A hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating and its preparation method and application

Abstract

The present invention relates to the field of biomedical materials, and discloses a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, and a preparation method and application thereof. Through the synergistic effect of hydrogen sulfide and nitric oxide, a coating with anti-inflammatory, anticoagulant and tissue repair-promoting properties is provided. The coating includes a hydrogen sulfide donor, which can release hydrogen sulfide or its precursor spontaneously or under external stimulation; the coating also includes a component that can catalyze the release of nitric oxide from an endogenous nitric oxide donor. The coating is covalently linked to the surface of the substrate by chemical means to form a strong interface bond; and the coating has a multi-crosslinked network structure, which gives the coating good stability and ensures its long-term functional application on the surface of biomedical materials. The present invention can be widely used in the surface modification of biomedical materials, especially in the functionalization of coatings of implantable devices such as valve clips, vascular stents, heart valves, occluders, vascular grafts, and myocardial patches.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating, and a preparation method and application thereof. Background Art

[0002] Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide. Common treatments include medication and surgical implants, such as cardiovascular stents, artificial blood vessels, valves, etc. However, the biocompatibility of traditional implant materials is insufficient, which often causes inflammatory reactions, thrombosis, and even restenosis. These problems seriously affect the long-term use of implants, and there is an urgent need to develop biomaterials with anti-inflammatory, anticoagulant and tissue repair functions.

[0003] Gasotransmitters, such as nitric oxide (NO) and hydrogen sulfide (H2S), have important physiological regulatory functions in the body. Studies have shown that NO has a strong anti-thrombotic effect, can inhibit platelet aggregation, promote vasodilation, and also promote endothelial cell proliferation. H2S also plays an important role in inflammation regulation and vascular protection. It can reduce oxidative stress, reduce inflammatory response, and play a positive role in tissue repair and vascular regeneration.

[0004] In recent years, the combined application of NO and H2S has received increasing attention. Studies have found that the two have a synergistic effect under certain conditions and can work together to exert more significant anti-inflammatory, anticoagulant and repair-promoting functions. Based on this, the development of a coating material that can continuously release NO and H2S can not only inhibit inflammatory response, reduce thrombosis, but also promote tissue repair, which has important clinical application value.

[0005] At present, although some coating materials can release NO or H2S alone, there are few materials that can release these two gas signal molecules at the same time, especially in-depth research and application in synergy. Therefore, providing a coating that releases NO and H2S at the same time and fully exerts its synergistic anti-inflammatory, anticoagulant and repair-promoting effects has become the key to solving the problems of the existing technology.

[0006] For example, the patent with publication number CN117398532A discloses an anticoagulant functional material based on the release of gas signal molecules and a preparation method thereof, in which the gas signal molecule donor in the material is combined with the polydopamine-polyethyleneimine coating through a covalent bond. However, although polydopamine has excellent adhesion properties and can attach the coating to the surface of the substrate material, this attachment mostly relies on physical adsorption or weak chemical action. Therefore, the binding force between the coating and the substrate is weak. Over time or in a complex physiological environment, external factors such as mechanical force and chemical changes may cause the stability and durability of the coating to decrease, affecting its long-term use effect. In addition, the NO in the material is a donor type, that is, spontaneous release is achieved by loading the NO donor in the coating, but the release rate of NO is difficult to accurately control, and the persistence of its release is also limited, which makes it difficult to maintain the anticoagulant effect at an ideal level, thereby affecting its effectiveness in long-term applications. Summary of the invention

[0007] Technical problems solved by the present invention:

[0008] The present invention solves the problems of insufficient anti-inflammatory, anticoagulant and repair-promoting performance of biomedical material surface coatings in the prior art, uncontrollable release of gas signal molecules, and poor coating adhesion and mechanical properties by developing a multi-crosslinked hydrogel coating with synergistic effects of H2S and NO. The coating has significant biocompatibility and multifunctional properties, provides an innovative solution for surface modification of biomedical materials, and has broad application prospects.

[0009] The technical solution adopted by the present invention is:

[0010] First, the present invention provides a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating, the raw materials of the coating include:

[0011] Component A, component B, component C, and metal ions capable of catalyzing the release of nitric oxide from an endogenous nitric oxide donor; component A is bonded to a substrate;

[0012] Component A at least includes: one or more double bonds, one or more groups I-1 that react chemically with component B, and 0 or more groups I-2 that react chemically with component C;

[0013] Component B at least includes: two or more groups II-1 that react chemically with component A, and zero or more groups II-2 that react chemically with component C;

[0014] Component C at least includes: one or more hydrogen sulfide donors, and one or more groups III-1 that react chemically with group I-2 or group II-2.

