CN110141679B - Slow-release myocardial patch material and application thereof - Google Patents
Slow-release myocardial patch material and application thereof Download PDFInfo
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Abstract
The invention provides a slow-release myocardial patch material, which adopts gelatin as a substrate substance, improves the electrical conductivity and mechanical ductility of the composition by adding gellan gum, graphene oxide, dopamine and methacrylic anhydride, more importantly, obtains a myocardial patch material capable of slowly releasing free radical scavenging substances in vivo by adding iloxan and Fmoc-FF and searching the mixture ratio and preparation process of different substances, can better recover the activity of myocardial infarction parts, and has good market prospect and social public value.
Description
Technical Field
The invention belongs to the technical field of biological medicines and medical instruments, and particularly relates to a slow-release myocardial patch material and application thereof.
Background
Ischemic heart disease is one of the main health problems in China, and brings heavy economic burden to China and other countries in the world. Cardiomyocytes are terminally differentiated cells that are difficult to regenerate after necrosis. Although current drugs, interventions and surgical treatments for ischemic heart disease can effectively improve myocardial blood supply and save dying myocardium, there is no repair effect on dead non-functional myocardium. Massive myocardial death can lead to severe arrhythmias, heart failure, and sudden cardiac arrest. For the treatment of advanced heart disease, the currently more effective method is myocardial transplantation. However, the number of donors for myocardial transplantation is small, and factors such as strong immune rejection increase medical costs and make it difficult to apply the method to a large scale clinically.
The implantation of stem cells or myocardial cells as a cell therapy provides a solution with great potential for treating myocardial infarction, and the implantation of stem cells as a cell therapy provides a solution with great potential for treating myocardial infarction. There are a number of clinical studies that have now fully demonstrated that stem cell engraftment can improve myocardial function in patients with myocardial infarction. However, the biggest bottleneck in the implantation treatment of stem cells or cardiac muscle cells is that the cells are easy to lose a large amount after being implanted, cannot be gathered at a treatment target, and have very low long-term survival rate, which seriously affect the clinical efficacy. In order to solve the problem, researchers think that stem cells are compounded with a certain biological material and then implanted into a myocardial infarction part or fixed in a myocardial infarction area, and then the concept of a myocardial patch is proposed. Therefore, the preparation of the myocardial patch product by the tissue engineering means draws attention of experts at home and abroad: the stem cells or the cardiac muscle cells are arranged according to a certain spatial sequence to form a cell sheet structure, so that the cell sheet structure can more accurately act on a myocardial infarction area, the cell retention rate and the survival rate are improved, the loss of excessive stem cells is avoided, the treatment effect is improved, and the treatment risk is reduced.
An ideal myocardial patch generally has the following characteristics: the cell support material used as the myocardial patch has good elasticity, extensibility, stability and plasticity and certain mechanical strength, and the scaffold cannot be damaged by the jumping of myocytes. The cell support material used as the myocardial patch has good biocompatibility, does not cause inflammatory reaction, toxic reaction and immunological rejection reaction in vivo, and can not be separated from a myocardial infarction part due to high-speed and high-strength beating of a heart when the myocardial patch is transplanted into an animal model body. The cell supporting material of the myocardial patch has the capability of synchronous contraction and good electric conductivity for biological electric conduction and avoids serious arrhythmia after being transplanted into a body.
Most current myocardial patches do not have all of the above properties. In the prior art, natural polymers are generally adopted as the myocardial repair materials and have good biocompatibility, such as gelatin, sodium alginate, type I collagen and fibrin, but the natural polymers have weak elasticity and are not easy to return to the original shape after being compressed. The macroporous chitosan-gelatin cryogel has good elastic property, but weak electric conductivity. The synthetic hydrogel comprises polyacrylic acid (PAA), polyethylene glycol (PEG) and polyvinyl alcohol (PVA), and is a cross-linked polymer system prepared by addition reaction, ring-opening polymerization and other reactions under artificial conditions. The synthesized hydrogel has the advantages of easy industrial production and chemical modification, accurate performance regulation and control and the like, and has good mechanical ductility, but compared with the natural hydrogel, the synthesized hydrogel lacks cell adhesion sites, and has poor biocompatibility and biodegradability.
Myocardial infarction is often accompanied by characteristics such as ischemia, hypoxia and inflammatory microenvironment, and further, a large amount of oxygen free Radicals (ROS) are generated in the infarcted part, so that the disease is aggravated by the loss of cells. Effectively eliminating ROS in microenvironment and better improving the treatment effect of the myocardial patch on myocardial infarction. In order to achieve the above purpose, in the prior art, a corresponding antioxidant or free radical scavenging substance is usually added into a myocardial patch to play a role in improving the microenvironment of a myocardial infarction part. For example, biomimetic pullulan-based hydrogels and silk fibroin hydrogels can be used to protect Mesenchymal Stem Cells (MSCs) or fibroblasts from damage in high oxidative stress microenvironments, or to add the antioxidant substance glutathione to the cardiac patch. Although, the method can improve the capacity of the myocardial patch to scavenge free radicals and repair microenvironment to a certain extent. However, the release rate of the above substances in the myocardial patch material is high, and the substances cannot be stable in vivo and achieve the radical scavenging effect for a long time.
Therefore, the problem that needs to be solved at present is to provide a myocardial patch composite material which has good mechanical ductility and cell compatibility, can exert the ability of scavenging ROS for a long time in vivo and can better repair myocardial infarction parts.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a sustained-release myocardial patch material which has good mechanical ductility and cell compatibility, can exert long-acting ROS scavenging capability in vivo and can better improve the microenvironment of a myocardial infarction part.
In one embodiment, the self-assembling peptide consists of 2 to 16 amino acid residues linked together, and under certain conditions, the self-assembling peptide can be self-assembled to form a nanofiber network scaffold and form a hydrogel. The self-assembly peptide is selected from ionic polypeptide, cyclic peptide and derivatives thereof, amphiphilic polypeptide or aromatic short peptide and derivatives thereof. The ionic polypeptide can be AGAGAKAKK2(EAK16), ion-complementing polypeptide RADA 16; the cyclic peptide and its derivatives may be Cyclo- ((L-Gln-D-Ala-L-Glu-D-Ala)2) (ii) a The amphiphilic polypeptide may be A6D、V6D、V6K2Etc.; the aromatic short peptide and the derivative thereof can be diphenylalanine (FF) or nonafluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF). Preferably, the self-assembling peptide is nonafluorenylmethoxycarbonylPhenyl-diphenylalanine (Fmoc-FF).
