Controlled release composition, its method of preparation and application thereof

Abstract

The present invention relates to a controlled release composition comprising at least an active ingredient, at least one polymer and at least one functionalized protein. The present invention also discloses a method of preparation of controlled release composition. The composition effectively provides wound healing properties in a controlled release manner having longer duration of action with improved biocompatibility and mechanical stability.

Description

[0001] CONTROLLED RELEASE COMPOSITION, ITS METHOD OF PREPARATION AND APPLICATION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to biomedical materials and particularly relates to a controlled release composition comprising at least two active ingredients i.e., dual active pharmaceutical ingredient (API), a protein such as functionalized egg albumin and a polymer such as polyvinyl alcohol designed for wound healing applications, providing improved biocompatibility, mechanical stability, and dual drug delivery functionalities.

[0004] BACKGROUND OF THE INVENTION

[0005] Generally, the wound healing process involves four main phases: hemostasis, inflammation, cell proliferation, and extracellular matrix (ECM) remodeling, which could be disrupted by certain conditions such as ischemia, systemic disorders, or corticosteroid use. Ongoing research targets these phases for better outcomes, and there is always a necessity to develop new wound dressing materials that can effectively maintain a moist environment, possess the required mechanical strength, and promote healing by addressing the conditions that disrupt natural healing.

[0006] In general, biomedical materials, and more specifically to film-based scaffolds designed for controlled delivery of multiple bioactive agents, are known for wound care applications. Such bioactive films incorporate proteinaceous and polymeric components to create a conformable dressing capable of adapting to irregular wound topographies. These materials aim to provide a hydrated microenvironment favorable for tissue repair while maintaining mechanical integrity suitable for clinical handling. Developments in film fabrication techniques, including solution casting and thermal crosslinking, facilitate the production of thin, uniform dressings that achieve a balance between flexibility and strength. Researchers have explored combinations of natural and synthetic polymers to impart films with desirable swelling behavior, cytocompatibility, and barrier functions against microbial ingress, all important considerations for effective wound management.

[0007] Effective wound dressings are designed to fulfill multiple roles: maintaining a moist environment, preventing infection, and modulating extracellular matrix formation. Hydrophilic polymer networks such as those based on poly (vinyl alcohol) (PVA) have been utilized for their high water uptake and biocompatibility, while protein-based components contribute cell-adhesive cues and biodegradability. There is a growing interest in incorporating more than one active molecule — such as an antimicrobial agent to limit bioburden and a growth-promoting factor to accelerate matrix deposition — into a single dressing format. By tailoring swelling kinetics and network architecture, dressings can be engineered to release actives in a phased or synchronized manner, thereby addressing multiple healing phases with a streamlined platform.

[0008] Despite the progress achieved with single-polymer and binary-blend films, conventional dressings often exhibit limitations in bioactivity, release control, and mechanical performance. Hydrophilic polymers alone lack antimicrobial or pro-regenerative functions, necessitating the addition of external agents whose release is prone to an initial burst followed by rapid depletion. Protein-polymer blends in bulk film form can suffer from phase separation, inconsistent drug loading, and insufficient tensile strength for handling. Moreover, many reported systems rely on chemical crosslinkers that may leave residual reagents or require extensive washing, complicating manufacturing and regulatory approval. There remains a need for a more refined approach to combine hydrophilicity, mechanical robustness, and multi-agent delivery in a single dressing.

[0009] Accordingly, various drug-loaded wound healing materials (bulk, micro, and nano forms) are being studied and known in the art. In particular, polymeric film dressings highlighted in the literature support multiple phases of wound healing with diverse characteristics (Chandika, P. et al., Int. J. Biol. Macromol. 2015, 77, pages 24-35). These characteristics are not feasible to achieve in a single polymer alone; hence, blends of two or more components are being investigated to complement their properties. Despite the availability of several wound healing materials, the low-cost FDA-approved poly(vinyl alcohol) (PVA) is being researched as a wound dressing material because it is hydrophilic, biocompatible, and can be fabricated in any form. Additionally, it possesses fluid retention and creates a moist environment favorable to nurture the proliferation of soft tissue. Nonetheless, PVA lacks self-bioactivity, so biopolymers with bioactivity are proposed for biological effectiveness. Natural polymers like animal-originated proteins including collagen, gelatin, human serum albumin (HSA), and bovine serum albumin (BSA) blended with, are expensive to produce due to their limited resources and ethical boundaries. Therefore, easily available proteins at low cost are recommended for the development of wound healing materials. For instance, egg albumin (EA), a Generally Recognized as Safe (GRAS) protein sourced from egg white, is a better choice compared to the expensive HSA or BSA due to its similarity in structure and properties, such as biodegradability, biocompatibility, and non-toxicity. In addition, EA possesses antibacterial and anti-inflammatory properties, and its ability to bind to angiogenic growth factors, favors cell growth promotion and forms irreversible crosslinks on annealing between 70° to 80°C.

[0010] The PVA-EA blend has already been reported for wound healing applications in the form of films and nanofibers, where the blends fabricated in bulk form have comparatively less hydrophilicity than the nanofibers due to their large surf ace-to- volume ratio (refer, Nour, S. et al., ACS Biomater. Sci. Eng. 2022, 8 (8), pages 3485-3497; and Wang, W. et al., Colloids Surfaces A. Physicochem. Eng. Asp. 2023, 658, 130658). However, these reports lack functionalized EA which actually may enhance the characteristics of the formulation specifically films.

[0011] In particular, protein-enhanced fdms based on unmodified egg albumin and PVA have demonstrated some antimicrobial effect and biocompatibility but fall short in sustaining dual-agent release and maintaining structural integrity over the healing period. The absence of tailored functional groups in the protein component limits film-water interactions and swelling capacity, reducing the ability to regulate drug diffusion and extend release beyond 24 hours. Concurrently, conventional films rarely integrate both antimicrobial and extracellular matrix-modulating actives at therapeutic levels without compromising film integrity. Addressing these shortcomings calls for a streamlined scaffold design that elevates protein hydrophilicity, enhances swelling, and provides controlled, sustained delivery of multiple actives within a mechanically resilient film.

[0012] Hence, a need still exists a need for a composition for wound healing applications that overcomes the above-mentioned problems of prior art. There is a need to have a composition that integrates both antimicrobial and extracellular matrix-modulating actives at therapeutic levels and a streamlined scaffold design that elevates protein hydrophilicity, enhances swelling, and provides controlled, sustained delivery of multiple actives within a mechanically resilient film.

[0013] OBJECTS OF THE INVENTION Main object of the present invention is to provide a controlled release composition for wound dressing that has antimicrobial and extracellular matrix-modulating actives.

[0014] A further object of the present invention is to provide a controlled release composition for wound dressing that has a streamlined scaffold design that elevates protein hydrophilicity, enhances swelling, and provides controlled, sustained delivery of multiple actives within a mechanically resilient film.

[0015] Another object of the present invention is to provide a mechanically stable and biocompatible composition, preferably in the form of films comprising functionalized egg albumin and poly(vinyl alcohol) (PVA), as a controlled release scaffold for the release of at least a bioactive molecule to enhance the efficient activity i.e. wound healing.

[0016] Yet another object of the present invention is to provide a process of preparation of a controlled release composition for wound dressing

[0017] SUMMARY OF THE INVENTION

[0018] Accordingly, the present invention provides a controlled release composition comprising: at least an active ingredients; at least one polymer; and at least one functionalized protein, wherein the amount of the active ingredient is 0.5 to 50 % w / v of the total weight of the composition, the amount of blend of at least one polymer and at least one functionalized protein is 5 to 30% w / v of the total weight of the composition.

[0019] In an embodiment, the weight range of the polymer and functionalized protein depends on the blend ratio and concentration of total polymer and functionalized protein present in the blend. In an embodiment, the concentration range of polymer and functionalized protein in the blend is 5, 10, 15, 20, 25, 30% w / w of the total weight of the composition.

[0020] In an embodiment, the weight ratio of at least one polymer: at least one functionalized protein in the blend is in the range of 5: 95 to 95: 5 w / w of the total weight of the blend comprising the polymer and the functionalized protein.

[0021] In an embodiment, the controlled release composition comprises of at least two active ingredients. In an embodiment of the present invention, the active ingredients may be selected from the group of antimicrobial agents, plant based phenolic agent, anti-septic agent, extracellular matrix (ECM) growth modulator, flavonoids, flavonols, flavanones, isoflavones, flavanols, flavonolignans, proanthocyanidins, alkaloids, essential oils, tannins, saponins and phenolic compounds, vitamins selected from vitamin A, vitamin C and vitamin E.

[0022] In an embodiment of the present invention, the antimicrobial agent may be selected from the group of metronidazole, nitazoxanide, amoxicillin, clindamycin, fidaxomicin, paromomycin, secnidazole, cefazolin, erythromycin, cefoxitin, cefotetan, trimethoprimsulfamethoxazole, tinidazole, vancomycin, graphene, graphene oxide (GO), curcumin, triclosan, ceftriaxone, chlortetracycline, tetracycline, doxycycline, chloramphenicol, linezolid, quinolones (fluoroquinolones), penicillin, cephalosporins, carbapenems, chlortetracycline, doxycycline, chloramphenicol, linezolid, sulfonamides, trimethoprim, and anthraquinone. The plant based phenolic agent may be selected from ketones, aliphatic alcohols, terpenes, isoflavonoids, aldehydes, and cinnamaldehyde.

[0023] In an embodiment, the polymer may be a synthetic polymer selected from the group of poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(c-caprolactone)(PCL), and polyurethane (PU); or a natural polymer selected from the group of cellulose, carboxymethyl cellulose (CMC), zein, hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), alginate, sodium alginate, chitosan, poly-L- lactide (PLA), guar gum, gum arabic and agar.

[0024] In an embodiment, the functionalized protein may be selected from the group of functionalized collagen, functionalized egg albumin (FEA), functionalized gelatin, functionalized gelatin methacrylate (GelMA), functionalized lactoferrin, functionalized casein, functionalized soy protein, functionalized whey protein, functionalized human serum albumin (HSA), functionalized Bovine serum albumin (BSA), functionalized zein, functionalized keratin, functionalized elastin, functionalized legumin, functionalized vicilin, functionalized lens culinaris agglutinin (LCA), functionalized lectin trypsin inhibitor (LTI), functionalized chenopodin, functionalized quinoa globulin 1 (QG1), functionalized quinoa globulin 2 (QG2) and functionalized quinoa vicilin-like proteins (QVLP). In yet another embodiment, the composition may be in the form of a film, aqueous film, hydrogel, hydro-film, ointment, gel, emollient, liniment, cream, lotion.

[0025] In an embodiment, the weight ratio of the active ingredient: blend of polymer and functionalized egg albumin is the range 0.5 to 50% w / w with respect to the total blend weight. In an embodiment, the weight ratio of the active ingredient: FEA blend may be 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0% w / w with respect to the total weight of the blend.

[0026] In an embodiment, the composition comprises 0.5 to 50.0% w / w metronidazole (Mtz) with respect to total weight of polymer and functionalized protein blend, 0.5 to 50.0% w / w ascorbic acid (AA) with respect to total polymer and functionalized protein blend weightand 5 to 30% w / w of a blend having poly(vinyl alcohol) (PVA) and functionalized egg albumin (FEA) wherein the weight ratio of PVA to FEA is 5: 95 to 95: 5 w / w of the total weight of the blend.

[0027] In yet another embodiment, the composition comprises 10% w / w metronidazole (Mtz), 10% w / w ascorbic acid (AA) and 10% w / v of a blend having functionalized egg albumin (FEA) and poly(vinyl alcohol) (PVA) wherein the weight ratio of FEA to PVA is 30:70 w / w of the total weight of the blend.

