CN114632194A

CN114632194A - Coating material with long-acting NO catalytic release function, preparation method and application

Info

Publication number: CN114632194A
Application number: CN202210374500.6A
Authority: CN
Inventors: 杨志禄; 杜泽煜; 黄楠; 牟小辉; 张文泰; 王颖; 杨梦毅
Original assignee: Dongguan Peoples Hospital
Current assignee: Dongguan Peoples Hospital
Priority date: 2022-04-11
Filing date: 2022-04-11
Publication date: 2022-06-17
Anticipated expiration: 2042-04-11
Also published as: CN114632194B

Abstract

The invention discloses a coating material with long-acting NO catalytic release, a preparation method and application thereof, and relates to the technical field of biological materials. Firstly, reacting carboxyl-or NHS-modified tetraazacyclic compound with nitric oxide catalytic release molecules, introducing the nitric oxide catalytic release molecules through chelation, and then reacting with amino compound to obtain modified amino compound; the modified amino compound and the phenolic compound containing carboxyl are reacted to obtain a phenol-amine-rich condensation compound, and then the base material is deposited in the phenol-amine-rich condensation compound solution under the alkaline condition to obtain the coating. The coating can realize the function of controllable nitric oxide catalytic release, and is a coating with high chemical stability; the coating has the advantages of improving the poor chemical stability of the existing phenolic amine coating, improving the efficiency of further functional modification of the coating, having excellent adhesion performance with the surface of a material, being not easy to deteriorate and damage when used under extreme conditions, and having excellent use safety.

Description

Coating material with long-acting NO catalytic release function and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a coating material with long-acting NO catalytic release, a preparation method and application thereof.

Background

In nature, marine mussels can strongly bind to the surfaces of various organic and inorganic materials by secreting byssus proteins containing various proteins. When the byssus protein is initially contacted with the surface, the catechol group and the interface form covalent and non-covalent interactions as main components, such as charge-charge interaction, pi-pi interaction, cation-pi interaction, coordination, hydrogen bond interaction, hydrophobic effect and the like. This time-scale dependent adhesion is regulated by factors such as pH and other ions in the environment. Due to the complex phenol-amine chemical reaction, the oxidation speed and the products of phenol can not be well regulated, and a plurality of oxidation products participate in the polymerization of the coating to generate a large amount of phenol oligomers. Although the adhesive coating can form a strong adhesive force with various organic and inorganic surfaces, the oligomer formed by non-covalent bonds in the polymerization process causes the chemical stability of the coating under the condition of partial acid-base to be poor, and the application of the coating under some extreme conditions cannot be met. Meanwhile, the surface of the coating retains primary amine groups with secondary reaction activity, which are mainly contributed by phenolic compounds, and the secondary functional modification of the surface of the coating is far from being satisfied.

NO is secreted by endothelial cells in natural blood vessels in the body and is an important factor in maintaining cardiovascular homeostasis and regulating vasodilation. NO plays an important role in promoting adhesion proliferation of endothelial cells, inhibiting platelet activation and smooth muscle proliferation in natural blood vessels. In recent years, the "metal-phenol-amine" surface chemistry modification strategyIs rapidly developed, and Cu with NO catalytic activity is selected²⁺Or the organic selenium compound can realize the long-term catalytic release of the coating surface.

However, the realization of the current NO-catalytic materials mainly depends on covalent grafting on the surface of the coating or chelating active molecules with catalytic release of NO through surface groups, the release amount of the materials is limited by the grafting amount of the surface active molecules and the catalytic efficiency, and the physiological environments with the requirements of large-dose NO and stable dose applied in different physiological environments are difficult to meet.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a coating material with long-acting NO catalytic release, a preparation method and application thereof, and aims to prepare a high-chemical-stability coating with nitric oxide catalytic release.

The invention is realized by the following steps:

in a first aspect, the present invention provides a method for preparing a coating material with long-acting NO catalytic release, comprising: reacting carboxyl-or NHS-modified tetraazacyclic compound with nitric oxide catalytic release molecules to obtain tetraazacyclic compound chelated with nitric oxide catalytic release molecules, and reacting the tetraazacyclic compound chelated with nitric oxide catalytic release molecules with amino compound to obtain modified amino compound;

reacting a carboxyl-containing phenolic compound with a modified amino compound to obtain a phenol-amine-rich condensation compound;

the substrate is placed in a solution of a phenol-amine rich condensation compound and the coating deposition is carried out under alkaline conditions.

In an optional embodiment, the carboxyl-or NHS-modified tetraazacyclic compound and the nitric oxide catalytic release molecule are subjected to ultrasonic reaction for 0.1h to 3h at the temperature of 0 to 240 ℃, and then subjected to centrifugal purification and then subjected to reaction with the amino compound for 0.001h to 240h at the temperature of 0 to 240 ℃;

preferably, the reaction temperature of the carboxyl-or NHS-modified tetraazacyclo compound and nitric oxide catalytic release molecules is 0-30 ℃, and the reaction time is 0.1-0.5 h; the reaction temperature of the amino compound and the amino compound is 0 to 30 ℃, and the reaction time is 0.01 to 24 hours.

