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CN115246910B - Polymer synthesis method and application thereof - Google Patents

Polymer synthesis method and application thereof Download PDF

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Publication number
CN115246910B
CN115246910B CN202210506228.2A CN202210506228A CN115246910B CN 115246910 B CN115246910 B CN 115246910B CN 202210506228 A CN202210506228 A CN 202210506228A CN 115246910 B CN115246910 B CN 115246910B
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CN115246910A (en
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请求不公布姓名
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Shanghai Weimi Medical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
    • C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/04—Acids; Metal salts or ammonium salts thereof
    • C08F220/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof

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  • Chemical & Material Sciences (AREA)
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  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Surgery (AREA)
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  • Heart & Thoracic Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The invention belongs to the technical field of A61K, and particularly relates to a synthetic method and application of a polymer. In a first aspect, a method for synthesizing a polymer is provided, comprising the steps of: (1) pretreating the polymerized monomer; (2) Dissolving the polymer monomer treated in the step (1) and the reaction monomer 1 in an organic solvent for reaction pretreatment; (3) Adding an initiator into the step (2), and then placing the reaction system into an oil bath for reaction; (4) Adding an initiator into the solution after the reaction in the step (3) to continue the reaction, and treating after the reaction to obtain a primary product; (5) Dialyzing the initial product of the step (4) to obtain a pure polymer; compared with the prior art, the synthesis method of the polymer provided by the invention can more conveniently and efficiently react phosphorylcholine substances serving as reaction monomers, and solves the problem that platelets and proteins possibly exist in the treatment of cardiovascular diseases adhere to the surface of the material.

Description

Polymer synthesis method and application thereof
Technical Field
The invention belongs to the technical field of A61K, and particularly relates to a synthetic method and application of a polymer.
Background
Cardiovascular disease has become an important cause of human health, and morbidity and mortality have seen a rising trend year by year. In recent years, stent implantation has become an effective means for treating cardiovascular diseases (e.g., atherosclerosis, coronary heart disease, etc.). Titanium and titanium alloys (e.g., nitinol) or cobalt-based alloys (e.g., cobalt-chromium-nickel-molybdenum alloys) are widely used for vascular stents due to their good mechanical properties, corrosion resistance, and excellent biocompatibility. However, the alloy materials commonly used today tend to cause platelet adhesion when in contact with blood, which can lead to thrombosis and restenosis within the stent. In order to improve the performance of implanted stents, drug Eluting Stents (DES) loaded with anticoagulant drugs have been developed. While DES improves the outcome of treatment, drugs loaded on DES inhibit endothelial cell growth and delay endothelialization of the stent surface. As an inner layer of a natural blood vessel, an inner cortex plays an important role in preventing thrombosis and intimal hyperplasia. Delayed endothelialization of vascular stents can lead to restenosis and advanced thrombosis, leading to treatment failure. Therefore, there is a need to develop vascular stents with good stain resistance and rapid endothelialization to increase the success rate of stent treatment.
The interaction between the implant material and the blood is mainly dependent on the surface properties of the implant material. Surface modification can improve the biocompatibility of the stent without changing the mechanical properties thereof, and the anti-fouling coating modification and the promotion of rapid endothelialization of the surface are effective methods for improving the blood compatibility of the vascular stent. The anti-fouling ability is critical to improve the biocompatibility of the blood contact material. The anti-fouling performance of the surface of the material is improved, and the adhesion of the vascular stent to proteins and platelets in blood at the initial stage of implantation can be effectively inhibited. Polyethylene glycol (PEG), zwitterionic polymer, heparin and the like are modified on the surface of the material, so that the blood compatibility of the material can be effectively improved. Phosphorylcholine (PC), a common zwitterion, is the major component of the erythrocyte membrane and has little interaction with proteins involved in the coagulation cascade. It is reported that modifying the polymer containing phosphorylcholine chain segment on the surface of the material can effectively reduce the adhesion of platelets and proteins on the surface of the material and prevent the occurrence of thrombus. However, after the polymer containing phosphorylcholine segments modifies the surface of the material, the adhesion of vascular Endothelial Cells (ECs) is reduced, and the endothelialization capacity of the surface of the material is impaired. The traditional phosphorylcholine monomer is synthesized in the last 70 th century, but has the defects of long synthetic route, low yield, severe reaction temperature requirement and the like, so the current method for synthesizing the phosphorylcholine monomer is still under continuous research.
