Antibacterial material as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial material and a preparation method and application thereof, and relates to the technical field of antibacterial materials.The preparation method comprises the following steps that S1, beta-cyclodextrin powder is dissolved in water, stirring and heating are conducted, after beta-cyclodextrin is completely dissolved in water, a colorless and transparent solution is formed, the colorless and transparent solution is placed at the room temperature, stirring and cooling are conducted, crystals are separated out, and a beta-cyclodextrin solution is obtained; a beta-cyclodextrin crystal is obtained; s2, activating the beta-cyclodextrin crystals by adopting ultrasonic waves to obtain the antibacterial material of the water free radical cations. The antibacterial material can basically kill bacteria in a short time, has high efficiency and broad spectrum, and overcomes the defects of narrow antibacterial spectrum and long antibacterial time of antibiotics. The antibacterial material does not have a killing effect on blood and normal cells, is high in safety, is a chitosan substance and is easy to metabolize in vivo, and the toxic and side effects of a photosensitizer and a sound-sensitive agent in vivo are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial materials, and particularly relates to an antibacterial material, a preparation method thereof, and an application thereof. Background Art

[0002] Superbugs are a type of drug-resistant bacteria, a general term for bacteria that are resistant to almost all antibiotics. Such superbugs can cause pustules and boils on the human body, and even gradually cause necrosis of human muscles. Even more terrifying is that antibiotic drugs have no effect on it. In recent years, multi-antibiotic-resistant bacteria have been growing at an alarming rate. Some Enterobacteriaceae bacteria producing carbapenemase are not only resistant to β-lactams and carbapenems, but also resistant to tigecycline, colistin, and fosfomycin. Therefore, there is an urgent need to develop new and effective strategies against bacterial resistance.

[0003] Currently, the treatment of superbugs mainly includes antibiotics, antibacterial agents, photodynamic therapy, and immunization. However, the long-term use of antibiotics and antibacterial agents will lead to stronger drug resistance of bacteria; the antibacterial strategy of photodynamic therapy has disadvantages such as weak penetration of short-wavelength light into biological tissues, easy detachment of photo-responsive nanoparticles NPs from the action site, and short release distance and short lifespan of ROS; immunization is not effective against all bacteria, and it takes a certain amount of time to generate sufficient immunity. In addition, vaccines may also pose risks of side effects and adverse reactions. Obviously, the defects of these technologies limit their application in the treatment of superbugs in reality. Therefore, there is an urgent need to develop new and effective drugs against bacterial resistance. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide an antibacterial material, a preparation method thereof, and an application thereof.

[0005] The technical solution of the present invention is as follows: A preparation method of an antibacterial material, comprising the following steps: S1: Dissolve β-cyclodextrin powder in water, stir and heat up. After β-cyclodextrin is completely dissolved in water to form a colorless and transparent solution, place it at room temperature and stir to cool and precipitate crystals to obtain β-cyclodextrin crystals; S2: Activate the β-cyclodextrin crystals by ultrasonic waves to obtain an antibacterial material of water free radical cations. In step S2, the ultrasonic frequency is 20 KHz - 20 MHz, the power is 1 - 300 W, and the duration is 1 - 80 min.

[0006] Preferably, in step S1, the temperature is raised to 25 - 100 °C.

[0007] Preferably, in step S1, the mass-volume ratio of β-cyclodextrin powder to water is 1.85 - 12:100.

[0008] Preferably, in step S2, the ultrasonic frequency is 40 KHz, the power is 105 W, and the duration is 80 min; or, it lasts for 3 min under the ultrasonic condition of 1 MHz and 1.5 W / cm².

[0009] The present invention also discloses an antibacterial material prepared by the above preparation method.

[0010] Preferably, it has a killing effect on Gram-negative bacteria, Gram-positive bacteria and their drug-resistant bacteria in vitro.

[0011] Preferably, it has a killing effect on Gram-negative bacteria, Gram-positive bacteria and their drug-resistant bacteria in vivo.

[0012] The present invention also discloses an application of an antibacterial material in the preparation of antibacterial products.

[0013] Preferably, the antibacterial product includes at least one of antibacterial dressings, antibacterial hydrogels, antibacterial drugs and disinfectants.

[0014] The beneficial effects of the present invention are as follows: (1) In the present invention, ultrasonic energy is imparted to β-cyclodextrin crystals to cause a phase change. During the phase change process, electrical energy is generated, and the electrical energy ionizes water into water radical cations, and the water radical cations are used for sterilization.

[0015] (2) The antibacterial material of the present invention can basically kill various bacteria in a short time, has high efficiency and broad spectrum, and overcomes the disadvantages of narrow antibacterial spectrum and long antibacterial time of antibiotics.

