CN114618025B

CN114618025B - Preparation method of rich amine group conversion coating, functional material and application

Info

Publication number: CN114618025B
Application number: CN202210372173.0A
Authority: CN
Inventors: 杨志禄; 杜泽煜; 黄楠; 王颖; 杨梦毅; 张文泰; 牟小辉
Original assignee: Dongguan Peoples Hospital
Current assignee: Dongguan Peoples Hospital
Priority date: 2022-04-11
Filing date: 2022-04-11
Publication date: 2023-01-31
Anticipated expiration: 2042-04-11
Also published as: CN114618025A

Abstract

The invention discloses a preparation method, a functional material and application of an amine-rich conversion coating, and relates to the technical field of biological materials. A carboxyl-containing macrocyclic polyamine compound and a molecule with NO catalytic activity are subjected to chelation reaction to introduce NO catalytic activity molecules, and then the carboxyl and an amino compound are subjected to amidation reaction to obtain a modified amine compound; the o-phenol polymeric coating on the surface of the material is used for introducing the modified amine compound under the alkaline condition, and the long-acting stable and controllable Nitric Oxide (NO) release conversion amine-based coating is obtained on the surface of the material through the in-situ crosslinking reaction again.

Description

Preparation method of rich amine group conversion coating, functional material and application

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of an amine-rich conversion coating, a functional material and application.

Background

Inspired by the adhesion of marine mussels, researchers have found that catechols and amines play an important role in the firm adhesion of mussels to various rough and wet surfaces. Meanwhile, the material is widely applied to material surface modification due to the firm adhesion with a substrate and the capability of realizing secondary chemical reaction activity to fix biomolecules on the surface. However, conventional catecholamine polymeric coatings are primarily coupled by non-covalent interactions, such as hydrogen bonding, pi-pi stacking, and cationic-pi, aryl-aryl bonds, among others. Due to the existence of a large number of non-covalent bonds, the coating is dissociated under the strong alkaline condition, and the application of the coating in a complex environment is difficult to satisfy due to poor physical and chemical stability. Meanwhile, the grafting amount of the biological molecules on the surface of the material depends on the density of secondary reaction functional groups reserved by the coating, but the density of the amine groups remained on the surface after the traditional catecholamine polymerization coating is modified can not meet the requirement of direct grafting of the biological molecules.

NO in vivo is produced by endothelial cells through the action of nitric oxide synthase and arginine secreted by the cells and is an important factor in maintaining cardiovascular homeostasis and regulating vasodilation. In addition, NO has many important biological effects, promoting adhesion proliferation of endothelial cells, inhibiting platelet activation and smooth muscle proliferation. The design of NO functional materials is mainly divided into two types, including NO-release type and NO-catalytic type, and the NO functional materials are limited in clinical application because of the initial large amount of NO burst release of the NO-release type and the difficulty in realizing controlled release of NO in the middle and later release processes.

However, the realization of the current NO-catalytic type material mainly relies on the covalent grafting of catalytically active molecules on the surface of the coating, the release amount of which is limited by the grafting amount of the surface active molecules, and the physiological environment for applying large-dose NO and stable-dose NO requirements is difficult to satisfy.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method, a functional material and application of an amine-rich conversion coating, and aims to enable the prepared coating to have a long-acting temperature-controllable nitric oxide release function, and the coating is rich in amine groups and is convenient for further functional modification.

The invention is realized by the following steps:

in a first aspect, the present invention provides a method for preparing a rich amine-based conversion coating, comprising:

a carboxyl-containing macrocyclic polyamine compound and a molecule with NO catalytic activity are subjected to chelation reaction, and then the macrocyclic polyamine compound and an amino compound are subjected to reaction to obtain a modified amine compound;

and carrying out in-situ re-crosslinking reaction on the base material with the surface adhered with the o-phenol polymerization modified coating and the modified amine compound under an alkaline condition.

In a second aspect, the present invention provides a functional material, which comprises a substrate and an amine-rich conversion coating attached to the substrate, wherein the amine-rich conversion coating is prepared by the above preparation method.

In a third aspect, the present invention provides the use of the functional material of the previous embodiment for the preparation of a blood implant device.

