Introduction

Diabetic foot ulcer (DFU) is a slow-healing open wound or sore and a common complication associated with diabetes.1 There are several pathological changes associated with diabetes that impair most of the wound healing processes including several intrinsic factors such as peripheral neuropathy, vascular problems and other complications, as well as extrinsic factors such as wound infection, callus formation and excessive pressure on the site.2–4 Untreated diabetic foot ulcers often become a source of infection, delaying wound healing and causing severe pain to the patient. This can result in hospitalization and amputation of the lower extremities.5 The most frequently isolated microorganism in the infected diabetic wound is an aerobic Gram-positive bacterium, Staphylococcus aureus (S. aureus). Streptococci, Enterococci, Enterobacteriaceae, and Pseudomonas are also common bacteria found in diabetic foot infections (DFI).6,7

Standard treatment involves cleansing the ulcer with saline every week, debriding or removing inactive tissue with a surgical blade to facilitate cell proliferation, and covering it with sterile gauze to keep it moist and to reduce the risk of infection and promote healing of the wound.8–10 Infections are treated with either systematic antibiotics, topical antibiotics, or a combination of the two, depending on the severity and type of infection.11 DFU wounds need dressing materials capable of promoting healing and isolating the wound site from pathogen microbes. An ideal wound dressing must be in close contact with the wound, absorb the exudates produced by the wound, protect against bacterial invasion, facilitate substance exchange, have exceptional mechanical performance, have a high porosity and swelling capacity, possibility of delivering bioactive agents and maintain the optimum moist environment to promote the healing process.12–16

As a wound dressing for DFU, a variety of natural and synthetic polymers have been utilized, including hydrocolloids, hydrogels, foams, and electrospun nanofibers.15,17 Among the distinctive features of electrospinning-based nanofiber dressings are the ability to be synthesized with different pore sizes which prevent microorganisms from invading, create an ideal moisture environment, can encapsulate or adsorb multiple bioactive molecules such as drugs and growth factors and have a large surface area, which is favorable for drug loading and sustained delivery.18,19 Moreover, nanofibers mimic the extracellular matrix in their porous structure, which encourages cell attachment and proliferation.20–22 Electrospun nanofibers have been fabricated and applied as wound dressing from a variety of natural polymers including chitosan, gelatin, collagen, and silk, as well as, synthetic polymers such as polycaprolactone, polyurethane, polyvinyl pyrrolidone (PVP), and polyvinyl alcohol (PVA).23 PVP is an FDA-approved hydrophilic polymer with easy spinnability, high porosity, biocompatibility and biodegradability, which makes it suitable for a wide variety of pharmaceutical and biomedical uses, including wound dressings.24–26 In addition, this polymer has a mucoadhesive property, making it suitable for use in biomedical applications and drug delivery systems. The large surface-to-volume ratio and ultrafast disintegration of the PVP nanofibers allow this fibrous system for the immediate release of the encapsulated drugs, making them particularly useful in drug delivery systems.27,28 It has been reported previously that nanofibers prepared from PVP exhibit a good mechanical property, which was measured as elongation at break (%), and ultimate tensile strength as 9.10 ± 0.2% and 2.30 ± 0.2 Mpa, respectively.29

Recently, the use of modified electrospun nanofiber strategies has gained attention in various biomedical applications, including skin wound healing. Zhang et al designed an innovative electrospun nanofiber that combined the properties of nanofibers with the high wound exudate-absorbing ability and moist healing-promoting environment of a hydrogel.30 Specifically, they developed a Polyasparthydrazide (PAHy) nanofibrous/nanoparticle hydrogel dressing containing 0.5 wt% silver nitrate (AgNO3). This combination has demonstrated antibacterial and wound healing-promoting capacities in a full-thickness wound model. Another recent study has shown the effectiveness of a modified electrospinning strategy that combined the strong mechanical properties of electrospun nanoyarn-woven textiles with nanofiber properties loaded with Chinese herbal extracts.31 This approach accelerated wound closure, enhanced collagen deposition, improved re-epithelialization and neovascularization, and increased hair follicle formation in a streptozotocin-induced diabetic mouse model with full-thickness skin wounds. These findings highlight the potential of using modified electrospun nanofiber strategies to develop advanced wound dressings with additive properties, such as improved exudate management, promotion of a moist healing environment, antibacterial activity, and enhanced wound healing outcomes.

Moxifloxacin (Moxi) is a broad-spectrum antibiotic effective against a variety of Gram-positive bacteria such as S. aureus. Since Moxi has broad antibacterial properties, high efficiency, and can promote wound healing in infected wounds, it is an ideal candidate for wound therapy.32–34 Fusidic acid (Fus) is another antibiotic that is available in different formulations including cream, ointments and gel and is widely used topically to treat skin infections.11,35 Pirfenidone (Pir), an FDA-approved therapeutic drug primarily used for idiopathic pulmonary fibrosis, has demonstrated various beneficial properties for potential wound healing treatments. It exhibits anti-inflammatory, antioxidative, and anti-scarring activities, as supported by previous studies.36–41 Notably, Pir has been shown to effectively reduce tissue inflammation by decreasing pro-inflammatory cytokines, chemokines, neutrophil infiltration, and collagen deposition within wounds. Additionally, it has been found to mitigate the generation of reactive oxygen species (ROS) in separate investigations.38

For the bioactive dressing, various types of drugs, such as antibiotics, growth factors, anti-inflammatories and anesthetics, have been delivered using nanofiber systems to enhance the treatment of chronic wound infections, prevent scarring, and enhance wound healing.11,42–45 For instance, cephradine-loaded gelatin/PVA nanofiber has been reported for its antibacterial activity against S. aureus in an induced chronic wound.46 Iqbal et al have developed chitosan (CS)/PVA nanofibers loaded with the broad-spectrum antibiotic cefadroxil monohydrate to accelerate the wound healing process and cost-effectively treat S. aureus-induced (resistant) skin infections.47 Similarly, Kamal et al have used the cephalexin antibiotic, which has been loaded into poly(3-hydroxy butyric acid-co-3-hydroxy valeric acid) nanofibers, demonstrating an antibacterial activity both in vitro and in vivo in diabetic mice model against methicillin-resistant S. aureus (MRSA).48

