Extracellular matrix / glycopeptide hydrogel for heart failure treatment and its preparation method

Abstract

The present invention discloses an extracellular matrix / glycopeptide hydrogel for heart failure treatment and its preparation method. The specific components of the hydrogel include cardiomyocyte extracellular matrix, polysaccharide and functional polypeptide. The hydrogel obtained by the present invention has a composition and structure imitating the extracellular matrix, can long-term replace the extracellular matrix of damaged tissues, and provide a microenvironment for cell survival, infiltration and proliferation; the extracellular matrix / glycopeptide hydrogel of the present invention has good injectability, can be directly injected into the myocardium, has good mechanical support characteristics, and has bioactivities of regulating immune inflammatory response and promoting angiogenesis, regulating cardiac remodeling and alleviating the heart failure process.

Description

Technical Field

[0001] The present invention relates to the technical field of research and development of biomedical materials and medical devices, and more particularly to an extracellular matrix / glycopeptide hydrogel for heart failure treatment and a preparation method thereof. Background Art

[0002] Heart failure, abbreviated as HF, is the end state of the development of various cardiovascular diseases including coronary artery disease (such as myocardial infarction), hypertension, myocarditis, and valvular heart disease, and is also a major health problem worldwide, causing serious social and economic burdens. For end-stage HF patients, the main methods to extend survival time are left ventricular assist device and heart transplantation, but both have problems such as high cost, complex surgery, and scarce heart transplantation donor sources. Most end-stage HF patients cannot receive effective treatment, and there is an urgent need to explore new treatment methods.

[0003] In recent years, with the development of material science and tissue engineering, new biomaterials have been widely used in the field of cardiovascular treatment. Representative biomaterials among them are extracellular matrix and polysaccharide hydrogels. The extracellular matrix hydrogel retains the original active components such as collagen, elastin, glycoprotein, and mucopolysaccharide in the cardiac extracellular matrix and the porous fibrous network structure, and can replace the abnormally degraded extracellular matrix after myocardial infarction, providing a three-dimensional framework for cell adhesion and differentiation. However, the mechanical stability and strength of the extracellular matrix hydrogel are relatively low, and it cannot provide sufficient mechanical support strength for the thinned ventricular wall after myocardial infarction. At the same time, the extracellular matrix hydrogel is still easily degraded rapidly by the matrix metalloproteinases continuously secreted by inflammatory cells after myocardial injury. In previous clinical trials, the extracellular matrix hydrogel failed to show effective ventricular remodeling efficacy. Polysaccharide hydrogels such as sodium alginate and hyaluronic acid hydrogel have good mechanical stability, injectability, and biocompatibility, and can reduce the myocardial wall tension after intramyocardial injection. However, the polysaccharide-based hydrogel has a relatively single structural component and lacks rich and special active components in the heart tissue, and cannot fully regulate the complex cell differentiation process, and also fails to improve the cardiac function and survival rate of patients in clinical trials.

[0004] Therefore, the currently used hydrogels cannot meet the treatment needs of heart failure, and the results in clinical trials are still not satisfactory. Based on this, developing a new type of multifunctional hydrogel that can meet the requirements of remodeling the cardiac extracellular matrix, providing mechanical support, regulating the inflammatory response, promoting angiogenesis, and improving the cardiac function and survival rate of HF patients has very important clinical significance. Summary of the Invention

[0005] In view of the above deficiencies of the prior art, the present invention provides a method for preparing an extracellular matrix / glycopeptide hydrogel for the treatment of heart failure. The obtained hydrogel not only has the composition and structure of an extracellular matrix mimicking cells, so as to replace the extracellular matrix of damaged tissues in the long term, reduce the ventricular wall stress and provide a favorable microenvironment, but also has the biological activities of regulating the inflammatory response and promoting angiogenesis, thereby inhibiting the progression of heart failure.

[0006] To achieve the above object, the object of the present invention can be realized by the following technical solutions:

[0007] An extracellular matrix / glycopeptide hydrogel for the treatment of heart failure, the main components of which are cardiac acellular matrix, polysaccharide and functional polypeptide, wherein the mass fraction of the cardiac acellular matrix in the total solid content of the system is 10-50%; the mass fraction of the polypeptide in the total solid content of the system is 0.5-20%; the mass fraction of the polysaccharide in the total solid content of the system is 10-90%.

