WO2023221152A1

WO2023221152A1 - Intelligent hydrogel with cardiac injury repair function, method for preparing same, and use thereof

Info

Publication number: WO2023221152A1
Application number: PCT/CN2022/094699
Authority: WO
Inventors: 王云兵; 杨立; 胡成; 龙林宇; 刘文琦; 杨霞; 张兴栋
Original assignee: 四川大学
Priority date: 2022-05-19
Filing date: 2022-05-24
Publication date: 2023-11-23
Also published as: CN117338697A; CN114767625A

Abstract

The present invention belongs to the technical field of medical materials. Provided are an intelligent hydrogel with a cardiac injury repair function, a method for preparing same, and use thereof. The method for preparing the hydrogel is as follows: an aldehyde group-containing polymer and an amino-containing molecule are subjected to a Schiff base reaction to prepare an intelligent hydrogel material with good biocompatibility, and meanwhile, a bioactive material is loaded to achieve the purposes of repairing cardiac injury and alleviating heart failure. The aldehyde group-containing polymer is at least one of an aldehyde-group PEG aldehyde group, a four-arm PEG aldehyde group, a six-arm PEG aldehyde group, an eight-arm PEG aldehyde group, an oxidized glucan polymer, an oxidized sodium alginate polymer, an oxidized hyaluronic acid polymer, and an oxidized methyl cellulose polymer. The amino-containing polymer is at least one of a water-soluble chitosan derivative, polylysine, polyethyleneimine, gelatin, an amino PEG amino, a four-arm PEG amino, a six-arm PEG amino, and an eight-arm PEG amino. The bioactive material is a recombinant I-type humanized collagen.

Description

Smart hydrogel with heart damage repair function and its preparation method and application

Technical field

This application relates to the technical field of medical materials, in particular to smart hydrogels with heart damage repair functions and their preparation methods and applications.

Background technique

Cardiovascular disease is currently the main cause of death in humans, and myocardial infarction and related heart failure are the main causes of death. Although direct percutaneous coronary intervention has improved the early survival rate of myocardial infarction, about 50% of patients will still develop myocardial infarction. Heart failure. Current effective treatments for heart failure include left ventricular assist devices and heart transplantation. However, the former method is limited by its reliance on long-term use of external equipment, and the latter method is limited by the severe shortage of donor organs, and both Both methods are extremely risky, so new clinical methods are urgently needed to repair the heart after myocardial infarction.

Existing treatment methods include direct injection of growth factors, small molecule drugs, stem cells and nucleic acids to treat myocardial infarction. However, they face low bioavailability, non-specific molecular delivery, limited cell proliferation, and the inability to form new functional heart tissue and genes. A series of issues such as retention difference. Injectable hydrogels can minimally invasively inject bioactive substances or cells with therapeutic effects into the infarction site to achieve targeted release, which greatly solves the above problems.

Stimulus-responsive hydrogels have attracted widespread attention due to their ability to change shape in response to environmental changes, such as reactive oxygen species (ROS), pH, temperature, enzymes, light, and ultrasound. In drug delivery, compared with traditional hydrogels, stimuli-responsive hydrogels can achieve smart local on-demand release of drugs at disease sites. Targeting the microenvironment of the myocardial infarction site, designing smart hydrogels that can respond accurately and quickly to multiple stimuli in the microenvironment plays an important role in repairing cardiac damage, promoting cardiac vascular regeneration, and improving cardiac function.

technical problem

The technical problem to be solved by the present invention is to improve the existing hydrogel formula, improve its injectable performance and controllable release ability of active substances, and be used to load recombinant type I humanized collagen bioactive materials, and provide a method that can be used A pH-responsive smart hydrogel that assists in the treatment of heart failure and promotes repair of damaged heart, making it have good biocompatibility under physiological conditions and promote cell proliferation and vascular regeneration in the damaged heart, and can respond to myocardial infarction The acidic inflammatory microenvironment at the site releases active substances to achieve functions such as remodeling of the damaged heart.

Technical solutions

In order to achieve the above objects, the technical solutions adopted by the present invention to solve the technical problems are:

A method for preparing a smart hydrogel with heart damage repair function, including the following steps under sterile conditions:

Step 1: Using PBS as a solvent, prepare an aldehyde group-containing polymer solution and an amino-containing polymer solution respectively;

Step 2, dissolving the biologically active substance in the aldehyde group-containing polymer solution;

The biologically active substance is recombinant type I humanized collagen;

Step 3: Mix an aldehyde group-containing polymer solution containing biologically active substances and an amino-containing polymer solution to obtain the smart hydrogel.

