Novel antibacterial medical silica gel catheter coating as well as preparation method and application thereof

Abstract

The invention discloses a novel antibacterial medical silica gel catheter coating and a preparation method and application thereof.The preparation method comprises the steps that a novel betaine polymer poly (GMA-co-SBMA-co-ST) is prepared and has the following structural formula, on the basis, poly (GMA-co-SBMA-co-ST) loaded nano-microspheres are prepared through a high-pressure emulsification-solvent evaporation method, and the nano-microspheres are used for preparing the antibacterial medical silica gel catheter coating; and spraying the nanospheres on the surface of the silica gel catheter by using a spraying technology to prepare the novel betaine polymer antibacterial silica gel catheter coating. An in-vitro activity result proves that the coating not only has an excellent antibacterial effect, but also can inhibit the formation of an acinetobacter baumannii biofilm. In addition, the in-vitro cytotoxicity to human embryo kidney cells (HEK293) shows that the coating has good biocompatibility.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a novel antibacterial medical silicone catheter coating and a preparation method and application thereof. Background Art

[0002] With the advancement of science and technology and huge clinical needs, biomedical materials are widely used in diagnosis and treatment. Among them, polymer medical devices used for indwelling or implantation in the human body, such as intravascular catheters and urinary catheters, have become an indispensable part of the medical field. However, the risk of biomaterial-related infections caused by bacteria and fungi is also increasing, especially catheter-related infections, which significantly increase the problem of nosocomial infections and lead to a serious medical burden. Studies have shown that indwelling catheter-related infections caused by bacterial biofilms account for the vast majority of medical implant device infections.

[0003] Clinical data suggest that catheters coated with drugs (minocycline-rifampin and chlorhexidine silver sulfadiazine) can play an important role in preventing infection. However, these devices containing antimicrobial agents provide limited protection against bacteria because the antimicrobial agents are rapidly washed out of these devices within a few days.

[0004] Zwitterionic polymers have been studied and applied in many fields such as surface antifouling coatings, implants and medical catheters. Compared with other types of zwitterions, betaine zwitterionic polymers have lower cytotoxicity, simpler synthesis and better environmental stability. Sulfathiazole (ST) is an antibacterial drug. Its advantages as a coating include broad antibacterial spectrum, low price and easy application.

[0005] In order to overcome these problems, the present invention aims to develop a new type of betaine polymer with antibacterial and anti-biofilm activity, and uses high-pressure emulsification-solvent evaporation technology to prepare nano-microspheres loaded with betaine polymer for application in silicone catheter coating to achieve long-lasting and strong antibacterial effect. Summary of the invention

[0006] In view of the deficiencies and needs of the prior art, one of the purposes of the present invention is to provide a new type of betaine polymer with antibacterial and anti-biofilm activity, and the second purpose is to provide a preparation method and application of the betaine polymer in antibacterial medical silicone catheter coating.

[0007] Specifically, the present invention is achieved through the following technical solutions:

[0008] In the first aspect of the present invention, the present invention provides a synthetic route of a betaine polymer represented by formula (I):

[0009]

[0010] In a second aspect of the present invention, the present invention provides a method for preparing a betaine zwitterionic polymer, characterized in that it comprises the following steps:

[0011] (1) GMA and SBMA monomers were used as starting reactants to prepare poly(GMA-co-SBMA); GMA and SBMA monomers were dissolved in a mixed solvent of methanol and water under a nitrogen atmosphere and stirred for reaction. AIBN was then added to the solution, the solution was reacted at 60°C, and the reaction was terminated in an ice bath.

[0012] (2) Dissolve poly(GMA-co-SBMA) and sulfathiazole (ST) in a reaction solvent, add an organic base to the reaction solution, stir and react at 65°C, and after the reaction is completed, remove the solvent under reduced pressure. The product is precipitated in acetone and purified three times with ethanol. The product is freeze-dried with a freeze dryer to obtain the final product.

