Thiazole-betaine zwitterionic polymer as well as preparation method and application thereof

Abstract

The invention discloses a thiazole-betaine zwitterionic polymer as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out free radical polymerization on a methacrylic acid 2-((2-(1-(3-chlorphenyl) carbamoyl) piperidine-4-formamido) thiazole-5-yl) amino) ethyl ester monomer, a sulphobetaine methacrylate monomer and a solution containing a small amount of a cross-linking agent, so as to obtain the thiazole-betaine zwitterionic polymer. And spraying the polymer solution on the titanium alloy by using an ultrasonic spraying technology to obtain the antibacterial coating. The orthopedic implant antibacterial coating obtained by modifying the surface of the titanium stent through the thiazole-betaine zwitterionic polymer has excellent antibacterial and anti-biofilm properties and good biocompatibility, so that the coating is expected to become an effective antibacterial material in orthopedic surgery.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical polymer materials, and in particular to a thiazole-betaine zwitterionic polymer and a preparation method and application thereof. Background Art

[0002] Titanium-based metal materials are commonly used internal fixation materials in orthopedics with excellent biocompatibility and mechanical properties. However, due to the lack of biological activity and antibacterial ability, titanium-based implants are very prone to poor bone integration and implant-related infections in the body. Related studies have shown that about 18% of implant failures are due to aseptic loosening, and 20% of failures are attributed to infection. Among them, implant site infections caused by bacteria will lead to the formation of fixatives on the surface of the implant. These fixatives not only provide an attachment surface for bacterial attachment and proliferation. But also will further form a biofilm, making it difficult to remove bacteria on the attachment surface. Therefore, there is a great demand for new methods to prevent such infections.

[0003] Thiazoles have antiviral and antibacterial activity. In in vitro studies, thiazoles can prevent biofilm formation of a variety of staphylococci, including Staphylococcus aureus and Staphylococcus epidermidis, two pathogens that cause approximately 80% of orthopedic implant-related infections. Antibacterial and anti-biofilm activity is shown against both Gram-positive and Gram-negative bacteria. Thiazoles have profound antibacterial properties, which makes them excellent candidates for orthopedic devices because these devices have a high infection rate. At the same time, sulfobetaine methacrylate (SBMA) is a zwitterionic compound with good biocompatibility and is often used for hydrophilic modification of membranes and coatings of blood processing equipment. Due to the superhydrophilicity generated by its unique chain structure, it can effectively prevent the adhesion of contaminants such as proteins and bacteria.

[0004] The present invention aims to develop a novel thiazole-betaine zwitterionic polymer with antibacterial and anti-biofilm properties and modify the surface of a titanium stent, thereby establishing an orthopedic implant antibacterial coating with the ability to kill bacteria and reduce biofilm formation. Summary of the invention

[0005] In view of the deficiencies and needs of the prior art, the object of the present invention is to provide a thiazole-betaine zwitterionic polymer and a preparation method and use thereof.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, the present invention provides a thiazole-betaine zwitterionic polymer, whose molecular structural formula is:

[0008]

[0009] In a second aspect of the present invention, the present invention provides a method for preparing a thiazole-betaine zwitterionic polymer, characterized in that it comprises the following steps:

[0010] (1) By an atom transfer radical method, 2-((2-(1-(3-chlorophenyl)carbamoyl)piperidin-4-carboxamido)thiazol-5-yl)amino)ethyl methacrylate monomer and sulfobetaine methacrylate monomer were mixed and stirred at 37° C. for reaction.

[0011] (2) After the reaction is completed, the reaction solution is evaporated to dryness to obtain the product, and the product is placed in a vacuum freeze dryer and dried for 24 hours.

[0012] Furthermore, the reaction in step (1) needs to be carried out in the presence of an initiator, ammonium persulfate.

[0013] Preferably, in step (1), the molar ratio of 2-((2-(1-(3-chlorophenyl)carbamoyl)piperidine-4-carboxamido)thiazol-5-yl)amino)ethyl methacrylate monomer to sulfobetaine methacrylate monomer is (1-2):(1-3), preferably 1.5:2.5.

[0014] Preferably, the amount of ammonium persulfate used as the initiator is 0.1%-5%, preferably 0.1%.

