Bioactive nanofiber composition for combating biofilm-associated wound infections

Abstract

A bioactive nanofiber composition for combating biofilm-associated wound infections, comprising: Nanofibers formed from a polymer matrix comprising polyacrylonitrile (PAN) and polyvinyl alcohol (PVA); one or more therapeutic agents selected from fluticasone, mupirocin, ciprofloxacin and silver sulfadiazine; optionally in combination with at least one bioamplifier selected from nanodiamond, graphene oxide and rhamnose; the nanofiber matrix is ​​manufactured in such a way that it achieves the following: a uniform fiber diameter in the range of 100 to 500 nanometers, thereby improving the efficiency of drug encapsulation and release kinetics; a porosity between 70% and 90% to facilitate oxygen exchange and exudate absorption at the wound site; a mechanical tensile strength sufficient to ensure integrity during application and dressing changes; and the composition, through the synergistic combination of ciprofloxacin, mupirocin and silver sulfadiazine, exhibits a broad-spectrum antimicrobial effect against gram-positive and gram-negative biofilm-forming pathogens, thereby promoting sustained drug release and improved wound healing.

Description

[0001] The present invention relates to the field of biomedical and pharmaceutical compositions, in particular a nanofiber-based bioactive composition developed for the effective treatment of chronic and infected wounds. It addresses infections caused by biofilm-forming, multi-resistant bacteria, which are frequently found in diabetic, surgical, and burn wounds. The invention particularly emphasizes a nanofiber matrix containing polymeric carriers, nanomaterials, and therapeutic agents to promote wound healing and inhibit biofilm formation.

[0002] Chronic wounds and burns are notoriously difficult to treat because they are often colonized by biofilm-forming, multidrug-resistant bacteria that create a protective extracellular matrix, shielding the microbial colonies from antibiotics and the host's immune response. These biofilms significantly delay wound healing by maintaining a persistent state of inflammation, preventing tissue regeneration, and contributing to recurrent infections. Conventional treatments, such as topical antibiotics and ointments, often fail to effectively penetrate biofilms or provide the optimal microenvironment required for tissue regeneration. The increasing prevalence of biofilm-associated infections, particularly among diabetic patients, underscores the urgent need for advanced therapeutic solutions that not only kill bacteria but also promote wound healing.

[0003] The challenge in treating such wounds lies in the inadequacy of conventional dressings, which lack multifunctional benefits such as moisture management, effective drug delivery, biofilm disruption, and tissue regeneration. Existing wound care solutions are generally unable to actively suppress inflammation, absorb wound exudate, and deliver multiple active ingredients sustainably. Furthermore, the inability of these treatments to adapt to the dynamic microenvironment of infected wounds limits their effectiveness in managing the complex pathophysiology of chronic and burn wounds.

[0004] To address these challenges, the present invention introduces an electrospun nanofiber-based composition of polyvinyl alcohol (PVA) and polyacrylonitrile (PAN), reinforced with graphene oxide, nanodiamonds, and rhamnose, and loaded with several pharmaceutical agents. This novel nanofiber composition combines anti-inflammatory, antibacterial, and regenerative properties, providing a multifunctional wound dressing capable of penetrating biofilms, controlling infections, reducing inflammation, and accelerating tissue repair. The invention thus offers a scientifically sound, synergistically effective, and clinically promising alternative for the treatment of chronic, diabetic, and burn wound infections.

[0005] One objective of the present disclosure is to provide a bioactive nanofiber composition comprising a polymeric matrix for the effective treatment of biofilm-associated wound infections.

[0006] Another objective of the present disclosure is to incorporate therapeutic agents such as fluticasone, mupirocin, ciprofloxacin and silver sulfadiazine into the nanofiber matrix to achieve a broad-spectrum antimicrobial effect.

[0007] Another objective of the present disclosure is to use bioenhancers such as nanodiamond, graphene oxide and rhamnose to improve the permeability, stability and overall therapeutic efficacy of the drug.