[0015] The coating obtained above, the hydrogen sulfide donor can release hydrogen sulfide or its precursor compound spontaneously or under external stimulation; and can catalyze the endogenous nitric oxide donor to release nitric oxide.

[0016] According to some preferred embodiments, the hydrogen sulfide donor contains a group, and the group includes at least one of thioether, mercapto, disulfide, polysulfide, thioamide, thiocarbamate, thiocarbonate, thiocarboxylic acid, thiourea, thioacetal, thioketone, and thioxime.

[0017] Some of the hydrogen sulfide donors containing the above-mentioned groups are listed here, specifically 4-aminothiobenzamide, O-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl) (4-(hydroxymethyl)phenyl)thiocarbamate, O-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl) (4-isothiocyanatophenyl)thiocarbamate, methacrylate-polyethylene glycol-thiobenzamide, 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester), cystamine dihydrochloride, and bis(2-hydroxyethyl)disulfide. Other hydrogen sulfide donors containing the above-mentioned groups can also be selected in the art according to actual needs.

[0018] According to some preferred embodiments, the metal ions include at least one of copper ions, iron ions, zinc ions, nickel ions, cobalt ions, manganese ions, cuprous ions, ferrous ions, stannous ions, trivalent cerium ions, and trivalent titanium ions.

[0019] According to some preferred embodiments,

[0020] Component A at least includes: 0 or more groups I-3 coordinated with metal ions; group I-3 includes at least one of carboxyl, amino, hydroxyl, thiol, ether bond, pyridyl, thioether, and imine;

[0021] Component B at least includes: 0 or more groups II-3 capable of coordinating with metal ions, and group II-3 includes at least one of carboxyl, amino, hydroxyl, thiol, ether bond, pyridine, thioether, and imine.

[0022] According to some preferred embodiments, the existence forms of the groups of component A, component B, and component C each independently include at least one of the following situations:

[0023] Scenario 1: It is composed of a mixture of compounds containing each group;

[0024] Case 2: It is composed of M1 compound and M2 compound, M1 compound has at least two groups selected arbitrarily, M2 compound is a compound containing a third group, and the third group is another group different from the two groups in M1 compound; when M1 compound has three groups, M2 compound is missing.

[0025] Here, taking component A as an example, a specific description is given:

[0026] In component A, the existence forms of group I-1, group I-2 and group I-3 include at least one of the following situations:

[0027] Case 1: Component A comprises a compound comprising group I-1, a compound comprising group I-2, and a compound comprising group I-3;

[0028] Case 2: Component A includes a compound grafted with group I-1 and group I-2 and a compound containing group I-3; or, component A includes a compound grafted with group I-2 and group I-3 and a compound containing group I-1; or, component A includes a compound grafted with group I-1 and group I-3 and a compound containing group I-2;

[0029] Case 3: Component A includes a compound grafted with group I-1, group I-2, and group I-3.

[0030] Second, the present invention provides a method for preparing the aforementioned hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising the following steps:

[0031] S1 covalently connects substance N1 to the surface of the substrate through a surface polymerization reaction to obtain a first treated body; the surface polymerization reaction refers to a surface-initiated free radical polymerization reaction of the carbon-carbon double bonds in N1. The surface free radicals can be obtained by introducing a free radical initiator on the surface of the material on the one hand, and on the other hand, by irradiating the material with high-energy rays to generate free radicals by homolytic cleavage of chemical bonds on the surface of the material.

[0032] S2 placing the first processed body in a solution containing substance N2, and soaking the solution to obtain a second processed body;

[0033] S3 placing the second processed body in a solution containing substance N3, and soaking the solution to obtain a third processed body;

[0034] S4 placing the third treated body in a metal ion solution for immersion to obtain a coating;

[0035] Substance N1, Substance N2, Substance N3 has any of the following limitations:

[0036] (1) Substance N1 is component A, substance N2 is component B, and substance N3 is component C;

[0037] (2) Substance N1 is the chemical reaction product of components A and C, and substance N2 is component B but lacks S3;

[0038] (3) When substance N1 is component A and substance N2 is the chemical reaction product of component B and component C, S3 is missing;

[0039] (4) Substance N1 is component A, lacking S2, and substance N3 is the chemical reaction product of components B and C.

[0040] That is to say: component C can be connected to component A in advance to form a substance containing a hydrogen sulfide donor, thereby omitting step S3; component C can be connected to component B in advance to form a substance containing a hydrogen sulfide donor, thereby omitting step S3; two or more groups II-1 in component B can chemically react with component A to form a covalently cross-linked network structure to enhance the mechanical properties of the coating.

