A self-assembled biomimetic human skin membrane ECM lyophilized sponge, a preparation method thereof and a wound repair application thereof

Abstract

The application relates to the technical field of biomedical materials, and discloses a self-assembled biomimetic human skin membrane ECM freeze-dried sponge, a preparation method and wound repair applications thereof. The sponge comprises recombinant human I / III type collagen, elastin, glycosaminoglycan, fibronectin and human antibacterial peptide LL-37 hydrophobic truncated peptide. In the preparation process, the carboxyl group of collagen is activated by EDC / NHS, and antibacterial peptides are covalently grafted to construct amphiphilic molecules, and a three-dimensional biomimetic hydrogel is formed by self-assembly of the amphiphilic molecules through hydrophobic and hydrophilic stacking, and the three-dimensional biomimetic hydrogel is prepared through gradient freeze-drying. The sponge highly simulates the microstructure of natural skin ECM, has the dual functions of broad-spectrum long-acting antibiosis and high-efficiency tissue repair, and all the components are homologous to the human body, have low immunogenicity and high biological safety. The application solves the technical problems of the split of the antibiosis and repair functions of traditional dressings, and the easy loss of antibacterial components, and is suitable for functional regenerative repair of difficult-to-heal wounds such as acute and chronic infected wounds and diabetic foot ulcers.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a self-assembled biomimetic human skin membrane (ECM) freeze-dried sponge, its preparation method, and its application in wound repair. Background Technology

[0002] In the process of acute and chronic skin wound repair, wound infection is a core factor leading to delayed healing, scar hyperplasia, and even disease exacerbation. There is a significant technological gap in biomimetic ECM dressings that combine highly effective antibacterial and tissue repair functions in clinical practice. Existing collagen-based ECM wound materials mostly use single collagen or physically blended antibacterial components, which suffer from problems such as easy component detachment, short antibacterial duration, and high cytotoxicity. Furthermore, conventional ECM materials often rely on chemical cross-linking or physical molding, resulting in structures that easily disintegrate upon contact with water, and difficulty in simultaneously achieving mechanical flexibility and stability, thus failing to simulate the microstructure and physiological functions of natural skin ECM.

[0003] Meanwhile, existing antimicrobial peptide-modified ECM technologies typically employ physical soaking, surface adsorption, or simple blending methods to load antimicrobial peptides into collagen or ECM materials. The peptide molecules are randomly distributed and irregularly arranged, exhibiting low biomimicry and differing significantly from the microstructure of natural skin ECM. Natural skin ECM is a highly ordered nanofiber network formed by collagen, glycosaminoglycans, and glycoproteins through molecular recognition and in-situ self-assembly. Existing antimicrobial peptide-modified ECMs are merely simple complexes of "peptides + collagen," exhibiting rapid peptide release, short-lasting antibacterial effects, and cytotoxicity at excessively high local concentrations. They lack molecular-level self-assembly behavior, fail to form a continuous biomimetic fiber network, and lack the microenvironment for cell adhesion, proliferation, and differentiation, making functional regeneration of chronic, difficult-to-heal wounds virtually impossible. Summary of the Invention

[0004] This invention provides a self-assembled bionic human skin membrane ECM freeze-dried sponge, its preparation method, and its application in wound repair. Through a hydrophilic and hydrophobic self-assembly mechanism, it achieves a deep synergy of antibacterial and repair functions, which reduces the infection rate of wounds, shortens the healing cycle, and reduces scar hyperplasia. It also has extremely high biosafety and mechanical stability.

[0005] This invention provides a self-assembled biomimetic human skin membrane (ECM) freeze-dried sponge, comprising recombinant human type I / III collagen, elastin, glycosaminoglycans, and fibronectin; After the recombinant human type I / III collagen was activated with EDC / NHS, the carboxyl group was covalently grafted with the human antimicrobial peptide LL-37 hydrophobic truncated peptide to construct an amphiphilic molecule containing a hydrophilic collagen backbone and a hydrophobic antimicrobial peptide side chain. The hydrophobic peptides of the amphiphilic molecules form physical cross-linking nodes through hydrophobic stacking, and spontaneously and orderly assemble with the elastin, glycosaminoglycans and fibronectin through intermolecular hydrogen bonds and hydrophilic-hydrophobic interactions to form a three-dimensional ECM hydrogel, which is then freeze-dried to obtain the sponge.

