Functionalized acellular matrix material and preparation method thereof

Abstract

The invention relates to an acellular matrix material with biological functional activity and a preparation method thereof. The preparation method comprises the following steps: 1) providing an acellular matrix material subjected to crosslinking treatment; 2) reacting the crosslinked acellular matrix material with a biocompatible polymer, wherein the biocompatible polymer has a reactive group and a first subject and / or guest molecule; the reactive group is covalently bonded to a functional group on the acellular matrix material; and 3) reacting the acellular matrix material obtained in the step 2) with a second subject and / or guest molecule modified by a biological functional active substance through subject-guest interaction. Through the mode, the anti-calcification performance and the biological activity of the acellular matrix material can be improved at the same time, and the acellular matrix material is flexibly endowed with various biological function activities.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a functionalized acellular matrix material and a preparation method thereof. Background Art

[0002] Decellularized matrix materials are made by decellularizing tissues / organs using appropriate methods to remove cells and other antigenic molecules in the tissues that may cause rejection reactions, while retaining the three-dimensional structure (collagen and elastin) and functional proteins (such as glycosaminoglycans, proteoglycans and growth factors, etc.). They have biological induction functions and can form specific functionalized tissues in vivo / in vitro. They are a new type of biomaterial that can be used for tissue damage repair / reconstruction.

[0003] Normally, chemical cross-linking modification is required to improve the thermal stability, mechanical strength, resistance to enzyme degradation and other physical and chemical properties of decellularized matrix materials. Currently, most of the more mature commercial cross-linking methods are based on glutaraldehyde cross-linking methods, but the materials cross-linked with glutaraldehyde have certain toxicity, poor anti-coagulant and anti-calcification properties, and can cause thrombosis, blockage and other problems after implantation in the body. Although other new cross-linking methods such as genipin are expected to become a solution to replace glutaraldehyde, they are still limited by challenges in material sterilization, disinfection and preservation processes.

[0004] Therefore, the key to solving the problem is to modify the decellularized matrix materials after cross-linking, especially to improve the anti-coagulation, anti-calcification and biocompatibility of the decellularized matrix materials. The required bioactive functions can be given to the materials by binding the bioactive functional molecules to the surface of the materials. Common methods include chemical bonding and ionic bonding, but the former will lead to the loss of activity of the functional molecules during the covalent bonding process, while the latter often lacks long-term stability.

[0005] Therefore, there is still a need for an improved functionalized decellularized matrix material and a method for preparing the same, wherein the bioavailability and stability of the bound bioactive functional molecules are improved. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] In order to overcome the deficiencies in the prior art, the present invention provides a functionalized decellularized matrix material and a preparation method thereof. The treatment method can improve the anti-calcification, anti-coagulation, biocompatibility and other properties of the biomaterial, and at the same time can improve the bioavailability of functional molecules, and flexibly endow the decellularized matrix material with various biological functional activities according to actual needs.

[0008] Solutions for solving problems

[0009] The present invention provides the following technical solutions

[0010] A decellularized matrix material having biological functional activity, the decellularized matrix material comprising: (a) a functionalized acellular matrix material surface; and (b) a grafted polymer layer, comprising a biocompatible polymer and a first host and / or guest molecule at the polymer side chain and / or terminal, and also comprising reactive groups at the polymer side chain and / or terminal, wherein the grafted polymer layer is covalently bonded to the surface of the functionalized decellularized matrix material through the reactive groups at the side chain and / or terminal; and (c) a biofunctional active surface layer, wherein the biofunctional active surface layer comprises a second host and / or guest molecule modified with a biofunctional active substance; and the biofunctional active surface layer is connected to the grafted polymer layer at least through a host-guest interaction.

[0011] In a feasible implementation, the biologically functional active substance is selected from at least one of anti-thrombotic molecules, antibacterial molecules, anti-crystallization molecules, anti-adhesion molecules, anti-inflammatory molecules, and molecules that promote cell proliferation and differentiation.

[0012] In a feasible implementation, the antithrombotic molecule is selected from at least one of heparin and its derivatives, hirudin, lysine, polylysine, lysine derivatives, heparinoids, antiplatelet agents, plasminogen activators, plasminogen, biotin and avidin; the antibacterial molecule is selected from at least one of antibacterial peptides, polycations, defensins, albumin and serum proteins.

