Multifunctional hydrogel patch and preparation method and application thereof

Abstract

The application discloses a multifunctional hydrogel patch, a preparation method and application thereof, and belongs to the field of tissue engineering and regenerative medicine. The biological patch comprises a decellularized freeze-dried tumor tissue (dFCT) and a hydrogel layer loaded with hair melanin nanoparticles (HNPs). The dFCT is obtained by decellularization and freeze-drying treatment of tumor tissue, and retains abundant natural extracellular matrix components. The HNPs are extracted from hair and have excellent active oxygen scavenging capacity and photothermal conversion performance. The hydrogel layer is preferably methacrylated gelatin (GelMA). The patch shows significant cell activity promotion and antibacterial capacity in vitro, and can accelerate wound healing by reducing oxidative stress, inhibiting inflammatory response and promoting angiogenesis in vivo. The patch has the advantages of simple preparation process, natural component source, multiple functions such as antibacterial, antioxidant and tissue regeneration promotion, and is suitable for wound repair treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a multifunctional hydrogel patch for promoting wound healing, and particularly to a composite biopatch comprising frozen decellularized tumor tissue and a hydrogel layer loaded with hair melanin nanoparticles, as well as its preparation method and application. Background Technology

[0002] Wound repair is a major challenge in the global public health field. Chronic wounds are often accompanied by a delayed healing process involving multiple stages such as coagulation, inflammation, proliferation and remodeling. They are also susceptible to bacterial infection, oxidative stress and nutritional deficiencies caused by aging, resulting in slow repair and a high risk of complications.

[0003] To promote wound healing, researchers have developed various types of wound dressings. Among them, hydrogel-based wound dressings are widely used to promote wound healing due to their excellent moisturizing properties and ability to load multiple functional molecules. For example, existing technologies disclose various functional hydrogel patches loaded with antibacterial drugs, growth factors, or nanomaterials, aiming to inhibit bacteria, regulate inflammation, and promote tissue regeneration. However, existing hydrogel patches typically rely on complex chemical synthesis processes, which can destroy the natural properties of the loaded bioactive ingredients, and their biocompatibility and long-term safety remain controversial. Furthermore, the nutrients in hydrogels are mostly exogenously added, making it difficult to mimic the microenvironment of natural tissues, thus limiting their effectiveness in tissue regeneration.

[0004] In recent years, biomaterials derived from natural tissues have shown great potential in promoting cell adhesion, migration, proliferation, and angiogenesis due to their rich extracellular matrix (ECM) components and various growth factors. Tumor tissue possesses unique advantages in rapid proliferation and angiogenesis; its ECM is rich in collagen, fibrin, and various growth factors. With effective decellularization and processing, it can serve as a natural nutrient scaffold for tissue regeneration. Meanwhile, melanin nanoparticles (HNPs), as naturally derived bioactive materials, exhibit excellent biocompatibility, biodegradability, reactive oxygen species (ROS) scavenging ability, and photothermal responsive antibacterial properties. They can inhibit bacterial infection and alleviate oxidative stress during wound healing.

[0005] Therefore, combining cryo-decellularized colon tumor tissue with HNPs and preparing a hydrogel patch via photoinitiated cross-linking with gelatin methacrylamide (GelMA) holds promise for providing multiple functions including natural nutrient supply, antibacterial properties, antioxidant activity, and angiogenesis promotion, significantly improving wound repair efficiency. This strategy has broad application prospects in the repair of various injuries such as chronic wounds, diabetic foot ulcers, burns, and surgical incisions. Summary of the Invention

[0006] To address the technical problems of insufficient bioactivity and difficulty in simulating natural microenvironments in existing hydrogel patches, this invention provides a multifunctional composite hydrogel patch comprising frozen decellularized tumor tissue and a hydrogel layer loaded with hair melanin nanoparticles, along with its preparation method and applications. This novel wound repair material is naturally derived, highly bioactive, and has a simple preparation process, while also possessing multiple functions such as nutritional support, antibacterial properties, antioxidant activity, and angiogenesis promotion. It has broad application prospects in various tissue repair fields, including chronic, difficult-to-heal wounds, burns, and trauma.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a multifunctional hydrogel patch comprising decellularized freeze-dried tumor tissue slices and a biocompatible hydrogel layer loaded with hair melanin nanoparticles.

[0009] The hydrogel layer is composited onto the surface of decellularized freeze-dried tumor tissue slices to form an integrated patch structure.

[0010] The composite biopatch of this invention exhibits a unique bilayer structure. The upper layer is a decellularized freeze-dried tumor tissue slice layer, which retains the porous three-dimensional structure of natural tissue and is rich in various bioactive factors, primarily functioning to provide a nutrient microenvironment and induce cell behavior. The lower layer is a biocompatible hydrogel layer loaded with hair melanin nanoparticles, which has a controllable porous network structure. This layer not only serves as a matrix to support the hair melanin nanoparticles, endowing the patch with antioxidant and photothermal response capabilities, but also provides the patch with good mechanical flexibility and adhesion.

[0011] Among them, hair melanin nanoparticles (HNPs) endow the patch with reactive oxygen species scavenging ability and photothermal responsive antibacterial properties, while decellularized freeze-dried tumor tissue sections (dFCT) provide extracellular matrix nutrients that promote cell proliferation, migration and angiogenesis.

[0012] Furthermore, the decellularized freeze-dried tumor tissue sections are prepared by decellularizing and freeze-drying tumor tissue, retaining at least one active component in the extracellular matrix; the active component is selected from growth factors, collagen, laminin, or fibronectin; the tumor tissue is selected from colon tumor tissue. The decellularized freeze-dried tumor tissue sections are cut using a mold and are approximately 0.5 mm thick.

