CN118949084A

CN118949084A - A conductive hydrogel microneedle patch for myocardial repair and preparation method thereof

Info

Publication number: CN118949084A
Application number: CN202411341946.4A
Authority: CN
Inventors: 郭瑞; 冯龙宝; 刘慧玲
Original assignee: Guangzhou Bioscience Co ltd
Current assignee: Guangzhou Bioscience Co ltd
Priority date: 2024-09-25
Filing date: 2024-09-25
Publication date: 2024-11-15
Anticipated expiration: 2044-09-25
Also published as: CN118949084B

Abstract

The present invention relates to a conductive hydrogel microneedle patch for myocardial repair and a preparation method thereof. The microneedle patch comprises a backing and a microneedle array, wherein the microneedle array is solidified from a hydrogel solution I and comprises methacrylylated dextran, epigallocatechin gallate, and basic fibroblast growth factor; the backing comprises an adhesion layer and an antifouling layer, wherein the adhesion layer comprises methacrylylated glycine and reduced graphene oxide; and the antifouling layer comprises polyethylene glycol diacrylate. The microneedle patch of the present invention has a needle tip loaded with a drug, a backing having a conductive adhesion layer, and an antifouling layer, which can provide mechanical support to the damaged cardiac tissue, effectively ensure the drug delivery depth, and can adhere well to the tissue surface, promote the conduction of electrical signals between myocardial cells, and avoid adhesion between the microneedle patch and other tissues and organs in the in vivo environment, and has a good cardiac repair effect and can be applied to the in vivo environment.

Description

Conductive hydrogel microneedle patch for myocardial repair and preparation method thereof

Technical Field

The invention relates to the field of medical biological materials, in particular to a conductive hydrogel microneedle patch for myocardial repair and a preparation method thereof.

Background

Myocardium is a tissue that contracts and relaxes in response to an electrical impulse. After myocardial infarction, there are many changes in the myocardial microenvironment, including fluctuations in the abundance of Reactive Oxygen Species (ROS) and inflammatory factors. ROS are overexpressed in infarcted myocardium, disrupting cellular homeostasis, leading to myocardial cell damage, while promoting inflammation and myocardial fibrosis. Excessive activation of inflammation can lead to inadaptation healing and ventricular remodeling. In addition, the reduced supply of oxygen and nutrients caused by coronary occlusion can lead to the development of fibrous scar tissue, thereby increasing the electrical resistivity of the myocardial tissue and impeding electrical signals between myocardial cells, causing abnormal simultaneous contraction between normal myocardial tissue and fibrous scar tissue, ultimately leading to ventricular dysfunction. Thus, to reverse this adverse microenvironment, reestablishing electrical signal conduction and/or promoting angiogenesis in the infarcted area may promote repair of cardiac function. Various biological materials have been used to promote cardiac functional recovery after myocardial infarction, including nanomaterials, hydrogels, cardiac patches, and the like.

In general, nanomaterials can achieve selective accumulation in infarcted areas by targeting to myocardial injury and inflammation sites; however, nanomaterial-based strategies are limited by potential toxicity and lack of mechanical support in myocardial infarction treatment. In addition, although intramyocardially injected hydrogels can reach the infarcted area directly to provide support for the ventricular wall, the resultant hydrogels tend to absorb water and swell, exerting pressure on surrounding tissue; and the injectable gel has low general modulus and high degradation speed in vivo, and cannot realize long-term treatment effect. In recent years, research on hydrogel myocardial patches in myocardial tissue engineering has also gained a great deal of attention, and some biological materials with adjustable structural properties can be prepared into cardiac patches with good biocompatibility, and the patches can provide mechanical support for cell growth and contribute to the repair of damaged hearts. However, in general, a single-layer hydrogel patch prepared from natural biological materials is advantageous for cell adhesion, and is easy to cause tissue adhesion problems in vivo. Thus, it remains a challenge to create a multi-functional cardiac patch that has both biomimetic mechanical properties to provide mechanical support, promote vascularization of infarcted tissue, scavenge reactive oxygen species, improve electrical signal conduction, and prevent tissue adhesions.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a conductive hydrogel microneedle patch for myocardial repair and a preparation method thereof.

