A polypeptide-based composite sponge dressing with antibacterial performance and a preparation method and application thereof

Abstract

The application belongs to the field of biomedical engineering, and particularly relates to a polypeptide-based composite sponge dressing with antibacterial performance and a preparation method and application thereof. The polypeptide-based composite sponge dressing with antibacterial performance comprises a polypeptide-based composite sponge dressing substrate and an antibacterial peptide loaded in the polypeptide-based composite sponge dressing substrate; the polypeptide-based composite sponge dressing substrate has a first network structure formed by cross-linking reaction of a bioactive macromolecule and a bioactive polypeptide, and also has a second network structure formed by topological polymerization between the bioactive polypeptides through a diacetylene group. The polypeptide-based composite sponge dressing with antibacterial performance not only has good mechanical strength, porosity and water vapor transmission rate, but also has good water absorption rate, and meanwhile, the bioactivity of the polypeptide is maintained; compared with the prior art, the physiological stability of the bioactive polypeptide in the dressing can be improved; the antibacterial peptide is loaded in the polypeptide-based composite sponge dressing substrate with the bioactive polypeptide, and has the functions of antioxidation, antibiosis, promotion of angiogenesis and promotion of cell migration, and can accelerate chronic wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically relating to a polypeptide-based composite sponge dressing with antibacterial properties, its preparation method, and its application. Background Technology

[0002] Chronic wounds are a major global health problem. These wounds are difficult to heal due to disrupted healing mechanisms. With the increasing aging of the global population, chronic wounds such as venous stasis ulcers, pressure ulcers, and diabetic wounds have become serious threats to human health, placing a huge burden on global healthcare systems.

[0003] The routine clinical treatment of chronic wounds relies on meticulous local care (including debridement, infection / inflammation control, and frequent dressing changes), but the overall cure rate is low, the recurrence rate is high, and it requires professional care. Long-term professional care not only places a financial and lifestyle burden on patients but also negatively impacts their mental health to some extent.

[0004] Chronic wounds, characterized by prolonged low-level inflammation and bacterial infection, accumulate excessive reactive oxygen species (ROS) at the wound surface, hindering angiogenesis and epithelialization, and delaying wound healing. Traditional wound dressings such as gauze, films, and foams can only act as physical barriers and absorb exudate, failing to meet all the needs of the entire chronic wound healing process. Therefore, improving treatment methods and developing bioactive, multifunctional wound dressings are crucial for chronic wound healing.

[0005] Peptides, as molecules with excellent bioactivity and biodegradability, have been proven to have significant effects in wound healing, antibacterial activity, and anti-inflammation. However, existing peptides have poor physiological stability, making it difficult to meet the high requirements of clinical wound dressings. The present invention aims to overcome the shortcomings of existing technologies by combining peptides with sponge dressing materials, developing a peptide-based composite sponge dressing with antioxidant, antibacterial, angiogenesis-promoting, and cell migration-promoting properties, which can accelerate the healing of chronic wounds. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a polypeptide-based composite sponge dressing with antibacterial properties, its preparation, and its application. This polypeptide-based composite sponge dressing with antibacterial properties not only possesses good mechanical strength, porosity, and water vapor permeability, but also good water absorption, while maintaining the bioactivity of the polypeptides. Compared to existing technologies, it can improve the physiological stability of bioactive polypeptides in the dressing. Furthermore, the antibacterial peptides of this invention are loaded into the polypeptide-based composite sponge dressing substrate containing bioactive polypeptides, exhibiting antioxidant, antibacterial, angiogenesis-promoting, and cell migration-promoting effects, and can accelerate the healing of chronic wounds.

[0007] The first aspect of the present invention is to provide a polypeptide-based composite sponge dressing with antibacterial properties, comprising a polypeptide-based composite sponge dressing substrate and an antibacterial peptide loaded in the polypeptide-based composite sponge dressing substrate;

[0008] The polypeptide-based composite sponge dressing substrate has a first network structure formed by cross-linking of bioactive macromolecules and bioactive polypeptides.

[0009] The bioactive polypeptide contains double bond functionalization modification and diacetylene group functionalization modification, and the bioactive macromolecule contains unsaturated groups. The double bond functionalization modification of the bioactive polypeptide and the unsaturated groups contained in the bioactive macromolecule are each independently methacryloyl groups. The first network structure is formed by covalent cross-linking of the methacryloyl groups side-linked to the bioactive macromolecule and the methacryloyl groups end-linked to the polypeptide through free radical polymerization.

[0010] The polypeptide-based composite sponge dressing substrate also has a second network structure formed by the topological polymerization of bioactive polypeptides through diacetylene groups.

[0011] Through research, the inventors of this invention have developed a polypeptide-based composite sponge dressing substrate by using bioactive polypeptides with double bond functionalization and diacetylene group functionalization to form a first network structure through cross-linking reactions with bioactive macromolecules with double bond functionalization. Furthermore, a second network structure is formed by topological polymerization of bioactive polypeptides through diacetylene groups. The polypeptides are covalently linked to bioactive macromolecules within the sponge dressing. This dressing has a sponge-like, porous structure and exhibits good mechanical strength, porosity, and water vapor permeability. It also possesses good water absorption and retention rates while maintaining the bioactivity of the polypeptides. Compared to existing technologies, this improves the physiological stability of the bioactive polypeptides in the dressing. Antimicrobial peptides loaded in the polypeptide-based composite sponge dressing substrate (with a dual network structure) possess antioxidant, antibacterial, angiogenesis-promoting, and cell migration-promoting properties, and can accelerate the healing of chronic wounds.

[0012] This invention uses a polypeptide-based composite sponge dressing as a base material. This base material itself contains bioactive polypeptides covalently bonded to the sponge dressing, exhibiting a sponge-like, porous structure. It possesses not only good mechanical strength, porosity, and water vapor permeability, but also good water absorption, while maintaining the bioactivity of the polypeptides. Compared to existing technologies, this improves the physiological stability of the bioactive polypeptides in the dressing. Furthermore, the polypeptide-based composite sponge dressing of this invention, based on this base material, loads antimicrobial peptides into the polypeptide-based composite sponge dressing base material, further exhibiting antioxidant, antibacterial, angiogenesis-promoting, and cell migration-promoting properties, and can accelerate the healing of chronic wounds.

[0013] According to the present invention, preferably, the bioactive polypeptide comprises two polypeptide chains, the double bond functionalization modification site is located on the amino group of the side chain of the C-terminal lysine of the polypeptide chain, and the diacetylene group couples the two polypeptide chains from the N-terminus of the polypeptide chain.

[0014] More preferably, the polypeptide chain is derived from a polypeptide raw material containing 2-20 amino acids (e.g., 2, 4, 6, 10, 14, 16, 18, 20 amino acids, or any two values ​​or any range of two values ​​of amino acids), and / or, one end of the polypeptide raw material is lysine, and / or, the polypeptide raw material is a synthetic polypeptide, preferably synthesized by the SPPS method.

[0015] More preferably, the polypeptide raw material has at least one of the properties of promoting wound healing, antibacterial, anti-inflammatory, and anti-wrinkle.

[0016] More preferably, the polypeptide raw material has wound healing properties and is preferably selected from at least one of thymosin β4, RGD cell adhesion peptide, sensory neuropeptide SP, hexapeptide, and antimicrobial tetrapeptide.

[0017] According to the present invention, preferably, the double bond functionalization modification is a methacryloyl group; and / or, the diacetylene group functionalization modification is derived from diacetic acid compounds, preferably from at least one of 3,5-octadiynediic acid, 5,7-dodecadiynediic acid, 7,9-hexadecadiynediic acid, 9,11-eicosadiynediic acid, 10,12-eicosadiynediic acid, and 4,4'-(but-1,3-diyne-1,4-diyl)dibenzoic acid; more preferably,

[0018] The bioactive polypeptide is at least one of the following compounds, where DA is a diacetyl group derived from diacetic acid compounds, and the side chain linked to Lys is a methacryloyl group:

[0019] Methacrylic acid

[0020] Lys(Methacrylic acid)-Asp-Gly-Arg-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproic acid-Arg-Gly-Asp-Lys(Methacrylic acid);

[0021] Lys(Methacrylic acid)-Arg-Arg-Gln-Met-Glu-Glu-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-6-Aminocaproic acid-Glu-Glu-Met-Gln-Arg-Arg-Lys(Methacrylic acid);

[0022] Methacrylic acid

[0023] Methacrylic acid

[0024] According to the present invention, preferably, the bioactive polypeptide is prepared by the following method: the C-terminal lysine side chain amino group of the polypeptide raw material is subjected to an amidation reaction with methacrylic acid to obtain a polypeptide raw material with double bond functionalization modification;

[0025] The carboxyl group of a diacetyl acid compound is amidated with the amino group of the N-terminal amino acid of a polypeptide raw material to couple the two polypeptide chains.