[0008] Another objective of the present disclosure is to produce the nanofiber matrix with a uniform fiber diameter in the range of 100 to 500 nanometers in order to improve the efficiency of drug encapsulation and release kinetics.

[0009] Another objective of the present disclosure is to achieve a controlled porosity between 70% and 90% in the nanofiber scaffold in order to facilitate oxygen exchange and exudate absorption at the wound site.

[0010] Another objective of the present disclosure is to ensure the mechanical integrity of the nanofiber composition through sufficient tensile strength, making it suitable for use in wound care and wound treatment.

[0011] Another objective of the present disclosure is to enable a sustained and controlled release of active substances from the nanofibers for a prolonged antimicrobial effect.

[0012] Another objective of the present disclosure is to enable a sustainable and controlled release of active substances from the nanofibers for a prolonged antimicrobial effect.

[0013] Another objective of the present disclosure is to inhibit the formation and persistence of biofilms at the wound site and thereby promote faster and more efficient wound healing.

[0014] Another objective of the present disclosure is to reduce the frequency of dressing changes and to improve patient compliance through improved stability and performance of the nanofiber matrix.

[0015] Another objective of the present disclosure is to offer a multifunctional wound dressing composition that combines antimicrobial, anti-inflammatory and wound-healing properties in a single platform.

[0016] The present invention relates generally to a bioactive nanofiber-based composition for the prevention and treatment of biofilm-associated wound infections, which contains a polymer matrix in which several therapeutic agents and bioenhancers are embedded.

[0017] One embodiment of the present invention comprises a nanofiber composition containing polymers such as polycaprolactone and chitosan, combined with antimicrobial agents such as fluticasone, mupirocin, ciprofloxacin and silver sulfadiazine to ensure broad efficacy.

[0018] Another embodiment of the invention integrates bioenhancers such as graphene oxide, nanodiamond and rhamnose into the nanofiber matrix to enable improved drug delivery, permeability and therapeutic activity at the wound site.

[0019] Another embodiment of the invention is the optimization of the fiber diameter and porosity within the nanofiber structure, which promotes a sustained release of active ingredients and a moist wound healing environment.

[0020] Another embodiment of the invention is the construction of a nanofiber scaffold with sufficient tensile strength and flexibility to withstand handling and the application of dressings without disintegrating.

[0021] Another embodiment of the invention is the provision of a multifunctional wound dressing composition that is non-toxic and biocompatible and suitable for the treatment of chronic and biofilm-associated wounds.

[0022] The present invention relates to a bioactive nanofiber composition specifically designed for the treatment and therapy of biofilm-associated wound infections. The composition comprises nanofibers made from a polymer matrix such as polyacrylonitrile (PAN) and polyvinyl alcohol (PVA) and integrated with one or more antimicrobial agents from the group consisting of fluticasone, mupirocin, ciprofloxacin, and silver sulfadiazine. To enhance drug release and therapeutic efficacy, the formulation may also contain bioenhancers such as nanodiamond, graphene oxide, or rhamnose. The nanofibers are engineered with controlled diameter and porosity to enable sustained drug release, optimal oxygen exchange, and effective exudate absorption.The invention also places particular emphasis on mechanical integrity to ensure that the nanofibers maintain their structure during application and dressing changes. This multifunctional composition addresses the challenges of drug resistance and persistent infections caused by biofilm-forming pathogens and offers a novel platform for modern wound care. EXAMPLE 1: Synthesis of nanofibers

[0023] Nanofiber Synthesis: Electrospinning of the nanofibers was carried out in three stages using different solutions based on the solubility of the ingredients. Layer 1 combined a 10% PAN solution (0.5 g PAN in 5 ml DMSO) with fluticasone (0.1 g in 1 ml DMSO) and mupirocin (0.1 g in 1 ml DMSO). Layer 2 used a 10% PVA solution (0.5 g in 5 ml deionized water) with ciprofloxacin (0.1 g in 1 ml) and GO (0.1 g in 1 ml), while Layer 3 mixed a 10% PAN solution with silver sulfadiazine (0.1 g in 1 ml DMSO). The solutions were sprayed sequentially at rates of 800 µl / h, 300 µl / h, and 400 µl / h at 15 kV and a distance of 14 cm between the needle and the collector. After evaporation of the solvent under controlled conditions, uniform nanofibers were obtained. Table 1 shows the formulation with the quantities of the various ingredients used to produce the nanofiber films. EXAMPLE 2: COMPOSITION

