Injectable colloidal gel material composed of polyphenol and protein composite particles, preparation method and application thereof

Abstract

The present invention belongs to the fields of materials science and biomedical materials, and specifically relates to an injectable colloidal gel material composed of polyphenol and protein composite particles, its preparation method, and its application. The hydrogel material is assembled and formed through hydrophobic interactions and hydrogen bonding between polyphenol / protein composite micro-nanoparticles; cross-linking between polyphenols can be achieved by adding an oxidant to induce covalent cross-linking between the polyphenols; the polyphenol / protein composite micro-nanoparticles are assembled and formed through hydrogen bonding, hydrophobic interactions, or electrostatic interactions between protein polymer chains and polyphenols; and stable micro-nanoparticles can be formed by covalent cross-linking of protein chains by adding a cross-linking agent. The composite particles and gel material prepared by the present invention have good stability and can be used in the biomedical field as drug sustained-release carriers, tissue engineering scaffolds, tissue adhesive hemostatic materials, and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of material science and biomedical materials, and specifically relates to a gel material composed of injectable and plastic protein particles formed by a composite of polyphenols and proteins, as well as a preparation method and application thereof. Background Art

[0002] Polymer micro-nanoparticles are a type of polymer material with unique physical properties, the main characteristics of which are small particle size and high specific surface area. Based on these characteristics, micro-nanoparticles are widely used in the biomedical field as microcarriers, microseparators, and microreactors. Micro-nanoparticles can be prepared through different manufacturing methods to obtain granular materials with desired shapes and sizes. In recent years, by controlling the interactions between particles, gel materials composed of particles can be obtained when the particles form a dense packing. Due to the reversibility of the interactions between particles, this type of gel material usually exhibits shear thinning and self-healing mechanical characteristics; at the same time, due to the differences in the functionality of different types of particles, micro-nanoparticles with different functions can be assembled to form a gel material with modular properties.

[0003] In biomedical applications, protein-based materials are widely used due to their excellent biocompatibility. Microparticles and nanoparticles synthesized from various naturally occurring or engineered proteins have become promising platforms for biomedical applications. Furthermore, the amphiphilic nature of proteins facilitates their interaction with both hydrophilic and hydrophobic drugs and solvents. The abundance of hydroxyl, amino, and carboxyl groups in them makes them amenable to chemical modification. Consequently, protein particles can be covalently or non-covalently linked to one or more different types of ligands and drug molecules, offering excellent surface modification properties. Over the past few decades, scientists have been exploiting the biomedical potential of proteins, including gelatin, silk fibroin, albumin, gliadin, and other materials from a wide range of sources, such as animals, plants, insects, and recombinant protein expression systems. However, the preparation of existing protein microparticles and nanoparticles is often cumbersome and results in low yields. Protein material synthesis methods primarily rely on antisolvent and emulsion methods. The antisolvent method typically requires the addition of large amounts of organic reagents to cause the protein to curl and form particles. The emulsion method typically requires the addition of oil as a surfactant, making the separation of the oil from the protein particles a tedious process. Therefore, it is of great significance to use a simple and universal protein particle preparation technology strategy to achieve mass production of protein nanoparticles. Summary of the Invention

[0004] The present invention provides a colloidal particle hydrogel material composed of polyphenols and proteins. The hydrogel material is assembled through hydrophobic interactions and hydrogen bonding interactions between polyphenol / protein composite micro-nano particles. Preferably, after the hydrogel material is assembled through hydrophobic interactions and hydrogen bonding interactions between polyphenol / protein composite micro-nano particles, an oxidant is added to covalently crosslink the polyphenols to achieve crosslinking between the polyphenol / protein composite micro-nano particles. The polyphenol / protein composite micro-nano particles are assembled through hydrogen bonding interactions, hydrophobic interactions, or electrostatic interactions between protein polymer chains and polyphenols. Preferably, after the polyphenol / protein composite micro-nano particles are assembled through hydrogen bonding interactions, hydrophobic interactions, or electrostatic interactions between protein polymer chains and polyphenols, a crosslinking agent is added to covalently crosslink the protein chains to form stable micro-nano particles.

[0005] Among them, the size of the composite microsphere particles is 10nm~500μm. When the particle size is 10nm~5μm, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 2~120v / v%; when the particle size is 5~500μm, the volume fraction of the composite micro-nano particles in the particle hydrogel material in the total volume of the hydrogel is 50~120v / v%.

[0006] When the particle size is 10 nm to 5 μm, the compressive elastic modulus of the hydrogel after covalent cross-linking is 0.5 kPa to 500 kPa;

[0007] When the particle size is 5 to 500 μm, the compressive elastic modulus of the hydrogel after covalent crosslinking is 0.5 kPa to 100 kPa.

[0008] The second aspect of the present invention provides a hydrogel material assembled from micro-nano colloidal particles composited with polyphenols, proteins and metal ions, wherein the hydrogel material is assembled and formed by hydrophobic interactions, hydrogen bonding interactions and metal coordination interactions between the polyphenol / protein composite micro-nano particles; preferably, after the hydrogel material is assembled and formed by hydrophobic interactions, hydrogen bonding interactions and metal coordination interactions between the polyphenol / protein composite micro-nano particles, an oxidant is added to covalently cross-link the polyphenols to achieve cross-linking between the polyphenol / protein composite micro-nano particles; the polyphenol / protein composite micro-nano particles are obtained by adding metal ions to the aforementioned polyphenol / protein composite micro-nano particles to form coordination interactions with the polyphenols in the particles.

[0009] Among them, the size of the composite microsphere particles is 10nm~500μm. When the particle size is 10nm~5μm, the volume fraction of the colloidal particles in the particle hydrogel material to the total volume of the hydrogel is 2~120v / v%, and the obtained particle hydrogel has shear thinning and self-healing properties; the compressive elastic modulus of the obtained particle hydrogel is 0.5kPa~5MPa.

[0010] When the particle size is 10 nm to 5 μm, the compressive elastic modulus of the hydrogel after covalent cross-linking is 0.5 kPa to 500 kPa;

[0011] When the particle size is 5 to 500 μm, the compressive elastic modulus of the hydrogel after covalent crosslinking is 0.5 kPa to 100 kPa.

[0012] The third aspect of the present invention provides a hydrogel material assembled from core-shell structured micro-nanoparticles of a rigid nanoparticle-polyphenol / protein composite, wherein the hydrogel material is assembled through hydrophobic interactions and hydrogen bonding interactions between the core-shell structured micro-nanoparticles; preferably, after the hydrogel material is assembled through hydrophobic interactions and hydrogen bonding interactions between the composite micro-nanocolloid particles, an oxidant is added to covalently crosslink the polyphenols to achieve crosslinking between the core-shell structured micro-nanoparticles; the core layer of the core-shell structured micro-nanoparticles is composed of a rigid nanoparticle material, and the shell layer is composed of polyphenol / protein composite particles, wherein the polyphenol / protein composite particles are formed through hydrogen bonding interactions, hydrophobic interactions, and electrostatic interactions between protein molecules and polyphenol molecules; preferably, after the protein molecules and polyphenol molecules are formed through hydrogen bonding interactions, hydrophobic interactions, and electrostatic interactions, a crosslinking agent is added to covalently crosslink the protein chains to form stable micro-nanoparticles.

[0013] Among them, the size of the composite microsphere particles is 10nm~500μm. When the particle size is 10nm~5μm, the volume fraction of the colloidal particles in the particle hydrogel material to the total volume of the hydrogel is 2~120v / v%, and the obtained particle hydrogel has shear thinning and self-healing properties; the compressive elastic modulus of the obtained particle hydrogel is 0.5kPa~5MPa.