In addition, the most common methods for modifying functional molecules to the surface of biological materials are physical and chemical methods. The modification of physical methods has focused on the physical adsorption of functional molecules to the surface of biological materials through van der waals forces, electrostatic interactions and hydrogen bonding. The method is simple and convenient, and is easy to implement. However, functional molecules are easily detached from the surface due to weak binding force, resulting in poor long-term stability of the modified surface. Modification by chemical means typically attaches functional molecules to the surface of biological materials via chemical bonds. The modified surface obtained by this method has a high long-term stability under physiological conditions, because of the relatively stable chemical bonds.
Thus two challenges facing researchers at this stage are: how to prepare a substance for modifying the material of the medical device and how to modify the substance to the surface of the material of the medical device.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a method for synthesizing a polymer, comprising the following steps:
(1) Pretreating a polymerized monomer;
(2) Dissolving the polymer monomer treated in the step (1) and the reaction monomer 1 in an organic solvent for reaction pretreatment;
(3) Adding an initiator into the step (2), and then placing the reaction system into an oil bath for reaction;
(4) Adding an initiator into the solution after the reaction in the step (3) to continue the reaction, and treating after the reaction to obtain a primary product;
(5) Dialyzing the initial product of the step (4) to obtain a pure polymer;
Preferably, the polymeric monomer comprises at least a carboxyl compound containing a carbon-carbon double bond.
Further preferably, the polymer monomer is at least one selected from the group consisting of alpha-methacrylic acid, acrylic acid, 2-ethacrylic acid, and 6-acrylamidocacetic acid.
More preferably, the polymer monomer is alpha-methacrylic acid.
Further preferably, the reaction monomer 1 is phosphorylcholine substance; more preferably, the phosphorylcholine is 2-Methacryloyloxyethyl Phosphorylcholine (MPC).
In some preferred embodiments, the polymer of step (5) has the formula:
Wherein: r is one of H and CH 3,C2H5; r 1 is one of- (CH 2)2-,-(CH2)3-,-(CH2)4 -, R 2 is one of CH 3,C2H5, m and n are the number of monomers, and m is n=1:99-99:1.
Further preferably, the molecular weight of the polymer is 5000-20000; more preferably, the molecular weight of the polymer is 9000-11000.
In some preferred embodiments, the pretreatment of step (1) comprises a polymerization inhibitor removal treatment, specifically comprising: distilling under reduced pressure at 105deg.C, collecting fraction to obtain purified MAA, and rapidly storing at-20deg.C.
In some preferred embodiments, the mass concentration of the polymer monomer of step (2) is from 0.01 to 0.2g/mL; the mass concentration of the reaction monomer 1 is 0.1-1g/mL.
Further preferably, the organic solvent in step (2) is selected from any organic solvent capable of dissolving the polymer monomer and the reaction monomer 1; more preferably, the organic solvent is anhydrous ethanol.
Further preferably, the specific operation of step (2) is: dissolving the polymer monomer and the reaction monomer 1 treated in the step (1) in absolute ethyl alcohol, magnetically stirring at room temperature for 5-30min, and bubbling N 2 for 30-60min.
In some preferred embodiments, the initiator described in step (3) is selected from peroxide initiators and/or azo-type initiators.
The type of the initiator to be added in the present application is not particularly limited as long as the radical polymerization reaction can be initiated; further preferably, the initiator is azobisisobutyronitrile.
In some preferred embodiments, the specific operation of step (3) is: and (3) adding an initiator into the reaction system in the step (2), and then placing the reaction system in an oil bath pot for 12-48 hours, wherein the temperature of the oil bath is 60 ℃.
In some preferred embodiments, the specific operation of step (4) is: and (3) adding an initiator into the solution obtained after the reaction in the step (3) to continue the reaction for 1-12h, and then putting the reacted solution into a refrigerator with the temperature of minus 20 ℃ to quench for 12-48h.
In some preferred embodiments, the specific operation of step (5) is: dialyzing the initial product after the reaction in the step (4), and then freeze-drying to obtain a pure polymer.
In some preferred embodiments, the dialysis control molecular weight of step (5) is from 5k to 15k.
In some preferred embodiments, the polymer monomer, reactive monomer 1, added in the present application is added in an amount of: 0.11g of polymer monomer and 0.89g of reaction monomer 1.
The adding proportion of the initiator in the step (3) and the step (4) is as follows: 3% initiator was added at a monomer concentration of 1 mol/L.
In a second aspect the invention provides the use of a method of synthesizing a polymer for surface treatment of a medical device in contact with body fluids or blood.
In some preferred embodiments, the medical device comprises at least a guidewire, catheter, stent, vascular prosthesis, cardiopulmonary prosthesis, ventricular assist device; the medical apparatus at least comprises the following materials: at least one of stainless steel, nickel titanium alloy, cobalt chromium alloy, platinum or platinum alloy.
