Composition of fluticasone / linezolid-loaded nanodiamond / polyacrylonitrile nanofibers

Abstract

A composition of fluticasone / linezolid-loaded nanodiamond / polyacrylonitrile nanofibers, consisting of: • Fluticasone propionate in a quantity of 25 mg to 100 mg; • Linezolid in a quantity of 25 mg to 100 mg; • Nanodiamonds dispersed in a concentration of approximately 1 mg / ml dimethylformamide (DMF); and • a polyacrylonitrile (PAN) polymer matrix prepared as a 10% w / v solution in DMF, wherein the drugs, nanodiamonds and PAN are uniformly combined to form an electrospinnable nanocomposite solution, and electrospinning is carried out under conditions suitable for producing nanofibers with controlled drug release, porosity and structural integrity for wound healing applications.

Description

[0001] The present invention relates to the field of pharmaceutical and biomedical compositions, in particular advanced wound care systems. More specifically, it relates to a nanofiber-based drug delivery composition containing linezolid and fluticasone integrated into nanodiamond / polyacrylonitrile fibers. The invention falls within the fields of nanomedicine, controlled drug delivery, and the treatment of infected burn wounds.

[0002] Burns are among the most severe forms of trauma and frequently lead to complex complications such as delayed healing, microbial infections, inflammation, and scarring. Conventional treatments for infected burn wounds rely on topical creams, dressings, or systemic antibiotics, which often have limitations such as poor drug penetration, short duration of action, high dosing frequency, and the risk of drug resistance. Furthermore, the concurrent treatment of infection and inflammation remains a critical unmet need in burn wound care.

[0003] Existing wound dressings are unable to deliver multiple active ingredients with synergistic effects sustainably and locally. Systemically administered antibiotics expose patients to side effects, while topical applications are quickly removed from the wound surface by exudates, resulting in suboptimal efficacy. Similarly, anti-inflammatory agents often fail to reach the wound microenvironment in effective concentrations, leaving inflammation uncontrolled and impairing tissue regeneration. These shortcomings underscore the need for an advanced wound dressing material that integrates both antimicrobial and anti-inflammatory functions.

[0004] To address these problems, the present invention provides a nanodiamond / polyacrylonitrile (ND / PAN) nanofiber composite containing linezolid (an antibiotic) and fluticasone (an anti-inflammatory agent). The nanofiber scaffold ensures controlled and prolonged drug release, improved wound adhesion, and mechanical stability, while nanodiamonds enhance biocompatibility and drug loading efficiency. This synergistic composition is intended not only to prevent infection but also to suppress inflammation, thereby promoting faster healing, reducing complications, and improving treatment outcomes in infected burn wounds.

[0005] One objective of the present disclosure is the development of a novel nanofiber-based composition containing fluticasone propionate and linezolid to improve wound healing.

[0006] Another objective of the present disclosure is the controlled and sustained release of drugs through nanodiamond / polyacrylonitrile nanofibers to improve therapeutic efficacy.

[0007] Another objective of the present disclosure is to improve the solubility and bioavailability of hydrophobic drugs such as fluticasone by incorporating nanodiamonds into the nanofiber matrix.

[0008] Another objective of the present disclosure is to provide a composition that exhibits high porosity and swelling capacity and facilitates the transfer of oxygen and nutrients to the wound site.

[0009] Another objective of the present disclosure is to ensure the biocompatibility of the nanofibers with minimal hemolytic activity and no cytotoxic effect on red blood cells.

[0010] Another objective of the present disclosure is to formulate nanofibers with antibacterial activity against pathogenic bacteria such as Staphylococcus aureus in order to prevent wound infections.

[0011] Another objective of the present disclosure is to provide a stable and structurally intact nanofiber matrix after electrospinning that preserves the chemical integrity of pharmaceuticals and nanodiamonds.

[0012] Another objective of the present disclosure is to develop an easily scalable electrospinning process for the production of reproducible and standardized drug-loaded nanofibers.

[0013] The present invention relates generally to a novel, nanofiber-based drug delivery system containing fluticasone propionate and linezolid applied to nanodiamond / polyacrylonitrile nanofibers to improve wound healing and control drug delivery.