[0013] Furthermore, the hair melanin nanoparticles were extracted from human black hair and were rod-shaped nanoparticles with an average particle size of 800-900 nm; the concentration of the hair melanin nanoparticles in the hydrogel layer was 0.5-4.0 mg / mL.

[0014] Furthermore, the biocompatible hydrogel layer loaded with hair melanin nanoparticles uses a gelatin methacrylamide crosslinking network as the hydrogel matrix.

[0015] The present invention also provides a method for preparing the above-mentioned multifunctional hydrogel patch, comprising the following steps:

[0016] S1. Preparation of decellularized freeze-dried tumor tissue sections:

[0017] Colon tumor tissue was decellularized and freeze-dried to retain the active components in the extracellular matrix, including growth factors, collagen, laminin and fibronectin.

[0018] S2. Preparation of hair melanin nanoparticles:

[0019] Extracting hair melanin nanoparticles from human black hair;

[0020] S3. Preparation of hair melanin nanoparticle-hydrogel prepolymer solution:

[0021] Hair melanin nanoparticles were dispersed in a prepolymer solution of a biocompatible hydrogel to obtain a hair melanin nanoparticle-hydrogel prepolymer solution.

[0022] S4, Composite Curing:

[0023] Hair melanin nanoparticles-hydrogel prepolymer was coated onto the surface of decellularized freeze-dried tumor tissue slices, and then cross-linked and cured to obtain a multifunctional hydrogel patch.

[0024] Further, the decellularization process in step S1 is as follows: the fresh colon tumor tissue sample is rinsed in phosphate buffer containing antibiotics to remove residual contaminants; then, it is decellularized using 0.5-2% Triton X-100 solution at 4°C for 4-8 hours to obtain transparent decellularized colon tumor tissue.

[0025] Colon tumor tissue is rich in ECM proteins such as collagen, laminin, and fibronectin, as well as various growth factors (e.g., EGF, HGF, VEGF). After decellularization and freeze-drying, these beneficial components are preserved to the greatest extent, providing an ideal natural nutritional scaffold for cell adhesion, proliferation, migration, and angiogenesis. During decellularization, non-ionic detergents (such as Triton X-100) are used to gently remove cellular components, minimizing damage to the active components of the ECM. Vacuum freeze-drying effectively preserves the three-dimensional structure and biological activity of the ECM.

[0026] Furthermore, the phosphate buffer contains penicillin and streptomycin at concentrations of 100 U / mL and 100 μg / mL, respectively.

[0027] Further, the freeze-drying process in step S1 is as follows: the decellularized colon tumor tissue is pre-frozen at -80°C for no less than 4 hours, and then freeze-dried under a vacuum of less than 10 Pa for 24-48 hours.

[0028] Furthermore, the preparation process of hair melanin nanoparticles is as follows: black hair was collected from volunteers, mixed with 1 mol / L NaOH solution, and alkali-dissolved at 60℃ for 10 h until completely dissolved; the resulting mixture was centrifuged at 8000 rpm for 15 min to separate the precipitate, the obtained hair melanin nanoparticles (HNPs) were collected, and the mixture was repeatedly rinsed with deionized water at least three times to remove residual alkali and impurities.

[0029] Hair melanin nanoparticles are natural biomaterials with excellent biocompatibility and biodegradability. Their molecular structure contains abundant active groups such as phenolic hydroxyl groups, giving them outstanding reactive oxygen species scavenging ability (antioxidant capacity). At the same time, hair melanin nanoparticles are also excellent photothermal conversion materials, which can efficiently convert light energy into heat energy under near-infrared light irradiation, thereby achieving non-invasive, spatiotemporally controllable photothermal antibacterial effects.

[0030] Furthermore, in step S3, the prepolymer solution of the biocompatible hydrogel is a gelatin methacrylamide prepolymer solution, which combines the biocompatibility of natural gelatin with the photocrosslinking processing properties, and can form a porous network structure to provide physical support for cell growth; in step S4, photocuring is used to crosslink and cure the prepolymer solution.

[0031] The present invention also provides the application of the above-mentioned multifunctional hydrogel patch in the preparation of drugs or medical materials that promote wound healing.

[0032] Preferably, the application further includes treating the wound under near-infrared light irradiation to activate the photothermal effect of HNPs and achieve photothermal antibacterial treatment of the wound. Specifically, after applying the patch of the present invention to the wound, the patch area is irradiated with 808nm near-infrared light of appropriate power density (e.g., 1.5W / cm²) for a certain period of time (e.g., 10min) to activate the photothermal effect of HNPs. The generated mild thermal effect (e.g., about 50°C) kills local bacteria in the wound, achieving efficient and controllable photothermal antibacterial treatment, thereby further promoting wound healing.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) This invention is the first to combine decellularized freeze-dried tumor tissue (dFCT) with a hydrogel layer loaded with hair melanin nanoparticles (HNPs) to construct a biomimetic multifunctional biopatch with a double-layer structure. This design cleverly utilizes the rich nutrient induction function of tumor tissue-derived ECM, the antioxidant and photothermal antibacterial functions of HNPs, and the cell adhesion and tissue regeneration support provided by the GelMA hydrogel network to achieve synergistic effect.

[0035] (2) The dFCT in this invention is derived from natural tumor tissue. Through a mild decellularization and freeze-drying process, it retains collagen, laminin, fibronectin, and various growth factors (such as EGF, HGF, and VEGF) in the natural ECM to the greatest extent. This natural and complex nutrient reservoir can better simulate the real physiological microenvironment than an artificial scaffold that simply adds a few factors from the outside, providing an ideal inducing environment for the adhesion, proliferation, migration, and angiogenesis of seed cells.

[0036] (3) The HNPs in this invention are extracted from natural hair, which is widely available and inexpensive. They not only have excellent antioxidant capacity, which can effectively remove excess ROS from wounds and reduce oxidative stress damage, but also have excellent photothermal conversion properties, which endow the patch with on-demand activated antibacterial ability. Through external infrared irradiation, precise, efficient and non-invasive control of wound infection can be achieved, avoiding the abuse of antibiotics.

