CN115054732B

CN115054732B - Suture-free multi-layer drug-loaded myocardial patch and preparation method thereof

Info

Publication number: CN115054732B
Application number: CN202210642108.5A
Authority: CN
Inventors: 毛吉富; 单梦琪; 李沂蒙; 王富军; 王璐
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2023-10-13
Anticipated expiration: 2042-06-07
Also published as: CN115054732A

Abstract

The invention relates to a suture-free multilayer drug-loaded cardiac muscle patch and a preparation method thereof, wherein the suture-free multilayer drug-loaded cardiac muscle patch comprises a cardiac muscle patch basal layer for loading statin drugs, an intermediate layer for loading curcumin and a microneedle layer for loading vascular endothelial growth factors; the myocardial patch basal layer and the middle layer are made of polymer gel, and the microneedle layer is made of polymer gel or degradable polymer material; the swelling rate of the myocardial patch basal layer > the swelling rate of the intermediate layer; after penetrating into the isolated pig heart, the myocardial patch is soaked in PBS buffer solution, and after 20 minutes, the swelling rate of the basal layer of the myocardial patch is more than 60 percent of the swelling rate of the middle layer; the preparation method comprises the following steps: sequentially preparing a microneedle layer, an intermediate layer and a myocardial patch basal layer, and demolding to obtain the suture-free multilayer drug-loaded myocardial patch. The preparation method is simple, the prepared multi-layer drug-loaded cardiac muscle patch has the effect of accurately positioning and releasing drugs after being implanted into cardiac muscle, and the unexpected suture-free effect can be achieved when the drug release is carried out.

Description

Suture-free multi-layer drug-loaded myocardial patch and preparation method thereof

Technical Field

The invention belongs to the technical field of drug-loaded cardiac muscle patches, and relates to a suture-free multilayer drug-loaded cardiac muscle patch and a preparation method thereof.

Background

Myocardial infarction is a common cardiovascular disease with high mortality rate in China, after myocardial infarction occurs, a large number of myocardial cells die due to ischemia and hypoxia, myocardial tissues undergo an inflammatory phase, a proliferation phase and a remodeling phase, and necrotic parts cure infarcted myocardium by forming fibrotic scar tissues and performing left ventricular remodeling, however, microenvironment of the infarcted myocardium is still bad, such as lack of oxygen and nutrition supply, inflammatory reaction and the like, which is unfavorable for development of the infarcted myocardium and surrounding normal myocardium, further myocardial fibrosis and hypertrophic growth of the normal myocardial cells are caused to compensate for heart functions, and heart failure is caused when serious. Thus, promoting angiogenesis at necrotic myocardium, reducing the occurrence of inflammatory reactions, and inhibiting myocardial fibrosis are currently effective treatments for heart failure.

The myocardial patch repair strategy is a promising alternative biological treatment method, and has the main functions of providing mechanical support for damaged myocardial parts and preventing ventricular rupture, and the myocardial patch can be used as a carrier for carrying medicines, proteins and cells.

Curcumin, cyclosporin a, puerarin, statins, recombinant human relaxin-2, vascular endothelial growth factor, basic fibroblast growth factor, interleukin 10, and other anti-inflammatory factors have all been shown to produce beneficial effects during myocardial repair (Biomaterials loaded with growth factors/cytokines and stem cells for cardiac tissue regeneration.international Journal of Molecular Sciences,2020, 21:5952.). The loading purpose can be realized by adding a certain proportion of drugs or factors in the myocardial patch forming process, such as adding the drugs or factors in the electrostatic spinning solution preparing process or uniformly mixing the drugs or factors after adding the polymer precursor solution (CN 201811636101.2, a myocardial repair hydrogel material and a preparation method thereof), and then curing or crosslinking to obtain the drug-loaded formed myocardial patch. However, the above methods are relatively single in terms of drug loading and delivery, and the epicardial myocardial patch typically delivers only one drug or growth factor, which is difficult to meet the complex requirements of myocardial repair. Hydrogels have a three-dimensional network structure similar to the natural extracellular matrix, and release of drugs can be performed by swelling, diffusion, etc., and growth factors and drugs can be controlled and sustained by designing parameters such as polymer materials, concentration, crosslinking reaction, structure, etc. Recently, a double-layer hydrogel system prepared by a team through material and structural design of hydrogels can be loaded with different drugs and then attached to the epicardium, so that the in-situ release time is prolonged, and the method is expected to reduce fibrosis and inhibit scar size (A Bi-Layer Hydrogel Cardiac Patch Made of Recombinant Functional proteins. Advanced Materials,2022, 34:2201411.). After myocardial infarction occurs, myocardial infarction parts and environments are very complex, epicardium plays an important role of bridging internal and external environments simultaneously, and the myocardial patch drug loading method and application mode only release drugs on the epicardium surface to repair damaged myocardium, ignores the characteristics of the environment in the epicardium and the damaged myocardium, and has short half-life period and rapid elimination in living environment, which are challenges in the field. In recent years, microneedle array patches composed of microneedles have become a promising tool for delivering drugs with high precision and efficiency, in addition to minimally invasive tissue penetration. If the Vascular Endothelial Growth Factor (VEGF) and interleukin 10 (IL-10) are wrapped in a microneedle array, the vascular remodeling of the damaged area of the heart can be promoted and inflammatory response can be inhibited (Induced cardiomyocytes-integrated conductive microneedle patch for treating myocardial research. Chemical Engineering Journal,2021, 414:128723.). The hydrogel and the microneedles are designed and combined, and epicardial positioning targeting treatment is carried out, so that the accurate release of the drugs and the growth factors in the myocardial repair process is hopeful to be realized, and the multiple requirements of repairing necrotic myocardium are met.

