Nano transdermal sustained-release biological scaffold as well as preparation method and application thereof

Abstract

The invention provides a nano transdermal sustained-release biological scaffold as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The nano transdermal sustained-release biological scaffold provided by the invention comprises the following components: a load and a nano carrier, the load comprises mussel mucin, trehalose and Ectoine; the mass ratio of the mussel mucin to the trehalose to the Ectoine is (4 to 6) to (0.5 to 1.5) to (0.5 to 2.0). Through the innovative design of a quaternary composite system of mussel mucin, trehalose, Ectoine and a nano-carrier, the three technical problems of short slow release period, insufficient targeting property and difficulty in large-scale production in a wound repair material in the prior art are successfully solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a nano-transdermal sustained-release biological scaffold and a preparation method and application thereof. BACKGROUND

[0002] Traditional medical dressings such as silicone gel, collagen dressing and the like generally have problems of low sustained-release efficiency (only 3-5 days), insufficient biocompatibility and the like, and cannot meet the long-term needs of chronic wound repair. Although the mussel adhesive protein has excellent wet adhesion and healing-promoting functions due to the DOPA group (3,4-dihydroxyphenylalanine), the mussel adhesive protein is easily deactivated by environmental pH value and oxidative stress when used alone, resulting in rapid degradation of active ingredients. Although existing targeting technologies (such as electrophoretic loading and genetic engineering modification) can improve the delivery efficiency, they depend on complex processes and are high in cost, and are difficult to mass-produce. Although similar technologies have been disclosed in recent years (such as CN119073590A and CN114767924B), the core problems have not been solved. The mussel adhesive protein nano-coating probiotic technology developed by CN119073590A realizes broad-spectrum coating of aerobic / anaerobic bacteria, but does not involve the design of a transdermal delivery system, and lacks sustained-release control of active ingredients. The mussel adhesive protein hydrogel proposed by CN114767924B introduces collagen peptide to enhance adhesion, but its sustained-release period is less than 7 days, and the targeting delivery function is not integrated. The sodium alginate scaffold developed by the team of Wuhan University promotes skin regeneration through a micro-nano gradient structure, but does not utilize the dynamic response characteristics of mussel adhesive protein, resulting in insufficient adaptability in a chronic inflammatory environment. SUMMARY

[0003] Therefore, the application aims to provide a nano-transdermal sustained-release biological scaffold, which solves the problems of short sustained-release period, poor adhesion stability, no targeting, and poor wound permeability of existing medical dressings in the art through a triple synergistic mechanism. The mussel adhesive protein provides a biological adhesion group (DOPA), trehalose protects it from oxidative inactivation, and ectoine adjusts the inflammatory microenvironment. The three are integrated in a nano-carrier to form a dynamically responsive scaffold. This design avoids the dependence on synthetic materials of CN112891623B (chitosan-sodium alginate scaffold), and has better biocompatibility than CN101773683A (disulfide bond degradable scaffold).

[0004] In order to achieve the above application purposes, the application provides the following technical solutions.

[0005] The application provides a nano-transdermal sustained-release biological scaffold, which comprises the following components: a load and a nano-carrier; the load comprises mytic mucus, trehalose and ectoine; and the mass ratio of the mytic mucus, the trehalose and the ectoine is (4-6):(0.5-1.5):(0.5-2.0).

[0006] Preferably, the mass ratio of the load and the nano-carrier is 1:1.

[0007] Preferably, the nano-carrier comprises polylactic acid-glycolic acid copolymer or superparamagnetic iron oxide nanoparticles.

[0008] Preferably, the surface of the nano-carrier is modified with a folate ligand; and the method for modifying the folate ligand on the surface of the nano-carrier is an EDC / NHS activation method.

[0009] Preferably, the loading amount of the folate ligand on the surface of the nano-carrier is 2.5-3.5 μg / mg.

[0010] Preferably, the particle size of the polylactic acid-glycolic acid copolymer is 150-250 nm, and the zeta potential is -20--30 mV; and the particle size of the superparamagnetic iron oxide nanoparticles is 10-30 nm, and the saturation magnetization is greater than or equal to 60 emu / g.

[0011] The application further provides a preparation method of the above biological scaffold, which comprises the following steps: mixing mytic mucus, trehalose and ectoine, adjusting the pH to 7.5-8.5 to obtain a load; and mixing the load with a nano-carrier, ultraviolet irradiation-thermal crosslinking to obtain the biological scaffold.

[0012] Preferably, the temperature of the ultraviolet irradiation-thermal crosslinking is 40-80 ℃, the time is 30-60 min, and the irradiation intensity is 8-12 mW / cm 2 .

[0013] The application further provides application of the above biological scaffold or the above preparation method in preparation of a wound repair product.

[0014] Preferably, the wound comprises a diabetic ulcer, a pressure ulcer, a post-medical and artistic wound, a nasal mucosa injury or an oral mucosa injury.

[0015] The application has the following beneficial effects:

[0016] The biological scaffold provided by the present application successfully solves the three technical problems of short slow-release period, insufficient targeting and difficulty in large-scale production in the wound repair material in the prior art through the innovative design of the four-component composite system of the mussel myoglobin, trehalose, ectoine and nanocarrier. The biological scaffold provided by the present application is a four-component composite system, and has very high adhesion strength, can still maintain strong adhesion force in harsh environments such as humidity, underwater and wound exudation, so as to realize good wound closure and reduce the invasion of external bacteria. The four-component composite system biological scaffold provided by the present application can promote angiogenesis, and has a significant promoting effect on wound healing and repair. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a pH response release mechanism schematic diagram of the biological scaffold described in the present application;

[0018] Figure 2 It is an adhesion test result, and from left to right, it is the adhesion result of glass, plastic and ceramic respectively;

[0019] Figure 3 It is an L929 cell migration test result;

[0020] Figure 4 It is a wound healing process of diabetic rats;

[0021] Figure 5 It is a HE staining result;

[0022] Figure 6 It is a ROS clearance rate result;

[0023] Figure 7 It is an immunohistochemical fluorescence map;

[0024] Figure 8 It is a cytokine fluorescence quantitative PCR experiment. DETAILED DESCRIPTION

[0025] The present application provides a nano-transdermal sustained-release biological scaffold, comprising the following components: a load and a nanocarrier; the load comprises mussel myoglobin, trehalose and ectoine; the mass ratio of the mussel myoglobin, trehalose and ectoine is (4-6):(0.5-1.5):(0.5-2.0).

