Sea squirt cellulose hydrogel as well as preparation method and application thereof

Abstract

The invention discloses sea squirt cellulose hydrogel as well as a preparation method and application thereof, and relates to the technical field of hydrogel preparation. In the sea squirt cellulose dispersion liquid or sea squirt cellulose hydrogel provided by the invention, PEI and AA are used as a hydrogel skeleton, and modified tannic acid provides adhesion and enhances interface interaction between tissues and the hydrogel. An ionization accelerator is introduced to improve the electrical coupling microenvironment of the infarction zone. An aldehyde modified product of the sea squirt cellulose has excellent performance in the aspects of adjusting the mechanical property and enhancing the adhesive force of the hydrogel, and is beneficial to providing mechanical support for the ventricular wall. The multifunctional hydrogel further comprises another hydrogel solution, and the hydrogel solution has the effects of resisting cell adhesion and preventing pericardium adhesion. The whole design provides a stable platform for a target small molecule drug, ensures that the target small molecule drug is effectively released in a heart environment, and provides good mechanical support and conductive recovery for injured heart tissues at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of hydrogel preparation, and in particular to an ascidian cellulose hydrogel, a preparation method and application thereof. Background Art

[0002] Myocardial infarction (MI) is one of the leading causes of disability and death worldwide. Irreversible damage to early cardiomyocytes and secondary cell death can lead to permanent loss of cardiac contractility, which in turn develops into heart failure. However, adult mammalian cardiomyocytes have limited proliferation and regeneration capacity after being damaged. Existing treatments, such as stem cell transplantation, exosomes, and myocardial patches, cannot effectively compensate for the loss of one's own cardiomyocytes after MI. In addition to myocardial damage, ventricular wall collapse and impaired electrical conductivity are also common phenomena after MI. In addition, ventricular wall collapse and severe impairment of electrical conductivity in damaged myocardial tissue affect normal cardiac function. Selecting materials with mechanical support and electrophysiological functions as drug delivery carriers will help to further optimize the therapeutic effect.

[0003] Traditional drug delivery methods (oral, topical, and systemic) have problems with low drug targeting and insufficient bioavailability, and may also cause a series of side effects, such as gastrointestinal irritation, thrombocytopenia, and liver damage. For example, oral administration is degraded by digestive enzymes, resulting in low drug bioavailability, incomplete absorption, and dependence on patient compliance. Local administration is limited to local effects and may cause allergic reactions and irritation. Systemic administration has uneven systemic distribution concentration, accumulation in non-target tissues, and uneven metabolism and excretion. The use of adapted biomaterials as carriers can improve local drug concentrations and maintain drug release. The use of drugs can be optimized by improving drug targeting and bioavailability while reducing side effects (such as gastrointestinal irritation, thrombocytopenia, and liver damage). Therefore, it is crucial to design and develop efficient biomaterial targeted drug delivery systems to maximize the in vivo regulatory effects of small molecule compounds.

[0004] As a soft and wet biomaterial with properties similar to those of the human extracellular matrix, hydrogels are widely used in the field of tissue engineering. Studies have shown that hydrogels not only provide mechanical support during myocardial tissue repair, but can also serve as carriers for cell or drug delivery [1]. With a deeper understanding of the changes in the myocardial microenvironment after myocardial infarction, hydrogels with intelligence and functionality have been synthesized for use in the cardiac repair process. For example, matrix metalloproteinase (MMP)-responsive hydrogels [2], ROS-scavenging hydrogels [3], and immunomodulatory hydrogels [4] are used to regulate MMP, ROS, and inflammation in the infarcted area, respectively. The development of these intelligent hydrogels provides a diverse and targeted approach to cardiac repair.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a sea squirt cellulose hydrogel, a preparation method and its application, which not only improves the bioavailability of small molecule compounds, but also provides necessary mechanical support and electrical conduction recovery for damaged myocardium, significantly improving the repair ability after myocardial infarction.

[0007] The present invention is achieved in that:

[0008] In the first aspect, the present invention provides a tunicate cellulose dispersion or a tunicate cellulose hydrogel. The tunicate cellulose dispersion comprises: a solution obtained by mixing the aldehyde-modified product of tunicate cellulose with polyethyleneimine, acrylic acid, modified tannic acid, an ionization promoter, and benzalkonium chloride. The tunicate cellulose hydrogel is prepared by adding an initiator and a cross-linking agent to the tunicate cellulose dispersion for chemical cross-linking reaction. In every 50 mL of the chemical cross-linking reaction system, the mixed mass ratio of the aldehyde-modified product of tunicate cellulose, polyethyleneimine, acrylic acid, modified tannic acid, ionization promoter, and benzalkonium chloride is (3-8g):(10-15g):(1-2g):(0.03-0.06g):(0.007-0.015g):(0.2-0.3g), and the solvent is water.

[0009] In a second aspect, the present invention also provides a method for preparing a tunicate cellulose dispersion or a tunicate cellulose hydrogel, which comprises: mixing corresponding proportions of the formaldehyde-modified product of tunicate cellulose, polyethyleneimine, acrylic acid, modified tannic acid, an ionization promoter, and benzalkonium chloride to obtain a tunicate cellulose dispersion, and obtaining a tunicate cellulose hydrogel through a chemical cross-linking reaction.

[0010] In a third aspect, the present invention further provides a multifunctional hydrogel, comprising: covering one surface of the above-mentioned ascidian cellulose hydrogel or the ascidian cellulose hydrogel prepared by the above-mentioned preparation method with another hydrogel; the hydrogel is prepared by a solution of a mixture of a carboxyl modified product of ascidian cellulose and acrylic acid and polyethylene glycol diacrylate, and a chemical cross-linking reaction initiated by an initiator;

[0011] For every 10 mL of chemical crosslinking reaction system, the mass ratio of the carboxyl modified product of ascidian cellulose to acrylic acid, polyethylene glycol diacrylate and initiator is (0.06-0.15g):(1.2-2g):(0.15-0.28g):(0.008-0.012g), and the solvent is water.

[0012] In a fourth aspect, the present invention further provides a drug-loaded multifunctional hydrogel, wherein the above-mentioned ascidian cellulose dispersion is mixed with Chrysin-7-O-glucuronide and / or its salt, an initiator and a cross-linking agent are added, a cross-linking reaction is carried out, and then another hydrogel is covered on one surface of the obtained hydrogel; the hydrogel is obtained by a chemical cross-linking reaction initiated by an initiator through a solution of a mixture of a carboxyl modified product of ascidian cellulose and acrylic acid and polyethylene glycol diacrylate;

[0013] For every 10 mL of chemical crosslinking reaction system, the mass ratio of the carboxyl modified product of the ascidian cellulose to acrylic acid, polyethylene glycol diacrylate and initiator is (0.06-0.15g):(1.2-2g):(0.15-0.28g):(0.008-0.012g), and the solvent is water.

[0014] In a fifth aspect, the present invention also provides a use of a drug-loaded multifunctional hydrogel in the preparation of a drug for treating ischemic heart disease and its complications, or the use of the above-mentioned ascidian cellulose hydrogel, the above-mentioned multifunctional hydrogel or the above-mentioned drug-loaded multifunctional hydrogel in the preparation of skin repair dressings, implants, biomaterials and medical devices;

[0015] In a preferred embodiment of the present invention, the biomaterial is selected from at least one of artificial skin, artificial bone, artificial cornea, tissue or organ scaffold and cell culture microcarrier;

[0016] In a preferred embodiment of the present invention, the skin repair dressing further comprises acellular matrix and collagen;

[0017] In a preferred embodiment of the present invention, the artificial bone is artificial cartilage.

[0018] The present invention has the following beneficial effects:

[0019] The tunicate cellulose dispersion or tunicate cellulose hydrogel provided by the present invention uses polyethyleneimine and acrylic acid as the hydrogel skeleton, and the modified tannic acid provides adhesion and enhances the interface interaction between the tissue and the hydrogel. In order to improve the electrical coupling microenvironment of the infarct area, an ionization promoter is introduced, so that the ion concentration can be increased and the charge can be neutralized, thereby promoting the conduction of electrical activity signals. The aldehyde-modified product of tunicate cellulose performs well in regulating mechanical properties and enhancing the adhesion of the hydrogel, which is beneficial to provide mechanical support for the ventricular wall.

[0020] In the multifunctional hydrogel, the above-mentioned sea squirt cellulose hydrogel is used as an adhesion layer. In order to resist cell adhesion and prevent pericardial adhesion, another non-adhesion layer is also provided, which is made from another hydrogel solution. Specifically, the carboxyl modified product of sea squirt cellulose is mixed with acrylic acid and anti-cell adhesion crosslinker polyethylene glycol diacrylate to prepare a hydrogel with anti-cell adhesion and pericardial adhesion prevention effects. The core goal of the entire design is to provide a stable platform for Chrysin-7-O-glucuronide to ensure its effective release in the cardiac environment, while providing good mechanical support and conductive recovery for damaged cardiac tissue. This carefully designed hydrogel system provides innovative treatment strategies for the treatment of heart diseases through layer-by-layer functional layers.

