of 80% of the drug within the first 30 minutes.⁵⁶ The enhanced dissolution rates of the fibrous formulation of the previously mentioned studies are attributable to the increased specific surface area of the nanofibers, the amorphous nature of the active ingredient after being electrospun, and the highly hydrophilic properties of the fiber-forming polymer. The rapid release of the second drug in the fibrous system, LD HCl, is align with Clitherow et al who reported a rapid release of LD HCl loaded on 10% (w/w) PVP, 12.5% (w/w) Eudragit RS100, and 10% (w/w) PEO (400 kDa) designed for oral mucosa delivery, with 80% released in the first hour, compared to 100% release observed in the present study.¹⁶ Differences in release rates are associated with variations in the release media used in the respective studies.

Overall, the observed release profile is clinically relevant for localized treatment of oral candidiasis characterized by burning, soreness, and discomfort. The rapid release of LD provides prompt local analgesia, reducing pain and discomfort related to oral lesions. Meanwhile, the more moderate effect of MZ facilitates early exposure to antifungal activity. Their combination further enhances antifungal efficacy.

Because conventional topical dosage forms are limited by salivary flow, tongue movement, and swallowing, electrospun and mucoadhesive platforms have been explored to deliver antifungal agents, alone or with analgesics, for the treatment of oral candidiasis.⁵⁷ For instance, Tonglairoum et al⁵⁸ loaded clotrimazole into electrospun fibers and observed faster antifungal activity than commercial clotrimazole lozenges; the fiber mats released approximately 64.81–74.15% of the drug within 4 hours and remained safe and mucoadhesive for at least 2 hours. Likewise, Tajeda et al¹⁷ demonstrated that buccal films containing both LD and MZ enabled rapid LD release for local anesthesia while sustaining MZ release over 24 hours, thereby providing prolonged antifungal activity. Similarly, our dual-drug nanofiber system was designed to enhance oral retention and sustain therapeutic drug levels at the mucosal surface. By combining rapid symptomatic relief with the moderate, localized antifungal effect, this platform represents a promising approach for managing pain associated with oral candidiasis.

It should be noted that release kinetic modeling was not performed in the present study. However, due to the rapid disintegration of the drug-loaded PVP nanofibers (≤12 seconds) and the molecular transformation of the loaded drugs after being electrospun (from crystalline to amorphous states) may contribute to the rapid release observed for both MZ and LD,^35,59. However, further studies incorporating appropriate kinetic models would be valuable for quantitatively characterizing the release mechanism and elucidating the contributions of matrix disintegration and drug solid-state properties to the observed release profiles. Moreover, evaluating drug release under more physiologically relevant, dynamic oral conditions, such as artificial saliva using dynamic-flow or flow-through models, would provide a more representative assessment of release behavior and its potential clinical relevance.

In Vitro Cytotoxicity Assessment of the Drugs

The percentage of viable HFF-1 cells for MZ, LD, and their combination was assessed using the MTS assay to ensure their biological relevance in mammalian cells, as shown in Figure 5. The MTS assay results indicated that MZ concentrations of 16 µg/mL or lower were nontoxic, confirming biocompatibility at this concentration. At the same time, the highest cell viability was observed after treatment with LD at 1250 µg/mL. The combination of LD and MZ at 78 and 16 µg/mL, respectively, maintained the highest cell viability among the tested combinations. The cell viability % of the combined drugs followed the MZ cell viability results, which showed safety at 16 µg/mL alone, indicating that despite using LD at a 5-fold higher concentration (5:1), there was still no improvement in MZ cell viability % (Figure 5).

Figure 5.

HFF-1 cells viability assessment of (A) MZ; (B) LD; and (C) LD:MZ combinations at a 5:1 ratio. Different concentrations and time intervals (24 hours and 48 hours) showed no toxic effects at concentrations of < 16 µg/mL for MZ, < 1250 µg/mL for LD, and < 16 and 78 µg/mL for MZ and LD, respectively, for their combination. Data are presented as mean ± SD (n = 3).

