Nanoparticle-charged Hydrogel Dressing for Wound Management

Abstract

Disclosed herein is a hydrogel patch designed to enhance wound healing through the integration of lipid-coated silver nanoparticles (AgNPs) with a novel blend of methi (fenugreek) seed mucilage and okra mucilage. The hydrogel is produced by synthesizing silver nanoparticles and applying a lipid coating to improve biocompatibility and stability. The hydrogel demonstrates significant anti-inflammatory properties and exceptional swelling behavior, ensuring effective water retention and moisture preservation at the wound site. It continuously releases silver lipid nanoparticles (Ag-LNPs) over a period of 24 hours, providing sustained antibacterial protection. These results indicate that the hydrogel patch offers promising potential for improving wound healing through prolonged antibacterial action and optimized moisture management.

Specification

Description:Nanoparticle-charged Hydrogel Dressing for Wound Management

Field of the Invention
The present invention relates to the preparation of composite hydrogel-based dressing. More particularly, the present invention relates to plant-based hydrogel preparation for wound management.

Background of the Invention
Wound healing can be hindered by factors such as chronic infections, poor blood circulation, and underlying conditions like diabetes or immune disorders. Excessive inflammation, inadequate moisture, or improper wound care can also delay healing, leading to complications like scarring, slow tissue regeneration, or chronic wounds. Hydrogels are highly effective in wound and ulcer management due to their high-water content which soothes wounds and supports the natural healing process. Their versatility and biocompatibility make them a popular choice for treating chronic wounds and ulcers.

Chitosan-based hydrogels are obtained from the shells of crustaceans and can be synthesized using fungi making it sustainable. However, synthetic hydrogels, while offering precise control over properties like strength and degradation rate, often lack the biocompatibility and bioactivity found in natural alternatives. They may also pose challenges in biodegradability, leading to potential issues with removal or disposal after use, and sometimes trigger immune responses or irritation in the body.

Plant-based hydrogels have gained attention in wound management due to their biocompatibility, sustainability, and unique ability to retain moisture, which is essential for tissue repair. These hydrogels create a moist environment that promotes cell migration and tissue regeneration, accelerating the healing process. Their high water-content helps in cooling the wound, alleviating pain, and reducing inflammation. Moreover, plant-based hydrogels offer a barrier against infections due to their natural antimicrobial properties.
Additionally, these hydrogels can be infused with bioactive compounds such as antioxidants, antimicrobial agents, or growth factors to further enhance wound healing. Their biodegradability also ensures that they break down naturally without causing harm, reducing the need for frequent dressing changes. Examples of plant-based hydrogels used in wound healing include alginate hydrogels derived from brown seaweed, alginate hydrogels; cellulose-based hydrogels using bacterial cellulose or plant cellulose; pectin hydrogels prepared from fruits and vegetables.

Despite their advantages, the plant-based hydrogels have relatively lower mechanical strength compared to synthetic hydrogels, which can limit their durability, especially in highly exuding or large wounds. Additionally, they may degrade faster than desired in some cases, leading to the need for more frequent dressing changes. Moreover, their production and purification processes can be complex and costly, which might limit widespread use.

