CN114632194B

CN114632194B - Coating material with long-acting NO catalytic release and preparation method and application thereof

Info

Publication number: CN114632194B
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: 2023-05-23
Anticipated expiration: 2042-04-11
Also published as: CN114632194A

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 a tetranitrogen heterocyclic compound modified by carboxyl or NHS with a nitric oxide catalytic release molecule, introducing the nitric oxide catalytic release molecule through chelation, and then reacting with an amino compound to obtain a modified amino compound; the modified amino compound reacts with the phenolic compound containing carboxyl to obtain a phenol-amine-rich condensation compound, and then the substrate is placed in a phenol-amine-rich condensation compound solution to be deposited under alkaline conditions 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 can improve the poor chemical stability of the existing phenolic amine coating, improves the efficiency of further functional modification of the coating, has excellent adhesion performance with the surface of the material, is not easy to be damaged by deterioration when being used under extreme conditions, and has excellent use safety.

Description

Coating material with long-acting NO catalytic release 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 surface of various organic and inorganic materials by secreting podoglobins containing various proteins. When podophyllotoxin is initially contacted with a surface, catechol groups and interfaces thereof form mainly covalent and non-covalent interactions, such as charge-charge interactions, pi-pi interactions, cation-pi interactions, coordination interactions, hydrogen bonding interactions, hydrophobic effects and the like. This time scale dependent adhesion is regulated by factors such as pH and other ions in the environment. Because of this complex phenolic amine chemical reaction, the oxidation rate and products of the phenol are not well regulated, and various oxidation products participate in the polymerization of the coating to form a large amount of phenolic oligomers. Although it can form adhesive coating with strong adhesion with various organic and inorganic surfaces, the oligomer formed by non-covalent bond in the polymerization process makes the chemical stability of the coating under the condition of meta-acid and alkali worse, and can not meet the application of the coating under some extreme conditions. Meanwhile, primary amino groups with secondary reactivity on the surface of the coating are mainly contributed by phenolic compounds, and the re-functionalization modification of the surface of the coating can not be met far.

NO is secreted by endothelial cells in natural blood vessels in vivo and is an important factor in maintaining cardiovascular homeostasis and regulating vasodilation. NO plays an important role in promoting the adhesion proliferation of endothelial cells, inhibiting platelet activation and smooth muscle proliferation in natural blood vessels. In recent years, the strategy of surface chemical modification of metal-phenol-amine is rapidly developed, and Cu with NO catalytic activity is selected ²⁺ Or the organic selenium compound can realize the long-term catalytic release of the surface of the coating.

However, the realization of the current NO-catalytic material mainly depends on covalent grafting on the surface of a coating or chelation of active molecules with catalytic release of NO through surface groups, the release amount of the active molecules is limited by the grafting amount and the catalytic efficiency of the surface active molecules, and the physiological environment of high dosage NO and stable dosage requirements applied to different physiological environments is difficult to meet.

In view of this, the present invention has been made.

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 coating with high chemical stability and nitric oxide catalytic release.

The invention is realized in the following way:

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

Reacting a phenolic compound containing carboxyl 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 performed under alkaline conditions.

In an alternative embodiment, the carboxylic group-containing or NHS modified tetraazaheterocyclic 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 are subjected to centrifugal purification and then are subjected to reaction for 0.001h to 240h with the amino compound at the temperature of 0 ℃ to 240 ℃;

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

In an alternative embodiment, the nitric oxide catalytic release molecule is selected from at least one of copper chloride, copper bromide, copper iodide, copper sulfate, selenocysteine, cystine, L-selenocysteine acetic acid, and cysteine and derivatives thereof;

preferably, the nitric oxide catalytic release molecule is a compound containing bivalent copper ions or monovalent copper ions, and the amount of the nitric oxide catalytic release molecule is controlled to control the molar ratio of the copper ions to the tetraazacyclo to be 0.01-1:5, a step of; preferably 0.01-1:2.