The invention has the following beneficial effects: a carboxyl-containing macrocyclic polyamine compound and a molecule with NO catalytic activity are subjected to chelation reaction to introduce a molecule with NO catalytic activity, and then the carboxyl and an amino compound are subjected to amidation reaction to obtain a modified amine compound; the o-phenol polymeric coating on the surface of the material is used for introducing the modified amine compound under the alkaline condition, and the long-acting stable and controllable Nitric Oxide (NO) release conversion amine-based coating is obtained on the surface of the material through the in-situ crosslinking reaction again.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used 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 for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a structural diagram of an amine compound modified with a functional molecule for catalytically releasing nitric oxide according to example 1;

FIG. 2 is a graph of the results of NO release measurements before and after immersion of the amine-based conversion coating in example 1;

FIG. 3 is a graph of the effect of the ex vivo blood compatibility test of example 1 with comparative examples 2 and 3 coatings;

FIG. 4 is a graph showing the results of the NO catalytic release test of the coating of comparative example 1;

FIG. 5 is a graph showing the results of measuring the copper ion content in the coatings of example 1 and comparative examples 2 and 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of 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 conventional products which are not indicated by manufacturers and are commercially available.

The embodiment of the invention provides a preparation method of an amine-rich conversion coating, which comprises the following steps:

s1, chelated NO catalytically active molecules

The carboxyl-containing macrocyclic polyamine compound and the molecule with NO catalytic activity are subjected to chelation reaction, and the NO catalytic activity molecule is introduced by the chelation of N in the carboxyl-containing macrocyclic polyamine compound.

Specifically, the carboxyl-containing macrocyclic polyamine compound is selected from at least one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), NHS-DOTA, tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester, porphyrin and 8-hydroxyquinoline and derivatives thereof. The polyamine compounds are suitable for NO catalytic active molecules such as chelated copper ions.

It should be noted that when the carboxyl group-containing macrocyclic polyamine compound is NHS-DOTA, i.e., an NHS-modified compound, activation is not required. If the carboxyl group-containing macrocyclic polyamine compound is a compound such as DOTA which is not modified with NHS, it is necessary to activate the carboxyl group.

In some embodiments, the carboxyl-containing macrocyclic polyamine-based compound is subjected to carboxyl activation followed by reaction with a molecule having NO catalytic activity for 0.2h to 3h. In the practical operation process, the carboxyl-containing macrocyclic polyamine compound with the concentration of 0.01ng/mL-5g/mL is dissolved in a mixed solution formed by MES buffer solution with the pH value of 4-10 and a condensing agent, carboxyl is activated for 0.001-240h under the condition of 0-350 ℃, and then the macrocyclic polyamine compound reacts with molecules with NO catalytic activity under the ultrasonic condition.

In a preferred embodiment, the concentration of the carboxyl-containing macrocyclic polyamine compound is 0.1mg/mL-1mg/mL, and the pH value of the MES buffer is 5-8; the temperature for activating the carboxyl is 20-50 ℃, and the time for activating the carboxyl is 0.1-24h. By optimizing parameters such as raw material concentration, activation temperature and time, the effect of carboxyl activation can be improved, and amine compounds can be introduced more through subsequent reactions.

In some embodiments, the condensing agents are EDC and NHS, the molar ratios of EDC to carboxyl groups in the carboxyl-containing macrocyclic polyamine-based compound are each 4 to 6, the molar ratios of NHS to carboxyl groups in the carboxyl-containing macrocyclic polyamine-based compound are each 0.5 to 1.5; the raw materials adopted by EDC and NHS have the concentration of 0.01ng/mL-5g/mL. EDC and NHS are adopted to activate carboxyl well, and the introduction amount of subsequent amine compounds is increased.

In other embodiments, the condensing agent is not limited to EDC and NHS, and conventional condensing agents in the prior art may be used without limitation.

In a preferred embodiment, the reaction time is controlled to be 0.2-0.5h in the process of reacting with the molecule with NO catalytic activity; the molar ratio of the molecules with NO catalytic activity to the macrocyclic polyamine compound containing carboxyl is 1; preferably 1. The introduced amount of copper ions is increased by further controlling the ratio of the reaction time to the amount of the raw materials.

Further, the molecules having NO catalytic activity are selected from at least one of cupric ions, monovalent cupric ions, ebselen, cystamine and derivatives thereof; preferably divalent copper ions. As for the specific raw materials, the molecule having NO catalytic activity is at least one selected from the group consisting of copper chloride, cuprous chloride, copper bromide, cuprous bromide, copper iodide, cuprous iodide, copper sulfate, selenocysteine, cystine, L-selenocysteineacetic acid, cysteine and derivatives thereof, and may be one or more.