[0021] In the drug-loaded multifunctional hydrogel, the in situ release of Chrysin-7-O-glucuronide in cardiac injury can be achieved with the help of the above multifunctional hydrogel. The drug-loaded multifunctional hydrogel not only achieves the sustained release of small molecule drugs in the damaged area, but also effectively adheres to the surface of the heart and inhibits pericardial adhesion. In addition, the good conductive properties of the hydrogel help to improve the conductive microenvironment of the infarct area, thereby enhancing the repair of myocardial infarction. The successful implementation of this study provides a reference strategy for the treatment of myocardial infarction and lays a theoretical foundation for further research and clinical application in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 FT-IR spectra of CNC, CNC-CHO and CNC-COOH;

[0024] Figure 2 FT-IR spectra of TA and mTA;

[0025] Figure 3 FT-IR spectra of CPA, CPAM, and CPAMm;

[0026] Figure 4 FT-IR spectra of CPAMm and PCA;

[0027] Figure 5 The brightness change diagram of the LED bulb in CPA hydrogel, CPAM hydrogel and CPAMm hydrogel;

[0028] Figure 6 This is the actual result diagram of the adhesion performance of CPAMm hydrogel to various materials and tissues and organs;

[0029] Figure 7 Analysis of the adhesion of the CPAMm side and PCA side of the CPAMm / PCA hydrogel to pig skin;

[0030] Figure 8 The stress-strain curves and elastic modulus statistical results of CPA / PCA hydrogel, CPAM / PCA hydrogel and CPAMm / PCA hydrogel (the data in the figure represent Mean±SD, significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0031] Fig. 9 Conductivity analysis of CPA / PCA hydrogel, CPAM / PCA hydrogel and CPAMm / PCA hydrogel (data in the figure represent Mean±SD. Significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0032] Fig.10 SEM images of CPAMm hydrogel and PCA hydrogel, scale bar = 20 μm;

[0033] Fig.11 This is the drug release curve of CPAMm / PCA loaded Chrysin-7-O-glucuronide hydrogel;

[0034] Fig.12 Biocompatibility test of CPAMm / PCA hydrogel and CPAMm / PCA drug-loaded hydrogel (the data in the figure represent Mean±SD, significant difference: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0035] Fig.13 M-mode echocardiographic images of different groups after 28 days of treatment (data in the figure represent Mean±SD; significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0036] Fig.14 Doppler echocardiographic images of different groups after 28 days of treatment (data in the figure represent Mean±SD; significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0037] Fig.15 This is a quantitative comparison of left ventricular function in different groups after 28 days of treatment (the data in the figure represent Mean±SD; significant difference: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0038] Fig.16 The results of Masson staining experiments in different groups ((a) Masson trichrome staining of cardiac area sections in different groups, scale = 1 mm (myocardium is red, fibrous tissue is blue); (b) Quantitative analysis of the thickness of the left ventricular anterior wall based on Masson trichrome staining images; the data in the figure represent Mean ± SD; significant differences: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0039] Fig.17 Figure 2. The restorative effect of C@CPAMm / PCA on MI rats after 28 days of treatment (n = 10 rats per group); (A) Optical mapping of action potential (AP) and ventricular Ca2+ transient initiation; (B) Active time of action potential phase; (C) Active time of Ca2+ transient initiation phase. Ventricular electrophysiological stability and VA susceptibility were assessed by electrocardiography (ECG), and (D) VA induction rate and (E) VF threshold were measured.

[0040] Fig.18 HE staining images of organs of rats in each group after 28 days of treatment, scale bar = 500 μm;

[0041] Fig.19 Comparison of (A) action potential duration at 90% depolarization (APD90) and (B) calcium transient duration at 90% recovery (CTD90) in the optical mapping field of view in different treatment groups; data are expressed as mean ± standard deviation; error bars represent standard deviation, **p<0.01, ***p<0.001, ****p<0.0001, n=3 independent samples. ns indicates no significant difference;

[0042] Fig. 20The effects of C@CPAMm / PCA intracardiac patch on improving cardiac function and restoring cardiac electrical conduction at 28 days after MI in the Bama miniature pig model. (A) Representative macroscopic images of the Bama miniature pig heart after MI injury (left panel) and images of the heart with C@CPAMm / PCA patch attached to it (right panel). (B) Quantitative analysis results of cTnI in the serum of each group. (C) Electrocardiogram (ECG) measurements of Bama miniature pigs. (D) ST-segment elevation ECG signals of each group. (E) Representative cross-sections of the hearts from the sham, saline, and C@CPAMm / PCA transplanted groups at 28 days. The heart was cut into five layers from the apex to the ligature level. (F) Quantitative analysis of infarct area in heart sections. (G) 2D and M-mode echocardiograms at day 28. (H,I) Statistical results of LVEF and LVFS measured by echocardiography in different groups. (J) Action potential (AP) and ventricular Ca 2+ Optical mapping images of transient firing. (K) Active time during the action potential phase. (L) Ca 2+ Active time during the transient start-up phase. Ventricular electrophysiological stability and VA susceptibility were assessed by electrocardiography (ECG), and (M)VA induction rate and (N)VF threshold were measured. Data are presented as mean ± SD. Error bars represent standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, p values ​​were generated by one-way analysis of variance (ANOVA) and tested by Tukey's post hoc multiple comparison test;

[0043] Fig.21 To evaluate left ventricular systolic function by heart cycle monitoring (HR) in a porcine model, C@CPAMm / PCA was used after treatment. The data in the figure are shown as mean ± standard deviation. Error bars represent standard deviation, *p<0.05, **p<0.01, ns indicates no significant difference;

[0044] Fig. 22 To compare (A) action potential duration at 90% depolarization (APD90) and (B) calcium transient duration at 90% recovery (CTD90) in the minipig model with different treatments, data are based on the average of the optical mapping field. Data are presented as mean ± SD. Error bars represent standard deviation, *p < 0.05, ***p < 0.001, based on n = 3 independent samples;

[0045] Fig.23 The stained images of the heart, liver, spleen, lung and kidney of miniature pigs in different treatment groups after 28 days of treatment (scale bar = 500 μm), data are based on n = 3 independent experiments. DETAILED DESCRIPTION

[0046] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0047] List of abbreviations:

[0048]

[0049] In a first aspect, the present invention provides a tunicate cellulose dispersion or a tunicate cellulose hydrogel, wherein the tunicate cellulose dispersion comprises: a solution obtained by mixing an aldehyde-modified product of tunicate cellulose with polyethyleneimine, acrylic acid, modified tannic acid, an ionization accelerator, and benzalkonium chloride, and the tunicate cellulose hydrogel is prepared by adding an initiator and a cross-linking agent to the tunicate cellulose dispersion to carry out a chemical cross-linking reaction;

[0050] In every 50 mL of chemical cross-linking reaction system, the mixing ratio of the aldehyde-modified product of the ascidian cellulose, polyethyleneimine, acrylic acid, modified tannic acid, ionization promoter and benzalkonium chloride is (3-8g):(10-15g):(1-2g):(0.03-0.06g):(0.007-0.015g):(0.2-0.3g), and the solvent is water.

[0051] The mixing ratio is: (3-5g):(10-13g):(1-2g):(0.03-0.06g):(0.007-0.015g):(0.2-0.3g), or (5-6g):(12-15g):(1-2g):(0.03-0.05g):(0.007-0.01g):(0.2-0.3g), or (3-8g):15g:(1-2g):(0.03-0.04g):(0.01-0.015g):(0.2-0.3g).

[0052] A one-pot method was used to prepare a cellulose dispersion or cellulose hydrogel (CPAMm) of ascidian cellulose (CNC), polyethyleneimine (PEI), acrylic acid (AA), an ionization promoter, and modified tannic acid (mTA). Polyethyleneimine (PEI) and acrylic acid (AA) were used as the hydrogel skeleton, and the modified tannic acid provided the adhesion of the hydrogel and enhanced the interfacial interaction between the tissue and the hydrogel. In order to improve the electrically coupled microenvironment of the infarct area, an ionization promoter was also introduced. The conductivity of the hydrogel was improved by the formation of an interpenetrating network after the introduction of the ionization promoter (especially MASEP); therefore, the hydrogel has the potential to be used to regulate cardiac tissue electrophysiology. In addition, the addition of ionization promoter also enhances the tissue adhesion of the hydrogel, which is due to the enhancement of multiple dynamic non-covalent interactions and physical entanglement between PEI and poly (AA-co-ionization promoter) polymer chains. The long alkyl chain of mTA destroys the hydration layer of the interface by absorbing water, and the exposed catechol, -COOH and -NH2 groups interact with the groups on the tissue surface to enhance the wet adhesion strength. CPAMm hydrogels show high adhesion on the surface of wet biological tissues, including various organs such as heart, lungs, kidneys, spleen and liver, which is of key significance for biomedical applications.

[0053] The aldehyde-modified products of ascidian cellulose performed well in regulating mechanical properties and enhancing the adhesion of hydrogels, which is beneficial for providing mechanical support to the ventricular wall.

[0054] By introducing the interpenetrating network formed by the ionization promoter, the elastic modulus and mechanical strength of the hydrogel are significantly improved. The elastic modulus of the hydrogel falls within the range of natural myocardium, meeting the elasticity requirements of cardiac tissue engineering scaffolds.

[0055] In a preferred embodiment of the present invention, the modified tannic acid refers to a precipitate obtained by mixing benzalkonium chloride with an alkaline solution of tannic acid. Further, the precipitate obtained by mixing benzalkonium chloride with an alkaline solution of tannic acid is freeze-dried.

[0056] The alkali solution of tannic acid refers to a sodium hydroxide solution of tannic acid, a potassium hydroxide solution of tannic acid, and the like.

[0057] In a preferred embodiment of the present invention, the ionization promoter is potassium 3-sulfonate propyl methacrylate.

[0058] In a preferred embodiment of the present invention, the chemical crosslinking reaction is carried out by adding an initiator and a crosslinking agent;

[0059] In a preferred embodiment of the present invention, the initiator is selected from any one of ammonium persulfate (APS), potassium persulfate (KPS), sodium persulfate, riboflavin (VB2) and azobisisobutyronitrile (AIBN); the crosslinking agent is selected from any one of TEMED (N,N,N',N'-tetramethylethylenediamine), N,N-methylenebisacrylamide, N,N-vinylbisacrylamide, methylenebisacrylamide and ethylene glycol dimethacrylate; TEMED has a better crosslinking effect.

[0060] In a preferred embodiment of the present invention, in every 50 mL of chemical crosslinking reaction system, the mass ratio of the aldehyde-modified product of the reaction monomer ascidian cellulose to the initiator and the crosslinker is (3-8 g): (0.04-0.06 g): (0.025-0.035 g).

[0061] In a second aspect, the present invention also provides a method for preparing a tunicate cellulose dispersion or a tunicate cellulose hydrogel, which comprises: mixing corresponding proportions of the formaldehyde-modified product of tunicate cellulose, polyethyleneimine, acrylic acid, modified tannic acid, an ionization promoter, and benzalkonium chloride to obtain a tunicate cellulose dispersion, and obtaining a tunicate cellulose hydrogel through a chemical cross-linking reaction.

