CN115521484A

CN115521484A - Injectable colloidal gel material composed of polyphenol and protein composite particles and preparation method and application thereof

Info

Publication number: CN115521484A
Application number: CN202211112129.2A
Authority: CN
Inventors: 王华楠; 陈楷文; 陈陶; 蹇光宇
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2022-09-13
Filing date: 2022-09-13
Publication date: 2022-12-27

Abstract

The invention belongs to the field of material science and the field of biomedical materials, and particularly relates to an injectable colloidal gel material consisting of polyphenol and protein composite particles, and a preparation method and application thereof. The hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction between polyphenol/protein composite micro-nano particles; the crosslinking among polyphenol/protein composite micro-nano particles can be realized by adding an oxidant to covalently crosslink among polyphenols; the polyphenol/protein composite micro-nano particles are formed by induced assembly through hydrogen bond action, hydrophobic action or electrostatic action between a protein polymer chain and polyphenol; and a crosslinking agent can be added to covalently crosslink protein chains to form stable micro-nano particles. The composite particles and the gel material thereof prepared by the invention have good stability, and can be used as drug sustained release carriers, tissue engineering scaffolds, tissue adhesion hemostatic materials and the like in the biomedical field.

Description

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

Technical Field

The invention belongs to the field of material science and the field of biomedical materials, and particularly relates to a gel material which is formed by compounding polyphenol and protein and consists of injectable and shapeable protein particles, and a preparation method and application thereof.

Background

The polymer micro-nano particles are a polymer material with special physical properties, and have the main characteristics of small particle size and high specific surface area. Based on the characteristics, the micro-nano particles are widely applied to microcarriers, micro separators and microreactors in the field of biomedicine. The micro-nano particles can be prepared by different manufacturing methods to obtain particle materials with expected shapes and sizes. In recent years, by controlling the interaction between particles, a gel material composed of particles can be obtained when close packing is formed between the particles. Due to the reversibility of the interaction between particles, the gel materials usually show the mechanical characteristics of shear thinning and self-repairing; meanwhile, due to the difference of the functionalities of different types of particles, micro-nano particles with different functions can be assembled to form a gel material with modular characteristics.

In the field of biomedical applications, protein materials are widely used because they generally have excellent biocompatibility. Micro-nano particles synthesized from various naturally occurring or engineered proteins have now become a promising platform for biomedical applications. In addition, the amphiphilic nature of the protein facilitates its interaction with hydrophilic and hydrophobic drugs and solvents. The large number of hydroxyl, amino and carboxyl groups present in them makes them susceptible to chemical modification. Thus, protein particles can be covalently or non-covalently linked to one or different types of ligands and drug molecules, which provides excellent surface modification properties. Over the past decades scientists have been using the biomedical potential of proteins, including gelatin, silk fibroin, albumin, gliadin and other substances from a wide variety of sources, such as animal, plant, insect and recombinant protein expression systems. However. The existing protein micro-nano particles are usually complex to prepare and low in yield. The protein material is prepared mainly by an anti-solvent method and an emulsion method. The preparation of nanoparticles by the anti-solvent method usually requires the addition of a large amount of organic reagents to cause protein to curl to form particles, and for the preparation of nanoparticles by the emulsion method, oil is usually required to be added as a surfactant, so that the separation process of oil and protein particles is very complicated. Therefore, it is important to use a simple and general protein particle preparation technical strategy to realize the mass production of protein nanoparticles.

Disclosure of Invention

The invention provides a colloidal particle hydrogel material compositely assembled by polyphenol and protein, which is formed by assembling through hydrophobic action and hydrogen bond action among polyphenol/protein composite micro-nano particles; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction between polyphenol/protein composite micro-nano particles, adding an oxidant to enable covalent crosslinking between polyphenols to realize crosslinking between polyphenol/protein composite micro-nano particles; the polyphenol/protein composite micro-nano particles are formed by induced assembly through hydrogen bond action, hydrophobic action or electrostatic action between a protein polymer chain and polyphenol; preferably, the polyphenol/protein composite micro-nano particles are formed by induced assembly through hydrogen bond interaction, hydrophobic interaction or electrostatic interaction between a protein polymer chain and polyphenol, and then the protein chain is covalently crosslinked by adding a crosslinking agent to form the stable micro-nano particles.

Wherein the size of the composite micro-nano particles is 10 nm-500 mu m, and when the size of the particles is 10 nm-5 mu m, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 2-120 v/v%; when the particle size is 5-500 mu m, the volume fraction of the composite micro-nano particles in the granular hydrogel material accounting for the total volume of the hydrogel is 50-120 v/v%.

When the particle size is 10nm to 5 μm, the compressive elastic modulus of the hydrogel after covalent crosslinking is 0.5kPa to 500kPa;

when the particle size is 5 to 500. Mu.m, the compressive modulus of elasticity of the hydrogel after covalent crosslinking is 0.5kPa to 100kPa.

The invention provides a hydrogel material assembled by micro-nano colloidal particles compounded by polyphenol, protein and metal ions, wherein the hydrogel material is formed by assembling the polyphenol/protein compounded micro-nano colloidal particles under the hydrophobic action, the hydrogen bond action and the metal coordination action; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction, hydrogen bond interaction and metal coordination between the polyphenol/protein composite micro-nano particles, an oxidant is added to enable covalent crosslinking between polyphenols to realize crosslinking between the polyphenol/protein composite micro-nano particles; the polyphenol/protein composite micro-nano particles are obtained by adding metal ions into the polyphenol/protein composite micro-nano particles to form coordination with polyphenol in the particles.

Wherein, the size of the composite microsphere particles is 10 nm-500 μm, when the size of the particles is 10 nm-5 μm, the volume fraction of the colloidal particles in the particle hydrogel material accounting for the total volume of the hydrogel is 2-120 v/v%, and the obtained particle hydrogel has the characteristics of shear thinning and self-repairing; the compression elastic modulus of the obtained granular hydrogel is 0.5 kPa-5 MPa.

The third aspect of the invention provides a hydrogel material assembled by rigid nanoparticle-polyphenol/protein composite core-shell structure micro/nano particles, wherein the hydrogel material is assembled by hydrophobic interaction and hydrogen bond interaction between the core-shell structure micro/nano particles; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction among the composite micro-nano colloidal particles, the crosslinking among the core-shell structure micro-nano particles is realized through covalent crosslinking among polyphenols by adding an oxidant; the core layer of the core-shell structure micro-nano particles is made of rigid nano particle materials, the shell layer is made of polyphenol/protein composite particles, and the polyphenol/protein composite particles are formed through hydrogen bond action, hydrophobic action and electrostatic action between protein molecules and polyphenol molecules; preferably, after the protein molecules and the polyphenol molecules are formed through hydrogen bond interaction, hydrophobic interaction and electrostatic interaction, a cross-linking agent is added to covalently cross-link protein chains to form the stable micro-nano particles.

