A biological valve with anti-inflammatory and endothelialization-promoting functions and a preparation method thereof

Abstract

The present invention discloses a biological valve with anti-inflammatory and endothelialization promoting functions and a preparation method thereof, and relates to the technical field of biomedical materials. The preparation method comprises the following steps: placing the valve material in a glutaraldehyde solution for cross-linking for 24-120 hours, taking it out and washing it with distilled water; soaking the valve material in a polyethyleneimine solution for reaction for 6-72 hours, taking it out and washing it with distilled water; soaking the valve material in a phospholipid polyethylene glycol active lipid solution for reaction for 6-72 hours, taking it out and washing it with a phosphate buffer, and sterilizing it; soaking the valve material in an exosome solution for reaction for 6-72 hours, taking it out and washing it with a sterile phosphate buffer. The present invention also discloses a biological valve with anti-inflammatory and endothelialization promoting functions obtained by the above method. The biological valve has good biocompatibility, long-term anti-inflammatory and endothelialization promoting capabilities, effectively improves the valve performance, and solves the problems of biological valve calcification and biological toxicity.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a biological valve with anti-inflammatory and endothelialization promoting functions and a preparation method thereof. Background Art

[0002] In recent years, the incidence and mortality of cardiovascular and cerebrovascular diseases have remained high worldwide, among which valvular heart disease has seriously threatened the lives and health of residents. Surgical open-chest valve replacement surgery and transcatheter minimally invasive valve replacement surgery are common treatments for valvular heart disease. However, whether the diseased valve is replaced by surgery or minimally invasive interventional surgery, patients need to receive anticoagulant drugs to reduce the incidence of adverse events. Among them, mechanical valves are commonly used in surgery, and patients need to take anticoagulants for life. In recent years, minimally invasive interventional surgery has gradually become the first choice for valve replacement due to its advantages of less trauma and faster recovery. The biological valves used clinically are mainly made of xenogeneic tissue (such as porcine pericardium or bovine pericardium) cross-linked with glutaraldehyde. However, due to the toxicity of residual aldehyde, biological valves are cytotoxic and may induce inflammatory reactions after implantation, leading to thrombosis and calcification, ultimately shortening their service life and increasing the risk of patients undergoing valve replacement surgery again. In recent years, a lot of research has been carried out on these issues. Among them, rapid endothelialization and anti-inflammation are considered to be the most effective strategies, because the existence of the endothelium and long-term anti-inflammation play a key role in preventing bioprosthetic valve thrombosis and calcification, and are expected to improve the durability of bioprosthetic valves.

[0003] Recent studies have focused on modifying bioprosthetic valves with small molecules or polymers to improve their endothelialization and anti-inflammatory properties. However, these small molecules or polymers fixed on bioprosthetic valves are often short-lived, and bioprosthetic valves may also face the risk of thrombosis. Modifying artificial bioprosthetic valves without affecting other properties of bioprosthetic valves has very important clinical significance. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a biological valve with anti-inflammatory and pro-endothelialization functions and a preparation method thereof. The biological valve has good biocompatibility, long-term anti-inflammatory and pro-endothelialization capabilities, effectively improves the valve performance, and solves the problems of biological valve calcification and biological toxicity.

[0005] The technical solution of the present invention to solve the above technical problem is as follows: a method for preparing a biological valve with anti-inflammatory and endothelialization promoting functions is provided, comprising the following steps:

[0006] (1) Place the valve material in glutaraldehyde solution and cross-link it at 4-37°C for 24-120 h, then remove it and wash it with distilled water;

[0007] (2) soaking the valve material treated in step (1) in a polyethyleneimine solution for 6-72 hours, then taking it out and washing it with distilled water;

[0008] (3) Soaking the valve material treated in step (2) in a phospholipid polyethylene glycol active lipid solution for 6-72 hours, taking it out, washing it with phosphate buffer, and then sterilizing it;

[0009] (4) The valve material treated in step (3) is immersed in the exosome solution for reaction for 6-72 hours, and then taken out and washed with sterile phosphate buffer to obtain a biological valve with anti-inflammatory and endothelial-promoting functions.

[0010] Furthermore, in step (1), the valve material is porcine pericardium or bovine pericardium.

[0011] Furthermore, in step (1), the concentration of the glutaraldehyde solution is 0.5-8 wt %, and the pH value is 2-9.

[0012] Furthermore, in step (2), the concentration of the polyethyleneimine solution is 0.1-50 mg / mL, and the pH value is 2-8.

[0013] Furthermore, in step (3), the concentration of the phospholipid polyethylene glycol active lipid solution is 0.1-50 mg / mL, and the pH value is 2-8.

[0014] Furthermore, in step (4), the concentration of the exosome solution is 10 4 -10 14 / mL, pH value is 2-8, wherein the exosomes are derived from at least one of vascular endothelial cells, endothelial progenitor cells and macrophages.

[0015] Furthermore, the exosomes were derived from human umbilical vein endothelial cells.

[0016] Furthermore, exosomes were derived from macrophage subtypes with an anti-inflammatory phenotype.