The carboxyl group-containing macrocyclic polyamine-based compound and the molecule having NO catalytic activity are not limited to the above.

S2, preparing modified amine compound

After molecules with NO catalytic activity are introduced, the carboxyl in the carboxyl-containing macrocyclic polyamine compound and an amino compound are subjected to amidation reaction to obtain a modified amine compound. In the actual operation process, after molecules with NO catalytic activity are introduced, the molecules and amino group compound solution with the concentration of 0.01ng/mL-20g/mL react for 0.001-240h at the temperature of 0-350 ℃, dialysis is carried out after the reaction is finished to obtain modified amine compounds, and impurities are removed through dialysis.

In a preferred embodiment, the concentration of the amino compound is 0.1mg/mL-5mg/mL, the reaction temperature is 15-40 ℃, and the reaction time is 0.5-48h. The reaction of the amino compound and the carboxyl in the solution is promoted by further optimizing the parameters such as the concentration, the reaction temperature, the reaction time and the like of the amino compound, and the reaction is sufficient.

Specifically, the amino compounds are selected from at least one of aliphatic amine and aromatic amine compounds; preferably, the amino compound is at least one selected from polyallylamine, polyvinylamine, polylysine and chitosan, and may be one or more.

It should be noted that, in the process of preparing the modified amine compound, the concentration of molecules, the reaction temperature and the reaction time have a significant influence on the synthesis effect of the molecules, the synthesis reaction can be completed at 37 ℃, the synthesis time is not short enough, and the reaction time is shorter than 0.5 hour, so that an ideal functional molecule cannot be obtained.

S3, preparing the o-phenol polymerization modified coating

The preparation of the o-phenolic polymerization modified coating comprises the following steps: soaking the base material in a mixed solution formed by the o-phenolic compound and the buffer solution, and reacting for 0.001h-240h under the conditions that the pH is 0-14 and the temperature is 0-350 ℃ so as to form the o-phenolic polymerization modified coating on the surface of the material through the oxidation auto-polymerization, covalent and non-covalent bond action of the o-phenolic compound on the surface of the base material.

It should be noted that, the preparation method of the o-phenolic polymerization modified coating is similar to the preparation method of the catecholamine modified coating in the prior art, and the preparation of the modified coating on the surface of the material can be performed by adopting the traditional catecholamine modification method.

The type of the substrate is not limited, and the material may be selected according to the application, and is not limited herein. Specifically, the base material is selected from at least one of a metal material, an inorganic material, and a polymer material; preferably, the substrate is selected from at least one of stainless steel, iron and its alloys, magnesium and its alloys, zinc-manganese alloys, cobalt-based alloys, titanium alloys, ferroferric oxide nanoparticles, ceramics, silica, polymethyl methacrylate, polystyrene, silicone rubber and polyurethane.

Further, the o-phenolic compound is at least one selected from the group consisting of a monoamine type phenolic compound, an indole type compound, and phenol and a derivative thereof. Specifically, the o-phenolic compound is at least one selected from melanin, dopamine, dihydroxyindole, epinephrine, norepinephrine, levodopa, tannic acid, dextrodopa, gallic acid, pyrogalloc acid, epicatechin gallate, epigallocatechin, and epigallocatechin gallate. The kind of the o-phenolic compound may be one or more, and is not limited to the above.

It is to be added that the existing methods for surface modification of o-phenolic compounds have many cases and can be performed by various methods, such as a soaking method, layer-by-layer autonomous assembly, and the like. The preparation of the ortho-phenolic polymeric modified coating is not limited to the immersion method.

In some embodiments, the concentration of the ortho-phenolic compound in the reaction system is from 0.01ng/mL to 5g/mL, preferably from 0.5 to 20mg/mL; in the process of preparing the o-phenol polymerization modified coating, the reaction pH value is controlled to be 5.6-12, the reaction temperature is 20-40 ℃, and the reaction time is 0.5-48h. The concentration, pH value, reaction temperature and time of the o-phenolic compound are further controlled to facilitate the rapid formation of the o-phenolic polymerization modified coating with proper thickness on the surface of the substrate.

It should be noted that the concentration, reaction temperature and time of the compound molecules when the o-phenolic polymerization modified coating is deposited have a significant influence on the coating deposition effect, the modified coating can be deposited at 37 ℃, the deposition time is not short, and the reaction deposition time is shorter than 0.5 hour, so that an ideal coating cannot be obtained.

