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INTRODUCTION
The incidence of Skin Cancer (SC) has increased dramatically over the past several decades.1 Skin cancers can be divided into melanoma and non-melanoma skin cancers, such as basal cell and squamous cell.2 Therapies available include surgery, chemotherapy, radiation therapy, immunotherapy and molecular targeted therapy as they depend on tumor histology and stage.3 Surgery is the primary treatment for early stage tumors, but it can lead to scarring, loss of function, and cosmetic issues, particularly in the areas of the body that are sensitive.4 There is a possibility of recurrence after successful surgery, and many patients are non-responsive, resistant to therapy, or have serious side effects.5 Local and topical delivery systems enable direct delivery to the tumor location with minimal systemic exposure.6 The stratum corneum, however, is a barrier to topical vehicles (creams, gels, and ointments) that restricts transdermal efficacy, release, and retention, reducing their effectiveness and compliance.
The high surface-to-volume ratio, tunable porosity, and extracellular-matrix-mimicking structure of electrospun nanofibers have attracted great interest in their application to topical and targeted therapy of skin cancer.7–9 The nanofibers can be used to encapsulate anticancer drugs and for controlled local delivery, and can have high skin adherence.10 They take advantage of the stratum corneum barrier by having high surface area for skin adhesion, a favorable concentration gradient for drug diffusion as well as having nanoscale dimensions that would enhance permeation through skin pores.11 Electrospinning allows the precise control of fiber composition, morphology and biofunctionalization, providing multi-functional constructs capable of simultaneous delivery of different drugs or photosensitizers and targeting moieties;12 encapsulation within a nanometric fiber prevents rapid inactivation of the drug by physiological or environmental factors.13 The polymers used in this study were Polycaprolactone (PCL) and Polyvinylpyrrolidone (PVP), and the functional molecules were itraconazole and quercetin. PCL is an FDA approved, biodegradable, synthetic polymer that can be dissolved in methanol, acetic acid, chloroform, benzene, trifluoroethanol and dichloromethane.14 High hydrophobicity and slow degradation are the limitation of its biomedical application; it is biocompatible in vitro and in vivo and has been blended with water soluble and biocompatible polymers like PVP to enhance its hydrophilicity and biocompatibility. PVP is a hydrophilic polymer that has excellent biocompatibility and low toxicity, has strong adhesion capabilities, and can interact with hydrophilic and hydrophobic materials.15 PCL and PVP are tissue compatible and non-toxic.16,17 Itraconazole is a broad-spectrum triazole antifungal drug which recently has been reported to have anticancer activity due to its antiangiogenic and anti-signaling effects in tumor growth and proliferation.18,19 Quercetin is a natural polyhydroxy flavone that has antioxidant, antitumoral, antimicrobial and antiviral properties.17–20 Itraconazole is a BCS Class II drug, with variable oral bioavailability, high plasma protein binding, poor dermal permeability, and a highly variable response to the drug when applied topically.21 Quercetin is also poorly soluble in water, has a short half-life, is light-sensitive and is rapidly metabolized prior to systemic circulation, thus reducing its stability and availability.22,23 Hence, an advanced drug delivery system is required for enhancing the solubility, stability, permeability and controlled release of both drugs.
Itraconazole and quercetin could be synergically used for the treatment of cancer and wounds, providing enhanced anticancer and antioxidant properties, when combined in the same nanofibrous matrix to enhance the management of postoperative skin cancers. The delivery of itraconazole and quercetin in PCL/ PVP nanofibers for the treatment of post-surgical skin cancer has not been extensively explored. The main goal of this study was to design, optimize and evaluate dual drug-loaded PCL/PVP nanofibers to be prepared using a Box-Behnken experimental design. The formulations were morphologically characterized, thermally evaluated, and assessed for crystallinity, wettability and drug release kinetics, followed by their in vitro antioxidant activity and cytotoxicity against A431 human epidermoid carcinoma cells, which proved them to be suitable as a sustained release localized therapeutic platform for managing post-surgical skin cancer treatment. Although individual studies on itraconazole and quercetin nano formulations have been reported, there is no previous work that has combined both agents in a single electrospun PCL/PVP nanofiber system optimized using Box-Behnken Design for the localized, sustained post-surgical delivery targeting residual tumour cells and oxidative stress simultaneously.
MATERIALS AND METHODS
Materials
Itraconazole (C35 H38 Cl2 N8 O4) and quercetin (C15 H10 O7) were procured as pharmaceutical-grade substances. Polycaprolactone (PCL, Mw ~9000-11000 Da) and Polyvinylpyrrolidone (PVP K30) were obtained from Sigma-Aldrich. Chloroform (CHCl3) and Dimethylformamide (DMF) (C3 H7 NO) were of analytical reagent grade. Phosphate buffer saline (pH 7.4) was prepared in the laboratory. A431 human epidermoid carcinoma cells were procured from the National Centre for Cell Science (NCCS), Pune, India. All other reagents were of analytical grade and used without further purification.
Preformulation Studies
Determination of λmax
The λmax of itraconazole and quercetin was determined by measuring the standard solution of both compounds (10 µg/mL) using a UV-visible spectrophotometer with a wavelength range of 200 to 400 nm. For this purpose, the standard solutions of both drugs were prepared in chloroform and DMF, respectively. It is important to mention here that the respective solvents served as the blanks.
Calibration Curve
s for itraconazole and quercetin were prepared by dissolving 10 mg of each drug separately itraconazole in 1 mL of chloroform and quercetin in 1 mL of DMF and making up to 10 mL with phosphate buffer to obtain stock solutions of 1000 µg/mL each. From each stock solution, 1 mL was pipetted and diluted to 100 mL with phosphate buffer to yield intermediate solutions of 10 µg/mL. Aliquots of 2, 4, 6, 8, and 10 mL from each intermediate solution were transferred into separate 10 mL volumetric flasks and made up to the mark with phosphate buffer, giving working standard concentrations ranging from 2 to 10 µg/ mL. Absorbance of itraconazole solutions was determined at λmax of 262 nm and quercetin solutions at λmax of 374 nm with the UV-vis Spectrophotometer using phosphate buffer as blank. Calibration curves were plotted between absorbance and concentration and the linearity was verified by obtaining regression equations and correlation coefficients with linear regression analysis (r²).24,25
Compatibility Study (FTIR)
To evaluate the chemical compatibility and identify possible interactions, Fourier Transform Infrared (FTIR) spectroscopy was used with a Shimadzu FTIR spectrophotometer (ATR mode), ranging from 4000-400 cm⁻¹ for pure drugs, individual polymers, physical mixtures (binary and multicomponent), and the final nanofiber formulation.