[0006] Furthermore, the mass fraction of each component is as follows: Recombinant human type I / III collagen accounts for 60%~72%, elastin accounts for 8%~12%, glycosaminoglycans account for 10%~16%, fibronectin accounts for 4%~8%, and human antimicrobial peptide LL-37 hydrophobic truncated peptide accounts for 1.5%~4%.

[0007] Furthermore, in the recombinant human type I / III collagen, the mass ratio of type I collagen to type III collagen is (2.5~3):1; The glycosaminoglycan is a mixture of hyaluronic acid and chondroitin sulfate in a mass ratio of 1:1.

[0008] Furthermore, the amino acid sequence of the human antimicrobial peptide LL-37 hydrophobic truncated peptide is selected from any one of sequence 1, sequence 2, sequence 3 or sequence 4 in the sequence listing.

[0009] The present invention also provides a method for preparing the self-assembled bionic human skin membrane ECM freeze-dried sponge as described above, comprising: S1. Recombinant human type I / III collagen was dissolved in sterile acetic acid solution to prepare a collagen solution and allowed to stand to remove bubbles. EDC and NHS were added under ice bath conditions to react and activate the carboxyl groups of the collagen side chain to form a highly active ester intermediate. S2. The human antimicrobial peptide LL-37 hydrophobic truncated peptide was dissolved in sterile PBS buffer to prepare an antimicrobial peptide solution. The antimicrobial peptide solution was slowly added dropwise to the activated collagen solution to undergo amide bond covalent coupling, thus completing the covalent grafting and obtaining an amphiphilic molecule solution. S3. Elastin solution, glycosaminoglycan solution and fibronectin solution are added sequentially to the amphiphilic molecular solution, and the mixture is stirred and mixed evenly to obtain ECM mixed mother liquor. The pH value and ionic strength of the ECM mixed mother liquor are adjusted, and the mixture is heated and incubated to carry out hydrophilic and hydrophobic self-assembly to form a three-dimensional ECM hydrogel. S4. The self-assembled three-dimensional ECM hydrogel is pre-frozen to completely solidify and shape the gel, then sublimated and dried to remove free water, and finally desorbed and dried to remove bound water, to obtain the sponge.

[0010] Furthermore, in S1, The concentration of the sterile acetic acid solution is 0.05 mol / L, and the concentration of the collagen solution is 6~10 mg / mL; The molar ratio of EDC to carboxyl groups on the side chains of collagen is controlled to be 1.2:1~2:1; The molar ratio of EDC to NHS should be controlled at 1:0.8 to 1:1. Adjust the pH of the reaction system to 4.5-5.5 and activate the reaction in the dark for 30-60 minutes.

[0011] Furthermore, in S2, The pH value of the sterile PBS buffer is 7.4, and the concentration of the antimicrobial peptide solution is 1~3 mg / mL; The mass ratio of antimicrobial peptides to recombinant human type I / III collagen was controlled at (1.5~4):(60~72); During the reaction, adjust the pH of the system to 6.5~7.0 and react at 4℃ in the dark for 10~24 hours.

[0012] Furthermore, in S3, Adjust the pH of the ECM mixed mother liquor to 7.2~8.0, adjust the ionic strength to 0.14~0.18 mol / L, raise the temperature to 25~30℃, and let it stand and incubate for 2~4 hours.

[0013] Furthermore, in S4, Pre-freezing stage: Pre-freeze at -40℃ for 6~8 hours to allow the gel to completely solidify and take shape; Sublimation drying stage: Dry at -20℃ and a vacuum of 10~15Pa for 10~24 hours; Analysis and drying stage: Drying at 25℃ and a vacuum of 5~8Pa for 6~10 hours.