[0016] (3) The antibacterial material of the present invention has no killing effect on blood and normal cells, has high safety, and its itself is a chitosan-like substance that is easily metabolized in the body, overcoming the toxic side effects of photosensitizers and sonosensitizers in the body. Description of the Drawings

[0017] Figure 1 Mass spectrometry detection results of ultrasonic generation of water radical cations from β-cyclodextrin crystals. Among them, A: Online real-time mass spectrometry detection of H2O + • Specific product reacting with nitrogen; B: Initial mass spectrometry diagram of ultrasonic treatment of β-cyclodextrin crystals using ND-EESI-MS; C: Secondary fingerprint spectrum for identifying m / z 64 using ND-EESI-MS; D: Secondary fingerprint spectrum for identifying m / z 33 using ND-EESI-MS.

[0018] Figure 2Test results of the anti-common bacteria effect of β-cyclodextrin crystal under ultrasound in vitro. A: Sterilization rates of Escherichia coli, Acinetobacter baumannii, Proteus spp., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Haemophilus influenzae among Gram-negative bacteria; B: Colony count of Escherichia coli; C: Sterilization rates of Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis, and Streptococcus pneumoniae among Gram-positive bacteria; D: Colony count of Staphylococcus aureus; In the figure, compared with US, indicates P < 0.0001.

[0019] Figure 3 For the bactericidal effects of β-cyclodextrin crystal under ultrasound on representative drug-resistant Gram-positive and Gram-negative bacteria, where A: Multidrug-resistant Escherichia coli; B: Methicillin-resistant Staphylococcus aureus; C: Multidrug-resistant Pseudomonas aeruginosa; D: Multidrug-resistant Klebsiella pneumoniae, Vancomycin-resistant Enterococcus, Multidrug-resistant Streptococcus pneumoniae, Acinetobacter baumannii; E: Under the ultrasound conditions of 1 MHz and 1.5 W / cm² for 3 minutes, H2O + • Bactericidal efficiency against multidrug-resistant Escherichia coli; F: Under the ultrasound conditions of 1 MHz and 1.5 W / cm² for 3 minutes, H2O + • Bactericidal efficiency against methicillin-resistant Staphylococcus aureus. In the figure, compared with US, indicates P < 0.05, indicates P < 0.01, indicates P < 0.001, indicates P < 0.0001; compared with the blank control group, # indicates P < 0.05.

[0020] Figure 4 For the results of the effect of β-cyclodextrin crystal under ultrasound on bacterial membranes; where A: SYTO9 / PI staining of multidrug-resistant Escherichia coli, red fluorescence represents dead cells with damaged membranes allowing propidium iodide (PI) to enter, and green represents live cells stained with SYTO 9; B: SYTO9 / PI staining of methicillin-resistant Staphylococcus aureus; C: Depolarization of the cell membrane of multidrug-resistant Escherichia coli with or without β-cyclodextrin crystal under ultrasound treatment; D: Depolarization of the cell membrane of MRSA with or without β-cyclodextrin crystal under ultrasound treatment; E: Scanning electron microscope characterization of multidrug-resistant Escherichia coli and MRSA with or without β-cyclodextrin crystal under ultrasound treatment; F: Transmission electron microscope characterization of multidrug-resistant bacteria and MRSA with or without β-cyclodextrin crystal under ultrasound treatment. In the figure, compared with US, indicates P < 0.05, indicates P < 0.01, indicates P < 0.001, indicates P < 0.0001.

[0021] Figure 5 These are the results of the ultrasonic safety experiment on β-cyclodextrin crystals. Among them, A: The effect of ultrasonic treatment on fresh rat red blood cells of β-CD crystals, and the cell rupture was quantified by measuring the optical density at 540 nm; B: The cytotoxicity of β-cyclodextrin crystals ultrasonic waves on NIH-3T3 cells; C: The cytotoxicity of β-cyclodextrin crystals ultrasonic waves on Raw 264.7 cells. Figure 6 These are the treatment results of β-cyclodextrin crystals ultrasonic waves on the full-thickness skin wound infection model of rats. Among them, A: The changes in the wound area and healing rate of rats; B: The changes in the body weight of rats; C: The reverse culture of bacteria at the wound; D: HE staining of rat wound tissues. In the figure, compared with US, It indicates P < 0.05, It indicates P < 0.01. Specific Embodiments

[0022] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0023] Example 1: Preparation of Antibacterial Material Preparation of β-cyclodextrin crystals: Add 12 g of β-cyclodextrin powder to a 100 mL beaker and dissolve it in 100 mL of ultrapure water. Place it on a magnetic stirring table and gradually heat it to 66 °C. After β-cyclodextrin is completely dissolved in water and the solution becomes colorless and transparent, let it cool with stirring at room temperature to precipitate crystals, and β-cyclodextrin crystals are obtained.