Preparation of Nanofibers
Preparation of Polymeric Solution
The polymeric solution was prepared by appropriate amount of Polycaprolactone (PCL) (0.84 g) was dissolved in chloroform (7 mL) under magnetic stirring of 400-500 rpm, controlled temperature of 30-40ºC, for 1-2 hr until a clear and homogeneous solution was obtained. Under similar stirring conditions, 0.36 g of Polyvinylpyrrolidone (PVP) was completely dissolved in 3 mL of Dimethylformamide (DMF) after 15-30 min. Once the polymers had completely dissolved, itraconazole was added into the PCL solution and stirred to complete dissolution. Simultaneously, quercetin was dissolved in DMF and added to the PVP solution while stirring. The PVP-quercetin solution was slowly added to the PCL-itraconazole solution, stirred for 3 hr at 400-500 rpm at 30-40ºC, and formed a clear and uniform viscous solution appropriate for electrospinning. The preparation of the polymeric solution is illustrated in Figure 1.
Electrospinning of nanofibers
The prepared polymeric solution was allowed to cool to room temperature and kept undisturbed for 30 min to ensure stabilization and removal of air bubbles. The solution was then transferred into a 10 mL syringe equipped with a 24-gauge needle. The electrospinning process was performed at an applied voltage of 17-19 kV and a controlled flow rate of 0.25 mL/hr. The distance between the needle tip and the collector was maintained at 15 cm. The collector was covered with aluminum foil to facilitate easy collection of nanofibers. The electrospinning process was continued for approximately 3 hr, after which the formed nanofibers were collected from the surface of the collector and stored for further characterization.26 A schematic representation of the electrospinning setup is shown in Figure 1.
Experimental Design and Optimization
The formulation optimization was done through three factor and three level Box-Behnken design, which was generated by Design-Expert® 13 software. The variables considered were the applied voltage (A: 12-18 kV), flow rate (B: 0.25-0.50 mL/hr) and the PCL:PVP ratio (C: 2.0-2.7). The responses were the diameter of the fibers (Y1: minimize), efficiency of encapsulation (Y2: maximize) and percentage drug release after 8 hr (Y3: maximize). The number of experimental runs generated was seventeen with five center point replicates. The data were fitted into a second order polynomial equation and analyzed using ANOVA (Design-Expert® 13). The independent variables and their coded levels used in the Box-Behnken Design are shown in Table 1.
Characterization
Morphological study
Scanning electron microscopy was used to characterize the morphology of the electrospun nanofiber scaffolds. Surface morphology and fiber diameter were evaluated using a ZEISS field-emission SEM. Samples were sputter-coated with gold under argon atmosphere and imaged at 10.00 kV, 11.0 mm working distance, at magnifications of 10,000×-45,000×. Fiber diameters were measured from three independent measurements using the ZEISS measurement tool.
Differential Scanning Calorimetry (DSC)
The DSC of pure components revealed the polymorphic transition (155.0ºC) and melting endotherm (168.2ºC) of itraconazole, the dehydration endotherm (105.3ºC) and melting endotherm (320.4ºC) of quercetin, the semicrystalline melting peak (60.3ºC, ΔHf=-81.5 J/g) of PCL, and the glass transition (~170ºC) without melting of PVP K30 which is amorphous. For the drug-polymer interaction, the PCL peak was broadened and suppressed (107.80ºC) in the ITZ/QCT-loaded nanofibers, confirming the amorphous solid dispersion formation, while both drug melting peaks were completely absent. The Purity analysis results confirmed enhancement in formulation integrity (99.34 mol%) without degradation during electrospinning, which led to improved solubility and dissolution (Table 5 and Figure 7).27,28
X-ray Diffraction (XRD)
Crystalline state and phase identification of pure drugs, polymers and nanofiber formulation were assessed using a PANalytical X'PERT PRO diffractometer equipped with Cu Kα radiation (λ=1.54060 Å) at 40 kV and 10 mA. Samples were scanned over a 2θ range of 10°-80° with a step size of 0.017° 2θ at a scan speed of 5.715 s/step. Peak positions, d-spacing values, and relative crystallinity index were analyzed using HighScore Plus software.27,28
Contact Angle Measurement
Water contact angles were measured by the sessile drop method using a goniometer equipped with a digital camera. Deionized water droplets (5 µL) were deposited on 2 × 2 cm nanofiber specimens and contact angles recorded immediately. Three independent measurements were taken and averaged.29
Encapsulation Efficiency
was determined in triplicate (n = 3), and the results are expressed as mean±Standard Deviation (SD). (). Encapsulation Efficiency ( EE%) is the fraction of entrapped drug loaded in the nanofiber matrix compared to the amount of drug added to the formulation during nanofiber fabrication. The drug loaded nanofibers (10 mg) were dissolved in phosphate buffer (pH 7.4) with gentle stirring of 10 mL. The solution was filtered through Whatman filter paper No. 1 and 1 mL of the clear filtrate was diluted to 100 mL using phosphate buffer (100 × dilution). Absorbance was measured spectrophotometrically using a UV-visible spectrophotometer (Shimadzu UV-1800, Japan) at 262 nm (Itraconazole) and 374 nm (Quercetin). Concentration of drugs was determined by fitting calibration equations (y = 0.031x [ITZ, R² = 0.999] and y = 0.059x [QCT, R² = 0.999]), and the total amount of drug recovered was calculated (mg). Encapsulation efficiency was calculated
In vitro Drug Release
The in vitro release of the drug from nanofiber formulations was determined using the Franz diffusion cell with a 0.45 µm cellulose acetate membrane that was soaked in glycerol for 12 hr. The receptor chamber was filled with 60 mL of phosphate buffer (pH 7.4) at 37±0.5ºC with stirring (50-100 rpm). Nanofiber formulation (10 mg) was placed on the membrane in the donor compartment. Samples (1 mL) were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hr and replaced with fresh buffer to ensure the sink conditions remained.
Samples were withdrawn (1 mL) and placed immediately into 10 mL volumetric flasks and diluted to the mark with the phosphate buffer (pH 7.4), giving a dilution of 10 × . Absorbance values of diluted samples of Itraconazole and Quercetin were determined using the double-beam UV-visible spectrophotometer at λmax = 262 nm and 374 nm with phosphate buffer as blank, respectively. Drug concentrations were determined using previously validated calibration equations and Cumulative percentage Drug Release (%CDR) was calculated with appropriate correction for volume replacement at each sampling time point. The optimized formulation. The release kinetics were analysed by DDSolver 1.0 (Zero-order, First-order, Higuchi and Korsmeyer-Peppas models).28
Physical Stability Study of Quercetin-Itraconazole Loaded Nanofibers
The optimized nanofiber formulation was packaged in airtight containers and kept at the refrigerated temperature (4-6ºC) and room temperature (25ºC/65±5% RH (Relative Humidity)). The physical appearance and the morphology of samples was determined by Scanning Electron Microscopy (SEM) and the percentage of ( Encapsulation Efficiency ( EE%) for quercetin and itraconazole was determined on Day 0 and Day 45. The study was conducted to evaluate the physical stability of nanofibers for different storage conditions.29
Antioxidant Activity (DPPH ASSAY)
The free radical scavenging activity of nanofibers was performed using the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. A freshly prepared 0.1 mM DPPH solution in methanol was added to serial dilutions of the formulation (50-250 µL) and ascorbic acid (1 mg/ mL) as a positive control. The reaction solutions were kept in the dark at room temperature for 30 min and absorbance was read at 517 nm on a UV-vis spectrophotometer with methanol as blank. The percentage inhibition of DPPH radicals was determined according to the following equation: % Inhibition=[(Ac−As)/Ac]×100. IC50 was calculated based on linear regression of the concentration versus % inhibition graph.