[0013] In a feasible implementation, the first and second host molecules are independently selected from at least one of β-cyclodextrin, β-cyclodextrin derivatives, α-cyclodextrin, α-cyclodextrin derivatives, γ-cyclodextrin, γ-cyclodextrin derivatives, cucurbituril, cucurbituril derivatives, crown ether, crown ether derivatives, calixarene, calixarene derivatives, columnar aromatics and columnar aromatics derivatives; the first and second guest molecules are independently selected from at least one of adamantane, adamantane derivatives, azobenzene, azobenzene derivatives, ferrocene, ferrocene derivatives, cholesterol and cholesterol derivatives.

[0014] In a feasible implementation, the biocompatible polymer is selected from one or more of poly-N-vinyl pyrrolidone, poly-N-vinyl pyrrolidone derivatives, polyethers, polyether derivatives, polyvinyl alcohol, polyvinyl alcohol derivatives, polysaccharides, and polyzwitterions.

[0015] In a feasible implementation, the number average molecular weight of the biocompatible polymer is 200-50000 Da, preferably 500-20000 Da.

[0016] The present invention also provides a method for modifying a cell-free matrix material, comprising the following steps: 1) Providing cross-linked decellularized matrix materials; 2) reacting the cross-linked decellularized matrix material with a biocompatible polymer, wherein the biocompatible polymer has a reactive group and a host and / or guest molecule; the reactive group is covalently bonded to a functional group on the decellularized matrix material; 3) reacting the acellular matrix material obtained in step 2) with a second host and / or guest molecule modified with a biologically functional active substance through a host-guest interaction.

[0017] In a feasible implementation, in step 2), the biocompatible polymer is prepared into a solution with a concentration of 0.01-20 mg / mL, preferably 1-10 mg / mL.

[0018] In a feasible implementation, in step 3), the host and / or guest molecules modified with the biological functional active substance are prepared into a solution with a concentration of 0.01-20 mg / mL, preferably 1-10 mg / mL.

[0019] In a feasible implementation, the cross-linking treatment is a glutaraldehyde cross-linking treatment.

[0020] Effects of the Invention

[0021] The present invention provides a decellularized matrix material with biological functional activity, which comprises a functionalized decellularized matrix material surface, a grafted polymer layer and a biological functional active surface layer, wherein the grafted polymer layer is covalently bonded to the functionalized decellularized matrix material surface via reactive groups, and the biological functional active surface layer is connected to the grafted polymer layer at least via a host-guest interaction.

[0022] The present invention provides various required biological functions for the decellularized matrix material through the biologically functional active surface layer, for example, it can reduce coagulation and thrombosis, reduce bacterial infection, etc. The functional surface layer is connected to the grafted polymer layer through the host-guest interaction, and the dynamic non-covalent binding advantage of the host-guest action is utilized to improve the bioavailability of the active molecules and reduce the activity loss of the molecules. At the same time, the biologically functional active molecules can be modularized to flexibly give the decellularized matrix material a single or composite biological functional activity. The grafted polymer layer of the present invention, as a mediating layer, not only shields the functional groups remaining on the surface of the decellularized matrix material, reduces the calcium crystallization sites, and reduces the calcification incidence of the decellularized matrix material. At the same time, its biocompatible segment characteristics can also be utilized to reduce the adsorption of proteins and further improve the anti-thrombotic performance of the material. In addition, the flexible segments provide more exposure possibilities for the surface biological functional active sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of the anti-calcification effects of Example 1 and Comparative Example 1.

[0025] Figure 2 The figure is a comparison chart of the anticoagulant effects of Example 2 and Comparative Example 1. DETAILED DESCRIPTION

[0027] In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0028] Unless defined otherwise, 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.

[0029] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoint numerical values ​​A and B.

[0030] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0031] It should be understood that the singular articles "a," "an," and "the," as used in this specification and the appended claims, include plural referents unless the context clearly dictates otherwise.

[0032] In this specification, the references to "one or some specific / preferred embodiments / schemes", "another or some other specific / preferred embodiments / schemes", "one or another embodiment / scheme", "one or another technical solution", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) related to the embodiment described are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0033] The term "comprising" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method or system, product or device comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or devices.

[0034] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0035] In the present invention the term "functionalization" and related terms include the process of treating a material to change its surface properties to meet specific requirements for a particular application, or the process of adding groups to a polymer to provide it with functionality that it does not normally have.