In an alternative embodiment, the amine-based compound is selected from at least one of a long chain aliphatic 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 amount of amine-based compound is controlled to be in the range of 0.001 to 2:1 molar ratio of carboxyl groups to amine groups; preferably 0.005-0.02:1.

In an alternative embodiment, the carboxylic group-containing or NHS-modified tetraazacyclocompound 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 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid tri-tert-butyl ester and derivatives thereof; preferably 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid;

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

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

In an alternative embodiment, the phenolic compound containing carboxyl is firstly mixed with a buffer solution with the pH value of 4-10, reacts with a condensing agent for 0.001-240 h at the temperature of 0-240 ℃ and then reacts with the modified amino compound, 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 with condensing agent is 0-30 ℃ and the reaction time is 0.5-48h; the reaction temperature with the modified amino compound is 0-30 ℃ and the reaction time is 0.5-48h;

preferably, the amount of the carboxyl-containing phenolic compound is controlled to be in a molar ratio of carboxyl groups to amine groups in the modified amine-based compound of 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 carboxyl group-containing phenolic compound is selected from at least one 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 condensing agent used in the reaction of the carboxyl group-containing tetrazaheterocyclic compound and the carboxyl group-containing phenolic compound with the condensing agent is at least one member selected from the group consisting 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 0.1-10:1 in terms of 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 group-containing tetrazaheterocyclic compound and the carboxyl group-containing phenolic compound with 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 of a phenol-amine rich condensation compound, an alkaline buffer is added to adjust the pH to 7-14, and the reaction is carried out for 0.001-240 hours at 0-240 ℃;

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

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

preferably, the alkaline buffer is sodium hydroxide solution with the concentration of 0.00001g/L to 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, polymethyl methacrylate, polystyrene, silicone rubber, and polyurethane.

In a second aspect, the present invention provides a coating material having a 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 the coating material with long-acting NO catalytic release of the foregoing embodiments in the manufacture of a blood implant device.

The invention has the following beneficial effects: firstly, reacting a tetranitrogen heterocyclic compound modified by carboxyl or NHS with a nitric oxide catalytic release molecule, introducing the nitric oxide catalytic release molecule through chelation, and then reacting with an amino compound to obtain a modified amino compound; the modified amino compound reacts with the phenolic compound containing carboxyl to obtain a phenol-amine-rich condensation compound, and then the substrate is placed in a phenol-amine-rich condensation compound solution to be deposited under alkaline conditions 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 can improve the poor chemical stability of the existing phenolic amine coating, improves the efficiency of further functional modification of the coating, has excellent adhesion performance with the surface of the material, is not easy to be damaged by deterioration when being used under extreme conditions, and has excellent use safety.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

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

FIG. 2 shows the results of the nitric oxide catalytic release assay in the coating of the comparative example;

FIG. 3 shows the results of the nitric oxide-releasing catalytic assay of the coatings prepared by this method.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

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 method comprises the steps of reacting a tetranitrogen heterocyclic compound modified by carboxyl or NHS with a nitric oxide catalytic release molecule to obtain a tetranitrogen heterocyclic compound chelated with the nitric oxide catalytic release molecule, and reacting the tetranitrogen heterocyclic compound chelated with the nitric oxide catalytic release molecule with an amino compound to obtain a modified amino compound.

It should be noted that the selection of the tetraazacyclo compound can realize Cu ²⁺ The efficient fixed chelation of the nitric oxide catalytic release molecules not only improves the long-term stable release efficiency of NO, but also changes the grafting amount of the catalytic active molecules according to the requirements of the actual application part on NO so as to realize controllable NO catalytic release.