When the fixation of the myocardial patch to the epicardium is effective, most studies have chosen to suture the patch to the epicardium using sutures. This fixation has problems, firstly, in that the suture needle passes through the heart, which tends to cause stress concentration at the puncture site and tissue damage at the site, thereby forming scar tissue. Second, after the epicardium is sutured, the heart beats again, which can cause some tissue damage and bleeding or infection, all of which can be secondary damage to the heart muscle. Thirdly, the difficulty of suturing the shaped patch to the dynamic heart is great, the time is long, and chest suturing is needed, so that the patch is not suitable for minimally invasive surgery.

Aiming at the defects of the epicardial patch application mode, the corresponding material and structural design are studied and focused on the suture-free performance of the myocardial patch. If a series of adhesive hydrogel myocardial patches with both adhesive and dissipative matrix portions were designed, the former attached to the myocardium by electrostatic interactions, covalent bonds and physical interpenetration, the latter energy dissipative by hysteresis amplification, compatible with dynamic myocardium (Tough adhesives for diverse wet surfaces. Science,2017,357: 378-381). Further, a conductive adhesive myocardial patch has been reported by the scholars to achieve rapid, stable, conformal and conductive integration between bioelectronics and various wet dynamic tissues and to successfully verify strong adhesion in pig in vivo models (Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration. Biomaterials,2021, 278:121171.).

Most of the research on the repair of cardiac muscle tissue with respect to microneedles has focused on drug and cell delivery, while less research has been conducted on the epicardial anchoring of myocardial patches with the raw materials, structural design of microneedles. Mainly because the patch for myocardial repair still has more requirements, the microneedle and the myocardial patch need to be constructed into an effective organic combination to meet the basic requirements and the stitching-free performance of the myocardial patch. At present, there is a study designed a crosslinked biphasic structured microneedle patch comprising an outer portion that absorbs water and produces tackiness and a rigid inner portion based on silk fibroin, and the outer layer of the microneedle absorbs water and expands when penetrating the epicardium, thereby achieving the mechanical interlocking (described in Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration). Patent CN202120534654.8 provides a cardiac muscle patch with microneedles for solving the technical problem of fixing the cardiac muscle patch to the heart surface, which can enable the cardiac muscle patch to be fixed at the target tissue quickly and effectively and atraumatically. However, the above researches all use complicated preparation methods, such as extraction of multiple raw materials and preparation for a long time or precise design of 3D printing parameters to obtain the barb structure on the surface of the microneedle, and the process is complicated and is not easy for mass production.

Therefore, a simple and repeatable biological manufacturing process is developed, the suture-free myocardial patch suitable for myocardial tissues is prepared, and the drug can be accurately positioned and released according to the requirements aiming at the characteristics of micro-environments after myocardial infarction so as to assist in repairing damaged cardiac muscle, so that important significance can be generated.

Disclosure of Invention

The invention aims to solve the problems that in the prior art, myocardial patches are single in drug loading, can not accurately position and release drugs and complex chest opening suturing is needed, and provides a multi-layer drug-loaded myocardial patch free of suturing and a preparation method thereof.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a suture-free multilayer drug-loaded myocardial patch comprises a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array and is uniformly distributed on the same side of the intermediate layer and is perpendicular to the plane of the intermediate layer, and the myocardial patch basal layer is positioned on the other side of the intermediate layer by an in-situ interface bonding method;

the basal layer of the cardiac muscle patch is loaded with statin drugs with functions of inhibiting fibrosis and protecting heart, the middle layer is loaded with curcumin with anti-inflammatory and left ventricle reconstruction inhibiting effects, the microneedle layer is loaded with Vascular Endothelial Growth Factor (VEGF) with blood vessel regeneration promoting effects, and the three layers respectively act on the environment where the myocardial infarction part is located, the epicardial surface and the epicardial interior;

The myocardial patch basal layer and the middle layer material are polymer gel, the microneedle layer material is polymer gel or degradable polymer material, under the mediation of in vivo moist environment, the middle layer and the myocardial patch basal layer are both swelled to release medicine, the microneedle layer slowly releases medicine according to the characteristics of the materials (namely, when the microneedle material is the degradable polymer material, the medicine is explained according to the slow drop of the material, when the microneedle material is the polymer gel, the medicine is released according to the swelling mechanism); the swelling rate of the myocardial patch basal layer is greater than the swelling rate of the middle layer, and when the material of the microneedle layer is polymer gel, the swelling rate of the myocardial patch basal layer is greater than the swelling rate of the middle layer and greater than the swelling rate of the microneedle layer; the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into isolated pig heart, and the swelling rate of the basal layer of the cardiac muscle patch is more than 60% of the swelling rate of the middle layer in 20 minutes, so that the middle layer and the basal layer can realize the suture-free effect in a short period while having larger traction force on the micro needle to incline when swelling;

the bonding strength of the myocardial patch basal layer and the middle layer is larger than 20kPa, and the micro needle deformation failure can be caused by the too small bonding strength.

