CN114585396A

CN114585396A - Injectable hydrogel with anti-inflammatory and repair promoting functions, preparation method thereof and application thereof in heart repair

Info

Publication number: CN114585396A
Application number: CN202180004209.3A
Authority: CN
Inventors: 王云兵; 胡成; 杨立; 刘文琦; 邵南
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-05-08
Filing date: 2021-05-08
Publication date: 2022-06-03
Anticipated expiration: 2041-05-08
Also published as: CN114585396B; WO2021223756A1; CN111437438A

Abstract

The application discloses hydrogel with anti-inflammatory and repair promoting functions and a preparation method and application thereof. The preparation method comprises the following steps: preparing a first polymer containing functional groups; mixing one or more of extracellular matrix, hydrophilic drug or hydrophobic drug, first polymer and polymer containing ortho-hydroxyl to prepare the injectable hydrogel.

Description

Injectable hydrogel with anti-inflammatory and repair promoting functions, preparation method thereof and application thereof in heart repair

Technical Field

The application relates to the technical field of biomedical materials, in particular to an injectable hydrogel with anti-inflammatory and repair promoting functions, a preparation method thereof and application thereof in heart repair.

Background

Myocardial Infarction (MI) is an occlusion of a coronary artery, a disruption of blood flow, resulting in local necrosis of a portion of the myocardium due to severe persistent ischemia, while a large area of myocardial infarction will develop Heart Failure (HF) and endanger the life of the patient, which is one of the leading causes of morbidity and mortality worldwide. Heart failure affects approximately 4000 million people worldwide, with an overall prevalence of 1-2% in developed countries, rising to over 10% in people over 65 years of age. Even with the best current treatment, the rate of readmission for heart failure is as high as 24.5%, with about 20% of heart failure patients dying within 1 year of diagnosis and about 50% of heart failure patients dying within 5 years of diagnosis. The investigation result in 2019 shows that the heart failure prevalence rate is 1.3% in residents of China who are more than or equal to 35 years old, namely about 1370 ten thousand of people suffer from heart failure diseases. With age, the prevalence of heart failure will increase year by year, resulting in a continuous rise in the medical burden on society. Until now, the treatment of heart failure alone remains a great challenge that has not been overcome in the cardiovascular field. The problems of high morbidity, high hospitalization rate and re-hospitalization rate, high mortality, poor living quality of patients, heavy economic burden and the like are still serious.

Acute myocardial infarction is one of the common critical conditions in cardiology, and seriously threatens the life of a patient. The main difficulties in the treatment of myocardial infarction are the limited ability of the heart tissue to regenerate itself, i.e. irreversibility of the heart injury and extremely short survival time of the heart tissue after ischemia. Myocardial cell death or myocardial tissue necrosis, which occurs due to decreased oxygen supply to infarcted tissue caused by insufficient blood flow, destroys collagen fibers connecting between myocardial cells, weakens extracellular matrix, and causes thinning and expansion of the ventricular wall. The granulation tissue formed by fibroblasts, endothelial cells and stem/progenitor cells of the infarcted tissue is gradually replaced by extracellular matrix, eventually forming scar tissue. Newly generated scar tissue leads to heart failure due to a lack of contractile properties required for heart pumping. Heart failure may be caused by many factors, but the most common risk factors are hypertension, coronary artery disease (heart artery blockage), diabetes, obesity, smoking, and genetics, among others. Currently, three methods of clinical treatment for heart failure include heart transplantation, interventional treatment of medical devices (including ventricular assist devices), and drug therapy. Heart transplantation remains the only effective treatment for replacing an infarcted heart with a healthy donor heart. However, the number of people who need heart transplantation is large, but the number of available donors is small, and heart transplantation is still an inefficient technique due to problems such as increase in the number of patients, decrease in the number of donors, and immune complications after heart transplantation. Although interventional therapy and drug therapy of medical equipment can improve the heart contractility, relieve the heart load, relieve pain and the like to a certain extent, the problem that the side effect of the drug is aggravated when a patient takes the drug for a long time is solved, and meanwhile, the heart assist device cannot fundamentally cure heart failure and relieve the development of heart failure diseases, so that the limited treatment efficiency is caused. Therefore, new treatments are urgently needed to more effectively address the problem of cardiac injury.

Cardiac repair by tissue engineering and regenerative medicine techniques is a promising treatment option. Among these, injectable hydrogels are of interest because they can be injected near infarcted heart tissue, providing mechanical support to the damaged heart tissue. Meanwhile, the injectable hydrogel can be used as a drug carrier to deliver drugs or active therapeutic substances to the infarcted part in situ, so that effective regeneration and repair of the infarcted part of the heart are realized. Currently, several injectable hydrogels have shown great potential for cardiac tissue repair, e.g., calcium alginate hydrogel has been used for MI treatment by providing mechanical support to the ventricular wall and has achieved some therapeutic effect. Meanwhile, other degradable hydrogels have also been used to load different drugs for MI treatment, such as cell growth factors, proteins, chemical and genetic drugs, etc. However, such drug-or active factor-loaded hydrogels fail to release the desired drug in response to different characteristics and stages of myocardial infarction disease, resulting in passive drug release behavior, and thus have limited therapeutic effects and limit further clinical transformations. In recent years, the intelligent response hydrogel constructed aiming at the microenvironment characteristics of the disease parts has been widely used for treating diseases such as cancer, rheumatoid arthritis, gastrointestinal diseases, chronic wounds and the like, and has achieved excellent treatment effect. Compared with traditional medicine-carrying hydrogel, the intelligent response hydrogel remarkably improves the treatment effect of patients by realizing accurate on-demand response and release of medicines at the disease parts, and can also reduce the administration frequency and reduce the side effect of the medicines. It is anticipated that the clinical transformation of smart responsive hydrogels will be greatly facilitated as precision drug delivery technologies mature.

In recent years, it has been found that the occurrence of post-myocardial failure is related to various factors such as inflammatory factors, signal transduction, genes, and the like. The necrotic myocardial cells after myocardial infarction can stimulate the body to generate local inflammatory reaction, generate a large amount of inflammatory factors, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like, increase the level of the inflammatory factors, continuously damage the heart muscle, and finally cause the apoptosis of the myocardial cells. Numerous experiments have demonstrated that a strong inflammatory response following myocardial infarction is one of the factors responsible for secondary damage to myocardial tissue. In the pathological process of myocardial infarction caused by severe ischemia, especially in the initial inflammation stage of myocardial infarction, oxidative stress and a weakly acidic environment are two main characteristics of the microenvironment for myocardial pathological tissue inflammation. However, currently, there are very few smart responsive hydrogels designed to address the inflammatory microenvironment characteristic of myocardial infarction sites. More importantly, how to inhibit the inflammatory reaction after myocardial infarction, save the myocardial cells damaged in the early stage of myocardial infarction, reduce the apoptosis of the myocardial cells and promote the regeneration of blood vessels in ischemic areas is the key for repairing the myocardium after myocardial infarction. Therefore, the construction of the multifunctional microenvironment response hydrogel has important significance for improving the myocardial infarction treatment, and widens the way for the application of the intelligent response hydrogel in the myocardial infarction treatment field.

Disclosure of Invention

Aiming at the clinical facts that the incidence rate of heart failure is continuously increased, the clinical existing method has poor treatment effect, and urgent needs to be improved, and the problems in the prior art, the application aims to provide a preparation method of an injectable hydrogel with anti-inflammatory and repair promoting functions and application thereof in heart injury repair, and can effectively solve the problems that the existing heart failure treatment method has poor effect and the like, and meanwhile, the hydrogel provided by the application has the advantages of simple preparation method and low cost.

An injectable hydrogel with anti-inflammatory and repair promoting functions, wherein the injectable hydrogel forms a gel through the interaction of functional groups of a polymer and adjacent hydroxyl groups of the polymer, and the hydrogel is disintegrated after the functional groups of the polymer and the adjacent hydroxyl groups of the polymer are released from the interaction in response to acidic conditions and/or active oxygen conditions.

Optionally, the injectable hydrogel is loaded with at least one of a hydrophilic drug, a hydrophobic drug, and an extracellular matrix.

Optionally, the polymer containing functional groups is at least one of sodium alginate containing phenylboronic acid groups, chitosan quaternary ammonium salt, polylysine, polyethyleneimine, gelatin, sodium alginate, hyaluronic acid, heparin, carboxymethyl cellulose, dextran, methyl cellulose, starch and cyclodextrin.

Under the alkalescent condition, the phenylboronic acid group and the adjacent hydroxyl group form gel through the interaction of the boroester bond, and under the acidic condition and/or the active oxygen condition, the boroester bond is broken and the gel is dissociated.

The injectable hydrogel is in a gel state under a weak alkaline condition, and under an acidic condition and/or an active oxygen condition, the gel is dissociated, and hydrophilic drugs, hydrophobic drugs, extracellular matrix and the like in the gel are released.

The phenylboronic acid group and the ortho-hydroxyl group can be present in the same polymer, namely the same polymer simultaneously contains a plurality of phenylboronic acid groups and ortho-hydroxyl groups, and the phenylboronic acid group and the ortho-hydroxyl group can also be respectively present in different polymers, namely one polymer contains a plurality of phenylboronic acid groups, and the other polymer contains a plurality of ortho-hydroxyl groups.

The acid condition and the alkalescent condition are relative concepts, when the number of the phenylboronic acid group and the ortho-hydroxyl is more, gel can be formed under the neutral condition, and when the number of the phenylboronic acid group and the ortho-hydroxyl is less, the gel needs to be formed under the alkalescent condition.

In the present application, the natural polymer is understood to include its modified product, i.e. chitosan includes unmodified chitosan and also includes the modified product of chitosan, and similarly, the quaternary ammonium salt of chitosan, polylysine, polyethyleneimine, gelatin, sodium alginate, hyaluronic acid, heparin, carboxymethyl cellulose, dextran, methyl cellulose, starch and cyclodextrin also include their corresponding modified products, and the modified products do not have adverse effect on the formation and dissociation of gel.

Optionally, the polymer containing ortho-hydroxyl is at least one of sodium alginate, polyvinyl alcohol, sodium alginate, hyaluronic acid, dextran and starch.

Optionally, the injectable hydrogel is loaded with extracellular matrix;

and hydrophilic drugs and/or hydrophobic drugs.

The injectable hydrogel is loaded with at least extracellular matrix, and at least one of hydrophilic drugs and hydrophobic drugs.

The substance loaded in the injectable hydrogel is selectively loaded in the hydrogel according to the requirements of the actual treatment.

Optionally, the extracellular matrix is at least one of collagen, non-collagen, elastin, proteoglycan, and aminoglycan.

Optionally, the extracellular matrix is a recombinant humanized collagen.

Optionally, the extracellular matrix is at least one of recombinant type I humanized collagen and recombinant type III humanized collagen.

Optionally, the recombinant type I humanized collagen and the recombinant type III humanized collagen comprise an amino acid sequence fragment capable of binding to a cell integrin.