The specific technical scheme of the invention is as follows:

In a first aspect of the present invention, there is provided a conductive hydrogel microneedle patch for myocardial repair, including a backing and a microneedle array, wherein the microneedle array is formed by curing a hydrogel solution I, and the hydrogel solution I contains methacryloylated dextran (DexMA), epigallocatechin gallate (EGCG) and basic fibroblast growth factor (bFGF); the back lining comprises an adhesion layer and an anti-fouling layer, the adhesion layer is connected with the microneedle array and positioned at the inner side of the back lining, the back lining is formed by solidifying a hydrogel solution II, and the hydrogel solution II comprises methacryloylglycine (ACG) and reduced graphene oxide (rGO); the anti-fouling layer is positioned on the outer side of the back lining and is formed by solidifying hydrogel solution III, wherein the hydrogel solution III comprises polyethylene glycol diacrylate (PEGDA).

Firstly, the microneedle array comprises methacryloyl dextran (DexMA), epigallocatechin gallate (EGCG) and basic fibroblast growth factor (bFGF), dexMA is a high-molecular hydrogel with good biocompatibility, a three-dimensional network structure formed by crosslinking can provide a platform for efficient loading and release of EGCG and bFGF, and bFGF can promote angiogenesis by promoting proliferation and migration of vascular endothelial cells and inducing transformation of surrounding fibroblasts into vascular endothelial cells. EGCG has various biological activities including antioxidation, anti-inflammatory, anti-tumor, anti-aging and the like; is helpful for reducing oxidative stress and inflammatory reaction in cells, promoting the repair and regeneration of cardiac muscle, and has a certain influence on the exertion of the oxidation resistance of EGCG by bFGF. Secondly, in the adhesive layer, an ACG forms a basic network, rGO is introduced, a stable conductive network can be formed, and the adhesive layer can be used as a conductive bracket of a cardiac patch, so that electric signal conduction among myocardial cells is promoted, and the normal function of the heart is facilitated. The two-dimensional structure and the high specific surface area of rGO enable the rGO to have good mechanical supporting performance, and the stability and the structural strength of the patch can be enhanced; the anti-fouling layer structure can prevent tissue from adhering and avoid adhesion.

Preferably, the hydrogel solution I comprises 1mg/mL-10mg/mL of epigallocatechin gallate, 100mg/mL-300mg/mL of methacryloyl dextran, and 0.001mg/mL-0.01mg/mL of basic fibroblast growth factor. In one preferred embodiment, the kit comprises 10mg/mL epigallocatechin gallate, 300mg/mL methacrylated dextran, and 0.01mg/mL basic fibroblast growth factor.

In some of these embodiments, the methacrylated dextran is prepared by the following method: mixing dextran, dimethyl sulfoxide and 4-dimethylaminopyridine, stirring, adding glycidyl methacrylate, stirring, dialyzing, and lyophilizing to obtain the methacryloylated dextran. Preferably, wherein the glucan: dimethyl sulfoxide: 4-dimethylaminopyridine: glycidyl methacrylate = 5.0g:50mL:1g:1.5mL.

Preferably, the hydrogel solution II contains 20wt% of methacryloyl glycine and 5mg/mL of reduced graphene oxide, calculated as the amount of water.

In some of these embodiments, the reduced graphene oxide is prepared by mixing L-ascorbic acid with an aqueous graphene oxide solution at ph=9 and 90 ℃. Preferably, L-ascorbic acid: graphene oxide = 0.5g:50mg of graphene oxide aqueous solution with a concentration of 1mg/mL.

In some embodiments, the graphene oxide is prepared by adding graphite into concentrated sulfuric acid, stirring at low temperature in an ice bath, adding potassium permanganate in batches, stirring for reaction in a water bath at 37 ℃ after fully and uniformly mixing, and stirring for reaction at 25 ℃; and then slowly adding deionized water I into the reaction liquid, heating the water bath to 90 ℃ for reaction after the generated gas is released, finally adding deionized water II to dilute the reaction liquid, naturally cooling to room temperature after the reaction is ended, adding 30% hydrogen peroxide solution until no bubbles are generated, standing until the reaction liquid is cooled, reacting with 10% dilute hydrochloric acid and barium chloride solution, repeatedly centrifuging until no precipitation is generated, and continuously using deionized water to wash the reaction liquid to be neutral to obtain the graphene oxide. Preferably, graphite: concentrated sulfuric acid: potassium permanganate: deionized water I: deionized water II = 2g:25mL:7g:120mL:200mL.