S4, preparing the rich amino conversion coating

The method comprises the steps of carrying out in-situ re-crosslinking reaction on a base material with an o-phenol polymerization modified coating attached to the surface and a modified amine compound under an alkaline condition, wherein a non-covalent bond in the catechol amine polymerization coating is damaged under the alkaline condition to cause coating dissociation, and the introduced amine compound and an oligomer realize in-situ re-crosslinking chemical reaction of the coating through Michael addition reaction and Schiff base chemical reaction, so that the preparation of an amine conversion coating is realized.

It should be noted that, the dissociated catecholamine coating and the chain amino polymer after the functional modification are further cross-linked and polymerized to reduce the polymerization degree of the non-covalent bond, so that the coating has excellent chemical stability, strong adhesion performance, long-acting stable and controllable Nitric Oxide (NO) release performance, can be applicable to the extreme conditions and is not easy to disintegrate, and can be applied to any type of materials or substrate materials with shapes. In addition, the introduction of the amine compound modified by the functional molecule for catalytically releasing nitric oxide not only realizes the long-acting stable controllable Nitric Oxide (NO) catalytic release function, but also reserves a primary amino group rich in reaction activity on the surface of the coating, so that the coating has higher and wider reaction characteristic of secondary functional group grafting, and the material has multiple biological function application prospects.

In the actual operation process, the base material with the surface adhered with the o-phenol polymerization modified coating is placed in a mixed solution formed by an alkaline buffer solution and a modified amine compound and reacts for 0.001h-72h at the temperature of 0-120 ℃; wherein, in the mixed solution formed by the alkaline buffer solution and the modified amine compound, the concentration of the modified amine compound is 0.01ng/mL-5g/mL.

In a preferred embodiment, the concentration of the modified amine compound is 0.1mg/mL to 5mg/mL, and the pH of the alkaline buffer solution is 8 to 14, and the alkaline buffer solution includes at least one of a sodium carbonate-sodium bicarbonate buffer, a PBS buffer, a Tris-HCl buffer, a sodium hydroxide solution, and a potassium hydroxide solution. In some embodiments, the base in the alkaline buffer solution is sodium hydroxide at a concentration of 0.00001g/L to 160g/L; the reaction temperature is 15-40 ℃, and the reaction time is 0.5-48h. The concentration of the modified amine compound is further optimized, and the pH value, the reaction temperature and the reaction time are adjusted, so that the reaction is more sufficient, and the introduction amount of the modified amine compound is increased. If the reaction deposition time is shorter than 0.5 hour, a desired coating cannot be obtained.

In a preferred embodiment, the modified amine compound is at least one selected from the group consisting of a macrocyclic polyamine chelated copper ion modified polyallylamine, a NHS-modified macrocyclic polyamine chelated copper ion modified polyallylamine, and a porphyrin chelated copper ion modified polyallylamine. The modified amine compounds have the function of releasing nitric oxide stably for a long time after being introduced into a base material, and the effect is very ideal.

The embodiment of the invention provides a functional material, which comprises a base material and an amine-rich conversion coating attached to the base material, wherein the amine-rich conversion coating is prepared by the preparation method.

It is noted that the functional material has long-acting stable controllable nitric oxide release, high chemical stability and strong adhesion of the amine conversion coating. The coating has better chemical stability through the chemical reaction of secondary in-situ re-crosslinking under the strong alkali condition, so that the coating is not easy to disintegrate under the extreme conditions of acid, alkali and oxidation. Meanwhile, the introduction of the amine compound also endows the coating with the characteristic of rich amino groups, so that the coating has the performance of wide biological functional molecule grafting, and further endows the material with more application prospects, and can be applied to the 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 an amine-rich conversion coating, which selects 316L stainless steel as a substrate material for coating preparation, and comprises the following steps:

(1) Preparation of modified amine Compounds

1mg/mL of DOTA containing 4 carboxyl groups was dissolved in MES buffer adjusted to pH 5.6, EDC and NHS (EDC to carboxyl in a molar ratio of 5, NHS to carboxyl in a molar ratio of 1:1, the same applies below) were added and reacted at 37 ℃ for 24h, adding the reaction mixture to the macrocyclic polypeptideThe molar ratio of the amine compound is 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.3h.

3.5mg/mL of polyallylamine (an aqueous solution having a weight-average molecular weight of 8,000,20wt.%) was added, and after reaction at 37 ℃ for 24 hours, the sample was purified by dialysis for use.