[0026] In the present application, the purity of mussel adhesive protein (MAP) is preferably ≥ 70%, higher than the industry standard (usually ≤ 50%), ensuring a DOPA group density of 15-20 μmol / g, significantly improving the cross-linking efficiency with nanocarriers. In the present application, trehalose not only protects DOPA groups as an antioxidant, but also forms a stable complex with ectoine through a hydroxyl hydrogen bond network, improving the stability of the scaffold in an oxidative stress environment (ROS level ≥ 500 μM) by 37%. In the present application, ectoine has a microenvironment regulating effect, can specifically inhibit the IL-17A / STAT3 pathway (inhibition rate ≥ 80%), and up-regulate miRNA-223 expression to 96% of the normal level, promoting the polarization of macrophages to M2 type, and fundamentally solving the delay of healing caused by chronic inflammation. In the present application, the mass ratio of the load and the nanocarrier is preferably 1:1. The mass ratio of the mussel adhesive protein, trehalose and ectoine is preferably 5:(1-1.2):(1-1.5), and the swelling rate of the three-dimensional network is preferably 1100%-1300%. In the present application, the nanocarrier includes polylactic acid-glycolic acid copolymer (PLGA) or ultra-ferromagnetic iron oxide nanoparticles (ufIONP). In the present application, the particle size of the polylactic acid-glycolic acid copolymer is preferably 150-250 nm, more preferably 180-220 nm, and the zeta potential is preferably -20 to -30 mV, more preferably -22 to -28 mV; the particle size of the ultra-ferromagnetic iron oxide nanoparticles is preferably 10-30 nm, more preferably 15-25 nm, and the saturation magnetization is preferably ≥ 60 emu / g. The present application does not have special limitations on the specific sources of the above components. In the present application, the DOPA groups and the carboxyl / amino groups on the surface of the nanocarrier are cross-linked by hydrogen bonds or covalent bonds to form a three-dimensional network, and trehalose and ectoine are wrapped in the nanocarrier, realizing a pH / oxidative stress dual-responsive gradient release, with a 24-hour release rate ≤ 15% in a pH 7.4 environment; in an inflammatory environment with pH ≤ 6.5 or reactive oxygen species (ROS) ≥ 500 μM, the 7-day cumulative release rate is ≥ 85%. The schematic diagram of the pH-responsive release mechanism of the biological scaffold of the present application is shown in Figure 1 .

[0027] The targeting and sustained release mechanism of the biological scaffold provided by the application comprises a physical and chemical double-path breakthrough, one of which can dynamically respond to release: the positive charge characteristic of MAP at physiological pH (7.4) is combined with the negative charge of PLGA nanoparticles through electrostatic action; when the pH of the wound surface decreases to 6.5 (inflammatory environment), the protonated DOPA group triggers bond rupture, realizing gradient release of active ingredients. The second is the design of a water-repellent adhesive layer: the catechol group of MAP forms a covalent bond with the lysine residues on the tissue surface, establishing a physical barrier on the wound surface, blocking the invasion of external microorganisms (bacteriostatic rate ≥ 99.99%) while reducing the loss of tissue fluid flushing (adhesion strength up to 1.5 ± 0.3 N / cm 2 ). In the application, the surface of the nanocarrier is preferably modified with folate ligands, and the loading amount of folate ligands on the surface of the nanocarrier is preferably 2.5-3.5 μg / mg, more preferably 2.8-3.2 μg / mg. By modifying the surface with folate ligands, the application can specifically recognize the high expression of folate receptor beta (FR-beta) around the wound, making the enrichment rate of the nanocarrier at the diabetic ulcer site increase by 4 times, and achieving active targeting synergism.

[0028] In the present application, the method for modifying folate ligand on the surface of nanocarriers is preferably the method of EDC / NHS activation. The specific steps of the method of EDC / NHS activation for modifying folate ligand on the surface of nanocarriers are preferably as follows: after the PLGA nanoparticles are cleaned with MES Buffer, the nanoparticles are re-dispersed in MES Buffer to obtain a re-suspension solution with a concentration of 5-10 mg / mL. In the continuously stirred re-suspension solution, aqueous solutions of NHS and EDC are sequentially added, wherein the PLGA surface carboxyl group: EDC: NHS is preferably 1:5:10, and the reaction is carried out at room temperature for 15-30 minutes to fully activate the PLGA surface carboxyl group into an NHS ester. Folic acid (FA) or folic acid-polyethylene glycol-amine (FA-PEG-NH2) (dissolved in a small amount of DMSO and then diluted with PBS buffer to ensure that the final concentration of DMSO is <5%) is quickly added to the above-mentioned activated nanoparticle reaction solution. The mass ratio of FA: PLGA is preferably 1:(2-5). The pH of the reaction system is adjusted to 7.0-7.4. The reaction is continuously carried out at room temperature and in the dark for 2-4 hours. After the reaction is completed, an excess of glycine or ethanolamine solution (50 mM, pH 8.0) is added to react for 15 minutes to quench the unreacted activated ester. The unreacted folic acid, EDC, NHS, urea by-products and the like are removed by repeatedly cleaning with PBS (pH 7.4) for 3-4 times. Finally, the purified folic acid-modified PLGA nanoparticles (FA-PLGA-NPs) are re-dispersed in PBS or ultrapure water, and stored at 4°C in the dark. In the present application, when the nanocarrier is a superparamagnetic iron oxide nanoparticle, the bulk surface does not naturally have a carboxyl group. By physical adsorption or covalent bonding, the surface is wrapped with a carboxyl-containing polymer (such as polyacrylic acid) to make the surface have a carboxyl group, which is convenient for subsequent polypeptide modification or binding of other biological molecules. In the present application, carboxylated superparamagnetic iron oxide nanoparticles can also be directly obtained by commercial purchase.

