CN110724426A

CN110724426A - Carboxylic betaine zwitterionic composite antibacterial functional coating material and preparation method and application thereof

Info

Publication number: CN110724426A
Application number: CN201911037575.XA
Authority: CN
Inventors: 李小杰; 罗静; 孟龙; 魏玮; 刘晓亚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2020-01-24
Anticipated expiration: 2039-10-29
Also published as: CN110724426B

Abstract

The invention discloses a carboxylic betaine zwitterionic composite antibacterial functional coating material and a preparation method and application thereof. The preparation method is simple, mild in condition and environment-friendly; the proportion of each functional group in the preparation process can be flexibly regulated, and the prepared coating material has excellent mechanical property, good stability and long-acting property for resisting bacterial adhesion and can regulate and control cell adhesion behavior; has potential application value in the aspects of bioengineering, medical equipment, medical instruments and implants.

Description

Carboxylic betaine zwitterionic composite antibacterial functional coating material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical high polymer materials, and particularly relates to a carboxylic acid betaine zwitterionic composite antibacterial functional coating material and a preparation method and application thereof.

Background

Medical equipment, medical instruments, implants and the like in various shapes and materials are widely applied clinically due to excellent comprehensive performance, but the implantation safety is seriously threatened by implantation-related infection because the implantation materials are mostly bio-inert materials. The surface of the implant material is the preferred site for adhesion of bacteria, the attached bacteria and the subsequent biofilm formation not only limit the useful life of the implant device, but can also lead to serious complications and even death, and the clinically commonly used thoroughly sterile and strictly sterile surgical protocols are still ineffective in avoiding the risk of implant infection. Whether implantation infection can be effectively controlled has become one of the key factors in determining the success rate of device implantation. From the viewpoint of research thinking, methods and effects, the polymer coating is undoubtedly an effective strategy for solving bacterial adhesion and biofilm formation due to the advantages of excellent structural function designability, antibacterial reliability, no influence on the overall mechanical properties and the like.

The zwitterionic polymer simultaneously contains anionic and cationic groups, the structured surface of the zwitterionic polymer can form a hydration layer by capturing free water molecules and form an electrostatic shielding layer by intermolecular or intramolecular electrostatic association, the two ways are combined to prevent adhesion of biological dirt such as bacteria, and the zwitterionic polymer has low cytotoxicity and is one of ideal materials for preparing surfaces for inhibiting adhesion of bacteria. However, there are two general problems with zwitterionic coating material research at present: one is that the fixing and preparation method of the super-hydrophilic Polymer coating is complicated and long, and the binding force between the coating and the substrate is insufficient (Polymer Chemistry,2017,8, 2309-; in addition, zwitterionic coating materials lack biocompatibility considerations (including cell adhesion and proliferation), and in many fields of implantation where antimicrobial requirements are required (e.g., bone replacement and repair materials), the ability of cells to adhere to the surface of the material is still required and is closely related to the therapeutic effect (Journal of coatings technology and Research,2018,15, 737-. Therefore, there is a need to try to fix zwitterionic polymer materials in a simple way to achieve coatings that are resistant to bacterial adhesion but are compatible and can modulate cell adhesion behavior.

Disclosure of Invention

Aiming at the problems and the defects in the prior art, the invention provides a carboxylic acid betaine zwitterionic composite antibacterial functional coating material, and a preparation method and application thereof, so as to realize the application of the coating material which can resist bacterial adhesion but is compatible with the bacterial adhesion and can regulate and control cell adhesion behavior in the aspects of bioengineering, medical equipment, medical instruments and implants.