In one embodiment, the free radical-scavenging substance is preferably itolein, which is also called itolein O (Itoside O), which is derived from Eleonia deciduous tree of the genus Gardenia of the family Hydnocarpi, and is an active extract of Eleocarpus orientalis, belonging to a new chain monoterpene glycoside component, and is identified as 3, 7-dimethyl-1, 6-octadien-3, 10-dihydroxy-10-O- α -L-arabinopyranosyl (1-6) -O- β -D-glucopyranoside (Chai XY, Xu ZR, Tang LY, et al. Itoside O, a new line monoterpene glycoside from the bark and twigs of Itoa orientalis origin. journal of Chinese medicinal materials (Phaseolus, 17; Scirkul 2008-79). The itonin has the effects of scavenging free radicals, inhibiting lipid peroxidation, and preventing and repairing cell injury caused by anoxia, ischemia or reperfusion.
The substance for removing free radicals can also be diazoxide, which is a selective mitoK + ATP channel opener, can obviously improve the recovery of cardiac function after ischemia reperfusion of isolated rat myocardium, reduces the release of lactate dehydrogenase (IDH), and has better myocardial protection effect.
The radical-removing substance may also be glutathione, which is a tripeptide composed of glutamic acid, cysteine and glycine, widely present in animals and plants, and involved in various reactions in the body. Modern pharmacological research shows that the sulfhydryl group enables glutathione to have stronger inoxidizability and free radical scavenging effect. In the reaction of scavenging free radicals, glutathione is involved in the redox reaction process in vivo, combines with peroxide, electrophilic groups and free radicals, protects the proteins and enzymes of the cell membrane from being damaged by the redox reaction, and has been shown to be involved in regulating cell proliferation, immune response of the body and functioning as neuromodulators and neurotransmitters in the nervous system.
In one embodiment, the hydrogel may be a natural polymer with good biocompatibility as a cardiac muscle repair material, such as gelatin, sodium alginate, type I collagen, and fibrin, or a synthetic hydrogel such as polyethylene glycol (PEG). Preferably, the hydrogel is gelatin, more preferably, the hydrogel is a mixture of glycerol, gellan gum and gelatin, and most preferably, the mixture of glycerol, gellan gum and gelatin is modified by methacrylic anhydride.
In one embodiment, the glycerin is glycerol, which absorbs moisture from the air, as well as hydrogen sulfide, hydrogen cyanide, and sulfur dioxide. Insoluble in benzene, chloroform, carbon tetrachloride, carbon disulfide, petroleum ether and oils. Glycerol is a backbone component of the triglyceride molecule. Relative density 1.26362. Melting point 17.8 ℃. The boiling point of 290.0 ℃ has plasticizing effect on the gelatin, and can improve the thermal stability and mechanical ductility of the gelatin.
In one embodiment, the gellan gum is a vegetable gel Gelzan gum prepared by microbial fermentation, has adjustable freezing point, melting point, elasticity and hardness, has good compatibility with additives, good thermal stability and other properties, and can improve the loading capacity of gelatin, and preferably is G3251 (a vegetable gel produced by Phytoechlab, CAS: 71010-52-1).
In one embodiment, the graphene oxide has good electrical conductivity and mechanical properties, which can improve the electrical conductivity and mechanical properties of the hydrogel.
The dopamine has the characteristics of super-strong adhesion, good biocompatibility, cell affinity and the like, and can enhance the adhesion of cells on the myocardial patch.
In one embodiment, the slow-release myocardial patch material comprises gelatin, glycerin, gellan gum, graphene oxide, dopamine, iloturin glycoside and Fmoc-FF, and preferably, the composition for preparing the myocardial patch comprises: 10-100 parts of gelatin, 4-100 parts of glycerol, 5-50 parts of gellan gum, 3-10 parts of graphene oxide, 0.6-5 parts of dopamine, 0.5-20 parts of itolein and 1-10 parts of Fmoc-FF; most preferably, the composition for preparing a myocardial patch comprises: 20 parts of gelatin, 8 parts of glycerol, 10 parts of gellan gum, 0.4 part of graphene oxide, 1.2 parts of dopamine, 3 parts of itonin and 0.8 part of Fmoc-FF.
In one embodiment, the preparation method of the sustained-release myocardial patch material comprises the following steps: (1) preparing transparent MA-Gelzan-Gly blending solution from gelatin, glycerol and gellan gum; (2) adding methacrylic anhydride into the blending liquid to obtain modified MA-Gelzan-Gly hydrogel; (3) mixing the modified MA-Gelzan-Gly hydrogel with graphene oxide to obtain a graphene oxide loaded MA-Gelzan-Gly-Go hydrogel; (4) mixing MA-Gelzan-Gly-Go hydrogel with dopamine and ilotoin, taking ammonium persulfate as an initiator and TEMED as a polymerization auxiliary agent, and simultaneously dripping Fmoc-FF solution to obtain the slow-release myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc; preferably, the method comprises
(1) Completely dissolving 2g of gelatin in Phosphate Buffer Solution (PBS) at 60 ℃ to obtain 10 w/v% gelatin solution, dissolving 0.8g of glycerol in distilled water to obtain 4 w/v% glycerol solution, dissolving 1g of gellan gum in distilled water at 60 ℃ to obtain 10 w/v% gellan gum solution, mixing the three solutions in a ratio of 1.2:1:1.5, and magnetically stirring for 1h to obtain MA-Gelzan-Gly blending solution;
(2) slowly dripping 4ml of methacrylic anhydride into the MA-Gelzan-Gly blending solution; stirring for 1-3 hours at 50 ℃ to gradually react, adding PBS to dilute and then stopping the reaction, adding the reactant into a dialysis bag, dialyzing for 5-9 days with deionized water, replacing the deionized water every 6-12 hours, and freeze-drying and storing to obtain the modified MA-Gelzan-Gly hydrogel;
(3) preparing 40mg of graphene into a 4mg/ml solution, preparing the modified MA-Gelzan-Gly hydrogel in the step (2) into a 10% solution, mixing the 10% MA-Gelzan-Gly hydrogel solution with the graphene solution in a ratio of 4:2, and placing the mixture in a refrigerator at 4 ℃ for 2-4h to obtain the MA-Gelzan-Gly-Go hydrogel loaded with graphene oxide;
(4) preparing or obtaining 4mg/mL Fmoc-FF solution, adding 0.12g dopamine and 0.3g ilotoin glycoside into the MA-Gelzan-Gly-Go hydrogel prepared in the step (3), uniformly mixing, adding 3mL 20% ammonium persulfate and 1mL TEMED solution, dropwise adding 20mL 4mg/mL Fmoc-FF solution, standing for 30min, and placing in a refrigerator at 4 ℃ for overnight to obtain MA-Gelzan-Gly-Go-Ito-DA-Fmoc.