[0017] The present invention also provides a biological valve with anti-inflammatory and endothelialization promoting functions obtained by the above-mentioned method for preparing the biological valve with anti-inflammatory and endothelialization promoting functions.

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

[0019] Exosomes are extracellular vesicles with a size of 30-150 nanometers, which are widely present in various tissues and biological fluids such as blood, milk and urine. Exosomes contain many functional biomolecules, including proteins, nucleic acids and lipids. Exosomes mediate cell-to-cell communication by transferring these signal molecules to receptor cells. Exosomes from different cell sources can play corresponding functions. For example, exosomes from macrophages have inherent inflammatory targeting properties, which are attributed to various chemokine receptors on their surfaces. The present invention introduces exosomes from different sources into glutaraldehyde-crosslinked bioprostheses by chemical grafting, and achieves better endothelialization and anti-inflammatory effects on the basis of improving biocompatibility. Compared with existing small molecule and polymer modified bioprostheses, this biomodified bioprostheses has better biocompatibility, long-term anti-inflammatory and endothelialization capabilities, and effectively improves the performance of valve use. In addition, the bioprostheses provided by the present invention can effectively seal the residual aldehyde groups on the bioprostheses, effectively reducing the calcification and biotoxicity problems of the bioprostheses. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Transmission electron microscopy observation of exosomes;

[0021] Figure 2 This is a scanning electron microscope observation of a bioprosthetic valve with anti-inflammatory and pro-endothelialization functions;

[0022] Figure 3 The fluorescence staining results of the biological valves of Comparative Example 1 and Example 1 are shown;

[0023] Figure 4 The graph shows the platelet adhesion results of the biological valves of Comparative Example 1 and Example 1. DETAILED DESCRIPTION

[0024] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0025] Example 1

[0026] A bioprosthetic valve with anti-inflammatory and endothelialization promoting functions, the preparation method of which comprises the following steps:

[0027] (1) The porcine pericardium was placed in a 1 wt% glutaraldehyde solution with a pH value of 7 and cross-linked at 20°C for 36 h, then removed and washed with distilled water;

[0028] (2) Soaking the valve material treated in step (1) in a 10 mg / mL polyethyleneimine solution with a pH value of 7 for 24 h, then taking it out and washing it with distilled water;

[0029] (3) Soaking the valve material treated in step (2) in a 2 mg / mL phospholipid polyethylene glycol active lipid solution at a pH of 7 for 24 h, taking it out and washing it with phosphate buffer, and then sterilizing it;

[0030] (4) Soak the valve material treated in step (3) in a 10% 12 The exosomes were reacted in a 100 μg / mL exosome solution for 12 h, and then taken out and washed with sterile phosphate buffer to obtain a biological valve with anti-inflammatory and endothelial-promoting functions; wherein the exosomes were extracted from human umbilical vein endothelial cells after density gradient centrifugation.

[0031] Example 2

[0032] A bioprosthetic valve with anti-inflammatory and endothelialization promoting functions, the preparation method of which comprises the following steps:

[0033] (1) The porcine pericardium was placed in an 8 wt% glutaraldehyde solution with a pH of 9 and cross-linked at 37°C for 120 h, then removed and washed with distilled water;

[0034] (2) soaking the valve material treated in step (1) in a 50 mg / mL polyethyleneimine solution with a pH value of 8 for 72 h, and then taking it out and washing it with distilled water;

[0035] (3) Soaking the valve material treated in step (2) in a 50 mg / mL phospholipid polyethylene glycol active lipid solution with a pH value of 8 for 72 h, taking it out and washing it with phosphate buffer, and then sterilizing it;

[0036] (4) Soak the valve material treated in step (3) in a 10% 14 The exosomes were reacted in a 100 μg / mL exosome solution for 72 h, and then taken out and washed with sterile phosphate buffer to obtain a biological valve with anti-inflammatory and endothelial-promoting functions; wherein the exosomes were extracted by density gradient centrifugation after culturing endothelial progenitor cells.

[0037] Example 3

[0038] A bioprosthetic valve with anti-inflammatory and endothelialization promoting functions, the preparation method of which comprises the following steps:

[0039] (1) The bovine pericardium was placed in a 0.5 wt% glutaraldehyde solution with a pH value of 2 and cross-linked at 4°C for 24 h, then removed and washed with distilled water;

[0040] (2) Soaking the valve material treated in step (1) in a 0.1 mg / mL polyethyleneimine solution with a pH value of 2 for 6 h, then taking it out and washing it with distilled water;

[0041] (3) Soak the valve material treated in step (2) in a 0.1 mg / mL phospholipid polyethylene glycol active lipid solution with a pH value of 2 for 6 h, remove it, wash it with sterile phosphate buffer, and then sterilize it;

[0042] (4) Soak the valve material treated in step (3) in a 10% 4 The exosomes were reacted in a 100 μg / mL exosome solution for 6 hours, taken out and washed with phosphate buffer to obtain a biological valve with anti-inflammatory and pro-endothelial functions; among them, the exosomes were extracted by density gradient centrifugation after culturing M2 macrophages.

