Novel antibacterial material without secondary drug resistance as well as preparation method and application of novel antibacterial material

Abstract

The invention belongs to the technical field of nano material preparation, and particularly relates to a novel antibacterial material without secondary drug resistance and a preparation method and application thereof. The method comprises the following steps: reacting boron nitride powder of which the particle size is reduced with an alkaline solution at 150-200 DEG C for 0.5-3 hours, hydroxylating boron nitride, cooling to room temperature, washing a product until the product is nearly neutral, dispersing the product in an ethanol-water mixed solution containing a silane coupling agent APTES, and stirring and refluxing for later use; and reacting the obtained product with a salt containing silver ions under a stirring condition to obtain the novel antibacterial boron nitride metal composite material without secondary drug resistance. The boron nitride / silver ion efficient catalytic antibacterial agent prepared by the method provided by the invention has the advantages of high antibacterial and antiviral efficiency, simple operation, low preparation difficulty and no drug resistance.

Description

Technical Field

[0001] This invention belongs to the technical field of nanomaterial preparation, specifically a novel antibacterial material without secondary drug resistance, its preparation method, and its application. Background Technology

[0002] Bacterial infections have become common diseases. The main difficulty in treatment lies in the fact that a bacterial infection in one part of the body can trigger infections in various organs and systems, leading to a wide variety of diseases. The widespread use of traditional antibiotics has led to increased bacterial resistance and the emergence of superbugs, making the treatment of infections extremely difficult. Antibacterial agents synthesized from inorganic materials have advantages such as not inducing drug resistance, non-volatility, and broad antibacterial spectrum. For example, metal nanoparticles have begun to be used in sterile materials due to their excellent antibacterial properties. However, given their unclear biosafety, the U.S. Food and Drug Administration (FDA) is currently taking a cautious approach to materials containing metal nanoparticles. Since the discovery of penicillin, the first-generation antibiotic, in 1928, scientists have discovered more than 100 antibiotics, but no new classes of antibiotics have been discovered since 1987. Statistics show that only a few new antibiotics have been introduced to the market in the past 40 years. For example, ertapenem, a new, potent, broad-spectrum carbapenem antibiotic, can be used to treat bacterial infections caused by *Pseudomonas aeruginosa*. Professor Kim Lewis's team at Northeastern University extracted darobactin, a novel antibiotic from the metabolites of *Bacillus nematodes* and *Bacillus luminifera*, which selectively kills bacteria by binding to the outer membrane protein bamA. Therefore, we may have already entered a post-antibiotic era. Worse still, bacteria are constantly evolving under antibiotic pressure, and drug resistance mechanisms are developing rapidly. The continuous emergence and widespread spread of superbugs has become one of the most challenging health problems facing the world. In 2015, researchers from China and abroad discovered a "superbug" gene, MCR-1, in livestock and humans that can resist potent antibiotics. Bacteria carrying this gene are resistant to all antibiotics (including polymyxins), meaning that the "last line of defense" among antibiotics used by humans is at risk of being breached. In 2016, American researchers discovered a second case of *E. coli* carrying this gene in clinical patient samples, indicating that the emergence and spread of "superbugs" far exceeds our imagination. Therefore, it is urgent to develop a safe, effective antibacterial agent that does not induce drug resistance.

[0003] Boron nitride-based nanomaterials exhibit good structural stability, but their smooth surfaces have relatively few active sites, resulting in weak interfacial interactions with the polymer matrix. Therefore, surface modification is necessary to increase the number of active sites. While boron nitride as a pure antibacterial material does not induce drug resistance, its antibacterial ability is poor. Therefore, modifying pure boron nitride to improve its antibacterial ability is an effective approach. Summary of the Invention

[0004] The purpose of this invention is to provide a novel antibacterial material that does not develop secondary drug resistance, as well as its preparation method and application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing a novel antibacterial material without secondary drug resistance.

[0007] Step 1) The boron nitride powder with reduced particle size is reacted with an alkaline solution at 150℃~200℃ for 0.5h~3h to hydroxylate the boron nitride. Then, the mixture is cooled to room temperature, washed until it is nearly neutral, and then dispersed in an ethanol-water mixed solution containing the silane coupling agent APTES. The mixture is then stirred and refluxed for later use.

[0008] Step 2) The product obtained above is reacted with a salt containing silver ions under stirring conditions to obtain a novel antibacterial boron nitride metal composite material without secondary drug resistance.

[0009] The boron nitride powder with reduced particle size is prepared by mixing zirconium oxide balls, boron nitride powder and distilled water in a mass ratio of 1:1:1 to 1000 and ball-milling for 0.5 to 4 hours, then set aside for use.