Fus is another antibiotic that has been integrated with polylactic glycolic acid (PLGA) nanofiber and showed activity against wound-colonized bacteria including Pseudomonas aeruginosa (P. aeruginosa), and MRSA.44 In addition, TGF-β1-inhibitor has been incorporated into poly(ε-caprolactone) (PCL)/gelatin (PG) co-electrospun nanofibrous scaffold, which has been shown to inhibit fibroblast generation and prevent scar formation in healing wounds.45 Moxi antibiotic was also incorporated in CS and polyethylene oxide (PEO) nanofibers, which showed antimicrobial activity against S. aureus, Escherichia coli (E. coli), and P. aeruginosa, and wound healing efficacy.49 Previous studies have explored the utilization of Pir in nanofiber applications for wound healing purposes. For example, a recent investigation conducted by Tottoli et al50 incorporated Pir into nanofiber systems consisting of PLA-PCL, which were used as dressings. Their findings demonstrated the drug’s antifibrotic effects on hypertrophic scar fibroblasts (HSFs), thus highlighting the potential of biofiber as an effective therapeutic approach for hypertrophic scar treatment within the context of wound healing. Furthermore, Tawfik et al demonstrated the antibacterial activity of a Moxi/Pir-loaded PLGA/PVP coaxial nanofiber system in the treatment of rabbit corneal infections.36

Considering that infection and inflammation can interfere with and delay normal wound healing, a combination of antibiotic and anti-inflammatory drugs can be an effective way of expediting wound healing. Thus, here we aim to develop coaxial electrospun nanofiber mats loaded with antibiotics, either Moxi, Fus, or Pir (as an anti-inflammatory) with the ability to be released directly on an injured site to prevent bacterial growth, minimize the inflammation, and thus accelerate wound closure.

Materials and Methods Materials

PVP with an average molecular weight of 1,300,000, Moxi hydrochloride, Fus sodium salt, formic acid (98–100% for LC-MS LiCropur) and ethanol (absolute, ≥99.8%) were all obtained from Sigma-Aldrich (St. Louis, MO, USA), while Pir was brought from TCI chemicals (Toshima, Tokyo, Japan). Acetonitrile (CHROMASOLV, ≥99.9% for LC-MS) was purchased from Fisher Scientific (Waltham, MA, USA), and distilled water was generated from Milli Q, Millipore (Billerica, MA, USA).

Fabrication of Core/Shell Fibers

The coaxial fibres were prepared using Spraybase® (Dublin, Ireland) based on a modified method of Alkahtani et al and Alshaya et al.51,52 The core polymer solution was prepared by dissolving 8% (w/v) of PVP in absolute ethanol and kept stirring at room temperature for 1 hour and then 0.5% (w/v) of Pir was added and stirred until complete dissolving. For the shell solution, 8% (w/v) PVP was also dissolved in ethanol with either 0.5% (w/v) Moxi or Fus and magnetically stirred at room temperature until completely dissolved. The prepared electrospun solutions were loaded into two 5 mL syringes and connected to a coaxial needle with an inner diameter of 0.45 mm and an outer diameter of 0.9 mm. The coaxial fibers of Moxi/Pir were produced at a constant voltage of 9.50 kV using a flow rate of 0.8 mL/hour for both core and shell solutions and a tip-to-collector distance of 15 cm. The coaxial fibers of Fus/Pir were produced using the same parameters but at a constant voltage of 9.00 kV. The electrospinning processes were carried out at room temperature with a relative humidity of 40%. Blank fibers were prepared with 8% (w/v) PVP solution both in the core and in the shell without the addition of any drug using the same electrospinning parameters but at a constant voltage of 7.00 kV.

Analysis of Fibers Morphology by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

The surface morphology of the electrospun nanofibers was analyzed using JEOL SEM (JSM-IT500HR, ASIA PTE. Ltd., Singapore). In preparation for imaging, samples were placed on SEM stubs using carbon tape and next coated with platinum (2 nm thickness) using the Auto Fine Coater (JEC-3000FC, JEOL, ASIA PTE. Ltd., Singapore). The average fiber diameters were measured from 50 different fibers using ImageJ software (National Institute of Health, MD, USA). The core and shell layers were distinguished via TEM (JEM-1400 TEM, JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV by collecting the fibers directly onto a copper grid while electrospinning.

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis

Thermo smart ATR IS20 Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) FTIR analyzer was used to determine the components and molecular structures of drug-loaded and blank fibers, as well as the pure PVP, Moxifloxacin, Pirfenidone, Fusidic Acid, and their physical mixture (PM). Both PM Moxi/Pir and PM Fus/Pir were prepared individually in a ratio of 16:1 from PVP: Moxi/Pir and PVP: Fus/Pir, respectively. The spectral resolution of 4 cm−1 was used to obtain the FTIR spectra of the samples at a wavenumber between 4000 and 600 cm−1, and 32 scans were taken for each sample.

X-Ray Diffraction (XRD) Analysis

Rigaku Miniflex 300/600 (Tokyo, Japan) with Cu Kα radiation (λ = 1.5148 227 Å) and a voltage of 40 kV and a current of 15 mA was used to evaluate the crystallinity of PVP, Moxi, Fus, Pir, PM Moxi/Pir, PM Fus/Pir, blank and drug-loaded nanofibers. After placing the samples on aluminum trays, the XRD patterns were scanned at a speed of 5°/minute across the diffraction angle (2θ) range between 2° and 60°.