In some of these embodiments, the methacryloylated glycine is prepared by the following method: under ice bath condition, sodium glycinate is dissolved in NaOH solution, then, acrylic chloride-tetrahydrofuran solution is added dropwise to the solution for reaction, then, the solution is washed for impurity removal, extracted, dried and concentrated, and the obtained product is dissolved in water and freeze-dried to obtain the methacryloyl glycine. Preferably, the concentration of the NaOH solution is 6M, and the concentration of the acryloyl chloride in the acryloyl chloride-tetrahydrofuran solution is as follows: tetrahydrofuran=5.5: 10 (volume ratio), sodium glycinate: naOH solution: acrylic chloride-tetrahydrofuran solution = 5g:25mL:15.5mL.

Preferably, in the hydrogel solution III, the content of polyethylene glycol diacrylate is 10% based on the amount of water.

In some of these embodiments, the polyethylene glycol diacrylate is prepared by: dissolving polyethylene glycol in anhydrous toluene at 40 ℃ under nitrogen, cooling to room temperature, adding triethylamine, stirring uniformly, then dropwise adding acryloyl chloride, stirring for reaction, precipitating the obtained polymer in n-hexane, purifying the precipitate, performing water-soluble dialysis, and freeze-drying. Preferably, polyethylene glycol: anhydrous toluene: triethylamine: acryloyl chloride = 8g:40mL:1.19mL:0.81mL.

The hydrogel solutions I, II and III of the present invention may further contain a photoinitiator, lemon yellow, etc. as required.

The invention also provides a preparation method of the conductive microneedle patch, wherein the conductive hydrogel microneedle patch can be prepared by 3D printing and curing, and the printing sequence is as follows: the anti-fouling layer, the adhesive layer and the microneedle array are immersed in water to remove completely polymerized monomers after printing is finished, and the conductive microneedle patch is obtained after drying and demolding.

The beneficial effects of the invention are as follows:

The invention designs the drug-loaded microneedle patch for myocardial repair, wherein the needle tip is loaded with drugs, the back lining is provided with the conductive adhesive layer and the anti-fouling layer, so that the drug-loaded microneedle patch can not only provide mechanical support for heart damaged tissues and effectively ensure the drug delivery depth, but also be well adhered to the tissue surface to promote the electric signal conduction between myocardial cells, and simultaneously avoid adhesion between the microneedle patch and other tissues and organs in an in-vivo environment, has good repairing effect, and can be applied to the in-vivo environment.

Drawings

FIG. 1 is a schematic diagram of a conductive hydrogel microneedle patch for myocardial repair according to the present invention;

FIG. 2 is a physical diagram of a conductive hydrogel microneedle patch for myocardial repair according to the present invention;

FIG. 3 is a graph showing the results of the antioxidant capacity analysis of a microneedle patch according to an embodiment of the present invention;

Fig. 4 is a graph showing the evaluation result of the anti-inflammatory effect of the microneedle patch according to the embodiment of the present invention.

Detailed Description

The invention provides a conductive hydrogel microneedle patch for myocardial repair, the structure of which is shown in figure 1, and the conductive hydrogel microneedle patch comprises a back lining and a microneedle array, wherein the microneedle array is formed by solidifying a hydrogel solution I, and the hydrogel solution I comprises methacryloyl dextran (DexMA), epigallocatechin gallate (EGCG) and basic fibroblast growth factor (bFGF); that is, EGCG and bFGF are introduced into DexMA hydrogel system, and bFGF can promote the proliferation and migration of vascular endothelial cells, induce the transformation of peripheral fibroblasts into vascular endothelial cells and promote angiogenesis. Secondly, EGCG has various biological activities including antioxidation, anti-inflammatory, anti-tumor, anti-aging and the like, is helpful for reducing oxidative stress and inflammatory reaction in cells and promoting repair and regeneration of cardiac muscle.

The back lining comprises an anti-fouling layer and an adhesion layer, the adhesion layer is connected with the microneedle array and is positioned at the inner side of the back lining, the hydrogel solution II is solidified to form the anti-fouling agent, polyethylene glycol diacrylate (ACG) and reduced graphene oxide (rGO) are contained in the hydrogel solution II, rGO has excellent conductivity, a stable conductive network can be formed by dispersing the anti-fouling agent in ACG gel, a continuous conductive path is formed, the anti-fouling agent can be used as a conductive support of a patch, electric signal conduction between myocardial cells is promoted, and normal functions of a heart are facilitated. In addition, the two-dimensional structure and the high specific surface area of the graphene oxide enable the graphene oxide to have good mechanical supporting performance, and the stability and the structural strength of the cardiac patch can be enhanced.

