1. A method of preparing drug-loaded polymeric wound healing scaffolds, the method comprising the steps of: • sulfonating polyether ether sulfone (PEES) by dissolving it in a solvent and subjecting it to a sulfonation reaction to produce sulfonated polyether ether sulfone (SPEES); • drying the resulting SPEES product; • dissolving the dried SPEES in a solvent selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), and N-methyl-2-pyrrolidone (NMP) to form a polymeric solution; • incorporating at least one therapeutic agent into the polymeric solution; and • fabricating the solution into a scaffold selected from a membrane via solvent evaporation or a nanofibrous mat via electrospinning.

2. The method as claimed in claim 1, wherein o the SPEES is prepared by varying the degree of sulphonation of polyether ether sulfone (PEES) subsequently membranes were made by dissolving in a solvent selected from DMF, DMSO, DMAc, or NMP; and o the ESPEES nanofibrous mats are fabricated by an electrospinning technique using SPEES dissolved in DMF at polymer concentrations of 30–40% (w/v);

3. The method as claimed in claim 1, wherein said sulfonation is performed at 10 °C for 24 hours to achieve optimal hydrophilicity and solubility.

4. The method as claimed in claim 1, wherein drying is performed for 1 day at 50 °C in a hot air oven or a vacuum oven.

5. A drug delivery system based on method as claimed in claim 1 comprising: • a polymer of sulfonated polyether ether sulphone (SPEES); and • drug loaded into the SPEES, wherein the scaffold is configured for sustained release of said class of drug, following zero-order kinetics.

6. The drug delivery system as claimed in claim 5, wherein the scaffold is a nanofibrous mats and/or membrane.

7. The drug delivery system as claimed in claim 5, wherein said drug -loaded membrane demonstrates a prolonged release of 12.87% in 1 hour, 46% in 24 hours, and 70-80% over 17 days.

8. The drug delivery system as claimed in claim 5, wherein said scaffold is a nanofibrous mat (ESPEES) demonstrates a sustained release of 14.18% in 1 hour, 42% in 24 hours, and 70-75% over 17 days.

9. The drug delivery system as claimed in claim 5, wherein the scaffolds exhibit sustained drug release over a period of 17–21 days following zero-order kinetics.

10. The drug delivery system as claimed in claim 5, wherein the scaffold exhibits a hemolysis rate below 8.9% and demonstrates excellent hemocompatibility.

11. A drug delivery system as claimed in claim 5, wherein the scaffold is a membrane for treating acute wounds and wherein the scaffold is a nanofibrous mat for treating chronic wounds.

12. A drug delivery system as claimed in claim 5, wherein said system demonstrates enhanced effectiveness in addressing aeration, angiogenesis, inflammation, prolonged drug release, and anti-infection properties and minimizes issues related to repeated dressing changes in wounds.

13. A drug delivery system as claimed in claim 5, wherein said drug is selected from wound healing drugs such as nalidixic acid and ciprofloxacin.

Description:FIELD OF THE INVENTION:
The present invention relates to the field of biomedical engineering and pharmaceutical, more particularly, the invention relates to a method of preparing sulfonated polyether ether sulfone (SPEES) membranes and electrospun sulfonated polyether ether sulfone (ESPEES) nanofibrous mats and a drug delivery system thereof. The invention provides controlled and sustained drug delivery system possessing antimicrobial properties, hemocompatibility, and biocompatibility for acute and chronic wound management.

BACKGROUND OF THE INVENTION:
Wound care is crucial in modern healthcare, especially post-surgery and in situations of infection. Global Health Management indicates that the demand for wound care is growing daily. Surgical wounds, burns, and diabetic foot ulcers necessitate expensive therapies, each characterized by distinct severity and limitations.

Conventional wound dressings such as gauze, cotton pads, and synthetic wound healing agents suffer from several drawbacks including poor drug release profiles, increased frequency of dressing changes, limited infection protection, and lack of compatibility with biological tissues. Chronic wounds, in particular, pose a significant challenge due to prolonged healing periods and high susceptibility to microbial infections.

Therefore, a rapid intervention is required to reduce dressing change frequency, minimize scarring, prevent infections, manage excessive fluid intake, and relieve the discomfort associated with wound healing.

Accordingly, the present invention provides effective wound dressing materials that concurrently address these challenges and provides a polymeric wound dressing scaffolds capable of delivering therapeutic agents in a controlled manner while simultaneously enhancing cell proliferation, preventing bacterial invasion, and maintaining hemocompatibility.

The disclosures available in the existing state of art are briefed below:

One such patent application is Chinese Patent application no.: CN-202210692870-A titled as “Lactic acid porous fiber membrane and application thereof” which relates to a porous fiber membrane consisting of sulphonated polylactic acid wrapped on a polydopamine (PDA) film self-assembled with gentamicin (GS). The prior art claims that GS plays a key role as an antibacterial agent and PDA functions towards wound healing.

While the present invention relates to use of sulfonated polyether ether sulphone to prepare a nanofibrous mat and a membrane to hold the drug. The present invention demonstrates enhanced effectiveness in addressing aeration, angiogenesis, inflammation, prolonged drug release, and anti-infection properties and minimizes issues related to repeated dressing changes in wounds when compared to case 1, of the prior art which addresses angiogenesis, inflammation, and moisture retention capacity.

Another reference is made to Indian Patent Application no.: IN-445DEN2012-A titled as “Electrospun silk material systems for wound healing”, the prior art relates to the process of preparing silk fibroin/polyethene oxide blended material and their usage in biomedical applications. They demonstrated suitable physical and biofunctional properties, such as fiber structure, topography, absorption, water vapor transmission rates, oxygen permeation, and biodegradability, relevant to biomaterial systems with utility for wound dressings.
In contrast to the silk fibroin/polyethene oxide blended material used in case 2 of the prior art to generate nanofibrous structure, the present invention have utilized sulfonated polyether ether sulphone (SPEES) to prepare nanofiber mats. The distinct topographies and fiber structures, presented in case 2 demonstrated that electrospun silk mats have a silk fibroin protein/PEO blend ratio ranging from 2:1 to 4:1, with silk fibroin having a thickness of roughly 20 to 80 microns. The present invention discloses electrospun nanofibrous mat, which is much superior to case 2 of prior art.