MZ produced a concentration-dependent reduction in HFF-1 cell viability, consistent with a recent study of human gingival fibroblasts, which found that 8 µg/mL of MZ was non-toxic, whereas 64 µg/mL increased apoptosis and exhibited cytotoxicity on this cell line. The observed differences between the current study and the recently reported study are attributable to cell line type, methodology, and exposure conditions. The reported safe concentration of MZ (8 µg/mL) in that recent study was also higher than the antifungal MIC (0.5 µg/mL) against Candida albicans.⁶⁰ Similarly, in this current study, the observation of high cell viability at 16 µg/mL indicates that HFF-1 cells maintained substantial viability at a concentration above the reported antifungal MIC (0.5 µg/mL). However, MIC values reflect antimicrobial activity under specific experimental conditions and should not be directly extrapolated to clinical therapeutic or tissue concentrations.

Based on a previous study evaluating LD cytotoxicity in human oral mucosa fibroblasts, cell viability was significantly reduced at concentrations >5% (5000 µg/mL).⁶¹ Whereas in the current study, concentrations >1250 µg/mL substantially reduced cell viability. Therefore, the concentrations that maintained high cell viability demonstrated biocompatibility under the experimental conditions used in this study, suggesting a safety profile for both drugs at concentrations above the reported therapeutic range; however, further investigation is required to determine the safest concentration for this combination.

Minimum Inhibitory Concentration (MIC) Assay

The MIC analysis results, as shown in Figure 6, reveal differences in the efficacy of the tested compounds. The MZ displayed an MIC value of 0.004 µg/mL against Candida albicans (ATCC 66027), confirming its high potency. This finding aligns with the results reported by Tejada et al, who observed an MIC of 0.004 µg/mL (4 ng/mL) for MZ.²⁴ Similarly, previous studies have reported MIC values for MZ ranging from 2 to 0.002 µg/mL, indicating its greater potency.^39,62 In contrast, LD did not markedly inhibit the growth of Candida albicans at the tested concentrations ranging from 2 to 0.001 µg/mL, compared with MZ. However, several studies have reported antifungal activity of LD against Candida albicans strains at much higher concentrations, from 1.25 to 40 mg/mL.^18–20 limitation: This study has a limitation in that MIC testing was performed in glucose-supplemented MHB rather than in RPMI 1640/MOPS as recommended by CLSI M27-A3; thus, the absolute MIC values cannot be directly extrapolated to standardized susceptibility breakpoints, and future studies will include CLSI-compliant MIC determinations to corroborate these in vitro findings.

Figure 6.

A graph of the MIC assay against Candida albicans (ATCC 66027) shows inhibition of the fungus when treated with serially diluted concentrations of MZ and LD, ranging from 2 to 0.001 μg/mL, as well as a 1:1 combination. The positive control is not treated with the fungus, and the negative control is the broth medium only. The results are shown as mean ± SD (n=3). MCZ=Miconazole, LDC: Lidocaine.

Moreover, the MZ:LD (1:1) combination yielded a lower MIC of 0.001 µg/mL (1 ng/mL) than MZ alone, indicating enhanced antifungal activity at the tested concentrations. A previous report has shown that LD has dose-dependent fungistatic and fungicidal activity against various Candida species. At lower concentrations, it induces metabolic impairment in yeast cells, whereas at higher concentrations, it directly compromises membrane integrity, leading to cell death.²⁰ This disruptive action likely enhances the penetration of co-administered MZ into fungal cells, thus lowering the concentration required to inhibit growth. In our study, this mechanism was not directly investigated; the exact molecular interactions between LD and fungal membranes, or between LD and antifungal drugs, remain hypothetical. Further mechanistic studies, including membrane integrity assays, fluorescence microscopy, and biophysical studies, are needed to investigate the observed enhancement in the antifungal effect of MZ: LD.