The anti-fouling layer is positioned on the outer side of the back lining and is formed by solidifying hydrogel solution III, and the hydrogel solution III comprises polyethylene glycol diacrylate (PEGDA) so as to avoid adhesion of the microneedle patch and other tissues and organs in an in-vivo environment.

The micro-needle structure of the embodiment of the invention is mutually matched and supported, not only can provide mechanical support for heart injury tissues, effectively ensure the drug delivery depth, but also can be well adhered to the tissue surface to promote the electric signal conduction between myocardial cells, and simultaneously avoid adhesion between the micro-needle patch and other tissues and organs in an in-vivo environment, thereby having good heart repairing effect.

In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are given below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The reagents, materials and equipment used in the embodiment of the invention are all commercially available sources unless specified; the test methods are conventional in the art unless otherwise specified.

Preparation of raw materials

(1) Preparation of methacryloylated dextran (DexMA)

First, 5.0g of dextran was weighed, added to 50mL of dimethyl sulfoxide (DMSO), stirred under magnetic stirring for 1h, and dissolved to a clear pale yellow homogeneous solution. Subsequently, 1g of 4-Dimethylaminopyridine (DMAP) was added to the dextran solution, and stirring was continued for 0.5 to dissolve to a clear pale green uniform solution; subsequently, 1.5mL Glycidyl Methacrylate (GMA) was added and stirred for 48h in the dark; finally, the reaction solution was transferred to a 14000Da dialysis bag placed in a cellulose dialysis bag having a molecular weight cut-off of 14000Da, and dialyzed in deionized water for 3 days to remove by-products. The dialyzed reaction solution was freeze-dried for 48 hours to obtain methacryloylated dextran (DexMA).

(2) Preparation of Graphene Oxide (GO)

Firstly, 2g of graphite is weighed, 25mL of concentrated sulfuric acid is added (stirring is carried out under a low-temperature ice bath), 7g of potassium permanganate powder is added in batches in 2h, the mixture is slowly stirred to be fully and uniformly mixed, then the mixture is continuously reacted for 1h under a water bath at 37 ℃, and then the mixture is stirred for 24h at 25 ℃. Subsequently, 120mL of deionized water was slowly added to the reaction solution, at which time the temperature of the reaction solution was instantaneously raised and a large amount of gas was generated, and then the water bath apparatus was heated to 90 c to allow the reaction to proceed for 30 minutes, and finally 200mL of deionized water was added to dilute the reaction solution. After the reaction was terminated, the reaction mixture was naturally cooled to room temperature, and a 30% hydrogen peroxide solution was added until no bubbles were generated. After the reaction liquid is cooled, 10% dilute hydrochloric acid reacts with the barium chloride solution, and the reaction liquid is repeatedly centrifuged until no sediment is generated, and is continuously and completely washed by deionized water until the reaction liquid is neutral. Finally, the reaction solution was freeze-dried in vacuo for 24 hours to obtain a brown yellow powder.

(3) Synthesis of reduced graphene oxide (rGO)

Taking 0.05g of graphene oxide powder, adding 50mL of deionized water, stirring for 30min, and performing ultrasonic treatment for 15min to obtain a GO dispersion liquid with the concentration of 1mg/mL for later use. 0.5g of L-ascorbic acid is added into 50mL of reduced graphene oxide suspension with the concentration of 1mg/mL, the mixture is stirred at 90 ℃, the pH value of the suspension is adjusted to 9 by using 25% ammonia water solution, the reaction time is 20min, the mixture is kept stand for 24h at normal temperature, the reduction reaction is thoroughly carried out, reduced graphene oxide dispersion is obtained, and the reaction solution is washed to be neutral by deionized water. Finally, the reaction solution was freeze-dried in vacuo for 48 hours to obtain a blackish brown powder.

(4) Synthesis of polyethylene glycol diacrylate (PEG)

8G of polyethylene glycol (PEG) was dissolved in 40mL of anhydrous toluene at 40℃under nitrogen, cooled to room temperature, 1.19mL of triethylamine was added thereto, and after stirring uniformly, 0.81mL of acryloyl chloride was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. The resulting polymer was precipitated in n-hexane, the precipitate was further purified 3 times by toluene and n-hexane, and then the purified polyethylene glycol diacrylate (PEGDA) was dissolved with deionized water and dialyzed (MW: 1000 Da) for 3 days, centrifuged at 10000rpm for 10 minutes, the impurities were removed, and the supernatant was lyophilized and stored at-20℃until use.