The present invention addresses aeration, angiogenesis, drug-releasing for prolonged days, anti-infections and reducing the repeated dressing of tissues in wounds. Accordingly, the present invention is able to overcome the limitations or drawbacks associated with said prior art.

Reference is also made to non-patent literature titled as “Design of an Antibiotic-Releasing Polymer: Physicochemical Characterization and Drug Release Patterns” by Himabindu Padinjarathil et al. The article discloses SPEEK, a biocompatible thermoplastic, was synthesized by sulfonating poly(ether ether ketone) (PEEK). While the present invention relates scaffolds from sulfonated polyether sulphone through solvent evaporation and electrospinning techniques with drug embedded

Reference is also made to non-patent literature titled as “Strategically designed SPEEK nanofibrous scaffold with tailored delivery of resveratrol for skin wound regeneration” by Rajalakshmi Ekambaram et al. The cited prior art relates to Electrospinnable sulphonated poly ether ether ketone polymeric materials for skin wound healing using and resveratrol as a 2D nano biomaterial in vitro and in vivo without cytotoxicity. Resveratrol-controlled drug release made nanofibers biocompatible for skin wound regeneration.

While the present invention relates to use of the sulphonated polyether ether sulphone, which belongs to the thermoplastic polymer family. The polymer and the results of present invention is completely different, the polymer and the release system are superior than to what is reported in the prior art.

In view of above drawbacks of the existing state of art, and to overcome the same, the present invention provides the application of modified PEES polymer and its fabrication into a fibrous mat or membrane for providing improved drug release for efficient wound treatment. The polymer backbone was modified by the sulfonation method.

OBJECT OF THE INVENTION:
The main object of the present invention is to provide a customized and sustained drug delivery system that effectively addresses the challenges in wound management.

Yet another object of the invention is to provide a wound dressing material that is bioinert, biocompatible, and hemocompatible, suitable for long-term biomedical applications.

Yet another object of the present invention is to provide a drug delivery system that can be fabricated into different forms (membranes and electrospun mats) to suit the requirements of various wound types, such as acute and chronic wounds.

Yet another object of the present invention is to provide a drug delivery scaffold that exhibits sustained drug release following zero-order kinetics, thereby reducing the need for repeated dressing changes and ensuring continuous therapeutic action.

Yet another object of the invention is to provide a delivery system available in both membrane (SS) and electrospun nanofibrous mat (ESS) forms to address the distinct requirements of acute and chronic wounds respectively.

Yet another object of the invention is to provide a membrane or mat that helps in cell proliferation and tissue regeneration at wound sites.

Yet another object of the invention is to provide a wound dressing material exhibiting high thermal and mechanical stability that remains non-degradable and bio-inert during the healing process.

Yet another object of the present invention is to provide transdermal bandages for effective and efficient wound healing.

SUMMARY OF THE INVENTION:
The present invention describes a method of preparing drug-loaded polymeric wound healing scaffolds comprising sulfonated polyether ether sulfone (SPEES) membranes and electrospun sulfonated polyether ether sulfone (ESPEES) nanofibrous mats and a system for drug delivery thereof. These scaffolds are incorporated with drug, which is gradually released in a sustained manner to provide antimicrobial activity and wound healing efficacy.

The invention utilizes SPEES, a modified form of PEES polymer. The modification is achieved through a sulfonation method.

The invention demonstrates that SPEES membranes are preferred in acute wound healing due to moderate yet efficient drug release and faster healing potential, whereas ESPEES nanofibrous mats are advantageous for chronic wound applications due to their prolonged drug release and reduced frequency of dressing changes. Both forms of scaffolds exhibit excellent hemocompatibility, biocompatibility, and antimicrobial barrier function, thereby making them suitable as advanced wound dressing biomaterials.

Accordingly, the present invention provides SPEES polymer's ability to provide sustained drug release over 17 to 21 days, avoiding an initial burst effect and ensuring a prolonged therapeutic effect. The membranes are suitable for acute wounds due to their relatively faster drug release, while the nanofibrous mats are preferred for chronic wounds, offering a slower release profile and requiring less frequent dressing changes. In vitro and in vivo studies have confirmed the scaffolds' excellent biocompatibility, hemocompatibility, and efficacy in inhibiting bacterial growth.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 depicts UV drug release profile of electrospun SPEES at different concentrations. A) initial 48-hour drug release pattern and B) 1-21 days drug release.
Figure 2 depicts UV spectra of drug release from SPEES membrane A) Various time intervals of drug release were studied B) Different polymer concentrations were studied to understand drug release
Figure 3 depicts Bar graph illustrating the Hemocompatibility of SPEES polymer in different forms (n=3)
Figure 4 depicts A) Indirect MTT assay of SPEES polymer in RAW264.7 cell lines (n=3). B) Indirect MTT assay on RAW 264.7 cell lines for 1, 3, and 7 days on SPEES membrane and electrospun mat with and without drug (Nalidixic sodium salt).
Figure 5 depicts the direct MTT assay on SPEES membrane and electrospun mat on RAW264.7 cell lines.
Figure 6 depicts the microscopic image of A) Electrospun SPEES with NA. B) Membrane SPEES with the NA were incubated for 7 days with RAW 264.7 cell lines at 4x and 20x magnification.
Figure 7 depicts the rate of wound closure by SPEES scaffolds with and without drug.
Figure 8 depicts the A) Photographic images of in vivo balb/c group with wound B) bar chart representation of in vivo balb/c group with wound from 0th day, 3rd, 7th and 14th days
Figure 9 depicts In vivo microbial swab studies on 3rd and 14th days of SPEES NA membrane and ESPEES NA electrospun mat.
Figure 10 depicts the histopathological specimens of groups showing fibro collagenous tissues with granulation tissue and infiltrated inflammation at 10x magnification.
Figure 11 depicts the morphological SEM images of (A) SPEES with nalidixic acid sodium salt. (B) Electrospun nanofibers of 35% w/v SPEES with nalidixic acid sodium salt
Figure 12 depicts the schematic representation of the present invention in a non-limiting manner.