[0024] Formulation No. Nanodiamond (mg) Graphene oxide (mg) Rhamnose (mg) PAN (mg) PVA (mg) Fluticasone (mg) Mupirocin (mg) Ciprofloxacin (mg) Silver sulfadiazine (mg) F1 - - - 10 10 100 100 100 100 F2 1 - - 10 10 100 100 100 100 F3 - 1 - 10 10 100 100 100 100 F4 - - 10 10 10 100 100 100 100 F5 1 1 1 10 10 100 100 100 100 F6 1 - - 10 10 - - - - F7 - 1 - 10 10 - - - - F8 - - 1 10 10 - - - - F9 - - - 10 10 - - - - Clinical sampling

[0025] The study was conducted from August to October 2023 at the University of Central Punjab (UCP) in Lahore, Pakistan, in collaboration with the microbiology laboratories of COMSATS University Islamabad (CUI), Lahore Campus. Participants or their accompanying persons provided written informed consent after receiving comprehensive information about the research. Wound samples were collected from 74 patients of varying ages and genders using sterile, pre-moistened swabs (Clinix, Lahore). After cleaning the wounds with normal saline solution, the swab was moved in a zigzag motion from the center to the edge of the wound, covering an area of ​​1–2 cm². The swabs were then placed in culture tubes containing transport medium. Sample processing, bacterial isolation and microbiological examination

[0026] Samples were labeled with all required markings and transported to the laboratory in insulated boxes with ice packs, in accordance with WHO guidelines. Incomplete, poorly labeled, or contaminated samples were excluded. A total of 74 bacterial strains were isolated, including 24 Gram-positive strains (Staphylococcus aureus) and 50 Gram-negative strains (Pseudomonas aeruginosa = 36, Klebsiella pneumoniae = 14) from burn, surgical, and diabetic wound samples taken at Jinnah Hospital and Lahore General Hospital. Samples were initially cultured for 24 hours at 37 °C on differential media such as blood agar and nutrient agar. Subsequently, selective media were used, such as mannitol salt agar for S. aureus, cetrimide agar for P. aeruginosa, and MacConkey agar for K. pneumoniae.The identification of the colonies was confirmed by Gram staining, macroscopic examination and biochemical tests, including catalase, indole, oxidase, motility, urease and TSI tests, according to Bergey's Manual of Determinative Bacteriology. EXAMPLE 3: Identification of Gram-negative bacteria using the QTS kit

[0027] Gram-negative isolates (n = 50) were identified using the QTS kit for Gram-negative bacteria identification. The isolates were suspended in sterile saline to achieve a turbidity of 0.5 McFarland and then inoculated into QTS strips with substrate-filled wells for enzymatic and metabolic profiling. Mineral oil was added to the ADH and H₂S wells to create anaerobic conditions, and water was added to the incubation box to maintain humidity. After sealing, the strips were incubated at 37 °C for 18 to 24 hours. Post-incubation color changes indicated the test results. Reagents were added to selected wells (TDA, IND, VP, GLU) according to the kit instructions. The results were interpreted by comparing the reaction patterns with the QTS database, generating a numerical profile for each isolate to ensure accurate identification. EXAMPLE 4: Biofilm Assays Tissue Culture Plate Method (TCP)