The invention provides a core-shell structure colloidal particle material compounded by rigid nano particles, polyphenol/protein/metal ions, wherein the hydrogel material is formed by assembling hydrophobic interaction, hydrogen bond interaction and metal coordination interaction among the micro-nano particles with the core-shell structure; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction, hydrogen bond interaction and metal coordination interaction between the core-shell structure micro-nano particles, an oxidant is added to enable covalent crosslinking between polyphenols to realize crosslinking between the core-shell structure micro-nano particles; the core layer of the core-shell structure micro-nano particle is made of rigid nano particle materials, the shell layer is made of polyphenol/protein/metal composite particles, and the polyphenol/protein/metal composite particles are obtained by adding metal ions into the polyphenol/protein composite particles of claim 3 to form coordination with polyphenol in the particles.

when the particle size is 5 to 500. Mu.m, the compressive elastic modulus of the hydrogel after covalent crosslinking is 0.5kPa to 100kPa.

In the above-mentioned technical solution, further,

the polyphenol material is selected from one or more of gallic acid, gallate, epigallocatechin, quercetin, curcumin, tannic acid, catechol, and dopamine;

the protein material is selected from pure structural protein, pure structural protein derivatives, pure structural protein and hydrophilic polymer material mixture; the pure structural protein is gelatin protein, sericin, silk fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, fluorescent protein GFP;

the oxidant in the polyphenol oxidation process is sodium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide and hydrogen peroxide;

the metal ions used for the coordination interaction of the metal ions 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 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, polystyrene nanoparticles;

when the size of the composite micro-nano particles is 10 nm-5 mu m, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 2-120 v/v%; when the size of the composite micro-nano particles is 5-500 mu m, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 50-120 v/v%.

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

(1) Dissolving protein in aqueous solution at 10-80 deg.c to obtain 0.1-10 w/v% concentration protein solution; dissolving polyphenol in water solution at 10-90 deg.c to obtain polyphenol water solution in the concentration of 0.1-10 w/v%;

(2) Adjusting the pH value of the protein solution to 3-7, adding a polyphenol solution into the protein solution to obtain a protein/polyphenol composite particle solution, and freeze-drying to obtain composite particle powder; wherein the mass ratio of the protein to the polyphenol is 0.1-20, preferably 1-20; preferably, adding a macromolecular cross-linking agent into the protein/polyphenol composite particle solution at normal temperature to stabilize the composite particles for reaction for 1-12 hrs, wherein the mass ratio of the protein to the macromolecular cross-linking agent is 0.1-100; cleaning to obtain a protein/polyphenol composite particle dispersion liquid, and freeze-drying the particle dispersion liquid to obtain composite particle powder;

(3) Mixing the protein/polyphenol composite particle powder with an aqueous solution to obtain an injectable self-repairing colloidal gel; preferably, the injectable and self-repairing colloidal gel is further added with an oxidant to polymerize polyphenol on the surfaces of the composite particles to obtain a crosslinked colloidal hydrogel;

in the step (2), the cross-linking agent is one or more of carbodiimide/N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, aromatic dialdehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, di-alkylene oxide, divinyl sulfones, polyfunctional aziridine and diisocyanate;

in the step (3), the oxidant is sodium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide and hydrogen peroxide;

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, silicon dioxide nanoparticles, lithium magnesium silicate nanoparticles, nano clay 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, alginic acid, hyaluronic acid, chitosan, chondroitin sulfate, pullulan, xanthan gum and starch.

In the above technical solution, further, the preparation method of the colloidal particle hydrogel material assembled by the polyphenol, the protein and the metal ions is characterized in that:

(1) Dissolving protein in aqueous solution at 10-80 deg.c to obtain 0.1-10 w/v% concentration protein solution; dissolving polyphenol in aqueous solution at 10-90 ℃ to obtain polyphenol aqueous solution with the concentration of 0.1-10 w/v%;

(2) Adjusting the pH value of the protein solution to 3-7, and adding a polyphenol solution into the protein solution to obtain a protein/polyphenol composite particle solution, wherein the mass ratio of the protein to the polyphenol is 0.1-20, preferably 1-20; adding metal ions into the particle solution, stirring to obtain a particle dispersion liquid, wherein the concentration of the metal ions is 1 mM-10M, centrifuging, washing and freeze-drying the particle dispersion liquid to obtain composite particle powder. Preferably, the particle solution is added with a macromolecular cross-linking agent at normal temperature to stabilize the composite particles, and then metal ions are added after the composite particles react for 1 to 12hrs, wherein the mass ratio of the protein to the macromolecular cross-linking agent is 0.1 to 100;

(3) Mixing the protein/polyphenol composite particle powder with an aqueous solution to obtain an injectable self-repairing colloidal gel; preferably, the injectable and self-repairing colloidal gel is further added with an oxidation cross-linking agent to polymerize polyphenol on the surfaces of the composite particles to obtain a cross-linked colloidal hydrogel;

in the step (2), the cross-linking agent is one or more of carbodiimide/N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, aromatic aldehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, dialkene oxide, divinyl sulfone, polyfunctional aziridine and diisocyanate;

in the step (3), the oxidative crosslinking agent is sodium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide and hydrogen peroxide; the metal ions used for the coordination crosslinking of the metal ions 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;

In the above technical solution, further, the preparation method of the core-shell structure colloidal particle material of the nanoparticle-polyphenol composite protein molecule is characterized in that the composite particle comprises the following preparation steps:

(1) Dissolving protein molecules in an aqueous solution at 10-80 ℃ to obtain a protein aqueous solution with the concentration of 0.1-10 w/v%; dissolving polyphenol in aqueous solution at 10-90 deg.c to obtain polyphenol water solution with concentration of 0.1-10 w/v%;

(2) Adding rigid nano particles into the protein aqueous solution, adjusting the pH value of the solution to 3-7, and adding a polyphenol solution to obtain a core-shell structure composite particle solution; freeze drying to obtain composite particle powder; wherein the mass ratio of the protein to the polyphenol is 0.1-20, and the mass ratio of the protein to the rigid nano particles is 0.1-30; preferably, at normal temperature, adding a cross-linking agent into the composite particle solution for further cross-linking and stabilizing the composite particles for reaction for 1-12 h, wherein the mass ratio of the protein to the macromolecular cross-linking agent is 0.1-100; (ii) a Cleaning to obtain a composite particle dispersion liquid of protein/polyphenol/nanoparticles, and freeze-drying the particle dispersion liquid to obtain composite particle powder;

(3) The composite particle powder and the aqueous solution are blended to obtain injectable self-repairing colloidal gel; preferably, the injectable self-repairing colloidal gel further comprises an oxidative crosslinking agent or metal ions to polymerize the polyphenol on the surface of the composite particles to obtain a crosslinked colloidal hydrogel;

in the step (2), the cross-linking agent is one or more of carbodiimide/N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, aromatic aldehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, dialkene oxide, divinyl sulfone, polyfunctional aziridine and diisocyanate; the rigid nanoparticles may be silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanoplatelets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanoplatelets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles;

in the step (3), the oxidation crosslinking agent is sodium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide and hydrogen peroxide; the metal ions used for the coordination crosslinking of the metal ions 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;

