CN116392633A - Injectable heart failure treatment hydrogel based on recombinant humanized collagen and preparation method thereof - Google Patents

Abstract

The invention discloses an injectable heart failure treatment hydrogel based on recombinant humanized collagen and a preparation method thereof, comprising the following steps: s1, preparing a functional polymer with a side chain containing a phenylboronic acid structure by adopting a polymer containing a carboxyl group and a compound containing an amino group and having the phenylboronic acid structure; s2, dissolving a water-soluble bioactive substance or a drug-loaded nano micelle in a compound solution containing two or more catechol structures to prepare a solution b; s3, preparing the functional polymer prepared in the step S1 into a solution a, and mixing the solution a with the solution b prepared in the step S2 to prepare the injectable heart failure treatment hydrogel based on the recombinant humanized collagen. The injectable heart failure treatment hydrogel based on the recombinant humanized collagen prepared by the invention not only can play a role in mechanical support, but also has obvious cell growth promoting function and antioxidant activity, and can effectively promote the generation of blood vessels at the damaged heart part and the repair of damaged myocardial tissues.

Description

An injectable hydrogel for treating heart failure based on recombinant humanized collagen and its Preparation

technical field

The invention belongs to the technical field of medical materials, and in particular relates to an injectable hydrogel for treating heart failure based on recombinant humanized collagen and a preparation method thereof.

Background technique

Heart failure, referred to as heart failure, is mainly a syndrome secondary to cardiovascular disease, including cardiac circulation disorders caused by some damage to the myocardium, cardiac systolic and diastolic function disorders, and a reduction in the effective ejection of the heart. symptoms. Myocardial infarction and related heart failure are the leading causes of death. Although direct percutaneous coronary intervention has improved the early survival rate of myocardial infarction, about 50% of patients still develop heart failure. The main difficulty in the treatment of heart failure comes from the limited ability of the heart tissue to regenerate itself. The current treatment options for heart failure are very limited, which can be divided into two categories: drug therapy and non-drug therapy. Drugs are currently the standard strategy. It can effectively improve myocardial contractility and reduce cardiac load, but it cannot curb the continuous decline of left ventricular function. Traditional non-drug treatments include cardiac resynchronization therapy, cardioverter-defibrillator, heart transplantation, etc. Although they can make up for the defects of drug treatment to a certain extent, they have the disadvantages of high price, short duration of curative effect, limited source of donors, and surgery. High risk, multiple clinical complications and other limitations, therefore, there is an urgent need for new therapeutic drugs to repair the heart after myocardial infarction.

Contents of the invention

The purpose of the present invention is to provide an injectable hydrogel for treating heart failure based on recombinant humanized collagen and its preparation method, which has good biocompatibility under physiological conditions, reduces the level of active oxygen and It has the advantages of promoting cell proliferation, migration, and angiogenesis in the damaged heart, so it can be used to achieve the purpose of functional repair of the damaged heart.

In order to achieve the above object, the present invention provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

S1: Using a carboxyl-containing polymer and an amino-containing compound with a phenylboronic acid structure to prepare a functional polymer with a phenylboronic acid structure in the side chain;

S2: Dissolving water-soluble bioactive substances or drug-loaded nano-micelles in a compound solution containing two or more catechol structures to prepare solution b;

S3: Prepare the functional polymer prepared in step S1 into solution a, and mix it with the solution b obtained in step S2 to prepare an injectable hydrogel for treating heart failure based on recombinant humanized collagen.

Further, step S1 specifically includes the following steps:

S1.1. Dissolving the polymer containing carboxyl groups in a buffer solution with a pH value of 4.6-6.0 to obtain a polymer solution, adding a mixed activation reagent to the polymer solution and stirring for 3-6 hours;

S1.2. Add a compound containing an amino group and a phenylboronic acid structure to the product prepared in step S1.1, and react for 24-48 hours at 30-40°C under the reaction conditions of inert gas protection, and pass the reaction product through deionized water After dialysis and freeze-drying, a functional polymer with a phenylboronic acid structure in the side chain can be prepared.

Further, the polymer containing carboxyl group is sodium alginate, hyaluronic acid, carboxymethyl chitosan, carboxymethyl cellulose, gelatin or their modified products; the compound containing amino group and having phenylboronic acid structure is 4- Aminophenylboronic acid, 3-aminophenylboronic acid or their derivatives; the mixed activating reagent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, containing The mass ratio of carboxyl polymer to 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is 10:1-3:0.5.

Further, in step S1, the mass ratio of the carboxyl group-containing polymer to the amino group-containing compound having a phenylboronic acid structure is 0.8-2:1.