Disintegration Test of Nanofiber Systems

A modified method described by Almuwallad et al53 was used to evaluate the disintegration of Moxi/pir nanofibers, Fus/pir nanofibers, and blank fibers. 2×2 cm of fibrous mats were placed in a petri dish containing 8 mL of distilled water at room temperature until detachment was completed. The results represent the average ± standard deviation (SD) of three replicates.

Drug Quantification by High-Performance Liquid Chromatography (HPLC)

The HPLC quantification of Moxi, Pir, and Fus was performed on a 1260 Infinity II HPLC system (Agilent, Santa Clara, CA, USA) comprising an autosampler with column oven compartment (G7129A), a binary solvent gradient flexible pump (G7111A), and a UV detector (G7114A). The HPLC separation of the drugs was accomplished using an Agilent HPLC column Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm) column, with a temperature set as ambient temperature. The mobile phase was an LC/MS grade water plus 0.1% formic acid (A) and acetonitrile plus 0.1% formic acid (B). At a flow rate of 0.40 mL/min, a linear gradient program with a total runtime of 15 minutes was used as follows: 0.0–2.0 min 5% (B), 2.0–10.0 min from 5% to 95% (B), 10.0–11.0 min 95% (B), 11.0–12.0 min from 95% to 5% (B) and finally 12.0–15.0 min 5% (B) and injection volume 10 µL. Moxi, Pir, and Fus were identified and detected at retention times Rt = 8.9 min (295 nm), 10.0 min (315 nm), and 14.2 min (223 nm), respectively, as shown in Supplementary Figure 1. Calibration curves were constructed using a series of concentrations ranging between (100–0.4 µg/mL) which is shown in Supplementary Figure 2A for Moxi and Pir and 2B for Fus and Pir. Data analysis was processed using OpenLab CDS software.

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

Certain amounts of the dual drug-loaded fibrous system were dissolved in 10 mL PBS pH 7 and kept at room temperature until complete dissolving. The actual amount of Moxi, Pir and Fus were determined using the above-developed HPLC method, and the EE% and DL were determined by the following equations:

(1)

(2)

The results represent the average ± SD of three replicates.

In vitro Drug Release by Franz Diffusion

In vitro, release of the drugs from the prepared nanofibers was studied using the Franz diffusion cell system (PermeGear, Hellertown, PA, USA). A piece of a semipermeable membrane (Spectra/Por®, Spectrum Laboratories Inc., Ranch Dominguz, CA, USA) with a molecular weight cut-off of 12–14 kD, was placed over each Franz cell. Phosphate-buffered saline PBS (pH 7) was used to fill the receptor compartments. One mL of PBS was added to each donor compartment. The system was maintained at 37 ± 0.02°C via a circulating water bath to mimic skin surface temperature, and the diffusion medium was stirred at 600 RPM. Certain amounts of the nanofiber samples were placed on the donor side, and the open ends of the Franz cells were covered with parafilm® to prevent evaporation. At predetermined time points up to 24 hours (0.5, 1, 2, 4, 6 and 24 hours), a 100 µL sample was withdrawn from the receptor compartment (containing 3 mL PBS pH 7) and replaced by fresh buffer to maintain the sink conditions. The withdrawn samples were analyzed by the developed HPLC method, and the cumulative amount of drug release was calculated according to the following equation:

(3)

The results represent the average ± SD of three replicates.

Determination of the Minimum Inhibitory Concentration (MIC) of the Pure Drugs

A total of four ATCC bacterial strains were used including S. aureus ATCC 29213 and ATCC BAA-977, P. aeruginosa ATCC 9721, and ATCC 27853 cultured in Muller-Hinton agar media and incubated at 37°C overnight. The MIC for Moxi, Fus and Pir were determined in Muller-Hinton broth using microdilution plates. Serial dilutions of drugs were prepared for Fus and Pir (1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg/mL) and (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg/mL) for Moxi in Muller-Hinton broth and added to 96-well plates. Then, the bacterial suspensions were added to each well to achieve a final inoculum of 1 × 106 CFU/mL. All 96-well plates were incubated at 37°C overnight with a continuous shaking speed at 140 RPM. The endpoint of MIC was measured at absorbance (600 nm) using CytationTM 3 Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, USA).

Determination of the Antibacterial Activity of the Dual Drug-Loaded Coaxial Fibers

The antibacterial effectiveness of the prepared coaxial nanofibrous systems was evaluated by the zone of inhibition assay. A certain weight of the dual drug-loaded fibers was placed in the Petri dishes that contained a final inoculum of 1 × 106 CFU/mL from each bacterium (S. aureus and P. aeruginosa). Drug-loaded discs were used as positive control, which contained a similar drug amount to the drug-loaded fibers, while blank (drug-free) fibers at a similar fibrous mat weight to the drug-loaded fibers were used as a negative control. The fibers and discs were loaded in the center of each Petri dish, and the dishes were incubated at 37°C overnight.

Determination of the Inhibitory Concentration (IC) of the Pure Drugs

Human dermal fibroblasts (HFF-1; ATCC SCRC-1041, Homo sapiens) were used to assess the IC20 and IC50 according to a modified method of Tawfik et al36 and Alshaya et al52 The cells were sub-cultured and maintained 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 obtained from Sigma-Aldrich (St. Louis, MO, USA). MTS reagent (cell titer 96® aqueous one solution cell proliferation assay, Promega, Southampton, UK) was used to conduct the MTS cytotoxicity assay on a living cellular model (passage = 12). HFF-1 cells were harvested with 0.25% Trypsin-EDTA, counted with trypan blue exclusion test, and seeded to a 96-well plate at a seeding density of 1 × 104 cells/well. The seeded cells were incubated overnight in a humified 5% CO2 cell culture incubator at a temperature of 37°C. After reaching around 50% to 60% confluency, 100 µL of DMEM that contained the tested drugs: Moxi, Fus alone or in combination with Pir and Pir only at a serial dilution range from 500 to 15.6 µg/mL, were then incubated with the cells for 24 hours. Untreated cells and 0.1% triton x-100 treated cells were the positive and negative controls used in this assay, respectively. Following the 24-hour incubation period, the mixture was aspirated from each well and a combination of DMEM and MTS reagent (4:1) with a total volume of 100 µL was added and incubated for 3 hours in a dark biosafety cabinet at 37°C. The color intensity of each well was measured at 490 nm via Cytation 3 absorbance microplate reader (BIOTEK Instruments Inc., Winooski, VT, USA). Finally, the cell viability percentage was calculated using the following equation:

(4)

whereas S, T, and H represent the absorbance of the cells treated with the tested drug, the absorbance of the cells treated with triton x-100, and the absorbance of the cells treated with DMEM. The results represent the average (± SD) of three replicates.

