A triple antibiotic nanofiber scaffold for wound dressings

Abstract

A triple antibiotic nanofiber scaffold for wound dressings, consisting of: (a) a polymer blend of polyacrylonitrile and pullulan in a weight ratio of about 10:1; (b) one or more fluoroquinolone active ingredients selected from levofloxacin, ciprofloxacin and ofloxacin in different concentrations; wherein the polymer-drug solution is electrospun using a voltage of about 15 kV and a flow rate of about 5 ml / h to form uniform nanofiber mats on a collector covered with aluminum foil, and where the nanofibers are configured to provide structural integrity, drug loading capacity, and potential wound healing properties.

Description

[0001] The present invention relates to the field of pharmaceutical compositions, in particular to drug-loaded nanofibrous scaffolds for wound healing. It comprises a composition of biocompatible polymers and therapeutic agents produced using electrospinning technology. The composition is designed to provide controlled drug release, increased porosity, and optimal swelling for improved wound healing.

[0002] Wound healing is a complex biological process involving multiple overlapping phases such as hemostasis, inflammation, proliferation, and remodeling. In chronic or infected wounds, these phases are often disrupted, leading to delayed healing, increased risk of infection, and poor tissue regeneration. Conventional wound dressings such as gauze and cotton pads do not provide a controlled environment, often resulting in inadequate moisture retention, lack of targeted drug delivery, and inadequate structural support for cell migration and tissue repair.

[0003] Current pharmaceutical interventions include topical applications of antibiotics, anti-inflammatory agents, or growth factors. However, these are often rapidly degraded, distribute unevenly, and have poor retention at the wound site. Furthermore, repeated administration is required to maintain therapeutic levels, which not only increases patient discomfort but also poses the risk of antimicrobial resistance. Therefore, there is an urgent need for advanced wound care systems that combine drug delivery with structural and biological support to accelerate healing while minimizing complications.

[0004] To overcome these limitations, the present invention proposes the development of nanofiber-based wound healing scaffolds fabricated using electrospinning technology. These scaffolds mimic the natural extracellular matrix and provide an ideal environment for cell adhesion and tissue regeneration. By incorporating therapeutic agents into the nanofibers, the system ensures controlled, localized drug release, maintaining effective concentrations at the wound site for an extended period. This approach significantly improves the efficacy of wound healing, reduces the risk of infection, and eliminates the need for frequent dressing changes or reapplication of medications.

[0005] One objective of the present disclosure is to develop a nanofiber-based wound dressing that enables sustained and controlled drug delivery.

[0006] Another object of the present disclosure is the production of a biocompatible and biodegradable scaffold using synthetic and natural polymers.

[0007] A further object of the present disclosure is the improvement of the antimicrobial efficacy of wound dressings by incorporating antibiotic active ingredients.

[0008] Another objective of the present disclosure is to promote faster wound healing through improved moisture retention and oxygen permeability.

[0009] Another objective of the present disclosure is to eliminate the need for frequent dressing changes due to the gradual erosion of the nanofibers.

[0010] Another object of the present disclosure is to provide a dressing with high porosity and high swelling ratio for effective fluid absorption.

[0011] Another objective of the present disclosure is to produce nanofibers with uniform morphology using optimized electrospinning parameters.

[0012] Another object of the present disclosure is to ensure patient comfort by reducing pain and trauma during application and removal of the dressing.

[0013] The present invention generally relates to a nanofiber-based wound dressing composition comprising synthetic and natural polymers associated with one or more antibacterial drugs for enhanced wound healing applications.

[0014] One embodiment of the present invention provides an electrospun nanofiber scaffold made of polyacrylonitrile (PAN) and pullulan that enables sustained drug release and promotes a moist wound environment.

[0015] Another embodiment of the invention includes the incorporation of antibiotics such as levofloxacin, ciprofloxacin or ofloxacin into the nanofiber matrix to provide antimicrobial protection directly at the wound site.

[0016] Another embodiment of the invention is the production of nanofibers using electrospinning technology under controlled parameters such as voltage, flow rate and solvent system to achieve uniform fiber morphology and mechanical stability.

