Anti-heart failure injectable hydrogel with myocardial tissue repair function and preparation method and application thereof

Abstract

The application discloses a heart failure treatment injectable hydrogel with myocardial tissue repair function and a preparation method and application thereof. The hydrogel is prepared by chelating alginic acid with multivalent cations, and active substances for repairing heart injury are loaded on the hydrogel. The active substances are recombinant humanized collagen type III. The hydrogel is injected into the left ventricular free wall through the endocardium, the left ventricular wall thickness is increased by implanting the hydrogel in the myocardium, the ventricular wall stress is adjusted, the left ventricular shape is influenced, the left ventricular enlargement is prevented or reversed, and meanwhile, the hydrogel is loaded with novel recombinant humanized collagen material with heart tissue injury repair function, so that the heart function is further improved and the heart repair is promoted.

Description

[0001] This application is a divisional application of the invention entitled "Injectable hydrogel with myocardial tissue repair function for heart failure and its preparation method and application", the original application was filed on May 7, 2022, and the original application number is 202210495091.5. Technical Field

[0002] This application belongs to the field of biomedical materials technology, specifically relating to an injectable hydrogel with myocardial tissue repair function for treating heart failure, its preparation method, and its application. Background Technology

[0003] Heart failure (HF) refers to impaired cardiac systolic / diastolic function, resulting in reduced blood supply to the heart and an inability to provide the body with the necessary blood flow. It is the end-stage clinical manifestation of almost all cardiovascular diseases, including coronary heart disease, hypertension, structural heart disease, and myocardial infarction. The treatment of heart failure remains a significant and unresolved challenge in the field of cardiovascular medicine.

[0004] Current treatment options for heart failure are very limited, mainly falling into two categories: pharmacological and non-pharmacological therapies. Pharmacological therapies can effectively improve myocardial contractility and reduce cardiac load, but they cannot curb the continuous decline in left ventricular function. Long-term use can cause side effects and has limited efficacy. Traditional non-pharmacological therapies include left ventricular assist devices, implantable cardioverter-defibrillators (ICDs), and heart transplantation. While these can compensate for the shortcomings of pharmacological therapies to some extent, preventing arrhythmias and increasing blood flow to alleviate symptoms, they cannot fundamentally cure heart failure. Heart transplantation, in particular, remains relatively rare due to donor scarcity and immune rejection. With the development of tissue engineering and regenerative medicine, implantable materials have become an effective treatment option for cardiac repair. Biomaterials such as hydrogels, collagen, extracellular matrix, and patches have been widely used in heart failure treatment research. Furthermore, by combining biomaterials with different active substances, they can effectively improve the treatment efficiency of heart failure, showing great promise for development and application in the biomedical field. Summary of the Invention

[0005] Implantable materials have become an effective treatment option for cardiac repair. This application provides an injectable hydrogel with myocardial tissue repair function for heart failure and its preparation method. The hydrogel is injected into the free wall of the left ventricle via the endocardium. By implanting the hydrogel into the myocardium, the thickness of the left ventricular wall is increased, the wall stress is adjusted, the morphology of the left ventricle is affected, and left ventricular enlargement is prevented or reversed. At the same time, the hydrogel is loaded with a novel recombinant humanized collagen material with cardiac tissue damage repair function, which further improves cardiac function and promotes cardiac repair.

[0006] The hydrogel was prepared by chelating alginate with multivalent cations, and the hydrogel was loaded with active substances for repairing heart damage.

[0007] Alginic acid is a natural polysaccharide with advantages such as low cost, easy availability, renewability, and good biocompatibility. It is a copolymer composed of α-L-mannuronic acid and β-D-guluronic acid linked by 1,4-glycosidic bonds. It contains a large number of carboxylic acid groups, which are chelated by polyvalent cations (such as divalent cations) to form hydrogels.

[0008] Hydrogels formed by alginate and polyvalent cations have good biocompatibility, are less likely to cause immune responses in the body, and have good injectability and a certain degree of bioinertness. They can effectively load bioactive substances and play a role in at least one aspect of cardiac damage repair, promoting cardiac angiogenesis, and improving cardiac function, depending on the different active substances loaded.

[0009] Hydrogels loaded with active substances have good biocompatibility under physiological conditions and can come into contact with the site of cardiac injury in various ways, including but not limited to: filling into a syringe or delivery system and injecting directly into the ventricular wall.

[0010] When hydrogels are injected into the site of cardiac injury, they not only provide effective mechanical support, but the active substances they carry also significantly promote cell growth, effectively promoting angiogenesis and repair in the heart.

[0011] Several alternative methods are provided below, but they are not intended as additional limitations on the overall solution above. They are merely further additions or optimizations. Provided there are no technical or logical contradictions, each alternative method can be combined individually with respect to the overall solution above, or multiple alternative methods can be combined with each other.

[0012] Appropriate water content ensures that the hydrogel has excellent rheological and injection properties, making it easy to fill into syringes or delivery systems and transport to the site of cardiac injury for treatment.

[0013] Optionally, the hydrogel is prepared by mixing an alginate solution and a calcium ion-containing solution, wherein the volume ratio of the alginate solution to the calcium ion-containing solution is 2:1 to 6:1.

[0014] Optionally, the concentration of alginate in the alginate solution is 0.5-5 wt%.

[0015] Optionally, the concentration of alginate in the alginate solution is 1-3 wt%.