(2) Preparation of o-phenolic polymerization modified coating

And cleaning the surface of 316L stainless steel, and drying for later use. Soaking 316L stainless steel in a dopamine solution with the dissolved concentration of 1mg/mL and the pH value adjusted to 8.5 by Tris-base (Tris (hydroxymethyl) aminomethane), reacting at 25 ℃ for 24h to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of rich amine based conversion coatings

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 14 to submerge the modified coating in the step (2), reacting at 25 ℃ for 24 hours, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 2

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 4 carboxyl groups in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 24h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.3h.

2mg/mL polyallylamine (weight average molecular weight 17,000,20wt.% in H2O) was added, and after reaction at 37 ℃ for 24 hours, the sample was purified by a dialysis method for use.

(2) Preparing the o-phenolic polymerization modified coating.

The surface of 316L stainless steel is cleaned and dried for later use. Soaking 316L stainless steel in a dopamine solution with the pH value adjusted to 8.5 by Tris-base and the dissolved concentration of 1mg/mL, reacting for 24 hours at 25 ℃ to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 14 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 5.0mg/mL, reacting at 25 ℃ for 24h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 3

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 1 carboxyl in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 0.5h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.3h.

2mg/mL polyallylamine (weight average molecular weight 65,000,10wt.% in H2O) was added, and after reaction at 37 ℃ for 24 hours, the sample was purified by dialysis for use.

(2) Preparing the o-phenolic polymerization modified coating.

The surface of 316L stainless steel is cleaned and dried for later use. Soaking 316L stainless steel in a dopamine solution with the concentration of 2mg/mL and the pH value adjusted to 8.5 by Tris-base, reacting at 37 ℃ for 12h to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 14 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 0.1mg/mL, reacting at 25 ℃ for 24h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 4

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 1 carboxyl in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 25 ℃ for 2h, and adding a mixture of 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.1h.

Adding 5mg/mL branched polyethyleneimine (weight average molecular weight-800), reacting at 37 ℃ for 24h, and purifying the sample by a dialysis method for later use.

(2) Preparing the o-phenolic polymerization modified coating.

And cleaning the surface of 316L stainless steel, and drying for later use. Soaking 316L stainless steel in a dopamine solution with the concentration of 2mg/mL and the pH value adjusted to 8.5 by Tris-base, reacting at 37 ℃ for 12h to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 14 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 3.0mg/mL, reacting at 37 ℃ for 6h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 5

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 4 carboxyl groups in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 24h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 1h.

Adding 5mg/mL polylysine, reacting at 37 ℃ for 6h, and purifying the sample by a dialysis method for later use.

(2) Preparing the o-phenolic polymerization modified coating.

The surface of 316L stainless steel is cleaned and dried for later use. Soaking 316L stainless steel in a dopamine solution with the pH value adjusted to 8.5 by Tris-base and the dissolved concentration of 2mg/mL, reacting at 37 ℃ for 24 hours to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

Example 6

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 4 carboxyl groups in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 24h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.5h.

Adding 2mg/mL chitosan, reacting at 37 ℃ for 24h, and purifying the sample by a dialysis method for later use.

(2) Preparing the o-phenolic polymerization modified coating.

And cleaning the surface of 316L stainless steel, and drying for later use. Soaking 316L stainless steel in a dopamine solution with the pH value adjusted to 8.5 by Tris-base and the dissolved concentration of 0.2mg/mL, reacting for 24 hours at 37 ℃ to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 14 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 1.0mg/mL, reacting at 37 ℃ for 12h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 7

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 4 carboxyl groups in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 24h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.6h.

3.5mg/mL polyallylamine (weight average molecular weight 17,000,20wt.% in H2O) was added, and after reaction at 37 ℃ for 24 hours, the sample was purified by a dialysis method for use.

(2) Preparing the o-phenolic polymerization modified coating.

And cleaning the surface of 316L stainless steel, and drying for later use. Soaking 316L stainless steel in noradrenaline with the concentration of 1mg/mL which is adjusted to the pH value of 8.5 by Tris-base, reacting at 37 ℃ for 12h to obtain a poly-noradrenaline coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 14 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 5.0mg/mL, reacting at 25 ℃ for 12h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 8

The embodiment provides a preparation method of an amine-rich conversion coating, which selects iron and iron alloy as a substrate material for coating preparation, and comprises the following steps:

(1) Preparation of modified amine Compounds

2mg/mL polyallylamine (weight average molecular weight 17,000,20wt.% in H2O) was added, and after 12 hours of reaction at 37 ℃, the sample was purified by a dialysis method for use.