[0014] When the particle size is 10 nm to 5 μm, the compressive elastic modulus of the hydrogel after covalent cross-linking is 0.5 kPa to 500 kPa;

[0015] When the particle size is 5 to 500 μm, the compressive elastic modulus of the hydrogel after covalent crosslinking is 0.5 kPa to 100 kPa.

[0016] The fourth aspect of the present invention provides a core-shell structured colloidal particle material composed of a composite of rigid nanoparticles-polyphenols / proteins / metal ions, wherein the hydrogel material is assembled and formed by hydrophobic interactions, hydrogen bonding interactions, and metal coordination interactions between the core-shell structured micro-nanoparticles; preferably, after the hydrogel material is assembled and formed by hydrophobic interactions, hydrogen bonding interactions, and metal coordination interactions between the core-shell structured micro-nanoparticles, an oxidant is added to covalently crosslink the polyphenols to achieve crosslinking between the core-shell structured micro-nanoparticles; the core layer of the core-shell structured micro-nanoparticles is composed of a rigid nanoparticle material, and the shell layer is composed of polyphenols / protein / metal composite particles, and the polyphenols / protein / metal composite particles are obtained by adding metal ions to the polyphenol / protein composite particles described in claim 3 to form a coordination effect with the polyphenols in the particles.

[0017] Among them, the size of the composite microsphere particles is 10nm~500μm. When the particle size is 10nm~5μm, the volume fraction of the colloidal particles in the particle hydrogel material to the total volume of the hydrogel is 2~120v / v%, and the obtained particle hydrogel has shear thinning and self-healing properties; the compressive elastic modulus of the obtained particle hydrogel is 0.5kPa~5MPa.

[0018] When the particle size is 10 nm to 5 μm, the compressive elastic modulus of the hydrogel after covalent cross-linking is 0.5 kPa to 500 kPa;

[0019] When the particle size is 5 to 500 μm, the compressive elastic modulus of the hydrogel after covalent crosslinking is 0.5 kPa to 100 kPa.

[0020] In the above technical solution, further,

[0021] The polyphenol material is selected from one or more of gallic acid, gallic acid ester, epigallocatechin, quercetin, curcumin, tannic acid, catechol, and dopamine;

[0022] The protein material is selected from pure structural protein, pure structural protein derivatives, and mixtures of pure structural protein and hydrophilic polymer materials; the pure structural protein is gelatin, sericin, fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, and fluorescent protein GFP;

[0023] The oxidant in the polyphenol oxidation process is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide, or hydrogen peroxide;

[0024] The metal ions used for the metal ion coordination interaction are one or more of aluminum, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, cadmium, cerium, europium, gadolinium and terbium ions;

[0025] The rigid nanoparticles are silicon dioxide nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles;

[0026] When the size of the composite micro-nano particles is 10 nm to 5 μm, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 2 to 120 v / v%; when the size of the composite micro-nano particles is 5 to 500 μm, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 50 to 120 v / v%.

[0027] In the above technical solution, further, the preparation method of the micro-nano particle hydrogel material assembled by the polyphenol and protein, the hydrogel comprises the following preparation steps:

[0028] (1) dissolving a protein in an aqueous solution at 10-80° C. to obtain a protein aqueous solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol aqueous solution with a concentration of 0.1-10 w / v%;

[0029] (2) adjusting the pH value of the protein solution to 3-7, adding a polyphenol solution to the protein solution to obtain a protein / polyphenol composite particle solution, and freeze-drying to obtain a composite particle powder; wherein the mass ratio of protein to polyphenol is 0.1-20, preferably 1-20; preferably, adding a macromolecular cross-linking agent to the protein / polyphenol composite particle solution at room temperature to stabilize the composite particles for 1-12 hours, wherein the mass ratio of protein to macromolecular cross-linking agent is 0.1-100; after washing, obtaining a protein / polyphenol composite particle dispersion, and freeze-drying the particle dispersion to obtain a composite particle powder;

[0030] (3) blending the protein / polyphenol composite particle powder with an aqueous solution to obtain an injectable, self-repairing colloidal gel; preferably, the injectable, self-repairing colloidal gel is further added with an oxidant to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel;

[0031] In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, fenvalerate, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, dialkylene oxide, divinyl sulfone, polyfunctional aziridine, and diisocyanate;

[0032] In step (3), the oxidant is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide, or hydrogen peroxide;

[0033] Preferably, the aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

[0034] In the above technical solution, further, the method for preparing the colloidal particle hydrogel material assembled from the polyphenols, proteins and metal ions is characterized by:

[0035] (1) dissolving a protein in an aqueous solution at 10-80° C. to obtain a protein aqueous solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol aqueous solution with a concentration of 0.1-10 w / v%;

[0036] (2) adjusting the pH of the protein solution to 3-7, adding a polyphenol solution to the protein solution to obtain a protein / polyphenol composite particle solution, wherein the mass ratio of protein to polyphenol is 0.1-20, preferably 1-20; adding metal ions to the above particle solution and stirring to obtain a particle dispersion, wherein the concentration of the metal ions is 1mM-10M, centrifuging the particle dispersion, washing, and freeze-drying to obtain a composite particle powder. Preferably, the particle solution is at room temperature, and a macromolecular cross-linking agent is added to stabilize the composite particles for 1-12 hours before adding the metal ions, wherein the mass ratio of protein to macromolecular cross-linking agent is 0.1-100;

[0037] (3) blending the protein / polyphenol composite particle powder with an aqueous solution to obtain an injectable, self-repairing colloidal gel; preferably, further adding an oxidative crosslinking agent to the injectable, self-repairing colloidal gel to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel;

[0038] In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, fenvalerate, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, dialkylene oxide, divinyl sulfone, polyfunctional aziridine, and diisocyanate;

[0039] In step (3), the oxidative crosslinking agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide, or hydrogen peroxide; the metal ions used for the metal ion coordination crosslinking are one or more of aluminum, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, cadmium, cerium, europium, gadolinium, and terbium ions;

[0040] Preferably, the aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

[0041] In the above technical solution, further, the method for preparing the core-shell structure colloidal particle material of the aforementioned nanoparticle-polyphenol composite protein molecule is characterized in that the composite particles include the following preparation steps:

[0042] (1) dissolving a protein molecule in an aqueous solution at 10-80° C. to obtain a protein aqueous solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol aqueous solution with a concentration of 0.1-10 w / v%;

[0043] (2) adding rigid nanoparticles to the above-mentioned protein aqueous solution and adjusting the pH of the solution to 3-7, and adding a polyphenol solution to obtain a core-shell structure composite particle solution; freeze-drying to obtain a composite particle powder; wherein the mass ratio of protein to polyphenol is 0.1-20, and the mass ratio of protein to rigid nanoparticles is 0.1-30; preferably, at room temperature, a cross-linking agent is added to the composite particle solution to further cross-link and stabilize the composite particles for 1-12 hours, wherein the mass ratio of protein to macromolecular cross-linking agent is 0.1-100; after washing, a protein / polyphenol / nanoparticle composite particle dispersion is obtained, and the above-mentioned particle dispersion is freeze-dried to obtain a composite particle powder;

[0044] (3) blending the composite particle powder with an aqueous solution to obtain an injectable, self-repairing colloidal gel; preferably, the injectable, self-repairing colloidal gel further comprises adding an oxidative crosslinking agent or metal ions to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel;

[0045] In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, oxalaldehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, diene oxide, divinyl sulfone, multifunctional aziridine, and diisocyanate; the rigid nanoparticles can be silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, and polystyrene nanoparticles;

[0046] In step (3), the oxidative crosslinking agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide, or hydrogen peroxide; the metal ions used for the metal ion coordination crosslinking are one or more of aluminum, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, cadmium, cerium, europium, gadolinium, and terbium ions;

[0047] Preferably, the aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

[0048] In the above technical solution, further, the method for preparing the core-shell structure colloidal particle material of the aforementioned nanoparticles-polyphenols, proteins and metal molecules is characterized in that the composite particles include the following preparation steps:

[0049] (1) dissolving a protein molecule in an aqueous solution at 10-80° C. to obtain a protein aqueous solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol aqueous solution with a concentration of 0.1-10 w / v%;

[0050] (2) adding rigid nanoparticles to the above protein aqueous solution and adjusting the pH of the solution to 3-7, and adding a polyphenol solution to obtain a core-shell structure composite particle solution; wherein the mass ratio of protein to polyphenol is 0.1-20, and the mass ratio of protein to hard nanoparticles is 0.1-30; adding metal ions to the particle dispersion and stirring, wherein the concentration of the metal ions is 1 mM-10 M, centrifuging the particle dispersion, washing, and freeze-drying to obtain a composite particle powder; preferably, at room temperature, adding a crosslinking agent to the core-shell structure composite particle solution to further crosslink and stabilize the composite particles for 1-12 hours, wherein the mass ratio of protein to macromolecular crosslinking agent is 0.1-100; after washing, a protein / polyphenol / nanoparticle composite particle dispersion is obtained;

[0051] (3) blending the composite particle powder with an aqueous solution to obtain an injectable, self-repairing colloidal gel; preferably, the injectable, self-repairing colloidal gel further comprises adding an oxidative crosslinking agent or metal ions to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel;

[0052] In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, oxalaldehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, diepoxide, divinyl sulfone, multifunctional aziridine, and diisocyanate; the nanoparticles can be silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, and polystyrene nanoparticles;

[0053] In step (3), the oxidative crosslinking agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide, or hydrogen peroxide; the metal ions used for the metal ion coordination crosslinking are one or more of aluminum, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, cadmium, cerium, europium, gadolinium, and terbium ions;

[0054] Preferably, the aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

[0055] A fifth aspect of the present invention provides applications of the aforementioned hydrogel material, which has the following aspects:

[0056] Application in the preparation of anti-inflammatory, antioxidant, and antibacterial scaffolds, which can slowly release polyphenols in the composite particles, and the scaffolds are used for repairing and filling wounds or defects of bone tissue, cartilage tissue, muscle, blood vessels, and skin;

[0057] or use in the preparation of a carrier or scaffold containing a drug component, wherein the drug component is one or more combinations of vitamins, amino acids, mineral elements, microecological regulators, growth factors, small molecule drugs, protein macromolecule drugs, antibiotics, hormones, anesthetics, antivirals, antibacterials, anticancer drugs, immunomodulatory drugs, nucleic acid drugs, or living cells;

[0058] Or it can be used in the preparation of superficial skin and subcutaneous fillers; preferably in plastic surgery; when used, the above gel is directly injected into the superficial skin or subcutaneous area, and mixed with a small amount of oxidant or metal ions. The colloidal particle gel can reversibly self-assemble under the action of non-covalent bonds to form a continuous porous particle network, which stays stably at the injection site, and the particle surface is further cross-linked to improve the stability of the gel and ensure the filling stability.

[0059] Or the application in the preparation of bio-ink, preferably bio-printing ink for printing with live cells; when used, colloidal particles are blended with an aqueous solution to obtain a colloidal particle gel, which is then mixed with a cell suspension to obtain a cell-loaded colloidal gel, i.e., bio-ink. The above ink is extruded or 3D-printed by inkjet to obtain a scaffold with a 3D structure, thereby obtaining a cell-loaded printed scaffold;

[0060] Or in the application of preparing tissue adhesive gel materials, wherein the composite microsphere particle size is <10μm, and the bonding strength between the colloidal gel and the tissue is 5-100kPa; the injectable, self-repairing colloidal gel material is injected into the damaged tissue site in the body, and waited for 1-30 minutes to achieve stable bonding between the colloidal gel and the tissue.

[0061] Or its application in the preparation of metal ion sustained-release carriers for use in wound tissue repair and anti-infection;

[0062] Or it can be used in rapid hemostatic sealing powder, which includes the composite microsphere particle powder prepared above. The particle powder is directly sprayed onto the defect wound on the tissue surface. After the powder fully absorbs the exuded blood, it will form an adhesion effect with the tissue and achieve effective hemostasis.

[0063] Beneficial effects of the present invention:

[0064] 1. This invention reports a universal method for preparing protein micro-nanoparticles. This method involves dissolving a protein material in an aqueous solution and then compounding it with an aqueous solution containing polyphenols. Polyphenols can rapidly bind to proteins through hydrogen bonds, hydrophobic interactions, and electrostatic interactions to form a polyphenol-protein network, thereby forming a polyphenol-protein composite particulate material. The protein particle preparation method reported in this invention does not require the use of organic reagents or surfactants used in traditional protein particle preparation processes, but rather utilizes the strong interaction between polyphenols and proteins to achieve particle synthesis. This significantly reduces the production cost of protein particles.

[0065] 2. The protein particle preparation method reported in this invention is applicable to different types of protein materials. Because polyphenols can interact with different types of proteins, the method reported in this invention can be used to prepare different types of protein particles. Compared with traditional protein particle preparation techniques that are generally only targeted at a specific protein material, the universal applicability of the current method greatly expands the protein particle production technology, as well as the particle preparation of some difficult-to-particle protein materials.

[0066] 3. The protein micro-nanoparticles reported in this paper reversibly assemble to form a gel material with shear-thinning and self-healing properties. Because the protein particles also contain polyphenols, the abundant phenolic hydroxyl units in the polyphenols can form stable interactions with tissue surfaces. Therefore, the particle gel material reported in this paper exhibits stable tissue adhesion and can be used as a tissue adhesive in wound repair.

[0067] 4. The protein micro-nanoparticles reported in this invention contain abundant phenolic hydroxyl groups, which can form complexes with various metal ions to rapidly prepare micro-nanoparticle units with different functions. Protein particles with different functions can be modularly assembled to form gel materials with different functions.

[0068] 5. The composite particles reported in this invention can also be used as therapeutic ingredients for antioxidant, anti-inflammatory, and antibacterial therapeutic applications. Polyphenols are also common hemostatic materials, so the colloidal gel prepared in this invention can have both hemostatic and tissue adhesive properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 1. This is a scanning electron micrograph of the metal ion-complexed gelatin / tannic acid colloidal gel prepared in Example 5; wherein, purple in the Cu composite group represents Cu ions; 2. yellow in the Fe composite group represents Fe ions; 3. orange in the Zn composite group represents Zn ions; 4. purple in the Ce composite group represents Ce ions.

[0070] Figure 2 3 is a scanning electron micrograph of the covalently cross-linked gelatin / tannic acid colloidal gel prepared in Example 7, wherein FIG. b is an enlarged view of FIG. a.

[0071] Figure 3 This is a transmission electron microscope image of the composite material particles with a silica core-gelatin / polyphenol shell structure prepared in Example 8.

[0072] Figure 4 This is the polyphenol release curve of the colloidal gel in Example 17.

[0073] Figure 5 This is a live-death staining image of cells cultured in a two-dimensional culture of the colloidal gel material obtained in Example 18. The cells survive well on the gel surface, indicating that the colloidal gel has excellent biocompatibility. The scale is 50 μm.

[0074] Figure 6 Figures 19 and 20 are graphs showing the antibacterial activity of the colloidal gel obtained in Example 19. In Figure A, bacteria grow normally around the yellow pure gelatin gel. In Figure B, the gelatin / tannic acid composite gel has no bacterial growth around it, indicating its antibacterial properties.

[0075] Figure 7 This is a graph showing the colloidal gel of Example 20 as a drug sustained-release carrier. Different types of protein drugs can be slowly released over 21 days.

[0076] Figure 8 This is a cell picture of the antioxidant effect of Ce ion composite colloidal gel in Example 21, where the green signal represents the reactive oxygen species DCFH and the red signal represents the reactive oxygen species Ru(dpp)Cl2.