[0028] Biofilm formation was quantified using the TCP method. LB broth was autoclaved and inoculated with bacterial cultures standardized to the 0.5 McFarland standard. A sterile 96-well plate was filled with 180 µl of LB broth and 20 µl of bacterial suspension per well; controls contained only LB broth. After 24, 48, and 72 hours of static incubation at 37 °C, non-adherent cells were removed by washing with PBS. The wells were fixed with methanol, stained with 0.1% crystal violet for 15 minutes, and washed again. The stain was dissolved with 33% glacial acetic acid, and the optical density (OD) was measured at 630 nm. The OD limit was calculated as ODavg + 3 (SD of ODavg). Based on the OD, the isolates were categorized as non-, weak, medium or strong biofilm producers. Tube method (crystal violet ring test)

[0029] The qualitative ability of bacteria to form biofilms at the air-liquid interface was assessed using the crystal violet ring test. Sterile glass tubes containing 5 ml of autoclaved LB broth were inoculated with fresh bacterial cultures and incubated at 37 °C for 24, 48, and 72 hours. After incubation, the broth was discarded, and the tubes were washed with PBS to remove non-adherent cells. Subsequently, 2 ml of a 0.1% crystal violet solution was added for 15–20 minutes. The tubes were rinsed with distilled water and air-dried. A visible violet ring at the air-liquid interface confirmed biofilm formation. The uniformity and intensity of the ring indicated the strength of the biofilm formation. Congo Red Test (CRA)

[0030] The CRA was used as a qualitative method to detect biofilm formation. Congo red agar was prepared by mixing 47 g of BHI medium, 36 g of sucrose, and 0.8 g of Congo red dye per liter of distilled water. BHI, Congo red dye, and sucrose were autoclaved separately and then mixed. The medium was poured into Petri dishes under aseptic conditions. The plates were inoculated with bacterial colonies and incubated for 24 hours at 37 °C. After incubation, colony morphology was assessed. Black, dry, crystalline colonies indicated biofilm producers, while pink colonies indicated weak or no biofilm producers.

[0031] The results show that a total of 74 wound samples were collected and processed during the study. Among the isolated bacteria were 24 Gram-positive Staphylococcus aureus and 50 Gram-negative species, including Pseudomonas aeruginosa (n = 36) and Klebsiella pneumoniae (n = 14). These isolates were identified using morphological, biochemical, and molecular methods. Biofilm formation was assessed using tissue culture plate (TCP), tube method (TM), and Congo red assay (CRA).

[0032] Using the TCP method, 16 isolates (21.6%) were classified as strong biofilm producers, 18 isolates (24.3%) as moderate producers, 24 isolates (32.4%) as weak producers, and 16 isolates (21.6%) as non-biofilm producers. This result demonstrated a significant prevalence of biofilm formation among clinical isolates, particularly among Gram-negative strains.

[0033] In the TM, 13 isolates (17.5%) showed strong ring formation, indicating strong biofilm activity; 21 isolates (28.3%) exhibited moderate ring intensity; 25 isolates (33.7%) had weak or pale rings; and 15 isolates (20.2%) did not form any rings. These qualitative observations were consistent with the TCP results and confirmed the biofilm-forming capacity.

[0034] The CRA test revealed that 18 isolates (24.3%) developed black, dry, crystalline colonies, indicating strong biofilm formation, while 20 isolates (27%) exhibited reddish-black colonies, indicating moderate biofilm formation. Pink colonies were observed in 36 isolates (48.6%), which were classified as weak or non-biofilm producers. EXAMPLE 5: Antibiotic susceptibility testing of bacterial isolates

[0035] Sensitivity to antimicrobial agents was determined using the Kirby-Bauer disk diffusion method. Bacterial suspensions standardized to 0.5 McFarland were plated onto Mueller-Hinton agar (MHA) plates. After drying, antibiotic disks were placed onto the agar surface using sterile forceps. The plates were incubated at 37 °C for 18 to 24 hours. Zone diameters were measured with a ruler, and susceptibility was interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. For Gram-negative isolates, the antibiotics included amikacin, ciprofloxacin, imipenem, piperacillin / tazobactam, colistin, cefepime, sulfamethoxazole, and ceftriaxone. For gram-positive isolates, the antibiotics included amikacin, ciprofloxacin, cefoxitin, levofloxacin, clindamycin, vancomycin, azithromycin, linezolid, erythromycin and ceftriaxone.