In vivo Diabetic Model

Thirty-two male Wistar rats weighing 250 ± 30 gm were obtained from the animal house, College of Pharmacy, King Saud University. Animals were maintained in a temperature-controlled room at 25°C and an average relative humidity of 50% in a 12:12 h light/dark cycle. All animal experiments strictly followed the guidelines of the Ethical Committee for Performing Studies on Animals, King Saud University, Riyadh, Saudi Arabia, protocol number KSU-SE-22-5. Rats were randomly divided into 8 groups (n = 4) and after an overnight fasting received a single 65 mg/kg intraperitoneal injection of streptozotocin (STZ; Sigma-Aldrich, Taufkirchen, Germany). After 72 hours, rats with a fasting blood glucose level of 250 mg/dL or higher were considered diabetic. Supplementary Figure 10 shows the rats’ blood sugar before and throughout the study.

Treatment Application and Measurement of Wound Size

On the day of wound excision, rats were anesthetized using 3% isoflurane, back hair was removed using an electric clipper, and the wound was excised using a sterilized scalpel. Rats were then randomly divided into 8 groups (n = 4) and either received blank fibers, Moxi/Pir fibers, Fus/Pir fibers, Pir fibers, Moxi fibers, or Fus fibers. Fibers were secured in place using 3M™ Tegaderm™ transparent film. One group of rats received the commercially available 10% Povidone Iodine (ie, PVP-I) non-adherent dressing, under the brand name of INADINE®, as a treatment-positive control, and one group of rats received no treatment and served as a disease control. Animals were treated once daily for 14 days or until total wound closure. Wound swab was taken before treatments and on days 0, 1, 3, 5, 7, 9, 11 and 13 post-treatments. Changes in wound appearance were monitored through taking photos of before and throughout the study and the wound sizes were measured using a micrometer-electronic digital caliper. Animal weight was also monitored, as shown in the Supplementary Figure 9. Statistical analysis was performed using one-way ANOVA and Tukey’s post-hoc test. GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis and graph plotting.

In vivo Microbial Total Count

Following the wounding of the rats participating in the experiment, and before the addition of treatments, standard bacterial swabs were taken from their wounds on the start day and then were collected from each group on days 0, 1, 3, 5, 7, 9, 11, and 13. The swabs were then placed into a sterile tube containing 1 mL of phosphate-buffered saline (PBS) to prepare a 1:10 dilution and spread on a Petri dish. The plates were incubated for 24 to 48 hours at 37 °C. Then, the transplanted colonies were counted and reported.

Histopathological Assessment of the Wounds

At the end of the study, animals were sacrificed using a CO2-filled chamber with a flow rate of 30–70% per minute for 5 minutes, and then wound or skin was trimmed and fixed in 10% formalin for 48 hours. To dehydrate the tissues, 70% ethanol was used, and tissues were then sent to the Department of Pathology, College of Medicine, King Saud University for Hematoxylin and Eosin staining (H&E). The histology of skin tissues for both treated and non-treated animals was examined by a pathologist, using light microscopy.

Statistical Analysis

The EE%, DL, drug release studies, and in vitro experiments were all conducted with three independent replicates, and the data are presented as the average ± SD. Statistical analysis was performed using OriginPro® 2021 (OriginLab Corporation, Northampton, MA, USA) and Microsoft Excel 2024 software. For the statistical analysis of in vivo experiments, one-way ANOVA and Tukey’s post-hoc test was employed, and GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA) was used for both statistical analysis and graph plotting, where the p-values of <0.05 were considered statistically significant.

Results and Discussions Analysis of Fibers Morphology by Scanning Electron Microscopy (SEM)

The surface morphologies of both the prepared coaxial fibers and blank nanofibers had a uniform and smooth surface without beads as shown in Figure 1. The blank nanofiber has a diameter of 760 ± 105 nm, while Moxi/pir fiber mats and Fus /pir mats were measured to be 443 ± 67 nm and 488 ± 92 nm, respectively. The larger diameter for the blank fibers was attributed to the lower voltage (ie, 7 kV) that was used compared to the dual drug-loaded fibers (ie, ≥9 kV) which stabilized the spinning jet and allowed for larger diameter fibers.54 A similar observation was demonstrated in the imeglimin-loaded PVP nanofibers of Alamer et al55

Figure 1 Surface morphologies of the drug-loaded coaxial fibers (A) blank, (B) Moxi/Pir, and (C) Fus/Pir nanofibers and their corresponding diameter distribution.

Abbreviations: Moxi, moxifloxacin; Fus, fusidic acid; Pir, pirfenidone.

Notes: The SEM images showed uniform and un-beaded fibers with a diameter of 760 ± 105 nm, 443 ± 67 nm and 488 ± 92 nm, respectively. The size distribution of the drug-loaded core/shell nanofibers was measured from 50 different fibers.