[0041] According to some preferred embodiments, the concentration of component A is 1-100 wt%, the concentration of component B is 0.2-20 wt%, the concentration of component C is 1-20 wt%, and the concentration of metal ions is 0.1-10 wt%; B / A=0.1-20; C / (A+B+C)=0.01-0.5.

[0042] According to some more preferred embodiments, B / A=0.2-2, C / (A+B+C)=0.1-0.3.

[0043] Third, the present invention provides an application of the aforementioned coating in the preparation of biomedical materials. Specifically, the present invention can be widely used in the surface modification of biomedical materials, especially in the functionalization of coatings of implantable devices such as valve clips, vascular stents, heart valves, occluders, vascular grafts, myocardial patches, etc., to provide highly effective anti-inflammatory, anticoagulant and repair-promoting properties.

[0044] The technical mechanism and beneficial effects adopted by the present invention are as follows:

[0045] (1) The present invention achieves the synergistic release of H2S and NO by designing a component containing a hydrogen sulfide donor and a component capable of catalyzing the release of endogenous nitric oxide. H2S and NO each have anti-inflammatory, anticoagulant and pro-repair effects in physiological processes. The synergistic release of the two can significantly enhance their biological functions. This synergistic mechanism can simultaneously reduce inflammatory responses, inhibit thrombosis, and accelerate the tissue repair process, thereby improving the overall efficacy of biomedical materials.

[0046] (2) The present invention firmly fixes the coating to the surface of the substrate through covalent bonds, and introduces a multi-crosslinked network structure combining covalent crosslinking and metal coordination crosslinking into the coating, which significantly improves the stability of the coating. This design not only greatly enhances the bonding strength between the coating and the substrate, but also improves the mechanical properties of the coating, ensuring its persistence and durability in a physiological environment, so that it can continue to function stably in long-term use.

[0047] (3) Both H2S and NO used in the present invention are endogenous gas signal molecules with natural biological activity and low toxicity. The coating material is safe and environmentally friendly, and can effectively function in vivo while reducing potential toxicity and side effects to surrounding tissues and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Water contact angles of the dual-release hydrogen sulfide / nitric oxide coating material PLLA-Alg / Cu / PEI-ABTA and the control sample;

[0049] Figure 2 The nitric oxide release behavior diagram of the coating material PLLA-Alg / Cu / PEI-ABTA for dual release of hydrogen sulfide / nitric oxide and the comparison sample;

[0050] Figure 3 The hydrogen sulfide release behavior diagram of the dual hydrogen sulfide / nitric oxide releasing coating material PLLA-Alg / Cu / PEI-ABTA and the comparative sample;

[0051] Figure 4 The platelet adhesion results of the dual hydrogen sulfide / nitric oxide-releasing coating material PLLA-Alg / Cu / PEI-ABTA and the control sample;

[0052] Figure 5 The activation results of the coating material PLLA-Alg / Cu / PEI-ABTA for dual release of hydrogen sulfide / nitric oxide and the comparison sample;

[0053] Figure 6 The graphs show the results of endothelial cell adhesion and proliferation of the dual hydrogen sulfide / nitric oxide-releasing coating material PLLA-Alg / Cu / PEI-ABTA and the control sample;

[0054] Figure 7 The endothelial cell activity results of the dual-release hydrogen sulfide / nitric oxide coating material PLLA-Alg / Cu / PEI-ABTA and the control sample;

[0055] Figure 8 This is a diagram showing the results of subcutaneous implantation of the dual hydrogen sulfide / nitric oxide-releasing coating material PLLA-Alg / Cu / PEI-ABTA and a control sample in SD rats. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0057] Example 1

[0058] This embodiment provides a method for preparing a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising:

[0059] (1) A benzophenone / ethanol solution with a concentration of 5 wt% was prepared and nitrogen was passed through the solution for 30 min; a polylactic acid (PLLA) substrate was placed in the benzophenone / ethanol solution and exposed to ultraviolet light for 30 min to obtain a polylactic acid substrate PLLA-BP containing a photoinitiator;

[0060] (2) preparing a monomer aqueous solution containing 3 wt% methacryloyl sodium alginate (MA-Alg), immersing the PLLA-BP obtained in step (1) in the monomer aqueous solution, passing nitrogen for 30 min, and then irradiating with ultraviolet light for 30 min. After washing with deionized water to remove unreacted monomers, a substrate PLLA-Alg with sodium alginate covalently bonded to the surface is obtained;

[0061] (3) Using the coupling reagent N,N'-carbonyldiimidazole (CDI) to connect 4-aminothiobenzamide (ABTA) to the polyethyleneimine (PEI) molecular chain, a polymer containing a hydrogen sulfide donor, PEI-ABTA, was obtained;