[0014] The present invention also provides an application of the self-assembled bionic human skin membrane ECM freeze-dried sponge as described above in the preparation of wound repair dressings, which are used for the repair of acute and chronic infected wounds, acute sensitive muscle repair, diabetic foot ulcers, burn wounds or pressure sores.

[0015] The beneficial effects of this invention are as follows: The self-assembled biomimetic human skin membrane extracellular matrix (ECM) freeze-dried sponge of this invention possesses significant multi-dimensional beneficial effects. In terms of antibacterial and anti-infection properties, this sponge exhibits long-lasting and excellent antibacterial performance, achieving inhibition rates of 93% or higher against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, significantly reducing the incidence of wound infection by more than 85%, and its antibacterial effect persists until complete wound healing. Regarding physical and mechanical properties, this sponge achieves a perfect balance between structural stability and flexibility. Its self-assembled scaffold has a compression rebound rate of over 90%, and it remains intact after immersion in phosphate-buffered saline (PBS) for 48 hours. It possesses both excellent structural support and good skin-adhesive flexibility, greatly improving the convenience of clinical operation. In clinical tissue repair, this sponge exhibits highly efficient healing-promoting capabilities, shortening the healing cycle of acute wounds by 68% and significantly increasing the healing rate of chronic, difficult-to-heal wounds such as diabetic ulcers by over 85%. Simultaneously, it effectively reduces post-repair skin scar hyperplasia by 70%, restoring the elasticity of new skin to near-normal levels. Regarding industrialization prospects, the preparation process of this invention is mild and controllable, requiring no complex equipment, and the raw materials are readily available commercially, resulting in significantly lower production costs compared to similar acellular dermal matrix materials, making it suitable for large-scale mass production. Attached Figure Description

[0016] Figure 1 This is a schematic flowchart of the preparation method of the self-assembled bionic human skin membrane ECM freeze-dried sponge of the present invention.

[0017] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] This invention provides a bifunctional freeze-dried sponge based on human homologous ECM components and covalently grafted with human antimicrobial peptide LL-37 hydrophobic core peptide. The amphiphilic molecules are constructed by grafting hydrophobic antimicrobial peptide onto collagen carboxyl groups activated by EDC / NHS, and then self-assembled into a stable and flexible ECM scaffold through hydrophilic-hydrophobic stacking. The invention also provides the application of the freeze-dried sponge in antibacterial and wound repair.

[0020] The technical problem solved by this invention is as follows: 1. To address the problems of fragmented repair function and easy loss of antibacterial components in traditional ECM wound dressings, existing materials either only inhibit bacteria and cannot promote tissue regeneration, or only repair without anti-infection effect. Especially for difficult-to-heal wounds such as infected wounds and diabetic ulcers, it is impossible to simultaneously achieve wound infection control and functional regeneration of skin tissue. This invention develops a new type of ECM biomimetic material with dual functions of antibacterial and repair.

[0021] 2. Overcoming the shortcomings of insufficient biomimicry in existing materials, which mostly use non-human homologous components or contain only a single collagen component, lacking core functional components of skin ECM such as elastin, glycosaminoglycans, and fibronectin, and do not use highly hydrophobic antimicrobial peptide sequences, and simple mixing cannot drive self-assembly. This invention requires the use of human homologous ECM components + human LL-37 hydrophobic truncated peptide to construct a biomimetic system that highly conforms to natural skin ECM, reducing immunogenicity and improving cell compatibility and tissue repair induction.

[0022] 3. This invention addresses the problem of unreasonable loading methods for existing modified antimicrobial peptide ECM structures. Traditional physical blending / adsorption methods easily lead to burst release of antimicrobial peptides, excessively high local concentrations causing cytotoxicity, short antimicrobial duration, and inability to provide long-term antibacterial inhibition. Furthermore, the binding force between antimicrobial peptides and the ECM matrix is ​​weak, making it difficult to achieve synergistic antimicrobial and repair functions. This invention achieves covalent grafting of antimicrobial peptides by activating collagen carboxyl groups through EDC / NHS, ensuring stable immobilization and uniform distribution of antimicrobial peptides, avoiding burst release toxicity, and achieving long-term broad-spectrum antimicrobial effects.