[0028] Another embodiment of the invention provides process of preparation of a controlled- release composition, the process comprising the steps of: a) preparing a blend of at least one polymer and at least one functionalized protein in a weight ratio of 5: 95 to 95:5 w / w of the total weight of the blend to obtain a homogenous mixture; or preparing individual solutions of at least one polymer and at least one functionalized protein in a suitable solvent; b) adding 0.5 to 50 % w / v of at least one active ingredient in said homogenous mixture of step a) to obtain a mixture or adding at least one active ingredient in the individual solutions of the polymer and functionalized protein respectively to obtain at least two solutions, and simple mixing of said at least two solutions to obtain a mixture; and c) crosslinking the mixture of step b) to obtain the composition.

[0029] In an embodiment, the process of preparation of the blend of the polymer and functionalized protein is carried out at a temperature of 20 to 35 °C for time period of 45 to 90 minutes followed by stirring the mixture for time period of 8 to 12 h to obtain a homogenous mixture; and the addition of the active ingredient in said homogenous mixture is carried out for 45 to 90 minutes followed by air drying at temperature of 25 to 35 °C to obtain a mixture; and the solution having at least one polymer is prepared at temperature between 70 to 110 °C for time period in the range of 2 to 4 h and the solution having at least one functionalized protein is prepared at temperature range of 20 to 35 °C for time period of 45 to 90 minutes; and adding active ingredient in said individual solutions and mixing the two solutions for time period of 45 to 90 minutes followed by air drying at temperature of 25 to 35 °C; and crosslinking the mixture in a vacuum oven at temperature range of 40 to 80 °C for time period of 2 to 4 h.

[0030] In yet another embodiment, the solution having at least one polymer is prepared by mixing and dissolving at least one polymer in a first solvent, the first solvent is selected from formic acid in water, distilled water or demineralized water, acetonitrile, acetone, methanol, ethanol, propanol, isopropanol; and the solution having at least one functionalized protein is prepared by mixing and dissolving at least one functionalized protein in a second solvent, the second solvent is selected from formic acid in water, distilled water or demineralized water, trifluoroacetic acid, glycerol, 3 -mercaptopropionic acid, dimethyl sulfoxide, triethanolamine, mercaptoethanol, DMF, acetic acid, isoamyl alcohol, chloroform, butyl chloride.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows (A) a reaction scheme for the functionalization of egg albumin with EDTAD, and (B) FTIR plot of EDTAD, egg albumin and functionalized egg albumin with percentage of lysine residue modification (PLRM) of 78%.

[0033] FIG. 2 shows SEM images of (A-C) surface morphology (4000x magnification) & (D-F) cross-section (lOOOx magnification) of the blank film dressings.

[0034] FIG. 3 shows XRD plots of (A) pristine and (B) film of blend of polymers [poly(vinyl alcohol) & functionalized egg albumin (78FEA)] and drugs [metronidazole (Mtz) & ascorbic acid (AA)], with and without drug loading. FIG. 4 shows mechanical properties of blank PVA film and 78FEP30 / 70 blend film under uniaxial tensile loading: (A) elongation at break, (B) tensile strength at break, (C) toughness, and (D) Young’s modulus.

[0035] FIG. 5A shows percentage swelling of diethyl ether treated and UV sterilized blank blend film dressings and FIG. 5B shows percentage swelling for the egg albumin-PVA (30:70 w / w) (EP 30 / 70), and functionalized egg albumin-PVA (30:70 w / w) (78FEP 30 / 70) films.

[0036] FIG. 6 shows cumulative percentage drug release (CPDR) studies: CPDR vs time plot for (A) the release of Mtz from diethyl ether treated and UV sterilized Mtz loaded (78FEP30 / 70_Mtzl0) film dressing, and (B) of Mtz & AA from Mtz & AA loaded (78FEP30 / 70_Mtzl0_AA10) blend film dressing.

[0037] FIG. 7 shows in vitro cell viability of pristine egg albumin (EA) and functionalized EA (78FEA), plain poly(vinyl alcohol) (PVA), egg albumin-PVA (30:70 w / w) (EP 30 / 70), functionalized egg albumin-PVA (30:70 w / w) (78FEP 30 / 70) films, diethyl ether treated and UV sterilized blank (78FEP30 / 70), Mtz loaded (78FEP30 / 70_Mtzl0), and Mtz & AA loaded (78FEP30 / 70_Mtzl0_AA10) blend film dressings in mouse fibroblast cell line L929 with incubation period of 24 h at 37 °C under CO2 incubator.

[0038] FIG. 8 shows anti-bacterial studies of plain 78FEP30 / 70, and metronidazole, Mtz loaded blend film dressings 78FEP30 / 70_Mtz5, & 78FEP30 / 70_Mtzl0 using OD method; where (A & B) represents the anti-bacterial effect of control [F+], 5% w / w Mtz loaded [FM5+], & 10% w / w Mtz loaded [FM10+] on Gram-positive bacteria US'. aurius)', and control [F-], 5% w / w Mtz loaded [FM5-], & 10% w / w Mtz loaded [FM10-] on Gram-negative bacteria (E. coli), respectively.

[0039] FIG. 9 shows In vivo wound healing studies in Wistar rats, (A) representative photographs of the wound area of GI (Control without treatment), GII (Standard treatment metronidazole gel 2% w / v), Gill (Plain film patch treatment, 78FEP30 / 70_DU), GIV (78FEP30 / 70_Mtzl0_DU) and GV (78FEP30 / 70_Mtzl0_AA10_DU) at different days of observations, and (B) plot of wound closure (%) vs time for GI, GII, Gill, GIV, and GV.

[0040] FIG. 10 shows comparative plot of re-epithelialization rate vs time post excision in the treatment groups, GI, GII, Gill, GIV, and GV. FIG. 11 illustrates microscopic representative images of (A) hematoxylin and eosin (H&E) and (B) Masson’s tri chrome stained wound tissues on the 15thday of experimental period under a light microscope from Wistar rats.

[0041] FIG. 12 represents the images of immunohistochemical sections indicating the CD31 expression level in wounds subjected to different treatments as compared to the control group GI.

[0042] FIG. 13 illustrates SDS-PAGE studies of HSA and functionalized human serum albumin (fHSA) confirming the modification of HSA by EDTA.

[0043] FIG. 14 shows that the comparative plot of FTIR spectra of HSA and fHSA confirming the modification of HSA by EDTA.

[0044] FIG. 15 shows FE-SEM images of films of various compositions: (a) blank film of zein- fHSA (75:25 w / w) and (b) 20% vit-E and 0.5% GO loaded film of zein-fHSA (75:25 w / w).

[0045] FIG. 16 shows FT-IR analysis plots of Zein, functionalized human serum albumin (fHSA), vitamin E (Vitamin E), blend film zein: fHSA (75:25 w / w) loaded with 0.5% w / w of graphene oxide (GO) (Z_fHSA), and blend film zein: fHSA (75:25 w / w) loaded with 0.5% w / w of GO and 20% w / w of Vitamin E (Z_fHSA_Vitamin E).

[0046] FIG. 17 is a graph representing the comparative swelling study of zein-HAS (75:25 w / w) and zein-fHSA (75:25 w / w) blend films in phosphate buffer saline (PBS, pH=7.4) over time.

[0047] FIG. 18 illustrates a bar chart representing the comparative hydrolytic degradation study of zein-HSA (75:25 w / w) and zein-fHSA (75:25 w / w) blend films in phosphate buffer saline (PBS, pH=7.4) over 25 days.

[0048] FIG. 19 is a comparative plot of vitamin E release from Zein: HSA (75:25 w / w) + 20% vit- E and Zein: fHSA (75:25 w / w) + 20% vit-E films in the release medium phosphate buffer saline, PBS (pH=7.4) at 37°C.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. As disclosed herein, the term “controlled release” when used to refer to a carrier system arranged to release one or more active ingredients agents including antimicrobials, ECM growth regulators, etc. In some embodiments, the formulation unit is arranged so as to release active ingredients over a period of up to 24 h, 48 h, 1 day, 4 days, 7 days, 15 days, or up to 1 month. Also, the controlled release can be generally defined as ‘control of rate of release’; it can be slow or fast depending on the target, route of administration, API selection, and selection of material make. Hence, the “Controlled release” can also be termed as “regulated release” for the present disclosure.

[0051] In an aspect, the present disclosure relates to a composition for controlled and sustained release of active ingredients, specifically for wound healing and antimicrobial effect.

[0052] In an embodiment, the present disclosure relates to a composition comprising: a. at least an active ingredient, b. at least one polymer, and c. at least one functionalized protein.

[0053] In an embodiment, the composition comprising: at least an active ingredient; at least one polymer; and at least one functionalized protein, wherein the amount of the active ingredient is 0.5 to 50% w / w of the total weight of the composition, the amount of blend of at least one polymer and at least one functionalized protein is 5 to 30% w / w of the total weight of the composition.

[0054] In an embodiment, the composition comprises at least two active ingredients. In an embodiment, the active ingredients may be selected from but not limited to antimicrobial agent, plant based phenolic agent, anti-septic agent, extracellular matrix (ECM) growth modulator, flavonoids, flavonols, flavanones, isoflavones, flavanols, flavonolignans, proanthocyanidins, alkaloids, essential oils, tannins, saponins, phenolic compounds and vitamins selected from vitamin A, vitamin C (ascorbic acid) and vitamin E (tocopherols).

[0055] In an embodiment, the active ingredients may be selected from vitamin A [1stgeneration such as retinol, retinaldehyde (retinal), retinoic acid (tretinoin), isotretinoin, and alitretinoin; (2ndgeneration) such as etretinate and its metabolite acitretin; 3rdgeneration such as bexarotene, tazarotene, and adapalene; 4thgeneration such as trifarotene], vitamin C (ascorbic acid), vitamin E [such as tocopherol, alpha-tocopherol, tocochromanol, D-a- tocopheryl polyethene glycol 1000 succinate (TPGS)].

[0056] In another embodiment, the antimicrobial agent is selected from but not limited to metronidazole, nitazoxanide, amoxicillin, clindamycin, fidaxomicin, paromomycin, secnidazole, cefazolin, erythromycin, cefoxitin, cefotetan, trimethoprim-sulfamethoxazole, tinidazole, vancomycin, graphene, graphene oxide, curcumin, triclosan, ceftriaxone, chlortetracycline, tetracycline, doxycycline, chloramphenicol, linezolid, quinolones (fluoroquinolones), penicillin, cephalosporins, carbapenems, chlortetracycline, doxycycline, chloramphenicol, linezolid, sulfonamides, trimethoprim, and anthraquinone.

[0057] In an embodiment, the plant based phenolic agent is selected from but not limited to ketones, aliphatic alcohols, terpenes, isoflavonoids, aldehydes, and cinnamaldehyde.

[0058] In another embodiment, the antiseptic is selected from but not limited to polyhexanide, poly(hexamethylene biguanide), iodine, and silver nanoparticles.

[0059] In another embodiment, the extracellular matrix (ECM) growth modulator is selected from but not limited to vitamin A [1stgeneration such as retinol, retinaldehyde (retinal), retinoic acid (tretinoin), isotretinoin, and alitretinoin; (2ndgeneration) such as etretinate and its metabolite acitretin; 3rdgeneration such as bexarotene, tazarotene, and adapalene; 4thgeneration such as trifarotene], vitamin C (ascorbic acid), vitamin E [such as tocopherol, alpha-tocopherol, tocochromanol, D-a-tocopheryl polyethene glycol 1000 succinate (TPGS)], and chitosan.