[0017] Another embodiment of the invention is the use of a biodegradable polymer blend that facilitates gradual erosion of the dressing material over time, thereby eliminating the need for removal and reducing patient discomfort.

[0018] Another embodiment of the invention is the development of a nanofiber scaffold with optimized porosity and swelling properties that improve exudate absorption and oxygen permeability and support faster tissue regeneration.

[0019] The present invention relates to a nanofibrous composition comprising a mixture of polyacrylonitrile and pullulan as a polymer matrix, which is admixed with one or more fluoroquinolone antibiotics selected from levofloxacin, ciprofloxacin, and ofloxacin. The polymer composition is prepared in precise weight ratios to ensure homogeneity and processability during electrospinning. The formulation enables the formation of uniform nanofiber mats by applying a solution electrospinning technique, in which the drug-loaded polymer solution is electrospun under optimized conditions to obtain nanofibers suitable for biomedical applications. In particular, the invention enables the incorporation of individual or multiple drugs into a nanostructured fiber scaffold for controlled-release applications while maintaining compatibility and stability within the polymer matrix. EXAMPLE 1: COMPOSITIONFormulation Code Quantity of polymer 1 (g) (Polyacrylonitrile) Amount of polymer 2 (g) Pullulan Amount of drug 1 (g) Levofloxacin Amount of the drug 2 (g) Ciprofloxacin Amount of the drug 3 (g) Ofloxacin Z1 1. 0 0.1 0.0 0 Z2 1.0 0.1 0.300 Z3 1.0 0.1 0.0 0.3 0 Z4 1.0 0.1 0.00.3 Z5 1.0 0.1 0.1 0.1 0.1 0.1 EXAMPLE 2: Electrospinning of a polymer solution

[0020] The nanocomposite solution was filled into a 5 mL BD syringe with an inner diameter of 11.99 mm. All electrospinning parameters were kept constant and previously optimized for uniform fiber formation. With increasing voltage, the polymer solution became increasingly charged. Electrospinning was performed using a FLUIDNATEK LE-10 electrospinning apparatus, with the solution dispensed at a controlled flow rate of 5 mL / h. The formation of a stable Taylor cone was observed at approximately 15 kV, indicating the onset of jet initiation. The charged polymer jet was directed onto a grounded collector covered with aluminum foil, where it deposited as nanofiber mats. The resulting nanofibers were collected as uniform, thin sheets. This procedure was consistently applied in the preparation of all sample sets. EXAMPLE 3: Fourier Transform Infrared Spectroscopy (FTIR)

[0021] Fourier transform infrared spectroscopy (FTIR) is a powerful technique used for both qualitative and quantitative analysis of chemical compounds. It provides valuable insights into the nature and type of bonds present and helps identify functional groups and reactive sites involved in chemical interactions. The FTIR spectrum is divided into two key regions: the functional group region and the fingerprint region, both of which help distinguish unique molecular features. As infrared radiation passes through a sample, specific bond vibrations absorb the radiation at characteristic frequencies, resulting in a unique absorption spectrum.This information provides insight into the nature of covalent bonds, facilitates the identification of molecular structures, and supports the evaluation of reaction kinetics and chemical changes in the sample.

[0022] Drug-loaded formulations containing levofloxacin (LVX), ciprofloxacin (CIP) and ofloxacin (OFL) showed a combination of peaks at 3262 cm- 1 (OH stretching), 2847 cm- 1 (CH stretch), 1727 cm -1 (C=O carbonyl stretching) and 1027 cm- 1 (OH bending). A clear peak at 2240 cm- 1 , corresponding to the C≡N stretch, was observed in LVX- and OFL-containing formulations, confirming the presence of the nitrile group. However, this peak was absent in the CIP-loaded nanofibers, likely due to the use of PVA as the polymer in the formulation. EXAMPLE 4: Thermogravimetric Analysis (TGA)

[0023] Thermogravimetric analysis (TGA) was performed to evaluate the thermal stability of nanocomposite sample Z5. The analysis was performed using a Q600 SDT thermogravimetric analyzer, which measures the change in sample weight as a function of temperature. The resulting thermogram shows significant weight losses at specific temperature intervals, indicating the material's degradation profile. These thermal degradation phases provide insight into the composition and stability of the nanofibers and help assess the material's suitability for various applications where thermal stability is critical.