In an alternative embodiment, the nitric oxide catalytically releasing molecule is selected from at least one of copper chloride, cuprous chloride, cupric bromide, cuprous bromide, cupric iodide, cuprous iodide, cupric sulfate, selenocysteine, cystine, L-selenocysteineacetic acid, and cysteine and derivatives thereof;

preferably, the nitric oxide catalytic release molecule is a compound containing cupric ions or monovalent copper ions, and the dosage of the nitric oxide catalytic release molecule is controlled by controlling the molar ratio of the cupric ions to the tetraazacyclo-nitrogen to be 0.01-1: 5; preferably 0.01 to 1: 2.

in an alternative embodiment, the amine-based compound is selected from at least one of an aliphatic long-chain amine-based compound and an aromatic polyamine-based compound;

preferably, the amine-based compound is selected from at least one of polyallylamine, polyvinylamine, polylysine, polyethylene glycol bis (amine), polyetheramine, polyamide, melamine, poly-N-isopropylacrylamide and chitosan and derivatives thereof; more preferably polyallylamine;

preferably, the amine compound is used in an amount to control the molar ratio of carboxyl groups to amine groups to be 0.001-2: 1; preferably 0.005-0.02: 1.

In an alternative embodiment, the carboxyl-containing or NHS-modified tetraazacyclo compound is selected from at least one of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, NHS-modified 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, tri-tert-butyl 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid and derivatives thereof; preferably 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid;

preferably, the carboxyl-containing tetraazacyclo compound solution is mixed with an acidic buffer solution with the pH value of 4-10, and the mixture reacts with a condensing agent for 0.001-240 h at the temperature of 0-240 ℃ and then reacts with nitric oxide catalytic release molecules;

preferably, the concentration of the carboxyl group-containing tetraazacyclic compound solution is from 0.01ng/mL to 5g/mL, more preferably from 0.1 to 1 mg/mL.

In an alternative embodiment, the phenolic compound containing carboxyl is firstly mixed with a buffer solution with the pH value of 4-10, and then reacts with the modified amino compound after reacting with the condensing agent for 0.001-240 h at the temperature of 0-240 ℃, and the reaction temperature is controlled to be 0-240 ℃ and the reaction time is controlled to be 0.001-240 h;

preferably, the reaction temperature of the condensation agent is 0-30 ℃, and the reaction time is 0.5-48 h; the reaction temperature of the modified amino compound is 0-30 ℃, and the reaction time is 0.5-48 h;

preferably, the carboxyl group-containing phenolic compound is used in an amount such that the molar ratio of the carboxyl group to the amine group in the modified amine group compound is controlled to 0.001-2:1, preferably 0.005-0.02: 1;

preferably, the carboxyl group-containing phenolic compound is a monoamine phenolic compound; more preferably, the phenolic compound having a carboxyl group is at least one selected from the group consisting of caffeic acid, hydrogenated caffeic acid, dihydroxyphenylalanine, gallic acid, tannic acid, ferulic acid, 2, 3-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid, and derivatives thereof.

In an alternative embodiment, the condensation agent used during the reaction of the carboxyl group-containing tetraazacyclic compound and the carboxyl group-containing phenolic compound with the condensation agent is selected from at least one of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, carbodiimide hydrochloride, 1, 3-diisopropylcarbodiimide, and dicyclohexylcarbodiimide;

preferably, the amount of the condensing agent is controlled so that the molar ratio of the condensing agent to the carboxyl groups is 0.1 to 10: 1;

preferably, the pH value of the buffer solution adopted in the reaction process of the carboxyl-containing tetraazacyclo compound and the carboxyl-containing phenolic compound and the condensing agent is 5-8;

preferably, the buffer solution is selected from at least one of 2- (N-morpholino) ethanesulfonic acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

In an alternative embodiment, the substrate is placed in a solution rich in the phenol-amine condensation compound, an alkaline buffer is added to adjust the pH value to 7-14, and the reaction is carried out for 0.001h-240h at the temperature of 0-240 ℃;

preferably, the reaction temperature of the base material and the rich phenol-amine condensation compound is 0-45 ℃, the reaction time is 0.1-48h, the reaction pH value is 7-11, and cleaning and drying are carried out after the reaction;

preferably, the functional modification is performed after the substrate has been reacted with the phenol-amine rich condensation compound, the functional modification being a reaction with a biologically active molecule with a carboxyl, thiol or NHS-group;

preferably, the alkaline buffer solution is a sodium hydroxide solution with the concentration of 0.00001g/L-40 g/L;

preferably, the substrate is selected from at least one of a metal material, an inorganic material, and a polymer material;

more preferably, the substrate is selected from at least one of stainless steel, iron and its alloys, magnesium alloys, zinc-manganese alloys, cobalt-based alloys, titanium alloys, ferroferric oxide nanoparticles, ceramics, silica, polymethylmethacrylate, polystyrene, silicone rubber, and polyurethane.