[0023] 4. Optimize the design of antimicrobial peptide sequences by using human LL-37 hydrophobic truncation of the core peptide to avoid the problems of excessive hydrophilicity and insufficient self-assembly driving force of full-length peptides.

[0024] 5. To avoid the biosafety issues caused by chemical crosslinking agents, existing materials use excessive amounts of chemical crosslinking agents to improve structural strength, which leads to increased material brittleness and cytotoxicity. This invention relies on the natural stacking of amphiphilic molecules to achieve self-assembly crosslinking, reducing the use of additional crosslinking agents and ensuring the biosafety of the material.

[0025] This invention provides a self-assembled biomimetic human skin membrane (ECM) freeze-dried sponge. Based on the natural ECM components of human skin, it uses recombinant human type I / III collagen, elastin, glycosaminoglycans, and fibronectin as core repair components. An EDC / NHS-activated collagen carboxyl group is covalently grafted with a human antimicrobial peptide, LL-37, to construct a collagen-hydrophobic antimicrobial peptide amphiphilic molecule. The hydrophilic end of this molecule consists of the collagen backbone and hydrophilic ECM components, while the hydrophobic end is the LL-37 hydrophobic truncated peptide side chain. Through intermolecular hydrophilic-hydrophobic interactions, hydrogen bonds, and hydrophobic stacking forces, it spontaneously and orderly assembles to form a stable yet flexible three-dimensional ECM hydrogel. This is then subjected to gradient freeze-drying to obtain a bifunctional freeze-dried sponge. The sponge possesses both broad-spectrum, long-lasting antibacterial properties and highly efficient tissue repair functions. All components are homologous to human tissue, exhibiting extremely low immunogenicity. Furthermore, by limiting the core sequence and conserved mutation forms of the LL-37 hydrophobic truncated peptide, it is clear that simple sequence variations still fall within the scope of this invention.

[0026] The biomimetic human skin membrane ECM freeze-dried sponge of this invention is composed of the following components by mass fraction, each component being a medical-grade human-derived or highly biocompatible raw material: Recombinant human type I / III collagen: 60%~72%, of which type I collagen: type III collagen = (2.5~3):1, which matches the proportion of collagen subtypes in natural human skin; Elastin: 8%~12%, imparts flexibility and elasticity to the scaffold, matching the mechanical properties of the skin; Glycosaminoglycans (GAG): 10%~16%, which are hyaluronic acid and chondroitin sulfate in a 1:1 mass ratio. They have moisturizing, growth factor binding, and anti-inflammatory functions. Fibronectin: 4%~8%, promotes cell adhesion, migration and proliferation in wounds, and accelerates epithelialization; Human antimicrobial peptide LL-37 hydrophobic truncated peptide: 1.5%~4%, with a highly hydrophobic core sequence, no hemolytic toxicity, and possesses both broad-spectrum antibacterial and angiogenesis-promoting activities; The remainder consists of sterile buffer components remaining after freeze-drying, with no toxic chemical residues.

[0027] In this invention, the core sequence (including the equivalent protected sequence) of the human antimicrobial peptide LL-37 hydrophobic truncated peptide is derived from the human antimicrobial peptide LL-37, whose full-length sequence is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES. A series of highly hydrophobic truncated peptides were obtained by truncating its hydrophobic core region and optimizing it. All the following sequences, as well as simple conserved mutations based on the core hydrophobic region and sequence variants with 1-2 amino acids added or removed from the terminal, are within the scope of protection of this invention. Specific sequences are as follows: (1) Core truncated peptide sequence 1 (LL-37(17–32), HC16, SEQ ID NO.1) Sequence: Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro (Single-letter abbreviation: KRIVQRIKDFLRNLVP) Sequence length: 16 amino acids; Core hydrophobic residues: Val, Ile, Phe, Leu, Pro, accounting for ≥60%, with strong hydrophobicity and excellent self-assembly driving force; Characteristics: Broad-spectrum anti-Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), non-cytotoxic, and retains complete repair-promoting activity.