[0024] Then, ultrasonic activation is carried out on the β-cyclodextrin crystals to generate aqueous free radical cations. The ultrasonic conditions are: ultrasonic treatment for 80 min under the conditions of 40 KHz and 105 W.

[0025] It should be noted that in specific applications, the ultrasonic conditions are not limited to this.

[0026] Test Example 1: Mass Spectrometry Detection of Aqueous Free Radical Cations Generated by Ultrasonic Waves of β-Cyclodextrin Crystals The ND-EESI-MS experiment was carried out using a neutral desorption electrospray ionization source, the structure of which is described in the reference [Huang XY, Fang XW, Zhang X, et al. Direct detection of chloramphenicol in honey by neutral desorption-extractive electrospray ionization mass spectrometry. Anal Bioanal Chem. 2014;406(29):7705-7714. doi:10.1007 / s00216-014-8176-y]. The device sequentially includes a reservoir containing a neutral desorption solution, a container containing a sample, a spray conduit, and an electrospray channel. During the experiment, nitrogen gas was introduced and passed through the conduit successively through the neutral desorption solution and the sample solution, and then ejected through the conduit to perform extractive ionization with the charged reagent released in the electrospray section, and then the ionized substances were detected by the LTQ-Tune system. The LTQ-Tune system automatically fine-tuned various detection parameters, including a mass scan range of m / z 50 to 800, an ionization voltage of 4 kV, the temperature of the ion transfer tube set at 200 °C, a capillary voltage of 35 V, a lens voltage of 110 V, the extraction solvent being methanol with a flow rate of 10 μL / min, a parent ion isolation width of 1.0 to 2.0 u, a collision energy between 15% and 40%, an activation Q value of 0.25, and a collision duration of 100 ms. Background subtraction plays a crucial role in mass spectrometry data analysis and can identify and eliminate ions from the background or sample matrix. This method can be used on full-scan data sets without making assumptions about the characteristics or behavior of the target ions. When the experiment was carried out under the same conditions, the mass spectrometry signal without the sample was the background signal. The background signal was subtracted from all mass spectrometry results.

[0027] The results are shown in Figure 1 , and the ultrasonic-activated β-CD crystals of Example 1 were detected using the above test method to determine the generation of H2O + •. Given the transient nature of H2O + •, nitrogen gas (N2) was introduced into the reaction system to observe the free radicals. The subsequent reaction between H2O + • and N2 generated N2O2H4 (m / z 64), which further decomposed into NH2OH (m / z 32) and [HNO + H] + (m / z 32) ( Figure 1 in A). These stable intermediates are easily detectable by mass spectrometry ( Figure 1In B - D). The detection of these products confirmed the successful and stable generation of H2O by this method + •. Mass spectrometry confirmed the presence of N2O2H4 (m / z 64, Figure 1 in C), [NH2OH] + (m / z 32, Figure 1 in D) and [HNO + H] + (m / z 32, Figure 1 in B). It shows that the method proposed in this example can reliably generate H2O + •.

[0028] Test Example 2: In vitro anti-superbacteria and common bacteria test of β-cyclodextrin crystals by ultrasound For representative Gram-negative bacteria: Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Proteus; Representative Gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, Bacillus subtilis; Representative drug-resistant bacteria: multidrug-resistant Escherichia coli, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, multidrug-resistant Klebsiella pneumoniae, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Streptococcus pneumoniae and Acinetobacter baumannii were used for antibacterial experiments.

[0029] First, β-cyclodextrin crystals were added to a centrifuge tube containing 200 μL of bacterial suspension with a concentration of 1×10 5 CFU·mL -1 . The groups subjected to ultrasound were sonicated for 80 min under the conditions of 40 KHz and 105 W. During sonication, constant-temperature flowing water at 25 °C was used to keep the temperature constant; at the same time, an ultrasound control group (US, only sonicating the bacteria without adding crystals) and a single crystal control group (β-CD, only adding crystals to the bacteria without sonication) were set up to exclude the effects of crystals and ultrasound itself on the bacteria; after sonication, the bacterial suspensions of each group were diluted to 1×10 3 CFU·mL -1 ; 50 μL of the diluted bacterial suspension was evenly dispersed on a nutrient agar plate and incubated in a 37 °C constant-temperature incubator for 24 h; the number of colonies on the plate was recorded and multiplied by the dilution factor to calculate the number of colonies in the original bacterial suspension; the untreated group was used as the blank control group (no sonication and no crystals, pure bacterial suspension group), and all bactericidal experiments were repeated in parallel 3 times. The bactericidal rate calculation formula: Bactericidal rate % = (B - A) / B × 100%.

[0030] Among them, A is the number of surviving bacteria after sample treatment, and B is the number of surviving bacteria in the blank control group.

