CN114432506A

CN114432506A - Zwitterion functionalized biological material, and preparation method and application thereof

Info

Publication number: CN114432506A
Application number: CN202210231786.2A
Authority: CN
Inventors: 杨志禄; 杜泽煜; 孟昊天; 牟小辉; 王颖; 张文泰
Original assignee: Dongguan Peoples Hospital
Current assignee: Dongguan Peoples Hospital
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2022-05-06

Abstract

The invention discloses a zwitterion functionalized biological material, a preparation method and application thereof, and belongs to the technical field of biological materials. A method for preparing zwitterion functionalized biological material comprises the following steps: reacting the substrate with the polyamine-phenol coating with a compound containing both a carbon-carbon double bond and a carboxyl group to form a polyamine-phenol coating containing a carbon-carbon double bond on the surface of the substrate; the polyamine-phenol coating containing carbon-carbon double bonds is reacted with a zwitterionic compound containing carbon-carbon double bonds to form a zwitterionic functionalized polyamine-phenol coating on the surface of the substrate. The polymerization of utilizing carbon-carbon double bond forms the macromolecular polymer long chain that has certain spatial structure and multi-functional, and hydrophilic end exposes in the outside and makes hydrophilicity increase, and the intermolecular aggregation degree increases, and then deposits gradually and forms firm coating on the material surface, prepares the material that obtains to have multiple biological function, like super hydrophilic, anti-freezing, antibiotic etc..

Description

Zwitterion functionalized biological material, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a zwitterion functionalized biological material, and a preparation method and application thereof.

Background

Blood-contact biomaterials are a widely used clinical implantable material that can diagnose, treat, repair or enhance the function of damaged tissues and organs in living beings. Although blood contact materials have good application prospects, in clinical practical applications, these medical devices often cause blood coagulation or infection directly or indirectly, which limits their clinical use. Such as blood contact and interventional devices and extracorporeal blood circulation devices, are subject to clinical complications such as thromboembolism, bacterial infection, local tissue damage, etc., which not only reduce the efficiency of treatment and shorten the normal life of the device, but also cause significant damage to the patient.

At present, clinically, in order to solve the problems of blood coagulation and infection, antibiotics, anticoagulation medicines and medicines for inhibiting bacterial infection are mainly used for adjuvant therapy in a systemic administration mode. However, long-term use of these drugs in humans can cause a series of side effects, resulting in persistent injuries such as antibiotic resistance, thrombocytopenia, bacterial resistance, tissue damage and bleeding. In conclusion, the development of the biomaterial is advanced, the biomaterial enters a brand new stage, and the diversified functional requirements and clinical practice require that the biomaterial has various biological functions while meeting the biocompatibility.

At present, strategies for improving the blood compatibility of the material by modifying the surface of the material by various physical and chemical methods mainly comprise zero interface surface tension, zero interface free energy, a negative charge surface, a flowing hydrophilic surface, a microphase separation structure, a heparinized surface, a biological surface and the like. However, most of the surface modification strategies have the problems of complex preparation process, fussy surface pretreatment, easy preparation of toxin, easy explosion of chemical reagent hazard and the like.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a zwitterion functionalized biological material and a preparation method thereof, aiming at introducing zwitterions into the surface of the material to realize various biological functions of the material, such as super-hydrophilicity, anticoagulation, antibiosis and the like.

The second purpose of the invention is to provide the application of the zwitterion functionalized biomaterial in the preparation of blood interventional instruments or extracorporeal blood circulation equipment.

The invention is realized by the following steps:

in a first aspect, the present invention provides a method for preparing a zwitterionic functionalized biomaterial, comprising:

reacting the substrate with the polyamine-phenol coating with a compound containing both a carbon-carbon double bond and a carboxyl group to form a polyamine-phenol coating containing a carbon-carbon double bond on the surface of the substrate;

the polyamine-phenol coating containing carbon-carbon double bonds is reacted with a zwitterionic compound containing carbon-carbon double bonds to form a zwitterionic functionalized polyamine-phenol coating on the surface of the substrate.

In a second aspect, the present invention provides a zwitterionic functionalized biomaterial produced by the method of any one of the preceding embodiments.

In a third aspect, the present invention provides the use of the zwitterionic functionalized biomaterial of the previous embodiment in the manufacture of a blood access device or an extracorporeal blood circulation apparatus.

The invention has the following beneficial effects: the carbon-carbon double bond is introduced to the substrate with the polyamine-phenol coating, then the substrate reacts with the zwitter-ion compound containing the carbon-carbon double bond, a macromolecular polymer long chain with a certain space structure and multiple functions is formed by utilizing the polymerization of the carbon-carbon double bond, the hydrophilicity is increased by exposing the hydrophilic end to the outside, the intermolecular aggregation degree is increased, and then the hydrophilic end is gradually deposited on the surface of the material to form a stable coating, so that the material with multiple biological functions, such as super-hydrophilicity, anticoagulation, antibiosis and the like, is prepared.