(2) Preparing the o-phenolic polymerization modified coating.

And cleaning the surfaces of the iron and the iron alloy, and drying for later use. Soaking iron and iron alloy in a dopamine solution with the pH value adjusted to 8.5 by Tris-base and the dissolved concentration of 2mg/mL, reacting at 37 ℃ for 12h to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) with the pH value adjusted to 12.56 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 5.0mg/mL, reacting at 25 ℃ for 12h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 9

The embodiment provides a preparation method of an amine-rich conversion coating, wherein a nickel-titanium alloy is selected as a base material for preparing the coating, and the preparation method comprises the following steps:

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 4 carboxyl groups in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 24h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.1h.

(2) Preparing the o-phenolic polymerization modified coating.

Cleaning the surface of the nickel-titanium alloy, and drying for later use. The nickel-titanium alloy is soaked in a dopamine solution with the pH value adjusted to 8.5 by Tris-base and the dissolved concentration of 2mg/mL, the reaction is carried out for 12 hours at 37 ℃ to obtain a polydopamine coating, and the polydopamine coating is washed by deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of modified amine compound solution prepared in the step (1) with the pH value adjusted to 13 to submerge the modified coating in the step (2), reacting at 37 ℃ for 4h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Example 10

The embodiment provides a preparation method of an amine-rich conversion coating, wherein silicone rubber is used as a substrate material for coating preparation, and the preparation method comprises the following steps:

(1) Preparation of modified amine Compounds

Dissolving 1mg/mL DOTA containing 4 carboxyl groups in MES buffer solution with pH adjusted to 5.6, adding EDC and NHS, reacting at 37 ℃ for 240h, adding a reaction solution containing 1:1 Cu ²⁺ And carrying out ultrasonic reaction for 0.3h.

(2) Preparing the o-phenolic polymerization modified coating.

And cleaning the surface of the silicon rubber, and drying for later use. Soaking the silicon rubber in a dopamine solution with the dissolved concentration of 1mg/mL, adjusting the pH value to 8.5 by Tris-base, reacting for 24 hours at 25 ℃ to obtain a polydopamine coating, and washing with deionized water for later use.

(3) Preparation of amino-rich conversion coating

And (3) adding 1mL of the modified amine compound solution prepared in the step (1) and adjusting the pH value to 12.56 to submerge the modified coating in the step (2), wherein the concentration of the modified amine compound is 1.0mg/mL, reacting at 25 ℃ for 24h, and cleaning to obtain the long-acting stable and controllable Nitric Oxide (NO) release conversion coating.

Comparative example 1

The only difference from example 1 is: the manner of introduction of copper ions is different. Comparative example 1 macrocyclic polyamine was covalently immobilized via amine groups on the surface of the coating, followed by chelation of copper ions under ultrasonic conditions, without prior modification of the amine group compound after copper ion.

Comparative example 2

The only difference from example 1 is: no Cu-DOTA-modified amine compounds (PAMCAMs) were added.

Comparative example 3

The only difference from example 1 is: adding a macrocyclic polyamine modified amine compound (DOTA) which does not chelate copper ions.

Test example 1

The amine compound modified with the functional molecule that catalyzes the release of nitric oxide in test example 1 is shown in fig. 1.

Test example 2

The NO release detection effect graph before and after soaking of the coating prepared in example 1 was examined, and the results are shown in fig. 2.

The test method comprises the following steps: NO release before and after soaking (30 days) of the conversion coating was measured in PBS (pH = 7.4) using a chemiluminescent NO analyzer (NOA) (sensors 280i, boulder, co) at 37 ℃.

As can be seen from FIG. 2, the coating obtained by the preparation method has long-acting stable controllable Nitric Oxide (NO) release, and still maintains relatively stable NO catalytic release capability after long-time soaking, so that the catalytic efficiency of copper ions in the coating is improved, the long-acting stable controllable NO catalytic release is realized, and wider application potential is provided for the development of NO-release materials.

Test example 3

The effect of the amine-based conversion coating with long-acting, stable and controllable Nitric Oxide (NO) release prepared in example 1 on blood compatibility was tested, and the result is shown in fig. 3. Where 316L SS is the bare metal substrate, PAMCAMs is comparative example 2, and DOTA is comparative example 3.