In the above technical solution, further, the preparation method of the core-shell structure colloidal particle material of nanoparticles-polyphenol, protein and metal molecule is characterized in that the composite particle comprises the following preparation steps:

(2) Adding rigid nano particles into the protein aqueous solution, adjusting the pH value 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 to the polyphenol is 0.1-20, and the mass ratio of the protein to the hard nano-particles is 0.1-30; adding metal ions into the particle dispersion liquid, stirring, centrifuging, washing and freeze-drying the particle dispersion liquid to obtain composite particle powder, wherein the concentration of the metal ions is 1 mM-10M; preferably, at normal temperature, adding a cross-linking agent into the core-shell structure composite particle solution for further cross-linking and stabilizing the composite particles for reaction for 1-12 h, wherein the mass ratio of the protein to the macromolecular cross-linking agent is 0.1-100; cleaning to obtain a composite particle dispersion liquid of protein/polyphenol/nanoparticles;

(3) The composite particle powder and the aqueous solution are blended to obtain injectable self-repairing colloidal gel; preferably, the injectable self-repairing colloidal gel further comprises an oxidative crosslinking agent or metal ions to polymerize the polyphenol on the surface of the composite particles, so as to obtain a crosslinked colloidal hydrogel;

in the step (2), the cross-linking agent is one or more of carbodiimide/N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, aromatic aldehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, dialkene oxide, divinyl sulfone, polyfunctional aziridine and diisocyanate; the nanoparticles may be silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanoplatelets, carbon nanotubes, bioglass nanoparticles, black phosphorus nanoplatelets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, polystyrene nanoparticles;

in the step (3), the oxidation crosslinking agent is sodium dichromate, potassium permanganate, sodium periodate, sodium peroxide, potassium peroxide and hydrogen peroxide; the metal ions used by 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;

In a fifth aspect, the present invention provides the use of the hydrogel material as described above, wherein the use comprises the following aspects:

the application of the compound granules in preparing anti-inflammatory, antioxidant and antibacterial scaffolds can slowly release polyphenol in the compound granules, and the scaffolds are used for repairing and filling wounds or defects of bone tissues, cartilage tissues, muscles, blood vessels and skin;

or in the preparation of a carrier or a bracket containing medicinal components, wherein the medicinal components are one or more of vitamins, amino acids, mineral elements, microecological regulators, growth factors, micromolecular medicaments, protein macromolecular medicaments, antibiotic medicaments, hormone medicaments, anesthetic medicaments, antiviral medicaments, antibacterial medicaments, anticancer medicaments, immunoregulation medicaments, nucleic acid medicaments or live cells;

or the application in preparing superficial skin and subcutaneous filler; preferably in cosmetic surgery; when the gel is applied, the gel is directly injected into superficial skin or subcutaneous region, a small amount of oxidant or metal ions are mixed, colloidal particle gel can be reversibly self-assembled under the action of non-covalent bonds to form a continuous porous particle network, the continuous porous particle network can stably stay at the injection position, the particle surface is further subjected to secondary crosslinking, the gel stability is improved, and the filling stability is ensured.

Or in the preparation of bio-inks, preferably bio-printing inks for printing with living cells; when the method is applied, colloidal particles and an aqueous solution are blended to obtain colloidal particle gel, then the colloidal particle gel is mixed with a cell suspension to obtain cell-loaded colloidal gel, namely biological ink, and the ink is extruded or subjected to ink-jet 3D printing to obtain a scaffold with a 3D structure, so that a cell-loaded printing scaffold is obtained;

or in the preparation of tissue adhesive gel material, wherein the composite microsphere particle size is less than 10 μm, and the adhesive strength between the colloidal gel and the tissue is 5-100 kPa; injecting the colloid gel material with injectable and self-repairing performance to the damaged part of the tissue in the body, and waiting for 1-30 min to realize the stable adhesion between the colloid gel and the tissue.

Or in the preparation of metal ion slow release carrier, used for wound tissue repair and anti-infection;

or the quick hemostasis sealing powder is applied to the quick hemostasis sealing powder, the quick hemostasis sealing powder comprises the prepared composite microsphere particle powder, the particle powder is directly sprayed to the wound surface defect position of the tissue surface, and after the powder fully absorbs exuded blood, the powder can form an adhesion effect with the tissue and realize effective hemostasis.

The invention has the beneficial effects that:

1. the invention discloses a preparation method of protein micro-nano particles with universality. The method dissolves protein materials in aqueous solution to be compounded with the aqueous solution containing polyphenol, and polyphenol can be rapidly combined with protein through hydrogen bond, hydrophobic interaction and electrostatic interaction to form polyphenol protein network, so that the polyphenol protein composite particle material is formed. The preparation method of the protein particles does not need to use organic reagents or surfactants used in the traditional protein particle preparation process, and only utilizes the strong interaction between polyphenol and protein to realize particle synthesis. This greatly reduces the production cost of the protein particles.

2. The preparation method of the protein particles is suitable for different kinds of protein materials. Since polyphenols can form interactions with different types of proteins, the method reported in the present invention can be used to prepare different types of protein particles. The general applicability of the current methods greatly expands protein particle manufacturing techniques, and in part particle manufacturing of difficult-to-granulate protein materials, as compared to conventional protein particle manufacturing techniques, which are typically directed only to a particular protein material.

3. The protein micro-nano particles reported by the invention are assembled to form a gel material with shear thinning and self-repairing performances through a reversible action; because the protein particles also contain polyphenol components, the abundant phenolic hydroxyl units in the polyphenol can form stable interaction with the tissue surface. Therefore, the particulate gel material reported by the invention has stable tissue adhesion and can be used as a tissue adhesion material for wound repair.

4. The protein micro-nano particles reported by the invention contain abundant phenolic hydroxyl groups, and can be coordinated and compounded with different types of metal ions to quickly realize the quick preparation of micro-nano particle units with different functions. Protein particles with different functions can be modularly assembled to form gel materials with different functions.

5. The composite particles reported in the present invention can also be used as therapeutic ingredients for antioxidant, anti-inflammatory and antibacterial therapeutic applications. Meanwhile, polyphenol is a common hemostatic material, so the colloidal gel prepared by the method has hemostatic and tissue adhesion properties.

Drawings

FIG. 1 is a scanning electron micrograph of a metal ion-complexed gelatin/tannic acid colloidal gel prepared in example 5; wherein purple in the Cu complex group represents Cu ions; yellow in the Fe complex group represents Fe ions; 3, orange color in the Zn complex group represents Zn ions; purple color in the Ce complex represents Ce ion.

FIG. 2 is a scanning electron micrograph of a covalently cross-linked gelatin/tannic acid colloidal gel prepared according to example 7, wherein panel b is an enlarged view of panel a.

Fig. 3 is a transmission electron microscope image of composite particles of the silica core-gelatin/polyphenol shell structure prepared in example 8.