Cytotoxicity Assay
The cell toxicity of ITZ/QCT co-loaded nanofibers was evaluated using MTT assay, which is based on the activity of mitochondrial dehydrogenase, as an indicator of cell viability. The A431 cells (NCCS, Pune) were cultured in the medium MEM containing 10% FBS, 1% sodium bicarbonate, 1% nonessential amino acids, and 1% sodium pyruvate at 37ºC, humidified 5% CO₂, and subcultured at 70-80% confluency. The nanofiber mat was dissolved in sterile DMSO to give stock concentrations of 100-1000 µg/mL (DMSO <0.1% v/v). 24 hr cells cultured in 96 well plate treated with the compound 24 hr, then MTT (0.5 mg/ mL) added for 5 hr; formazan crystals dissolved in DMSO and absorbance read at 570/650 nm to determine viability and IC₅₀. The percentage cell viability was determined by:
The dose-response curve was used to graphically determine the IC₅₀ with GraphPad Prism software.
Ethical statement
No human participants or experimental animals were involved in this study. The A431 human epidermoid carcinoma cell line was procured from the National Centre for Cell Science (NCCS), Pune, India, and all experimental procedures complied with institutional laboratory biosafety guidelines.
Statistical analysis
was performed using Design-Expert® version 13.0 software for optimization and GraphPad Prism version 9.0 for cytotoxicity analysis. Experimental data were expressed as mean±Standard Deviation (SD) from triplicate measurements (n=3). Analysis of Variance (ANOVA) was used to determine the significance of model terms, and p<0.05 was considered statistically significant. Box-Behnken design were analysed using Design-Expert® software. Quadratic polynomial models were generated and evaluated by Analysis of Variance (ANOVA). Model adequacy was assessed using the F-value, p-value, coefficient of determination (R²), adjusted R², predicted R2, adequate precision, Coefficient of Variation (CV), and lack-of-fit test. Statistical significance was considered at p<0.05.
| Factor | Symbol | Low (−1) | Mid (0) | High (+1) |
|---|---|---|---|---|
| Voltage (kV) | A | 12 | 15 | 18 |
| Flow Rate (mL/hr) | B | 0.25 | 0.375 | 0.50 |
| PCL: PVP Ratio | C | 2.0 | 2.35 | 2.7 |
| Conc. (µg/ mL) | ITZ Absorbance (262 nm) | QCT Absorbance (374 nm) |
|---|---|---|
| 0 | 0.000 | 0.000 |
| 2 | 0.061 | 0.115 |
| 4 | 0.123 | 0.239 |
| 6 | 0.185 | 0.353 |
| 8 | 0.248 | 0.478 |
| 10 | 0.311 | 0.591 |
RESULTS
λmax Determination and Calibration Curve
UV spectral scanning established λmax at 262 nm for itraconazole (chloroform; principal peak at 262.00 nm, absorbance 0.571; secondary peak at 236.50 nm) and at 374.5 nm for quercetin (DMF; Band I cinnamoyl system at 374.50 nm, absorbance 0.638; Band II benzoyl system at 256.50 nm, absorbance 0.668), consistent with the characteristic two-band flavonoid UV absorption profile. Calibration curves exhibited excellent linearity (Beer-Lambert law compliance): ITZ: y=0.031x, R²=0.999; QCT: y=0.059x, R²=0.999, over 0-10 µg/mL. The UV-visible absorption spectra of itraconazole and quercetin are presented in Figure 2. The calibration data for itraconazole and quercetin are presented in Table 2.
FTIR Compatibility Studies
FTIR spectra of pure itraconazole revealed characteristic N-H/O-H stretching at 3383.47 cm⁻¹, C=O stretching of piperazinone ring at 1696.05 cm⁻¹, C-N stretching of triazole ring at 1329.84 cm⁻¹, and C-O-C stretching of dioxolane ring at 1223.57 and 1044.05 cm⁻¹. Pure quercetin exhibited phenolic O-H stretching at 3275.77 cm⁻¹, C=O ketone stretching at 1660.14 cm⁻¹, and aromatic C=C stretching at 1602.70 and 1507.91 cm⁻¹.
PCL showed characteristic ester C=O stretching at 1723.33 cm⁻¹ and PVP displayed lactam C=O stretching at 1642.91 cm⁻¹.
Physical mixture spectra of all components demonstrated co-existence of characteristic peaks from all individual ingredients without appearance of new peaks or significant peak shifts, confirming absence of chemical incompatibility. The nanofiber formulation spectrum retained the PCL ester C=O at 1726.20 cm⁻¹, PVP lactam C=O at 1654.40 cm⁻¹, and ITZ C=O at 1586.90 cm⁻¹, with no new peaks, confirming physical compatibility and successful drug encapsulation without chemical interaction during electrospinning.25,30–32 The FTIR spectra of pure drugs, polymers, physical mixtures, and optimized nanofibers are shown in Figure 3.
Box-Behnken Design optimization of Itraconazole-quercetin Loaded Pcl/Pvp Electrospun Nanofibers
A three-factor, three-level Box-Behnken Design (BBD) employing Design-Expert® 13 software generated 17 experimental runs spanning the design space defined by voltage (12-18 kV), flow rate (0.25-0.50 mL/hr), and PCL:PVP ratio (2.00-2.70 mg). Across these runs, fiber diameter ranged from 391.9 to 397.8 nm, encapsulation efficiency from 91.0 to 94.2%, and drug release from 84.8 to 88.4% (Table 3), confirming a wide and informative response surface across the selected factor ranges. Centre-point replicates (Runs 9-13 corresponding to Std 5, 8, 15, 16, 17) showed low variability, supporting the reproducibility and adequacy of the experimental model.