[0014] One embodiment of the present invention provides for nanofibers that have a high porosity and swelling capacity, thereby enabling efficient absorption of wound exudates and facilitating the transfer of oxygen and nutrients to the wound site.

[0015] Another embodiment of the invention relates to the sustained and controlled release of hydrophilic and hydrophobic drugs through the nanofiber matrix in order to improve therapeutic efficacy over a longer period of time.

[0016] Another embodiment of the invention relates to nanofibers that are biocompatible, non-hemolytic and non-cytotoxic, thus ensuring safe application on human skin.

[0017] Another embodiment of the invention is the antibacterial effectiveness of the nanofibers against pathogenic bacteria, thereby reducing the risk of infection at the wound site.

[0018] Another embodiment of the invention is a method for producing uniform and reproducible drug-loaded nanofibers using an optimized electrospinning process that maintains the chemical integrity of the drugs and nanodiamonds.

[0019] The present invention relates to a novel, nanofiber-based drug delivery composition containing fluticasone propionate and linezolid, integrated into a nanodiamond / polyacrylonitrile (PAN) matrix. The nanofibers are manufactured using an optimized electrospinning process, ensuring uniform morphology, structural stability, and reproducibility. The composition offers controlled and sustained drug release, improved porosity and swelling capacity, which promotes oxygen and nutrient transfer to the wound site. The nanofibers exhibit excellent biocompatibility with minimal hemolytic activity and significant antibacterial activity against pathogenic microorganisms. This invention provides a multifunctional wound care system that accelerates healing, prevents infection, and maintains a moist wound environment. EXAMPLE 1: Composition Formulations Fluticasone propionate (mg) Linezolid (mg) F1 0 0 F2 0 100 F3 100 0 F4 100 100 F5 75 25 F6 25 75 F7 50 50 EXAMPLE 2: Preparation of the electrospinning solution

[0020] A polymeric PAN solution (10% w / v) was prepared by dissolving 1 g of PAN in 10 ml of DMF. For complete dissolution, the solution was heated to 45–60 °C for one hour with continuous stirring. ND was dispersed at a concentration of 1 mg in 1 ml of DMF with a magnetic stirrer at 300 rpm and 71 °C for 2–3 hours with continuous stirring. The solution was then ultrasonically treated twice for 20 minutes at 37 kHz (Elmasonic S 30H from Elma, Germany) to ensure efficient dispersion of ND. The drug solutions were prepared separately in 2 ml of DMF. The prepared PAN and ND solutions were then combined with the drug solutions with continuous stirring for 8–10 hours to produce a standardized nanocomposite solution for the electrospinning process. EXAMPLE 3: Electrospiders

[0021] A 5 ml syringe (inner diameter: 11.99 mm) with a 21G needle was attached to the electrospinning machine and filled with the prepared solution. Electrospinning was performed under optimized conditions applicable to all nanofiber fabrication. The charged solution was dispensed at a controlled flow rate of 400 µl / h and formed a stable Taylor cone under an applied voltage of 17 kV. The distance between the syringe tip and the aluminum collector was maintained at 15 cm. The aluminum foil-covered collector was rotated at a constant speed using a DC motor to ensure uniform fiber deposition. Process conditions were maintained at 20–25 °C and 30–35% relative humidity. After electrospinning, the nanofiber mats were air-dried and carefully peeled from the aluminum foil for further use. EXAMPLE 4: Source ratio

[0022] The swelling behavior of FLNPNFs (fluticasone / linezolid-loaded nanodiamond / polyacrylonitrile nanofibers) was evaluated to determine their drug delivery and exudate absorption capacity. The nanofiber sheets were prepared in 3 × 3 cm format. 2 Large patches were cut, weighed in their dry state (Wd) using an analytical balance (Biobase, BE 30002), and immersed in freshly prepared PBS solution. At predetermined intervals (10, 20, 30, 60 minutes, 1.5, 2, 3, 4, 6, 8, 12, 16, 20, and 24 hours), the swollen fibers (Ws) were removed, patted dry, and weighed to calculate the swelling ratio using the formula SR = Ws / Wd. The investigation was performed three times, and the mean values ​​were recorded. EXAMPLE 5: Erosion examination

[0023] The erosion of the nanofibers was assessed using a gravimetric method. Following the swelling studies, the moist nanofibers were air-dried at room temperature and weighed at defined time intervals until a constant weight was reached. The erosion percentage was calculated using the following formula: Film erosion (%) = (W° - Wf / W°) × 100, where W° is the initial weight of the swollen film and Wf is the final weight of the dry film. All experiments were performed in triplicate to ensure reproducibility.