(5) Synthesis of methacryloylated glycine (ACG)

5G of sodium glycinate was dissolved in 6M NaOH solution (25 ml) and stirred under ice bath for 20min. 5.5ml of acryloyl chloride was added to 10ml of tetrahydrofuran, and after stirring, the solution was slowly added dropwise thereto for 2 hours, and after the completion of the dropwise addition, the ice bath was continued for 1 hour. The resulting solution was added with 10ml of water, washed three times with diethyl ether to remove impurities, the pH of the aqueous phase was adjusted to 2, saturated with NaCl and finally extracted multiple times with ethyl acetate. After drying and concentration, the obtained product was dissolved in 10ml of water and lyophilized to obtain ACG.

Microneedle patch preparation

Table 1 raw material compositions of the different examples and comparative examples

Example 1

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution III: 2g of PEGDA was dissolved in 20mL of deionized water to give a 10% PEGDA solution, and 12mg of lemon yellow and 20mg of LAP photoinitiator were added to give hydrogel solution III for curing to prepare an anti-fouling layer.

Hydrogel solution II: 100mg of reduced graphene oxide was added to 20mL of deionized water to obtain an aqueous dispersion of reduced graphene oxide having a dispersion concentration of 5 mg/mL. The solution was sonicated for 1h to give a stable and uniform reduced graphene oxide/water dispersion, then 4g of ACG was added to 20mL of the reduced graphene oxide water dispersion, and 12mg of lemon yellow and 20mg of LAP photoinitiator were added to give hydrogel solution II for curing to prepare an adhesive layer.

Hydrogel solution I: 1mg of EGCG and 300mg of DexMA are dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.01mg of bFGF is added to EGCG/DexMA solution, then 0.6mg of lemon yellow and 1mg of photoinitiator are added to obtain hydrogel solution I, and the hydrogel solution I is used for curing to prepare a microneedle array.

S2 3D curing printing

The prepared hydrogel solution is sequentially added into a trough, the printing sequence is an anti-fouling layer, an adhesive layer and a microneedle array, and after each layer of printing is finished, new solution is added into the trough.

Curing conditions of the outer anti-fouling layer as microneedle backing: the light intensity was 15mW/cm ², the exposure time was 15s, and the base layer exposure time was 17s.

Curing conditions of the inner adhesive layer as microneedle backing: the light intensity was 16mW/cm ², the exposure time was 16s, and the base layer exposure time was 18s.

Curing conditions of microneedle arrays: the light intensity was 13mW/cm ², the exposure time was 16s, and the base layer exposure time was 16s.

After printing was completed, the mixture was immersed in water for 5 minutes to remove incompletely polymerized monomers. And drying and demolding to obtain the conductive microneedle patch.

Example 2

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 10mg of EGCG and 300mg of DexMA were dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.01mg of bFGF was added to EGCG/DexMA solution, and then 0.06wt% of lemon yellow and 0.1wt% of LAP photoinitiator were added to obtain hydrogel solution I for curing preparation of microneedle array.

S2 3D curing printing

Curing conditions of microneedle arrays: the light intensity was 13mW/cm ², the exposure time was 16s, and the base layer exposure time was 16s. After printing was completed, the mixture was immersed in water for 5 minutes to remove incompletely polymerized monomers. And drying and demolding to obtain the conductive microneedle patch.

Example 3

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 1mg of EGCG and 100mg of DexMA were dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.001mg of bFGF was added to EGCG/DexMA solution, and then 0.06wt% of lemon yellow and 0.1wt% of LAP photoinitiator were added to obtain hydrogel solution I for curing preparation of microneedle array.

S2 3D curing printing

Example 4

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 5mg of EGCG and 200mg of DexMA% were dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.005mg of bFGF was added to EGCG/DexMA solution, and then 0.06% by weight of lemon yellow and 0.1% by weight of LAP photoinitiator were added to obtain hydrogel solution I for curing preparation of microneedle array.

S2 3D curing printing

Example 5

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 10mg of EGCG and 300mg of DexMA were dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.001mg of bFGF was added to EGCG/DexMA solution, and then 0.06wt% of lemon yellow and 0.1wt% of LAP photoinitiator were added to obtain hydrogel solution I for curing preparation of microneedle array.

S2 3D curing printing

Example 6

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 5mg of EGCG and 300mg of DexMA% were dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.01mg of bFGF was added to EGCG/DexMA solution, and then 0.06% by weight of lemon yellow and 0.1% by weight of LAP photoinitiator were added to obtain hydrogel solution I for curing preparation of microneedle array.