The invention provides a technical scheme that a preparation method of a carboxylic acid betaine zwitterionic composite antibacterial functional coating material comprises the following steps:

s1: preparing a tert-butyl protected carboxylic betaine polymer comprising the steps of:

s1-1: dissolving a carboxylic acid betaine monomer containing tert-butyl protection, (methyl) acrylate monomer and a (methyl) acrylamide monomer of dopamine in a DMF solvent to obtain a monomer mixed solution;

s1-2: dissolving an initiator AIBN in a DMF solvent to obtain an initiator solution;

s1-3: uniformly mixing the monomer mixed solution in the step S1-1 and the initiator solution in the step S1-2, continuously reacting for 24 hours under the condition of nitrogen, and purifying to obtain a tert-butyl protected carboxylic acid betaine polymer;

the reaction formula is as follows:

wherein R is₁、R₂And R₄Is H or CH₃；R₃The substituent is an alkyl chain formed by 1-18 carbon atoms; m, p, q are the number of repeating units of each monomer unit;

s2: preparing a polymer-metal ion coating solution using the tert-butyl protected carboxylic betaine polymer obtained in S1 and a metal ion;

s3: coating the polymer-metal ion coating liquid on the surface of a base material by adopting a coating technology to form a film, and volatilizing and drying a solvent to obtain a tert-butyl protected carboxylic betaine zwitterionic composite antibacterial functional coating material;

s4: and (3) placing the carboxylic acid betaine zwitterionic composite antibacterial functional coating material protected by the tert-butyl group into trifluoroacetic acid to immerse and remove the tert-butyl group, then washing with PBS buffer solution and deionized water, and volatilizing and drying a solvent to obtain the carboxylic acid betaine zwitterionic composite antibacterial functional coating material capable of regulating and controlling cell adhesion behaviors.

Further, in the step S1-1, the molar ratio of the tert-butyl protected carboxylic betaine monomer, the (meth) acrylate monomer and the (meth) acrylamide monomer of dopamine is (1-99): (9-99): (9-99); the concentration of the reaction liquid of the reaction monomer is lower than 10 g/mL;

in the step S1-2, the initiator AIBN is added in an amount of 0.1-5% of the total mole of the monomers.

Further, the metal ion is Ag⁺,Cu²⁺,Zn²⁺One or more of the metal ions are metal ions which can generate chelation with catechol group (catechol structure) and have antibacterial and bactericidal functions.

Further, the S2 includes the following steps:

s2-1: self-assembling the tert-butyl protected carboxylic betaine polymer obtained in S1 in a mixed solvent of ethanol and isobutanol to form colloidal particles;

s2-2: and adding the metal ion solution with the same volume into the colloidal particle solution formed by self-assembly in S2-1 to obtain the metal ion-loaded polymer gel-metal ion coating liquid.

Further, in S2, the tert-butyl protected carboxylic acid betaine polymer obtained in S1 and the metal ion are directly and uniformly mixed in a mixed solvent of ethanol and DMF to prepare a polymer-metal ion coating solution.

Further, the coating technique includes dip coating, spin coating, spray coating or electrophoresis.

Further, the base material is one or more of glass, stainless steel, titanium alloy, silicon rubber, polypropylene, polytetrafluoroethylene, polyvinyl chloride, polycarbonate or polydimethylsiloxane.

Further, after the carboxylic acid betaine zwitterionic composite antibacterial functional coating sample is soaked in PBS for 60 days, the residual mass of the coating is still more than 90%, the solution stability time is far longer than 60 days, and the solution stability is better; the cytotoxicity grade is grade I, and the antibacterial metal ion carrier has good cell compatibility and antibacterial metal ion loading safety; the coating has good leveling property, excellent adhesive force of 0 grade, high hardness and excellent mechanical property.

As a second aspect of the invention, the carboxylic acid betaine zwitterionic composite antibacterial functional coating material prepared by the method is provided. The surface of the coating material can obviously inhibit the initial contact of bacteria, and the quaternary ammonium salt and the released Cu²⁺The surface synergistic effect of the antibacterial coating has excellent bactericidal performance on gram-positive model bacteria and gram-negative model bacteria, almost no bacteria exist on the surface of the coating after 72 hours, and the antibacterial coating has long-term antibacterial performance.

As a third aspect of the invention, the application of the carboxylic acid betaine zwitterionic composite antibacterial functional coating material in the aspects of bioengineering, medical equipment, medical instruments and implants is provided.

The invention has the following beneficial effects:

1. the carboxylic acid betaine polymer provided by the invention simultaneously contains an anti-adhesion zwitterion functional group, a quaternary ammonium cation group with a sterilization function, a catechol group which interacts with the surface of a substrate, and an alkyl chain which maintains the flexibility and the leveling property of a coating. The polymer has simple preparation method and process, and the proportion of each functional group can be flexibly regulated and controlled.