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 graph showing the antibacterial effect of an unmodified silicone rubber coating (SR), a polyamine-Phenol Coating (PCPA), a polyamine-phenol coating containing carbon-carbon double bonds (E-PCPA), and a zwitterionic functionalized polyamine-phenol coating (MPC);

FIG. 2 is a graph of an in-half animal experiment of an unmodified silicone rubber coating (SR), a polyamine-Phenolic Coating (PCPA), a polyamine-phenolic coating containing carbon-carbon double bonds (E-PCPA), a zwitterionic functionalized polyamine-phenolic coating (MPC);

FIG. 3 is a schematic diagram showing the water contact angles of the surfaces of the materials with zwitterionic 2-Methacryloyloxyethyl Phosphorylcholine (MPC) grafting concentrations of 25mg/mL and 100mg/mL respectively;

FIG. 4 is a schematic diagram showing the water contact angles of the surfaces of the materials with zwitterionic 2-Methacryloyloxyethyl Phosphorylcholine (MPC) grafting concentrations of 50mg/mL and 100mg/mL respectively;

FIG. 5 is a schematic diagram showing the water contact angles of the surface of a material with zwitterionic 2-Methacryloyloxyethyl Phosphorylcholine (MPC) grafting times of 12h and 24h, respectively;

FIG. 6 is a schematic diagram showing the water contact angles of the surfaces of the materials with zwitterionic 2-Methacryloyloxyethyl Phosphorylcholine (MPC) grafting concentrations of 5mg/mL and 100mg/mL, respectively.

Detailed Description

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

The embodiment of the invention provides a preparation method of a zwitterion functionalized biological material, which comprises the following steps:

s1 preparation of polyamine-phenol coating

The preparation process of the polyamine-phenol coating comprises the following steps: activating carboxyl of a biological functional molecule containing carboxyl and phenolic hydroxyl, reacting the biological functional molecule with a water-soluble polymer rich in amino, and dialyzing to obtain a polyamine-phenol solution; the substrate is placed in a polyamine-phenol solution to react to form a poly-aminophenol coating on the surface of the substrate.

Wherein, the process of activating the carboxyl of the biological functional molecule comprises the following steps: mixing the biological functional molecules with EDC and NHS, and incubating for 0.5-1 hr at pH 5-7 and 30-45 deg.C; preferably, the incubation time is 30-60min, the molar ratio of EDC to the carboxyl content of the bio-functional molecule is 0.5-10:1, and the molar ratio of NHS to the carboxyl content of the bio-functional molecule is 0.5-10: 1. EDC and NHS can be used for activating carboxyl in the biological functional molecule under weak acid condition to promote the subsequent amidation reaction, and the dosage of EDC and NHS is determined according to the carboxyl content in the biological functional molecule.

In particular, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) is a water-soluble carbodiimide that is used as an activator of carboxyl groups in amide synthesis, often in conjunction with NHS (N-hydroxysuccinimide), and the addition of NHS results in a stable intermediate to improve coupling efficiency.

Wherein, in the preparation process of the polyamine-phenol solution, the reaction temperature of the biological functional molecules and the water-soluble polymer rich in amino is controlled to be 4-10 ℃, the reaction time is 12-24h, and dialysis is carried out for 48-72h after the reaction is finished to remove small molecules. The carboxyl group on the biological functional molecule and the amine group are subjected to amidation reaction to form polyamine-phenol, and the reaction temperature and time are preferably controlled within the above range to promote the amidation reaction.

Further, in an amidation reaction system, the concentration of the biological functional molecules is 0.1-10mg/mL, and the concentration of the water-soluble polymer rich in amino groups is 0.01-10 mg/mL; preferably, in the reaction system, the concentration of the biological functional molecules is 0.5-5mg/mL, and the concentration of the water-soluble polymer rich in amine groups is 0.01-1 mg/mL. The concentration of the biological functional molecules and the concentration of the water-soluble polymer rich in amine groups are further controlled to promote the amidation reaction.

Specifically, the concentration of the biofunctional molecule, the amine group-rich water-soluble polymer, means the concentration in the reaction system, that is, the concentration in the mixed solution formed after the raw materials are mixed.

Further, the bio-functional molecule is selected from at least one of 3, 4-dihydroxyphenylpropionic acid, ethyl dihydrocaffeate, 3,4, 5-trihydroxybenzoic acid, caffeic acid, methyl caffeate, caftaric acid, 2, 3-dihydroxyphenylalanine, 5-O-caffeoylshikimic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, L-DOPA, pelargonidin, cyanidin, gallic acid, (-) -epicatechin, (-) -epigallocatechin 3-gallate, isoquercetin, salvianolic acid, rosmarinic acid, tannic acid, etc. Specifically, the types of the biofunctional molecules are not limited to the above, and include derivatives produced from the above raw materials, which may be one or more.

Further, the water-soluble polymer rich in amine groups is selected from at least one of polyallylamine and hydrochloride thereof, polyallylhydrazine, polylysine, polyethyleneimine and derivatives thereof, polyacrylamide, 3-pyrroline, hexamethylenediamine, ethylenediamine, chitosan and crotylamine. The kind of the water-soluble polymer rich in amine groups is not limited to the above, and the raw materials may be one or a mixture of a plurality of the above.

Further, the base material is selected from at least one of a metal material, an inorganic non-metal material and a polymer material, and the kind of the base material may be selected according to the application of the material. Specifically, the substrate is selected from stainless steel, pure titanium, titanium alloy, iron, TiO₂、Si、SiO₂At least one of PVC, silicon rubber and metal oxide, which can be one or more.

Wherein the polyamine-phenol coating is prepared by placing the base material in polyamine-phenol solution, reacting at pH of 8-11, controlling the reaction temperature at 25-45 deg.C, and reacting for 12-36 h. The base material is put into polyamine-phenol solution and alkaline solution to react, so that the adhesion of phenolic hydroxyl groups can be utilized to perform firmer adhesion on the surface of the base material.