Preferably, the method further comprises: and (3) dripping the MA-Gelzan-Gly-Go-Ito-DA-Fmoc onto the surface of the PDMS template, and standing at room temperature for one day or placing at 4 ℃ to form gel. And (3) after the hydrogel is gelatinized and air-dried, repeating the steps for 2-5 times to obtain a gel layer with 2-5 layers, and stripping the gel layer from the PDMS template to obtain the slow-release myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc.
In one embodiment, the composition for preparing a myocardial patch may be used for preparing a myocardial patch, preferably, a cardiomyocyte or a pluripotent stem cell is seeded to the composition, preferably, the pluripotent stem cell is an adipose stem cell, a mesenchymal stem cell, a bone marrow stem cell.
Compared with the prior art, the invention has the following advantages: the invention provides a slow-release myocardial patch material, which adopts gelatin as a substrate substance, improves the electrical conductivity and mechanical ductility of the composition by adding gellan gum, graphene oxide, dopamine and methacrylic anhydride, more importantly, obtains a myocardial patch material capable of slowly releasing free radical scavenging substances in vivo by adding iloxan and Fmoc-FF and searching the mixture ratio and preparation process of different substances, can better recover the activity of myocardial infarction parts, and has good market prospect and social public value.
Drawings
FIG. 1: the elastic modulus test result graphs of MA-Gelzan-Gly-Go-Ito-DA-a, MA-Gelzan-Gly-Go-Ito-DA-b and MA-Gelzan-Gly-Go-Ito-DA-c;
FIG. 2: the elastic modulus test result graphs of MA-Gelzan-Gly-Go-Ito-DA-c, MA-Gelzan-Gly-Go-Ito-DA-d, MA-Gelzan-Gly-Go-Ito-DA-e and MA-Gelzan-Gly-Go-Ito-DA-f;
FIG. 3: the slow release performance test result chart of the myocardial patch material containing the self-assembly peptide is shown in the figure, wherein FF is the release curve of MA-Gelzan-Gly-Go-Ito-DA-Fmoc to ilongin, and h is the release curve of MA-Gelzan-Gly-Go-Ito-DA-h to ilongin;
FIG. 4: the proliferation conditions of cardiomyocytes of MA-Gelzan-Gly-Go-Ito-DA-Fmoc and MA-Gelzan-Gly-Go-Ito-DA-h, wherein FF is MA-Gelzan-Gly-Go-Ito-DA-Fmoc, and h is MA-Gelzan-Gly-Go-Ito-DA-h
FIG. 5: HE staining patterns of the transplanted group, the myocardial infarction control group and the sham operation group, wherein a is the sham operation group, b is the myocardial infarction control group, and c is the transplanted group.
Detailed Description
The present invention will be described in detail below with reference to the best embodiments thereof, but the following description should not be construed as limiting the scope of the present invention.
EXAMPLE 1 preparation of composition MA-Gelzan-Gly-Go-Ito-DA for preparing myocardial Patch
1. Preparation of MA-Gelzan-Gly blending solution
Weighing 2g of gelatin (Sigma), putting into 20ml of PBS solution, heating in a water bath at 60 ℃, and fully dissolving by magnetic stirring for 40min to obtain 10 w/v% gelatin solution; dissolving 0.8ml of glycerol in 20ml of distilled water, and uniformly mixing to obtain a 4 v/v% glycerol solution; weighing 1G of G3251 (a plant gel produced by Phytoechlab, CAS:71010-52-1), placing into 10ml of PBS solution, heating in water bath at 60 deg.C, magnetically stirring for 30min, and dissolving to obtain 10 w/v% G3251 solution; mixing the 10 w/v% gelatin solution, 4 w/v% glycerol solution and 10 w/v% G3251 solution at a volume ratio of 1:1:1, heating in water bath at 60 deg.C, and magnetically stirring for 1h to obtain MA-Gelzan-Gly blend.
2. Preparation of modified MA-Gelzan-Gly hydrogel
Slowly dripping 4ml of methacrylic anhydride into the MA-Gelzan-Gly blending solution; stirring for 1-3 hours at 50 ℃ to gradually react, then adding 100ml PBS to dilute and terminate the reaction, adding the reactant into a dialysis bag, dialyzing for 7 days with deionized water, replacing the deionized water every 8 hours, and freeze-drying and storing to obtain modified MA-Gelzan-Gly hydrogel;
3. preparation of MA-Gelzan-Gly-Go hydrogel loaded with graphene oxide
Under the condition of ice water bath, 1g of natural crystalline flake graphite and 2g of NaNO3 are slowly added into 25mL of concentrated sulfuric acid under stirring, the mixture is uniformly stirred, 3g of potassium permanganate is slowly added, the reaction temperature is controlled below 5 ℃ for reaction for 3 hours, and the reaction is further stirred at 35 ℃ for reaction for 2 hours. Then 46mL of deionized water is dripped for high-temperature reaction for 0.5h, the reaction temperature is controlled not to exceed 85 ℃, then 40mL of deionized water with the temperature of 60 ℃ is added, the mixture is stirred uniformly, and 30% hydrogen peroxide is added until no gas is generated, and the solution turns bright yellow. Filtering, washing, dialyzing to neutrality, and freeze drying. Preparing 40mg of graphene oxide into a solution with the concentration of 4mg/mL, and performing ultrasonic treatment for 30min (the frequency is 40kHz and the power is 200W) to prepare a uniform graphene oxide solution.
Preparing a 10% solution from the modified MA-Gelzan-Gly hydrogel, mixing the 10% MA-Gelzan-Gly hydrogel solution with the graphene solution in a ratio of 4: 1, placing the mixture in a refrigerator at 4 ℃ for 2 to 4 hours to obtain the MA-Gelzan-Gly-Go hydrogel loaded with the graphene oxide;
4. preparation of composition MA-Gelzan-Gly-Go-Ito-DA
Adding 0.12g of dopamine and 0.16g of iloturin glycoside into the MA-Gelzan-Gly-Go hydrogel prepared in the step 3, uniformly mixing, adding 3ml of 20% ammonium persulfate and 1ml of TEMED solution, and placing in a refrigerator at 4 ℃ for overnight to obtain the composition MA-Gelzan-Gly-Go-Ito-DA for preparing the myocardial patch.