2. In the process of preparing the coating, the multi-interaction between the catechol, the metal ions and the base material is utilized to obtain the universal coating with various construction modes and strong base material,

3. after the coating is dried and leveled, on one hand, the composite metal ions with antibacterial performance can be chelated with catechol groups to enhance the hardness of the coating and can be slowly released to provide the long-term antibacterial performance of the coating; on the other hand, the adhesion behavior of cells is regulated by changing the hydrophilicity and hydrophobicity of the coating surface.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the relationship between P532 and P433 in tert-butyl protected carboxylate betaine zwitterionic polymer P (tert-butyl protected carboxylate betaine-co-isooctylacrylate-co-dopamine methacrylamide) abbreviated as P (CB-tBu-co-EHA-co-DOPA-Aa)¹H NMR spectrum.

FIG. 2 is a graph of the OD values of bacteria of Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) after exposure for 72h on the surface of Bare titanium Bare Ti, Ti-CP433, Ti-CP532, Ti-CP433-ZW, Ti-CP532-ZW samples.

FIG. 3 is a graph of cell adhesion morphology of L929 cells after being cultured on the surface of samples of Ti-CP433(A1, A2), Ti-CP433-ZW (B1, B2), Ti-CP532(C1, C2), Ti-CP532-ZW (D1, D2) for 24h (A1, B1, C1, D1) and 48h (A2, B2, C2, D2).

FIG. 4 shows the results of experiments on Escherichia coli (A1-D1) and Staphylococcus aureus (A2-D2) in Bare SS (A1, A2), SS-CP532(B1, B2), SS-CP532-ZW (C1, C2) and unloaded Ag⁺The surface of the SS-CP532-ZW (D1, D2) sample is exposed for 72h, and then the fluorescence microscope photograph shows that the staining reagent is SYTO 9/PI.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the following detailed description of the present invention is made with reference to the accompanying drawings and examples, it is to be noted that the following examples are only for explaining and illustrating the present invention, and are not to be construed as limiting the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Example 1

According to the preparation process of the step S1, using DMF as a solvent, certain amounts of tert-butyl protected carboxylic acid betaine (CB-tBu), isooctyl acrylate (EHA), dopamine methacrylamide (DOPA-Aa) are added according to a molar ratio n [ CB-tBu ]]:n[EHA]:n[DOPA-Aa]The ratio of the reactant to the reaction solution is 5:3:2, and the reaction solution is put into a round-bottom flask, wherein the concentration of the reaction solution is 1.0 g/mL; adding initiator AIBN according to 1.5 percent of the total mole number of the monomers, introducing N₂Deoxidizing, vacuumizing, sealing, placing in oil bath at 68 ℃ for magnetic stirring reaction for 24 hours, cooling to room temperature after the reaction is finished, repeatedly purifying for 3 times by using anhydrous ether as a precipitator, finally drying in vacuum to constant weight to obtain a polymer P (CB-tBu-co-EHA-co-DOPA-Aa), referred to as P532 for short, and storing the product in a drying oven. Of the polymer P532¹The HNMR spectrum is shown in FIG. 1.

Then preparing 20mg/mL polymer P532 ethanol solution, and dropwise adding isobutanol into the polymer ethanol solution at the speed of 20 mu L/min under the stirring condition, wherein the P532 polymer shows dilution, polymer self-assembly and aggregation processes along with the addition of the isobutanol. In order to prepare a stable dispersed colloid system, a CP532 colloid particle solution is formed by self-assembly in a mode that the volume ratio of V (isobutyl alcohol) to V (ethanol) is 1: 2. The particle size and the distribution of the colloidal particles are measured by a nanometer particle size and Zeta potential analyzer to represent the surface charge condition of the particles, wherein the particle size of the CP532 colloidal particles is relatively increased to 110.0nm, and the charge capacity is 48.2 mV.