[0016] Optionally, the concentration of calcium ions in the calcium ion-containing solution is 0.5-2 wt%.

[0017] Optionally, the concentration of calcium ions in the calcium ion-containing solution is 1-2 wt%.

[0018] Optionally, the alginate is sodium alginate, and the solute in the calcium ion-containing solution is at least one of calcium lactate, calcium carbonate, calcium gluconate, calcium citrate, and calcium chloride.

[0019] Optionally, the loading concentration of the active substance in the hydrogel is 1 to 10 g / L.

[0020] Optionally, the loading concentration of the active substance in the hydrogel is 1 to 5 g / L.

[0021] Optionally, the active substance is a protein containing an amino acid sequence fragment that can bind to cytokines.

[0022] The active substance has at least one of the following functions:

[0023] a) Promotes the repair of damaged heart;

[0024] b) Adjunctive treatment for heart failure;

[0025] c) Promotes cell proliferation in damaged areas of the heart;

[0026] b) Promotes angiogenesis;

[0027] e) Damaged cardiac remodeling.

[0028] Optionally, the active substance is recombinant type III humanized collagen.

[0029] The recombinant type III humanized collagen was prepared through screening and biosynthesis. It has the same amino acid sequence and structure as human type III collagen, and has the effect of promoting cell growth. Compared with animal collagen, it can reduce the immunogenicity of animal tissues and repair heart damage.

[0030] Optionally, the loading concentration of recombinant type III humanized collagen in the hydrogel is 1–10 g / L.

[0031] Optionally, the recombinant type III humanized collagen contains an amino acid sequence fragment that can bind to cytokines.

[0032] Recombinant type III humanized collagen refers to: full-length or partial amino acid sequence fragments encoded by specific type genes of human type III collagen prepared by DNA recombination technology, or combinations containing functional fragments of human collagen.

[0033] Optionally, the amino acid sequence of the recombinant type III humanized collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0034] The recombinant type III humanized collagen has no significant cytotoxicity and low immune rejection in the human body. Due to the presence of hydrophilic functional groups such as carboxyl, amino and guanidinyl groups in its structure, as well as the concentration of positive and negative charges in its structure, it has high water solubility and cell adhesion activity, and also has high endothelial cell affinity. It is a customized collagen that can be used for the modification of cardiovascular materials.

[0035] The recombinant type III humanized collagen has a significant cell growth promoting effect. Compared with animal collagen, it can reduce the immunogenicity of animal tissues and repair heart damage.

[0036] Recombinant type III human collagen is an important component of the human extracellular matrix, widely distributed in the vascular system and internal organs. Studies have found that type III collagen is a key component for wound healing and tissue regeneration, and it is widely used in tissue engineering research and tissue regeneration. Animal-derived type III collagen has limitations such as immunogenicity, batch-to-batch variability, and poor water solubility. In contrast, recombinant type III human collagen has the same amino acid sequence as collagen derived from human tissue, making it safer. Therefore, recombinant type III human collagen is the preferred choice for tissue engineering applications. However, recombinant type III human collagen has lower mechanical properties and is easily enzymatically broken down in the human body, which greatly weakens its efficacy in tissue repair and regeneration.

[0037] Optionally, the storage modulus of the hydrogel is 100-7000 Pa.

[0038] Optionally, the hydrogel has a three-dimensional network structure with a pore size of 30–100 μm.

[0039] The polyvalent cation is calcium ion. As a natural, non-toxic, and biocompatible biomaterial, calcium alginate hydrogel will not be hydrolyzed by enzymes in the human body. Its unique 3D network microstructure not only transports nutrients to tissues, but also has good mechanical properties, which can support cell proliferation and migration. However, the repair and regeneration function of calcium alginate hydrogel for cardiac cells still needs to be improved.

[0040] Optionally, the loading of recombinant type III humanized collagen in the hydrogel is 0.1-1 wt%.

[0041] Optionally, the loading concentration of recombinant type III humanized collagen in the hydrogel is 1–10 g / L.

[0042] This application also provides a method for preparing the aforementioned injectable hydrogel with myocardial tissue repair function for treating heart failure, comprising:

[0043] Alginate solution is mixed with a polyvalent cation solution containing active substances to obtain the injectable hydrogel with myocardial tissue repair function for treating heart failure.

[0044] Operate at room temperature, meaning no additional heating or cooling is required. Room temperature is typically between -10°C and 40°C. Dissolution and mixing must be thorough and complete.

[0045] The calcium alginate hydrogel for treating heart failure provided in this application is produced by uniformly mixing an alginate solution containing bioactive substances with a calcium ion-containing solution under sterile conditions to form a gel with high water content. The gel is then filled into a syringe or delivery system and injected directly into the ventricular wall for treatment.

[0046] The hydrogel was prepared under sterile conditions, where alginate was chelated by polyvalent cations to form a hydrogel.

[0047] The active substance can be premixed in an alginate solution or a polyvalent cation solution, and then the alginate solution and the polyvalent cation solution are mixed.

[0048] Optionally, an alginate solution containing active substances can be mixed with a polyvalent cation solution to obtain the injectable hydrogel with myocardial tissue repair function for treating heart failure.

[0049] Optionally, the alginate is sodium alginate, and the solute in the calcium ion-containing solution is at least one of calcium lactate, calcium carbonate, calcium gluconate, calcium citrate, and calcium chloride.