5. Composition MA-Gelzan-Gly-Go-Ito-DA for preparing myocardial patch
Dropping 300 μ l of MA-Gelzan-Gly-Go-Ito-DA onto the surface of PDMS template, lightly covering the surface of hydrogel solution with a slide, standing at room temperature for one day or standing at 4 deg.C to form gel. And (3) after the hydrogel is gelatinized and dried, repeating the steps for 2-5 times to obtain a gel layer with 2-5 layers, and peeling the gel layer from the PDMS template to prepare the MA-Gelzan-Gly-Go-Ito-DA myocardial patch material.
Example 2 influence of different compounding ratios of gelatin, glycerin and gellan gum on mechanical properties of MA-Gelzan-Gly-Go-Ito-DA myocardial patch material
1. Preparation of myocardial patch materials of different models
In order to investigate the effect of different compounding ratios of gelatin, glycerol and gellan gum on the mechanical properties of the MA-Gelzan-Gly-Go-Ito-DA myocardial patch material, 3 different kinds of gelatin, glycerol and gellan gum were designed in the examples, as specifically described in table 1:
TABLE 1 preparation of myocardial patch materials with gelatin, glycerin, gellan gum in different volume ratios
| Myocardial patch material type | Volume ratio of |
| MA-Gelzan-Gly-Go-Ito-DA-a | 1:1:1 |
| MA-Gelzan-Gly-Go-Ito-DA-b | 1:1.2:1 |
| MA-Gelzan-Gly-Go-Ito-DA-c | 1.2:1:1.5 |
The preparation method of MA-Gelzan-Gly-Go-Ito-DA myocardial patch materials was as described in example 1, wherein the volume ratio of 10 w/v% gelatin solution, 4 w/v% glycerin solution, and 10 w/v% G3251 solution in step 1 was adjusted as described in Table 1, to obtain the myocardial patch materials MA-Gelzan-Gly-Go-Ito-DA-a, MA-Gelzan-Gly-Go-Ito-DA-b, and MA-Gelzan-Gly-Go-Ito-DA-c, respectively.
2. Mechanical performance test of myocardial patch materials of different models
(1) Measurement of modulus of elasticity
The elastic modulus of MA-Gelzan-Gly-Go-Ito-DA-a, MA-Gelzan-Gly-Go-Ito-DA-b and MA-Gelzan-Gly-Go-Ito-DA-c was measured by an Instron universal material testing machine. The sample size was 6mm in height and 12mm in diameter. The measurement mode was compression mode, and the compression rate was 0.02 mm/s. The stress-strain curve was analyzed to calculate the elastic modulus of the hydrogel. Each set of samples was set up in 5 replicates.
3. Test results
As shown in FIG. 1, the elastic modulus of MA-Gelzan-Gly-Go-Ito-DA-a, MA-Gelzan-Gly-Go-Ito-DA-b and MA-Gelzan-Gly-Go-Ito-DA-c are 189KPa, 167KPa and 258KPa respectively, and it can be seen from the results that glycerin as a plasticizer can improve the mechanical elongation of the hydrogel, but excessive glycerin can excessively dilute the gelatin and reduce the elastic modulus, and the gellan gum has good elasticity, so that the increase of the content of the gellan gum in the hydrogel is helpful to improve the mechanical properties of the hydrogel. Therefore, when the ratio of the gelatin to the glycerin to the gellan gum is 1.2:1:1.5, the mechanical property of the hydrogel is optimal.
Example 3 Effect of different content of graphene oxide on mechanical Properties and conductivity of MA-Gelzan-Gly-Go-Ito-DA myocardial Patch Material
1. Preparation of myocardial patch materials of different models
In order to investigate the influence of graphene oxide on the mechanical properties and the electrical conductivity of the MA-Gelzan-Gly-Go-Ito-DA myocardial patch material, when the ratio of gelatin, glycerol and gellan gum is 1.2:1:1.5, 4 different graphene oxide contents are designed, specifically as shown in Table 2:
TABLE 2 myocardial patch materials prepared with different contents of graphene oxide
| Myocardial patch material type | Proportion of MA-Gelzan-Gly hydrogel solution to graphene oxide solution |
| MA-Gelzan-Gly-Go-Ito-DA-c | 4:1 |
| MA-Gelzan-Gly-Go-Ito-DA-d | 4:1.5 |
| MA-Gelzan-Gly-Go-Ito-DA-e | 4:2 |
| MA-Gelzan-Gly-Go-Ito-DA-f | 4:2.5 |
The preparation method of the MA-Gelzan-Gly-Go-Ito-DA myocardial patch material is as described in example 1, wherein the volume ratio of the 10 w/v% gelatin solution, the 4 w/v% glycerin solution, and the 10 w/v% G3251 solution in step 1 is adjusted to 1.2:1:1.5, and the ratio of the MA-Gelzan-Gly hydrogel solution to the graphene solution in step 3 is adjusted according to the ratio in table 2, so as to obtain the myocardial patch materials MA-Gelzan-Gly-Go-Ito-DA-c, MA-Gelzan-Gly-Go-Ito-DA-d, MA-Gelzan-Gly-Go-Ito-DA-e, and MA-Gelzan-Gly-Go-Ito-DA-f, respectively.
2. Mechanical performance test of myocardial patch materials of different models
(1) Measurement of modulus of elasticity
The modulus of elasticity of the above materials was measured using an Instron universal materials testing machine. The sample size was 6mm in height and 12mm in diameter. The measurement mode was compression mode, and the compression rate was 0.02 mm/s. The stress-strain curve was analyzed to calculate the elastic modulus of the hydrogel. Each set of samples was set up in 5 replicates.
3. Conductivity test of myocardial patch materials of different models
Connecting iron wires or copper wires on the upper and lower surfaces of the myocardial patch materials of different types, and covering a thin layer of conductive adhesive on the surface to manufacture the electrode. The conductivity of five groups of samples was measured at room temperature using an electrochemical workstation. Each sample was tested 3 times and each set was replicated 5 times.