In some embodiments, the carboxylic 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 are subjected to centrifugal purification and then are subjected to reaction for 0.001h to 240h with the amino compound at the temperature of 0 ℃ to 240 ℃; preferably, the reaction temperature of the tetrazacyclic compound containing carboxyl or NHS modification and the nitric oxide catalytic release molecule is 0-30 ℃ and the reaction time is 0.1-0.5 h; the reaction temperature with the amino compound is 0-30 ℃ and the reaction time is 0.01-24 h; the dosage of the amino compound is controlled to be 0.001-2:1 of the molar ratio of carboxyl to amino; preferably 0.005-0.02:1. Chelating the tetrazacyclic compound and the nitric oxide catalytic release molecule under the ultrasonic condition, thereby realizing the immobilization of the molecule with NO catalytic activity. Then adding amino compound, after the reaction is finished, purifying the synthesized molecule by a dialysis method for standby. 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.

In the molecular synthesis process, the concentration, reaction temperature and time of the molecule have significant influence on the synthesis effect of the molecule, the synthesis reaction can be generally completed at 37 ℃, the synthesis time is not too short, and the ideal functional molecule cannot be obtained after the reaction is shorter than 0.5 hour.

In some embodiments, the carboxylic group-containing or NHS-modified tetraazacyclocompound 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 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid tri-tert-butyl ester 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 according to the embodiment of the invention is not limited to the above-mentioned functional molecules.

In some embodiments, the nitric oxide catalytic release 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-selenocysteine acetic acid, cysteine and derivatives thereof, and may be one or more. The above-mentioned several NO catalytic active substances are relatively common and easy to obtain, and the nitric oxide catalytic release molecules according to 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 cupric ion or cupric ion, and the amount of the nitric oxide catalytic releasing molecule is controlled to control the molar ratio of cupric ion to tetraazacyclo to be 0.01-1:5, a step of; preferably 0.01-1:2. the nitric oxide catalytic release molecules are optimized, so that 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 the group consisting of aliphatic long chain amine-based compounds and aromatic polyamine-based compounds, and may be one or more. 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. The amino compound is optimized, so that the introduction amount of the amino is improved, and other functional molecules can be introduced further.

If the tetrazacyclic compound used contains only carboxyl groups without modification of NHS, the tetrazacyclic compound needs to be subjected to carboxyl activation and then reacts with the nitric oxide catalytic release molecule. In the actual operation process, the carboxylic group-containing tetraazaheterocyclic compound solution is mixed with an acidic buffer solution with the pH value of 4-10, reacts with a condensing agent at the temperature of 0-240 ℃ for 0.001-240 h, and then reacts with nitric oxide catalytic release molecules; the concentration of the carboxyl group-containing tetrazaheterocyclic compound solution is 0.01ng/mL-5g/mL.

In a preferred embodiment, the concentration of the solution of the carboxylic group-containing tetraazaheterocyclic compound 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 controlled to be 0.1-10:1 by controlling the molar ratio of the condensing agent to the carboxyl group so as to achieve the effect of full activation and increase the introduction amount of the amino group.

In some embodiments, the condensing agent used in the reaction of the carboxyl group-containing tetraazaheterocyclic compound and 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 (EDC), N-hydroxysuccinimide (NHS), 4-Dimethylaminopyridine (DMAP), carbodiimide hydrochloride (EDCI), 1, 3-Diisopropylcarbodiimide (DIC) and Dicyclohexylcarbodiimide (DCC), and may be one or more. EDC and NHS are preferred.

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

In preferred embodiments, the modified amine-based compound may be a macrocyclic polyamine copper ion-chelating modified polyallylamine (DOTA-Cu/pPAM), a NHS-modified macrocyclic polyamine copper ion-chelating modified polyallylamine (DOTA-Cu/pPAM), and derivatives thereof.

S2 preparation of phenol-rich amine condensation compound

The phenol compound containing carboxyl is reacted with the modified amino compound to obtain the phenol-amine-rich condensation compound, the combination efficiency of phenolic hydroxyl and a substrate interface is increased through condensation of carboxyl phenols and the amino compound, and the introduction of the amino compound containing more carbon is also increased, so that the crosslinking polymerization of the coating is improved, the coating has more excellent chemical tolerance, and can be suitable for polar acid, polar alkali and strong oxidizing environments.