Figure 4 is a graph of the release of polyphenols from the colloidal gel of example 17.

FIG. 5 is a two-dimensional cell culture dead-live staining pattern of the colloidal gel material obtained in example 18, and the survival condition of the cells on the surface of the gel is good, indicating that the colloidal gel has excellent biocompatibility. Wherein the ruler is 50 μm.

FIG. 6 is a graph showing the bacteriostatic test of the colloidal gel obtained in example 19. Wherein the yellow pure gelatin colloid gel in the A picture normally grows bacteria, the gel in the B picture is gelatin/tannin composite colloid gel, and no bacteria growth around the gel indicates that the gel has bacteriostasis.

FIG. 7 is a graph showing the colloidal gel of example 20 as a drug sustained-release carrier. Different kinds of protein drugs can be slowly released within 21 days.

FIG. 8 is a photograph of the cells of Ce ion composite colloidal gel against oxidation in example 21, in which the green signal represents active oxygen DCFH and the red signal represents active oxygen Ru (dpp) Cl ₂ 。

FIG. 9 is a photograph showing the staining of osteogenic factor alkaline phosphatase after co-culturing Zn ion complex colloidal gel with osteoblasts MC-3T3 in example 21, wherein the bluish purple signal represents the signal of alkaline phosphatase.

FIG. 10 is a photograph showing the co-culture of the Cu ion composite colloidal gel and endothelial cells in example 23.

Figure 11 is a sample picture of the colloidal gel 3D printed stent of example 22.

Detailed Description

The invention is further illustrated but is not in any way limited by the following specific examples.

Example 1

A gelatin solution was obtained by dissolving 5g of gelatin powder in 100mL of deionized water at 50 ℃ and adjusting the pH to 3, 5 and 7, and a tannic acid solution was obtained by dissolving 0.5g of tannic acid powder in 100mL of deionized water at 50 ℃. 100mL of tannic acid solution was slowly added to the gelatin solution over 30min and the solution was stirred at 1500rpm to rapidly bind the protein to the tannic acid molecules to form particles. Then 165. Mu.L of glutaraldehyde, a crosslinking agent, was added and stirred for 12hrs. And (3) cleaning the composite particles by using deionized water, and freeze-drying the composite particles to obtain gelatin/tannic acid nano-particle powder. The size and charge of the resulting nanoparticles are shown in table 1.

TABLE 1

	Particle size	Surface charge
			pH＝3	214.6nm	-14.7mV
pH＝5	239.5nm	-10.9mV
			pH＝7	306.5nm	-6.1mV

Repeatedly blowing and beating 0.3g of gelatin/tannic acid nano-particle powder and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable self-repairing composite particle gel. The gel was tested for storage modulus G' using a time-sweep mode of a rotational rheometer and the self-healing efficiency is shown in table 2. Where the frequency was 1Hz and the strain was 0.5%. The data in table 2 shows that as the mass fraction of particles increases, the storage modulus of the gel formed increases and the gel has self-healing properties.

TABLE 2

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

Comparative example 1

5g of gelatin powder was dissolved in 100mL of deionized water at 50 ℃ to give a gelatin solution and the pH was adjusted to 9, 11. 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50 ℃ to obtain a tannic acid solution. The 100mL tannic acid solution is added into a gelatin solution within 30min, the color of the solution is brown and clear, and the particle size in the solution is further tested to show that micro-nano particles are not formed in the test.

Comparative example 2

A gelatin solution was obtained by dissolving 5g of gelatin powder in 100mL of deionized water at 50 ℃ and adjusting the pH to 3, 5 and 7, and a tannic acid solution was obtained by dissolving 0.5g of tannic acid powder in 100mL of deionized water at 50 ℃. 100mL of gelatin solution was slowly added to the tannic acid solution over 30min, whereupon a brown flocculent precipitate formed, which failed to give an emulsified particle suspension.

Example 2

A gelatin solution was obtained by dissolving 5g of gelatin powder in 100mL of deionized water at 50 ℃ and adjusting the pH to 3, 5 and 7, and a tannic acid solution was obtained by dissolving 0.5g of tannic acid powder in 100mL of deionized water at 50 ℃. 100mL of tannic acid solution was slowly added to the gelatin solution over 30min and the solution was stirred at 1500rpm to rapidly bind the protein to the tannic acid molecules to form particles. And (3) cleaning the composite particles by using deionized water, and freeze-drying the composite particles to obtain gelatin/tannic acid nano-particle powder. The size and charge of the resulting nanoparticles are shown in table 3.

TABLE 3

	Particle size	Surface charge
			pH＝3	331.9nm	-3.7mV
pH＝5	409.7nm	-5.9mV
			pH＝7	426.9nm	-4.3mV

Repeatedly blowing and beating 0.3g of gelatin/tannic acid nano-particle powder and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable self-repairing composite particle gel. The gel was tested for storage modulus G' using a time-sweep mode of a rotational rheometer and the self-healing efficiency is shown in table 3. Where the frequency was 1Hz and the strain was 0.5%. The data in table 4 shows that as the mass fraction of particles increases, the storage modulus of the formed gel increases and the gel has self-healing properties.

TABLE 4

	Storage modulus	Efficiency of self-repair
			pH＝3	6.2kPa	88.1％
pH＝5	4.7kPa	94.6％
			pH＝7	4.5kPa	95.4％

Example 3

5g of gelatin powder was dissolved in 100mL of deionized water at 50 ℃ to give a gelatin solution and the pH was adjusted to 5. 0.25, 0.5, 2, 5g of tannic acid powder was dissolved in 100mL of deionized water at 50 ℃ to obtain a tannic acid solution. The above 100mL tannic acid solution was added to the gelatin solution within 30min and stirred at 1500rpm to bind protein molecules and tannic acid molecules into particles. 165. Mu.L of glutaraldehyde, a crosslinking agent, was then added and stirred for 12hrs. And cleaning the composite particles by using deionized water, and freeze-drying the composite particles to obtain composite particle powder. The size and charge of the resulting composite particles are shown in table 5.

TABLE 5

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

Repeatedly blowing and beating 0.3g of gelatin/tannic acid composite particle powder and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable self-repairing particle gel. The storage modulus G' of the particle gel was obtained using the time-scan mode of the rotational rheometer and the self-healing efficiency is shown in table 6. Where the frequency was 1Hz and the strain was 0.5%. Rheological experiments show that the highest storage modulus is shown when the ratio of the tannic acid to the gelatin is 0.1, and the highest self-repairing efficiency is shown when the ratio of the tannic acid to the gelatin is 1.