4. Test results
As shown in FIG. 2, the elastic moduli of MA-Gelzan-Gly-Go-Ito-DA-c, MA-Gelzan-Gly-Go-Ito-DA-d, MA-Gelzan-Gly-Go-Ito-DA-e, and MA-Gelzan-Gly-Go-Ito-DA-f were 258KPa, 264KPa, 279KPa, and 212KPa, respectively. The resistance values of the 4 prepared materials with different ratios are shown in table 3,
TABLE 3 myocardial patch materials prepared with different contents of graphene oxide
| Myocardial patch material type | Resistance value (M omega) |
| MA-Gelzan-Gly-Go-Ito-DA-c | 0.12±0.09 |
| MA-Gelzan-Gly-Go-Ito-DA-d | 0.1±0.07 |
| MA-Gelzan-Gly-Go-Ito-DA-e | 0.084±0.09 |
| MA-Gelzan-Gly-Go-Ito-DA-f | 0.082±0.06 |
As can be seen from the results of table 3 and fig. 2, although increasing the content of graphene oxide in the hydrogel can improve the conductivity, when the content of graphene oxide in the hydrogel is conductive to a certain amount, the increase of the conductivity is limited, and accordingly, graphene itself has good mechanical properties, and the addition of a proper amount of graphene helps to improve the mechanical properties, but too much graphene may reduce the mechanical ductility of the hydrogel, which may be due to the fact that too much graphene hinders the cross-linking between hydrogel molecules. As can be seen, the optimal ratio of MA-Gelzan-Gly-Go-Ito-DA hydrogel solution to graphene oxide solution is 4: 2.
EXAMPLE 4 Effect of different levels of Ilicis Cornuta on the ability of MA-Gelzan-Gly-Go-Ito-DA myocardial patch Material to scavenge free radicals
1. Preparation of myocardial patch materials of different models
In order to explore the influence of ilouside on the radical scavenging capacity of the MA-Gelzan-Gly-Go-Ito-DA myocardial patch material, 4 different ilouside contents were designed, and are specifically shown in Table 4:
TABLE 4 myocardial patch materials prepared with different contents of iloturin glycoside
| Myocardial patch material type | Content of ilouside (g) |
| MA-Gelzan-Gly-Go-Ito-DA-e | 0.16 |
| MA-Gelzan-Gly-Go-Ito-DA-g | 0.2 |
| MA-Gelzan-Gly-Go-Ito-DA-h | 0.25 |
| MA-Gelzan-Gly-Go-Ito-DA-i | 0.3 |
The preparation method of the MA-Gelzan-Gly-Go-Ito-DA myocardial patch material is as described in example 1, wherein the volume ratio of 10 w/v% gelatin solution, 4 w/v% glycerin solution and 10 w/v% G3251 solution in the step 1 is adjusted to be 1.2:1:1.5, the ratio of MA-Gelzan-Gly hydrogel solution and graphene solution in the step 3 is adjusted to be 4:2, the addition amount of itonin in the step 4 is adjusted according to the following table 4, respectively obtaining myocardial patch materials MA-Gelzan-Gly-Go-Ito-DA-e, MA-Gelzan-Gly-Go-Ito-DA-g, MA-Gelzan-Gly-Go-Ito-DA-h and MA-Gelzan-Gly-Go-Ito-DA-i.
2. Measurement of hydroxyl radical scavenging ability
Hydroxyl radical is the radical with the strongest reactivity among many active oxygen radicals, which can induce severe damage to proteins, nucleic acids, unsaturated fatty acids, etc., and is one of the major factors causing cell damage in myocardial infarction. The evaluation of the clearing capability of the myocardial patch materials of different models on the hydroxyl radicals is one of the main indexes for evaluating whether the materials can reduce cell damage caused by oxygen radicals in vivo. The method for evaluating the hydroxyl radical scavenging capacity of the myocardial patch material by using the fading degree of crocus sativus O dye comprises the following specific steps:
2mM FeSO was prepared4The solution, 6 wt% hydrogen peroxide solution and 360ug/mL crocus sativus solution are prepared for standby, 10mg of different types of myocardial patch materials are weighed and put into an EP tube, and 1mL of water and 1.2mL of Fe SO are sequentially added into the EP tube4Mixing the solution and 1ml of crocus sativus solution uniformly, standing for 12min, adding 1.6ml of hydrogen peroxide solution, heating in a water bath at 55 ℃ for 35min, measuring the absorbance value at 492nm after the reaction is finished, and calculating the scavenging capacity of the material to hydroxyl radicals. The above experiments were repeated at least 3 times, with 5 replicates per experimental group.
3. Test results
The hydroxyl radical scavenging capacity of 4 myocardial patch materials with different addition amounts of iloposide is shown in table 5
TABLE 5 hydroxy radical scavenging Capacity of myocardial patch materials prepared with different amounts of ilotoin glycoside
| Myocardial patch material type | Scavenging ability of hydroxyl radical (%) |
| MA-Gelzan-Gly-Go-Ito-DA-e | 86.14±1.23 |
| MA-Gelzan-Gly-Go-Ito-DA-g | 95.46±1.59 |
| MA-Gelzan-Gly-Go-Ito-DA-h | 98.23±1.74 |
| MA-Gelzan-Gly-Go-Ito-DA-i | 98.41±1.62 |
As can be seen from the results in table 5, increasing the addition amount of iloturin in the myocardial patch material significantly improved the scavenging ability of the material for hydroxyl radicals, but when the amount of iloturin in the material reached a certain level, the scavenging ability thereof for improving hydroxyl radicals tended to be uniform, based on which the addition amount of 0.25g of iloturin was considered to be optimal.
Example 5 preparation and Performance characterization of myocardial patch materials containing self-assembling peptides
1. Preparation of myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing self-assembly peptide
(1) Preparation of MA-Gelzan-Gly blending solution
Weighing 2g of gelatin (Sigma), putting into 20ml of PBS solution, heating in a water bath at 60 ℃, and fully dissolving by magnetic stirring for 40min to obtain 10 w/v% gelatin solution; dissolving 0.8ml of glycerol in 20ml of distilled water, and uniformly mixing to obtain a 4 v/v% glycerol solution; weighing 1G of G3251 (a plant gel produced by Phytoechlab, CAS:71010-52-1), placing into 10ml of PBS solution, heating in water bath at 60 deg.C, magnetically stirring for 30min, and dissolving to obtain 10 w/v% G3251 solution; mixing the 10 w/v% gelatin solution, 4 w/v% glycerol solution and 10 w/v% G3251 solution according to the volume ratio of 1.2:1:1.5, heating in a water bath at 60 ℃, and magnetically stirring for 1h to obtain MA-Gelzan-Gly blending solution.