[0026] More preferably, the method for preparing the bioactive polypeptide includes:

[0027] (1) The polypeptide raw material was subjected to an amidation reaction with methacrylic acid in the presence of benzotriazol-1-yl-oxytripyrrolylphosphine (PyBOP) and diisopropylethylamine (DIPEA) to obtain a polypeptide raw material whose C-terminal lysine side chain amino group was functionalized by double bond.

[0028] (2) The double bond functionalized peptide raw material is then subjected to an amidation reaction with diacetyl acid compounds in the presence of PyBOP and DIPEA. The two peptide chains are coupled from the N-terminus of the peptide raw material through the diacetyl group to obtain a bioactive peptide.

[0029] More preferably, the diacetic acid compound is selected from at least one of 3,5-octadiynediic acid, 5,7-dodecadiynediic acid, 7,9-hexadecadiynediic acid, 9,11-eicosadiynediic acid, 10,12-docosadiynediic acid, and 4,4'-(but-1,3-diyne-1,4-diyl)dibenzoic acid; and / or,

[0030] More preferably, the mass ratio of the polypeptide raw material to methacrylic acid is 1:(1-8); and / or,

[0031] More preferably, the mass ratio of the C-terminal double-bond functionalized peptide raw material to the diacetic acid compound is 1:(0.4-0.6); and / or,

[0032] More preferably, the reaction conditions include: in steps (1) and (2), the ratio of the amount of PyBOP to the molar amount of the reactant based on carboxyl groups is 1:(0.9-1.1); and / or, in steps (1) and (2), the ratio of the amount of DIPEA to the molar amount of the reactant based on carboxyl groups is 2:(0.9-1.1).

[0033] Regarding the preparation method of bioactive peptides, the specific conditions for double bond functionalization modification in this invention can be referred to the method described in "Nat.Comm.2024,15,2038"; the specific conditions for diacetylene group functionalization modification can be referred to the method described in "J.Am.Chem.Soc.2012,134,2028-2031".

[0034] According to the present invention, preferably, the bioactive macromolecule is selected from at least one of methacrylamide gelatin, methacrylamide silk fibroin, methacrylamide hyaluronic acid, methacrylamide chondroitin sulfate, methacrylamide sodium alginate, methacrylamide chitosan, methacrylamide dextran, methacrylamide heparin, polyether F127 diacrylate, polyethylene glycol diacrylate, tetra-arm polyethylene glycol acrylate, and methacrylamidized polyvinyl alcohol.

[0035] The above-mentioned bioactive macromolecules are commercially available. Preferably, the degree of substitution of the bioactive macromolecules with methacrylylation is 70% or more, and more preferably 70%-95%.

[0036] According to the present invention, preferably, the mass ratio of bioactive macromolecules and bioactive peptides is (1-10):1, for example, 1, 3, 5, 7, 9, 10, and the ratio of any two values ​​or any range of any two values ​​to 1, preferably (4-6):1.

[0037] According to the present invention, preferably, the polypeptide-based composite sponge dressing with antibacterial properties and the polypeptide-based composite sponge dressing substrate each have at least one of the following characteristics:

[0038] The compression modulus is tested using a compression method and is not less than 50 kPa, preferably 270-450 kPa.

[0039] The porosity was determined using the ethanol immersion method to be 40%-95%, preferably 40%-75%.

[0040] The water vapor transmission rate, determined by gravity method, was 700-2000 g / m³ after 48 hours. 2 Preferably 800-1200g / m 2 ;

[0041] The water absorption rate, measured by specific gravity at 37°C and 48 hours, was 300%-1100%; preferably 300%-600%.

[0042] The water retention rate at 37℃ and 24 hours was determined to be 40%-75% using the specific gravity method.

[0043] The detection methods for the above items are all existing technologies. For example, specific detection methods are described in the embodiments of the present invention.

[0044] According to the present invention, preferably, the preparation method of the polypeptide-based composite sponge dressing substrate includes:

[0045] The bioactive macromolecules and bioactive peptides are subjected to a first polymerization in solution in the presence of an initiator to obtain a gel-like intermediate.

[0046] The gel-like intermediate was freeze-dried to obtain a sponge-like intermediate.

[0047] The sponge-like intermediate is subjected to a second polymerization by light irradiation to obtain the polypeptide-based composite sponge dressing substrate.

[0048] According to the present invention, preferably, the antimicrobial peptide is loaded in the network structure of the polypeptide-based composite sponge dressing substrate.

[0049] According to the present invention, preferably, the antimicrobial peptide has antimicrobial properties, preferably contains 5-50 amino acids, and / or preferably the antimicrobial peptide is a synthetic polypeptide, preferably synthesized by the SPPS method.

[0050] More preferably, the antimicrobial peptide is selected from at least one of the following antimicrobial peptides:

[0051] Antimicrobial peptide a, with the sequence Lys-Lys-Glu (dopamine)-Phe-Glu (dopamine)-Phe-Glu (dopamine)-Phe-Glu (dopamine)-Phe-Lys-Lys;

[0052] Lysozyme, with the sequence Tyr-Cys-Ser-Asp-Val-Gly-Val-Ala-Glu-Pro-Ala-Ser;

[0053] Human Defensin-1 has the sequence Gly-Cys-Cys-Gly-Gly-Gly-Cys-Cys-Gly.

[0054] Bactenecin, with the sequence Gly-Gly-Gly-Phe-Gly-Gly-Ala-Ser-Lys-Phe-Leu-Ala-Ser;

[0055] Magaiinin-2, a metalloproteinase inhibitory peptide, has the sequence Gly-Gly-Ser-Tyr-Ser-Gly-Cys-Glu-Val-Ser-Tyr-Ser-Lys;

[0056] Human antimicrobial peptide LL-37, sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES;

[0057] Cecropin A, sequence: AAAACATTCAGGAGAGCACTAAAGGAGGGA;

[0058] Batmin-1, with the sequence TGLKRKVRKKSAAKKWKKLKS.

[0059] According to the present invention, preferably, based on 100 parts of the total mass of the polypeptide-based composite sponge dressing substrate, the content of antimicrobial peptides in the polypeptide-based composite sponge dressing with antibacterial properties is 1-20 parts.

[0060] According to the present invention, preferably, the preparation method of the polypeptide-based composite sponge dressing with antibacterial properties includes: performing a first polymerization of the bioactive macromolecule and the bioactive polypeptide in an aqueous solution containing an antimicrobial peptide in the presence of an initiator to obtain a gel-like intermediate;

[0061] The gel-like intermediate was freeze-dried to obtain a sponge-like intermediate.

[0062] The sponge-like intermediate is subjected to a second polymerization by light irradiation to obtain the polypeptide-based composite sponge dressing with antibacterial properties.

[0063] A second aspect of the present invention is to provide a method for preparing the polypeptide-based composite sponge dressing with antibacterial properties as described above, comprising: performing a first polymerization of the bioactive macromolecule and the bioactive polypeptide in an aqueous solution containing an antimicrobial peptide in the presence of an initiator to obtain a gel-like intermediate;

[0064] The gel-like intermediate was freeze-dried to obtain a sponge-like intermediate.

[0065] The sponge-like intermediate is subjected to a second polymerization by light irradiation to obtain the polypeptide-based composite sponge dressing with antibacterial properties.

[0066] According to the present invention, preferably, the preparation method of the polypeptide-based composite sponge dressing with antibacterial properties includes the following steps:

[0067] (1) The raw materials, including bioactive macromolecules, bioactive peptides, antimicrobial peptides and initiators, are mixed in water and degassed to obtain a prepolymer solution.

[0068] (2) The prepolymer liquid is subjected to a first polymerization under free radical polymerization conditions to obtain a gel-like intermediate;

[0069] (3) Freeze-dry the gel-like intermediate to obtain a sponge-like intermediate;

[0070] (4) The sponge-like intermediate is subjected to a second polymerization under ultraviolet light irradiation: the diacetylene unit in the bioactive polypeptide undergoes topological polymerization to obtain a polypeptide-based composite sponge dressing with antibacterial properties.

[0071] Preferably, based on parts by weight, relative to 1000 parts of water, the prepolymer solution contains: 30-150 parts of bioactive macromolecules, for example, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 parts, or any two values ​​or any range of any two values, preferably 50-100 parts; and 5-30 parts of bioactive peptides, for example, 5, 8, 10, 13, 15, 20, 25, 30 parts, etc. The dosage of the antimicrobial peptide is 5-30 parts, such as 5, 8, 10, 13, 15, 20, 25, or 30 parts, and any two values ​​or any range of two values, preferably 10-20 parts; the dosage of the initiator is 1-10 parts, such as 1, 2, 3, 4, 5, 6, 8, or 10 parts, and any two values ​​or any range of two values, preferably 4-6 parts.