[0050] Optionally, the active substance is recombinant type III humanized collagen.

[0051] Optionally, the alginate solution and the multivalent cation solution use the same solvent, namely water or PBS buffer.

[0052] Optionally, the active substance is dissolved in the alginate solution and then mixed with a calcium ion-containing solution.

[0053] Optionally, the alginate solution is a sodium alginate PBS solution, wherein the concentration of sodium alginate in the sodium alginate PBS solution is 0.5-5 wt%.

[0054] The molecular formula of sodium alginate is (C6H7NaO6). x Under sterile conditions at room temperature, sterile and pyrogen-free sodium alginate is dissolved in PBS until fully dissolved to obtain a sodium alginate PBS solution. The dissolution time is determined based on the actual dosage, for example, 12 hours or 24 hours.

[0055] Optionally, the polyvalent cation solution is a calcium ion PBS solution, wherein the concentration of calcium ions in the calcium ion PBS solution is 0.5-2% wt%.

[0056] Under sterile conditions at room temperature, sterile, pyrogen-free calcium salts are dissolved in PBS until fully dissolved, yielding a PBS solution containing calcium ions. The dissolution time is determined based on the actual dosage, for example, 12 hours or 24 hours.

[0057] Optionally, the volume ratio of the sodium alginate PBS solution to the calcium ion PBS solution is 2:1 to 6:1.

[0058] When alginate solution is mixed with calcium ion solution, a hydrogel with high water content is formed, which improves injection performance and makes it easier to fill into syringes or delivery systems and deliver to the site of cardiac injury for treatment.

[0059] This application provides an injectable hydrogel for treating heart failure with myocardial tissue repair function, prepared using the aforementioned preparation method.

[0060] This application also provides a method for preparing calcium alginate hydrogel loaded with active substances, comprising the following steps:

[0061] Step (1) Preparation of sodium alginate system: Under aseptic conditions, a calculated amount of sterile, pyrogen-free sodium alginate was dissolved in sterile PBS and allowed to dissolve completely at room temperature;

[0062] Step (2) Preparation of calcium ion system: Under sterile conditions, sterile and pyrogen-free calcium salt is uniformly dissolved in sterile PBS and fully dissolved at room temperature;

[0063] Step (3) Loading active substances into the sodium alginate system: Under sterile conditions, dissolve the active substances in the sodium alginate system prepared in step (1) and allow it to dissolve completely at room temperature;

[0064] Step (4) Crosslinking of sodium alginate system and calcium ion system: Under sterile conditions, the sodium alginate system loaded with active substances prepared in step (3) and the calcium ion system in step (2) are thoroughly mixed in a certain proportion and uniformly gelled.

[0065] Optionally, the active substance can be mixed first with a calcium ion-containing solution, and then mixed with a sodium alginate solution.

[0066] Optionally, at least one of calcium alginate and calcium chloride can be dissolved in water to obtain the aqueous solution containing calcium ions.

[0067] Optionally, the calcium-containing aqueous solution is a calcium alginate aqueous solution, and the concentration of calcium alginate in the calcium alginate aqueous solution is 0.01-0.1 wt%.

[0068] Optionally, the calcium-containing aqueous solution is a calcium chloride aqueous solution, and the concentration of calcium chloride in the calcium chloride aqueous solution is 1-7 wt%.

[0069] Optionally, the active substance is an aqueous solution of recombinant type III humanized collagen, wherein the concentration of recombinant type III humanized collagen in the aqueous solution is 0.05 to 0.3 wt%.

[0070] Optionally, the sodium alginate content in the sodium alginate solution is 2-7 wt%.

[0071] Optionally, the mass ratio of the calcium ion-containing aqueous solution and the recombinant type III humanized collagen aqueous solution is 0.1–0.9:0.1–0.8.

[0072] Optionally, an aqueous solution containing calcium ions is mixed with an aqueous solution of recombinant type III humanized collagen to obtain a mixed solution, wherein the mass ratio of the mixed solution to the sodium alginate solution is 0.01–0.05: 0.1–0.5.

[0073] This application also provides a method for preparing the aforementioned injectable hydrogel for treating heart failure with myocardial tissue repair function, comprising the following steps performed under aseptic conditions:

[0074] Step 1: Prepare an aqueous solution containing calcium ions, denoted as solution A;

[0075] Prepare an aqueous solution of recombinant type III humanized collagen, denoted as solution B;

[0076] Step 2: Mix solution A and solution B thoroughly to obtain solution C;

[0077] Step 3: Mix solution C thoroughly with sodium alginate solution to obtain the hydrogel.

[0078] The amino acid sequence of the recombinant type III humanized collagen is as follows:

[0079] GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0080] The composite hydrogel prepared by this method not only improves the mechanical properties and biodegradability of type III humanized collagen, but also enhances the ability of calcium alginate to repair and regenerate tissues. Therefore, this hydrogel can maintain the normal function of cardiomyocytes and tissues, and also promote the repair and regeneration of damaged tissues, which is expected to achieve a complete cure for heart failure.

[0081] The composite hydrogel prepared by this method is not easily hydrolyzed by enzymes, can repair tissues, and induce normal proliferation and growth of cells and tissues.

[0082] Optionally, at least one of calcium alginate and calcium chloride can be dissolved in water to obtain the aqueous solution containing calcium ions.