In step 1, the aldehyde-containing polymer, the solute of the amino-containing polymer, and the PBS solvent must meet the requirements of sterility and pyrogen-free to meet the application scenario of repairing heart damage.

In steps 1 to 3, all operations are performed at room temperature, that is, no additional heating or cooling is required. The room temperature is usually between -10°C and 40°C, and dissolution and mixing must be sufficient and complete.

The hydrogel provided in this application is prepared by cross-linking an aldehyde group-containing polymer and an amino-containing polymer through a Schiff base reaction.

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

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

GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS

The above-mentioned recombinant humanized collagen is a systematic study of the full-length sequence of human type I collagen, screening out high cell adhesion activity gene sequences with concentrated positive and negative charges, and computer-assisted protein structure prediction and verification screening through genetic engineering and fermentation engineering. The collagen material obtained through biosynthesis has the same sequence as human type I collagen. It has high water solubility and good biocompatibility. It can promote cell adhesion and proliferation without significant cytotoxicity and immunogenicity.

The recombinant type I humanized collagen used in this application has the effect of promoting cell proliferation. Compared with animal collagen, it greatly reduces the immunogenicity of animal-derived tissues and can effectively treat heart damage after myocardial infarction.

The hydrogel used to repair heart damage provided by this application is to uniformly mix an aldehyde-containing polymer solution loaded with biologically active substances and an amino-containing polymer solution in a sterile environment to form a gel, resulting in a higher water content. The hydrogel is then filled into a syringe or delivery system and injected directly into the ventricular wall for treatment.

Several optional methods are also provided below, but they are not used as additional limitations on the above-mentioned overall plan. They are only further additions or preferences. On the premise that there are no technical or logical contradictions, each optional method can be independently implemented for the above-mentioned overall plan. Combination can also be a combination between multiple optional methods.

Optionally, in the aldehyde group-containing polymer solution, the mass concentration of the aldehyde group-containing polymer is: 0.5 to 12% w/w.

Optionally, in the aldehyde group-containing polymer solution, the mass concentration of the aldehyde group-containing polymer is: 0.5 to 10% w/w.

Optionally, in the aldehyde group-containing polymer solution, the mass concentration of the aldehyde group-containing polymer is: 2.5 to 10% w/w.

Optionally, in the amino-containing polymer solution, the mass concentration of the amino-containing polymer is: 0.5 to 15% w/w.

Optionally, in the amino-containing polymer solution, the mass concentration of the amino-containing polymer is: 0.5 to 10% w/w.

Optionally, in the amino-containing polymer solution, the mass concentration of the amino-containing polymer is: 5 to 10% w/w.

Optionally, the aldehyde group-containing polymer is at least one of the following substances:

Aldehyde PEG aldehyde group, four-arm PEG aldehyde group, six-arm PEG aldehyde group, eight-arm PEG aldehyde group, oxidized dextran polymer, oxidized sodium alginate polymer, oxidized hyaluronic acid polymer, oxidized methylcellulose polymer.

In order to form a hydrogel, the aldehyde group-containing polymer used must be at least soluble in the PBS solvent to form a solution with uniform properties. Therefore, the molecular weight must be selected to at least satisfy the aldehyde group-containing polymer to have good solubility in PBS. Generally, the weight average molecular weight of aldehyde PEG aldehyde group, four-arm PEG aldehyde group, six-arm PEG aldehyde group, and eight-arm PEG aldehyde group is not greater than 10,000.

The aldehyde group-containing polymer described in step (1) is an oxidized ortho-hydroxyl-containing polymer. The ortho-hydroxyl-containing polymer includes: dextran, sodium alginate, hyaluronic acid, and methylcellulose. Or its modified product, the oxidizing agent is sodium periodate.

Optionally, the amino-containing polymer is at least one of the following substances:

Water-soluble chitosan derivatives, polylysine, polyethylenimine, gelatin, amino PEG amino, four-arm PEG amino, six-arm PEG amino, eight-arm PEG amino.

In order to form a hydrogel, the amino-containing polymer used must be at least soluble in the PBS solvent to form a solution with uniform properties. Therefore, the molecular weight must be selected so that at least the amino-containing polymer has good solubility in PBS. Generally, the weight average molecular weight of amino PEG amino, four-arm PEG amino, six-arm PEG amino, and eight-arm PEG amino is not greater than 10,000.

Water-soluble chitosan derivatives include: carboxymethyl chitosan and hydroxypropyl chitosan.