[0062] In a preferred embodiment of the present invention, the formaldehyde-modified product of ascidian cellulose, polyethyleneimine, acrylic acid, modified tannic acid, an ionization accelerator, and benzalkonium chloride are mixed, and then an initiator and a crosslinking agent are added to carry out a crosslinking reaction.

[0063] In a preferred embodiment of the present invention, in every 50 ml of chemical cross-linking reaction system, the reaction monomer (the mass ratio of tannic acid to the initiator and the cross-linking agent is (0.03-0.06 g): (0.04-0.06 g): (0.025-0.035 g) and the solvent is water.

[0064] In the third aspect, the present invention also provides a multifunctional hydrogel, which comprises: another hydrogel is covered on one surface of the above-mentioned tunicate cellulose hydrogel or the tunicate cellulose hydrogel prepared by the above-mentioned preparation method; the other hydrogel is prepared by a solution of a mixture of a carboxyl modified product of tunicate cellulose with acrylic acid and polyethylene glycol diacrylate, and a chemical cross-linking reaction initiated by an initiator.

[0065] Another setting of hydrogel solution has the function of resisting cell adhesion and preventing pericardial adhesion, which is beneficial to promoting the repair of myocardial damage.

[0066] The multifunctional hydrogel has tissue adhesion properties on one side of the ascidian cellulose hydrogel, and anti-cell adhesion and pericardial adhesion prevention functions on the other side.

[0067] For every 10 mL of chemical crosslinking reaction system, the mass ratio of the carboxyl modified product of ascidian cellulose to acrylic acid, polyethylene glycol diacrylate and initiator is (0.06-0.15g):(1.2-2g):(0.15-0.28g):(0.008-0.012g), and the solvent is water.

[0068] In a fourth aspect, the present invention further provides a drug-loaded multifunctional hydrogel, wherein the above-mentioned ascidian cellulose dispersion is mixed with Chrysin-7-O-glucuronide and / or its salt, an initiator and a cross-linking agent are added, a cross-linking reaction is carried out, and then another hydrogel is covered on one surface of the obtained hydrogel; the hydrogel is obtained by a chemical cross-linking reaction initiated by an initiator through a solution of a mixture of a carboxyl modified product of ascidian cellulose and acrylic acid and polyethylene glycol diacrylate;

[0069] For every 10 mL of chemical crosslinking reaction system, the mass ratio of the carboxyl modified product of ascidian cellulose to acrylic acid, polyethylene glycol diacrylate and initiator is (0.06-0.15 g):(1.2-2 g):(0.15-0.28 g):(0.008-0.012 g), the total volume used is 10 mL of the system, and the solvent is water.

[0070] In a preferred embodiment of the present invention, the mixing ratio of the ascidian cellulose dispersion and Chrysin-7-O-glucuronide and / or its salt is (40-60 ml): (1 g-2 g).

[0071] The present invention utilizes biomaterials such as algae cellulose (CNC), polyethyleneimine (PEI), acrylic acid (AA), ionization promoter, modified tannic acid (mTA) and polyethylene glycol diacrylate (PEGDA) to encapsulate the screened small molecule compound Chrysin-7-O-glucuronide in an asymmetric adhesive conductive hydrogel (named CPAMm / PCA hydrogel) provided by the present invention to achieve delivery and sustained release in the myocardial infarction area, which can not only improve the bioavailability of small molecule drugs, but also provide a microenvironment with a certain mechanical support strength and conductive repair for the infarction area, thereby promoting the self-repair process after heart damage.

[0072] The present invention proves that the CPAMm / PCA hydrogel has a significant sustained release effect through a 12-day continuous drug sustained release experiment, and the drug sustained release rate on the 12th day reaches 98.78%.

[0073] In addition, the drug-loaded multifunctional hydrogel has good biocompatibility in the biomedical field, which means that the hydrogel and the drug-loaded hydrogel show low toxicity in the interaction with biological systems, providing a reliable basis for its medical application.

[0074] In a fifth aspect, the present invention also provides an application of a drug-loaded multifunctional hydrogel in the preparation of a drug for treating ischemic heart disease and its complications, or an application of the above-mentioned tunicate cellulose hydrogel, the above-mentioned multifunctional hydrogel or the above-mentioned drug-loaded multifunctional hydrogel in the preparation of skin repair dressings, implants, biomaterials and medical devices.

[0075] In a preferred embodiment of the present invention, the biomaterial is selected from at least one of artificial skin, artificial bone, artificial cornea, tissue or organ scaffold and cell culture microcarrier;

[0076] In a preferred embodiment of the present invention, the skin repair dressing further comprises acellular matrix and collagen;

[0077] In a preferred embodiment of the present invention, the artificial bone is artificial cartilage.

[0078] In a preferred embodiment of the present invention, the above-mentioned ischemic heart disease and its complications are selected from any one of myocardial infarction, coronary heart disease, angina pectoris, arrhythmia, ventricular fibrosis, heart failure, myocardial ischemia, myocarditis and atherosclerosis;

[0079] In a preferred embodiment of the present invention, the application includes at least one of the following application modes:

[0080] (1) Promote the proliferation of cardiomyocytes;

[0081] (2) Reduce cardiomyocyte apoptosis;

[0082] (3) inhibiting the expression of apoptosis-related proteins;

[0083] (4) Regulate the cell cycle;

[0084] (5) Promote the repair of ischemic myocardial tissue;

[0085] (6) Reduce myocardial infarction area;

[0086] (7) Reduce cardiac fibrosis.

[0087] In a preferred embodiment of the present invention, regulating the cell cycle includes: promoting the transition of the cell cycle G1-S phase of cardiomyocytes;

[0088] In a preferred embodiment of the present invention, regulating the cell cycle includes regulating in the following ways: by affecting the expression level of cyclin;

[0089] In a preferred embodiment of the present invention, influencing the expression level of cyclin includes at least one of the following application modes:

[0090] (1) Downregulate the expression of ALDH7A1 gene;

[0091] (2) upregulating the expression of TSPAN5 gene;

[0092] (3) down-regulating the expression of at least one protein selected from the group consisting of Cyclin D1, CDK4, pRB and E2F1 proteins;

[0093] (4) Upregulation of CDK2 protein expression;

[0094] (5) downregulate the expression of ACOT7 protein;

[0095] (6) downregulate the expression of LAM5 protein;

[0096] (7) Upregulate the expression of ERBB2 gene.

[0097] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0098] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0099] The initial in vitro experimental results showed that Chrysin-7-O-glucuronide, a specific small molecule compound obtained through high-throughput screening, can effectively regulate the function of LRP6 protein to promote cardiomyocytes to re-enter the cell cycle and inhibit apoptosis. However, the traditional drug delivery method has the problems of low drug targeting and insufficient bioavailability, and may also cause a series of side effects, such as gastrointestinal irritation, thrombocytopenia and liver damage. In addition, the collapse of the ventricular wall and severe damage to the conductive function of the damaged myocardial tissue affect normal cardiac function. Therefore, using hydrogels with mechanical support and electrophysiological functions as drug delivery carriers can help further optimize the therapeutic effect. This not only improves the bioavailability of small molecule compounds, but also provides the necessary mechanical support and electrical conduction recovery for the damaged myocardium, significantly improving the repair ability after myocardial infarction.

[0100] The following example provides a multifunctional hydrogel system to achieve in situ release of Chrysin-7-O-glucuronide in cardiac injury. The hydrogel system contains two key layers, the bottom layer uses AA and PEI as the hydrogel skeleton, and the modified mTA provides adhesion and enhances the interfacial interaction between tissue and hydrogel. In order to improve the electrical coupling microenvironment of the infarct area, the ionization promoter MASEP is introduced. CNC-CHO performs well in regulating mechanical properties and enhancing the adhesion of hydrogels, which is beneficial for providing mechanical support to the ventricular wall. In contrast to the bottom layer, the top layer is composed of AA and CNC-COOH, and the anti-cell adhesion PEGDA is used as a crosslinker to prepare a hydrogel with anti-cell adhesion and pericardial adhesion prevention effects. The core goal of the entire design is to provide a stable platform for Chrysin-7-O-glucuronide to ensure its effective release in the cardiac environment, while providing good mechanical support and conductive recovery for damaged cardiac tissue. This carefully designed hydrogel system provides an innovative therapeutic strategy for the treatment of cardiac diseases through layer-by-layer functional layers.

[0101] The experimental materials involved in the embodiment are as follows:

[0102] 1. Experimental cell lines

[0103] The human cardiomyocyte cell line AC16 was purchased from the American Type Culture Collection (ATCC).

[0104] 2. Experimental Reagents

[0105] Main experimental materials and reagents

[0106]

[0107]

[0108] 3. Main experimental instruments and equipment

[0109]

[0110] 4. Preparation of common reagents

[0111] (1) Cell culture medium: 1% penicillin-streptomycin mixture, 10% fetal bovine serum, 89% culture medium (DMEM / F-12).

[0112] 5. Statistical Analysis

[0113] All data were presented as mean ± standard deviation, and Fisher's test, nonparametric Mann-Whitney rank sum test and one-way analysis of variance were used for relevant experimental statistical analysis. In all statistical analyses, P<0.05 indicated that the difference was statistically significant, and significant differences were marked as *p<0.05, **p<0.01, ***p<0.001.

[0114] Example 1

[0115] This example is used to prepare a cellulose dispersion and a cellulose hydrogel (CPAMm hydrogel).

[0116] 1. Preparation of Ascidian Cellulose

[0117] (1) Prepare the tunicate sample: remove the internal organs and tissues of the tunicate, retain the tunicate cyst, and wash it repeatedly to ensure that its surface is clean.

[0118] (2) Pretreatment of the ascidian cysts: Cut the ascidian cysts into small pieces, pre-hydrolyze them in a 5 wt% NaOH aqueous solution at a ratio of (1 g:100 ml), mechanically stir them in a 60°C water bath for 3 h, and repeat 3 times. After the reaction is completed, filter them, wash them with water until they are neutral, and dry them in an oven at 60°C.