[0077] Figure 9 This is a picture of alkaline phosphatase staining of osteogenic factors after co-culture of the Zn ion composite colloidal gel and osteoblasts MC-3T3 in Example 21, wherein the blue-purple signal represents the alkaline phosphatase signal.

[0078] Figure 10 This is a picture of the co-culture of Cu ion composite colloidal gel and endothelial cells in Example 23.

[0079] Figure 11 This is a sample picture of the colloidal gel 3D printed scaffold in Example 22. DETAILED DESCRIPTION

[0080] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0081] Example 1

[0082] At 50°C, 5g of gelatin powder was dissolved in 100mL of deionized water to obtain a gelatin solution, and the pH was adjusted to 3, 5, and 7. At 50°C, 0.5g of tannic acid powder was dissolved in 100mL of deionized water to obtain a tannic acid solution. 100mL of tannic acid solution was slowly added to the gelatin solution over 30 minutes and stirred at 1500rpm to allow the protein and tannic acid molecules to rapidly bind to form particles. Subsequently, 165μL of the crosslinking agent glutaraldehyde was added and stirred for 12 hours. The composite particles were washed with deionized water and freeze-dried to obtain gelatin / tannic acid nanoparticle powder. The size and charge of the resulting nanoparticles are shown in Table 1.

[0083] Table 1

[0084] Particle size surface charge pH=3 214.6nm -14.7mV pH = 5 239.5nm -10.9mV pH = 7 306.5nm -6.1mV

[0085] An injectable, self-healing composite particle gel was prepared by repeatedly pipetting 0.3 g of gelatin / tannic acid nanoparticle powder and 1 mL of deionized water through a Luer adapter syringe 10 times at room temperature. The storage modulus G' of the gel was measured using a rotational rheometer in time-sweep mode. The self-healing efficiency is shown in Table 2. The frequency was 1 Hz and the strain was 0.5%. The data in Table 2 show that the storage modulus of the gel increases with increasing particle mass fraction, indicating that the gel exhibits self-healing properties.

[0086] Table 2

[0087] Storage modulus Self-repair efficiency pH=3 8.9kPa 73.1％ pH = 5 9.7kPa 75.2％ pH = 7 7.2kPa 79.7％

[0088] Comparative Example 1

[0089] 5g of gelatin powder was dissolved in 100mL of deionized water at 50°C to obtain a gelatin solution, and the pH was adjusted to 9 or 11. 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50°C to obtain a tannic acid solution. This 100mL tannic acid solution was added to the gelatin solution over 30 minutes. The solution then turned brown and clear. Further particle size testing revealed no micro- or nanoparticles were formed.

[0090] Comparative Example 2

[0091] 5 g of gelatin powder was dissolved in 100 mL of deionized water at 50°C to obtain a gelatin solution, and the pH was adjusted to 3, 5, and 7. 0.5 g of tannic acid powder was dissolved in 100 mL of deionized water at 50°C to obtain a tannic acid solution. 100 mL of the gelatin solution was slowly added to the tannic acid solution over 30 minutes. A brown flocculent precipitate formed, and an emulsified particle suspension could not be obtained.

[0092] Example 2

[0093] 5 g of gelatin powder was dissolved in 100 mL of deionized water at 50°C to obtain a gelatin solution, and the pH was adjusted to 3, 5, and 7. 0.5 g of tannic acid powder was dissolved in 100 mL of deionized water at 50°C to obtain a tannic acid solution. This 100 mL tannic acid solution was slowly added to the gelatin solution over 30 minutes while stirring at 1500 rpm to allow the protein and tannic acid molecules to rapidly bind to form particles. The composite particles were washed with deionized water and freeze-dried to obtain gelatin / tannic acid nanoparticle powder. The size and charge of the resulting nanoparticles are shown in Table 3.

[0094] Table 3

[0095] Particle size surface charge pH=3 331.9nm -3.7mV pH = 5 409.7nm -5.9mV pH = 7 426.9nm -4.3mV

[0096] An injectable, self-healing composite particle gel was prepared by repeatedly pipetting 0.3 g of gelatin / tannic acid nanoparticle powder and 1 mL of deionized water through a Luer adapter syringe 10 times at room temperature. The storage modulus G' of the gel was measured using a rotational rheometer in time-sweep mode. The self-healing efficiency is shown in Table 3. The frequency was 1 Hz and the strain was 0.5%. The data in Table 4 show that the storage modulus of the gel increased with increasing particle mass fraction, indicating the gel exhibits self-healing properties.

[0097] Table 4

[0098] Storage modulus Self-repair efficiency pH=3 6.2kPa 88.1％ pH = 5 4.7kPa 94.6％ pH = 7 4.5kPa 95.4％

[0099] Example 3

[0100] At 50°C, 5g of gelatin powder was dissolved in 100mL of deionized water to obtain a gelatin solution and the pH was adjusted to 5. At 50°C, 0.25, 0.5, 2, and 5g of tannic acid powder were dissolved in 100mL of deionized water to obtain a tannic acid solution. The above 100mL of tannic acid solution was added to the gelatin solution within 30min and stirred at 1500rpm to allow the protein molecules to combine with the tannic acid molecules to form particles. Subsequently, 165μL of the cross-linking agent glutaraldehyde was added and stirred for 12hrs. After washing the composite particles with deionized water, the composite particles were freeze-dried to obtain composite particle powder. The size and charge of the obtained composite particles are shown in Table 5.

[0101] Table 5

[0102] Tannic acid / gelatin (w / w) Particle size surface charge 0.05 254.6nm -12.7mV 0.1 219.5nm -16.9mV 0.4 116.5nm -23.1mV 1 85.6nm -33.5mV

[0103] 0.3 g of gelatin / tannic acid composite particle powder and 1 mL of deionized water were repeatedly pipetted 10 times through a Luer adapter syringe at room temperature to produce an injectable self-healing particle gel. The storage modulus G' of the particle gel was determined using a rotational rheometer in time-sweep mode. The self-healing efficiency is shown in Table 6. The frequency was 1 Hz and the strain was 0.5%. Rheological experiments showed that a tannic acid to gelatin ratio of 0.1 exhibited the highest storage modulus, while a tannic acid to gelatin ratio of 1 exhibited the highest self-healing efficiency.

[0104] Table 6

[0105] Tannic acid / gelatin (w / w) Storage modulus Self-repair efficiency 0.05 6.9kPa 73.5％ 0.1 7.6kPa 76.9％ 0.4 5.5kPa 83.9％ 1 4.9kPa 89.4％

[0106] Comparative Example 3

[0107] 5g of gelatin powder was dissolved in 100mL of deionized water at 50°C to obtain a gelatin solution and the pH was adjusted to 5. 0.1g of tannic acid powder was dissolved in 100mL of deionized water at 50°C to obtain a tannic acid solution. The above 100mL of tannic acid solution was added to the gelatin solution within 30 minutes and stirred at 1500rpm. At this time, the solution was transparent and clear, and no emulsification occurred. Further testing of the particle size in the solution showed that no micro-nanoparticles were formed, indicating that the gelatin / tannic acid mass ratio of 0.02 was too low to form nanoparticles.

[0108] 5g of gelatin powder was dissolved in 100mL of deionized water at 50°C to obtain a gelatin solution, and the pH was adjusted to 5. 150g of tannic acid powder was dissolved in 100mL of deionized water at 50°C to obtain a tannic acid solution. This 100mL tannic acid solution was added to the gelatin solution over 30 minutes with stirring at 1500rpm. A brown precipitate quickly formed, indicating that a tannic acid mass ratio of 30 resulted in particle agglomeration and the inability to obtain dispersed granular material.