[0062] (4) preparing a PBS buffer solution containing 3 wt% PEI-ABTA and 0.5 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), immersing the PLLA-Alg obtained in step (2) in the buffer solution, and reacting at room temperature for 12 h to obtain a substrate PLLA-Alg / PEI-ABTA of a double cross-linked network hydrogel coating material;

[0063] (5) Prepare a 1 wt% copper sulfate (CuSO4) aqueous solution, soak the PLLA-Alg / PEI-ABTA obtained in step (4) in the copper sulfate aqueous solution for 24 h, and then soak and wash in deionized water for 24 h to obtain a coating material PLLA-Alg / Cu / PEI-ABTA that dually releases hydrogen sulfide / nitric oxide.

[0064] Example 2

[0065] This embodiment provides a method for preparing a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising:

[0066] (1) The polyurethane substrate (PU) was immersed in an isopropanol solution containing 1 wt% tert-butyl peroxycarbonate-2-ethylhexyl ester (TBEC, CAS: 34443-12-4) at room temperature for 3 hours to obtain a substrate PU-TBEC containing an initiator on the surface;

[0067] (2) preparing an aqueous solution containing 5 wt% methacryloyl sodium alginate (MA-Alg) and 0.5 wt% ferrous chloride (FeCl2), immersing the PU-TBEC obtained in step (1) in the aqueous solution, reacting at room temperature for 12 hours, and obtaining a substrate PU-Alg with sodium alginate hydrogel covalently bonded to the surface through a redox-initiated polymerization reaction;

[0068] (3) preparing a PBS buffer solution containing 3 wt% PEI and 0.5 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), immersing the PU-Alg obtained in step (2) in the buffer solution, and reacting at room temperature for 12 h to obtain a substrate PLLA-Alg / PEI of a double cross-linked network hydrogel coating material;

[0069] (4) An anhydrous dimethyl sulfoxide solution containing 3 wt% 4-(hydroxymethyl)phenylboronic acid pinacol ester, 20 wt% 1,4-phenylene diisocyanate, and 0.1 wt‰ di-n-butyltin dilaurate (DBTDL) was prepared and reacted at 60 °C for 12 h. After ether precipitation, the product was separated and purified on a silica gel column to obtain a donor compound NCS-PeroxyTCM containing a thioisocyanate (NCS) functional group and a reactive oxygen species that releases hydrogen sulfide.

[0070] (5) preparing an aqueous solution containing 1 wt% NCS-PeroxyTCM and 30 wt% dimethyl sulfoxide, immersing the PLLA-Alg / PEI obtained in step (3) in the aqueous solution, and utilizing the click chemistry reaction between the amino group and the thioisocyanate to covalently bind the hydrogen sulfide donor to the hydrogel coating to obtain PLLA-Alg / PEI-PeroxyTCM;

[0071] (6) Prepare a 5 wt% copper chloride (CuCl2) aqueous solution, soak the PLLA-Alg / PEI-PeroxyTCM obtained in step (5) in the copper chloride aqueous solution for 30 min, and then soak and wash it in deionized water for 3 times, each time for 10 min, to obtain a coating material PLLA-Alg / Cu / PEI-PeroxyTCM that dually releases hydrogen sulfide / nitric oxide.

[0072] Example 3

[0073] This embodiment provides a method for preparing a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising:

[0074] (1) A hydrogen sulfide donor MA-PEG2000-ABTA containing a double bond was prepared by using a nucleophilic substitution reaction between an active ester of methacrylate polyethylene glycol (MA-PEG2000-NHS) and an active ester and an amino group in 4-aminothiobenzamide (ABTA). Specifically, an anhydrous tetrahydrofuran solution containing 10 wt% MA-PEG2000-NHS, 3 wt% triethylamine, and 3 wt% ABTA was prepared, and the mixture was stirred at room temperature for 24 h, precipitated in ether, and dried to obtain MA-PEG2000-ABTA.

[0075] (2) Immersing a polyvinyl chloride substrate (PVC) in an acetone solution containing 1 wt% tert-butyl peroxycarbonate-2-ethylhexyl ester (TBEC, CAS: 34443-12-4) at room temperature for 30 minutes to obtain a substrate PVC-TBEC containing an initiator on the surface;

[0076] (3) preparing an aqueous solution containing 3 wt% methacryloyl sodium alginate (MA-Alg), 3 wt% MA-PEG2000-ABTA and 0.5 wt% ferrous gluconate, immersing the PVC-TBEC obtained in step (2) in the aqueous solution, reacting at room temperature for 12 hours, and obtaining a substrate PVC-Alg / ABTA with a sulfide donor and sodium alginate hydrogel covalently bonded to the surface through a redox-initiated polymerization reaction;