S2 3D curing printing

Comparative example 1

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 10mg of EGCG and 300mg of DexMA were dissolved in 1mL of deionized water to form EGCG/DexMA solution, and then 0.06wt% of lemon yellow and 0.1wt% of LAP photoinitiator were added to obtain hydrogel solution I for curing to prepare a microneedle array.

S2 3D curing printing

Comparative example 2

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution I: 0.01mg of bFGF and 300mg of DexMA were dissolved in 1mL of deionized water to form a bFGF/DexMA solution, and then 0.06wt% of lemon yellow and 0.1wt% of LAP photoinitiator were added to obtain hydrogel solution I for curing to prepare a microneedle array.

S2 3D curing printing

Comparative example 3

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution II: 4g of ACG was added to 20mL of deionized water, and 12mg of lemon yellow and 20mg of LAP photoinitiator were added to give hydrogel solution II for curing to prepare an adhesive layer.

Hydrogel solution I:10 mg of EGCG and 300mg of DexMA are dissolved in 1mL of deionized water to form EGCG/DexMA solution, 0.01mg of bFGF is added to EGCG/DexMA solution, then 0.6mg of lemon yellow and 1mg of photoinitiator are added to obtain hydrogel solution I, and the hydrogel solution I is used for curing to prepare a microneedle array.

S2 3D curing printing

Comparative example 4

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution II: 100mg of reduced graphene oxide was added to 20mL of deionized water to obtain an aqueous dispersion of reduced graphene oxide having a dispersion concentration of 5 mg/mL. The solution was sonicated for 1h to give a stable and uniform reduced graphene oxide/water dispersion, then 1g of ACG was added to 20mL of the reduced graphene oxide water dispersion, and 12mg of lemon yellow and 20mg of LAP photoinitiator were added to give hydrogel solution II for curing to prepare an adhesive layer.

S2 3D curing printing

Comparative example 5

A microneedle patch is prepared by the following steps:

s1 preparing hydrogel solution

Hydrogel solution II: 100mg of reduced graphene oxide was added to 20mL of deionized water to obtain an aqueous dispersion of reduced graphene oxide having a dispersion concentration of 5 mg/mL. The solution was sonicated for 1h to give a stable and uniform reduced graphene oxide/water dispersion, then 6g of ACG was added to 20mL of the reduced graphene oxide water dispersion, and 12mg of lemon yellow and 20mg of LAP photoinitiator were added to give hydrogel solution II for curing to prepare an adhesive layer.

S2 3D curing printing

Performance testing

The microneedle patches prepared in examples and comparative examples were subjected to performance test, and the results are as follows.

1. Microneedle appearance

Fig. 2 shows an image of a conductive microneedle patch according to an embodiment of the present invention.

2. Mechanical properties of single needle

The testing method comprises the following steps: and placing the microneedle patch sample on a sample table, enabling the tip to be upward, and adjusting the height of the sample table to enable the upper surface and the lower surface to be in contact with the clamp. Compression at a constant rate of 0.05mm/s, compression displacement (L) and load (P) were recorded until the set displacement end point. The mechanical strength of a single needle is the ratio of the load to the number of needle points.

The test results are shown in Table 3:

TABLE 3 Single needle mechanical Strength value test results for examples and comparative examples

Since myocardial tissue has certain toughness and elasticity, penetration of myocardial tissue requires a certain mechanical strength of the microneedle. From the test results of Table 3, it is shown that the main factor affecting the strength of the tip is the mass concentration of DexMA in the tip, the higher the mass concentration, the higher the mechanical strength of a single needle. The addition of bFGF and EGCG has little effect on the mechanical strength of the needle tip.

3. Conductivity test

The testing method comprises the following steps: the microneedle patches obtained in examples and comparative examples were subjected to conductivity testing with a four-probe conductivity meter, and each set of tests was repeated three times.

Table 4 conductivity test of hydrogel microneedles

From table 4, it can be seen that the microneedle patch without added reduced graphene oxide has a rather low conductivity (comparative example 3), and an appropriate amount of ACG helps to form a stable conductive network, helps rGO to form a continuous conductive path, and improves the conductive performance. Too low or too high an ACG content can affect the conductivity of the patch. This is probably due to the presence of a small amount of oxygen-containing functional groups on the surface of the reduced graphene oxide, large pi bonds on the surface, and improved electron transport, resulting in better conductivity of the reduced graphene oxide itself; in addition, the proper amount of ACG can improve the dispersibility of rGO in a solution or a matrix, prevent rGO sheets from agglomerating, so that the conductivity is maintained or slightly improved, but the dispersibility is possibly insufficient when the content is small, and when the content of ACG is too high, the ACG can excessively cover the surface of rGO, block the electron transmission channels among the rGO sheets, increase the overall resistance of the material and reduce the conductivity. In summary, ACG and rGO interact with each other, and their proportions are adjusted to adjust the conductivity of the material.