[0008] Preferably, the source of the cardiac acellular matrix includes pigs, cows, sheep, mice, rabbits or transplanted organs.

[0009] Preferably, the polysaccharide is one or more of alginic acid, chitosan, carboxymethyl cellulose, dextran, hyaluronic acid, chondroitin sulfate and heparin.

[0010] Preferably, the polypeptide is one or more of Q11 polypeptide (QQKFQFQFEQQ), RADA polypeptide (RADARADARADARADA), KK polypeptide (KKSLSLSLSLSLSLKK), KEF9 polypeptide (KKFKFEFEF).

[0011] Another object of the present invention is to provide a method for preparing the above extracellular matrix / glycopeptide hydrogel for the treatment of heart failure, comprising the following steps:

[0012] (1) Prepare an extracellular matrix hydrogel: Cut fresh heart tissue into pieces, stir in sodium dodecyl sulfate (SDS) with a concentration of 0.1-3 wt% at a speed of 20-100 r / min for 5-10 days until the heart tissue becomes white and transparent, completing the decellularization process. Continue to stir the decellularized tissue in deionized water for 2-3 days to thoroughly remove the residual SDS, freeze-dry and grind it into powder. Dissolve the extracellular matrix powder in 0.1-1 M hydrochloric acid solution (the pH of the hydrochloric acid solution is 1-2) and digest it with pepsin for 12-72 hours. Titrate with 1-10 M sodium hydroxide solution until the pH value of the extracellular matrix solution is 7-8 to terminate the digestion, and let it stand to form an extracellular matrix hydrogel;

[0013] (2) Preparation of maleimide-polysaccharide: Dissolve polysaccharide with a concentration of 1-10 wt% in dimethyl sulfoxide, add maleimide butyric acid or maleimide propionic acid, and react for 48-72 h under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP). The maleimide-glucomannan powder is obtained through dialysis and lyophilization.

[0014] (3) Preparation of extracellular matrix / glycopeptide hydrogel: Under sterile conditions, mix the polypeptide solution, extracellular matrix hydrogel solution and maleimide-polysaccharide solution, stir evenly, crosslink through Michael addition reaction, and stand for 5-60 min to form the glycopeptide hydrogel.

[0015] Preferably, in step (1), the mass ratio of the powdered extracellular matrix to pepsin is 5-20:1 to obtain extracellular matrix degradation products with appropriate molecular weight.

[0016] Preferably, in step (1), the concentration of the dissolved extracellular matrix powder is 10-100 mg / mL to obtain a gel precursor solution with moderate viscosity.

[0017] Preferably, in step (2), the molar ratio of the polysaccharide to maleimide butyric acid or maleimide propionic acid is 1:1-100, the molar ratio to EDC is 1:1-10; and the molar ratio to DMAP is 1:1-10.

[0018] Preferably, the final solid content of the hydrogel is 5-50% to meet the injectability and mechanical strength requirements of the hydrogel for heart failure treatment.

[0019] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0020] The glycopeptide hydrogel composite with extracellular matrix of the present invention can retain the three-dimensional network structure and active components of the cardiac extracellular matrix. At the same time, glycopeptide components are introduced through chemical crosslinking to enhance the mechanical stability of the hydrogel and endow it with special biological functions of immune regulation and promoting angiogenesis, so as to regulate the myocardial remodeling process after myocardial infarction in multiple aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0022] Figure 1 Flow chart for the preparation of decellularized extracellular matrix (dECM)

[0023] Figure 2 For glucomannan (GM) and glucomannan conjugated with maleimide groups (MAL-GM) 1 1H-NMR spectrum

[0024] Figure 3 For the rheological properties of extracellular matrix / glycopeptide hydrogel (dECM / GP), extracellular matrix hydrogel (dECM) and glycopeptide hydrogel (GP)

[0025] Figure 4 For the scanning electron microscopy microstructure of decellularized extracellular matrix and extracellular matrix / glycopeptide hydrogel

[0026] Figure 5 For the Masson staining results of extracellular matrix / glycopeptide hydrogel (dECM / GP), extracellular matrix hydrogel (dECM) and glycopeptide hydrogel (GP) in treating rats with acute myocardial infarction