The recombinant I type humanized collagen and the recombinant III type humanized collagen are amino acid sequence fragments which are coded by specific type genes of the human collagen prepared by a DNA recombination technology and can be combined with cell integrins.

Optionally, the extracellular matrix is a recombinant type III humanized collagen.

Optionally, at least one of the hydrophobic drugs naproxen, sulvastatin, curcumin and aspirin.

Optionally, the hydrophobic drug is loaded in the injectable hydrogel by taking an amphiphilic polymer as a carrier.

Optionally, the injectable hydrogel is loaded with: curcumin and recombinant humanized collagen.

Optionally, the injectable hydrogel is loaded with: curcumin and recombinant type III humanized collagen.

The application also provides a hydrogel for repairing cardiac injury, which is the injectable hydrogel acting on the cardiac injury part.

The curcumin is used for diminishing inflammation of damaged parts, the recombinant human type III collagen promotes the regeneration of myocardial cells, and the recombinant human type III collagen interact with each other to effectively promote the proliferation and the growth of the myocardial cells and repair heart damage.

The application also provides a hydrogel for treating heart failure, which is the injectable hydrogel acting on the pathological change part of the heart.

The application also provides a hydrogel for treating myocardial infarction, which acts on the myocardial infarction part and is the injectable hydrogel.

The application also provides application of the injectable hydrogel with the anti-inflammatory and repair promoting functions in repairing cardiac injuries.

The application also provides application of the injectable hydrogel with the anti-inflammatory and repair promoting functions in treating heart failure.

The application also provides application of the injectable hydrogel with the anti-inflammatory and repair promoting functions in treatment of myocardial infarction.

The application also provides a heart injury repair method, and the injectable hydrogel is acted on the heart injury part.

The application also provides a method for treating heart failure, and the injectable hydrogel is applied to a heart pathological change part.

The application also provides a method for repairing the heart injury, and the injectable hydrogel is injected at the heart lesion site.

The present application also provides a method of treating heart failure by injecting the injectable hydrogel at the site of myocardial infarction.

The present application also provides a method for treating myocardial infarction by injecting the injectable hydrogel at the site of myocardial infarction.

The application also provides a preparation method of the injectable hydrogel, which comprises the following steps:

preparing a first polymer containing phenylboronic acid groups;

mixing at least one of hydrophilic drugs, extracellular matrix and hydrophobic drugs, a first polymer and an ortho-hydroxyl-containing polymer to prepare the injectable hydrogel.

The polymer containing the ortho-hydroxyl contains a plurality of ortho-hydroxyl groups, and after the first polymer and the polymer containing the ortho-hydroxyl are mixed, gel can be formed without additionally adjusting the pH value.

Optionally, the first polymer is prepared by any one of the following raw material combinations in the presence of a condensing agent and a catalyst:

a) an amino-or hydroxyl-containing polymer and a carboxyl-containing phenylboronic acid;

b) carboxyl-containing polymers and amino-or hydroxyl-containing phenylboronic acids.

The introduction of a phenylboronic acid group into a polymer may be carried out by amidation of an amino group and a carboxyl group, or esterification of a hydroxyl group and a carboxyl group, and therefore, both the polymer and the phenylboronic acid, one of which contains an amino group or a hydroxyl group and the other of which contains a carboxyl group, may be introduced into the polymer.

The condensing agent is at least one of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, O-benzotriazole-tetramethylurea hexafluorophosphate, benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate and dicyclohexylcarbodiimide, and the catalyst is at least one of 4-dimethylaminopyridine, N-hydroxysuccinimide and 1-hydroxybenzotriazole.

Optionally, the preparation method of the first polymer is as follows:

after the raw materials are dissolved, reacting for 30-60h at 30-40 ℃ to prepare the first polymer containing the phenylboronic acid group.

After the raw materials are reacted, appropriate post-treatment is required, including dialysis in deionized water, freeze drying and the like, and the side chain of the first polymer contains phenylboronic acid groups.

The amount of phenylboronic acid groups in the polymer will affect gel formation and dissociation, and the amount of phenylboronic acid groups introduced will be appropriate.

Optionally, the mass ratio of the polymer containing amino or hydroxyl, the phenylboronic acid containing carboxyl, the condensing agent and the catalyst is 7 (4-5) to (2-3) to 1; or the mass ratio of the carboxyl-containing polymer, the phenyl boric acid containing amino or hydroxyl, the condensing agent and the catalyst is 7 (4-5) to (2-3) to 1.

Optionally, the preparation method of the first polymer is as follows:

dissolving the raw materials, reacting for 48 hours at 37 ℃, dialyzing in deionized water for 3 days, and then performing freeze drying to prepare a polymer with a side chain grafted phenylboronic acid group; wherein the mass ratio of the polymer containing amino or hydroxyl, the phenylboronic acid containing carboxyl, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-N-hydroxysuccinimide is 7:4.5:2.5: 1; or the mass ratio of the carboxyl-containing polymer to the phenyl boric acid containing amino or hydroxyl, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to N-N-hydroxysuccinimide is 7:4.5:2.5: 1.

The hydrophilic drug, the extracellular matrix and other substances with better water solubility can be directly mixed with the first polymer and the polymer containing the ortho-hydroxyl to obtain the injectable hydrogel, and after the hydrophobic drug needs to be specially treated, the injectable hydrogel is mixed with the first polymer and the polymer containing the ortho-hydroxyl.

Optionally, the hydrophobic drug is prepared into a drug-loaded nano-micelle by taking an amphiphilic polymer as a carrier, and the drug-loaded nano-micelle is mixed with the first polymer and the polymer containing the ortho-hydroxyl to prepare the injectable hydrogel containing the hydrophobic drug.

The drug-loaded nano-micelle is prepared between the amphiphilic polymer and the hydrophobic drug in a self-assembly mode, the amphiphilic polymer is a drug carrier, and the hydrophobic drug is the wrapped drug.

The amphiphilic polymer is composed of a hydrophilic chain segment and a hydrophobic chain segment, the hydrophilic chain segment is at least one of polyethylene glycol, polyvinyl ether, polyvinyl alcohol, polyethyleneimine, polyvinylpyrrolidone and polyacrylamide, and the hydrophobic chain segment is at least one of polypropylene oxide, polystyrene, polysiloxane, polybutadiene, polymethyl methacrylate, polymethyl acrylate and polybutyl acrylate.

Optionally, the preparation of the drug-loaded nano-micelle comprises:

dissolving amphiphilic polymer and hydrophobic drug in benign solvent, slowly dripping into water under the condition of continuous stirring, and dialyzing to obtain drug-loaded nano micelle solution with the concentration of 1-2 mg/mL.

The benign solvent is at least one of DMSO, DMF, methanol and acetone. Further preferably, the benign solvent is DMSO and/or acetone.

Specifically, the amphiphilic polymer and the hydrophobic drug are dissolved in a benign solvent, slowly dropped into deionized water under the condition of continuous stirring, stirred for 3-6 hours, and dialyzed in the deionized water to prepare the drug-loaded nano micelle.

Optionally, the mass ratio of the amphiphilic polymer to the hydrophobic drug is 4-8: 1.

Specifically, the amphiphilic polymer and the hydrophobic drug are dissolved in a benign solvent, slowly added into deionized water in a dropwise manner under the condition of continuous stirring, stirred for 4 hours and dialyzed in the deionized water to prepare the drug-loaded nano micelle, wherein the mass ratio of the amphiphilic polymer to the hydrophobic drug is 5: 1.

Optionally, an aqueous solution of a first polymer is mixed with an aqueous solution of a polymer containing an ortho-hydroxyl group to obtain the injectable hydrogel, the aqueous solution of the first polymer contains at least one of a hydrophilic drug, an extracellular matrix and a drug-loaded nano micelle, and the mass concentration of the first polymer in the aqueous solution of the first polymer is 0.5-10% w/v.

Optionally, an aqueous solution of a first polymer is mixed with an aqueous solution of a polymer containing an ortho-hydroxyl group to obtain the injectable hydrogel, the aqueous solution of the first polymer contains at least one of a hydrophilic drug, an extracellular matrix and a drug-loaded nano micelle, and the mass concentration of the first polymer in the aqueous solution of the first polymer is 0.5-5% w/v.

Optionally, an aqueous solution of a first polymer is mixed with an aqueous solution of a polymer containing an ortho-hydroxyl group to obtain the injectable hydrogel, the aqueous solution of the first polymer contains at least one of a hydrophilic drug, an extracellular matrix and a drug-loaded nano micelle, and the mass concentration of the first polymer in the aqueous solution of the first polymer is 0.5-3% w/v.

Optionally, the mass concentration of the hydrophilic drug in the first polymer aqueous solution is 1-1000 μ g mg/mL.

Optionally, the mass concentration of the hydrophilic drug in the first polymer aqueous solution is 1-500 μ g mg/mL.

Optionally, in the first polymer aqueous solution, the mass concentration of the extracellular matrix is 1-6 mg/mL.

Optionally, in the first polymer aqueous solution, the mass concentration of the extracellular matrix is 1-3 mg/mL.

Optionally, in the first polymer aqueous solution, the mass concentration of the drug-loaded nano-micelle is 30-200 μ g/mL.

Optionally, in the first polymer aqueous solution, the mass concentration of the drug-loaded nano-micelle is 50-150 μ g/mL.

Optionally, in the aqueous solution of the polymer containing the ortho-hydroxyl, the mass concentration of the polymer containing the ortho-hydroxyl is 0.5-10% w/v.

Optionally, in the aqueous solution of the polymer containing the ortho-hydroxyl, the mass concentration of the polymer containing the ortho-hydroxyl is 0.5-5% w/v.

Optionally, in the aqueous solution of the polymer containing the ortho-hydroxyl, the mass concentration of the polymer containing the ortho-hydroxyl is 0.5-3% w/v.

Optionally, the volume ratio of the first aqueous polymer solution to the aqueous solution of the polymer containing the ortho-hydroxyl is 1: 0.5-1.5.

Optionally, the volume ratio of the first aqueous polymer solution to the aqueous solution of the polymer containing the ortho-hydroxyl is 1: 0.8-1.2.

Optionally, the volume ratio of the aqueous solution of the first polymer to the aqueous solution of the polymer containing an ortho-hydroxyl group is 1: 1.

Optionally, an aqueous solution of a first polymer is mixed with an aqueous solution of a polymer containing an ortho-hydroxyl group to obtain the injectable hydrogel, the aqueous solution of the first polymer contains at least one of a hydrophilic drug, an extracellular matrix and a drug-loaded nano micelle, the mass concentration of the first polymer in the aqueous solution of the first polymer is 1% w/v, the mass concentration of polyvinyl alcohol in the aqueous solution of polyvinyl alcohol is 1% w/v, and the aqueous solution of the first polymer and the aqueous solution of polyvinyl alcohol are mixed in equal volume to obtain the hydrogel.

Optionally, the polymer containing amino groups is at least one of chitosan, chitosan quaternary ammonium salt, polylysine, polyethyleneimine and gelatin.

Optionally, the polymer containing carboxyl is at least one of sodium alginate, hyaluronic acid, heparin and carboxymethyl cellulose.