[0028] (2) Core truncated peptide sequence 2 (LL-37(14–34), HC21, SEQ ID NO.2) Sequence: Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe LeuArg Asn Leu Val Pro Arg (Single-letter abbreviation: IGKEFKRIVQRIKDFLRNLVPR) Sequence length: 21 amino acids; Features: The hydrophobic regions extend continuously, resulting in stronger intermolecular stacking forces and a more stable ECM structure formed by self-assembly, while retaining hydrophilic sites and not affecting cell adhesion.

[0029] (3) Core truncated peptide sequence 3 (LL-37(16–32), HC14, SEQ ID NO.3) Sequence: Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val (Single-letter abbreviation: RIVQRIKDFLRNLV) Sequence length: 14 amino acids; Characteristics: Maximized hydrophobicity, most prominent amphiphilic molecular characteristics, obvious separation of hydrophilic and hydrophobic phases after grafting collagen, and more uniform pores in the self-assembled three-dimensional network.

[0030] (4) Conserved hydrophobic mutant peptide sequence 4 (HC16 mutant, SEQ ID NO.4) Sequence: Lys Arg Ile Val Gln Arg Ile Val Asp Phe Leu Val Asn Leu Val Pro (Single-letter abbreviation: KRIVQRIVDFLVNLVP) Sequence length: 16 amino acids; Mutation method: Replace Lys and Arg in sequence 1 with hydrophobic Val and Phe with Leu. This is only a simple change in hydrophobic conservation and does not change the core hydrophobic framework and function. Features: Hydrophobicity is further improved, self-assembly stability is stronger, antibacterial activity is not reduced, and it belongs to the equivalent substitution sequence of the core sequence of this invention.

[0031] Any sequence that uses the above four sequences of this invention as its core and involves only 1-2 hydrophobic conservative substitutions of amino acids (such as Leu and Ile interchange, Val and Ala interchange, Phe and Tyr interchange) or the deletion / addition of one non-charged amino acid at the end, falls within the protection scope of this invention because it is a simple sequence change that can be obtained by those skilled in the art without creative effort.

[0032] like Figure 1 As shown, the present invention also provides a method for preparing a self-assembled bionic human skin membrane ECM freeze-dried sponge (EDC / NHS activation grafting + hydrophilic and hydrophobic self-assembly), comprising the following steps: S1, EDC / NHS activation of recombinant human type I / III collagen carboxyl groups 1) Dissolve recombinant human type I / III collagen in 0.05 mol / L sterile acetic acid solution to prepare a collagen solution with a concentration of 6~10 mg / mL. Stir at 4℃ until completely dissolved and let stand for 30 min to remove bubbles. 2) Under ice bath conditions, add EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to the collagen solution, and control the molar ratio of EDC to collagen side chain carboxyl groups to be (1.2~2):1; then add NHS (N-hydroxysuccinimide), and control the molar ratio of EDC:NHS to be 1:(0.8~1); 3) Adjust the pH of the system to 4.5~5.5, and activate the reaction in an ice bath in the dark for 30~60 minutes to activate the carboxyl groups of the collagen side chains to form highly active ester intermediates, providing reaction sites for subsequent covalent grafting.

[0033] S2, covalently grafted with LL-37 hydrophobic truncated peptides, to construct collagen-antimicrobial peptide amphiphilic molecules. 1) Dissolve the above four human LL-37 hydrophobic truncated peptides in sterile PBS buffer (pH=7.4) to prepare antimicrobial peptide solutions with a concentration of 1~3 mg / mL, and store at 4℃. 2) Slowly add each antimicrobial peptide solution to the activated collagen solution, controlling the mass ratio of antimicrobial peptide to collagen to be (1.5~4):(60~72), so that the N-terminal amino group of the antimicrobial peptide and the activated carboxyl group of the collagen undergo amide bond covalent coupling; 3) Adjust the pH of the system to 6.5~7.0, react at 4℃ in the dark for 12~24h to complete covalent grafting and obtain a solution of collagen-LL-37 hydrophobic truncated peptide amphiphilic molecule. This molecule has both a hydrophilic collagen backbone and a hydrophobic antibacterial peptide side chain, laying the foundation for subsequent self-assembly.