As a preferable technical scheme:

according to the suture-free multilayer drug-loaded cardiac muscle patch, after the multilayer drug-loaded cardiac muscle patch is penetrated into an isolated pig heart, the multilayer drug-loaded cardiac muscle patch is soaked in PBS buffer solution, and after 20 minutes, the included angle between the axis direction of the micro needle and the plane of the middle layer is measured, wherein the included angle between the axis direction of the micro needle and the plane of the middle layer is 30-70 degrees;

fixing a myocardial patch on a pig myocardial tissue sample with the thickness of 10mm, immersing the pig myocardial tissue sample in PBS buffer solution, testing the obtained myocardial patch after 20 minutes to obtain the maximum pull-out force of 10-50 cN, and evaluating the bonding strength of the patch and the tissue with the maximum pull-out force, thereby representing the stitching-free effect, wherein the testing method of the maximum pull-out force comprises the following steps: the myocardial patch is stuck on the upper sensor probe of the electronic universal material tester, the pig myocardial tissue sample is fixed on the lower clamp, and the multi-layer myocardial patch is driven upwards at the speed of 0.05mm/s until the micro needle is completely separated from the pig myocardial tissue.

The suture-free multilayer drug-loaded myocardial patch comprises a myocardial patch basal layer material and an interlayer material which are respectively selected from one of methacrylic acid hyaluronic acid, methacrylic acid acylated gelatin and polyethylene glycol diacrylate, and preferably methacrylic acid acylated gelatin or polyethylene glycol diacrylate; the microneedle layer material is silk fibroin, polyethylene glycol diacrylate, methacrylic acid hyaluronic acid, polyvinyl alcohol or methacrylic acid acylated gelatin (silk fibroin is degradable polymer material, and the rest material is polymer gel), preferably polyvinyl alcohol or polyethylene glycol diacrylate.

The suture-free multilayer drug-loaded myocardial patch has the shape of a microneedle which is bullet-headed, conical or quadrangular; the distribution density of the micro-needles is 0.5-2 needles/mm;

when the microneedle is bullet-shaped, the radius of the bottom is 150-400 micrometers, the height of the cylinder is 600-1000 micrometers, and the height of the cone is 300-500 micrometers;

when the microneedle is conical, the radius of the bottom is 150-400 micrometers, and the length of the microneedle is 1000-1500 micrometers;

when the microneedle is in the shape of a rectangular pyramid, the side length of the bottom is 150-400 micrometers, and the length of the microneedle is 1000-1500 micrometers.

The multilayer drug-loaded myocardial patch without sewing has the thickness of the basal layer of the myocardial patch of 500-1200 micrometers; the thickness of the intermediate layer is 400-800 micrometers.

The suture-free multi-layer drug-loaded cardiac muscle patch has the specification of 0.8cm multiplied by 0.8 cm-1.5 cm multiplied by 1.5cm.

The invention also provides a preparation method of the suture-free multilayer drug-loaded myocardial patch, which comprises the following steps:

(1) Preparation of microneedle layer: uniformly dispersing Vascular Endothelial Growth Factor (VEGF) in a solution of a microneedle layer material to obtain casting solution A, casting the casting solution A on a microneedle part of a microneedle mould (the microneedle mould is a PDMS mould with a microneedle hole and a substrate groove), drying/ultraviolet curing to obtain the microneedle layer A, and temporarily not demoulding;

(2) Preparation of an intermediate layer: uniformly dispersing curcumin in a solution of an intermediate layer material to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet light curing to obtain the intermediate layer B, wherein the intermediate layer B is not demoulded;

(3) Preparation of myocardial patch basal layer: uniformly dispersing statin drugs in a solution of a myocardial patch basal layer material to obtain casting solution C, casting the casting solution C to the rest part in a basal groove of a microneedle mould, and performing ultraviolet curing to obtain a myocardial patch basal layer C;

(4) And (3) demolding to obtain the myocardial patch A-B-C aggregate, namely the suture-free multilayer drug-loaded myocardial patch.

The method comprises the steps that (1) the solution of the microneedle layer material is silk fibroin solution, polyethylene glycol diacrylate solution, methacrylic acid hyaluronic acid solution, methacrylic acid acylated gelatin solution or polyvinyl alcohol solution;

the concentration of the silk fibroin solution is 3% (w/v) to 7% (w/v), and the solvent is water;

the concentration of the polyethylene glycol diacrylate solution is 60% (v/v) to 80% (v/v), and the solvent is water;

the concentration of the methacrylic acid hyaluronic acid solution is 5% (w/v) to 10% (w/v), and the solvent is water;

the concentration of the methacrylic acid acylated gelatin solution is 30% (w/w) to 50% (w/w), and the solvent is water;

The concentration of the polyvinyl alcohol solution is 15% (w/w) to 30% (w/w), and the solvent is water;

the solution of the intermediate layer material in the step (2) is methacrylic acid hyaluronic acid solution, polyethylene glycol diacrylate solution or methacrylic acid acylated gelatin solution;

the concentration of the methacrylic acid hyaluronic acid solution is 4% (w/v) to 8% (w/v), and the solvent is water;

the concentration of the polyethylene glycol diacrylate solution is 40% (v/v) to 60% (v/v), and the solvent is water;

the concentration of the methacrylic acid acylated gelatin solution is 20% (w/w) to 40% (w/w), and the solvent is water;

the solution of the central muscle patch basal layer material in the step (3) is methacrylic acid hyaluronic acid solution, polyethylene glycol diacrylate solution or methacrylic acid acylated gelatin solution;

the concentration of the methacrylic acid hyaluronic acid solution is 3% (w/v) to 7% (w/v), and the solvent is water;

the concentration of the polyethylene glycol diacrylate solution is 30% (v/v) to 50% (v/v), and the solvent is water;

the concentration of the methacrylic acid acylated gelatin solution is 15% (w/w) to 30% (w/w), and the solvent is water.