[0024] TGA analysis of sample Z5 revealed an initial weight loss of 6.5% due to evaporation of moisture and solvents. A second weight loss of 28% occurred between 225 and 320 °C, due to degradation of the active ingredient and degradation of the polymer chain. A final loss of 16.8% was due to the formation of ash residue. EXAMPLE 5: X-ray diffraction spectroscopy (XRD)

[0025] X-ray diffraction (XRD) analysis was performed on the drug-loaded nanofiber samples to evaluate their crystalline nature. This technique involves directing X-rays onto the sample, absorbing some of the rays and diffracting others, resulting in a diffraction pattern. The diffractogram allows for the determination of the degree of crystallinity, semi-crystallinity, or amorphous nature of the nanofibers, which is crucial for understanding drug dispersion, compatibility, and release behavior within the polymer matrix.

[0026] XRD analysis revealed that the pure drugs and polymers exhibited sharp peaks indicating crystallinity, while sample Z5 exhibited semi-crystalline properties with moderately sharp peaks. EXAMPLE 6: Drug release studies

[0027] Drug release studies were conducted using a dissolution apparatus with 0.1 N HCl as the dissolution medium, maintained at a physiological temperature of 37°C. Approximately 4 mg of the nanofiber samples were added to the designated compartments of the apparatus. At predetermined intervals of 15, 30, 60, 90, and 120 minutes, 2 mL aliquots were withdrawn with a micropipette, transferred to a cuvette, and analyzed for absorbance using a UV spectrophotometer. The absorbance values ​​were measured at specific wavelengths corresponding to the individual drugs: CIP at 278 nm, LVX at 298 nm, and OFL at 294 nm. All measurements were performed in triplicate to ensure the reproducibility and statistical reliability of the release profile data.

[0028] The drug release results showed rapid release from the nanofiber scaffolds. Samples Z5 and Z2 released approximately 70% of the drug within the first 15 minutes and achieved complete release after 120 minutes. In contrast, Z3 and Z4 exhibited a slower release profile, with 39% and 59% release after 15 minutes, respectively. Their drug release continued gradually over the next 120 minutes. The faster release in Z5 and Z2 was attributed to the lower drug loading, which led to faster counterion exchange and less competition within the release medium. Example 7: Swelling ratio

[0029] The swelling behavior of the nanofiber composite formulations was evaluated by measuring their water absorption capacity at room temperature in a medium with a pH of 6.8. Uniform square pieces (1 × 1 cm) of each composite film were carefully cut with scissors and weighed to obtain the initial dry weight (Wd). These samples were then immersed in Petri dishes containing 10 mL of distilled water and allowed to swell for 24 hours. After predetermined time intervals of 15, 30, 60, 90, and 120 minutes, the films were removed, gently blotted with filter paper to remove surface water, and weighed again to determine the swollen weight (Ws). The equilibrium swelling ratio (SR) was calculated using the following formula: SR=Ws / Wd where SR is the swelling ratio, Ws is the swollen weight, and Wd is the dry weight. The entire experiment was performed in triplicate to ensure reproducibility.

[0030] All formulations were prepared with a uniform dry weight of 15 mg. Among the tested samples, formulation Z1 exhibited the highest swelling ratio of 3.33 ± 0.02, with a swollen weight of 50.3 mg, indicating excellent water absorption capacity. Similarly, formulation Z5 exhibited a high swelling ratio of 3.2 ± 0.2 with a swollen weight of 48.1 mg, comparable to that of Z1, indicating effective hydrophilicity.

[0031] Formulation Z2 exhibited a moderate swelling ratio of 2.68 ± 0.01, corresponding to a swollen weight of 36.9 mg. Z4 and Z3 exhibited relatively lower swelling behavior, with a swelling ratio of 2.26 ± 0.03 and 2.0 ± 0.1, respectively, and a corresponding swollen weight of 30 mg and 34 mg, respectively. These results indicate that formulations Z1 and Z5 have better water absorption capacity, which may be beneficial for wound healing applications where moisture retention is important. EXAMPLE 8: Erosion studies

[0032] To determine the erosion profile of the nanofiber films, the swollen samples from the swelling study were air-dried at room temperature until they reached a constant weight. The degree of film erosion was then calculated using the following formula: ES=(Wi−Wf) / Wi×100 where ES denotes the percentage of erosion, Wi denotes the initial wet weight of the swollen film, and Wf denotes the final dry weight after complete drying. This measurement helped evaluate the structural stability of the nanofibers under aqueous conditions.