In some preferred embodiments, the use of a method of synthesizing a polymer for the treatment of a surface of a medical device material, said treatment comprising the specific steps of:
s1: cleaning the surface of the medical instrument material;
S2: hydroxylating the metal treated by the S1 to obtain a treated sample 1;
s3: preparing an amination solution;
s4: immersing the treated sample 1 obtained in the step S2 into the solution prepared in the step S3 for amination treatment to obtain a treated sample 2;
S5: cleaning and drying the sample 2 after the treatment of S4 again to obtain a treated sample 3;
S6: activating the polymer in the solution;
S7: placing the sample 3 treated by the S5 in a PBS/DMSO mixed solution for incubation;
S8: adding the solution in the step S6 into the step S7, mixing the two solutions, and continuing the reaction of the treated sample 3 to obtain a treated sample 4;
s9: cleaning and drying the treated sample 4 to obtain a treated sample 5;
S10: immersing the treated sample 5 into an anhydrous DMSO solution of EDC/NHS for activation;
s11: placing the polypeptide in PBS/DMSO mixed solution for incubation;
s12: adding the solution after the incubation of the S11 into the S10, mixing the two solutions, and continuously reacting the treated sample 5 in the solution to obtain a treated sample 6;
S13: and cleaning and drying the treated sample 6 to obtain the treated material.
In some preferred embodiments, the cleaning process described in step S1 employs a cleaning agent; further preferably, the cleaning agent is selected from one or more of acetone, ethanol, propanol, isopropanol, isoamyl alcohol and ultrapure water.
Further preferably, the cleaning agent in step S1 is selected from a group consisting of acetone, isopropyl alcohol, and ultrapure water.
More preferably, the cleaning agent is used in the following method: in a specific cleaning process, acetone is used, then isopropanol is used, and finally ultrapure water is used for cleaning.
Through a great deal of creative experimental researches of the applicant, the cleaning step of the surface of the medical instrument material and the type of the cleaning agent selected to be used have great influence on the reaction of the prepared polymer, the stability of the coating of the modified surface of the medical instrument material can be directly influenced, the applicant finds that the cleaning process is carried out by using a great deal of random researches and experiments, the cleaning process is carried out by using acetone preferentially, the cleaning process is carried out by using isopropanol, and finally the cleaning process is carried out by selecting ultrapure water, so that the impurities and grease on the surface of the medical instrument material can be ensured to be completely cleaned, the condition that the coating of the surface of the medical instrument material is easy to fall off in the subsequent use process is avoided, and the applicant speculates that the reason for the phenomenon is as follows: after the acetone is adopted for cleaning, fat-soluble and water-soluble substances on the surface of the material can be further dissolved, then the isopropanol is used for cleaning again, organic substances and inorganic substances on the surface of the material can be further dissolved, the surface of the material is thoroughly cleaned, and finally the acetone and the isopropanol are prevented from remaining on the surface of the material after the ultrapure water is used for cleaning, so that the cleanliness in the cleaning process is ensured.
In addition, the applicant found that when isopropanol is replaced by ethanol or the cleaning sequence is changed, the adhesion of the coating on the surface of the medical device material after subsequent modification is reduced, and the problem of ensuring the stability of the polymer on the surface of the medical device material and the stability in the long-term use process can not be solved.
In some preferred embodiments, the cleaning mode is selected from one or a combination of rinsing and ultrasonic cleaning, and the cleaning time can be 5-10min, and the cleaning times are 1-3.
In some preferred embodiments, the agent for the hydroxylation treatment described in step S2 is aqueous sodium hydroxide.
Further preferably, the molar concentration of the sodium hydroxide aqueous solution is 3mol/L to 20mol/L; the hydroxylation treatment time is 12-48h; the reaction temperature is 50-100 ℃.
Further preferably, the hydroxylation treatment temperature is 60 ℃.
Through a great deal of creative experimental researches of applicant, the effect of hydroxylation treatment can be greatly improved by controlling the temperature of hydroxylation treatment in experiments in a body system, and the subsequent amination treatment is ensured to be easier to operate, and through a great deal of careful experiments of applicant, the hydroxylation reaction on the surface of a medical instrument material in the process can be smoothly carried out by controlling the hydroxylation treatment temperature to 60 ℃ in the body system, and side reactions can be caused in the system when the temperature exceeds 60 ℃, so that stable coatings cannot be formed on the surface of the medical instrument material in the subsequent amination or other reactions, and the phenomenon of greatly reducing the modification result is caused.
In some preferred embodiments, the amination solution described in step S3 is configured in the following manner: preparing an amination solution with the volume concentration of 1% -20% by using ethanol with the volume fraction of 95%, wherein the operation conditions are as follows: stirring at room temperature for 0.5-10h.