The core and shell layers were distinguished using the TEM as shown in Figure 2. This was in agreement with Alkahtani et al and Alshaya et al who demonstrated a similar observation for their PVP/PVP coaxial nanofibrous systems.51,52 Due to the miscibility of the core and shell polymer solutions, which were both at the same concentration (8% w/v) and the flow rate was kept constant at 0.8 mL/hour, the thickness of the inner layer appeared considerably large (as shown in Figure 2). The reason that the fibers were prepared using the coaxial needle was to avoid loading a high concentration of drugs (1% w/v, ie, equally divided for each drug) into single-layered (monoaxial) fibers, which would reduce the drug-to-polymer ratio, preventing the encapsulation of the drugs and accelerate their release. In addition, owing to the ultrarapid disintegration of the PVP fibers, this delivery system will be used as a drug delivery system and not as a medicated fibrous scaffold. Overall, the successful preparation of this coaxial system was satisfying to further test the fibers using in vitro and in vivo tests.

Figure 2 A representative TEM image showing the two distinctive layers of the inner and outer layers of this PVP/PVP nanofibrous system.

Fourier-Transform Infrared Spectroscopy (FTIR) Analysis

The FTIR is considered one of the easiest and fastest techniques used to detect the important functional groups for each sample and gives an initial indication of any specific molecular interactions between the samples. Thus, the compatibility of the drugs and the polymer was tracked using FTIR spectroscopy to assess any structural changes that might have occurred due to the drugs’ incorporation with PVP and to assure the stability of the resulting formulation. Table 1 summarizes the most characteristic FTIR peaks of all pure materials (PVP, Moxi, Fus, Pir) based on the FTIR spectrum in Figure 3. The presence of C-N vibrations from pyrrolidone around the 1280 cm−1 region and the strong C=O band at 1638 cm−1 in the FTIR spectra of PVP, blank fiber, drug-loaded fibers, and the PMs led to the inference that PVP polymer was present. In addition, the presence of a broad O-H stretching peak at 3430 cm−1, owing to the hygroscopic nature of PVP, provides additional evidence for the presence of the polymer. The PVP findings align with what was found by Alshaya et al52

Table 1 FTIR Characteristic Peaks of the Pure PVP (Polyvinyl Pyrrolidone), Moxi (Moxifloxacin); Pir (Pirfenidone); and Fus (Fusidic Acid)

Figure 3 FTIR spectrum of PVP, Moxi, Fus, Pir, PM Moxi/Pir, PM Fus/Pir, blank and drug-loaded nanofibers.

Abbreviations: Moxi, moxifloxacin; Fus, fusidic acid; Pir, pirfenidone; PM, physical mixture; DL, drug-loaded.

The PM Moxi/Pir spectrum in Figure 3 displayed low-intensity peaks related to the PVP and the drugs. The PVP polymer was indicated by peaks at 1652, 1423, and 1279 cm−1, while Moxi and Pir peaks appeared at a lower intensity within the fingerprint region, corresponding to the polymer-to-drug ratio (16:1). Moreover, the drug-loaded fibers in Figure 3 exhibited peaks related to Moxi and Pir, including a C=C peak at around 1500 cm−1, stretching vibration of phenyl fluoride at 1181 cm−1, and intense peaks that were detected in the fingerprint region at approximately 1003, 932, 891, and 649 cm−1 corresponding the C–H from substituted benzene. These results are consistent with the previously published findings of Tawfik et al.59

The FTIR of the second PM of Fus/Pir in Figure 3 showed high-intensity peaks that represent the PVP at 1651, 1495, 1456,1422, 1370, 1283, and 1271 cm−1, while less-intense peaks represented the drugs at 1557 and 1338 cm−1 as a result of C=C stretching of and C-H bending, respectively. On the other hand, the intensity increase of the peaks, especially at 3650–3100 cm−1 and 1650 cm−1 in the drug-loaded fibers in Figure 3 due to the stretching vibrations of O-H and C=O for PVP and Fus, in addition to the peaks presented in the PVP polymer and the blank fibers at 1492, 1459, 1436, and 1421 cm−1 for the C-H bending vibration and 1286 cm−1 for the pyrrolidone stretching vibration. Fus and Pir in the DL fibers also exhibited many low-intensity peaks at 1018, 1001, and 930 cm−1 in the fingerprint region, which could correspond to the C-H from substituted benzene and C=C-H vibrations.

As described in numerous studies, these observations for both drug-loaded fibers demonstrate the compatibility of the drugs with PVP without any chemical interactions. Aburayan et al demonstrated the absence of any changes in the chemical structure or interaction between the Halicin and PVP nanofibers after the electrospinning process.62 Furthermore, the PVP nanofibers preserved the characteristic properties after loading albendazole sulfoxide and praziquantel drugs by Gültekin et al, which indicates the compatibility between drugs and PVP through electrospinning.63

X-Ray Diffraction (XRD) Analysis

XRD was used to determine the drug’s crystallinity and to assess the amorphous state of the solid dispersion as illustrated in Figure 4. The XRD pattern of PVP demonstrated a broad-halo peak due to the amorphous nature of the PVP polymer, with no crystalline peaks expected to be observed, as well as in the blank fibers. This is consistent with the findings published by Alkahtani et al.51 The XRD of the pure Pir, Moxi, and Fus drugs exhibited crystallinity forms, as evidenced by the presence of several narrow and intense Bragg reflection peaks in their XRD patterns. The Pir pattern (Figure 4) showed distinctive Bragg reflections that confirm its crystalline nature at 2θ values of 8.85°, 14.43°, 15.4°, 18.63°, 18.97°, 22.99°, 24.59°, 26.86°, 27.44°, 37.38°, 45.57°, and 48.03°. These findings align with published results of Borhade et al.64 Additionally, Moxi pattern also exhibited intense peaks at 5.84°, 8.48°, 10.13°, 17.37°, and 29.22° indicating its crystallinity nature, which is consistent with Tawfik et al59 and Shah et al.65 As shown in PM Moxi/Pir pattern (Figure 4), Moxi and Pir peaks were observed at diffraction angles of 5.66°, 8.80°, 9.93°, 15.04°, 17.21°, and 24.49°.