Aldehyde PEG aldehyde group, four-arm PEG aldehyde group, six-arm PEG aldehyde group, eight-arm PEG aldehyde group;

The amino-containing polymer is carboxymethyl chitosan or hydroxypropyl chitosan.

Optionally, the aldehyde group-containing polymer is a four-arm PEG aldehyde group;

The amino-containing polymer is at least one of the following substances:

Polylysine, polyethylenimine, gelatin, amino PEG amino, four-arm PEG amino, six-arm PEG amino, eight-arm PEG amino.

Oxidized dextran polymer, oxidized sodium alginate polymer, oxidized hyaluronic acid polymer, oxidized methylcellulose polymer;

Optionally, in the aldehyde group-containing polymer solution containing biologically active substances, the concentration of the biologically active substances is 1 to 8g/L.

Optionally, in the aldehyde group-containing polymer solution containing biologically active substances, the concentration of the biologically active substances is 1~5g/L.

Optionally, in step 3, the volume ratio of the aldehyde group-containing polymer solution and the amino group-containing polymer solution is: 6:1~1:6.

Optionally, in step 3, the volume ratio of the aldehyde group-containing polymer solution and the amino group-containing polymer solution is: 6:1~1:1.

Optionally, in step 3, the volume ratio of the aldehyde group-containing polymer solution and the amino group-containing polymer solution is: 1:1~1:6.

This application also provides a hydrogel for repairing heart damage, which is prepared using the preparation method.

The pH-responsive smart hydrogel used to repair heart damage is to uniformly mix an aldehyde-containing polymer solution loaded with bioactive substances and an amino-containing polymer solution in a sterile environment to form a gel, resulting in a higher water content. The hydrogel is then filled into a syringe or delivery system and injected directly into the ventricular wall for treatment.

This application also provides a hydrogel for repairing heart damage, where the hydrogel is the smart hydrogel that acts on the site of heart disease.

This application also provides the application of the smart hydrogel in treating cardiac injury.

beneficial effects

The beneficial effects produced by this application include at least one of the following:

1. The smart hydrogel loaded with active substances in this application uses the Schiff base reaction between aldehyde groups and amino groups to cross-link, and gels quickly;

2. The preparation process of the hydrogel in this application is simple. The hydrogel has excellent rheological properties and injectable properties, and has various dynamic functions such as self-healing and injectability;

3. The hydrogel of the present application has a pH response mechanism and can quickly respond and release active substances in the acidic inflammatory microenvironment at the site of myocardial infarction.

3. The hydrogel of the present application not only functions as a mechanical support, but also has an obvious function of promoting cell growth, and can effectively promote the formation and repair of blood vessels in the damaged heart.

Description of the drawings

Figure 1 is an image of the hydrogel in Example 1 of the present application;

Figure 2a is a scanning electron microscope image of the blank hydrogel;

Figure 2b is a scanning electron microscope image of the hydrogel prepared in Example 2;

Figure 2c is a scanning electron microscope image of the hydrogel prepared in Example 1;

Figure 3 is a graph showing the injectability results of the hydrogel in Example 1 of the present application;

Figure 4a is a frequency scan chart of the hydrogels of Example 1 and Example 2 of the present application;

Figure 4b is an experimental diagram of alternating step strain scanning of the hydrogels of Example 1 and Example 2 of the present application;

Figure 5 shows the results of live and dead staining of H9C2 cells in Example 1 and Example 2 of the present application;

Figure 6 is the cytotoxicity results of hydrogels on H9C2 cells in Example 1 and Example 2 of the present application;

Figure 7 is the results of cardiac ultrasound on 14d and 28d of the hydrogel in Example 1 and Example 2 of the present application.

Embodiments of the invention

Specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

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

In the following examples, the sequence of recombinant type I humanized collagen used is as follows:

GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS

Example 1

A method for preparing a smart hydrogel with heart damage repair function. The preparation steps are as follows:

(1) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg four-arm PEG aldehyde group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS, stirred at room temperature overnight to fully dissolve;

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 mg carboxymethyl chitosan was dissolved in 1 mL sterile PBS and stirred at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of bioactive substance recombinant type I humanized collagen in 1 mL of four-arm PEG aldehyde-based solution and fully dissolve at room temperature;

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 2

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

(3) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 1 mg of bioactive substance recombinant type I humanized collagen in 1 mL of four-arm PEG aldehyde-based solution and fully dissolve at room temperature;

(4) Preparation of gel

Example 3

(1) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg aldehyde PEG aldehyde group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS and stirred at room temperature overnight to fully dissolve;

(2) Preparation of amino-containing polymer solution

(3) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of bioactive substance recombinant type I humanized collagen in 1 mL of aldehyde-based PEG aldehyde-based solution and fully dissolve at room temperature;

(4) Preparation of gel

At room temperature, 1 mL of aldehyde-based PEG aldehyde-based solution loaded with active substances was slowly added to 1 mL of stirred carboxymethyl chitosan solution, which was immediately cross-linked to form a hydrogel.