[0036] In the present study, 50% of the bacterial isolates showed the ability to form biofilms, which is lower compared to previous studies reporting prevalences of 73% to 78.2% [22-24]. This discrepancy may be due to differences in the antibiotic resistance profiles of the isolates. Among the Gram-positive isolates, particularly Staphylococcus aureus, 42% were biofilm producers. The antibiotic resistance profile of the biofilm-forming S. aureus strains showed high resistance rates: 100% resistance to levofloxacin, vancomycin, azithromycin, and ceftriaxone; 90% to ciprofloxacin and erythromycin; 80% to amikacin and cefoxitin; and 60% to clindamycin. In comparison, non-biofilm-forming strains showed 100 percent sensitivity to clindamycin, vancomycin and ceftriaxone, and somewhat lower resistance rates to other antibiotics, suggesting a higher antibiotic sensitivity.

[0037] Among the Gram-negative isolates, 54% were identified as biofilm producers, with Pseudomonas aeruginosa and Klebsiella pneumoniae exhibiting particularly high resistance profiles. Biofilm-forming P. aeruginosa showed complete (100%) resistance to ciprofloxacin, imipenem, cefepime, sulfamethoxazole, and ceftriaxone, while resistance to amikacin, piperacillin / tazobactam, and colistin was 87%, 87%, and 95%, respectively. Similarly, biofilm-forming K. pneumoniae showed 100% resistance to ciprofloxacin, imipenem, sulfamethoxazole, and ceftriaxone; 92% resistance to amikacin; 83% resistance to cefepime and piperacillin / tazobactam; and 42% resistance to colistin. Notably, non-biofilm-forming K. pneumoniae isolates exhibited significantly lower resistance, particularly only 50% resistance to most antibiotics.These results suggest a strong association between biofilm formation and multidrug resistance (MDR), which is consistent with previous studies. In our results, 50% of the MDR isolates were biofilm producers, while Swarna et al. reported 80.39% of the MDR isolates as biofilm producers, and another study found an even higher percentage of 88.88%.

[0038] The increased resistance observed in biofilm producers is likely due to the close cell-to-cell contact within the biofilm matrix, which facilitates horizontal gene transfer, particularly of plasmids carrying MDR genes. Furthermore, the nutrient composition of the culture medium plays a crucial role in promoting biofilm formation. Tryptone, a pancreatic digestive enzyme derived from casein and rich in peptides and amino acids, has been identified as a key component of Luria Bertani (LB) broth that promotes biofilm formation. These nutrients enhance bacterial growth and metabolic processes, thereby supporting the development of the biofilm matrix. While both tryptone and yeast extract contribute to biofilm formation, tryptone has a more direct effect due to its influence on protein synthesis and biofilm matrix structure. EXAMPLE 7: Material and formulation studies for nanofiber films

[0039] The surface morphology of the nanofiber films was investigated after carbon coating using scanning electron microscopy (SEM) at 15 kV. Swelling behavior was assessed by incubating the films in phosphate buffer (pH 6.8) at 37 °C for 24 hours. The swelling index was calculated based on the weight differences between dry and wet samples. Hydrophilicity was evaluated by contact angle measurements taken 10 seconds after the application of distilled water. Bioelectrical interaction (BET) analysis was performed to predict tissue interaction and drug release behavior. Drug loading efficiency was determined spectrophotometrically after dissolving the nanofibers in phosphate buffer.

[0040] The nanofiber films manufactured for wound healing applications contained a variety of additives—rhamnose, nanodiamond, and graphene oxide—as well as various active pharmaceutical ingredients (APIs) such as fluticasone, mupirocin, ciprofloxacin, and silver sulfadiazine. These components were embedded in a polymer blend of polyvinyl alcohol (PVA) and polyacrylonitrile (PAN). Scanning electron microscopy (SEM) of all nanofiber films revealed the formation of nanofibers with visible bead structures, most likely resulting from the deposition of the polymer matrix during the electrospraying process.