[0029] The biological scaffold provided by the present application has a synergistic effect, specifically including: (1) Anti-oxidation-anti-inflammatory linkage: trehalose removes excess ROS on the wound surface (removal rate ≥ 90%), protects ecdyson to maintain anti-inflammatory activity, and increases the inhibition rate of ecdyson to TNF-α to 70%. (2) Adhesion-regeneration synergy: the DOPA group of MAP provides immediate adhesion (the initial adhesion strength is ≥ 0.8 N / cm 2), and simultaneously activates the integrin-FAK signal pathway to accelerate the migration of fibroblasts (the migration speed is increased by 40%). (3) Adhesion test: the bio-scaffold can form strong chemical bonding with various material surfaces, including metals, ceramics, plastics and biological tissues, etc., specifically, the covalent bond is formed with the material surface through oxidation reaction, and the non-covalent bond such as hydrogen bond and ionic bond can also be formed with the polar groups on the material surface, so that firm adhesion is achieved. The bio-scaffold provided by the application is a quaternary composite system, and the adhesion strength is very high, which can still maintain strong adhesion in harsh environments such as moisture, underwater and wound exudate, so as to achieve good wound closure and reduce the invasion of external bacteria. (4) Microenvironment remodeling: the quaternary composite system bio-scaffold provided by the application can promote angiogenesis (the expression amount of VEGF is 368.3 pg / mL), and cooperates with Ekdin to shorten the inflammation period to 3 days (7 days are required for traditional dressings).

[0030] The core difference comparison between the bio-scaffold and the existing product is shown in Table 1. The bio-scaffold provided by the application can realize the antioxidant-anti-inflammatory linkage: the trehalose removes the excess ROS on the wound (the removal rate is greater than or equal to 90%), protects the Ekdin to maintain the anti-inflammatory activity, and the inhibition rate of Ekdin to TNF-α is increased to 70%. The bio-scaffold provided by the application can promote angiogenesis (the expression amount of VEGF is 368.3 pg / mL), and cooperates with Ekdin to shorten the inflammation period to 3 days (7 days are required for traditional dressings).

[0031] Table 1 Core difference comparison between the bio-scaffold and the existing product

[0032] Technical features Traditional medical dressings Existing applications of mussel proteins The present invention Sustained-release period 3-5 days ≤7 days 7-14 days Adhesion stability Easy to fall off in wet state Strong pH sensitivity pH / oxidative stress dual response Targeting ability None Local adhesion without targeting Folic acid / antibody active targeting Production process Simple Genetic engineering complex UV / thermal crosslinking scale Wound permeability Only epidermis Partial dermis Full skin layer gradient permeation

[0033] The application further provides a preparation method of the bio-scaffold, which comprises the following steps: mixing the mussel myoglobin, trehalose and Ekdin, adjusting the pH to 7.5-8.5 to obtain a loading material; and mixing the loading material with a nano-carrier, ultraviolet irradiation-thermal crosslinking to obtain the bio-scaffold.

[0034] In the application, the temperature of the ultraviolet irradiation-thermal crosslinking is preferably 40-80 DEG C, more preferably 50-70 DEG C, the time is preferably 30-60 min, more preferably 40-50 min, and the irradiation intensity is preferably 8-12 mW / cm 2 , more preferably 9-11 mW / cm 2The application adopts ultraviolet irradiation-heat double induction to crosslink, solves the bottleneck of large-scale production, and specifically, low-temperature and high-efficiency crosslinking: ultraviolet irradiation (wavelength 365 nm) is performed at 40-80 DEG C for 30-60 min, compared with traditional EDC / NHS chemical crosslinking (24 hours are needed), the time is shortened to 1 / 48, and toxic crosslinking agent residues (residual amount is less than or equal to 0.01 ppm) are avoided; nano-carrier self-assembly: PLGA or ufIONP is self-assembled into a three-dimensional network (PDI is less than or equal to 0.2) with a particle size of 200-400 nm through DOPA-carboxyl / amino hydrogen bond, and a complex mixed process depending on collagen peptides in CN114767924B is broken through; modular production design: by adjusting the mass ratio of trehalose to ectoine (1:0.5-2), the scaffold is flexibly switched from a rapid anti-inflammatory type to a long-acting repair type, and different wound needs are met.

[0035] In the application, the ultraviolet irradiation is preferably performed under nitrogen protection, and the oxygen concentration is preferably less than or equal to 100 ppm.

[0036] The application also provides application of the above biological scaffold or the above preparation method in preparation of a wound repair product. In the application, the wound preferably includes diabetic ulcers, pressure ulcers, post-medical and artistic wounds, nasal mucosa injuries or oral mucosa injuries (oral ulcers are treated by using the wet adhesion of the scaffold), and the nasal mucosa injury preferably includes rhinitis or nosebleed. In the application, the dosage form of the biological scaffold preferably includes a transdermal patch, a gel (for wound repair), a nasal spray or a targeted injection (ufIONP carrier type, for tumor or kidney diseases).

[0037] The biological scaffold provided by the application has high biocompatibility and almost no cytotoxicity: after being co-cultured with human mesenchymal stem cells for 7 days, the survival rate is greater than or equal to 98% (the survival rate of the control group of silicone dressings is 75%); immunogenicity: there is no significant difference (p>0.05) in IgE antibody level in the mouse body compared with the physiological saline group. The biological scaffold provided by the application has significant advantages in treating chronic wounds: diabetic ulcers: the healing time is shortened to 14±2 days (the traditional dressing needs 28±3 days); pressure ulcers: the closure rate reaches 95.3% on the 10th day (the positive control group is 68.7%), and the regenerated skin contains hair follicles and nerve endings. In addition, the biological scaffold provided by the application has a long shelf life, and the activity is retained by more than or equal to 90% after being stored at 4 DEG C for 12 months; and has a low production cost, which is reduced by 60% compared with a genetically engineered load type scaffold, and thus has high industrialization performance.

[0038] The application first proposes an antioxidant mechanism of using trehalose to protect DOPA groups, breaks through the stability bottleneck of MAP application, develops an ultraviolet-thermal double-induced crosslinking process, realizes efficient and non-toxic production, and cooperates with pH response release through folate-mediated active targeting, and improves the efficiency of chronic wound repair. The biological scaffold can be applied to wound repair, post-care of medical art, tumor photothermal synergistic treatment and transdermal delivery of skin wounds, such as magnetic targeting and local hyperthermia realized through ufIONP carriers.

[0039] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they cannot be understood as limitations to the protection scope of the application.

[0040] In the following examples, if not specifically stated, they are all conventional methods.

[0041] In the following examples, the materials, reagents and the like used, if not specifically stated, can be obtained from commercial channels.

[0042] In the following examples, the mussel adhesive protein (MAP) is purchased from Jiangsu Jinpinuo'an Biotechnology Co., Ltd.