[0060] In an embodiment, the at least one polymer is selected from but not limited to synthetic polymer and natural polymer.

[0061] In an aspect, the synthetic polymer is selected from but not limited to poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(c-caprolactone) (PCL), and polyurethane (PU). In an embodiment, the natural polymer is selected from but not limited to cellulose, carboxymethyl cellulose (CMC), zein, hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), alginate, sodium alginate, chitosan, poly-L-lactide (PLA), guar gum, gum arabic and agar.

[0062] In an embodiment, the at least one functionalized protein is selected from but not limited to functionalized collagen, functionalized egg albumin (FEA), functionalized gelatin, functionalized gelatin methacrylate (GelMA), functionalized lactoferrin, functionalized casein, functionalized soy protein, functionalized whey protein, functionalized human serum albumin (HSA), functionalized Bovine serum albumin (BSA), functionalized zein, functionalized keratin, functionalized elastin, functionalized legumin, functionalized vicilin, functionalized lens culinaris agglutinin (LCA), functionalized lectin trypsin inhibitor (LTI), functionalized chenopodin, functionalized quinoa globulin 1 (QG1), functionalized quinoa globulin 2 (QG2), functionalized quinoa vicilin-like proteins (QVLP), and so on.

[0063] In an embodiment, the at least one polymer is selected from but not limited to synthetic polymer and natural polymer.

[0064] In an embodiment, the at least one polymer in the composition may be poly(vinyl alcohol) (PVA).

[0065] In an embodiment, the at least one polymer in the composition may be zein. In an embodiment, the amount of the active ingredients is in the range of but not limited to 0.5 to 50% w / w of the total weight of the composition. In an embodiment, the amount of the two active ingredients is in the range of but not limited to 0.5 to 50% w / w, 0.5 to 45% w / w, 0.5 to 40% w / w, 0.5 to 35% w / w, 0.5 to 30% w / w, 0.5 to 25% w / w, 0.5 to 20% w / w, 0.5 to 15% w / w or 0.5 to 10% w / w of the total weight of the composition.

[0066] In an embodiment, the amount of the at least two active ingredients is 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% w / v.

[0067] In an embodiment, the amount of at least one polymer and the at least one functionalized protein together is in the range of but not limited to 5 to 30% w / w of the total weight of the composition. In an embodiment, the amount of the at least one polymer and the at least one functionalized protein together is in the range of but not limited to 5 to 25% w / w of the total weight of the composition.

[0068] In an embodiment, the amount or concentration of the at least one polymer and the at least one functionalized protein together is in the range of but not limited to 5 to 30% w / w, 5 to 20% w / w, 5 to 15% w / w or 5 to 10% w / w.

[0069] In an embodiment, the amount or concentration of the at least one polymer and the at least one functionalized protein together is but not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% w / w.

[0070] In an embodiment, the weight ratio of the active ingredient: blend of polymer and functionalized egg albumin is the range 0.5 to 50% w / w with respect to the total blend weight. In an embodiment, the weight ratio of active ingredient: FEA blend is the 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 20.0, 30.0% w / w with respect to the weight of total polymer and functionalized protein blend. In an embodiment, the weight ratio of at least one polymer: at least one functionalized protein in the blend is in the range of 5: 95 to 95: 5 w / w of the total weight of the blend comprising polymer and functionalized protein.

[0071] In an embodiment, the weight ratio of the at least one polymer: the at least one functionalized protein in said composition is in the range of 5: 95 to 95: 5 w / w or 25: 75 to 75: 25 w / w or 50: 50 to 75: 25 w / w of the total weight of the blend comprising polymer and functionalized protein.

[0072] In an embodiment, the weight ratio of the at least one polymer: the at least one functionalized protein in said composition is 50: 50 w / w, 55:45 w / w, 60: 40 w / w, 65: 35 w / w, 70: 30 w / w or 75: 25 w / w of the total weight of the blend comprising polymer and functionalized protein.

[0073] In an embodiment, the % modification in said at least one functionalized protein is in the range of but not limited to 70 to 90%.

[0074] In an embodiment, the % modification in said at least one functionalized protein may be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90%. In an embodiment, the composition or formulation is in the form of a film, aqueous film, hydrogel, or hydro-film.

[0075] In an embodiment, the at least one functionalized protein may be functionalized egg albumin.

[0076] In another embodiment, the at least one functionalized protein may be functionalized human serum albumin.

[0077] In yet another embodiment, the composition comprises metronidazole (Mtz), ascorbic acid (AA) and a blend having functionalized egg albumin (FEA) and poly(vinyl alcohol) (PVA).

[0078] In an embodiment, the composition comprises 10% w / w metronidazole (Mtz), 10% w / w ascorbic acid (AA) and 10% w / w of a blend having functionalized egg albumin (FEA) and poly(vinyl alcohol) (PVA) wherein the weight ratio of FEA to PVA is 30:70 w / w of the total weight of the blend.

[0079] In an embodiment, the composition comprises of two active ingredients which may be graphene oxide and vitamin-E, the polymer may zein and functionalized protein may be functionalized human serum albumin.

[0080] In an embodiment, the present disclosure provides a process of preparation of the composition comprising steps of: a) preparing a blend of at least one polymer and at least one functionalized protein in a weight ratio of 5: 95 to 95:5 w / w of the total weight of the blend to obtain a homogenous mixture; or preparing individual solutions of at least one polymer and at least one functionalized protein in a suitable solvent; b) adding 0.5 to 50 % w / w of at least one active ingredient in said homogenous mixture of step a) to obtain a mixture or adding at least one active ingredient in the individual solutions of the polymer and functionalized protein respectively to obtain at least two solutions, and simple mixing of said at least two solutions to obtain a mixture; and c) crosslinking the mixture of step b) to obtain the composition. In an embodiment, the present disclosure provides a process of preparation of the composition or formulation, comprising steps of: a) preparing a blend of at least one polymer and at least one functionalized protein by mixing a solution of the at least one polymer with a solution of the at least one functionalized protein in a specific weight ratio at temperature in the range of 20 to 35 °C for time period in the range of 45 to 90 minutes followed by stirring the mixture for time period in the range of 8 to 12 h to obtain a homogenous mixture; b) adding at least two active ingredients in said homogenous mixture of step a) by simple mixing for time period in the range of 45 to 90 minutes followed by air drying at temperature in the range of 25 to 35 °C to obtain a mixture; or c) adding at least two active ingredients in said solution of the at least one polymer and / or the solution of the at least one functionalized protein to obtain at least two solutions, and simple mixing said at least two solutions for time period in the range of 45 to 90 minutes followed by air drying at temperature in the range of 25 to 35 °C to obtain a mixture; and d) crosslinking the mixture of step b) or c) in a vacuum oven at temperature in the range of 40 to 80 °C for time period in the range of 2 to 4 h to obtain the composition or formulation for the controlled and sustained release of the active ingredients.

[0081] In an embodiment, the solution of the at least one polymer is prepared by mixing and dissolving the at least one polymer in a first solvent at temperature in the range of 70 to 110 °C for time period in the range of 2 to 4 h.

[0082] In an embodiment, the solution of the at least one functionalized protein is prepared by mixing and dissolving the at least one functionalized protein in a second solvent at temperature in the range of 20 to 35 °C for time period in the range of 45 to 90 minutes.

[0083] In an embodiment, the first solvent may be selected from formic acid in water, distilled water or demineralized water, acetonitrile, acetone, methanol, ethanol, propanol, isopropanol and so on.

[0084] In an embodiment, the second solvent may be selected from formic acid in water, distilled water or demineralized water, trifluoroacetic acid, glycerol, 3 -mercaptopropionic acid, dimethyl sulfoxide, triethanolamine, mercaptoethanol, DMF, acetic acid, isoamyl alcohol, chloroform, butyl chloride and so on.

[0085] In an embodiment, a concentration of the first solvent or second solvent is in the range of 1- 100 N / N%.

[0086] In an embodiment, the process additionally comprises step of removing a first solvent or second solvent from the final composition obtained by treating the composition with diethyl ether to obtain first and / or second solvent free composition.

[0087] In an embodiment, the process of preparation of said composition is in the form of a film, aqueous film, hydrogel, or hydro -film. In an embodiment, the composition may in the form of ointment, gel, emollient, liniment, cream and lotion.

[0088] In an embodiment, the functionalization of protein is done based on chemical group selected from ethylenediaminetetraacetic dianhydride, maleic anhydride, glycolic acid, 1 ,2,4,5- benzene tetracarboxylic dianhydride, 3,3'4,4'-biphenyl tetracarboxylic dianhydride, 3,3 '4, 4 '-benzophenone tetracarboxylic dianhydride, 2,3,6,7-naphthalene tetracarboxylic dianhydride, cyclobutane tetracarboxylic dianhydride, diethylenetriaminepentaacetic dianhydride, citraconic anhydride, succinic anhydride, 3 -hydroxyphthalic anhydride, trimellitic anhydride, methyltetrahydrophthalic anhydride, czs-aconitic anhydride, fatty acid anhydrides, hexahydrophthalic anhydride, phthalic anhydride, and so on.

[0089] In an embodiment, the at least one functionalized protein comprises replacement of at least three carboxylic groups for each amino or aminoacyl group in the peptide chain of the protein.

[0090] In an embodiment, the composition has a patterned with gap and lean coating of the active ingredient(s) onto the surface of the polymer and functionalized protein blend (or film). Also, there is a presence of carved spaces and holes (according to SEM analysis) in said composition.

[0091] Specifically, the active ingredients are embedded inside the polymer and functionalized protein blend (or film) as well as on the surface. In an embodiment, the composition is in the semi-crystalline or amorphous form.

[0092] In an embodiment, the composition possesses high swelling % due to chemistry of the composition (e.g. film) and due to increase in hydrophilic and polar functional groups caused by the functionalization of protein material contained therein. When compared to conventional compositions containing protein without functionalization, the present composition provides around 50-90% increase in swelling which is useful for healing purpose.

[0093] Specifically, the composition or formulation of the present disclosure has better or enhanced swelling ratio around -600 times when compared with the conventional formulation / film containing PVA+EA, due to which the release rate of the drugs / APIs is reduced thereby extending the time of release. The developed formulation or film provides high swellability and thus maintain the moist conditions at the site of wound for optimum healing. Apart from the wound healing, it can also be used as scaffold for bone regeneration and tissue engineering applications.

[0094] In an embodiment, the thickness of the composition is in the range of 160 to 300 pm.

[0095] In an embodiment, the thickness of the composition is in the range of 230 ± 58 pm.

[0096] In a nutshell, the present disclosure provides a dual drug loaded film of functionalized egg albumin (FEA) -poly (vinyl alcohol) (PVA) blend housed with metronidazole (Mtz)- and ascorbic acid (AA).

[0097] EXAMPLES

[0098] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

[0099] Example 1: Preparation of Composition (film) containing two active ingredients (metronidazole and ascorbic acid), polymer (PVA) and functionalized polymer (functionalized egg albumin):

[0100] A) Purification, functional modification, and fabrication of EA films: Procurement details of Chemicals and materials used:

[0101] Egg albumin (EA) was purchased from Otto Chemika-Biochemika-Reagents Pvt. Ltd, Mumbai, India. Poly (vinyl alcohol) (PVA) ((C2H4O)n, Mw~ 125000) LR was obtained from S.D. Fine-Chem Limited, India. Formic acid (98%) was purchased from RANKEM, Chemi Shoppe, India. Ethylenediaminetetraacetic dianhydride (EDTAD), trinitrobenzene sulfonic acid (TNBS), Metronidazole (Mtz), 3-(4,5-dimethyl thiazol-2-yl)-5-diphenyl tetrazolium bromide (MTT), fetal bovine serum (FBS), phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were procured from Sigma-Aldrich, Bangalore, India. L-Ascorbic Acid (Vitamin C) extra pure (99.7%) AR was obtained from Sisco Research Laboratories Pvt. Ltd., SRL, Mumbai, India. Dialysis bag of molecular weight cut-off (MWCO) ~12 kD was obtained from Sigma- Aldrich, Mumbai, India. L929 fibroblast cell line was purchased from National Centre for Cell Science (NCCS), Pune, India.