DETAILED DESCRIPTION OF THE INVENTION
Some embodiments of the present disclosure, illustrating all its features, will now be discussed in detail. It must also be noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of the ordinary skills in art will readily recognize that the present disclosure including the definitions listed here below are not intended to be limited to the embodiments illustrated but is to be accorded with the widest scope consistent with the principles and features described herein.

A person of ordinary skill in art will readily ascertain that the illustrated steps detailed in the figures and here below are set out to explain the exemplary embodiments shown, and it should be anticipated that ongoing technological development will change the way functions are performed. These examples are presented herein for purposes of illustration, and not limitation. Further, the boundaries of the functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternative boundaries defined as the specified functions and relationships thereof are appropriately performed. Alternatives (including equivalents, extensions, variations, deviations, etc., of those described herein) will be apparent to persons skilled in the relevant art(s) based on the teachings contained herein. Such alternatives fall within the scope and spirit of the disclosed embodiments.

Before discussing example, embodiments in more detail, it is to be noted that the drawings are to be regarded as being schematic representations and elements that are not necessarily shown to scale. Rather, the various elements are represented such that their function and general purpose becomes apparent to a person skilled in the art.

Any connection or coupling between functional blocks, devices, components, or other physical or functional units shown in the drawings or described herein may also be implemented by an indirect connection or coupling. A coupling between components may also be established over a wireless connection. Functional blocks may be implemented in hardware, firmware, software, or a combination thereof.

Further, the flowcharts provided herein, describe the operations as sequential processes. Many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of operations re-arranged. The processes may be terminated when their operations are completed but may also have additional steps not included in the figured. It should be noted, that in some alternative implementations, the functions/acts/ steps noted may occur out of the order noted in the figured. For example, two figures shown in succession may, in fact, be executed concurrently, or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.

Further, the terms first, second etc… may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer or section from another region, layer, or a section. Thus, a first element, component, region layer, or section discussed below could be termed a second element, component, region, layer, or section without departing form the scope of the example embodiments.

The terminology used herein is for the purpose of describing example embodiments only and is not intended to be limiting. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

As used herein, the singular forms “a,” “an,” and “the,” are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the terms “and/or” and “at least one of” include all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises,” “comprising,” “includes,” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

The present invention provides a sulphonated poly(ether ether sulfone) (SPEES) composition characterized by enhanced biocompatibility and hemocompatibility, specifically configured for use as a sustained-release drug delivery vehicle in wound management applications. Whereas native poly(ether ether sulfone) (PEES) exhibits high solvent resistance and inherent hydrophobicity—limiting its physiological utility. The present invention utilizes a sulphonation process to increase the hydrophilic character of the polymer backbone. In one embodiment, the invention relates to the structural analysis and performance of said SPEES in the form of film-forming matrices (SS) and electrospun nanofibrous mats (ESS), each providing a scaffold for the continuous and prolonged release of therapeutic agents to a wound site.

The sulphonation reaction was performed at low temperatures for a longer duration had resulted in extremely poor hydrophilicity and solubility. By increasing temperatures from 25 °C to 50 °C, for a period of 24 hours the product showed a high sulphonation which resulted in low yield due to high solubility in water. The reaction was optimised to a temperature of 10 °C for a period of 24 hours, which produced a significant yield of sulfonated polymer, demonstrating optimal hydrophobicity and solubility. The product was dried under a vacuum oven for 24 hours.

Polymer Modification and Synthesis of Scaffold
The modes of scaffold were prepared from SPEES
a) membranes through the solvent evaporation method (SS), and
b) a nanofiber mat through the electrospinning technique (ESS).

Formulation of Membrane and Incorporation of Drugs
Optimization of the SPEES membrane (SS) was carried out by dissolving a minimal amount of polymer (50-500 mg) in different solvents to check the solubility and membrane formation. It was then casted into a membrane in a culture petric plate and then dried at 50 ˚C – 80 ˚C overnight in a vacuum oven. The SS membranes were prepared by using solvent evaporation technique and taken for characterisation studies. In the case of drug embedding, the drug-to-polymer ratio was obtained by determining the solubility of the drugs in the polymer. The drugs nalidixic acid sodium salt and ciprofloxacin were weighed (0.05 - 5 mg) and dissolved in a few drops of DMF solvent. The polymer and drug solution were agitated, the resultant solution was further sonicated to attain a homogeneous mixture. It was then cast into a culture plate by drop-wise addition and was then dried at 50 ˚C overnight in a vacuum oven.
The solubility of SPEES depends on the solvent (DMF, NMP, DMAc, and DMSO) used for dissolution. This variation could be attributed to differences in their form and nature. Both the PEES pellet and the amorphous pretreatment powder were found to be insoluble in solvents (DMF). The sulphonated versions of PEES (SPEES), showed solubility in nearly all the solvents that they were tested in, eliminating the need for extensive heating or sonication. A SS membrane was created by casting polymer in different weights, namely 50-500 mg, drying 50-100 mg of SPEES in a small volume of solvent for 1 day at 50 °C in a hot air oven or a vacuum oven produced the best results. Starting from 70 mg polymer, a thin and dedicated membrane was developed in comparison to other weights. Different solvents, including DMF, NMP, and DMSO, were used to optimize casting process towards membranes preparation.