In the actual operation process, the phenolic compound containing carboxyl is firstly mixed with buffer solution with pH value of 4-10, then reacts with condensing agent for 0.001-240 h at 0-240 ℃ and then reacts with modified amino compound, the reaction temperature is controlled to be 0-240 ℃ and the reaction time is controlled to be 0.001-240 h. The dosage of the phenolic compound containing carboxyl is controlled to be 0.001-2:1 of the molar ratio of carboxyl to amino in the modified amino compound. The reaction can be completed by controlling the temperature, time and raw material dosage 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 hours; the reaction temperature with the modified amino compound is 0-30 ℃, the reaction time is 0.5-48h, and the synthesized molecules are purified in anaerobic water for standby by adopting a dialysis method after the reaction is completed. The amount of the phenolic compound containing carboxyl is controlled to be 0.005-0.02:1 of the molar ratio of carboxyl to amino in the modified amino compound. The reaction is more fully carried out by optimizing the reaction temperature, time and the dosage ratio of raw materials.

In the process of the condensation reaction of the phenol-amine, the concentration of the molecules, the oxygen content in the solution, the reaction temperature and the reaction time have significant influence on the synthesis effect of the molecules, and generally, the molecules can be synthesized at 25 ℃, the synthesis time is not too short, and the ideal molecules cannot be obtained in the reaction time shorter than 0.5 hour.

In some embodiments, the carboxyl group-containing phenolic compound may be a monoamine phenolic compound, the specific type being 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 and 3, 4-dihydroxybenzoic acid and derivatives thereof, and can be one or more. The above-mentioned several carboxyl-containing phenolic compounds are relatively common and easy to obtain, and the carboxyl-containing phenolic compounds according to the embodiments of the present invention are not limited to the above-mentioned several.

Similar to step S1, the condensing agent used in the reaction of the carboxyl-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; may be one or more, and is preferably EDC and NHS. The amount of the condensing agent is preferably controlled so that the molar ratio of the condensing agent to the carboxyl group is 0.1-10:1. Preferably, the pH value of the buffer solution adopted in the reaction process of the carboxyl group-containing tetrazaheterocyclic compound and the carboxyl group-containing phenolic compound with the condensing agent is 5-8; the buffer solution is selected from at least one of 2- (N-morpholino) ethanesulfonic acid (MES) buffer, citric acid-sodium hydroxide-hydrochloric acid buffer and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

S3, coating deposition

And (3) placing the substrate in a solution rich in phenol-amine condensation compound, and performing coating deposition under alkaline conditions to obtain a coating which continuously catalyzes and stably releases nitric oxide, and has higher chemical stability and strong adhesive property.

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

The kind of the base material is not limited, and may be selected according to specific requirements, for example, a metal material, an inorganic material, a polymer material, and the like. Specifically, the substrate may be at least one selected from stainless steel, iron and alloys thereof, magnesium alloy, zinc-manganese alloy, cobalt-based alloy, titanium alloy, ferroferric oxide nanoparticle, ceramic, silicon dioxide, 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 sodium hydroxide solution with a concentration of 0.00001g/L to 40g/L, and other alkaline solutions are also possible.

The surface of the coating retains rich primary amine groups, so that the coating has the reaction characteristic of extensive secondary reaction grafting functional groups, and the material has multiple biological function application prospects. In some embodiments, the substrate is subjected to a functionalization modification after completion of the reaction with the phenol-amine rich condensation compound, the functionalization modification being a reaction with a biologically active molecule bearing a carboxyl, sulfhydryl 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 continuous catalytic and stable release of nitric oxide, higher chemical stability and strong adhesive property, and can be applied to the preparation of blood implantation devices.