[0013] Furthermore, the reaction in step (1) needs to be carried out in the presence of a solvent and an initiator.

[0014] Preferably, in step (1), the molar ratio of GMA, SBMA and initiator AIBN is 50-100:50-100:0.5-3, preferably 50:50:1.

[0015] Preferably, the ratio of the mixed solvent is 1:1-1:3; preferably 1:3.

[0016] Preferably, in step (2), the mass volume ratio of poly(GMA-co-SBMA) and sulfathiazole (ST) to the organic base is 1:0.1-0.4:0.5-2, preferably 1:0.3:1.

[0017] Preferably, in step (2), poly(GMA-co-SBMA) and sulfathiazole (ST) are dissolved in a solvent to participate in the reaction, and the solvent is selected from one or more of tetrahydrofuran, dichloromethane, 1,4-dioxane, and methanol; preferably methanol.

[0018] Furthermore, step (2) is carried out in the presence of a base, and the base is an organic base.

[0019] Preferably, the organic base is selected from one or more of triethylamine, methylamine, ethylamine and pyridine, preferably triethylamine.

[0020] In the third aspect of the present invention, the present invention provides a method for preparing the antibacterial silicone catheter coating of the polymer of formula (I), characterized in that it comprises the following steps:

[0021] (1) immersing silica gel in an ethanol solution for surface pretreatment;

[0022] (2) The betaine polymer was dissolved in ethanol and dispersed uniformly, and then mixed into an aqueous solution containing poloxam 407 and stirred for 5 minutes.

[0023] (3) A high pressure homogenizer was then used to prepare a homogenous pre-emulsion at a pressure of 400 bar. The obtained nanoemulsion was stirred uniformly at room temperature until nanospheres were formed and then centrifuged.

[0024] (4) Finally, the obtained nanospheres are washed with purified water for multiple times. The nanosphere suspension is repeatedly and evenly sprayed onto the silicone catheter using a spraying technique.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The betaine polymer structure provided by the present invention contains the antibacterial drug sulfathiazole monomer, and uses the advantages of spraying technology to prepare the antibacterial silicone catheter coating, avoiding the complicated manufacturing process and shortening the production cycle.

[0027] 2. In addition, the in vitro activity results confirmed that the silicone catheter coating has a broad-spectrum antibacterial activity and can inhibit the formation of biofilm of Acinetobacter baumannii. At the same time, in vitro cytotoxicity showed that the coating has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0029] Figure 1 The anti-biofilm activity of the silicone catheter coating of the present invention was evaluated.

[0030] Figure 2 The biocompatibility of the silicone catheter coating of the present invention was studied. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] Example 1 Preparation of polymer of formula (I)

[0034] The synthetic route is:

[0035]

[0036] Reagents and reaction conditions used in the synthetic route: (a) SBMA, GMA, methanol, purified water, 60°C, about 6 h; (b) ST, methanol, acetone, ethanol, 65°C, about 6 h.

[0037] The specific preparation process includes the following steps:

[0038] (1) Preparation of poly(GMA-co-SBMA)

[0039] First, 24.87 g of GMA and 48.88 g of SBMA monomers were added to a mixed solvent of methanol and water in a volume ratio of 1:1, and then stirred for 10 min. The mixed solution was then purged with N2 gas for 15 min. Then 10 mL of AIBN with a 1% equivalent was added to the solution and mixed for another 15 min. The solution was reacted at 60 °C for 6 h, and the reaction was terminated in an ice bath.

[0040] The solution was cooled for more than 3 h, and a white precipitate was found in the reaction solution. The white product was then redissolved in water and purified three times with methanol.

[0041] (2) Preparation of poly(GMA-co-SBMA-co-ST)

[0042] 20.00g poly(GMA-co-SBMA) and 6.00g sulfathiazole (ST) were weighed and dissolved in 150mL methanol solution. Finally, triethylamine was added to the reaction solution and stirred at 65°C for 6h. After the reaction was completed, the solvent was evaporated under reduced pressure. The product was precipitated in acetone and purified three times with ethanol. The product was freeze-dried in a freeze dryer to obtain 23g of light yellow powder.