In some embodiments, the substrate is washed and dried after the reaction is completed, and then placed in the polyamine-phenol solution to repeat the reaction 1-3 times. Specifically, the cleaning can remove particles and solution remained on the surface, and the drying can be dried by nitrogen.

S2 preparation of polyamine-phenol coatings containing carbon-carbon double bonds

Reacting the base material with polyamine-phenol coating with compound containing carbon-carbon double bond and carboxyl group to form polyamine-phenol coating containing carbon-carbon double bond on the surface of the base material, and introducing the carbon-carbon double bond into the polyamine-phenol coating by amidation reaction of carboxyl group and amine group on the polyamine-phenol coating.

In the actual operation process, firstly, a compound containing a carbon-carbon double bond and a carboxyl is dissolved, and then the compound reacts with the polyamine-phenol coating on the base material under the condition that the pH value is 6.21-8.21, the reaction temperature is controlled to be 25-37 ℃, and the reaction time is 6-24 hours. Specifically, the solvent used for dissolving the compound containing both the carbon-carbon double bond and the carboxyl group may be dimethyl sulfoxide (DMSO), and may also be other organic solvents, which are not limited herein.

In a preferred embodiment, the reaction temperature of the compound containing both a carbon-carbon double bond and a carboxyl group with the polyamine-phenol coating is 30 to 37 ℃, the reaction time is 6 to 12 hours, and the concentration of the compound containing both a carbon-carbon double bond and a carboxyl group in the reaction system is 0.5 to 10 mg/mL. The pH value of the reaction can be adjusted by adopting PBS buffer solution, and the introduction amount of carbon-carbon double bonds can be increased by optimizing reaction conditions to promote amidation reaction.

In order to further increase the introduction amount of the carbon-carbon double bond, the material is dried by blowing after the reaction is completed, and the reaction is repeated for 1 to 3 times.

In some embodiments, the compound containing both a carbon-carbon double bond and a carboxyl group is an ester, such as an NHS ester, to impart multifunctional properties to the material.

In a preferred embodiment, the compound containing both carbon-carbon double bond and carboxyl group is at least one selected from the group consisting of N-hydroxysuccinimide methacrylate, N-succinimidyl acrylate and PEG-N-hydroxysuccinimide acrylate, and may be one or more.

S3 preparation of zwitterionic functionalized polyamine-phenol coatings

The polyamine-phenol coating containing carbon-carbon double bonds is reacted with a zwitterionic compound containing carbon-carbon double bonds to form a zwitterionic functionalized polyamine-phenol coating on the surface of the substrate. In practice, the process of forming a zwitterionic functionalized polyamine-phenol coating comprises: activating the carbon-carbon double bond on the polyamine-phenol coating containing the carbon-carbon double bond, and then reacting with a mixed solution formed by an initiator, a pro-initiator and a zwitterionic compound for 12-36 h.

It is to be noted that, in the process of introducing the zwitterionic compound, an initiator and an initiator accelerator are added, the carbon-carbon double bond on the surface of the pretreated coating is activated, then the zwitterionic compound, which also contains carbon-carbon double bonds, is added to polymerize with the activated double bond to form a macromolecular polymer long chain with a certain spatial structure and multiple functions, the hydrophilic end is exposed outside to increase the surface hydrophilicity, the intermolecular aggregation degree is increased, and the hydrophilic end is gradually deposited on the surface of the material to form a stable coating.

In an alternative embodiment, the process of activating the carbon-carbon double bond comprises: mixing an initiator and an initiator accelerator, and carrying out contact reaction with a polyamine-phenol coating containing carbon-carbon double bonds for 5-20min at the reaction temperature of 20-30 ℃; preferably, the reaction time for activating the carbon-carbon double bond is 10-20min, and the reaction temperature is 23-27 ℃. The subsequent polymerization reaction with the zwitterionic compound is promoted by activating the carbon-carbon double bond in the coating.

In a preferred embodiment, the reaction time with the zwitterionic compound is 24-36h, the concentration of the zwitterionic compound in the reaction system is 50-200mg/mL, and the reaction process is carried out in the absence of oxygen to prevent oxidation. After the reaction is finished, blow-drying is carried out to obtain a clean product.

Preferably, the zwitterionic compound is at least one selected from 2-methacryloyloxyethyl phosphorylcholine, methacryloyloxyethyl trimethyl ammonium chloride, acryloyloxyethyl trimethyl ammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid, methacryloyloxyethyl sulfobetaine and 3- [ [2- (methacryloyloxy) ethyl ] dimethyl ammonium ] propionate, and may be one or a mixture of several thereof.

In some embodiments, the initiators used in forming the zwitterionic functionalized polyamine-phenol coating are each selected from at least one of sodium persulfate, ammonium persulfate, and potassium persulfate, and the concentration of the initiator in the reaction system solution is from 1 to 10 mg/mL. The variety of the initiator can be selected from the above several types, and can be one or a mixture of several types, and the activation effect on carbon-carbon double bonds is improved by controlling the dosage of the initiator.