[0119] (3) Bleaching and removal of impurities: Use 500 ml of 1.2 wt% H 2 O 2 The solution was stirred in a water bath at 80°C for 3 hours for bleaching reaction, then filtered, washed with water until neutral, and dried in an oven at 60°C, and repeated 3 times. Then, 500 ml of 5wt% HCl was stirred at room temperature for 6 hours to remove impurities such as minerals. The bleached sample was soaked in anhydrous ethanol for 24 hours to better dissolve the residual color. Finally, it was washed with water until neutral and dried in an oven at 60°C.

[0120] (4) Acid hydrolysis: Add 50 ml of 65 wt% H 2 SO 4 Then heat to 50℃, stir and react for 2h, add 10 times distilled water to terminate the reaction, centrifuge. Finally, put the sample in a dialysis bag and dialyze in distilled water for about 1 week to remove excess acid.

[0121] (5) Freeze drying: The dialyzed sample is freeze dried to obtain white CNC.

[0122] 2. Modification of Ascidian Cellulose

[0123] (1) Aldehydation of ascidian cellulose: CNC was placed in a solution containing 5 wt% sodium periodate at a ratio of (1 g:100 mL) and stirred for 6 h in the dark. Then, ethylene glycol was added to terminate the reaction. Finally, it was washed with deionized water and anhydrous ethanol several times and then freeze-dried to obtain CNC-CHO (1000 KDa).

[0124] (2) Carboxylated tunicate cellulose: CNC was added to a 3 mol / L citrate solution in a ratio of (1 g: 100 mL), and 20 wt% hydrochloric acid was added as a catalyst (the ratio was 1: 10 ml, i.e., 1 ml was added to a 10 ml system), and the mixture was placed in a beaker and mechanically stirred for hydrolysis. The reaction conditions were 80 °C and the stirring time was 3.75 h. The resulting suspension was washed by centrifugation with distilled water several times until it was neutral. Subsequently, the suspension was treated with an ultrasonic cell disruptor to obtain a CNC-COOH aqueous solution. Finally, the aqueous solution was freeze-dried in vacuum to obtain CNC-COOH.

[0125] 3. Tannic acid modification

[0126] TA was dissolved in 0.1 mol / L NaOH solution at 25°C in a ratio of (1 mg: 30 mL). At the same time, benzalkonium chloride (BAC) was dissolved in deionized water in a ratio of (1 mg: 5 mL). The BAC solution was added dropwise to the TA solution to induce the formation of a white precipitate. The mixture was stirred at room temperature for 3 h, and then the precipitate was collected by vacuum filtration. Subsequently, it was washed three times with deionized water to remove NaOH, residual TA and BAC. Finally, the precipitate was freeze-dried to obtain mTA.

[0127] 4. Preparation of Ascidian Cellulose Hydrogel (CPAMm Hydrogel)

[0128] (1) Preparation of CNC-CHO solution: NaOH, urea and deionized water were mixed in a volume ratio of 7:12:81 to form a uniform NaOH / urea solution, and then 5 g of CNC-CHO was dissolved in 30 ml of the NaOH / urea solution. The CNC-CHO solution was prepared by ultrasonic dispersion.

[0129] (2) Preparation of CPAM hydrogel: CNC-CHO solution (30 ml), PEI solution (10 g PEI dissolved in 15 ml deionized water), 1.5 g AA, 0.01 g MASEP, and 0.25 g BAC were mixed, and deionized water was added to 50 ml and stirred to form a uniform solution. Subsequently, APS (0.05 g) and TEMED (0.025 g) were added and stirred uniformly to form a mixture. Finally, the mixture (50 ml) was poured into a PTFE mold and placed at 50 ° C for 4 h to prepare CPAM hydrogel.

[0130] (3) Preparation of CPAMm hydrogel: CNC-CHO solution (30 ml), PEI solution (10 g PEI dissolved in 15 ml deionized water), 1.5 g AA, 0.045 g mTA, 0.01 g MASEP, and 0.25 g BAC were mixed, and deionized water was added to 50 ml and stirred to form a uniform solution. Subsequently, APS (0.05 g) and TEMED (0.025 g) were added and stirred uniformly to form a mixture. Finally, the mixture (50 ml) was poured into a PTFE mold and placed at 50 ° C for 4 h to prepare CPAMm hydrogel.

[0131] Example 2

[0132] This example is about the preparation of CPAMm / PCA multifunctional hydrogel.

[0133] First, an adhesion layer hydrogel was pre-prepared according to the CPAMm hydrogel preparation method in Example 1, and then a non-adhesion layer hydrogel solution was prepared according to the PCA hydrogel. Finally, the non-adhesion layer hydrogel solution was added to the adhesion layer pre-gel and kept at 50°C for 4 hours to prepare the CPAMm / PCA multifunctional hydrogel.

[0134] The preparation method of the non-adhesive layer hydrogel solution is as follows:

[0135] 0.1 g CNC-COOH, 1.5 g AA, 0.2 g PEGDA and an appropriate amount of APS (0.01 g) were mixed in deionized water and stirred to obtain a mixed solution (10 mL).

[0136] When preparing the non-adhesive layer hydrogel, the mixture was poured into a PTFE mold and placed at 50° C. for 4 h to prepare the PCA hydrogel.

[0137] Example 3

[0138] This example is used to prepare drug-loaded hydrogel.

[0139] CNC-CHO solution (30 ml), PEI solution (10 g PEI dissolved in 15 ml deionized water), 1.5 g AA, 0.045 g mTA, 0.01 g MASEP, 0.25 g BAC were mixed, and deionized water was added to 50 ml and stirred to form a uniform solution. Subsequently, 1.5 g of the small molecule compound Chrysin-7-O-glucuronide was added and stirred again. APS (0.05 g) and TEMED (0.025 g) were added to the solution, stirred evenly to form a mixture, and the mixture was poured into a PTFE mold for prefabrication to maintain the shape and state. PCA hydrogel was added to the surface of the prefabricated hydrogel, and then kept at 50 ° C for 4 hours to make it a drug-loaded multifunctional hydrogel.

[0140] Comparative Example 1

[0141] Preparation of CPA hydrogel: CNC-CHO solution (30 ml), PEI solution (10 g PEI dissolved in 15 ml deionized water), 1.5 g AA, 0.25 g BAC were mixed, and deionized water was added to 50 ml and stirred. Subsequently, ammonium persulfate (0.05 g) (ammonium persulphate, APS) and tetramethylethylenediamine (N, N, N', N'-Tetramethylethylenediamine, TEMED) (0.025 g) were added and stirred evenly to form a 50 mL system mixture. Finally, the mixture was poured into a PTFE mold and placed at 50 ° C for 4 hours to prepare CPA hydrogel.

[0142] Comparative Example 2

[0143] Preparation of PCA hydrogel: 0.1 g CNC-COOH, 1.5 g AA, 0.2 g PEGDA and an appropriate amount of APS (0.01 g) were mixed and stirred to obtain a mixed solution (10 mL). Finally, the mixture was poured into a PTFE mold and placed at 50° C. for 4 h to prepare PCA hydrogel.

[0144] Experimental Example 1

[0145] Fourier transform infrared spectroscopy characterization:

[0146] A small amount of CNC, CNC-CHO, CNC-COOH, TA, mTA, CPAMm, PCA and CPAMm / PCA hydrogel was freeze-dried to ensure the stability of the sample. The dried sample was then placed in the sample cell of a Fourier transform infrared spectrometer. When performing Fourier transform infrared spectroscopy (FT-IR) analysis, the scan range was set to 525-4000 cm-1 , by scanning the sample spectrum, the FT-IR spectrum is obtained.

[0147] (1) FT-IR spectra of CNC, CNC-CHO, and CNC-COOH refer to Figure 1 As shown in the figure, cellulose is widely used for its outstanding mechanical strength, high surface activity and biodegradability. Ascidian cellulose, as a unique form of animal cellulose in nature, can be prepared into nanofibers with high crystallinity and modulus and extremely high aspect ratio, namely CNC, by acid hydrolysis. The physical entanglement interaction and hydrogen bonding between CNCs help to improve the mechanical properties of hydrogels. In addition, modification of CNCs can further optimize the properties of hydrogels, among which aldehyde- and carboxyl-modified CNCs are particularly helpful for the cross-linking effect of hydrogels.

[0148] First, CNC was successfully prepared by acid hydrolysis. The FT-IR spectrum of the sample is shown in Figure 1 As shown. In the ascidian cellulose, the stretching vibrations of OH, CH and CO were observed to appear near 3341cm-1, 2903cm-1 and 1054cm-1, respectively, while the two peaks at 3273cm-1 and 706cm-1 represented the Iβ absorption characteristics of cellulose. Further comparison of the FT-IR spectra of pure CNC and CNC-CHO revealed that CNC-CHO exhibited a significant aldehyde absorption peak at 1725cm-1, indicating that CNC-CHO was successfully synthesized. In addition, in the spectrum of CNC-COOH, a new absorption peak was observed to appear at 1733cm-1, corresponding to the stretching vibration of C=O, further confirming the formation of CNC-COOH.

[0149] (2) The structures of TA and mTA were characterized by FT-IR spectroscopy. Figure 2 shown.

[0150] The polyelectrolyte complex hydrogel formed by the composite of PAA and PEI with opposite charges exhibits certain adhesion on the wet substrate, but its adhesion strength is relatively weak, and there is also the problem of insufficient cohesive strength. In order to improve this situation, inspired by the adhesion of mussels, TA containing a catechol structure was introduced to effectively enhance the adhesion of the hydrogel to the tissue. In order to solve the problem of cohesive strength of the hydrogel, TA was combined with a hydrophobic long alkyl chain to prepare mTA. In order to characterize the structure of mTA, FT-IR spectroscopy analysis ( Figure 2 The FT-IR spectrum of mTA shows the characteristic absorption peak of CH of BAC (located at 2925 cm -1 ) and the C=O characteristic absorption peak of TA (located at 1714 cm -1). Therefore, FT-IR spectroscopy demonstrated that mTA was synthesized via the electrostatic assembly of TA and BAC.