[0043] Example 2. Preparation method and application of betaine polymer in antibacterial medical silicone catheter coating

[0044] (1) Place the medical silicone catheter in an ethanol solution for surface pretreatment for 5 minutes;

[0045] (2) The betaine polymer was dissolved in 15 mL of ethanol and dispersed evenly, and then mixed into an aqueous solution containing poloxam 407 and stirred for 5 min.

[0046] (3) A homogenized pre-emulsion was prepared using a high-pressure homogenizer at a pressure of 400 bar. The obtained nanoemulsion was stirred evenly at room temperature until the ethanol was completely evaporated to form nanospheres and then centrifuged.

[0047] (4) Wash the nanospheres slowly and repeatedly with purified water, and use a spraying technique to repeatedly and evenly spray the nanosphere suspension onto the silicone catheter using an air pump spray gun.

[0048] Example 3. Determination of in vitro antibacterial activity of the compound of formula (I)

[0049] Experimental methods:

[0050] The minimum inhibitory concentration (MIC) of the compound of formula (I) against several common pathogenic bacteria was detected by microbroth dilution method. The concentration of bacterial cells was diluted to about 5×10 5 CFU / mL, and then add these diluted bacterial solutions to a 96-well plate containing the compound of formula (I), and use a culture medium without the compound of formula (I) as a control. The 96-well plate was placed in an incubator and cultured at 37°C for 24 hours, and the OD was finally measured. 600 The MIC value was defined as the lowest concentration with no significant increase compared to the control group. The experimental results are listed in Table 1.

[0051] Table 1 In vitro antibacterial activity of the tested compounds (unit: μg / mL)

[0052]

[0053] Note: a Sa: Staphylococcus aureus ATCC 29213, b Ef: Enterococcus faecalis ATCC 29212, c Bs: Bacillus subtilis ATCC 6051, d Ab: Acinetobacter baumannii ATCC 19606, e Ec: Escherichia coli ATCC 25922, f Pa: Pseudomonas aeruginosa PAO1.

[0054] As can be seen from Table 1, the polymer of formula (I) not only exhibits good antibacterial activity against Gram-positive bacteria (MIC=1-2), but also has antibacterial activity against Gram-negative bacteria (MIC=1-8).

[0055] Example 4. Anti-biofilm activity study

[0056] In order to evaluate the inhibitory ability of the antibacterial catheter coating of the present invention on biofilm formation, we used crystal violet staining to perform quantitative analysis of biofilm. A single colony of Acinetobacter baumannii ATCC 19606 was picked up in TSB liquid culture medium and cultured overnight at 37°C and 200 rpm. The OD value of the bacterial solution was measured and the above bacterial solution was diluted to OD 600nm=0.01. In the presence of samples of different concentrations, diluted Acinetobacter baumannii bacterial solution was added to the 96-well plate. The wells without added samples were used as the control group. The 96-well plate was placed in a constant temperature incubator and cultured at 37°C for 24 hours. Then the culture solution was aspirated with a spray gun, and PBS was added to gently rinse the plate, trying not to touch the wall of the well. Then, it was stained with 0.1% crystal violet for 30 minutes, and then the crystal violet was removed and repeatedly rinsed with PBS until the aspirated liquid was colorless. Dissolved with 33% acetic acid solution for 20 minutes, and then the OD was measured using an enzyme-labeled detector. 590nm Fresh TSB medium was used as a control.

[0057] As shown in Figure 1, compared with the control group, the antibacterial catheter coating of the present invention can effectively inhibit the formation of biofilm of Acinetobacter baumannii ATCC 19606. The inhibition rate shows concentration dependence. As the sample concentration gradually increases, the inhibition rate gradually increases. When the concentration is 32 mg / L, the inhibition rate of biofilm is 88.3±2.4%.