[0024] The investigation of the percentage erosion rate of FLNPNFs revealed significant differences between the formulations, reflecting the influence of the drug type and the interactions between the polymers on the drug release behavior. The drug-free nanofibers (F1) exhibited the highest erosion rate at 40.4 ± 0.01%, which is attributed to increased water flow and greater relaxation of the polymer chains in the absence of drugs. Among the drug-loaded nanofibers, formulations containing linezolid showed comparatively higher erosion, with F2 at 38.3 ± 0.0% and F6 at 28.5 ± 0.04%. This is due to the hydrophilic nature of linezolid, which makes the solvents miscible and accelerates erosion.In contrast, the nanofibers loaded with fluticasone showed significantly lower erosion, with values ​​of 21.2 ± 0.02% (F3), 18.9 ± 0.02% (F4), and 15.3 ± 0.03% (F5), indicating their hydrophobic nature, which limited water uptake and erosion. A moderate erosion rate was observed for F7 (25.6 ± 0.02%), which contained both active ingredients, suggesting a balanced effect of the hydrophilic and hydrophobic components. The erosion trend followed the order F1 > F2 > F6 > F7 > F3 > F4 > F5, and the differences between all formulations were statistically significant (p < 0.05). EXAMPLE 6: Porosity test

[0025] The porosity of the FLNPNFs was analyzed using the liquid displacement method. Samples were immersed in solvent, and the volume change was measured before and after immersion. The porosity was calculated using the following formula: Porosity (%) = (W2 - W1) / p(V1 - V2) × 100, where W1 and W2 are the weights of the dry and wet samples, respectively, and V1 and V2 are the solvent volumes before and after displacement. The experiment was performed three times to obtain mean values.

[0026] The porosity of the FLNPNFs, determined using the fluid displacement method, showed a strong correlation with their swelling behavior, as higher swelling ratios were consistently associated with greater porosity. This property is particularly advantageous for wound dressings, since increased porosity improves the absorption of wound exudates and facilitates the diffusion of oxygen, nutrients, and other fluids to the wound site, thereby accelerating healing. The drug-free nanofiber (F1) exhibited the highest porosity at 82.4 ± 2.3%, which is attributed to the presence of a greater number of cavities within the fiber network.In contrast, drug-loaded nanofibers showed reduced porosity; however, formulations containing the hydrophilic drug linezolid exhibited relatively higher values, with F2 at 76.8 ± 1.9%, F6 at 74.5 ± 2.1%, and F7 at 72.6 ± 2.4%, compared to the fluticasone-loaded formulations F3 (65.7 ± 2.0%), F4 (63.9 ± 1.8%), and F5 (61.5 ± 2.2%). These variations in porosity values ​​were statistically significant (p < 0.05), confirming that the drug solubility and its hydrophilic properties play a crucial role in regulating the porous architecture of nanofiber films. EXAMPLE 7: In-vitro study on drug release

[0027] The release profile of linezolid (LIN) and fluticasone propionate (FTP) from FLNPNFs was investigated using a paddle-type dissolution device (DIS-09). Nanofiber patches (10 × 10 cm) 2Samples were immersed in 300 ml of PBS (pH 5.5), which was maintained at 37 ± 0.5 °C and stirred at 50 rpm. At predetermined time intervals (10, 20, 30, 60, 90, 120, and 150 minutes), 5 ml aliquots were taken and replaced with the same amount of fresh PBS to maintain the settling conditions. The samples were analyzed using a UV spectrophotometer (Shimadzu UV-1800) at 250 nm for LIN and 240 nm for FTP. The study was performed in triplicate.