[0083] Optionally, the calcium-containing aqueous solution is a calcium alginate aqueous solution, and the concentration of calcium alginate in the calcium alginate aqueous solution is 0.01-0.1 wt%.

[0084] Optionally, the calcium-containing aqueous solution is a calcium chloride aqueous solution, and the concentration of calcium chloride in the calcium chloride aqueous solution is 1-7 wt%.

[0085] Optionally, the recombinant type III humanized collagen aqueous solution has a concentration of 0.05–0.3 wt%.

[0086] Optionally, the sodium alginate content in the sodium alginate solution is 2-7 wt%.

[0087] Optionally, the mass ratio of solution A to solution B when mixed is 0.1–0.9:0.1–0.8.

[0088] Optionally, the mass ratio of solution A to solution B when mixed is 1 to 1.125:1.

[0089] Optionally, the mass ratio of solution C to sodium alginate solution is 0.01–0.05: 0.1–0.5.

[0090] Optionally, the mass ratio of solution C to sodium alginate solution is 1:2 to 50.

[0091] This application also provides a method for preparing a hydrogel that can promote the repair and regeneration of myocardial tissue, the steps of which are as follows: (1) Prepare a medical-grade CaCl2 solution A with a concentration of 0.01-0.1wt% and set aside; (2) Weigh 0.001-0.006g of recombinant type III humanized collagen powder into a 15mL centrifuge tube, add 2g of distilled water, shake and mix well to obtain collagen aqueous solution B; (3) Take 0.1-0.9g of solution A and 0.1-0.8g of solution B respectively. (4) Mix solution B to obtain mixed solution C; (5) Transfer mixed solution C to 3 mL syringe 1 for later use; weigh 0.1-0.7 g of medical grade sodium alginate solution into 3 mL syringe 2; (6) Connect syringe 1 and syringe 2 using a three-way valve, and mix solution A and solution B by 20-40 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product; (7) Store the hydrogel product in an environment of 0-10℃.

[0092] A calcium alginate / recombinant type III humanized collagen hydrogel was prepared by physical mixing. This hydrogel has strong mechanical strength, is not easily degraded by enzymes, has a unique three-dimensional network microstructure, and can repair damaged tissue and induce tissue regeneration.

[0093] The advantage of this method is that it can prepare calcium alginate / recombinant type III humanized collagen composite hydrogel through simple physical mixing. The hydrogel has a three-dimensional network structure with a network pore size of 50 μm and a storage modulus of 100-7000 Pa, providing a new material for the treatment of heart failure.

[0094] This application also provides a method for preparing a hydrogel that can promote the repair and regeneration of myocardial tissue, the steps of which are as follows: (1) Prepare a 0.01-0.1wt% medical-grade calcium alginate aqueous solution A for later use; (2) Weigh 0.001-0.006g of recombinant type III humanized collagen powder into a 15mL centrifuge tube, add 2g of distilled water, shake and mix well to obtain collagen aqueous solution B; (3) Take 0.1-0.9g of solution A and 0.1-0.8g of solution B respectively. (4) Mix solution B to obtain mixed solution C; (5) Transfer mixed solution C to 3 mL syringe 1 for later use; weigh 0.1-0.7 g of medical grade sodium alginate solution into 3 mL syringe 2; (6) Connect syringe 1 and syringe 2 using a three-way valve, and mix solution A and solution B by 20-40 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product; (7) Store the hydrogel product in an environment of 0-10℃.

[0095] The hydrogel prepared in this application has the advantages of repairing damaged tissues, promoting cell and tissue proliferation and normal growth, being resistant to enzymatic hydrolysis, possessing excellent mechanical strength, and maintaining its repair and regeneration properties for a long time. This hydrogel provides new hope for achieving a complete cure for heart failure and has important potential value in the biomedical field.

[0096] This application also provides a hydrogel for repairing cardiac damage, wherein the hydrogel is an injectable hydrogel for treating heart failure that acts on the site of cardiac damage and has myocardial tissue repair function.

[0097] This application also provides a hydrogel for treating heart failure, wherein the hydrogel is an injectable hydrogel for treating heart failure that acts on the site of cardiac lesions and has myocardial tissue repair function.

[0098] This application also provides a hydrogel for treating myocardial infarction, wherein the hydrogel is an injectable hydrogel for treating heart failure that has myocardial tissue repair function and acts on the site of myocardial infarction.

[0099] This application also provides the application of the aforementioned injectable hydrogel for heart failure treatment with myocardial tissue repair function in the repair of cardiac injury.

[0100] This application also provides the application of the aforementioned injectable hydrogel with myocardial tissue repair function in the treatment of heart failure.

[0101] This application also provides the application of the aforementioned injectable hydrogel with myocardial tissue repair function in the treatment of myocardial infarction.

[0102] This application also provides a method for repairing cardiac damage, in which the injectable hydrogel for treating heart failure with myocardial tissue repair function is applied to the site of cardiac damage.

[0103] This application also provides a method for repairing heart damage, which involves injecting the aforementioned injectable hydrogel for treating heart failure with myocardial tissue repair function into the site of heart lesion.

[0104] This application also provides a method for treating heart failure, wherein the injectable hydrogel for treating heart failure with myocardial tissue repair function is applied to the site of cardiac lesions.

[0105] This application also provides a method for treating heart failure by injecting the aforementioned injectable hydrogel with myocardial tissue repair function into the myocardial infarction site.