[0072] Preferably, the initiator is selected from at least one of per-2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0073] Preferably, the conditions for the first polymerization include:

[0074] The irradiation is performed under ultraviolet or blue light, and / or for a time of 1-15 minutes, such as 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 15 minutes, or any two values ​​or any range of any two values; preferably, irradiation under ultraviolet or blue light for 5-15 minutes. For example, irradiation can be performed under ultraviolet light (e.g., 365 nm) with a radiation power of 1-100 mW; or under blue light (e.g., 405 nm) with a radiation power of 1-100 mW.

[0075] Preferably, the conditions for the second polymerization include:

[0076] The procedure is performed under ultraviolet light irradiation for 1-15 minutes, such as 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes, 15 minutes, or any two values ​​or any range of any two values; preferably, the ultraviolet light irradiation is performed for 5-15 minutes.

[0077] The preparation method of the above-mentioned polypeptide-based composite sponge dressing with antibacterial properties differs in that no antibacterial peptides are added to the raw materials in the first polymerization, thus obtaining the polypeptide-based composite sponge dressing substrate.

[0078] A third aspect of the present invention is to provide the aforementioned polypeptide-based composite sponge dressing with antibacterial properties, or the polypeptide-based composite sponge dressing with antibacterial properties prepared by the aforementioned preparation method, as a wound dressing for the healing of chronic wounds, preferably for the healing of venous congestion ulcers, pressure ulcers, and diabetic wounds.

[0079] According to the present invention, the porous structure of sponge dressings plays several key roles in wound care, mainly in the following aspects:

[0080] Absorbing exudate: The porous structure of sponge dressings effectively absorbs wound exudate, which is crucial for maintaining a moist wound environment and accelerating the healing process. Accumulated exudate provides a breeding ground for bacteria; proper absorption can prevent infection.

[0081] Promoting gas exchange: The porous structure of sponge dressings provides ventilation space for the wound, allowing it to breathe normally. Sufficient oxygen supply helps promote cell proliferation and angiogenesis, thereby accelerating healing.

[0082] Reducing pressure and friction: The softness and porosity of sponge dressings can disperse external pressure to some extent, reducing friction and pressure on the wound. This is crucial for protecting the wound from external irritation and alleviating patient discomfort.

[0083] Promoting Cell Growth and Adhesion: Porous structures provide a supporting surface for cell attachment and growth. This is especially important during wound healing, when new cells need to grow directionally on the dressing surface. Porous structures enhance the interaction between cells and the dressing, thereby promoting healing.

[0084] Dispersed drug release: If the dressing contains medications or bioactive substances, the pores of the sponge can help release these substances slowly, allowing them to exert their therapeutic effect continuously. This controlled release mechanism helps maintain the long-lasting effect of the medication on the wound surface and reduces the inconvenience of frequent dressing changes.

[0085] Reduced risk of infection: The porous sponge dressing of the present invention is used as the base material and is specially designed to contain antibacterial components. Its porous structure helps these antibacterial components to be evenly distributed on the wound surface and improves their antibacterial effect, thereby effectively reducing the risk of wound infection.

[0086] The polypeptide-based composite sponge dressing substrate developed in this invention has significant clinical application value and market prospects. Its main innovations are reflected in the following aspects:

[0087] This invention preserves the bioactivity of polypeptides. For example, using the preferred polypeptides of this invention improves antibacterial properties: the above polypeptides have natural antibacterial properties, and by combining them with sponge materials, they can effectively reduce bacterial infection, prevent secondary infection of wounds, and promote wound healing; promoting healing: polypeptides play an important role in promoting cell proliferation and wound healing, and can accelerate the wound repair process.

[0088] Biocompatibility and biodegradability: As a natural biomolecule, polypeptides have good biocompatibility and biodegradability, which can reduce irritation and allergic reactions to the human body. At the same time, composite sponge dressings can degrade on their own after wound healing, reducing the risk of secondary surgery.

[0089] Adaptability to specific wounds: The polypeptide-based composite sponge dressing substrate can be customized according to different wound types (such as diabetic foot, chronic wounds, etc.), which has strong targeting and therapeutic effect.

[0090] The polypeptide-based composite sponge dressing substrate developed in this invention not only improves the performance of traditional dressings but also addresses some shortcomings of existing products in clinical use, such as insufficient bioactivity, slow healing speed, and inability to meet personalized needs. It possesses significant innovation and market demand. The research and application of this technology will bring new breakthroughs to the field of wound care and provide more effective solutions for clinical treatment.

[0091] In summary, the porous structure of the sponge dressing of this invention provides an ideal physical and chemical environment for wound healing, enabling it to absorb exudate, promote gas exchange, support cell growth, and potentially reduce the risk of infection. Furthermore, the cross-linking of bioactive peptides into the sponge dressing in this invention can increase the tissue retention time of the bioactive peptides and improve their physiological stability during application.

[0092] Compared with the prior art, the advantages of the present invention are:

[0093] This invention crosslinks specially designed bioactive macromolecules and specially designed bioactive peptides (the bioactive peptides contain double bond functionalization modification and diacetylene group functionalization modification, and the bioactive macromolecules contain unsaturated groups), and then performs topological polymerization of the bioactive peptides themselves. In this process, preferably, the antimicrobial peptides of this invention and the peptide-based composite sponge dressing substrate are molded together, and the antimicrobial peptides are loaded in the substrate to obtain a peptide-based composite sponge dressing. This peptide-based composite sponge dressing not only has good mechanical properties and suitable water vapor permeability, but also excellent moisturizing ability. On the basis of the above physical properties, the peptide-based composite sponge dressing of this invention still retains the cellular activity of the bioactive peptides and antimicrobial peptides. By selecting the preferred type of bioactive peptides of this invention, it can also promote angiogenesis and cell migration, achieving unexpected technical effects.

[0094] Furthermore, this invention crosslinks bioactive peptides into the sponge dressing and molds antimicrobial peptides together with the substrate, which can improve the tissue retention time of bioactive peptides and antimicrobial peptides, thereby enhancing the physiological stability of the bioactive peptides and antimicrobial peptides in the dressing during application. The peptide-based composite sponge dressing with antibacterial properties, through the synergistic effect of antimicrobial peptides and bioactive peptides, exhibits further superior antibacterial and wound-healing-promoting properties.

[0095] The advantages of the antibacterial polypeptide-based composite sponge dressing of this invention in the treatment of chronic wounds are mainly reflected in promoting wound healing, enhancing antibacterial properties, good biocompatibility and degradability, accelerating the repair process, controlling drug release, and strong adaptability. Its innovative and comprehensive effects make it a promising treatment option for chronic wound care.

[0096] Overall, compared with the prior art, the above technical solutions of the present invention can achieve at least the following beneficial effects.

[0097] (1) The polypeptide-based composite sponge dressing with antibacterial properties provided by the present invention has good ability to absorb tissue exudate, water retention and moisturizing ability, as well as good mechanical strength, porosity and water vapor permeability.

[0098] (2) The polypeptide-based composite sponge dressing with antibacterial properties provided by the present invention can promote angiogenesis and epidermal cell migration through wound healing peptides.

[0099] (3) The polypeptide-based composite sponge dressing with antibacterial properties provided by this invention has good antibacterial properties. The antimicrobial peptides loaded in the sponge dressing can damage the cell membranes of bacteria and fungi, causing leakage of cell contents (DNA, RNA). It kills bacteria and fungi and has excellent antibacterial properties against drug-resistant bacteria.

[0100] (4) The polypeptide-based composite sponge dressing with antibacterial properties provided by the present invention has good ROS removal performance. Preferably, ROS reacts with catechol groups on the side chains of antibacterial peptides to form quinones, thereby reducing the overexpressed ROS in wound tissues and cells and alleviating oxidative stress damage to cells and tissues.

[0101] (5) The polypeptide-based composite sponge dressing provided by the present invention can accelerate wound healing and reduce inflammatory response in a rat diabetic wound model. Attached Figure Description

[0102] Figure 1 The molecular structure of the antimicrobial peptide in the polypeptide-based composite sponge dressing of this invention is shown.

[0103] Figure 2 This invention demonstrates the antibacterial activity of the polypeptide-based composite sponge dressing.

[0104] Figure 3 This invention relates to the antioxidant properties of the polypeptide-based composite sponge dressing.

[0105] Figure 4 This is a schematic diagram illustrating the effect of the polypeptide-based composite sponge dressing of the present invention on promoting wound healing in diabetic rats. Detailed Implementation

[0106] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0107] In this embodiment, the polypeptide raw material, such as the wound healing peptide, is synthesized using the standard solid-phase synthesis (SPPS) method. The effective sequence of the wound healing peptide is derived from thymosin β4 (Tβ4), which has biological activities such as promoting angiogenesis and cell migration.

[0108] The bioactive peptides in this invention are modified by SPPS or SPPS-combined liquid-phase synthesis (LPPS) to perform double bond functionalization and diacetylene functionalization on the peptide raw materials. The double bond functionalization is performed according to the method described in Nat. Comm. 2024, 15, 2038. The diacetylene functionalization is performed according to the method described in J. Am. Chem. Soc. 2012, 134, 2028-2031.