The material and the concentration of the material in preparation of the myocardial patch basal layer and the interlayer are at least one point different.

The method comprises the following steps that (1) the concentration of the vascular endothelial growth factor in the casting solution A is 0.5-3 mug/mL;

The concentration of curcumin in the casting solution B in the step (2) is 1-3 mg/mL;

in the step (3), the statin medicine is pravastatin or atorvastatin, and the concentration of the statin medicine in the casting solution C is 5-10 mg/kg.

The method refers to drying and curing at 25-60 ℃ for 12-24 hours;

the ultraviolet curing time is 10 s-24 h, the photoinitiator is 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone, and the dosage of the photoinitiator is 0.05-1% (v/v) of the casting solution A, the casting solution B or the casting solution C.

When the polymer in the casting mould liquid is silk fibroin, the molding condition is drying and curing, the drying temperature is 25-60 ℃, and the drying time is 12-24 hours;

when the polymer in the casting mould liquid is polyvinyl alcohol, the molding condition is drying and curing, the drying temperature is 25-40 ℃, and the drying time is 12-24 hours;

when the macromolecule in the casting solution is methacrylic acid hyaluronic acid or methacrylic acid acylated gelatin, the molding condition is ultraviolet light curing, the photoinitiator is 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone, the dosage of the photoinitiator is 0.05% (v/v) to 0.5% (v/v) of the casting solution, and the light curing time is 20min to 6h;

when the macromolecule in the casting solution is polyethylene glycol diacrylate, the molding condition is ultraviolet light curing, the photoinitiator is 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone, the dosage of the photoinitiator is 0.5% (v/v) to 1% (v/v) of the casting solution, and the light curing time is 10s to 40s.

The principle of the invention is as follows:

for the stitching-free performance of the myocardial patch, the prior art generally performs material design (e.g., the synthetic adhesive patch is directly adhered to the epicardium), or structural design (e.g., complex methods such as fine processing techniques, 3D printing, laser cutting, ferrofluid control, etc.) to design the microneedle into a barb shape, a bee sting shape, an arrow shape, a pagoda shape, etc. The invention provides an unexpected stitching-free effect of the myocardial patch when the drug release is carried out: namely, when the patch is implanted into epicardium, under the mediation of in vivo moist environment, the middle layer and the basal layer of the myocardial patch are swelled to release medicine, and the release speed of the microneedle layer is slowest, so that the purpose of releasing vascular endothelial growth factor for a long time to promote angiogenesis is achieved; the swelling effect can lead to the volume expansion of the middle layer at various directions, and the micro-needles are connected with myocardial tissues because the middle layer is connected with the micro-needles, so that the micro-needle array can be triggered to be converted into an angle clamping structure from a vertical structure under the action of the expansion traction force of the middle layer and the resistance of the myocardial tissues to the micro-needles, thereby achieving the self-fixing effect; meanwhile, the drug release rate and the swelling rate of the outermost layer (the myocardial patch basal layer) are fastest, and the volume expansion is faster than that of the middle layer, so that a certain pressure effect can be generated on the microneedle layer and the middle layer, the myocardial patch can generate a self-tightening effect on the epicardium surface, and is tightly attached to the epicardium surface, and the stitching-free effect can be further enhanced.

The beneficial effects are that:

(1) The multilayer drug-loaded myocardial patch prepared by the invention has accurate positioning release effect after being implanted into cardiac muscle, namely, corresponding drugs or growth factors can be released respectively according to the characteristics of the epicardium inner surface, the epicardium surface and the environment where cardiac muscle tissues are located; the microneedle array layer releases Vascular Endothelial Growth Factor (VEGF) to promote revascularization, the intermediate layer releases curcumin to resist inflammation and inhibit left ventricular remodeling, and the myocardial patch basal layer releases statins to inhibit fibrosis and protect cardiac function. The invention can release corresponding medicines according to different treatment requirements after myocardial infarction, is beneficial to promoting angiogenesis at necrotic myocardium, reducing inflammatory reaction, inhibiting myocardial fibrosis and promoting infarcted myocardial repair.

(2) When the multilayer drug-loaded myocardial patch prepared by the invention is implanted into damaged myocardial tissue, the microneedle array is perpendicular to the damaged myocardial tissue, after the damaged myocardial tissue is penetrated, the middle layer and the basal layer are instantly swelled to release drugs under the mediation of a wet environment in vivo, the macro appearance is that the volume expansion occurs, so that the microneedle array is triggered to be converted into an angle clamping structure from a vertical structure, meanwhile, the expansion force of the basal layer of the myocardial patch can cause certain pressure effect on the microneedle layer and the middle layer, so that the myocardial patch generates a self-tightening effect similar to the epicardial surface, is tightly attached to the epicardial surface, and realizes the stitching-free effect. The operation can be performed through thoracoscopic surgery without suture fixation, so that the surgery difficulty and postoperative complications are reduced, and the surgery time is shortened.