ANOVA and Model Statistics
All three response models were highly significant (p<0.0001). Flow rate (B) was determined to be the most important variable influencing all three responses (p<0.0001), where increase in flow rate leads to an increase in diameter of the fibers but reduction in encapsulation efficiency and drug release rate. The effect of PCL:PVP ratio (C) was highly significant on all three responses, whereas the effect of applied voltage (A) was significant on fiber diameter only. Interaction between BC was significant in all three responses, suggesting a synergistic interaction of flow rate and composition of the polymers. Quadratic effects were significant on EE and drug release models, and hence, quadratic models had to be used. The model fit statistics obtained from ANOVA are summarized in Table 3.
The high R² values (≥0.9669 for all responses) indicated that over 96% of response variation was explained by the respective models. The strong agreement between adjusted R² and predicted R² (difference<0.2) confirmed absence of overfitting. Adequate precision values well above 4 confirmed a sufficient signal to noise ratio for design space navigation is reliable.
The effect of the three independent variables on the response was quantified by obtaining a regression equation for each response in the Design-Expert® software. The regression equations can give information about the effect of the independent variables like the voltage (A), flow rate (B) and PCL:PVP ratio (C).
Y₁: Fiber Diameter (2FI Model)
Y 1 = 394.17 − 0.3243 A + 1.29 B + 0.8970 C + 0.1519 AB + 0.0715 AC + 1.24 BC
Interpretation
Flow rate (B) and PCL:PVP ratio (C) were found to be positive factors affecting the fiber diameter while voltage (A) was negative factor. The most significant factor was the flow rate, followed by the polymer ratio. The BC interaction was highly significant, the AB interaction was not significant and the AC interaction was not significant, verifying the appropriateness of the 2FI model.
Y₂: Encapsulation Efficiency (Quadratic Model)
Y 2 = 92.86 + 0.0792 A − 0.6738 B − 0.5517 C + 0.0479 AB − 0.1615 AC − 0.3309 BC − 0.0324 A² + 0.3308 B² − 0.2403 C²
Interpretation
Flow rate (B) and PCL:PVP ratio (C) significantly affected the encapsulation efficiency with the flow rate having the most significant effect. The voltage (A) had a small and insignificant positive effect. There were significant AC and BC interactions which further reduced EE, and significant quadratic terms (A2, B2, and C2) that gave justification to the quadratic model.
Y₃: Drug Release (Quadratic Model)
Y 3 = 87.48 + 0.0373 A − 0.6658 B − 0.6402 C − 0.0081 AB − 0.1538 AC − 0.5617 BC − 0.0677 A² + 0.0644 B² − 0.1630 C²
Interpretation
Flow rate (B) and PCL:PVP ratio (C) showed significant reduction in drug release, while voltage (A) did not significantly affect the drug release alone. The BC interaction was the strongest negative interaction, followed by the AC interaction, which suggested that both flow rate and polymer ratio had an interaction effect on the drug release. The high value of the A² term was significant, indicating curvature in the response and thus validating the quadratic model.
quadratic model.
Response Surface Analysis
The combined effects of voltage, flow rate, and PCL:PVP ratio on each of the responses were shown in three-dimensional response surface plots (Figure 4). The flow rate and PCL:PVP ratio showed an increase in fiber diameter (391.9-397.8 nm) due to increased delivery of polymer and solution viscosity in the presence of strong electrostatic force. The results of EE indicated that the surface was concave at low voltage and low flow rate and PCL:PVP ratio, and that the surface was dome-shaped at high voltage and high flow rate and PCL:PVP ratio because the viscous overloading effect and incomplete solvent removal effect affected the stability of the encapsulation interface. The release of the drug was greater for higher voltage but lower for higher flow rate and lower PCL:PVP ratio, because thicker and less porous membranes prevented diffusion and less cumulative release of the drug.
Perturbation, Interaction, And Contour Plot Analysis
Perturbation plots revealed that flowrate was the most significant factor for fiber diameter, encapsulation efficiency, and drug release while PCL:PVP ratio had the strongest influence on the three responses due to its effect on solution viscosity, fiber formation, and drug entrapment. The interaction plots showed that there were synergistic and quadratic interactions for flowrate, PCL:PVP ratio and voltage for all responses, with BC (flowrate × PCL:PVP ratio) being most significant and AC (voltage × PCL:PVP ratio) being significant for encapsulation efficiency and drug release. The optimum region (high voltage, low flowrate, low PCL:PVP ratio) was verified by contour plots, which showed the nonlinear variable interactions and confirmed the Box-Behnken design (Figure 5).
Numerical Optimization and Validation
A numerical optimization was performed simultaneously considering minimum fiber diameter, maximum EE, and maximum drug release within the range (all desirable) that yielded 76 feasible solutions. The top-ranked optimal formulation with composite desirability D=0.927 was identified at Voltage=17.0 kV, Flow Rate=0.25mL/hr, and PCL: PVP=2.29, predicting Diameter=392.459 nm, EE=93.921%, and DR=88.220%. The optimized formulation parameters obtained from numerical optimization are presented in Table 4.
The optimized formulation was experimentally prepared and characterized, and was found to be having fiber diameter of 389.8±35.0 nm, EE of 93.95% (ITZ) and 92.89% (QCT) and cumulative drug release of 88.00% (ITZ) and 87.50% (QCT) at 8 hr. The BBD-RSM models showed high predictive accuracy and validity for formulation scale up as indicated by the high correlation between the predicted and experimental values (prediction error<1.5%).
Scanning Electron Microscopy
The optimized nanofibers showed a well-dispersed and randomly oriented fibrous matrix with smooth and continuous fibre morphology, which confirmed that there was no drug aggregation or precipitation in the polymer matrix. A porous interconnected structure was observed at 10,000 × , which facilitated moisture balance and long-term drug permeation. At higher magnifications (20,000 × -25,000 × ), ribbon-like or flattened fiber morphology was seen, which was typical of PCL-PVP electrospun systems and was thought to come from the differential evaporation of the solvent during fiber formation. At high magnification (45,000 × ), precise fiber diameter measurements yielded Pa1 = 351.7 nm, Pa2 = 396.8 nm, and Pa3 = 421.0 nm, giving a mean fiber diameter of 389.8±35.0 nm (n = 3), which is close to the value predicted by BBD-RSM (392-394 nm). High surface area and sub-400 nm diameter are predicted to improve drug dissolution and maintain localized drug delivery at the wound interface. The surface morphology of the optimized nanofibers was examined by SEM (Figure 6).
Differential Scanning Calorimetry
DSC thermograms of pure components showed characteristic events: itraconazole exhibited a polymorphic transition at 155.0ºC and a sharp crystalline melting endotherm at 168.2ºC; quercetin displayed a dehydration endotherm at 105.3ºC and a sharp melt at 320.4ºC; PCL showed a single sharp semicrystalline melting peak at 60.3ºC (ΔHf=-81.5 J/g); and PVP K30 exhibited a glass transition at ~170ºC with no melting endotherm, confirming its fully amorphous character.