[0036] Decellularized matrix materials with biological functional activity

[0037] The present invention provides a decellularized matrix material having biological functional activity, comprising: (a) Functionalized surface of decellularized matrix materials; (b) grafted polymer layer; (c) biologically functional active surface; Wherein, the grafted polymer layer comprises a biocompatible polymer and a first host and / or guest molecule at the polymer side chain and / or the end, and also comprises reactive groups at the polymer side chain and / or the end, and the grafted polymer layer is covalently bonded to the surface of the functionalized decellularized matrix material through the reactive groups at the side chain and / or the end; The biologically functional active surface layer comprises a second host and / or guest molecule modified with a biologically functional active substance; and the biologically functional active surface layer is connected to the grafted polymer layer at least through a host-guest interaction.

[0038] <Decellularized Matrix Materials>

[0039] In the present invention, the decellularized matrix material is a decellularized tissue taken from an organism or the human body, for example, a decellularized animal tissue taken from a pig, sheep or cattle, and the animal tissue includes blood vessels, heart valves, pericardium, dermis, small intestinal submucosa, etc. After decellularization, the above tissue retains active ingredients and some non-antigenic components, such as polysaccharides, collagen, glycoproteins, fibronectin, etc., to maintain the activity, shape and structure of natural tissues and organs.

[0040] Usually, chemical cross-linking modification is further adopted to improve the thermal stability, mechanical strength, resistance to enzyme degradation and other physical and chemical properties of decellularized matrix materials. For example, cross-linking agents such as glutaraldehyde or other aldehyde-containing substances, carbodiimide, polyepoxy resin, genipin, 1,6-hexamethylene diisocyanate, proanthocyanidin, tannic acid, etc. are used. Among them, glutaraldehyde is the most mature commercial means, but aldehyde groups will remain on the surface after cross-linking. The free aldehyde groups cause increased toxicity, higher calcification incidence and participation in immunogenic response, and further produce potential calcium binding sites in the tissue that can cause calcification in vivo; carbodiimide promotes the reaction of amino and carboxyl groups of proteins to form amide bonds through nucleophilic substitution, but because it can only cross-link adjacent amino and carboxyl groups, there are still many active functional groups remaining on the surface of the matrix material after cross-linking. Polyepoxides react with amino, carboxyl and hydroxyl groups through their epoxy groups, but the chemical bonds are easily hydrolyzed, which also causes poor cross-linking stability. Therefore, functional groups such as aldehyde, amino, and carboxyl groups will remain on the surface of the decellularized matrix material after chemical cross-linking treatment, thereby causing certain toxicity, calcification, and thrombogenicity.

[0041] <Graft Polymer Layer>

[0042] In the present invention, the grafted polymer layer comprises a biocompatible polymer and a host and / or guest molecule at the polymer side chain and / or terminal, and the grafted polymer layer is covalently bonded to the surface of the functionalized decellularized matrix material via the reactive groups at the side chain and / or terminal.

[0043] Biocompatible polymers refer to high molecular synthetic polymers that can interact with the cells of an organism, and their surface characteristics and structural characteristics are compatible with the organism. The most common are hydrophilic polymers, which refer to high molecular weight linear, branched or cross-linked polymers composed of macromolecules. The polymer has affinity for water or other polar liquids and can provide hydrophilicity for the coating. The hydrophilic polymer can be synthetic or derived from natural products. The hydrophilic polymer can be a homopolymer, a copolymer or a blend. For example, it can be selected from one or more of poly-N-vinyl pyrrolidone, poly-N-vinyl pyrrolidone derivatives, polyethers, polyether derivatives, polyvinyl alcohol, polyvinyl alcohol derivatives, polysaccharides, and polyzwitterions.

[0044] The biocompatible polymer in the present invention includes a main chain of the polymer, and a terminal and / or a side chain. At least one terminal or side chain is a reactive group, such as an amino group, an aldehyde group, a carboxyl group, a thiol group, a hydroxyl group, an epoxy group, an azide group, a double bond, an alkynyl group, etc. These reactive groups can correspond to the functional groups on the surface of the decellularized matrix material and undergo a chemical bonding reaction. On the one hand, the active functional groups on the surface of the decellularized matrix material can be shielded. On the other hand, the biocompatibility / hydrophilicity of the polymer can further improve the biocompatibility of the decellularized matrix material, and provide an anchor point for subsequent biofunctional active molecules, thereby enhancing the connection strength between the grafted layer and the subsequent biofunctional active surface layer.