[0041] Regarding swelling behavior, formulation F4 showed the highest swelling score of 11. This is likely due to the addition of rhamnose, a sugar rich in hydroxyl groups that improves hydrophilicity and water uptake. In contrast, F8, which also contained rhamnose but no other additives, showed no swelling. This could be due to the formation of a dense polymer network in the PVA / PAN matrix, which restricts water penetration. F9, lacking all three additives (rhamnose, nanodiamond, and graphene oxide), exhibited a relatively low swelling score of 4.5, possibly due to the hydrophobic nature of the active ingredients it contains. The overall swelling score, from highest to lowest, was as follows: F4 > F1 > F7 > F2 > F3 > F9 > F5 > F6 > F8.These results underline the role of additives, especially rhamnose, in improving the hydrophilic properties and water absorption capacity of nanofiber films, while hydrophobic agents and dense polymer structures tend to hinder swelling.

[0042] Contact angle measurements provided insights into the surface wettability and the hydrophobic / hydrophilic properties of the films. Formulation F3, containing graphene oxide, exhibited the highest contact angle (56.83°) and was thus the most hydrophobic of all samples. This is consistent with the known surface properties of graphene oxide, whose layered structure reduces surface energy. In contrast, formulations containing nanodiamond (F2, F5, and F6) showed significantly lower contact angles, indicating increased hydrophilicity, likely due to the oxygen-containing functional groups and the large surface area of ​​the nanodiamond particles. Films containing rhamnose (F4 and F8) showed lower contact angles than F1, highlighting the contribution of rhamnose to hydrophilicity.Interestingly, F1 showed a moderate contact angle (55.535°) without any additives, suggesting that the PAN / PVA mixture naturally balances hydrophobic and hydrophilic properties.

[0043] The Brunauer-Emmett-Teller (BET) surface analysis further illustrated how additives influence porosity. F8 exhibited the largest surface area (1759.566 m²). 2 / g), followed by F4 (262.803 m 2 / g), both containing rhamnose. This suggests that rhamnose significantly improves porosity and surface area, potentially enhancing drug loading and release. F3, which contained graphene oxide, also exhibited a high surface area (187.695 m²). 2 / g). In contrast, the formulations loaded with nanodiamonds (F2, F5 and F6) showed negligible surface areas, indicating minimal porosity. F9, which contained no additives, exhibited a small surface area but a high micropore volume, indicating limited but fine porosity.

[0044] Biological evaluation of the nanofiber films revealed hemolysis values ​​well within acceptable limits for biocompatibility. The highest hemolysis was observed with F1 (1.37%), while the lowest was found with F8 (0.11%). Cell viability, assessed using the MTT assay, showed that F3 (graphene oxide) exhibited the best cytocompatibility with an inhibition rate of 86.29%, followed by F2 (60.78%) and F5 (35.88%). The anti-inflammatory effect was also highest with F5 (47%) and F3 (34.4%), which can be attributed to the presence of nanodiamond and graphene oxide, respectively.

[0045] Single-component tests showed that silver sulfadiazine exhibited the highest anti-inflammatory potential (90.55%), followed by mupirocin (45.52%) and fluticasone (48.34%). In contrast, ciprofloxacin had the lowest anti-inflammatory effect (8.96%). Rhamnose, graphene oxide, and nanodiamond alone contributed moderately to the anti-inflammatory effect, with values ​​of 13.35%, 33.68%, and 13.35%, respectively. EXAMPLE 8: Studies on biological and functional activity

[0046] Biocompatibility and therapeutic efficacy were evaluated using several assays. Hemocompatibility was measured using a hemolysis assay on human red blood cells. Cytotoxicity was assessed using the MTT assay on NIH 3T3 fibroblast cells compared to doxorubicin and a control group. Anti-inflammatory activity was evaluated by quantifying ROS inhibition using ibuprofen as a reference. Angiogenic potential was tested using a chicken chorioallantoic membrane (CAM) assay, observing the development of blood vessels around nanofiber discs. In vivo wound-healing potential was investigated using rabbit models with burn wounds infected with S. aureus, P. aeruginosa, and K. pneumoniae, followed by 14 days of treatment with nanofiber films.The healing was observed visually and confirmed by histopathological analysis of fibroblast activity, inflammation, and collagen formation.