In a second aspect, the present invention provides a coating material with long-acting NO catalytic release, prepared by the preparation method of any one of the preceding embodiments.

In a third aspect, the present invention provides the use of a coating material with long-acting NO catalytic release according to the previous embodiments for the preparation of a blood implant device.

The invention has the following beneficial effects: firstly, carboxyl-or NHS-modified tetraazacyclic compound is utilized to react with nitric oxide catalytic release molecules, the nitric oxide catalytic release molecules are introduced through chelation, and then the nitric oxide catalytic release molecules react with amino compound to obtain modified amino compound; the modified amino compound and the phenolic compound containing carboxyl are reacted to obtain a phenol-amine-rich condensation compound, and then the base material is deposited in the phenol-amine-rich condensation compound solution under the alkaline condition to obtain the coating. The coating can realize the function of controllable nitric oxide catalytic release, and is a coating with high chemical stability; the phenolic amine coating has the advantages of improving the poor chemical stability of the existing phenolic amine coating, improving the efficiency of further functionalization and modification of the coating, having excellent adhesion performance with the surface of a material, being not easy to deteriorate and damage under extreme conditions, and having excellent use safety.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 shows the results of chemical stability tests of the coating prepared by the method;

FIG. 2 shows the results of the nitric oxide catalyzed release test in the coating of the comparative example;

FIG. 3 shows the detection result of the catalytic release of nitric oxide by the coating prepared by the method.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides a preparation method of a coating material with long-acting NO catalytic release, which comprises the following steps:

s1 preparation of modified amino compound

The carboxyl-or NHS-modified tetraazacyclo compound is reacted with nitric oxide catalytic release molecules to obtain the tetraazacyclo compound chelated with the nitric oxide catalytic release molecules, and the tetraazacyclo compound chelated with the nitric oxide catalytic release molecules is reacted with an amino compound to obtain the modified amino compound.

It should be noted that the selection of the tetranitrogen heterocyclic compound can realize Cu²⁺The high-efficiency fixation and chelation of the nitric oxide catalytic release molecules can not only improve the long-term stable release efficiency of NO, but also can be carried out according to the actual application partThe NO demand changes the grafting amount of the catalytically active molecules to realize controllable NO catalytic release.

In some embodiments, carboxyl-or NHS-modified tetraazacyclic compounds are subjected to an ultrasonic reaction with nitric oxide-catalyzed releasing molecules at 0-240 ℃ for 0.1h-3h, and after centrifugal purification, are subjected to a reaction with amine-based compounds at 0-240 ℃ for 0.001h-240 h; preferably, the reaction temperature of the carboxyl-or NHS-modified tetraazacyclo compound and the nitric oxide catalytic release molecule is 0-30 ℃, and the reaction time is 0.1-0.5 h; the reaction temperature of the reaction product and the amino compound is 0-30 ℃, and the reaction time is 0.01h-24 h; the dosage of the amino compound is that the molar ratio of the carboxyl to the amino is controlled to be 0.001-2: 1; preferably 0.005-0.02: 1. Chelating a tetraazacyclo compound and nitric oxide catalytic release molecules under an ultrasonic condition, and further realizing the fixation of molecules with NO catalytic activity. And then adding an amino compound, and after the reaction is finished, purifying the synthesized molecule by a dialysis method for later use. The reaction temperature, the reaction time and the dosage ratio of the two-step reaction are optimized to ensure that the reaction is sufficient, and the introduction amount of copper ions and amino compounds is increased.

It should be noted that, in the process of synthesizing molecules, the concentration of molecules, the reaction temperature and the reaction time have a significant influence on the synthesis effect of molecules, the synthesis reaction can be completed at 37 ℃, the synthesis time is not too short, and the reaction time shorter than 0.5 hour cannot obtain ideal functional molecules.

In some embodiments, the carboxyl-containing or NHS-modified tetraazacyclododecane compound is selected from at least one of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), NHS-modified 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (NHS-DOTA), tri-tert-butyl 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid and derivatives thereof; preferably 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid. The functional molecules for fixing the catalytic activity of NO are common and easy to obtain, and the tetraazacyclic compound in the embodiment of the invention is not limited to the above functional molecules.

In some embodiments, the nitric oxide-catalytically releasing molecule is selected from at least one of copper chloride, cuprous chloride, cupric bromide, cuprous bromide, cupric iodide, cuprous iodide, cupric sulfate, selenocysteine, cystine, L-selenocysteineacetic acid, cysteine and derivatives thereof, and may be one or more. The above-mentioned several NO catalytic active substances are relatively common and readily available, and the nitric oxide catalytic release molecules referred to in the embodiments of the present invention are not limited to the above-mentioned several substances.