[0043] Example 1

[0044] A nano-transdermal sustained-release biological scaffold is made of a load and a polylactic acid-glycolic acid copolymer in a mass ratio of 1:1, the load is composed of mussel adhesive protein, trehalose and ekdine; the mass ratio of the mussel adhesive protein, trehalose and ekdine is 4:0.5:0.5; the particle size of the polylactic acid-glycolic acid copolymer is 150 nm, and the zeta potential is -20 mV.

[0045] The preparation method is as follows:

[0046] The mussel adhesive protein, trehalose and ekdine are mixed, the pH is adjusted to 7.5, and the load is obtained; the load and the polylactic acid-glycolic acid copolymer are mixed, the irradiation intensity is 8 mW / cm 2 The biological scaffold is obtained by ultraviolet irradiation-thermal crosslinking for 60 min.

[0047] Example 2

[0048] A nano-transdermal sustained-release biological scaffold is made of a load and a polylactic acid-glycolic acid copolymer in a mass ratio of 1:1, the load is composed of mussel adhesive protein, trehalose and ekdine; the mass ratio of the mussel adhesive protein, trehalose and ekdine is 4:0.5:0.5; the particle size of the polylactic acid-glycolic acid copolymer is 150 nm, and the zeta potential is -20 mV.

[0049] The preparation method is as follows:

[0050] The mussels mucin, trehalose and ectoine are mixed, the pH is adjusted to 8.5 to obtain a loading material; the loading material is mixed with polylactic acid-glycolic acid copolymer, 80℃, the irradiation intensity is 12mW / cm 2 Ultraviolet irradiation-thermal crosslinking for 30min to obtain the biological scaffold.

[0051] Example 3

[0052] A nano-transdermal sustained-release biological scaffold is prepared from a loading material and polylactic acid-glycolic acid copolymer in a mass ratio of 1:1, wherein the loading material is composed of mussels mucin, trehalose and ectoine; the mass ratio of the mussels mucin, trehalose and ectoine is 5:1:1.5; and the particle size of the polylactic acid-glycolic acid copolymer is 200nm and the zeta potential is -25mV.

[0053] The preparation method is as follows:

[0054] The mussels mucin, trehalose and ectoine are mixed, the pH is adjusted to 8.0 to obtain a loading material; the loading material is mixed with polylactic acid-glycolic acid copolymer, 60℃, the irradiation intensity is 10mW / cm 2 Ultraviolet irradiation-thermal crosslinking for 50min to obtain the biological scaffold.

[0055] Example 4

[0056] A nano-transdermal sustained-release biological scaffold is prepared from a loading material and polylactic acid-glycolic acid copolymer modified with a folic acid ligand on the surface in a mass ratio of 1:1, wherein the loading material is composed of mussels mucin, trehalose and ectoine; the mass ratio of the mussels mucin, trehalose and ectoine is 5:1:1; and the particle size of the polylactic acid-glycolic acid copolymer is 220nm and the zeta potential is -28mV.

[0057] The preparation method of the polylactic acid-glycolic acid copolymer surface modified with folate ligand is as follows: taking the PLGA nanoparticle suspension, washing twice with MES Buffer (0.1M, pH 5.5) by ultracentrifugation (20000rpm, 30min, 4℃). Redispersed in an appropriate amount of MES Buffer, the concentration is 5mg / mL, and a resuspension is obtained. In the continuously stirred PLGA nanoparticle MES resuspension, freshly prepared aqueous solutions of NHS and EDC are sequentially added, and the mass ratio (molar ratio) of PLGA surface carboxyl: EDC: NHS is 1:5:10. The carboxyl group on the surface of PLGA is fully activated to NHS ester at room temperature for 15 minutes. Folic acid (FA) is dissolved in DMSO and then diluted with PBS buffer to ensure that the final concentration of DMSO is <5%, and then quickly added to the above-mentioned activated nanoparticle reaction solution. The mass ratio of FA: PLGA is 1:2. The pH of the reaction system is adjusted to 7.0 with dilute NaOH. The reaction is continued at room temperature and in the dark for 2 hours. After the reaction is completed, an excess of glycine solution (50mM, pH 8.0) is added and reacted for 15 minutes to quench the unreacted activated ester. The unreacted folic acid, EDC, NHS, urea by-products and the like are removed by repeatedly washing with PBS (pH 7.4) using ultracentrifugation method for 3 times. Finally, the purified folic acid modified PLGA nanoparticles are redispersed in PBS or ultrapure water, and stored at 4℃ in the dark. It is detected that the loading amount of folate ligand on the surface of polylactic acid-glycolic acid copolymer is 2.6μg / mg.

[0058] The preparation method of the nano-transdermal sustained-release biological scaffold is as follows:

[0059] The mytilus mucin, trehalose and ectoine are mixed, and the pH is adjusted to 8.0 to obtain a loading material; the loading material is mixed with the polylactic acid-glycolic acid copolymer surface modified with folate ligand, and the mixture is irradiated at 60℃ and an irradiation intensity of 10mW / cm 2 UV irradiation-thermal crosslinking for 50min to obtain the biological scaffold.

[0060] Example 5

[0061] A nano-transdermal sustained-release biological scaffold is made from a loading material and superparamagnetic iron oxide nanoparticles surface modified with folate ligand at a mass ratio of 1:1, wherein the loading material is composed of mytilus mucin, trehalose and ectoine; the mass ratio of the mytilus mucin, trehalose and ectoine is 5:1.2:1.5; and the superparamagnetic iron oxide nanoparticles have a particle size of 10nm and a saturation magnetization intensity ≥60emu / g.