Further, the water-soluble biologically active substance is recombinant type I humanized collagen, recombinant type III humanized collagen, anti-inflammatory drugs, growth factors, gene drugs or drugs for promoting angiogenesis.

Preferably, the recombinant type II humanized collagen refers to: the full-length or partial amino acid sequence fragment encoded by the human type III collagen specific type gene prepared by DNA recombinant technology, or a combination containing functional fragments of human collagen.

The amino acid sequence of the recombinant type III humanized collagen is as follows:

GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

Preferably, the recombinant type I humanized collagen refers to: a full-length or partial amino acid sequence fragment encoded by a specific type gene of human type I collagen prepared by DNA recombinant technology, or a combination containing functional fragments of human collagen.

The amino acid sequence of recombinant type I humanized collagen is as follows:

GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS.

Preferably, the compound containing two or more catechol structures is proanthocyanidin, tannic acid, epigallocatechin gallate, ellagic acid, epigallocatechin, epicatechin gallate, cotton phenols or their derivatives.

Further, drug-loaded nanomicelles were prepared by the following method:

Dissolve the amphiphilic polymer and hydrophobic drug in a benign solvent, add deionized water dropwise while stirring continuously, stir for 2 to 4 hours, and dialyze in deionized water to obtain it;

The mass ratio of the amphiphilic polymer to the hydrophobic drug is 5-7:1.

The above-mentioned hydrophobic drugs are anti-inflammatory drugs, drugs that promote cell proliferation or drugs that promote cell migration; among them, curcumin, MMP-9 agonist pyrazolone derivatives, etc. are preferred.

The above-mentioned good solvent is dimethyl sulfoxide, N,N dimethylformamide, methanol or acetone.

Preferably, during the preparation of drug-loaded nanomicelles, the amphiphilic polymer is prepared by the following method: the hydrophilic polymer and the hydrophobic molecule are completely dissolved in a mixed solvent of water and dimethyl sulfoxide at room temperature, Add N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to it, stir continuously for 45-50 hours, dialyze the mixture in water for 2-3 days, and finally lyophilize it for later use; wherein, the hydrophilic The mass ratio of the polymer, the hydrophobic molecule, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is 4-6:3-5:2-4:1.

In the preparation method of the above-mentioned amphiphilic polymer, the hydrophilic polymer is hyaluronic acid, sodium alginate, dextran, chitosan, sodium carboxymethyl cellulose, polyethylene glycol, polylysine or poly Ethyleneimine; the hydrophobic molecule is cholesterol, p-aldehyde, p-hydroxymethylbenzaldehyde, polylactic acid, phospholipids or hydrophobic polypeptides.

Further, in the mixed solution in step S2, the concentration of water-soluble bioactive substances or drug-loaded nano-micelles is 0.1-8.0 g/L, and the concentration of compounds containing two or more catechol structures is 5-20 wt %.

Further, the mixing volume ratio of solution a to solution b in step S3 is 2-6:1.

Further, in the mixed solution in step S3, the mass concentration of the functional polymer containing phenylboronic acid structure is 2-15 wt%.

The present invention also provides an injectable hydrogel for treating heart failure based on recombinant humanized collagen prepared by the above preparation method. The above-mentioned hydrogel is uniformly mixed with a compound solution containing two or more catechol structures containing biologically active substances and a functional polymer solution containing phenylboronic acid in a sterile environment to form a gel, and then filled into a syringe Or in the delivery system, direct multi-site injection into the ventricular wall for treatment.

In summary, the present invention has the following advantages:

1. In the preparation method of the present invention, natural polyphenol compounds containing two or more catechol structures such as proanthocyanidins have good antioxidant activity, and can be cross-linked with functional polymers containing phenylboronic acid to prepare injectable Hydrogel, and can be loaded with a variety of active substances, has the potential to promote the repair of damaged cardiac muscle tissue.

In the preparation method of the present invention, the hydrogel based on boronic acid ester bond and containing catechol structure has good biocompatibility, is not easy to cause immune reaction of the body, and has good injectability and antioxidant activity, and can Effectively loaded with biologically active substances (including recombinant type I humanized collagen, recombinant type III humanized collagen and growth factors), it plays an important role in repairing cardiac damage, promoting cardiac angiogenesis, and improving cardiac function.

2. The preparation method of the present invention utilizes the dynamic borate bond cross-linking between the phenylboronic acid group and the catechol group, and the gelation is rapid; and the preparation method is simple, and the hydrogel has superior rheological properties and Injectable properties.