In a preferred embodiment, the nitric oxide catalytic releasing molecule is a compound containing divalent copper ions or monovalent copper ions, and the amount of the nitric oxide catalytic releasing molecule is controlled by controlling the molar ratio of the copper ions to the tetraazacyclo ring to be 0.01-1: 5; preferably 0.01 to 1: 2. by optimizing nitric oxide catalytic release molecules, the stability of NO release of the prepared coating material is improved.

In some embodiments, the amine-based compound is at least one selected from aliphatic long-chain amine-based compounds and aromatic polyamine-based compounds, and may be one or more. Preferably, the amine-based compound is at least one selected from polyallylamine, polyvinylamine, polylysine, polyethylene glycol bis (amine), polyetheramine, polyamide, melamine, poly-N-isopropylacrylamide and chitosan and derivatives thereof; more preferably polyallylamine. By optimizing the amino compound, the introduction amount of the amino is favorably improved, and other functional molecules are conveniently introduced subsequently.

If the adopted tetraazacyclo compound does not have NHS modification and only contains carboxyl, the carboxyl needs to be activated and then reacted with nitric oxide catalytic release molecules. In the actual operation process, the carboxyl-containing tetraazacyclo compound solution is mixed with an acidic buffer solution with the pH value of 4-10, and reacts with a condensing agent for 0.001-240 h at the temperature of 0-240 ℃ and then reacts with nitric oxide catalytic release molecules; the concentration of the carboxyl-containing tetraazacyclic compound solution is 0.01ng/mL-5 g/mL.

In a preferred embodiment, the concentration of the carboxyl-containing tetraazacyclic compound solution is 0.1-1mg/mL, the pH value of the buffer solution adopted in the reaction process is 5-8, and the dosage of the condensing agent is to control the molar ratio of the condensing agent to the carboxyl to be 0.1-10:1 so as to achieve the effect of full activation and increase the introduction amount of the amine group.

In some embodiments, the condensation agent used during the reaction of the carboxyl group-containing tetraazacyclic compound and the carboxyl group-containing phenolic compound with the condensation agent is selected from at least one, and may be one or more, of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4-Dimethylaminopyridine (DMAP), carbodiimide hydrochloride (EDCI), 1, 3-Diisopropylcarbodiimide (DIC), and Dicyclohexylcarbodiimide (DCC). Preferably EDC and NHS.

In some embodiments, the buffer solution is selected from at least one of a 2- (N-morpholino) ethanesulfonic acid (MES) buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, and a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably a MES buffer solution.

In a preferred embodiment, the modified amine compound may be polyallylamine (DOTA-Cu/pam) modified with macrocyclic polyamine chelated copper ions, polyallylamine (DOTA-Cu/pam) modified with NHS-modified macrocyclic polyamine chelated copper ions, and derivatives thereof.

S2 preparation of Rich phenol-amine condensation Compound

The carboxyl-containing phenolic compound and the modified amino compound are reacted to obtain the rich phenol-amine condensation compound, the condensation of the carboxyl phenol and the amino compound increases the combination efficiency of a phenolic hydroxyl group and a substrate interface, and the introduction of the amino compound with more carbon content also increases the cross-linking polymerization of the coating, so that the coating has more excellent chemical resistance and can be suitable for environments with extreme acid, extreme alkali and strong oxidizing property.

In the actual operation process, the phenolic compound containing carboxyl is firstly mixed with the buffer solution with the pH value of 4-10, and then reacts with the modified amino compound after reacting with the condensing agent for 0.001-240 h at the temperature of 0-240 ℃, and the reaction temperature is controlled to be 0-240 ℃ and the reaction time is controlled to be 0.001-240 h. The amount of the phenolic compound containing carboxyl is controlled to be 0.001-2:1 of the molar ratio of the carboxyl to the amino in the modified amino compound. The reaction can be completed by controlling the temperature, time and the amount of the raw materials within the above ranges.

In a preferred embodiment, the reaction temperature with the condensing agent is 0-30 ℃, and the reaction time is 0.5-48 h; the reaction temperature of the modified amino compound and the modified amino compound is 0-30 ℃, the reaction time is 0.5-48h, and the molecules synthesized by the reaction are purified in oxygen-free water by a dialysis method for later use. The amount of the phenolic compound containing carboxyl is controlled to be 0.005-0.02:1 of the molar ratio of the carboxyl to the amino in the modified amino compound. The reaction temperature, the reaction time and the dosage ratio of the raw materials are optimized so as to ensure that the reaction is carried out more fully.