Antimicrobial Activity of MZ/LD–Loaded PVP Nanofiber Patches

The diffusion assay results demonstrated that the fabricated drug-loaded nanofiber patch system effectively inhibited Candida albicans growth. As shown in Figure 7, the free drugs of MZ and LD (used as positive controls) produced a zone of Inhibition (ZOI) measuring 21 ± 1 mm at a concentration of 23 µg/mL. In comparison, 0.5 mg of MZ/LD-loaded PVP nanofiber patches (loaded with 23 µg of MZ and 20.5 µg of LD) produced a ZOI of 20 ± 1 mm. The blank PVP nanofiber patches, used as a negative control, exhibited a ZOI of 5 mm, likely due to mechanical displacement of the patch during placement on the agar surface, as shown in Figure 7. These findings confirm that the drug-loaded nanofiber patches retained MZ’s antifungal efficacy against Candida albicans after electrospinning.³⁹

Figure 7.

Antimicrobial diffusion assay against Candida albicans (ATCC 66027). The fungal strain was treated with MZ/LD-loaded discs (containing 20 µg of LD and 23 µg of MZ; left image, D), MZ/LD—loaded nanofibers (similar concentrations to the loaded discs; middle image, F), and blank nanofibers (similar weight to the drug-loaded nanofibers; right image, B). After overnight incubation, the inhibition zones were measured as 21 ± 1 mm and 20 ± 1 mm for the loaded discs and the drug-loaded fibers, respectively. Results are the mean ± SD (n=3).

Abbreviations: MZ, Miconazole; LD, Lidocaine.

Previous studies have also investigated the antimicrobial efficacy of novel drug delivery systems incorporating MZ and LD for the treatment of oral candidiasis. For instance, one study evaluated buccal films containing microencapsulated MZ nitrate embedded in a hydroxypropyl methylcellulose (HPMC-GEL) matrix. The results demonstrated that these buccal films provided sustained drug release and effectively maintained antifungal activity over time.¹⁷ In another study, nanoparticles encapsulated with both MZ and LD were developed. This delivery system prolonged the release of MZ nitrate, maintaining antifungal effectiveness against Candida albicans for at least 24 hours.²⁴ Furthermore, in a study by Santos et al, PVP/PMMA polymer nanofiber disks were loaded with the antiseptic cetylpyridinium chloride (CPC) to evaluate their antifungal activity. The results showed that nanofibers containing 5% CPC produced larger inhibition zones against Candida albicans than those containing 5% MZ in the CPC/PVP/PMMA system. This enhanced antifungal effect was attributed to the faster release of CPC from the PVP/PMMA nanofibers, in contrast to the slower release of MZ, due to its larger molecular weight, rather than to MZ’s lower potency.⁶³ These findings suggest that localized drug delivery systems offer a new approach to improving the therapeutic efficacy of oral fungal infection treatment.

In Vivo Evaluation of the Antifungal Activity Against the Oral Candidiasis Mouse Model

An animal model was used to evaluate the efficacy of dual-drug-loaded nanofiber patches compared with the commercial oral MZ gel in treating oral candidiasis. Additionally, a single drug-loaded nanofiber patch and drug-free (blank) nanofiber patches were assessed as experimental controls. Kinetically, we monitored the level of Candida infection in the mice’s oral cavities up to day 7. Candida albicans swabs were collected from the mice’s oral cavities between days 1 and 7 post-inoculation. As shown in Figure 8, untreated and blank nanofiber patch groups showed no significant reduction in CFU over time, indicating no antifungal activity. This confirms that nanofiber patches alone do not contribute to antifungal activity. Starting on days 5–7, animals given MZ nanofiber patches and the MZ market gel showed a moderate reduction in Candida albicans CFU in their mouths. The group treated with LD nanofiber patches also exhibited a modest reduction in CFU, suggesting antifungal activity that warrants further investigation. A previous study reported that LD exhibits fungistatic activity at low concentrations (5.0–40.0 mg/mL) and fungicidal activity at higher concentrations (50 mg/mL).²⁰ Notably, from day 3 to day 7, the animals treated with the MZ/LD nanofiber patch exhibited a sharp, significant (P < 0.001) reduction in CFU at day 3, followed by a marked reduction in fungal burden by day 7 (Figure 8). Moreover, groups treated with MZ/LD nanofiber patches showed a significant reduction (P < 0.0001) in the number of CFU (22 CFU/mL) compared with groups treated with MZ nanofiber patches, LD nanofiber patches, and the MZ market gel at day 7 (Figure 9).