[0031] The bactericidal effects against various Gram-negative bacteria were experimentally tested under optimized conditions: ultrasonic frequency of 40 KHz, power of 105 W, duration of 80 minutes, with 2 mg of β-cyclodextrin crystals added, and an additional 0.1 mg of β-cyclodextrin crystals added every 10 minutes (2 mg + 0.7 mg).

[0032] The test results are shown in Figure 2 , and the results showed that compared with US, the bactericidal effects against typical Gram-negative bacteria such as Escherichia coli (MDRE. coli) reached 99.43% (P < 0.0001), Acinetobacter baumannii 99.21% (P < 0.0001), Proteus 99.33% (P < 0.0001), Klebsiella pneumoniae 98.92% (P < 0.0001), Pseudomonas aeruginosa 98.99% (P < 0.0001), Haemophilus influenzae 99.42% (P < 0.0001), as specifically shown in Figure 2 A and B in

[0033] For Gram-positive bacteria, under the action of ultrasonic waves with a frequency of 40 KHz, power of 105 W, and duration of 80 minutes, 1 mg of β-cyclodextrin crystals was added, and an additional 0.1 mg of β-cyclodextrin crystals was added every 10 minutes (1 mg + 0.7 mg). The bactericidal effect of ultrasound-induced β-cyclodextrin crystals against Gram-positive bacteria was the best. Under these conditions, compared with US ( Figure 2 C and D in + • The bactericidal rate against Staphylococcus aureus (S. aureus) was 99.03% (P < 0.0001), against Enterococcus faecalis (E. faecalis) was 94.90% (P < 0.0001), against Staphylococcus epidermidis (S. epidermidis) was 96.02% (P < 0.0001), against Bacillus subtilis (B. subtilis) was 91.89% (P < 0.0001), and against Streptococcus pneumoniae (S. pneumoniae) was 96.07% (P < 0.0001). In summary, these results indicate that H2O + • has a broad-spectrum bactericidal effect.

[0034] Using the above conditions for Gram-positive and Gram-negative bacteria, the bactericidal effects against representative drug-resistant Gram-positive and Gram-negative bacteria were tested. The results showed that H2O +• The bactericidal rate against multidrug-resistant Escherichia coli reached 99.84% (P < 0.0001). It is worth noting that when the clinically commonly used antibiotic cefepime for multidrug-resistant Escherichia coli at 10 μg / mL was added to the bacterial solution and acted for 80 minutes, the bactericidal rate against multidrug-resistant Escherichia coli was only 19.50% (P < 0.05), significantly lower than that of H2O + • ( Figure 3 A in + ). In addition, H2O + • The bactericidal rate against methicillin-resistant Staphylococcus aureus (MRSA) reached 9.60% (P < 0.0001), while when the clinically commonly used MRSA antibiotic vancomycin at 105 μg / mL was used for 80 minutes, the bactericidal rate was only 26.62%, and the effect was significantly inferior to that of H2O + • (B in Figure 3). H2O + • Although the bactericidal efficacy against multidrug-resistant Pseudomonas aeruginosa was only 89.78% (P < 0.001), it far exceeded 13.5% of cefepime (10 μg / mL) under the same action time (C in Figure 3). In addition, H2O + • The bactericidal rates against multidrug-resistant Klebsiella pneumoniae, vancomycin-resistant Enterococcus (VRE), multidrug-resistant Streptococcus pneumoniae, and Acinetobacter baumannii were 100% (P < 0.0001), 95.88% (P < 0.0001), 96.00% (P < 0.0001), and 96.17% (P < 0.0001) respectively (D in Figure 3). In summary, H2O + • showed significant bactericidal effects against drug-resistant bacteria that are difficult to eliminate with antibiotics. It is worth noting that H2O

[0035] To explore the therapeutic effect of H2O + • on in vivo bacterial infections and ensure the consistency of in vivo and in vitro conditions, the ultrasonic parameters that can be used to generate H2O in vivo were adopted. First, β-cyclodextrin crystals were added to a 96-well plate containing 200 μL of bacterial solution with a concentration of 1×10 + CFU·mL 5 . For the groups undergoing ultrasound, a coupling agent was applied to the bottom of the 96-well plate, and the ultrasonic treatment instrument (WED-100) was used to perform ultrasound for 3 minutes under the ultrasonic conditions of 1 MHz and 1.5 W / cm². The subsequent operations were the same as before, and used to evaluate its bactericidal effects against multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus. As -1 . Figure 3As shown in E and F in [reference], under ultrasonic conditions of 1 MHz and 1.5 W / cm² for 3 minutes, the bactericidal efficiencies against multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) were 93.12% (P < 0.0001) and 100% (P < 0.0001), respectively. These results indicate that the bactericidal effect produced by this ultrasonic energy level is comparable to that of 40 KHz, 105 W, and 80-minute ultrasonic energy.