In the ITZ/QCT-loaded PCL-PVP nanofiber formulation, the characteristic melting peaks of both itraconazole (168.2ºC) and quercetin (320.4ºC) were completely absent, providing unequivocal evidence of amorphous solid dispersion formation. The PCL melting peak was broadened and suppressed (107.80ºC), indicating disruption of the polymer crystalline lattice by drug-polymer interactions. Purity analysis confirmed formulation integrity at 99.34 mol%, with no evidence of drug degradation during electrospinning. Conversion of crystalline drugs to amorphous dispersions within the polymer matrix is expected to substantially enhance drug solubility and dissolution rate. The DSC thermograms of the pure components and optimized formulation are shown in Figure 7. The thermal parameters obtained from DSC analysis are summarized in Table 5.
X-ray Diffraction
The XRD diffractograms of the nanofiber formulation showed Dominant peaks at 2θ=21.78° (100% relative intensity) and 22.44° (45.42% relative intensity) corresponding to the characteristic diffraction planes of the semicrystalline PCL polymer lattice. The PVP K30 showed no sharp crystalline peaks, in accordance with its amorphous state. Most importantly, the characteristic peaks of pure itraconazole (9.0°, 17.4°, and 18.5° 2θ) and of pure quercetin (11.5° and 27.0° 2θ) were completely missing in the nanofiber formulation. The disappearance of the crystalline reflections of both drugs, analyzed by HighScore Plus software, is definite evidence of complete amorphization (molecular dispersion) of both drugs in the PCL/PVP matrix. This amorphous transformation is in accordance with the DSC results and is anticipated to enhance the dissolution kinetic and therapeutic bioavailability. The XRD diffraction patterns are presented in Figure 7.
Contact Angle and Wettability
The water contacts angle of the nanofiber membrane made of PCL/PVP/ITZ/QCT was 89.49±0.29°, which is a mildly hydrophilic surface (contact angle < 90°). This is a significant improvement compared to the pure PCL membranes which generally have contact angles in the range of 105°-120°. This increased hydrophilicity is because of the amphiphilic character of the PVP K30 and surface-active properties of the loaded drug molecules. The scaffold's slightly hydrophilic property is ideal for post-surgical wound dressing applications, as it allows sufficient drug elution and management of wound exudates and maintains scaffold structural integrity. The wettability of the nanofibers was evaluated by contact angle measurement (Figure 8). The contact angle values are summarized in Table 6.
Encapsulation Efficiency
For the optimized nanofiber formulation, encapsulation efficiency was repeated three times (n=3), to assess the reproducibility of the result. The three independent batches encapsulation efficiency is given below in Table 7. The optimized formulation had an EE of 93.95±0.38% and 92.89±0.42% (mean±SD, n=3) for itraconazole and quercetin, respectively, showing high drug entrapment in the nanofibers. The high encapsulation efficiency in all three batches shows that the optimized formulation is reproducible. The good encapsulation efficiency can be explained by the excellent compatibility between the drugs and the polymer matrix, which was confirmed by FTIR analysis, and by the optimized PCL:PVP ratio and the optimized electrospinning process parameters, which reduced the loss of the drug during the solvent evaporation process, allowing the incorporation of the hydrophilic drug quercetin into the hydrophilic PVP matrix and the hydrophobic drug itraconazole into the hydrophobic PCL matrix. Moreover, the experimental encapsulation efficiency was also found to be close to the predicted value by Box-Behnken Design-Response Surface Methodology (BBD-RSM) which was 93.92%, confirming the accuracy of the optimized model and the robustness of optimized formulation.
In vitro Drug Release and Release Kinetics
In vitro drug release
The in vitro drug release study of the optimized ITZ/QCT-loaded PCL/PVP electrospun nanofibers were repeated three times (n=3), and the values are represented as mean±SD. Itraconazole (ITZ) and Quercetin (QCT) showed a biphasic drug release pattern during the 8 hr evaluation period. Itraconazole release increased from 10.71±0.18% at 0.5 hr to 88.00±0.39% at 8 hr, while quercetin release increased from 12.76±0.17% to 87.50±0.31% over the same period. A small amount of initial burst release was seen in the first 2 hr, followed by a slow and controlled release up to 8 hr. The low values of SD at each sampling point suggest good reproducibility of optimized formulation. The cumulative drug release is summarized in (Table 8).
The in vitro drug release study was performed in triplicate (n=3). At each sampling time, cumulative drug release was calculated and expressed as mean±Standard Deviation (SD).
Release kinetics
Release kinetic analysis was performed using DDSolver 1.0 by fitting the release data to Zero-order, First-order, Higuchi, and Korsmeyer-Peppas models. The cumulative drug release data obtained during the 8 hr study period were fitted to Zero-order, First-order, Higuchi, and Korsmeyer-Peppas kinetic models using DDSolver 1.0. Model fitting revealed that the Korsmeyer-Peppas model provided the best statistical fit for both itraconazole (R²=0.9918, R²adj=0.9908, AIC=−45.11, MSC=4.07) and quercetin (R²=0.9955, R²adj=0.9949, AIC=−51.69, MSC=4.64), as determined by the highest R², adjusted R², and MSC values alongside the lowest MSE and most negative AIC. The Higuchi model also showed good fit (ITZ: R²=0.9679; QCT: R²=0.9803), indicating a significant contribution of diffusion from the polymer matrix, whereas the Zero-order and First-order models demonstrated comparatively inferior fit statistics.
The release exponents (n) of 0.61 for itraconazole and 0.58 for quercetin (both between 0.45 and 0.89), confirmed a non-Fickian (anomalous) transport mechanism, which suggested that drug release had taken place by an interplay between Fickian diffusion through the PCL polymer network and polymer relaxation/ swelling of the hydrophilic PVP. This mechanism is in good agreement with the composite matrix structure of the PCL: PVP system which is composed of a heterogeneous blend of PCL and PVP, in which diffusion in the hydrophobic PCL phase is slowed, and swelling of the PVP phase upon hydration gives rise to a swelling-mediated release component. The results from the goodness of fit for the kinetic models are presented in Table 9.
Best-fit model. n=0.61 (ITZ) and 0.58 (QCT): Non-Fickian anomalous transport.
Physical Stability Study of Quercetin-Itraconazole Loaded Nanofibers
The nanofibers were physically stable during the study period, and the appearance was very clear at both Day 0 and Day 45 under refrigerated and room-temperature conditions. The Day 45 sample was stored at room temperature and SEM results showed that there were very little structural changes in the fiber shape. However, the sample kept under refrigerated conditions exhibited slight changes in its structure, which could be due to the uptake of moisture during storage. The encapsulation efficiency slightly decreased after 45 days, suggesting good retention of quercetin and itraconazole. Overall, the room temperature storage proved to be more stable physically than the refrigerated storage. The physical stability results are presented in Table 10. The SEM images after the stability study are shown in Figure 9.