Figure 8.

Effect of locally administered groups 2–7 on the oral candidiasis model in mice, showing the number of viable Candida albicans in the oral cavity over 7 days following infection. Data are presented as mean ± SD (n = 4). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple-comparison test. **P < 0.001 for MZ/LD nanofibers compared with MZ nanofibers and the marketed MZ gel.

Figure 9.

Effect of locally administered groups 4–7 on the oral candidiasis model in mice, showing the number of viable Candida albicans in the oral cavity on day 7 after infection. Data are presented as mean ± SD (n = 4). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05 for MZ/LD nanofibers compared with MZ nanofibers and the marketed MZ gel. ****P < 0.0001 for MZ/LD nanofibers compared with LD nanofibers.

The combination of MZ and LD in the nanofiber patches was the most effective group, exhibiting enhanced antifungal activity, as previously demonstrated in Figure 6. This activity may be attributed to LD disrupting the Candida membrane, enhancing MZ penetration. The nanofiber ensures the localization and sustains the drug release at the infection site. According to Tejada et al, an LD-loaded buccal film co-loaded with MZ nitrate microbubbles has demonstrated antifungal activity for up to 24 hours. In this system, LD contributed to rapid symptom relief through its anesthetic effect and enabled the immediate, localized action of MZ. This implies that, beyond its symptomatic benefits, LD may also help modify the mucosal environment.¹⁷

Furthermore, we evaluated the lesion score in an experiment assessing the therapeutic efficacy of locally administered MZ, LD, and MZ/LD nanofibers compared with the MZ market gel in a mouse model of oral candidiasis (Figure 10). The lesion score, assessed on day 7 post-infection, served as a clinical indicator of disease severity based on the extent of visible white plaques, erythema, and mucosal damage on the tongue. The combination treatment group (MZ/LD nanofiber patches) demonstrated the lowest lesion score, suggesting enhanced antifungal activity and mucosal healing compared to either MZ nanofibers, LD nanofibers, or the marketed MZ gel. Meanwhile, the LD nanofiber-treated group showed only a modest reduction in tongue lesion score, which was not statistically significant compared with the infected untreated group. A limitation of this proof-of-concept study is the relatively small sample size, with four animals per treatment group. Although this number was selected in accordance with institutional ethical guidelines and the principles of the 3Rs to minimize animal use, future studies should include larger cohorts with an a priori sample size calculation to increase statistical power and provide more precise estimates of treatment effects.

Figure 10.

Effect of locally administered groups 4 to 7 on the oral candidiasis model in mice, showing the score of tongue lesions on day 7 after infection. The data are presented as mean ± SD (n = 4).

Additionally, both the MZ-loaded nanofibers and the marketed MZ gel demonstrated comparable reductions in tongue lesion scores, consistent with the findings of Alfassam et al, who reported similar outcomes using MZ-loaded centrifugal-spun sugar fibers, with a drug loading of 52 ± 4.5 μg/mg.³⁹ In contrast, the MZ/LD co-loaded nanofiber patches in our study yielded a lower lesion score than both the MZ nanofibers and the market MZ gel, suggesting that the co-delivery of LD may enhance the therapeutic effect beyond that achieved with MZ alone and beyond that reported by Alfassam et al³⁹ While this observation shows enhanced antifungal activity of MZ/LD co-loaded nanofiber patches, it should be regarded as a potential enhancing effect rather than definitive evidence of pharmacodynamic synergy, as formal synergy assays were not performed. Clitherow et al demonstrated that electrospun mucoadhesive patches containing PVP in the polymeric matrix can improve local retention at the application site and promote localized therapeutic delivery, with faster release of LD HCL compared to its base form.¹⁶ Similarly, the rapid release from our MZ/LD co-loaded nanofiber patches provides rapid exposure of the mucosa to both MZ and LD, which may be beneficial for achieving high local concentrations. However, the present data do not demonstrate prolonged release or extended residence time, and future work will explore whether the formulation engineered for extended delivery confers clinical advantages in the treatment of oral candidiasis. In addition, a limitation of this study is the exclusive use of male mice; future studies should include both male and female animals to evaluate potential sex-related differences in therapeutic response and to improve the generalizability of the findings.