[0027] Figure 6 For the immunofluorescence staining results of CD68 / CD206 in the myocardial tissue of the infarcted area after 28 days of treatment

[0028] Figure 7 For the immunofluorescence staining results of α-SMA / cTNT in the myocardial tissue of the infarcted area after 28 days of treatment. Detailed implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] Select fresh porcine left ventricular free wall tissue and cut it into small pieces of 2 mm 3 , put them into 1 wt% SDS solution and stir rapidly for 5 days. During this period, replace the SDS solution every other day until the tissue becomes transparent. After decellularization, the tissue is put into deionized water and stirred for 2 days. During this period, continuously replace the deionized water to remove the residual SDS. Freeze-dry the tissue for 48 h, grind it into powder with a cryogenic grinder, dissolve the extracellular matrix powder at 50 mg / mL in 0.1 M hydrochloric acid solution, add porcine pepsin at a mass ratio of 10:1, titrate with 1 M sodium hydroxide solution after digestion for 24 h to make the pH value of the solution 7.5, and let it stand to form an extracellular matrix hydrogel.

[0032] Dissolve 2 g of hyaluronic acid (20 kDa) in 100 mL of deionized water, add 2.2 g of maleimidobutyric acid, 7 g of EDC and 0.5 g of DMAP, react at room temperature for 48 h, dialyze the obtained product for 5 days, and freeze-dry for 48 h to obtain maleimide-modified hyaluronic acid, which is dissolved at a concentration of 8 wt% to form a polysaccharide solution.

[0033] Prepare a solution of the angiogenesis-promoting polypeptide KK at a concentration of 4 wt%. Mix the extracellular matrix hydrogel (5 wt%), the polysaccharide solution (8 wt%), and the polypeptide solution in a volume ratio of 2:1:1, vortex thoroughly and then let stand, and crosslink through Michael addition reaction to form an extracellular matrix / glycopeptide hydrogel.

[0034] Example 2

[0035] Select fresh porcine left ventricular free wall tissue and cut it into small pieces of 22 mm 3 Put it into 1 wt% SDS solution and stir rapidly for 5 days, changing the SDS solution every other day until the tissue becomes transparent. Put the decellularized tissue into deionized water and stir for 2 days, continuously changing the deionized water during this period to remove the residual SDS. Freeze-dry the tissue for 48 h and grind it into powder with a cryogenic grinder. Dissolve the extracellular matrix powder at 20 mg / mL in 0.1 M hydrochloric acid solution, add porcine pepsin in a mass ratio of 10:1, digest for 24 h, and then titrate with 1 M sodium hydroxide solution to make the pH value of the solution 7.5, and let it stand to form an extracellular matrix hydrogel. Figure 1 The preparation process of the extracellular matrix hydrogel is shown as follows.

[0036] Dissolve 2 g of glucan (100 kDa) in 100 mL of deionized water, add 2.2 g of maleimidobutyric acid, 7 g of EDC and 0.5 g of DMAP, react at room temperature for 48 h, dialyze the obtained product for 5 days, and freeze-dry for 48 h, and dissolve it at a concentration of 10% to form a polysaccharide hydrogel. Figure 2 Shown is the 1 1H-NMR of maleimide-modified glucan, proving the successful synthesis of glucan grafted with maleimide groups.

[0037] Prepare a solution of the angiogenesis-promoting polypeptide RADA at a concentration of 2 wt%. Mix the extracellular matrix hydrogel (2 wt%), the polysaccharide (10 wt%), and the polypeptide (2 wt%) in a volume ratio of 1:1:1, vortex thoroughly and then let stand, and crosslink through Michael addition reaction to form an extracellular matrix / glycopeptide hydrogel.

[0038] Example 3

[0039] Rheological tests were performed on the extracellular matrix / glycopeptide hydrogel, extracellular matrix hydrogel, and glycopeptide hydrogel prepared in Example 2. The glycopeptide hydrogel used was the product of mixing the polysaccharide solution and the polypeptide solution in Example 1 at a volume ratio of 1:1.