Fibrous Nanomat Preparation and Incorporation of Antibacterial Drugs
Electrospinning-based method was employed for synthesizing ESS (nanofibrous mat). The optimization was done by using polymer dissolved in different solvents like DMF, DMSO and NMP for better mat formation, thereby, the concentration of polymer and solvent (w/v ratio), voltage, flow rate and distance were varied to produce mat. The polymeric solution was prepared to dissolve in DMF and was taken for further synthesis. Each solution was filled into a 5 mL sterile disposable syringe with a blunt end and mounted on a syringe pump with a “21G x 1.5” (0.80 x 40 mm) needle. The pump’s flow rate was 0.1 -1 mL/hr. An aluminium sheet collector plate was positioned at 12-14 cm from the needle tip. The spinning process was performed for approximately 3 hours to reach a layer thickness for handle comfortably. In drug-embedded mat synthesis, nalidixic acid sodium salt was weighed 0.05 - 5 mg, respectively, agitated in 1 mL of polymer solution and loaded to electrospun to obtain drug-loaded mats. Nanofiber of SPEES which was synthesized using DMF as a solvent to enhance viscosity, and concentrations 10% w/v to 40% w/v were tried. A homogeneous solution from a 30% w/v solution at a voltage of 18–19 kV and a flow rate of 0.05–0.1 mL/hr produces the desired fibrous mat. The optimization parameter of 40% w/v concentration was used to produce drug-loaded nanofibers. The UV drug release profile of electrospun SPEES at different concentrations. A) initial 48-hour drug release pattern and B) 1-21 days drug release is shown in Figure 1.

In vitro Drug Release Kinetics

Electrospun Mat
In the realm of electrospinning, SPEES (ESS) demonstrated controlled drug release, effectively mitigating the occurrence of an initial burst release of the drug was ruled out. The 40% w/v ESPEES mat containing 5 mg drug showed the highest rate of drug release when compared to a low concentration of 0.5 mg as shown in Figure 1. However, the pattern of drug release was the same despite concentration of drug and the rate of drug release increases with time. Swelling was observed in polymer mat with the increase of time, by following diffusion mechanism of drug release. At first, a minimum drug release was observed from the mat and increased rates were observed after 24 hr; 14 % to 88 % on the 21st day at which the drug was exhausted in the polymer mat as shown in Figure 1.

Membrane
The kinetics of drug release from polymer membranes containing ciprofloxacin and nalidixic acid sodium salt were investigated. The drug-containing samples were immersed in simulated body fluids and left undisturbed for 45 mins before being gently agitated and analysed using UV absorbance at different intervals. From the absorbance values obtained, the drug distribution coefficient in the polymer/SBF system was estimated and the pattern of drug release over time was examined. The process was repeated until saturation was reached, and readings were taken after 96 hours to determine the release pattern over a prolonged period to confirm with mathematical modelling as shown in Figure 2.

About 1 mg of nalidixic acid was introduced in different polymer concentrations (ESS NA) such as 100 mg and 30 mg. The membranes of ESS NA showed the same drug release but as the concentration of drug is decreased there observed an increase in drug release as shown in Figure 2. There was a steady release, and the rate of release increase was observed after 24 hours till 408 hours. The drug interactions of the membrane decreases significantly with the high concentration of nalidixic acid, resulting in weaker bonds and a higher release rate. Drug release for the 100 mg dosage shows a clear trend over time; an impressive 74% of the drug is released over 17 days, with 13% already released within the first hour. This demonstrates the effectiveness of formulation in facilitating drug release.

The experimental concentration of drug release was determined by recording the absorbance of the standard solution of both drugs at different concentrations. The drugs nalidixic acid sodium salt incorporated into the polymer matrix showed an absorbance of 313 nm and 331 nm respectively in SBF. The initial 6 hours saw a modest increase in the absorption value, which then remained constant for the following 24 hours. In general, membranes created using the solvent evaporation approach display the drug release's diffusion mechanism. In the investigation, a diffusion control mechanism was observed for the drug release for both membrane and nanofibrous mat. The absorbance versus time plot for nalidixic acid shows a period of moderate increment in the amount of drug release, along with a phase of uniform release. Similarly, both drug graphs do not show a rapid efflux of the drug from the polymer membrane, ruling out burst release and confirming prolonged release. UV spectra of drug release from SPEES membrane A) Various time intervals of drug release were studied B) Different polymer concentrations were studied to understand drug release are shown in Figure 2.

SPEES membranes and electrospin nanofibrous mat concerning with time of drug release were carried out for mathematical modelling. Time periods showed a uniform release along with steady raise in rate of release was observed. Both scaffolds release the drug and obeys zero-order kinetics. This is also justified because the pattern of drug release does not change with change in the concentration of the drug initially loaded. It was reconfirmed by the Ritger-Peppas model, it was found that the drug release pattern obeys non-Fickian (Type 2) transport, i.e. drug release happens via fast diffusion of the solvent and swelling. The type 2 non-Fickian diffusion and the anomalous non-Fickian diffusion differ from each other as the magnitude of swelling is greater than the magnitude of diffusion in the former while the magnitude of relaxation is equal to the magnitude of relaxation in the latter. The polymer does not undergo degradation or erosion during the process of drug release. This can be established by the determination of the erosion grade constant from the mathematical equation corresponding to the Hopfenberg model. A low value of the erosion grade constant indicates that the erosion observed is insignificant. The erosion constant of the carrier in the Hopfenberg Model was observed to be in the order of 10-12 cm/sec, indicating that erosion plays an insignificant role in the drug release predominately controlled by diffusion and swelling.

Pharmaco-Physicochemical Studies

The distribution coefficients of the drug molecules in the simulated body fluid (SBF)– SPEES membrane system and SPEES fibrous nano mat were considered by employing the amount of drug released after 24 hours and the total amount of drug loaded into the membrane. The diffusion coefficients of the polymer-SBF system can be determined by the equation of Fick’s law of diffusion.

Membrane:
The distribution coefficient of the nalidixic acid sodium salt-loaded SPEES membrane was observed to be 0.1-0.2 while that of the ciprofloxacin-loaded SPEES membrane was observed to be 0.1- 0.2.

Electrospun mat:
Electrospin SPEES loaded with nalidixic sodium salt release drug for 48 hours, to study the physicochemical studies. The distribution coefficient is 1.46. From the values obtained in both cases, it is understood that the amount of drug present in the aqueous layer, after a period of 6 hours, is greater than the amount of drug left in the membrane matrix after the same time, indicating that a significant amount of drug has been displaced from the membrane to deionized water.