[0033] The results of the erosion study showed a clear relationship between the swelling behavior and the extent of erosion in the nanofiber formulations. Formulation Z1 exhibited the highest erosion percentage (94.0 ± 0.2%), consistent with its highest swelling ratio. This is likely due to its all-polymer composition and larger surface area, leading to increased water absorption and structural collapse. Among the drug-loaded formulations, Z5 exhibited a relatively high erosion percentage (91.6 ± 0.2%), consistent with its increased swelling ratio and improved drug release profile. In comparison, formulations Z2, Z3, and Z4 exhibited lower erosion percentages of 85.9 ± 0.3%, 82.66 ± 0.01%, and 84 ± 0.1%, respectively. EXAMPLE 9: Porosity

[0034] The porosity of the nanofiber films was assessed using a water absorption-based method. As in the swelling study, film samples (1 × 1 cm) were prepared and first weighed to determine their dry weight (W1). Each film was then immersed in 10 ml of distilled water (pH 6.0–6.5) and removed at hourly intervals to measure the absorbed weight (W2) until the films reached saturation. This process continued for up to 24 hours. Porosity was calculated using the following equation: Porosity=[(W2−W1) / (ρ(V1−V2))]×100% where W2 and W1 represent the wet and dry weights, respectively, V1 and V2 are the initial and final volumes of water, and ρ is the density of distilled water at 25 °C (0.9970 g / ml). All tests were performed three times to ensure consistency.

[0035] Porosity analysis of various nanofiber formulations revealed a direct relationship between porosity and swelling behavior. Formulation Z1 exhibited the highest porosity (44.29 ± 0.02%), consistent with its high swelling ratio and indicating greater water absorption capacity due to the presence of pores. Similarly, formulation Z5, a drug-loaded nanofiber, exhibited a relatively high porosity (41.53 ± 0.03%), supporting its enhanced swelling and drug release potential. In contrast, formulations Z2, Z3, and Z4 exhibited lower porosity values ​​of 36.62 ± 0.01%, 30.0 ± 0.2%, and 31.0 ± 0.5%, respectively. It is noteworthy that Z3 with the lowest swelling ratio also had the lowest porosity, indicating a denser fiber structure with reduced liquid absorption capacity. EXAMPLE 10: Antibacterial Study

[0036] The antibacterial activity of the developed nanofiber formulations was evaluated against the Gram-negative bacterial strain Klebsiella pneumoniae. The main materials included nutrient agar, standard antibiotics, sterile filter paper discs, micropipettes, Petri dishes, and sterile tools operated under a laminar flow hood. The experiment followed aseptic protocols and was performed in triplicate for statistical reliability. Media preparation

[0037] To prepare the growth medium, 12 mg of nutrient agar was dissolved in 40 ml of distilled water and sterilized together with the Petri dishes in an autoclave at 121 °C for approximately 2.5 hours. After sterilization, the hot agar medium was poured into Petri dishes and allowed to solidify at room temperature. After solidification, a lawn culture of Klebsiella pneumoniae was prepared by evenly spreading the bacterial suspension over the surface with a sterilized cotton swab.

[0038] After sterilization, 5 ml of broth containing Klebsiella pneumoniae was incubated at 37°C for 24 hours to promote bacterial growth. Nutrient agar was poured into sterilized Petri dishes and allowed to solidify. Then, 10 µl of the bacterial suspension was carefully spread onto the solidified agar. Sterile filter paper discs soaked with various sample solutions were placed at specific locations on the agar surface. The plates were incubated at 37°C for 24 hours. After incubation, the zones of inhibition around each disc were measured with a millimeter ruler to assess antibacterial activity. All experiments were performed in triplicate to ensure consistency and accuracy.