[0036] To prove that the effects observed under ultrasonic conditions were not caused by changes in cyclodextrin crystals, a negative potential of -2V was applied while under ultrasonic conditions of 1 MHz and 1.5 W / cm² for 3 minutes. Under these conditions, H2O + • preferentially binds to the negative charges in the electric field, thus reducing the concentration of H2O + •. Then, the bactericidal effects on bacteria before and after reducing the concentration of H2O + • were evaluated. The results showed that after applying a -2V voltage, the bactericidal efficiency against multidrug-resistant Escherichia coli decreased from 93.13% to 2.84% ( Figure 3 E in [reference]), and the bactericidal efficiency against MRSA decreased from 100% to 23.17% ( Figure 3 F in [reference]). At the same time, it can be seen that the -2V voltage itself has no killing effect on bacteria. These findings indicate that the decrease in the concentration of H2O + • leads to a corresponding decrease in bactericidal activity, which confirms that H2O + • plays a key role in the process of killing bacteria.

[0037] Test Example 3: Effect of Ultrasonic Treatment of β-Cyclodextrin Crystals on Bacterial Cell Membrane Permeability MRSA and MDR E. coli were selected. The bacteria cultured to the logarithmic growth phase were collected by centrifugation at 8000 r / min for 3 minutes, washed twice with PBS, and the bacterial cells were resuspended to 10 5Bacterial suspension of CFU / mL; 1 mg of β-cyclodextrin crystal was added to MRSA, and 2 mg of β-cyclodextrin crystal was added to MDR E. coli. Ultrasonic treatment was carried out at 40 KHz, 105 W for 80 minutes. At the same time, an ultrasonic control group (US, only ultrasonic treatment of the bacterial solution without adding crystals) and a blank control group (only containing the bacterial solution) were set up. After the ultrasonic treatment, the bacterial solution was aspirated, and 50 μL of SYTO 9 (0.5 μM) and 50 μL of propidium iodide (PI) (15 μM) were used to incubate in the dark for 30 min; after the incubation was completed, the bacteria were collected by centrifugation, washed twice with PBS and resuspended. 20 μL of the bacterial solution was aspirated and smeared on a glass slide, and the staining of bacteria by PI was observed using the green excitation group module of a confocal microscope. The excitation maximum and emission maximum of SYTO 9 were 483 nm and 503 nm respectively. The excitation maximum and emission maximum of PI were at 493 nm and 636 nm respectively. The stronger the red fluorescence, the greater the permeability of the bacteria.

[0038] The change of bacterial cell membrane permeability was evaluated using SYTO9 / PI dye. As Figure 4 shown in A of + •, H2O + • significantly enhanced the red fluorescence of multidrug-resistant Escherichia coli, indicating that H2O Figure 4 • damaged the bacterial cell membrane structure and changed its permeability. Similar results were obtained for methicillin-resistant Staphylococcus aureus (MRSA) (

[0039] Test Example 4: Effect of ultrasonic treatment of β-cyclodextrin crystal on bacterial membrane depolarization MRSA and MDR E. coli were selected, and the bacteria were cultured to the logarithmic phase. The bacteria were collected by centrifugation at 8000 r / min for 3 min using a centrifuge. Washed three times with buffer A (5 mM HEPES, 20 mM glucose), and then resuspended in buffer B (for Gram-positive bacteria, 5 mM HEPES, 20 mM glucose and 100 mM KCl) or buffer C (for Gram-negative bacteria, 5 mM HEPES, 20 mM glucose, 100 mM KCl and 2 mM EDTA). KCl was used to balance the K in the cytoplasm and the outside + ; the bacteria were diluted to 10 7 CFU / mL, 100 µL was taken and added to a 96-well plate (the bacterial solution was used as the blank control group), and mixed with 50 μL of 1 μM DiSC3(5) staining solution in an opaque 96-well plate, and incubated at room temperature for 90 min; using a multimode microplate reader λ 激发 = 622 nm, λ 发射Detect the fluorescence intensity at 673 nm, measure the fluorescence intensity at 2-min intervals for 10 min; take another 200 μL of the bacterial solution and add it to a centrifuge tube, mix it with 100 μL of 1 μM DiSC3(5) staining solution, keep it away from light, add 1 mg of β-cyclodextrin crystal to MRSA, add 2 mg of β-cyclodextrin crystal to MDR E. coli, and perform ultrasonic treatment at 40 KHz, 105 W for 80 minutes; at the same time, set up an ultrasonic control group (US, only perform ultrasonic treatment on the bacterial solution without adding crystals), and every 5 min during ultrasonic treatment, aspirate 150 μL of the bacterial solution into a 96-well plate to detect the fluorescence intensity, after inspection, aspirate it back into the centrifuge tube for ultrasonic treatment, and continuously record the time for 80 min. Use 0.1% Triton X-100 as the positive control.