The diffusion coefficient measures the extent to which the osmotic movement of the drug has taken place from the polymer matrix to the mainstream solvent (water in this case) measured. The diffusion coefficient, in both cases, was observed to be 2.411x10-9cm2/s. This is higher than the value of the erosion grade coefficient indicating that the phenomenon of diffusion overrides erosion.

The permeability coefficient of the nalidixic acid sodium salt-loaded membrane was observed to be 6.3639x10-8 cm/s while that of the ciprofloxacin-loaded membrane was observed to be 7.2947x10-8 cm/s. The values of the permeability coefficient and diffusion coefficient of both drug-loaded membranes lie in the range of 10-8-10-10. This serves as an indication of the fact that drug release from the membranes in consideration occurs chiefly via diffusion.

Anti-Microbial Properties of SPEES

The zone of inhibition method was used to assess the antibacterial efficacy of the membranes SPEES with CF and NA drug and electrospun SPEES mat, against S. aureus and E. coli, C. albicans and dermatophytes (patient sample). Gram-positive bacteria are represented by S. aureus, whereas Gram-negative bacteria are represented by E. coli were cultures in LB nutrient broth. In this study, we used the bacterial zone of inhibition to assess the effectiveness of the antibacterial property.

The LB agar plates are incubated with bacterial suspensions for 24 hours. The test membranes and mat were prepared by patching machine (6 mm in diameter) and Azithromycin and ciprofloxacin strips were used as positive controls. The Kirby-Bauer disc diffusion method was used to assess the dressings' antibacterial activity.

A digital Vernier calliper was used to measure the zone of inhibition (ZOI) in millimetres after circular films (6 mm in diameter) were placed over the colonized agar plates and incubated for 24 hours (at a temperature of 37 °C). There was clear significant zone of inhibition of 17 mm observed in nalidixic acid sodium salt loaded membrane and electrospun mat in E coli. In the case of membrane and electrospun mat without drug showed no zone but slight clearance was seen. This indicates that polymer has no role as anti-microbial agent but good drug delivers and barrier to growth of microbes. In case of S. aureus the ciprofloxacin loaded membrane and mat showed 12~13 mm zone of inhibition. Besides, the polymer scaffold was tested against dermatophytes by taking the patient samples to isolate the dermatophytes and culture in saline water for 72 hours then the swab was taken and cultured in a Sabourose dextrose agar plate for 72 hours. Then samples were placed to examine the anti-dermatophyte properties. Membrane type of SPEES with drug showed zone of inhibition (10~13 mm) at the same time electrospin SPEES with drug (15 mm) respectively as shown in Table 1. This claims that the drug from the polymer in both modes is released and the polymer is an excellent barrier and drug carrier.

Table 1: Zone of inhibition of SPEES membrane and electrospun mat

Hemocompatibility Studies
A photometrical analysis was performed to investigate PEES, sulfonated PEES, membranes, and fibrous nano mat forms of SPEES to know the hemorrhaging (hemolysis). The erythrocyte pellet was diluted (1:9) after being washed three times in phosphate buffer solution (PBS). The polymer samples were incubated in 0.8 mL of PBS at 37 °C for 30 min then add 0.2 mL of RBC diluted pellets kept for 2 hours of incubation. Thereafter the samples were centrifugated at 1600 rpm for 10 mins followed by determining the absorbance of the supernatants at 570 nm in the UV spectrophotometer and % hemolysis was calibrated. To understand the degree of toxicity of sulphonated PEES which are made into electrospun mat and membrane. It is crucial since they will encounter blood either directly when used as a bandage or indirectly when injected into the body. Studies on hemolysis were done on erythrocytes that had been exposed to the samples. With the use of Spectrophotometry, the lysis caused by the membrane was examined. PEES and SPEES both showed low hemolytic activity (0.35 and 2.30 %) less than 5 %, although SPEES membrane form and electrospun form showed slightly 7 % hemolysis (which is still below FDI values) as shown in Figure 3. Deionized water and PBS were employed as positive (PC) and negative (NC) controls, respectively. Less erythrocyte lysis and hemoglobin spills can be caused by the membrane and mat, which results in less hemolytic and is highly compatible.

In vitro Biocompatibility Studies
Cell viability tests was performed to investigate the cytocompatibility of PEES, sulfonated PEES, membranes, and fibrous nano mat. About 5 x 104 cells per well count were taken to seed Vero cell line cells and 3T3L1 cell line to culture initially in 96 well plates in the presence of Dulbecco's modified Eagle's medium (DMEM, Sigma), 10% FBS, and incubated at 37 °C for 24 hour as shown in Figure 4A. The IC50 of the SPEES membrane was found to be more than 100 µg/mL (102.55 and 130.86 µg/mL) which is less significant towards toxicity proving that the membrane is excellent biocompatible in nature.

Indirect MTT assay of SPEES polymer in RAW264.7 cell lines (n=3) and Indirect MTT assay on RAW264.7 cell lines for 1, 3, and 7 days on SPEES membrane and electrospun mat with and without drug (Nalidixic sodium salt) is shown in Figure 4A

The indirect and direct MTT assays are carried out to understand the cell cytotoxicity and cell viability for 24 hours as shown in figure 4B. In indirect the scaffold is solubilized then cells are incubated. Whereas the scaffold directly encounters cells (Direct MTT assay). To understand biocompatibility, it is necessary to study for long periods. These studies were carried out on RAW264.7 cell lines. The cytotoxicity activity of the electrospun mat and membrane against normal cell line RAW264.7 (macrophage cells) based on their initial inhibitory results. According to Figure 4B, indirect MTT assay of various concentrations was shown to have excellent inhibitory effects against the target cell line in both membrane and electrospun mat above 100 µg/mL. The standard protocol of testing recommends a scaffold of 10 µg/mL for in vitro studies and 100 µg/mL for in vivo studies should be performed. However, the cumulative growth of cells for 1,3, and 7 days of the RAW 264.7 cell line proved highly active and proliferative in electrospun mat than membrane (200 % and 150% respectively).