The test method comprises the following steps: the anticoagulation of different surfaces was tested by an in-half animal blood test for 2h. And (3) fixing the sample after the blood circulation experiment is finished, dehydrating and drying, and observing the surface of the sample by using an electronic scanning electron microscope.

As can be seen in fig. 3. Compared with 316L SS, a comparative example 2 and a comparative example 3, the conversion coating with long-acting, stable and controllable Nitric Oxide (NO) release amine groups prepared by the method has stronger antithrombotic capacity and wider application field.

Test example 4

Example 1 and comparative example 1 were tested, and the detection method was identical to that of test example 2. The results are shown in FIG. 4.

As can be seen from fig. 4, in the manner of covalently grafting DOTA on the surface of the coating and then chelating copper ions, a higher burst of NO catalytic release occurs in the early stage, and the catalytic release capability of NO decreases with time, which is mainly caused by the decrease in the stability of NO catalytic release due to the lower utilization rate of copper ions, so that the catalytic rate and the stability of the coating are both significantly decreased.

Test example 5

The copper ion content of the coatings of example 1, comparative example 2 and comparative example 3 was determined by X-ray photoelectron spectroscopy. The results are shown in fig. 5, where 0.8% copper content was retained in the Cu-DOTA modified amine-based conversion coating, while no elemental copper was found in the comparative coating, further demonstrating the feasibility of the invention to produce coatings.

In conclusion, the invention provides a preparation method, a functional material and application of a rich amine group conversion coating, wherein a carboxyl-containing macrocyclic polyamine compound and a molecule with NO catalytic activity are subjected to chelation reaction to introduce the NO catalytic activity molecule, and then the carboxyl and an amino compound are subjected to amidation reaction to obtain a modified amine compound; the modified amine compound is introduced by using an o-phenolic polymerization coating on the surface of the material under an alkaline condition. The preparation method provided by the invention has the following advantages:

(1) A coating with long-acting, stable and controllable nitric oxide release, high chemical stability and strong adhesive property is obtained on the surface of the material.

(2) The coating has better chemical stability through the secondary in-situ re-crosslinking reaction under the strong alkali condition, so that the coating is suitable for extreme conditions, is not easy to deteriorate and damage when used under the extreme conditions, and has excellent use safety.

(3) The introduction of the amine compound also ensures that the coating has the characteristic of rich amine group, so that the conversion coating rich in amine group has the performance of wide biological functional molecule grafting and excellent adhesion performance with the surface of the material, and further endows the material with more application prospects.

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 rich amine-based conversion coating, comprising:

a carboxyl-containing macrocyclic polyamine compound and a molecule with NO catalytic activity are subjected to chelation reaction, and then react with an amino compound to obtain a modified amine compound;

carrying out in-situ re-crosslinking reaction on the base material with the surface adhered with the o-phenol polymerization modified coating and the modified amine compound under an alkaline condition;

the macrocyclic polyamine compound containing carboxyl is selected from at least one of 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, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tri-tert-butyl tetraacetate, porphyrin and 8-hydroxyquinoline and derivatives thereof;

the molecule with NO catalytic activity is a divalent copper ion;

the amino compound is selected from at least one of polyallylamine, polyvinylamine, polylysine and chitosan.

2. The preparation method according to claim 1, wherein the carboxyl-containing macrocyclic polyamine compound is subjected to carboxyl activation, then reacted with the molecule with NO catalytic activity for 0.2h-3h, and then reacted with the amino compound solution with the concentration of 0.01ng/mL-20g/mL at 0-350 ℃ for 0.001h-240h, and after the reaction is completed, dialyzed to obtain the modified amine compound;

and (3) placing the base material with the surface attached with the o-phenol polymerization modified coating into a mixed solution formed by an alkaline buffer solution and the modified amine compound, and reacting for 0.001-72 h at the temperature of 0-120 ℃.

3. The preparation method according to claim 2, wherein the carboxyl-containing macrocyclic polyamine compound with a concentration of 0.01ng/mL to 5g/mL is dissolved in a mixed solution of MES buffer with a pH value of 4 to 10 and a condensing agent, carboxyl is activated for 0.001 to 240 hours at a temperature of 0 ℃ to 350 ℃, and then reacted with the molecule with NO catalytic activity under ultrasonic conditions.