[0106] This application also provides a method for treating myocardial infarction, wherein the injectable hydrogel for treating heart failure with myocardial tissue repair function is injected into the site of myocardial infarction.

[0107] The hydrogel loaded with active material provided in this application for the treatment of heart failure has at least one of the following beneficial effects:

[0108] 1) The calcium alginate hydrogel loaded with active substances utilizes the chelation of polycarboxylic acid groups and polyvalent cations of alginate to form a gel rapidly;

[0109] 2) The hydrogel preparation process is simple, and it has excellent rheological properties and injectability.

[0110] 3) Hydrogels not only provide mechanical support, but also have a significant cell growth-promoting function, which can effectively promote the formation and repair of blood vessels in damaged heart areas. Attached Figure Description

[0111] Figure 1a Image of sodium alginate PBS solution;

[0112] Figure 1b Image of hydrogel group 1;

[0113] Figure 1c Image of hydrogel group 2;

[0114] Figure 2a This is a scanning electron microscope image of hydrogel group 1 in Experimental Example 1 of this application;

[0115] Figure 2b This is a scanning electron microscope image of hydrogel group 2 in Experimental Example 1 of this application;

[0116] Figure 3a This is a graph showing the injectability results of hydrogel group 1 in Experimental Example 1 of this application;

[0117] Figure 3b This is a graph showing the injectability results of hydrogel group 2 in Experimental Example 1 of this application;

[0118] Figure 4 This is a scan diagram of the alternating step strain of the hydrogel in Experimental Example 1 of this application;

[0119] Figure 5a This is the cytotoxicity result of the hydrogel on HUVECs cells in Experiment Example 1 of this application;

[0120] Figure 5b This is the cytotoxicity result of the hydrogel on H9C2 cells in Experiment Example 1 of this application;

[0121] Figure 6 These are the echocardiogram results of the hydrogel at 7 days and 14 days in Experiment Example 1 of this application;

[0122] Figure 7 This describes the preparation process of the calcium alginate / recombinant type III humanized collagen composite hydrogels in Examples 5-9;

[0123] Figure 8 This is a rheological and mechanical property diagram of the calcium alginate / recombinant type III humanized collagen composite hydrogel of Example 5;

[0124] Figure 9 This is a rheological and mechanical property diagram of the calcium alginate / recombinant type III humanized collagen composite hydrogel of Example 6;

[0125] Figure 10 This is a rheological and mechanical property diagram of the calcium alginate / recombinant type III humanized collagen composite hydrogel of Example 7;

[0126] Figure 11 This is a rheological and mechanical property diagram of the calcium alginate / recombinant type III humanized collagen composite hydrogel of Example 8;

[0127] Figure 12 This is a rheological and mechanical property diagram of the calcium alginate / recombinant type III humanized collagen composite hydrogel of Example 9;

[0128] Figure 13 This is the infrared spectrum of the calcium alginate / recombinant type III humanized collagen composite hydrogel of Example 9;

[0129] Figure 14 This is a SEM image of the calcium alginate / recombinant type III humanized collagen composite hydrogel from Example 9. Detailed Implementation

[0130] In the following examples, all reagents are chemically pure unless otherwise stated.

[0131] The core amino acid sequence of the recombinant type III humanized collagen used in the following examples is GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0132] Example 1

[0133] A method for preparing an injectable hydrogel for the treatment of heart failure with myocardial tissue repair function, the preparation steps of which are as follows:

[0134] (1) Preparation of sodium alginate system

[0135] Under aseptic conditions, accurately weigh 20 mg of sodium alginate and dissolve it in 2 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0136] (2) Preparation of calcium lactate system

[0137] Under aseptic conditions, accurately weigh 10 mg of calcium lactate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0138] (3) Calcium alginate system loaded with active substances

[0139] Under aseptic conditions, 10 mg of recombinant type III humanized collagen was dissolved in 2 mL of sodium alginate solution and allowed to dissolve completely at room temperature.

[0140] (4) Sodium alginate and calcium lactate form a gel

[0141] At room temperature, 350 μL of calcium lactate solution was slowly added to 2 mL of stirred sodium alginate solution, and the solution immediately crosslinked to form a hydrogel.

[0142] Example 2

[0143] A method for preparing an injectable hydrogel for the treatment of heart failure with myocardial tissue repair function, the preparation steps of which are as follows:

[0144] (1) Preparation of sodium alginate system

[0145] Under aseptic conditions, accurately weigh 10 mg of sodium alginate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0146] (2) Preparation of calcium carbonate system

[0147] Under aseptic conditions, accurately weigh 10 mg of calcium carbonate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0148] (3) Calcium alginate system loaded with active substances

[0149] Under aseptic conditions, 5 mg of recombinant type III humanized collagen was dissolved in sodium alginate solution and allowed to dissolve completely at room temperature.

[0150] (4) Sodium alginate and calcium carbonate form a gel

[0151] At room temperature, 0.5 mL of calcium carbonate solution was slowly added to 1 mL of sodium alginate solution under stirring, and the solution immediately crosslinked to form a hydrogel.