Example 4

(1) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg six-arm PEG aldehyde group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS, stirred at room temperature overnight to fully dissolve;

(2) Preparation of amino-containing polymer solution

(3) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of bioactive substance recombinant type I humanized collagen in 1 mL of six-arm PEG aldehyde-based solution and fully dissolve at room temperature;

(4) Preparation of gel

Slowly add 1 mL of the six-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 5

(1) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg eight-arm PEG aldehyde group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS, stirred at room temperature overnight to fully dissolve;

(2) Preparation of amino-containing polymer solution

(3) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of bioactive substance recombinant type I humanized collagen in 1 mL of eight-arm PEG aldehyde-based solution and fully dissolve at room temperature;

(4) Preparation of gel

Slowly add 1 mL of the eight-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 6

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 Dissolve mg hydroxypropyl chitosan in 1 mL sterile PBS and stir at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL hydroxypropyl chitosan solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 7

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 Dissolve mg polylysine in 1 mL sterile PBS and stir at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL polylysine solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 8

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 Dissolve mg polyethylenimine in 1 mL sterile PBS and stir at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL polyethyleneimine solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 9

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 Dissolve mg gelatin in 1 mL sterile PBS and stir at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde-based solution loaded with active substances into the stirring 1 mL gelatin solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 10

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 mg aminoPEG amino (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS and stirred at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde solution loaded with active substances into the stirring 1 mL amino PEG amino solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 11

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 mg four-arm PEG amino group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS and stirred at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde solution loaded with active substances into the stirring 1 mL four-arm PEG amino solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 12

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 mg six-arm PEG amino group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS, stirred at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde solution loaded with active substances into the stirring 1 mL six-arm PEG amino solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 13

(1) Preparation of polymer solution containing aldehyde groups

(2) Preparation of amino-containing polymer solution

Under sterile conditions, accurately weigh 50 mg eight-arm PEG amino group (weight average molecular weight 2000) was dissolved in 1 mL sterile PBS, stirred at room temperature overnight to fully dissolve;

(3) Aldehyde-containing polymer solution loaded with active substances

(4) Preparation of gel

Slowly add 1 mL of the four-arm PEG aldehyde solution loaded with active substances into the stirring 1 mL eight-arm PEG amino solution at room temperature, and it will be immediately cross-linked to form a hydrogel.

Example 14

(1) Synthesis of oxidized dextran polymer (OD)

Precisely weigh 10.00 g of dextran and dissolve it in 200 mL of deionized water, and add 8.00 g of sodium periodate to it. Then, stir at 37°C for 6 h, add 3 mL of ethylene glycol and stir for 2 h to terminate the reaction. Finally, it was dialyzed in deionized water (pH 7.4) for 3 days. After 3 days, it was freeze-dried with a freeze dryer to obtain purified OD;

(2) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg OD was dissolved in 1 mL sterile PBS and stirred at room temperature overnight to fully dissolve;

(3) Preparation of amino-containing polymer solution

(4) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of bioactive substance recombinant type I humanized collagen in 1 mL OD solution and fully dissolve at room temperature;

(5) Preparation of gel

1 mL of OD solution loaded with active substances was slowly added to the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it was immediately cross-linked to form a hydrogel.

Example 15

(1) Synthesis of oxidized sodium alginate polymer (OSA)

Precisely weigh 10.00 g of sodium alginate and dissolve it in 200 mL of deionized water, and add 8.00 g of sodium periodate to it. Then, stir at 37°C for 6 h, add 3 mL of ethylene glycol and stir for 2 h to terminate the reaction. Finally, it was dialyzed in deionized water (pH 7.4) for 3 days. After 3 days, it was freeze-dried with a freeze dryer to obtain purified OSA;

(2) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg OSA was dissolved in 1 mL of sterile PBS and stirred at room temperature overnight to fully dissolve;

(3) Preparation of amino-containing polymer solution

(4) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of bioactive substance recombinant type I humanized collagen in 1 mL of OSA solution and fully dissolve at room temperature;

(5) Preparation of gel

1 mL of OSA solution loaded with active substances was slowly added to the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it was immediately cross-linked to form a hydrogel.