[0039] The results show that the yellow colonies of Klebsiella pneumoniae confirmed bacterial growth, and the drug-loaded formulations exhibited distinct zones of inhibition compared to the pure drug and the empty scaffolds. Notably, the empty formulation showed no inhibition, confirming that the antibacterial activity was solely due to the incorporated antibiotics. All formulations demonstrated activity against Gram-negative bacteria, confirming the potential of these antibiotic-loaded nanofibers as effective wound dressings. EXAMPLE 11: In vivo wound healing studies

[0040] An in vivo wound healing study is used to evaluate the efficacy of drug-loaded nanofibers compared to drug-free nanofibers, a commercially available formulation (Quench), and untreated open wounds. Three rabbits weighing between 1 and 2 kg participated in the experiment. The dorsal area of ​​each rabbit was carefully shaved and cleaned with ethanol to avoid skin irritation.

[0041] After administering anesthesia, four uniform wounds were created on the back using a red-hot iron rod. Each wound was assigned a specific treatment: one was treated with drug-loaded nanofibers, another with drug-free nanofibers, the third received the commercially available wound healing formulation, and the fourth remained untreated as a control. The healing progress of the wounds was monitored over a period of time, and the percentage of wound closure was calculated using the following formula: Wound area (%) = (A / Ao) × 100 where Ao is the original wound area and A is the wound area at a given time.

[0042] The results showed that wounds treated with drug-loaded nanofibers exhibited significantly accelerated healing, with complete wound closure observed by day 15. The empty nanofibers also promoted healing better than the commercially available cream and the untreated wounds, likely due to the intrinsic antibacterial effect of the PU polymer. By day 21, all wounds in the treated groups showed hair growth and complete epithelialization, indicating successful tissue regeneration and the wound-healing potential of nanofiber scaffolds. Examples 1. A triple antibiotic nanofiber scaffold for wound dressings, consisting of: (a) a polymer blend of polyacrylonitrile and pullulan in a weight ratio of about 10:1; (b) one or more fluoroquinolone active ingredients selected from levofloxacin, ciprofloxacin and ofloxacin in different concentrations; wherein the polymer-drug solution is electrospun using a voltage of about 15 kV and a flow rate of about 5 ml / h to form uniform nanofiber mats on a collector covered with aluminum foil, and where the nanofibers are configured to provide structural integrity, drug loading capacity, and potential wound healing properties.

Claims

[1] A triple antibiotic nanofiber scaffold for wound dressings, consisting of: (a) a polymer blend of polyacrylonitrile and pullulan in a weight ratio of about 10:1; (b) one or more fluoroquinolone active ingredients selected from levofloxacin, ciprofloxacin and ofloxacin in different concentrations; wherein the polymer-drug solution is electrospun using a voltage of about 15 kV and a flow rate of about 5 ml / h to form uniform nanofiber mats on a collector covered with aluminum foil, and where the nanofibers are configured to provide structural integrity, drug loading capacity, and potential wound healing properties. [2] The composition of claim 1, wherein the nanofibers are prepared using a polymer blend containing 1.0 g of polyacrylonitrile and 0.1 g of pullulan. [3] The composition of claim 1, wherein the fluoroquinolone drug is selected from 0.3 g of levofloxacin, 0.3 g of ciprofloxacin or 0.3 g ofloxacin. [4] The composition of claim 1, wherein the composition comprises a combination of 0.1 g each of levofloxacin, ciprofloxacin and ofloxacin embedded in the nanofiber matrix. [5] The composition of claim 1, wherein the polymer solution is dispensed through a syringe having an inner diameter of about 11.99 mm. [6] The composition of claim 1, wherein the nanofibers have been collected in the form of a thin, uniform, non-woven web suitable for use as a wound dressing. [7] The composition of claim 1, wherein the nanofibers have a swelling ratio in the range of 2.0 to 3.33, determined by water absorption tests at a pH of 6.

8. [8] The composition of claim 1, wherein the nanofibers have a porosity in the range of 30% to 44.29%, which facilitates moisture retention at the wound site. [9] The composition of claim 1, wherein the nanofibers, after swelling and drying under standard conditions, have an erosion rate between 82% and 94%, indicating biodegradability. [10] The composition of claim 1, wherein the drug release profile achieves complete release within 120 minutes, promoting rapid therapeutic action.