(3) The preparation method is simple, has high repeatability and is suitable for large-scale production.

Drawings

FIG. 1 is a schematic diagram of the structure and application of a suture-free multi-layer drug-loaded myocardial patch;

FIG. 2 is a flow chart of the preparation of a suture-free multi-layer drug-loaded myocardial patch.

Detailed Description

The application is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the contents of the present application, and such equivalents are also within the scope of the present application as defined in the appended claims.

The microneedle mould adopted by the application is a PDMS mould with microperforations and a substrate groove.

The application adopts the following test method:

(1) Bond strength: the layer to be tested is 10-20 mm longer than the other layer, the longer part is arranged on an upper clamp of a peeling tester, the rest part is fixed on a 90-degree test platform, a peeling strength test is carried out, and the interface bonding strength of the myocardial patch basal layer and the middle layer as well as the interface bonding strength of the middle layer and the microneedle layer are tested;

(2) Maximum pull-out force: and fixing the myocardial patch on a pig myocardial tissue sample with the thickness of 10mm, immersing in PBS buffer solution for 20 minutes, taking out, adhering the myocardial patch on an upper sensor probe of an electronic universal material tester, fixing the pig myocardial tissue sample on a lower clamp, driving the multi-layer myocardial patch upwards at the speed of 0.05mm/s until the micro needle is completely separated from pig myocardial tissue, and measuring the maximum pulling-out force of the myocardial patch.

Example 1

As shown in fig. 2, a preparation method of the suture-free multilayer drug-loaded cardiac muscle patch comprises the following steps:

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in silk fibroin solution (water is used as a solvent) with the concentration of 3% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, drying and curing at 60 ℃ for 12 hours to obtain a microneedle layer A, and temporarily demolding; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 0.5 mug/mL; the microneedle is bullet-shaped, the radius of the bottom is 150 micrometers, the height of the cylinder is 800 micrometers, the height of the cone is 300 micrometers, and the length of the microneedle is 1100 micrometers; the microneedle distribution density was 2 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in a methacrylic acid acylated gelatin solution (water is used as a solvent) with the concentration of 40% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 6 hours under the condition that a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.05% (v/v) of the casting solution B is added to obtain an intermediate layer B with the thickness of 500 micrometers, and performing temporary demoulding; wherein the concentration of curcumin in the casting solution B is 2mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin (pravastatin) in a methacrylic hyaluronic acid solution (water as a solvent) with the concentration of 7% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 4 hours under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.5% (v/v) of the casting solution C to obtain a myocardial patch substrate layer C with the thickness of 500 micrometers; wherein, the concentration of statin drugs in the casting solution C is 10mg/kg;

As shown in FIG. 1, the prepared suture-free multilayer drug-loaded myocardial patch has a specification of 0.8cm multiplied by 0.8cm and comprises a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array and is uniformly distributed on the same side of the intermediate layer and is perpendicular to the plane of the intermediate layer, and the myocardial patch basal layer is positioned on the other side of the intermediate layer by an in-situ interface bonding method;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are respectively 350 percent and 130 percent when the time is 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 60 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 30kPa, and the bonding strength of the middle layer and the microneedle layer is 25kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 30cN after 20 minutes.

Example 2

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in a silk fibroin solution (water is used as a solvent) with the concentration of 7% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, drying and curing at 25 ℃ for 18 hours to obtain a microneedle layer A, and temporarily demolding; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 3 mug/mL; the microneedle is bullet-shaped, the radius of the bottom is 400 microns, the height of the cylinder is 600 microns, the height of the cone is 500 microns, and the length of the microneedle is 1100 microns; the microneedle distribution density was 0.5 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in a methacrylic acid solution (water serving as a solvent) with the concentration of 8% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 20min under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.5% (v/v) of the casting solution B to obtain an intermediate layer B with the thickness of 600 micrometers, wherein the intermediate layer B is not demoulded temporarily; wherein the concentration of curcumin in the casting solution B is 1mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin (pravastatin) in a methacrylic hyaluronic acid solution (water as a solvent) with the concentration of 6% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 6 hours under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.2% (v/v) of the casting solution C to obtain a myocardial patch substrate layer C with the thickness of 600 micrometers; wherein, the concentration of statin drugs in the casting solution C is 5mg/kg;

As shown in FIG. 1, the prepared suture-free multilayer drug-loaded myocardial patch has a specification of 1.5cm×1.5cm and comprises a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array and is uniformly distributed on the same side of the intermediate layer and is perpendicular to the plane of the intermediate layer, and the myocardial patch basal layer is positioned on the other side of the intermediate layer by an in-situ interface bonding method;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are 400 percent and 300 percent respectively in 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 40 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 35kPa, and the bonding strength of the middle layer and the microneedle layer is 30kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 45cN after 20 minutes.