In some embodiments, the coinitiators used in the formation of the zwitterionic functionalized polyamine-phenolic coating are each selected from at least one of N, N "-tetramethyldiethylamine, cobalt octoate, cobalt naphthenate and tertiary amines, and the concentration of the coinitiators in the reaction system solution is 0.1 to 10 μ L/mL. The variety of the initiator can be selected from the above several types, and can be one or a mixture of several types, and the activation effect on the carbon-carbon double bond is improved by controlling the dosage of the initiator.

The embodiment of the invention provides a zwitterion functionalized biomaterial, which is prepared by the preparation method and has the performances of super-hydrophilicity, anticoagulation, antibiosis and the like. The zwitterionic functionalized biomaterial can be further prepared into blood interventional instruments or extracorporeal blood circulation equipment, and has a good application prospect.

It should be noted that the coating in the zwitterionic functionalized biomaterial provided by the embodiment of the invention is easy to deposit, has high strength, strong adhesion and high compactness, and can be widely applied to various materials.

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

Example 1

This example provides a method of preparing a polyamine-phenolic coating on the principle of: firstly, EDC and NHS are used for activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA), then polyallylamine is added, after amidation reaction, polyamine-phenol solution is obtained, and then the coating is obtained by the base material by combining the adhesive property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid.

The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 70mg HCA powder, mixing well, adding 100mL of oxygen-free MES buffer solution with pH of 5.6, incubating at 37 deg.C for 40 min, adding 0.5mL of polyallylamine stock solution, mixing well, introducing N₂And reacting at 8 ℃ for 24 hours.

B. And (3) taking out the reaction solution in the step A, putting the reaction solution in a 7000Da dialysis bag for dialysis for 48 hours, and replacing water every 6-8 hours to obtain the polyamine-phenol solution.

C. Equal volumes of polyamine-phenol solution and 10mg/mL sodium hydroxide solution were added to the surface of the clean silicone rubber and reacted at 37 ℃ for 24 hours. Cleaning to remove residual solution or particles on the surface, drying by using nitrogen, and repeating the drying for three times to obtain the polyamine-phenol coating.

Example 2

This example provides a method of preparing a polyamine-phenolic coating on the principle of: firstly, activating carboxyl of 3,4, 5-trihydroxybenzoic acid by EDC and NHS, then adding polyallylamine, obtaining polyamine-phenol solution after amidation reaction, and then combining the phenol hydroxyl adhesion performance of the 3,4, 5-trihydroxybenzoic acid to make the substrate material obtain the coating.

The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 65.3mg 3,4, 5-trihydroxybenzoic acid powder, mixing well, adding 100mL oxygen-free MES buffer solution with pH of 5.6, incubating at 37 deg.C for 30 min, adding 0.5mL polyallylamine stock solution, mixing well, introducing N₂And reacting at 8 ℃ for 24 hours.

Example 3

This example provides a method of preparing a polyamine-phenolic coating on the principle of: firstly, activating caffeic acid carboxyl by EDC and NHS, then adding polyallylamine, obtaining polyamine-phenol solution after amidation reaction, and then combining the phenol hydroxyl adhesion property of caffeic acid to make the base material obtain the coating.

The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 69.2mg caffeic acid powder, mixing well, adding 100mL oxygen-free MES buffer solution with pH 5.6, incubating at 37 deg.C for 45 min, adding 0.5mL polyallylamine stock solution, mixing well, introducing N₂And reacting at 8 ℃ for 24 hours.

B. And taking out the reaction solution in the step A, putting the reaction solution in a 7000Da dialysis bag for dialysis for 48 hours, and changing water every 6-8 hours to obtain the polyamine-phenol solution.

Example 4

This example provides a method of preparing a polyamine-phenolic coating on the principle of: firstly activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA) by EDC and NHS, then adding polyallylhydrazine, obtaining polyamine-phenol solution after amidation reaction, and then combining the phenol hydroxyl adhesive property of the 3, 4-dihydroxyphenyl propionic acid to make the substrate material obtain the coating.

The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 70mg HCA powder, mixing well, adding 100mL of anaerobic MES buffer solution with pH of 5.6, incubating at 37 deg.C for 45 min, adding 63mg polyallylamine powder, mixing well, and introducing N₂And reacting at 8 ℃ for 24 hours.

Example 5

This example provides a method of preparing a polyamine-phenolic coating, the principle of which is: firstly, EDC and NHS are used for activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA), then crotylamine is added, polyamine-phenol solution is obtained after amidation reaction, and then the coating is obtained by the base material by combining the phenol hydroxyl adhesive property of the 3, 4-dihydroxyphenyl propionic acid.

The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 70mg HCA powder, mixing well, adding 100mL oxygen-free MES buffer solution with pH of 5.6, incubating at 37 deg.C for 45 min, adding 62.2mg crotonamine powder, mixing well, and introducing N₂And reacting at 8 ℃ for 24 hours.

Example 6

The present example provides a method for preparing a polyamine-phenol coating containing carbon-carbon double bonds, which is based on the following principle: firstly, EDC and NHS are used for activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA), then polyallylamine is added, after amidation reaction, polyamine-phenol solution is obtained, and then the adhesion property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid is combined, so that the base material obtains the polyamine-phenol coating. Further adding acrylic acid-N-succinimide ester solution, and soaking to obtain polyamine-phenol coating containing carbon-carbon double bond.

The method comprises the following specific steps:

A. weighing 135mg of EDC and 62mg NHS and 70mg HCA powder, mixing well, adding 100mL of oxygen-free MES buffer solution with pH of 5.6, incubating at 37 deg.C for 45 min, adding 0.5mL of polyallylamine stock solution, mixing well, and introducing N₂And reacting at 8 ℃ for 24 hours.