Selecting antibacterial Cu²⁺Introducing CP433 colloidal particles by CuSO₄Preparation concentration of 80. mu.g/mL Cu²⁺Under the condition of continuous stirring, the CuSO solution is slowly added at a constant speed₄The aqueous solution is dripped into the CP532 colloidal solution with the same volume of 10mg/mL,stirring and stabilizing for a period of time after finishing the dropwise adding to obtain the loaded Cu²⁺The colloidal particle solution of (5) was found to have a concentration of CP532 of 5 mg/mL. At this time, Cu²⁺The coordination complexation with DOPA-Aa occurs, and the typical Cu appears in the polymer solution²⁺Color development after interaction with DOPA-Aa.

The CP532-Cu colloidal particles with positive charge points are utilized to prepare the antibacterial multifunctional coating material on the surface of the medical titanium metal by adopting a cathode electrophoretic deposition technology. Selection of Cu²⁺The loaded colloidal particle solution with the addition of 80 mug/mL is used as a deposition solution, the concentration is 5mg/mL, ethanol/isobutanol mixed solution is used as electrolyte, and a coating material is prepared by using the conditions of 170V deposition voltage and 20min deposition time and is marked as Ti-CP 532-Cu. After the coating is dried, a coating sample is placed in a certain amount of trifluoroacetic acid to be immersed for 15min to remove tert-butyl groups, then PBS buffer solution and deionized water are used for washing for 3 times, and the carboxylic acid betaine zwitterionic functional coating material with tert-butyl protection is obtained after drying and is named as Ti-CP 532-ZW-Cu. Wherein the water contact angle value of the bare titanium substrate is 69.1 +/-1.7 degrees, the water contact angle of the Ti-CP532-Cu sample is 84.7 +/-1.7 degrees, and the water contact angle of the Ti-CP532-ZW-Cu sample is 48.9 +/-1.9 degrees.

And (3) determining the cytotoxicity of the sample by using an indirect method, wherein after the Ti-CP532-Cu and Ti-CP532-ZW-Cu coating samples are soaked in the PBS solution for 60 days in the leaching liquor, L929 cells are cultured for 48 hours, the corresponding cell density of all samples is basically consistent with that of DMEM, and the samples all present a healthy growth form and show good cell compatibility. The relative activity of the cells in each leach solution was further determined by the MTT method. The cell activity of the cells in the leaching liquor of each sample after being cultured for 24 hours is higher than 90%, the activity is still good after being cultured for 48 hours, and the cell activity of the Ti-CP532 and Ti-CP532-ZW leaching liquors is 96.8% and 106.2% respectively, which shows that no toxic substances are released or lower than a safety critical value in the leaching liquors, and no obvious influence is generated on the growth and proliferation of the cells. This also indirectly indicates that the coating has cell compatibility and Cu²⁺The safety of the load.

In the next antibacterial experiment, after the sample is exposed in the bacterial suspension for 24 hours, a large number of bacterial clusters are observed on the surface of naked titanium, and the existence of living bacteria is hardly observed on the surfaces of the Ti-CP532-Cu and Ti-CP532-ZW-Cu samples; after the contact time of the bacteria and the samples is prolonged to 72 hours, the OD values of escherichia coli and staphylococcus aureus adhered to the surfaces of the samples after 72 hours of contact are measured and analyzed, and the results are shown in figure 2, it can be seen that the OD value of the naked titanium sample is extremely high, which indicates that a large amount of bacteria are adhered to and grow on the naked titanium surface, and for Ti-CP532-Cu and Ti-CP532-ZW-Cu coating samples, the OD value of the bacteria after 72 hours of contact with the bacteria is extremely low, which indicates that almost no living bacteria exist on the surface. The results fully show that the coating before and after the tert-butyl protection effectively inhibits the adhesion of escherichia coli and staphylococcus aureus bacteria on the surface and generates a biofilm, and has long-acting antibacterial activity.

After the Ti-CP532-Cu and Ti-CP532-ZW-Cu coating samples are soaked in PBS for 60 days, the residual mass of the coating is still more than 90%, and the solution stability time is far longer than 60 days, which shows that the coating has better solution stability.