[0109] In the following implementation, the methacrylamide gelatin has a substitution degree of 75% to 90% and is manufactured by EFL, namely Suzhou Yongqinquan Intelligent Equipment Co., Ltd.

[0110] Methacrylamide hyaluronic acid, with a substitution degree of 75%–80%, manufactured by EFL;

[0111] Sodium methacrylate, with a substitution degree of 70%–80%, is manufactured by EFL.

[0112] Unless otherwise specified, the equipment and reagents used in this embodiment are all commercially available products. Unless otherwise specified, the preparation temperature of this invention is at room temperature (25°C).

[0113] Preparation Example 1

[0114] Preparation of wound healing peptides (i.e., bioactive polypeptides), method (1): SPPS

[0115] 1) Weigh 1.0 g of Rink Amide-MBHA resin into the peptide synthesis apparatus, add analytical grade N,N-dimethylformamide (DMF) to swell the resin for 2 hours, and then remove the solvent under argon pressure. Then, deprotect the resin with 10 mL of DMF solution containing 20% ​​piperidine, reacting twice, 20 min each time. After the reaction, wash the resin three times with DMF. Next, take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin color changes from white to purple. This indicates successful deprotection and the amino acid coupling reaction can proceed. Otherwise, the deprotection reaction needs to be continued.

[0116] 2) Weigh 0.8914 g of Fmoc-Lys(Alloc)-OH and 1.0252 g of benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBOP), dissolve them in 10 mL of DMF, and then transfer them to the peptide synthesis apparatus containing the treated resin from step 1). Add 690 μL of diisopropylethylamine (DIPEA) to the system and react at room temperature for 4 h. Then wash the resin twice with 10 mL of DMF, and react again for 4 h using the same proportion to ensure complete coupling to the resin. Finally, wash the resin three times with 10 mL of DMF, and place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling. If the resin does not change color, the first amino acid has been successfully coupled to the resin. Otherwise, the coupling reaction needs to be continued.

[0117] 3) Deprotect the first amino acid with 10 mL of DMF solution containing 20% ​​piperidine. Repeat the reaction twice, each time for 20 minutes. After the reaction, wash the resin three times with DMF. Then, place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin color changes from white to purple. This indicates successful deprotection of the first amino acid, allowing subsequent amino acid coupling reactions to proceed. Otherwise, the deprotection reaction needs to be continued.

[0118] 4) Referring to steps 2) and 3), condense Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-6-aminohexanoic acid, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Val-OH, and Fmoc-Gly-OH in sequence.

[0119] 5) Weigh 0.0758 g of tetra(triphenylphosphine)palladium, dissolve it in 10 mL of dichloromethane (DCM), and then transfer the solution to the peptide synthesis apparatus containing the treated resin from step 4). Add 1943 μL of phenylsilane to the system to remove the protecting group of the lysine side chain. React twice, 20 min each time. After the reaction, wash the resin three times with DMF. Then, take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe the resin color change from white to purple. This indicates that the lysine side chain has been successfully deprotected, and the subsequent amino acid coupling reaction can proceed. Otherwise, the deprotection reaction needs to be continued.

[0120] 6) Weigh 170 μL of methacrylic acid and 1.0252 g of PyBOP, dissolve them in 10 mL of DMF, and then transfer them to the peptide synthesis apparatus containing the treated resin from step 5). Add 690 μL of DIPEA to the system and react at room temperature for 4 h. Then wash the resin twice with 10 mL of DMF, and react again for 4 h using the same proportion to ensure complete coupling to the resin. Finally, wash the resin three times with 10 mL of DMF, and place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling. If the resin does not change color, the amino acid and lysine side chain have been successfully coupled. Otherwise, the coupling reaction needs to be continued.

[0121] 7) Deprotect the resin obtained in step 6) as in step 3).

[0122] 8) Weigh 0.0720 g of DA (4,4'-(butyryl-1,3-diyne-1,4-diyl)benzoic acid) and 0.260 g of PyBOP, dissolve them in 10 mL of DMF, and then transfer them to the peptide synthesis apparatus containing the treated resin from step 7). Add 175 μL of DIPEA to the system and react at room temperature for 8 h. Finally, wash the resin three times with 10 mL of DMF. Take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat it to boiling, and observe whether the resin changes color. If so, the amino acid and lysine side chain have been successfully coupled. Otherwise, the coupling reaction needs to be continued.

[0123] 9) Wash the resin three times each with 10 mL of DCM, methanol, and DCM in sequence, and then dry the resin with a vacuum oil pump for 1 hour.

[0124] 10) Add 9.5 mL of trifluoroacetic acid (TFA), 0.25 mL of triisopropylsilane (TIS), and 0.25 mL of deionized water to the peptide reaction apparatus. React at room temperature for 3 h to cleave and separate the peptide from the resin. Slowly add the cleavage solution dropwise into ice-cold ether, and a white precipitate will appear. The crude product was then separated by centrifugation at 3000 rpm for 10 min. The supernatant was discarded, and the precipitate was dissolved in an appropriate amount of methanol. The product was purified by a medium-pressure purification chromatography system, and finally freeze-dried to collect the white powder, which yielded the sequence Lys(Methacrylic acid)-Gln-Thr-Glu-Thr-Lys-Lys-Leu-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproicacid-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Lys(Methacrylic acid), where DA refers to 4,4'-(but-1,3-diyne-1,4-diyl)benzoyl, and Methacrylic acid refers to methacrylic acid. This product is a wound-healing peptide that promotes angiogenesis and cell migration.

[0125] Preparation Example 2

[0126] Preparation of wound healing peptides, method (2): SPPS combined with LPPS

[0127] 1) Weigh 1.0 g of Sieber Amide resin into the peptide synthesis apparatus, add analytical grade DMF to swell the resin for 2 hours, and then purge the solvent under argon pressure. Then, deprotect the resin with 10 mL of DMF solution containing 20% ​​piperidine, reacting twice, 20 min each time. After the reaction, wash the resin three times with DMF. Next, take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin color changes from white to purple. If so, the resin deprotection is successful and the amino acid coupling reaction can proceed. Otherwise, the deprotection reaction needs to be continued.

[0128] 2) Refer to steps 2)-7) in method (1) to couple amino acids.

[0129] 3) Wash and dry the resin according to step 9) in method (1).

[0130] 4) Add 0.5 mL TFA and 9.5 mL DCM to the peptide reaction apparatus and react at room temperature for 3 h to cleave and separate the peptide from the resin. Slowly add the cleavage solution dropwise into ice-cold ether, and a white precipitate will appear. Then centrifuge at 3000 rpm for 10 min to separate the crude product, discard the supernatant, and collect the white solid intermediate 1, which is the crude product with the sequence Lys(Methacrylicacid)-Gln(Trt)-Thr(tBu)-Glu(OtBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Leu-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly.

[0131] 5) Weigh 0.0720g DA (4,4'-(butyryl-1,3-diyne-1,4-diyl)dibenzoic acid) and 0.260g PyBOP, dissolve them in 10mL DMF, then transfer them to intermediate 1 obtained in step 4), and add 175μL DIPEA to the system. React at room temperature for 8h.

[0132] 6) The reaction solution was slowly added dropwise to ice-cold ether, and a white precipitate immediately appeared. The crude product was then separated by centrifugation at 3000 rpm for 10 min. The supernatant was discarded to obtain intermediate 2, with the sequence Lys(Methacrylicacid)-Gln(Trt)-Thr(tBu)-Glu(OtBu)-Thr(tBu)-Lys(Boc)-Lys(Boc)-Leu-6-Aminocaproic

[0133] acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproic

[0134] acid-Leu-Lys(Boc)-Lys(Boc)-Thr(tBu)-Glu(OtBu)-Thr(tBu)-Gln(Trt)-Lys(Methacrylic acid).

[0135] 7) Add 9.5 mL TFA, 0.25 mL TIS, and 0.25 mL deionized water to intermediate 2 and react at room temperature for 3 h to deprotect the polypeptide side chains. Slowly add the reaction solution dropwise to ice-cold ether, and a white precipitate will appear. Then centrifuge at 3000 rpm for 10 min to separate the crude product, discard the supernatant, dissolve the precipitate in an appropriate amount of methanol, purify the product using a medium-pressure purification chromatography system, and finally freeze-dry to collect the white powder, which is the product with the sequence Lys(Methacrylic acid)-Gln-Thr-Glu-Thr-Lys-Lys-Leu-6-Aminocaproic

[0136] Methacrylic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproicacid-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Lys (Methacrylic acid) is a wound-healing peptide that promotes angiogenesis and cell migration.