Further preferably, the amination solution is a 3-aminopropyl triethoxysilane solution.
Further preferably, the stirring time is 2 hours.
In some preferred embodiments, nitrogen protection and reflux agitation are required during the amination treatment in step S4; the treatment temperature is 50-100 ℃ and the reaction time is 12-48h.
In some preferred embodiments, the solution in step S6 may be exemplified by dimethyl sulfoxide and tetrahydrofuran.
Further preferably, the solvent is dimethyl sulfoxide.
Further preferably, the specific operation of the step S6 is as follows: mixing and activating the prepared polymer, EDS and NHS in dimethyl sulfoxide for 1-8h at 25-40deg.C; wherein the mass concentrations of polymer, EDS and NHS may preferably be respectively: 2-15mg/mL, 0.3-10mg/mL, 0.5-12mg/mL.
In some preferred embodiments, the PBS/DMSO mixture solution in step S7 is in a volume ratio of 1:1, mixing; the specific operation of the incubation is as follows: controlling the pH value of the solution to 7.4, the temperature to 30-60 ℃ and the incubation time to 1-8h.
In some preferred embodiments, the reaction described in step S8 controls the reaction temperature to 30-100 ℃.
As an example, the reaction temperature may be 30 ℃, 35 ℃, 40 ℃, 45 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃.
Further preferably, the reaction temperature in step S8 is 35 ℃.
Further preferably, the reaction time is 12 to 20 hours.
As an example, the reaction time may be selected from 12h, 13h, 15h, 16h, 18h, 20h.
Through a great deal of creative research by the applicant in the experimental process, the temperature of the reaction needs to be strictly controlled in the S8 step reaction process, and the applicant in the experimental process finds that the reaction of the polymer in the reaction solvent is insufficient at the temperature lower than 35 ℃, and the applicant speculates that the probability of mutual collision reaction is greatly reduced because the movement frequency between molecules in the system is greatly reduced when the temperature is lower than 35 ℃; however, the applicant found that in the system, when the temperature is higher than 100 ℃, the molecular interaction strength between the polymer chain segments in the system is affected, so that the polymer is decomposed, and the modification effect of the surface of the medical device material is reduced. However, when the reaction temperature is controlled to be 35 ℃, the reaction time is required to be prolonged, the preparation is obtained through a great amount of experimental investigation and design, the best modification result can be ensured when the reaction time is 24 hours, the coating is stable, and the adhesiveness is greatly improved.
In some preferred embodiments, the conditions of activation described in step S10 are defined as: the temperature used for activation is 30-60 ℃, the activation time is generally 0.5-8h, and the concentration of EDS and NHS can be preferably 0.3-10mg/mL and 0.5-12mg/mL respectively.
It should be noted that the cleaning involved in the experimental process may include rinsing and flushing, and the number of times of cleaning may be 1-3.
As an example, the number of times of washing may be selected from 1,2, or 3 times.
The drying related to the invention can be realized by drying inert gases such as nitrogen and argon.
In some preferred embodiments, in step S11, the PBS/DMSO mixture solution is present in a volume ratio of 1:1, mixing; the specific operation of the incubation is as follows: the pH value of the solution is controlled to be 7.4, the incubation temperature can be selected to be between 30 and 60 ℃, and the incubation time is 1 to 8 hours for better reaction.
In some preferred embodiments, the temperature of the reaction described in step S12 is 30-100 ℃.
The beneficial effects are that:
Compared with the prior art, the synthesis method of the polymer provided by the invention can more conveniently and efficiently react phosphorylcholine substances as reaction monomers, and the polymer containing phosphorylcholine chain segments is used for modifying the surface of the subsequent medical instrument material, so that the problem that platelets and proteins possibly exist in the treatment of cardiovascular diseases adhere to the surface of the material, and thrombus is caused; in addition, the polymer containing the phosphorylcholine chain segment prepared by the method can be modified on the surface of a medical instrument material, especially on the surface of a nickel-titanium alloy material by a chemical method, compared with the traditional physical modification, the method provided by the invention can enable the polymer and endothelial cell selective adhesion peptide REDV to be grafted on the surface of the nickel-titanium alloy (NiTi-PAMPC/REDV) and fixed on the surface of an alloy bracket, the modified polymer functional molecules are not easy to fall off from the surface, the modified coating is ensured to have better adhesive force, and the modified medical instrument can maintain long-term stability under physiological conditions.