Example 2

According to the preparation process of the step S1, using DMF as a solvent, certain amounts of tert-butyl protected carboxylic acid betaine (CB-tBu), isooctyl acrylate (EHA), dopamine methacrylamide (DOPA-Aa) are added according to a molar ratio n [ CB-tBu ]]:n[EHA]:n[DOPA-Aa]Putting the mixture into a round-bottom flask at a ratio of 4:3:3, wherein the concentration of a reaction solution is 1.0 g/mL; adding AIBN as initiator in 1.5% of total mole of monomer and introducing N₂Deoxidizing, vacuumizing, sealing, placing in oil bath at 68 ℃ for magnetic stirring reaction for 24 hours, cooling to room temperature after the reaction is finished, repeatedly purifying for 3 times by using anhydrous ether as a precipitator, finally drying in vacuum to constant weight to obtain a polymer P (CB-tBu-co-EHA-co-DOPA-Aa), referred to as P433 for short, and storing the product in a drying oven. Of the polymer P433¹The H NMR spectrum is shown in FIG. 1.

Preparing 20mg/mL polymer P433 ethanol solution, dropwise adding isobutanol into the polymer ethanol solution at the speed of 20 mu L/min under the stirring condition, and with the addition of the isobutanol, the P433 polymer shows the processes of dilution, polymer self-assembly and aggregation. In order to prepare a stable dispersed colloid system, a CP433 colloid particle solution is formed by self-assembly in a mode that the volume ratio of V (isobutyl alcohol) to V (ethanol) is 1: 2. The particle size and the distribution of the colloidal particles are measured by a nanometer particle size and Zeta potential analyzer to represent the surface charge condition of the particles, wherein the hydrodynamic diameter of the CP433 colloidal particles is 133.4nm, the surface Zeta potential value is 42.6mV positive charge, and the charge quantity is reduced compared with the CP532 because the quaternary ammonium salt (CB-tBu) component in the polymer chain is reduced.

Selecting antibacterial Cu²⁺Introducing CP433 colloidal particles by CuSO₄Preparation concentration of 80. mu.g/mL Cu²⁺Under the condition of continuous stirring, the CuSO solution is slowly added at a constant speed₄Dropwise adding the aqueous solution into an isovolumetric 10mg/mL CP433 colloidal solution, and stirring and stabilizing for a period of time after dropwise adding to obtain the loaded Cu²⁺The colloidal particle solution CP433-Cu of (1), concentration was 5 mg/mL. At this time, Cu²⁺The coordination complexation with DOPA-Aa occurs, and the typical Cu appears in the polymer solution²⁺Color development after interaction with DOPA-Aa.

The CP433-Cu colloidal particles with positive charge points are utilized, and a cathode electrophoretic deposition technology is adopted to prepare the antibacterial multifunctional coating material on the surface of the medical titanium. With Cu²⁺The loaded colloidal particle solution with the addition of 80 mug/mL is used as a deposition solution, the concentration is 5mg/mL, ethanol/isobutanol mixed solution is used as electrolyte, and a coating material is prepared by using the conditions of 170V deposition voltage and 20min deposition time and is marked as Ti-CP 433-Cu. After the coating is dried, a coating sample is placed in a certain amount of trifluoroacetic acid to be immersed for 15min to remove tert-butyl groups, then PBS buffer solution and deionized water are used for washing for 3 times, and the carboxylic acid betaine zwitterionic functional coating material with tert-butyl protection is obtained after drying and is named as Ti-CP 433-ZW-Cu. Wherein the water contact angle of the Ti-CP433-Cu sample is 76.3 +/-3.6 degrees, and the water contact angle of the Ti-CP433-ZW-Cu sample is 54.9 +/-1.0 degrees.

Similarly, after Ti-CP433-Cu and Ti-CP433-ZW-Cu coating samples are soaked in PBS solution for 60 days in the leaching liquor, L929 cells are cultured for 48 hours, the corresponding cell density of all samples is basically consistent with that of DMEM, and the samples all show a healthy growth form and good cell compatibility. The relative activity of the cells in each leach solution was further determined by the MTT method. The activity of the cells in the leaching liquor of each sample is higher than 90% after the cells are cultured for 24h, the activity is still good after the cells are cultured for 48h,the cell activities of the Ti-CP433 and TiCP433-ZW leaching solutions are 96.5% and 100.9% respectively, which shows that no toxic substances are released in the leaching solutions or are lower than a safety critical value, and no obvious influence is generated on the growth and proliferation of cells. This also indirectly indicates that the coating has cell compatibility and Cu²⁺The safety of the load.