[0137] Preparation Example 3

[0138] A second bioactive polypeptide, a cell adhesion peptide, was synthesized using the same method as in Preparation Example 1, except that the amino acid sequence was different. The resulting bioactive polypeptide had the sequence Lys(Methacrylic acid)-Asp-Gly-Arg-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproicacid-Arg-Gly-Asp-Lys(Methacrylic acid).

[0139] Preparation Example 4

[0140] A third bioactive polypeptide, a hexapeptide, was synthesized using the same method as in Preparation Example 1, except that the amino acid sequence was different. The resulting bioactive polypeptide had the sequence Lys(Methacrylic acid)-Arg-Arg-Gln-Met-Glu-Glu-6-Aminocaproicacid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproicacid-Glu-Glu-Met-Gln-Arg-Arg-Lys(Methacrylic acid).

[0141] Preparation Example 5

[0142] A fourth bioactive polypeptide was synthesized using the same method as in Preparation Example 1. The difference was that 3,5-octadiyndiic acid replaced 4,4'-(but-1,3-diyn-1,4-diyl)dibenzoic acid, resulting in a bioactive polypeptide with the sequence Lys(Methacrylic acid)-Phe-Phe-Gly-Arg-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-3,5-octadiyndiic acid-Gly-Val-Val-Ala-Ala-6-Aminocaproic acid-Arg-Gly-Phe-Phe-Lys(Acrylic acid).

[0143] The antimicrobial peptides in the following examples were synthesized using the SPPS method and exhibit bioactivity against drug-resistant bacteria and fungi.

[0144] Preparation Example 1B

[0145] The preparation of antimicrobial peptides includes the following sub-steps:

[0146] 1) Weigh 1.0 g of Rink Amide-MBHA resin into the peptide synthesis apparatus, add analytical grade DMF to swell the resin for 2 hours, and then remove the solvent under argon pressure. Then, deprotect the resin with 10 mL of DMF solution containing 20% ​​piperidine, reacting twice, 20 min each time. After the reaction, wash the resin three times with DMF. Next, take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin color changes from white to purple. This indicates successful deprotection and the amino acid coupling reaction can proceed. Otherwise, the deprotection reaction needs to be continued.

[0147] 2) Weigh 0.9230g Fmoc-Lys(Boc)-OH and 1.0252g PyBOP, dissolve them in 10mL DMF, and then transfer them to the peptide synthesis apparatus containing the treated resin from step 1). Add 690μL DIPEA to the system and react at room temperature for 4 hours. Then wash the resin twice with 10mL DMF, and react again for 4 hours using the same proportion to ensure complete coupling to the resin. Finally, wash the resin three times with 10mL DMF, and place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling. If the resin does not change color, the first amino acid has been successfully coupled to the resin. Otherwise, the coupling reaction needs to be continued.

[0148] 3) Deprotect the first amino acid with 10 mL of DMF solution containing 20% ​​piperidine. Repeat the reaction twice, each time for 20 min. After the reaction, wash the resin three times with DMF. Then, place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin color changes from white to purple. This indicates successful deprotection of the first amino acid, allowing subsequent amino acid coupling reactions to proceed. Otherwise, the deprotection reaction needs to be continued.

[0149] 4) Referring to steps 2) and 3), sequentially condense Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Glu(OAll)-OH, Fmoc-Phe-OH, Fmoc-Glu(OAll)-OH, Fmoc-Phe-OH, Fmoc-Glu(OAll)-OH, Fmoc-Phe-OH, Fmoc-Glu(OAll)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH.

[0150] 5) Weigh 0.0462 g of tetra(triphenylphosphine)palladium, dissolve it in 10 mL of DCM, and then transfer the solution to the polypeptide synthesis apparatus containing the treated resin in step 4). Add 1455 μL of benzylsilane to the system to remove the lysine side chain protecting group. React twice, 30 min each time. After the reaction is completed, wash the resin three times with DMF.

[0151] 6) Weigh 0.9307 g of dopamine and 2.5520 g of PyBOP, dissolve them in 10 mL of DMF, and then transfer them to the peptide synthesis apparatus containing the treated resin from step 5). Add 820 μL of DIPEA to the system and react at room temperature for 4 h. Then wash the resin twice with 10 mL of DMF, and react again for 4 h with the same proportion of reactants to ensure complete coupling to the resin. Finally, wash the resin three times with 10 mL of DMF.

[0152] 7) Deprotect the resin obtained in step 6) as in step 3).

[0153] 8) Wash the resin three times each with 10 mL of DCM, methanol, and DCM in sequence, and then dry the resin with a vacuum oil pump for 1 hour.

[0154] 9) Add 9.5 mL TFA, 0.25 mL TIS, and 0.25 mL deionized water to the peptide reaction apparatus and react at room temperature for 3 h to separate the peptide from the resin. Slowly add the cleavage solution dropwise to ice-cold ether, and a white precipitate will appear. Then centrifuge at 3000 rpm for 10 min to separate the crude product, discard the supernatant, dissolve the precipitate in an appropriate amount of methanol, purify the product using a medium-pressure purification chromatography system, and finally freeze-dry to collect the white powder, which is the antibacterial and antioxidant antimicrobial peptide with the sequence Lys-Lys-Glu(Dopamine)-Phe-Glu(Dopamine)-Phe-Glu(Dopamine)-Phe-Glu(Dopamine)-Phe-Lys-Lys. The molecular structure of the antimicrobial peptide is as follows. Figure 1 As shown.

[0155] Example 1

[0156] The preparation of a polypeptide-based composite sponge dressing with antibacterial properties includes the following sub-steps:

[0157] 1) Weigh 50 mg of methacrylamide gelatin, 10 mg of wound healing peptide (prepared using Preparation Example 1), 5 mg of Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone), and 10 mg of antimicrobial peptide (prepared using Preparation Example 1B), dissolve them in 1 mL of deionized water, mix them evenly, and degas to obtain a prepolymer solution.

[0158] 2) The prepolymer solution was subjected to free radical polymerization under ultraviolet light (365nm, 3mW) irradiation to obtain a gel-like intermediate. Specifically, the irradiation was carried out for 10 minutes.

[0159] 3) The gel-like intermediate was freeze-dried to obtain the sponge-like intermediate.

[0160] 4) The sponge-like intermediate was irradiated with ultraviolet light (254nm, 3mW) to induce topological polymerization of the diacetylene units, thereby obtaining a polypeptide-based composite sponge dressing with antibacterial properties. Specifically, the irradiation was performed for 10 minutes.

[0161] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 1-JC.

[0162] Example 2

[0163] The polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the irradiation time in steps 2) and 4) was 1 min each.

[0164] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 2-JC.

[0165] Example 3

[0166] The polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the irradiation time in steps 2) and 4) was 3 min each.

[0167] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that antimicrobial peptides are not added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 3-JC.

[0168] Example 4

[0169] The polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the irradiation time in steps 2) and 4) was 5 min each.

[0170] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 4-JC.

[0171] Example 5

[0172] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method in Example 1, except that the amount of bioactive macromolecules used was 100 mg, the amount of bioactive polypeptides used was 20 mg, and the amount of antimicrobial peptides used was 20 mg.

[0173] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 5-JC.

[0174] Example 6

[0175] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method in Example 1, except that the initiator was changed to lithium phenyl-2,4,6-trimethylbenzoyl phosphite.

[0176] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 6-JC.

[0177] Example 7

[0178] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the bioactive polypeptide obtained in Preparation Example 3 was used to replace the bioactive polypeptide in Example 1.

[0179] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that antimicrobial peptides are not added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 7-JC.

[0180] Example 8

[0181] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the bioactive polypeptide obtained in Preparation Example 4 was used to replace the bioactive polypeptide in Example 1.

[0182] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that antimicrobial peptides are not added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 8-JC.

[0183] Example 9

[0184] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the bioactive polypeptide obtained in Preparation Example 5 was used to replace the bioactive polypeptide in Example 1.

[0185] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 9-JC.

[0186] Example 10

[0187] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that methacrylamide gelatin in Example 1 was replaced with methacrylamide-modified hyaluronic acid.

[0188] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 10-JC.

[0189] Example 11

[0190] A polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that sodium methacrylamide alginate was used instead of methacrylamide gelatin in Example 1.

[0191] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 11-JC.

[0192] Example 12

[0193] The polypeptide-based composite sponge dressing with antibacterial properties was prepared according to the method of Example 1, except that the irradiation time in steps 2) and 4) was 15 min each.

[0194] The steps and conditions for preparing a polypeptide-based composite sponge dressing with antibacterial properties in this embodiment are followed, except that no antimicrobial peptides are added in step 1). Samples are prepared in parallel to obtain a polypeptide-based composite sponge dressing substrate, which is referred to as Example 12-JC.