Drawings
FIG. 1 is a 1 H NMR spectrum of PAMPC prepared in example 1;
FIG. 2 is XPS spectra of NiTi, niTi-OH, niTi-NH 2, niTi-PAMPC and NiTi-PAMPC/REDV surfaces;
FIG. 3 is a graph of platelet adhesion for NiTi, niTi-OH, niTi-NH 2, niTi-PAMPC and NiTi-PAMPC/REDV surfaces;
FIG. 4 is a graph of fibrinogen adhesion on NiTi, niTi-OH, niTi-NH 2, niTi-PAMPC and NiTi-PAMPC/REDV surfaces;
FIG. 5 is a graph of adhesion of NiTi, niTi-OH, niTi-NH 2, niTi-PAMPC and NiTi-PAMPC/REDV surfaces HUVECs;
FIG. 6 depicts proliferation of NiTi, niTi-OH, niTi-NH 2, niTi-PAMPC and NiTi-PAMPC/REDV surfaces HUVECs;
FIG. 7 migration patterns of NiTi, niTi-OH, niTi-NH 2, niTi-PAMPC and NiTi-PAMPC/REDV surfaces HUVECs.
Detailed Description
Examples
Example 1
A method of synthesizing a polymer comprising the steps of:
(1) Pretreatment of the polymerized monomers: distilling under reduced pressure at 105deg.C, collecting the fraction to obtain purified polymerized monomer, and rapidly storing at-20deg.C;
(2) Dissolving the polymer monomer treated in the step (1) and the reaction monomer 1 in an organic solvent for reaction pretreatment, wherein the reaction pretreatment comprises the following steps: dissolving the polymer monomer (0.11 g) and the reaction monomer 1 (0.89 g) treated in the step (1) in 4.3mL of absolute ethyl alcohol, magnetically stirring at room temperature for 15min, and bubbling N 2 for 30min;
(3) Adding an initiator into the step (2), and then placing the reaction system into an oil bath pot for reaction for 24 hours, wherein the temperature of the oil bath is 60 ℃;
(4) Adding an initiator into the solution after the reaction in the step (3), continuing the reaction for 2 hours, and then putting the reacted solution into a refrigerator with the temperature of minus 20 ℃ for quenching for 24 hours to obtain an initial product;
(5) Dialyzing the initial product obtained in the step (4), and then freeze-drying to obtain a pure polymer, wherein the dialysis control molecular weight is 10K.
The polymer is as follows: alpha-methacrylic acid; the reaction monomer 1 is 2-methacryloyloxyethyl phosphorylcholine; the initiator is azodiisobutyronitrile; the organic solvent is absolute ethyl alcohol;
in addition, the addition ratio of the initiator in the step (3) and the step (4) is as follows: the monomer concentration of 1mol/L requires the addition of 3% by weight of initiator.
In a second aspect the invention provides the use of a method of synthesizing a polymer for surface treatment of a medical device in contact with body fluids or blood.
The medical apparatus is a nickel-titanium alloy bracket.
The application of the polymer synthesis method is used for treating the surface of the nickel-titanium alloy bracket, and the specific steps of the treatment are as follows:
s1: cleaning the surface of the nickel-titanium alloy bracket material: the specific process uses a cleaning agent, wherein the cleaning agent is selected from the combination of acetone, isopropanol and ultrapure water; the use method of the cleaning agent comprises the following steps: in the specific cleaning process, acetone is firstly used, then isopropanol is used, and finally ultrapure water is used for cleaning, wherein the cleaning mode is ultrasonic cleaning, the cleaning time is 10min, and the cleaning times are 3 times;
s2: hydroxylation treatment is carried out on the metal treated by the S1 to obtain a treated sample 1, which is marked as TiNi-OH: the reagent for hydroxylation treatment is sodium hydroxide aqueous solution, and the molar concentration of the sodium hydroxide aqueous solution is 5mol/L; the hydroxylation treatment time is 24 hours; the reaction temperature is 60 ℃;
S3: preparing an amination solution: preparing a 5% volume concentration 3-aminopropyl triethoxysilane amination solution by using 95% ethanol by volume fraction, wherein the operation conditions are as follows: stirring for 2h at room temperature;
s4: immersing the treated sample 1 obtained in the step S2 into the solution prepared in the step S3 for amination treatment, wherein nitrogen protection and reflux stirring are needed in the process; the treatment temperature is 60 ℃ and the reaction time is 24 hours, so that a treated sample 2 is obtained;
s5: cleaning and drying the sample 2 after the S4 treatment again to obtain a treated sample 3, and marking the treated sample as TiNi-NH 2;
S6: the polymer is activated in the solution, and the specific operation is as follows: mixing and activating the prepared polymer, EDS and NHS in dimethyl sulfoxide for 4 hours at 35 ℃; wherein the mass concentrations of the polymer, EDS and NHS are respectively as follows: 10mg/mL, 1mg/mL, 2mg/mL (180 mgPAMPC, 27.54, mgEDC, 25.14, mgNHS in 24mL anhydrous DMSO).