[0109] Example 4

[0110] 5g of gelatin powder was dissolved in 100mL of deionized water at 50°C to obtain a gelatin solution and the pH was adjusted to 5. 0.5g of EGCG (epigallocatechin gallate), gallic acid, quercetin, and catechin powder were dissolved in 100mL of deionized water at 50°C to obtain the corresponding polyphenol solutions. The above-mentioned different types of polyphenol solutions were added to the gelatin solution and stirred rapidly over 30 minutes to allow the gelatin molecules to combine with the different types of polyphenols to form micro-nanoparticles. Subsequently, 165μL of the crosslinking agent glutaraldehyde was added and stirred for 12 hours. After washing the composite particles with deionized water, the composite particles were freeze-dried to obtain different polyphenols and gelatin composite nanoparticle powders. The size and charge of the obtained nanoparticles are shown in Table 7.

[0111] Table 7

[0112] Particle type Particle size surface charge EGCG / gelatin 317.9nm -17.7mV Gallic acid / gelatin 313.9nm -13.9mV Quercetin / Gelatin 269.4nm -19.8mV Catechin / Gelatin 295.7nm -9.3mV

[0113] 0.3g of EGCG / gelatin, gallic acid / gelatin, quercetin / gelatin, and catechin / gelatin nanoparticle powders were repeatedly pipetted 10 times with 1mL of deionized water at room temperature through a Luer adapter syringe to obtain injectable self-healing particle gels. The storage modulus G' of the particle gels was obtained using a rotational rheometer in time sweep mode, and the self-healing efficiency is shown in Table 8. The frequency was 1Hz and the strain was 0.5%. Rheological experiments revealed that the use of different types of polyphenol materials had no significant effect on the storage modulus and self-healing properties of the final gel.

[0114] Table 8

[0115] Particle type Storage modulus Self-repair efficiency EGCG / gelatin 6.3kPa 81.3％ Gallic acid / gelatin 7.1kPa 75.9％ Quercetin / Gelatin 7.2kPa 78.9％ Catechin / Gelatin 5.9kPa 74.2％

[0116] Example 5

[0117] 5g of sericin, fibroin, albumin, serum proteins, keratin, elastin, hemoglobin, immunoglobulins, fibrin, fluorescent protein (GFP), and the gelatin derivative gelatin methacrylate were dissolved in 100mL of deionized water at 37°C to obtain a protein solution, and the pH was adjusted to 5. 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50°C to obtain a tannic acid solution. The 100mL tannic acid solution was added over 30 minutes with rapid stirring. The silk fibroin molecules combined with the tannic acid molecules to form nanospheres, which were then stirred for 12 hours. The nanoparticles were rinsed with deionized water and freeze-dried to obtain a silk fibroin / tannic acid nanoparticle powder. The size and charge of the resulting nanoparticles are shown in Table 9.

[0118] Table 9

[0119] Protein type Particle size surface charge Sericin 227.8nm -27.7mV Silk fibroin 279.2nm -31.3mV albumin 173.4nm -17.2mV serum proteins 149.0nm -19.2mV Keratin 219.3nm -9.2mV Elastin 293.5nm -4.7mV Hemoglobin 102.2nm 5.8mV Immunoglobulins 52.3nm -7.2mV Fibrin 71.2nm -12.8mV Fluorescent proteins 49.2nm 2.9mV Gelatin methacrylate 284.5nm -17.9mV

[0120] 0.4 g of silk fibroin / tannic acid, albumin / tannic acid, keratin / tannic acid, and fluorescent protein / tannic acid granular powders were repeatedly pipetted 10 times with 1 mL of deionized water at room temperature through a Luer adapter syringe to produce injectable self-healing particle gels. The storage modulus G' of the particle gels was obtained using a rotational rheometer in time-sweep mode, as shown in Table 4. The frequency was 1 Hz and the strain was 0.5%. The different types of proteins exhibited different mechanical strengths when combined with tannic acid, as shown in Table 10.

[0121] Table 10

[0122] Protein type Storage modulus Silk fibroin 28.6kPa albumin 14.1kPa Keratin 11.2kPa Fluorescent proteins 7.9kPa Gelatin methacrylate 12.9kPa

[0123] Example 6

[0124] Dissolve 5g of gelatin powder in 100mL of deionized water at 50°C and adjust the pH to 5. Dissolve 0.5g of tannic acid powder in 100mL of deionized water at 50°C to obtain a tannic acid solution. Add the above 100mL of tannic acid solution to the gelatin solution within 30min and stir rapidly to combine the gelatin and tannic acid to form micro-nano composite particles. Then add 165μL of cross-linking agent glutaraldehyde and stir for 12hrs. After washing the composite particles with deionized water, dilute the particle concentration to 10mg / mL with deionized water, add copper chloride, ferric chloride, zinc chloride, and europium chloride to the solution respectively until the final solution concentration reaches 10mM, stir for 12hrs, and repeatedly wash the composite particles 3 times with deionized water to obtain metal composite protein particles. The size and charge of the obtained nanoparticles are shown in Table 11, where the scanning electron microscope image of the particles is shown in Table 11. Figure 1 As shown, the corresponding metal elements have been successfully complexed in the particles.

[0125] Table 11

[0126] Chelated metal ion types Particle size surface charge Cu 214.6nm -14.7mV Fe 235.9nm -19.3mV Zn 275.1nm -21.3mV Ce 224.7nm -19.2mV

[0127] 0.4g of different metal ion-chelated protein / polyphenol nanoparticle powders and 1mL of deionized water were repeatedly pipetted 10 times through a Luer adapter syringe at room temperature to produce an injectable self-healing particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-sweep mode, and the self-healing efficiency is shown in Table 12. The frequency was 1Hz and the strain was 0.5%. Compared with Example 1, it was found that the mechanical strength of the gel was significantly increased after the particle surface was chelated by metal ions.

[0128] Table 12

[0129] Metal Type Storage modulus Cu 28.6kPa Fe 24.1kPa Zn 21.2kPa Ce 17.9kPa

[0130] Example 7

[0131] Using the gelatin / tannic acid micro-nanoparticle powder prepared in Example 1 (pH value is 5), 0.4 g of the particle powder was repeatedly blown 10 times with 5 mg of different oxidative crosslinking agents, sodium periodate, sodium dichromate, sodium peroxide, hydrogen peroxide and 1 mL of deionized water at room temperature through a Luer adapter syringe to obtain an injectable particle gel. The storage modulus G' of the particle gel was measured using the time scanning mode of the rotational rheometer, and the self-healing efficiency is shown in Table 13. The frequency is 1 Hz and the strain is 0.5%. Since the oxidative crosslinking agent will induce covalent crosslinking of tannic acid molecules on the surface of the particles, the composite particle gel can be further enhanced. The storage modulus G' of the particle gel was obtained using the time scanning mode of the rotational rheometer. The frequency is 1 Hz and the strain is 0.5%. Compared with the storage modulus of the uncrosslinked gelatin / tannic acid nanoparticles in Example 1, it can be obtained that the mechanical strength of the gel with the same mass fraction is significantly improved after the addition of the oxidative crosslinking agent. The microstructure of the gel after covalent crosslinking is as shown in Table 13. Figure 2 As shown in the scanning electron microscope image.

[0132] Table 13

[0133]

[0134]

[0135] Example 8

[0136] 5g of gelatin powder was dissolved in 100mL of deionized water at 50°C and the pH of the solution was adjusted to 5. 1g of silica nanoparticles, graphene nanosheets, hydroxyapatite nanoparticles, silk nanoparticles, and bioglass nanoparticles were added to the solution to obtain a gelatin solution containing nanoparticles. 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50°C to obtain a tannic acid solution. Over 30 minutes, the 100mL tannic acid solution was added to the gelatin solution containing silica nanoparticles, graphene nanosheets, hydroxyapatite nanoparticles, silk nanoparticles, and bioglass nanoparticles, respectively, with rapid stirring. This allowed the gelatin molecules to bind to the tannic acid molecules, forming micro-nanoparticles and encapsulating the nanoparticles. Subsequently, 165μL of the crosslinking agent glutaraldehyde was added and stirred for 12 hours. The nanoparticles were rinsed with deionized water and freeze-dried to obtain a gelatin / tannic acid / nanoparticle powder. The size and charge of the resulting nanoparticles are shown in Table 14. The transmission electron microscopy of the composite particles wrapped with silica is as follows Figure 3 shown.