[0195] Performance testing

[0196] The prepared peptide-based composite sponge dressing substrate and the antibacterial peptide-based composite sponge dressing demonstrated bioactivity in promoting angiogenesis, thereby promoting chronic wound healing, using the following method: Human umbilical vein endothelial cells (HUVECs) were seeded onto a matrix adhesive plate, and then the cells were stimulated with the peptide-based composite sponge dressing substrate Example 1-JC (with the concentration of wound-healing peptides controlled at 100 μg / mL) prepared in Example 1. Angiogenesis was recorded using an optical microscope at different time points (as the experimental group) and compared with untreated cells. It was found that the experimental group (i.e., the Example group) promoted the faster formation of more tubular structures in HUVECs in vitro. Therefore, the peptide-based composite sponge dressing substrate obtained in Example 1 possesses bioactivity in promoting angiogenesis, forming important channels for transporting nutrients, oxygen, and other substances.

[0197] The following method was used to verify that the prepared peptide-based composite sponge dressing substrate and the antibacterial peptide-based composite sponge dressing promote cell migration: Human epithelial keratinocytes (HaCaT) were cultured to a density close to 100%, and 500-micrometer scratches were created in the cell culture plate. The cells were then stimulated with the peptide-based composite sponge dressing substrate prepared in Example 1 (with the concentration of wound-healing peptides controlled at 100 μg / mL). Cell migration was recorded using an optical microscope at different time points and compared with untreated cells. The results showed that the experimental group accelerated HaCaT migration and wound closure, indicating that the peptide-based composite sponge dressing substrate prepared in Example 1 has bioactivity that promotes cell migration, thereby achieving tissue re-epithelialization.

[0198] The performance of the polypeptide-based composite sponge dressing substrates obtained in Examples 2-12 and the polypeptide-based composite sponge dressings with antibacterial properties obtained in Examples 1-12 were tested using the same method as above. It was found that both the polypeptide-based composite sponge dressing substrates and the polypeptide-based composite sponge dressings with antibacterial properties have good bioactivity in promoting angiogenesis and good bioactivity in promoting cell migration. The addition of antimicrobial peptides resulted in even better antibacterial properties.

[0199] Water Absorption Capacity Test: As an example, the water absorption capacity of the polypeptide-based composite sponge dressing substrate prepared in the embodiment was tested according to the following method to evaluate the absorption capacity of the wound dressing provided by the present invention for wound tissue exudate. Specifically, the dry mass of the polypeptide-based composite sponge dressing substrate prepared in the embodiment was recorded. Then, it was placed in physiological saline and kept at 37°C for 24 hours to allow it to reach a saturation equilibrium state for water absorption. After removal, the surface liquid was wiped off with filter paper, and the wet mass was recorded. The water absorption rate was calculated as follows: Water absorption rate = (Wet mass - Dry mass) / Dry mass × 100%. The test was performed in parallel three times.

[0200] It was found that the polypeptide-based composite sponge dressing substrate prepared in the examples has good water absorption properties, and can bind nearly 10 times its own weight in water, which can absorb wound exudate and reduce the possibility of infection caused by exudate to the wound.

[0201] Water Retention Capacity Test: As an example, the water retention capacity of the polypeptide-based composite sponge dressing substrate prepared in the embodiment was tested according to the following method to evaluate the moisturizing ability of the wound dressing provided by the present invention for wound tissue. Specifically, the polypeptide-based composite sponge dressing substrate prepared in the embodiment was placed in physiological saline and placed at 37°C for 24 hours to allow it to absorb water to reach saturation equilibrium. After removal, the surface liquid was wiped off with filter paper, and the mass was recorded as 0. Subsequently, the saturated sponge dressing was placed in an environment of 37°C and 60% relative humidity, and the mass was recorded at different times. t Calculate the water retention rate: Water retention rate = (mass) / (volume)t / mass 0)×100%, three parallel tests.

[0202] The tests show that the polypeptide-based composite sponge dressing substrate prepared in the examples has good moisturizing ability, which avoids excessive loss of wound exudate and excessive dehydration of the wound, thereby delaying wound healing.

[0203] The method for detecting compressive modulus is as follows: The photocrosslinked sample is placed on a stainless steel plate of a mechanical testing machine, and the sensor compresses it at a rate of 1 mm / min, with a maximum loading force of 100 N. Throughout the test, the relationship between the applied stress and the sample strain is recorded.

[0204] The water vapor transmission rate was determined using a specific gravity method over 48 hours. Specifically, the prepared sample was placed in the mouth of a bottle containing 10 mL of deionized water, the bottle mouth was sealed with sealing film, and the initial weight (W0) was recorded. The bottle was then placed in a desiccator at 37°C and 79% relative humidity, with an uncoated bottle serving as a control. The bottle weight (W0) was recorded at 6, 12, 24, and 48 hours. t The water vapor transmission rate is calculated according to the formula, where A is the area of ​​the bottle opening.

[0205] Water vapor transmission rate = (W0 - W) t ) / A

[0206] The porosity was determined using the ethanol immersion method. Specifically, a 10mm diameter disc was prepared from the sample and immersed in ethanol until saturation. The mass (W0, W1) and apparent volume (V) of the sample before and after ethanol immersion were recorded. The porosity of the sample was calculated using the formula, where ρ is the density of ethanol (0.789 g / mL).

[0207] Porosity (%) = (W1 - W0) / ρV × 100%

[0208] The test results of the polypeptide-based composite sponge dressing substrates prepared in each embodiment are shown in Table 1.

[0209] Table 1

[0210]

[0211]

[0212] Testing revealed that the compressive modulus, water vapor transmission rate, porosity, and water retention rate of the polypeptide-based composite sponge dressing substrate JC obtained in Examples 6-10 were similar to those in Examples 1-JC. This demonstrates that the above 12 polypeptide-based composite sponge dressing substrates possess good compressive modulus, suitable water vapor transmission rate, and excellent moisturizing ability.

[0213] Testing confirmed that the physical properties of the antibacterial polypeptide-based composite sponge dressing prepared in parallel with those of the parallel-prepared polypeptide-based composite sponge dressing substrate were very similar, with no significant difference. Specifically, the properties of the antibacterial polypeptide-based composite sponge dressing obtained by adding antimicrobial peptides using the same method were all within 95%-105% of the properties of the polypeptide-based composite sponge dressing substrate in Table 1 above, indicating that the addition of antimicrobial peptides had little impact on the physical properties of the sponge dressing.

[0214] Using the same method as in Example 2, except that the bioactive peptides containing double bond functionalization and diacetylene group functionalization modifications as described in this invention are not used, even though the rest of the preparation method is the same, the overall performance of the resulting peptide-based composite sponge dressing substrate and the parallel-prepared peptide-based composite sponge dressing with antibacterial properties is not suitable as a medical sponge excipient; for example, the compression modulus is significantly reduced. As an example, the peptide-based composite sponge dressing substrate is prepared using the same method as in Example 2, except that the bioactive peptide used is: Lys(Methacrylic acid)-Gln-Thr-Glu-Thr-Lys-Lys-Leu-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly (the preparation method of this peptide is as follows: the method of preparation example 1 is used, but the acetylene group functionalization modification in step 8 is not performed), to obtain peptide-based composite sponge dressing substrate sample D1-JC; or, the peptide-based composite sponge dressing substrate is prepared using the same method as in example 2, except that the bioactive peptide used is: Lys-Gln-Thr-Glu-Thr-Lys-Lys-Leu-6-Aminocaproicacid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproicacid-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Lys (the preparation method of this peptide is as follows: the method of preparation example 1 is used, but the methacryloyl group functionalization modification in step 6 is not performed), to obtain peptide-based composite sponge dressing substrate sample D2-JC. The compressive modulus of samples D1-JC and D2-JC is only 20 kPa.

[0215] This invention crosslinks specially designed bioactive macromolecules and specially designed bioactive peptides (the bioactive peptides contain double bond functionalization modification and diacetylene group functionalization modification, and the bioactive macromolecules contain unsaturated groups), and obtains a peptide-based composite sponge dressing substrate through the topological polymerization of the bioactive peptides themselves. Preferably, antimicrobial peptides are added together with the polymerization raw materials, and the antimicrobial peptides are loaded into the network structure of the peptide-based composite sponge dressing substrate during the polymerization process. This substrate and the peptide-based composite sponge dressing with antibacterial properties not only have good mechanical properties and suitable water vapor permeability, but also excellent moisturizing ability. Based on the above physical properties, the peptide-based composite sponge dressing substrate and the peptide-based composite sponge dressing with antibacterial properties of this invention still retain the cellular activity of bioactive peptides and antimicrobial peptides. By using the preferred peptide type of this invention, it can also promote angiogenesis and cell migration, achieving unexpected technical effects.

[0216] Furthermore, compared to existing technologies and samples D1-JC and D2-JC, this invention, by incorporating bioactive peptides into a double-network cross-linked sponge dressing formed through chemical bonds, can increase the tissue retention time of the bioactive peptides and improve their physiological stability during application. Preferably, loading the antimicrobial peptides into the network structure of the peptide-based composite sponge dressing substrate during polymerization can further enhance the physiological stability of the antimicrobial peptides.