Prior arts:
A biocompatible aloe vera-based hydrogels for wound healing are prepared which shows good cell viability, biocompatibility, and antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Okra-based hydrogels for diabetic wound healing are prepared with antioxidant-rich composition and have potential to promote cell migration, angiogenesis, and wound remodeling in diabetic rats. However, the prior arts lack comprehensive in vivo testing, long-term stability data, and comparative analysis with existing products. A composite polysaccharide hydrogel from fenugreek gum and cellulose is designed to enhance wound healing and hemostasis. The hydrogel shows excellent biocompatibility, thermal stability, and sustained release properties, with strong in vitro and in vivo performance. However, challenges include optimizing stability, mechanical strength, scalability, and cost-effectiveness for commercial use. A psyllium seed mucilage-based hydrogels for drug delivery have been developed, modified through grafting and crosslinking. These hydrogels exhibit strong thermal, mechanical, and biomedical properties, with sustained release of a drug. Tamarind seed mucilage-collagen aerogels have been fabricated for tissue engineering, showing enhanced 3D structural topography, thermal stability, and cytocompatibility. These aerogels exhibited reduced degradation, pro-angiogenic potential, and anti-oxidative properties, suggesting they could be a promising alternative to traditional collagen biomaterials for soft tissue repair. A novel green hydrogel (PGCO) reinforced with okra mucilage and cross-linked with citric acid, incorporating nanocurcumin (NC) as a model drug has been prepared. The hydrogel exhibits improved mechanical and thermal properties and demonstrates sustained drug release over 100 hours. Another fenugreek seed gum (FSG)/clay nanocomposite films using the solution casting method, are developed. The films demonstrate enhanced oxygen barrier, thermal properties, and high tensile strength, alongside strong antimicrobial activity against foodborne pathogens [Chelu, et al. Int Jour Mol Sci, 24, 3893, 2023; Xin, et al., Chin Chem Lett, 34, 108125, 2023; Hussain, et al., Int Jour Biol Macromol, 202, 332-344, 2022; Singh, et al., Food Hydrocoll Health, 2, 100059, 2022; Kumari, et al., Mater Today Commun, 34, 105426, 2023; Deka. et al., Int J Biol Macromol, 234, 123618, 2023; Memiş, et al., Int J Biol Macromol, 103, 669-675, 2017].

However, these formulations provide no information about effectiveness on the target condition in vivo and lacks clear application as drug delivery agent. Furthermore, the biodegradability of the prepared hydrogels being an essential has not been established.
US8288347B2U describes purified silk fibroin, methods for its purification, and hydrogels made from it. These hydrogels, with or without amphiphilic peptides, are used in medical applications such as tissue fillers, scaffolds for tissue engineering, disease models, medical device coatings, and drug delivery platforms. CN101293110B describes a medical hydrogel wound dressing made from sodium polyacrylate, polyvinyl alcohol, and water. The dressing is created through casting, radiation cross-linking, and sterilization. It has high swelling capacity, making it ideal for treating burns, scalds, and traumatic wounds. A prior art US20110052695A1 discloses the extraction of refined silk fibroin, the techniques employed for its purification, and the production of hydrogels using this material. Hydrogels find application in various medical fields, including tissue augmentation, tissue engineering scaffolds, disease modeling, medical device coatings, and drug delivery systems. However, there is no report on improvement or better management of wound healing using the above-mentioned formulations. This lack of in vivo effect evidence fails to define safety, relative advantages, and market positions of these formulations.

It is required to develop an efficient hydrogel which not only absorbs exudates but also can help in control bleeding, making it ideal for highly exuding wounds. The hydrogel should be eco-friendly and offer moisture retention, infection prevention, and potential for bioactive enhancement, making them a promising alternative in modern wound healing applications.


Summary of the Invention
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter. Nor is this summary intended to be used to limit the claimed subject matter's scope.

One objective of the present invention is to prepare a hydrogel using two plant sources having bioactive potential.

Another objective of the present invention is to prepare a hydrogel patch for tissue regeneration in shorter duration.

Another objective of the present invention is to prepare a nanoparticle-based hydrogel patch for tissue regeneration to improve wound healing.

According to one embodiment of the present invention, there is provided a composite hydrogel dressing, comprises two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1; a cross-linkinhg agent; and lipid-infused metal nanoparticles, for enhanced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing.

According to one exemplary embodiment of the present invention, there is provided a composite hydrogel dressing, comprises two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1; a cross-linkinhg agent; and lipid-infused metal nanoparticles, for enhanced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing, wherein said hydrogel dressing can be prepared in any form of dressing, preferably a patch form.

According to another exemplary embodiment of the present invention, there is provided a composite hydrogel dressing, comprises two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1; a cross-linkinhg agent; and lipid-infused metal nanoparticles, for enhanced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing, wherein said two or more natural polysaccharrides are obtained from seeds, fruits, roots, stems, or leaves of a plant.