(2) Preparation of modified MA-Gelzan-Gly hydrogel
Slowly dripping 4ml of methacrylic anhydride into the MA-Gelzan-Gly blending solution; stirring for 1-3 hours at 50 ℃ to gradually react, then adding 100ml PBS to dilute and terminate the reaction, adding the reactant into a dialysis bag, dialyzing for 7 days with deionized water, replacing the deionized water every 8 hours, and freeze-drying and storing to obtain modified MA-Gelzan-Gly hydrogel;
(3) preparation of MA-Gelzan-Gly-Go hydrogel loaded with graphene oxide
Under the condition of ice water bath, 1g of natural crystalline flake graphite and 2g of NaNO3 are slowly added into 25mL of concentrated sulfuric acid under stirring, the mixture is uniformly stirred, 3g of potassium permanganate is slowly added, the reaction temperature is controlled below 5 ℃ for reaction for 3 hours, and the reaction is further stirred at 35 ℃ for reaction for 2 hours. Then 46mL of deionized water is dripped for high-temperature reaction for 0.5h, the reaction temperature is controlled not to exceed 85 ℃, then 40mL of deionized water with the temperature of 60 ℃ is added, the mixture is stirred uniformly, and 30% hydrogen peroxide is added until no gas is generated, and the solution turns bright yellow. Filtering, washing, dialyzing to neutrality, and freeze drying. Preparing 40mg of graphene oxide into a solution with the concentration of 4mg/mL, and performing ultrasonic treatment for 30min (the frequency is 40kHz and the power is 200W) to prepare a uniform graphene oxide solution.
Preparing a 10% solution from the modified MA-Gelzan-Gly hydrogel, mixing the 10% MA-Gelzan-Gly hydrogel solution with the graphene solution in a ratio of 4:2, placing the mixture in a refrigerator at the temperature of 4 ℃ for 2 to 4 hours to obtain the MA-Gelzan-Gly-Go hydrogel loaded with the graphene oxide;
(4) preparation of composition MA-Gelzan-Gly-Go-Ito-DA-Fmoc
80mg of nonafluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) was weighed out and added to 20mL of dd H2O, performing ultrasonic dispersion for 30min, then adding 120 mu L of 0.5mol/L NaOH solution, stirring and dissolving to obtain 4mg/ml Fmoc-FF solution; adding 0.12g of dopamine and 0.3g of iloturin glycoside into the MA-Gelzan-Gly-Go hydrogel prepared in the step 3, uniformly mixing, adding 3ml of 20ml% ammonium persulfate and 1ml TEMED solution, 4mg/ml Fmoc-FF solution was slowly and uniformly added dropwise to the surface of the solution 20m L using a syringe. Shaking while dropping to allow the formed microcapsule to sink into the solution, standing for 30min, and placing in refrigerator at 4 deg.C overnight to obtain MA-Gelzan-Gly-Go-Ito-DA-Fmoc.
(5) Preparation of myocardial patch by MA-Gelzan-Gly-Go-Ito-DA-Fmoc
And (3) dripping 300 mu l of MA-Gelzan-Gly-Go-Ito-DA-Fmoc onto the surface of the PDMS template, lightly covering the surface of the hydrogel solution with a slide, and standing at room temperature for one day or placing at 4 ℃ for gelling. And (3) after the hydrogel is gelatinized and air-dried, repeating the steps for 2-5 times to obtain a gel layer with 2-5 layers, and stripping the gel layer from the PDMS template to obtain the slow-release myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc.
2. Performance characterization of myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing self-assembly peptide
To test the mechanical properties and electrical conductivity of the myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing the self-assembling peptide, MA-Gelzan-Gly-Go-Ito-DA-Fmoc was tested using the test method of elastic modulus and the electrical conductivity test in example 3, using MA-Gelzan-Gly-Go-Ito-DA-h as a control.
3. Test results
The elastic modulus of MA-Gelzan-Gly-Go-Ito-DA-Fmoc and MA-Gelzan-Gly-Go-Ito-DA-h are 257KPa and 268KPa respectively. The resistance values are as described in table 6,
TABLE 6 conductivity of myocardial patch materials containing self-assembling peptides
| Myocardial patch material type | Resistance value (M omega) |
| MA-Gelzan-Gly-Go-Ito-DA-Fmoc | 0.097±0.06 |
| MA-Gelzan-Gly-Go-Ito-DA-h | 0.086±0.05 |
From the results, the mechanical property and the conductivity of the myocardial patch material added with the self-assembly peptide of the nonafluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) are reduced, which is probably caused by that the Fmoc-FF forms a micro-capsule loaded with a medicament in gel, but the comprehensive comparison of the mechanical property and the conductivity of the MA-Gelzan-Gly-Go-Ito-DA-Fmoc still belongs to an excellent level.
Example 6 determination of free radical scavenging Capacity of myocardial Patch Material containing self-assembling peptides
To test the radical scavenging ability of the myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing the self-assembled peptide, the scavenging ability of MA-Gelzan-Gly-Go-Ito-DA-Fmoc to hydroxyl radicals was examined by the method of example 4. MA-Gelzan-Gly-Go-Ito-DA-h was used as a control.
Test results, as shown in table 7:
TABLE 7 hydroxy radical scavenging Capacity of myocardial Patch materials containing self-assembling peptides
| Myocardial patch material type | Scavenging ability of hydroxyl radical (%) |
| MA-Gelzan-Gly-Go-Ito-DA-Fmoc | 96.24±0.98 |
| MA-Gelzan-Gly-Go-Ito-DA-h | 97.56±1.68 |
As can be seen from the results in Table 5, the myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing the self-assembling peptide had similar effects to those of MA-Gelzan-Gly-Go-Ito-DA-h having the best hydroxyl radical scavenging ability in example 4.