[0151] (3) The structures of CPA, CPAM, CPAMm, and CPAMm and PCA were characterized by FT-IR spectroscopy.

[0152] from Figure 3 It can be observed that a series of hydrogels have -1 The formation of CO-NH at 1040 cm-1 indicated that the dynamic imine bond was successfully formed between CNC-CHO and PEI in the hydrogel network. -1 There is a strong absorption peak at 733 cm, which is attributed to the sulfonic acid group, indicating that MASEP is successfully introduced into the hydrogel. -1 An absorption peak is shown nearby, corresponding to the stretching vibration of the phenolic hydroxyl group (Ph-OH) of caffeic acid, indicating that the catechol structure was successfully introduced into the hydrogel polymer network.

[0153] Unlike CPAMm hydrogel, PCA hydrogel does not contain catechol, amino and aldehyde groups, but contains a large number of carboxyl groups and polyethylene glycol chains, which enables PCA hydrogel to reduce adhesion and significantly exhibit anti-cell adhesion properties. Figure 4 ), PCA hydrogel was observed at 920 cm -1 and 1710cm -1 The absorption peak at is significantly stronger than that of CPAMm hydrogel, indicating that the carboxyl content of PCA hydrogel is higher than that of CPAMm group.

[0154] Experimental Example 2

[0155] Characterization of conductivity of adhesion layer hydrogel:

[0156] In order to verify the conductivity of the adhesion layer hydrogel, a constant voltage circuit was constructed, which included a light emitting diode (LED). The hydrogel was connected in series with devices such as the LED, and the conductivity of the hydrogel was evaluated by monitoring the brightness changes of the LED.

[0157] The experimental results are as follows Figure 5As shown, although CPA hydrogel can light up LED, its brightness is relatively weak. Subsequently, when MASEP (ionic agent) was added, CPAM hydrogel showed stronger brightness, indicating that the introduction of ionic agent and the formation of interpenetrating network significantly improved the conductivity of hydrogel. Further observations showed that CPAMm hydrogel containing mTA exhibited excellent conductivity, and the addition of mTA did not affect the conductive properties of hydrogel. The comprehensive analysis results show that CPAMm hydrogel has the potential to be used to regulate cardiac tissue electrophysiology.

[0158] Experimental Example 3

[0159] Hydrogel Adhesion Characterization:

[0160] In this experiment, different material samples were prepared, including plastic, rubber, glass, tissue organs, and pig skin to verify the adhesion of the hydrogel. The prepared hydrogel samples were contacted with each material separately, and the adhesion of the hydrogel on the surface of different materials was recorded to evaluate its adhesion performance.

[0161] To gain a deeper understanding of the adhesion properties of the hydrogel, a detailed study was conducted on the needs of myocardial infarction repair. Strong adhesion between cardiac tissue and hydrogel is essential for providing appropriate mechanical support and achieving electrophysiological bonding to promote cardiac function recovery. To enhance the adhesion properties of the hydrogel, the ionization promoter MASEP and mTA containing catechol groups were introduced. Such improvements make CPAMm hydrogels perform better in tissue adhesion, which is mainly attributed to the enhancement of multiple dynamic non-covalent interactions and physical entanglements between PEI and poly (AA-co-MASEP) polymer chains. The long alkyl chains of mTA destroy the interfacial hydration layer by absorbing water, and the exposed catechol, -COOH and -NH2 groups interact with the groups on the tissue surface to enhance the wet adhesion strength. Figure 6 The adhesion properties of CPAMm hydrogel on different surfaces were demonstrated. The experimental results showed that CPAMm hydrogel could firmly adhere to a 50g weight. In addition, CPAMm hydrogel exhibited excellent adhesion and could tightly adhere to hydrophilic and hydrophobic surfaces, such as rubber and glass. More importantly, CPAMm hydrogel showed high adhesion on the surface of wet biological tissues, including various organs such as the heart, lungs, kidneys, spleen and liver, which is of key significance for biomedical applications.

[0162] In order to effectively prevent adverse tissue adhesion, CPAMm / PCA hydrogels were prepared by adding an anti-tissue adhesion layer to the top layer of the adhesive bottom layer hydrogel. Unlike CPAMm hydrogels, PCA hydrogels lack catechol, amino, and aldehyde groups, but are rich in carboxyl groups and polyethylene glycol chains, which enable PCA hydrogels to significantly reduce adhesion and exhibit excellent anti-cell adhesion properties. The CPAMm side and PCA side of the CPAMm / PCA hydrogel exhibit completely opposite adhesion properties, as shown in Figure 7 . It can be clearly seen from the figure that the CPAMm side of the CPAMm / PCA hydrogel exhibits high adhesion to pig skin, while the PCA side exhibits negligible adhesion. Due to the high adhesion of the CPAMm hydrogel layer, the CPAMm / PCA hydrogel can be stably fixed on the heart, achieving long-term effective anti-adhesion. Compared with a single anti-adhesion physical barrier that is easy to fall off, the CPAMm / PCA hydrogel can continue to remain on the surface of the heart, effectively preventing adhesion between the heart and the chest wall, thereby achieving a long-term anti-adhesion effect.

[0163] Experimental Example 4

[0164] The mechanical properties of the multifunctional hydrogel were analyzed.

[0165] An electronic universal testing machine was used to measure the stress-strain curve and compression modulus to test the mechanical properties of the multifunctional hydrogel. In the experiment, the strain rate was set to 10% / min and a preload of 0.05N was applied to ensure that the sample was in a suitable test state.

[0166] The mechanical properties of hydrogel scaffolds have a crucial impact on the function of engineered tissue patches. An ideal patch scaffold should have two key mechanical properties. First, its elastic modulus should be close to that of natural heart tissue to promote synchronized contraction of cardiomyocytes in vitro; second, the scaffold should have excellent fatigue resistance to maintain stable cardiac contraction and relaxation after transplantation into the infarcted heart. To this end, three different hydrogels were subjected to compression tests. Figure 8The compressive stress-strain curve of the hydrogel is shown, and the elastic modulus can be calculated from this figure. It is worth noting that after compression to 60% strain, none of the samples broke. The elastic modulus and mechanical strength of the hydrogel were significantly improved by introducing the interpenetrating network formed by MASEP. At 60% compressive strain, the elastic modulus increased from 49.29±4.54kPa to 154.89±23.98kPa after the addition of MASEP. However, after the introduction of mTA, the elastic modulus of the CPAMm hydrogel decreased to 25.27±2.53kPa. The elastic moduli of all hydrogels fall within the range of natural myocardium, meeting the elasticity requirements of cardiac tissue engineering scaffolds. This shows that by regulating the composition of the hydrogel, especially the introduction of an interpenetrating network structure, the mechanical properties of the scaffold can be effectively adjusted to better meet the engineering needs of cardiac tissue.

[0167] Experimental Example 5

[0168] Analysis of the conductive properties of multifunctional hydrogels:

[0169] In order to evaluate the conductivity of the hydrogel, a resistivity tester was used for a systematic experiment. First, the hydrogel sample was prepared to ensure that its shape and size met the experimental requirements. Then, the electrodes of the resistivity tester were precisely placed on both sides of the hydrogel sample to ensure that the electrodes were in full contact with the sample, so that the resistance could be accurately measured.

[0170] The conductivity of native myocardium ranges from 10 -4 To 10S / cm, which can be used as a reference value for preparing conductive hydrogels for cardiac tissue engineering repair after myocardial infarction. In order to evaluate the conductive properties of these hydrogels, the conductivity of the multifunctional hydrogels was tested using a resistivity tester. Fig. 9 In the experiment, the CPA / PAC hydrogel without MASEP showed a relatively low conductivity of 0.063±0.004S / m. In contrast, the conductivity of the CPAM / PAC hydrogel with the addition of MASEP increased to 0.110±0.006S / m. The CPAMm / PAC hydrogel containing MASEP and mTA showed the best conductivity of 0.127±0.004S / m. This conductivity value is completely consistent with the electrical signal conduction level of natural myocardial tissue. These results show that with the addition of MASEP and mTA, the conductivity of the hydrogel is significantly improved, especially the CPAMm / PAC hydrogel shows conductive properties comparable to those of natural myocardial tissue.

[0171] Experimental Example 6

[0172] Characterization of multifunctional hydrogel morphology.

[0173] CPAMm hydrogel and PCA hydrogel were freeze-dried to ensure the stability of the samples. Subsequently, the freeze-dried samples were taken out and immediately fractured with liquid nitrogen to maintain the original morphology of the samples. Finally, the freeze-dried samples were characterized by scanning electron microscope (SEM) and the samples on the copper sheet were placed in the SEM instrument for observation to obtain detailed information on the surface morphology and microstructure of the samples.

[0174] The hydrogels of the CPAMm layer and PCA layer were observed using a scanning electron microscope. The relevant results are shown in Fig.10 . It is obvious from the cross-sectional electron micrograph that both hydrogels exhibit obvious pore structures, and the number of pores is relatively large. The CPAMm layer, as a material with adhesion properties, exhibits a relatively dense pore structure. In contrast, the PCA layer, as an anti-adhesion material, exhibits a larger pore structure. This differential pore structure helps to better achieve close adhesion of the hydrogel to the site of myocardial injury, thereby providing more favorable conditions for treatment.

[0175] Experimental Example 7

[0176] Determination of drug release curve of drug-loaded hydrogel:

[0177] Following the experimental process of Example 3, a drug-loaded hydrogel containing 0.1 mg Chrysin-7-O-glucuronide was synthesized. It was placed in a brown glass bottle and 20 mL of PBS buffer with a pH of 7.4 was added. Subsequently, the brown glass bottle containing the hydrogel was placed in a constant temperature shaking incubator at 37°C. In the first two days, 100 μL of sample was extracted every 12 hours and supplemented with 100 μL PBS, and the sample was extracted every 24 hours for the next 10 days. The detection was carried out at a wavelength of 310 nm using a full-wavelength microplate reader equipped with a UV-Vis spectrophotometer, and the absorbance value was recorded. The absorbance value was converted into drug concentration, and a release curve was prepared to evaluate the sustained release performance of the drug-loaded hydrogel.