[0010] The boron nitride powder with reduced particle size is mixed with an alkaline solution at a mass ratio of 1:10 to 1:100, wherein the alkaline solution is potassium hydroxide and sodium hydroxide, and the mass ratio of the two is 1:1 to 100.

[0011] After the reaction, the product was cooled to room temperature for 3-5 hours. Then, the product was repeatedly washed with ethanol and deionized water until the pH of the filtrate was close to neutral. The product was then ultrasonically dispersed in an ethanol-water mixture containing the silane coupling agent APTES for 1-3 hours. After ultrasonication, the mixture was refluxed and stirred for 6 hours. The mixture was then filtered to obtain a pale yellow powder (NH2-BNNS), which was finally dried in a vacuum oven.

[0012] The volume ratio of the silane coupling agent APTES to ethanol is 1:9 to 10000; the volume ratio of ethanol to water is 1:10 to 1:1.

[0013] The obtained pale yellow powder (NH2-BNNS) is dried in a vacuum oven at 60–100°C.

[0014] The NH2-BNNS was mixed with a silver ion-containing salt at a mass ratio of 0.1:1 to 100. After mixing, the mixture was stirred at 25°C for 2-24 hours. The mixture was then washed after the reaction was completed.

[0015] A novel antibacterial material prepared by a method without secondary drug resistance, wherein the method prepares a novel antibacterial material of sheet-like boron nitride without secondary drug resistance.

[0016] The application of the novel antibacterial material that does not develop secondary drug resistance, and the application of the material in the preparation of antibacterial agents that do not develop secondary drug resistance.

[0017] The bacteria are Escherichia coli and / or Staphylococcus aureus.

[0018] This invention achieves successful hydroxylation of boron nitride using a chemical method. The hydroxylation of boron nitride can be achieved through a chemical environment, which facilitates the breaking of the BN bond, thereby exposing the B atom sites and enabling them to react with the hydroxyl groups. Then, under anhydrous conditions, APTES first adsorbs onto the boron nitride surface through hydrogen bonds formed between the amino group and the hydroxyl group on the boron nitride surface. Some of these hydrogen bonds undergo proton transfer, resulting in stronger ionic interactions. In subsequent processing, APTES condenses with the silanol groups on the boron nitride surface to form covalent bonds, releasing the amino group. After the APTES undergoes a flipping process, the amino group is exposed on the outer surface.

[0019] This invention utilizes a coordination method to allow the lone pair electrons on the amino group to coordinate with the empty orbitals on the silver ion. The amino group has a strong electron-donating ability and readily combines with the empty outer electron orbitals of metal atoms to form chemical bonds; in other words, it provides a stronger binding force to the metal atoms, significantly reducing the aggregation between metal atoms and allowing them to exist in single-atom form. This greatly improves the utilization efficiency of silver atoms, reduces the amount of antibacterial nanomaterials needed, and significantly enhances antibacterial efficiency.

[0020] The boron nitride / silver ion highly efficient catalytic antibacterial agent prepared by the method proposed in this invention has high antibacterial and antiviral efficiency, is simple to operate, has low preparation difficulty, and does not produce drug resistance. Attached Figure Description

[0021] Figure 1 SEM images of h-BNNSs@Ag and h-BNNSs provided in this embodiment of the invention.

[0022] Figure 2 The antibacterial results were obtained using national standards.

[0023] Figure 3 The minimum bactericidal concentration (MBC) of the boron nitride / silver ion highly efficient catalytic antibacterial agent prepared according to the present invention against Escherichia coli and Staphylococcus aureus.

[0024] Figure 4 To investigate the secondary drug resistance of the boron nitride / silver ion highly efficient catalytic antibacterial agent prepared using the present invention. Detailed Implementation

[0025] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0026] Example 1

[0027] First, boron nitride was ball-milled for 2 hours to reduce its particle size. Then, sodium hydroxide (2.0600 g) and potassium hydroxide (2.7160 g) were ground until homogeneous. Next, 0.248 g of boron nitride powder was added. The mixture was further ground into uniform boron nitride nanosheets (approximately 500 nm in diameter) and transferred to a PTFE-lined stainless steel reactor. The system was reacted at a specific temperature (180°C) for a specific time (2 hours), and then cooled to room temperature. A strong ammonia odor was detected when the reactor was opened.

[0028] The reaction vessel was removed, and the solid product was collected. It was repeatedly washed with ethanol and deionized water until the pH of the filtrate was close to neutral. The hydroxylated material was then placed in a 7:3 volume ratio of the hydroxylated material to the mixture in a mixed solution of silane coupling agent APTES and ethanol. The mixture was ultrasonically dispersed for 1 hour. The solution was then refluxed and stirred for 6 hours. During this time, APTS in the silane coupling agent was mainly adsorbed onto the surface of OH-BNNSs via physical adsorption. To form covalent bonds between APTS and OH-BNNSs, the refluxed solution was vacuum-dried at 120°C for 24 hours. After cooling to room temperature, the resulting solid powder was washed with anhydrous ethanol to remove unreacted silane coupling agent. The powder was then filtered to obtain a pale yellow powder (NH2-BNNSs) (i.e., h-BNNSs), which was finally dried in a vacuum oven at 80°C.