[0217] Antibacterial ability of peptide-based composite sponge dressing with antibacterial properties: In this embodiment, it was verified that the peptide-based composite sponge dressing with antibacterial properties prepared in the embodiment has good antibacterial ability, thereby avoiding or treating wound infection.

[0218] Specifically, the bacterial cultures of methicillin-resistant Staphylococcus aureus, multidrug-resistant Escherichia coli, pan-drug-resistant Pseudomonas aeruginosa, and Candida albicans, which had been passaged twice, were adjusted to 10. 6 CFU / mL, 1 mL was inoculated onto the antibacterial polypeptide-based composite sponge dressing prepared in Example 1 (the antimicrobial peptide concentration was controlled at 100 μg / mL). Methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Escherichia coli (MRC), and drug-resistant Pseudomonas aeruginosa groups were incubated at 37°C for 2 h, and Candida albicans group was incubated at 28°C for 2 h. A control group was set up without interaction with the sponge dressing. Subsequently, the bacterial suspension was diluted 100-fold, and 50 μL of the bacterial suspension was evenly spread onto agar solid medium. The MRSA, MRC, and drug-resistant Pseudomonas aeruginosa groups and their corresponding control groups were incubated at 37°C for 24 h, and the Candida albicans group and its corresponding control group were incubated at 28°C for 24 h.

[0219] Depend on Figure 2The results show that the polypeptide-based composite sponge dressing with antibacterial properties prepared in Example 1 can effectively kill methicillin-resistant Staphylococcus aureus, multidrug-resistant Escherichia coli, pan-drug-resistant Pseudomonas aeruginosa, and Candida albicans. It can be seen that the polypeptide-based composite sponge dressing with antibacterial properties prepared in Example 1 has good antibacterial properties and can effectively deal with wound infection.

[0220] Antioxidant properties of the peptide-based composite sponge dressing with antibacterial properties: In this embodiment, it was verified that the peptide-based composite sponge dressing with antibacterial properties prepared in the embodiment has excellent antioxidant properties, thereby promoting wound healing.

[0221] Specifically, a 0.04 mg / mL 2,2-diphenyl-1-picrylhydrazine (DPPH·) ethanol solution was prepared. 1 mL of this solution was mixed with 1 mL of an ethanol solution containing different concentrations of the antibacterial polypeptide-based composite sponge dressing prepared in Example 1 (based on antimicrobial peptide concentration), and incubated at room temperature for 30 min. The absorbance at 517 nm was measured using a UV-Vis spectrophotometer. The DPPH· scavenging rate (%) was calculated as: [Absorbance value of blank tube - (Absorbance value of sample test tube - Absorbance value of sample control tube)] / Absorbance value of blank tube × 100%. Simultaneously, a 100 μg / mL vitamin C solution was used as a positive control.

[0222] Depend on Figure 3 The results show that the polypeptide-based composite sponge dressing with antibacterial properties prepared in Example 1 has excellent antioxidant properties, thereby reducing the overexpression of ROS in wound tissues and cells and alleviating oxidative stress damage to cells and tissues.

[0223] An antibacterial peptide-based composite sponge dressing was used to test chronic wound healing in diabetic animals. Specifically, a full-thickness skin defect model with a diameter of 10 mm was created on the back skin of diabetic SD rats using a punch. The antibacterial peptide-based composite sponge dressing (10 mm in diameter, 2 mm thick) prepared in the example was placed on the skin defect site, and a new dressing was applied every three days. The control group's skin defect sites were left untreated. Photographs of the wound sites were taken on days 0, 3, 5, 10, 15, and 20.

[0224] Depend on Figure 4 The results showed that rat skin wounds covered with the antibacterial polypeptide-based composite sponge dressing prepared in Example 1 healed faster than untreated rat wounds, indicating that the polypeptide-based composite sponge dressing prepared in Example 1 has a good function in promoting the repair of chronic wounds.

[0225] Verification showed that the polypeptide-based composite sponge dressings with antibacterial properties prepared in Examples 2-12 also had good antibacterial ability, good antioxidant properties, and good function in promoting chronic wound repair.

[0226] In summary, this invention crosslinks specially designed bioactive macromolecules and specially designed bioactive peptides (the bioactive peptides contain double bond functionalization modification and diacetylene group functionalization modification, and the bioactive macromolecules contain unsaturated groups), and through the topological polymerization of the bioactive peptides themselves, preferably, the antimicrobial peptides of this invention and the peptide-based composite sponge dressing substrate are molded together and the antimicrobial peptides are loaded in the substrate to obtain a peptide-based composite sponge dressing. This peptide-based composite sponge dressing not only has good mechanical properties and suitable water vapor permeability, but also excellent moisturizing ability. On the basis of the above physical properties, the peptide-based composite sponge dressing of this invention still retains the cellular activity of the bioactive peptides and antimicrobial peptides. By selecting the preferred type of bioactive peptides of this invention, it can also promote angiogenesis and cell migration, achieving unexpected technical effects.

[0227] Furthermore, this invention crosslinks bioactive peptides into the sponge dressing and molds antimicrobial peptides together with the substrate, which can improve the tissue retention time of bioactive peptides and antimicrobial peptides, thereby enhancing the physiological stability of the bioactive peptides and antimicrobial peptides in the dressing during application. The peptide-based composite sponge dressing with antibacterial properties, through the synergistic effect of antimicrobial peptides and bioactive peptides, exhibits further superior antibacterial and wound-healing-promoting properties.

[0228] The advantages of the antibacterial polypeptide-based composite sponge dressing of this invention in the treatment of chronic wounds are mainly reflected in promoting wound healing, enhancing antibacterial properties, good biocompatibility and degradability, accelerating the repair process, controlling drug release, and strong adaptability. Its innovative and comprehensive effects make it a promising treatment option for chronic wound care.

[0229] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A polypeptide-based composite sponge dressing with antibacterial properties, comprising a polypeptide-based composite sponge dressing substrate and an antibacterial peptide loaded in the polypeptide-based composite sponge dressing substrate; The polypeptide-based composite sponge dressing substrate has a first network structure formed by cross-linking of bioactive macromolecules and bioactive polypeptides. The bioactive polypeptide contains double bond functionalization modification and diacetylene group functionalization modification, and the bioactive macromolecule contains unsaturated groups. The double bond functionalization modification of the bioactive polypeptide and the unsaturated groups contained in the bioactive macromolecule are each independently methacryloyl groups. The polypeptide-based composite sponge dressing substrate also has a second network structure formed by the topological polymerization of bioactive polypeptides through diacetylene groups.

2. The polypeptide-based composite sponge dressing with antibacterial properties according to claim 1, characterized in that: The bioactive polypeptide comprises two polypeptide chains, with the double bond functionalization modification site located on the amino group of the C-terminal lysine residue of the polypeptide chain, and the diacetylene group coupling the two polypeptide chains from the N-terminus; preferably, Preferably, the polypeptide chain is derived from a polypeptide raw material containing 2-20 amino acids, and / or, one end of the polypeptide raw material is lysine, and / or, the polypeptide raw material is a synthetic polypeptide, preferably synthesized by the SPPS method; More preferably, the polypeptide raw material has at least one of the properties of promoting wound healing, antibacterial, anti-inflammatory, and anti-wrinkle. More preferably, the polypeptide raw material has wound healing properties and is preferably selected from at least one of thymosin β4, RGD cell adhesion peptide, sensory neuropeptide SP, hexapeptide, and antimicrobial tetrapeptide.

