Preparation method and application of efficient antibacterial material based on bionic design

Abstract

The invention relates to a preparation method of an efficient antibacterial material based on bionic design. The method comprises the following steps: step A, synthesizing a piezoelectric antibacterial material; and step B, combining the piezoelectric antibacterial material and a targeting material through a liposome carrier to prepare a bionic bacteriophage. According to the scheme, an innovative bionic bacteriophage concept is provided, a simple and intelligent antibacterial system is developed, reactive oxygen species (ROS) in a bacterial microenvironment is stimulated to be improved through energy conversion, bacterial cell membranes, protein and DNA are destroyed, and therefore antibacterial treatment is carried out.

Description

Technical Field

[0001] The present invention relates to a preparation method of an efficient antibacterial material designed based on bionics, belonging to the field of medical material applications, and specifically relates to the application of a liposome assembly designed by bionically mimicking natural bacteria-invading viruses - bacteriophages in the treatment of bacterial infections. Background Art

[0002] The bacterial drug resistance and side effects on the human body caused by antibiotics widely used clinically are attracting increasing attention.

[0003] More and more studies focus on antibacterial treatment, including iterative modification of antibiotics, development of new antibiotics, etc. However, the drug treatment method mainly based on antibiotics has problems such as large side effects and easy increase of bacterial drug resistance. Therefore, there is an urgent need for a simple and effective method to treat bacterial infections in clinical practice. Bacteriophages are natural bacteria-invading viruses. Due to the molecular structure complementarity between their adsorption organs and the surface receptors of host bacteria, bacteriophages have host specificity and can kill specific bacteria. Referring to the structure and function of bacteriophages, the present invention designs an efficient antibacterial liposome material based on bionics, namely barium titanate hydroxide-vancomycin-liposome (BTOH-Vanco-liposome). The uniformly distributed vancomycin molecules on the bionically mimicked bacteriophage membrane can help the antibacterial material target and locate bacteria and inhibit the synthesis of bacterial cell walls. At the same time, the combination of ultrasonic stimulation and barium titanate hydroxide piezoelectric material catalyzes the bacterial microenvironment and releases a large amount of reactive oxygen species (ROS), further destroying the bacterial integrity, thereby completely killing bacteria. The solutions disclosed in the prior art are as follows: Patent Publication No. CN119499372A uses ultrasonic activation of metal-organic framework piezoelectric materials to release reactive oxygen for antibacterial purposes, and Patent Publication No. CN119433835A uses a piezoelectric gain photocatalytic antibacterial film formed by the composite of photocatalytic nanoparticles / polymer piezoelectric materials, both of which do not have a targeting effect; Patent Publication No. CN116392454A uses a protein bionic membrane to coat the ultrasonic piezoelectric catalytic material barium titanate to target melanoma tumors. This method is exquisitely and complexly designed, but has problems such as high cost, low yield, and uncertain in vivo targeting effect. The present invention uses antibiotics as targeting materials, reduces the dosage of antibiotics, reduces the generation of bacterial drug resistance; more uses the binding sites of antibiotics themselves to bacteria, reduces the R & D cost; and finally cooperates with the physical destruction effect of piezoelectric materials to completely kill bacteria.

[0004] The experimental results of the present invention show that the antibacterial rate of the bionically mimicked bacteriophage against Staphylococcus aureus reaches 82%, and the antibacterial rate against Escherichia coli is close to 100%, and the biocompatibility is above 85%, indicating the successful preparation of this efficient antibacterial material. Summary of the Invention

[0005] In view of the problems existing in the current antibacterial technology, the present invention provides a method for preparing an efficient antibacterial material based on bionics design. This technical solution develops a simple, effective and intelligent system, which combines antibiotic targeting and inherent antibacterial properties. At the same time, ultrasound activation releases reactive oxygen species to destroy the integrity and physiological processes of bacteria, kill bacteria, improve bacterial infection, and thus conduct antibacterial treatment.