[0152] Example 3

[0153] A method for preparing an injectable hydrogel for the treatment of heart failure with myocardial tissue repair function, the preparation steps of which are as follows:

[0154] (1) Preparation of sodium alginate system

[0155] Under aseptic conditions, accurately weigh 10 mg of sodium alginate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0156] (2) Preparation of calcium gluconate system

[0157] Under aseptic conditions, accurately weigh 10 mg of calcium gluconate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0158] (3) Calcium alginate system loaded with active substances

[0159] Under aseptic conditions, 8 mg of recombinant type III humanized collagen was dissolved in sodium alginate solution and allowed to dissolve completely at room temperature.

[0160] (4) Sodium alginate and calcium gluconate form a gel

[0161] At room temperature, 1 mL of calcium gluconate solution was slowly added to 1 mL of sodium alginate solution under stirring, and the mixture immediately crosslinked to form a hydrogel.

[0162] Example 4

[0163] A method for preparing an injectable hydrogel for the treatment of heart failure with myocardial tissue repair function, the preparation steps of which are as follows:

[0164] (1) Preparation of sodium alginate system

[0165] Under aseptic conditions, accurately weigh 20 mg of sodium alginate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature until fully dissolved.

[0166] (2) Preparation of calcium citrate system

[0167] Under aseptic conditions, accurately weigh 10 mg of calcium citrate and dissolve it in 1 mL of sterile PBS. Stir overnight at room temperature to ensure complete dissolution.

[0168] (3) Calcium alginate system loaded with active substances

[0169] Under aseptic conditions, recombinant type III humanized collagen, a bioactive substance, was dissolved in sodium alginate solution and allowed to dissolve completely at room temperature.

[0170] (4) Sodium alginate and calcium citrate form a gel

[0171] At room temperature, 1 mL of calcium citrate solution was slowly added to 1 mL of sodium alginate solution under stirring, and the mixture immediately crosslinked to form a hydrogel.

[0172] Example 5

[0173] The specific steps for preparing the calcium alginate / recombinant type III humanized collagen composite hydrogel in this embodiment are as follows:

[0174] (1) Prepare a 0.01wt% medical-grade CaCl2 solution A for later use;

[0175] (2) Weigh 0.004g of recombinant type III humanized collagen powder and put it into a 15mL centrifuge tube. Add 2g of distilled water and shake to mix well to obtain collagen aqueous solution B.

[0176] (3) Take 0.2g of solution A and 0.1g of solution B respectively and mix them to obtain mixed solution C;

[0177] (4) Transfer the mixed solution C into a syringe, and label it syringe 1;

[0178] Weigh 0.4g of medical-grade sodium alginate solution into a 3mL syringe 2;

[0179] like Figure 7As shown, a three-way valve is used to connect syringe 1 and syringe 2. The liquids in syringe 1 and syringe 2 are mixed evenly through 20 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product. The hydrogel product is stored in an environment of 0-10℃.

[0180] The prepared composite hydrogel has a three-dimensional network structure and a storage modulus of 161 Pa.

[0181] Rheological and mechanical properties of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown in the figure. Figure 8 As shown, when the shear stress increases to above 1 Pa, the storage modulus of the hydrogel begins to decrease, while the loss modulus gradually increases. When the shear stress exceeds 62 Pa, the storage modulus of the hydrogel is less than the loss modulus, at which point the structure of the hydrogel is destroyed. This indicates that the hydrogel exhibits the shear-thinning property of injectable hydrogels.

[0182] Example 6

[0183] (1) Prepare a 0.01wt% medical-grade CaCl2 solution A for later use;

[0184] (2) Weigh 0.004g of recombinant type III humanized collagen powder and put it into a 15mL centrifuge tube. Add 2g of distilled water and shake to mix well to obtain collagen aqueous solution B.

[0185] (3) Take 0.2g of solution A and 0.3g of solution B respectively and mix them to obtain mixed solution C;

[0186] (4) Transfer the mixed solution C into a syringe, and label it syringe 1;

[0187] Weigh 0.4g of medical-grade sodium alginate solution into a 3mL syringe;

[0188] Using a three-way valve to connect syringe 1 and syringe 2, the liquids in syringe 1 and syringe 2 are mixed evenly through 30 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product; the hydrogel product is stored in an environment of 0-10℃.

[0189] The prepared composite hydrogel has a three-dimensional network structure and a storage modulus of 462 Pa.

[0190] Rheological and mechanical properties of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown in the figure. Figure 9 As shown.

[0191] Example 7

[0192] (1) Prepare a 0.01wt% medical-grade CaCl2 solution A for later use;

[0193] (2) Weigh 0.004g of recombinant type III humanized collagen powder and put it into a 15mL centrifuge tube. Add 2g of distilled water and shake to mix well to obtain collagen aqueous solution B.

[0194] (3) Measure 0.2g of solution A and 0.4g of solution B respectively and mix them to obtain mixed solution C;

[0195] (4) Transfer the mixed solution C into a syringe, and label it syringe 1;

[0196] Weigh 0.4g of medical-grade sodium alginate solution into a 3mL syringe 2;

[0197] Using a three-way valve to connect syringe 1 and syringe 2, the liquids in syringe 1 and syringe 2 are mixed evenly through 40 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product; the hydrogel product is stored in an environment of 0-10℃.

[0198] The prepared composite hydrogel has a three-dimensional network structure and a storage modulus of 648 Pa.

[0199] Rheological and mechanical properties of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown in the figure. Figure 10 As shown.