Optionally, the hydroxyl-containing polymer is at least one of starch, cellulose (methyl cellulose), gellan gum, konjac gum, gum arabic, lignin, dextran, and cyclodextrin.

Optionally, the polymer containing ortho-hydroxyl is at least one of polyvinyl alcohol, sodium alginate, hyaluronic acid, dextran, and starch.

Optionally, the polymer containing ortho-hydroxyl groups is polyvinyl alcohol.

Optionally, the phenylboronic acid containing carboxyl is at least one of 4-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyl-3-fluorophenylboronic acid, 3-carboxyl-4-fluorophenylboronic acid, 5-carboxyl-2-chlorophenylboronic acid and 4-carboxyl-2-chlorophenylboronic acid;

the amino-containing phenylboronic acid is at least one of 4-aminophenylboronic acid, 2-aminophenylboronic acid, 3-carbamoylphenylboronic acid, 3-amino-4-fluorobenzeneboronic acid and 3-amino-4-methylbenzeneboronic acid;

the hydroxyl-containing phenylboronic acid is at least one of 4-hydroxyphenylboronic acid, 3-fluoro-4-hydroxyphenylboronic acid, 2-fluoro-3-hydroxyphenylboronic acid, 2-fluoro-5-hydroxyphenylboronic acid, 3-hydroxy-4-chlorophenylboronic acid and 3-fluoro-4-hydroxyphenylboronic acid.

The extracellular matrix is different from the medicine, and has the effects of promoting cell proliferation and growth and accelerating the repair process.

Optionally, the extracellular matrix is at least one of basic fibroblast growth factor (bFGF), Vascular Endothelial Growth Factor (VEGF), recombinant humanized collagen, and Desferoxamine (DFO).

Optionally, the extracellular matrix is a recombinant humanized collagen.

Optionally, the hydrophobic drug is at least one of an anti-inflammatory drug, an analgesic, a pro-angiogenic drug, a diuretic, an angiotensin converting enzyme inhibitor, a beta blocker, a digitalis drug, an aldosterone antagonist, an angiotensin-two-receptor antagonist, an anticoagulant, and an antiplatelet drug.

Optionally, the hydrophobic drug is at least one of naproxen, sulvastatin, curcumin and aspirin.

The application also provides the injectable hydrogel with anti-inflammatory and repair promoting functions prepared by the preparation method.

The preparation method and the prepared injectable hydrogel have at least one of the following beneficial effects:

1. in terms of materials, the material for preparing the hydrogel carrier with the anti-inflammatory and repair promoting functions is a natural polymer material, has wide sources and low cost, and simultaneously has good biocompatibility;

2. the used hydrophilic extracellular matrix has small immune and rejection reactions and good solubility, and is a very safe biological extracellular matrix;

3. the dual-response hydrogel is simple in preparation process and excellent in physicochemical property, such as short gelling time, good injectability and the like;

4. the dual-response hydrogel has pH and ROS response performances at the same time, and can realize the response release of the hydrophobic drug and the hydrophilic extracellular matrix as required;

5. the dual-response hydrogel promotes rapid repair of the damaged heart through antioxidant, anti-inflammatory and pro-angiogenic mechanisms.

In conclusion, the strategy of stimulating drug release on demand by the micro-environmental response of the dual-response hydrogel and the good biocompatibility of the dual-response hydrogel can realize the rapid repair of the myocardial infarction part.

The application also provides an injectable hydrogel with anti-inflammatory and repair promoting functions, which comprises the following raw material components: biomacromolecules containing amino and ortho-hydroxyl groups, biomacromolecules containing carboxyl and ortho-hydroxyl groups and phenylboronic acid containing amino, hydroxyl or carboxyl groups.

Optionally, the following components are also included: hydrophilic drugs and/or hydrophobic drugs.

The application also provides an injectable hydrogel with anti-inflammatory and repair promoting functions, which comprises the following raw material components: hydrophilic drugs, hydrophobic drugs, amino-and ortho-hydroxyl-containing biomacromolecules, carboxyl-and ortho-hydroxyl-containing biomacromolecules, and amino-, hydroxyl-or carboxyl-containing phenylboronic acids.

Further, hydrophilic drugs include growth factors, polypeptide drugs, gene drugs, and water-soluble protein drugs.

Further, hydrophobic drugs include anti-inflammatory drugs, pro-angiogenic drugs, pro-cell proliferative drugs, and pro-cell migratory drugs.

Further, the anti-inflammatory agent includes aspirin, paracetamol, amoxicillin, phenylbutazone and other lipid soluble drugs.

Further, the biomacromolecule containing amino and ortho-hydroxyl comprises at least one of chitosan, sodium alginate modified product and hyaluronic acid modified product.

The sodium alginate modified product and the hyaluronic acid modified product are obtained by introducing amino groups into sodium alginate and hyaluronic acid, and the natural polymer can be theoretically used as a raw material of injectable hydrogel if the amino groups, carboxyl groups or ortho-hydroxyl groups for reaction can be introduced into the sodium alginate and the hyaluronic acid through chemical reaction.

Further, the biomacromolecule containing carboxyl and o-hydroxyl includes sodium alginate, hyaluronic acid and modified products thereof, carboxymethyl cellulose, or carboxymethyl cellulose modified products.

Further, the phenylboronic acid containing an amino group, a hydroxyl group or a carboxyl group is o-aminophenylboronic acid, m-aminophenylboronic acid, p-aminophenylboronic acid, o-hydroxyphenylboronic acid, m-hydroxyphenylboronic acid, p-hydroxyphenylboronic acid, o-carboxyphenylboronic acid, m-carboxyphenylboronic acid or p-carboxyphenylboronic acid.

The application also provides application of the injectable hydrogel with anti-inflammatory and repair promoting functions in cardiac injury repair.

The preparation method of the injectable hydrogel with the anti-inflammatory and repair promoting functions comprises the following steps:

(1) synthesizing an amphiphilic polymer as a carrier for entrapping the hydrophobic drug;

(2) the drug-loaded nano-micelle is prepared by dissolving amphiphilic polymer and hydrophobic drug in benign solvent together and then slowly dropping the solution into poor solvent (such as deionized water) in the process of continuously stirring;

(3) preparing a polymer grafted with a phenylboronic acid group through amidation reaction of carboxyl and amino;

(4) dissolving the polymer grafted with the phenylboronic acid group and the hydrophilic drug in a drug-loaded micelle solution, and adding a proper amount of alkaline solution to adjust the pH value of the mixed solution to 8.5 to obtain the modified drug-loaded micelle.

Furthermore, the drug-loaded nano-micelle in the step (2) is spherical and uniform in particle size, and has high drug loading rate and encapsulation rate.

Further, the carrier of the drug-loaded nano-micelle in the step (2) is an amphiphilic polymer.

Further, the drug-loaded nano-micelle in the step (2) is used for encapsulating the hydrophobic drug.

Further, the drug-loaded nano-micelle in the step (2) can be disintegrated at the inflammation part structure, and the release of the drug is accelerated.

Further, the injectable hydrogel having anti-inflammatory and repair promoting functions as described above can be prepared only by changing the pH of the solution.

Furthermore, the injectable hydrogel with anti-inflammatory and repair promoting functions has multiple dynamic functions of self-healing, remodeling, injectability and the like.

Further, the injectable hydrogel having anti-inflammatory and repair promoting functions as described above can work by a controlled drug release system mediated by pH and Reactive Oxygen Species (ROS) response.

Furthermore, the injectable hydrogel with anti-inflammatory and repair promoting functions has good cell compatibility and no adverse reaction in vivo.

The injectable hydrogel with anti-inflammatory and repair promoting functions is applied to preparation or application of the hydrogel in heart repair.

The hydrogel has at least one of the following beneficial effects:

1. the multi-responsive hydrogel is prepared from a single and defined polymer component.

2. The multi-responsive hydrogel is successfully prepared by a minimum of synthetic steps and procedures;

3. injectable hydrogel formulations still have good rheological properties and retained structure after loading with two different drugs

Integrity;

4. the injectable hydrogel formula has multiple dynamic functions of self-healing, remodeling, injection and the like;

5. the injectable hydrogel formulation has super-strong responsive drug release characteristics in inflammatory areas;

7. the injectable hydrogel formulation has good cell compatibility and no adverse reaction in vivo.

9. The injectable hydrogel with the anti-inflammatory and repair promoting functions, which is prepared by the method, can increase the drug loading amount, the drug loading types and the like in the hydrogel dressing. Not only can different polymers be replaced to form various hydrogels, but also different medicaments, including hydrophilic medicaments or hydrophobic medicaments, can be replaced to endow the hydrogels with different active functions.

10. The hydrogel provided by the application has good injectability, is injected to a damaged part of a heart, has small immune and rejection reactions, and promotes the quick repair of the damaged heart through various actions of oxidation resistance, anti-inflammation, angiogenesis promotion and the like of a hydrophilic drug and/or a hydrophobic drug loaded on the hydrogel at the damaged part of the heart.

The application also provides a preparation method of the injectable hydrogel with anti-inflammatory and repair promoting functions, which comprises the following steps:

step 1, reacting a functional polymer containing ortho hydroxyl with phenylboronic acid containing amino, hydroxyl or carboxyl to prepare a polymer of side chain grafted phenylboronic acid; the functional polymer containing the ortho-hydroxyl is a biological macromolecule containing amino and ortho-hydroxyl or a biological macromolecule containing carboxyl and ortho-hydroxyl;

and 2, dissolving the phenylboronic acid polymer in water, and adjusting the pH value of the mixed solution to 8-9 to obtain the hydrogel.

Optionally, the hydrophilic drug and/or the micelle coated with the hydrophobic drug is mixed with the phenylboronic acid polymer aqueous solution, and the pH value of the mixed solution is adjusted to 8-9, so that the compound is prepared.

Optionally, a benign solvent is used for dissolving the amphiphilic drug carrier and the hydrophobic drug, water is slowly added into the benign solvent in a stirring state to prepare a drug-loaded micelle solution, the hydrophilic drug and/or the drug-loaded micelle solution is mixed with the phenylboronic acid polymer aqueous solution, and the pH value of the mixed solution is adjusted to 8-9. The application also provides a preparation method of the injectable hydrogel with anti-inflammatory and repair promoting functions, which comprises the following steps:

(1) reacting functional polymer containing ortho hydroxyl with phenylboronic acid containing amino, hydroxyl or carboxyl to prepare phenylboronic acid polymer;

(2) preparing an amphiphilic drug carrier;

(3) dissolving the amphiphilic drug carrier and the hydrophobic drug prepared in the step (2) by using a benign solvent, and then adding water into the mixture under a stirring state to prepare a drug-loaded micelle solution;

(4) and (2) dissolving the hydrophilic drug and the phenylboronic acid polymer prepared in the step (1) into the drug-loaded micelle solution prepared in the step (3), and then adjusting the pH value of the mixed solution to 8-9 to obtain the drug-loaded micelle solution.