[0006] To achieve the above object, the technical solution of the present invention is as follows. A method for preparing an efficient antibacterial material based on bionics design, characterized in that the method comprises the following steps:

[0007] Step A: Preparation of piezoelectric material, that is, preparation of hydroxylated barium titanate;

[0008] Step B: Assembly of bionic phage, that is, preparation of hydroxylated barium titanate - vancomycin - liposome. The bionic phage is assembled by wrapping a piezoelectric nanomaterial and an antibiotic drug with a liposome, and a liposome assembly material is designed by imitating the structure and function of the virus - phage that naturally invades bacteria. A phage consists of a head wrapped with a protein shell and a genetic material and a tail with specific adsorption sites. While specifically adsorbing and binding to the host bacteria, the tail penetrates into the host bacteria and releases the genetic material inside the host bacteria to interfere with the biosynthesis of the host bacteria; due to the dissolution of phage synthase, when the number of progeny phages increases to a certain amount, it will cause the host bacteria to rupture and the phages to mature and release. The liposome assembly designed by the bionic mechanism is assembled by wrapping a piezoelectric nanomaterial and an antibiotic drug with a liposome.

[0009] As a further improvement of the present invention, in step A, the barium source for preparing the piezoelectric material in the bionic phage includes one or more of barium sulfate, barium nitrate, barium chloride, barium carbonate, barium hydroxide, preferably hydrated barium hydroxide; the titanium source includes one or more of titanic acid, titanium hydroxide, titanium trichloride, titanium tetrachloride, titanium dioxide, tetrabutyl titanate, preferably tetrabutyl titanate; the oxidant is selected from one or more of hydrogen peroxide, peracetic acid, sodium dichromate, chromic acid, nitric acid, potassium permanganate, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, preferably hydrogen peroxide solution; the solvent is selected from one or more of deionized water, ethanol, diethanolamine, ammonia water, acetone and chloroform, preferably deionized water, ethanol, ammonia water and diethanolamine; the molar ratio of the titanium source to the barium source is 10:1 to 1:1.

[0010] As a further improvement of the present invention, in step B, the piezoelectric material in the bionic phage is selected from one of barium titanate, barium calcium zirconate titanate, sodium bismuth titanate, sodium potassium niobate, titanium dioxide, zinc oxide, strontium titanium oxide and other applications in the biological field.

[0011] As a further improvement of the present invention, in step B, the liposome preparation method is selected from one of the thin film hydration method, reverse phase evaporation method, multiple emulsion method, solvent injection method, hot melt method, and extrusion method.

[0012] As a further improvement of the present invention, the structure of the bionic phage is as follows: the materials selected in the bionic phage are tetragonal barium titanate hydroxide nanoparticles and vancomycin molecules. The morphology of the bionic phage shows that barium titanate hydroxide is uniformly encapsulated in spherical liposomes, and vancomycin molecules are uniformly modified on the liposome membrane surface. The design of spherical liposomes can increase the specific surface area. On the one hand, it increases the modification ratio of vancomycin to increase the contact area with bacteria. On the other hand, it increases the action area of reactive oxygen species released after ultrasonic catalysis of barium titanate hydroxide piezoelectric materials, further enhancing the ability to kill bacteria.

[0013] As a further improvement of the present invention, the average particle size of the barium titanate hydroxide is 50 - 200 nm; the average molecular weight of the vancomycin molecule is about 1450 Da; the vancomycin-barium titanate hydroxide-liposome is 150 - 300 nm. Hydroxylated tetragonal barium titanate is formed by reducing tetragonal barium titanate with hydrogen peroxide and is encapsulated in liposomes. Vancomycin molecules are uniformly distributed on the liposome membrane to achieve the preparation of the bionic phage.

[0014] As a further improvement of the present invention, barium titanate hydroxide is prepared by oxidizing barium titanate with hydrogen peroxide, and vancomycin is doped in the liposome membrane. Barium titanate hydroxide is encapsulated in the liposome to achieve the preparation of the bionic phage. Specifically as follows:

[0015] Step A: Preparation of the piezoelectric material, that is, preparation of barium titanate hydroxide. Specifically as follows: Dissolve the barium source and the titanium source in a solvent respectively, then put them into a reaction kettle and stir evenly, and then place them in an oven for reaction. After the experiment is over, wait for the reaction kettle to cool to room temperature and then open it. The product is repeatedly washed with deionized water and centrifuged until the pH of the supernatant is neutral. The product is placed in a vacuum drying oven for drying to obtain tetragonal barium titanate. After grinding the tetragonal barium titanate evenly, take 1 g of barium titanate powder and add it to 400 mL of oxidant solution and stir for 4 h, and then filter and separate the solid product by filtration. Wash it 3 times with deionized water and acetone in turn, and place the solid product in a vacuum drying oven overnight to obtain barium titanate hydroxide nanoparticles.