[0102] In the following examples, solution cast films of FEA and PVA in different ratios (25 / 75, 30 / 70, and 40 / 60 w / w) were prepared. The optimized blend ratio of 30 / 70 w / w was selected for further studies. The films were characterized via. SEM, FTIR, UTM. The optimized blend ratio of 30 / 70 w / w was subsequently chosen for further examination. The surface morphology and physicochemical properties of the films were analyzed to understand the influence of FEA in terms of morphology, hydrophilicity, crystallinity, mechanical and thermal properties. Furthermore, to evaluate the potential of the film as wound healing material in vitro drug release, cell viability, and antimicrobial activity were tested in vivo wound healing, and histopathological studies were done on Wistar rat models.

[0103] Experiments:

[0104] A.l) Purification: The egg albumin (EA) was purified by dissolution, centrifugation and freeze-drying method. Briefly, a 5 % w / v solution of EA was prepared in distilled water (DW) and then centrifuged @ 5000 rpm. The supernatant was collected and freeze dried to attain pure EA.

[0105] A.2) Functional modification of lyophilized EA (FEA): The lysyl residues (-NH2) of egg albumin were modified with carboxylic groups by reacting it with EDTAD (Ethylenediaminetetraacetic Dianhydride) as reported in the literature [Rathna, G. V. N. et al., Polym. hit. 2004, 53 (12), 1994-2000]. The percentage of lysine residue modification (PLRM) is the measurement of the proportion of lysine amino acid residues within a protein that has been chemically altered. Briefly, a 5 % (w / v) egg albumin solution was prepared in distilled water, and the pH was adjusted to 10.5 using IM NaOH. Weighed amount of EDTAD was added incrementally to the above solution while stirring and maintaining the pH = 10.5. After addition and 3-4 h of stirring at the same pH, the solution mixture was neutralized and dialyzed with deionized water using an activated dialysis bag of MWC0- 12 kDa for 48 h and later, the functionally modified egg albumin (EA) solution was lyophilized to obtain dry powder. Following the similar procedure, four different modifications were attained by adding 0.15 g, 0.25 g, 0.35 g and 0.50 g of EDTAD to 1.0 g of EA respectively. The modified lysyl contents of functionally modified egg albumin (FEA) was determined by the trinitrobenzene sulfonic acid (TNBS) method in triplicates according to the procedure of Hall et.al., Analyst 1973, 98 (1170), 673-686. It is found that the PLRM increased linearly (R2= 0.997) with increasing EDTAD to EA ratio.

[0106] FIG. 1(A) describes the functionalization reaction scheme, where reaction with EDTAD, introduced three carboxylic groups for each lysyl residue in the peptide chain. FIG. 1(B) shows the FTIR spectra of EDTAD, EA, and 78FEA samples. EDTAD showed two distinctive characteristic peaks at 1806 cm1and 1762 cm1, which were attributed to the high and low-frequency absorption peaks of -C=O in the acid anhydride groups, respectively. EA spectra showed characteristic peaks at 3364 cm1(-NH stretching; secondary amide); 1665 cm1(-C=O stretching; amide-I), 1538 cm1(-NH bending; amide II), 1233 cm1(-CN stretching; amine), and 1080 cm1(-CO stretching; carbonyl). Similar peaks were observed for 78FEA, however, due to functionalization, peaks at 1655 cm1for amide I and 1544 cm1for amide II were slightly shifted. The shifting occurred due to the merging of the carboxylic acid peak by EDTAD.

[0107] A.3) Fabrication of FEA-PVA blend film dressings with metronidazole (Mtz) and ascorbic acid (AA) loading: The polymeric blend solutions (10 % w / v) were prepared by dissolving FEA (with percentage lysyl residue modification (PLRM) of 72 %, 78 %, 83 %, and 89 %, respectively) and PVA in formic acid (98 %) in the optimized ratio of 30 / 70 w / w (refer, Table 1-3). Briefly, the homogeneous and degassed solutions of PVA and FEA were prepared separately in formic acid. PVA powder was dissolved at 90 °C for 3 h, while FEA was dissolved at 25 °C for 1 h. PVA solution at room temperature was mixed gently with the FEA solutions for about 1 h and later kept on the motor shaker (DLAB, SK-R1807-E) P_W0 100780 for overnight homogenous blending. To fabricate drug-loaded films, FEA with PLRM of 78% was selected and blended with PVA at the optimized ratio of 30 / 70 w / w. For Mtz loaded (PVA-FEA) films, the respective 5 and 10% of Mtz (w / w) with respect to total polymer was initially dissolved in the FEA solution for 1 h and later mixed with PVA solution. Similarly, for Mtz and AA loaded (PVA-FEA) films, 10% of Mtz and AA each

[0108] (w / w) wrt total polymer weight were dissolved separately in the FEA and PVA solution (The AA in PVA solution was always protected from direct exposure to light due to the oxidative nature of AA). The respective solution mixture (5 mL) was poured onto the separate Teflon (PTFE) petri dishes with -6.85 cm of internal diameter and kept for overnight air drying in the chemical fume hood at room temperature. Later, the casted films were thermally crosslinked in a vacuum oven at 60 °C for 3 h. The crosslinked fdms, with thickness of 230 ± 58 pm were transferred in the plastic pouches and stored in a desiccator.

[0109] Table 1. Polymer blend compositions of egg albumin (EA) and poly(vinyl alcohol) (PVA)

[0110] Table 2. Polymer blend compositions of functionalized egg albumin (FEA) of various PLRM and poly (vinyl alcohol) (PVA) at FEA: PVA ratio of 30 / 70 w / w wrt total polymer weight

[0111]

[0112] Table 3. Polymer blend compositions of functionalized egg albumin with PLRM - 78% (78FEA) and poly(vinyl alcohol) (PVA)

[0113] Before going for the actual fabrication of the FEA-PVA blend film as dressings, the blend ratio EA: PVA was optimized as explained below:

[0114] The polymeric blend fdms were prepared in various ratios, as given in Table 1, following the same procedure as described for plain FEP and PVA blend fdms. Similar methodology was followed to prepare the blend films of FEA of different PLRM (0%, 73%, 78%, 83%, and 89%) and PVA at blend ratio of FEA / PVA (30 / 70 w / w) for selection of suitable PLRM for further experiments. Further, a suitable PLRM of FEA was selected for blending with PVA and casted films at a ratio of FEA: PVA of 30: 70 w / w. During experiments, it was observed that at an EDTAD / EA ratio of 0.50, was gelled, which later caused cracks after air drying. For further experiments, EDTAD / EA of 0.25 (PLRM = 78%) was selected, since it produced the most transparent and smooth textured films.

[0115] A.4) Removal of formic acid from the blend films prepared in A.3) and estimation of drug losses: The blend films of circular pieces of diameter - 2.5 cm were treated with diethyl ether (dipped twice in 8 mL of diethyl ether for 60 s per cycle) to remove the residual formic acid present in the films. The leftover diethyl ether solution was used for the estimation of Mtz and AA losses using a UV-visible spectrophotometer by measuring its absorbance at km ax of 320 nm and 266 nm, respectively. The percentage of drug loss from 78FEP30 / 70_Mtzl0 after diethyl ether treatment was -38.00% (w / w wrt initial Mtz weight loaded), while -0.78 % (w / w wrt initial Mtz weight loaded) of Mtz and -6.05% (w / w wrt initial AA weight loaded) of AA was lost from 78FEP30 / 70_Mtzl0_AA10.

[0116] A.5) Characterization of the FEA-PVA film dressing:

[0117] SEM analysis: The surface morphology and cross-sectional view of the blend film dressings were analyzed by environmental scanning electron microscopy (ESEM) (FEI Quanta 200 3D, Netherlands). The samples were mounted on the SEM stub with the help of carbon adhesive tape and sputtered with gold using an SC7620 sputter coating unit (Quorum Technologies Ltd., Judges House, Lewes Road, Laughton, East Sussex, UK). The surface morphology and the microstructural properties of the films affect the drug release pattern, cellular behavior, and function significantly. FIG. 2(A-C) showed the respective SEM images of the surface morphology of the diethyl ether treated and UV sterilized blank (78FEP30 / 70), Mtz loaded (78FEP30 / 70_Mtzl0) & Mtz and AA loaded (78FEP30 / 70_Mtzl0_AA10) film dressings. The surface morphology of the blank film dressing was observed to be rough and slightly smoother as compared to the drug-loaded films. With Mtz loading, a patterned coating of the drug was observed throughout the surface of the film. Further, the introduction of AA into the Mtz-loaded film dressing leads to a similar pattern on the surface with gapping and lean coating. FIG. 2(D-F) showed the cross-sectional SEM images of the respective treated 78FEP30 / 70, 78FEP30 / 70_Mtzl0 & 78FEP30 / 70_Mtzl0_AA10 films. Cross-sectional images showed that the internal structure of the films was not smooth instead there were the presence of carved spaces and holes. The presence of the crystal- shaped structure in the cross-sections of 78FEP30 / 70_Mtzl0 & 78FEP30 / 70_Mtzl0_AA10 indicates that the drugs are embedded inside the films as well as on the surface.

[0118] FTIR analysis: Fourier transform infrared (FT-IR) spectra of the drug-loaded Mtz and AA films were recorded on PerkinElmer spectrometer I, FT-IR attenuated total reflectance (ATR) mode, USA in the wavenumber ranging from 4000 to 500 cm1with a resolution of 4 cm1and an average of 12 scans. The blending of 78FEA with PVA caused a shift in the characteristic peaks of PVA, for instance, the peaks at 3456 (-OH stretching), 2940 (-CH stretching), 1735 & 1453 (-CO and -C=O stretching) and 1096 cm1(-CH bending) in PVA were shifted to 3280, 2920, 1706, 1420 and 1080 cm1in the blend; similarly, for 78FEA, the peaks at 1654 ( -C=O stretching, amide I), and 1516 cm1(-NH bending, amide II) were shifted to 1644, and 1544 cm1. The IR spectrum of 78FEP30 / 70_Mtzl0 contains peaks at 1535 (-C=N stretching), 1425 (-C-C stretching), and 1265 cm-1(-CO stretching) which were shifted from original Mtz peaks positions at 1523, 1479, and 1275 cm1, due to interaction occurred between the 78FEA and Mtz during the fabrication of the film. It confirms the loading of Mtz in the 78FEP30 / 70 blend film dressing. Eikewise, the peaks obtained from the spectrum of 78FEP30 / 70 _Mtzl0_AA10 confirm the presence of both Mtz and AA in the film dressing 78FEP30 / 70. The peaks with a slight shift in positions at 1276 cm1(C-O-C stretching) from 1277 cm1, and 960 cm1(-CH & -OH bending) from 990 cm1came from AA. Therefore, the FTIR analysis confirms the successful loading of Mtz and AA in the blend film dressing 78FEP30 / 70.

[0119] TGA analysis: Thermal degradation profiles of the pristine polymers and the blend film dressing samples were recorded using a simultaneous thermal analyzer (STA) 6000 (Perkin- Elmer, USA) in the temperature range of 50 - 900 °C with a controlled heating rate of 10 °C min1, and the nitrogen flushing at 50 mL min1. Table 4 gives the characteristics comparison of thermal degradation profile of the native constituents, their blends and active ingredients Mtz and AA loaded blend films.