3. The polypeptide-based composite sponge dressing with antibacterial properties according to claim 2, characterized in that: The double bond functionalization modification is a methacryloyl group; and / or, the diacetylene group functionalization modification is derived from diacetic acid compounds, preferably from at least one of 3,5-octadiynediic acid, 5,7-dodecadiynediic acid, 7,9-hexadecadiynediic acid, 9,11-eicosadiynediic acid, 10,12-eicosadiynediic acid, and 4,4'-(but-1,3-diyne-1,4-diyl)dibenzoic acid; more preferably, The bioactive polypeptide is at least one of the following compounds, where DA is a diacetyl group derived from diacetic acid compounds, and the side chain linked to Lys is a methacryloyl group: Methacrylic acid Lys(Methacrylic acid)-Asp-Gly-Arg-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproic acid-Arg-Gly-Asp-Lys(Methacrylic acid); Lys(Methacrylic acid)-Arg-Arg-Gln-Met-Glu-Glu-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproic acid-Glu-Glu-Met-Gln-Arg-Arg-Lys(Methacrylic acid); Lys(Methacrylic acid)-Ser-Val-Val-Val-Arg-Thr-6-Aminocaproic acid-Ala-Ala-Val-Val-Gly-DA-Gly-Val-Val-Ala-Ala-6-Aminocaproic acid-Thr-Arg-Val-Val-Val-Ser-Lys(Methacrylic acid); Methacrylic acid 4. The polypeptide-based composite sponge dressing with antibacterial properties according to claim 1, characterized in that: The bioactive polypeptide was prepared by the following method: the C-terminal lysine side chain amino group of the polypeptide raw material was subjected to an amidation reaction with methacrylic acid to obtain a polypeptide raw material with double bond functionalization. The carboxyl group of a diacetyl acid compound is amidated with the amino group of the N-terminal amino acid of a polypeptide raw material to couple the two polypeptide chains. Preferably, the method for preparing the bioactive polypeptide includes: (1) The polypeptide raw material was subjected to an amidation reaction with methacrylic acid in the presence of benzotriazol-1-yl-oxytripyrrolylphosphine (PyBOP) and diisopropylethylamine (DIPEA) to obtain a polypeptide raw material whose C-terminal lysine side chain amino group was functionalized by double bond. (2) The double bond functionalized peptide raw material is then subjected to an amidation reaction with diacetyl acid compounds in the presence of PyBOP and DIPEA. The two peptide chains are coupled from the N-terminus of the peptide raw material through the diacetyl group to obtain a bioactive peptide. More preferably, the diacetic acid compound is selected from at least one of 3,5-octadiynediic acid, 5,7-dodecadiynediic acid, 7,9-hexadecadiynediic acid, 9,11-eicosadiynediic acid, 10,12-docosadiynediic acid, and 4,4'-(but-1,3-diyne-1,4-diyl)dibenzoic acid; and / or, More preferably, the mass ratio of the polypeptide raw material to methacrylic acid is 1:(1-8); and / or, More preferably, the mass ratio of the C-terminal double-bond functionalized peptide raw material to the diacetic acid compound is 1:(0.4-0.6); and / or, More preferably, the reaction conditions include: in steps (1) and (2), the ratio of the amount of PyBOP to the molar amount of the reactant based on carboxyl groups is 1:(0.9-1.1); and / or, in steps (1) and (2), the ratio of the amount of DIPEA to the molar amount of the reactant based on carboxyl groups is 2:(0.9-1.1).

5. The polypeptide-based composite sponge dressing with antibacterial properties according to claim 1, characterized in that: The bioactive macromolecule is selected from at least one of the following: methacrylamide gelatin, methacrylamide silk fibroin, methacrylamide hyaluronic acid, methacrylamide chondroitin sulfate, methacrylamide sodium alginate, methacrylamide chitosan, methacrylamide dextran, methacrylamide heparin, polyether F127 diacrylate, polyethylene glycol diacrylate, tetra-arm polyethylene glycol acrylate, and methacrylamide polyvinyl alcohol; and / or, The mass ratio of bioactive macromolecules to bioactive peptides is (1-10):1, preferably (4-6):

1.

6. The polypeptide-based composite sponge dressing with antibacterial properties according to claim 1, characterized in that: The polypeptide-based composite sponge dressing with antibacterial properties and the polypeptide-based composite sponge dressing substrate each have at least one of the following characteristics: The compression modulus is tested using a compression method and is not less than 50 kPa, preferably 270-450 kPa. The porosity was determined using the ethanol immersion method to be 40%-95%, preferably 40%-75%. The water vapor transmission rate, determined by gravity method, was 700-2000 g / m³ after 48 hours. 2 The preferred value is 800-1200 g / m³. 2 ; The water absorption rate, measured by specific gravity at 37°C and 48 hours, was 300%-1100%; preferably 300%-600%. The water retention rate at 37℃ for 24 hours was determined to be 40%-75% using the specific gravity method. Preferably, the preparation method of the polypeptide-based composite sponge dressing substrate includes: The bioactive macromolecules and bioactive peptides are subjected to a first polymerization in solution in the presence of an initiator to obtain a gel-like intermediate. The gel-like intermediate was freeze-dried to obtain a sponge-like intermediate. The sponge-like intermediate is subjected to a second polymerization by light irradiation to obtain the polypeptide-based composite sponge dressing substrate.

7. The polypeptide-based composite sponge dressing with antibacterial properties according to claim 1, characterized in that: The antimicrobial peptide is loaded into the network structure of the polypeptide-based composite sponge dressing substrate; And / or, the antimicrobial peptide has antimicrobial properties, preferably contains 5-50 amino acids, and / or, preferably, the antimicrobial peptide is a synthetic polypeptide, preferably synthesized by the SPPS method; more preferably, The antimicrobial peptide is selected from at least one of the following antimicrobial peptides: Antimicrobial peptide a, with the sequence Lys-Lys-Glu (dopamine)-Phe-Glu (dopamine)-Phe-Glu (dopamine)-Phe-Glu (dopamine)-Phe-Lys-Lys; Lysozyme, with the sequence Tyr-Cys-Ser-Asp-Val-Gly-Val-Ala-Glu-Pro-Ala-Ser; Human Defensin-1 has the sequence Gly-Cys-Cys-Gly-Gly-Gly-Cys-Cys-Gly. Bactenecin, with the sequence Gly-Gly-Gly-Phe-Gly-Gly-Ala-Ser-Lys-Phe-Leu-Ala-Ser; Magaiinin-2, a metalloproteinase inhibitory peptide, has the sequence Gly-Gly-Ser-Tyr-Ser-Gly-Cys-Glu-Val-Ser-Tyr-Ser-Lys; Human antimicrobial peptide LL-37, sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; Cecropin A, sequence: AAAACATTCAGGAGAGCACTAAAGGAGGGA; Batmin-1, with the sequence TGLKRKVRKKSAAKKWKKLKS.

8. The polypeptide-based composite sponge dressing with antibacterial properties according to any one of claims 1-7, characterized in that: Based on a total mass of 100 parts for the polypeptide-based composite sponge dressing substrate, the content of antimicrobial peptides in the polypeptide-based composite sponge dressing with antibacterial properties is 1-20 parts. And / or, The preparation method of the polypeptide-based composite sponge dressing with antibacterial properties includes: The bioactive macromolecules and bioactive peptides are subjected to a first polymerization in an aqueous solution containing antimicrobial peptides in the presence of an initiator to obtain a gel-like intermediate. The gel-like intermediate was freeze-dried to obtain a sponge-like intermediate. The sponge-like intermediate is subjected to a second polymerization by light irradiation to obtain the polypeptide-based composite sponge dressing with antibacterial properties.

9. A method for preparing a polypeptide-based composite sponge dressing with antibacterial properties according to any one of claims 1-8, comprising: The bioactive macromolecules and bioactive peptides are subjected to a first polymerization in an aqueous solution containing antimicrobial peptides in the presence of an initiator to obtain a gel-like intermediate; The gel-like intermediate was freeze-dried to obtain a sponge-like intermediate. The sponge-like intermediate is subjected to a second polymerization by light irradiation to obtain the polypeptide-based composite sponge dressing with antibacterial properties.

10. The preparation method according to claim 9, characterized in that, The preparation method of the polypeptide-based composite sponge dressing with antibacterial properties includes the following steps: (1) The raw materials, including bioactive macromolecules, bioactive peptides, antimicrobial peptides and initiators, are mixed in water and degassed to obtain a prepolymer solution. (2) The prepolymer liquid is subjected to a first polymerization under free radical polymerization conditions to obtain a gel-like intermediate; (3) Freeze-dry the gel-like intermediate to obtain a sponge-like intermediate; (4) The sponge-like intermediate is subjected to ultraviolet light irradiation for a second polymerization: the diacetylene unit in the bioactive polypeptide undergoes topological polymerization to obtain a polypeptide-based composite sponge dressing with antibacterial properties. Preferably, based on parts by weight, relative to 1000 parts of water, the prepolymer solution contains: 30-150 parts of bioactive macromolecules, preferably 50-100 parts; 5-30 parts of bioactive peptides, preferably 10-20 parts; 5-30 parts of antimicrobial peptides, preferably 10-20 parts; 1-10 parts of initiator, preferably 4-6 parts; and / or, Preferably, the initiator is selected from at least one of per-2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and phenyl-2,4,6-trimethylbenzoyl phosphite; and / or, Preferably, the conditions for the first polymerization include: The procedure is performed under ultraviolet or blue light irradiation for 1-15 minutes; preferably, irradiation under ultraviolet or blue light irradiation for 5-15 minutes; and / or, Preferably, the conditions for the second polymerization include: The procedure is performed under ultraviolet light irradiation for 1-15 minutes; preferably 5-15 minutes.

11. The use of a polypeptide-based composite sponge dressing with antibacterial properties as described in any one of claims 1-8, or a polypeptide-based composite sponge dressing with antibacterial properties prepared by the preparation method described in claim 9 or 10, as a wound dressing for the healing of chronic wounds, preferably for the healing of venous congestion ulcers, pressure ulcers, and diabetic wounds.

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