According to one preferred embodiment of the present invention, there is provided a composite hydrogel dressing, comprises two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1; a cross-linkinhg agent; and lipid-infused metal nanoparticles, for enhannced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing, wherein said two or more natural polysaccharrides are obtained from seeds, fruits, roots, stems, or leaves of a plant, wherein said two or more natural polysaccharrides are obtained from fenugreek seeds and okra mucilage.

According to another exemplary embodiment of the present invention, there is provided a composite hydrogel dressing, comprises two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1; a cross-linkinhg agent; and lipid-infused metal nanoparticles, for enhanced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing, wherein said cross-linkinhg agent is a free radical initiator such as ammonium per sulphate, Glyceraldehyde, Citric acid.
According to another exemplary embodiment of the present invention, there is provided a composite hydrogel dressing, comprises two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1; a cross-linkinhg agent; and lipid-infused metal nanoparticles, for enhanced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing, wherein said lipid-infused metal nanoparticles are prepared as gold, sillver, zinc, or copper nanoparticles, preferably silver nanoparticles.

According to another embodiment of the present invention, there is provided a method comprising soaking small pieces of okra and fenugreek seeds, separately, in distilled water in a ratio of 1:10 - 1:20 (w/v) for 24-48 hours at room temperature; heating the soaked okra sample at a high temperature between 50-60°C for 1-2 hours; filtering the heated sample using muslin cloth to extract okra mucilage; mechanically blending the soaked seeds / boiling the soaked seeds; filtering the blended / boiled fenugreek seeds using muslin cloth to extract fenugreek seed mucilage; washing the extracted mucilage from okra and fenugreek seeds using a non-polar solvent to obtain purified mucilage; mixing the obtained okra and fenugreek mucilage in a ratio of 1:4 to 4:1 at room temperature followed by adding optimized concentration of a crosslinking agent; adjusting the pH in a range from 6.5 to 8.0 to obtain a composite;preparing silver nanoparticles (NPs) and applying a layer of lipid to it to obatin lipid-infused silver NPs; and combining the lipid-infused silver NPs with the composite, for preparing a highly moist, stable, biocompatible, and antibacterial hydrogel patch for wound dressing.
Both the foregoing summary and the following detailed description provide examples and are explanatory only. Accordingly, the foregoing summary and the following detailed description should not be considered to be restrictive. Further, features or variations may be provided in addition to those set forth herein. For example, embodiments may be directed to various feature combinations and sub-combinations described in the detailed description.

Brief Description of the Drawings
Figure 1 shows different formulation preparation.
Figure 2 shows the EDS% at different pH.
Figure 3 shows the antibiotic efficacy of Ag LNP hydrogel against (A) Escherichia coli, (B) Bacillus spp., and (c) Staphylococcus aureus.
Figure 4 shows the X-ray diffraction analysis representing crystalline structures of four different samples (a) Combined Hydrogel, (b) fenugreek polysaccharide, (c) Okra polysaccharide, and (d) Ag LNP.
Figure 5 shows the FTIR spectroscopy of prepared Ag incorporated Hydrogel.
Figure 6 shows the FESEM analysis of Silver Lipid Nanoparticles (NPs).
Figure 7 shows the FESEM analysis of Prepared Hydrogel.
Figure 8 shows the EDAX is performed in conjunction with FESEM to determine the elemental composition of the hydrogel and confirm the presence of silver NPs.
Figure 9 shows wound healing in mice at different days.
Figure 10 shows the histopathological analysis which shows growth of fibroblast cells.
Figure 11 shows the hemolysis test (a) Positive Control having distilled water which causes complete hemolysis, resulting in a clear red solution; (b) Negative Control having saline solution shows no hemolysis and maintains intact red cells; and (c) Test Sample showing substance's hemolytic potential as observed and compared to controls.

Detailed Description of the Invention
In the present invention, hydrogel is made by mixing plant mucilage from different sources and combining lipid-coated nanoparticles (NPs) with the mucilage composite of the plant-based mucilage. The process of preparing hydrogel patch entails applying a lipid layer on metal NPs to augment biocompatibility and stability. The NPs are evenly distributed across the hydrogel matrix.

The efficacy of the hydrogel patch is evaluated which reveals a significant reduction in wound healing time from the usual 15 days to just 7 days, accompanied by total hair regrowth.