In the next antibacterial experiment, after the sample is exposed in the bacterial suspension for 24 hours, a large number of bacterial clusters are observed on the surface of naked titanium, and the existence of living bacteria is hardly observed on the surfaces of Ti-CP433-Cu and Ti-CP433-ZW-Cu samples; after the contact time of the bacteria and the samples is prolonged to 72 hours, the OD values of escherichia coli and staphylococcus aureus adhered to the surfaces of the samples after 72 hours of contact are measured and analyzed, and the results are shown in figure 2, and the OD values of the bacteria after 72 hours of contact of the Ti-CP433-Cu, Ti-CP433-ZW-Cu and the coating samples with the bacteria are extremely low, which indicates that almost no live bacteria exist on the surfaces. The results fully show that the coating before and after deprotection can effectively inhibit the adhesion of escherichia coli and staphylococcus aureus bacteria on the surface and generate a biological film, and has long-acting antibacterial activity.

After Ti-CP433-Cu and Ti-CP433-ZW-Cu coating samples are soaked in PBS for 60 days, the residual mass of the coating is still more than 90%, and the solution stability time is far longer than 60 days, which shows that the coating has better solution stability.

In addition, a polymer structure with the same structure and different hydrophilic-hydrophobic monomer ratios, namely P532 and P433, is selected, and the cell adhesion behavior can be adjusted by adjusting the surface hydrophilicity and hydrophobicity, which is specifically as follows:

after the coating is firstly deposited on the surface of the substrate, the water contact angle values of Ti-CP433-Cu and Ti-CP532-Cu are respectively increased to 76.3 +/-3.6 degrees and 84.7 +/-1.7 degrees, the coating presents certain hydrophobicity, and the relatively higher water contact angle value of Ti-CP532 is caused by less DOPA-Aa groups and more CB-tBu groups. After tert-butyl protection is removed, the surface hydrophilicity of the coating is obviously increased, the water contact angles of Ti-CP433-ZW-Cu and Ti-CP532-ZW-Cu samples are reduced to 54.9 +/-1.0 degrees and 48.9 +/-1.9 degrees, the tert-butyl is removed to generate a zwitterionic SBMA component, and the solid hydrophilicity is increased; and the content of the SBMA component in the Ti-CP532-ZW-Cu sample is higher than that of the Ti-CP433-ZW-Cu sample, the water contact angle is relatively lower, and the excessive increase of the hydrophilicity is inhibited by the existence of the EHA component. The above results show that the hydrophilic and hydrophobic properties of the coating surface can be changed by adjusting the hydrophilic and hydrophobic monomer ratio and the subsequent tert-butyl protecting group removing behavior.

FIG. 3 is a fluorescent microscope photograph of L929 cells inoculated on the surfaces of Ti-CP433-Cu, Ti-CP433-ZW-Cu, Ti-CP532-Cu and Ti-CP532-ZW-Cu samples and cultured for 24h and 48h, respectively. After 24h of culture, a large amount of cells are adhered to the Ti-CP433 and the Ti-CP532 which are not subjected to deprotection coating, and the cells are in a healthy spindle shape, and the number and the density of the cells are greatly increased after 48h of culture, which shows that the Ti-CP433 and the Ti-CP532 have good biocompatibility, and the cells can be adhered to and proliferate on the surfaces of the cells; for Ti-CP433-ZW and Ti-CP532-ZW after the tert-butyl protection is removed, the cell adhesion is obviously inhibited no matter 24h and 48h of culture. Wherein, the zwitter-ion component in the Ti-CP532-ZW is higher, and the solid inhibition effect is more obvious. In general, Ti-CP433 and Ti-CP532 have good cell compatibility, and Ti-CP433-ZW and Ti-CP532-ZW have excellent ability of inhibiting cell adhesion, that is, the cell adhesion can be adjusted by adjusting the proportion of hydrophilic and hydrophobic functional groups, so that the coating has potential application in different fields.