[0047] The biological and functional evaluation of the manufactured nanofiber films - including studies on antibacterial activity, hemolysis, cytotoxicity (MTT), anti-inflammatory activity, angiogenesis and wound healing - provided important insights into their therapeutic potential, which was particularly influenced by the addition of nanodiamond, graphene oxide and rhamnose. Antibacterial activity

[0048] The antibacterial study revealed significant differences in activity based on the additive composition of each formulation. Formulation F1, containing nanodiamond, demonstrated strong antibacterial activity against Staphylococcus aureus with an inhibition zone (Zol) of 17.78 mm. This suggests that the large surface area and inherent antibacterial properties of nanodiamond enhance its efficacy against Gram-positive bacteria. Conversely, formulation F8, containing graphene oxide, showed strong antibacterial activity against Pseudomonas aeruginosa (13 mm) and Klebsiella pneumoniae (16.11 mm), highlighting the antimicrobial potential of graphene oxide due to its layered structure and large surface area. The rhamnose-containing formulation F4 showed moderate antibacterial activity with a Zol of 19 mm against Staphylococcus aureus, but lower activity against the other two strains.The moderate antibacterial effect of F4 could be due to the fact that rhamnose promotes swelling and release of the drug, but has no intrinsic antibacterial activity, especially against gram-negative bacteria. Hemolysis and cytotoxicity (MTT assay)

[0049] The hemolysis test confirmed the biocompatibility of most formulations, with values ​​well within the limits permitted for biomedical applications. F8, containing rhamnose, exhibited the lowest hemolysis (0.11 ± 0.07%), indicating high compatibility with red blood cells. F7 also showed low hemolysis (0.68 ± 0.04%), indicating good biocompatibility. Slightly higher, but still acceptable, hemolysis values ​​were observed for F4 and F9 (1.47% and 1.42%, respectively), further confirming the overall safety profile of the films for wound healing purposes.

[0050] The MTT assay revealed the cytotoxicity profiles of the individual formulations. F3, containing graphene oxide, exhibited the highest cytotoxicity (86.29% inhibition), likely due to the strong bioactivity and potential oxidative stress induced by graphene oxide. Similarly, F2 (nanodiamond) also showed significant cytotoxicity (60.78%). Formulations F1, F4, F5, and F6 showed moderate cytotoxicity (24.8% to 35.88%), indicating a balanced interaction between bioactive components and cellular systems. The most biocompatible formulation was F8, with the lowest inhibition rate (9.6%), highlighting the beneficial role of rhamnose in reducing cytotoxicity. anti-inflammatory effect

[0051] The evaluation of anti-inflammatory activity revealed that formulation F5, containing all three additives—nanodiamond, graphene oxide, and rhamnose—exhibited the highest inhibition (47%), suggesting a synergistic effect contributing to the suppression of inflammation. F3 also demonstrated a notable anti-inflammatory effect (34.4%) due to the presence of graphene oxide. Moderate activity was observed with F8 and F9 (19.7% and 17.6%, respectively), while F1 and F4 exhibited relatively low inhibition (9.4% and 10.5%, respectively). This suggests that while rhamnose contributes to swelling reduction and biocompatibility, it plays a less significant role in inflammatory modulation compared to nanodiamond and graphene oxide. Among the independently tested active ingredients, silver sulfadiazine showed the highest anti-inflammatory potential (90.55%), while ciprofloxacin showed the lowest (8.96%). Angiogenesis and wound healing