[0077] (4) preparing a PBS buffer solution containing 3 wt% gelatin and 0.5 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), immersing the PVC-Alg / ABTA obtained in step (3) in the buffer solution, and reacting at room temperature for 12 h to obtain the substrate PVC-Alg / ABTA / PEI of the double cross-linked network hydrogel coating material;

[0078] (5) Prepare a 5 wt% zinc chloride (ZnCl2) aqueous solution, soak the PVC-Alg / ABTA / PEI obtained in step (4) in the ZnCl2 aqueous solution for 24 h, and then soak and clean it in deionized water for 24 h to obtain a coating material PVC-Alg / Zn / ABTA / PEI that dually releases hydrogen sulfide / nitric oxide.

[0079] Example 4

[0080] This embodiment provides a method for preparing a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising:

[0081] (1) A hydrogen sulfide donor MA-PEG2000-ABTA containing a double bond was prepared by using a nucleophilic substitution reaction between an active ester of methacrylate polyethylene glycol (MA-PEG2000-NHS) and an active ester and an amino group in 4-aminothiobenzamide (ABTA). Specifically, an anhydrous tetrahydrofuran solution containing 10 wt% MA-PEG2000-NHS, 3 wt% triethylamine, and 3 wt% ABTA was prepared, and the mixture was stirred at room temperature for 24 h, precipitated in ether, and dried to obtain MA-PEG2000-ABTA.

[0082] (2) The polypropylene substrate (PP) was immersed in an aqueous solution containing 5 wt% MA-PEG2000-ABTA and 5 wt% 2-aminoethyl methacrylate hydrochloride (CAS: 2420-94-2), and nitrogen was bubbled for 30 min. The substrate was sealed and subjected to 20 kGy electron beam treatment to obtain a coating material PP-ABTA / NH2 containing hydrogen sulfide donors and amino groups on the surface.

[0083] (3) using sodium periodate (NaIO4) to oxidize sodium alginate to obtain aldehyde-modified sodium alginate Alg-CHO, preparing an aqueous solution containing 5 wt% Alg-CHO, immersing the PP-ABTA / NH2 obtained in step (2) in the aqueous solution, and utilizing the reaction between the aldehyde group and the amino group to form a covalently cross-linked hydrogel coating material PP-ABTA / Alg;

[0084] (4) Prepare a 1 wt% ferric chloride aqueous solution, soak the PP-ABTA / Alg obtained in step (3) in the ferric chloride aqueous solution for 10 h, and then soak and clean it in deionized water for 12 h to obtain a coating material PP-ABTA / Alg / Fe that dually releases hydrogen sulfide / nitric oxide.

[0085] Example 5

[0086] This embodiment provides a method for preparing a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising:

[0087] (1) MA-PEG2000-PDITC was obtained by reacting methacrylate polyethylene glycol (MA-PEG2000) with p-phenylene diisothiocyanate (PDITC), and then reacted with 4-(hydroxymethyl)phenylboronic acid pinacol ester to obtain MA-PEG2000-PeroxyTCM, a donor monomer that releases hydrogen sulfide in response to active oxygen. Specifically, an anhydrous tetrahydrofuran solution containing 10 wt% MA-PEG2000, 3 wt% dibutyltin dilaurate (DBTDL), and 5 wt% PDITC was prepared, stirred at 60 °C for 24 h, precipitated in ether, and dried to obtain MA-PEG2000-PDITC.

[0088] (2) Utilizing the esterification reaction between the carboxyl group in disodium ethylenediaminetetraacetate (EDTA) and the hydroxyl group in hydroxyethyl methacrylate (HEMA), a methacrylated metal ligand compound MA-EDTA is obtained; specifically, an aqueous solution containing 10 wt% EDTA, 5 wt% HEMA, 5 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 0.5 wt% 4-dimethylaminopyridine (DMAP) is prepared, stirred at room temperature for 24 h, precipitated in tetrahydrofuran, and dried to obtain MA-EDTA;

[0089] (3) The polyether block polyamide substrate (PEBAX) was immersed in an aqueous solution containing 5 wt% MA-PEG2000- PeroxyTCM and 5 wt% MA-EDTA, and nitrogen was bubbled into the solution for 30 min. The solution was sealed and irradiated with 15 kGy γ-rays to obtain a coating material PEBAX-PeroxyTCM / EDTA containing hydrogen sulfide donors and metal ligands on the surface.