Antioxidant Activity (DPPH ASSAY)
The results of the DPPH free radical scavenging assay showed that both the standard (ascorbic acid) and the test sample (the nanofiber) exhibited concentration-dependent increases in % inhibition. Ascorbic acid showed % inhibition from 11.1% (2 µg/mL) to 72.3% (10 µg/mL) with excellent linearity (y=7.540x-4.503, R²=0.997). The ITZ/QCT-loaded nanofiber formulation exhibited % inhibition values of 19.44%, 33.33%, 51.38%, 62.50%, and 72.22% % at 50, 100, 150, 200, and 250 µL, respectively, yielding an IC50 of approximately 158.3 µL. The phenolic hydroxyl groups of quercetin, a well characterized free radical scavenger, are the main contributors to the Strong anti-oxidant activity that is effectively trapped in the nanofiber matrix and bioactively released. The DPPH antioxidant activity of the standard is presented in Table 11. The DPPH inhibition plots are shown in Figure 10. The antioxidant activity of the nanofiber formulation is presented in Table 12.
In vitro Cytotoxicity (MTT ASSAY)
The MTT assay against A431 cells showed the viability was significantly decreased in a time and dose dependent manner. Cytotoxicity was not seen at 100-200 µg/mL, showing good biocompatibility at sub-therapeutic doses. At 300-500 µg/mL, there were signs of progressive cell rounding, shrinkage of the cytoplasm, membrane blebbing and detachment, consistent with apoptosis, while at 600-1000 µg/mL there was widespread detachment and nuclear condensation consistent with advanced apoptosis/necrosis. The IC50 was 520 µg/mL at 24 hr, which was significantly less than that of free itraconazole (800-1200 µg/mL) and free quercetin (650-900 µg/mL), indicating an additive-tosynergistic antiproliferative effect from co-delivery. This increased cytotoxicity is believed to be due to the fact that both ITZ and QCT are released continuously, allowing for the inhibition of both the Hedgehog/angiogenic (ITZ) and the PI3K/Akt/NF-κB (QCT) pathways (Figure 11 and Table 13).
| Parameter | Fiber Diameter (nm) | EE (%) | Drug Release (%) |
|---|---|---|---|
| Model Type | 2FI | Quadratic | Quadratic |
| R² | 0.9669 | 0.9938 | 0.9889 |
| Adjusted R² | 0.9470 | 0.9859 | 0.9746 |
| Predicted R² | 0.8162 | 0.9743 | 0.8384 |
| Adequate Precision | 22.911 | 35.179 | 24.538 |
| C.V. (%) | 0.1011 | 0.1235 | 0.2029 |
| Lack of Fit (p) | 0.4193 (NS) | 0.9539 (NS) | 0.2010 (NS) |
| Parameter | Optimized Value | Goal |
|---|---|---|
| A: Voltage (kV) | 17.00 kV | In range |
| B: Flow Rate (mL/hr) | 0.250 mL/hr | In range |
| C: PCL: PVP Ratio | 2.290 | In range |
| Y1: Fiber Diameter (nm) | 392.459 nm | Minimize |
| Y2: Encapsulation Efficiency (%) | 93.921% | Maximize |
| Y3: Drug Release (%) | 88.220% | Maximize |
| Overall Desirability | 0.927 | — |
| Sample | Tg (ºC) | Tm (ºC) | ΔHf (J/g) | Key Observation |
|---|---|---|---|---|
| Pure Itraconazole | — | 168.2 | — | Crystalline; polymorphic transition at 155ºC |
| Pure Quercetin | — | 320.4 | — | Crystalline; dehydration at 105.3ºC |
| Pure PCL | ~−60 | 60.3 | −81.5 | Semicrystalline; sharp endotherm |
| PVP K30 | ~170 | None | — | Amorphous; hygroscopic Tg |
| Nanofiber Formulation | ~170 (PVP) | 313.72 | 93.12 | No drug peaks; amorphous dispersion confirmed |
| Measurement | Left Angle (°) | Right Angle (°) | Average (°) |
|---|---|---|---|
| Sample 1 | 88.87 | 90.77 | 89.82 |
| Sample 2 | 90.77 | 88.08 | 89.42 |
| Sample 3 | 89.23 | 89.23 | 89.23 |
| Overall Mean±SD | — | — | 89.49±0.29 |
| Batch | Itraconazole EE (%) | Quercetin EE (%) |
|---|---|---|
| Batch 1 | 93.57 | 92.47 |
| Batch 2 | 93.95 | 92.89 |
| Batch 3 | 94.33 | 93.31 |
| Mean±SD | 93.95±0.38 | 92.89±0.42 |
| Time (h) | ITZ Batch 1 (%CDR) | ITZ Batch 2 (%CDR) | ITZ Batch 3 (%CDR) | ITZ Mean±SD | QCT Batch 1 (%CDR) | QCT Batch 2 (%CDR) | QCT Batch 3 (%CDR) | QCT Mean±SD |
|---|---|---|---|---|---|---|---|---|
| 0.0 | 0.00 | 0.00 | 0.00 | 0.00±0.00 | 0.00 | 0.00 | 0.00 | 0.00±0.00 |
| 0.5 | 10.54 | 10.89 | 10.70 | 10.71±0.18 | 12.60 | 12.94 | 12.74 | 12.76±0.17 |
| 1.0 | 20.18 | 20.61 | 20.32 | 20.37±0.22 | 23.08 | 23.48 | 23.25 | 23.27±0.20 |
| 2.0 | 36.78 | 37.39 | 37.07 | 37.08±0.31 | 38.42 | 39.00 | 38.72 | 38.71±0.29 |
| 3.0 | 50.21 | 50.89 | 50.49 | 50.53±0.34 | 50.20 | 50.96 | 50.58 | 50.58±0.38 |
| 4.0 | 61.28 | 61.94 | 60.67 | 61.30±0.64 | 60.88 | 61.63 | 61.12 | 61.21±0.38 |
| 5.0 | 70.25 | 71.06 | 70.61 | 70.64±0.41 | 69.42 | 70.04 | 69.67 | 69.71±0.31 |
| 6.0 | 77.74 | 78.43 | 78.07 | 78.08±0.35 | 76.82 | 77.42 | 77.03 | 77.09±0.30 |
| 7.0 | 82.54 | 83.28 | 82.91 | 82.91±0.37 | 82.10 | 82.64 | 82.31 | 82.35±0.27 |
| 8.0 | 87.62 | 88.39 | 87.99 | 88.00±0.39 | 87.22 | 87.84 | 87.44 | 87.50±0.31 |
| Model | ITZ R² | ITZ R²adj | ITZ AIC | ITZ MSC | QCT R² | QCT R²adj | QCT AIC | QCT MSC |
|---|---|---|---|---|---|---|---|---|
| Zero-order | 0.9151 | 0.9151 | −23.76 | 1.94 | 0.8962 | 0.8962 | −22.36 | 1.71 |
| First-order | 0.9163 | 0.9163 | −23.90 | 1.95 | 0.8975 | 0.8975 | −22.48 | 1.72 |
| Higuchi | 0.9679 | 0.9679 | −33.49 | 2.91 | 0.9803 | 0.9803 | −39.00 | 3.37 |
| Korsmeyer-Peppas* | 0.9918 | 0.9908 | −45.11 | 4.07 | 0.9955 | 0.9949 | −51.69 | 4.64 |
| Storage | Day | Appearance | Quercetin EE (%) | Itraconazole EE (%) | SEM Observation |
|---|---|---|---|---|---|
| 4-6ºC | 0 | Clear | 93.35 | 92.89 | Normal fiber morphology |
| 4-6ºC | 45 | Clear | 92.93 | 91.57 | Slight structural changes due to moisture |
| 25ºC | 0 | Clear | 93.35 | 92.89 | Normal fiber morphology |
| 25ºC | 45 | Clear | 93.12 | 92.63 | Fiber morphology retained; stable |
| Concentration (µg/mL) | % Inhibition |
|---|---|
| 2 | 11.1 |
| 4 | 25.6 |
| 6 | 40.6 |
| 8 | 53.9 |
| 10 | 72.3 |