[0178] In the in vitro sustained release experiment, a multifunctional hydrogel loaded with 0.1 mg Chrysin-7-O-glucuronide was selected to study its drug sustained release ability. The experiment lasted for 12 days, and the drug release curve obtained by continuous sampling and testing was as follows: Fig.11 As shown. The multifunctional hydrogel exhibited a slow release property under a neutral environment, with the sustained release rate reaching 49.09% in the first 48 hours. After 12 days of the experiment, the drug sustained release rate further increased to 98.78%. These results show that the hydrogel successfully achieved the drug sustained release effect.

[0179] Experimental Example 8

[0180] Multifunctional hydrogel biocompatibility testing:

[0181] (1) Preparation of hydrogel extract: The multifunctional hydrogel and the drug-loaded multifunctional hydrogel were soaked in DMEM / F12 culture medium for 24 h to obtain a hydrogel extract with a concentration of 10 mg / mL.

[0182] (2) Cell culture preparation: AC16 cells were cultured in a special culture medium, seeded at a density of 5000 cells / well on a 96-well plate, and cultured for 12 h.

[0183] (3) Experimental treatment: The cell culture medium was replaced with the previously prepared hydrogel extract and culture was continued for 24 hours. After the culture was completed, the culture medium was removed and CCK-8 mixed solution was added to each well.

[0184] (4) Cell viability determination: Place the 96-well plate in the incubator for 2 h to allow the CCK-8 mixture to fully act. Place the 96-well plate in a microplate reader and select a wavelength of 450 nm for absorbance reading. Calculate the cell viability based on the absorbance value obtained by the microplate reader to evaluate the biocompatibility of the hydrogel with AC16 cells.

[0185] The biocompatibility of hydrogels is an important criterion for evaluating their use as biomedical materials. In order to fully understand the cytotoxicity of hydrogels, the CCK-8 method was used for evaluation. The specific results are as follows: Fig.12 As shown. For CPAMm / PCA hydrogel, a relatively low cell proliferation rate was observed, with a proliferation rate of 77.75±3%. It should be noted that despite the low cell proliferation rate, its cytotoxicity rating reached level 1. In comparison, the cytotoxicity of CPAMm / PCA drug-loaded hydrogel is still level 1, but the cell proliferation rate is increased by 6%. The results of the study showed that the cytotoxicity of CPAMm / PCA hydrogel and its drug-loaded hydrogel were both lower than the national standard level 2, confirming that they have good biocompatibility in the biomedical field. This means that this type of hydrogel and drug-loaded hydrogel show low toxicity in their interactions with biological systems, providing a reliable basis for their medical applications. Therefore, the research results emphasize the potential application of CPAMm / PCA hydrogel and its drug-carrying hydrogel in the treatment of myocardial ischemia-reperfusion diseases, providing strong support for subsequent animal experiments.

[0186] Experimental Example 9

[0187] By performing batch screening of Taoshu Biotechnology's small molecule compound library, a variety of small molecule compounds were successfully screened out, which can effectively bind to LRP6 protein in three-dimensional structure. According to the final docking score results, the most potential binding compounds were selected for subsequent biological verification. Table 1 lists the names, molecular formulas and docking scores of the top 10 compounds.

[0188] Table 1 Top 10 small molecule compounds in high throughput screening

[0189]

[0190] In the early stage of small molecule screening research, the small molecule compound Chrysin-7-O-glucuronide was successfully identified. This compound can specifically bind to the LRP6 protein and effectively inhibit the expression of LRP6.

[0191] Based on this, this embodiment designs a multifunctional drug-loaded hydrogel system based on the physiological conditions of ventricular wall collapse and electrical coupling microenvironment in the infarcted area. The bottom layer of the system not only provides adhesion, enhances the interface interaction between tissue and hydrogel, effectively supports the ventricular wall, but also introduces ionization promoters to promote the conduction of myocardial cells, while the top layer is designed with the functions of anti-cell adhesion and preventing pericardial adhesion, which is conducive to promoting myocardial damage repair.

[0192] By constructing a rat myocardial ischemia-reperfusion model, the treatment group was compared with the sham group to evaluate the therapeutic effect of the multifunctional drug-loaded hydrogel. The performance of the composite material in the treatment process will be comprehensively examined through multiple evaluation methods such as rat echocardiography, cardiac function indicators, Masson staining, and HE staining.

[0193] 1. Experimental Materials

[0194] (1) Experimental animals

[0195] SPF Sprague Dawley (SD) rats (200-230 g), of either sex, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. To ensure the hygiene standards of the animals, these rats were kept under conditions with a 12-h light-dark cycle and provided with adequate water and food. All experiments involving animals were reviewed and approved by the Experimental Animal Ethics Committee of Sichuan Provincial People's Hospital.

[0196] (2) Experimental materials and reagents

[0197] Table 2 Main experimental materials and reagents

[0198]

[0199] (3) Experimental instruments

[0200] Table 3 Main experimental instruments and equipment

[0201]

[0202]

[0203] 2. Experimental Methods

[0204] A. Construction of myocardial ischemia-reperfusion model

[0205] Myocardial ischemia-reperfusion model was established in SD rats according to the following experimental method. After 40 minutes, CPAMm / PCA and CPAMm / PCA loaded with Chrysin-7-O-glucuronide hydrogel were transplanted in the ischemic lesion of the heart. The sham operation group only underwent thoracotomy without ligation of the left anterior descending branch, and the PBS group and the drug-treated group were injected with PBS and Chrysin-7-O-glucuronide by tail vein injection, respectively.

[0206] The method for establishing the rat myocardial ischemia-reperfusion model is as follows:

[0207] (1) Anesthesia and endotracheal intubation surgery preparation: SD rats were anesthetized with isoflurane and endotracheal intubation was performed using a small animal ventilator to ensure respiratory stability.

[0208] (2) Chest exposure and thoracotomy: The pleura was exposed between the third and fourth ribs of the left chest of the rat.

[0209] (3) Coronary artery slip knot ligation: Use 6-0 suture to slip knot ligature the left anterior descending branch of the coronary artery 2 to 3 mm away from the left atrial appendage. Ensure that there is no obvious bleeding in the heart during the operation and keep the surgical area clean.

[0210] (4) Layer-by-layer suturing: The surgical area is sutured layer by layer using sutures to ensure that each tissue layer is tightly combined.

[0211] (5) Establishment of myocardial ischemia-reperfusion model: After 40 minutes of ischemia, the coronary artery ligature was removed.

[0212] B. Myocardial infarction physiological index detection

[0213] Echocardiography was used to detect cardiac function indices after different treatment measures. After the myocardial ischemia-reperfusion model was established and different materials were transplanted in the infarct area, SD rats were anesthetized with isoflurane 28 days later. After the SD rats entered deep anesthesia, they were tested using the Visualsonics ultra-high resolution small animal ultrasound imaging system. Pulsed Doppler ultrasound and M-mode ultrasound were used to detect mitral blood flow and cardiac function indices.

[0214] C. Organ tissue collection, pathological HE staining and heart tissue Masson staining

[0215] (1) Tissue collection: After the 28-day treatment period, the rats were euthanized and the organ tissues were collected. In order to fix the tissues, 4% paraformaldehyde was used for fixation for more than 24 hours.

[0216] (2) Paraffin embedding and sectioning: Take an appropriate amount of tissue, dehydrate it, and then embed it in paraffin. Use a paraffin sectioning machine to cut the tissue into 5 μm slices, unfold the slices in warm water, and then place them on a slide.

[0217] (3) HE staining: The collected tissue samples are placed in a fully automatic staining machine for staining. After staining, the stained tissue sections are sealed with neutral gum to ensure the fixation and preservation of the samples. Finally, images are collected under a microscope.

[0218] (4) Masson staining: The heart tissue was placed in an automatic staining machine for dewaxing, and then stained according to the kit instructions provided by the Solebo manufacturer. After completion, the slides were sealed with neutral gum. Images were collected under an upright microscope.

[0219] D. Cardiac troponin-t (cTnT) detection

[0220] According to the kit instructions, a quantitative ELISA kit (Abcam) was used to detect cTnT levels. After each model was successfully established, 0.5 mL of venous blood was collected to detect cTnT concentration.

[0221] E. Physiological index detection

[0222] Physiological parameters were measured by echocardiography. SD rats were mechanically ventilated under isoflurane anesthesia. Echocardiograms were recorded using the Visualsonics Ultra-High Resolution Small Animal Ultrasound Imaging System (Visualsonics, Toronto, Canada). Pulsed-wave Doppler echocardiography and M-mode echocardiography were used to detect mitral valve blood flow.

[0223] F. Histological Analysis of Heart Sections

[0224] Four weeks later, all animals were euthanized. The hearts were removed and cut into paraffin sections with a thickness of 5 μm. The histological characteristics of each organ were obtained according to the manufacturer's instructions. The histological characteristics of myocardial infarction were obtained by Masson's trichrome staining and HE staining according to the manufacturer's instructions. The thickness of the infarct area was measured using Image J software.

[0225] G. Electrical integration testing

[0226] Electrical integration testing was performed as described previously [1,2]. Briefly, an electrical stimulator and an electrocardiogram (ECG) recorder were used to assess the propagation of electrical signals in scar tissue. Scar tissue was harvested from rats after euthanasia, and 3 mm of healthy tissue was retained at each end of the scar. The two ends of the tissue were connected to the electrical stimulator and ECG recorder, respectively. The electrical stimulator parameters were set at 10 mV and 1 Hz. Four weeks later, signals were recorded for 5 min using a three-lead ECG and analyzed using Lab Chart software to determine the duration of the QRS complex. Arrhythmia induction was assessed according to a standard clinical electrical stimulation (PES) protocol. The epicardial activation between healthy and infarcted myocardium was mapped using a 64-channel electrophysiological mapping system (MappingLab). After the heart was isolated and Langendorff perfused, a 64-channel electrode array (8 × 8 grid, 0.45 mm pitch) was positioned at the border zone of healthy and infarcted tissue. Electrical stimulation (2 mV, 6 Hz) was applied to the epicardial surface beneath the left atrium. Action potential and Ca2+ activation time were determined by identifying the point of maximum negative slope. The inducibility of ventricular arrhythmias and ventricular fibrillation threshold were assessed using a bipolar electrode catheter sutured to the epicardium of the right ventricle. Programmed electrical stimulation was performed at an intensity twice the diastolic threshold using 8 consecutive S1-S1 stimuli with a cycle length of 330 ms, followed by additional stimulation (S2-S3-S4).