D. Dissolving the acrylic acid-N-succinimidyl ester by DMSO, adding PBS buffer solution, and uniformly mixing to obtain acrylic acid-N-succinimidyl ester solution with the concentration of 3 mg/mL.

E. The above-mentioned N-succinimidyl acrylate solution was added to the polyamine-phenol coating in C and reacted at 37 ℃ for 12 hours. Cleaning to remove residual solution or particles on the surface, drying by using nitrogen, and repeating twice to obtain the polyamine-phenol coating containing carbon-carbon double bonds.

Example 7

The present example provides a method for preparing a polyamine-phenol coating containing carbon-carbon double bonds, which is based on the following principle: firstly, EDC and NHS are used for activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA), then polyallylamine is added, after amidation reaction, polyamine-phenol solution is obtained, and then the adhesion property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid is combined, so that the base material obtains the polyamine-phenol coating. And further adding methacrylic acid N-hydroxysuccinimide ester solution, and soaking to obtain the polyamine-phenol coating containing carbon-carbon double bonds. The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 70mg HCA powder, mixing well, adding 100mL of oxygen-free MES buffer solution with pH of 5.6, incubating at 37 deg.C for 45 min, adding 0.5mL of polyallylamine stock solution, mixing well, and introducing N₂And reacting at 8 ℃ for 24 hours.

D. Dissolving N-hydroxysuccinimide methacrylate in DMSO, adding PBS buffer solution, and mixing to obtain 3mg/mL acrylic acid-N-succinimide ester solution.

E. The above N-hydroxysuccinimide methacrylate solution was added to the polyamine-phenol coating layer in C and reacted at 37 ℃ for 12 hours. Cleaning to remove residual solution or particles on the surface, drying by using nitrogen, and repeating for three times to obtain the polyamine-phenol coating containing carbon-carbon double bonds.

Example 8

The embodiment provides a preparation method of a zwitterion functionalized biological material, which comprises the following steps: firstly, activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA) by EDC and NHS, then adding polyallylamine, obtaining polyamine-phenol solution after amidation reaction, and combining the adhesive property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid to ensure that the base material obtains the polyamine-phenol coating. And adding acrylic acid-N-succinimide ester solution, and soaking to obtain the polyamine-phenol coating containing carbon-carbon double bonds. Further, TEMED and NaS were used first₂O₈Activating carbon-carbon double bonds on the coating by the mixed solution, adding 2-Methacryloyloxyethyl Phosphorylcholine (MPC) aqueous solution, and polymerizing the double bonds to obtain the zwitterion functionalized polyamine-phenol coating.

The method comprises the following specific steps:

E. The above-mentioned N-succinimidyl acrylate solution was added to the polyamine-phenol coating in C and reacted at 37 ℃ for 12 hours. Cleaning to remove residual solution or particles on the surface, drying by using nitrogen, and repeating for three times to obtain the polyamine-phenol coating containing carbon-carbon double bonds.

F. 10mg/mL NaS was prepared₂O₈And 4. mu.L/mL TEMED mixture, added to the coating surface in E, and activated the double bond for 10 minutes in an oxygen-free environment. A further 100mg/mL MPC solution was added and reacted for 24 hours, again in the absence of oxygen, to form a zwitterionic functionalized polyamine-phenol coating by double bond polymerization.

Example 9

The embodiment provides a preparation method of a zwitterion functionalized biological material, which comprises the following steps: firstly, activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA) by EDC and NHS, then adding polyallylamine, obtaining polyamine-phenol solution after amidation reaction, and combining the adhesive property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid to ensure that the base material obtains the polyamine-phenol coating. And adding acrylic acid-N-succinimide ester solution, and soaking to obtain the polyamine-phenol coating containing carbon-carbon double bonds. Further, TEMED and NaS were used first₂O₈Activating carbon-carbon double bonds on the coating by the mixed solution, adding 2-acrylamido-2-methylpropane sulfonic Acid (AMPS) aqueous solution, and polymerizing the double bonds to obtain the zwitterion functionalized polyamine-phenol coating.

The method comprises the following specific steps:

A. 135mg EDC, 62mg NHS and 70mg HCA powder were weighed, mixed well, added 100mL of pH 5.6 anaerobic MES buffer solution, incubated at 37 ℃ for 45 minutesAdding 0.5mL of polyallylamine stock solution, mixing uniformly, and continuously introducing N₂And reacting at 4 ℃ for 24 hours.

F. 10mg/mL NaS was prepared₂O₈And 4. mu.L/mL TEMED mixture, added to the coating surface in E, and activated the double bond for 10 minutes in an oxygen-free environment. Then 100mg/mL AMPS aqueous solution is added, and the reaction is carried out for 24 hours in the oxygen-free environment, and the zwitterion functionalized polyamine-phenol coating is formed through double bond polymerization.