| Concentration (µL) | % Inhibition |
|---|---|
| 50 | 19.44 |
| 100 | 33.33 |
| 150 | 51.38 |
| 200 | 62.50 |
| 250 | 72.22 |
| Concentration (µg/mL) | % Cell Viability (Mean±SD) | Remarks |
|---|---|---|
| Control (0) | 100.00±5.03 | Untreated baseline |
| 100 | 100.08±5.62 | No cytotoxicity |
| 200 | 100.97±2.99 | No cytotoxicity |
| 300 | 92.26±3.84 | Mild growth inhibition |
| 400 | 68.79±3.80 | Moderate cytotoxicity |
| 500 | 53.91±1.75 | Near IC₅₀ |
| 600 | 33.56±2.82 | Marked cytotoxicity |
| 700 | 22.26±1.56 | High cytotoxicity |
| 800 | 13.80±1.90 | Severe cytotoxicity |
| 900 | 6.63±1.10 | Near-complete inhibition |
| 1000 | 4.83±1.40 | Near-complete inhibition |
DISCUSSION
Process-Property Relationships in the Optimized Electrospinning System
Flow rate had the highest effect on all three responses in the BBD model (p<0.0001), which reflects the general electrospinning principle that the shear force generated by flow rate is responsible for the mass of polymer solution fed to the Taylor cone in a given amount of time and therefore, for the jet mass, the rate of solvent evaporation and the fiber diameter.9 The high influences of PCL:PVP ratio on all three responses are associated with the solution viscosity and phase behaviour during the blend electrospinning, which have been reported in the literature to be miscible and spinnable throughout a range of PCL:PVP ratios that includes the ratios used in this study,16 and are also biocompatible with both polymers. The dome-shaped surface of the response surface plot for EE, with peaks at high voltage and low flow rate, is in agreement with the expected decrease of residence time for the solvent evaporation and jet thinning at high voltage and low flow rate, which would in turn reduce the amount of drug diffusing out of the fiber into the surrounding bulk matrix and therefore preserve the encapsulated drug in the fiber core.
Solid-State Transformation and Drug-Polymer Compatibility
FTIR analysis did not reveal any new characteristic peaks which would signal the presence of chemical interactions between Itraconazole (ITZ) and Quercetin (QCT), confirming that there was no chemical interaction between the two drugs when they were physically incorporated into the PCL/PVP nanofiber matrix.17 The absence of the melting endothermic peaks in DSC and crystalline peaks in XRD also suggested a conversion of both drugs from crystalline to amorphous state, probably due to hydrogen bonding between the carbonyl group of the PVP and the N-H/O-H group of the drugs in the rapid evaporation of the solvent during electrospinning.33,34 No direct interaction between the two drugs was detected on the ITZ-QCT composite suggesting that both drugs dispersed independently in the polymer matrix. This amorphous transformation helped achieve high encapsulation efficiency (>93.95% for ITZ and >92.89% for QCT) and improved the cumulative drug release (>87.5%).
Surface Wettability and Fiber Morphology
The optimized membrane also exhibited a significantly reduced contact angle (89.49±0.29°) compared to the 105-120° which is generally reported for unmodified PCL nanofibers, in consistent with the known effect of PVP as a hydrophilic, amphiphilic co-polymer that migrates toward the surface during electrospinning, decreasing the surface energy, and electrospinning parameters and solvent selection that have also been shown to affect PCL nanofiber contact angle in a similar manner.35 A moderately hydrophilic surface of 80°-90° is typically regarded as desirable for post-surgical wound dressings, because it allows proper wetting for drug elution and exudate absorption, but does not leave the surface too water-permeable to cause excessive swelling that may compromise the scaffold's integrity. This morphology, characterized by beads that are smooth, randomly oriented and not seen in other systems that have been electrospun, has been reported in PCL/PVP systems and has been cited as being caused by differential evaporation rates between chloroform and DMF during the jet drying process instead of drug-related defects.
Drug Release Mechanism
The optimized ITZ/QCT-loaded PCL/PVP electrospun nanofibers exhibited a reproducible biphasic drug release profile, characterized by an initial burst release followed by a controlled release during the 8 hr evaluation period. The initial burst release is attributed to the dissolution of drug molecules located near the fiber surface and the rapid hydration of the hydrophilic PVP phase, whereas the subsequent release was predominantly governed by diffusion through the hydrophobic PCL matrix. Release kinetic analysis demonstrated that the Korsmeyer-Peppas model provided the best fit for both drugs, indicating a non-Fickian (anomalous) transport mechanism involving both Fickian diffusion and polymer relaxation. These findings suggest that the optimized nanofibers provide controlled drug release suitable for localized topical delivery following skin cancer surgery. It was decided to use the 8 hr release period to represent the early post-operative period following skin cancer surgery, when the first dressing change is usually undertaken and when the risk of local recurrence and wound infection is greatest. Thus, it was more important to ensure sustained release during this timeframe than prolonged multi-day release. This helps the intended use of the nanofiber as a dress-for-surgical-wounds, as opposed to a long-term implant, in which the drug delivery can be repeated with every dressing change. However, the in vitro release study was carried out for only 8 hr and more studies with longer duration are needed for extended delivery of the drug.