S7: sample 3 after S5 treatment was incubated in PBS/DMSO mixture: the volume ratio of the mixed solution is 1:1, mixing; the specific operation of the incubation is as follows: controlling the pH value of the solution to 7.4, the temperature to 35 ℃ and the incubation time to 4 hours;
S8: adding the solution in the step S6 into the step S7, mixing the two solutions, continuing to react the treated sample 3, controlling the reaction temperature to be 35 ℃ and the reaction time to be 24 hours, and obtaining a treated sample 4;
S9: cleaning and drying the treated sample 4 to obtain a treated sample 5, which is marked as TiNi-PAMPC;
s10: the treated sample 5 was immersed in an anhydrous DMSO solution of EDC/NHS for activation: the temperature used for activation is 35 ℃, the activation time is 4 hours, and the concentration of EDS and NHS is 1mg/mL and 2mg/mL respectively (37.12 mgEDS and 33.52mgNHS are dissolved in 15mL anhydrous DMSO);
S11: the polypeptides were incubated in a PBS/DMSO mixture: the volume ratio of the mixed solution is 1:1, mixing; the specific operation of the incubation is as follows: controlling the pH value of the solution to 7.4, the temperature to 35 ℃ and the incubation time to 4 hours;
s12: adding the solution after the incubation of the S11 into the S10, mixing the two solutions, and continuously reacting the treated sample 5 in the solution at the temperature of 35 ℃ to obtain a treated sample 6;
s13: and (3) cleaning and drying the treated sample 6 to obtain the polymer polypeptide modified nickel-titanium alloy material, which is named TiNi-PAMPC/REDV.
Performance testing
1) The polymer (PAMPC) prepared in example 1 was subjected to nuclear magnetic characterization, and the specific results are shown in FIG. 1.
Successful synthesis of PAMPC can be demonstrated by the presence of a peak at position b (. Apprxeq.1.60 ppm) and a peak at position c (. Apprxeq.1.90 ppm) in FIG. 1.
2) XPS test is carried out on the polymer polypeptide modified nickel-titanium alloy material prepared in the embodiment 1, and specific results are shown in FIG. 2.
Compared with NiTi, the O content of NiTi-OH is increased from 48.77% to 52.21%. This is mainly due to the introduction of hydroxyl groups, which increases the O content in NiTi-OH, indicating successful preparation of NiTi-OH. After amination, the N and Si content of the NiTi-NH 2 surface increases from 1.73% and 1.73% to 3.74% and 2.55%, respectively, in NiTi-OH.
The main reason is that the amination solution has a higher N and O content, resulting in an increased N and O content in NiTi-NH 2. Grafting PAMPC onto NiTi-NH 2 through amidation reaction to obtain NiTi-PAMPC. The significant increase in P content on NiTi-PAMPC compared to NiTi-NH 2 indicates successful grafting of PAMPC to the surface of NiTi-NH 2. The N content in CREDVW is higher, so that the N content is improved from 3.12% to 4.77% after CREDVW is grafted on the surface of NiTi-PAMPC.
In addition, the surface element compositions of NiTi, niTi-OH, niTi-NH 2, the polymer modified nickel-titanium alloy (NiTi-PAMPC) prepared by the application and the polymer polypeptide modified nickel-titanium alloy (NiTi-PAMPC/REDV) are shown in the following table:
3) The polymer polypeptide modified nickel-titanium alloy material and nickel-titanium alloy (NiTi) prepared in example 1 and the polymer modified nickel-titanium alloy prepared in the application are subjected to the following performance tests.
A. Blood compatibility test
A) Platelet adhesion
Whole blood from healthy donors was centrifuged at 1500rpm for 15min to obtain Platelet Rich Plasma (PRP). Nickel-titanium alloy (NiTi), polymer modified nickel-titanium alloy prepared according to the application (NiTi-PAMPC) and polymer polypeptide modified nickel-titanium alloy (NiTi-PAMPC/REDV) were placed in 48-well plates, 50. Mu.L PRP was added to each sample, and incubated for 2h at 37 ℃. PRP was removed and the sample was washed 3 times with physiological saline. All samples were fixed with 2.5% glutaraldehyde for 1h at room temperature. The samples were then washed with physiological saline and dehydrated with gradient volume concentration ethanol (50%, 75%, 85%, 95%, 100%). After the metal spraying, the adhesion of platelets on the surface of the sample was observed by a Scanning Electron Microscope (SEM), and the specific results are shown in fig. 3.