[0090] (4) preparing a PBS buffer solution containing 3 wt% gelatin (Gel) and 0.5 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), immersing the PEBAX-PeroxyTCM / EDTA obtained in step (3) in the buffer solution, and reacting at room temperature for 12 h to obtain a covalently cross-linked network hydrogel coating material PEBAX-PeroxyTCM / EDTA / Gel;

[0091] (5) Prepare a 1 wt% nickel chloride (NiCl2) aqueous solution, soak the PEBAX-PeroxyTCM / EDTA / Gel obtained in step (4) in the NiCl2 aqueous solution for 8 h, and then rinse with deionized water three times to obtain the dual hydrogen sulfide / nitric oxide-releasing coating material PEBAX-PeroxyTCM / EDTA / Ni / Gel.

[0092] Example 6

[0093] This embodiment provides a method for preparing a hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating, comprising:

[0094] (1) The stainless steel substrate (SS) was immersed in a 3M NaOH aqueous solution for 3 hours, and then rinsed with deionized water and ethanol to obtain a hydroxylated stainless steel substrate SS-OH;

[0095] (2) preparing a solution containing 5 wt% 3-(trimethoxymethylsilyl)propyl methacrylate (TMSPMA), 5 wt% H2O, and 90 wt% ethanol, and immersing the SS-OH obtained in step (1) in the solution, reacting at room temperature for 1 hour, and curing in a nitrogen atmosphere at 70°C for 1 hour to obtain a substrate SS-MA containing double bonds;

[0096] (3) Prepare an aqueous solution containing 5 wt % MA-PEG2000-ABTA (the preparation method is the same as that in Example 1), 5 wt % 2-aminoethyl methacrylate hydrochloride and 0.1 wt % I2959 (photoinitiator), immerse the SS-MA obtained in step (2) in the aqueous solution, bubble argon for 30 min, seal and expose to ultraviolet light, and then wash with deionized water three times to obtain a substrate SS-ABTA / NH2 containing hydrogen sulfide donors and amino groups on the surface.

[0097] (4) preparing a PBS buffer solution containing 5 wt% sodium alginate and 0.8 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), immersing the SS-ABTA / NH2 obtained in step (3) in the buffer solution, and reacting at room temperature for 12 h to obtain a covalently cross-linked network hydrogel coating material SS-ABTA / Alg;

[0098] (5) Prepare a 1 wt% copper chloride (CuCl2) aqueous solution, soak the SS-ABTA / Alg obtained in step (4) in the CuCl2 aqueous solution for 12 h, and then rinse with deionized water three times to obtain a coating material SS-ABTA / Alg / Cu that dually releases hydrogen sulfide / nitric oxide.

[0099] Comparative Example 1

[0100] This comparative example provides a method for preparing a comparative sample of a coating that releases only nitric oxide, comprising:

[0101] (1) A benzophenone / ethanol solution with a concentration of 5 wt% was prepared and nitrogen was passed through the solution for 30 min; a polylactic acid (PLLA) substrate was placed in the benzophenone / ethanol solution and exposed to ultraviolet light for 30 min to obtain a polylactic acid substrate PLLA-BP containing a photoinitiator;

[0102] (2) preparing a monomer aqueous solution containing 3 wt% methacryloyl sodium alginate (MA-Alg), immersing the PLLA-BP obtained in step (1) in the monomer solution, passing nitrogen for 30 min, and then irradiating with ultraviolet light for 30 min. After washing with deionized water to remove unreacted monomers, a substrate PLLA-Alg with sodium alginate covalently bonded to the surface is obtained;

[0103] (4) preparing a PBS buffer solution containing 3 wt% PEI and 0.5 wt% 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), immersing the PLLA-Alg obtained in step (2) in the buffer solution, and reacting at room temperature for 12 h to obtain a substrate PLLA-Alg / PEI of a double cross-linked network hydrogel coating material;

[0104] (5) Prepare a 1 wt% copper sulfate (CuSO4) aqueous solution, soak the PLLA-Alg / PEI obtained in step (4) in the copper sulfate aqueous solution for 24 h, and then soak and wash in deionized water for 24 h to obtain a nitric oxide-releasing coating comparison sample PLLA-Alg / Cu / PEI.

[0105] Test example

[0106] 1. Water contact angle

[0107] The water contact angle test was performed on the coating material PLLA-Alg / Cu / PEI-ABTA for dual release of hydrogen sulfide / nitric oxide obtained in Example 1, the coating comparison sample PLLA-Alg / PEI-ABTA for only releasing hydrogen sulfide, the coating comparison sample PLLA-Alg / Cu / PEI for catalytic release of nitric oxide obtained in Comparative Example 1, the coating comparison sample PLLA-Alg / PEI for only covalently binding hydrogel, and the blank substrate PLLA without coating treatment. The results are shown in FIG. Figure 1 .