[0043] Comparative Example 1

[0044] A glutaraldehyde cross-linked biological valve, the preparation method of which comprises the following steps: placing porcine pericardium in a 1 wt% glutaraldehyde solution with a pH value of 7, cross-linking at a temperature of 20°C for 36 hours, and then taking it out and washing it with distilled water.

[0045] Test Example 1

[0046] (1) The exosome morphology of Example 1 was analyzed and identified using a transmission electron microscope. The exosomes were stained with 1% uranyl acetate, and any excess dye solution was removed using filter paper. The resulting mixture was then deposited on a 200-mesh carbon-coated copper grid for observation. The results are shown in FIG. Figure 1 As shown, exosomes with uniform morphology and size and typical vesicle structure were observed.

[0047] (2) The bioprosthetic valve with anti-inflammatory and endothelialization functions prepared in Example 1 was cut into samples with a diameter of 6 mm and then freeze-dried. The fiber structure and morphology of the samples were examined using a scanning electron microscope. The results were as follows: Figure 2 As shown, there is a dense exosome coating on the surface of the valve material, indicating that the exosomes have been successfully grafted onto the valve material.

[0048] Test Example 2

[0049] The bioprosthetic valve with anti-inflammatory and endothelialization functions of Example 1 and the bioprosthetic valve cross-linked with glutaraldehyde of Comparative Example 1 were sterilized and then placed in a well plate. Human umbilical vein endothelial cells were inoculated at 2×10 4 The density of cells was inoculated into each well. After incubation at 37°C for 24 h, the biocompatibility of these materials was evaluated using a cell live-dead staining assay. The live cells on the surface of these materials were labeled with calcein AM and observed using a fluorescence microscope. The results are shown in Figure 2. Figure 3shown.

[0050] Depend on Figure 3 It can be seen that compared with the biological valve cross-linked with glutaraldehyde, the number of living cells adhered to the surface of the biological valve with anti-inflammatory and pro-endothelialization functions was significantly increased, indicating that the valve modified with exosomes has good biocompatibility and good pro-endothelialization function.

[0051] Test Example 3

[0052] Fresh rabbit blood was centrifuged to obtain the upper platelet-rich plasma. The bioprostheses of Comparative Example 1 and Example 1 were sterilized and placed in a well plate. 100 μL of platelet-rich plasma was added to each well and incubated at 37°C for 2 h. The samples were then washed 3 times with PBS and fixed with 2.5% glutaraldehyde solution for 2 h. Gradient dehydration and freeze-drying were then performed. Finally, the adhesion of platelets on the samples was observed by scanning electron microscopy. The results are shown in Figure 2. Figure 4 shown.

[0053] Depend on Figure 4 It can be seen that compared with the biological valve of Comparative Example 1, the platelets adhered to the surface of the biological valve of Example 1 are significantly reduced, indicating that the valve modified with exosomes has good anti-platelet adhesion performance, proving that the biological valve with anti-inflammatory and pro-endothelialization functions has good anti-coagulation function.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a bioprosthetic valve with anti-inflammatory and endothelialization-promoting functions, characterized in that: The following steps are involved: (1) Place the valve material in glutaraldehyde solution and cross-link it at 4-37°C for 24-120 h, then remove it and wash it with distilled water; (2) soaking the valve material treated in step (1) in a polyethyleneimine solution for 6-72 hours, then taking it out and washing it with distilled water; (3) Soaking the valve material treated in step (2) in a phospholipid polyethylene glycol active lipid solution for 6-72 hours, taking it out and washing it with phosphate buffer, and then sterilizing it; the concentration of the phospholipid polyethylene glycol active lipid solution is 0.1-50 mg / mL, and the pH value is 2-8; (4) Soaking the valve material treated in step (3) in an exosome solution for 6-72 h, taking it out and washing it with a sterile phosphate buffer to obtain a bioprosthetic valve with anti-inflammatory and endothelial-promoting functions; the concentration of the exosome solution is 10 4 -10 14 / mL, pH value is 2-8, wherein the exosomes are derived from at least one of vascular endothelial cells, endothelial progenitor cells and macrophages.

2. The method for preparing a bioprosthetic valve with anti-inflammatory and endothelialization promoting functions according to claim 1, characterized in that: In step (1), the valve material is porcine pericardium or bovine pericardium.

3. The method for preparing a bioprosthetic valve with anti-inflammatory and endothelialization promoting functions according to claim 1, characterized in that: In step (1), the concentration of the glutaraldehyde solution is 0.5-8 wt %, and the pH value is 2-9.

4. The method for preparing a bioprosthetic valve with anti-inflammatory and endothelialization promoting functions according to claim 1, characterized in that: In step (2), the concentration of the polyethyleneimine solution is 0.1-50 mg / mL, and the pH value is 2-8.

5. The method for preparing a bioprosthetic valve with anti-inflammatory and endothelialization promoting functions according to claim 1, characterized in that: In step (4), the exosomes in the exosome solution are derived from human umbilical vein endothelial cells.

6. A biological valve with anti-inflammatory and endothelialization promoting functions obtained by the method for preparing a biological valve with anti-inflammatory and endothelialization promoting functions as described in any one of claims 1 to 5.

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