Figure 4 XRD patterns of PVP, Moxi, Fus, Pir, PM Moxi/Pir, PM Fus/Pir, blank and drug-loaded nanofibers.

Abbreviations: Moxi, moxifloxacin; Fus, fusidic acid; Pir, pirfenidone; PM, physical mixture; DL, drug-loaded.

Furthermore, Figure 4 presents the diffractogram of the second formulation, which signified the crystalline structure of Fus at 5.05°, 6.93°, 8.17°, 14.30°, 15.02°, 15.67°, 19.55°, 21°, and 24.81°, similarly to Thakur et al,66 and Gilchrist et al.67 Both Fus and Pir peaks were observed at the PM Fus/Pir at 8.08°, 8.81°, 14.95°, 21.13°, and 43.06° but in a very low intensity. The presence of the low-intensity peaks in both PMs, while they were lacking in the drug-loaded fibers (displayed broad-halo), confirms the molecular transformation of the drugs after being. These findings agreed with the results in numerous studies that confirm the transformation of the drug’s crystallinity to an amorphous state due to the electrospinning process.62,68,69

Disintegration Test of Nanofiber Systems

The disintegration was measured to be ≤2 seconds for all tested nanofiber systems, ie, blank, Moxi/Pir, and Fus/Pir nanofibers, as demonstrated in Figure 5. Both Alkahtani et al and Bai et al exhibited a similar ultrafast disintegration of the drug-loaded PVP nanofibers, which is attributed to the hydrophilic and hygroscopic nature of the PVP polymer.51,70

Figure 5 Disintegration test of (A) blank nanofibers, (B) Moxi/Pir nanofibers, and (C) Fus/Pir nanofibers.

Notes: All nanofiber systems disintegrated ≤2 seconds (n = 3).

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

The amount of encapsulated drugs and the drug loading of the prepared coaxial nanofibers were determined using a developed HPLC-UV system, using predetermined calibration curves, as shown in Supplementary Figure 2A for Moxi and Pir and 2B for Fus and Pir. The EE% and DL for the Moxi/Pir nanofibers were measured to be 70 ± 3% and 20 ± 1 µg/mg, respectively, for Moxi, and 96 ± 6% and 28 ± 2 µg/mg, respectively, for Pir.

The EE% of Pir was very close to the theoretical amount (100%), whereas the EE% of Moxi was lower, which needs further investigation. Different studies have shown a highly encapsulated Moxi in hydrophilic polymer systems including Hameed et al who showed about 100% encapsulation of the Moxi loaded into CS: PEO nanofiber system.49 Tawfik et al have also shown a high EE% of 87.5 ± 3% and 80 ± 2%, respectively, for the Moxi/Pir-loaded PVP/PLGA nanofiber system.59 The corresponding EE% and DL for the Fus/Pir-loaded into coaxial PVP fibers were 95 ± 6% and 28 ± 2 µg/mg, respectively, for Fus, and 102 ± 5% and 30 ± 2 µg/mg, respectively, for Pir. Both drugs were highly incorporated into the nanofiber probably due to the high solubility and miscibility of the drug solutions in the preparatory step.

In vitro Release Franz Diffusion Studies

The in vitro drug release profiles of the dual drug-loaded nanofiber systems were performed using a Franz diffusion cell system using PBS (pH 7). In the first coaxial system, PVP: Moxi (shell)/PVP:Pir (core), both drugs had a similar release pattern as shown in Figure 6A. The released amount of Moxi and Pir at the first 30 minutes were 19% and 26%, respectively, reaching to 34% and 40%, respectively, after the first 2 hours. Pir, however, reached a higher release amount of 71% in 6 hours compared to 57% for Moxi. Both drugs were released completely after 24 hours. PVP is a hydrophilic polymer, which is highly soluble in hydrophilic solutions. Also, Moxi and Pir are highly soluble drugs. Therefore, the drug release profile of both drugs was similar owing to the miscibility of nanofiber system components, in addition to the use of the same concentration of polymers and drugs in the preparation of this nanofiber system. Different studies have incorporated multiple drugs in the coaxial nanofiber system including Tawfik et al59 who incorporated Moxi as a core and Pir as a shell on PVP/PLGA nanofiber system. They had a high-release profile, which reached 100% of Pir and 70% Moxi in 24 hours. This study showed a lower release of Moxi than this current study, possibly due to the location of the drug in the core and the outer layer of PLGA could decrease the amount of drug released. In another study, the use of a hydrophilic nanofiber system such as the combination of CS and PEO effectively delivered Moxi, demonstrating a release rate of 79.8 ± 4.2% within 48 hours at pH 7.4. Although their sustained-release profile persisted for a longer duration, the amount of encapsulated drugs in their system exceeded this current study, falling within the range of 99–101%. Comparatively, our nanofiber system achieved 100% drug release within 24 hours at pH 7, but with an encapsulation efficiency of 67%, while, the study achieved approximately 60% drug release within the first 24 hours but with an EE% of 99–101%.49

Figure 6 In vitro release profile of (A) Moxi/Pir-loaded coaxial nanofibers and (B) Fus/Pir-loaded coaxial nanofibers.

Abbreviations: Moxi, moxifloxacin; Fus, fusidic acid; Pir, pirfenidone.

Notes: Results represent the average ± SD (n = 3).

On the other hand, the release of the other coaxial nanofibrous system, ie, PVP:Fus (shell)/PVP:Pir (core) demonstrated a similar release profile for both drugs, as shown in Figure 6B. After 4 hours, 23% of Fus and 37% of Pir were released, followed by 29 and 44%, respectively, after 6 hours, and finally, 65% and 80%, respectively, after 24 hours. To the best of our knowledge, this study represents the first attempt to incorporate Fus into a hydrophilic nanofiber system. Considering the hydrophilic nature of both PVP and the drugs, it is anticipated that they will rapidly dissolve under in vitro sink conditions. However, a slower release of Fus was shown in previous studies by Almostafa et al71 and Kinani et al,49 for Fus-loaded hydrogel and nanoemulgel and Fus-loaded PVA/CS nanofibers, respectively, using the Franz diffusion system. It was reported in both studies that the solubility and Fus release rate might be affected owing to the degree of polymer matrix swelling, the polymer and/or excipient ratio, higher viscosity of the formulation and the polymer being behaved like a drug reservoir might slow down the release and diffusion of the loaded drugs.