[0016] Step 2: Assembly of the bionic phage, i.e., preparation of hydroxyapatite barium titanate-vancomycin-liposome, is as follows: Dissolve 20 mg of soy lecithin, 5 mg of DSPE, 5 mg of cholesterol, and 1 mg of vancomycin molecules in 10 ml of chloroform in a round-bottom flask, and water-bath at 55 °C for 1 h. Use a rotary evaporator to remove chloroform to form a lipid film. Add 5 mL of PBS (0.01 M, pH 7.4) solution and an equal volume of glass beads, and add hydroxyapatite barium titanate in a ratio of 10% (w / w) of liposome, and rotary evaporate at 55 °C for 2 h. Ultrasonicate the hydrated liposome solution in an ultrasonic cell disruptor (power 80 W) for 10 min (under ice-bath conditions) to obtain the product hydroxyapatite barium titanate-vancomycin-liposome, i.e., the bionic phage.

[0017] Application of the bionic phage. The bionic phage can be applied to a series of diseases caused by bacterial infection, such as bacterial infection diseases like skin trauma, deep wound, diabetic foot, etc.

[0018] Compared with the prior art, the present invention has the following advantages: By mimicking the structure and function of the virus - phage that invades bacteria in nature, a simple and effective liposome material is designed. Structurally: The tetragonal barium titanate nanoparticles are wrapped by liposomes to form the "phage" head, and vancomycin molecules are loaded outside the liposomes as specific recognition molecules; functionally: The targeting of vancomycin molecules and the biocompatibility of liposomes enable the material to attach to bacteria. By ultrasonic stimulation of barium titanate with piezoelectric effect, the concentration of reactive oxygen species in the bacterial microenvironment is changed, and substances such as bacterial cell walls, proteins, and DNA are damaged, killing bacteria, including drug-resistant bacteria, inhibiting the continued exchange of drug-resistant genes in the bacterial population, and completely preventing the further spread of drug resistance. In terms of the technical implementation path, the synergistic effect of physical destruction (piezoelectric effect) and chemical killing (antibiotic release) can be achieved, and spatio-temporal precise treatment can also be realized by controlling the application site, time, and power of ultrasonic stimulation, etc.; a large amount of reactive oxygen species are locally and in-situ released at the infected site to damage the bacterial cell membrane and physiological processes, thereby achieving the antibacterial purpose and reducing the damage to normal tissues, and it is expected to be applied in the system of bacterial infection diseases. At the same time, due to the limited biocompatibility of piezoelectric materials such as barium titanate in the body, which restricts its application, the bionic phage uses liposomes to wrap barium titanate, and the hydroxyl groups in hydroxyapatite barium titanate interact with the hydrophilic groups in liposomes, enhancing the uniform wrapping effect and also enhancing the biocompatibility of the piezoelectric nanomaterials. In addition, the bionic phage product is a white suspension, and can be administered by injection, topical application, combined with dressings such as cotton yarn and hydrogel, etc. There are many administration routes and wide application scenarios. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the antibacterial mechanism of the bionic phage.

[0020] Figure 2 It is a diagram of a bionic phage under a transmission electron microscope.

[0021] Figure 3 It is the antibacterial rate results of the bionic phage against Staphylococcus aureus and Escherichia coli.

[0022] Figure 4 It is the biocompatibility result of the bionic phage. The bionic phage at a concentration of 60% (far higher than the working concentration of the present invention) still maintains a cell survival rate of more than 85%. Detailed implementation manners

[0023] To deepen the understanding of the present invention, the following will make a detailed description of this example in conjunction with the accompanying drawings.

[0024] As Figure 1 shown, by imitating phages in nature, a liposome material, barium titanate hydroxide-vancomycin-liposome, which can specifically bind to bacteria and release a large amount of reactive oxygen species to kill bacteria, is designed. This liposome material can greatly increase the bactericidal effect of vancomycin antibiotic molecules, thereby effectively preventing the increase of bacterial drug resistance and achieving the purpose of completely sterilizing and antibacterial.