[0029] In a beaker equipped with a magnetic stirrer, add the NH2-BNNS obtained in the above steps, then add 10 mL of deionized water, and finally add a certain mass of silver nitrate (0.16 g). Stir magnetically (the concentration of silver nitrate in the system is 0.02 mol / L). After stirring for 2 hours, wash three times each with deionized water and anhydrous ethanol to obtain h-BNNSs (see [link to product description]). Figure 1 ).

[0030] Depend on Figure 1 As can be seen, the surface of h-BNNSs is relatively smooth, and small metal particles are attached to the boron nitride nanosheets of h-BNNSs@Ag.

[0031] The boron nitride / silver ion materials obtained in Example 1 and the comparative example were tested for their bactericidal rates against *Escherichia coli* and *Staphylococcus aureus*, respectively. The test method followed GB / T21510-2008, "Test Method for Antibacterial Properties of Nano-Inorganic Materials." The test first required...

[0032] Escherichia coli and Staphylococcus aureus glycerol bacteria were removed from an environment of -80℃. A small amount of ice residue was scraped off using a sterilized inoculation loop and allowed to thaw at room temperature. A small amount of the bacterial solution was dipped into the agar plate and inoculated using a zigzag pattern. The plates were then incubated at 37℃ for 24 hours. Then, a single colony of E. coli was picked using a sterilized inoculation loop and placed in 20 mL of LB medium, while Staphylococcus aureus was placed in nutrient broth. The plates were incubated at 37℃ for 220 rpm. -1 The culture was incubated overnight for 20 hours in a constant temperature shaking incubator to obtain primary bacterial suspension. 1% of the bacterial suspension was inoculated into 25 mL of LB medium and incubated at 37°C and 220 rpm. -1 After culturing for 2-4 hours, the OD600 was 0.4-0.6, yielding a secondary bacterial culture. Then, a negative control was prepared (5.0 mL of pre-prepared secondary bacterial culture was added to 95 mL of phosphate buffer containing 0.1% Tween-80) and different material experiments were conducted (0.5 g ± 0.05 g of the materials obtained in each step of Example 1 were placed in an Erlenmeyer flask, 95 mL of phosphate buffer containing 0.1% Tween-80 was added, mixed well, and then 5.0 mL of the pre-prepared secondary bacterial culture was added). The mixture was then shaken and incubated. Finally, 100 μL of both the shaken control and experimental solutions were transferred to nutrient agar medium, spread evenly, and placed in a 37°C incubator for viable cell counting.

[0033] The minimum bactericidal concentration (MBC) was determined using 250 mL Erlenmeyer flasks. The Erlenmeyer flasks containing the negative control group and the material test group were fixed on the shaker of a constant temperature shaking incubator and shaken at 220 r·min⁻¹ for 12 h at an operating temperature of 37℃±1℃.

[0034] The concentration of the test sample stock solution was 2 mg·mL⁻¹. Seven Erlenmeyer flasks were numbered 1, 2, 3…7 sequentially. First, 12 mL of PBS solution was added to flask #1, followed by 10 mL of PBS solution to flasks #2, #3, #4…7. Then, 8 mL of the sample stock solution was added to flask #1, and the mixture was thoroughly mixed by pipetting. 10 mL of the PBS suspension was then added to flask #2, and so on. Finally, 10 mL of diluted bacterial suspension was added to each flask, with the concentrations from left to right being 500, 250, 125, 64, 32, 16, and 8 μg·mL⁻¹, respectively. For the negative control, 10 mL of PBS was added first, followed by 10 mL of diluted bacterial suspension.

[0035] Deep-well plates containing negative control and material test samples were fixed on a shaker in a constant-temperature shaking incubator. After incubation at 37℃±1℃ and a shaking speed of 220 r·min⁻¹ for 16–20 h, 100 μL of each sample was transferred to nutrient agar medium, evenly spread, and then placed in a 37℃ constant-temperature incubator for viable cell counting. The results are as follows: Figure 2 , Figure 3 As shown.