[0137] Table 14

[0138] Types of core layer nanoparticles Particle size surface charge Silicon dioxide 514.6nm -14.7mV graphene 435.9nm -19.3mV Hydroxyapatite 775.1nm -21.3mV Silk fibroin 624.7nm -19.2mV Bioglass 724.7nm -14.7mV

[0139] 0.4 g of core-shell particle powder containing different core-layer particles was repeatedly pipetted 10 times with 1 mL of deionized water through a Luer adapter syringe at room temperature to produce an injectable self-healing particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-sweep mode, as shown in Table 15. The frequency was 1 Hz and the strain was 0.5%. Core-shell composite particles with different core-layer particle types exhibited different mechanical strengths.

[0140] Table 15

[0141]

[0142]

[0143] Example 9

[0144] Using the protein polyphenol core-shell particle suspension prepared in Example 8, taking the composite particle suspension in which the core layer nanoparticles are silica as an example, copper chloride, ferric chloride, zinc chloride, and europium chloride were added to the solution respectively to a final solution concentration of 10 mM, stirred for 12 hrs, and the nanoparticles were repeatedly washed three times with deionized water to obtain metal composite protein polyphenol core-shell particles.

[0145] 0.4 g of protein / polyphenol core-shell structured particles with different metal ions were mixed with 1 mL of deionized water and pipetted 10 times through a Luer adapter syringe at room temperature to produce an injectable composite particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time sweep mode, as shown in Table 16. The frequency was 1 Hz and the strain was 0.5%.

[0146] Table 16

[0147] Metal Type Storage modulus Cu 58.6kPa Fe 64.1kPa Zn 51.2kPa Ce 47.9kPa

[0148] Example 10

[0149] Using the core-shell particle powder containing particles of different core layers prepared in Example 8, 0.4 g of the particle powder, 5 mg of sodium periodate, and 1 mL of deionized water were repeatedly pipetted 10 times through a Luer adapter syringe at room temperature to obtain an injectable particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time-sweep mode, as shown in Table 17. The frequency was 1 Hz and the strain was 0.5%. Because sodium periodate induces covalent crosslinking of tannic acid molecules on the particle surface, the storage modulus G' of the composite particle gel is further increased.

[0150] Table 17

[0151] Types of core layer nanoparticles Storage modulus Silicon dioxide 57.6kPa graphene 51.9kPa Hydroxyapatite 65.0kPa Silk fibroin 54.3kPa Bioglass 53.1kPa

[0152] Example 11

[0153] Using the gelatin / tannic acid nanoparticle powder prepared in Example 1, 0.4 g of the granular powder was mixed with 1 mL of deionized water containing 20 mg of each of silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, magnetic iron oxide nanoparticles, silk fibroin nanoparticles, polylactic acid nanoparticles, alginic acid, and pullulan. The mixture was pipetted 10 times at room temperature using a Luer adapter syringe to produce an injectable particle gel. The storage modulus G' of the particle gel was obtained using a rotational rheometer in time sweep mode, as shown in Table 18. The frequency was 1 Hz and the strain was 0.5%.

[0154] Table 18

[0155] Ingredients in aqueous solution Storage modulus Silica nanoparticles 28.9kPa Lithium magnesium silicate nanoparticles 25.1kPa Nanoclay particles 30.6kPa Hydroxyapatite nanoparticles 20.7kPa Magnetic iron oxide nanoparticles 19.8kPa Silk fibroin nanoparticles 23.9kPa Polylactic acid nanoparticles 21.7kPa alginic acid 15.2kPa Pullulan 14.9kPa

[0156] Example 12

[0157] Compression tests were conducted using the gelatin / tannic acid granular gel obtained at a pH of 5 in Example 1, the colloidal gel cross-linked with metal ions in Example 6, and the covalently cross-linked gelatin / tannic acid colloidal gel obtained in Example 7. The colloidal gels were formed into cylinders (diameter: 6.4 mm, height: 6 mm) and compression tests were performed at a loading rate of 0.0002 mm / s. The compressive strain and compressive strength of the samples are shown in Table 19. Covalent cross-linking significantly increased the compressive strength and compressive strain of the gels.

[0158] Table 19

[0159]

[0160] Example 13

[0161] Compression tests were performed using the gelatin / tannic acid / silica colloidal gel obtained in Example 6, the core-shell colloidal gel after metal ion crosslinking in Example 9, and the covalently crosslinked gelatin / tannic acid / silica colloidal gel obtained in Example 10. The colloidal gel was formed into a cylinder (diameter: 6.4 mm, height: 6 mm). The compression test was performed at a loading rate of 0.0002 mm / s. The compressive strain and compressive strength of the samples are shown in Table 20. After covalent crosslinking, the mechanical strength of the gel was significantly increased.

[0162] Table 20

[0163]

[0164]

[0165] Example 14

[0166] Tissue adhesion testing was performed using the gelatin / tannic acid particle gel obtained in Example 1 at a pH of 5. 0.1 mL of the particle gel was overlapped and bonded to two glass surfaces (5.0 cm × 2.0 cm rectangles), with the overlap area being a 1.5 cm × 2.0 cm rectangle. After standing for 10 minutes, the overlapped samples were subjected to shear peeling (peel rate: 10 mm / min) using a tensile tester with a 50 N force transducer. The tissue adhesion strength is shown in Table 21.

[0167] Table 21

[0168] 20% (w / v) 40% (w / v) Adhesive strength 12.8kPa 32.1kPa

[0169] Example 15

[0170] Tissue adhesion testing was performed using the gelatin / tannic acid / silica colloidal gel obtained in Example 6. 0.1 mL of the colloidal gel was injected onto the surfaces of two glass panels (5.0 cm × 2.0 cm rectangles) overlapped and bonded together. The overlapped area was a 1.5 cm × 2.0 cm rectangle. After standing for 10 minutes, the overlapped samples were subjected to shear peeling (peel rate: 10 mm / min) using a tensile tester with a 50 N load cell. The tissue adhesion strength is shown in Table 22.

[0171] Table 22

[0172] Adhesive strength 15.8kPa

[0173] Example 16

[0174] Tissue adhesion testing was performed using the gelatin / tannic acid / metal ion colloidal gel obtained in Example 7. 0.1 mL of the colloidal gel was injected onto the surfaces of two glass panels (5.0 cm × 2.0 cm rectangles) bonded together, with the overlap area being a 1.5 cm × 2.0 cm rectangle. After standing for 10 minutes, the overlapped samples were subjected to shear peeling (peel rate: 10 mm / min) using a tensile tester equipped with a 50 N load cell. A stress-strain curve was obtained during the peeling process, and the bond strength was defined as the maximum stress point on the curve. The bond strengths are shown in Table 23.

[0175] Table 23

[0176] Adhesive strength 7.1kPa

[0177] Example 17

[0178] The gelatin / tannic acid colloidal gel obtained in Example 1 was placed on an oscillator (30 rpm) at an ambient temperature of 37°C to simulate the dynamic environment in vivo. 1 ml of PBS supernatant was absorbed at 1 day, 3 days, 7 days, 14 days, and 21 days, and then an equal amount of fresh PBS solution was added. The tannic acid content was measured at each time point using a high-performance liquid chromatography kit. HPLC detection revealed that tannic acid was slowly released from the colloidal gel at a relatively constant concentration. This uniform release could still be detected on the 21st day. The release amount was as follows: Figure 4 The above results indicate that the composite colloidal gel can sustainably release polyphenols and can be used as a carrier of bioactive substances.