[0120] Table 4. TGA Analysis: stages of degradation, moisture content (%), char (%), onset temperature (°C), and temperature at 10 % degradation for pristine polymers, drugs, and film dressings of their blends with or without drug loading.

[0121] DSC analysis: Other thermal properties of the samples were investigated using a differential scanning calorimeter (Model Q10 DSC, TA Instruments, New Castle, DE, USA). Briefly, 3-5 mg of each sample was weighed in DSC pan with the lid and was sealed by applying pressure and exposed it to two cycles of heating (200 °C at the ramp of 10 °C min1) and one cycle of cooling (-70 °C at a rate of 100°C min1). The respective samples were initially equilibrated to -70 °C for 2 min. DSC thermograms of the film dressings of the plain blends, loaded with metronidazole (Mtz), and loaded with both Mtz and ascorbic acid (AA) after diethyl ether treatment and UV sterilization was studied. Table 5 showed the comparison between native constituents, their blends and active ingredients Mtz and AA loaded blend films for Tg, Tm, and water evaporation temperature. It was observed that, the loading of Mtz and AA in the 78FEP30 / 70_DU, causes loss in their crystallinity, but brings back crystallinity of the PVA in the drug loaded film dressings. Therefore, Mtz and AA become amorphous when loaded in the film dressings of 78FEA and PVA blends. Table 5: Tg, Tm, and water evaporation temperature for pristine polymers, drugs, and film dressings of their blends with or without drug loading.

[0122] *‘NA’ means ‘not applicable’.

[0123] XRD analysis: The crystallinity of the pristine polymers (EA, 78FEA and PVA) and their blend films (78FEP30 / 70, 78FEP30 / 70_Mtzl0, and 78FEP30 / 70_Mtzl0_AA10) was studied using Xray diffractometer (PANalytical X'Pert PRO diffractor) with Cu Ka radiation ( = 1.5418 A) as the X-ray source and the samples were scanned in the 29 range of 5° - 50°. The crystallinity index (CI) was also evaluated for those samples using XRD deconvolution method where a Gaussian curve-fitting model was employed and the crystallinity index was evaluated by the equation (1).

[0124] Sum of Area of the crystalline peaks

[0125] Crystallinity Index (CI)

[0126] Sum of areas of both crystalline peaks and. amorphous peaks 100. (1)

[0127] FIG. 3 showed that the blending of 78FEA into PVA leads to loss of crystallinity of PVA (CI -53%) and later the treatment with diethyl ether and UV sterilization further reduces it to CI -36%. However, with the incorporation of drugs Mtz and AA in the diethyl ether treated and UV sterilized blend film dressing 78FEP30 / 70_DU, the CI enhanced to -47%. The characteristics peaks of Mtz as well as AA were observed in the blend film indicating that their crystallinity in the blend film was still preserved due to intercalation of the drug molecules in the polymer blend matrix. Overall, the crystallinity of drug molecules reduced from their pristine forms and they attained amorphous structure due to the molecular interaction between them and the polymer chains.

[0128] Mechanical analysis: The uniaxial mechanical characterization of films was estimated by the Instron 5943 tensile test machine (Instron Corporation, MA, USA) with a maximum load of 1 kN. Dog-bone samples were carved out following modified ASTM D882 - 18 standard for tensile testing. The tensile testing speed was set to 5 mm min1with a gauge length of 10 mm and the measurement was carried out at room temperature (n = 5). The tensile strength, strength at break, elongation at break, and toughness were calculated based on the generated tensile stress-strain curve. FIG. 4 (A-D) summarizes the mechanical properties of tensile strength, elastic modulus, elongation at break and toughness of PVA and 78FEP30 / 70 blend films respectively. PVA showed a tensile strength at break of ~ 6 MPa which increased slightly to ~7 MPa after blending with 78FEA. Similarly, the elastic modulus was enhanced from ~22 to ~35 MPa. Further, the reduction in the elongation at break, toughness and ultimate tensile strength was observed due to uneven surface roughness which prevents the uniform spread of the stress load through the blend film during the tensile testing. The absence of any chemical cross-linking or covalent bond formation suggests that the surface morphology and the physiochemical properties of the individual components of the blend played crucial role in the mechanical outcomes.

[0129] Swelling Studies in Phosphate Buffer Saline (PBS, pH = 7.4): The respective blend films of various compositions, 78FEP30 / 70, 78FEP30 / 70_Mtzl0, and

[0130] 78FEP30 / 70_Mtzl0_AA10 of dimension 1 x 1 cm2were taken in triplicates. Each film sample was immersed in a plastic vial (10 mL) containing 5 mL of PBS (pH = 7.4) at ambient temperature for 24 h. The initial dry and swollen weights of the films were measured gravimetrically after wiping off any excess water on the surfaces with filter paper. The percentage swelling was calculated using the equation: 100 . (2)

[0131] Where, Ws= Equilibrium swollen weight of the film, and Wd = Dry weight of the film. The swelling ability of a wound dressing material is a peculiar feature that shows its ability to absorb exudates and wound secretions. FIG. 5 shows the swelling behavior of the prepared blend film dressings. All samples showed high swelling capacity and reached equilibrium at 24 h. Such high-swelling % was associated with the chemistry of the film and due to increase in hydrophilic and polar functional groups caused by the functionalization of EA. The blank film 78FEP30 / 70 showed 1618 ± 0.40% swelling and with incorporation of Mtz, it increases to 2143 ± 80.07%.

[0132] Moreover, there was a drastic increase in % swelling observed when both Mtz and AA were loaded in the film dressing, 6081 ± 513.13% of swelling. To summarize, the parameters of the film prepared by the present invention are different and better than conventionally known PVA+EA film, as proved in below table 6. It was observed that the parameters such as elongation at break (%), relative cell viability (%), relative cytotoxicity (%) and swelling ratio (%) were observed to be superior compared to conventional composition without functionalization. Table 6: Comparison of physiochemical parameters between EA+PVA and FEA+PVA compositions

[0133] *The properties of FEA-PVA film are significant over EA-PVA film. The obtained mechanical properties of the developed FEA-PVA fall under the desirable mechanical requirements to be used as a wound dressing material (Kalra et al., J. of Mat. Sci. & Eng. 2016, 5; Wang et. al., J. of Cell, and Mol. Med., 2013 17 (7), 823-32).

[0134] A.6) In vitro drug release. The amount of the drug released at regular intervals was evaluated in duplicates using a temperature -regulated thermal shaker (Julabo SW23). In brief, 2 mg of the respective drug loaded films (78FEP30 / 70 [Control], 78FEP30 / 70_Mtzl0, and 78FEP30 / 70_Mtzl0_AA10) were placed in wide mouth glass tubes containing 10 mL of PBS (pH = 7.4) and were placed in the shaking water bath operated at 37 °C with 50 rpm min1. At regular intervals of time, 1 mL of buffer solution was removed from the sample bottle to estimate the drug released and was replaced with 1 mL of fresh buffer solution to maintain the sink condition. A calibration curve of Mtz and AA recorded at 320 and 266 nm respectively was used to determine the amount of the drug released at those intervals. The obtained results were plotted in terms of cumulative percent release as a function of time. The cumulative percent of drug released (CPDR) was calculated based on the equation: 100 . (3)

[0135] Where, Mtis the amount of drug released at time t and M*, is the amount of drug present in the film dressing samples after preparation and estimation of drug losses on treating those with diethyl ether.

[0136] The release of Mtz and AA with respect to blank film dressing (78FEP30 / 70) was evaluated to check their suitability as a wound healing material. FIG. 4 represents the plots for the release of Mtz from 78FEP30 / 70_Mtzl0 and of both Mtz & AA from 78FEP30 / 70_Mtzl0_AA10. The CPDR for Mtz from 78FEP30 / 70_Mtzl0 at the 24th hour was observed to be -81% following the non-Fickian diffusion (0.45 < n < 0.89), and from 78FEP30 / 70_Mtzl0_AA10 was -41% following Fickian diffusion (n < 0.45) (refer Table 7). Further, AA followed the same trend as in case of Mtz release from 78FEP30 / 70_Mtzl0. The CPDR for AA from 78FEP30 / 70_Mtzl0_AA10 was -77%. This indicates that there was some competitive interaction of Mtz and AA happened with PBS and since AA was dissolved first in PVA before blending to FEA, PVA being more hydrophilic than FEA, allows faster release of AA compared to Mtz. Table 7: Percentage loading and release kinetic mechanism

[0137] *K is kinetic constant; N is the release exponent and R2is coefficient of linear regression

[0138] A.7) In vitro cell viability study. The in vitro cell viability of pristine polymers (EA, 78FEA, PVA), untreated films (EP30 / 70 and 78FEP30 / 70) and diethyl ether treated films (78FEP30 / 70, 78FEP30 / 70_Mtzl0, and 78FEP30 / 70_Mtz_AA) were investigated using standard MTT assay. In brief, the monolayer cell culture (Mouse fibroblast cell line (E929), p = 26) was trypsinized and the cell count was adjusted to 1.0 x 105cells mF1using Dulbecco’s minimal essential media (DMEM) containing 10% fetal bovine serum (FBS). Cell seeding was done by adding 0.1 mL of the diluted cell suspension -10,000 cells per mL per well to the 96-well microtiter plate. After 24 h, when a partial monolayer was formed, the spent media from each well was removed, and the monolayer was washed with sterilized PBS (pH = 7.4). The cells were maintained in DMEM with 10% FBS at 37 °C in a humidified CO2 atmosphere. The film dressing samples were cut into small discs (diameter - 0.5 cm) & were sterilized by UV irradiation and then placed in 1 mL of complete media for 24 h of leaching. Later, the pristine sterilized EA, 78FEA, PVA, and the leached solutions of sterilized film dressing were added into the wells at the concentration of 1 mg mL1. After 24 h of post incubation at 37 °C in a humidified CO2 atmosphere, the supernatants in the wells were pipetted out and the filtered sterilized MTT reagent was added. 10 pL of MTT solution (5 mg mL1in PBS) and 100 pL of culture medium were introduced into the wells. After 4 h of incubation in the dark, the precipitates were solubilized in 100 pL of dimethyl sulfoxide (DMSO). The absorbance intensity was measured using a microplate reader (Multiskan Ex. 51118170 (200-240 V), Thermo Scientific, Finland) at 540 nm. The percent of relative cell viability to the positive control (only cells) was expressed (n = 3) as 100 . (4)

[0139] Cellular biocompatibility is a prerequisite for any biomaterial that can support tissue regeneration and wound healing. Therefore, the biomaterial surfaces must possess desirable cell viability and adhesion to promote tissue regeneration and healing. Based on ISO 10993- 5 guidelines, a non-cytotoxic feature can be attributed to a material with a cell viability percentage above 70%. FIG. 7 represents the comparison of the relative cell viability of EA before and after functionalization. As anticipated, functionalization improves the hydrophilicity of EA, thus increasing cell compatibility and proliferation; EA and functionalized EA (78FEA) showed -76% and -88% relative cell viability respectively, compared to the untreated L929 fibroblast cells. From FIG. 7, it can be observed that both 78FEP30 / 70 and 78FEP30 / 70_Mtzl0 did not cause any cytotoxic effect since the relative cell viability was greater than 70%. The introduction of AA in the blend film improves cell adhesion since, it acts as a cofactor for the synthesis of collagen, an important protein in the formation of extracellular matrix (ECM). It was observed that after 24 h, the relative cell viability was greater than 100% compared to the control. It indicates that 78FEP30 / 70_Mtzl0_AA10 has cell proliferation ability as well. The statistical analysis proved that the addition of AA improved cell viability and proliferation in the 78FEP30 / 70_Mtzl0_AA10 film compared to the control.