Example 10

The embodiment provides a preparation method of a zwitterion functionalized biological material, which comprises the following steps: firstly, activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA) by EDC and NHS, then adding polyallylamine, obtaining polyamine-phenol solution after amidation reaction, and combining the adhesive property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid to ensure that the base material obtains the polyamine-phenol coating. And adding acrylic acid-N-succinimide ester solution, and soaking to obtain the polyamine-phenol coating containing carbon-carbon double bonds. Further, TEMED and NaS were used first₂O₈Activating carbon-carbon double bonds on the coating by the mixed solution, adding a methacryloyl ethyl Sulfobetaine (SBMA) aqueous solution, and polymerizing the double bonds to obtain zwitterion functionalized polyAmine-phenol coatings. The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 70mg HCA powder, mixing well, adding 100mL of oxygen-free MES buffer solution with pH of 5.6, incubating at 37 deg.C for 45 min, adding 0.5mL of polyallylamine stock solution, mixing well, and introducing N₂And reacting at 10 ℃ for 24 hours.

C. Equal volumes of polyamine-phenol solution and 10mg/mL sodium hydroxide solution were added to the surface of the clean silicone rubber and reacted at 37 ℃ for 24 hours. Washing to remove residual solution or particles on the surface, drying by using nitrogen, and repeating the drying for three times to obtain the polyamine-phenol coating.

F. 10mg/mL NaS was prepared₂O₈And 4. mu.L/mL TEMED mixture, added to the coating surface in E, and activated the double bond for 15 minutes in an oxygen-free environment. Then 100mg/mL of SBMA aqueous solution is added, and the reaction is carried out for 24 hours in the absence of oxygen, and the zwitterion functionalized polyamine-phenol coating is formed through the double bond polymerization.

Example 11

The embodiment provides a preparation method of a zwitterion functionalized biological material, which comprises the following steps: firstly, activating carboxyl of 3, 4-dihydroxyphenyl propionic acid (HCA) by EDC and NHS, then adding polyallylamine, obtaining polyamine-phenol solution after amidation reaction, and combining the adhesive property of the phenolic hydroxyl of the 3, 4-dihydroxyphenyl propionic acid to ensure that the base material obtains the polyamine-phenol coating. Adding acrylic acid-N-succinimide ester solution, and soaking to obtain polyamine containing carbon-carbon double bond-a phenol coating. Further, TEMED and NaS were used first₂O₈Activating carbon-carbon double bond on the coating by the mixed solution, and adding 3- [ [2- (methacryloyloxy) ethyl ] group]Dimethyl ammonium salt]The zwitterion functionalized polyamine-phenol coating is obtained by double bond polymerization of a propionate (CBMA) aqueous solution.

The method comprises the following specific steps:

A. weighing 135mg EDC, 62mg NHS and 70mg HCA powder, mixing well, adding 100mL of anaerobic MES buffer solution with pH of 5.6, incubating at 37 deg.C for 45 min, adding 0.5mL of polyallylamine stock solution, mixing well, and introducing N₂The reaction was carried out for 24 hours.

F. 10mg/mL NaS was prepared₂O₈And 4. mu.L/mL TEMED mixture, added to the coating surface in E, and activated the double bond for 20 minutes in an oxygen-free environment. A further 100mg/mL CBMA solution was added and reacted for 24 hours, again in the absence of oxygen, to form a zwitterionic functionalized polyamine-phenol coating by double bond polymerization.

Example 12

The only difference from example 8 is: the concentration of the aqueous MPC solution was 50 mg/mL.

Example 13

The only difference from example 8 is: the reaction time for grafting MPC was 12 h.

Comparative example 1

The only difference from example 8 is: the concentration of the aqueous MPC solution was 25 mg/mL.

Comparative example 2

The only difference from example 8 is: the concentration of the aqueous MPC solution was 5 mg/mL.

Test example 1

The zwitterion-functionalized biomaterial prepared in experimental example 8 was subjected to a bacterial experiment.

Preparation of experimental samples: the preparation method and conditions of the experimental sample were the same as those of example 8, and a catheter sample was prepared by the above preparation method.

The numbers of the experimental samples are as follows: SR, PCPA, E-PCPA, MPC.

Strain: gram-positive bacteria (staphylococcus epidermidis s) and gram-negative bacteria (escherichia coli e.

The specific process is as follows:

(1) activating bacteria: the solid medium was poured into a petri dish and irradiated with uv light for 10 minutes, and ventilated for 5 minutes. 5mL of physiological saline was drawn by a syringe and placed in a cell vial to dilute the strain. One loop from the bacterial strain was taken with the loop and placed in a cell vial containing 5mL of physiological saline. After bacteria are uniformly distributed, dipping a loopful of bacteria liquid by using an inoculating loop, scribing on a solid culture medium by adopting a scribing method, inverting the solid culture medium after the scribing is finished, and putting the solid culture medium into an incubator at 37 ℃ for incubation for 24 hours.

(2) Shaking the bacteria: 5mL of liquid medium was placed in the cell flask. And after the liquid culture medium is cooled, selecting a ring of smaller bacteria from the solid culture medium for activating the bacteria by using an inoculating ring, putting the smaller bacteria into the liquid culture medium, shaking the bacteria uniformly, and putting the cell culture bottle into an incubator at 37 ℃ for incubation for 24 hours after the culture is finished.

(3) Inoculating bacteria: 0.5mL of the liquid medium was poured into 500mL of physiological saline, and 9mL of the solution was taken and added to a centrifuge tube. Escherichia coli was diluted 109-fold and Staphylococcus epidermidis was diluted 105-fold. Adding 1mL of bacterial liquid into the first centrifugal tube through a Pasteur tube, uniformly blowing, adding 1mL of bacterial liquid into the first centrifugal tube into the second centrifugal tube, and uniformly blowing. And (5) sequentially diluting. And inoculating 100 mu L of the finally diluted bacterial liquid to the surface of the sample. The well plate with the inoculated bacteria is put into an incubator at 37 ℃ for incubation for 24 h.