The phenylboronic acid polymer contains both phenylboronic acid groups and o-hydroxyl groups, and the pH value needs to be adjusted to form gel due to the number of the o-hydroxyl groups and the phenylboronic acid groups and the steric hindrance.

Further, the specific reaction process in the step (1) is as follows: dissolving a functional polymer containing ortho hydroxyl to obtain a functional polymer solution containing ortho hydroxyl, adding a condensing agent and phenylboronic acid containing amino, hydroxyl or carboxyl into the solution, stirring the solution for 20 to 30 hours at the temperature of between 30 and 40 ℃, dialyzing the solution in deionized water, and then carrying out freeze drying to obtain the purified phenylboronic acid polymer.

Furthermore, the mass ratio of the functional polymer with ortho-position hydroxyl, the condensing agent and the phenylboronic acid with amino, hydroxyl or carboxyl is 4-6:4-6: 1-2.

Further, the specific reaction process in the step (1) is as follows: dissolving a functional polymer containing ortho hydroxyl to obtain a functional polymer solution containing the ortho hydroxyl, adding a condensing agent and phenylboronic acid containing amino, hydroxyl or carboxyl into the solution, stirring the solution at 37 ℃ for 24 hours, dialyzing the solution in deionized water for 3 days, and then carrying out freeze drying to obtain a purified phenylboronic acid polymer, wherein the mass ratio of the functional polymer containing the ortho hydroxyl, the condensing agent and the phenylboronic acid containing the amino, the hydroxyl or the carboxyl is 5:4.8: 1.95.

Further, the condensing agent includes 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.

Further, the specific reaction process in the step (2) is as follows: completely dissolving a hydrophilic high molecular polymer and a hydrophobic molecule in dimethyl sulfoxide at 80 ℃, adding N, N '-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, continuously stirring for 48 hours at 80 ℃, dialyzing a reactant in water for 2 days, removing unreacted hydrophobic molecule, and finally freeze-drying for later use, wherein the mass ratio of the hydrophilic high molecular polymer to the hydrophobic molecule to the N, N' -dicyclohexylcarbodiimide to the 4-dimethylaminopyridine is 2:1.5:1: 0.5.

Further, the hydrophilic high molecular polymer includes hyaluronic acid, starch, cellulose, polyacrylic acid, polyacrylamide, polyvinyl alcohol, glycolic acid, and polylysine.

Further, hydrophobic molecules include cholesterol, polyolefins, polycarbonates, polyamides, polyacrylonitriles, polyesters, polylactic acids, and acrylates.

Further, the specific reaction process in the step (3) is as follows: dissolving the amphiphilic drug carrier and the hydrophobic drug prepared in the step (2) in a benign solvent, heating to 70-90 ℃, dropwise adding water into the solvent under the stirring condition, and dialyzing to obtain the drug-loaded micelle solution with the concentration of 0.5-1.5 mg/mL.

Further, the specific reaction process in the step (3) is as follows: and (3) dissolving 0.04 part of the amphiphilic drug carrier prepared in the step (2) and 0.008 part of hydrophobic drug in a benign solvent, heating to 80 ℃, dropwise adding water into the mixture under the stirring condition, and dialyzing to prepare a drug-loaded micelle solution with the concentration of 1 mg/mL.

Further, the benign solvent in step (3) includes DMSO, DMF, methanol, and acetone.

Further, the specific reaction process in the step (4) is as follows: and (2) dissolving the hydrophilic drug and the phenylboronic acid polymer prepared in the step (1) into the drug-loaded micelle solution prepared in the step (3) to ensure that the concentration of the phenylboronic acid polymer in the drug-loaded micelle solution is 7-11% w/v.

Further, adding an alkaline solution into a phenylboronic acid polymer aqueous solution containing the drug-loaded micelle and/or the hydrophilic drug, and adjusting the pH value to 8.5 to prepare the injectable hydrogel.

Further, the concentration of the phenylboronic acid polymer in the micelle loading solution in step (4) is 9% w/v.

Further, the functional polymer containing ortho hydroxyl is a biological macromolecule containing amino and ortho hydroxyl or a biological macromolecule containing carboxyl and ortho hydroxyl.

Further, the biomacromolecule containing carboxyl and ortho-hydroxyl is sodium alginate, hyaluronic acid or a modified product thereof, and phenylboronic acid containing amino and hydroxyl groups is grafted to a biomacromolecule side chain containing carboxyl groups and ortho-hydroxyl groups on the side chain through amidation reaction or esterification reaction; the biomacromolecule containing amino and ortho-hydroxyl is chitosan or a modified product thereof, and phenylboronic acid with carboxyl groups is grafted to a biomacromolecule side chain containing amino groups and ortho-hydroxyl on the side chain through amidation reaction.

Further, the hydrophilic drug includes one of a growth factor, a gene and a water-soluble protein drug.

Further, the hydrophobic drug includes one of an anti-inflammatory drug, an angiogenesis promoting drug, a cell proliferation promoting drug and a cell migration promoting drug.

Further, the anti-inflammatory agent comprises one of aspirin, paracetamol, amoxicillin and phenylbutazone.

The hydrogel provided by the application can be used for injecting into the heart, and the selection of hydrophilic drugs, hydrophobic drugs and extracellular matrix is not completely the same based on different use scenes, and the selection is appropriately carried out according to actual needs.

The hydrogel prepared by the method has at least one of the following beneficial effects:

1. the hydrogel is prepared from a single and definite polymer component, has simple synthesis steps, convenient operation and mild reaction conditions, still has good rheological property and complete structure after loading two different drugs, has multiple dynamic functions of self-healing, remodeling, injection and the like, has good cell compatibility and has no adverse reaction in vivo.

3. The phenylboronic acid ester bond in the injectable hydrogel is rapidly broken under the conditions of high active oxygen and low pH value, the hydrogel structure is destroyed, and therefore the hydrophilic drug and the hydrophobic drug are released in a rapid response mode.

4. The injectable hydrogel is loaded with extracellular matrix, hydrophilic drugs and/or hydrophobic drugs at the same time, generally, molecules of the hydrophilic drugs and the hydrophobic drugs are smaller, molecules of the extracellular matrix are larger, and due to different steric hindrance sizes, the hydrophilic drugs and the hydrophobic drugs with the smaller molecules are firstly released to diminish inflammation of injured tissues, and then the extracellular matrix is released to promote cell regeneration, so that tissue repair is facilitated.

Since the hydrophobic drug takes the amphiphilic polymer as a carrier, the size of the whole micelle of the hydrophobic drug loaded on the amphiphilic polymer can be adjusted by adjusting parameters such as the structure, the dosage and the like of the amphiphilic polymer, and the order of the hydrophobic drug released from the hydrogel relative to other substances can be adjusted.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of CMC and CMC-BA in example 1 of the present application;

FIG. 2 is a graph of the particle size of the drug-loaded nanoparticles of example 1 of the present application;

FIG. 3 is a gelation diagram of a hydrogel in example 1 of the present application;

FIG. 4 is a graph of injectability results for the hydrogels of example 1 of the present application;

FIG. 5 is a graph showing the results of cell viability of endothelial cells at 24, 48 and 72 hours after treatment with different hydrogels according to example 1 of the present application;

FIG. 6 is a graph showing the results of cell viability at 24, 48 and 72 hours for cardiomyocytes treated with different hydrogels according to example 1 of the present application;

FIG. 7 is a graph showing the results of IL-6 and TNF- α expression in macrophages after different hydrogel treatments in example 1 of the present application at 24 and 48 hours;

FIG. 8 is a graph of H & E staining and Masson staining of rat hearts in example 1 of the present application;

FIG. 9 is a nuclear magnetic hydrogen spectrum of ALG, BA and ALG-BA described in example 6 of the present application;

FIG. 10 is a nuclear magnetic hydrogen spectrum of CHOL, HA and HA-CHOL described in example 6 of the present application;

FIG. 11 is a schematic diagram of the reaction process of phenylboronic acid grafting carboxymethyl cellulose functional polymer.

Detailed Description

The following detailed description of embodiments of the present application refers to the accompanying drawings.

In the following examples, the chemical agents other than the matrix are chemically pure unless otherwise stated.

Example 1

The reaction equation and the preparation steps of the preparation method of the injectable hydrogel with anti-inflammatory and repair promoting functions are shown in fig. 11.

(1) Preparation of phenylboronic acid grafted carboxymethyl cellulose functional polymer (CMC-BA)

Carboxymethyl cellulose (CMC, 10.00g) and 3-aminophenylboronic acid (BA, 6.50g) were precisely weighed and dissolved in 500mL of MES buffer (0.1mol, pH5.0), to which was added 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl, 4.00g) and N-hydroxysuccinimide (NHS, 1.5 g). Then stirring for 48h at 37 ℃, finally dialyzing in deionized water (pH7.4) for 3 days, and freeze-drying the solution by a freeze dryer after 3 days to obtain purified CMC-BA;

(2) preparation of drug-loaded nanoparticles (PLGA @ Cur)

Completely dissolving polylactic-co-glycolic acid (PLGA, 60mg) and curcumin (Cur, 12mg) in DMSO (5mL) at 37 ℃, then dropwise adding into 15mL deionized water under stirring, and continuously stirring for 4h at 37 ℃; dialyzing the solution in water for 3 days to obtain a PLGA @ Cur solution, and storing the solution at 4 ℃ in a dark place after freeze-drying;

(3) preparation of hydrogels

An injectable hydrogel was immediately prepared by mixing an aqueous CMC-BA (1% w/v) solution containing human recombinant type III collagen (2mg/mL) and PLGA @ Cur (100. mu.g/mL) with an equal volume of 1% by weight polyvinyl alcohol solution.

Example 2

A preparation method of injectable hydrogel with anti-inflammatory and repair promoting functions comprises the following steps:

(1) preparation of phenylboronic acid grafted hyaluronic acid functional polymer (HA-BA)

Hyaluronic acid (HA, 10.00g) and 4-aminophenylboronic acid (BA, 6.50g) were precisely weighed and dissolved in 500mL of MES buffer (0.1mol, pH5.0), to which 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl, 4.00g) and N-hydroxysuccinimide (NHS, 1.5g) were added. Then stirring for 48h at 37 ℃, finally dialyzing in deionized water (pH7.4) for 3 days, and freeze-drying the solution by a freeze dryer after 3 days to obtain purified HA-BA;

(2) preparation of drug-loaded nanoparticles (PLGA @ Nap)

Completely dissolving polylactic-co-glycolic acid (PLGA, 60mg) and naproxen (Nap, 12mg) in DMSO (5mL) at 37 ℃, then dropwise adding into 15mL deionized water under stirring, and continuously stirring for 4h at 37 ℃; dialyzing the solution in water for 3 days to obtain a PLGA @ Nap solution, and storing the solution at 4 ℃ in a dark place after freeze-drying;

(3) preparation of hydrogels

An injectable hydrogel was prepared immediately by mixing a solution of HA-BA (1% w/v water) containing human recombinant type I collagen (2mg/mL) and PLGA @ Nap (100. mu.g/mL) in equal volumes with a 1% polyvinyl alcohol solution.