Determination of the Minimum Inhibitory Concentration (MIC) of the Pure Drugs

Numerous factors can hinder the healing process in diabetic foot wounds, mainly bacterial colonization and wound infection.72 Here, nanofibers loaded with antibiotics were used as wound dressings to treat the infection and help expedite the healing processes. The MIC assay was used to determine the antibacterial activity of the pure drugs, ie, Moxi, Fus and Pir against the most common pathogenic bacteria in diabetic foot wounds, the Gram-positive S. aureus and the Gram-negative P. aeruginosa.73,74 The MIC values of Moxi against two different strains of S. aureus, ie, ATCC 29213 and ATCC BAA-977, were <0.5 μg/mL, whereas the MICs against two different strains of P. aeruginosa, ie, ATCC 9721 and ATCC 27853, were measured as 2 μg/mL and 4 μg/mL, respectively (Supplementary Figure 3 and Supplementary Table 1). Pir has shown no antibacterial activity against the pathogenic bacteria (Supplementary Figure 3 and Supplementary Table 1), which was in agreement with the result of Tawfik et al.36 The MIC of Moxi was similar or within ± two-fold dilution to that of Tawfik et al,36 which was 0.125 μg/mL against S. aureus and 4 μg/mL against P. aeruginosa, respectively. Speciale et al75 also reported Moxi activity against different strains of S. aureus in a range of 0.015–0.25 μg/mL or 1–2 μg/mL. In addition, Masadeh et al76 reported an activity of Moxi against S. aureus ase 0.05 ± 0.02 μg/mL and against P. aeruginosa as 1.7 ± 0.7 μg/mL. However, the bacterial strains were different from those used in this current study.

The MIC of Fus was measured as <1 μg/mL against the two strains of S. aureus (ie, ATCC 29213 and ATCC BAA-977) and recorded a weaker activity of >512 μg/mL against the two strains of P. aeruginosa (Supplementary Figure 3 and Supplementary Table 1). These results were consistent with the results of Jones et al,77 which reported a MIC ranging between 0.06 and 0.25 μg/mL against S. aureus ATCC 29213. Moreover, according to previous studies, Staphylococci are normally susceptible to Fus at MICs of ≤0.25, ≤0.5, or ≤1 μg/mL.78–80 In addition, McGhee et al81 have reported the MIC activity of 40 different MRSA strains isolated from cystic fibrosis patients to be 0.125 to 0.5 μg/mL, 0.5 μg/mL against S. aureus (HMC2230) and 0.25 μg/mL against S. aureus (HMC2232). Their MIC results against P. aeruginosa were also similar to this study, which were recorded to be >512 μg/mL against P. aeruginosa HMC461 and 256 μg/mL against P. aeruginosa HMC468. O’Brien et al82 have also reported a more potent activity of Fus against S. aureus 25,923, which was 0.0156 μg/mL and a weaker activity of >256 μg/mL against P. aeruginosa PAO1.

Determination of the Antibacterial Activity of the Dual Drug-Loaded Coaxial Fibers

The antibacterial activity of the two dual drug-loaded coaxial nanofiber systems was evaluated using the zone of inhibition assay against the similar bacterial strains that were used in the MIC study, as shown in Table 2. Moxi is a broad-spectrum antibiotic that is effective against Gram-positive and Gram-negative bacteria.33,34 Here, the antibacterial activity of Moxi/Pir nanofibers was tested using 1 mg of the fibers that contained 20 µg Moxi and 28 µg Pir compared to the blank fibers as a negative control and a similar amount of the drugs loaded into a microbiological disc, as a positive control. Similarly, 1 mg of the Fus/Pir nanofibers that contained 28 µg Fus and 30 µg Pir was tested.

Table 2 Zone of Inhibition of Moxi/Pir Nanofibers, Fus/Pir Nanofibers (Fus/Pir), and the Positive Controls (Drugs Loaded into Discs in a Similar Dose) Against Different Bacteria Strains (S. aureus ATCC 29213, S. aureus ATCC BAA-977, P. aeruginosa ATCC 27853 and P. aeruginosa ATCC 9721)

The calculated zones of inhibition of Moxi/Pir-loaded nanofibers against S. aureus were 31 mm (ATCC 29213) and 38 mm (ATCC BAA-977), while they were 40 mm (ATCC 29213) and 41 mm (ATCC BAA-977) for the positive control (Table 2). On the other hand, a zone of inhibition of 25 mm and 30 mm were observed for this nanofiber system and positive control, respectively, against P. aeruginosa (ATCC 27853), and 10 mm for both the drug-loaded fibers and positive control against P. aeruginosa (ATCC 9721), as shown in Table 2. The blank fibers did not show any activity against the tested bacterial strains. This nanofiber system demonstrated a nearly identical zone of inhibition as the positive control (discs), which confirms the effectiveness of Moxi after being electrospun. The variation in the results between the fibers and the disc might be attributed to the deviation of the drug loading within the nanofibers. Various studies have incorporated Moxi antibiotics into different nanofiber systems to treat infections including Tawfik et al36 who have incorporated this antibiotic into a PVP/PLGA coaxial nanofibrous system, and the study demonstrated a consistent result to this current study. Also, Hameed et al49 have shown the effectiveness of CS and PEO nanofiber systems incorporated with Moxi in inhibiting the activity of S. aureus, E. coli and P. aeruginosa using the zone of inhibition assay, which were recorded to be 32.33 ± 1.15 mm, 35.67 ± 1.53 mm, and 36.83 ± 2.56 mm, respectively. Liu et al83 have also demonstrated an activity of Moxi loaded into PVA/CS nanofibers prepared at different ratios of both polymers. They have reported a zone of inhibition between 41 and 42 mm against S. aureus and between 8 and 31 mm against P. aeruginosa.