According to one embodiment of the present invention, mucilage is extracted from a plant source, preferably from Okra and Fenugreek, in different processing conditions as solvent ratio and duration of extraction, followed by mixing the extracted mucilage of okra and fenugreek in different concentrations to prepare different formulations for the nanoparticle-charged hydrogel dressing, particularly a nanoparticle-based patch.

Extraction of Mucilage:
Fresh okra pods are thoroughly washed and cut into small pieces. The pieces are then soaked in distilled water at a ratio of 1:2 (w/v) for 24-48 hours at room temperature. The soaked pieces are macerated with hand and heated at 50-60°C for 1-2 hours, followed by filtering through muslin cloth to extract the mucilage, and the resulting filtrate is purified using a non-polar solvent. Similarly, fenugreek seeds are soaked in distilled water at a ratio of 1:20 (w/v) for 24-48 hours. The soaked seeds are boiled at high temperature for 30-60 minutes followed by blending mechanically and filtering the mixture through muslin cloth. The resulting filtrate is purified using a non-polar solvent.

EXAMPLE 1
Fresh okra pods are thoroughly washed and cut into small pieces. The pieces are then soaked in distilled water at a ratio of 1:2 (w/v) for 24 hours at 27oC. After soaking, the soaked pods are macerated by hand and heated at 50oC for 1 hour followed by filtering the mixture through muslin cloth to extract the mucilage, and the resulting filtrate is added to acetone for purification.

EXAMPLE 2
Fenugreek seeds are soaked in distilled water at a ratio of 1:20 (w/v) for 24 hours. The soaked seeds are boiled at 60oC for 1 hour and mechanically blended, and the mixture is filtered through muslin cloth. The resulting filtrate is added to acetone for purification.

According to one embodiment of the present invention, nanoparticles (NPs) are prepared as one of the ingredients in bioactive hydrogel formulation, wherein said NPs are lipid- or water-based metal NPs, wherein said metal is selected from copper, gold, silver, or zinc.

Preparation of lipid-based silver nanoparticles:
0.5-2.0 mM silver salt solution is prepared and reduced using a suitable reducing agent in the presence of a lipid stabilizer to form lipid-encapsulated silver NPs.

EXAMPLE 3
A 1 mM solution of silver nitrate (AgNO₃) is prepared in distilled water. Palmitic acid is dissolved in ethanol to form a lipid solution. The silver nitrate solution is then added to the lipid solution under constant stirring. Sodium borohydride (NaBH₄) is added dropwise to the mixture to reduce the silver ions into silver nanoparticles. The mixture is then ultrasonicated to ensure uniform dispersion of the nanoparticles.

EXAMPLE 4
Nyctanthes arbor-tristis is used to green synthesize silver nanoparticles (AgNPs) by mixing 1% aqueous leaf extract with 1 mM AgNO3 solution. The reaction mixture is exposed to light for 10 minutes at room temperature, leading to the formation of AgNPs. The synthesis is confirmed by a UV-Vis spectrum showing a peak at 460 nm, indicating surface plasmon resonance. The synthesized NPs are centrifuged and washed 3-4 times with ethanol. Then dried in hot air oven at 60oC then the lipid and AgNP are dissolved in ethanol and then this mixture is added dropwise in hot surfactant (Tween 80) solution under constant stirring at 1000 rpm and left under stirring at temperature 70oC until the solvent evaporated. Then the mixture obtained is probe sonicated in ice bath for 15 minutes.

Preparation of hydrogel mix:
Table 1 shows various combinations of the ingredients of the hydrogel including extracted mucilage from okra and fenugreek, and a free radical based cross-linking agents such as ammonium per sulphate (APS), Glyceraldehyde, or Citric acid.

EXAMPLE 5
The hydrogel includes mixing of extracted mucilage from okra and fenugreek in a ratio from 1:4 to 4:1 [Table 1], and addition of a cross-linking agent to form 5 different formulations of the hydrogel matrix.