[0028] Drug release from FLNPNFs was evaluated in PBS at a wound pH of 5.5, and the results showed different release patterns for the various formulations. Formulation F4 exhibited the highest initial burst release of 14.18 ± 0.01% within the first 10 minutes, which is attributed to the rapid release of linezolid, which is loosely bound to the fiber surface. This was followed by a substantial release of 65.95 ± 0.04% within 60 minutes, which can be explained by polymer swelling, reduced drug-polymer interactions, and the inherently higher solubility of linezolid. The presence of nanodiamonds further enhanced solubility through interactions between their hydroxyl / carboxyl groups and the hydroxyl / amino groups of linezolid, resulting in a synergistic effect.In comparison, the drug release order after 60 minutes (t60) for the other formulations was F2 (62.3 ± 0.02%) > F7 (58.7 ± 0.03%) > F6 (55.4 ± 0.02%) > F5 (42.1 ± 0.01%), with F5 exhibiting the lowest release due to the reduced drug content and the hydrophobic effect of fluticasone. These results confirm that both the drug solubility and the polymer-drug interactions strongly influence the release kinetics of FLNPNFs.

[0029] The release profile of fluticasone (FTP) at a wound pH of 5.5 showed a rapid initial increase, with F4 exhibiting a release of 14.2 ± 0.007% in the first 10 minutes. This is attributed to weak ionic interactions and loose binding of the drug to the fiber surface. After 60 minutes, the FTP release from F4 reached 57.30 ± 0.10%, which was lower than that of linezolid. This is likely due to deeper encapsulation and longer diffusion pathways, promoting sustained release for wound healing. Among the other formulations at t60, the release order was F7 (54.8 ± 0.03%) > F5 (49.6 ± 0.02%) > F6 (45.2 ± 0.01%) > F3 (13.19 ± 0.01%). The slower release in F3 was attributed to the exclusive presence of FTP and fewer polar functional groups. EXAMPLE 8: Kinetics and Mechanism of Drug Release

[0030] The release data of LIN and FTP were fitted into kinetic models, including zeroth-order, first-order, Higuchi, and Hixson-Crowell equations, using the DD Solver software. The drug release mechanism was further investigated using the Korsmeyer-Peppas model, with the diffusion exponent (n) used to distinguish between Fickian (n ≤ 0.45), non-Fickian or anomalous (0.5 < n < 1), and case II transport (n > 1). The best-fitting model was determined based on the regression coefficients (R). 2 ) determined.

[0031] Kinetic modeling of drug release from FLNPNFs revealed that both linezolid (LIN) and fluticasone (FTP) followed diffusion-controlled mechanisms with contributions from polymer relaxation. For linezolid, the Higuchi model provided the best fit for all formulations with the highest regression coefficients (R). 2) and Korsmeyer-Peppas exponent values ​​(n > 0.5), which suggests non-Fickian diffusion. For example, F2 showed a value of R 2 = 0.9591 (Higuchi) and n = 0.552, while F4 has a value of R 2 = 0.9012 (first order) and n = 0.531, confirming the combined effect of diffusion and polymer swelling. Similarly, F5 and F6 showed strong Higuchi correlations with R 2 = 0.8942 and 0.8464 with n-values ​​of 0.720 and 0.706 respectively, reflecting anomalous transport. F7 showed the most pronounced non-Fick release with R 2 = 0.9468 (first order), R 2 = 0.9412 (Korsmeyer-Peppas) and n = 0.829, which confirmed the drug release driven by diffusion and polymer relaxation.

[0032] A similar trend was observed for the release of fluticasone, with all formulations best fitting the Higuchi model and exhibiting non-Fickian diffusion (n > 0.5). F3 showed R 2 = 0.9050 (first order), R 2 = 0.9000 (Korsmeyer-Peppas) and n = 0.750, indicating strong anomalous diffusion. F4 showed a higher Higuchi dependence with R 2 = 0.9349 and n = 0.590, while F5 with R 2 F6 showed a relatively lower correlation with Higuchi (R) = 0.8375 and n = 0.583. 2 = 0.8551) and n = 0.642, while F7 with R 2 The n = 0.8732 (Higuchi) and n = 0.593 followed the same pattern. Overall, the release kinetics of both active ingredients from FLNPNFs confirmed a diffusion-dominant mechanism in conjunction with polymer relaxation, which supports their suitability for sustained drug delivery in wounds. EXAMPLE 9: Hemolysis Study

[0033] The hemocompatibility of FLNPNFs was assessed by a hemolysis assay. Fresh blood samples drawn from a healthy donor were mixed with EDTA and centrifuged at 3000 rpm for 6 minutes to separate the hematocrit. Nanofibers dissolved in PBS (pH 7.4) were diluted with an erythrocyte suspension (1 mL hematocrit in 99 mL saline and PBS). The negative control contained hematocrit diluted with saline and PBS, while the positive control used hematocrit mixed with distilled water. The samples were incubated at 37 °C for 30 minutes, centrifuged, and the supernatant was analyzed at 542 nm. The hemolysis (%) was calculated using the following formula: (TS - NC) / (PC - NC) × 100, where TS, NC and PC represent the absorption of the test, negative and positive controls.