[0108] 2. Nitric Oxide Release Rate

[0109] The nitric oxide release rate was determined using the Griess reagent method. Figure 2 shown.

[0110] 3. Hydrogen sulfide release rate

[0111] The hydrogen sulfide release rate was determined by the methylene blue method. Figure 3 shown.

[0112] 4. Platelet Adhesion and Activation

[0113] About 10 mL of rabbit venous blood was taken and placed in a centrifuge, and centrifuged at 2000 rpm for 15 minutes. Subsequently, the supernatant obtained was centrifuged again at 3500 rpm for 10 minutes, and platelet-rich plasma (PRP) was collected from the bottom of the centrifuge tube with a syringe. The PRP samples were divided into 48-well plates, and 500 μL of PRP, 20 μM S-nitroso-N-acetylpenicillamine (SNAP) and 20 μM glutathione (GSH) were added to each well, and incubated in a cell culture incubator at 37 °C for 1 hour. After incubation, the number of platelets adhered to the surface of the material was observed and counted by scanning electron microscopy (SEM), and the amount of P-selectin in PRP was measured using a P-selectin ELISA kit (Solarbio, Beijing) to reflect the activation of platelets. The platelet number results are shown in the following table. Figure 4 As shown in Figure 5 shown.

[0114] 5. Endothelial cell adhesion and proliferation

[0115] Human umbilical vein endothelial cells (ECs, from West China Hospital, Sichuan University) were cultured at 37 °C and 5% CO2. The culture medium was Dulbecco's modified DMEM containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 20 μM S-nitroso-N-acetylpenicillamine (SNAP) and 20 μM glutathione (GSH). 4 ECs were directly inoculated on the sample surface at a density of cells / mL. After incubation for 1 day and 3 days, the adhesion and proliferation of cells were observed using a fluorescence microscope. The results are as follows: Figure 6 As shown, the CCK-8 method was used to quantitatively evaluate cell activity and proliferation. Figure 7 shown.

[0116] 6. Subcutaneous implantation experiment

[0117] Healthy SD rats were selected, and the samples of the experimental group and the control group were implanted subcutaneously in the back of the rats under anesthesia. After 15 days and 30 days, the implanted samples and surrounding tissues were removed, rinsed with PBS solution, and fixed with 2.5% paraformaldehyde. The fixed tissues were stained with hematoxylin-eosin (H&E) and histologically analyzed. The results are shown in Figure 8 shown.

[0118] The experimental results show that H2S and NO exhibit good synergistic biological effects and can effectively improve the anticoagulant, anti-inflammatory and endothelialization properties of the material.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating, characterized in that: The raw materials of the coating include: Component A, component B, component C, and metal ions capable of catalyzing the release of nitric oxide from an endogenous nitric oxide donor; component A is bonded to a substrate; Component A at least includes: one or more double bonds, one or more groups I-1 that react chemically with component B, 0 or more groups I-2 that react chemically with component C, and 0 or more groups I-3 that coordinate with metal ions; Component B at least includes: two or more groups II-1 that react chemically with component A, zero or more groups II-2 that react chemically with component C, and zero or more groups II-3 that coordinate with metal ions; Component C comprises at least: one or more hydrogen sulfide donors, one or more groups III-1 that react chemically with group I-2 or group II-2; The number of groups I-2 and II-2 cannot be 0 at the same time; The number of groups I-3 and II-3 cannot be 0 at the same time.

2. The hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating according to claim 1, characterized in that: The coating comprises at least one of the features (1) to (2): (1) The hydrogen sulfide donor contains a group, and the group includes at least one of thioether, mercapto, disulfide, polysulfide, thioamide, thiocarbamate, thiocarbonate, thiocarboxylic acid, thiourea, thioacetal, thioketone, and thioxime; (2) The metal ion includes at least one of copper ion, iron ion, zinc ion, nickel ion, cobalt ion, manganese ion, cuprous ion, ferrous ion, stannous ion, trivalent cerium ion, and trivalent titanium ion.

3. The hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating according to claim 1, characterized in that: The coating includes at least one of features (3) to (4): (3) Group I-3 includes at least one of a carboxyl group, an amino group, a hydroxyl group, a thiol group, an ether bond, a pyridyl group, a thioether group, and an imine group; (4) Group II-3 includes at least one of a carboxyl group, an amino group, a hydroxyl group, a thiol group, an ether bond, a pyridyl group, a thioether group, and an imine group.

4. The hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating according to claim 1, characterized in that: The existence forms of the groups of component A, component B and component C independently include at least one of the following situations: Scenario 1: It is composed of a mixture of compounds containing each group; Case 2: It is composed of M1 compound and M2 compound, M1 compound has at least two groups selected arbitrarily, M2 compound is a compound containing a third group, and the third group is another group different from the two groups in M1 compound; when M1 compound has three groups, M2 compound is missing.