Example 3

(1) preparation of phenylboronic acid grafted starch functional polymer

Starch (10.00g) and 4-carboxyphenylboronic acid (6.50g) were precisely weighed, dissolved in 500mL of deionized water (pH7.4), and dicyclohexylcarbodiimide (DCC, 1.00g) and 4-dimethylaminopyridine (DMAP, 0.50g) were added thereto. Then stirring for 48h at 37 ℃, finally dialyzing in deionized water (pH7.4) for 3 days, and freeze-drying the mixture by using a freeze dryer after 3 days to obtain a purified functional polymer;

(2) preparation of drug-loaded nanoparticles

Completely dissolving polyethylene glycol phospholipid (DSPE-PEG, 60mg) and sulvastatin (12mg) in DMSO (5mL) at 37 ℃, then dropwise adding into 15mL of deionized water under stirring, and continuously stirring for 4h at 37 ℃; dialyzing in water for 3 days to obtain drug-loaded nanoparticle solution, lyophilizing, and storing at 4 deg.C in dark place;

(3) preparation of hydrogels

An injectable hydrogel was immediately prepared by mixing an aqueous solution of a functional polymer (1% w/v) containing basic fibroblast growth factor (1mg/mL) and drug-loaded nanoparticles (100. mu.g/mL) prepared above with an equal volume of 1% polyvinyl alcohol solution.

Example 4

(1) preparation of phenylboronic acid grafted polylysine functional polymer

Polylysine (10.00g) and 3-carboxyphenylboronic acid (6.50g) were weighed accurately, dissolved in 500mL of MES buffer (0.1mol, pH5.0), and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl, 4.00g) and N-hydroxysuccinimide (NHS, 1.5g) were added thereto. Then stirring for 48h at 37 ℃, finally dialyzing in deionized water (pH7.4) for 3 days, and freeze-drying the mixture by using a freeze dryer after 3 days to obtain a purified functional polymer;

(2) preparation of drug-loaded nanoparticles

Completely dissolving phospholipid polyethylene glycol polylactic acid-glycolic acid copolymer (DSPE-PEG-PLGA,60mg) and aspirin (12mg) in DMSO (5mL) at 37 ℃, then dropwise adding into 15mL of deionized water under the stirring condition, and continuously stirring for 4 hours at 37 ℃; dialyzing in water for 3 days to obtain drug-loaded nanoparticle solution, lyophilizing, and storing at 4 deg.C in dark place;

(3) preparation of hydrogels

An injectable hydrogel was immediately prepared by mixing an aqueous solution of a functional polymer (1% w/v) containing vascular endothelial growth factor (1mg/mL) and drug-loaded nanoparticles (100. mu.g/mL) prepared above with a 1% polyvinyl alcohol solution in equal volume.

Example 5

(1) preparation of phenylboronic acid grafted sodium alginate functional polymer

Sodium alginate (10.00g) and 3-hydroxyphenylboronic acid (6.50g) were precisely weighed, dissolved in 500mL of deionized water (pH7.4), and dicyclohexylcarbodiimide (DCC, 1.00g) and 4-dimethylaminopyridine (DMAP, 0.50g) were added thereto. Then stirring for 48h at 37 ℃, finally dialyzing in deionized water (pH7.4) for 3 days, and freeze-drying the solution by using a freeze dryer after 3 days to obtain a purified functional polymer;

(2) preparation of drug-loaded nanoparticles

Completely dissolving polyethylene glycol polylactic acid copolymer (PEG-PLA, 60mg) and verapamil (12mg) in DMSO (5mL) at 37 ℃, then dropwise adding the mixture into 15mL of deionized water under the stirring condition, and continuously stirring the mixture for 4 hours at 37 ℃; dialyzing in water for 3 days to obtain drug-loaded nanoparticle solution, lyophilizing, and storing at 4 deg.C in dark place;

(3) preparation of hydrogels

The injectable hydrogel can be prepared immediately by mixing an aqueous solution of a functional polymer (1% w/v) containing mangiferin (1mg/mL) and the drug-loaded nanoparticles (100. mu.g/mL) prepared above with a 1% polyvinyl alcohol solution in equal volume.

Test example 1

Taking the substance prepared in the example 1 as an example, the detection is carried out, and the specific operation process and the result are as follows:

firstly, detecting the CMC-BA prepared in the step (1), wherein the specific result is shown in figure 1, and the detailed result is shown in figure 1, and the CMC-BA polymer is prepared¹The presence of an aromatic proton peak of phenylboronic acid groups (. delta. -7.5 ppm) in the H NMR spectrum, evidencing the successful preparation of CMC-BA polymers.

Secondly, particle size detection is carried out on the drug-loaded nanoparticles prepared in the step (2), as shown in fig. 2, the particle size distribution result shows that the particle size of the PLGA nanoparticles before drug loading is 126.4nm, the particle size after drug loading is 133.8nm, and the PDI of the nanoparticles before and after drug loading is less than 0.2, so that good uniform dispersibility is proved, and successful preparation of the drug-loaded nanoparticles is proved.

In the following experimental illustrations, the hydrogel groups 1 to 4 represent the following combinations, respectively, unless otherwise specified: hydrogel group 1(Hydrogel 1): blank hydrogel; hydrogel group 2(Hydrogel 2): loading PLGA @ Cur hydrogel; hydrogel group 3(Hydrogel 3): loading hydrogel of recombinant human III-type collagen; hydrogel group 4(Hydrogel 4): hydrogel loaded with PLGA @ Cur and recombinant human type III collagen.

And thirdly, detecting the gelling performance and the injection performance of the hydrogel prepared in the step (3).

FIG. 3 is a gelation diagram of a hydrogel demonstrating the successful preparation of the hydrogel; FIG. 4 is a graph showing injectability of a hydrogel that can be injected from a 1mL syringe and into the HEART using the English letter "HEART", demonstrating good injectability of the hydrogel.

Fourth, detection of biocompatibility of hydrogel

Biocompatibility of the hydrogels was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs). The hydrogel prepared under sterile conditions was at pH5.0 and contained 1mM H₂O ₂The cells were soaked in the cell culture medium (0.2g/mL) for 24 hours. HUVECs cells were seeded in 96-well plates at a density of 8000 cells per well. After 12h, the cell culture medium was removed and different hydrogel leachates were added to continue incubation of the cells. The proliferation rates of 24h, 48h and 72h HUVECs were measured by CCK-8. After 24h, 48h, 72h incubation, 10% CCK-8 in fresh medium was added to each well. After 2h, the cell proliferation rate was calculated by measuring the absorbance at 450nm with a microplate reader.

The survival rate results of the hydrogel on endothelial cells are shown in fig. 5, and the results show that all hydrogel groups show no toxicity to the endothelial cells in 24h, 48h and 72h, which indicates that the hydrogel has good cell compatibility. In addition, after the hydrogel is loaded with the recombinant human type III collagen, the cell survival rate is higher than that of a control group and a blank hydrogel group, and the fact that the recombinant human type III collagen effectively promotes the proliferation of endothelial cells is shown.

Fifth, the capacity of hydrogel to promote proliferation of cardiomyocytes

Cardiomyocytes H9C2 were seeded in 96-well plates at a density of 8000 cells per well. After 12h, the cell culture medium was removed and different hydrogel leachates were added to continue incubation of the cells. The proliferation rates of 24H, 48H and 72H H9C2 cells were measured using CCK-8. The survival rate of the hydrogel on the myocardial cells is shown in fig. 6, and the result shows that the cell proliferation of the hydrogel is the most after the hydrogel is loaded with the recombinant human type III collagen compared with that of a control group and a blank hydrogel group, and the recombinant human type III collagen effectively promotes the proliferation of the myocardial cells.

Sixthly, the hydrogel inhibits the expression of inflammation-related protein

ELISA experiments

Press 10⁶The density macrophages of each cell hole are respectively inoculated on a 6-hole plate, and lipopolysaccharide is added to be pre-incubated with the macrophages for 2 h. Cells were then incubated with 2mL of hydrogel extract. At 24h, 48h, cell culture supernatants were aspirated and the tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) concentrations in the supernatants were determined using a TNF-alpha and interleukin-6 (IL-6) ELISA kit. As a result, as shown in FIG. 7, the macrophages treated with hydrogel 4 exhibited lower expression levels of TNF-. alpha.and IL-6 than those treated with lipopolysaccharide and hydrogel 1. The above results indicate that hydrogel 4 group can effectively inhibit the inflammatory response of macrophages.

Seventhly, detection of in-vivo heart repair effect of hydrogel

In order to research the influence of the hydrogel on the in-vivo heart repair effect, a rat myocardial infarction disease model is established. Hematoxylin and eosin (H & E) staining results and Masson staining results on day 28 are shown in fig. 8, the ventricular wall thickness of the heart tissue of the hydrogel 4 group was the highest, and the scar area was the smallest, indicating that the hydrogel treatment group 4 effectively reduced the scar area at the myocardial infarction site, demonstrating that the cardiac repair function was good.

The preparation process of the hydrogel and the mechanism for promoting myocardial repair in example 1 are as follows:

first, curcumin is efficiently loaded into PLGA nanoparticles by hydrophilic-hydrophobic interactions. The preparation method comprises the steps of grafting 3-aminophenylboronic acid to a carboxymethyl cellulose side chain by utilizing an amide reaction to obtain a functional polymer, finally preparing hydrogel by utilizing the characteristic that a boric acid group of the functional polymer and a hydroxyl group of polyvinyl alcohol are easy to form a boron ester bond, and meanwhile, adding PLGA nano particles for encapsulating curcumin and recombinant human type III collagen to successfully obtain multifunctional hydrogel in the gelation process, wherein the hydrogel is degraded in an acid environment and high ROS, and releases anti-inflammatory drug curcumin and recombinant human type III collagen in an 'on-demand' response controllable manner. The released curcumin inhibits the expression of inflammation-related factors and can effectively reduce the inflammatory reaction of myocardial infarction parts; the recombinant human III-type collagen can promote the proliferation of endothelial cells and cardiac muscle cells and the expression of relevant factors for angiogenesis, thereby promoting the generation of new vessels at infarct positions; the repair of the damaged heart under the inflammatory environment is accelerated by an anti-inflammatory and pro-vascular combined treatment strategy, and the cardiac function after the myocardial infarction is effectively improved.

The following examples:

the drug-loaded nano-micelle can be prepared into a micelle by covalent bonding of hydrophilic and hydrophobic high molecular polymers. If hyaluronic acid and the like are used as hydrophilic ends, cholesterol and the like can be selected as hydrophobic ends to synthesize the drug-carrying micelle.

The biological macromolecule with amino and ortho-hydroxyl is one of chitosan and other natural macromolecules.

The biomacromolecule with carboxyl and ortho-hydroxyl is one of sodium alginate, hyaluronic acid and modified products thereof.

A compound providing a phenylboronic acid group such as one of ortho-aminobenzeneboronic acid, meta-aminobenzeneboronic acid, para-aminobenzeneboronic acid, ortho-carboxyphenylboronic acid, meta-carboxyphenylboronic acid, and para-carboxyphenylboronic acid, ortho-hydroxyphenylboronic acid, meta-hydroxyphenylboronic acid, and para-carboxyphenylboronic acid.