Formulation Okra Fenugreek APS Incubation time
G1 250mg 250 mg 100mg 18hrs
G2 150mg 350mg 100mg 18hrs
G3 100mg 400mg 100mg 18hrs
G4 350mg 150mg 100mg 18hrs
G5 400 mg 100mg 100mg 18hrs


According to one embodiment of the present invention, a hydrogel patch, (i.e., hydrogel encapsulated silver lipid nanoparticle) is prepared for improved wound healing, wherein okra and fenugreek mucilage are combined in a ratio ranging from 1:4 to 4:1 followed by incorporation of 5 mg/ml lipid-based silver nanoparticles into the mucilage mixture; addition of a cross-linking agent to form said hydrogel; and the pH is adjusted to 6.5-8.0.

Encapsulation and Hydrogel Patch Formation:
The hydrogel is mixed with the lipid-infused silver NPs solution under continuous stirring. Ammonium per sulphate is added to the mixture to form a stable hydrogel network to obtain a patch form. The hydrogel is then allowed to set at 60°C until gelation occurs [ Figure 1].

EXAMPLE 6
A hydrogel is prepared followed by preparation of a hydrogel-based patch for wound healing, wherein okra and fenugreek mucilage are combined in a ratio of 1:4 followed by incorporation of 5 mg/ml lipid-based silver NPs into the mucilage mixture under constant stirring; addition of ammonium persulfate as a crosslinking agent to form said hydrogel; and the pH is adjusted to 7. The mixture is left to stand at 60°C until gelation occurs.

EXAMPLE 7
A hydrogel is prepared followed by preparation of a hydrogel-based patch for wound healing, wherein okra and fenugreek mucilage are combined in a ratio of 7:3 followed by incorporation of 5 mg/ml lipid-based silver NPs into the mucilage mixture under constant stirring; addition of ammonium persulfate as a crosslinking agent to form said hydrogel; and the pH is adjusted to 7. The mixture is left to stand at 60°C until gelation occurs.

EXAMPLE 8
A hydrogel is prepared followed by preparation of a hydrogel-based patch for wound healing, wherein okra and fenugreek mucilage are combined in a ratio of 1:1 followed by incorporation of 5 mg/ml lipid-based silver NPs into the mucilage mixture under constant stirring; addition of ammonium persulfate as a crosslinking agent to form said hydrogel; and the pH is adjusted to 7. The mixture is left to stand at 60°C until gelation occurs.
1:1 ratio (250 mg okra and 250 mg fenugreek mucilage)