[0052] The chicken chorioallantoic membrane (CAM) assay showed significant new blood vessel formation with most formulations, highlighting their proangiogenic potential. However, formulations F2, F3, and F6, each containing either nanodiamond or graphene oxide, showed a reduced angiogenic response. This could be due to the potential cytotoxic effects of these additives at higher concentrations. In contrast, additive-free formulations and those containing rhamnose showed remarkable vascular density, confirming their beneficial interaction with vascular tissue. Statistical analysis

[0053] Statistical analyses were performed using paired t-tests in SPSS version 22.0. A p-value of <0.05 was considered statistically significant. Examples 1. A bioactive nanofiber composition for combating biofilm-associated wound infections, comprising: Nanofibers formed from a polymer matrix comprising polyacrylonitrile (PAN) and polyvinyl alcohol (PVA); one or more therapeutic agents selected from fluticasone, mupirocin, ciprofloxacin and silver sulfadiazine; optionally in combination with at least one bioamplifier selected from nanodiamond, graphene oxide and rhamnose; the nanofiber matrix is ​​manufactured in such a way that it achieves the following: a uniform fiber diameter in the range of 100 to 500 nanometers, thereby improving the efficiency of drug encapsulation and release kinetics; a porosity between 70% and 90% to facilitate oxygen exchange and exudate absorption at the wound site; a mechanical tensile strength sufficient to ensure integrity during application and dressing changes; and wherein the composition, through the synergistic combination of ciprofloxacin, mupirocin and silver sulfadiazine, exhibits a broad-spectrum antimicrobial effect against gram-positive and gram-negative biofilm-forming pathogens, thereby promoting sustained drug release and improved wound healing.

Claims

[1] A bioactive nanofiber composition for combating biofilm-associated wound infections, comprising: Nanofibers formed from a polymer matrix comprising polyacrylonitrile (PAN) and polyvinyl alcohol (PVA); one or more therapeutic agents selected from fluticasone, mupirocin, ciprofloxacin and silver sulfadiazine; optionally in combination with at least one bioamplifier selected from nanodiamond, graphene oxide and rhamnose; the nanofiber matrix is ​​manufactured in such a way that it achieves the following: a uniform fiber diameter in the range of 100 to 500 nanometers, thereby improving the efficiency of drug encapsulation and release kinetics; a porosity between 70% and 90% to facilitate oxygen exchange and exudate absorption at the wound site; a mechanical tensile strength sufficient to ensure integrity during application and dressing changes; and the composition, through the synergistic combination of ciprofloxacin, mupirocin and silver sulfadiazine, exhibits a broad-spectrum antimicrobial effect against gram-positive and gram-negative biofilm-forming pathogens, thereby promoting sustained drug release and improved wound healing. [2] Composition according to claim 1, wherein the nanofibers are produced using an electrospinning process to achieve a diameter in the nano range and a large surface area for improved drug delivery. [3] Composition according to claim 1, wherein the polymer matrix comprises PAN and PVA in a weight ratio of 1:

1. [4] Composition according to claim 1, wherein the nanodiamond is present in an amount of about 1 mg per 100 mg of the total formulation. [5] Composition according to claim 1, wherein the graphene oxide is present in an amount of about 1 mg per 100 mg of the total formulation. [6] Composition according to claim 1, wherein the rhamnose acts as a quorum-sensing inhibitor to suppress the formation of biofilms by pathogenic bacteria. [7] Composition according to claim 1, wherein the therapeutic agents are contained in the following approximate concentrations: fluticasone (100 mg), mupirocin (100 mg), ciprofloxacin (100 mg) and silver sulfadiazine (100 mg) per formulation. [8] Composition according to claim 1, wherein the combined use of nanodiamond, graphene oxide and rhamnose with antimicrobial agents achieves a synergistic effect against biofilm-forming bacteria. [9] Composition according to claim 1, wherein the nanodiamond, the graphene oxide and the rhamnose are each present individually or in combination to modulate the release profile of the therapeutic agents and to improve their penetration through biofilm matrices. [10] Composition according to claim 1, wherein the composition is configured as a wound dressing plaster or topical nanofiber mat suitable for direct application to diabetic wounds, burns or infections at surgical sites.