Hydrophobic drugs can be classified into one of anti-inflammatory drugs, angiogenesis promoting drugs, cell proliferation promoting drugs, migration promoting drugs and the like according to pharmacological activity.

The hydrophilic drugs can be classified into growth factors and one of DNA, RNA, and protein drugs, etc. according to pharmacological activities.

The structural unit of the injectable hydrogel is that phenylboronic acid with amino groups is grafted to a polymer side chain containing carboxyl groups and ortho-hydroxyl groups on the side chain through amidation reaction, and the polymer can be one of sodium alginate, hyaluronic acid and modified products thereof.

The injectable hydrogel structural unit can also be prepared by grafting phenylboronic acid with carboxyl groups onto a polymer side chain containing amino groups and ortho-hydroxyl groups through amidation reaction, wherein the polymer is chitosan or one of other natural macromolecules.

Example 6

1) synthesis of phenylboronic acid modified alginate (ALG-BA)

Sodium alginate (ALG, 5.00g) was precisely weighed, dissolved in 500mL of MES buffer (0.1mol, pH5.0), and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl, 4.80g, 25.0mmol) and 3-aminophenylboronic acid (BA, 1.95g, 12.5mmol) were added thereto. Then, the mixture was stirred at 37 ℃ for 24 hours and finally dialyzed against deionized water (pH7.4) for 3 days. After 3 days, freeze-drying with a freeze dryer to obtain purified ALG-BA.

2) Synthesis of cholesterol-modified hyaluronic acid (HA-CHOL)

HA (2.00g) and cholesterol (CHOL, 1.50g) were completely dissolved in dimethyl sulfoxide (DMSO, 30mL) at 80 deg.C, N' -dicyclohexylcarbodiimide (DCC, 1.00g) and 4-dimethylaminopyridine (DMAP, 0.50g) were added, and stirring was continued at 80 deg.C for 48 h. The mixture was then dialyzed against water for 2 days, excess cholesterol was removed by centrifugation, and the final product was lyophilized for use.

3) Preparation of drug-loaded Micelles (MICs)

HA-CHOL (40.0mg) and naproxen (Nap, 8.00mg) were dissolved in DMSO (10mL) and heated to 80 ℃. Then in the case of slow stirringIn this case, 10mL of H₂And O is added into the mixed solution dropwise. Finally, the mixture was dialyzed in water for 2 days to prepare a drug-loaded micelle solution.

4) Preparation of hydrogels

Respectively dissolving ALG-BA (1.00g) and amikacin (AM, 100mg) prepared in the step (1) into the drug-loaded micelle solution (1mg/mL) prepared in the step (3) to enable the final concentration of the ALG-BA in the drug-loaded micelle solution to be 9% w/v, and adjusting the pH of the drug-loaded micelle solution to 8.5 by using an alkaline solution to rapidly gelatinize the drug-loaded micelle solution to form hydrogel.

Example 7

A preparation method of injectable hydrogel with anti-inflammatory and repair promoting functions comprises the following preparation steps:

(1) synthesis of phenylboronic acid modified hyaluronic acid (HA-BA)

Precisely weighing 5g of hyaluronic acid, dissolving in 500mL of MES buffer (0.1mol, pH5.0), adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl, 4.80g, 25.0mmol) and 3-aminophenylboronic acid (BA, 1.95g, 12.5mmol), stirring at 37 ℃ for 24h, finally dialyzing in deionized water (pH7.4) for 3 days, and freeze-drying with a freeze dryer to obtain purified HA-BA;

(2) amphiphilic drug carrier

Completely dissolving sodium alginate (2.00g) and cholesterol (CHOL, 1.50g) in a mixed solution of dimethyl sulfoxide and water (DMSO, 30mL), and adding N, N' -dicyclohexylcarbodiimide (DCC, 1.00g) and 4-dimethylaminopyridine (DMAP, 0.50g) thereto, and continuously stirring for 48 hours; dialyzing the mixture in water for 2 days, and removing the excessive cholesterol by centrifugation; finally, freeze-drying the finished product for later use;

(3) preparation of drug-loaded micelles

Dissolving 10mg of the amphiphilic drug carrier in step (2) and 1mg of the anti-inflammatory drug (piroxicam) in DMSO (1mL), then adding it dropwise to 10mL of water with slow stirring, and finally, dialyzing the mixture in water for 2 days;

(4) preparation of hydrogels

When preparing injectable hydrogel, 1g of HA-BA polymer and 100mg of cell growth factor are respectively dissolved in a drug-loaded micelle solution (1mg/mL), and a proper amount of sodium hydroxide solution is added to adjust the pH value of the solution to 8.5, so that the solution can be rapidly gelatinized.

Example 8

(1) synthesis of phenylboronic acid modified chitosan

5g of chitosan was precisely weighed and dissolved in 500mL of MES buffer (0.1mol, pH5.0), and EDC. HCl (4.80g, 25.0mmol) and 2-carboxyphenylboronic acid (BA, 1.95g, 12.5mmol) were added. Then, stirred at 37 ℃ for 24h and finally dialyzed against deionized water (pH7.4) for 3 days. Freeze-drying with freeze dryer.

(2) Amphiphilic drug carrier

The amphiphilic drug carrier is directly selected from polylactic-co-glycolic acid (PLGA) purchased from the company, and is polymerized by lactic acid and glycolic acid according to the proportion of 50: 50.

(3) Preparation of drug-loaded micelles

Dissolving 10mg of PLGA and an anti-inflammatory drug (ibuprofen, 1mg) in DMSO (10mL), then adding it dropwise to 10mL of water with slow stirring, and finally dialyzing the mixture in water for 2 days to obtain;

(4) preparation of hydrogels

Respectively dissolving the phenylboronic acid modified chitosan polymer (1g) and the deferoxamine (100 mg) in the step (1) into a drug-loaded micelle solution (1mg/mL), and then adding a proper amount of sodium hydroxide solution to adjust the pH value of the solution to 8.5, so that the solution can be rapidly gelatinized.

Example 9

(1) synthesis of phenylboronic acid modified hyaluronic acid (HA-BA)

(2) amphiphilic drug carrier

(3) preparation of drug-loaded micelles

Dissolving 10mg of the amphiphilic drug carrier of step (2) and 1mg of the anti-inflammatory drug (sulvastatin) in DMSO (1mL), then adding it drop by drop into 10mL of water with slow stirring, and finally dialyzing the mixture in water for 2 days;

(4) preparation of hydrogels

When preparing injectable hydrogel, 1g of HA-BA polymer and 100mg of Epidermal Growth Factor (EGF) are respectively dissolved in a drug-loaded micelle solution (1mg/mL), and a proper amount of sodium hydroxide solution is added to adjust the pH value of the solution to 8.5, so that the solution can be rapidly gelled.

Example 10

(1) synthesis of phenylboronic acid modified chitosan

5g of chitosan was precisely weighed and dissolved in 500mL of MES buffer (0.1mol, pH5.0), and EDC. HCl (4.80g, 25.0mmol) and 3-carboxyphenylboronic acid (BA, 1.95g, 12.5mmol) were added. Then, stirred at 37 ℃ for 24h and finally dialyzed against deionized water (pH7.4) for 3 days. Freeze-drying with freeze dryer.

(2) Amphiphilic drug carrier

The amphiphilic carrier is directly selected from polylactic-co-glycolic acid (PLGA) which is purchased from the company.

(3) Preparation of drug-loaded micelle

(4) preparation of hydrogels

Respectively dissolving the phenylboronic acid modified chitosan polymer (1g) and the angiogenesis promoting drug (deferoxamine mesylate) of 100mg in the step (1) into a drug-loaded micelle solution (1mg/mL), and then adding a proper amount of sodium hydroxide solution to adjust the pH of the solution to 8.5, so that the solution can be rapidly gelatinized.

Example 11

(1) synthesis of phenylboronic acid modified hyaluronic acid (HA-BA)

Precisely weighing 5g of hyaluronic acid, dissolving the hyaluronic acid in 500mL of deionized water, adding N, N' -dicyclohexylcarbodiimide (DCC, 3.00g) and one of 4-dimethylaminopyridine (DMAP, 1.50g) and 2-hydroxyphenylboronic acid (BA, 1.95g, 12.5mmol) into the deionized water, stirring the mixture at 37 ℃ for 24 hours, dialyzing the mixture in deionized water (pH7.4) for 3 days, and freeze-drying the mixture by using a freeze dryer to obtain purified HA-BA;

(2) amphiphilic drug carrier

(3) preparation of drug-loaded micelles

(4) preparation of hydrogels

Example 12

(1) synthesis of phenylboronic acid modified chitosan

5g of chitosan was precisely weighed and dissolved in 500mL of deionized water, and one of N, N' -dicyclohexylcarbodiimide (DCC, 3.00g) and 4-dimethylaminopyridine (DMAP, 1.50g) and 4-hydroxyphenylboronic acid (BA, 1.95g, 12.5mmol) was added thereto. Then, stirred at 37 ℃ for 24h and finally dialyzed against deionized water (pH7.4) for 3 days. Freeze-drying with freeze dryer.

(2) Amphiphilic drug carrier

(3) Preparation of drug-loaded micelles

(4) preparation of hydrogels

Test example 2

firstly, detecting the ALG-BA prepared in the step (1), wherein the specific result is shown in figure 9, and the nuclear magnetic result of figure 9 proves that the ALG-BA polymer is successfully prepared.

Secondly, detecting the HA-CHOL prepared in the step (2), particularly referring to FIG. 10, as shown in FIG. 10, and proving that the HA-CHOL is successfully prepared; the degree of grafting of the CHOL was 13.5% as determined by nuclear magnetic resonance.

Test example 3

In vivo cardiac repair assay with hydrogel prepared in example 9

Hydrogel groups 1-4 represent the following combinations, respectively: hydrogel group 1(Hydrogel 1): blank hydrogel; hydrogel group 2(Hydrogel 2): a hydrogel loaded with anti-inflammatory drug (piroxicam) loaded micelles; hydrogel group 3(Hydrogel 3): an Epidermal Growth Factor (EGF) -loaded hydrogel; hydrogel group 4(Hydrogel 4): hydrogel loaded with anti-inflammatory drug (sulvastatin) drug-loaded micelle and Epidermal Growth Factor (EGF).

In order to research the influence of the hydrogel on the in-vivo heart repair effect, a rat myocardial infarction disease model is established. The heart ultrasonic results of the rats at 14 days and 28 days show that the heart function recovery result of the rat of the hydrogel group 4 is obviously faster than that of the rats of the other groups, the ejection fraction and the left ventricle shortening fraction of the rat of the hydrogel group 4 are respectively obviously improved compared with the MI group at 28 days, and the systolic end ventricle volume and the diastolic end ventricle volume are obviously reduced compared with the MI group, which shows that the hydrogel group 4 can effectively repair the myocardium and recover the myocardial function, and the hematoxylin and eosin (H & E) staining result, the Masson staining result and the quantitative result of the infarct area at 28 days show that the ventricular wall thickness of the heart tissue of the hydrogel group 4 is the highest and the area of the scar area is the smallest, which shows that the hydrogel group 4 effectively reduces the scar area of the myocardial infarction part and proves that the heart repair function of the rat is good.