[0062] The preparation method of the superparamagnetic iron oxide nanoparticles modified with folate ligands is as follows: the superparamagnetic iron oxide nanoparticles are taken, and the MES Buffer (0.1M, pH 5.5) is washed for 3 times by high-speed centrifugation (20000rpm, 30min, 4℃). The superparamagnetic iron oxide nanoparticles are coated with polyacrylic acid to have carboxyl groups on the surface, and the nanoparticles are re-dispersed in a proper amount of MES Buffer to obtain a suspension with a concentration of 10mg / mL. Then, the freshly prepared aqueous solutions of NHS and EDC are sequentially added to the continuously stirred MES suspension of the superparamagnetic iron oxide nanoparticles, and the mass ratio (molar ratio) of the carboxyl groups on the surface of the superparamagnetic iron oxide nanoparticles, EDC and NHS is 1:5:10. The carboxyl groups on the surface of the superparamagnetic iron oxide nanoparticles are activated to NHS esters at room temperature for 30min. The folate-polyethylene glycol-amine (FA-PEG-NH2) is dissolved in DMSO, then diluted with PBS buffer to ensure that the final concentration of DMSO is less than 5%, and then quickly added to the above-mentioned activated nanoparticle reaction solution. The mass ratio of folate-polyethylene glycol-amine to superparamagnetic iron oxide nanoparticles is 1:5. The pH of the reaction system is adjusted to 7.4 with dilute NaOH. The reaction is continuously carried out at room temperature and in the dark for 4 hours. After the reaction is completed, an excess of ethanolamine solution (50mM, pH 8.0) is added for reaction for 15min to quench the unreacted activated ester. The unreacted folate, EDC, NHS, urea by-products and the like are removed by repeatedly washing with PBS (pH 7.4) using high-speed centrifugation for 4 times. Finally, the purified folate-modified superparamagnetic iron oxide nanoparticles are re-dispersed in PBS or ultrapure water, and stored at 4℃ in the dark. It is detected that the loading amount of the folate ligands on the surface of the superparamagnetic iron oxide nanoparticles is 3.0μg / mg.

[0063] The preparation method of the nano-transdermal sustained-release biological scaffold is as follows:

[0064] The mussel myoglobin, trehalose and ectoine are mixed, and the pH is adjusted to 8.0 to obtain a loading material; the loading material is mixed with the superparamagnetic iron oxide nanoparticles modified with folate ligands, and the mixture is irradiated at 60℃ and an irradiation intensity of 10mW / cm 2 The mixture is ultraviolet irradiated and heat crosslinked for 50min to obtain the biological scaffold.

[0065] Example 6

[0066] A nano-transdermal sustained-release biological scaffold is prepared from a loading material and superparamagnetic iron oxide nanoparticles in a mass ratio of 1:1, wherein the loading material is composed of mussel myoglobin, trehalose and ectoine; the mass ratio of the mussel myoglobin, trehalose and ectoine is 6:1.5:2; and the particle size of the superparamagnetic iron oxide nanoparticles is 30nm, and the saturation magnetization is ≥60emu / g.

[0067] The preparation method is as follows:

[0068] The mussels mucin, trehalose and ectoine are mixed, and the pH is adjusted to 8.5 to obtain a loading; the loading is mixed with superparamagnetic iron oxide nanoparticles, and the mixture is irradiated at 70°C and an irradiation intensity of 11 mW / cm 2 Ultraviolet irradiation-thermal crosslinking for 50 min to obtain the biological scaffold.

[0069] Test Example 1

[0070] Adhesion test of the biological scaffold obtained in Example 3

[0071] Since the skin structure of Bama miniature pigs is similar to that of humans, and the genetic stability and individual differences are small, the Bama miniature pigs are often used as model animals for studying human skin. In this test, the abdominal skin of a 31-day-old male Bama miniature pig was used to test the adhesion of the biological scaffold obtained in Example 3: The pig skin was cut into a size of 1.5×1.5 cm, and the quaternary composite system material was uniformly applied to the outer surface (A surface) of the pig skin. The corresponding test materials (glass (10 mL of a lincomycin bottle, 10 g), polypropylene (PP centrifuge tube, 10 g), and ceramic (ceramic crucible, 50 g)) were placed on the A surface of the pig skin coated with the biological scaffold obtained in Example 3 for 3 min, and then the test materials were lifted, and the pig skin was adhered to the test materials and could be lifted. After standing for another 5 min, a 100 g weight was pressed on the other side (B surface) of the pig skin, and the weight was rotated and lifted, and no test materials and pig skin were detached (as shown in FIG. 2), indicating that the biological scaffold (quaternary composite system material) obtained in Example 3 had penetrated the pig skin (from the A surface to the B surface) and had good adhesion to the metal weight. Figure 2

[0072] After the test, physiological saline was sprayed to rinse and coat the adhesion position, and the adhesion still had a certain viscosity. The above results show that the biological scaffold (quaternary composite system) of the present application has good adhesion to pig skin tissue and various substrates. The pig skin under the surface without coating the hydrogel also has good adhesion to the materials. The adhesion is strong and can resist physiological saline flushing.

[0073] Test Example 2

[0074] L929 cell migration test

[0075] Plating: ① Prepare a low serum medium (99% MEM + 1% serum + 0.1% double antibody); ② Digest the adherent cells (L929, the fifth generation) in the logarithmic growth phase using 0.25% trypsin to prepare a single cell suspension and perform cell counting; ③ After cell counting, inoculate the six-well culture plate with 0.158 mL per well, and 5.69×10 5 ​Cells were cultured in 6-well plates at 37℃, 5% CO2 incubator for 24 hours. The culture medium was removed, and the cells were washed with PBS twice. The cells were scraped with a 200-mL gun, and the medium was removed. The cells were washed with PBS twice, and the floating cells were removed. The cells were placed in a 37℃, 5% CO2 incubator for 24 hours.

[0076] Scratch: ①Use a ruler to compare, and use a 200-mL gun to make a cell scratch (the plane of the gun tip is perpendicular to the plane of the culture plate and is tightly scraped through the cell layer; the medium is removed, and the cells are washed with PBS twice; and the floating cells are washed off completely; ②Set up a blank group, an H2O2 group, a biological scaffold group (a four-component composite system group) obtained in Example 4, and a commercially available similar product (mussel myelin repair dressing (containing mussel myelin, trehalose)), and two parallel groups (i.e., two wells) are prepared for each group; after the liquid is changed, the cells are placed in a 37℃, 5% CO2 incubator for culture.

[0077] Sample addition: 200-mL test solution is added to the scratch using a pipette, and the low-serum culture medium is added to a volume of 2 mL.

[0078] Observation: The cells are observed and photographed at 0 h, 12 h, and 24 h, respectively, and the observation results are recorded.

[0079] The results are shown in Table 1, which show that the biological scaffold obtained in the application can promote the migration of fibroblasts, and the effect of promoting the migration of fibroblasts is better than that of a commercially available product. Figure 3 Example 7

[0080] A nano-transdermal sustained-release biological scaffold is prepared from a load and a polylactic acid-glycolic acid copolymer at a mass ratio of 1:1, the load is composed of mussel myelin, trehalose, and ikkduin, the mass ratio of the mussel myelin, trehalose, and ikkduin is 5:1:1, and the particle size of the polylactic acid-glycolic acid copolymer is 200 nm, and the zeta potential is -28 mV.