The features and capabilities of the present invention are described in further detail below in connection with the 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. Nitric oxide catalytic activity molecule modified amino compound

1mg/mL of a DOTA solution containing carboxyl groups was mixed with MES acidic buffer solution adjusted to pH 5.6, EDC and NHS (molar ratio of EDC to carboxyl groups: 5:1, molar ratio of NHS to carboxyl groups: 1:1, the same applies hereinafter) were added, and after reaction at 37℃for 0.5h, the molar ratio with DOTA was 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.01:1 (weight average molecular weight 8,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL of hydrogenated caffeic acid was dissolved in MES buffer solution which was completely oxygen-free and pH was adjusted to 5.6, EDC and NHS (molar ratio of EDC to carboxyl was 5:1, molar ratio of NHS to carboxyl was 1:1, the same applies hereinafter) were added, and after 0.5h of reaction at 37 ℃, the molar ratio of carboxyl to amino groups of DOTA-Cu/pPAM was 0.01:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 24 hours at 10 ℃.

C. Coating preparation

And (3) placing the sample to be modified in the molecular solution obtained in the step (B), adding an alkaline anaerobic buffer solution to adjust the pH to 9, reacting for 24 hours at 37 ℃, and washing and drying to obtain the coating.

Example 2

A. nitric oxide catalytic activity molecule modified amino compound

Mixing 1mg/mL DOTA solution containing 4 carboxyl groups with MES acidic buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37deg.C for 0.5 hr, addingMolar ratio to DOTA is 1:1 CuCl ₂ Ultrasonic reaction for 0.1h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.01:1 (weight average molecular weight 8,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL caffeic acid was dissolved in MES buffer solution which was completely oxygen-free and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h reaction at 37℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.01:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 24 hours at 4 ℃.

C. Coating preparation

Example 3

A. nitric oxide catalytic activity molecule modified amino compound

1mg/mL of carboxyl-containing DOTA solution was mixed with MES acidic buffer solution adjusted to pH 5.6, EDC and NHS were added, and after 0.5h reaction at 37℃the molar ratio to DOTA was 1:1, ultrasonically reacting for 0.5h, centrifugally purifying, and then adding a molar ratio of carboxyl to amino of 0.01:1 (weight average molecular weight 8,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL of 2, 3-dihydroxybenzoic acid was dissolved in MES buffer solution which was completely anaerobic and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h of reaction at 25℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.01:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 24 hours at 10 ℃.

C. Coating preparation

Example 4

A. nitric oxide catalytic activity molecule modified amino compound

2mg/mL of a carboxyl group-containing DOTA solution was mixed with MES acidic buffer solution adjusted to pH 5.6, EDC and NHS were added, and after 0.5 hour of reaction at 37℃the molar ratio to DOTA was 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.01:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL of 2, 3-dihydroxybenzoic acid was dissolved in MES buffer solution which was completely anaerobic and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h of reaction at 25℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.01:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 24 hours at 25 ℃.

C. Coating preparation

Example 5

A. nitric oxide catalytic activity molecule modified amino compound

2mg/mL carboxyl groupMixing DOTA with MES acidic buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding the mixture with DOTA in a molar ratio of 1:1 CuCl ₂ Ultrasonic reaction for 0.1h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.01:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL of hydrogenated caffeic acid was dissolved in MES buffer solution which was completely oxygen-free and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5 hour of reaction at 25℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.01:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 12 hours at 4 ℃.

C. Coating preparation

Example 6

A. nitric oxide catalytic activity molecule modified amino compound

1mg/mL of carboxyl-containing DOTA solution was mixed with MES acidic buffer solution adjusted to pH 5.6, EDC and NHS were added, and after 0.5h reaction at 37℃the molar ratio to DOTA was 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.19:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL caffeic acid was dissolved in MES buffer solution which was completely oxygen-free and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h reaction at 37℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.01:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 12 hours at 10 ℃.

C. Coating preparation

And (3) placing the sample to be modified in the molecular solution obtained in the step (B), adding an alkaline anaerobic buffer solution to adjust the pH to 9, reacting for 12 hours at 25 ℃, and washing and drying to obtain the coating.