[0040] The depolarization of the cytoplasmic membrane potential of Escherichia coli and Staphylococcus aureus was evaluated using the 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5)) probe. Figure 4 C and D in + showed that under the stimulation of H2O

[0041] Test Example 5: Scanning electron microscopy characterization of the morphology of bacteria after ultrasonic treatment with β-cyclodextrin crystals Select MRSA and MDR E. coli, culture the bacteria to the logarithmic phase, use a centrifuge to collect the bacteria at 8000 r / min for 3 min, and redisperse them in PBS buffer; dilute the bacteria to 10 6 CFU / mL, add β-cyclodextrin crystals to the diluted bacterial solution and perform ultrasonic treatment at 40 KHz, 105 W for 80 minutes; at the same time, set up an ultrasonic control group (US, only perform ultrasonic treatment on the bacterial solution without adding crystals) and a blank control group (only add the bacterial solution). After ultrasonic treatment, aspirate the bacterial solution and remove the β-cyclodextrin crystals; then fix it overnight with 2.5% glutaraldehyde in deionized water at 25°C. Wash it 3 times with PBS to remove the fixing solution, perform gradient dehydration with 20%, 50%, 80% and 100% ethanol solutions by volume, mix and let it stand for 10 min at each concentration, and centrifuge for 7 min to remove the eluent; finally, wash it three times with tert-butanol, let it stand for 30 min and then disperse it; drop the dispersed bacterial suspension on a clean silicon wafer to dry, spray gold on the surface, and observe the morphological changes of the bacteria with SEM.

[0042] The morphological changes of multi-drug resistant Escherichia coli and MRSA after treatment with H2O + • were observed by scanning electron microscopy (SEM). Figure 4The E in [description] showed that the bacteria in the control group and the ultrasound group were in normal rod or spherical shapes. In sharp contrast, after exposure, the morphology of the bacteria was distorted and atrophied.

[0043] Test Example 6: Transmission Electron Microscopy Characterization of the Effect of β-Cyclodextrin Crystal Ultrasound on Bacterial Morphology MRSA and MDR E. coli were selected. The bacteria were cultured to the logarithmic phase, and the bacteria were collected by centrifugation at 8000 r / min for 3 min using a centrifuge and redispersed into PBS buffer; the bacteria were diluted to 10 8 CFU / mL. β-Cyclodextrin crystals were added to the diluted bacterial solution for ultrasound at 40 KHz, 105 W for 80 minutes; at the same time, an ultrasound control group (US, only the bacterial solution was ultrasonically treated without adding crystals) and a blank control group (only containing the bacterial solution) were set up. After the ultrasound was completed, the bacterial solution was aspirated to remove the β-cyclodextrin crystals; then it was fixed overnight in 2.5% glutaraldehyde in deionized water at 25 °C; rinsed 3 times with PBS to wash away the fixative, then fixed with 1% osmium tetroxide solution for 2 h, rinsed again with PBS, and dehydrated with graded ethanol (30% - 100%); each concentration was treated for 15 min, 100% ethanol was treated for 20 min, then treated with a mixture of embedding agent and acetone (v / v = 1 / 1) for 1 h and treated with a mixture of embedding agent and acetone (v / v = 3 / 1) for 3 h; finally, the sample was treated with pure embedding agent and heated overnight at 70 °C. The embedded samples were sectioned on a LEICA EM UC7 ultramicrotome to obtain sections of 70 - 90 nm; after staining with lead citrate solution and 50% saturated uranyl acetate - 15 acetate solution for 5 min, the sections were observed under a transmission electron microscope.

[0044] Observed under a transmission electron microscope (F in Figure 4). In the blank control group and the ultrasound group (US), the cell walls and cell membranes of the bacteria were intact, and the nucleic acids were evenly distributed within the bacterial cells. After treatment with H2O + •, obvious signs of necrosis appeared in multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus, manifested as the appearance of vacuoles inside the bacteria, rupture of the cell membrane, and leakage of bacterial contents, ultimately leading to the death of the bacteria. Based on these results, it can be reasonably speculated that H2O + • binds to anions on the bacterial cell membrane through electrostatic interaction, resulting in membrane depolarization, changing membrane permeability, and ultimately causing the death of the bacteria.

[0045] Test Example 7: Therapeutic Effect of β-Cyclodextrin Crystal Ultrasound on Infected Full-Thickness Skin Wounds in Rats (1) Blood Compatibility Experiment Rat whole blood was centrifuged at 1500 r / min for 15 min to collect red blood cells (RBCs). The collected RBCs (1.0 mL) were further diluted with 0.9% NaCl solution (9.0 mL). β-cyclodextrin crystals were sonicated with the diluted red blood cells at 40 KHz, 105 W for 80 min. 100 μL of 0.9% NaCl solution and 100 μL of 0.1% Triton-X solution were added as negative and positive controls respectively. After centrifugation at 3500 rpm for 10 min, 200 μL of the supernatant was taken and placed in a 96-well plate, and the OD value at 540 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate was calculated by the formula: Hemolysis rate (%) = (ODs - ODnc) / (ODpc - ODnc) × 100%, where ODs, ODnc, and ODpc represent the OD values of the sample group, negative control, and positive control group respectively.