[0045] At least the other end or side chain of the biocompatible polymer of the present invention is a first host and / or guest molecule.

[0046] The first host molecule includes a macrocyclic host, which has a repeating structure bonded into a ring, has a cavity structure, and can selectively bind to certain guest molecules of suitable size, for example, it can be selected from at least one of β-cyclodextrin, β-cyclodextrin derivatives, α-cyclodextrin, α-cyclodextrin derivatives, γ-cyclodextrin, γ-cyclodextrin derivatives, cucurbituril, cucurbituril derivatives, crown ether, crown ether derivatives, calixarene, calixarene derivatives, pillararene and pillararene derivatives. Further preferably, the host molecule includes cyclodextrin, cucurbituril, calixarene and / or pillararene. More preferably, cyclodextrin and its derivatives are selected.

[0047] The first guest molecule refers to a molecule that can bind to the host molecule through non-covalent interaction. Examples of non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. Due to the difference in the structure and number of the bonded repeating units of the host molecule, the cavity space sizes of different host molecules are different, which makes the host-guest pairing have certain specificity, and the guest molecule includes molecules and ions that match the cavity shape of the corresponding host molecule. In some cases, the guest molecule is non-cyclic, and the guest molecule specifically includes adamantane, azobenzene, ferrocene, lithocholic acid, cholesterol, polyethylene glycol, polypropylene glycol, polycaprolactone, diaminoalkane, benzene, tetrahydrofuran, methylbenzylamine, naphthalene, methylviologen, choline, and derivatives thereof. Further, the guest molecule is preferably adamantane, azobenzene, cholesterol, ferrocene, polyethylene glycol, and derivatives thereof.

[0048] In a preferred embodiment of the present invention, the biocompatible polymer comprises a main chain of the polymer and at least two ends. At least one of the ends is a reactive group, such as an amino group, an aldehyde group, a carboxyl group, a sulfhydryl group, a hydroxyl group, an epoxy group, an azide group, a double bond, an alkynyl group, etc. More preferably, the reactive group is selected from an amino group, an aldehyde group, a carboxyl group, a sulfhydryl group, a hydroxyl group, and an epoxy group. At least one of the ends is a first host and / or a guest molecule.

[0049] In a preferred embodiment of the present invention, the biocompatible polymer is selected from linear hydrophilic polymers, and illustratively can be selected from polyethylene glycol, poloxamer, polyvinyl pyrrolidone and the like.

[0050] Preferably, the number average molecular weight of the biocompatible polymer is 200-50000Da, more preferably, the number average molecular weight of the biocompatible polymer is 500-20000Da. The molecular weight is controlled within a reasonable range to maintain the flexibility of the mediating layer while ensuring covalent bonding with the surface of the acellular matrix material.

[0051] <Biologically active surface>

[0052] In the present invention, the biologically functional active surface layer comprises a second host and / or guest molecule modified with a biologically functional active substance.

[0053] The biologically functional active substance is selected from at least one of anti-thrombotic molecules, anti-bacterial molecules, anti-crystallization molecules, anti-adhesion molecules, anti-inflammatory molecules, and molecules that promote cell proliferation and differentiation.

[0054] In a preferred embodiment of the present invention, the antithrombotic molecule is selected from at least one of heparin and its derivatives, hirudin, lysine, polylysine, lysine derivatives, heparinoids, antiplatelet agents, plasminogen activators, plasminogen, biotin and avidin; the antibacterial molecule is selected from at least one of antibacterial peptides, polycations, defensins, albumin and serum proteins.

[0055] The biologically functional active substance can be bonded to the host and / or guest molecules to form the host and / or guest molecules modified with the biologically functional active substance in a manner commonly used by those skilled in the art, through the reactive groups in its structure or by modifying the reactive groups with chemical bonds.