According to one embodiment of the present invention, said hydrogel formulation is characterized based on various parameters including swelling study; structure analysis through Optical Contact Angle (OCA) measurements to assess hydrophilicity; Energy Dispersive X-ray Spectroscopy (EDS) to determine elemental composition; X-Ray Diffraction (XRD) to analyze crystalline structure; Fourier-transform infrared spectroscopy (FTIR) spectrum to identify chemical composition; and Field Emission Scanning Electron Microscopy (FESEM) analysis for surface morphology, followed by in vitro antibacterial and in vivo wound healing properties determination.
For swelling analysis, the prepared hydrogel samples are dried and weighed (W₀). The samples are then immersed in distilled water at room temperature. At specific time intervals from 10 minutes to 60 minutes the samples are removed, blotted to remove excess water, and weighed (Wₜ). The swelling ratio is calculated using following formula:
Swelling %= (Wt-Wo)/Wt* 100,
where Wt = final weight of Hydrogel; and Wo= initial weight of Hydrogel
Swelling capacity of the prepared patch is checked in terms of water absorption capacity at different pH of different formulations G1-G5. Group G1 shows significant swelling at pH 8.4 but less consistent behavior at pH 4 and 7; Group G2 shows peaks sharply at pH 4 and 7 but declines, showing less stability; Group G3 demonstrates stable and moderate swelling across all pH levels as, at pH 4- stable around 150% EDS, at pH 7- peaks around 100% EDS, then stabilizes, and at pH 8.4- stable around 1000% EDS; Group G4 shows moderate swelling but less consistency; and Group G5 shows minimal swelling, indicating low responsiveness [Figure 2]. Based on the EDS % graphs at pH levels 4, 7, and 8.4, the best polymer for biomedical applications is likely Group G3. This group shows consistent and moderate swelling across all pH levels, which is crucial for biomedical applications that require stability in varying environments, such as different parts of the human body. Group G3's consistent and moderate swelling behavior made it a reliable choice for applications like drug delivery systems or tissue engineering, where stability in varying pH environments is essential.
The efficacy of the silver NPs and NPs-based hydrogel is assessed through antibacterial assays. An agar well diffusion assay is employed to evaluate antibacterial activity. Bacterial strains are cultured in nutrient broth at 37°C for 24 hours. Wells are created in agar plates inoculated with bacterial suspension. Test compounds are introduced into the wells, and the plates are incubated at 37°C for 24 hours. Zones of inhibition are measured to determine antibacterial efficacy [Figure 3].
The hydrogel exhibits significant antibacterial activity against three bacterial strains: Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, as evidenced by the inhibition zones observed for each bacterial strain. The zones of inhibition are measured for E. coli, S. aureus, and B. subtilis as 3, 3, and 4 cm, respectively. These results indicate that the hydrogel is effective in inhibiting the growth of both Gram-positive and Gram-negative bacteria, with the highest efficacy against B. subtilis.
For XRD analysis, the hydrogel samples are dried and ground into a fine powder. XRD analysis is performed using an X-ray diffractometer to determine the crystalline structure of the hydrogel and the presence of silver nanoparticles. The combined hydrogel (a) sample shows sharp peaks at around 16.59°, 21.28°, and 31.24° in 2-theta, indicating a well-ordered crystalline structure within the hydrogel composite; the fenugreek polysaccharide (b) shows the peaks at approximately 16.99° and 21.49° suggest some degree of crystalline order within this polysaccharide sample; okra Polysaccharide (c) sample displays broader peaks around 16.99° and a broad hump near 22-24° in 2-theta, indicating a more amorphous structure compared to the other samples; and Ag-LNP (d) shows a broad peak cantered around approximately 41.22° in 2-theta suggests that these silver NPs have very small crystal domains or are largely amorphous due to liquid Ag LNP [Figure 4].
To confirm the presence of functional groups in the prepared hydrogel and the successful incorporation of mucilage and silver NPs in it, FTIR analysis is performed [Figure 5]. The FTIR spectrum of the hydrogel containing Ag-LNP exhibits multiple significant functional groups. The presence of hydroxyl or amine groups can be inferred from a prominent peak at 3325.85 cm⁻¹, which suggests O-H or N-H stretching vibrations. The peak observed at 2952.22 cm⁻¹ corresponds to the stretching vibrations of C-H bonds, which are characteristic of aliphatic hydrocarbons. The peak observed at 1653.00 cm⁻¹ is caused by the stretching vibrations of C=O bonds in carbonyl groups. On the other hand, the peak at 1541.41 cm⁻¹ is related to the bending vibrations of N-H bonds, which are characteristic of amide groups. The signal observed at 1026.89 cm⁻¹ corresponds to the stretching vibrations of the C-O bond, indicating the presence of alcohols, ethers, or esters. The presence of a peak at 476.00 cm⁻¹ suggests that there are Ag-O stretching vibrations, which confirms that there is a connection between the silver nanoparticles and the hydrogel matrix. These findings emphasize the intricate molecular interactions occurring within the hydrogel.
The surface morphology of the prepared silver lipid nano particle is examined using FESEM [Figure 6]. The samples are coated with a thin layer of gold before imaging. The FESEM micrograph of the prepared hydrogel reveals a highly porous structure with uniformly distributed pores of varying sizes [Figure 7]. The interconnected pores are clearly visible, indicating a well-developed porous network. This architecture is crucial for applications requiring high surface area and fluid absorption, such as drug delivery systems and tissue engineering scaffolds. The porous structure facilitates efficient nutrient and oxygen diffusion, which is essential for cell growth and proliferation. Additionally, the presence of these pores enhances the mechanical flexibility and strength of the hydrogel, making it suitable for various biomedical applications. The FESEM analysis confirms the successful synthesis of a hydrogel with desirable structural characteristics, highlighting its potential for advanced biomedical use.
EDAX analysis of silver lipid NP incorporated hydrogel is done. The EDAX spectrum shows peaks corresponding to carbon (C), oxygen (O), sulfur (S), and silver (Ag). The prominent silver peak confirms the successful incorporation of silver lipid NPs into the hydrogel. The presence of carbon and oxygen indicates the organic components of the hydrogel, while sulfur may be from the lipid component [Figure 8].