Example 3

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in 5% (w/v) silk fibroin solution (water as solvent) to obtain casting solution A, casting the casting solution A on the microneedle part of a microneedle mould, drying and curing at 40 ℃ for 24 hours to obtain a microneedle layer A, and temporarily demolding; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 1 mug/mL; the microneedle is bullet-shaped, the radius of the bottom is 200 microns, the height of the cylinder is 1000 microns, the height of the cone is 400 microns, and the length of the microneedle is 1400 microns; the microneedle distribution density was 0.8 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in 30% (w/v) methacrylic acid acylated gelatin solution (water is used as a solvent) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet light curing for 3 hours under the condition of adding 0.3% (v/v) of photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of the casting solution B to obtain an intermediate layer B with the thickness of 400 micrometers, wherein the intermediate layer B is not demoulded temporarily; wherein the concentration of curcumin in the casting solution B is 3mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin (pravastatin) in a methacrylic acid acylated gelatin solution (water as a solvent) with the concentration of 20% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 40min under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.3% (v/v) of the casting solution C to obtain a myocardial patch substrate layer C with the thickness of 500 micrometers; wherein, the concentration of statin drugs in the casting solution C is 9mg/kg;

As shown in FIG. 1, the prepared suture-free multilayer drug-loaded myocardial patch has a specification of 1.2cm multiplied by 1.2cm and comprises a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array and is uniformly distributed on the same side of the intermediate layer and is perpendicular to the plane of the intermediate layer, and the myocardial patch basal layer is positioned on the other side of the intermediate layer by an in-situ interface bonding method;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are 280% and 180% respectively in 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 50 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 40kPa, and the bonding strength of the middle layer and the microneedle layer is 25kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 30cN after 20 minutes.

Example 4

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in a polyethylene glycol diacrylate solution (water is used as a solvent) with the concentration of 70% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, and carrying out ultraviolet curing for 10s under the condition that a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 1% (v/v) of the casting solution A is added to obtain a microneedle layer A, so that demoulding is not carried out temporarily; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 1.5 mug/mL; the shape of the microneedle is conical, the radius of the bottom is 150 microns, and the length of the microneedle is 1000 microns; the microneedle distribution density was 1.5 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in 50% (w/v) polyethylene glycol diacrylate solution (water is used as a solvent) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 40s under the condition of adding 0.5% (v/v) photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone of the casting solution B to obtain an intermediate layer B with the thickness of 500 micrometers, and performing demolding temporarily; wherein the concentration of curcumin in the casting solution B is 1.5mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin drugs (atorvastatin) in 30% (w/v) methacrylic acid acylated gelatin solution (water as solvent) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 20min under the condition of adding 0.5% (v/v) of photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of the casting solution C to obtain myocardial patch basal layer C with the thickness of 600 microns; wherein, the concentration of statin drugs in the casting solution C is 8mg/kg;

As shown in FIG. 1, the prepared suture-free multilayer drug-loaded myocardial patch has a specification of 1cm multiplied by 1cm and comprises a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array and is uniformly distributed on the same side of the intermediate layer and is perpendicular to the plane of the intermediate layer, and the myocardial patch basal layer is positioned on the other side of the intermediate layer by an in-situ interface bonding method;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are respectively 230 percent and 100 percent when the time is 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 65 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 35kPa, and the bonding strength of the middle layer and the microneedle layer is 45kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 25cN after 20 minutes.

Example 5

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in a polyethylene glycol diacrylate solution (water is used as a solvent) with the concentration of 80% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, and carrying out ultraviolet curing for 20s under the condition that a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 1% (v/v) of the casting solution A is added to obtain a microneedle layer A, so that demoulding is not carried out temporarily; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 1.2 mug/mL; the shape of the microneedle is conical, the radius of the bottom is 300 microns, and the length of the microneedle is 1200 microns; the microneedle distribution density is 1 needle/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in a polyethylene glycol diacrylate solution (water is used as a solvent) with the concentration of 40% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 10s under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 1% (v/v) of the casting solution B to obtain an intermediate layer B with the thickness of 400 micrometers, wherein the intermediate layer B is temporarily not demoulded; wherein the concentration of curcumin in the casting solution B is 1.8mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin drugs (atorvastatin) in a methacrylic acid acylated gelatin solution (water as a solvent) with the concentration of 25% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 3h under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.1% (v/v) of the casting solution C to obtain a myocardial patch basal layer C with the thickness of 500 micrometers; wherein, the concentration of statin drugs in the casting solution C is 7mg/kg;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are respectively 250 percent and 120 percent when the time is 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 60 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 30kPa, and the bonding strength of the middle layer and the microneedle layer is 40kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 28cN after 20 minutes.

Example 6

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in a methacrylic acid solution (water is used as a solvent) with the concentration of 8% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, and carrying out ultraviolet curing for 20min under the condition that a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.05% (v/v) of the casting solution A is added to obtain a microneedle layer A, so that demoulding is not carried out temporarily; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 1.8 mug/mL; the shape of the micro needle is a quadrangular pyramid, the side length of the bottom is 400 microns, and the length of the micro needle is 1500 microns; the microneedle distribution density was 1.2 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in a methacrylic acid solution (water serving as a solvent) with the concentration of 6% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 1h under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.4% (v/v) of the casting solution B to obtain an intermediate layer B with the thickness of 500 micrometers, wherein the intermediate layer B is not demoulded temporarily; wherein the concentration of curcumin in the casting solution B is 2.2mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin (pravastatin) in a methacrylic hyaluronic acid solution (water as a solvent) with the concentration of 5% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 5h under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.05% (v/v) of the casting solution C to obtain a myocardial patch substrate layer C with the thickness of 500 micrometers; wherein, the concentration of statin drugs in the casting solution C is 6mg/kg;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are respectively 600 percent and 450 percent when the time is 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 30 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 40kPa, and the bonding strength of the middle layer and the microneedle layer is 40kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 50cN after 20 minutes.