[0037] (4) The main raw materials used in this invention (dFCT, HNPs, GelMA) are all derived from natural biomaterials or bioacceptable materials. The preparation process is mild and avoids the use of toxic chemical reagents. Hemolysis and cytotoxicity experiments have confirmed that the composite biopatch prepared in this invention has good blood compatibility and cell compatibility, meeting the safety requirements as a medical implant material.

[0038] (5) The patch can significantly promote cell activity and angiogenesis in vitro, and accelerate wound healing in vivo by reducing oxidative stress, inhibiting inflammatory response and promoting angiogenesis. It is suitable for various tissue repair scenarios such as chronic wounds, diabetic foot, burns and surgical incisions. Attached Figure Description

[0039] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0040] Figure 1Characterization results of HNPs: (a) Schematic diagram of HNP extraction process and corresponding optical images; (b) SEM images of HNP morphology, scale bar 500 μm; (c) Particle size distribution of HNPs, scale bar 200 μm; (d) SEM images of HNPs after soaking in PBS, sweat and DDW for 3 days; stability of HDPs in different media over 3 days; (e) Absorbance of HNPs at 500 nm, reflecting the total amount of melanin in HNPs; (f) Ratio of absorbance of HNPs at 650 nm and 500 nm, reflecting the ratio of eumelanin to total melanin.

[0041] Figure 2 Characterization results of dFCT and bio-patterns: (a) Quantitative analysis of collagen content in raw CT and dCT (n=4); (b) Quantitative analysis of DNA in raw CT and dCT (n=3); (d) i. Optical image of raw CT after in vitro treatment, ii. Optical image of raw CT after freeze-drying, and iii. SEM image of raw CT, scale bar 10 μm; (e) i. Optical image of dCT, ii. Optical image of dCT after freeze-drying, and iii. (f) SEM images of dCT, scale bar 5 μm; (h) Representative images of CT and dCT, including H&E staining (i, ii), Masson staining (iii, iv), laminin staining (v, vi) and fibronectin staining (vii, viii); (g) ELISA quantitative detection of epidermal growth factor, hepatocyte growth factor and vascular endothelial growth factor (n=4), scale bar 100 μm; (h) Representative SEM images of dFCT-HNPs-GelMA biopatch: cross-section i and magnified view ii, scale bars 60 and 6 μm (magnified view); (i) Antioxidant capacity of different concentrations of dFCT-HNPs-GelMA biopatch quantified by DPPH assay.

[0042] Figure 3 For proteomics analysis of dFCT, the bar graph shows the log2-fold change in protein abundance in dFCT relative to CT as determined by mass spectrometry analysis. Red bars represent proteins that are retained or relatively enriched after decellularization, while blue bars represent proteins that are significantly reduced or removed (n=5).

[0043] Figure 4 Scanning electron microscopy image of a multifunctional patch derived from colon tumor tissue.

[0044] Figure 5 (a) SEM image of GelMA, (b) representative SEM image of dFCT layer, (c) representative SEM image of HNPs-GelMA layer.

[0045] Figure 6Schematic diagrams of the photothermal and antibacterial properties of bio-patterns: (a) Schematic diagram of the photothermal and antibacterial properties of the bio-pattern; (b) GelMA, dFCT-GelMA, and dFCT-HNPs-GelMA at 1.50 W / cm² (c) Temperature change curves under NIR irradiation (HNPs concentration in dFCT-HNPs-GelMA is 2.0 mg / mL); (d) Temperature change curves of dFCT-HNPs-GelMA with different HNPs concentrations under 1.50 W / cm² laser irradiation; (e) Temperature change curves of dFCT-HNPs-GelMA under different NIR laser intensities (HNPs concentration is 2.0 mg / mL); (f) Photothermal heating-cooling cycle of dFCT-HNPs-GelMA with HNPs concentration of 2.0 mg / mL under 1.50 W / cm² laser irradiation; (g) Live / dead fluorescence images of bacteria treated by different methods, with the dFCT-HNPs-GelMA@NIR group mainly showing red fluorescence (representing bacterial death); (h) Representative bacterial colony images after different treatments; Scale bar: (g) 200 μm, (h) 0.5 cm.

[0046] Figure 7 In vitro assays for cell-induced functional and antioxidant activity: (a) Images of hemolysis assay results; (b) Statistical analysis of hemolysis assay results (n=5); (c) Statistical analysis of cell viability percentage (n=3); (d) Representative images of four wound scratch assays; (e) Representative images of four angiogenesis assays; (f) dFCT-HNPs-GelMA-induced reduction of H2O2-induced cell damage; (g) Quantitative analysis of cell migration rate (n=3); (h) Quantitative analysis of tube formation ability (n=3); (i) Cell viability measured in negative control, positive control, and dFCT-HNPs-GelMA groups (n=3); Scale bars: (d) 300 μm, (e) 100 μm, (f) 150 μm.

[0047] Figure 8 To evaluate the wound healing effect of dFCT-HNPs-GelMA in a skin defect model: (a) Schematic diagram of the wound healing process; (b) Thermal image of the wound in group G5; (c) Photographs of representative wounds of rats treated with four different methods; (d) H&E staining of wound tissue, with blue arrows indicating the wound edges; (e) Temperature change curves of the wound surface in groups G4 and G5 (n=3); (f) Quantitative assessment of wound area (n=5); (g) Quantitative assessment of wound edges on day 9 (n=5); Scale bars: (c) 0.5 cm, (d) 0.5 mm and 100 μm (magnified views).