The electrical conductivity of the myocardium is typically between 0.1 and 0.6S/m, a range of conductivity sufficient to support the propagation of electrical activity of the heart, ensure efficient conduction of electrical signals, and coordinate the contraction of the heart. It can be seen that the conductivity of the microneedle patches of examples 1-6 of the present invention is similar to that of the myocardium, indicating that it has great potential for modulating the conduction of electrical signals to myocardial tissue.

4. Antioxidant Capacity test

The testing method comprises the following steps: the antioxidant properties of the different examples and comparative examples were evaluated by scavenging 1, 1-diphenyl-2-pyridine hydrazide (DPPH) free radical. The mixture of the different examples and comparative examples with DPPH reagent was incubated in the dark for 30 minutes with stirring and the remaining DPPH was analyzed by uv-vis spectroscopy. The clearance formula for determining DPPH is:

DVC (%) = [ (a blank-a positive control)/a blank ] ×100%

D (%) = [ [ a blank- (a assay-a control) ]/a blank ] ×100%

As can be seen from fig. 3, the efficiency of scavenging free radicals increases with increasing concentration of EGCG, which indicates that EGCG can effectively scavenge free radicals and has good antioxidant effect. In addition, in combination with comparative example 1, it can be seen that bFGF also has a certain effect on the exertion of the oxidation resistance of EGCG, and can promote EGCG to exert its oxidation resistance to a certain extent, and has a certain synergistic effect with EGCG.

5. Evaluation of anti-inflammatory Effect in rats

According to the test results of 1-4, the microneedle patch of example 2 was selected for animal experiments, and the application effect of the microneedle patch of the present invention was evaluated.

The testing method comprises the following steps: rats were anesthetized with 3% sodium pentobarbital (30 mg/kg), anesthetized by intraperitoneal injection, the chest and underarm hairs of the rats were shaved with a small animal shaver (fully exposing the operating field), and sterilized with iodine and 75% ethanol operating field. Tracheal cannula: after anesthesia, MI operation can be performed without reaction in toe clamping detection. The external light source and the microscope switch are turned on, the breathing machine is turned on, various parameters (the breathing ratio is 2:1, the tidal volume is 6-8mL, the frequency is 70 times/min) are set, the tracheal intubation is inserted into the trachea along the glottis, the rat is taken down and connected with the breathing machine, the breathing condition of the rat is observed, and the thoracic cavity fluctuation and the breathing machine frequency are consistent to indicate that the intubation is successful, so that the MI operation can be performed. The rat adopts the right lateral position, cuts in left anterior limb armpit with ophthalmology, opens the thorax fully exposes heart with micro-shearing in three, four intercostals, micro-straight tweezers gently hold a small amount of pericardium and tear a small amount of pericardium in left auricle, fully exposes left coronary anterior descending branch (LAD) or place region. Ligating the coronary arteries: the LAD trend or possible location is found under a microscope, a needle holder holds a 5-0 needle suture, and a 5-0 noninvasive suture is used to pass through the anterior descending branch (LAD) of the left coronary artery below the root of the left heart auricle beside the pulmonary artery so as to completely block the LAD blood flow. Closing chest: after ligation was completed, the microneedle patch of example 2 was attached to myocardial tissue at the infarcted site, the thoracic cavity was closed by completely suturing the thoracic cavity opening with 5-0 sutures (ensuring no gap, no dislocation), and layers of muscle and skin were sutured layer by layer from inside to outside.

Taking a heart tissue sample 3 days after operation, cleaning and removing blood in precooled PBS (0.02 mol/L, pH 7.0-7.2), and weighing for later use; 1.0g of tissue pieces were transferred into a glass homogenizer, and 5mL of pre-chilled PBS was added for sufficient grinding, which was performed on ice; the resulting homogenate was repeatedly freeze-thawed 2 times, and the prepared homogenate was centrifuged at 5000 Xg for 5 minutes, and the supernatant was collected and assayed using ELISA kit.

And simultaneously, setting a myocardial infarction model group and a false operation group for comparison, wherein the comparison result is shown in figure 4.