[0140] A.8) Antibacterial activity. The antibacterial activity of films of 78FEP30 / 70 loaded with metronidazole, Mtz at concentrations of 0, 5, 10 (% w / w wrt total polymer) was studied against the Gram-positive bacteria Staphylococcus aureus NCIM 5257 and Gram-negative Escherichia coli NCIM 2065. The study was performed following the optical density method (OMD) over the zone of inhibition method (ZOI). In brief, overnight grown cultures of both Gram-positive and Gram-negative were taken, and the cell number was adjusted to 0.5 McFarland (~lxl08cells mL1) using the Muller Hinton broth (HiMedia Laboratories Pvt Ltd. Pune, Maharashtra, India). The films were carved out into circular pieces of diameter ~ 0.5 cm and were placed into each well of microtiter plate and later, the respective culture was inoculated into each well. The microtiter plate was incubated at 37 °C with the orbital shaking rate of 567 cpm. The antimicrobial kinetics for 24 h was observed by measuring the OD at 600 nm using the Synergy™ Hl, a flexible monochromator-based multi-mode microtiter plate reader. FIG. 8 (A - B) showed that the OD was maximum for the control and due to the inherent anti-bacterial property of EA, a decrease in OD was observed for 78FEP30 / 70 (F+ / F-) blend film in both Gram-positive and Gram-negative bacteria respectively. With the introduction of Mtz, in the films of the blends, concentrationdependent anti-bacterial activity was observed for both Gram-positive and Gram-negative bacteria.

[0141] A.9) In vivo wound healing. In vivo wound healing study was performed on male adult Wistar albino rats (150 - 200 g) to investigate the efficacy of developed functionalized EA- PVA films loaded with metronidazole (Mtz) and ascorbic acid (AA). Rats were procured from the National Institute of Biosciences, Pune, India and were subjected to acclimatization for 10 days in an animal house approved by CCSEA (Committee for Control and Supervision of Experiments on Animals). Rats were given a standard diet supplied by Nutrivet Life Sciences, Pune. They were provided ad libitum access to water and food. The study protocol was approved by the Institutional Animal Ethics Committee (Ref. no.: SOPMITWPU / IAEC / 2022-23 / M2 / 09; Dated 24 / 04 / 2023). Rats were anesthetized with an intraperitoneal injection of ketamine (60 mg kg1) followed by shaving of dorsal hairs in the area of the wound incision by keeping a coin (area -2.5 cm2), later the marked area was cleaned with a disinfectant. A full skin thickness circular excision wound of initial average area (-2.5 cm2) was formed on the dorsum of each rat by excising the dorsal skin. The polymer blend films with and without wound healing formulation were applied on excised wounds every alternate day, covered and tied with absorbent gauze for 12 h of the day, and then kept open for the remaining time of the day. This procedure was followed throughout the wound healing studies of 14 days. Images of the wounds were taken at different time intervals (3, 6, 8, 10, 12, and 14 post-incision days) before applying polymer films to the rats for continuous monitoring of the wound healing progression. The wound closure rate was monitored by planimetric measurement of the wound area on the mentioned post- excision days. The wound area was measured by tracing the wound area on transparent plastic sheets. The reduction in the wound area was expressed as a percentage wound closure of the original wound size by the following equation 100 . (5) Where, Ao- wound area on day zero; Ad- wound area on the day of observation.

[0142] The animals were divided into five groups, containing six rats per group: GI(Control without treatment), GII (Standard treatment metronidazole gel 2% w / v), Gill (Plain film treatment, 78FEP30 / 70_DU), GIV (film loaded with 10% (w / w of total polymer) of metronidazole, 78FEP30 / 70_Mtzl0_DU) and GV (film loaded with 10% (w / w of total polymer) of metronidazole, Mtz and ascorbic acid, AA each, 78FEP30 / 70_Mtzl0_AA10_DU) were made with six rats per group; as shown in FIG. 9 (A) at different days of observations viz., 0, 3, 6, 8, 10, 12, and 14thday. FIG. 9(B) represents the comparative plot of percentage wound closure against time of observation.

[0143] Table 8: Effect of formulated functionalized EA-PVA blend films on wound closure in Wistar rat modelsa. It was observed that the control group GI showed a comparatively better wound closure than the other treatment groups on all days of observation. The probable reason was the changing of dressing material at each alternate day for all the treatment groups except the GI. Due to the repeated changing of the dressing material, the medical tape used adhered to the wound exudates. Therefore, in the process of changing the dressing, the tissues near the wound edge were damaged and caused increase in the open wound area, resulting in disrupted wound closure. On the last day of observation, i.e. 14thday, the wound closure percentage for the control group GI was -96%, while GII, Gill, GIV, and GV had -91%, -92%, -89%, and -85% respectively (Table 8). It was expected that GIV and GV should achieve a higher percentage wound closure compared to GI, however, here it was observed that both achieved non-significant comparable percentages of wound closure to the GI.

[0144] Table 9. Effect of formulated functionalized EA-PVA blend films on re-epithelialization rate in Wistar rat modela.

[0145] The re-epithelialization rate and changes in its pattern during the wound healing experiment in all the treatment groups were studied. FIG. 10 showed the comparative plot of re- epithelialization rate vs time post excision in the treatment groups GI, GII, Gill, GIV, and GV. On day 8thof observation, GV showed significantly higher re-epithelialization rate (16.20 ± 5.36 mm day1) compared to the control GI (6.06 ± 1.03 mm day1) (Table 9). On day 10thof observation, maximum re-epithelialization rate of 20.48 ± 5.60 mm day1and 25.91 ± 5.66 mm day1was achieved by Gill and GV respectively with significance level of p < 0.01 and p < 0.0001 which was significantly higher than control GI. However, at later phases of wound healing, the re-epithelialization process decreased and ECM remodeling occurred. The data showed similar behaviour, where the rate of re- epithelialization reduced non- significantly at the 12thand 14thday of observations.

[0146] The above results were obtained by physical observation and digital image processing, and were correlated with the tissue surface (epidermal layer) visualization, and understanding superficial wound healing. Therefore, for more insights of the wound healing at the dermal tissue level (inner skin layers such as dermis, hypodermis, etc.), histological studies were performed.

[0147] A.10) Histopathological studies:

[0148] The rats were sacrificed on the 15th day, and regenerated wound tissue samples were collected for histological examination. The histopathological evaluation was performed using H&E and Masson’s trichrome staining. The stained tissue slides were examined under a light microscope (ten randomly selected fields) for the healing process with the proliferation of granulation tissue composed of fibroblasts and the neovascularization process, collagen deposition, the presence of inflammatory cells, etc.

[0149] FIG. 11 illustrates microscopic representative images of (A) hematoxylin and eosin (H&E) and (B) Masson’s tri chrome stained wound tissues on the 15th day of experimental period under a light microscope (n = 3; multiplier: lOOx; scale bar: 100 pm) from Wistar rats of the (GO) natural skin without wound, (GI) wound-induced control group; (GII) standard metronidazole gel 2% treated group; (Gill, GIV, and GV) plain 78FEP30 / 70_DU, 78FEP30 / 70_Mtzl0_DU, & 78FEP30 / 70_Mtzl0_ AA10_DU blend films treated groups, respectively. Comparative estimation of (C) percentage collagen and (D) coherency plot of collagen orientation from Masson Trichrome staining images of different treatment groups GI, GII, Gill, GIV, and GV at 14th Day post excision.

[0150] FIG. 11(A) represents that in the formulation treated groups, the GV (78FEP30 / 70_Mtzl0_AA10_DU) showed the most wound tissue recovery and the wound morphology was similar to the normal skin tissue with an intact epithelial layer, presence of hair follicles, blood vessels, and a broad collagen layer in the dermis layer. Although the wound closure percent of GV was less significant than the open wound of the control group, GI, the internal healing of the wound i.e. tissue regeneration was better than the GI. The wound tissue of the experimental groups Gill, GIV, and GV showed the presence of fewer inflammatory cells, a greater number of new small blood vessels (black arrow), complete regeneration of epithelial tissues, increased production of hair follicles (green thick arrow), and sebaceous glands (yellow star mark), with denser collagen arrangement (red arrow). The groups, Gill, GIV and GV showed progressive wound healing, where an increase in blood vessel and collagen formation with a smaller number of inflammatory cells was observed with the influence of Mtz and AA (GV). The standard treatment group GII, being treated with only Mtz gel 2%, caused inhibition of bacterial growth but the inflammation was sustained. The Gill showed similar healing ability as compared to the standard treatment because of the inherent antimicrobial property of the functionalized EA.

[0151] FIG. 11(B) described the Masson’s tri chrome staining images for the treatment groups. Similar to the H&E staining, these images described the formation of collagen and its layer deposition in the wound tissue and supported the results obtained in the H&E staining. The formation of collagen fibrils corresponds to accelerated remodeling of the target tissue and organization of repair tissue for faster healing of the wound. The wound tissue of the treatment group GV showed more collagen content with densely packed fiber morphology (green broad arrows) as compared to other treatment groups. [Note: (A) Blue broad arrow indicates re-epithelization of the epidermal layer of skin, the black arrow indicates new blood vessels formed in wound healing granulation tissue, the red color dotted double pointed arrow indicates old punctured blood vessels, the yellow star mark indicates sebaceous gland, the green color thick arrow indicates hair follicles, and the red arrow indicates collagen deposits in healing wound with fibrosis; (B) Bright red arrow indicates re-epithelization of the epidermal layer of skin, orange broad arrow indicates blood vessels in wound healing granulation tissue, and green broad arrow indicates collagen deposits in healing wound with fibrosis.]

[0152] FIG. 11(C) showed the significant collagen % deposition (71.75 ± 2.67%) present in GV as compared to GI (59.94 ± 1.54%) at the end of 14 days post excision. Further, FIG. 11(D) showed that the coherency of local collagen fibers orientation was significantly higher in GV (0.243 ± 0.035) as compared to the control GI (0.107 ± 0.012); which suggested better healing in GV. The possible reason for achieving better healing in GV was the presence of AA as one of the constituents in its composition. AA acts as a cofactor for non-heme iron a-ketoglutarate-dependent dioxygenases, for instance; prolyl 4-hydroxylase and lysyl hydroxylase play a role in the synthesis of collagen. As an electron donor, AA can keep iron in the ferrous state, thereby maintaining the activity of collagen hydroxylase. Hence, promotes the hydroxylation of proline and lysine residues, allowing pro-collagen to correct intracellular folding giving rise to a stable triple-helix conformation. Also, AA being a powerful antioxidant helps in scavenging deleterious reactive oxygen species that are responsible for cell apoptosis during the inflammation stage of wound healing.

[0153] All) Immunohistochemistry staining:

[0154] Using immunohistochemical assay, it was proved that the GV is able to promote angiogenesis, formation of new blood vessels in the wound region during wound healing. Angiogenesis helps in the transport of nutrients and oxygen required for the repair and reconstruction of tissues. It was estimated CD31 (also known as platelet / endothelial cell adhesion molecule- 1, PECAM-1) expression level in the wound region to prove the existence of neo-vascularization. CD31 is a highly expressed biomarker in endothelial cells, which plays crucial role in the maintenance of the vascular integrity and normal functions of vascular endothelial cells.