[0048] Figure 9Histological and immunofluorescence assessment of the wound site: (a) Representative histological and immunofluorescence images of Masson staining and expression of IL-6, CD31 (red) and α-SMA (green) markers in wound sections; (b) Quantitative analysis of collagen content (n=5), (c) IL-6 level (n=5) and (d) CD31 positive vessel density (n=5); Scale bar: (a) 50 μm. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The materials and cell lines used in these embodiments are as follows: Gelatin and methacrylic anhydride were purchased from Sigma-Aldrich (USA) and used in the laboratory to synthesize GelMA hydrogels. Sodium hydroxide was purchased from Macklin (China). Diphenylpicrylhydrazine was purchased from Macklin (China). SYTO dye and propidium iodide were purchased from Thermo Fisher Scientific. NIH3T3 and HUVEC cell lines were provided by the Wenzhou Institute of the University of Chinese Academy of Sciences. Antibodies targeting IL-6 and α-SMA were purchased from Boster Biological Technology (China), and CD31 antibody was purchased from Abcam (UK).

[0051] Example 1

[0052] Extraction of hair melanin nanoparticles (HNPs):

[0053] (1) Raw material acquisition

[0054] Natural, untreated black hair was collected from healthy volunteers. The collected hair was rinsed with running deionized water to remove surface dust and loose impurities; then soaked in a 2% (w / v) sodium dodecyl sulfate (SDS) solution for 15 minutes to initially remove surface oil and protein residue, followed by rinsing with deionized water until the shampoo was clear and foam-free. The washed hair was then laid out on a clean tray and dried in a 60°C oven to constant weight for later use.

[0055] (2) Alkaline extraction

[0056] Weigh a sample of dried black hair, add 1 mol / L NaOH solution, place it in an alkali-resistant flask, seal it, and then place it in a constant temperature water bath at 60℃ for 10 hours. During the reaction, gently shake or stir at low speed with a magnetic stirrer every 2 hours to promote the dissolution of melanin. At the end of the reaction, the mixture is dark brown or nearly black, and the hair is visibly completely dissolved.

[0057] (3) Separation and purification

[0058] Cool the alkaline hydrolysis reaction solution to room temperature (20-25℃) and centrifuge at 8000 rpm for 15 min to separate the melanin nanoparticle precipitate from the supernatant. Discard the supernatant, collect the precipitate, and resuspend it in a large amount of deionized water. Perform at least three centrifugation-wash cycles to thoroughly remove residual NaOH and soluble impurities. To further reduce ion residue, ultrapure water can be used in the final wash. Resuspend the purified HNPs in deionized water and store at 4℃ in the dark.

[0059] (4) Characterization

[0060] Morphological analysis was performed on the obtained HNPs. First, the HNP suspension was dropped onto the surface of a clean silicon wafer or conductive tape, allowed to dry naturally, and then metal sputtered (e.g., gold / palladium). The morphology and structure were observed using a scanning electron microscope (SEM), and the particle size distribution was further evaluated using a Malvern Zetasizer NanoZS for dynamic light scattering analysis.

[0061] Hair melanin nanoparticles were extracted from human black hair. The hair was washed with sterile water and then dissolved in sodium hydroxide solution. After centrifugation, HNPs were successfully prepared and collected, such as... Figure 1 As shown in Figure a. Scanning electron microscopy analysis revealed that the HNPs were rod-shaped, with an average length of approximately 800 nm and a width of approximately 250 nm. Figure 1 b). Meanwhile, the average size of the HNPs was 882.7 nm, showing a uniform distribution. Figure 1 c). It is worth noting that the stability of HNPs is crucial for practical applications. To test this, HNPs were immersed in different solutions for three days, including deionized water (DDW), phosphate-buffered saline (PBS), and sweat. SEM observations showed that even in different solution environments, HNPs maintained their structural integrity and morphological consistency. Figure 1 d). The corresponding absorption peaks at 500 nm and 650 nm were measured, and it was found that the absorption peak of HNPs at 500 nm remained almost unchanged with the extension of immersion time, which further verified the stability of HNPs. Figure 1e). Furthermore, the ratio of the absorption peaks at 650 nm and 500 nm can reflect the proportion of eumelanin in total melanin. Detection revealed that after three days of immersion in deionized water (DDW), phosphate-buffered saline (PBS), and sweat, the content of HNPs remained stable. Figure 1 f). These results indicate that HNPs exhibit excellent stability, and their structure and content are unaffected by sweat or other common solvents.

[0062] Example 2

[0063] Preparation of decellularized freeze-dried tumor tissue sections:

[0064] (1) Raw material pretreatment: With the approval of the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (KY2021-063), surgically removed human colon tumor tissue was collected and processed within 30 minutes after excision. Fresh tissue was cut into pieces of approximately 1 cm. 3 Small pieces were placed in a phosphate-buffered saline (PBS) solution containing antibiotics, including penicillin (100 U / mL) and streptomycin (100 μg / mL), and gently shaken three times at 4°C to remove blood and residual contaminants.

[0065] (2) Decellularization: The washed tissue was immersed in a 1% (v / v) Triton X-100 solution and magnetically stirred at 4°C for 48 h to promote decellularization. The solution was changed every 12 h until the tissue became translucent. After the washing treatment was completed, the sample was washed three times with PBS (10 min each time) to remove residual decellularization reagent, and decellularized tumor tissue (dCT) was obtained.

[0066] (3) Vacuum freeze-drying: The decellularized colon tumor tissue was pre-frozen at -80°C for at least 4 hours, and then freeze-dried under vacuum (<10 Pa) at -55°C for 24 hours to obtain decellularized freeze-dried tumor tissue (dFCT). The dFCT tissue was stored under sterile conditions (-20°C) and sectioned for subsequent use.

[0067] (4) DNA content detection: To assess decellularization efficiency, DNA content in colon tumor tissue (CT) and decellularized tumor tissue (dCT) was measured. Tissue samples were first freeze-dried and weighed to determine dry weight, and then DNA was extracted using a genomic DNA extraction kit (KeyGEN BioTECH, China). DNA content was quantified using a spectrophotometer, and results were normalized to tissue dry weight and reported in nanograms per milligram.