[0056] The second host molecule includes a macrocyclic host, which has a repeating structure bonded into a ring and a cavity structure, and can selectively bind to certain guest molecules of suitable size, for example, it can be selected from at least one of β-cyclodextrin, β-cyclodextrin derivatives, α-cyclodextrin, α-cyclodextrin derivatives, γ-cyclodextrin, γ-cyclodextrin derivatives, cucurbituril, cucurbituril derivatives, crown ether, crown ether derivatives, calixarene, calixarene derivatives, pillararene and pillararene derivatives. Further preferably, the host molecule includes cyclodextrin, cucurbituril, calixarene and / or pillararene. More preferably, cyclodextrin and its derivatives are selected.

[0057] The second guest molecule refers to a molecule that can bind to the host molecule through non-covalent interaction. Examples of non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. Due to the difference in the structure and number of the bonded repeating units of the host molecule, the cavity space sizes of different host molecules are different, which makes the host-guest pairing have certain specificity, and the guest molecule includes molecules and ions that match the cavity shape of the corresponding host molecule. In some cases, the guest molecule is non-cyclic, and the guest molecule specifically includes adamantane, azobenzene, ferrocene, lithocholic acid, cholesterol, polyethylene glycol, polypropylene glycol, polycaprolactone, diaminoalkane, benzene, tetrahydrofuran, methylbenzylamine, naphthalene, methylviologen, choline, and derivatives thereof. Further, the guest molecule is preferably adamantane, azobenzene, cholesterol, ferrocene, polyethylene glycol, and derivatives thereof.

[0058] In the present invention, the biologically functional active surface layer and the grafted polymer layer are connected at least through host-guest interaction. Other interactions are not excluded, such as electrostatic interaction, hydrophobic interaction, hydrogen bond, biospecific interaction, etc.

[0059] The present invention also provides a method for modifying a cell-free matrix material, comprising: 1) Providing cross-linked decellularized matrix materials; 2) reacting the cross-linked decellularized matrix material with a biocompatible polymer, wherein the biocompatible polymer has a reactive group and a first host and / or guest molecule; the reactive group is covalently bonded to a functional group on the decellularized matrix material; 3) reacting the acellular matrix material obtained in step 2) with a second host and / or guest molecule modified with a biologically functional active substance through a host-guest interaction.

[0060] The decellularized matrix material is a decellularized tissue taken from an organism or the human body, for example, a decellularized animal tissue taken from a pig, sheep or cattle, and the animal tissue includes blood vessels, heart valves, pericardium, dermis, small intestinal submucosa, etc. The method for decellularizing the above tissue can be selected from one or more combinations of hypotonic oscillation treatment, surfactant method, repeated freezing and thawing method and enzyme treatment method.

[0061] The cross-linking treatment may be performed by immersing the decellularized matrix material in a collagen cross-linking agent solution for cross-linking. The collagen cross-linking agent may generally be selected from one or more combinations of glutaraldehyde or other aldehyde-containing substances, 1,6-hexamethylene diisocyanate, carbodiimide, polyepoxy resin, genipin, proanthocyanidins, and tannic acid solution.

[0062] Preferably, the cross-linking agent is glutaraldehyde. Treatment of biomaterials with glutaraldehyde has the advantages of being fast, flexible, reducing immunogenicity and being able to be fixed with common amino acid residues, so most biological heart valves currently use glutaraldehyde for fixation to reduce immunogenicity. Further preferably, the glutaraldehyde solution is heat treated before cross-linking with the cleaned biological tissue. Further preferably, the reaction temperature of the heat treatment is 40-90°C. The mild heat treatment step can remove the unstable glutaraldehyde portion after glutaraldehyde fixation, further reducing the occurrence of calcification.

[0063] The biocompatible polymer in step 2) of the present invention includes a main chain of the polymer, as well as a terminal and / or a side chain. At least one terminal or side chain is a reactive group, such as an amino group, an aldehyde group, a carboxyl group, a thiol group, a hydroxyl group, an epoxy group, an azide group, a double bond, an alkynyl group, etc. These reactive groups can correspond to the functional groups on the surface of the decellularized matrix material and undergo a chemical bonding reaction. On the one hand, the active functional groups on the surface of the decellularized matrix material can be shielded. On the other hand, the biocompatibility / hydrophilicity of the polymer can further improve the biocompatibility of the decellularized matrix material, and provide an anchor point for subsequent biofunctional active molecules, thereby enhancing the connection strength between the grafted layer and the subsequent biofunctional active surface layer.