[0040] Figure 3 The test results showed that the extracellular matrix / glycopeptide hydrogel had higher storage modulus (G′) and loss modulus (G″) than the extracellular matrix hydrogel and glycopeptide hydrogel alone, demonstrating its better mechanical properties.

[0041] Figure 4 The three-dimensional microscopic structure diagrams of porcine acellular extracellular matrix and the extracellular matrix / glycopeptide hydrogel obtained in Example 1 were shown. It could be seen from the figure that the prepared extracellular matrix / glycopeptide hydrogel had a three-dimensional porous nanofiber network structure similar to that of the acellular extracellular matrix, thus being expected to replace the extracellular matrix of damaged tissues and provide a favorable microenvironment for cell growth, proliferation, and infiltration.

[0042] Example 4

[0043] To investigate the therapeutic effect of the extracellular matrix / glycopeptide hydrogel on heart failure, the extracellular matrix / glycopeptide hydrogel, extracellular matrix hydrogel, glycopeptide hydrogel prepared in Example 3 were used for animal experiments. Sprague Dawley (SD) rats at 6 weeks of age with a body weight between 180 - 200 g were used to prepare an acute myocardial infarction model. After anesthesia, tracheal intubation was performed, and the ventilator parameters were adjusted. The rats were fixed on the operating board, and a longitudinal incision about 2 cm long was made 2 mm to the left of the sternum. The skin and fascia were incised successively, and the pectoralis major and serratus anterior muscles were bluntly separated using a hemostat. A transverse incision about 1 cm long was made parallel to the ribs between the 3rd and 4th intercostals, and the intercostals were retracted to expose the heart. The pericardium was torn open using forceps to fully expose the heart structure. A 6-0 suture was used to ligate the left anterior descending coronary artery 2 - 3 mm below the midpoint of the line connecting the arterial cone and the left atrial appendage. After ligation, it could be observed that the color of the anterior wall myocardium of the left ventricle became pale and the wall motion weakened, indicating successful ligation. After 30 min of ligation and ischemia, 4 evenly spaced injection points were taken in the infarct border zone, and 25 μl of the extracellular matrix / glycopeptide hydrogel, extracellular matrix hydrogel, glycopeptide hydrogel, or PBS solution was injected at each point, totaling 100 μl. After injection, sterile gauze was used to stop bleeding thoroughly. The chest cavity was closed layer by layer using interrupted sutures with 3-0 sutures, and thoracic cavity decompression was performed to avoid pneumothorax. The serratus anterior and pectoralis major muscles were approximated, and the skin was sutured. The tracheal intubation was removed after the rats resumed spontaneous breathing. Penicillin 200,000 U / rat was injected intraperitoneally to prevent infection.

[0044] Figure 5These are the results of Masson staining of heart sections at 7 days and 28 days after surgery. The results showed that, compared with other groups, the extracellular matrix / glycopeptide hydrogel could significantly reduce the infarct area, and the wall thickness at the injury site was thicker. In addition, the Masson staining results at 28 days also indicated that extensive and dense collagen deposition was observed to varying degrees in other treatment groups, indicating pathological changes in heart failure, while the extracellular matrix / glycopeptide hydrogel group effectively inhibited this adverse ventricular remodeling process.

[0045] Example 5

[0046] Through immunofluorescence staining, the matrix of the extracellular matrix / glycopeptide hydrogel regulating heart remodeling and healing was investigated. For macrophage polarization studies, the sections were incubated with rabbit anti-CD68 (1:200), mouse anti-CD206 (1:200), mouse anti-α-SMA (1:200), and rabbit anti-cardiac troponin T (1:200) respectively. After overnight incubation, the sections were incubated with AlexaFlour488 donkey anti-mouse IgG (1:200) and AlexaFlour594 donkey anti-rabbit IgG (1:200) corresponding to the primary antibodies for 2 h. After washing with PBS, the sections were mounted with a mounting medium containing DAPI and observed and analyzed by confocal microscopy.