[0068] Patient-derived colon tumor tissue was used as the raw material for preparing the biopatch because it is rich in extracellular matrix nutrients. Typically, colon tumor tissue undergoes decellularization immediately after ex vivo; initially, due to the presence of red blood cells, the original colon tumor tissue has a pinkish appearance. Figure 2 di and ii), SEM imaging confirmed the presence of tumor cells ( Figure 2 d iii). Compared to the original colon tumor tissue, the decellularized colon tumor tissue appears translucent ( Figure 2 (ei and ii), SEM images revealed its unique three-dimensional structure, with no cellular residue ( Figure 2 e iii). Hematoxylin and eosin staining of decellularized colon tumor tissue (dCT) showed no obvious residual cell nuclei compared to colon tumor tissue (CT), indicating that tumor cells were effectively removed. Figure 2 (fi and ii) This is because the decellularization process uses the mild, non-ionic detergent Triton X-100, which removes the nuclear components from the original colon tumor tissue while minimizing damage to the bioactive ECM components. Histochemical staining further confirmed that key ECM proteins, including laminin and fibronectin, were preserved in dCT (fi and ii). Figure 2 fiii-viii). As a major structural ECM component, collagen was also well preserved, as shown in the quantitative analysis ( Figure 2 a) Proteomics analysis further characterized the ECM components ( Figure 3 Specifically, key structural proteins were significantly preserved, including various collagen subtypes, laminin subunits, fibronectin, and proteoglycans. Cytoskeletal and intracellular proteins were significantly reduced, confirming effective decellularization and enrichment of ECM components. Furthermore, ELISA results showed that although some growth factors were lost during decellularization, dCT retained considerable levels of essential factors such as EGF, HGF, and VEGF (…). Figure 2 g), supporting its role as a bioactive nutrient reservoir.

[0069] To ensure the safety of dCT, we performed quantitative DNA analysis to assess the degree of decellularization. The residual DNA level in the dCT tissue was found to be below 50 ng / mg, demonstrating the effectiveness of the decellularization process. Figure 2 (b) This low level of nuclear material indicates a reduced risk of immune responses from residual cellular components, thus addressing potential biosafety concerns associated with dCT. Subsequently, the dFCT was cut into blocks using a specific tissue mold for further use.

[0070] Example 3

[0071] Preparation of dFCT-HNPs-GelMA multifunctional hydrogel patch:

[0072] (1) Preparation of GelMA solution

[0073] Weigh 1g of gelatin methacrylamide (GelMA) powder and place it in a clean beaker. Add an appropriate amount of highly purified water and stir in a constant temperature water bath at 40-50℃ until completely dissolved to obtain a 10% (w / v) transparent solution. Then add the photoinitiator HMPP (2-hydroxy-2-methyl-1-phenyl-1-propanone) at a concentration of 10% (w / w), stir to dissolve, and continue stirring until completely dissolved. Store in the dark for later use.

[0074] (2) Mixture of HNPs and GelMA

[0075] The melanin nanoparticle (HNPs) suspension obtained in Example 1 was subjected to low-power (100W) intermittent ultrasonic cleaning (5s on, 3s off, 3min cycle) to disperse the particle agglomeration. The HNPs were added to the above GelMA pregel solution and mixed for 10min under magnetic stirring to ensure that the HNPs were uniformly distributed in the pregel matrix, resulting in a homogeneous GelMA-HNPs pregel.

[0076] (3) Combined with dFCT

[0077] Take the decellularized freeze-dried colon tumor tissue (dFCT) sections prepared in Example 2 and gently wet them with sterile PBS to facilitate subsequent infiltration. Apply GelMA-HNPs pregel uniformly to the surface of the dFCT sections, ensuring the pregel completely infiltrates their porous structure. To avoid air bubbles affecting the composite quality, vacuum degassing can be performed for 30 seconds to promote pregel penetration into the internal pores of the dFCT.

[0078] (4) Photocrosslinking curing

[0079] dFCTs pre-gelled with GelMA-HNPs were placed on sterile plates and irradiated with a 365nm UV light source for 60s (light intensity approximately 10mW / cm²) to induce photo-initiated cross-linking and curing of the GelMA network, forming a structurally stable, multifunctional hydrogel patch with certain mechanical strength and flexibility. After curing, the patch was removed from the plate and immediately stored under sterile conditions for use in subsequent experiments.

[0080] (5) Morphological observation

[0081] Take the prepared patch sample, freeze-dry it, cut out small pieces, and observe its surface and cross-sectional micromorphology using SEM. For example... Figure 4 As shown, the patch surface exhibits a uniform three-dimensional porous network structure.

[0082] Given its high similarity in composition and structure to natural ECM, methacrylamide gelatin was selected as the main component of the biopatch. SEM analysis revealed the porous network structure in the GelMA hydrogel. Figure 5 a) This is considered an ideal candidate material for biomedical patches for wound healing. Based on this, HNPs are incorporated into a GelMA prepolymer solution, which can be polymerized by UV curing. Due to the flexible processability of hydrogels, HNPs-GelMA hydrogels with complex geometries (e.g., circular, lace-shaped, pentagonal, and heart-shaped) can be produced, and this shape-adaptive ability may be suitable for irregular wounds. dFCT is integrated during the preparation process to enhance its nutritional properties. Specifically, the HNPs-GelMA prepolymer solution is coated onto the surface of a dFCT block, and then a dFCT-HNPs-GelMA biopatch is prepared by UV curing. The resulting biopatch exhibits a bilayer structure, consisting of an upper dFCT layer and a lower HNPs-GelMA layer. Figure 4 The upper dFCT layer retains its natural three-dimensional structure. Figure 5 b), while the HNPs-GelMA layer retains the characteristic porous network ( Figure 5 c).