TABLE 6

Tannic acid/gelatin (w/w)	Storage modulus	Efficiency of self-repair
			0.05	6.9kPa	73.5％
0.1	7.6kPa	76.9％
			0.4	5.5kPa	83.9％
1	4.9kPa	89.4％

Comparative example 3

5g of gelatin powder was dissolved in 100mL of deionized water at 50 ℃ to give 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 ℃ to obtain a tannic acid solution. Adding the 100mL tannic acid solution into gelatin solution within 30min, and stirring at 1500rpm, wherein the solution is transparent and clear, and no emulsification occurs. Further testing the particle size in the solution shows that no micro-nano particles are formed in the test, which indicates that the gelatin/tannic acid mass ratio is too low to form nano particles at 0.02.

5g of gelatin powder was dissolved in 100mL of deionized water at 50 ℃ to give a gelatin solution and the pH was adjusted to 5. A tannic acid solution was obtained by dissolving 150g of tannic acid powder in 100mL of deionized water at 50 ℃. When the above 100mL of tannic acid solution was added to the gelatin solution within 30min and stirred at 1500rpm, a brown precipitate was rapidly produced, indicating that the tannic acid mass ratio of 30 resulted in agglomeration of particles and failure to obtain a dispersed particulate material.

Example 4

5g of gelatin powder was dissolved in 100mL of deionized water at 50 ℃ to give a gelatin solution and the pH was adjusted to 5. Respectively dissolving 0.5g EGCG (epigallocatechin gallate), gallic acid, quercetin and catechin powder in 100mL deionized water at 50 deg.C to obtain corresponding polyphenol solution. Adding the polyphenol solutions into the gelatin solution within 30min, and rapidly stirring to combine gelatin molecules with different polyphenols to form micro-nano particles. 165. Mu.L of glutaraldehyde, a crosslinking agent, was then added and stirred for 12hrs. And (3) cleaning the composite particles by using deionized water, and freeze-drying the composite particles to obtain different polyphenol and gelatin composite nano-particle powder. The size and charge of the resulting nanoparticles are shown in table 7.

TABLE 7

Type of particle	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

Repeatedly blowing and beating 0.3g of EGCG/gelatin, gallic acid/gelatin, quercetin/gelatin and catechin/gelatin nano-particle powder and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable self-repairing particle gel. The storage modulus G' of the particulate gel was obtained using the time-sweep mode of the rotational rheometer and the self-healing efficiency is shown in table 8. Where the frequency was 1Hz and the strain was 0.5%. Rheological experiments show that the use of different kinds of polyphenol materials has no obvious influence on the storage modulus and the self-repairing performance of the final gel.

TABLE 8

Type of particle	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％

Example 5

5g of sericin, silk fibroin, albumin, serum protein, keratin, elastin, hemoglobin, immunoglobulin, fibrin, fluorescent protein GFP and gelatin derivative methacrylate gelatin were dissolved in 100mL of deionized water at 37 ℃ to obtain a protein solution and pH was adjusted to 5. 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50 ℃ to obtain a tannic acid solution. Adding the above 100mL tannic acid solution within 30min, stirring rapidly, combining with the tannic acid molecules using fibroin molecules to form nanoscale spheres, and stirring for 12hrs. And (3) washing the nano particles by using deionized water, and freeze-drying the nano particles to obtain fibroin/tannin nano particle powder. The size and charge of the resulting nanoparticles are shown in table 9.

TABLE 9

Protein species	Particle size	Surface charge
			Sericin	227.8nm	-27.7mV
Silk fibroin	279.2nm	-31.3mV
			Albumin	173.4nm	-17.2mV
Serum protein	149.0nm	-19.2mV
			Keratin protein	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

Repeatedly blowing 0.4g of silk fibroin/tannin, albumin/tannin, keratin/tannin, fluorescent protein/tannin particle powder and 1mL of deionized water through a luer adapter injector at normal temperature for 10 times to obtain the injectable self-repairing particle gel. The storage modulus G' of the particulate gel was obtained using the time-sweep mode of the rotational rheometer as shown in table 4. Where the frequency was 1Hz and the strain was 0.5%. Different types of proteins showed different mechanical strengths after complexing with tannic acid as shown in table 10.

Watch 10

Protein type	Storage modulus
		Silk fibroin	28.6kPa
Albumin	14.1kPa
		Keratin protein	11.2kPa
Fluorescent proteins	7.9kPa
		Gelatin methacrylate	12.9kPa

Example 6

5g of gelatin powder was dissolved in 100mL of deionized water at 50 ℃ and pH was adjusted to 5, and 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50 ℃ to obtain a tannic acid solution. Adding the 100mL of tannic acid solution into the gelatin solution within 30min, and rapidly stirring to combine the gelatin and tannic acid to form micro-nano composite particles. 165. Mu.L of glutaraldehyde, a crosslinking agent, was then added and stirred for 12hrs. After the composite particles are washed by deionized water, the concentration of the particles is diluted to 10mg/mL by the deionized water, copper chloride, ferric chloride, zinc chloride and europium chloride are respectively added into the solution until the final solution concentration reaches 10mM, the mixture is stirred for 12hrs, and the composite particles are repeatedly washed by the deionized water for 3 times, so that the metal composite protein particles are obtained. The size and charge of the obtained nanoparticles are shown in table 11, wherein the scanning electron micrograph of the particles is shown in fig. 1, and the corresponding metal elements have been successfully compounded in the particles.

TABLE 11

Chelating metal ion species	Particle size	Surface charge
			Cu	214.6nm	-14.7mV
Fe	235.9nm	-19.3mV
			Zn	275.1nm	-21.3mV
Ce	224.7nm	-19.2mV

Repeatedly blowing 0.4g of different metal ion chelating protein/polyphenol nanoparticle powder and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable self-repairing particle gel. The storage modulus G' of the particulate gel was obtained using a time-sweep mode of a rotational rheometer, and the self-healing efficiency is shown in table 12. Where the frequency was 1Hz and the strain was 0.5%. In comparison with example 1, it can be seen that the mechanical strength of the gel is significantly increased when the surface of the particles is chelated by the metal ions.

TABLE 12

Type of metal	Storage modulus
		Cu	28.6kPa
Fe	24.1kPa
		Zn	21.2kPa
Ce	17.9kPa

Example 7

Using the gelatin/tannic acid micro-nano particle powder prepared in example 1 (pH 5), 0.4g of the particle powder was repeatedly blown and beaten with 5mg of sodium periodate, sodium dichromate, sodium peroxide, hydrogen peroxide, and 1mL of deionized water at normal temperature through a luer adapter syringe for 10 times to obtain injectable particle gel. The storage modulus G' of the particulate gel was measured using a time-sweep mode of a rotational rheometer and the self-healing efficiency is shown in table 13. Where the frequency was 1Hz and the strain was 0.5%. The composite particulate gel can be further enhanced because the oxidative crosslinking agent induces covalent crosslinking of the tannin molecules on the surface of the particles. Where the frequency was 1Hz and the strain was 0.5%. The storage modulus of the uncrosslinked gelatin/tannic acid nanoparticles of comparative example 1 was obtained, and the mechanical strength of the same mass fraction gel was significantly improved when the oxidative crosslinking agent was added. The microstructure of the covalently cross-linked gel is shown in the scanning electron micrograph of FIG. 2.