As can be seen from FIG. 4, compared with the myocardial infarction model group, the micro-needle patch loaded with EGCG and bFGF can effectively reduce the expression levels of inflammatory factors TNF-alpha, IL-6 and IL-beta, and provide a stable microenvironment for myocardial repair.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A conductive hydrogel microneedle patch for myocardial repair, comprising a backing and a microneedle array, characterized in that:

The microneedle array is solidified by a hydrogel solution I, wherein the hydrogel solution I contains methacrylylated dextran, epigallocatechin gallate, and basic fibroblast growth factor;

The backing comprises an adhesive layer and an anti-fouling layer, wherein the adhesive layer is connected to the microneedle array, is located on the inner side of the backing, and is solidified from a hydrogel solution II, wherein the hydrogel solution II comprises methacrylylated glycine and reduced graphene oxide;

The anti-fouling layer is located on the outer side of the backing and is formed by solidifying the hydrogel solution III, wherein the hydrogel solution III includes polyethylene glycol diacrylate.

2. The conductive hydrogel microneedle patch according to claim 1, characterized in that the hydrogel solution I comprises 1 mg/mL-10 mg/mL of epigallocatechin gallate, 100 mg/mL-300 mg/mL of methacryloyl dextran, and 0.001 mg/mL-0.01 mg/mL of basic fibroblast growth factor.

3. The conductive hydrogel microneedle patch according to claim 1, characterized in that the methacryloylated dextran is prepared by the following method:

Dextran, dimethyl sulfoxide and 4-dimethylaminopyridine are mixed and stirred, glycidyl methacrylate is added, stirred, dialyzed and freeze-dried to obtain the methacryloylated dextran.

4. The conductive hydrogel microneedle patch according to claim 1, characterized in that the hydrogel solution II, based on the amount of water, contains 20 wt% methacryloylated glycine and 5 mg/mL reduced graphene oxide.

5. The conductive hydrogel microneedle patch according to claim 1, characterized in that the reduced graphene oxide is prepared by mixing L-ascorbic acid and a graphene oxide aqueous solution at pH = 9 and 90°C.

6. The conductive hydrogel microneedle patch according to claim 5 is characterized in that the graphene oxide is prepared by adding graphite to concentrated sulfuric acid, stirring in a low-temperature ice bath, adding potassium permanganate in batches, mixing well and then stirring in a 37°C water bath, and then stirring at 25°C; then, slowly adding deionized water I to the reaction solution, heating the water bath to 90°C for reaction after the generated gas is released, and finally adding deionized water II to dilute the reaction solution. After the reaction is terminated, it is naturally cooled to room temperature, 30% hydrogen peroxide solution is added until no bubbles are generated, and the reaction solution is left to stand for cooling, and then 10% dilute hydrochloric acid reacts with barium chloride solution, and centrifugation is repeated until no precipitation is generated, and deionized water is continued to be used to wash until the reaction solution is neutral to obtain the graphene oxide.

7. The conductive hydrogel microneedle patch according to claim 1, characterized in that the methacryloylated glycine is prepared by the following method:

Under ice bath conditions, sodium glycine is dissolved in a NaOH solution, and then acryloyl chloride-tetrahydrofuran solution is added dropwise to the above solution for reaction. After the reaction is completed, the solution is washed and impurities are removed, extracted, dried and concentrated, and the obtained product is dissolved in water and freeze-dried to obtain the methacryloylated glycine.

8. The conductive hydrogel microneedle patch according to claim 1, characterized in that the content of polyethylene glycol diacrylate in the hydrogel solution III is 10 wt% based on the amount of water.

9. The conductive hydrogel microneedle patch according to claim 1, characterized in that the polyethylene glycol diacrylate is prepared by the following method:

Polyethylene glycol is dissolved in anhydrous toluene at 40° C. under a nitrogen environment, triethylamine is added after cooling to room temperature, acryloyl chloride is added dropwise after stirring evenly, and the reaction is stirred. Then the obtained polymer is precipitated in n-hexane. After the precipitate is purified, it is dissolved in water, dialyzed, and freeze-dried to obtain the polyethylene glycol diacrylate.

10. The method for preparing a conductive hydrogel microneedle patch according to any one of claims 1 to 9, characterized in that the conductive hydrogel microneedle patch is prepared by 3D printing and curing, and the printing order is: anti-fouling layer, adhesion layer, microneedle array. After printing is completed, the incompletely polymerized monomers are removed by water immersion, and the conductive hydrogel microneedle patch is obtained after drying and demolding.