[0034] S3, Multi-component mixing and hydrophilic / hydrophobic self-assembly molding 1) Add elastin, glycosaminoglycan, and fibronectin solutions sequentially to the amphiphilic molecular solution, and stir at low speed (200~300r / min) to mix evenly to obtain ECM mixed mother liquor; 2) Adjust the pH of the mixed mother liquor to 7.2~8.0, the ionic strength to 0.14~0.18 mol / L, raise the temperature to 25~30℃, and let it stand and incubate for 2~4 hours; 3) Self-assembly mechanism: The hydrophobic peptide segments in the collagen-antimicrobial peptide amphiphilic molecules form physical cross-linking nodes through hydrophobic stacking. The hydrophilic collagen chains, glycosaminoglycans, and fibronectin extend through hydrogen bonds and electrostatic attraction to form a flexible network framework. Without the addition of additional chemical cross-linking agents, a three-dimensional biomimetic ECM hydrogel with a stable structure, uniform pores, and excellent flexibility can be formed, achieving the structural characteristics of "stability without losing flexibility".

[0035] S4. Gradient freeze-drying to prepare sponges 1) Pre-freezing stage: Transfer the self-assembled ECM hydrogel to a freeze dryer and pre-freeze at -40℃ for 6~8h to allow the gel to fully solidify and form; 2) Sublimation drying stage: Dry at -20℃ and vacuum degree 10~15Pa for 12~24h to remove free water from the gel; 3) Drying stage: Drying at 25℃ and vacuum degree 5~8Pa for 6~10h to remove bound moisture and obtain a porous, highly resilient, antibacterial and repair-functional bionic human skin membrane ECM freeze-dried sponge. 4) Post-processing: The sponge is aseptically cut and packaged, and then sterilized with 25kGy γ-rays to obtain the finished product.

[0036] The freeze-dried sponge prepared using the above method possesses a dual-function mechanism of antibacterial and repair properties, as detailed below: 1. Antibacterial function: The covalently grafted LL-37 hydrophobic truncated peptide is evenly distributed in the ECM scaffold framework. The hydrophobic sequence can target and destroy the bacterial cell membrane, kill pathogenic bacteria on the wound surface in a broad spectrum, and inhibit the formation of bacterial biofilm. The covalent grafting method avoids the burst release of antimicrobial peptides, achieves long-lasting antibacterial effect, and does not produce drug resistance. It is non-toxic to normal human cells.

[0037] 2. Repair function: Recombinant human type I / III collagen provides an adhesion scaffold for skin cells, elastin ensures skin elasticity after repair, glycosaminoglycan provides long-lasting moisturizing, regulates wound inflammation, and binds endogenous growth factors, fibronectin promotes the migration and proliferation of fibroblasts and epidermal stem cells, and hydrophobic truncated peptides have angiogenic activity. The multi-component synergistic effect accelerates wound epithelialization and tissue regeneration, and reduces scar hyperplasia.

[0038] 3. Structural advantages: The three-dimensional network formed by hydrophilic and hydrophobic self-assembly has a porosity of 85%~95%, forming a continuous and interconnected multi-level channel. The micropores between fibers are 2~30μm in diameter, and the macroscopic functional pores are 50~200μm in diameter. It can efficiently support cell infiltration, nutrient / metabolite diffusion and angiogenesis. It does not disintegrate or collapse when exposed to water, fits various irregular wounds, and its mechanical properties are highly matched with those of natural skin.

[0039] The core innovations of this invention are mainly reflected in three dimensions: dual-function integrated design, innovative self-assembly mechanism, and high biocompatibility. In terms of product configuration, this invention is the first to covalently combine human-derived extracellular matrix repair components with human-derived antimicrobial peptides and hydrophobic truncated peptides, achieving a synergistic effect of antibacterial and anti-infection properties and tissue repair and regeneration, effectively overcoming the technical shortcomings of traditional dressings with only one function. In terms of structural forming, this invention uses specific activating reagents for covalent grafting to construct amphiphilic molecules containing collagen and hydrophobic antimicrobial peptides, which naturally stack to form a three-dimensional network structure simply through intermolecular hydrophilic-hydrophobic interactions. This self-assembly mechanism does not require excessive addition of chemical cross-linking agents, successfully overcoming the technical bottleneck of the trade-off between scaffold structure stability and flexibility. Regarding clinical safety, all core components in the system are human-derived substances with extremely low immunogenicity, and the degradation products are all natural substances such as amino acids and polysaccharides, ensuring no toxic chemical residues or foreign body reactions, and enabling safe adaptation to various acute and chronic infected wounds.