Example 7 sustained Release Performance assay of myocardial Patch Material containing self-assembling peptides
In order to explore the drug slow release performance of the myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing the self-assembly peptide, the in vitro release performance test of ilotoin glycoside of MA-Gelzan-Gly-Go-Ito-DA-Fmoc is designed in the embodiment, and the specific steps are as follows:
taking 2ml of MA-Gelzan-Gly-Go-Ito-DA-Fmoc hydrogel, putting the MA-Gelzan-Gly-Go-Ito-DA-Fmoc hydrogel into a 10ml centrifuge tube, adding 6ml of PBS solution, recording the light absorption values at different moments by using a spectrophotometer, measuring the light absorption values of different amounts of ilongin solution (PBS solution) by using an ultraviolet spectrophotometer as reference, drawing a standard curve, measuring and calculating the concentration of ilongin based on the light absorption values of ketoprofen at different sampling moments, and converting the recorded data into the drug release amount for analysis. The same procedure as described above was used to test the in vitro release amount of iloturin glycoside using MA-Gelzan-Gly-Go-Ito-DA-h as a test substance.
And (3) testing results:
as shown in FIG. 3, the total release rate of the MA-Gelzan-Gly-Go-Ito-DA-Fmoc to the ilongin is close to 95%, which is much higher than the release rate of the MA-Gelzan-Gly-Go-Ito-DA-h to the ilongin, from the release rate, the MA-Gelzan-Gly-Go-Ito-DA-h rapidly releases approximately 60% of the ilongin at 15h, and then the release rate is gentle, while the MA-Gelzan-Gly-Go-Ito-DA-Fmoc continuously releases the ilongin all the time, and the release rate reaches 95% at 35 h. Therefore, the self-assembly peptide-containing myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc is obviously due to other myocardial patch materials in both overall release rate and slow release performance.
Example 8 Effect of myocardial Patch Material containing self-assembling peptides on myocardial cell proliferation
In order to influence the myocardial patch material containing the self-assembly peptide on myocardial cell proliferation, the myocardial cells were inoculated with MA-Gelzan-Gly-Go-Ito-DA-Fmoc and MA-Gelzan-Gly-Go-Ito-DA-h in the examples of the field, and the proliferation of the myocardial cells was observed.
1. Isolation and inoculation of cardiomyocytes
Newborn SD rats one to two days after birth are removed from the neck and sacrificed, and the myocardium is removed and then washed 2-3 sides with pre-cooled HBSS. A portion of the ventricles was cut into pieces and digested overnight with 0.125% trypsin containing EDTA. Removing pancreatin, adding collagenase solution, and treating with CO at 37 deg.C2The culture box is used for 10-15 minutes. Taking the cell suspension, filtering with 200 mesh copper wire mesh, adding a-MEM cell culture solution into the filtered cell suspension to terminate digestion, centrifuging at 2000rpm for 3-5 min. Removing supernatant, adding a-MEM cell culture medium, resuspending, and adding CO at 37 deg.C2The culture box of (1.5) allows fibroblasts to be attached to the wall in a large quantity, and the cardiomyocytes to be left in suspension.
Placing MA-Gelzan-Gly-Go-Ito-DA-Fmoc and MA-Gelzan-Gly-Go-Ito-DA-h in a sterilized culture dish, sterilizing the myocardial patch material with alcohol, removing alcohol, adding PBS, washing for 2 times, and removing residual alcohol. At 4.5X 104One seed was inoculated into the myocardial patch material described above, with 5 replicates for each type of material.
2. Proliferation status detection
After 1, 2 and 4 days of cell culture, the cells were tested by using a cell counting kit CCK-8, which was carried out according to the instructions of the CCK-8 kit.
3. The result of the detection
As can be seen from FIG. 4, the proliferation of cardiomyocytes on MA-Gelzan-Gly-Go-Ito-DA-Fmoc, MA-Gelzan-Gly-Go-Ito-DA-h was similar, and as a result, the cardiomyocyte patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc containing the self-assembling peptide was also suitable for the proliferation of cardiomyocytes.
Example 9 myocardial patch Material containing self-assembling peptides repair in vivo myocardial infarction
1. Preparation of rat myocardial infarction model
Healthy male SD rats, weighing 200-.
Pentobarbital sodium hydrochloride is injected into the abdominal cavity for anesthesia. The rat respirator assisted respiration is performed through the oral trachea cannula, and the electrocardiogram is continuously monitored. Opening the chest layer by layer between the 3 rd and 4 th intercostals of the left chest of the myocardial infarction group, opening the pericardium, penetrating the myocardial surface layer of the anterior descending branch of the left coronary artery at the position of 1mm of the right lower edge of the left auricle and the left edge of the pulmonary artery cone by using a non-invasive suture needle, and ligating the proximal end of the anterior descending branch of the left coronary artery by using silk threads to prepare the myocardial infarction model. Sham operated rats underwent the procedure described above, with the silk thread passing under the coronary artery but not ligated.
2. Experimental groups and myocardial patch transplantation
10 days after myocardial infarction was established, 11 living rats were co-existed, 10 rats were selected from 11 rats and randomly divided into a transplant group and an myocardial infarction control group, 5 rats were each group, and the myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc was cut into squares having a diameter of 8mm and a height of 1.5mm, and the squares were divided into 4.5X 10 blocks4And inoculating into the myocardial patch material. The myocardial patch is transplanted to the myocardial infarction part, and the myocardial infarction control group and the pseudo-operation group adopt the same operation without the patch transplantation.
3. Determination of phosphocreatine kinase isoenzyme (CK-MB)
After 3 weeks of myocardial patch transplantation, blood is taken from the inner canthus of the rat eyes of a myocardial infarction control group, a sham operation group and a transplantation group, a blood sample is placed in a thermostatic water bath at 37 ℃ for standing for 2 hours, then the blood sample is centrifuged at 3500 r/min for 10min, supernatant is sucked into an EP tube, and the content of phosphocreatine kinase isoenzyme (CK-MB) is uniformly determined by a full-automatic biochemical analyzer to be used as an index for evaluating myocardial damage.
4. HE staining detection of myocardial tissue morphology and myocardial infarction area
After 3 weeks of myocardial patch transplantation, the myocardium was excised after sacrifice. Repeatedly washing myocardium with 4 deg.C physiological saline, collecting tissue of myocardial infarction part, fixing with 4% paraformaldehyde solution for 24 hr, dehydrating, embedding in paraffin, preparing tissue section with thickness of 4 μm, performing HE staining, and observing general morphological change of myocardial tissue after myocardial infarction. Infarct area was determined by image analysis software and averaged, and the infarct area was (inner arc length of scar + outer arc length of scar)/(outer circumference + inner circumference) × 100%.