Example 7

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in 50% (w/v) methacrylic acid acylated gelatin solution (water is used as a solvent) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, and carrying out ultraviolet curing for 6 hours under the condition that a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.5% (v/v) of the casting solution A is added to obtain a microneedle layer A, so that demoulding is not carried out temporarily; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 2 mug/mL; the microneedle is bullet-shaped, the radius of the bottom is 300 microns, the height of the cylinder is 1000 microns, the height of the cone is 400 microns, and the length of the microneedle is 1400 microns; the microneedle distribution density was 1.8 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in a polyethylene glycol diacrylate solution (water is used as a solvent) with the concentration of 40% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 20s under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.5% (v/v) of the casting solution B to obtain an intermediate layer B with the thickness of 400 micrometers, wherein the intermediate layer B is not demoulded temporarily; wherein the concentration of curcumin in the casting solution B is 2.5mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin drugs (atorvastatin) in a methacrylic acid acylated gelatin solution (water as a solvent) with the concentration of 20% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 1h under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.5% (v/v) of the casting solution C to obtain a myocardial patch basal layer C with the thickness of 800 micrometers; wherein, the concentration of statin drugs in the casting solution C is 9mg/kg;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are respectively 200 percent and 100 percent when the time is 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 65 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 30kPa, and the bonding strength of the middle layer and the microneedle layer is 30kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 22cN after 20 minutes.

Example 8

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in a polyvinyl alcohol solution (water is used as a solvent) with the concentration of 20% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, drying and curing at 25 ℃ for 24 hours to obtain a microneedle layer A, and temporarily demolding; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 2.5 mug/mL; the shape of the microneedle is conical, the radius of the bottom is 400 microns, and the length of the microneedle is 1500 microns; the microneedle distribution density was 1.5 needles/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in polyethylene glycol diacrylate solution (water is used as a solvent) with the concentration of 60% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 20s under the condition of adding photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 1% (v/v) of the casting solution B to obtain an intermediate layer B with the thickness of 400 micrometers, and temporarily not demolding; wherein the concentration of curcumin in the casting solution B is 2.8mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin (pravastatin) in 30% (w/v) methacrylic acid acylated gelatin solution (water as solvent) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 2h under the condition of adding 0.4% (v/v) of photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone of the casting solution C to obtain myocardial patch substrate layer C with the thickness of 600 microns; wherein, the concentration of statin drugs in the casting solution C is 8mg/kg;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are respectively 260% and 80% when 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 70 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 35kPa, and the bonding strength of the middle layer and the microneedle layer is 25kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 12cN after 20 minutes.

Example 9

(1) Preparation of microneedle layer:

uniformly dispersing vascular endothelial growth factor in a polyethylene glycol diacrylate solution (water is used as a solvent) with the concentration of 80% (w/v) to obtain casting solution A, pouring the casting solution A on the microneedle part of a microneedle mould, and carrying out ultraviolet curing for 20s under the condition that a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 1% (v/v) of the casting solution A is added to obtain a microneedle layer A, so that demoulding is not carried out temporarily; wherein, the concentration of the vascular endothelial growth factor in the casting solution A is 2.2 mug/mL; the shape of the micro needle is a quadrangular pyramid, the side length of the bottom is 150 microns, and the length of the micro needle is 1000 microns; the microneedle distribution density is 1 needle/mm;

(2) Preparation of an intermediate layer:

uniformly dispersing curcumin in a methacrylic acid solution (water serving as a solvent) with the concentration of 7% (w/v) to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet curing for 4 hours under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.2% (v/v) of the casting solution B to obtain an intermediate layer B with the thickness of 500 micrometers, wherein the intermediate layer B is not demoulded temporarily; wherein the concentration of curcumin in the casting solution B is 2mg/mL;

(3) Preparation of myocardial patch basal layer:

uniformly dispersing statin drugs (atorvastatin) in a methacrylic hyaluronic acid solution (water is used as a solvent) with the concentration of 6% (w/v) to obtain casting solution C, pouring the casting solution C into the rest part in a substrate groove of a microneedle mould, and carrying out ultraviolet curing for 4 hours under the condition of adding a photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone with the content of 0.45% (v/v) of the casting solution C to obtain a myocardial patch substrate layer C with the thickness of 800 micrometers; wherein, the concentration of statin drugs in the casting solution C is 7mg/kg;

the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into the isolated pig heart, the swelling rate of the basal layer of the cardiac muscle patch and the swelling rate of the middle layer are 400 percent and 350 percent respectively in 20 minutes, and the included angle between the axis direction of the micro needle and the plane of the middle layer is 35 degrees;

The bonding strength of the myocardial patch basal layer and the middle layer is 35kPa, and the bonding strength of the middle layer and the microneedle layer is 25kPa; the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 48cN after 20 minutes.