[0155] FIG. 12 represents the images of immunohistochemical sections indicating the CD31 expression level in wounds subjected to different treatments as compared to the control group GI. Microscopic representative images of immunohistochemical sections of Wistar rats’ skin tissues representing different treatment groups after euthanization on the 15thday after wound creation under a light microscope (n = 3; multiplier: lOOx; scale bar: 25 pm). The positive expression of CD31 in the endothelial cells was represented by red color arrows indicating neo-vascularization.

[0156] The treatment groups were, GO- normal skin without wound (negative control), GI- wound- induced control group; GII- standard metronidazole gel 2% treated group; Gill, GIV, and GV- plain 78FEP30 / 70_DU, 78FEP30 / 70_Mtzl0_DU, & 78FEP30 / 70_Mtzl0_AA10_DU blend films, respectively. The treatment with plain Mtz ointment (GII) showed slightly improved expression of CD31 while with the application of the plain blend film (Gill) enhanced expression was observed. This suggested that neo-vascularization was promoted in Gill. The reason could be the inherent property of EA to capture vascular endothelial growth factor (VEGF) during the initial stages of wound healing due to the presence of ovalbumin as one of EA constituent. Similar results were shown by GIV, indicating that Mtz has no direct role in neo-angiogenesis, moreover with the introduction of AA, GV drastically enhanced the CD31 expression demonstrating that the final film composition 78FEP30 / 70_Mtzl0_ AA10_DU promoted neo-angiogenesis and led to a stable vascular network development. This observation can be supported by the fact that AA in a given environment can modulate VEGF expression by DNA methylation. It may be inferred that AA doesn’t directly influence angiogenesis but it helps in controlling ROS and inflammation at the later phases of wound healing.

[0157] Example 2: Preparation of Composition (film) containing two active ingredients (graphene oxide (GO) and vitamin-E (alpha tocopherol)), polymer (zein) and functionalized protein - (functionalized human serum albumin):

[0158] Procurement details of chemicals and materials used: The zein from com kernels (strain: Suwan 5) was obtained from Sigma Aldrich, Mumbai, India. Other chemical used where sodium bicarbonate was purchased from SD Fine, Mumbai, India. The model drugs used were Vitamin E (Sigma Aldrich, Bangalore, India) and Graphene oxide (Platonic nanotech, Jharkhand, India). The human serum albumin (HAS) protein was procured from the Sigma Aldrich, Bangalore, India. Ethylenediamine tetraacetic dianhydride (EDTAD), trinitrobenzene sulfonic (TNBS) acid, sodium potassium tartrate and dialysis membranes (molecular weight cutoff, 6000-8000 g mol-1) were obtained from the Sigma Aldrich company (St Louis, MO, USA). The organic solvents purchased were formic acid, ethanol of analytical reagent. The organic solvents formic acid, ethanol purchased were of analytical grade.

[0159] Experiment:

[0160] A) Purification of zein, functional modification of HSA, and fabrication of zein-fHSA films:

[0161] A.l) Purification of zein: Zein is a natural, biodegradable, and biocompatible polymer extracted from com. For purification, 10 grams of zein was taken and mixed with 70% ethanol in a ratio of 1:10 (w / v). The mixture was stirred at room temperature (25-30°C) for 12 hours to dissolve the zein. Afterward, the zein-ethanol mixture was centrifuged at 10,000 RPM for 15-20 minutes at 4°C to separate the insoluble material. The supernatant, containing the dissolved zein, was then collected. The pH of the supernatant was adjusted to 5-6 using a mild base, sodium bicarbonate. Next, an equal amount of water was added to the mixture in a separating funnel, and the mixture was vortexed horizontally to precipitate the zein out of the ethanol. The lower pellet was collected and air-dried in a hood. The supernatant was then collected and centrifuged at 10,000 RPM for 20 minutes. The pellet obtained after this centrifugation was air-dried in the hood.

[0162] A.2) Functional modification of HSA: The lysyl residues (-NH2) of human serum albumin were modified with carboxylic groups by reacting it with EDTAD as reported in the literature [refer, Rathna, G. V. N. el al., Polym. Int. 2004, 53 (12), 1994-2000] similar to the functionalization of egg albumin as mentioned in Example 1 (refer Experiment A2). The percentage lysyl residue modified (PLRM) obtained was 87.35%.

[0163] A.2.1) Confirmation of functionalization of HSA: The functionalization of HSA was confirmed using sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FT-IR) spectroscopy.

[0164] A.2.1-1) SDS-PAGE: All the gels used in this study were lab-made with either 12% or 15% resolving gel or 5% stacking gel in 1.0 mm mini gel cassettes (Bio-Rad, USA). To perform SDS-PAGE, gels were first secured in a cassette with IX SDS running buffer filled in both the anode and cathode chambers. Before loading, protein samples were mixed with the loading dye (7 l) and denatured at 90°C for 2 min. Electrophoresis was performed at 150 V, 130 mA for 90 minutes. Gels were then stained with Coomassie Brilliant Blue dye to visualize the protein bands.

[0165] The structural integrity and molecular characteristics of HSA and fHSA were evaluated using SDS-PAGE (12% resolving gel), with 40 pg of protein loaded in each well. The molecular weight marker lane exhibited distinct bands ranging from 10 to 250 kDa, confirming the proper resolution capacity of the gel. Both HSA and fHSA displayed a prominent protein band at approximately 66 kDa, which corresponds to the molecular weight of serum albumin. The HSA lane exhibited a sharp and well-defined band, while the fHSA lane revealed a comparatively broader and more intense band at the same molecular weight region as shown in FIG. 13. This difference in band sharpness and intensity indicates successful functionalization of HSA, which is expected to slightly alter its electrophoretic mobility and produce a heterogeneous population of protein species. Importantly, no additional significant lower molecular weight bands were observed for either HS A or fHS A, suggesting the absence of major protein degradation or fragmentation during the modification process. The preservation of the major albumin band around 66 kDa demonstrates that the functionalization procedure did not disrupt the protein’s primary structural framework. These results collectively confirm that the HSA was effectively functionalized to form fHSA without losing its structural integrity.

[0166] A.2.1-2) FT-IR: Fourier Transform Infrared Spectroscopy (FT-IR) studies were performed to confirm the chemical modification of HSA. FT-IR spectra of both HSA and fHSA were recorded on PerkinElmer spectrometer I, FT-IR attenuated total reflectance (ATR) mode, USA in the wavenumber ranging from 4000 to 400 cm1with a resolution of 4 cm1and an average of eight scans. FIG. 14 showed the comparative plot of FTIR spectra of HSA and fHSA. The FTIR spectra of native HSA exhibited characteristic amide bands, with the broad amide A band at -3307 cm corresponding to N-H stretching vibrations, and aliphatic C- H stretching peaks at 2959 and 2868 cm The amide I band, primarily associated with C=O stretching of the peptide backbone, was observed at 1661 while the amide II band corresponding to N-H bending and C-N stretching appeared at 1535 These bands confirm the preservation of the protein secondary structure.

[0167] In the spectrum of fHSA, the characteristic amide bands were still observed, but with slight shifts in their positions. The amide A peak appeared at -3292 cm suggesting changes in hydrogen bonding interactions and partial substitution of amino groups by EDTAD-derived carboxyl functionalities. Similarly, the C-H stretching vibrations were detected at 2955 and 2873 with a minor shift compared to HSA reflecting alterations in the local hydrophobic environment. The amide I band was shifted to 1656 cm1and appeared more sharper and intense suggesting alterations in the protein backbone conformation. The decreased percentage transmittance intensity can be ascribed to changes in the dipole environment of carbonyl groups, consistent with covalent modification of lysine residues by EDTAD while preserving the overall protein framework. The amide II band was shifted to 1525 supporting modification at amine sites due to the introduction of additional carboxyl groups from EDTAD. This enhancement reflects modifications in the N-H bending and C-N stretching environment, further supporting successful functionalization.

[0168] Overall, the FT-IR spectra comparison confirms that the fundamental secondary structure of HSA remains intact after modification to fHSA, while subtle spectral shifts provide evidence of successful chemical modification and altered microenvironment of specific functional groups.

[0169] A.3) Fabrication of zein-fHSA blend film dressings with graphene oxide (GO) and vitamin-E (vit-E) loading: The polymeric blend solutions (30% w / v) were prepared by dissolving fHSA (functionalized human serum albumin) (PLRM = 87.35%) and purified zein separately in formic acid (98%) in the optimized ratio of zein / fHSA = 75 / 25 w / w. Briefly, the homogeneous and degassed solutions of zein and fHSA were prepared separately in formic acid at 25 °C. fHSA solution at room temperature was mixed gently with the zein solution for about 1 h and later kept on the motor shaker (DLAB, SK-R1807- E) for overnight homogenous blending. To fabricate drug-loaded films, two Eppendorf tubes containing GO (0.5% and 1% w / w wrt total polymer weight) and vit-E (10% and 20% w / w wrt total polymer weight) was added in absolutely ethanol and kept on roller mixture for 24 h to mixed properly. After 24 h suspended GO particles were verted for 5 min and added in complete dissolved solution of fHSA in formic acid and vit-E in zein solution. Again, both the Eppendorf tube kept on roller mixture for 24h to mix properly. At last, vit- E with zein suspension was vertexing it for 5 min so that it was mixed properly and added in fHSA solution with suspended GO particles and kept on roller mixture for 48 h to mixed properly. After 48h the final solution was probe sonicated for 15 min to mix both the immiscible hydrophilic and hydrophobic layer. Later on, solution mixture (5 mL) was immediately poured into Teflon (PTFE) Petri plate with -6.85 cm of internal diameter and kept for drying in the chemical fume hood at RT for 48 h. Later, the casted films were thermally crosslinked in a vacuum oven at 60 °C for 3 h. The crosslinked films, with thickness of 230 ± 58 pm were transferred in the plastic pouches and stored in a desiccator.

[0170] Table 10. Polymer blend compositions of zein and functionalized human serum albumin with PLRM - 87% (fHSA)

[0171] B. Characterization of the zein-fHSA blend film:

[0172] Bl. SEM analysis: The surface morphology and cross-sectional view of the blend film were analyzed using field emission scanning electron microscopy (FE-SEM) (NOVA NANOSEM-450, FEI, USA) at an acceleration voltage of 20 kV. FIG. 15 shows the morphologies of the respective compositions. FIG. 15(a) shows blank film zein-fHSA (75:25 w / w) appeared to be highly porous (pores present in the film) with thickness between 369.7 to 401.5 pm. The 20% vit-E and 0.5% GO loaded fdm of zein-fHSA (75:25 w / w) shows a dense structure with very few pores and the thickness vary from 101.5 um to 118.2 pm as shown in FIG. 15(b).

[0173] B2. FTIR analysis: Fourier transform infrared (FT-IR) spectra of the vit-E and GO loaded zein-fHSA films were recorded on PerkinElmer spectrometer I, FT-IR attenuated total reflectance (ATR) mode, USA in the wavenumber ranging from 4000 to 400 cm1with a resolution of 4 cm1. Each spectrum was composed of an average of eight scans. FT-IR studies were done to understand the functional changes of protein after the fabrication of films.