Example 3

According to the preparation process of the step S1, using DMF as a solvent, certain amounts of tert-butyl protected carboxylic acid betaine (CB-tBu), isooctyl acrylate (EHA), dopamine methacrylamide (DOPA-Aa) are added according to a molar ratio n [ CB-tBu ]]:n[EHA]:n[DOPA-Aa]The mixture is put into a round-bottom flask at a ratio of 5:3:2, and the concentration of a reaction solution is 1.5 g/mL; adding initiator AIBN according to 1.5 percent of the total mole number of the monomers, introducing N₂Deoxidizing, vacuumizing, sealing, placing in oil bath at 68 ℃ for magnetic stirring reaction for 24 hours, cooling to room temperature after the reaction is finished, repeatedly purifying for 3 times by using anhydrous ether as a precipitator, finally drying in vacuum to constant weight to obtain a polymer P (CB-tBu-co-EHA-co-DOPA-Aa), referred to as P532 for short, and storing the product in a drying oven.

Preparing 10mg/mL polymer ethanol solution, and dropwise adding AgNO with concentration of 20 mug/mL into the polymer ethanol solution with the same volume at the speed of 20 mug/mL under the stirring condition₃Ethanol solution, drippingStirring and stabilizing for a period of time after the addition is finished to obtain Ag⁺The concentration of the supported polymer solution P532-Ag is 5 mg/mL. Stainless steel 316L (SS) was immersed in the P532-Ag polymer solution for 20s, taken out and dried for 10s, and repeated 3 times. After the coating is dried, obtaining a tert-butyl protected carboxylic betaine composite antibacterial functional coating P532-Ag, immersing a coating sample in a certain amount of trifluoroacetic acid for 15min to remove tert-butyl, washing for 3 times by using PBS buffer solution and deionized water, and drying to obtain a tert-butyl protected carboxylic betaine zwitterionic functional coating material named as SS-P532-ZW.

In addition, the surface of the coating material can obviously inhibit the initial contact of bacteria, and has excellent bactericidal performance on gram-positive and gram-negative model bacteria through the synergistic action of the quaternary ammonium salt and the surface releasing metal ions, and the specific steps are as follows:

escherichia coli (E.coli ATCC 8739) and Staphylococcus aureus (S.aureus, ATCC 6538) were inoculated into 10mL Tryptone Soy Broth (TSB) Erlenmeyer flasks (Journal of materials chemistry B,2015,3(32):6676-⁶CFU/mL of bacterial suspension. Bare titanium, SS-P532-Ag SS-P532-ZW-Ag and SS-P532-ZW- (without Ag)⁺) The coated samples were placed in 12-well plates, 1mL of the bacterial TSB suspension obtained above was added, and cultured in an incubator at 37 ℃ for 72 hours. After incubation, taking out the sample, gently cleaning the surface of the coating by using 0.9% NaCl solution, transferring the sample to a new 12-hole culture plate, adding 1mL of TSB culture medium and a proper amount of SYTO 9/PI dye solution, culturing for 15min, and observing and shooting the bacterial adhesion condition on the surface of the sample by using a Nikon 80i type upright fluorescence microscope.