Storage Stability
The optimized nanofibers retained acceptable morphology and encapsulation efficiency after 45 days under both refrigerated (4-6ºC) and room-temperature (25ºC /65±5% RH) storage conditions, with only a slight decline in encapsulation efficiency (QCT: 93.35% to 92.93% at 4-6ºC and to 93.12% at 25ºC /65±5% RH; ITZ: 92.89% to 91.57% at 4-6ºC and to 92.63% at 25ºC /65±5% RH). The stability samples were stored in airtight containers in accordance with ICH Q1A(R2) long-term and intermediate storage recommendations. The greater structural changes observed under refrigerated conditions were most likely due to moisture condensation during repeated removal from cold storage, which may plasticize the hygroscopic PVP phase and cause local disruption of the nanofiber structure. In contrast, room-temperature storage showed better preservation of fiber morphology, suggesting that avoiding condensation-related moisture uptake is beneficial for maintaining structural integrity. For future scale-up, packaging the nanofibers in heat-sealed aluminium foil pouches with a desiccant sachet is recommended to minimize moisture exposure, particularly because of the hygroscopic nature of PVP, and to help preserve fiber integrity during storage.
Antioxidant and Cytotoxic Performance in Context
The DPPH IC50 value of 158.3 µL for the nanofiber formulation indicates that significant antioxidant activity of quercetin was maintained after electrospinning and encapsulation within the PCL/PVP nanofibers. The co-loaded formulation had an IC50 of 520 µg/mL against A431 cells, which is lower than the IC50 ranges of 800-1200 µg/mL for free itraconazole and 650-900 µg/mL for free quercetin individually against squamous carcinoma lines. The increased efficacy of the combined formulation over either drug alone is reminiscent of the synergistic action described for both agents alone, which include itraconazole, targeting the Hedgehog/Smoothened signaling pathway, and endothelial or tumour cell proliferation in various models of solid tumours,18,36 and quercetin, inhibiting cell proliferation via modulation of the Bcl-2/Bax and PI3K/Akt signaling pathways in several tumour cell lines.19 The complementary intracellular drug levels resulting from the sustained release of both agents from a single nanofiber matrix (as shown in Section 3.13) suggest a possible reason for the improved cytotoxic activity when the nanofiber matrix was loaded with both agents. In the present study, formal combination-index analysis was not performed and would be necessary to determine true synergism between the co-delivered agents.
Although cytotoxicity against normal skin cells was not evaluated in this study, previous reports suggest that the formulation is likely to have a favourable safety profile. Quercetin has been shown to produce greater cytotoxicity in cancer cells than in normal skin cells, with minimal effects on normal skin cell viability at concentrations effective against skin cancer cells.37 Similarly, topical itraconazole nanocarrier formulations have demonstrated good local tolerability and non-irritant behaviour in skin irritation studies.38 In addition, PCL and PVP are widely used electrospun polymers for wound dressings and are generally considered biocompatible and non-cytotoxic to keratinocytes and fibroblasts.4 These findings indicate that the developed formulation is likely to be well tolerated by the surrounding healthy skin. However, direct cytotoxicity studies using normal skin cells and therapeutic-index evaluation are still required to confirm its safety.
Comparison with Existing Topical Delivery Systems
Unlike conventional topical carriers like liposomes, transferosomes, ethosomes, and nanoemulgels, electrospun nanofibers offer the dual advantage of drug delivery and wound dressing. While vesicular systems typically need a secondary dressing, the PCL/PVP nanofibrous mat can provide mechanical support, an ECM-like structure,37 and sustained non Fickian drug release.39 Both itraconazole and quercetin have been studied alone,17,40 in the previous studies, while the current study successfully co-loaded both drugs into a single Box-Behnkenoptimized PCL/PVP nanofiber. The optimized formulation exhibited high encapsulation efficiency (93.95% for ITZ and 92.89% for QCT) and the drug release was controlled for 8 hr. However, additional ex vivo skin permeation and in vivo studies are warranted to support its therapeutic efficacy and to compare its efficacy with available topical delivery systems.
STUDY LIMITATIONS AND FUTURE DIRECTIONS
There are some limitations in this study. Only the human epidermoid carcinoma (A431) cell line in vitro was used to assess the anticancer activity of the ITZ/QCT-loaded nanofibers while no in vivo tumour or wound-healing model or ex vivo dermal permeation was performed. Although PCL and PVP are known to be biocompatible, cytotoxicity was examined only toward A431 tumour cells, while selectivity towards normal skin cells was not evaluated and both drugs exhibit dose-dependent cytotoxicity at higher concentrations. Therefore, future studies should include normal keratinocyte or fibroblast cell lines with therapeutic index determination, along with in vivo post-surgical wound models and ex vivo human or porcine skin permeation studies to confirm the cytotoxicity, release kinetics, stability, local toxicity, skin irritation, biocompatibility, and long-term safety before translational or clinical conclusions can be drawn.
CONCLUSION
This study successfully formulated and optimized itraconazole and quercetin co-loaded PCL/PVP electrospun nanofibers for the management of skin cancer after surgery topically by Box-Behnken (BBM) response surface methodology. The optimized formulation electrospun at 17.0 kV, 0.25 mL/hr flow rate and PCL:PVP ratio of 2.29 had a fiber diameter of 389.8±35.0 nm, encapsulation efficiencies of 93.95% (ITZ) and 92.89% (QCT), and cumulative release of > 87.5% in 8 hr, with high composite desirability (D=0.927).
Amorphous solid dispersion in the nanofiber matrix was confirmed by physicochemical analysis (DSC and XRD), and there was no chemical interaction between the two drugs (FTIR) or bead-free morphology of the nanofiber matrix (SEM) which was slightly hydrophilic (contact angle 89.49°). Nanofibers were found stable for 45 days at 4-6ºC and (25 ºC /65±5% RH, with a slightly higher stability at room temperature than in the refrigerated state because of moisture uptake on fibers. The release was found to be non-Fickian (n=0.58-0.61), indicating the presence of both diffusion and polymer relaxation. The formulation exhibited strong cytotoxicity to A431 cells (IC50= 520 µg/mL) and antioxidant activity (DPPH IC50 =158.3 µL) which is greater than antioxidant activity of free drugs, which is concentration dependent.
The results indicate the dual-drug PCL/PVP nanofiber platform as a promising post-surgical drug delivery system featuring a combination of Itraconazole's anti-angiogenic, Hedgehog-inhibitory activity with Quercetin's antioxidant, pro-apoptotic effects. The next steps involve scale-up, in vivo wound healing, and ex vivo permeation studies.
ADDITIONAL STATEMENT
The authors confirm that no paper mill was used in the preparation of this manuscript and that all data were generated and analyzed by the authors.
AUTHORS CONTRIBUTION
P.P.: Conceptualization, Methodology, Writing -Original Draft. A.S.: Investigation, Writing -Review and Editing. V.A.N.: Formal Analysis, Data Curation. S.K.: Supervision, Project Administration. All authors approved the final manuscript.