Watch 13

Example 8

Dissolving 5g of gelatin powder in 100mL of deionized water at 50 ℃, adjusting the pH value of the solution to 5, and respectively adding 1g of silicon dioxide nanoparticles, graphene nanosheets, hydroxyapatite nanoparticles, fibroin nanoparticles and bioglass nanoparticle powder to obtain the gelatin solution containing nanoparticles. 0.5g of tannic acid powder was dissolved in 100mL of deionized water at 50 ℃ to obtain a tannic acid solution. Adding 100mL of tannic acid solution into gelatin solution containing silica nanoparticles, graphene nanosheets, hydroxyapatite nanoparticles, fibroin nanoparticles and bioglass nanoparticles within 30min, rapidly stirring to combine gelatin molecules and tannic acid molecules to form micro-nano particles, and coating the nano particles. 165. Mu.L of glutaraldehyde, a crosslinking agent, was then added and stirred for 12hrs. After washing the nanoparticles with deionized water, the nanoparticles were freeze-dried to obtain gelatin/tannic acid/nanoparticle powder. The size and charge of the resulting nanoparticles are shown in table 14. The transmission electron microscope of the composite particles in which the silica is wrapped is shown in fig. 3.

TABLE 14

Core layer nanoparticle species	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

And repeatedly blowing 0.4g of core-shell particle powder containing different core-layer particles and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable self-repairing particle gel. The storage modulus G' of the particulate gel was obtained using the time-sweep mode of the rotational rheometer as shown in table 15. Where the frequency was 1Hz and the strain was 0.5%. Core-shell composite particles of different core-layer particle types exhibit different mechanical strengths.

Watch 15

Example 9

Using the suspension of protein polyphenol core-shell particles prepared in example 8, and taking the suspension of composite particles 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 until the final solution concentration was 10mM, and the mixture was stirred for 12hrs, and the nanoparticles were repeatedly washed 3 times using deionization, to obtain metal composite protein polyphenol core-shell particles.

Repeatedly blowing 0.4g of protein/polyphenol core-shell structure particle powder compounded by different metal ions and 1mL of deionized water for 10 times through a luer adapter injector at normal temperature to obtain the injectable composite particle gel. The storage modulus G' of the particulate gel obtained using the time-sweep mode of the rotational rheometer is shown in table 16. Where the frequency was 1Hz and the strain was 0.5%.

TABLE 16

Type of metal	Storage modulus
		Cu	58.6kPa
Fe	64.1kPa
		Zn	51.2kPa
Ce	47.9kPa

Example 10

Using the core-shell particle powder containing different core layer particles prepared in example 8, 0.4g of the particle powder, 5mg of sodium periodate, and 1mL of deionized water were repeatedly blown through a luer adapter syringe at normal temperature for 10 times to obtain an injectable particle gel. The storage modulus G' of the particulate gel obtained using the time-sweep mode of the rotational rheometer is shown in table 17. Where the frequency was 1Hz and the strain was 0.5%. The storage modulus G' of the composite particle gel is further improved because sodium periodate can induce covalent crosslinking of tannin molecules on the surface of the particles.

TABLE 17

Core layer nanoparticle species	Storage modulus
		Silicon dioxide	57.6kPa
Graphene	51.9kPa
		Hydroxyapatite	65.0kPa
Silk fibroin	54.3kPa
		Bioglass	53.1kPa

Example 11

Using the gelatin/tannic acid nanoparticle powder prepared in example 1, 0.4g of the particle powder was repeatedly beaten with 1mL of deionized water containing 20mg of silica nanoparticles, lithium magnesium silicate nanoparticles, nanoclay particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, silk fibroin nanoparticles, polylactic acid nanoparticles, alginic acid and pullulan through a luer adapter syringe at normal temperature for 10 times to obtain an injectable particle gel. The storage modulus G' of the particulate gel obtained using the time-sweep mode of the rotational rheometer is shown in table 18. Where the frequency was 1Hz and the strain was 0.5%.

Watch 18

Components in aqueous solutions	Storage modulus
		Silica nanoparticles	28.9kPa
Lithium magnesium silicate nanoparticles	25.1kPa
		Nano clay 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 polysaccharide	14.9kPa

Example 12

Compression testing was performed using the gelatin/tannic acid particle gel obtained in example 1 at pH5, the colloidal gel after metal ion crosslinking in example 6, and the covalently crosslinked gelatin/tannic acid colloidal gel obtained in example 7. The colloidal gel was formed into a cylinder (diameter: 6.4mm, height: 6 mm) and the compression test was conducted at a loading rate of 0.0002 mm/s. The compressive strain and compressive strength of the samples are shown in table 19, and after covalent crosslinking, both the compressive strength and the compressive strain of the gel are significantly increased.

Watch 19

Example 13

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: 6.4mm, 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, and the mechanical strength of the gel is significantly increased after covalent cross-linking.

Watch 20

Example 14

The gelatin/tannin particle gel obtained in example 1 at pH5 was used for the tissue adhesion test. After 0.1mL of the granular gel was lap-bonded to the surfaces of two pieces of glass (5.0 cm. Times.2.0 cm rectangles) in which the lap-overlapped area was (1.5 cm. Times.2.0 cm rectangles) and left to stand for 10 minutes, the lap-bonded sample was subjected to shear peeling (peeling rate: 10 mm/min) using a tensile tester having a 50N mechanical sensor, and the tissue adhesive strength was as shown in Table 21.

TABLE 21

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

Example 15

The gelatin/tannic acid/silica colloidal gel obtained in example 6 was used for the tissue adhesion test. 0.1mL of colloidal gel was injected to the surface of two pieces of glass (5.0 cm. Times.2.0 cm rectangle) bonded in an overlapping region (1.5 cm. Times.2.0 cm rectangle) by lap bonding for 10 minutes, and then the lap samples were subjected to shear peeling (peeling rate: 10 mm/min) using a tensile tester with a 50N load cell, and the tissue adhesive strength was as shown in Table 22.

TABLE 22

Adhesive strength

15.8kPa

Example 16

The gelatin/tannic acid/metal ion colloidal gel obtained in example 7 was used to perform a tissue adhesion test. 0.1mL of colloidal gel was injected to the surfaces of two pieces of glass (5.0 cm. Times.2.0 cm rectangles) bonded in an overlapping manner in which the overlapping region was (1.5 cm. Times.2.0 cm rectangles) and left to stand for 10min, and then the overlapped samples were subjected to shear peeling (peeling rate: 10 mm/min) using a tensile tester with a 50N load cell to obtain a peeling process stress-strain curve, and the adhesive strength was defined by the stress maximum point of the curve as shown in Table 23.