Example 16

(1) Synthesis of oxidized hyaluronic acid polymer (OHA)

Precisely weigh 10.00 g of hyaluronic acid and dissolve it in 200 mL of deionized water, and add 8.00 g of sodium periodate to it. Then, stir at 37°C for 6 h, add 3 mL of ethylene glycol and stir for 2 h to terminate the reaction. Finally, it was dialyzed in deionized water (pH 7.4) for 3 days. After 3 days, it was freeze-dried with a freeze dryer to obtain purified OHA;

(2) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 Dissolve mg OHA in 1 mL sterile PBS and stir at room temperature overnight to fully dissolve;

(3) Preparation of amino-containing polymer solution

(4) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of the bioactive substance recombinant type I humanized collagen in 1 mL of OHA solution and fully dissolve at room temperature;

(5) Preparation of gel

1 mL of OHA solution loaded with active substances was slowly added to the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it was immediately cross-linked to form a hydrogel.

Example 17

(1) Synthesis of oxidized methylcellulose polymer (OMC)

Precisely weigh 10.00 g of methylcellulose and dissolve it in 200 mL of deionized water, and add 8.00 g of sodium periodate to it. Then, stir at 37°C for 6 h, add 3 mL of ethylene glycol and stir for 2 h to terminate the reaction. Finally, it was dialyzed in deionized water (pH 7.4) for 3 days. After 3 days, it was freeze-dried with a freeze dryer to obtain purified OMC;

(2) Preparation of polymer solution containing aldehyde groups

Under sterile conditions, accurately weigh 50 mg OMC was dissolved in 1 mL sterile PBS and stirred at room temperature overnight to fully dissolve;

(3) Preparation of amino-containing polymer solution

(4) Aldehyde-containing polymer solution loaded with active substances

Under sterile conditions, dissolve 4 mg of the bioactive substance recombinant type I humanized collagen in 1 mL of OMC solution and fully dissolve at room temperature;

(5) Preparation of gel

1 mL of OMC solution loaded with active substances was slowly added to the stirring 1 mL carboxymethyl chitosan solution at room temperature, and it was immediately cross-linked to form a hydrogel.

Test example

Taking the substances prepared in Example 1 and Example 2 as an example, the specific operation process and results are as follows:

Unless otherwise specified, Hydrogel: blank hydrogel; 4 mg/ml I: hydrogel loaded with 4 mg/ml recombinant humanized type I collagen; 1 mg/ml I: loaded with 1 mg/ml recombinant type I collagen Hydrogels of humanized collagen.

1. Test the hydrogel prepared in step (4), and use the MCR302 rheometer to test the rheological properties of the hydrogel. A dual concentric cylinder geometry with a gap of 4 mm was used for steady shear flow at 37°C. Frequency sweep uses 1% strain and ranges from 0.1 to 100 Oscillation frequency in rad/s. The oscillation frequency of strain scanning is 1Hz, and the strain ranges from 0.01 to 1000%. In the self-healing experiment, alternating step strain scanning experiments (large strain: 1000%, 60s and small strain: 1%, 60s) were used. See picture for details.

Figure 1 is an image of 4 mg/ml I hydrogel; Figure 2a is a scanning electron microscope image of a blank hydrogel; Figure 2b is a scanning electron microscope image of the hydrogel prepared in Example 2; Figure 2c is a scanning electron microscope image of the hydrogel prepared in Example 1 Scanning electron microscope images of hydrogels; all hydrogels have a uniform porous structure; Figure 3 shows the injectability diagram of 4 mg/ml I hydrogel. The hydrogel can be injected from a 27G needle, proving that the hydrogel has Injectability; Figure 4a shows the frequency scan chart of the hydrogel. The results show that the storage modulus of the three hydrogels is between 550-580 Pa, and the storage modulus is greater than the loss modulus, and has stable Hydrogel structure. Figure 4b shows the results of the alternating step strain scan of hydrogels. The results show that after the hydrogel structure was destroyed three times, more than 85% of the storage modulus of the three groups of hydrogels was restored, proving that the hydrogels have relatively high performance. Strong self-healing performance.