Example 7

A. nitric oxide catalytic activity molecule modified amino compound

To a 1mg/mL NHS-DOTA solution mixed with PBS buffer adjusted to pH 8.5, a molar ratio to DOTA of 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.2:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

2.3mg/mL of hydrogenated caffeic acid was dissolved in MES buffer solution which was completely oxygen-free and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h of reaction at 37℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.19:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 12 hours at 10 ℃.

C. Coating preparation

And (3) placing the sample to be modified in the molecular solution obtained in the step (B), adding an alkaline anaerobic buffer solution to adjust the pH to 9, reacting for 12 hours at 37 ℃, and washing and drying to obtain the coating.

Example 8

A. nitric oxide catalytic activity molecule modified amino compound

1mg/mL of NHS-DOTA solution was mixed with PBS buffer adjusted to pH 8.5 and added in a molar ratio to DOTA of 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.1:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

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

C. Coating preparation

And (3) placing the sample to be modified in the molecular solution obtained in the step (B), adding an alkaline anaerobic buffer solution to adjust the pH to 9, reacting for 24 hours at 25 ℃, and washing and drying to obtain the coating.

Example 9

A. nitric oxide catalytic activity molecule modified amino compound

1mg/mL of NHS-DOTA solution was mixed with PBS buffer adjusted to pH 8.5 and added in a molar ratio to DOTA of 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.5:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL of 2, 3-dihydroxybenzoic acid was dissolved in MES buffer solution which was completely anaerobic and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h of reaction at 25℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.3:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 24 hours at 4 ℃.

C. Coating preparation

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 substrate material for coating preparation and comprises the following steps:

A. nitric oxide catalytic activity molecule modified amino compound

1mg/mL of NHS-DOTA solution was mixed with PBS buffer adjusted to pH 8.5 and added in a molar ratio to DOTA of 1:1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.01:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

3mg/mL caffeic acid was dissolved in MES buffer solution which was completely oxygen free and pH was adjusted to 5.6, EDC and NHS were added, and after 0.2h reaction at 37℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.1:1, and purifying the synthesized molecules in anaerobic water by a dialysis method after reacting for 24 hours at 5 ℃ for later use.

C. Coating preparation

Example 11

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

A. nitric oxide catalytic activity molecule modified amino compound

Adjusting the pH of a 1mg/mL NHS-DOTA solutionTo 8.5 in PBS buffer, 1 was added in a molar ratio to DOTA: 1 CuCl ₂ Ultrasonic reaction for 0.2h, centrifugal purification, and subsequent addition of carboxyl to amine molar ratio of 0.5:1 (weight average molecular weight 17,000,20wt.% aqueous solution), and purifying the synthesized molecules (DOTA-Cu/pPAM) by dialysis after 24 hours of reaction at 37 ℃.

B. Phenol-amine rich compound condensation

1mg/mL caffeic acid was dissolved in MES buffer solution which was completely oxygen-free and pH was adjusted to 5.6, EDC and NHS were added, and after 0.5h reaction at 37℃the molar ratio of carboxyl groups to amine groups of DOTA-Cu/pPAM was 0.5:1, and purifying the synthesized molecules in oxygen-free water by a dialysis method after reacting for 12 hours at 25 ℃.

C. Coating preparation

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 compounds.

Comparative example 2

The difference from example 1 is that: changing the introduction mode of copper ions, in comparative example 2, macrocyclic polyamine is covalently immobilized through amine groups on the surface of the coating, and then copper is chelated; the method of the invention grafts the macrocyclic polyamine chelating copper ions on the long chain of the amino compound to be introduced into the coating, thereby realizing the fixation of copper ions in the coating.

Test example 1

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

The detection method comprises the following steps: the coating obtained in example 1 was immersed in a strong acid (ph=0), a strong base (ph=14) and a strong oxidizing property (H ₂ O ₂ After 24h, 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 present method has a relatively strong chemical stability, and can still maintain a complete coating under extremely strong acid, strong alkali and strong oxidation conditions. Compared with the traditional catechol coating, the coating prepared by the method has more excellent chemical stability, can not damage the integrity of the coating even in extreme environments, and has wider application fields.