The result of direct contact MTT assays showed similar results as indirect MTT assay on RAW264.7 cell lines as shown in Figure 5. High proliferation rate was observed after 3 days, which lead to a conclusion that the cells takes time to adhere initially but thereafter they proliferate after seven days. This property demonstrate that SPEES is biocompatible in nature as shown in Figure 6. The proliferation rate was high in the electrospin mat this might be due to the porous structure and mimic ECM gives the cell a good environment to grow.

Direct MTT assay on SPEES membrane and electrospun mat on RAW264.7 cell lines is very clearly shown in Figure 5.

To validate these results, morphological studies on membrane and mat was performed by observing them in a confocal microscope after incubation for 7 days as shown in Figure 6. The morphological evaluation of normal cells using phase-contrast microscopy, and acridine orange-ethidium bromide staining, displayed progressive proliferative and live features by both scaffolds.

Wound healing studies
To determine the effect of the membrane and electrospun mat of SPEES on cell migration, a wound-healing scratch assay in normal cell lines was performed. The SPEES electrospun (ESS) mat as demonstrated a greater response with a significant wound closure compared to membranes (SS). The wound healing assay demonstrated that the control cells achieved confluency on the “wound” created throughout 72 hours. The angiogenic and migration activities exhibited by cells with the help of a scaffold which mimics extra cellular matrix (ECM) property. The polymer SPEES successfully diminished the crucial steps of wound healing by initializing different cell cascades: angiogenesis, migration and colonization of neoplastic cells in vitro as shown in Figure 7.

In vivo Studies

Healthy adult Balb/c mice of either sex were randomly selected for the assessment of wound healing properties. The study was conducted at the Regional Cancer Centre in Thiruvananthapuram, India, following the guidelines of the Committee for Control and Supervision of Experiments on Animals (CPCSEA) with clearance from the Institutional Animal Ethical Committee (IAEC) (Reg No: RCC/IAEC/02/2023). Four-week-old mice weighing 22–25 grams were used for the experiment. Each mouse was kept in a polypropylene cage on the floor with controlled temperatures ranging from 19 to 25 ˚C, a 12-hour light and 12-hour dark cycle, and free access to a conventional pellet diet. About 18 male BALB/c mice were subjected to an incision wound model for investigation. Mice were anesthetized by injecting intraperitoneally with xylazine (10 mg/kg) and ketamine (80 mg/kg). The dorsal hair was eliminated with Veet sensitive skin hair removal product after administering aesthetic, then the area was cleansed with 70% ethanol. Biopsy punches were utilized to create full-thickness excisions with a diameter of 5 mm. The scaffolds of identical dimensions were washed with saline and immediately positioned on the respective animals' surgical wounds. Bactergras, a commercially available product, was used to treat the wound identified as the positive control. Following the study, animals were housed in cages and given access to commercially available water for drinking. Photos were taken at time points of 0, 3, 7, and 14 days after the injury using a digital camera as shown in Figure 8.

Accordingly, Photographic images of in vivo balb/c group with wound and bar chart representation of in vivo balb/c group with wound from 0th day, 3rd, 7th and 14th days is shown in Figure 8

The mice were euthanised either by cervical dislocation or in a chloroform chamber at regular intervals. The skin from the wound site was excised, fixed in 10% formaldehyde, and stained with hematoxylin and eosin (H&E) for histological analysis.

Advances in developing a biocompatible scaffold using SPEES polymer have shown promising results for conducting experiments in animal models. The effectiveness of nalidixic acid sodium salt was evaluated as a medicine for creating drug-loaded scaffolds in preclinical cutaneous wound healing models. Bactigras (by Ranbaxy) was utilized as the positive control. Prior research has documented the utilization of sulfonated polyether ether ketone (SPEEK) containing active compounds to assess its antibacterial and wound healing characteristics. In this work, wounds were fully covered by both the control and drug-loaded membrane and electrospun mat. Gross pictures were captured on days 0, 3, 7, and 14 as shown in Figure 8. It was noted that all the wounds were healed within 14 days. A superior healing response was seen for membrane and electrospun mat (84% and 83% closure rate on the 14th day) compared to the positive control (81% closure rate) in Figure 8. The presence of the medication has a notable impact on the healing capability at 3 days in the SPEES NA membrane, as shown by the results. Figure 10 displays the percentage of wound healing determined by measuring the wound area on various days. Both membrane and electrospun mats made of SPEES with the medication exhibited more substantial wound closure than the other options. The scaffolds of SPEES are proven to be barrier towards infections as shown in Figure 9. The swabs collected wound surface showed a significant reduction of microbial culture in the SPEES membrane from 3rd day itself when compared to control and Positive control. A significant barrier for microbial infection was noted in electrospun mat ESS.

In vivo microbial swab studies on 3rd and 14th days of SS NA membrane and ESS NA electrospun mat was performed and the results are shown in Figure 9.

Each day, skin samples were gathered for histopathological analyses, the histological findings included analysis of ulceration, granulation quantity, collagen fibre orientation, inflammatory infiltrates, necrosis, epithelialization, and vascularization. Healing was evaluated based on these observed characteristics. The healing score improved from day three to fourteen in all groups of ESS NA with superior outcomes compared to the negative and positive control groups as shown in Table 2. We have developed an excision wound model in a controlled setting where wound infections are restricted. Antibiotic medication is useful for treating infected wounds. Fibrocollagenous stroma and mature granulation with re-epithelization were seen in PC, SS, SS NA, ESS, and ESS NA prominently on 14 days but ESS NA was shown fibrocollagenous and early epithelization on 3rd day itself. Masson trichrome staining was used to analyse wound collagen alignment and vascularisation as shown in Figure 10. The treated models SS and SS NA showed clear fine and coarse collagen deposition and angiogenesis on the 14th day. The staining is a reliable method used to distinguish collagen and is commonly used in wound healing research. Swabs from the wound region was taken and performed a spread plate technique to quantify the number of colonies generated to confirm the antibacterial activity of the scaffold. The drug-loaded membrane exhibited substantial antibacterial efficacy compared to the membrane SS. Fewer colonies were seen on day 14th compared to the first day due to slow drug release, as confirmed by the in vitro studies in membranes compared to nanofibrous mat.
Table no 2: Histopathological score chart of SPEES scaffolds on 3rd and 14th day
Parameters Control PC SS SS NA ESS ESS NA
Time period (days) 3rd 14th 3rd 14th 3rd 14th 3rd 14th 3rd 14th 3rd 14th
Amount of
granulation ++ +++ +++ - +++ ++++ +++ ++++ +++ ++++ +++ +++
Fibrocollagenous
stroma - +++ - +++ - +++ - +++ + +++ ++ +++
Cogestion of blood vessel - ++ - - - - - - - - - -
Inflammatory infiltrates +++ + +++ + +++ + + + +++ + + +
Migration of keratinocytes - - - ++ - ++ - ++ + + + +
Necrosis +++ - - - ++ - - - - - - -
Ulceration +++ + +++ - +++ + + + + - + -
Reepithelialzation - - - +++ - +++ - +++ + +++ + +++
Exudates - - + - - - - - - - ++