It should be noted that, in the process of the phenol-amine-rich condensation reaction, the concentration of molecules, the oxygen content in the solution, the reaction temperature and the reaction time have significant influence on the molecular synthesis effect, and the synthesis can be performed at 25 ℃, the synthesis time is not short enough, and the ideal molecules cannot be obtained when the reaction time is shorter than 0.5 hour.

In some embodiments, the carboxyl group-containing phenolic compound may be a monoamine phenolic compound, and the specific kind is not limited. Specifically, the phenolic compound containing carboxyl is at least one selected from caffeic acid, hydrogenated caffeic acid, dihydroxyphenylalanine, gallic acid, tannic acid, ferulic acid, 2, 3-dihydroxybenzoic acid, 3, 4-dihydroxybenzoic acid and their derivatives, and can be one or more. The above mentioned several kinds of phenolic compounds containing carboxyl are relatively common and easily available, and the phenolic compounds containing carboxyl in the embodiments of the present invention are not limited to the above mentioned ones.

Similarly to step S1, the condensing agent used in the reaction of the carboxyl group-containing phenolic compound with the condensing agent is at least one selected from the group consisting of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, carbodiimide hydrochloride, 1, 3-diisopropylcarbodiimide, and dicyclohexylcarbodiimide; may be one or more, preferably EDC and NHS. The amount of the condensing agent to be used is preferably such that the molar ratio of the condensing agent to the carboxyl group is 0.1 to 10: 1. Preferably, the pH value of the buffer solution adopted in the reaction process of the carboxyl-containing tetraazacyclo compound and the carboxyl-containing phenolic compound and the condensing agent is 5-8; the buffer solution is at least one selected from 2- (N-morpholino) ethanesulfonic acid (MES) buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.

S3, coating deposition

The base material is put into a solution rich in a phenol-amine condensed compound, and coating deposition is carried out under an alkaline condition, so that a coating which can continuously catalyze and stably release nitric oxide, has higher chemical stability and strong adhesive property is obtained.

In the actual operation process, the base material is placed in a solution of a phenol-amine-rich condensation compound, an alkaline buffer solution is added to adjust the pH value to 7-14, and the reaction is carried out for 0.001-240 h at the temperature of 0-240 ℃; preferably, the reaction temperature of the base material and the rich phenol-amine condensation compound is 0-45 ℃, the reaction time is 0.1-48h, the reaction pH value is 7-11, and the base material and the rich phenol-amine condensation compound are cleaned and dried after the reaction. The reaction temperature, time and pH value are optimized to ensure the deposition amount on the substrate.

The kind of the base material is not limited, and can be selected according to specific requirements, such as a metal material, an inorganic material, a polymer material, and the like. Specifically, the base material may be at least one selected from stainless steel, iron and its alloy, magnesium alloy, zinc-manganese alloy, cobalt-based alloy, titanium alloy, ferroferric oxide nanoparticles, ceramic, silica, polymethyl methacrylate, polystyrene, silicone rubber, and polyurethane, and may be one or more. The materials are suitable for preparing the o-phenol polymerization modified layer and are widely used for preparing biological materials.

In some embodiments, the alkaline buffer is a sodium hydroxide solution with a concentration of 0.00001g/L to 40g/L, and other alkaline solutions are also possible.

The coating surface retains abundant primary amine groups, so that the coating has the reaction characteristic of wide secondary reaction grafting functional groups, and the material has multiple biological function application prospects. In some embodiments, the functionalized modification is performed after the substrate has been reacted with the phenol-amine rich condensation compound, and the functionalized modification is reacted with a biologically active molecule having a carboxyl, thiol, or NHS group, such as heparin, bivalirudin, hyaluronic acid, and the like.

The embodiment of the invention provides a coating material with long-acting NO catalytic release, which is prepared by the preparation method, is a coating with sustained catalytic stable release of nitric oxide, high chemical stability and strong adhesion performance, and can be applied to preparation of blood implantation devices.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a preparation method of a coating material with long-acting NO catalytic release, which selects 316L stainless steel as a substrate material for coating preparation, and comprises the following steps:

A. amino compound modified by nitric oxide catalytic active molecules

Mixing 1mg/mL carboxyl group-containing DOTA solution with MES acidic buffer solution adjusted to pH 5.6, adding EDC and NHS (EDC to carboxyl group molar ratio of 5:1, NHS to carboxyl group molar ratio of 1:1, the same below), reacting at 37 deg.C for 0.5h, adding 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.01: polyallylamine (weight average molecular weight 8,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL hydrogenated caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS (EDC and carboxyl in a molar ratio of 5:1, NHS and carboxyl in a molar ratio of 1:1, the same applies below), reacting at 37 ℃ for 0.5h, adding carboxyl and DOTA-Cu/pPAM in an amine group molar ratio of 0.01: 1, reacting at 10 ℃ for 24h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

And (4) placing the sample to be modified in the molecular solution obtained in the step B, adding an alkaline oxygen-free buffer solution to adjust the pH value to 9, reacting for 24 hours at 37 ℃, and cleaning and drying to obtain the coating.