[0200] Example 8

[0201] (1) Prepare a 0.01wt% medical-grade CaCl2 solution A for later use;

[0202] (2) Weigh 0.004g of recombinant type III humanized collagen powder and put it into a 15mL centrifuge tube. Add 2g of distilled water and shake to mix well to obtain collagen aqueous solution B.

[0203] (3) Measure 0.9g of solution A and 0.4g of solution B respectively and mix them to obtain mixed solution C;

[0204] (4) Transfer the mixed solution C into a syringe, and label it syringe 1;

[0205] Weigh 0.4g of medical-grade sodium alginate solution into a 3mL syringe 2;

[0206] Using a three-way valve to connect syringe 1 and syringe 2, the liquids in syringe 1 and syringe 2 are mixed evenly through 30 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product; the hydrogel product is stored in an environment of 0-10℃.

[0207] The prepared composite hydrogel has a three-dimensional network structure and a storage modulus of 1663 Pa.

[0208] Rheological and mechanical properties of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown in the figure. Figure 11 As shown.

[0209] Example 9

[0210] (1) Prepare a 0.01wt% medical-grade CaCl2 solution A for later use;

[0211] (2) Weigh 0.004g of recombinant type III humanized collagen powder and put it into a 15mL centrifuge tube. Add 2g of distilled water and shake to mix well to obtain collagen aqueous solution B.

[0212] (3) Measure 0.8g of solution A and 0.1g of solution B respectively and mix them to obtain mixed solution C;

[0213] (4) Transfer the mixed solution C into a syringe, and label it syringe 1;

[0214] Weigh 0.4g of medical-grade sodium alginate solution into a 3mL syringe 2;

[0215] Using a three-way valve to connect syringe 1 and syringe 2, the liquids in syringe 1 and syringe 2 are mixed evenly through 30 cycles of injection to obtain the final calcium alginate / recombinant type III humanized collagen hydrogel product; the hydrogel product is stored in an environment of 0-10℃.

[0216] The prepared composite hydrogel has a three-dimensional network structure and a storage modulus of 7000 Pa.

[0217] Rheological and mechanical properties of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown in the figure. Figure 12 As shown.

[0218] Infrared spectrum of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown in the figure. Figure 13 As shown. The characteristic amide peak of collagen can be observed: amide I (1649–1659 cm⁻¹). -1 ), Amide II (1550~1561cm) -1 ), Amide III (1237~1242cm) -1 ), Amide A (3409~3426cm) -1 ) and amide B (2932~2961cm) -1 ).

[0219] SEM image of calcium alginate / recombinant type III humanized collagen composite hydrogel, as shown. Figure 14 As shown, the hydrogel has a porous structure.

[0220] Test case

[0221] Taking the substance obtained in Example 1 as an example, the detection was performed, and the specific operation process and results are as follows:

[0222] Unless otherwise specified, hydrogel group 1 and group 2 represent the following combinations: Hydrogel group 1: blank hydrogel; Hydrogel group 2: hydrogel loaded with recombinant type III humanized collagen.

[0223] 1. The hydrogel prepared in step (4) was tested, and its rheological properties were measured using an MCR302 rheometer. Steady-state shear flow was performed at 37℃ using a double concentric cylindrical geometry with a gap of 4 mm. Frequency scanning was performed using 1% strain and oscillation frequencies of 0.1–100 rad / s. The strain scan oscillation frequency was 1 Hz, and the strain ranged from 0.01% to 1000%. In the self-healing experiment, alternating step strain scanning experiments were used (large strain: 1000%, 60 s and small strain: 1%, 60 s).

[0224] Figure 1a Image of sodium alginate PBS solution; Figure 1b Image of hydrogel group 1; Figure 1c Image of hydrogel group 2; Figure 2a , Figure 2b Scanning electron microscope image of the hydrogel; Figure 3a , Figure 3b The figure shows the injectability of the hydrogel, which can be injected through a 27G needle, proving that the hydrogel is injectable. Frequency and amplitude scans of the hydrogel show that the storage modulus (G') is greater than the loss modulus (G"), proving the successful preparation of the hydrogel. Figure 4 The image shows the alternating step strain scanning results of the hydrogel. The results indicate that after the hydrogel structure was damaged three times, 89.4% of the storage modulus was still recovered, proving that the hydrogel has strong self-healing properties.

[0225] 2. Biocompatibility testing of the hydrogel obtained in step (4)

[0226] The biocompatibility of the hydrogels was evaluated using human umbilical vein endothelial cells (HUVECs). The UV-sterilized hydrogels were extracted in cell culture medium (0.1 g / mL) for 48 h to prepare the extraction buffer. HUVECs were seeded into 96-well plates at a density of 8000 cells per well. After 24 h, the cell culture medium was removed, and the hydrogel extraction buffer was added to the wells instead of the different hydrogel samples. The proliferation rate and morphology of HUVECs cultured for 24 h and 72 h were detected using CCK-8 and FDA / PI staining, respectively. After staining HUVECs with FDA (30 μg / mL) and PI (10 μg / mL), the cells were incubated for 5 min and then observed under a fluorescence microscope. The results showed that the fluorescence intensity of hydrogel group 1 and hydrogel group 2 was 1.4 and 1.8 times that of the control group, respectively. After incubation for 24 h and 72 h, 90 μL of fresh culture medium and 10 μL of diluted CCK-8 solution were added to each well. After 2 h, the cell proliferation rate was calculated by measuring the absorbance at 450 nm using a microplate reader.