[0174] The FT-IR spectrum of Z_fHSA_Vitamin E blend film is presented in FIG. 16. The FT-IR spectrum of Z_fHSA_Vitamin E exhibited a broad band at 3288 cm1corresponding to overlapping -OH and -NH stretching vibrations (Amide A 3307 cm ') present in zein and fHSA and -OH (3457 indicating hydrogen bonding interactions among zein, fHSA, and vit-E. The characteristic C-H stretching vibrations (2926 and 2869 cm ') associated with aliphatic chains confirm the presence of vit-E in the blend film. The amide I and II bands, observed at 1638 cm1and 1532 cm1respectively, corresponds to C=O stretching and N-H bending vibrations from the protein components (zein and fHSA), and their retention in the blend film indicates that the protein structure remained intact during film formation. Notably, the appearance of a distinct peak at 1087 cm1in the composite film, absent in both zein and fHSA spectra but prominent in the vit-E spectrum, corresponds to the ether (R-O-R) stretching vibration, confirming the successful incorporation of vit-E. Additional bands at 1262 cm1and 1378-1448 associated with C-0 and C-H deformation modes, further support the presence of vit-E. Overall, the FTIR data confirm the formation of a physically entrapped, hydrogen-bonded blend film integrating zein, fHSA, and vit-E.

[0175] C. Swelling study in PBS (pH=7.4): Swelling study was performed to evaluate how much water the materials absorb and swell over a period, which is critical for drug delivery applications, particularly in topical systems. Briefly, two films were taken, one nonfunctionalized zein-HSA (75:25 w / w) and the other functionalized zein-fHSA (75:25 w / w), both in duplicates with the same weight. The films were weighed and added to empty Eppendorf tubes. Then, 1 mL of phosphate buffer solution (PBS, pH=7.4) was added to each tube. The four Eppendorf tubes were left undisturbed for 24 h. After 24 h, the buffer in each tube was removed, and the weight of all the tubes was recorded. The initial dry and swollen weights of the films were measured gravimetrically. The percentage swelling was calculated using the Equilibrium swelling (%) as mentioned above in Example 1. This process was repeated until consistent readings of the swollen samples were obtained.

[0176] As shown in the FIG. 17, initially both films started at a swelling ratio of zero. Later, zein: fHSA showed a faster and higher initial swelling, reaching ~3.5 at 24th hour while zein: HSA showed a lower swelling ratio ~1.7 at the same point. The zein: fHSA film reached swelling plateau of -4.3 between 24-72 h while zein: HSA showed a much slower swelling, reaching only about -2.3. Further, long-term swelling (72-200 h) resulted into achieving a swelling ratio of -7 by zein: fHSA whereas zein: HSA swelling increases gradually to -4 by 200 h. In conclusion, zein: fHSA film swells significantly more and faster than zein: HSA, indicating higher water uptake capacity. This could be due to functionalization of HSA, enhancing hydrophilicity or network porosity. For topical drug delivery, higher swelling may correlate with better fluid absorption, prolonged drug release, and enhanced skin adherence.

[0177] D. Hydrolytic degradation study: Briefly, two films were taken, one non-functionalized zein-HAS (75:25 w / w) and the other functionalized zein-fHSA (75:25 w / w), both in duplicates with the same weight. The films were weighed and added to empty Eppendorf tubes. Then, 1 mL of phosphate buffer solution (PBS, pH=7.4) was added to each tube. The four Eppendorf tubes were left undisturbed for 25 days. After 25 days, the buffer in each tube was removed, and the films were dried in vacuum oven till constant weight.

[0178] FIG. 18 presents the comparative weight loss percentages for the zein-HSA (75:25 w / w) and zein-fHSA (75:25 w / w) blend films due to hydrolytic degradation in phosphate buffer saline (PBS, pH=7.4). Since, functionalization of HSA improves its hydrophilicity, the zein- fHSA (75:25 w / w) blend film showed higher weight loss of 46% than the zein-HSA (75:25 w / w) blend film showing weight loss of 23%. It suggests that, the film zein-fHSA (75:25 w / w) would completely degrade by 55 days. The average chronic wound usually takes 30 to 50 days to heal, and hence the zein-fHSA (75:25 w / w) is an ideal match for wound healing patch where the rate of degradation will match up with rate of healing.

[0179] E. In vitro drug release: The amount of the drug released at regular intervals was evaluated in duplicates using a temperature-regulated thermal shaker (Julabo SW23). In brief, 2 mg of the respective drug loaded films (Zn / HSA75 / 25, Zn / fHSA 75 / 25 [Controls], Zn / HSA75 / 25_0.5%GO_20%vit-E [ZeimHSA (75:25 w / w) + 20% vit-E], and Zn / fHSA75 / 25_0.5%GO_20%vit-E [ZeimfHSA (75:25 w / w) + 20% vit-E]) in duplicates were placed in wide mouth glass tubes containing 10 mL of PBS (pH = 7.4) each and were placed in the shaking water bath operated at 37 °C with 50 rpm min1. At regular intervals of time, 1 mL of buffer solution was removed from the sample bottle to estimate the drug released and was replaced with 1 mL of fresh buffer solution to maintain the sink condition. A calibration curve of vitamin E recorded at 292 nm was used to determine the amount of the vitamin E released at those intervals. The obtained results were plotted in terms of cumulative percent release as a function of time. The cumulative percent of drug released (CPDR) was calculated based on the equation as mentioned in Example 1.

[0180] The release of vitamin E from Zein: HSA (75:25 w / w) + 20% vit-E and Zein: fHSA (75:25 w / w) + 20% vit-E films with respect to blank films (Zein: HSA (75:25 w / w) and Zein: fHSA (75:25 w / w)) were evaluated to check their suitability as a wound healing material. FIG. 19 represents the plots for the release of vit-E from Zein: HSA (75:25 w / w) + 20% vit-E and Zein: fHSA (75:25 w / w) + 20% vit-E films. The CPDR for vitamin E from Zein: HSA (75:25 w / w) + 20% vit-E and Zein: fHSA (75:25 w / w) + 20% vit-E films at the 24th hour was observed to be -99.75% and -71.30% respectively. The release of vitamin E was comparatively slower in case of functionalized HSA, since vitamin E is hydrophobic in nature and fHSA is more hydrophilic than HSA; and due to hydrophobic -hydrophobic interactions vitamin E diffusion from the blend was restricted.

[0181] Considering all the properties of the developed composition above, the formulated film can be deemed as a suitable wound dressing material.

Claims

We claim:

1. A controlled-release composition comprising: a. at least an active ingredient; b. at least one polymer; and c. at least one functionalized protein, wherein the amount of the active ingredient is 0.5 to 50 % w / w of the total weight of the composition, the amount of the blend of at least one polymer and at least one functionalized protein is 5 to 30% w / w of the total weight of the composition.

2. The composition as claimed in claim 1 , wherein the weight ratio of at least one polymer: at least one functionalized protein in the blend is in the range of 5:95 to 95:5 w / w of the total weight of the blend.

3. The composition as claimed in claim 1, wherein it comprises at least two active ingredients and the active ingredients are selected from the group consisting of antimicrobial agents, plant based phenolic agent, anti-septic agent, extracellular matrix (ECM) growth modulator, flavonoids, flavonols, flavanones, isoflavones, flavanols, flavonolignans, proanthocyanidins, alkaloids, essential oils, tannins, saponins, phenolic compounds and vitamins selected from vitamin A, vitamin C, and vitamin E.

4. The composition as claimed in claim 3, wherein the antimicrobial agent is selected from the group consisting of metronidazole, nitazoxanide, amoxicillin, clindamycin, fidaxomicin, paromomycin, secnidazole, cefazolin, erythromycin, cefoxitin, cefotetan, trimethoprim-sulfamethoxazole, tinidazole, vancomycin, graphene, graphene oxide (GO), curcumin, triclosan, ceftriaxone, chlortetracycline, tetracycline, doxycycline, chloramphenicol, linezolid, quinolones (fluoroquinolones), penicillin, cephalosporins, carbapenems, chlortetracycline, doxycycline, chloramphenicol, linezolid, sulfonamides, trimethoprim, and anthraquinone, and the plant based phenolic agent is selected from the group of ketones, aliphatic alcohols, terpenes, isoflavonoids, aldehydes, and cinnamaldehyde.

5. The composition as claimed in claim 1, wherein the polymer is a synthetic polymer selected from the group consisting of poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(c-caprolactone) (PCL), and polyurethane (PU); or a natural polymer selected from the group consisting of cellulose, zein, carboxymethyl cellulose (CMC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), alginate, sodium alginate, chitosan, poly-L-lactide (PLA), guar gum, gum arabic and agar.

6. The composition as claimed in claim 1, wherein the functionalized protein is selected from the group consisting of functionalized collagen, functionalized egg albumin (FEA), functionalized gelatin, functionalized gelatin methacrylate (GelMA), functionalized lactoferrin, functionalized casein, functionalized soy protein, functionalized whey protein, functionalized human serum albumin (HSA), functionalized Bovine serum albumin (BSA), functionalized zein, functionalized keratin, functionalized elastin, functionalized legumin, functionalized vicilin, functionalized lens culinaris agglutinin (LCA), functionalized lectin trypsin inhibitor (LTI), functionalized chenopodin, functionalized quinoa globulin 1 (QG1), functionalized quinoa globulin 2 (QG2) and functionalized quinoa vicilin-like proteins (QVLP).

7. The composition as claimed in claim 1, wherein the composition is for wound dressing and is in the form of a film, aqueous film, hydrogel, hydro-film, ointment, gel, emollient, liniment, cream and lotion.

8. The composition as claimed in claim 1, wherein the composition comprises 10% w / w metronidazole (Mtz), 10% w / w ascorbic acid (A A) and 10% w / v of the blend having the functionalized egg albumin (FEA) and the poly(vinyl alcohol) (PVA), wherein the ratio of the functionalized egg albumin (FEA) and the poly(vinyl alcohol) (PVA) is 30:70 w / w of the total weight of the blend.

9. A process of preparation of a controlled-release composition, the process comprising the steps of:a) preparing a blend of at least one polymer and at least one functionalized protein in a weight ratio of 5: 95 to 95:5 w / w of the total weight of the blend to obtain a homogenous mixture; or preparing individual solutions of at least one polymer and at least one functionalized protein in a suitable solvent; b) adding 0.5 to 50 % w / w of at least one active ingredient in said homogenous mixture of step a) to obtain a mixture or adding at least one active ingredient in the individual solutions of the polymer and functionalized protein respectively to obtain at least two solutions, and mixing of said at least two solutions to obtain a mixture; and c) crosslinking the mixture of step b) to obtain the composition.

10. The process as claimed in claim 9, wherein the preparation of the blend of the polymer and functionalized protein is carried out at a temperature of 20 to 35 °C for time period of 45 to 90 minutes followed by stirring the mixture for time period of 8 to 12 h to obtain a homogenous mixture; and the addition of the active ingredient in said homogenous mixture is carried out for 45 to 90 minutes followed by air drying at temperature of 25 to 35 °C to obtain a mixture; and the solution having at least one polymer is prepared at temperature of 70 to 110 °C for time period of 2 to 4 h, and the solution having at least one functionalized protein is prepared at temperature of 20 to 35 °C for time period of 45 to 90 minutes; and adding active ingredient in said individual solutions and mixing the two solutions for time period of 45 to 90 minutes followed by air drying at temperature in the range of 25 to 35 °C; and crosslinking the mixture in a vacuum oven at temperature range of 40 to 80 °C for time period of 2 to 4 h.

11. The process as claimed in claims 9 and 10, wherein the solution having at least one polymer is prepared by mixing and dissolving at least one polymer in a first solvent, the first solvent is selected from formic acid in water, distilled water or demineralized water, acetonitrile, acetone, methanol, ethanol, propanol, isopropanol, and the solution having at least one functionalized protein is prepared by mixing and dissolving at least one functionalized protein in a second solvent, the second solvent isselected from formic acid in water, distilled water or demineralized water, trifluoroacetic acid, glycerol, 3 -mercaptopropionic acid, dimethyl sulfoxide, triethanolamine, mercaptoethanol, DMF, acetic acid, isoamyl alcohol, chloroform, butyl chloride.

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