TABLE 23

Adhesive strength

7.1kPa

Example 17

The gelatin/tannin colloidal gel obtained in example 1 was used to simulate an in vivo dynamic environment by placing it on a shaker (30 rpm) at an ambient temperature of 37 ℃.1ml of PBS supernatant was taken up at 1d,3d,7d,14d and 21d, respectively, and then an equal amount of fresh PBS solution was added. The content of tannic acid is measured at each time point by using a high performance liquid chromatography kit, and the high performance liquid chromatography detection shows that tannic acid is slowly released in the colloidal gel at a relatively constant concentration, the uniform release can still be detected at the 21 st day, and the release amount is shown in figure 4, which indicates that the composite colloidal gel can continuously and slowly release polyphenol and can be used as a carrier of bioactive substances.

Example 18

Taking mouse primary mesenchymal stem cells as an example, the cell culture medium was changed every two days in proliferation culture (DMEM, containing 10% fetal bovine serum (FBS, gibco)) at 37 ℃,95% relative humidity and 5% carbon dioxide. Before use, cells were dissociated in Phosphate Buffered Saline (PBS) using a trypsin/EDTA solution (0.25% trypsin/0.02% EDTA) for 5 minutes and suspended in the medium for use. The cell suspension was directly dropped onto the surface of the colloidal gel exemplified in example 1 (pH 5) at a cell concentration of 5000 cells/cm ² Inoculating, standing for 1hr, and adding culture medium.

The cytotoxicity of the gel material was examined by using Live/Dead fluorescent staining (Live/Dead assay). 2mM calcein (green labeled live cells) and 4mM ethidium homodimer (red fluorescence labeled dead cells) were added at room temperature and confocal laser scanning microscopy was used. The results are shown in FIG. 5, where green fluorescence represents live cells, red fluorescence represents dead cells, and 3T3 cells cultured on the hydrogel particles for a long time are all live cells, indicating excellent biocompatibility.

Example 19

Using the gelatin/tannic acid colloidal gel of example 1 (pH 5), the colloidal gel was prepared into a cylinder (diameter 8mm, height 2 mm). Staphylococcus aureus or Escherichia coli was cultured overnight in LB medium and then plated on the surface of an agar plate for 6 hours. The colloidal gel was added to the surface of the agar plate of the inoculated bacteria, and the mixture was placed in an incubator at 37 ℃ and incubated at relative humidity for 12 hours. The agar plate was photographed and the growth of bacteria around the cylindrical colloidal gel was observed. FIG. 6 shows no bacterial growth around the colloidal gel, indicating that the gel of the present invention is bacteriostatic.

Example 20

Taking the particle gel prepared in example 1 (pH 5) as an example, the particle gel is respectively blended with a natural active factor VEGF, an anticancer drug adriamycin, a protein drug immunoglobulin and a nucleic acid drug mRNA and placed on a shaker (30 r/min) at the ambient temperature of 37 ℃ so as to simulate the in vivo dynamic environment. 1ml of PBS supernatant was taken up at 1d,3d,7d,14d and 21d, respectively, and then an equal amount of fresh PBS solution was added. The amount of drug released was measured at each time point using ELISA kit, high performance liquid chromatography with three samples per group. Through detection, fig. 7 shows that each type of drug molecules in the colloidal gel are slowly released at a relatively constant concentration, and the uniform release can still be detected at 21 days, which indicates that the covalently crosslinkable colloidal gel has drug slow release performance and can be used as a carrier of bioactive substances.

Example 21

Treating the cells with an ROS inducer to obtain cells containing high ROS as a positive control; normal cells as negative control; the Ce ion-containing composite particles of example 6 were used for co-culture with cells with high ROS content. By using a probe for active oxygen DCFH-DA and Ru (dpp) ₃ Cl ₂ The above different groups were stained, and the content of active oxygen in the cells was observed. Stained cells were observed using the EVOS system and readings were taken using a multimode plate reader (PerkinElmer, USA) at 485nm excitation wavelength and 525nm emission wavelength. Wherein the green and red fluorescence signals are DCFH-DA and Ru (dpp) ₃ Cl ₂ And all the probes can represent ROS content. The fluorescence image of FIG. 8 shows that the red and green fluorescence intensities in normal cells are low, indicating that the ROS content is low (negative control group in the image). The green and red fluorescence signals were evident in cells treated with the ROS inducer (positive control in the figure), indicating that the cells contain high levels of ROS. After incubation treatment of the Ce ion composite particles and cells with high ROS content, the ROS content in the cells is remarkably reduced, and the surface Ce ion composite particles have anti-oxidation property (Ce ion composite particle groups in the figure).

Example 22

Mesenchymal stem cells were cultured in contact with 10mg/mL of the gelatin/tannin colloid gel of example 1 (pH 5), the Zn ion complex colloid gel of example 6 for 7 days, and then their osteogenic differentiation behavior was evaluated by alkaline phosphatase staining (ALP). Fig. 9 shows that the cell group treated with the composite protein particles and the Zn ion composite particles showed a higher degree of osteogenic differentiation characteristics compared to the blank control group. And the Zn ion compounded colloidal gel shows higher osteogenic differentiation performance than the pure colloidal gel.

Example 23

Endothelial cells HUVEC were co-cultured with 10mg/mL of the Cu ion complex colloidal gel of example 6, and proliferation and migration of endothelial cells were assessed by cell staining. FIG. 10 shows that co-culture with colloidal gel promotes endothelial cell proliferation compared to the blank control.

Example 24

Using the colloidal gel prepared in example 1 (pH 5), the non-covalently crosslinked particulate gel was loaded into a syringe and extrusion printed using a 3D bioprinter through a needle having a gauge of G16-23. And printing the material layer by layer according to a route designed by a set program to obtain the 3D biological printing support with the fine structure.

Example 25

Using the colloidal gel described in example 1 (pH 5), a total of 100. Mu.L of gel material was injected subcutaneously on both sides of SD rats: the subcutaneous filling effect is observed at time points of 1, 4, 8 and 12 weeks respectively, and experiments show that the particle gel can be stably filled in rats subcutaneously for 21 weeks, and the physiological conditions of the rats are stable.

It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Claims

1. A hydrogel material assembled by polyphenol and protein composite micro-nano particles is characterized in that:

the hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction between polyphenol/protein composite micro-nano particles; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction between the polyphenol/protein composite micro-nano particles, an oxidant is added to enable covalent crosslinking between polyphenols to realize crosslinking between the polyphenol/protein composite micro-nano particles; the polyphenol/protein composite micro-nano particles are formed by induced assembly through hydrogen bond action, hydrophobic action or electrostatic action between a protein polymer chain and polyphenol; preferably, the polyphenol/protein composite micro-nano particles are formed by induced assembly through hydrogen bond interaction, hydrophobic interaction or electrostatic interaction between a protein polymer chain and polyphenol, and then the protein chain is covalently crosslinked by adding a crosslinking agent to form the stable micro-nano particles.

2. A hydrogel material assembled by micro-nano colloidal particles compounded by polyphenol, protein and metal ions is characterized in that:

the hydrogel material is formed by assembling through hydrophobic interaction, hydrogen bond interaction and metal coordination interaction among polyphenol/protein composite micro-nano particles; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction, hydrogen bond interaction and metal coordination between the polyphenol/protein composite micro-nano particles, an oxidant is added to enable covalent crosslinking between polyphenols to realize crosslinking between the polyphenol/protein composite micro-nano particles; the polyphenol/protein composite micro-nano particles are obtained by adding metal ions into the polyphenol/protein composite micro-nano particles of claim 1 to form coordination with polyphenol in the particles.