Histopathological specimens of groups showing fibro collagenous tissues with granulation tissue and infiltrated inflammation at 10x magnification is shown in Figure 10.

Structural characterization
The degree of sulphonation of SPEES was determined by two methods NMR instrumental and volumetric by IEC (ion exchange method) and NMR spectrophotometric analysis was used to confirm the PEES's sulfonation and quantify the number of sulfonic acid groups substituted onto the polymer chain (degree of sulfonation). The degree of sulfonation was calculated from the peak 7.5 and 7.6 ppm. Characteristic aromatic proton resonance was exhibited at 7-7.75 ppm in all the samples. In which doublet proton at 7 ppm, singlet at 7.15-7.26 ppm and multiplet at 7.8 ppm respectively was exhibited. Singlet at 7.51 ppm indicates the presence of sulfuric acid. The degree of sulfonation can be evaluated through NMR with the help of an equation and is calculated at 72 %.

In the FTIR spectra, Chemical Interactions were obtained, and SPEES showed a peak corresponding to carbonyl stretching observed around 2200-2100 cm-1. The presence of sulphonic acid groups was observed to correspond to asymmetric stretching of S=O frequency at 1216 cm-1. The peak corresponding to O=S=O vibration was observed around 1073 cm-1. The aromatic ring between ether groups has spilt board peaks in between 1650 cm-1. A board band at 3500 cm-1 was observed which corresponds to –OH stretch.
Thermal stability was studied by TGA. The subsequent weight loss was more significant because, as a result of the breakdown of the sulfonate groups that were added to the polymer matrix during the sulphonation process, sulfur trioxide was ejected from the matrix. The sulfonic acid's negative charge, which is very vulnerable to hydrolytic heat destruction, was therefore prevented by polymeric amine counter ions. Yet again, O=S=O causes the polymer's backbone to begin to break down. The decomposition product at 500 °C, though in very minute amounts, can be attributed to the polymer chain disintegrating into 4-phenoxy phenol, and the gradual decline up to 800 °C resulted in the creation of carbon monoxide and carbon dioxide. The thermogravimetric curves of Electrospun SPEES fibres at different initial polymer concentrations in the organic solvent. A change in the thermal behaviour of SPEES fibres was recorded with the initial polymer concentration changed. However, this result cannot be related to the different polymer concentrations but rather to changes that probably occurred in the preparation stage of pure SPEES. The effect of different concentrations of ciprofloxacin (1%, 5%, and 10%) on the thermal properties of SPEES fibres (40 w/v in DMF) is increased thermal stability when compared with SPEES alone. The morphology determination is studied of SPEES along with membrane and electrospun nanofibrous mat. The average thickness of the membrane was found to be 62.5 ± 7.4 microns of SPEES membrane blank as well as drug-loaded membranes. It gets significantly different upon sulphonation, due to which other factors like hydrophilic nature and thermal stability were noted. The membrane morphology of SPEES was the rod-like structures leading to asymmetric membranes, similar observations were seen in the different drug-embedded sulfonated poly (ether ether sulfone) revealing distinctly different morphology due to the sulfonation. The electrospun threads were analysed using SEM to evaluate their structural dimensions using ImageJ software. Electrospun nanofibrous were thread-like structures having a rough texture visible, due to the surface negative charge of the polymer led to aggregation enhancing the mechanical strength and high surface area. The fibrous was in a dimension with 42-81nm range reconfirming the formation of nanofibrous but aggregation leads to the micron level. The range of size and morphology was the same in both the drugs nalidixic acid sodium salt and ciprofloxacin effect of different initial polymer concentration in the organic solvent on the morphology of SPEES fibres. Increasing the initial polymer concentration in the organic solvent (DMF) promotes the formation of electrospun fibers with a more uniform morphology as shown in Figure 11.
Morphological SEM images of SPEES with nalidixic acid sodium salt and Electrospun nanofibers of 35% w/v SPEES with nalidixic acid sodium salt is very clearly shown in Figure 11.

Water-Uptake Capacity

Water-uptake investigations conducted for SPEES yielded findings that were correlated with the level of sulphonation. The water absorption of the sulphonated polymer rises with time. Excessive sulfonation resulted in uncontrolled swelling, causing inadequate mechanical stability. An 80 ±5% water absorption after 2 hours benefits the membrane by facilitating diffusive drug release when pharmaceuticals are incorporated into the membrane matrix. The water absorption values reported provide evidence of the increased hydrophilic nature of the polymer following sulphonation. Several experiments were conducted until the technique was perfected, and the findings were consistently repeatable. The water absorption rate of SPEES is influenced by the quantity of sulphonic acid groups present on the polymer's backbone.
Therefore, the invention relates to a new member of biocompatible materials, from the class of thermoplastic polymer. Polyether ether sulphone was subjected to sulphonation, to improve the hydrophilic nature, and solubility. The resultant sulphonated PEES (SPEES) was made into membrane (SS), membrane with nalidixic acid (SS NA), membrane with ciprofloxacin (SS CF), nano mat (ESS), and nano mat with NA (ESS NA). The drug release experiments proved that release was good in mat when compared to membranes. The release kinetics follows zero order with Fickian diffusion. The scaffold enables the spread of biocompatibility and inertness to cell by in vitro studies. The nanofibrous mat is excellent wound management revealed by in vivo excision wound studies. In connection with membranes, the sustained release was high compared with nanofiberous mat which can be comfortable for chronic wounds which demands a high drug concentration at the initial stage. In case of acute wound, a sustained and prolonged drug release is expected for an effective treatment. In the present invention, the nanofibrous mat loaded with NA is capable of releasing the drug is a sustained fashion for a period of 21 days, which is highly suitable for the acute wound management.