5. Myocardial cell in situ apoptosis assay
After the myocardial patch is transplanted for 3 weeks, after a rat is killed, the myocardium is cut to be fixed by formaldehyde, after the routine paraffin embedding treatment of the myocardial tissue is carried out in a pathology department, a thick section is made from a wax block, the section is gently searched by a polylysine glass slide, the section is placed in a 37 ℃ baking machine to be baked, the section is placed in a 60 ℃ baking oven to be baked for about 2 hours before detection, the apoptosis condition of the myocardial cell is determined by adopting a TdT-media dUTP nickend labeling (TUNEL) method, and the Apoptosis Index (AI) of the myocardial cell is calculated.
6. Statistical treatment
SPSS 13.0 statistical software is used for data processing, and single-factor variance is adopted for mean comparison among multiple groups. P < 0.05 is statistically significant.
7. Results of the experiment
As shown in figure 5, myocardial cell envelopes of the rats in the myocardial infarction group are incomplete, the arrangement is disordered, the cytoplasm of the myocardial tissue is colored and deepened, myocardial fibers are broken and are infiltrated by a large number of inflammatory cells, the myocardial cell envelopes of the rats in the pseudo-operation group are complete, the myocardial fibers run regularly, inflammatory cell infiltration is avoided, and the myocardial tissue has no obvious pathological change. Compared with a pseudo-operation, the degeneration degree of the rat myocardial cells in the transplanted group is reduced, and the myocardial patch prepared by MA-Gelzan-Gly-Go-Ito-DA-Fmoc has a positive effect on the morphological repair of myocardial tissues.
The myocardial apoptosis index, myocardial infarct area and CK-MB content of the transplanted, control and sham-operated groups are shown in Table 6
TABLE 6 myocardial apoptosis index, myocardial infarct area and CK-MB content of each group
As can be seen from table 8, after the myocardial patch prepared from the self-assembling peptide-containing myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc prepared by the present application is transplanted into a body, due to its excellent ilotoin release performance, the myocardial patch material can well scavenge free radicals at the myocardial infarction part, improve the cell microenvironment, and has a positive therapeutic effect on myocardial infarction.
The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. The slow-release myocardial patch material is characterized by comprising 10-100 parts of gelatin, 4-100 parts of glycerol, 5-50 parts of gellan gum, 3-10 parts of graphene oxide, 0.6-5 parts of dopamine, 0.5-20 parts of itonin, 0.10-10 parts of Fmoc-FF1 and a proper amount of methacrylic anhydride.
2. The sustained-release myocardial patch material according to claim 1, wherein the gellan gum is G3251, a plant gel produced by Phytoechlab, CAS: 71010-52-1.
3. A method for preparing a sustained-release myocardial patch material, which is characterized by comprising the following steps of: (1) preparing transparent MA-Gelzan-Gly blending solution from gelatin, glycerol and gellan gum; (2) adding methacrylic anhydride into the blending liquid to obtain modified MA-Gelzan-Gly hydrogel; (3) mixing the modified MA-Gelzan-Gly hydrogel with graphene oxide to obtain a graphene oxide loaded MA-Gelzan-Gly-Go hydrogel; (4) mixing MA-Gelzan-Gly-Go hydrogel with dopamine and ilotoin, taking ammonium persulfate as an initiator and TEMED as a polymerization auxiliary agent, and simultaneously dripping Fmoc-FF solution.
4. The method of claim 3, wherein the step (1) is: completely dissolving 2g of gelatin in Phosphate Buffered Saline (PBS) at 60 ℃ to obtain 10 w/v% gelatin solution, dissolving 0.8g of glycerol in distilled water to obtain 4 w/v% glycerol solution, dissolving 1g of gellan gum in distilled water at 60 ℃ to obtain 10 w/v% gellan gum solution, mixing the three solutions in a ratio of 1.2:1:1.5, and magnetically stirring for 1h to obtain MA-Gelzan-Gly blending solution.
5. The method of claim 3, wherein the step (2) is: slowly dripping 4mL of methacrylic anhydride into the MA-Gelzan-Gly blending solution, stirring for 1-3 hours at 50 ℃ to enable the mixture to react gradually, then adding PBS to dilute the mixture and stopping the reaction; adding the reactant into a dialysis bag, dialyzing with deionized water for 5-9 days, replacing the deionized water every 6-12 hours, and freeze-drying and storing to obtain modified MA-Gelzan-Gly hydrogel; the step (3) is as follows: preparing 40mg of graphene oxide into a 4mg/mL solution, preparing the modified MA-Gelzan-Gly hydrogel obtained in the step (2) into a 10% solution, mixing the 10% MA-Gelzan-Gly hydrogel solution with the graphene oxide solution in a ratio of 4:2, and placing the mixture in a refrigerator at 4 ℃ for 2-4h to obtain the graphene oxide loaded MA-Gelzan-Gly-Go hydrogel.
6. The method of claim 3, wherein the step (4) is: preparing an Fmoc-FF solution of 4mg/mL, adding 0.12g of dopamine and 0.3g of ilottoside into the MA-Gelzan-Gly-Go hydrogel prepared in the step (3), uniformly mixing, adding 3mL of 20% ammonium persulfate and 1mL of TEMED solution, dropwise adding 20mL of Fmoc-FF solution of 4mg/mL, standing for 30min, and placing in a refrigerator at 4 ℃ for overnight to obtain MA-Gelzan-Gly-Go-Ito-DA-Fmoc.
7. The method according to any one of claims 3 to 6, wherein the method further comprises step (5): and (3) dripping 300 mu L of the product obtained in the step (4) onto the surface of a PDMS template, slightly covering a glass slide on the surface of a hydrogel solution, standing at room temperature for one day or placing at 4 ℃ for gelling, repeating the steps for 2-5 times after the hydrogel is gelled and air-dried to obtain a gel layer with 2-5 layers, and stripping the gel layer from the PDMS template to obtain the slow-release myocardial patch material MA-Gelzan-Gly-Go-Ito-DA-Fmoc.
8. A slow release myocardial patch material prepared by the method of any one of claims 3 to 7.
9. Use of the sustained-release myocardial patch material of any one of claims 1-2 and 8 in preparing a myocardial patch, wherein primary myocardial cells or pluripotent stem cells are inoculated in the sustained-release myocardial patch material, and the pluripotent stem cells are adipose-derived stem cells, mesenchymal stem cells and bone marrow stem cells.
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