Example 2

A. amino compound modified by nitric oxide catalytic active molecules

Mixing 1mg/mL solution of DOTA containing 4 carboxyl groups with MES acidic buffer solution adjusted to pH 5.6, adding EDC and NHS, reacting at 37 deg.C for 0.5h, adding a mixture of DOTA and water in a molar ratio of 1:1 of CuCl₂Ultrasonic reaction for 0.1h, centrifugal purification, and then adding a mixture of carboxyl and amino with the molar ratio of 0.01: polyallylamine (weight average molecular weight 8,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding carboxyl and DOTA-Cu/pPAM with amine group molar ratio of 0.01: 1, reacting at 4 ℃ for 24h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

Example 3

A. amino compound modified by nitric oxide catalytic active molecules

Mixing 1mg/mL of carboxyl-containing DOTA solution with MES acidic buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding a catalyst with a molar ratio of 1:1, carrying out ultrasonic reaction for 0.5h, carrying out centrifugal purification, and then adding a mixture of CuCl2 with a carboxyl-amine group molar ratio of 0.01: polyallylamine (weight average molecular weight 8,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL of 2, 3-dihydroxybenzoic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 25 ℃ for 0.5h, and adding carboxyl and DOTA-Cu/pPAM with the amino molar ratio of 0.01: 1, reacting at 10 ℃ for 24h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

Example 4

A. amino compound modified by nitric oxide catalytic active molecules

Mixing 2mg/mL of carboxyl-containing DOTA solution with MES acidic buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding a catalyst with a molar ratio of 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.01: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL of 2, 3-dihydroxybenzoic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 25 ℃ for 0.5h, and adding carboxyl and DOTA-Cu/pPAM with the amino molar ratio of 0.01: 1, reacting at 25 ℃ for 24h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

Example 5

A. amino compound modified by nitric oxide catalytic active molecules

Mixing 2mg/mL of carboxyl-containing DOTA solution with MES acidic buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding a catalyst with a molar ratio of 1:1 of CuCl₂Ultrasonic reaction for 0.1h, centrifugal purification, and then adding a mixture of carboxyl and amino with the molar ratio of 0.01: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL hydrogenated caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 25 ℃ for 0.5h, adding carboxyl and the amine group of DOTA-Cu/pPAM in a molar ratio of 0.01: 1, reacting at 4 ℃ for 12h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

Example 6

A. amino compound modified by nitric oxide catalytic active molecules

Mixing 1mg/mL of carboxyl-containing DOTA solution with MES acidic buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding a catalyst with a molar ratio of 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.19: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding carboxyl and DOTA-Cu/pPAM with amine group molar ratio of 0.01: 1, reacting at 10 ℃ for 12h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

And (4) placing the sample to be modified in the molecular solution obtained in the step B, adding an alkaline oxygen-free buffer solution to adjust the pH value to 9, reacting for 12 hours at 25 ℃, and cleaning and drying to obtain the coating.

Example 7

A. amino compound modified by nitric oxide catalytic active molecules

Mixing a 1mg/mL NHS-DOTA solution with a PBS buffer solution with the pH adjusted to 8.5, adding a 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.2: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 2.3mg/mL hydrogenated caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 37 deg.C for 0.5h, adding carboxyl and DOTA-Cu/pPAM with amine group molar ratio of 0.19: 1, reacting at 10 ℃ for 12h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

And (4) placing the sample to be modified in the molecular solution obtained in the step B, adding an alkaline oxygen-free buffer solution to adjust the pH value to 9, reacting for 12 hours at 37 ℃, and cleaning and drying to obtain the coating.

Example 8

A. amino compound modified by nitric oxide catalytic active molecules

Mixing a 1mg/mL NHS-DOTA solution with a PBS buffer solution with the pH adjusted to 8.5, adding a 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.1: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding carboxyl and DOTA-Cu/pPAM with amine group molar ratio of 0.19: 1, reacting at 4 ℃ for 36h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

And (4) placing the sample to be modified in the molecular solution obtained in the step B, adding an alkaline oxygen-free buffer solution to adjust the pH value to 9, reacting for 24 hours at 25 ℃, and cleaning and drying to obtain the coating.