According to one of the above embodiments of the present invention, the synergistic healing properties of fenugreek and okra mucilage, combined with silver NPs, results in accelerated and enhanced wound healing, wherein the hydrogel demonstrates significant efficacy, achieving complete recovery within 7 days in a mouse model, marking a notable improvement over existing treatments; encouraging hair regrowth in the treated area, highlighting its enhanced regenerative properties, wherein the lipid coating on the silver nanoparticles ensures controlled and sustained release, which reduces cytotoxicity and enhances therapeutic outcomes.

Furthermore, the silver nanoparticles provide significant antibacterial and anti-inflammatory effects, crucial for effective wound healing and infection prevention. This invention represents a sustainable and effective approach to wound management by utilizing natural and biocompatible materials. The novel composite material has the potential to transform current wound care practices, offering a new and effective solution for wound healing.

According to one embodiment of the present invention, the combination of silver NPs with fenugreek and okra mucilage offers several advantages for wound healing, wherein silver NPs are renowned for their strong antimicrobial properties, providing a broad-spectrum effect that significantly reduces the risk of infection compared to many commercial creams; the synergy of lipid coatings with fenugreek and okra mucilage enhances the wound healing process by allowing sustained release of the nanoparticles while creating a moist environment that promotes faster tissue regeneration and minimizes scarring, achieving complete healing in just 7 days-much quicker than typical commercial wound healing products.

Additionally, biogenic synthesis of silver NPs using natural extracts results in lower cytotoxicity, making them safer for prolonged use on wounds. The natural anti-inflammatory properties of fenugreek and okra mucilage help alleviate inflammation and pain at the wound site, further enhancing patient comfort. Furthermore, utilizing these natural extracts for nanoparticle synthesis and hydrogel formation is more environmentally friendly and sustainable than the synthetic chemicals commonly found in many commercial wound healing products.
According to one embodiment of the present invention, healthy Wistar mice are used for in vivo evaluation of properties, wherein the study is divided into control and experimental groups, wherein a uniform wound is introduced on the dorsal side of each mouse and the experimental group receives hydrogel administration at the wound site, while the control group undergoes routine treatment or no treatment; the wound healing process is monitored over a specified period of 14 days at intervals and recording of parameters such as wound size reduction, healing time, and any signs of infection or inflammation is done [Figure 9].
During observations, the measurements are taken for wound closure, re-epithelialization, and tissue regeneration [Figure 10].
According to the above embodiment of the present invention, at the end of the experiment, the mice are euthanized, and tissue samples are collected from the wound site followed by histopathological analysis to evaluate tissue regeneration, quality of healing, any adverse effects, and the biocompatibility of the hydrogel, as shown in Figure 11.
According to one embodiment of the present invention, fresh human blood is collected and centrifuged to obtain red blood cells (RBCs); the RBCs are exposed to the hydrogel, and hemolysis is measured by the release of hemoglobin, wherein percentage of hemolysis is calculated to determine the biocompatibility of the hydrogel.