[0227] The results of hydrogel on the survival rate of human umbilical vein endothelial cells (HUVECs) are as follows: Figure 5a As shown, the results indicate that all hydrogel groups showed no toxicity to cells at 24h and 72h. Furthermore, the cell survival rate of the hydrogel group loaded with recombinant type III humanized collagen was higher than that of the blank hydrogel group, indicating that recombinant type III humanized collagen effectively promoted cell proliferation and that the hydrogel has good biocompatibility.

[0228] III. Hydrogels protect cardiomyocytes from oxidative stress damage.

[0229] The effects of hydrogels on protecting cardiomyocytes from oxidative stress and reducing the expression of apoptosis-related proteins were evaluated using rat cardiomyocytes (H9C2) and Western blot analysis. The hydrogel, sterilized by UV, was extracted in cell culture medium (0.1 g / mL) for 48 h to prepare the extract. H9C2 cells were seeded into 96-well plates at a density of 8000 cells per well. After 24 h, the cell culture medium was removed, and the H9C2 cells were pretreated with 200 μM H2O2 for 1 h to induce oxidative stress. Then, the hydrogel extract was added to the wells instead of different hydrogel samples. The proliferation rate and morphology of H9C2 cells after 24 h and 72 h of culture 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 then incubated for 5 min before being observed under a fluorescence microscope. The results showed that, compared to the control group, the cell viability of the H2O2 group was significantly reduced, with its fluorescence intensity only 0.565 times that of the control group, indicating that cardiomyocytes suffered oxidative stress damage and reduced viability. However, the addition of hydrogels restored cell viability to some extent; the fluorescence intensities of hydrogel group 1 and hydrogel group 2 were 0.84 and 1.16 times that of the control group, respectively. 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. Two h later, the cell proliferation rate was calculated by measuring the absorbance at 450 nm using a microplate reader.

[0230] The results of hydrogel on survival rate are as follows Figure 5b As shown, the results indicate that all hydrogel groups showed good biocompatibility with cells at 24h and 72h. Furthermore, the cell survival rate of the hydrogel group loaded with recombinant type III humanized collagen was higher than that of the blank hydrogel group, indicating that recombinant type III humanized collagen protects cardiomyocytes from oxidative stress damage.

[0231] IV. In vivo cardiac repair effect test of hydrogel

[0232] To investigate the effect of hydrogels on in vivo cardiac repair, a rat model of myocardial infarction was established. M-mode echocardiography was performed on the successfully modeled rats on days 7 and 14. The results are as follows: Figure 6 As shown, the ventricular wall contraction and relaxation movements were significantly better in the hydrogel group 2. On days 7 and 14, the ventricular wall thickness, fibrosis degree and collagen deposition in the MI group, hydrogel group 1 and hydrogel group 2 were assessed by hematoxylin and eosin (H&E) staining and Masson staining. Figure 7Histological H&E staining results showed that, compared with the MI control group, the ventricular walls of hydrogel groups 1 and 2 were thicker and the degree of fibrosis was lower. Furthermore, the ventricular wall thickness of hydrogel group 2 was the greatest, significantly different from hydrogel group 1 and the MI control group. The collected cardiac tissue was then stained with Masson staining, where blue represents collagen-rich fibrotic tissue caused by MI and red represents normal myocardial tissue. Some residual gel was observed in myocardial tissue sections at 7 and 14 days in both hydrogel groups 1 and 2. Compared with the MI control group, hydrogel group 2 had less fibrotic tissue and more normal myocardial tissue. The fibrotic area in hydrogel group 2 (5.9%) was significantly smaller than that in the MI control group (23.8%) and hydrogel group 1 (12.3%). In conclusion, hydrogels can promote myocardial tissue regeneration and cardiac function recovery after myocardial infarction.

Claims

1. An injectable hydrogel for treating heart failure with myocardial tissue repair function, characterized in that, The hydrogel was prepared by chelating sodium alginate solution with a calcium ion-containing solution, and the hydrogel was loaded with active substances for repairing heart damage. The active substance is recombinant type III humanized collagen, and the amino acid fragment sequence of the recombinant type III humanized collagen is GERGAPGFRGPAGPNGIPGEKGPAGERGAP; The storage modulus of the hydrogel is 100-7000 Pa; The hydrogel has a three-dimensional network structure with a pore size of 30-100μm; The method for preparing the injectable hydrogel for heart failure treatment with myocardial tissue repair function includes the following steps performed under aseptic conditions: Step 1: Prepare an aqueous solution containing calcium ions, denoted as solution A; The calcium-containing aqueous solution is a calcium chloride aqueous solution, and the concentration of calcium chloride in the calcium chloride aqueous solution is 1-7 wt%. Prepare an aqueous solution of recombinant type III humanized collagen, denoted as solution B; The concentration of recombinant type III humanized collagen in the aqueous solution is 0.05–0.3 wt%. Step 2: Mix solution A and solution B thoroughly to obtain solution C; The mass ratio of solution A to solution B when mixed is 0.1–0.9:0.1–0.8; Step 3: Mix solution C thoroughly with sodium alginate solution to obtain the hydrogel; The sodium alginate solution contains 2-7 wt% sodium alginate. The mass ratio of solution C to sodium alginate solution is 0.01–0.05: 0.1–0.5.

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