3. The injectable hydrogel for treating heart failure based on recombinant humanized collagen prepared by the present invention has good injection performance and anti-oxidation performance, and is loaded with biologically active substances such as recombinant humanized collagen, which can be used under physiological conditions. With good biocompatibility, it can reduce the level of reactive oxygen species and promote the proliferation, migration and angiogenesis of related cells in the damaged heart, and finally achieve the purpose of functional repair of the damaged heart.

4. The injectable hydrogel for the treatment of heart failure based on recombinant humanized collagen prepared by the present invention can not only play the role of mechanical support, but also has obvious cell growth-promoting function and antioxidant activity, which can effectively promote the recovery of damaged hearts. Partial angiogenesis and repair of damaged myocardial tissue.

Description of drawings

Fig. 1 is the gel image of hydrogel in the embodiment of the present invention 1;

Fig. 2 is the cardiomyocyte toxicity result of hydrogel in the embodiment of the present invention 1;

Fig. 3 is the ultrasonic experiment result of animal treatment of hydrogel in the embodiment 1 of the present invention;

Fig. 4 is the slice result of animal treatment of the hydrogel in Example 1 of the present invention.

Detailed ways

The principles and features of the present invention are described below in conjunction with the examples, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Example 1

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

4 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (2 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 2

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

5 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 3

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

Preparation of proanthocyanidin solution loaded with vascular endothelial growth factor in S2

1.0 mg of vascular endothelial growth factor was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of proanthocyanidin solution loaded with vascular endothelial growth factor under stirring, and immediately cross-linked to form a hydrogel.

Example 4

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

Preparation of proanthocyanidin solution loaded with deferoxamine (pro-angiogenic drug) in S2

1.0 mg of deferoxamine was dissolved in 1 mL of proanthocyanidin solution (10 wt%), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt%) solution was slowly added to 1 mL of deferoxamine-loaded proanthocyanidin solution under stirring, and immediately cross-linked to form a hydrogel.

Example 5

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

Preparation of S2 drug-loaded nanomicelles

10 mg of polylactic-co-glycolic acid (PLGA) and 1 mg of curcumin were dissolved in DMSO (dimethyl sulfoxide 10 mL), and then added dropwise to 10 mL of water with slow stirring, and finally, the mixture was Dialyze in water for 2 days to prepare drug-loaded nanomicelles. The PLGA in this example is commercially available and does not need to be prepared separately.

Preparation of proanthocyanidin solution loaded with drug-loaded nanomicelles in S3

Dissolve 0.1 mg of drug-loaded nano-micelles in 1 mL of proanthocyanidin solution (10 wt%), and fully dissolve at room temperature.

Preparation of S4 hydrogel

At room temperature, 1 mL of ALG-BA (10 wt%) solution was slowly added to 1 mL of proanthocyanidin solution loaded with drug-loaded nanomicelles under stirring, and the hydrogel was immediately cross-linked.

Example 6

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

S1 Synthesis of Phenylboronic Acid Modified Hyaluronic Acid (HA-BA)

S1.1 Dissolve 1 g of hyaluronic acid in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the hyaluronic acid.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain HA-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of HA-BA (10 wt%) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 7

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

S1 Synthesis of phenylboronic acid modified carboxymethyl chitosan (CMCS-BA)

S1.1 Dissolve 1 g of carboxymethyl chitosan in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 In the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in carboxymethyl chitosan.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain CMCS-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of CMCS-BA (10 wt %) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 8

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

S1 Synthesis of phenylboronic acid modified carboxymethyl cellulose (CMC-BA)

S1.1 Dissolve 1 g of carboxymethyl cellulose in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 Into the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in carboxymethyl cellulose.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain CMC-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of CMC-BA (10 wt %) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 9

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

S1 Synthesis of phenylboronic acid modified gelatin (GL-BA)

S1.1 Dissolve 1 g of gelatin in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl groups in the gelatin.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain GL-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of GL-BA (10 wt%) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 10

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 4-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Proanthocyanidin Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of proanthocyanidin solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of proanthocyanidin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 11

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

Preparation of tannic acid solution loaded with recombinant type III humanized collagen in S2

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of tannic acid solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of tannic acid solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 12

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Epigallocatechin Gallate Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant humanized collagen type III was dissolved in 1 mL of epigallocatechin gallate solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of recombinant type III humanized collagen-loaded epigallocatechin gallate solution under stirring, and immediately cross-linked to form a hydrogel.