(4) Coating a plate: adding 1mL of physiological saline into each sample, blowing bacteria by using a 1mL liquid transfer gun, adding 20-50 mu L of bacterial liquid into each culture dish, marking, coating a plate, inverting, and putting into an incubator for culturing for 24 hours.

(5) Counting: (4) after 24 hours of medium culture, the cells were taken out from the incubator and counted by photographing. See fig. 1.

As can be seen from FIG. 1, the unmodified Silicone Rubber (SR) and the polyamine-Phenol Coating (PCPA) were found to be present in a larger amount in both E.coli and S.epidermidis, and the polyamine-phenol coating (E-PCPA) containing carbon-carbon double bonds and the zwitterionic modified coating sample (MPC) were found to be present in a smaller amount in bacteria than in the former two. It can be seen that the hydrophilicity and super-lubricity of the zwitterion endow the sample with an excellent antibacterial function.

Test example 2

The zwitterionic functionalized biomaterial prepared in Experimental example 8 was subjected to an in vivo animal experiment.

The numbers of the experimental samples are as follows: SR, PCPA, E-PCPA, MPC.

The specific process is as follows:

(1) the four groups of samples are firmly connected and assembled with the semi-body internal circulation catheter device, physiological saline is injected into one end of the semi-body internal circulation catheter device, the light elastic sample tube is connected with the three-way tube to ensure that air bubbles are completely discharged, and the two ends of the light elastic sample tube are clamped by hemostatic forceps.

(2) The rabbit white New Zealand was anesthetized by 30mg/ml pentobarbital sodium solution injection at the ear margin, the skin of the neck was dissected and the left and right jugular veins of the rabbit were carefully dissected out, leaving the blood vessels bare and fixed.

(3) The heparin-soaked indwelling needle was removed and inserted by puncture into the rabbit carotid artery and jugular vein, and then the assembled sample catheters were connected to the rabbit carotid artery and vein, respectively. After the hemostat is taken down, the blood is returned to the heart to form a blood circulation loop in a half body. And record the start time of the experiment. The change of the blood color in the catheter is continuously observed in the experimental process, and the flow detection is carried out once every 30 minutes to check whether the catheter is blocked.

(4) After circulating for 2-4 h, the experiment is stopped, the catheter is taken down, and the catheter is washed by normal saline. After the sample has dried, a picture of the sample and the catheter cross-section is taken, and the anticoagulant function is evaluated by a cross-sectional picture of the experimental catheter, see fig. 2.

As can be seen from fig. 2, the unmodified silicone rubber catheter (SR) and the polyamine-phenol coated catheter (PCPA) were almost completely blocked, the polyamine-phenol coated catheter containing carbon-carbon double bonds (E-PCPA) was partially blocked, and the zwitterionic modified catheter sample (MPC) was almost unblocked, demonstrating that the hydrophilicity and super-lubricity of the zwitterion imparted the sample an excellent anticoagulant function.

Test example 3

The hydrophilicity of the products obtained in example 8, example 12, and comparative example 1 was tested, and the results are shown in fig. 3 and 4.

As can be seen from FIGS. 3 and 4, when the zwitterionic 2-Methacryloyloxyethyl Phosphorylcholine (MPC) grafting concentration is 25mg/mL, the material surface water contact angle is 42 ° ± 3 °, the water contact angle is 26 ± 3 when the grafting concentration is 50mg/mL, and the water contact angle is 8 ° ± 3 ° when the grafting concentration is 100mg/mL, thus demonstrating that the hydrophilicity is more significant when the MPC grafting concentration is 100mg/mL in example 8.

The hydrophilicity of the products obtained in example 8 and example 13 were tested and compared, and the results are shown in fig. 5. As can be seen from the figure, the 12h water contact angle is 15 +/-3 degrees, and the effect of the embodiment 8 is more ideal.

The hydrophilicity of the products obtained in example 8 and comparative example 2 were tested and compared, and the results are shown in fig. 6. As can be seen from the figure, the modified water contact angle of 70. + -. 3 ℃ at the MPC grafting concentration of 5mg/mL is much different from the hydrophilicity of example 8.

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 zwitterion functionalized biomaterial, which is characterized by comprising the following steps:

the polyamine-phenol coating containing the carbon-carbon double bond is reacted with a zwitterion compound containing the carbon-carbon double bond to form a zwitterion functionalized polyamine-phenol coating on the surface of the substrate.

2. The method according to claim 1, wherein the compound having both a carbon-carbon double bond and a carboxyl group is dissolved and then reacted with the polyamine-phenol coating layer on the substrate at a pH of 6.21 to 8.21, the reaction temperature is controlled to be 25 to 37 ℃ and the reaction time is controlled to be 6 to 24 hours;

preferably, the reaction temperature of the compound containing both carbon-carbon double bonds and carboxyl groups and the polyamine-phenol coating is 30-37 ℃, and the reaction time is 6-12 h;

preferably, PBS buffer solution is adopted to adjust the pH value of the reaction;

preferably, the material is blow dried after the reaction is completed, and the reaction is repeated 1-3 times.