[0081] The preparation method is as follows:

[0082] 0.5 g of high-purity mussel myelin (70%, containing DOPA 18 μmol / g), 0.1 g of trehalose, and 0.1 g of ikkduin are dissolved in PBS, and the pH is adjusted to 8.0 to obtain a load; the polylactic acid-glycolic acid copolymer is added to the load, and the biological scaffold is obtained by ultraviolet (365 nm) irradiation-thermal crosslinking at 45℃ and an irradiation intensity of 10 mW / cm 2 for 30 min.

[0083] One, antioxidant stability detection:

[0084] Test method:

[0085] Experimental group: three biological scaffold samples (containing PLGA nanoparticles) prepared by the method of this example are taken, and each sample has a mass of 20 mg.

[0086] The results are shown in Table 1, which show that the biological scaffold obtained in the application can promote the migration of fibroblasts, and the effect of promoting the migration of fibroblasts is better than that of a commercially available product.

[0087] Control group: Prepare 3 portions of pure mussel adhesive protein (70%, containing DOPA 18 pmol / g) in PBS (pH 7.4) solution with a concentration of 5 mg / mL.

[0088] Oxidative stress treatment: Place the experimental group scaffolds and the control group protein solution in 5 mL of hydrogen peroxide (H2O2) solution with a concentration of 500 mM, respectively. (Note: This concentration is used to simulate the moderate inflammatory oxidative environment in the wound site).

[0089] Place the samples in a 37°C constant temperature shaker and incubate at a speed of 60 rpm for 72 hours. Take samples at 0 hours (initial) and 72 hours after incubation, respectively.

[0090] For the experimental group (scaffolds): Take out the samples, gently rinse the surface with ultrapure water to remove residual H2O2, then dissolve them in PBS containing 100 mM sodium borohydride (NaBH4, as a reducing agent and stabilizer), and vortex thoroughly to ensure complete dissolution.

[0091] For the control group (protein solution): Directly take 100 mL of protein solution and add 900 mL of PBS containing 100 mM NaBH4 for dilution and stabilization.

[0092] DOPA content determination: Quantitative analysis was performed using high performance liquid chromatography (HPLC).

[0093] The results are shown in Table 2. After incubation in 500 mM H2O2 for 72 hours, the DOPA oxidation rate of the experimental group scaffolds containing PLGA nanoparticles was significantly lower than that of the pure protein control group (14.2% vs 85.3%), demonstrating excellent antioxidant stability. This indicates that PLGA nanoparticles effectively protect the active DOPA groups in mussel adhesive protein.

[0094] Table 2 Determination results of three parallel samples (n = 3)

[0095]

[0096]

[0097] II. Sustained-release performance:

[0098] Test method: Accurately weigh 3 portions of the biological scaffold samples prepared in this example (each containing about 10 mg of ekdine), and place them in 50 mL centrifuge tubes. Comparative samples: Take 3 portions of commercially available traditional silicone dressings, and load the same amount of ekdine (10 mg) using the same method.

[0099] Add 25 mL of phosphate buffered saline (PBS, pH 7.4) to each centrifuge tube and place it in a constant temperature shaker at 37°C and 100 rpm.

[0100] At the preset time point, 1 mL of release medium was taken out, and 1 mL of fresh preheated PBS was immediately supplemented into the tube to maintain the leak tank condition.

[0101] Ectoine concentration determination: the removed sample liquid was analyzed by ultraviolet-visible spectrophotometry (UV-Vis).

[0102] A standard curve was drawn by ectoine standard solution, and the sample concentration was calculated according to the absorbance value.

[0103] The results are shown in Table 3, and the release kinetics data show that the biological scaffold prepared in this embodiment exhibits a significant sustained release effect. It releases 82.3% of ectoine within 7 days, and nearly completely releases (96.5%) within 14 days, and the release curve is flat. While the traditional silicone dressing as a control has more than 95% of the drug burst released within 3 days. This shows that the biological scaffold (composite scaffold) of the application can effectively delay the release of ectoine, and is expected to provide more persistent moisturizing and cell protection in application.

[0104] Table 3 Ectoine release rate

[0105]

[0106]

[0107] Test Example 3

[0108] Chronic inflammation treatment

[0109] Diabetic foot ulcer repair

[0110] A full-thickness skin defect (5 mm in diameter) was made on the foot of a streptozotocin (STZ)-induced diabetic rat model.

[0111] (1) Experimental animal grouping and establishment of diabetic model: 8-10 week old male SD rats were selected, and after adaptive feeding for one week, they were fasted for 12 hours without water.

[0112] Diabetes induction: a single intraperitoneal injection of streptozotocin (STZ, dissolved in 0.1 mol / L citric acid buffer, pH 4.5) was performed at a dose of 55 mg / kg body weight. The control group (normal group) was injected with the same volume of citric acid buffer.

[0113] Modeling standard: 72 hours after injection, blood was taken from the tail vein to measure the fasting blood glucose value. Rats with blood glucose concentration ≥16.7 mmol / L for two consecutive times were identified as successful establishment of a diabetic model and included in the subsequent experiments.

[0114] (2) Wound model preparation

[0115] Procedure: After anesthetizing diabetic rats with isoflurane inhalation, the skin on their backs was prepared and disinfected. A full-thickness skin defect with a diameter of 5 mm (deep to the fascia layer) was created on the dorsum of the foot using a sterile biopsy puncture instrument. Rats with successful modeling were randomly divided into the following three groups (n=6):

[0116] Experimental group (quaternary composite system group): The biological scaffold (hydrogel dressing) containing the active ingredients obtained in Example 4 was applied / covered to the wound.

[0117] Positive control group (commercially available control group): The wound was treated with alginate dressings, which are commonly used in clinical practice.

[0118] Negative control group (blank control group): The wound was covered only with sterile PBS-moistened gauze. This group was used to simulate the natural healing process and served as a baseline control.

[0119] (3) Dosing regimen

[0120] Administration frequency: Change the dressing once a day to maintain a clean wound environment and an effective concentration of medication.

[0121] Dosage / method of administration: For hydrogel dressings (experimental group), each time the dressing is changed, take an appropriate amount of hydrogel (about 100-200 μL, which is enough to evenly cover the entire wound and form a film about 2 mm thick) and gently apply it to the wound.

[0122] The positive control group used the appropriate dressings according to the instructions.

[0123] The negative control group had their gauze moistened with PBS replaced.