Example 9

A. amino compound modified by nitric oxide catalytic active molecules

Mixing a 1mg/mL NHS-DOTA solution with a PBS buffer solution with the pH adjusted to 8.5, adding a 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.5: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL of 2, 3-dihydroxybenzoic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 25 ℃ for 0.5h, and adding carboxyl and DOTA-Cu/pPAM with an amino molar ratio of 0.3: 1, reacting at 4 ℃ for 24 hours, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

Example 10

The embodiment provides a preparation method of a coating material with long-acting NO catalytic release, which selects nickel-titanium alloy as a base material for coating preparation, and comprises the following steps:

A. amino compound modified by nitric oxide catalytic active molecules

Mixing a 1mg/mL NHS-DOTA solution with a PBS buffer solution with the pH adjusted to 8.5, adding a 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.01: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 3mg/mL caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.2h, adding carboxyl and DOTA-Cu/pPAM with amino molar ratio of 0.1: 1, reacting at 5 ℃ for 24h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

Example 11

The embodiment provides a preparation method of a coating material with long-acting NO catalytic release, iron and an alloy thereof are selected as a substrate material for coating preparation, and the preparation method comprises the following steps:

A. amino compound modified by nitric oxide catalytic active molecules

The 1mg/mL NHS-DOTA solution was mixed with PBS buffer adjusted to pH 8.5, and the 1:1 of CuCl₂Ultrasonic reaction for 0.2h, centrifugal purification, and then adding a mixture of carboxyl and amino with a molar ratio of 0.5: polyallylamine (weight average molecular weight 17,000,20 wt.% aqueous solution) of 1, after reaction at 37 ℃ for 24h, the synthesized molecule (DOTA-Cu/pam) was purified by dialysis for use.

B. Phenol-rich amine compound condensation

Dissolving 1mg/mL caffeic acid in MES buffer solution completely free of oxygen and adjusted to pH 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding carboxyl and DOTA-Cu/pPAM with amine group molar ratio of 0.5: 1, reacting at 25 ℃ for 12h, and purifying the synthesized molecules in oxygen-free water by a dialysis method for later use.

C. Preparation of the coating

And (4) putting a sample to be modified into the molecular solution obtained in the step B, adding an alkaline oxygen-free buffer solution to adjust the pH value to 9, reacting for 24 hours at 37 ℃, and cleaning and drying to obtain the coating.

Comparative example 1

The difference from example 1 is that: the coating is formed by oxidative self-polymerization using conventional catecholamine compounds, without the introduction of amine-based compounds.

Comparative example 2

The difference from example 1 is that: changing the introduction mode of copper ions, comparative example 2 covalently fixes macrocyclic polyamine through the surface amine group of the coating, and then chelates copper; according to the method, macrocyclic polyamine chelated with copper ions is grafted to the long chain of an amino compound to be introduced into the coating, so that the copper ions in the coating are fixed.

Test example 1

The chemical stability of the coatings of example 1 and comparative example 1 was examined and the results are shown in figure 1.

The detection method comprises the following steps: the coating obtained in example 1 was immersed in a strong acid (pH 0),Strong base (pH 14) and strong oxidizing property (H)₂O₂Mass fraction 30%) 24h later, the morphology of the coating surface was compared with the polymerized coating under neutral conditions (pH 8.5) and recorded by photographing.

As can be seen from FIG. 1, the NO catalytic release coating obtained by the preparation method has strong chemical stability, and can still maintain a complete coating under the conditions of extreme strong acid, strong base and strong oxidation. The coating prepared by the method is proved to have more excellent chemical stability compared with the traditional catechol coating, the integrity of the coating can not be damaged even under extreme environment, and the method has wider application field.

Test example 2

The NO catalytic release detection effect of the coating of comparative example 2 is shown in FIG. 2.

The test method comprises the following steps: the effect of the NO catalytic release rate was measured in PBS at 37 ℃ using a chemiluminescent NO analyzer (NOA) (sensors 280i, Boulder, CO).

FIG. 2 shows that the coating prepared by the method of comparative example 2 has a relatively obvious burst release during early NO catalytic release, and the NO catalytic release capacity of the coating gradually decreases with the lapse of time; the coating is pulled out and then inserted into the reaction solution again, the catalytic NO release capacity of the coating is obviously reduced compared with that of the coating which is firstly obviously reduced, and the result also suggests that the mode of adopting the step-by-step grafting functional molecules to chelate copper ions has certain limitation on the stable catalytic NO release. The low utilization rate of copper ions reduces the stability of NO catalytic release, so that the catalytic rate and the stability of the copper ions are obviously reduced.

Test example 3

The effect of the NO catalytic release test of the coating of example 1 is shown in FIG. 3.

The test method comprises the following steps: this was the same as in test example 2.

As can be seen from fig. 3, the burst release of the coating obtained by the preparation method in the early NO catalytic release process is significantly reduced compared with the coating of comparative example 2, and the burst release of NO is reduced by about 5 times. The NO catalytic release capacity of the coating does not show a descending trend in the process of catalytically releasing NO by the coating of the comparative example 2 over time, and the stable catalytic release capacity is always maintained and the initial NO catalytic release capacity is also maintained over time. The detection result directly proves that the coating prepared by the method has the performance of long-acting stable catalytic release of NO.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a coating material with long-acting NO catalytic release is characterized by comprising the following steps: reacting carboxyl-or NHS-modified tetraazacyclo compound with nitric oxide catalytic release molecules to obtain a tetraazacyclo compound chelated with nitric oxide catalytic release molecules, and reacting the tetraazacyclo compound chelated with nitric oxide catalytic release molecules with an amino compound to obtain a modified amino compound;

reacting a carboxyl-containing phenolic compound with the modified amino compound to obtain a phenol-amine-rich condensation compound;

the substrate is placed in the solution of the phenol-amine rich condensation compound and the coating deposition is carried out under alkaline conditions.