Test example 2

The NO catalytic release test effect of the coating of comparative example 2 is shown in fig. 2.

The testing method comprises the following steps: the effect of NO catalytic release rate was measured in PBS at 37 ℃ using a chemiluminescent NO analyzer (NOA) (receivers 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 passage of time; the ability of the coating to catalyze and release NO is obviously reduced compared with that of the first time after the coating is pulled out and reinserted into the reaction solution, and the result also suggests that the mode of chelating copper ions by adopting the step-by-step grafting functional molecules has certain limitation on the stable catalysis and release of NO. The lower utilization rate of copper ions reduces the stability of NO catalytic release, so that the catalytic rate and stability of the NO catalytic release are obviously reduced.

Test example 3

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

The testing method comprises the following steps: the same as in detection example 2.

As can be seen from fig. 3, the coating obtained by the present preparation method has significantly reduced burst release at early NO catalytic release compared to the coating of comparative example 2, which is about 5-fold reduced burst release of NO. The NO catalytic release capability did not show a decrease in the catalytic release of NO by the coating of comparative example 2 over time, and the stable catalytic release capability was maintained at all times and the initial NO catalytic release capability was maintained over time. The detection result directly proves that the coating prepared by the method has long-acting and stable performance of catalyzing and releasing NO.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. 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 having a long-acting NO catalytic release, comprising: reacting a carboxyl-containing or NHS-modified tetraazacycle compound with a nitric oxide catalytic release molecule to obtain a tetraazacycle compound chelated with the nitric oxide catalytic release molecule, and reacting the tetraazacycle compound chelated with the nitric oxide catalytic release molecule with an amino compound to obtain a modified amino compound;

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

placing a substrate in the solution of the phenol-amine-rich condensation compound, and performing coating deposition under alkaline conditions;

the tetrazacyclic compound containing carboxyl or NHS modification is selected from at least one of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, NHS modification 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 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid tri-tert-butyl ester and derivatives thereof;

Mixing a carboxyl-containing tetraazaheterocyclic compound solution with an acidic buffer solution with a pH value of 4-10, reacting with a condensing agent at the temperature of 0-240 ℃ for 0.001-240 h, and then reacting with the nitric oxide catalytic release molecule; the carboxyl-or NHS-modified tetraazacyclo compound and the nitric oxide catalytic release molecule are subjected to ultrasonic reaction for 0.1h-3h at the temperature of 0-240 ℃, and then are subjected to centrifugal purification and reaction for 0.001h-240h with the amino compound at the temperature of 0-240 ℃;

mixing a phenolic compound containing carboxyl with a buffer solution with the pH value of 4-10, reacting with a condensing agent at the temperature of 0-240 ℃ for 0.001-240 h, and then reacting with the modified amino compound, wherein the reaction temperature is controlled to be 0-240 ℃ and the reaction time is controlled to be 0.001-240 h;

placing a substrate in the solution of the phenol-amine-enriched condensation compound, adding an alkaline buffer solution to adjust the pH value to 7-14, and reacting for 0.001-240 h at the temperature of 0-240 ℃;

the phenolic compound containing carboxyl is selected from at least one 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;

the nitric oxide catalytic release molecule is a compound containing bivalent copper ions or monovalent copper ions, and the dosage of the nitric oxide catalytic release molecule is that the molar ratio of the copper ions to the tetraazacyclo is controlled to be 0.01-1:5.

2. The method according to claim 1, wherein the reaction temperature of the carboxylic group-containing or NHS-modified tetraazacyclic compound and the nitric oxide-catalyzing and releasing molecule is 0-30 ℃ and the reaction time is 0.1-0.5 h; the reaction temperature with the amino compound is 0-30 ℃ and the reaction time is 0.01-24 h.