[0036] Figure 2 The antibacterial results were obtained according to national standards. The left h-BNNSs@Ag inhibited *Escherichia coli*, and the right h-BNNSs@Ag inhibited *Staphylococcus aureus*, with no bacterial colony growth observed. However, h-BNNSs alone was completely covered in bacteria. Meanwhile... Figure 3 In the figure, h-BNNSs@Ag represents the minimum bactericidal concentration (MBC) of Escherichia coli and Staphylococcus aureus. The leftmost image shows h-BNNSs confluent with bacteria. The number of colonies of h-BNNSs@Ag at 125 μg / mL in Escherichia coli is less than 5, indicating that the MBC is 125 μg / mL. Similarly, the number of colonies of h-BNNSs@Ag at 16 μg / mL in Escherichia coli is less than 5, indicating that the MBC is 16 μg / mL.

[0037] Establishment of secondary drug resistance methods

[0038] The minimum bactericidal concentration of the material group obtained in Example 1 was obtained from the above experimental results. Its sub-MBC was used to conduct a secondary drug resistance test. The test was conducted using a 250mL conical flask. The conical flask containing the negative control group and the material test group was fixed on the shaker of the constant temperature shaking incubator and shaken at 220r·min-1 for 12h under the condition of 37℃±1℃.

[0039] First, following the method for investigating MBC (Medium-to-Microbial Concentration), the material was diluted to sub-MBC level and incubated at 37℃±1℃ with shaking at 220 rpm for 16–20 h. Then, 100 μL of both the negative control and the test material were transferred to nutrient agar medium, spread evenly, and incubated at 37℃ for viable cell count. This was repeated for four weeks. The results are as follows: Figure 4 As shown.

[0040] Depend on Figure 4 The results showed no secondary drug resistance for four consecutive weeks, with no bacterial colony growth, proving that there was no secondary drug resistance.

Claims

1. A method for preparing a novel antibacterial material without secondary drug resistance, characterized in that: Step 1) The boron nitride powder with reduced particle size is reacted with an alkaline solution at 150℃~200℃ for 0.5h~3h to hydroxylate the boron nitride. Then, the mixture is cooled to room temperature, washed until it is nearly neutral, and then dispersed in an ethanol-water mixed solution containing the silane coupling agent APTES. The mixture is then stirred and refluxed for later use. Step 2) The product obtained above is reacted with a salt containing silver ions under stirring conditions to obtain a novel antibacterial boron nitride metal composite material without secondary drug resistance.

2. The method for preparing the novel antibacterial material without secondary drug resistance according to claim 1, characterized in that: The boron nitride powder with reduced particle size is prepared by mixing zirconium oxide balls, boron nitride powder and distilled water in a mass ratio of 1:1:1 to 1000 and ball-milling for 0.5 to 4 hours, then set aside for use.

3. The method for preparing the novel antibacterial material without secondary drug resistance according to claim 1, characterized in that: The boron nitride powder with reduced particle size is mixed with an alkaline solution at a mass ratio of 1:10 to 1:100, wherein the alkaline solution is potassium hydroxide and sodium hydroxide, and the mass ratio of the two is 1:1 to 100.

4. The method for preparing the novel antibacterial material without secondary drug resistance according to claim 1, characterized in that: After the reaction, the product was cooled to room temperature for 3-5 hours. Then, the product was repeatedly washed with ethanol and deionized water until the pH of the filtrate was close to neutral. The product was then ultrasonically dispersed in an ethanol-water mixture containing the silane coupling agent APTES for 1-3 hours. After ultrasonication, the mixture was refluxed and stirred for 6 hours. The mixture was then filtered to obtain a pale yellow powder (NH2-BNNS), which was finally dried in a vacuum oven.

5. The method for preparing the novel antibacterial material without secondary drug resistance according to claim 1 or 4, characterized in that: The volume ratio of the silane coupling agent APTES to ethanol is 1:9 to 10000; the volume ratio of ethanol to water is 1:10 to 1:

1.

6. The method for preparing the novel antibacterial material without secondary drug resistance according to claim 4, characterized in that: The obtained pale yellow powder (NH2-BNNS) is dried in a vacuum oven at 60–100°C.

7. The method for preparing the novel antibacterial material without secondary drug resistance according to claim 1, characterized in that: The NH2-BNNS was mixed with a silver ion-containing salt at a mass ratio of 0.1:1 to 100. After mixing, the mixture was stirred at 25°C for 2-24 hours. The mixture was then washed after the reaction was completed.

8. A novel antibacterial material prepared by the method of claim 1 without secondary drug resistance, characterized in that: A novel antibacterial material with no secondary drug resistance was prepared according to the method described in claim 1.

9. The application of the novel antibacterial material according to claim 8 that does not develop secondary drug resistance, characterized in that: Application of the material in the preparation of antibacterial agents that do not develop secondary drug resistance.

10. The application of the novel antibacterial material without secondary drug resistance according to claim 9, characterized in that: The bacteria are Escherichia coli and / or Staphylococcus aureus.

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