The foregoing is merely exemplary and illustrative of the structure of the present application and it is contemplated that modifications and additions to the specific embodiments described or substitutions in a similar manner, may be made by those skilled in the art without inventive faculty, while remaining within the scope of the invention.

Claims

An injectable hydrogel having anti-inflammatory and repair promoting functions, wherein the injectable hydrogel forms a gel through the interaction of functional groups of a polymer and adjacent hydroxyl groups of the polymer, and the hydrogel disintegrates after the functional groups of the polymer and the adjacent hydroxyl groups of the polymer are released from the interaction in response to acidic conditions and/or active oxygen conditions.
The injectable hydrogel having anti-inflammatory and repair-promoting effects of claim 1, wherein the injectable hydrogel is loaded with one or more of extracellular matrix, hydrophilic drug, and hydrophobic drug.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 1, wherein the polymer having functional groups is at least one of sodium alginate containing phenylboronic acid groups, chitosan, quaternary ammonium salt of chitosan, polylysine, polyethyleneimine, gelatin, hyaluronic acid, heparin, carboxymethylcellulose, dextran, methylcellulose, starch, and cyclodextrin.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 1, wherein the polymer containing ortho-hydroxyl groups is at least one of sodium alginate, polyvinyl alcohol, hyaluronic acid, dextran, and starch.
The injectable hydrogel having anti-inflammatory and repair-promoting effects of claim 1, wherein said injectable hydrogel is loaded with: an extracellular matrix;

and hydrophilic drugs and/or hydrophobic drugs.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 1, wherein the extracellular matrix is at least one of collagen, non-collagens, elastin, proteoglycans, and aminoglycans.
The injectable hydrogel having anti-inflammatory and repair-promoting effects of claim 1, wherein the extracellular matrix is a recombinant humanized collagen.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 1, wherein the extracellular matrix is at least one of a recombinant type I humanized collagen and a recombinant type III humanized collagen.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 1, wherein the recombinant type I humanized collagen and the recombinant type III humanized collagen comprise amino acid sequence fragments capable of binding to cytokinin.
The injectable hydrogel having anti-inflammatory and repair-promoting properties according to claim 1, wherein the extracellular matrix is a recombinant type III humanized collagen.
The injectable hydrogel with anti-inflammatory and repair-promoting functions of claim 1, wherein the hydrophobic drug is at least one of naproxen, sulvastatin, curcumin, aspirin.
The injectable hydrogel having anti-inflammatory and repair-promoting effects of claim 1, wherein the hydrophobic drug is loaded in the injectable hydrogel with an amphiphilic polymer as a carrier.
The injectable hydrogel having anti-inflammatory and repair-promoting effects of claim 1, wherein said injectable hydrogel is loaded with: curcumin and recombinant humanized collagen.
The injectable hydrogel having anti-inflammatory and repair-promoting effects of claim 1, wherein said injectable hydrogel is loaded with: curcumin and recombinant type III humanized collagen.
A hydrogel for repairing cardiac injury, wherein the hydrogel is the injectable hydrogel according to any one of claims 1 to 14 applied to the site of cardiac injury.
A hydrogel for treating heart failure, wherein the hydrogel is the injectable hydrogel according to any one of claims 1 to 14 applied to a diseased region of a heart.
A hydrogel for treating myocardial infarction, which is the injectable hydrogel according to any one of claims 1 to 14 acting on a myocardial infarction site.
Use of the injectable hydrogel with anti-inflammatory and repair promoting functions according to any one of claims 1 to 14 in cardiac injury repair.
Use of the injectable hydrogel with anti-inflammatory and repair promoting effects of any one of claims 1 to 14 for treating heart failure.
The use of the injectable hydrogel with anti-inflammatory and repair-promoting effects of any one of claims 1 to 14 in the treatment of myocardial infarction.
A method for repairing a cardiac injury, comprising applying the injectable hydrogel of any one of claims 1 to 14 to a cardiac injury site.
A method of treating heart failure by applying an injectable hydrogel according to any one of claims 1 to 14 to a site of a heart disorder.
A method for repairing a damaged heart, characterized by injecting the injectable hydrogel according to any one of claims 1 to 14 at the site of a heart lesion.
A method of treating heart failure by injecting the injectable hydrogel of any one of claims 1 to 14 at a site of myocardial infarction.
A method of treating myocardial infarction, characterized in that an injectable hydrogel according to any one of claims 1 to 14 is injected at the site of myocardial infarction.
A method of preparing an injectable hydrogel according to any one of claims 1 to 14 comprising:

preparing a first polymer containing phenylboronic acid groups;

mixing at least one of extracellular matrix, hydrophilic drug and hydrophobic drug, first polymer and polymer containing ortho-hydroxyl to prepare the injectable hydrogel.
The method of preparing an injectable hydrogel of claim 26 wherein said first polymer is prepared by reacting any one of the following raw material combinations in the presence of a condensing agent and a catalyst:

a) an amino-or hydroxyl-containing polymer and a carboxyl-containing phenylboronic acid;

b) carboxyl-containing polymers and amino-or hydroxyl-containing phenylboronic acids.
The method of preparing an injectable hydrogel of claim 27, wherein said first polymer is prepared by:

after the raw materials are dissolved, the reaction is carried out for 30-60h at the temperature of 30-40 ℃ to prepare the first polymer containing the phenylboronic acid group.
The method for preparing an injectable hydrogel according to claim 27, wherein the mass ratio of the amino-or hydroxyl-containing polymer, the carboxyl-containing phenylboronic acid, the condensing agent and the catalyst is 7 (4-5): 2-3): 1; or the mass ratio of the carboxyl-containing polymer, the phenyl boric acid containing amino or hydroxyl, the condensing agent and the catalyst is 7 (4-5) to (2-3) to 1.
The method for preparing the injectable hydrogel according to claim 26, wherein the hydrophobic drug is prepared as a drug-loaded nano-micelle by using an amphiphilic polymer as a carrier, and the drug-loaded nano-micelle is mixed with the first polymer and the polymer containing the ortho-hydroxyl group to prepare the injectable hydrogel containing the hydrophobic drug.
The method of preparing an injectable hydrogel of claim 30, wherein the preparation of drug-loaded nanomicelles comprises:

dissolving amphiphilic polymer and hydrophobic drug in benign solvent, slowly dripping into water under the condition of continuous stirring, and dialyzing to obtain drug-loaded nano micelle solution with the concentration of 1-2 mg/mL.
The method of preparing an injectable hydrogel of claim 31 wherein the mass ratio of amphiphilic polymer to hydrophobic drug is 4-8: 1.
The method for preparing the injectable hydrogel according to claim 26, wherein the injectable hydrogel is obtained by mixing an aqueous solution of a first polymer with an aqueous solution of a polymer containing an ortho-hydroxyl group, wherein the aqueous solution of the first polymer contains at least one of a hydrophilic drug, an extracellular matrix and a drug-loaded nano micelle, and the mass concentration of the first polymer in the aqueous solution of the first polymer is 0.5-10% w/v.
The method for preparing an injectable hydrogel of claim 33 wherein the mass concentration of the hydrophilic drug in the aqueous first polymer solution is 1 to 1000 μ g/mL.
The method of preparing an injectable hydrogel of claim 33, wherein the first aqueous polymer solution has an extracellular matrix concentration of 1 to 6 mg/mL.
The method for preparing the injectable hydrogel of claim 33, wherein the mass concentration of the drug-loaded nanomicelle in the first aqueous polymer solution is 30-200 μ g/mL.
The method for preparing an injectable hydrogel according to claim 33, wherein the mass concentration of the ortho-hydroxyl group-containing polymer in the aqueous solution of the ortho-hydroxyl group-containing polymer is 0.5 to 10% w/v.
The method of preparing an injectable hydrogel of claim 27 wherein the amino group-containing polymer is at least one of chitosan, quaternary ammonium salt of chitosan, polylysine, polyethyleneimine, and gelatin.
The method of preparing an injectable hydrogel according to claim 27 wherein the carboxyl-containing polymer is at least one of sodium alginate, hyaluronic acid, heparin, carboxymethyl cellulose.
The method of preparing an injectable hydrogel of claim 27 wherein the hydroxyl group-containing polymer is at least one of starch, cellulose, gellan gum, konjac gum, gum arabic, lignin, dextran, and cyclodextrin.
The method of preparing an injectable hydrogel of claim 26 wherein the polymer containing ortho-hydroxyl groups is at least one of polyvinyl alcohol, sodium alginate, hyaluronic acid, dextran, starch.
The method for preparing an injectable hydrogel according to claim 27, wherein the carboxyl group-containing phenylboronic acid is at least one of 4-carboxyphenylboronic acid, 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyl-3-fluorophenylboronic acid, 3-carboxyl-4-fluorophenylboronic acid, 5-carboxyl-2-chlorophenylboronic acid and 4-carboxyl-2-chlorophenylboronic acid;

the amino-containing phenylboronic acid is at least one of 4-aminophenylboronic acid, 2-aminophenylboronic acid, 3-carbamoylphenylboronic acid, 3-amino-4-fluorobenzeneboronic acid and 3-amino-4-methylbenzeneboronic acid;