[0015] In the third aspect of the present invention, the present invention provides a method for preparing a thiazole-betaine zwitterionic polymer antibacterial coating, characterized in that it comprises the following steps:

[0016] The thiazole-betaine zwitterionic polymer obtained above was prepared into a solution, and the solution was evenly coated on the surface of the titanium alloy using an ultrasonic spraying process, and then placed at room temperature and under sterile conditions for 30 minutes to obtain an antibacterial coating.

[0017] Furthermore, the concentration of the prepared polymer solution in the step is 10-100 mg / mL, preferably 30 mg / mL.

[0018] The beneficial effects produced by the present invention are:

[0019] The present invention provides a thiazole-betaine zwitterionic polymer and a preparation method and use thereof. The preparation method is simple in process; in addition, the coating not only has excellent bactericidal performance, but also has the ability to inhibit the formation of biofilm and has good biocompatibility. The thiazole-betaine zwitterionic polymer antibacterial coating placed on the surface of titanium-based orthopedic implants is expected to solve the infection problem of orthopedic implants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0021] Figure 1 The inhibitory ability of the antibacterial coating of the present invention on the biofilm of Staphylococcus aureus was evaluated.

[0022] Figure 2 The biocompatibility of the antibacterial coating of the present invention was evaluated. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0024] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0025] Example 1 Preparation of the compound of formula (I)

[0026] 1. Dissolve 2-((2-(1-(3-chlorophenyl)carbamoyl)piperidin-4-carboxamido)thiazol-5-yl)amino)ethyl methacrylate (1500 mmol) and sulfobetaine methacrylate (2500 mmol) in a round-bottom flask containing 25 mL (purified water: tetrahydrofuran = 1:1) and sonicate for 10 min until the solid is completely dissolved.

[0027] 2. Add the initiator ammonium persulfate (1.2 mmol) to the above reaction solution and start stirring.

[0028] 3. React under nitrogen protection, set the reaction temperature to 37°C, and stir the reaction time for 24 hours.

[0029] 4. Stop the reaction at room temperature and place the reaction solution in a freeze dryer at -60°C and dry for 24 hours.

[0030] Example 2. Preparation of antibacterial coating for orthopedic implants

[0031] The dried sample was prepared into a 30 mg / mL aqueous solution, which was then sprayed on the surface of the titanium alloy and then placed at room temperature under sterile conditions for 30 minutes to obtain an antibacterial coating.

[0032] Example 3. In vitro antibacterial activity determination of antibacterial coating

[0033] Experimental methods:

[0034] The minimum inhibitory concentration (MIC) of the betaine polymer antibacterial coating against Staphylococcus aureus was tested by the microbroth dilution method. The concentration of the bacterial cell suspension was adjusted to about 5×10 5 CFU / mL. The bacterial cell suspension was incubated with the betaine polymer antibacterial coating material at 37°C for 18-24 hours and then the OD was measured. 600 The MIC value was defined as the lowest concentration without significant increase compared with the control group. The test results are shown in Table 1.

[0035] Table 1 Bactericidal properties of betaine polymer antibacterial coatings (unit: μg / mL)

[0036]

[0037] Note: a Sa: Staphylococcus aureus ATCC 29213, b Ef: Enterococcus faecalis ATCC 29212, c Ab: Acinetobacter baumannii ATCC 19606, d Ec: Escherichia coli ATCC 25922, e Pa: Pseudomonas aeruginosa PAO1.

[0038] As can be seen from Table 1, the antibacterial coating prepared by the present invention exhibits excellent antibacterial activity against five strains of Staphylococcus aureus, Enterococcus faecalis, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa, and the bactericidal effect against Staphylococcus aureus and Acinetobacter baumannii is more obvious.

[0039] Example 4. Inhibition experiment of biofilm formation

[0040] Experimental methods:

[0041] Crystal violet staining was used to determine the inhibitory ability of the samples on the biofilm formation of Staphylococcus aureus. The concentration of Staphylococcus aureus was adjusted to 1×10 6 CFU / mL. In the presence of different concentrations of samples, the diluted bacterial solution was transferred to a 96-well plate and incubated at 37°C for 24 hours. The plate was gently washed to remove planktonic bacteria. The biofilm was stained with 0.1% crystal violet for 30 minutes, and then the excess dye was rinsed off with PBS. The dye bound to the attached bacterial cells was dissolved with 33% acetic acid solution, and the absorbance was measured at a wavelength of 590nm using an enzyme-labeled detector. The culture medium without the sample was used as a control. The inhibition rate of the sample on the biofilm was calculated by comparing with the value of the control group.