The hemolysis study involves three groups: a positive control (PC), a negative control (NC), and a test sample. The PC, treated with Triton X-100, exhibits 100% hemolysis, confirming the assay's effectiveness. The NC, treated with phosphate-buffered saline (PBS), shows negligible hemolysis, validating the baseline. The test sample demonstrates 10% hemolysis, indicating a moderate level of RBCs disruption. These results suggest that the test sample has relatively low hemolytic activity, making it potentially safe for biomedical applications. Statistical analysis confirms significant differences between the groups (p < 0.05).
% Hemolysis = (OD(Test)-OD(Negetive control))/(OD(Positive control)-OD(Negetive Control))
According to one exemplary embodiment of the present invention, wherein said hydrogel has potential of securing its applications in Military and Defense, Pharmaceutical companies, Hospitals and clinics.
The qualities of said hydrogel in terms of its anti-inflammatory and swelling effects are examined. The findings demonstrate that the hydrogel displays notable anti-inflammatory characteristics and exceptional swelling behavior, suggesting effective water retention and moisture preservation at the location of the wound. The hydrogel exhibits a continuous discharge of LNPs (lipid-infused NPs) for a duration of 24 hours, guaranteeing an extended period of antibacterial effectiveness at the wound location.
According to one embodiment of the present invention, the developed hydrogel patch combines fenugreek seeds mucilage and okra mucilage with silver NPs offering enhanced wound healing compared to conventional treatments like Soframycin, while traditional dressings or artificial hydrogels often lack biocompatibility and extended antibacterial effects.
This instant hydrogel promotes faster healing by improving moisture retention and reducing inflammation additionally its natural mucilage components provide prolonged antibacterial action and contribute to better overall healing outcomes making the hydrogel a promising alternative for both medical and veterinary applications
While the invention is amenable to various modifications and alternative forms, some embodiments have been illustrated by way of example in the drawings and are described in detail above. The intention, however, is not to limit the invention by those examples and the invention is intended to cover all modifications, equivalents, and alternatives to the embodiments described in this specification.
The embodiments in the specification are described in a progressive manner and the focus of description in each embodiment is the difference from other embodiments. For same or similar parts of each embodiment, reference may be made to each other.
It will be appreciated by those skilled in the art that the above description is in respect of preferred embodiments and that various alterations and modifications are possible within the broad scope of the appended claims without departing from the spirit of the invention with the necessary modifications. , Claims:We Claim:
1. A composite hydrogel dressing comprises:
two or more natural polysaccharides mixed in a ratio of 1:4 to 4:1;
a cross-linkinhg agent; and
lipid-infused metal nanoparticles,
for enhanced biocompatibility, moisture content, stability, and bioactivity of hydrogel dressing to improve wound healing.

2. The composite hydrogel dressing as claimed in claim 1, wherein said hydrogel dressing can be prepared in any form of dressing, preferably a patch form.

3. The composite hydrogel dressing as claimed in claim 1, wherein said two or more natural polysaccharrides are obtained from seeds, fruits, roots, stems, or leaves of a plant.

4. The composite hydrogel dressing as claimed in claims 1 and 3, wherein said two or more natural polysaccharrides are obtained from fenugreek seeds and okra mucilage.

5. The composite hydrogel dressing as claimed in claim 1, wherein said cross-linkinhg agent is a free radical initiator such as ammonium per sulphate (APS), Citric acid, glyceryl aldehyde, preferably APS.

6. The composite hydrogel dressing as claimed in claim 1, wherein said lipid-infused metal nanoparticles are prepared as gold, sillver, zinc, or copper nanoparticles, preferably silver nanoparticles.

7. A method comprising:
soaking small pieces of okra and fenugreek seeds, separately, in distilled water in a ratio of 1:10 - 1:20 (w/v) for 24-48 hours at room temperature;
heating the soaked okra sample at a high temperature between 50-60°C for 1-2 hours;
filtering the heated sample using muslin cloth to extract okra mucilage;
mechanically blending the soaked seeds after boiling it at 60°C;
filtering the blended fenugreek seeds using muslin cloth to extract fenugreek seed mucilage;
washing the extracted mucilage from okra and fenugreek seeds using a non-polar solvent like acetone, chloroform, diethyl ether, or hexane, to obtain purified mucilage;
mixing the obtained okra and fenugreek mucilage in a ratio of 1:4 to 4:1 at room temperature followed by adding optimized concentration of a crosslinking agent;
adjusting the pH in a range from 6.5 to 8.0 to obtain a composite;
preparing silver nanoparticles (NPs) and applying a layer of lipid to it to obatin lipid-infused silver NPs; and
combining the lipid-infused silver NPs with the composite,
for preparing a highly moist, stable, biocompatible, and antibacterial hydrogel patch for wound dressing.