[0040] The LL-37 hydrophobic truncated peptides described in this invention can all be prepared on a large scale using solid-phase synthesis, with a purity ≥98%, meeting the standards for medical biomaterials. This invention has been verified through laboratory-scale trials, performance testing, and animal experiments; the technical solution is reproducible, the effects are stable, and it possesses potential for clinical translation and industrial mass production. Specific examples are used below to illustrate this invention: Example 1: Preparation of ECM sponges grafted with LL-37(17–32) hydrophobic truncated peptide (SEQ ID NO.1) 1) Prepare an 8 mg / mL recombinant human I / III collagen solution (I:III = 2.5:1), add activation reagent under ice bath at a ratio of EDC:carboxyl = 1.5:1 and EDC:NHS = 1:0.9, and activate at pH 5.2 for 40 min; 2) Add the antimicrobial peptide solution of SEQ ID NO.1 to make the antimicrobial peptide mass ratio 2.5%, react at 4°C in the dark for 18 hours to obtain collagen-antimicrobial peptide amphiphilic molecules; 3) Add 10% elastin, 12% glycosaminoglycan, and 5.5% fibronectin in sequence, stir and mix well, adjust the pH to 7.3, and allow to self-assemble at room temperature for 3 hours; 4) Gradient freeze drying and sterilization to obtain bifunctional ECM freeze-dried sponge.

[0041] Example 2: Preparation of ECM sponge grafted with HC16 mutant (SEQ ID NO.4) Using the hydrophobic mutant peptide of SEQ ID NO.4, the remaining component ratios, EDC / NHS activation grafting process, self-assembly and freeze-drying process were the same as in Example 1. The resulting sponge structure had 15% higher stability than that in Example 1, and no difference in antibacterial activity.

[0042] Example 3: Performance Testing Structural characterization: Porosity 92%~94%, pore size 120~180μm, infrared spectroscopy detected characteristic peaks of amide bonds, proving that covalent grafting was successful; Mechanical properties: tensile strength 0.4~0.45MPa, compression resilience 92%, intact morphology after PBS soaking for 48h; Antibacterial properties: The sponges in Examples 1 and 2 both showed an inhibition rate of ≥93% against Staphylococcus aureus and Escherichia coli; Cell experiments: L929 fibroblasts showed an adhesion rate ≥90%, a 50% increase in proliferation rate, and grade 0 cytotoxicity; Animal experiments: In rats, the infected wound healed completely in 14 days, and the scar area was reduced by 70% compared with ordinary collagen sponge.

[0043] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, apparatus, article, or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, apparatus, article, or method. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, apparatus, article, or method that includes that element.

[0044] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A self-assembled bionic human skin membrane ECM freeze-dried sponge, characterized in that, Including recombinant human type I / III collagen, elastin, glycosaminoglycans, and fibronectin; After the recombinant human type I / III collagen was activated with EDC / NHS, the carboxyl group was covalently grafted with the human antimicrobial peptide LL-37 hydrophobic truncated peptide to construct an amphiphilic molecule containing a hydrophilic collagen backbone and a hydrophobic antimicrobial peptide side chain. The hydrophobic peptides of the amphiphilic molecules form physical cross-linking nodes through hydrophobic stacking, and spontaneously and orderly assemble with the elastin, glycosaminoglycans and fibronectin through intermolecular hydrogen bonds and hydrophilic-hydrophobic interactions to form a three-dimensional ECM hydrogel, which is then freeze-dried to obtain the sponge.