2. Biocompatibility of hydrogels and their ability to protect cardiomyocytes from oxidative stress damage

Rat cardiomyocyte (H9C2) culture method was used to evaluate the hydrogel's protection of cardiomyocytes from oxidative stress damage and reduction of cardiomyocyte apoptosis. The sterilized hydrogel was extracted in cell culture medium (0.1 g/mL) for 48 h to prepare a material extraction solution. H9C2 was inoculated into a 96-well plate at a density of 8,000 cells per well. After 24 h, the cell culture medium was removed, and 200 μL H ₂ O ₂ was used to pretreat H9C2 for oxidative stress damage for 1 h, and then hydrogel extract was added to the well plate instead of different hydrogel samples. The proliferation rate and morphology of H9C2 cells cultured for 24 h and 72 h were detected using CCK-8 and FDA/PI staining, respectively. H9C2 cells were stained with FDA (30 μg/mL) and PI (10 μg/mL) and allowed to stand for 5 minutes. The cells were then observed with a fluorescence microscope, see Figure 5. After incubation for 24 h and 72 h, fresh culture 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 absorption value at 450 nm with a microplate reader.

The hydrogel survival rate results are shown in Figure 6. The results show that all hydrogel groups have good biocompatibility for cells at 24h and 72h. In addition, the hydrogels loaded with bioactive substances recombinant type I humanized After collagen administration, the cell survival rate was higher than that of the blank hydrogel group, indicating that recombinant type I humanized collagen protects cardiomyocytes from oxidative stress damage.

3. In vivo heart repair effect testing of hydrogel

In order to study the effect of hydrogel on cardiac repair in vivo, a rat myocardial infarction disease model was established. M-mode echocardiography was performed on the successfully modeled myocardial infarction rats on the 14th and 28th days. The results are shown in Figure 7. The ventricles of the hydrogel group loaded with the bioactive substance recombinant type I humanized collagen The wall contraction and relaxation movements were significantly better, indicating that the hydrogel loaded with the bioactive substance recombinant type I humanized collagen has a certain promoting effect on the repair of cardiac function after myocardial infarction.

The above-described embodiments only express several implementation modes of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all fall within the protection scope of the present application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

A method for preparing a smart hydrogel with heart damage repair function, which is characterized by including the following steps under sterile conditions:

Step 1: Using PBS as a solvent, prepare an aldehyde group-containing polymer solution and an amino-containing polymer solution respectively;

Step 2, dissolving the biologically active substance in the aldehyde group-containing polymer solution;

The biologically active substance is recombinant type I humanized collagen;

Step 3: Mix an aldehyde group-containing polymer solution containing biologically active substances and an amino-containing polymer solution to obtain the smart hydrogel.
The method for preparing a smart hydrogel with heart damage repair function according to claim 1, wherein the mass concentration of the aldehyde group-containing polymer in the aldehyde group-containing polymer solution is: 0.5 to 12 %w/w.
The method for preparing a smart hydrogel with heart damage repair function according to claim 2, wherein the mass concentration of the amino-containing polymer in the amino-containing polymer solution is: 0.5 to 15%w /w.
The method for preparing a smart hydrogel with heart damage repair function according to any one of claims 1 to 3, wherein the aldehyde group-containing polymer is at least one of the following substances:

Aldehyde PEG aldehyde group, four-arm PEG aldehyde group, six-arm PEG aldehyde group, eight-arm PEG aldehyde group, oxidized dextran polymer, oxidized sodium alginate polymer, oxidized hyaluronic acid polymer, oxidized methylcellulose polymer.
The method for preparing a smart hydrogel with heart damage repair function according to any one of claims 1 to 3, wherein the amino-containing polymer is at least one of the following substances:

Water-soluble chitosan derivatives, polylysine, polyethylenimine, gelatin, amino PEG amino, four-arm PEG amino, six-arm PEG amino, eight-arm PEG amino.
The method for preparing a smart hydrogel with heart damage repair function as claimed in claim 3, wherein the concentration of the bioactive substance in the aldehyde group-containing polymer solution containing the bioactive substance is 1 to 8 g/L. .
The method for preparing a smart hydrogel with heart damage repair function according to claim 3, characterized in that in step 3, the volume ratio of the aldehyde group-containing polymer solution and the amino-containing polymer solution is: 6:1~1:6.
A hydrogel for repairing heart damage, characterized in that it is prepared by the preparation method according to any one of claims 1 to 7.
A hydrogel for repairing heart damage, characterized in that the hydrogel is a smart hydrogel for repairing heart damage as claimed in claim 8 that acts on the heart injury site.
Use of the hydrogel for repairing heart damage as claimed in claim 8 in treating heart damage.