[0124] Intervention period: Continuous administration until the experimental endpoint (days 3, 7, and 14). Key effect statistics (such as tactile threshold and tissue collection) were performed on days 7 and 14 after intervention to observe changes in the inflammatory and proliferative phases.

[0125] (4) Sample collection and testing

[0126] On days 3, 7, and 14 after the intervention, rats in each group were sacrificed, and the wounds and surrounding tissues were collected.

[0127] A portion of the tissue was fixed with 4% paraformaldehyde for use in paraffin sections (for H&E staining, immunohistochemistry, etc.).

[0128] Another portion of the tissue was frozen at -80°C for the extraction of proteins (ELISA detection of cytokines) or RNA.

[0129] In vivo testing: Behavioral tests such as tactile threshold (neurological function) were measured multiple times on days 1, 3, 7, 10, and 14 to plot recovery curves.

[0130] (5) Test results

[0131] 1. The wound healing process of each group was observed at 0, 7 and 14 days after intervention, and the results are shown in Table 1, indicating that the biological scaffold of the application has a significant promoting effect on the healing of diabetic wounds. Figure 4

[0132] On day 14, the rats were sacrificed, and the wounds of the negative control group and the experimental group were collected for paraffin section and H&E staining, and the results are shown in Table 2. Figure 5

[0133] 2. Inflammation regulation: ELISA method was used to detect the concentration of cytokines in the supernatant of wound tissue homogenate. The results of the 7th day are shown in Table 4, indicating that compared with the two control groups, the experimental group can significantly reduce the levels of pro-inflammatory factors IL-17A and TNF-α, while significantly increase the expression of anti-inflammatory factors IL-13 and IL-10 (*p<0.05), effectively reversing the chronic inflammatory microenvironment of diabetic wounds.

[0134] Table 4: Results on the 7th day (regulation of inflammation peak)

[0135] Group (Group) IL-17A (pg / mL) IL-13 (pg / mL) IL-10 (pg / mL) TNF-α (pg / mL) Experimental group 36.67±3.21 62.00±5.18 45.23±4.10 58.45±6.32 Positive control group 60.15±5.87* 50.41±4.65* 35.67±3.54* 95.68±8.91* Negative control group 74.00±6.95* 45.50±4.02* 28.45±2.87* 125.41±12.36*

[0136] Note: * indicates p<0.05 compared with the experimental group

[0137] 3. Angiogenesis:

[0138] Method 1: ELISA was used to detect the concentration of VEGF in the tissue homogenate.

[0139] Method 2: Immunohistochemistry (IHC): CD31 (vascular endothelial cell marker) antibody was used to stain the wound tissue sections, and the microvessel density was calculated.

[0140] The results are shown in Table 5, indicating that the experimental group has the highest VEGF expression, which is 3.1 times that of the negative control group. CD31 immunohistochemical quantitative analysis shows that the microvessel density of the experimental group is significantly higher than that of the positive control group and the negative control group (*p<0.05), which is about 2 times that of the negative control group, indicating that the biological scaffold dressing of the application can promote angiogenesis in diabetic wounds and improve local blood supply.

[0141] Table 5: Results on the 14th day (key period of angiogenesis)

[0142] Group (Group) VEGF (pg / mL) Microvessel density (number / field) Experimental group 368.3±32.5 28.4±3.2 Positive control group 210.7±25.4* 18.1±2.3* Negative control group 118.5±15.8* 14.2±1.9*

[0143] Note: * indicates p<0.05 compared with the experimental group

[0144] 4. Recovery of nerve function

[0145] ​​Detection method: behavioral test-mechanical tactile threshold: the newly formed skin of the wound was stimulated with a series of Von Frey filaments of different stiffness, and the minimum force (gram, g) that caused the rat to withdraw its paw was recorded to evaluate the recovery of sensory nerves. The lower the threshold, the more sensitive the sensation.

[0146] The results shown in Table 6 indicate that the tactile threshold of the experimental group was significantly lower than that of the control group from the 7th day. By the 14th day, the threshold of the experimental group had approached the level of normal skin (the tactile threshold of the normal rat foot was about 2-4 g), while the control group recovered slowly. This indicates that the biological scaffold dressing of the application not only accelerates wound closure, but also promotes the functional regeneration of sensory nerves, achieving high-quality healing.

[0147] Table 6 Tactile threshold curve over time

[0148] Time (Day) Experimental group (g) Positive control group (g) Negative control group (g) 1 >15 >15 >15 7 8.5±1.2 12.3±1.5* >15* 14 3.2±0.4 5.1±0.7* 6.8±1.1*

[0149] Note: where * indicates p < 0.05 compared with the experimental group.

[0150] Test Example 4

[0151] ROS clearance rate test

[0152] Principle: 1,1-diphenyl-2-trinitrobenzene hydrazine radical (·DPPH) is a stable free radical, which is purple and has a maximum absorption peak at 517 nm. Antioxidants can provide electrons to quench it, causing the solution to fade and the absorbance to decrease.

[0153] Method: Glutathione (a recognized antioxidant), mussel mucin, trehalose, and the biological scaffold obtained in Example 4 (quaternary composite system) were each prepared into a 1.0 mg / mL solution with purified water, and then mixed gently with 2.5 mL of 1 x 10 -4 mol / L DPPH ethanol solution. After 1 h of reaction in the dark, each test tube was centrifuged at 4000 rpm for 10 min, and 1 mL of supernatant was transferred to a cuvette with a pipette. The cuvette was placed in a UV-visible spectrophotometer, and the absorbance value at 517 nm was measured after zeroing with anhydrous ethanol. The absorbance data of each group was recorded. The clearance rate was calculated.

[0154] Clearance rate (%) = [(blank group absorbance - sample group absorbance) / blank group absorbance] x 100%

[0155] The results are as follows: Figure 6As shown, it is shown that: the mussel mucin itself is a very efficient natural antioxidant. This provides a solid foundation for the high efficiency of the quaternary composite system. Glutathione as the core antioxidant in cells, its 79.2% clearance rate proves the effectiveness of the experimental model. The ROS clearance rate (98.6%) of the quaternary composite system is much higher than that of each single component (mussel mucin 82.4%, trehalose 56.7%) and the control (glutathione 79.2%), which shows that there is a significant synergistic effect between the components, rather than the simple addition of single components. It shows that the biological scaffold provided by the application is expected to be used as a high-efficiency antioxidant formula, which can be applied in the field of skin care, anti-aging, etc.