The present invention provides following advantages:
1. Controlled and Sustained Drug Release
• Zero-Order Kinetics: The scaffolds (both membranes and mats) release medications, such as antibiotics, following zero-order kinetics, which ensures a steady and predictable release pattern.
• Avoidance of Burst Release: The system effectively rules out "burst release"—the sudden initial efflux of medication—thereby providing a prolonged and consistent therapeutic effect.
• Diffusion-Driven Mechanism: Drug release is primarily controlled by diffusion and swelling rather than polymer degradation or erosion, ensuring the structural integrity of the dressing during treatment.
• Controlled and steady drug release for 17 days by both forms.

2. Superior Biocompatibility and Safety
• Enhanced Hydrophilicity: The sulfonation process transforms the naturally hydrophobic PEES polymer into a hydrophilic material, which is essential for biomedical applications and effective drug integration.
• Non-Toxic Nature (Biocompatible): In vitro studies on various cell lines (such as RAW 264.7 and Vero) demonstrate high cell viability and low cytotoxicity, with high proliferation rates observed over seven days.
• Hemocompatibility: The material shows excellent compatibility with blood, with hemolysis levels below 7%, which is within acceptable regulatory standards for medical devices.

3. Enhanced Wound Healing Performance
• Faster Closure Rates: In vivo studies on mouse models showed that SPEES scaffolds achieved superior wound closure rates (up to 84% in 14 days) compared to standard commercial controls like Bactigras (81%).
• Effective Microbial Barrier: The polymer acts as a physical barrier against infections, and drug-loaded versions show significant zones of inhibition against common pathogens like E. coli and S. aureus.
• ECM Mimicry: The electrospun nanofibrous mats (ESS) mimic the extracellular matrix (ECM), providing an ideal environment for cell adhesion, migration, and angiogenesis (new blood vessel formation).

4. Versatility and Customization
• Multiple Forms: The polymer can be engineered into two distinct forms based on the clinical need: a thin membrane (SS) for acute wounds or an electrospun nanofibrous mat (ESS) for chronic wounds.
• Structural Stability: The materials possess exceptional strength and longevity, making them non-degradable and well-suited for both wound dressings and sutures.
• Scalability: The sulfonation method used to modify the polymer is easy to scale up for larger-scale production.

Therefore, the present invention offers a significant step forward in wound care by providing a highly effective, safe, and customizable solution for managing complex wounds.
, C , Claims:We claim
1. A method of preparing drug-loaded polymeric wound healing scaffolds, the method comprising the steps of:
• sulfonating polyether ether sulfone (PEES) by dissolving it in a solvent and subjecting it to a sulfonation reaction to produce sulfonated polyether ether sulfone (SPEES);
• drying the resulting SPEES product;
• dissolving the dried SPEES in a solvent selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), and N-methyl-2-pyrrolidone (NMP) to form a polymeric solution;
• incorporating at least one therapeutic agent into the polymeric solution; and
• fabricating the solution into a scaffold selected from a membrane via solvent evaporation or a nanofibrous mat via electrospinning.
2. The method as claimed in claim 1, wherein
o the SPEES is prepared by varying the degree of sulphonation of polyether ether sulfone (PEES) subsequently membranes were made by dissolving in a solvent selected from DMF, DMSO, DMAc, or NMP; and
o the ESPEES nanofibrous mats are fabricated by an electrospinning technique using SPEES dissolved in DMF at polymer concentrations of 30–40% (w/v);
3. The method as claimed in claim 1, wherein said sulfonation is performed at 10 °C for 24 hours to achieve optimal hydrophilicity and solubility.
4. The method as claimed in claim 1, wherein drying is performed for 1 day at 50 °C in a hot air oven or a vacuum oven.
5. A drug delivery system based on method as claimed in claim 1 comprising:
• a polymer of sulfonated polyether ether sulphone (SPEES); and
• drug loaded into the SPEES,
wherein the scaffold is configured for sustained release of said class of drug, following zero-order kinetics.
6. The drug delivery system as claimed in claim 5, wherein the scaffold is a nanofibrous mats and/or membrane.
7. The drug delivery system as claimed in claim 5, wherein said drug -loaded membrane demonstrates a prolonged release of 12.87% in 1 hour, 46% in 24 hours, and 70-80% over 17 days.
8. The drug delivery system as claimed in claim 5, wherein said scaffold is a nanofibrous mat (ESPEES) demonstrates a sustained release of 14.18% in 1 hour, 42% in 24 hours, and 70-75% over 17 days.
9. The drug delivery system as claimed in claim 5, wherein the scaffolds exhibit sustained drug release over a period of 17–21 days following zero-order kinetics.
10. The drug delivery system as claimed in claim 5, wherein the scaffold exhibits a hemolysis rate below 8.9% and demonstrates excellent hemocompatibility.
11. A drug delivery system as claimed in claim 5, wherein the scaffold is a membrane for treating acute wounds and wherein the scaffold is a nanofibrous mat for treating chronic wounds.
12. A drug delivery system as claimed in claim 5, wherein said system demonstrates enhanced effectiveness in addressing aeration, angiogenesis, inflammation, prolonged drug release, and anti-infection properties and minimizes issues related to repeated dressing changes in wounds.
13. A drug delivery system as claimed in claim 5, wherein said drug is selected from wound healing drugs such as nalidixic acid and ciprofloxacin.