FIG. 4 shows Escherichia coli (A1-D1) and Staphylococcus aureus (A2-D2) in bare stainless steel 316L (SS) (A1, A2), SS-P532(B1, B2), SS-P532-ZW (C1, C2) and unloaded Ag⁺The surface of the SS-P532-ZW (D1, D2) sample was exposed for 72h and then subjected to fluorescence microscopy, wherein green fluorescence is live bacteria and red fluorescence is dead bacteria. As can be seen, the surface of the stainless steel 316L substrate is easy to adhere with a large amount of bacteria and grow into a biological filmAlmost all bacteria exhibit green fluorescence; after the surface of the stainless steel 316L substrate is covered with SS-P532, the substrate is in a quaternary ammonium salt type and releases Ag⁺Surface effect, excellent bactericidal property to two model bacteria, almost only red fluorescence display on the surface of the sample (B1, B2); compared with the SS-P532 sample, the SS-P532-ZW surface has no green fluorescence, and the red fluorescence intensity is greatly reduced (C1 and C2), which shows that the SS-P532-ZW coating surface can obviously inhibit the initial contact of bacteria and has a bactericidal effect. From FIGS. 4D1 and D2, it was confirmed that Ag was not supported⁺The total amount of bacteria adhered to the surface of the SS-P532-ZW sample was still significantly reduced, but a small number of sporadic red and green fluorescence spots were observed, indicating that a small amount of bacteria adhered to the surface of the coating, the residual CB-tBu component only provided partial bactericidal power, and the Ag-loading component⁺Thereafter, the live bacteria substantially disappeared (compare C1, C2). The result shows that the SS-P532 coating constructs a quaternary ammonium salt bactericidal surface, and after the protective group is removed, the SS-P532-ZW coating inhibits the initial adhesion of bacteria through a shielding layer and then further inhibits the initial adhesion of bacteria through Ag⁺The method effectively solves the problem of stubborn adhesion of bacteria by releasing and killing the bacteria, and proves the reliability of the method.

It should be understood that the above description is only an example of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent flow transformations that may be applied to the present invention as described in the specification, or applied to other related fields, directly or indirectly, are included in the scope of the present invention.

Claims

1. A preparation method of a carboxylic acid betaine zwitterionic composite antibacterial functional coating material is characterized by comprising the following steps:

the reaction formula is as follows:

s2, preparing a polymer-metal ion coating solution by using the tert-butyl protected carboxylic betaine polymer obtained in S1 and metal ions;

and S4, placing the carboxylic acid betaine zwitterionic composite antibacterial functional coating material protected by the tert-butyl group into trifluoroacetic acid to immerse and remove the tert-butyl group, then washing with PBS buffer solution and deionized water, and volatilizing and drying a solvent to obtain the carboxylic acid betaine zwitterionic composite antibacterial functional coating material.

2. The preparation method of the carboxylic betaine zwitterionic composite antibacterial functional coating material according to claim 1, characterized in that:

the molar ratio of the tert-butyl protected carboxylic betaine monomer, the (meth) acrylate monomer and the (meth) acrylamide monomer of dopamine in the step S1-1 is (1-99): (1-99): (1-99); the concentration of the reaction liquid of the reaction monomer is lower than 10 g/mL; in the step S1-2, the initiator AIBN is added in an amount of 0.1-5% of the total mole of the monomers.

3. The carboxylic betaine of claim 2The preparation method of the amphoteric ion type composite antibacterial functional coating material is characterized in that the metal ions are Ag⁺，Cu²⁺，Zn²⁺One or more of them.

4. The preparation method of the carboxylic betaine zwitterionic composite antibacterial functional coating material according to claim 2, characterized in that: the S2 includes the steps of:

5. The method for preparing the carboxylic betaine zwitterionic composite antibacterial functional coating material according to claim 2, wherein the step S2 is to directly and uniformly mix the tert-butyl protected carboxylic betaine polymer obtained in the step S1 and metal ions in a mixed solvent of ethanol and DMF to prepare the polymer-metal ion coating solution.

6. The carboxylic betaine zwitterionic complex antibacterial functional coating material according to claim 1, wherein the coating technique comprises dip coating, spin coating, spray coating or electrophoresis.

7. The carboxylic betaine zwitterionic composite antibacterial functional coating material as claimed in claim 1, wherein the substrate is one or more of glass, stainless steel, titanium alloy, silicone rubber, polypropylene, polytetrafluoroethylene, polyvinyl chloride, polycarbonate or polydimethylsiloxane.

8. The carboxylic betaine zwitterionic composite antibacterial functional coating material as claimed in claim 1, wherein after the carboxylic betaine zwitterionic composite antibacterial functional coating sample is soaked in PBS for 60 days, the residual mass of the coating is greater than 90%, and the cytotoxicity grade is grade I.

9. A carboxylic betaine zwitterionic composite antibacterial functional coating material, which is characterized by being prepared by the method of any one of claims 1-8.

10. The carboxylic betaine zwitterionic composite antibacterial functional coating material as claimed in claim 9, is applied to bioengineering, medical equipment, medical instruments and implants.