[0179] Example 18

[0180] Taking primary mouse mesenchymal stem cells as an example, they were cultured in DMEM (containing 10% fetal bovine serum (FBS, Gibco)) at 37°C, 95% relative humidity and 5% carbon dioxide, with the cell culture medium replaced every two days. Before use, the cells were separated in phosphate-buffered saline (PBS) using a trypsin / EDTA solution (0.25% trypsin / 0.02% EDTA) for 5 minutes and suspended in the culture medium for use. The cell suspension was directly added dropwise to the surface of the colloidal gel as in Example 1 (pH 5), at a cell concentration of 5000 cells / cm 2 After inoculation and 1 hrs of rest, culture medium was added.

[0181] The cytotoxicity of the gel material was investigated by using a live / dead assay. 2 mM calcein (green to mark live cells) and 4 mM ethidium homodimer (red fluorescence to mark dead cells) were added at room temperature and analyzed using a confocal laser scanning microscope. Figure 5 As shown in the figure, green fluorescence represents living cells, and red fluorescence represents dead cells. 3T3 cells cultured on the particle hydrogel for a long time are all living cells, indicating that it has excellent biocompatibility.

[0182] Example 19

[0183] The gelatin / tannic acid colloidal gel described in Example 1 (pH 5) was prepared into cylinders (8 mm diameter, 2 mm height). Staphylococcus aureus or Escherichia coli were cultured overnight in LB medium and then applied to agar plates for 6 hours. The colloidal gel was then added to the inoculated agar plates and placed in an incubator at 37°C and relative humidity for 12 hours. The agar plates were photographed, and bacterial growth around the cylinders was observed. Figure 6 It was shown that no bacteria grew around the colloidal gel, indicating that the gel of the present invention has antibacterial properties.

[0184] Example 20

[0185] Taking the particle gel prepared in Example 1 (pH 5) as an example, the particles were mixed with the natural active factor VEGF, the anticancer drug doxorubicin, the protein drug immunoglobulin, and the nucleic acid drug mRNA, and placed on an oscillator (30 rpm) at an ambient temperature of 37°C to simulate the dynamic environment in the body. 1 ml of PBS supernatant was absorbed at 1d, 3d, 7d, 14d, and 21d, and then an equal amount of fresh PBS solution was added. The content of drug component release was measured at each time point using an ELISA kit and high-performance liquid chromatography, with three samples in each group. After testing, Figure 7 It shows that all kinds of drug molecules in the colloidal gel are slowly released at a relatively constant concentration. This uniform release can still be detected on the 21st day, indicating that the covalently cross-linked colloidal gel has drug sustained-release properties and can be used as a carrier of bioactive substances.

[0186] Example 21

[0187] The cells were treated with ROS inducers to obtain cells containing high ROS as a positive control; normal cells were used as a negative control; and the composite particles containing Ce ions in Example 6 were co-cultured with cells with high ROS content. The above different groups were stained with reactive oxygen species probes DCFH-DA and Ru(dpp)3Cl2 to observe the content of reactive oxygen species in the cells. The stained cells were observed using the EVOS system, and the readings were read using a multimode plate reader (PerkinElmer, USA) at an excitation wavelength of 485nm and an emission wavelength of 525nm. The green and red fluorescence signals were DCFH-DA and Ru(dpp)3Cl2 probes, respectively, both of which can represent ROS content. Figure 8 Fluorescence images show low red and green fluorescence intensities in normal cells, indicating low ROS levels (negative control group in the figure). Cells treated with ROS inducers showed significant green and red fluorescence signals (positive control group in the figure), indicating high ROS levels in the cells. Incubation of Ce ion composite particles with cells containing high ROS levels significantly reduced intracellular ROS levels, demonstrating that the surface Ce ion composite particles exhibit antioxidant properties (Ce ion composite particle group in the figure).

[0188] Example 22

[0189] After culturing the mesenchymal stem cells with 10 mg / mL of the gelatin / tannic acid colloidal gel in Example 1 (pH 5) and the Zn ion composite colloidal gel in Example 6 for 7 days, the osteogenic differentiation behavior of the stem cells was evaluated by alkaline phosphatase (ALP) staining. Figure 9The results showed that compared with the blank control group, the cell groups treated with composite protein particles and Zn ion composite particles showed a higher degree of osteogenic differentiation characteristics. The Zn ion composite colloidal gel showed higher osteogenic differentiation performance than the simple colloidal gel.

[0190] Example 23

[0191] After endothelial cells HUVEC were co-cultured with 10 mg / mL of the Cu ion composite colloidal gel of Example 6, the proliferation and migration of the endothelial cells were evaluated by cell staining. Figure 10 It was shown that co-culture with colloidal gel could promote the proliferation of endothelial cells compared with the blank control group.

[0192] Example 24

[0193] Using the colloidal gel prepared in Example 1 (pH 5), the uncovalently cross-linked particle gel was loaded into a syringe and extruded through a G16-23 caliber needle using a 3D bioprinter. The material was printed layer by layer according to a pre-programmed route, resulting in a 3D bioprinted scaffold with a fine structure.

[0194] Example 25

[0195] The colloidal gel described in Example 1 (pH 5) was injected subcutaneously on both sides of SD rats, totaling 100 μL of the gel material. The subcutaneous filling effect was observed at time points of 1, 4, 8, and 12 weeks. The experiment found that the particle gel could stably fill the rats' subcutaneous tissue for 21 weeks, and the rats' physiological conditions were stable.

[0196] Anyone skilled in the art will be able to utilize the above-disclosed technical content to make many possible changes and modifications to the technical solution of the present invention, or to modify it into equivalent embodiments with equivalent changes, without departing from the scope of the technical solution of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A hydrogel material assembled from polyphenol and protein composite micro-nano colloidal particles, characterized in that: The preparation method of the hydrogel material comprises the following preparation steps: (1) dissolving a protein material in an aqueous solution at 10-80° C. to obtain a protein solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol solution with a concentration of 0.1-10 w / v%; (2) adjusting the pH value of the protein solution to 3-7, adding a polyphenol solution to the protein solution to obtain a protein / polyphenol composite particle solution, wherein the mass ratio of the protein material to the polyphenol is 0.1-20; adding a crosslinker to the protein / polyphenol composite particle solution at room temperature to stabilize the composite particles for 1-12 hours, washing to obtain a protein / polyphenol composite particle dispersion, and freeze-drying the particle dispersion to obtain a composite particle powder, wherein the mass ratio of the protein material to the crosslinker is 0.1-100; (3) blending the protein / polyphenol composite particle powder with an aqueous solution to obtain an injectable, self-healing colloidal gel; In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, fenvalerate, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, divinyl sulfone, polyfunctional aziridine, and diisocyanate; The protein material is selected from pure structural protein, pure structural protein derivatives, and mixtures of pure structural protein and hydrophilic polymer materials; the pure structural protein is gelatin, sericin, fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, and fluorescent protein GFP; The polyphenol is selected from one or more of gallic acid, gallic acid ester, epigallocatechin, quercetin, curcumin, tannic acid, catechol, and dopamine.

2. The hydrogel material according to claim 1, characterized in that The injectable, self-repairing colloidal gel of step (3) is further prepared by adding an oxidant to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel; The oxidizing agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide or hydrogen peroxide.

3. The hydrogel material according to claim 1, wherein The mass ratio of the protein material to the polyphenols in step (2) is 1-20.