The effect of the suture-free multi-layer drug-loaded myocardial patch of examples 1-9 on myocardial repair was investigated as follows:

(1) Rats were anesthetized with medium diethyl ether, left chest incisions were made to expose the heart, and then left coronary arteries were ligated with 8-0# suture at 2mm below the left atrial appendage to establish a rat myocardial infarction model;

(2) Dividing the mice with myocardial infarction into two groups, wherein one group is implanted with a multi-layer drug-carrying myocardial patch as an experimental group, and the other group is not subjected to any operation;

(3) After 2 weeks, rat heart function was observed using echocardiography, rats were sacrificed, hearts were collected and fixed with 4% paraformaldehyde at 4 ℃ and dehydrated in ethanol, and myocardial tissue sections were Masson trichromatized to observe changes in fibrotic tissue, infarct area and left chamber wall thickness;

experimental results show that implantation of the multi-layer drug-loaded myocardial patch can improve the ejection fraction and short-axis shortening rate of the left ventricle of the rat infarcted heart, and in addition, the internal size of the left ventricle in the systole is obviously reduced, and the fibrotic tissue and the infarcted area are reduced.

Claims

1. A suture-free multi-layer drug-loaded myocardial patch, which is characterized in that: comprises a myocardial patch basal layer, an intermediate layer and a microneedle layer; the microneedle layer is a microneedle array and is uniformly distributed on the same side of the intermediate layer and is perpendicular to the plane of the intermediate layer, and the myocardial patch basal layer is positioned on the other side of the intermediate layer by an in-situ interface bonding method;

the basal layer of the cardiac muscle patch is loaded with statin drugs, the middle layer is loaded with curcumin, and the microneedle layer is loaded with vascular endothelial growth factors;

the myocardial patch basal layer and the middle layer are made of polymer gel, and the microneedle layer is made of polymer gel or degradable polymer material; the myocardial patch basal layer material and the middle layer material are respectively selected from one of methacrylic acid hyaluronic acid, methacrylic acid acylated gelatin and polyethylene glycol diacrylate; the microneedle layer material is silk fibroin, polyethylene glycol diacrylate, methacrylic acid hyaluronic acid, polyvinyl alcohol or methacrylic acid acylated gelatin;

the swelling rate of the myocardial patch basal layer is greater than the swelling rate of the middle layer, and when the material of the microneedle layer is polymer gel, the swelling rate of the myocardial patch basal layer is greater than the swelling rate of the middle layer and greater than the swelling rate of the microneedle layer; the multi-layer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after being penetrated into isolated pig heart, and the swelling rate of the basal layer of the cardiac muscle patch is more than 60% of the swelling rate of the middle layer in 20 minutes;

The bonding strength of the myocardial patch substrate layer and the intermediate layer is greater than 20kPa.

2. The suture-free multilayer drug-loaded cardiac muscle patch according to claim 1, wherein the multilayer drug-loaded cardiac muscle patch is immersed in PBS buffer solution after penetrating into the isolated pig heart, and an included angle between the axis direction of the microneedle and the plane of the middle layer is measured after 20 minutes, and the included angle between the axis direction of the microneedle and the plane of the middle layer is 30-70 degrees;

the myocardial patch is fixed on a pig myocardial tissue sample with the thickness of 10mm, then is soaked in PBS buffer solution, and the maximum pulling-out force of the myocardial patch is 10-50 cN after 20 minutes of testing.

3. The suture-free multilayered drug-loaded cardiac patch of claim 1, wherein the microneedle is bullet-shaped, conical or quadrangular; the distribution density of the micro-needles is 0.5-2 needles/mm;

4. The suture-free multilayered drug-loaded myocardial patch of claim 1, wherein the myocardial patch base layer thickness is 500-1200 microns; the thickness of the intermediate layer is 400-800 micrometers.

5. The suture-free multi-layer drug-loaded cardiac patch of claim 1, wherein the multi-layer drug-loaded cardiac patch has a gauge of 0.8cm x 0.8cm to 1.5cm x 1.5cm.

6. The method for preparing the suture-free multilayer drug-loaded cardiac muscle patch according to any one of claims 1 to 5, which is characterized by comprising the following steps:

(1) Preparation of microneedle layer: uniformly dispersing vascular endothelial growth factor in a solution of a microneedle layer material to obtain casting solution A, casting the casting solution A on a microneedle part of a microneedle mould, and drying/ultraviolet curing to obtain a microneedle layer A;

(2) Preparation of an intermediate layer: uniformly dispersing curcumin in a solution of an intermediate layer material to obtain casting solution B, pouring the casting solution B into a substrate groove of a microneedle mould, and performing ultraviolet light curing to obtain an intermediate layer B;

7. The method of claim 6, wherein the solution of microneedle layer material in step (1) is a silk fibroin solution, a polyethylene glycol diacrylate solution, a methacrylic acid hyaluronic acid solution, a methacrylic acid acylated gelatin solution, or a polyvinyl alcohol solution;

8. The method according to claim 6, wherein the concentration of vascular endothelial growth factor in the casting solution A in the step (1) is 0.5 to 3. Mu.g/mL;

9. The method according to claim 6, wherein drying and curing means drying at 25 to 60 ℃ for 12 to 24 hours;