[0227] H. Statistical analysis

[0228] All data were presented as mean ± standard deviation, and Fisher's test, nonparametric Mann-Whitney rank sum test and one-way analysis of variance were used for relevant experimental statistical analysis. In all statistical analyses, P<0.05 indicated that the difference was statistically significant, and significant differences were marked as *p<0.05, **p<0.01, ***p<0.001.

[0229] 3. Experimental results and discussion

[0230] (1) Rat cardiac physiological indicators

[0231] To evaluate the effect of multifunctional drug-loaded hydrogel on the recovery of cardiac function in rats with myocardial ischemia-reperfusion, a myocardial ischemia-reperfusion model was established in SD rats. Subsequently, PBS and Chrysin-7-O-glucuronide groups were respectively given by tail vein injection, and the CPAMm / PCA group and CPAMm / PCA loaded with Chrysin-7-O-glucuronide group were transplanted into the infarcted myocardium of rats with myocardial ischemia-reperfusion to explore the effect of composite drug-loaded hydrogel on the repair of myocardial damage in rats. Ultrasound detection was performed on the 28th day after treatment.

[0232] M-mode echocardiography detects cardiac systolic and diastolic function. Fig.13 The results showed that compared with the normal contraction of the left ventricular wall in the sham group, the contraction amplitude of the ventricular anterior wall in the PBS group was weak, and a certain contraction of the ventricular anterior wall was observed in the Chrysin-7-O-glucuronide group and the CPAMm / PCA group, while the contraction amplitude of the left ventricular anterior wall in the CPAMm / PCA group loaded with Chrysin-7-O-glucuronide was significantly increased.

[0233] Doppler ultrasound was used to detect cardiac function. The results showed that the E / A peak ratio could be improved in the Chrysin-7-O-glucuronide injection group and the CPMm / PCA transplantation group, but the E / A peak ratio was higher in the Chrysin-7-O-glucuronide-loaded CPAMm / PCA transplantation group, showing a more significant therapeutic effect ( Fig.14 In addition, typical echocardiographic parameters such as ejection fraction (EF), fractional shortening (FS), E / A peak ratio, and heart rate (HR) were analyzed ( Fig.15 ), it can be seen from the results that the CPAMm / PCA-loaded Chrysin-7-O-glucuronide transplantation group has the best therapeutic effect. In summary, the results of echocardiography show that the CPAMm / PCA-loaded Chrysin-7-O-glucuronide transplantation group can significantly improve the cardiac function of rats after myocardial ischemia-reperfusion.

[0234] (2) Masson staining of the myocardial ischemia-reperfusion area confirmed the repair effect of the multifunctional drug-loaded hydrogel.

[0235] To further evaluate the role of CPAMm / PCA loaded with Chrysin-7-O-glucuronide in the repair of myocardial injury, five groups of different heart samples were collected 28 days after treatment, namely: sham group (sham operation group), PBS group (ischemia-reperfusion group), Chrysin-7-O-glucuronide group (drug group), CPAMm / PCA group (material group), CPAMm / PCA loaded with Chrysin-7-O-glucuronide group (multifunctional drug-loaded hydrogel group). Myocardial sections were processed by Masson staining to evaluate the repair effects of different groups. The staining results showed (see Fig.16 In a), due to the presence of a large amount of ischemic necrotic tissue in the myocardial ischemia-reperfusion zone, a large amount of fibrous tissue appeared blue in the heart sections of the PBS group, and there was almost no red myocardial tissue in the anterior wall of the ventricle. However, some red myocardial tissue was observed in the injured area of ​​the Chrysin-7-O-glucuronide and CPAMm / PCA groups, and more myocardial tissue was observed in the Chrysin-7-O-glucuronide-loaded CPAMm / PCA group. The Masson staining images were processed using ImageJ software to calculate the thickness of the infarct wall, and there were significant differences among the groups (see Fig.16 b). The thickness of the left ventricular wall in the CPAMm / PCA loaded Chrysin-7-O-glucuronide group reached 1.34±0.19mm, while that in the PBS group was only 0.67±0.05mm. These results indicate that the multifunctional drug-loaded hydrogel based on CPAMm / PCA loaded Chrysin-7-O-glucuronide can enhance the cardiac function and reduce scar formation in rats with myocardial ischemia-reperfusion, indicating that CPAMm / PCA loaded Chrysin-7-O-glucuronide has potential repair ability in myocardial injury repair.

[0236] (3) To evaluate the effect of C7Og (i.e., Chrysin-7-O-glucuronide) on cardiac repair in a rat MI (MI) model, different experimental groups were treated with CPAMm / PCA hydrogel, C7Og-loaded CPAMm / PCA (C@CPAMm / PCA), PBS, or C7Og via transplantation or tail vein injection. Myocardial function was evaluated 28 days after treatment.

[0237] Optical mapping was used to assess transmembrane action potential (Vm) and calcium ions (Ca) in Langendorff-perfused hearts. 2+ ) dynamics. Experimental results using RH237 and Rhod-2 AM dyes under 6 Hz stimulation conditions showed that MI injury disrupted action potential propagation and Ca2+ Regulation Fig.17 C@CPAMm / PCA transplantation significantly improved action potential propagation in the infarcted ventricle ( Fig.17 B) and Ca 2+ dynamic( Fig.17 Middle C). MI injury leads to a significant prolongation of APD90 and CTD90 ( Fig.19 ), however, after 4 weeks of C@CPAMm / PCA treatment, these durations were significantly shortened, repolarization was restored, and the risk of arrhythmias was reduced. We also assessed ventricular arrhythmia (VA) susceptibility by programmed stimulation to determine the VA induction rate and ventricular fibrillation (VF) threshold by burst stimulation. MI significantly increased VA susceptibility, as manifested by a higher VA induction rate ( Fig.17 D) and a lower VF threshold ( Fig.17 (center E). Notably, C@CPAMm / PCA treatment effectively reversed these pathological changes, significantly reduced the VA induction rate and increased the VF threshold, thereby reducing the risk of arrhythmic death.

[0238] (4) Effects of multifunctional drug-loaded hydrogel on organ tissue pathology after treatment:

[0239] In order to further explore the safety of the multifunctional drug-loaded hydrogel system in vivo, after 28 days of treatment, the main organs (heart, liver, spleen, lung, and kidney) of rats in different treatment groups were stained with HE to observe their pathological changes. In the hematoxylin stain, the cell nucleus appears dark blue, while in the eosin solution, the cytoplasm appears red, which helps to observe the pathological changes of different organs through color changes. The experimental results are shown in Fig.18 , the heart sections of the five groups showed that the cardiomyocytes were neatly arranged, and no characteristic changes of cardiac injury such as inflammation and fibrosis were observed in the interstitium. The hepatocytes were arranged divergently with the central vein as the center, and the nuclei were large and round. The spleen sections showed that the white bones were stained blue-purple, the dense red tissue formed trabeculae by the spleen capsule, and the rest was red pulp. The overall structure was intact and there was no abnormality. The lung tissue structure was intact, the glomerular structure was good, the blood vessels were not ruptured, and there were no obvious abnormalities. The kidney sections showed that the glomerular structure was clear and complete, and no fibrosis was observed. There were no obvious abnormalities in the main organs of SD rats in each group, indicating that the multifunctional drug-loaded hydrogel therapeutic system is a relatively safe drug delivery system with excellent in vivo safety and biocompatibility.

[0240] In summary, the CPAMm / PCA-loaded Chrysin-7-O-glucuronide hydrogel prepared by the present invention has good electrical conductivity and mechanical properties. The conductivity of the CPAMm / PCA hydrogel is 0.127±0.004S / m, which meets the requirements of cardiac electrophysiological signal conduction, and its Young's modulus is 25.27±2.53kPa, which meets the requirements of cardiac tissue engineering scaffolds for elasticity. The multifunctional hydrogel has an asymmetric porous microstructure, which helps to achieve close adhesion to the site of myocardial infarction and prevent adhesion of the pericardium. The effect of the multifunctional hydrogel in slowly releasing drugs was verified by a 12-day sustained release experiment, and the biocompatibility experiment proved that it has good biocompatibility. This study provides an important basis for the preparation and performance verification of multifunctional hydrogels.

[0241] CPAMm / PCA-loaded Chrysin-7-O-glucuronide hydrogel can promote the repair of damaged myocardium in vivo. CPAMm / PCA and Chrysin-7-O-glucuronide played a certain role in reducing myocardial infarction area and cardiac fibrosis, thereby improving the cardiac function of rats. However, the rats in the CPAMm / PCA-loaded Chrysin-7-O-glucuronide hydrogel group had the smallest cardiac scar and the best cardiac function recovery effect. In addition, HE staining of the organ tissues of different dosing groups showed that there were no abnormalities in the heart, liver, spleen, lung, and kidney of the five groups of rats, indicating that CPAMm / PCA-loaded Chrysin-7-O-glucuronide hydrogel has good safety and biocompatibility in vivo. These findings provide substantial support for the application of multifunctional hydrogel systems in the treatment of cardiovascular diseases.

[0242] Example 10

[0243] C@CPAMm / PCA improves cardiac function and electrical conduction after MI in Bama miniature pigs.