[0064] In a preferred embodiment of the present invention, the main chain of the compatible polymer is selected from a linear hydrophilic polymer, which can be exemplarily selected from polyethylene glycol, poloxamer, polyvinyl pyrrolidone, etc. The hydrophilic polymer segment has good hydrophilicity and biocompatibility, and covalently connecting it to the decellularized matrix material can reduce protein adsorption, further improve the biocompatibility of the material, and reduce the occurrence of infection and coagulation; in addition, the introduction of the linear hydrophilic polymer segment can form a spacer arm between the biological valve material and the subsequent biological functional active surface layer, provide a flexible segment, and better expose the active sites of the active molecules in the physiological environment.

[0065] In some embodiments, the number average molecular weight of the biocompatible polymer is 200-50000Da, or 500-20000Da, or 1000Da, 2000Da, 3000Da, 4000Da, 5000Da, 6000Da, 7000Da, 8000Da, 9000Da, 10000Da, 12000Da, 14000Da, 16000Da, 18000Da or 20000Da. As well as specific values ​​between the above values, the present invention will not list the specific values ​​included in the range due to space limitations and for simplicity.

[0066] At least the other end or side chain of the biocompatible polymer of the present invention is a first host and / or guest molecule.

[0067] The first host molecule includes a macrocyclic host, which has a repeating structure bonded into a ring, has a cavity structure, and can selectively bind to certain guest molecules of suitable size, for example, it can be selected from at least one of β-cyclodextrin, β-cyclodextrin derivatives, α-cyclodextrin, α-cyclodextrin derivatives, γ-cyclodextrin, γ-cyclodextrin derivatives, cucurbituril, cucurbituril derivatives, crown ether, crown ether derivatives, calixarene, calixarene derivatives, pillararene and pillararene derivatives. Further preferably, the host molecule includes cyclodextrin, cucurbituril, calixarene and / or pillararene. More preferably, cyclodextrin and its derivatives are selected.

[0068] The first guest molecule refers to a molecule that can bind to the host molecule through non-covalent interaction. Examples of non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. Due to the difference in the structure and number of the bonded repeating units of the host molecule, the cavity space sizes of different host molecules are different, which makes the host-guest pairing have certain specificity, and the guest molecule includes molecules and ions that match the cavity shape of the corresponding host molecule. In some cases, the guest molecule is non-cyclic, and the guest molecule specifically includes adamantane, azobenzene, ferrocene, lithocholic acid, cholesterol, polyethylene glycol, polypropylene glycol, polycaprolactone, diaminoalkane, benzene, tetrahydrofuran, methylbenzylamine, naphthalene, methylviologen, choline, and derivatives thereof. Further, the guest molecule is preferably adamantane, azobenzene, cholesterol, ferrocene, polyethylene glycol, and derivatives thereof.

[0069] In a preferred embodiment of the present invention, it is desirable to react the biocompatible polymer solution prepared in step 2) with the decellularized matrix material after cross-linking treatment. Preferably, the biocompatible polymer is dissolved in one or a combination of the following selected solutions: an aqueous solution such as an aqueous buffer solution, water, a short-chain alcohol, glycerol or a plasticizer; an organic solvent, and an organic buffer solution.

[0070] It is understood that the type of solvent used and the concentration of the solution vary depending on the composition and molecular weight of the biocompatible polymer and the type and properties of the decellularized matrix material to be modified.

[0071] In a preferred embodiment of the present invention, the concentration of the biocompatible polymer solution is 0.01-20 mg / mL, preferably 1-10 mg / mL, and may also be 2 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL or 8 mg / mL. As well as specific values ​​between the above values, the present invention will not list the specific values ​​included in the range due to space limitations and for simplicity.

[0072] In a preferred embodiment of the present invention, it is desirable to prepare a solution of the host and / or guest molecules modified with the biofunctional active substance in step 3) and react with the decellularized matrix material treated in step 2). Preferably, the biocompatible polymer is dissolved in one of the following selected solutions or a combination of the following selected solutions: an aqueous solution such as an aqueous buffer solution, water, a short-chain alcohol, glycerol or a plasticizer; an organic solvent, and an organic buffer solution.

[0073] It is understood that the type of solvent used and the concentration of the solution vary depending on the composition and molecular weight of the biofunctionally active substance and the type and properties of the decellularized matrix material to be modified.