[0046] (2) Cell culture and toxicity experiment Mouse embryonic fibroblasts (NIH-3T3) were purchased from Haixing and cultured using Haixing complete medium. 100 μL of cell suspension was added to a sterile 96-well plate, ensuring that there were no less than 10 4 cells per well, and incubated at 37 °C for 24 h. Different concentrations of β-cyclodextrin crystals were added to the 96-well plate and sonicated at 40 KHz, 105 W for 80 min. At the same time, a blank control group (culture medium without cells, CCK-8) and a positive control group (culture medium with cells, CCK-8, without the sample to be tested) were set up. All the culture media were aspirated, and the cells were washed with PBS. The cell counting reagent (CCK-8 reagent) was diluted 10 times with cell culture medium, and 100 μL of CCK-8 solution was added to each well and incubated at 37 °C for 1 - 4 h. The cell optical density at 450 nm was measured with a multifunctional ELISA reader. The samples were set up in triplicate to obtain the cell survival rate. The formula for calculating the cell survival rate is: H% = (As - An) / (Ap - An) × 100%. Where H: cell survival rate; As: optical density of the experimental well (culture medium with cells, CCK-8, sample to be tested); An: absorbance of the blank control (culture medium without cells, CCK-8); Ap: absorbance of the positive control (culture medium with cells, CCK-8, without the sample to be tested).

[0047] (3) Treatment of the full-thickness skin wound infection model in rats One day before model establishment, the hair on the animal's back was shaved with a hair clipper, and the shaved area was completely removed with depilatory cream. 24 h after hair removal on the animal's back, it was washed with normal saline, dried, and disinfected. After anesthesia by intraperitoneal injection of 0.5% sodium pentobarbital (0.1 mL / 10 g), a circular mark with a diameter of 1.5 cm was made on the hairless area of the back, and the skin was cut along the edge. Immediately, 0.2 mL of 108 9 kinds of bacteria at CFU / mL. After anesthetizing the rats, place them on the experimental tabletop. After the rats wake up, put them into the rat cages. After successful modeling the next day, randomly divide them into groups: blank control group (administer normal saline to the wound after modeling), ultrasound group (US, perform ultrasound treatment on the wound without adding crystals), crystal control group (only add crystals to the wound without performing ultrasound treatment), 1 mg + US group (add crystals and perform ultrasound), 2 mg + US group (add crystals and perform ultrasound), positive control group (vancomycin at 1.56 mg / mL), with 10 rats in each group. Administer the drug once a day. The β-cyclodextrin crystal ultrasound group uses an ultrasound therapy instrument (WED-100) for ultrasound stimulation. The ultrasound conditions are 1 MHz, 1.50 W / cm 2 , 3 min, and continuously administer the drug for 14 days. Record the weight changes of the mice in each group every day.

[0048] From the successful modeling to the end of the experiment, take pictures and record the wound healing area of the rats in each group every day, and calculate the healing area rate of each group. Wound healing rate formula: Rn (%) = (S0 - Sn) / S0 × 100, Rn (%) - the wound healing rate on the nth day; S0 - the initial wound area; Sn - the wound area on the nth day, n = 1 - 14 days. After 14 days of drug administration, excise the wound site, weigh it, and homogenize it in PBS containing 0.1% TX-100. Then serially dilute the homogenate and evenly spread it on LB agar.

[0049] On the 3rd, 8th, and 14th days after drug administration, sacrifice the mice. Use a surgical blade to remove the diseased part and the surrounding normal skin on the back of the rats together. After sampling, fix it with 4% paraformaldehyde solution for more than 24 h, embed it in paraffin, and obtain 8 µm pathological sections, and stain them with hematoxylin and eosin (H&E).

[0050] Antibacterial effect in vivo. Since the ultrasound power used in in vitro experiments is as high as 300 W and cannot be used in in vivo experiments. Therefore, an ultrasound therapy instrument is used to deliver H2O in in vivo experiments + •. The ultrasound conditions are set to 1 MHz, 1.5 W / cm², and 3 minutes. In view of the necessity of ensuring drug safety in in vivo experiments, hemolysis and cytotoxicity (CCK-8) experiments are carried out. As Figure 5 shown in A below, the hemolysis rates caused by β-CD crystals themselves and β-CD crystals under ultrasound stimulation are both lower than 5%, indicating that H2O + • is safe in vivo. Similarly, Figure 5 shown in B and C below, the cytotoxicities of β-CD crystals and β-CD crystals under ultrasound stimulation to mouse embryonic fibroblasts (NIH-3T3) and mouse mononuclear macrophages (Raw 264.7) are both lower than 10%.