[0156] Test Example 5

[0157] During wound healing, new tissue regeneration requires a variety of nutrients and cytokines, so blood vessels that transport these substances play a crucial role in the process. A large number of vascular endothelial cells are produced during the process of angiogenesis, and vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) are the main biomarkers that can promote vascular endothelial cell proliferation and vascular formation.

[0158] According to the method of Test Example 3, the modeling successful rats were randomly divided into the following four groups (n=6):

[0159] Experimental group (biological scaffold (quaternary composite system group) obtained in Example 4): wound smearing / covering with the biological scaffold (hydrogel dressing) containing active ingredients obtained in Example 4.

[0160] MAP group: using 1.0 mg / mL MAP solution.

[0161] Ecdyson group: using 0.2 mg / mL ecdyson solution.

[0162] Negative control group (blank control group): only using sterile saline wet gauze to cover the wound. This group is used to simulate the natural healing process as a baseline control.

[0163] Dosing regimen:

[0164] Dosing frequency: replacing the dressing once a day to maintain the cleanliness of the wound environment and the effective concentration of the drug.

[0165] Dosing dose / method: when replacing each time, take an appropriate amount of hydrogel (sufficient to evenly cover the entire wound and form a thin film with a thickness of about 2 mm) and gently smear it on the wound.

[0166] The negative control group replaces the new saline wet gauze.

[0167] Intervention period: continue dosing until day 14.

[0168] On day 14 after intervention, rats in each group were sacrificed respectively, and wound and surrounding tissue were collected.

[0169] Part of the tissue was fixed with 4% paraformaldehyde for paraffin section (immunohistochemical test). Another part of the tissue was frozen at -80℃ for protein extraction (cytokine fluorescence quantitative PCR).

[0170] VEGF level was measured by immunohistochemical test to monitor angiogenesis at the wound. The results are shown in Figure 7 (immunohistochemical fluorescence image), the positive rate of VEGF in the quaternary complex system group was significantly higher than that in other groups, indicating that the blood vessel formation in the group was the most. At the same time, platelet-endothelial cell adhesion molecule (CD31) was also detected, mainly to prove the existence of endothelial cell organization, and also used to evaluate the density and distribution of angiogenesis. From Figure 7 It was observed that even if the wound was in the inflammation period, the content of CD31 in the wound of the quaternary complex system group was the most compared with the other three groups of dressings, indicating that the density of the newly formed blood vessels was significantly increased. Interleukin-17A (IL-17A) is a strong pro-inflammatory factor, which can recruit inflammatory cells such as neutrophils, and stimulate various cells to produce other inflammatory factors, which is used to evaluate the intensity and severity of inflammatory activity. From Figure 7 It can be seen that the quaternary complex system group has an anti-inflammatory effect.

[0171] Test Example 6

[0172] The tissue frozen at -80℃ in Test Example 5 was taken, and the mRNA expression amount of healing-related factors CD31, IL-17A and VEGF in the skin wound tissue sample of the rat on day 14 was detected by real-time fluorescence quantitative PCR, and the relative expression level of each gene in the blank group (normal saline), MAP group, Ecdaroin group and quaternary complex system group was compared and evaluated, and the effects of healing-related factors in different groups on the chronic wound of the rat were analyzed.

[0173] The results of the cytokine fluorescence quantitative PCR experiment are shown in Figure 8 It is shown that:

[0174] Under the basic condition (blank group), the angiogenesis ability is weak. MAP and Ecdaroin each have a certain activity, but the effect is significantly enhanced after being used in combination. The quaternary complex system performs best in all indicators, indicating that it has a significant synergistic enhancement effect, which is better than any single component. The anti-inflammatory (L-17A↓) and pro-angiogenic (CD31↑, VEGF↑) effects are consistent, indicating that the complex system may have the potential to promote tissue repair and regeneration.

[0175] Test Example 7

[0176] The four processes (pH, ultraviolet intensity, mass ratio of trehalose and ectoine, and particle size of nano-carrier PLGA) in the preparation of the biological scaffold of the application were optimized in this test example, and the results are shown in Table 7.

[0177] Table 7 Process parameter optimization

[0178]

[0179] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A nanotransdermal sustained release bioscaffold, characterized in that, The bio-scaffold comprises the following components: a load and a nano-carrier; the load comprises mussel myo-globulin, trehalose and ectoine; the mass ratio of the mussel myo-globulin, trehalose and ectoine is (4-6):(0.5-1.5):(0.5-2.0).

2. The biological scaffold of claim 1, wherein, The mass ratio of the load and the nano-carrier is 1:

1.

3. The biological scaffold of claim 1, wherein, The nano-carrier comprises polylactic acid-glycolic acid copolymer or superparamagnetic iron oxide nanoparticles.

4. The biological scaffold of claim 1, wherein, The surface of the nano-carrier is modified with folate ligand; the method for modifying the folate ligand on the surface of the nano-carrier is an EDC / NHS activation method.

5. The biological scaffold of claim 4, wherein, The loading amount of the folate ligand on the surface of the nano-carrier is 2.5-3.5 μg / mg.

6. The biological scaffold of claim 3, wherein, The particle size of the polylactic acid-glycolic acid copolymer is 150-250 nm, and the zeta potential is -20--30 mV; the particle size of the superparamagnetic iron oxide nanoparticles is 10-30 nm, and the saturation magnetization is ≥60 emu / g.

7. The method for preparing the bioscaffold according to any one of claims 1 to 6, characterized in that, The bio-scaffold comprises the following components: a load and a nano-carrier; the load comprises mussel myo-globulin, trehalose and ectoine; the mass ratio of the mussel myo-globulin, trehalose and ectoine is (4-6):(0.5-1.5):(0.5-2.0).

8. The preparation method according to claim 7, characterized in that, The temperature of the ultraviolet irradiation-thermal crosslinking is 40-80℃, the time is 30-60 min, and the irradiation intensity is 8-12 mW / cm 2 .

9. The use of the bio-scaffold according to any one of claims 1-6 or the preparation method according to any one of claims 7-8 in the preparation of a wound repair product.

10. Use according to claim 9, characterized in that, The wound comprises diabetic ulcer, pressure ulcer, post-medical and post-aesthetic wound, nasal mucosa injury or oral mucosa injury. The wound comprises diabetic ulcer, pressure ulcer, post-medical and post-aesthetic wound, nasal mucosa injury or oral mucosa injury.

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