[0074] In a preferred embodiment of the present invention, the concentration of the host and / or guest molecule modified by the biological functional active substance in the solution is 0.01-20 mg / mL, preferably 1-10 mg / mL, and can also be 2 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL or 8 mg / mL. As well as specific point values ​​between the above values, the present invention will not list the specific point values ​​included in the range due to space limitations and for simplicity.

[0075] Of course, in other preferred embodiments, after the above step 1), or after the above step 2), or after the above step 3), the processing method provided in the present application further comprises: cleaning the decellularized matrix material.

[0076] Of course, in other preferred embodiments, after the above step 3), the treatment method provided by the present application further comprises: washing the decellularized matrix material with a mixed solution, wherein the mixed solution comprises at least formaldehyde, Tween and ethanol. The use of alcohol and surfactant can enhance the anti-calcification ability of valve tissue and promote the improvement of durability.

[0077] The artificial bioprosthetic valve prepared by the above treatment method has excellent biocompatibility, anticoagulation, antithrombosis, antibacterial, tissue endothelialization and long-term durability. The required different biological functional molecules can be modified to the surface of the decellularized matrix material in a modular way, and the damage to the active function of the active molecules in the conventional chemical modification process is greatly reduced by the host-guest interaction of non-covalent binding. The flexible biocompatible polymer chain segment is used as a grafting layer, and the dynamic host-guest interaction exposes more active sites on the surface, providing technical support for the functional design of decellularized matrix materials.

[0078] With reference to the above implementation contents, in order to make the technical solution of the present application more specific, clear and easy to understand, the technical solution of the present application is now exemplified. However, it should be noted that the contents to be protected by the present application are not limited to the following embodiments. Example

[0079] Example 1

[0080] A method for modifying a decellularized matrix material having biological functional activity comprises the following steps:

[0081] Obtain bovine pericardial tissue that has been stripped of fat, trimmed, cleaned, and decellularized.

[0082] The cleaned decellularized bovine pericardial tissue was immersed in 0.3wt% glutaraldehyde solution for cross-linking for 3 days.

[0083] The bovine pericardial tissue after the cross-linking treatment in step 2 was cleaned, immersed in a PBS solution (pH 7.4) containing 5 mg / mL NH2-PEG (1000)-cyclodextrin for 4 hours, and then the tissue was taken out.

[0084] After washing the tissue treated in step 3, immerse it in a PBS solution (pH 7.4) containing 5 mg / mL heparin-adamantane for 1 hour, remove the tissue and wash it. The preparation method of heparin-adamantane can refer to the preparation method of the public paper "Construction of polysaccharide-based hydrogel dressing based on skin wound microenvironment and its application in injury repair".

[0085] Example 2 A modification method for a decellularized matrix material with biological functional activity comprises the following steps.

[0086] Obtain bovine pericardial tissue that has been stripped of fat, trimmed, cleaned, and decellularized.

[0087] The cleaned decellularized bovine pericardial tissue was immersed in 0.3wt% glutaraldehyde solution for cross-linking for 3 days.

[0088] The tissue cross-linked in step 2 was cleaned, immersed in a PBS solution (pH 7.4) containing 5 mg / mL NH2-PEG (1000)-adamantane for 4 hours, and then the tissue was taken out.

[0089] After cleaning the tissue treated in step 3, immerse it in a PBS solution (pH 7.4) containing 5 mg / mL heparin-cyclodextrin for 0.5 h, remove the tissue and clean it. The preparation method of heparin-cyclodextrin can refer to the content of the applicant's prior patent technology (202111674445.4).

[0090] Comparative Example 1 A modification method for a decellularized matrix material with biological functional activity comprises the following steps.

[0091] Obtain bovine pericardial tissue that has been stripped of fat, trimmed, cleaned, and decellularized.

[0092] The cleaned decellularized bovine pericardial tissue was immersed in 0.3wt% glutaraldehyde solution for cross-linking for 3 days.

[0093] Test example Taking the decellularized matrix materials prepared in Example 1 and Comparative Example 1 as examples, the materials were implanted subcutaneously for 3 weeks, then taken out for pathological section processing, Vankossa staining to observe calcification, and calcium content analysis. Figure 1 and Table 1.

[0094] Calcium ion content (μg / mg) Example 1 24 Comparative Example 1 162 Table 1 Calcium content data of Example 1 and Comparative Example 1

[0095] Taking the acellular matrix materials prepared in Example 2 and Comparative Example 1 as an example, the anticoagulant performance test was performed according to the partial thromboplastin time (PTT) test in the national standard GBT14233.2. Figure 2 .