3. The preparation method according to claim 2, wherein the compound containing both a carbon-carbon double bond and a carboxyl group is an ester;

preferably, the compound containing both the carbon-carbon double bond and the carboxyl group is selected from at least one of N-hydroxysuccinimide methacrylate, N-succinimidyl acrylate and PEG-N-hydroxysuccinimide acrylate;

preferably, the concentration of the compound containing both a carbon-carbon double bond and a carboxyl group in the reaction system is 0.5 to 10 mg/mL.

4. The method of claim 1, wherein forming the zwitterionic functionalized polyamine-phenol coating comprises: activating the carbon-carbon double bond on the polyamine-phenol coating containing the carbon-carbon double bond, and then reacting with a mixed solution formed by an initiator, a pro-initiator and the zwitterionic compound for 12-36 h;

preferably, the reaction time is 24-36h, and the reaction process is carried out under the condition of no oxygen;

preferably, the zwitterionic compound is selected from at least one of 2-methacryloyloxyethyl phosphorylcholine, methacryloyloxyethyl trimethylammonium chloride, acryloyloxyethyl trimethylammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid, methacryloylethyl sulfobetaine, and 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate;

preferably, the concentration of the zwitterionic compound in the reaction system is 50-200 mg/mL;

preferably, blow drying is performed after the reaction is completed.

5. The method according to claim 4, wherein the step of activating the carbon-carbon double bond comprises: mixing an initiator and an initiator accelerator, and carrying out contact reaction with the polyamine-phenol coating containing carbon-carbon double bonds for 5-20min at the reaction temperature of 20-30 ℃;

preferably, the reaction time for activating the carbon-carbon double bond is 10-20min, and the reaction temperature is 23-27 ℃;

preferably, the initiators used in the process of forming the zwitterion-functionalized polyamine-phenol coating are all selected from at least one of sodium persulfate, ammonium persulfate and potassium persulfate, and the concentration of the initiators in the reaction system solution is 1-10 mg/mL;

preferably, the coinitiators used in the formation of the zwitterionic functionalized polyamine-phenol coating are each selected from at least one of N, N' -tetramethyldiethylamine, cobalt octoate, cobalt naphthenate and tertiary amines, and the concentration of the coinitiators in the reaction system solution is 0.1-10 μ L/mL.

6. The method of claim 1, wherein the polyamine-phenolic coating is prepared by a process comprising: activating carboxyl of a biological functional molecule containing carboxyl and phenolic hydroxyl, reacting the biological functional molecule with a water-soluble polymer rich in amino, and dialyzing to obtain a polyamine-phenol solution; placing a substrate in the polyamine-phenol solution for reaction;

preferably, the base material is placed in the polyamine-phenol solution and reacts under the condition that the pH value is 8-11, the reaction temperature is controlled to be 25-45 ℃, and the reaction time is 12-36 h;

preferably, the substrate is washed and dried after the reaction is completed, and then placed in the polyamine-phenol solution to repeat the reaction 1 to 3 times.

7. The method according to claim 6, wherein the temperature of the reaction between the bio-functional molecule and the amino-rich water-soluble polymer is controlled to be 4-10 ℃ and the reaction time is 12-24h during the preparation of the polyamine-phenol solution, and dialysis is performed for 48-72h after the reaction is completed;

preferably, in the reaction system, the concentration of the biological functional molecules is 0.1-10mg/mL, and the concentration of the water-soluble polymer rich in the amine groups is 0.01-10 mg/mL; more preferably, in the reaction system, the concentration of the biological functional molecules is 0.5-5mg/mL, and the concentration of the water-soluble polymer rich in the amine groups is 0.01-1 mg/mL;

preferably, the biofunctional molecule is selected from at least one of 3, 4-dihydroxyphenylpropionic acid, ethyl dihydrocaffeate, 3,4, 5-trihydroxybenzoic acid, caffeic acid, methyl caffeate, caffeoyltartaric acid, 2, 3-dihydroxyphenylalanine, 5-O-caffeoylshikimic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, L-DOPA, pelargonidin, cyanidin, gallic acid, (-) -epicatechin, (-) -epigallocatechin 3-gallate, isoquercitin, salvianolic acid, rosmarinic acid, tannic acid and the like;

preferably, the water-soluble polymer rich in amine groups is selected from at least one of polyallylamine and hydrochloride thereof, polyallylhydrazine, polylysine, polyethyleneimine and derivatives thereof, polyacrylamide, 3-pyrroline, hexamethylenediamine, ethylenediamine, chitosan and crotylamine;

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

more preferably, the substrate is selected from stainless steel, pure titanium, titanium alloys, iron, TiO₂、Si、SiO₂At least one of PVC, silicone rubber and metal oxide.

8. The method according to claim 6, wherein the step of activating the carboxyl group of the biofunctional molecule comprises: mixing the biological functional molecules with EDC and NHS, and incubating for 0.5-1 hour under the conditions that the pH value is 5-7 and the temperature is 30-45 ℃; preferably, the incubation time is 30-60 min;

preferably, the molar ratio of EDC to the carboxyl content of the biofunctional molecule is 0.5-10:1 and the molar ratio of NHS to the carboxyl content of the biofunctional molecule is 0.5-10: 1.

9. A zwitterionic functionalized biomaterial, prepared by the preparation method of any one of claims 1-8.

10. Use of the zwitterionic functionalized biomaterial of claim 9 in the manufacture of a blood access device or an extracorporeal blood circulation apparatus.