[0047] Figure 6 The results showed that there was less infiltration of inflammatory cells in the myocardial tissue of the extracellular matrix / glycopeptide hydrogel group, and there was a greater distribution of M2 macrophages, indicating that the extracellular matrix / glycopeptide hydrogel could effectively promote the polarization of M2 macrophages and reduce the inflammatory response after myocardial injury. Figure 7 The results showed that there was more α-SMA+ angiogenesis in the myocardial tissue of the extracellular matrix / glycopeptide hydrogel group and more myocardial tissue appeared around the newly formed blood vessels, demonstrating that the extracellular matrix / glycopeptide hydrogel could promote angiogenesis and cardiomyocyte survival, thereby inhibiting the progression of heart failure.

[0048] In this specification, each example is described in a progressive manner. The key point of each example is to illustrate the differences from other examples. The same or similar parts among the examples can be referred to each other. For the devices disclosed in the examples, since they correspond to the methods disclosed in the examples, the description is relatively simple, and the relevant parts can be referred to the description of the method part.

[0049] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An extracellular matrix / glycopeptide hydrogel for the treatment of heart failure, characterized in that: The hydrogel comprises a cardiac acellular matrix, a polysaccharide, and a functional polypeptide; Among them, the mass fraction of the cardiac acellular matrix in the total solid content of the system is 10-50%; the mass fraction of the functional polypeptide in the total solid content of the hydrogel system is 0.5-20%; the mass fraction of the polysaccharide in the total solid content of the system is 10-90%; The polypeptide is one or more of Q11 polypeptide, RADA polypeptide, and KK polypeptide; The solid content of the hydrogel is 5-50%; The preparation method of the extracellular matrix / glycopeptide hydrogel for heart failure treatment comprises the following steps: (1) Prepare an extracellular matrix hydrogel: Cut the cardiac tissue into small pieces, add it to sodium dodecyl sulfate and stir until the cardiac tissue becomes white and transparent, completing the decellularization process. Continue to stir the decellularized tissue in water to remove the residual sodium dodecyl sulfate, then freeze-dry and grind it into powder. Dissolve the extracellular matrix powder in hydrochloric acid solution with a pH of 1-2 and digest it with pepsin for 12-72 h, then titrate with sodium hydroxide solution until the pH value of the extracellular matrix solution reaches 7-8 to terminate digestion, and let it stand to form an extracellular matrix hydrogel; (2) Prepare maleimide-polysaccharide: Dissolve the polysaccharide in dimethyl sulfoxide, add maleimide butyric acid or maleimide propionic acid, and react under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine for 48-72 h. Obtain maleimide-polysaccharide powder through dialysis and freeze-drying; (3) Prepare an extracellular matrix / glycopeptide hydrogel: Under sterile conditions, mix the polypeptide solution, the extracellular matrix hydrogel solution, and the maleimide-polysaccharide solution, stir evenly, and crosslink through a Michael addition reaction. Let it stand for 5-60 min to form a glycopeptide hydrogel.

2. The extracellular matrix / glycopeptide hydrogel for heart failure treatment according to claim 1, wherein: The polysaccharide is one or more of alginic acid, chitosan, carboxymethyl cellulose, dextran, hyaluronic acid, chondroitin sulfate, and heparin.

3. The extracellular matrix / glycopeptide hydrogel for heart failure treatment according to claim 1, characterized in that, In step (1), the mass concentration of the sodium dodecyl sulfate is 0.1-3 wt%; the concentration of the hydrochloric acid solution is 0.1-1 M; the concentration of the sodium hydroxide solution is 1-10 M; the stirring speed is 20-100 r / min.

4. The extracellular matrix / glycopeptide hydrogel for heart failure treatment according to claim 1, wherein In step (1), the mass ratio of the powdered extracellular matrix to pepsin is 5-20:

1.

5. The extracellular matrix / glycopeptide hydrogel for heart failure treatment according to claim 1, characterized in that, In step (1), the dissolution concentration of the extracellular matrix powder is 10-100 mg / mL.

6. The extracellular matrix / glycopeptide hydrogel for heart failure treatment according to claim 1, wherein, In step (2), the mass concentration of the polysaccharide solution is 1-10 wt%.

7. The extracellular matrix / glycopeptide hydrogel for heart failure treatment according to claim 1, wherein In step (2), the molar ratio of the polysaccharide to maleimide butyric acid or maleimide propionic acid is 1:1-100; the molar ratio of the polysaccharide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1-10; the molar ratio of the polysaccharide to 4-dimethylaminopyridine is 1:1-10.

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