[0096] from Figure 1 and Figure 2 The results of the experiment showed that the anti-calcification performance and anti-coagulation performance of the embodiment of the present invention were higher than those of the control group, which proved that the biocompatibility of the decellularized matrix material obtained by the treatment of the embodiment of the present invention was improved and the biological activity function was increased.

[0097] However, the above is only the preferred embodiment of the present invention. After understanding the technical means of the present invention, professionals familiar with this technology can naturally make changes according to actual needs under the guidance of the present invention. Therefore, all equivalent changes and modifications made according to the scope of the patent application of the present invention should still be within the scope of the patent of the present invention.

Claims

1. A decellularized matrix material having biological functional activity, characterized in that: The decellularized matrix material comprises: (a) a functionalized acellular matrix material surface; and (b) a grafted polymer layer, comprising a biocompatible polymer and a first host and / or guest molecule at the polymer side chain and / or terminal, and also comprising reactive groups at the polymer side chain and / or terminal, wherein the grafted polymer layer is covalently bonded to the surface of the functionalized decellularized matrix material through the reactive groups at the side chain and / or terminal; and (c) a biofunctional active surface layer, wherein the biofunctional active surface layer comprises a second host and / or guest molecule modified with a biofunctional active substance; and the biofunctional active surface layer is connected to the grafted polymer layer at least through a host-guest interaction.

2. The acellular matrix material according to claim 1, characterized in that: The biologically functional active substance is selected from at least one of anti-thrombotic molecules, antibacterial molecules, anti-crystallization molecules, anti-adhesion molecules, anti-inflammatory molecules, and molecules promoting cell proliferation and differentiation.

3. The acellular matrix material according to claim 2, characterized in that: The antithrombotic molecule is selected from at least one of heparin and its derivatives, hirudin, lysine, polylysine, lysine derivatives, heparinoids, antiplatelet agents, plasminogen activators, plasminogen, biotin and avidin; the antibacterial molecule is selected from at least one of antibacterial peptides, polycations, defensins, albumin and serum proteins.

4. The decellularized matrix material according to claim 1, characterized in that: The first and second host molecules are independently selected from at least one of β-cyclodextrin, β-cyclodextrin derivatives, α-cyclodextrin, α-cyclodextrin derivatives, γ-cyclodextrin, γ-cyclodextrin derivatives, cucurbituril, cucurbituril derivatives, crown ether, crown ether derivatives, calixarene, calixarene derivatives, columnararene and columnararene derivatives; the first and second guest molecules are independently selected from at least one of adamantane, adamantane derivatives, azobenzene, azobenzene derivatives, ferrocene, ferrocene derivatives, cholesterol and cholesterol derivatives.

5. The acellular matrix material according to claim 1, characterized in that: The biocompatible polymer is selected from one or more of poly-N-vinyl pyrrolidone, poly-N-vinyl pyrrolidone derivatives, polyethers, polyether derivatives, polyvinyl alcohol, polyvinyl alcohol derivatives, polysaccharides, and polyzwitterions.

6. The decellularized matrix material according to claim 1, characterized in that: The number average molecular weight of the biocompatible polymer is 200-50000 Da, preferably 500-20000 Da.

7. A method for modifying a decellularized matrix material, characterized in that: The following steps are involved: 1) Providing cross-linked decellularized matrix materials; 2) reacting the cross-linked decellularized matrix material with a biocompatible polymer, wherein the biocompatible polymer has a reactive group and a first host and / or guest molecule; the reactive group is covalently bonded to a functional group on the decellularized matrix material; 3) reacting the acellular matrix material obtained in step 2) with a second host and / or guest molecule modified with a biologically functional active substance through a host-guest interaction.

8. The modification method according to claim 7, characterized in that: In step 2), the biocompatible polymer is prepared into a solution with a concentration of 0.01-20 mg / mL, preferably 1-10 mg / mL.

9. The modification method according to claim 7, characterized in that: In step 3), the host and / or guest molecules modified with the biologically active substance are prepared into a solution with a concentration of 0.01-20 mg / mL, preferably 1-10 mg / mL.

10. The modification method according to any one of claims 7 to 9, characterized in that: The cross-linking treatment is glutaraldehyde cross-linking treatment.

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