[0244] In this embodiment, a Bama miniature pig weighing 15-20 kg was used to establish an MI model. The pigs were fasted for 12 hours before surgery to reduce the risk of aspiration. Anesthesia was induced by isoflurane and maintained by continuous administration of drugs through endotracheal intubation (1-2% isoflurane). Vital signs such as heart rate, respiratory rate and blood oxygen saturation were continuously monitored by electrocardiogram (ECG). The surgical area (especially the left chest) was shaved and disinfected with povidone-iodine solution. A median thoracotomy was performed to expose the heart. Before ligation, the left anterior descending coronary artery (second diagonal branch) was pre-clamped for 15 minutes. The chest cavity was closed in layers using absorbable sutures. Antibiotics (cefazolin, 25 mg / kg) and analgesics (lidocaine, 2 mg / kg) were given postoperatively, and the pigs were closely monitored during recovery. Myocardial infarction was confirmed by ECG immediately after surgery. The extent of myocardial damage was further assessed by echocardiography after surgery. At the end of the study, Bama miniature pigs were humanely killed using an overdose of pentobarbital, and heart tissues were obtained for histological analysis to confirm myocardial infarction.

[0245] Given the structural and functional similarities between the porcine heart and the human heart, the C@CPAMm / PCA myocardial patch was evaluated in the Bama miniature pig MI model ( Fig. 20 After application of the patch to damaged myocardium, a significant decrease in serum cTnI levels was observed compared with the control saline group ( Fig. 20 Middle B), and ST segment elevation was significantly reduced ( Fig. 20 In addition, the patch resulted in a 50% reduction in infarct size ( Fig. 20 E and F), and significantly improved left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) ( Fig. 20 Middle G, H and I; Fig.21 ), these indicators are close to normal levels.

[0246] Optical mapping results showed that C@CPAMm / PCA significantly enhanced action potential (AP) propagation and calcium ion (Ca2+) dynamics, leading to a significant decrease in APD90 and CaD90, while effectively preventing pathological ventricular remodeling ( Fig. 20 Middle J, 6K and 6L; Fig. 22 We assessed susceptibility to ventricular arrhythmias (VA) using programmed stimulation and measured ventricular fibrillation (VF) threshold using burst stimulation. MI significantly increased VA susceptibility, as evidenced by an increase in VA induction rate ( Fig. 20 M) and a decrease in VF threshold ( Fig. 20 N); however, C@CPAMm / PCA treatment effectively reversed these pathological changes. Histological analysis four weeks after transplantation showed no significant pathological changes in important organs, further confirming the biosafety of the C@CPAMm / PCA patch ( Fig.23 ).

[0247] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0248] References:

[0249] [1]LI M,LIU Y,HUANG B,etc.A Self-Homing and Traceable Cardiac PatchLeveraging Ferumoxytol for Spatiotemporal Therapeutic Delivery[J].ACS nano,2024,18(4):3073-86.

[0250] [2]CHENG P, CHENG L, HAN H, etc.A pH / H(2)O(2) / MMP9 Time-Response GelSystem with Sparc(high)TregsDerived Extracellular Vesicles Promote RecoveryAfter Acute Myocardial Infarction[J]. Advanced healthcare materials, 2022, 11(22):e2200971.

[0251] [3] MARTIN JR, PATIL P, YU F, etc. Enhanced stem cell retention and antioxidative protection with injectable, ROS-degradable PEG hydrogels[J]. Biomaterials, 2020, 263: 120377.

[0252] [4]MONGUIó-TORTAJADA M,PRAT-VIDAL C,MORON-FONT M,etc.Localadministration of porcineimmunomodulatory,chemotactic and angiogenicextracellular vesicles using engineered cardiac scaffolds formyocardialinfarction[J].Bioactive materials,2021,6(10):3314-27.

Claims

1. A tunicate cellulose dispersion or a tunicate cellulose hydrogel, characterized in that: The tunicate cellulose dispersion comprises: a solution obtained by mixing an aldehyde-modified product of tunicate cellulose with polyethyleneimine, acrylic acid, modified tannic acid, an ionization promoter, and benzalkonium chloride; the tunicate cellulose hydrogel is prepared by adding an initiator and a cross-linking agent to the tunicate cellulose dispersion for chemical cross-linking reaction; in every 50 mL of the chemical cross-linking reaction system, the mixing ratio of the aldehyde-modified product of tunicate cellulose, polyethyleneimine, acrylic acid, modified tannic acid, ionization promoter, and benzalkonium chloride is (3-8g):(10-15g):(1-2g):(0.03-0.06g):(0.007-0.015g):(0.2-0.3g), and the solvent is water.

2. The ascidian cellulose dispersion or ascidian cellulose hydrogel according to claim 1, characterized in that: The modified tannic acid refers to: The precipitate after the reaction of benzalkonium chloride and tannic acid in an alkaline solution.

3. The ascidian cellulose dispersion or ascidian cellulose hydrogel according to claim 1, characterized in that: The ionization promoter is potassium 3-sulfonate propyl methacrylate.

4. The ascidian cellulose dispersion or ascidian cellulose hydrogel according to claim 1, characterized in that: The chemical cross-linking reaction is carried out by adding an initiator and a cross-linking agent to perform chemical cross-linking; Preferably, the initiator is selected from any one of ammonium persulfate (APS), potassium persulfate (KPS), sodium persulfate, riboflavin (VB2) and azobisisobutyronitrile (AIBN); the crosslinking agent is selected from any one of tetramethylethylenediamine, N,N-methylenebisacrylamide, N,N-vinylbisacrylamide, methylenebisacrylamide and ethylene glycol dimethacrylate; Preferably, in every 50 mL of the chemical crosslinking reaction system, the mass ratio of the aldehyde-modified product of ascidian cellulose to the initiator and the crosslinking agent is (3-8 g): (0.04-0.06 g): (0.025-0.035 g).

5. The method for preparing the ascidian cellulose dispersion or ascidian cellulose hydrogel according to any one of claims 1 to 4, characterized in that: The method comprises: mixing corresponding proportions of the aldehyde-modified product of ascidian cellulose, polyethyleneimine, acrylic acid, modified tannic acid, an ionization accelerator and benzalkonium chloride to obtain an ascidian cellulose dispersion, and obtaining an ascidian cellulose hydrogel through a chemical cross-linking reaction.

6. The method for preparing the ascidian cellulose hydrogel according to claim 5, characterized in that: The aldehyde modified product of ascidian cellulose, polyethyleneimine, acrylic acid, modified tannic acid, an ionization accelerator, and benzalkonium chloride are mixed, and then an initiator and a cross-linking agent are added to carry out a cross-linking reaction; Preferably, in every 50 ml of chemical cross-linking reaction system, the mass ratio of the modified tannic acid to the initiator and the cross-linking agent is (0.03-0.06 g): (0.04-0.06 g): (0.025-0.035 g).

7. A multifunctional hydrogel, characterized in that: It includes: One surface of the ascidian cellulose hydrogel described in any one of claims 1 to 4 or the ascidian cellulose hydrogel prepared by the preparation method described in any one of claims 5 to 6 is covered with another hydrogel; the other hydrogel is prepared by a chemical cross-linking reaction initiated by an initiator through a solution of a mixture of a carboxyl modified product of ascidian cellulose and acrylic acid and polyethylene glycol diacrylate; In every 10 mL of chemical crosslinking reaction system, the mass ratio of the carboxyl modified product of the ascidian cellulose to acrylic acid, polyethylene glycol diacrylate and initiator is (0.06-0.15g):(1.2-2g):(0.15-0.28g):(0.008-0.012g), and the solvent is water.

8. A drug-loaded multifunctional hydrogel, characterized in that: The ascidian cellulose dispersion according to any one of claims 1 to 4 is mixed with Chrysin-7-O-glucuronide and / or its salt, an initiator and a cross-linking agent are added, a cross-linking reaction is carried out, and then another hydrogel is covered on one surface of the obtained hydrogel; the other hydrogel is obtained by a chemical cross-linking reaction initiated by an initiator through a solution of a mixture of a carboxyl modified product of ascidian cellulose and acrylic acid and polyethylene glycol diacrylate; For every 10 mL of chemical crosslinking reaction system, the mass ratio of the carboxyl modified product of ascidian cellulose to acrylic acid, polyethylene glycol diacrylate and initiator is (0.06-0.15 g):(1.2-2 g):(0.15-0.28 g):(0.008-0.012 g); the solvent is water; Preferably, the mixing ratio of the ascidian cellulose dispersion and Chrysin-7-O-glucuronide and / or its salt is (40-60 ml): (1 g-2 g).

9. Use of the drug-loaded multifunctional hydrogel according to claim 8 in the preparation of a drug for treating ischemic heart disease and its complications, or use of the ascidian cellulose hydrogel according to any one of claims 1 to 4, the multifunctional hydrogel according to claim 7, or the drug-loaded multifunctional hydrogel according to claim 8 in the preparation of skin repair dressings, implants, biomaterials and medical devices; Preferably, the biomaterial is selected from at least one of artificial skin, artificial bone, artificial cornea, tissue or organ scaffold and cell culture microcarrier; Preferably, the skin repair dressing further comprises acellular matrix and collagen; Preferably, the artificial bone is artificial cartilage.

10. The use according to claim 9, characterized in that: The ischemic heart disease and its complications are selected from any one of myocardial infarction, coronary heart disease, angina pectoris, arrhythmia, ventricular fibrosis, heart failure, myocardial ischemia, myocarditis and atherosclerosis; Preferably, the application includes at least one of the following application modes: (1) Promote the proliferation of cardiomyocytes; (2) Reduce cardiomyocyte apoptosis; (3) inhibiting the expression of apoptosis-related proteins; (4) Regulate the cell cycle; (5) Promote the repair of ischemic myocardial tissue; (6) Reduce myocardial infarction area; (7) Reduce cardiac fibrosis; Preferably, the regulating cell cycle comprises: promoting the transition of the cell cycle G1-S phase of cardiomyocytes; Preferably, the cell cycle regulation includes the following methods: by affecting the expression level of cyclin; preferably, the influencing the expression level of cyclin includes at least one of the following application methods: (1) Downregulate the expression of ALDH7A1 gene; (2) upregulating the expression of TSPAN5 gene; (3) down-regulating the expression of at least one protein selected from the group consisting of Cyclin D1, CDK4, pRB and E2F1 proteins; (4) Upregulation of CDK2 protein expression; (5) downregulate the expression of ACOT7 protein; (6) downregulate the expression of LAM5 protein; (7) Upregulate the expression of ERBB2 gene.

Images