Conclusion

Oral candidiasis remains a common opportunistic infection, particularly affecting individuals with compromised immune systems, and is associated with considerable discomfort and potential complications. In this study, a dual drug-loaded electrospun nanofiber system composed of PVP encapsulating MZ and LD was successfully fabricated and evaluated for its antifungal efficacy. The fibers exhibited smooth, uniform morphology with an average diameter of 490 ± 122 nm. Both drugs demonstrated high EE%, with MZ at approximately 100% and LD at 89%. The drug release study indicated rapid dissolution of LD (within 30 minutes) and MZ (within 120 minutes), resulting in rapid local exposure of the oral mucosa to both analgesic and antifungal agents rather than a prolonged, sustained release. The MIC of the MZ/LD was 0.001 μg/mL, compared to 0.004 μg/mL for MZ only, while the dual drug-loaded nanofibers demonstrated an inhibition zone of 20 ± 1 mm, equivalent to that of the dual drug-loaded disc (21 ± 1 mm), which was used as a control, against Candida albicans. Importantly, no cytotoxic effects were observed at concentrations < 16 µg/mL for MZ, < 1250 µg/mL for LD, and < 16 and 78 µg/mL for MZ and LD, respectively, for their combination, confirming the biocompatibility of these concentrations for the drugs used. The in vivo results further confirmed the enhanced antifungal performance of the MZ/LD co-loaded nanofiber patches, with an enhanced reduction in fungal burden (22 CFU/mL) and improved tissue healing compared to both MZ-only nanofiber patches and a commercial MZ formulation.

Overall, these findings suggest that the dual drug-loaded nanofibrous system offers a promising therapeutic strategy for treating oral candidiasis. Future studies should investigate the combined antifungal activity of MZ/LD co-loaded nanofibers using checkerboard/fractional inhibitory concentration index (FICI) assays, as well as time-kill, membrane integrity, and permeability assays, to determine whether the observed effects are synergistic, additive, or coincidental. In addition, further studies should evaluate the fibers’ mucoadhesive strength, long-term storage stability, and mechanical properties. In addition, clinical models mimicking salivary flow and mucosal dynamics could provide deeper insights into the long-term efficacy and retention of the nanofiber system. Finally, larger sample sizes would increase statistical power and provide more precise estimates of treatment effects. Collectively, these investigations will strengthen the evidence supporting the clinical translation of this innovative oral delivery platform.

Funding Statement

This study received funding from a research grant funded by the Research, Development, and Innovation Authority (RDIA) - Kingdom of Saudi Arabia - with grant number (12932-KACST-2023-KACST-R-3-1-HW).

Data Sharing Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Ethics Statement

The in vivo study protocol was approved by the Local Committee of Ethics of Research on Living Creatures in King Abdulaziz City for Science and Technology- Institutional Review Board (KACST-IRB#25007), in adherence with the Declaration of Helsinki, the Guiding Principle in Care and Use of Animals (DHEW production NIH 80-23) and the Standards of Laboratory Animal Care (NIH distribution #85-23, reconsidered in 1985).

Disclosure

This work was submitted to the Saudi Authority for Intellectual Property under submission number (1020256425) on (01 September 2025). The mention of a patent application is solely to disclose the intellectual property status of the formulation and does not affect the scientific design, data acquisition, analysis, or interpretation. Also, the experimental work and conclusions are based entirely on the data presented in the manuscript, independent of any patent considerations. The authors declare no other conflicts of interest in this work.