[0083] Example 4

[0084] Bioperformance testing:

[0085] Antioxidant effect of dFCT-HNPs-GelMA biopatches: To investigate antioxidant performance, biopatches incorporating different concentrations of HNPs (0.1-0.5 mg / mL) were prepared. The antioxidant efficiency of melanin nanoparticles was tested using DPPH. Specifically, 2 mg of DPPH was dissolved in 50 mL of ethanol to prepare a stock solution, which was then adjusted to an absorbance of approximately 0.9 at 517 nm to obtain the working solution. The prepared dFCT-HNPs-GelMA was then immersed in the DPPH working solution and gently shaken in the dark for 30 min. After incubation, the supernatant was collected for digital imaging, and its absorbance was quantified by spectrophotometric analysis.

[0086] Photothermal performance of dFCT-HNPs-GelMA biopatches: dFCT-GelMA and dFCT-HNPs-GelMA biopatches were irradiated with an 808 nm near-infrared beam for 3 min. The irradiation intensity and HNPs concentration were adjusted, and the temperature change of the biopatches was monitored using an infrared thermal imager (FLIR E5-XT, Germany) to evaluate their photothermal response.

[0087] Antibacterial efficacy evaluation: The suspensions of Escherichia coli and Staphylococcus aureus were adjusted to 1×10⁻⁶. 6CFU / mL was used to transfer 1 mL of each bacterial culture to a single well of a 24-well plate for co-culturing with the biopatches. In the dFCT-HNPs-GelMA@NIR group, the biopatches were exposed to NIR (808 nm 1.50 W / cm²) for 10 min, resulting in a rapid temperature rise and activation of the photothermal antibacterial effect. After culturing, the bacterial samples were incubated with SYTO and PI dyes for 15 min. Subsequently, the stained solution was transferred to a microscope slide for fluorescence imaging. Simultaneously, the bacterial suspension was diluted 10-fold with sterile PBS. Then, 50 μL of the diluted suspension was spread on an agar surface and incubated overnight. Bacterial colony formation was subsequently recorded by photography.

[0088] Hemolysis analysis: Fresh rat blood was first centrifuged at 3000 rpm for 20 min, and the supernatant was gently aspirated using a pipette. Hemolysis was assessed by mixing 100 μL of erythrocyte suspension with 900 μL of PBS containing various extracts (GelMA, HNPs-GelMA, or dFCT-HNPs-GelMA) in a 2 mL centrifuge tube. After incubation for 2 h, the sample was centrifuged, and the resulting supernatant was collected. The centrifuge tubes were then photographed for visual assessment of the hemolysis effect.

[0089] Biocompatibility assessment: NIH 3T3 fibroblasts were cultured in complete medium and seeded into 24-well plates for further analysis. GelMA, HNPs-GelMA, or dFCT-HNPs-GelMA biopatches were gently transferred into the wells of the Transwell chambers. On days 1, 3, and 5, the used medium was replaced with fresh medium containing 10% CCK-8 reagent. After 2 hours of incubation, 100 μL of the supernatant was collected for absorbance analysis. Simultaneously, Calcein-AM and PI were added to label live / dead cells, followed by fluorescence imaging using a fluorescence microscope.

[0090] Scratch healing test: A concentration of 2×10 5 HUVEC suspension at a concentration of cells / mL was aliquoted into 24-well plates. After cell adhesion, uniform scratches were made using a sterile P200 pipette tip, and unattached cells were washed with PBS to remove them. Transwell chambers were then inserted into the wells, and GelMA, dFCT-GelMA, or dFCT-HNPs-GelMA biopatches were placed in the upper chamber for co-culture. Cell migration across the scratched areas was observed and recorded at predetermined time intervals.

[0091] Matrigel tube formation assay: Before inoculation, 24-well plates were pre-coated with Matrigel diluted 1:1 with the culture medium. Then, 5 × 10⁶ tubes were inoculated per well. 4HUVECs were seeded at a density of 1,000 cells. After cell adhesion, Transwell chambers containing different biopatches were placed in well plates and incubated for 6 hours. The formation of capillary-like tubular networks was then observed and imaged under a fluorescence microscope.

[0092] Cellular oxidative stress relief: Cell culture conditions and seeding density were the same as those used in the biocompatibility assessment. After 24 h of incubation, the original medium was replaced with serum-free medium (as a negative control), H2O2-rich medium (as a positive control), or medium containing H2O2 and simultaneously inserting a Transwell chamber loaded with dFCT-HNPs-GelMA biopatches. After another 24 h, cell morphology was assessed under an inverted bright-field microscope. Subsequently, cell viability was quantified and analyzed.

[0093] In vivo assessment of wound healing: Sprague-Dawley rats used in the in vivo experiments were provided by Zhejiang Vital River Laboratory Animal Technology Co., Ltd. All animal procedures were approved by the WIUCAS Animal Ethics Committee (No. WIUCAS25010805). A full-thickness skin wound model was established in 30 healthy SD rats to evaluate the therapeutic potential of the dFCT-HNPs-GelMA biopatch. Animals were randomly assigned to 5 groups: Group 0 (G0, PBS), Group 1 (G1, NIR), Group 2 (G2, GelMA), Group 3 (G3, dFCT-GelMA), Group 4 (G4, dFCT-HNPs-GelMA), and Group 5 (G5, dFCT-HNPs-GelMA@NIR). After anesthesia, the hair on the back of the rats was shaved, and a full-thickness circular wound with a diameter of 1.0 cm was surgically created on the back of the rats. The biopatch prepared by the method described above was gently applied to the wound surface. In Group G4, the wound was irradiated with near-infrared light (808 nm). Wound healing progress was documented by photographs on days 0, 3, 5, 7, and 9. At the designated endpoints, granulation tissue and adjacent skin were collected and fixed in 4 wt% paraformaldehyde for subsequent histological evaluation. Histochemical and immunofluorescence analyses were performed on the collected tissue samples to assess wound regeneration, collagen deposition, and cellular activity at the injury site.