5. The hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating according to any one of claims 1 to 4, characterized in that: The group I-1 includes at least one of a carboxyl group, anhydride, acid chloride, active ester, isocyanate, thioisocyanate, epoxy, carbon-carbon triple bond, carbon-carbon double bond, azide, hydroxyl, amino, amino hydrochloride, halide, and carbonyl group; The definitions of group I-1 and group II-1 include at least one of features (5) to (9): (5) When group I-1 includes at least one of carboxyl, anhydride, acyl chloride, active ester, carbonyl, isocyanate, thioisocyanate, and epoxy, group II-1 includes at least one of hydroxyl, thiol, amino, and amino hydrochloride; (6) When group I-1 includes at least one of a carbon-carbon triple bond and a carbon-carbon double bond, group II-1 includes at least one of a thiol group, an amino group, an amino hydrochloride, an azide group, a carbon-carbon triple bond, and a carbon-carbon double bond; (7) When group I-1 includes azide, group II-1 includes a carbon-carbon triple bond; (8) When group I-1 includes at least one of hydroxyl, amino, and amino hydrochloride, group II-1 includes at least one of carboxyl, anhydride, acyl chloride, active ester, isocyanate, thioisocyanate, and epoxy; (9) When the group I-1 includes a halogen group, the group II-1 includes at least one of a hydroxyl group and an amino group.

6. The hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anti-coagulation and repair-promoting coating according to any one of claims 1 to 4, characterized in that: The group III-1 includes at least one of a carboxyl group, anhydride, acid chloride, active ester, isocyanate, thioisocyanate, epoxy, carbon-carbon triple bond, carbon-carbon double bond, azide, hydroxyl, amino, amino hydrochloride, halide, and carbonyl group; The definition of group III-1 and group I-1, and the definition of group III-1 and group II-2 include at least one of features (10) to (14): (10) When group III-1 includes at least one of carboxyl, anhydride, acyl chloride, active ester, carbonyl, isocyanate, thioisocyanate, and epoxy, group I-2 and group II-2 each independently include at least one of hydroxyl, thiol, amino, and amino hydrochloride; (11) When group III-1 includes at least one of a carbon-carbon triple bond and a carbon-carbon double bond, group I-2 and group II-2 each independently include at least one of a thiol group, an amino group, an amino hydrochloride, an azide group, a carbon-carbon triple bond, and a carbon-carbon double bond; (12) When group III-1 includes azide, group I-2 and group II-2 include a carbon-carbon triple bond; (13) When group III-1 includes at least one of hydroxyl, amino, and amino hydrochloride, group I-2 and group II-2 each independently include at least one of carboxyl, anhydride, acyl chloride, active ester, isocyanate, thioisocyanate, and epoxy; (14) When the group III-1 includes a halogen group, the group I-2 and the group II-2 each independently include at least one of a hydroxyl group and an amino group.

7. A method for preparing the hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating according to any one of claims 1 to 6, characterized in that: The steps include: S1 covalently connects substance N1 to the surface of the substrate through a surface polymerization reaction to obtain a first treated body; S2 placing the first processed body in a solution containing substance N2, and soaking the solution to obtain a second processed body; S3 placing the second processed body in a solution containing substance N3, and soaking the solution to obtain a third processed body; S4 placing the third treated body in a metal ion solution for immersion to obtain a coating; Substance N1, Substance N2, Substance N3 has any of the following limitations: (1) Substance N1 is component A, substance N2 is component B, and substance N3 is component C; (2) Substance N1 is the chemical reaction product of components A and C, and substance N2 is component B, lacking S3; (3) When substance N1 is component A and substance N2 is the chemical reaction product of component B and component C, S3 is missing. In this case, the third processed body in S4 is the second processed body.

8. The method for preparing the hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating according to claim 7, characterized in that: The concentration of component A is 1-100 wt%, the concentration of component B is 0.2-20 wt%, the concentration of component C is 1-20 wt%, and the concentration of metal ions is 0.1-10 wt%; B / A=0.1-20; C / (A+B+C)=0.01-0.

5.

9. The method for preparing the hydrogen sulfide / nitric oxide synergistic anti-inflammatory, anticoagulant and repair-promoting coating according to claim 8, characterized in that: B / A=0.2-2, C / (A+B+C)=0.1-0.

3.

10. Use of the coating according to any one of claims 1 to 6, or the coating obtained by the preparation method according to any one of claims 7 to 9 in the preparation of biomedical materials.

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