2. The preparation method of claim 1, wherein the carboxyl-or NHS-modified tetraazacyclic compound is subjected to an ultrasonic reaction with the nitric oxide-catalyzed releasing molecule at 0-240 ℃ for 0.1h-3h, and after centrifugal purification is subjected to a reaction with the amine-based compound at 0-240 ℃ for 0.001h-240 h;

preferably, the reaction temperature of the carboxyl-or NHS-modified tetraazacyclic compound and the nitric oxide catalytic release molecule is 0-30 ℃, and the reaction time is 0.1-0.5 h; the reaction temperature of the amino compound is 0-30 ℃, and the reaction time is 0.01-24 h.

3. The method of claim 2, wherein the nitric oxide catalytically releasing molecule is selected from at least one of copper chloride, cuprous chloride, cupric bromide, cuprous bromide, cupric iodide, cuprous iodide, cupric sulfate, selenocysteine, cystine, L-selenocysteineacetic acid, and cysteine, and derivatives thereof;

4. the production method according to claim 2, wherein the amine-based compound is at least one selected from the group consisting of an aliphatic long-chain amine-based compound and an aromatic polyamine-based compound;

preferably, the dosage of the amine compound is to control the molar ratio of carboxyl to amine to be 0.001-2: 1; preferably 0.005-0.02: 1.

5. The method according to claim 2, wherein the carboxyl-or NHS-modified tetraazacyclo compound is at least one selected from the group consisting of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, NHS-modified 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, tri-tert-butyl-1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, and derivatives thereof; preferably 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid;

preferably, carboxyl-containing tetraazacyclic compound solution is mixed with acidic buffer solution with the pH value of 4-10, and the mixture reacts with the condensing agent for 0.001-240 h at the temperature of 0-240 ℃ and then reacts with the nitric oxide catalytic release molecule;

preferably, the concentration of the carboxyl-containing tetraazacyclic compound solution is 0.01ng/mL to 5g/mL, more preferably 0.1 to 1 mg/mL.

6. The preparation method of claim 5, wherein the phenolic compound containing carboxyl is mixed with the buffer solution with the pH value of 4-10, and reacts with the condensing agent for 0.001h-240h at 0-240 ℃, and then reacts with the modified amino compound, wherein the reaction temperature is controlled to be 0-240 ℃ and the reaction time is controlled to be 0.001h-240 h;

preferably, the amount of the phenolic compound containing carboxyl is controlled to be 0.001-2:1, preferably 0.005-0.02:1 in molar ratio of carboxyl to amine in the modified amine compound;

7. The method according to claim 6, wherein the condensing agent used in the reaction of the carboxyl group-containing tetraazacyclic compound and the carboxyl group-containing phenolic compound with the condensing agent is selected from at least one of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, carbodiimide hydrochloride, 1, 3-diisopropylcarbodiimide, and dicyclohexylcarbodiimide;

preferably, the amount of the condensing agent is controlled to be 0.1 to 10:1 in terms of the molar ratio of the condensing agent to the carboxyl group;

preferably, the pH value of the buffer solution adopted in the reaction process of the carboxyl-containing tetraazacyclic compound and the carboxyl-containing phenolic compound and the condensing agent is 5-8;

8. The method according to claim 1, wherein the substrate is placed in the solution of the phenol-amine rich condensed compound, an alkaline buffer is added to adjust the pH value to 7-14, and the reaction is carried out at 0-240 ℃ for 0.001-240 h;

preferably, the reaction temperature of the base material and the rich phenol-amine condensation compound is 0-45 ℃, the reaction time is 0.1-48h, the reaction pH value is 7-11, and the base material and the rich phenol-amine condensation compound are cleaned and dried after reaction;

preferably, the functionalized modification is carried out after the reaction of the substrate with the phenol-amine rich condensation compound is completed, the functionalized modification being a reaction with a biologically active molecule bearing a carboxyl group, a thiol group or a NHS-functionalization;

more preferably, the substrate is selected from at least one of stainless steel, iron and its alloys, magnesium alloys, zinc-manganese alloys, cobalt-based alloys, titanium alloys, ferroferric oxide nanoparticles, ceramics, silica, polymethyl methacrylate, polystyrene, silicone rubber, and polyurethane.

9. Coating material with long-lasting NO catalytic release, characterized in that it is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a coating material according to claim 9 with long-acting NO catalytic release for the preparation of a blood implant device.