Example 13

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Ellagic Acid Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant humanized collagen type III was dissolved in 1 mL of ellagic acid solution (10 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of ellagic acid solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 14

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Epigallocatechin Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of epigallocatechin solution (12 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of epigallocatechin solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 15

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of epicatechin gallate solution loaded with recombinant type III humanized collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of epicatechin gallate solution (12 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of epicatechin gallate solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Example 16

This embodiment provides a method for preparing an injectable hydrogel for treating heart failure based on recombinant humanized collagen, comprising the following steps:

Synthesis of S1 Phenylboronic Acid Modified Sodium Alginate (ALG-BA)

S1.1 Dissolve 1 g of sodium alginate in 100 mL of distilled water, and keep the solution in a protective atmosphere of _N2 .

S1.2 Add 575 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 345 mg of NHS (N-hydroxysuccinimide) to MES buffer at pH 5.0 To the solution, add the solution prepared in step S1.1 and stir for 3 h to activate the carboxyl group in the sodium alginate.

S1.3 Then, 569mg of 3-aminophenylboronic acid was added to the mixed solution prepared in step 1.2, and the reaction was continued at room temperature for 36h.

S1.4 After the reaction, the solution was dialyzed under acidic conditions for 3 days and freeze-dried to obtain ALG-BA.

S2 Preparation of Gossypol Solution Loaded with Recombinant Type III Humanized Collagen

4.0 mg of recombinant type III humanized collagen was dissolved in 1 mL of gossypol solution (12 wt %), and fully dissolved at room temperature.

Preparation of S3 hydrogels

At room temperature, 1 mL of ALG-BA (10 wt %) solution was slowly added to 1 mL of gossypol solution loaded with recombinant type III humanized collagen under stirring, and immediately cross-linked to form a hydrogel.

Performance Testing

Taking the substance prepared in Example 1 as an example, the specific operation process and results are as follows (unless otherwise specified, the hydrogel groups 1-4 represent the following combinations respectively):

Hydrogel group 1 (Hydrogel 1): blank hydrogel;

Hydrogel group 2 (Hydrogel 2): hydrogel entrapped curcumin;

Hydrogel group 3 (Hydrogel 3): Hydrogel encapsulates recombinant humanized type III collagen;

Hydrogel group 4 (Hydrogel 4): hydrogel entrapped curcumin and recombinant humanized type III collagen.

1. Test the gelation performance of hydrogel

Figure 1 is a diagram of the preparation and injectability of the hydrogel. The hydrogel can be injected from a 1mL syringe, which proves that the hydrogel has good injectability.

2. Biocompatibility testing of hydrogels

The biocompatibility of the hydrogel was evaluated using rat cardiomyocytes (H9C2). The hydrogel sterilized by ultraviolet light was leached in the cell culture medium (0.2g/mL) for 48h to prepare material extract, and H9C2 cells were inoculated in 96-well plates at a seeding density of 8000 cells per well. After 24 hours, the cell culture solution was removed, and the hydrogel extract was added to the well plate instead of different hydrogel samples. The proliferation rate of H9C2 cells cultured for 72 hours was detected by CCK-8. After incubation for 72 h, fresh medium (90 μL) and diluted CCK-8 solution (10 μL) were added to each well. After 2 h, the cell proliferation rate was calculated by measuring the absorbance at 450 nm with a microplate reader.

The results of the survival rate of the hydrogel on rat cardiomyocytes are shown in Figure 2. The results showed that all the hydrogel groups showed no toxicity to the cells at 72 hours, indicating that the hydrogel had good cytocompatibility. In addition, after the hydrogel was loaded with biologically active substances, the recombinant type III humanized collagen, the cell survival rate was higher than that of the control group and the blank hydrogel group, indicating that the recombinant type III humanized collagen effectively promoted the proliferation of cells , indicating that the hydrogel has good biocompatibility.

3. In vivo effectiveness testing of hydrogels

A disease model of acute myocardial infarction in rats was constructed by permanently ligation of the left anterior descending branch of the heart. Immediately after ligation, physiological saline or hydrogel was injected into the myocardial infarction area, 3-5 sites were injected, and 20 μL was injected at each point. On the 14th and 28th day after operation, the rats with myocardial infarction were detected by echocardiography (directly observe the movement of the ventricular wall by using the M-mode image); the rat heart samples were obtained on the 14th and 28th day for section staining : Observe the wall thickness and fibrosis degree of left ventricle by Masson staining.