[0051] After confirming its safety, the antibacterial effect in vivo and wound healing of H2O + • were detected in a wound model rat infected with nine different bacteria. The rats had circular full-thickness skin wounds (about 1.5 cm in diameter) on their backs. After 24 hours of bacterial infection, low-concentration 1 mg and high-concentration 2 mg of β-CD crystal + US treatment were applied to the infected wound area, and the wound repair was monitored and compared with the blank control group, the ultrasound group (US), the β-CD crystal group, and the vancomycin positive group (A in Figure 6). As Figure 6 shown in A, the wound healing rate of the control group was the slowest, with obvious scars, the largest relative wound area on the 14th day, and the wound healing rate was only 89.59%. In contrast, the 1mg + US group had the fastest wound healing rate, the smallest wound area on the 14th day, and the wound healing rate was 99.00%, even exceeding the treatment effect of the positive drug vancomycin (96.22%). The healing rates of the ultrasound group and the crystal group were similar to those of the normal saline group, about 90%. These results indicate that H2O + • produced by ultrasound-induced β-CD crystals also showed significant antibacterial activity in vivo and promoted wound healing. In addition, the body weight of the rats decreased slightly at the initial stage of bacterial infection, but then increased steadily during the subsequent experimental process ( Figure 6 B). On the last day of treatment, the results of reverse bacterial culture of the rat wounds showed that there were almost no viable bacteria in the wound tissues of the 1mg + US group, and the number of viable bacteria in the 2mg + US group and the vancomycin group was also low ( Figure 6 C). However, there were still a large number of bacteria in the wounds of the blank control group, the US group, and the crystal group. HE staining of the rat wound tissues showed ( Figure 6 D) that on the 3rd day after wound formation, a large number of neutrophils (blue arrows) and lymphocytes (red arrows) infiltrated all infected wounds, indicating that the wounds were experiencing a severe inflammatory reaction at this time. By the 8th day, more granulation tissue and capillary formation, as well as the appearance of fibroblasts (green arrows), were observed in the 1mg+US group, 2mg+US group, and vancomycin group. In contrast, there were still many inflammatory cells in the wounds of the control group, the US group, and the crystal group. By the 14th day, new epidermis (black arrows) grew in the wounds of the 1mg+US group, 2mg+US group, and vancomycin group, and the inflammatory cells basically disappeared. These results clearly indicate that H2O + • also has a strong bactericidal effect in vivo and can promote the healing of infected wounds.

[0052] The above-described embodiments merely represent the preferred embodiments of the present invention. The description is relatively specific and detailed, but it should not be construed as limiting the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, various other corresponding changes and deformations can be made based on the technical solutions and concepts described above, and all such changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A preparation method of an antibacterial material, characterized in that, Comprising the following steps: S1: Dissolve β-cyclodextrin powder in water, stir and heat up. Wait until β-cyclodextrin is completely dissolved in water to form a colorless and transparent solution, then place it at room temperature and stir to cool and precipitate crystals to obtain β-cyclodextrin crystals; S2: Activate the β-cyclodextrin crystals by ultrasonic waves to obtain an antibacterial material of water free radical cations; In step S2, the ultrasonic frequency is 20 KHz - 20 MHz, the power is 1 - 300 W, and the duration is 1 - 80 min.

2. The preparation method of an antibacterial material according to claim 1, characterized in that, In step S1, heat up to 25 - 100 °C.

3. The preparation method of an antibacterial material according to claim 1, characterized in that, In step S1, the mass-volume ratio of β-cyclodextrin powder to water is 1.85 - 12:

100.

4. The preparation method of an antibacterial material according to claim 1, characterized in that, In step S2, the ultrasonic frequency is 40 KHz, the power is 105 W, and the duration is 80 min; or; under the ultrasonic condition of 1 MHz and 1.5 W / cm², continue for 3 min.

5. An antibacterial material, characterized in that, Prepared by the preparation method described in any one of claims 1 - 4.

6. An antibacterial material according to claim 5, characterized in that, It has a killing effect on Gram-negative bacteria, Gram-positive bacteria and their drug-resistant bacteria in vitro.

7. An antibacterial material according to claim 5, characterized in that, It has a killing effect on Gram-negative bacteria, Gram-positive bacteria and their drug-resistant bacteria in vivo.

8. Use of an antibacterial material according to claim 5 in the preparation of antibacterial products.

9. The application according to claim 8, characterized in that, The antibacterial product includes at least one of antibacterial dressings, antibacterial hydrogels, antibacterial drugs and disinfectants.

Images