[0094] Statistical analysis: GraphPad Prism was used for statistical processing. Variables are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups.

[0095] The test results are as follows:

[0096] With active groups such as phenolic hydroxyl and amino groups, HNPs exhibit high antioxidant activity, which endows the dFCT-HNPs-GelMA biopatch with corresponding properties. To mechanistically verify this property, this invention employs a 2,2-diphenyl-1-trinitrophenylhydrazine free radical scavenging assay based on the titanium sulfate method and a hydrogen peroxide decomposition assay. DPPH is a stable free radical; when reduced by an antioxidant, its color changes from deep purple to light yellow. Increasing the HNP content in the biopatch leads to a gradual lightening of the solution color and a decrease in absorbance at 517 nm, indicating a dose-dependent scavenging effect. Figure 2 i), indicating that the dFCT-HNPs-GelMA biopatch has strong antioxidant capabilities, supporting its potential in wound healing applications.

[0097] Due to the photothermal conversion capability of HNPs, the dFCT-HNPs-GelMA biopatch was endowed with photothermal response properties. To investigate this, materials including GelMA, dFCT-GelMA, and dFCT-HNPs-GelMA were irradiated with 808nm near-infrared light, and temperature changes were monitored. Figure 6 a). The results showed that the dFCT-HNPs-GelMA biopatch exhibited a significant increase in temperature change, reaching equilibrium at 150 s, while the dFCT-GelMA and GelMA groups showed very small temperature changes (a). Figure 6 b). The photothermal conversion process of the dFCT-HNPs-GelMA biopatch was captured using an infrared thermal imager, showing that its temperature gradually increased to approximately 52.4°C within 150 seconds, consistent with the temperature curve results. Figure 6 c). Since the photothermal conversion capability depends on the concentration of doped HNPs, biopatches with different HNP concentrations (0, 0.50, 1.00, 2.00, and 4.00 mg / mL) were prepared. With increasing HNP concentration, the temperature of the biopatches increased accordingly under 1.50 W / cm² NIR irradiation. Figure 6 d). Simultaneously, increased irradiation power contributes to the photothermal response ( Figure 6 e). Considering that the optimal temperature is crucial for the antibacterial efficacy and skin safety of the patch, an HNP concentration of 2.0 mg / mL was selected at 1.50 W / cm². Furthermore, after 5 laser-switched cycles, the biopatch exhibited excellent heating and cooling performance, confirming the stability of its photothermal response. Figure 6 f).

[0098] Severe infections often lead to refractory wounds. The photothermal effect of bio-patches generates sufficient heat to disrupt bacterial membranes, thereby achieving effective sterilization. Representative strains of Staphylococcus aureus and Escherichia coli were used to evaluate the antibacterial efficacy of the bio-patches. Live / dead fluorescence images showed that the dFCT-HNPs-GelMA patch exhibited antibacterial activity under NIR irradiation. Figure 6 g). In contrast, the control group (treated with PBS), the dFCT-GelMA group, and the dFCT-HNPs-GelMA group (without NIR irradiation) showed green fluorescence, indicating that they had almost no antibacterial activity. Furthermore, agar plate assays also showed that the dFCT-HNPs-GelMA@NIR group significantly inhibited bacterial growth ( Figure 6 h). The dFCT-HNPs-GelMA biopatch, combined with controllable NIR-induced heating, effectively inhibits bacterial growth and has the potential to maintain a sterile wound environment.

[0099] Blood compatibility and biocompatibility are crucial for the effective tissue repair support of dFCT-HNPs-GelMA biopatch. The hemolysis rates of GelMA, dFCT-GelMA, and dFCT-HNPs-GelMA were all below 5%, indicating that their blood compatibility met the required standards. Figure 7 a and 7b). After co-culturing with NIH 3T3 cells for 3 days, the GelMA, dFCT-GelMA, and dFCT-HNPs-GelMA groups all maintained good cell viability. Figure 7 c). Scratch assays showed that the dFCT-HNPs-GelMA group accelerated wound closure and promoted cell migration within 12 hours. Figure 7 d and 7g). Due to nutrient retention, biopatch containing dFCT also showed the ability to promote angiogenesis ( Figure 7 e and 7h). Fluorescence images showed that, compared with other groups, the dFCT-HNPs-GelMA group formed more vascular-like structures (e and 7h). Figure 7 e).

[0100] To assess the ability of HNPs-GelMA to alleviate oxidative stress, the viability of three cell groups was evaluated: a negative control group (treated with blank culture medium), a positive control group (treated with 400 μM H2O2), and a dFCT-HNPs-GelMA group (treated with dFCT-HNPs-GelMA and 400 μM H2O2). Figure 7As shown in f and 7i, compared with the positive control group, cells in the dFCT-HNPs-GelMA group exhibited higher viability and cell morphology more similar to the negative control group. These results indicate that dFCT-HNPs-GelMA can protect cells from oxidative stress damage. The dFCT-HNPs-GelMA biopatch constructed in this invention not only exhibits good blood compatibility and biocompatibility, but also possesses multiple functions beneficial to wound healing.