The results of M-mode echocardiography in rats with myocardial infarction on the 14th and 28th days after operation are shown in Figure 3. The left ventricular wall systolic and diastolic of the rats in the sham operation group (SHAM) was normal and showed a standard M-mode; the rats in the myocardial infarction group (MI ) of the ventricular wall stopped contraction and relaxation, and was in a straight line; the ventricular wall of the blank hydrogel (Hydrogel 1) was slightly improved compared with the myocardial infarction group, which was related to the mechanical support of the hydrogel; the hydrogel loaded with curcumin, recombinant human Derived type III collagen (Hydrogel 2, 3, 4) also had a greater improvement compared with the blank hydrogel group, among which Hydrogel 4 had the most obvious therapeutic effect.

The results of MASSON staining of rats with myocardial infarction on the 14th and 28th day after operation are shown in Figure 4. The left ventricular wall thickness of the rats in the SHAM group was normal; the left ventricle in the MI group was negatively dilated and the ventricular wall was thinned; In the group containing curcumin in gel and recombinant humanized type III collagen, the ventricular wall thickened and the fiber area decreased sequentially, and the treatment effect of Hydrogel 4 was the most obvious.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

Although the specific implementation of the present invention has been described in detail, it should not be construed as limiting the protection scope of this patent. Within the scope described in the claims, various modifications and deformations that can be made by those skilled in the art without creative work still belong to the protection scope of this patent.

Claims (10)

1. A preparation method for an injectable heart failure treatment hydrogel based on recombinant humanized collagen, characterized in that, comprising the following steps: S1: Using a carboxyl-containing polymer and an amino-containing compound with a phenylboronic acid structure to prepare a functional polymer with a phenylboronic acid structure in the side chain; S2: Dissolving water-soluble bioactive substances or drug-loaded nano-micelles in a compound solution containing two or more catechol structures to prepare solution b; S3: Prepare the functional polymer prepared in step S1 into solution a, and mix it with the solution b obtained in step S2 to prepare an injectable hydrogel for treating heart failure based on recombinant humanized collagen. 2. the preparation method as claimed in claim 1 is characterized in that, specifically comprises the following steps in the described step S1: S1.1. Dissolving the polymer containing carboxyl groups in a buffer solution with a pH value of 4.6-6.0 to obtain a polymer solution, adding a mixed activation reagent to the polymer solution and stirring for 3-6 hours; S1.2. Add a compound containing an amino group and a phenylboronic acid structure to the product prepared in step S1.1, and react for 24-48 hours at 30-40°C under the reaction conditions of inert gas protection, and pass the reaction product through deionized water After dialysis and freeze-drying, a functional polymer with a phenylboronic acid structure in the side chain can be prepared. 3. preparation method as claimed in claim 2, is characterized in that, the polymer containing carboxyl is sodium alginate, hyaluronic acid, carboxymethyl chitosan, carboxymethyl cellulose, gelatin or their modification Sexual product; The compound containing amino and phenylboronic acid structure is 4-aminophenylboronic acid, 3-aminophenylboronic acid or their derivatives; The mixed activating reagent is 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide and N-hydroxysuccinimide, the carboxyl-containing polymer and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N -The mass ratio of hydroxysuccinimide is 10:1-3:0.5. 4. The preparation method according to claim 1, characterized in that, in the step S1, the mass ratio of the carboxyl-containing polymer to the amino-containing compound having a phenylboronic acid structure is 0.8-2:1. 5. The preparation method according to claim 1, wherein the water-soluble bioactive substance is recombinant type I humanized collagen, recombinant type III humanized collagen, anti-inflammatory drugs, growth factors, gene Drugs or pro-angiogenic drugs; The compound containing two or more catechol structures is proanthocyanidin, tannic acid, epigallocatechin gallate, ellagic acid, epigallocatechin, epicatechin gallate, gossypol or their derivatives. 6. The preparation method according to claim 1, wherein the drug-loaded nano-micelle is prepared by the following method: Dissolve the amphiphilic polymer and hydrophobic drug in a benign solvent, add deionized water dropwise while stirring continuously, stir for 2 to 4 hours, and dialyze in deionized water to obtain it; The mass ratio of the amphiphilic polymer to the hydrophobic drug is 5-7:1. 7. The preparation method according to claim 1, characterized in that, in the mixed solution of step S2, the concentrations of water-soluble bioactive substances or drug-loaded nano-micelles are both 0.1-8.0 g/L, containing two The concentration of the compound with the above catechol structure is 5-20wt%. 8. The preparation method according to claim 1, characterized in that, in the step S3, the mixing volume ratio of solution a to solution b is 2-6:1. 9. The preparation method according to claim 1 or 8, characterized in that, in the mixed solution in step S3, the mass concentration of the functional polymer containing phenylboronic acid structure is 2-15 wt%. 10. The injectable hydrogel for treating heart failure based on recombinant humanized collagen prepared by the preparation method according to any one of claims 1-9.

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