Fus/Pir coaxial nanofiber mat was also tested for its antibacterial activity against S. aureus and P. aeruginosa. The nanofibers and the positive control (discs) showed zones of inhibition of 31 mm and 40 mm, respectively, against S. aureus (ATCC 29213), and 33 mm and 36 mm, against S. aureus (ATCC BAA-977), as shown in Table 2. Both systems did not show any activity against the two strains of P. aeruginosa, which was expected due to the low Fus dose used in this study compared to the drug MIC (>512 μg/mL). The blank fibers did not report any antibacterial activity against both bacteria. This nanofiber system has also been proven to maintain the efficacy of Fus after being electrospun. Said et al44 exhibited that Fus-incorporated PLGA nanofibers were able to inhibit pathogenic bacteria, with inhibition zones of 46.35 mm against S. aureus and 33.5 mm against P. aeruginosa. In addition, Gilchrist et al84 have shown an antibacterial activity of combining Fus and rifampicin-loaded PLGA against four Gram-positive Staphylococcus bacterial strains. Supplementary Figures 4–8 show the zone of inhibitions of this current study’s nanofiber systems.

Determination of the Inhibitory Concentration (IC) of the Pure Drugs

It is essential to determine the ideal concentrations of the drugs that can be applied in vitro without resulting in any cytotoxicity to the cultured cells to assess the biocompatibility of the dual drug-loaded nanofiber systems with the normal cells. Cultured skin fibroblasts (ie, HFF-1) were exposed to solutions of Pir, Moxi, and Fus and their combination for 24 hours to determine the IC20 and IC50. 80% viability (represents the IC20) was considered the maximum accepted growth inhibition in this study. Figure 7 depicts the cell viability after 24-hour exposure to the drugs alone or in combination with Pir. In Figure 7A, Pir showed a cell viability of >90% with all tested concentrations up to 500 µg/mL. In contrast, based on the charts in (Figure 7B and C), Moxi and Fus exhibited ≥80% cell viability in concentrations below 250 µg/mL. A similar pattern was observed with both Moxi/Pir and Fus/Pir (charts in Figure 7D and E, respectively) indicating that the drugs have no interaction concerning the cell viability. Accordingly, a concentration of ≤150 µg/mL was selected for further biological experiments with the dual drug-loaded nanofiber systems.

Figure 7 Cell viability % of (A), Pir; (B), Moxi; (C), Fus; (D), Moxi/Pir; and (E), Fus/Pir against HFF-1 cell line.

Abbreviations: Moxi, moxifloxacin; Fus, fusidic acid; Pir, pirfenidone.

Notes: Results represent the average ± SD (n = 3).

In vivo Efficacy Testing of the Dual Drug-Loaded Nanofibers in Diabetic Wound Healing

A 14-day trial was conducted to determine whether the nanofibers loaded with either single or combinational therapies were effective in treating an induced wound in diabetic rats. The rats were administered with no treatments as a negative control, PVP-I dressing as a positive control, blank fibers, Moxi/Pir fibers, Fus/Pir fibers, Pir fibers, Moxi fibers, or Fus fibers once a day. The blank and the single drug-loaded fibers were tested as experimental controls. It is expected that the PVP drug-loaded nanofibers rapidly disintegrate upon contact with the wound site owing to the hydrophilic nature of the polymer and drugs. Consequently, it is necessary to change the nanofiber formulations daily. The size and status of the wound were monitored throughout the study. The rats’ weight blood glucose and wound swabs were also collected to monitor the rats’ health and to test the ability of dual drug-loaded nanofiber systems to prevent wound infection and the results are shown in the Supplementary Figures 9, 10 and 13, respectively. Overall, all treatment groups showed a reduction in the wound area over time, as demonstrated in Figure 8 and the Supplementary Figure 11, and representative images of the healing progression to 13 days are shown in Supplementary Figure 12.

Figure 8 Representation of daily wound size treated with drug-loaded fibers in comparison with Control non-treated, positive control, and blank fibers.

Abbreviations: Moxi, moxifloxacin; Fus, fusidic acid; Pir, pirfenidone.

Notes: Data are represented as average ± SD (n = 4).

Results indicated that on day 2, an initial reduction in the wound size was seen with PVP-I dressing, Fus fibers and Moxi fibers and no significant difference in the wound size between the three groups was apparent. There was, however, a significant reduction in the wound size between PVP-I dressing and Pir, Moxi/Pir and Fus/Pir fibers as well as the Fus fibers and Fus/Pir fibers (Figure 8, Supplementary Figures 11 and 12 and Supplementary Table 3). On day 4, the reduction in the wound size was more significant in animals treated with Fus fibers compared to the control, ie, PVP-I dressing, and fiber combinational therapy (Figure 8, Supplementary Figures 11 and 12 and Supplementary Table 3). Throughout day 6, the fibers with the combination therapy and the Fus-only fibers started to take the lead in wound closer with Fus/Pir fibers showing the most significant difference and a complete wound closer on day 8. All drug-loaded fibers were able to show a significant difference in the wound size on day 9 compared to PVP-I dressing and the blank fibers (Figure 8 and Supplementary Figures 11 and 12). Both single-loaded Fus and Moxi fibers showed complete wound closure on day 11. All other animal groups showed wounds closer on day 13 (Figure 8 and Supplementary Figures 11 and 12). No significant difference between animal weights was found between different treatment groups as shown in Supplementary Figure 9. In summary, although PVP-I dressing seemed to initiate wound healing earlier