[0101] Based on the above findings, a 1.0 cm diameter full-thickness skin incision was created on the back of rats to evaluate the in vivo wound healing performance of the dFCT-HNPs-GelMA biopatch. Figure 8 a). Based on different treatment strategies, rats were randomly divided into four groups: G0 (PBS treatment), G1 (NIR treatment), G2 (GelMA), G3 (dFCT-GelMA), G4 (dFCT-HNPs-GelMA), and G5 (dFCT-HNPs-GelMA@NIR). Temperature changes in the G5 group were recorded using thermal imaging. Figure 8 b). The results showed that the wound surface temperature in this group gradually rose to 49.4°C within 150 seconds, and then tended to stabilize. Figure 8 (b and 8e). A stable temperature profile ensured sustained antibacterial effects throughout the irradiation period. Wound closure progress was monitored at multiple time points during the healing process ( Figure 5 c), Notably, the introduction of dFCT accelerated the wound healing process, attributed to its rich nutrient supply. The G5 group exhibited the fastest wound closure and the smallest remaining wound area observed on day 9. Figure 8 f). This enhanced healing effect is attributed to NIR-induced photothermal antibacterial action. Quantitative analysis based on H&E images further supports this, showing that this group had the smallest wound width (f). Figure 8 (d and 8g). These results demonstrate that biopatches with NIR-dependent antimicrobial properties can significantly enhance wound healing.

[0102] To further elucidate the wound regeneration process, a series of in-depth histological and immunological analyses were performed. Masson trichrome staining showed that the G5 group exhibited the highest level of collagen accumulation at the wound site, indicating enhanced extracellular matrix remodeling and improved tissue regeneration. Figure 9 (a and 9b). Additionally, analyses were performed on day 9 to assess interleukin-6 expression. (e.g., ...) Figure 9As shown in a and 9c, the G5 group exhibited significantly reduced IL-6 expression compared to other groups. Elevated IL-6 levels in the control group reflected a significant inflammatory response, while its downregulation in the treatment group indicated effective antibacterial and anti-inflammatory activity. To further assess in vivo angiogenesis, dual immunofluorescence staining was performed targeting CD31 (an endothelial cell marker) and α-smooth muscle actin (an indicator of mature blood vessel development). Figure 9 As shown in a and 9d, the control group exhibited relatively low expression of both biomarkers, indicating limited angiogenesis. In contrast, the G5 group showed significantly increased expression of CD31 and α-SMA, which may be attributed to reduced inflammation due to bacterial clearance and the presence of bioactive components that support angiogenesis. In summary, these findings suggest that the dFCT-HNPs-GelMA biopatch effectively promotes tissue remodeling, reduces inflammation, and enhances angiogenesis without causing significant immune damage, thereby accelerating wound healing.

[0103] This invention develops a hydrogel biopatch made from decellularized colon tumor tissue and human hair-derived melanin, designed for the management of refractory wounds. The biopatch aims to retain ECM components, such as growth factors, cytokines, and fibrin, which are crucial for tissue regeneration. Due to the flexible processing properties of the hydrogel, its morphology can be adapted to various wound types. Furthermore, the incorporation of HNPs imparts antioxidant capacity and NIR-induced antibacterial properties. These unique characteristics help reduce local inflammation, promote tissue remodeling and healing of damaged skin. Combined with its safety and applicability, the proposed hydrogel biopatch shows great potential for wound healing.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multifunctional hydrogel patch, characterized in that, This includes decellularized freeze-dried tumor tissue sections and a biocompatible hydrogel layer loaded with hair melanin nanoparticles. The hydrogel layer is composited onto the surface of decellularized freeze-dried tumor tissue slices to form an integrated patch structure. The hair melanin nanoparticles were extracted from human black hair. They are rod-shaped nanoparticles with an average particle size of 800-900 nm. The concentration of the hair melanin nanoparticles in the hydrogel layer was 2 mg / mL. When using hydrogel patches, irradiation with 808nm near-infrared light is applied, with an irradiation power of 1.50W / cm². The decellularized freeze-dried tumor tissue section is prepared by decellularizing and freeze-drying tumor tissue, which retains at least one active component in the extracellular matrix; the active component is selected from growth factors, collagen, laminin or fibronectin; the tumor tissue is selected from colon tumor tissue; The biocompatible hydrogel layer loaded with hair melanin nanoparticles uses a gelatin methacrylamide crosslinking network as the hydrogel matrix.

2. A method for preparing the multifunctional hydrogel patch as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of decellularized freeze-dried tumor tissue sections: Colon tumor tissue was decellularized and freeze-dried to retain the active components in the extracellular matrix, including growth factors, collagen, laminin and fibronectin. S2. Preparation of hair melanin nanoparticles: Extracting hair melanin nanoparticles from human black hair; S3. Preparation of hair melanin nanoparticle-hydrogel prepolymer solution: Hair melanin nanoparticles were dispersed in a prepolymer solution of a biocompatible hydrogel to obtain a hair melanin nanoparticle-hydrogel prepolymer solution. S4, Composite Curing: Hair melanin nanoparticles-hydrogel prepolymer was coated onto the surface of decellularized freeze-dried tumor tissue slices, and then cross-linked and cured to obtain a multifunctional hydrogel patch.

3. The preparation method according to claim 2, characterized in that, The decellularization process in step S1 is as follows: the fresh colon tumor tissue sample is rinsed in antibiotic-containing phosphate buffer to remove residual contaminants; Subsequently, decellularization was performed using 0.5-2% Triton X-100 solution at 4°C for 48 hours to obtain clear, decellularized colon tumor tissue.

4. The preparation method according to claim 3, characterized in that, Phosphate buffer contains penicillin and streptomycin at concentrations of 100 U / mL and 100 μg / mL, respectively.

5. The preparation method according to claim 2, characterized in that, The freeze-drying process in step S1 is as follows: the decellularized colon tumor tissue is pre-frozen at -80°C for no less than 4 hours, and then freeze-dried under vacuum conditions below 10 Pa for 24-48 hours.

6. The preparation method according to claim 2, characterized in that, In step S3, the prepolymer solution for the biocompatible hydrogel is a gelatin methacrylamide prepolymer solution; in step S4, photocuring is used to cross-link and cure the prepolymer solution.

7. The use of the multifunctional hydrogel patch as described in claim 1 in the preparation of drugs or medical materials that promote wound healing.

Images