The performance test results show that: niTi surface adheres more platelets than NiTi-PAMPC and NiTi-PAMPC/REDV surfaces. The surface hydrophilicity can be increased mainly because the surfaces of NiTi-PAMPC and NiTi-PAMPC/REDV are grafted with PAMPC, which is favorable for inhibiting the adhesion of platelets.
B) Fibrinogen adhesion
Fibrinogen in bovine plasma was dissolved in deionized water at a concentration of 0.5mg mL -1. The sample was then placed in fibrinogen solution and incubated at 37℃for 2h. The fibrinogen solution was removed and the sample was washed 3 times with physiological saline. All samples were fixed with 2.5% glutaraldehyde for 1h at room temperature. The samples were then washed with physiological saline and dehydrated with gradient volume concentrations of ethanol (50%, 75%, 85%, 95%, 100%). After the metal spraying, the adhesion of fibrinogen on the surface of the sample was observed by SEM, and the specific results are shown in FIG. 4.
The performance test results show that: the amount of fibrinogen adhering to the surfaces of NiTi-PAMPC and NiTi-PAMPC/REDV was significantly reduced compared to the NiTi group. The metal ions on the surface of the metal alloy can bind to proteins, resulting in more fibrinogen adhering to the pure NiTi surface. After the hydrophilic PAMPC is grafted, the surface hydrophilicity is increased, and the water molecules adsorbed on the surface are favorable for preventing the adhesion of fibrinogen.
B. cell experiment
A) Adhesion test of modified surface Human Umbilical Vein Endothelial Cells (HUVECs)
The nickel-titanium alloy (NiTi), the polymer modified nickel-titanium alloy (NiTi-PAMPC) prepared by the application and the polymer polypeptide modified nickel-titanium alloy (NiTi-PAMPC/REDV) are immersed in 75% alcohol for 30min and then dried. After drying, HUVECs were seeded onto the sample surface at a density of 1 x 10 4 cells/well and incubated for 2h at 37 ℃ under 5% co 2. After washing the samples 3 times with PBS, cells on the surface of nickel-titanium alloy (NiTi), polymer modified nickel-titanium alloy prepared according to the application (NiTi-PAMPC) and polymer polypeptide modified nickel-titanium alloy (NiTi-PAMPC/REDV) were photographed using a Leica DMRX fluorescence microscope (DMRX, leica, germany) and the number of cells adhered to the surface was counted, as shown in FIG. 5.
The performance test results show that: the adhesion of NiTi-PAMPC/REDV surfaces to HUVECs is increased compared to NiTi-PAMPC. This is primarily because REDV peptides can bind to the alpha 4β1 integrin on HUVECs, thereby enhancing the interaction of ECs with surfaces and thus promoting the adhesion of HUVECs.
B) Proliferation performance test of modified surface HUVECs
HUVECs were inoculated at a density of 1X 10 4 cells/well onto the surface of sterilized nickel-titanium alloy (NiTi), polymer modified nickel-titanium alloy prepared by the present application (NiTi-PAMPC) and polymer polypeptide modified nickel-titanium alloy (NiTi-PAMPC/REDV) samples, and incubated at 37℃under 5% CO 2 for 1d and 2d. After washing the samples with PBS, 0.1mL of serum-free medium containing CFDA-SE was added to each well and incubated for an additional 2h. Cells were photographed using a Leica DMRX fluorescence microscope (DMRX, leica, germany) and the cell numbers on the surface were counted, and the specific results are shown in fig. 6.
The performance test results show that: after 2d of culture, the cell densities of the surfaces of NiTi-PAMPC and NiTi-PAMPC/REDV are higher than those of NiTi. The adhesion and proliferation of cells on the surface of biological materials are affected by the surface hydrophilicity, and the moderate increase of the hydrophilicity is beneficial to the growth and proliferation of cells, so that the number of cells on the surfaces of NiTi-PAMPC and NiTi-PAMPC/REDV is more. In addition, the cell density on NiTi-PAMPC/REDV is higher than that on NiTi-PAMPC. This will be attributed to the fact that REDV peptides can promote the adhesion and proliferation of ECs.
C) Migration performance test of modified surface HUVECs
Wound healing test inserts were used to form scratches of the same width on the sample surface. Briefly, inserts were placed on samples sterilized with 75% ethanol by volume. Endothelial cells (1.4X10 4 cells/well) were added to the wells of the inserts, incubated at 37℃at 5% CO 2 until the cells were completely adherent, then the inserts were removed, 500. Mu.L of medium containing 1% serum was added and the culture was continued at 37℃at 5% CO2 for 0, 12 and 48h. The scratch width of the sample surface was 500 μm. Cells were labeled with CFDA-SE at 0h, 12h and 48h, respectively. Healing of the scratches was observed using a Leica DMRX fluorescence microscope (DMRX, leica, germany), and the specific results are shown in fig. 7.