[0034] The hemolysis assay confirmed that all FLNPNF formulations exhibited hemolysis percentages below the acceptable limit of 5%, demonstrating excellent biocompatibility and the absence of cytotoxic effects on red blood cells. The hemolysis index values ​​were recorded as follows: F1: 0.29 ± 0.09%, F3: 0.07 ± 0.08%, F5: 0.10 ± 0.01%, and F7: 0.04 ± 0.00%, indicating negligible hemolysis. Formulations F4 and F6 showed slightly higher values ​​compared to the other groups but remained well within the non-hemolytic range, likely due to the moderate effect of fluticasone despite its lower concentration. The differences in hemolytic values ​​between the formulations can be attributed to differences in their composition.Statistical analysis confirmed a significant difference between the groups (p < 0.05), highlighting the safety and biocompatibility of FLNPNFs for therapeutic applications. EXAMPLE 10: Antimicrobial test

[0035] The antibacterial efficacy of FLNPNFs against Staphylococcus aureus was evaluated using the agar-disc diffusion method. Mueller-Hinton agar (MHA) was prepared, sterilized, and poured into Petri dishes. After solidification, the bacterial culture was evenly distributed on the agar surface. Nanofiber films (10 × 10 mm) 2 The samples were placed on the inoculated plates and incubated for 24 hours at 37 °C. The zone of inhibition (ZOI) was measured in millimeters to assess the antibacterial potential. The study was performed three times, and the results are presented as mean ± standard error.

[0036] The antimicrobial evaluation of FLNPNFs against S. aureus revealed that formulations F2, F4, and F6 exhibited measurable antibacterial activity, with inhibition zones of 8 ± 1.73 mm, 7.6 ± 1.15 mm, and 6.3 ± 0.57 mm, respectively, compared to the standard LIN, which showed 17.6 ± 0.57 mm. F2 showed slightly higher activity than F4, likely due to faster drug release from the surface nanofiber zones. In contrast, formulations F1, F3, and F5 showed no inhibitory effect, indicating negligible antibacterial activity, while the standard FTP also showed no activity. The significant reduction in bacterial growth (p < 0.05) underlines the antimicrobial potential of the drug-loaded nanofiber formulations, with ND acting synergistically with antibiotics to enhance the effect. EXAMPLE 11: In-vivo study on wound healing

[0037] The wound-healing potential of FLNPNFs was investigated in male rabbits (6-8 weeks old, 1-2 kg) approved by the Research Ethics Committee of UVAS Lahore. The rabbits were divided into four groups: control, standard (treated with commercially available Cutivate-M ointment), drug-loaded FLNPNFs, and empty FLNPNFs. After anesthesia with ketamine (75 mg / kg) and xylene (15 mg / kg), wounds measuring 1.3 cm were examined. 2 A 0.5 cm deep wound was created on the back of the skin, disinfected with ethanol, and infected with S. aureus. Treatments were performed daily for three consecutive days, and the wound areas were measured on days 0, 2, 3, 4, 6, 11, 15, and 18. Wound healing (%) was calculated using the following formula: (A / A°) × 100, where A° is the original wound area and A is the wound area after treatment.