3. A hydrogel material assembled by rigid nanoparticle-polyphenol/protein composite core-shell structure micro-nano particles is characterized in that:

the hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction between core-shell structure micro-nano particles; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction and hydrogen bond interaction among the composite micro-nano colloidal particles, the crosslinking among the core-shell structure micro-nano particles is realized through covalent crosslinking among polyphenols by adding an oxidant; the core layer of the core-shell structure micro-nano particles is made of rigid nano particle materials, the shell layer is made of polyphenol/protein composite particles, and the polyphenol/protein composite particles are formed through hydrogen bond action, hydrophobic action and electrostatic action between protein molecules and polyphenol molecules; preferably, after the protein molecules and the polyphenol molecules are formed through hydrogen bond interaction, hydrophobic interaction and electrostatic interaction, a cross-linking agent is added to covalently cross-link protein chains to form the stable micro-nano particles.

4. A core-shell structured colloidal particle material complexed by rigid nanoparticles-polyphenols/proteins/metal ions, characterized in that:

the hydrogel material is formed by assembling hydrophobic interaction, hydrogen bond interaction and metal coordination interaction among the core-shell structure micro-nano particles; preferably, after the hydrogel material is formed by assembling through hydrophobic interaction, hydrogen bond interaction and metal coordination interaction between the core-shell structure micro-nano particles, an oxidant is added to enable covalent crosslinking between polyphenols to realize crosslinking between the core-shell structure micro-nano particles; the core layer of the core-shell structure micro-nano particle is composed of rigid nano particle materials, the shell layer is composed of polyphenol/protein/metal composite particles, and the polyphenol/protein/metal composite particles are obtained by adding metal ions into the polyphenol/protein composite particles of claim 3 to form coordination with polyphenol in the particles.

5. The hydrogel material according to any one of claims 1 to 4, wherein the polyphenol material is selected from one or more of gallic acid, gallic acid esters, epigallocatechin, quercetin, curcumin, tannic acid, catechol, dopamine;

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, polystyrene nanoparticles; when the size of the composite micro-nano particles is 10 nm-5 mu m, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 2-120 v/v%; when the size of the composite micro-nano particles is 5-500 mu m, the volume fraction of the composite micro-nano particles in the total volume of the hydrogel is 50-120 v/v%.

6. The preparation method of the polyphenol and protein assembled micro-nano particle hydrogel material of claim 1, wherein the hydrogel comprises the following preparation steps:

7. The method for preparing a polyphenol, protein and metal ion assembled colloidal particle hydrogel material as set forth in claim 2, characterized in that:

(1) Dissolving protein in aqueous solution at 10-80 ℃ to obtain protein aqueous solution with the concentration of 0.1-10 w/v%; dissolving polyphenol in water solution at 10-90 deg.c to obtain polyphenol water solution in the concentration of 0.1-10 w/v%;

8. The method for preparing the colloidal particle material with the core-shell structure of the nanoparticle-polyphenol compound protein molecule as claimed in claim 3, wherein the compound particle comprises the following preparation steps:

(2) Adding rigid nano particles into the protein aqueous solution, adjusting the pH value of the solution to 3-7, and adding a polyphenol solution to obtain a core-shell structure composite particle solution; freeze drying to obtain composite particle powder; wherein the mass ratio of the protein to the polyphenol is 0.1-20, and the mass ratio of the protein to the rigid nano-particles is 0.1-30; preferably, at normal temperature, adding a cross-linking agent into the composite particle solution for further cross-linking and stabilizing the composite particles for reaction for 1-12 h, wherein the mass ratio of the protein to the macromolecular cross-linking agent is 0.1-100; cleaning to obtain a composite particle dispersion liquid of protein/polyphenol/nanoparticles, and freeze-drying the particle dispersion liquid to obtain composite particle powder;

in the step (2), the cross-linking agent is one or more of carbodiimide/N-hydroxysuccinimide, formaldehyde, acetaldehyde, glyceraldehyde, aromatic aldehyde, glutaraldehyde, succinaldehyde, genipin, diglycidyl ether, dialkene oxide, 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, polystyrene nanoparticles;

9. The method for preparing the core-shell structure colloidal particle material composed of nanoparticles, polyphenol, protein and metal molecules as claimed in claim 3, wherein the composite particles comprise the following preparation steps:

(1) Dissolving protein molecules in an aqueous solution at 10-80 ℃ to obtain a protein aqueous solution with the concentration of 0.1-10 w/v%; dissolving polyphenol in water solution at 10-90 deg.c to obtain polyphenol water solution in the concentration of 0.1-10 w/v%;

(2) Adding rigid nano particles into the protein aqueous solution, adjusting the pH value 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 to the polyphenol is 0.1-20, and the mass ratio of the protein to the rigid nano-particles is 0.1-30; adding metal ions into the particle dispersion liquid, stirring, centrifuging, washing and freeze-drying the particle dispersion liquid to obtain composite particle powder, wherein the concentration of the metal ions is 1 mM-10M; preferably, at normal temperature, adding a cross-linking agent into the core-shell structure composite particle solution for further cross-linking and stabilizing the composite particles for reaction for 1-12 h, wherein the mass ratio of the protein to the macromolecular cross-linking agent is 0.1-100; cleaning to obtain a composite particle dispersion liquid of protein/polyphenol/nanoparticles;

10. Use of the hydrogel material according to any one of claims 1 to 4 for the preparation of an anti-inflammatory, antioxidant, antibacterial scaffold for the repair filling of wounds or defects in bone tissue, cartilage tissue, muscle, blood vessels, skin;

or the application in preparing superficial skin and subcutaneous filler; preferably in medical and cosmetic fillings;

or in the preparation of bio-inks, preferably bio-printing inks for printing with living cells; preferably, when the method is applied, the colloidal particles and the aqueous solution are blended to obtain colloidal particle gel, then the colloidal particle gel is mixed with the cell suspension to obtain cell-loaded colloidal gel, namely biological ink, and the ink is extruded or subjected to ink-jet 3D printing to obtain a scaffold with a 3D structure, so as to obtain a cell-loaded printing scaffold;

or in the preparation of tissue adhesive gel material, wherein the composite microsphere particle size is less than 10 μm, and the adhesive strength between the colloidal gel and the tissue is 5-100 kPa; injecting the gel to the tissue injury part in vivo, and waiting for 1-30 min to realize stable adhesion between the colloidal gel and the tissue;

or the application in the hemostatic sealing powder; preferably, when applied to the hemostatic sealing powder, the hemostatic sealing powder comprises the composite micro-nano particle powder prepared according to claims 6 to 9.

Or as a metal ion slow release carrier to realize wound repair.