3. The method according to claim 2, wherein the amount of the nitric oxide catalytic releasing molecule is controlled to control the molar ratio of copper ions to tetraazacyclo to be 0.01-1:2.

4. the method according to claim 1, wherein the amine-based compound is at least one selected from the group consisting of aliphatic long-chain amine-based compounds and aromatic polyamine-based compounds.

5. The method according to claim 4, wherein the amine-based compound is at least one selected from the group consisting of polyallylamine, polyvinylamine, polylysine, polyethylene glycol diamine, polyetheramine, polyamide, melamine, poly-N-isopropylacrylamide, chitosan and derivatives thereof.

6. The method according to claim 5, wherein the amine-based compound is polyallylamine.

7. The method of claim 6, wherein the amine-based compound is used in an amount such that the molar ratio of carboxyl groups to amine groups is controlled to be 0.001-2:1.

8. The method of claim 7, wherein the amine-based compound is used in an amount such that the molar ratio of carboxyl groups to amine groups is controlled to be 0.005-0.02:1.

9. The method of claim 2, wherein the carboxylic or NHS modified tetraazacyclocompound is 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid.

10. The method according to claim 2, wherein the concentration of the solution of the carboxyl group-containing tetrazacyclic compound is 0.01ng/mL to 5g/mL.

11. The method according to claim 10, wherein the concentration of the solution of the carboxyl group-containing tetrazacyclic compound is 0.1 to 1mg/mL.

12. The process according to claim 11, wherein the reaction temperature with the condensing agent is 0 to 30 ℃ and the reaction time is 0.5 to 48 hours; the reaction temperature with the modified amino compound is 0-30 ℃ and the reaction time is 0.5-48h.

13. The method of claim 12, wherein the amount of the carboxyl group-containing phenolic compound is controlled to be 0.001-2:1 in terms of the molar ratio of carboxyl groups to amine groups in the modified amine group compound.

14. The method of claim 13, wherein the amount of the carboxyl group-containing phenolic compound is controlled to be 0.005-0.02:1 molar ratio of carboxyl groups to amine groups in the modified amine group compound.

15. The method according to claim 14, wherein the condensing agent used in the reaction of the carboxyl group-containing tetrazacyclic compound and 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.

16. The method of claim 15, wherein the condensing agent is used in an amount such that the molar ratio of condensing agent to carboxyl is controlled to be 0.1-10:1.

17. The method according to claim 16, wherein the pH of the buffer solution used in the reaction of the carboxylic group-containing tetraazacyclic compound and the carboxylic group-containing phenolic compound with the condensing agent is 5 to 8.

18. The method of preparing according to claim 17, wherein 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.

19. The method according to claim 1, wherein the reaction temperature of the substrate and the phenol-amine-rich condensed compound is 0 to 45 ℃, the reaction time is 0.1 to 48 hours, the reaction pH is 7 to 11, and the substrate is washed and dried after the reaction.

20. The method of claim 19, wherein the substrate is subjected to a functionalization modification after completion of the reaction with the phenol-amine rich condensation compound, the functionalization modification being a reaction with a biologically active molecule bearing a carboxyl, sulfhydryl or NHS group.

21. The method according to claim 20, wherein the alkaline buffer is sodium hydroxide solution having a concentration of 0.00001g/L to 40 g/L.

22. The method according to claim 21, wherein the base material is at least one selected from an inorganic material and a polymer material.

23. The method of claim 22, wherein the substrate is selected from at least one of iron and its alloys, magnesium alloys, zinc manganese alloys, cobalt-based alloys, titanium alloys, ferroferric oxide nanoparticles, ceramics, polymethyl methacrylate, polystyrene, silicone rubber, and polyurethane.

24. A coating material having a long-lasting NO catalytic release, characterized in that it is prepared by the preparation method according to any one of claims 1-23.

25. Use of the coating material with long-acting NO catalytic release according to claim 24 for the preparation of a blood implant device.