[0038] The wound healing study with FLNPNFs (F4) showed progressive wound closure over 18 days. The percentage of wound closure was assessed on days 0, 3, 7, 11, 15, and 18, with complete closure (0% wound area) achieved by day 18. On day 7, the wound area in F4 was 0.19 cm². 2 , compared to 0.58 cm 2 in the positive control and 0.85 cm 2in the negative control, highlighting the faster healing of F4. On day 15, F4 continued to exhibit superior healing conditions compared to both controls. Although the results were not statistically significant (p > 0.05), F4 showed accelerated healing in the first 10 days, attributable to the antibacterial effect of LIN, the penetration capability of NDs, and the high surface area to volume ratio of the polymer matrix, supporting its potential to prevent secondary infections and improve early wound healing. EXAMPLE 12: Histopathological examination

[0039] At the end of the in vivo wound healing study, wound tissue samples were taken for histopathological examination. The samples were fixed in 10% formalin, sectioned after 24 hours, and embedded in paraffin. Thin sections (5–6 µm) were prepared, stained with hematoxylin and eosin (H&E), and analyzed under a digital microscope. The tissue architecture was examined for parameters such as granulation tissue formation, necrosis, edema, and calcification to confirm the wound-healing efficacy of FLNPNFs.

[0040] Histopathological findings revealed distinct tissue reactions in the different groups. The negative control showed disorganized epithelial cells with granular degeneration, cell debris in the acini, and focal inflammatory infiltrates. The positive control exhibited dermal infiltration of inflammatory cells and hemorrhages, but no necrosis or major structural disorganization. In contrast, wounds treated with F4 showed hyperkeratinization, limited inflammatory infiltration, and a compact collagen structure without necrosis, suggesting improved tissue repair. The reduced inflammatory migration may be related to the modulating effect of FTP. Although the study was limited to four rabbits due to ethical 3R principles, it provides strong preliminary evidence for the wound-healing efficacy of F4 nanofiber films. Examples 1. A composition of fluticasone / linezolid-loaded nanodiamond / polyacrylonitrile nanofibers, consisting of: • Fluticasone propionate in a quantity of 25 mg to 100 mg; • Linezolid in a quantity of 25 mg to 100 mg; • Nanodiamonds dispersed in a concentration of approximately 1 mg / ml dimethylformamide (DMF); and • a polyacrylonitrile (PAN) polymer matrix prepared as a 10% w / v solution in DMF, wherein the drugs, nanodiamonds and PAN are uniformly combined to form an electrospinnable nanocomposite solution, and electrospinning is carried out under conditions suitable for producing nanofibers with controlled drug release, porosity and structural integrity for wound healing applications.

Claims

[1] A composition of fluticasone / linezolid loaded nanodiamond / polyacrylonitrile nanofibers, consisting of: • Fluticasone propionate in a quantity of 25 mg to 100 mg; • Linezolid in a quantity of 25 mg to 100 mg; • Nanodiamonds dispersed in a concentration of approximately 1 mg / ml dimethylformamide (DMF); and • a polyacrylonitrile (PAN) polymer matrix prepared as a 10% w / v solution in DMF, wherein the drugs, nanodiamonds and PAN are uniformly combined to form an electrospinnable nanocomposite solution, and electrospinning is carried out under conditions suitable for producing nanofibers with controlled drug release, porosity and structural integrity for wound healing applications. [2] Composition according to claim 1, wherein the ratio of fluticasone propionate to linezolid is selected from the group consisting of 1:0, 0:1, 1:1, 3:1, 1:3 and 1:2 to modulate drug release and wound healing performance. [3] Composition according to claim 1, wherein the nanofibers exhibit a swelling ratio in the range of 4.2 to 4.9 after 24 hours in phosphate-buffered saline solution, which facilitates the absorption of wound exudates. [4] Composition according to claim 1, wherein the nanofibers have a porosity in the range of 70% to 90%, which supports the oxygen and nutrient transfer to the wound sites. [5] Composition according to claim 1, wherein the nanofibers exhibit an erosion percentage in the range of 15% to 40% over 24 hours, enabling controlled drug release. [6] Composition according to claim 1, wherein the release of linezolid from the nanofibers follows the Higuchi kinetic model with non-Fick diffusion and the release of fluticasone also follows the Higuchi model with non-Fick diffusion. [7] Composition according to claim 1, wherein the nanofibers have a hemolysis percentage of less than 5%, indicating excellent biocompatibility with red blood cells. [8] Composition according to claim 1, wherein the nanofibers exhibit antibacterial activity against Staphylococcus aureus with an inhibition zone of 6.3 mm to 8 mm, demonstrating antimicrobial efficacy. [9] Composition according to claim 1, wherein the nanofibers promote wound closure in vivo and achieve complete closure within 18 days as well as improved healing during the first 10 days.