[0058] Example 5. Biocompatibility study

[0059] This experiment detected the cytotoxicity of betaine polymer coating on human embryonic kidney cells (HEK293) by CCK-8 method. The samples were sterilized by UV for 30 minutes before use. The cells were seeded at a density of 5000 per well on a 48-well plate. After the cells grew and adhered to the wall, different concentrations of materials were added to the culture medium and co-cultured with human embryonic kidney cells. After 24 hours, CCK-8 reagent was added to each well, and the absorbance was measured at 450nm after incubation for 4 hours. All measurements were performed at least twice. Figure 2 As shown, the catheter coating sample of the present invention has little effect on the metabolic activity of human embryonic kidney cells (HEK293), showing very good biocompatibility. When its concentration is 16 mg / L, the relative metabolic activity of the cells is still maintained at a high level (93.1±2.1%). Therefore, the betaine polymer antibacterial catheter coating has great application potential in the field of antibacterial surfaces of implant materials.

Claims

1. A novel betaine polymer, characterized in that: Its molecular structure is:

2. A method for preparing a polymer of formula (I), characterized in that: The steps include: (1) Under nitrogen atmosphere, GMA and SBMA monomers are dissolved in a mixed solvent of methanol and water in a certain proportion, and stirred for reaction. AIBN is then added to the above solution, and the solution is reacted at 60° C. to prepare poly(GMA-co-SBMA), and the reaction is terminated in an ice bath. (2) Dissolve poly(GMA-co-SBMA) and sulfathiazole (ST) in a reaction solvent, add an organic base to the reaction solution, stir and react at 65°C, and after the reaction is completed, remove the solvent under reduced pressure. The product is precipitated in acetone and purified three times with ethanol. The product is freeze-dried with a freeze dryer to obtain a compound of formula (I).

3. The method according to claim 2, characterized in that The reaction of step (1) needs to be carried out in the presence of a solvent and an initiator; Preferably, in step (1), the molar ratio of GMA, SBMA and initiator AIBN is 50-100:50-100:0.5-3, preferably 50:50:

1. Preferably, the ratio of the mixed solvent is 1:1-1:3; preferably 1:

3. Preferably, in step (2), the mass volume ratio of poly(GMA-co-SBMA) and sulfathiazole (ST) to the organic base is 1:0.1-0.4:0.5-2, preferably 1:0.3:

1. Preferably, in step (2), poly(GMA-co-SBMA) and sulfathiazole (ST) are dissolved in a solvent to participate in the reaction, and the solvent is selected from one or more of tetrahydrofuran, dichloromethane, 1,4-dioxane, and methanol; preferably methanol. Preferably, step (2) is carried out in the presence of a base, and the base is an organic base. Preferably, the organic base is selected from one or more of triethylamine, methylamine, ethylamine and pyridine, preferably triethylamine.

4. A method for preparing a polymer antibacterial silicone catheter coating of formula (I), characterized in that: The steps include: (1) Soaking the medical silicone catheter in ethanol for surface pretreatment; (2) The betaine polymer was dissolved in ethanol and dispersed uniformly, and then mixed into an aqueous solution containing poloxam 407 and stirred for 5 minutes. (3) Then, a high pressure homogenizer was used to prepare a homogenous pre-emulsion at a pressure of 400 bar. The obtained nanoemulsion was stirred uniformly at room temperature until nanospheres were formed and then centrifuged. (4) Finally, the nanospheres are washed slowly and repeatedly with purified water. Using the spraying technology, the nanosphere suspension is vertically sprayed onto the rotating catheter with an air pump spray gun, and the spraying is repeated evenly.

5. An antibacterial silicone catheter prepared by the method according to any one of claims 1 to 4, characterized in that: The antibacterial silicone catheter has strong and lasting antibacterial effect and inhibits the formation of biofilm.

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