[0042] The results are as follows Figure 1As shown, the inhibition rate of the sample on the formation of Staphylococcus aureus biofilm is concentration-dependent. As the sample concentration gradually increases, the inhibition rate gradually increases. When the concentration is 8 mg / L, the inhibition rate of the biofilm is 79.8±2.6%. Therefore, the coating sample can effectively inhibit the formation of Staphylococcus aureus biofilm.

[0043] Example 5. Biocompatibility study of antibacterial coating

[0044] Experimental methods:

[0045] We used the CCK-8 experiment to determine the biocompatibility of the samples to human embryonic kidney cells (HEK293). First, HEK293 was subcultured. The samples were first sterilized by UV for 30 minutes, and then the cells were seeded on a 48-well plate at a density of 5000 cells / well. After the cells grew and adhered to the wall, samples of different concentrations were added to the culture medium and cultured with human embryonic kidney cells for 24 hours. Wells without samples served as controls. Then, culture medium containing 10 μL of CCK-8 solution was added, and after culturing in the dark at 37°C for 4 hours, the absorbance was measured at 450 nm. All measurements were performed with at least two biological replicates. The results are shown in the following table. Figure 2 Compared with the control, the coating sample had less effect on the metabolic activity of human embryonic kidney cells (HEK293) within 24 hours. When its concentration was 4 mg / L, the relative metabolic activity of the cells was still maintained at a high level (95.1±1.4%), which indicated that the sample had good biocompatibility.

Claims

1. A thiazole-betaine zwitterionic polymer, characterized in that: Its molecular structure is:

2. The method according to claim 1, characterized in that Taking advantage of the functionalizable characteristics of sulfobetaine methacrylate monomer, the introduction of 2-((2-(1-(3-chlorophenyl)carbamoyl)piperidin-4-carboxamido)thiazol-5-yl)amino)ethyl methacrylate monomer not only improves the mechanical properties of the zwitterionic polymer coating, but also enhances its excellent antibacterial and anti-biofilm properties.

3. A method for preparing a thiazole-betaine zwitterionic polymer, characterized in that: The steps include: (1) By an atom transfer radical method, 2-((2-(1-(3-chlorophenyl)carbamoyl)piperidin-4-carboxamido)thiazol-5-yl)amino)ethyl methacrylate monomer and sulfobetaine methacrylate monomer were mixed and stirred at 37° C. for reaction. (2) After the reaction is completed, the reaction solution is evaporated to dryness, and the product is precipitated in acetone. The product is dried in a vacuum freeze dryer for 24 hours.

4. The method according to claim 3, characterized in that The reaction in step (1) needs to be carried out in the presence of an initiator, ammonium persulfate. Preferably, in step (1), the molar ratio of sulfobetaine methacrylate monomer to 2-((2-(1-(3-chlorophenyl)carbamoyl)piperidin-4-carboxamido)thiazol-5-yl)amino)ethyl methacrylate monomer is (1-2):(1-3), preferably 1.5:2.

5. Preferably, the amount of ammonium persulfate used as the initiator is 0.1%-5%, preferably 0.1%.

5. A method for preparing a thiazole-betaine zwitterionic polymer antibacterial coating, characterized in that: The steps include: The thiazole-betaine zwitterionic polymer is configured into a solution, and the solution is evenly coated on the surface of the titanium alloy using an ultrasonic spraying process, and then placed at room temperature and under sterile conditions for 30 minutes to obtain an antibacterial coating. The concentration of the prepared polymer solution is 10-100 mg / mL, preferably 30 mg / mL.

6. An antibacterial coating for orthopedic implants prepared by the method according to any one of claims 1 to 5, characterized in that: The orthopedic implant antimicrobial coating is used to inhibit bacterial infection or inhibit the formation of biofilm to eliminate the chance of infection around the implant.

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