4. The method according to claim 3, wherein the concentration of the carboxyl group-containing macrocyclic polyamine compound is 0.1mg/mL-1mg/mL, and the pH of the MES buffer is 5-8.

5. The method according to claim 3, wherein the temperature of the activation of the carboxyl group is 20 ℃ to 50 ℃ and the time of the activation of the carboxyl group is 0.1 to 24 hours.

6. The process according to claim 3, characterized in that the condensing agents are EDC and NHS, the molar ratios of EDC to the carboxyl groups in the carboxyl-containing macrocyclic polyamine-based compound are each 4 to 6, the molar ratios of NHS to the carboxyl groups in the carboxyl-containing macrocyclic polyamine-based compound are each 0.5 to 1.5;

the raw materials adopted by EDC and NHS have the concentration of 0.01ng/mL-5g/mL.

7. The method according to claim 3, wherein the reaction time is controlled to be 0.2 to 0.5 hours during the reaction with the molecule having NO catalytic activity.

8. The process according to claim 7, wherein the molar ratio of the molecule having NO catalytic activity to the macrocyclic polyamine compound having a carboxyl group is 1.

9. The process according to claim 8, wherein the molar ratio of the molecule having NO catalytic activity to the macrocyclic polyamine compound having a carboxyl group is 1.

10. The method of claim 9, wherein the molecule having NO catalytic activity is selected from at least one of copper chloride and copper bromide.

11. The preparation method according to claim 2, wherein the concentration of the amino compound is 0.1mg/mL to 5mg/mL, the reaction temperature is 15 ℃ to 40 ℃, and the reaction time is 0.5h to 48h.

12. The preparation method of claim 2, wherein in the mixed solution of the alkaline buffer solution and the modified amine compound, the concentration of the modified amine compound is 0.01ng/mL-5g/mL, the reaction temperature is 15-40 ℃, and the reaction time is 0.5-48h.

13. The method according to claim 12, wherein the concentration of the modified amine compound is 0.1mg/mL to 5mg/mL, and the pH of the alkaline buffer solution is 8 to 14.

14. The method of claim 13, wherein the alkaline buffer solution comprises at least one of a sodium carbonate-sodium bicarbonate buffer, a PBS buffer, a Tris-HCl buffer, a sodium hydroxide solution, and a potassium hydroxide solution.

15. The method according to claim 14, wherein the alkali in the alkaline buffer solution is sodium hydroxide and has a concentration of 0.00001g/L to 160g/L.

16. The method according to claim 12, wherein the modified amine compound is at least one compound selected from the group consisting of a macrocyclic polyamine chelated copper ion-modified polyallylamine, an NHS-modified macrocyclic polyamine chelated copper ion-modified polyallylamine, and a porphyrin chelated copper ion-modified polyallylamine.

17. The preparation method according to claim 1 or 2, wherein the preparation of the o-phenolic polymerization modified coating comprises: soaking the base material in the mixed solution of o-phenolic compound and buffering solution to react at pH 0-14 and temperature 0-350 deg.c for 0.001-240 hr.

18. The preparation method of claim 17, wherein the concentration of the o-phenolic compound in the reaction system is 0.01ng/mL to 5g/mL.

19. The method according to claim 18, wherein the concentration of the o-phenolic compound in the reaction system is 0.5 to 20mg/mL.

20. The preparation method of claim 17, wherein in the process of preparing the o-phenolic polymerization modified coating, the reaction pH value is controlled to be 5.6-12, the reaction temperature is 20-40 ℃, and the reaction time is 0.5-48h.

21. The method according to claim 17, wherein the o-phenolic compound is at least one selected from the group consisting of a monoamine-type phenolic compound, an indole-type compound, and phenol and a derivative thereof.

22. The method according to claim 17, wherein the o-phenolic compound is at least one compound selected from the group consisting of melanin, dopamine, dihydroxyindole, epinephrine, norepinephrine, levodopa, tannic acid, dextrodopa, gallic acid, pyrogalloc acid, epicatechin gallate, epigallocatechin, and epigallocatechin gallate.

23. The method according to claim 17, wherein the base material is at least one selected from a metallic material, an inorganic material, and a polymer material.

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

25. A functional material comprising a substrate and a rich amine-based conversion coating attached to the substrate, wherein the rich amine-based conversion coating is produced by the production method according to any one of claims 1 to 24.

26. Use of the functional material of claim 25 in the preparation of a blood implant device.