the hydroxyl-containing phenylboronic acid is at least one of 4-hydroxyphenylboronic acid, 3-fluoro-4-hydroxyphenylboronic acid, 2-fluoro-3-hydroxyphenylboronic acid, 2-fluoro-5-hydroxyphenylboronic acid, 3-hydroxy-4-chlorophenylboronic acid and 3-fluoro-4-hydroxyphenylboronic acid.
The method of preparing an injectable hydrogel of claim 26 wherein said extracellular matrix is at least one of collagen, non-collagens, elastin, proteoglycans, aminoglycans.
The method of making an injectable hydrogel of claim 26 wherein said extracellular matrix is a recombinant humanized collagen.
The method of preparing an injectable hydrogel according to claim 26 wherein said hydrophobic drug is at least one of an anti-inflammatory drug, an analgesic, a pro-angiogenic drug, a diuretic, an angiotensin converting enzyme inhibitor, a beta blocker, a digitalis drug, an aldosterone antagonist, an angiotensin-two-receptor antagonist, an anticoagulant, and an antiplatelet drug.
The injectable hydrogel with anti-inflammatory and repair promoting effects prepared by the preparation method according to any one of claims 26-45.
A hydrogel for repairing a cardiac injury, wherein the hydrogel is the injectable hydrogel of claim 46 acting at the site of the cardiac injury.
A hydrogel for treating heart failure, wherein the hydrogel is the injectable hydrogel of claim 46 applied to a diseased site of the heart.
A hydrogel for treating myocardial infarction, wherein the hydrogel is the injectable hydrogel according to claim 46 acting on the site of myocardial infarction.
Use of the injectable hydrogel with anti-inflammatory and repair-promoting functions as claimed in claim 46 in the repair of cardiac injuries.
Use of the injectable hydrogel with anti-inflammatory and repair-promoting functions of claim 46 for the treatment of heart failure.
Use of the injectable hydrogel with anti-inflammatory and repair promoting effects of claim 46 in the treatment of myocardial infarction.
A method for repairing a cardiac injury by applying the injectable hydrogel of claim 46 to the site of the cardiac injury.
A method of treating heart failure, wherein the injectable hydrogel of claim 46 is applied to a site of a cardiac disorder.
A method of repairing a damaged heart by injecting the injectable hydrogel of claim 46 at the site of a heart lesion.
A method of treating heart failure by injecting the injectable hydrogel of claim 46 at a site of myocardial infarction.
A method of treating myocardial infarction, wherein the injectable hydrogel of claim 46 is injected at the site of myocardial infarction.
An injectable hydrogel with anti-inflammatory and repair promoting functions is characterized by comprising the following raw material components: biomacromolecules containing amino and ortho-hydroxyl groups, biomacromolecules containing carboxyl and ortho-hydroxyl groups and phenylboronic acid containing amino, hydroxyl or carboxyl groups.
The injectable hydrogel having anti-inflammatory and repair-promoting properties according to claim 58, further comprising the following components: hydrophilic drugs and/or hydrophobic drugs.
The injectable hydrogel having anti-inflammatory and repair-promoting properties according to claim 59, wherein said hydrophilic drug is at least one of a growth factor, a gene drug, a water-soluble protein drug.
The injectable hydrogel having anti-inflammatory and pro-reparative properties according to claim 59, wherein said hydrophobic drug is at least one of an anti-inflammatory agent, a pro-angiogenic agent, a pro-cell proliferative agent, and a pro-cell migratory agent.
The injectable hydrogel having anti-inflammatory and pro-reparative properties according to claim 61, wherein said anti-inflammatory agent is at least one of aspirin, paracetamol, amoxicillin and phenylbutazone.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 58, wherein the amino and o-hydroxyl containing biomacromolecule is at least one of chitosan, sodium alginate modified product, hyaluronic acid modified product.
The injectable hydrogel having anti-inflammatory and repair promoting effects of claim 58, wherein the biomacromolecule having carboxyl and o-hydroxyl groups is sodium alginate, a sodium alginate-modified product, hyaluronic acid, a hyaluronic acid-modified product, carboxymethyl cellulose, or a carboxymethyl cellulose-modified product.
The injectable hydrogel having anti-inflammatory and repair-promoting properties according to claim 58, wherein said amino, hydroxyl or carboxyl group-containing phenylboronic acid is ortho-aminobenzeneboronic acid, meta-aminobenzeneboronic acid, para-aminobenzeneboronic acid, ortho-hydroxyphenylboronic acid, meta-hydroxyphenylboronic acid, para-hydroxyphenylboronic acid, ortho-carboxyphenylboronic acid, meta-carboxyphenylboronic acid or para-carboxyphenylboronic acid.
A hydrogel for repairing a cardiac injury, wherein the hydrogel is an injectable hydrogel according to any one of claims 58 to 65 for acting at the site of the cardiac injury.
A hydrogel for the treatment of heart failure, wherein said hydrogel is an injectable hydrogel according to any one of claims 58 to 65 for application to a site of a cardiac disorder.
A hydrogel for use in the treatment of myocardial infarction, wherein said hydrogel is an injectable hydrogel according to any one of claims 58 to 65 that acts on a site of myocardial infarction.
Use of the injectable hydrogel with anti-inflammatory and repair-promoting functions according to any one of claims 58 to 65 for cardiac injury repair.
Use of an injectable hydrogel with anti-inflammatory and repair promoting effects of any of claims 58-65 in the treatment of heart failure.
Use of an injectable hydrogel with anti-inflammatory and pro-reparative effects according to any of claims 58-65 for the treatment of myocardial infarction.
A method for repairing a cardiac injury by applying the injectable hydrogel of any one of claims 58 to 65 to the site of the cardiac injury.
A method for treating heart failure by applying the injectable hydrogel of any one of claims 58 to 65 to a site of heart failure.
A method of repairing a damaged heart by injecting the injectable hydrogel of any one of claims 58 to 65 at the site of a heart lesion.
A method of treating heart failure by injecting the injectable hydrogel of any one of claims 58 to 65 at the site of myocardial infarction.
A method of treating myocardial infarction, wherein the injectable hydrogel of any one of claims 58 to 65 is injected at the site of myocardial infarction.
A preparation method of injectable hydrogel with anti-inflammatory and repair promoting functions is characterized by comprising the following steps:

step 1, reacting a functional polymer containing ortho hydroxyl with phenylboronic acid containing amino, hydroxyl or carboxyl to prepare a polymer of side chain grafted phenylboronic acid; the functional polymer containing the ortho-hydroxyl is a biological macromolecule containing amino and ortho-hydroxyl or a biological macromolecule containing carboxyl and ortho-hydroxyl;

and 2, dissolving the phenylboronic acid polymer in water, and adjusting the pH value of the mixed solution to 8-9 to obtain the hydrogel.
The method for preparing an injectable hydrogel having anti-inflammatory and repair promoting effects as claimed in claim 77, wherein the injectable hydrogel is prepared by mixing a hydrophilic drug and/or a micelle encapsulating a hydrophobic drug with a phenylboronic acid polymer aqueous solution, and adjusting the pH of the mixed solution to 8-9.
The method of preparing an injectable hydrogel having anti-inflammatory and repair promoting effects as claimed in claim 77, wherein the amphiphilic drug carrier and the hydrophobic drug are dissolved in a benign solvent, water is slowly added thereto under stirring to prepare a drug-loaded micelle solution, the hydrophilic drug and/or the drug-loaded micelle solution is mixed with the phenylboronic acid polymer aqueous solution, and the pH of the mixed solution is adjusted to 8 to 9.
The method for preparing an injectable hydrogel with anti-inflammatory and repair promoting effects as claimed in claim 77, wherein the specific reaction process of step 1 is as follows: dissolving the functional polymer containing the ortho-position hydroxyl to obtain a functional polymer solution containing the ortho-position hydroxyl, adding a condensing agent and phenylboronic acid containing amino or carboxyl into the solution, stirring the solution for 20 to 30 hours at the temperature of between 30 and 40 ℃, dialyzing the solution in deionized water, and then carrying out freeze drying to obtain the purified phenylboronic acid polymer.
The method of claim 80, wherein the weight ratio of the functional polymer with ortho-position hydroxyl group, the condensing agent and the phenylboronic acid with amino group, hydroxyl group or carboxyl group is 4-6:4-6: 1-2.
The method of making an injectable hydrogel with anti-inflammatory and repair promoting properties as claimed in claim 80 wherein the condensing agent comprises 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.
The method of claim 79, wherein the amphiphilic drug carrier is prepared by the steps of: completely dissolving a hydrophilic high molecular polymer and a hydrophobic molecule in dimethyl sulfoxide at 80 ℃, adding N, N '-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, continuously stirring for 48 hours at 80 ℃, dialyzing a reactant in water for 2 days, removing unreacted hydrophobic molecule, and finally freeze-drying for later use, wherein the mass ratio of the hydrophilic high molecular polymer to the hydrophobic molecule to the N, N' -dicyclohexylcarbodiimide to the 4-dimethylaminopyridine is 2:1.5:1: 0.5.
The method of claim 83, wherein the hydrophilic polymer comprises hyaluronic acid, starch, cellulose, polyacrylic acid, polyacrylamide, polyvinyl alcohol, glycolic acid, and polylysine.
The method of claim 83, wherein the hydrophobic molecules comprise cholesterol, polyolefin, polycarbonate, polyamide, polyacrylonitrile, polyester, polylactic acid and acrylate.
The method of preparing an injectable hydrogel having anti-inflammatory and repair promoting effects of claim 83, wherein the amphiphilic drug carrier and the hydrophobic drug are dissolved in a benign solvent, then heated to 70-90 ℃, water is added dropwise thereto under stirring, and after dialysis, a drug-loaded micelle solution with a concentration of 0.5-1.5mg/mL is obtained.
The method of claim 86, wherein the hydrophilic drug and the phenylboronic acid polymer are dissolved in the drug-loaded micellar solution such that the concentration of the phenylboronic acid polymer in the drug-loaded micellar solution is 7-11% w/v.
The method of claim 87, wherein the injectable hydrogel is prepared by adding an alkaline solution to the aqueous solution of phenylboronic acid polymer containing the drug-loaded micelle and/or the hydrophilic drug, and adjusting the pH to 8.5.
The method for preparing an injectable hydrogel with anti-inflammatory and repair promoting effects of claim 87, wherein the mass ratio of the hydrophilic drug to the phenylboronic acid polymer is 1: 10.
The method of claim 77, wherein the amino, hydroxy or carboxy group-containing phenylboronic acid is ortho-aminobenzeneboronic acid, meta-aminobenzeneboronic acid, para-aminobenzeneboronic acid, ortho-hydroxyphenylboronic acid, meta-hydroxyphenylboronic acid, para-hydroxyphenylboronic acid, ortho-carboxyphenylboronic acid, meta-carboxyphenylboronic acid or para-carboxyphenylboronic acid.
The method of claim 78, wherein the hydrophilic drug is at least one of a growth factor, a gene, and a water-soluble protein drug.
The method of claim 78, wherein the hydrophobic drug is at least one of an anti-inflammatory agent, an angiogenesis promoting agent, a cell proliferation promoting agent, and a cell migration promoting agent.
The preparation method according to any one of claims 77 to 92, wherein the injectable hydrogel has anti-inflammatory and repair-promoting functions.
A hydrogel for repairing a cardiac injury, wherein the hydrogel is the injectable hydrogel of claim 93 acting at the site of the cardiac injury.
A hydrogel for treating heart failure, wherein the hydrogel is the injectable hydrogel of claim 93 for application to a diseased site in the heart.
A hydrogel for treating myocardial infarction, wherein the hydrogel is the injectable hydrogel according to claim 93 acting on the site of myocardial infarction.
Use of the injectable hydrogel with anti-inflammatory and repair-promoting effects of claim 93 for repairing cardiac injuries.
Use of an injectable hydrogel with anti-inflammatory and pro-reparative effects according to claim 93 for the treatment of heart failure.
Use of the injectable hydrogel with anti-inflammatory and repair-promoting effects of claim 93 for the treatment of myocardial infarction.
A method for repairing a cardiac injury by applying the injectable hydrogel of claim 93 to the site of the cardiac injury.
A method of treating heart failure by applying the injectable hydrogel of claim 93 to a site of a cardiac disorder.
A method of repairing a cardiac injury by injecting the injectable hydrogel of claim 93 at the site of the cardiac lesion.
A method of treating heart failure by injecting the injectable hydrogel of claim 93 at a site of myocardial infarction.
A method of treating myocardial infarction, wherein the injectable hydrogel of claim 93 is injected at the site of myocardial infarction.