4. The hydrogel material according to claim 1, characterized in that The aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

5. A hydrogel material composed of micro-nano colloidal particles composited with polyphenols, proteins and metal ions, characterized in that: The preparation method of the hydrogel material comprises the following preparation steps: (1) dissolving a protein material in an aqueous solution at 10-80° C. to obtain a protein solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol solution with a concentration of 0.1-10 w / v%; (2) adjusting the pH of the protein solution to 3-7, adding a polyphenol solution to the protein solution to obtain a protein / polyphenol composite particle solution, wherein the mass ratio of the protein material to the polyphenol is 0.1-20; adding a crosslinker to the solution at room temperature to stabilize the composite particles and react for 1-12 hours before adding metal ions, wherein the concentration of the metal ions is 1 mM-10 M; centrifuging the particle dispersion, washing, and freeze-drying to obtain a composite particle powder; wherein the mass ratio of the protein material to the crosslinker is 0.1-100; (3) blending the protein / polyphenol composite particle powder with an aqueous solution to obtain an injectable, self-healing colloidal gel; In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, fenvalerate, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, divinyl sulfone, polyfunctional aziridine, and diisocyanate; The metal ions used are one or more of aluminum, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, cadmium, cerium, europium, gadolinium and terbium ions; The protein material is selected from pure structural protein, pure structural protein derivatives, and mixtures of pure structural protein and hydrophilic polymer materials; the pure structural protein is gelatin, sericin, fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, and fluorescent protein GFP; The polyphenol is selected from one or more of gallic acid, gallic acid ester, epigallocatechin, quercetin, curcumin, tannic acid, catechol, and dopamine.

6. The hydrogel material according to claim 5, characterized in that The injectable, self-repairing colloidal gel of step (3) is further prepared by adding an oxidant to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel; The oxidizing agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide or hydrogen peroxide.

7. The hydrogel material according to claim 5, wherein The mass ratio of the protein material to the polyphenols in step (2) is 1-20.

8. The hydrogel material according to claim 5, characterized in that The aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

9. A hydrogel material composed of rigid nanoparticles-polyphenol / protein composite core-shell structured micro-nano colloidal particles, characterized in that: The preparation method of the hydrogel material comprises the following preparation steps: (1) dissolving a protein material in an aqueous solution at 10-80° C. to obtain a protein solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol solution with a concentration of 0.1-10 w / v%; (2) adding rigid nanoparticles to the protein solution and adjusting the pH of the solution to 3-7, and then adding a polyphenol solution to obtain a core-shell structure composite particle solution; wherein the mass ratio of the protein material to the polyphenol is 0.1-20, and the mass ratio of the protein material to the rigid nanoparticles is 0.1-30; adding a crosslinking agent to the composite particle solution at room temperature to further crosslink and stabilize the composite particles for 1-12 hours, wherein the mass ratio of the protein material to the crosslinking agent is 0.1-100; after washing, obtaining a composite particle dispersion of protein / polyphenol / nanoparticles, and freeze-drying the particle dispersion to obtain a composite particle powder; (3) blending the composite particle powder with an aqueous solution to obtain an injectable, self-healing colloidal gel; In step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, fenvalerate, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, divinyl sulfone, polyfunctional aziridine, and diisocyanate; The rigid nanoparticles are silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, and polystyrene nanoparticles; The protein material is selected from pure structural protein, pure structural protein derivatives, and mixtures of pure structural protein and hydrophilic polymer materials; the pure structural protein is gelatin, sericin, fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, and fluorescent protein GFP; The polyphenol is selected from one or more of gallic acid, gallic acid ester, epigallocatechin, quercetin, curcumin, tannic acid, catechol, and dopamine.

10. The hydrogel material according to claim 9, characterized in that The injectable, self-repairing colloidal gel of step (3) is further prepared by adding an oxidant to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel; The oxidizing agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide or hydrogen peroxide.

11. The hydrogel material according to claim 9, wherein The mass ratio of the protein material to the polyphenols in step (2) is 1-20.

12. The hydrogel material according to claim 9, characterized in that The aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

13. A core-shell structured colloidal particle hydrogel material composed of rigid nanoparticles, polyphenols, proteins, and metal ions, characterized in that: The preparation method of the hydrogel material comprises the following preparation steps: (1) dissolving a protein material in an aqueous solution at 10-80° C. to obtain a protein solution with a concentration of 0.1-10 w / v%; dissolving a polyphenol in an aqueous solution at 10-90° C. to obtain a polyphenol solution with a concentration of 0.1-10 w / v%; (2) adding rigid nanoparticles to the above protein solution and adjusting the pH of the solution to 3-7, and adding a polyphenol solution to obtain a core-shell structure composite particle solution; wherein the mass ratio of the protein material to the polyphenol is 0.1-20, and the mass ratio of the protein material to the rigid nanoparticles is 0.1-30; adding metal ions to the particle dispersion and stirring, wherein the concentration of the metal ions is 1 mM-10 M; adding a cross-linking agent at room temperature to further cross-link and stabilize the composite particles for 1-12 hours, wherein the mass ratio of the protein material to the cross-linking agent is 0.1-100; washing to obtain a protein / polyphenol / nanoparticle composite particle dispersion, centrifuging, washing, and freeze-drying the dispersion to obtain a composite particle powder; (3) blending the composite particle powder with an aqueous solution to obtain an injectable, self-healing colloidal gel; in step (2), the crosslinking agent is one or more of carbodiimide / N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, fenvalerate, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, divinyl sulfone, polyfunctional aziridine, and diisocyanate; The rigid nanoparticles are silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, and polystyrene nanoparticles; The metal ions used are one or more of aluminum, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, cadmium, cerium, europium, gadolinium and terbium ions; The protein material is selected from pure structural protein, pure structural protein derivatives, and mixtures of pure structural protein and hydrophilic polymer materials; the pure structural protein is gelatin, sericin, fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, and fluorescent protein GFP; The polyphenol is selected from one or more of gallic acid, gallic acid ester, epigallocatechin, quercetin, curcumin, tannic acid, catechol, and dopamine.

14. The hydrogel material according to claim 13, characterized in that The injectable, self-repairing colloidal gel of step (3) is further prepared by adding an oxidant to polymerize the polyphenols on the surface of the composite particles to obtain a cross-linked colloidal hydrogel; The oxidizing agent is sodium dichromate, potassium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide or hydrogen peroxide.

15. The hydrogel material according to claim 13, wherein The mass ratio of the protein material to the polyphenols in step (2) is 1-20.

16. The hydrogel material according to claim 13, characterized in that The aqueous solution is water or an aqueous solution blended with other substances; the other substances are one or more of vitamins, amino acids, mineral elements, microecological regulators, silica nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles, alginate, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum, and starch.

17. Use of the hydrogel material according to any one of claims 1 to 16, characterized in that: Application in the preparation of anti-inflammatory, anti-oxidative and antibacterial scaffolds, which are used to repair and fill wounds or defects of bone tissue, cartilage tissue, muscle, blood vessels and skin; or use in the preparation of a carrier containing a pharmaceutical ingredient, wherein the pharmaceutical ingredient is one or more combinations of vitamins, amino acids, mineral elements, microecological regulators, small molecule drugs, protein macromolecule drugs, hormone drugs, anesthetic drugs, antiviral drugs, antibacterial drugs, anticancer drugs, immunomodulatory drugs, nucleic acid drugs, or living cells; or in the preparation of superficial skin and subcutaneous fillers; or application in the preparation of bio-ink; or in the preparation of tissue adhesive gel materials, wherein the colloidal particle size is <10 μm and the adhesive strength between the gel and the tissue is 5-100 kPa; or use in the preparation of hemostatic sealing powder; Or it can be used in the preparation of a metal ion sustained-release carrier, wherein the carrier is used to achieve wound repair.

18. The use according to claim 17, characterized in that The superficial skin and subcutaneous fillers are medical cosmetic fillers.

19. The use according to claim 17, characterized in that The bio-ink is a bio-printing ink for printing living cells.

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