The performance test results show that: HUVECs on NiTi-PAMPC/REDV exhibit a stronger migration capacity than NiTi and NiTi-PAMPC. This is mainly due to the fact that the NiTi-PAMPC/REDV surface after grafting REDV provides a more suitable growth environment for HUVECs and promotes migration of HUVECs. The improvement of the migration capacity of HUVECs on the surface of NiTi-PAMPC/REDV is beneficial to promoting endothelialization of vascular stents and keeping the vascular stents smooth for a long time.

Claims (2)

1. Use of a method for the synthesis of polymers, characterized by surface treatment of medical devices intended to come into contact with body fluids or blood, said treatment comprising the following specific steps:
s1: cleaning the surface of the medical instrument material;
S2: hydroxylating the metal treated by the S1 to obtain a treated sample 1;
s3: preparing an amination solution;
s4: immersing the treated sample 1 obtained in the step S2 into the solution prepared in the step S3 for amination treatment to obtain a treated sample 2;
S5: cleaning and drying the sample 2 after the treatment of S4 again to obtain a treated sample 3;
S6: activating the polymer in the solution;
S7: placing the sample 3 treated by the S5 in a PBS/DMSO mixed solution for incubation;
S8: adding the solution in the step S6 into the step S7, mixing the two solutions, and continuing the reaction of the treated sample 3 to obtain a treated sample 4;
s9: cleaning and drying the treated sample 4 to obtain a treated sample 5;
S10: immersing the treated sample 5 into an anhydrous DMSO solution of EDC/NHS for activation;
s11: placing the polypeptide REDV in a PBS/DMSO mixed solution for incubation;
s12: adding the solution after the incubation of the S11 into the S10, mixing the two solutions, and continuously reacting the treated sample 5 in the solution to obtain a treated sample 6;
S13: cleaning and drying the treated sample 6 to obtain a treated material;
The cleaning agent in the step S1 is selected from the combination of acetone, isopropanol and ultrapure water; the use method of the cleaning agent comprises the following steps: in the specific cleaning process, acetone is firstly used, then isopropanol is used, and finally ultrapure water is used for cleaning;
S2, the reagent for hydroxylation treatment is sodium hydroxide aqueous solution, the molar concentration of the sodium hydroxide aqueous solution is 3mol/L-20mol/L, the hydroxylation treatment time is 12-48h, and the hydroxylation treatment temperature is 60 ℃; the amination solution is a 3-aminopropyl triethoxysilane solution;
The reaction temperature in the step S8 is controlled to be 35 ℃; the reaction time is 24 hours;
The synthesis method of the polymer comprises the following steps:
(1) Pretreatment of the polymerized monomers: distilling under reduced pressure at 105deg.C, collecting the fraction to obtain purified polymerized monomer, and rapidly storing at-20deg.C;
(2) Dissolving the polymer monomer treated in the step (1) and the reaction monomer 1 in an organic solvent for reaction pretreatment, wherein the reaction pretreatment comprises the following steps: dissolving 0.11g of the polymer monomer treated in the step (1) and 0.89g of the reaction monomer 1 in 4.3mL of absolute ethyl alcohol, magnetically stirring at room temperature for 15min, and bubbling N 2 for 30min;
(3) Adding an initiator into the step (2), and then placing the reaction system into an oil bath pot for reaction for 24 hours, wherein the temperature of the oil bath is 60 ℃;
(4) Adding an initiator into the solution after the reaction in the step (3), continuing the reaction for 2 hours, and then putting the reacted solution into a refrigerator with the temperature of minus 20 ℃ for quenching for 24 hours to obtain an initial product;
(5) Dialyzing the initial product in the step (4), and then freeze-drying to obtain a pure polymer, wherein the molecular weight of the dialysis control is 10K;
the polymer monomer is as follows: alpha-methacrylic acid; the reaction monomer 1 is 2-methacryloyloxyethyl phosphorylcholine.
2. Use of a method of synthesizing a polymer according to claim 1, wherein the medical device comprises at least a guidewire, a catheter, a stent, an artificial blood vessel, an artificial heart-lung, a ventricular assist device; the medical apparatus at least comprises the following materials: at least one of stainless steel, nickel titanium alloy, cobalt chromium alloy, platinum or platinum alloy metal.
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