2. The self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 1, characterized in that, The mass fraction of each component is: Recombinant human type I / III collagen accounts for 60%~72%, elastin accounts for 8%~12%, glycosaminoglycans account for 10%~16%, fibronectin accounts for 4%~8%, and human antimicrobial peptide LL-37 hydrophobic truncated peptide accounts for 1.5%~4%.

3. The self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 2, characterized in that, In the recombinant human type I / III collagen, the mass ratio of type I collagen to type III collagen is (2.5~3):1; The glycosaminoglycan is a mixture of hyaluronic acid and chondroitin sulfate in a mass ratio of 1:

1.

4. The self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 1, characterized in that, The amino acid sequence of the human antimicrobial peptide LL-37 hydrophobic truncated peptide is selected from any one of sequence 1, sequence 2, sequence 3 or sequence 4 in the sequence listing.

5. A method for preparing a self-assembled bionic human skin membrane (ECM) freeze-dried sponge as described in any one of claims 1 to 4, characterized in that, include: S1. Recombinant human type I / III collagen was dissolved in sterile acetic acid solution to prepare a collagen solution and allowed to stand to remove bubbles. EDC and NHS were added under ice bath conditions to react and activate the carboxyl groups of the collagen side chain to form a highly active ester intermediate. S2. The human antimicrobial peptide LL-37 hydrophobic truncated peptide was dissolved in sterile PBS buffer to prepare an antimicrobial peptide solution. The antimicrobial peptide solution was slowly added dropwise to the activated collagen solution to undergo amide bond covalent coupling, thus completing the covalent grafting and obtaining an amphiphilic molecule solution. S3. Elastin solution, glycosaminoglycan solution and fibronectin solution are added sequentially to the amphiphilic molecular solution, and the mixture is stirred and mixed evenly to obtain ECM mixed mother liquor. The pH value and ionic strength of the ECM mixed mother liquor are adjusted, and the mixture is heated and incubated to carry out hydrophilic and hydrophobic self-assembly to form a three-dimensional ECM hydrogel. S4. The self-assembled three-dimensional ECM hydrogel is pre-frozen to completely solidify and shape the gel, then sublimated and dried to remove free water, and finally desorbed and dried to remove bound water, to obtain the sponge.

6. The method for preparing the self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 5, characterized in that, In S1, The concentration of the sterile acetic acid solution is 0.05 mol / L, and the concentration of the collagen solution is 6~10 mg / mL; The molar ratio of EDC to carboxyl groups on the side chains of collagen is controlled to be 1.2:1~2:1; The molar ratio of EDC to NHS should be controlled at 1:0.8 to 1:

1. Adjust the pH of the reaction system to 4.5-5.5 and activate the reaction in the dark for 30-60 minutes.

7. The method for preparing the self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 5, characterized in that, In S2, The pH value of the sterile PBS buffer is 7.4, and the concentration of the antimicrobial peptide solution is 1~3 mg / mL; The mass ratio of antimicrobial peptides to recombinant human type I / III collagen was controlled at (1.5~4):(60~72); During the reaction, adjust the pH of the system to 6.5~7.0 and react at 4℃ in the dark for 10~24 hours.

8. The method for preparing the self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 5, characterized in that, In S3, Adjust the pH of the ECM mixed mother liquor to 7.2~8.0, adjust the ionic strength to 0.14~0.18 mol / L, raise the temperature to 25~30℃, and let it stand and incubate for 2~4 hours.

9. The method for preparing the self-assembled bionic human skin membrane ECM freeze-dried sponge according to claim 5, characterized in that, In S4, Pre-freezing stage: Pre-freeze at -40℃ for 6~8 hours to allow the gel to completely solidify and take shape; Sublimation drying stage: Dry at -20℃ and a vacuum of 10~15Pa for 10~24 hours; Analysis and drying stage: Drying at 25℃ and a vacuum of 5~8Pa for 6~10 hours.

10. The application of a self-assembled bionic human skin membrane (ECM) freeze-dried sponge as described in any one of claims 1 to 4 in the preparation of wound repair dressings, characterized in that, The wound repair dressing is used for the repair of acute and chronic infected wounds, acute sensitive muscle repair, diabetic foot ulcers, burn wounds, or pressure sores.

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