[0025] Example 1:

[0026] A preparation method of a bionic phage specifically has the following steps:

[0027] Step A: Weigh 2.57 g of barium hydroxide hydrate and dissolve it in 25 mL of deionized water; separately weigh 3.40 g of tetrabutyl titanate and dissolve it in 20 mL of ethanol and 7 mL of ammonia water; mix the two in a reaction kettle, add 5 mL of diethanolamine, stir and mix, put it in an oven, and react at 200 °C for 48 h. After the experiment, wait for the reaction kettle to cool to room temperature and then open it. The product is repeatedly washed with deionized water and centrifuged until the pH of the supernatant is neutral. The product is placed in a vacuum drying oven for drying to obtain tetragonal barium titanate. After grinding the tetragonal barium titanate evenly, take 1 g of barium titanate powder and add it to 400 mL of hydrogen peroxide solution and stir for 4 h. Then, the solid product is separated by filtration, washed 3 times with deionized water and acetone in sequence, and the solid product is placed in a vacuum drying oven overnight to obtain barium titanate hydroxide nanoparticles.

[0028] Step B: Dissolve 20 mg of soy lecithin, 5 mg of DSPE, 5 mg of cholesterol, and 1 mg of vancomycin molecule in 10 mL of chloroform in a round-bottom flask, and water-bath at 55 °C for 1 h. Remove chloroform using a rotary evaporator to form a lipid film. Add 5 mL of PBS (0.01 M, pH 7.4) solution and an equal volume of glass beads, and add barium titanate hydroxide in a ratio of 10% liposome (w / w). Rotate and evaporate for 2 h at 55 °C. Ultrasonicate the hydrated liposome solution in an ultrasonic cell disruptor (power 80 W) for 10 min (ice bath) to obtain the product barium titanate hydroxide-vancomycin-liposome, i.e., the biomimetic phage.

[0029] The prepared barium titanate hydroxide-vancomycin-liposome is a liposome material with a concentration of 600 mg / mL, and the therapeutic working concentration used in the experiment is 100 mg / mL.

[0030] Example 2:

[0031] Characterization of the morphology of the biomimetic phage:

[0032] Dilute the barium titanate hydroxide-vancomycin-liposome into a dilute solution of 50 mg / mL, and prepare samples for scanning electron microscopy and transmission electron microscopy on silicon wafers and copper grids respectively, and dry them in an oven at 37 °C. Use scanning electron microscopy and transmission electron microscopy to take pictures and observe the morphology of the prepared biomimetic phage: barium titanate hydroxide is evenly wrapped inside the liposome, and vancomycin molecules are distributed on the surface of the spherical liposome.

[0033] Example 3:

[0034] Evaluation of the antibacterial ability of the biomimetic phage:

[0035] Use Staphylococcus aureus (S. aureus) as a representative Gram-positive bacterium and Escherichia coli (E. coli) as a representative Gram-negative bacterium to construct an in vitro bacterial infection model. Set up a blank group, a blank plus liposome material group, and a liposome material plus ultrasound group as control groups. Add barium titanate hydroxide-vancomycin-liposome (100 mg / mL) to the two groups of the liposome material plus group, and set the ultrasound conditions of the ultrasound stimulation group to a power of 1.5 W·cm -2 、10 min. After placing the liposome material in a water bath at 20 °C ± 1 °C for 5 min, add 0.1 mL of S. aureus and E. coli bacterial suspensions cultured to the exponential growth phase, mix quickly and ultrasonicate, and aspirate the treatment solution and dilute it with PBS by 1×10 4 , then aspirate 1.0 mL of the test bacteria and sample mixture and inoculate 2 petri dishes respectively for viable bacteria culture counting. At the same time, use PBS instead of the sample for a parallel test as a positive control. The number of recovered colonies in the positive control is within 1.0×10 4CFU / mL to 9.0×10 4 CFU / mL. Take the same batch of PBS and culture medium as negative controls. All test samples and control samples were cultured at 36°C ± 1°C, and the final results were observed after 48 h of incubation of bacterial vegetative cells. The experimental results showed that the antibacterial rate of the barium titanate hydroxide-vancomycin-liposome material under ultrasonic stimulation reached more than 85%, indicating excellent antibacterial ability.

[0036] Example 4:

[0037] Evaluation of the leakage of soluble proteins and nucleic acids of bacteria by the bionic phage:

[0038] Prepare 100 μL of 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg / mL BSA standard solutions respectively. Take 25 μL of each dilution standard solution and mix it evenly with 200 μL of BCA staining working solution in a 96-well plate. Place it at room temperature for 20 min and measure the OD595nm value in an enzyme-linked immunosorbent assay (ELISA) reader. Use the standard protein concentration (μg / mL) as the abscissa and the OD595 absorbance value as the ordinate to plot a graph, which is the standard curve. Set the sample groups as the blank group, barium titanate hydroxide group, vancomycin group, barium titanate hydroxide-vancomycin-liposome group, and the corresponding ultrasonic stimulation groups. Each group was treated with Staphylococcus aureus and Escherichia coli. This experiment needs to be carried out in an ice bath. The ultrasonic group was stimulated for 1 min with an ultrasonic power of 1.5 W·cm -2 . Take 20 μL of the sample after ultrasonic treatment and mix it evenly with 200 μL of BCA solution. Place it at room temperature for 20 min and measure the OD595 value in an ELISA reader. According to the OD595 value of the sample and the standard curve, the protein concentration of the unknown sample was determined. Since proteins and nucleic acids are closely related and both have absorption in the ultraviolet region, to reduce mutual interference, the method of measuring the mixed solution was used to further calculate the concentrations of soluble proteins and nucleic acids respectively according to the empirical formula. After ultrasonic stimulation for 1 min in an ice bath, the bacterial suspension was centrifuged at 12,000 rpm for 5 min, and the OD260nm and OD280nm of the supernatant were detected using a UV-visible spectrophotometer. Through calculation using the empirical formula, it was determined that the amounts of proteins and nucleic acids released by the liposome material under ultrasonic stimulation increased significantly, indicating that the integrity of the bacterial cell membrane was damaged, causing its DNA and proteins to leak extracellularly, interfering with the normal physiological activities of bacteria, inhibiting bacterial growth, and causing bacterial death.

[0039] Example 5:

[0040] Effect of the bionic phage on in vitro cell compatibility

[0041] An in vitro cell model was constructed using mouse embryonic fibroblasts (MEF). 100 μL of cells were inoculated in a 96-well plate and cultured at 37°C and 5% CO2 , culture for 24 h under the condition of 90% humidity. Set sample groups with different gradient concentrations, namely blank group, barium titanate hydroxide group, vancomycin group, barium titanate hydroxide - vancomycin - liposome group and corresponding ultrasonic stimulation groups. The concentration gradients are 0, 20%, 40%, 60%, 80%, 100%. For each group, both non - ultrasonic and ultrasonic stimulation controls are set. Both Staphylococcus aureus and Escherichia coli are treated, and 5 replicate wells are set for each treatment. Add different samples into the corresponding well plates and culture at 37 °C, 5% CO 2 , culture for 24 h under the condition of 90% humidity. Add 15 μL of MTT solution (0.5 mg / mL) to each well and culture at 37 °C, 5% CO 2 , culture for 24 h under the condition of 90% humidity. After aspirating the supernatant, add 100 μL of DMSO solution to each well. After shaking the 96 - well plate for 1 min, measure OD570nm with an enzyme - linked immunosorbent assay (ELISA) reader. The experiment finds that the biocompatibility of the barium titanate hydroxide - vancomycin - liposome group is better than that of the barium titanate hydroxide group, and the liposome assembly technology shows excellent biocompatibility.

[0042] It should be noted that the above - mentioned embodiments are not used to limit the protection scope of the present invention. Equivalent transformations or substitutions made on the basis of the above - mentioned technical solutions all fall within the scope protected by the claims of the present invention.

Claims

1. A method for preparing a highly effective antibacterial material based on bionics design, characterized in that: The method adopts piezoelectric material and targeting material antibiotic to combine and assemble through liposome carrier.

2. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 1, characterized in that: The piezoelectric material is selected from one of the piezoelectric nanomaterials used in the biological field, namely, barium titanate, barium calcium zirconate titanate, sodium bismuth titanate, potassium sodium niobate, titanium dioxide, zinc oxide, and strontium titanium oxide.

3. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 1, characterized in that: The antibiotic is selected from one of vancomycin, doxorubicin, penicillin, gentamicin and tetracycline.

4. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 1, characterized in that: The following steps are involved: Step A: Preparation of piezoelectric material; Step B: Synthesis of biomimetic phage, i.e., preparation of hydroxylated barium titanate-vancomycin-liposomes: phospholipids, cholesterol and vancomycin molecules are dissolved in a solvent in a round-bottom flask, and the solvent is removed by rotary evaporation to form a lipid film; 5 mL of PBS solution and an equal volume of glass beads are added, and hydroxylated barium titanate particles are added at a ratio of 10% (w / w) liposomes, and rotary evaporation is performed for 2 h. The hydrated liposome solution is ultrasonically disrupted in an ultrasonic cell disruptor to obtain the product hydroxylated barium titanate-vancomycin-liposomes.

5. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 4, characterized in that: The preparation method of the piezoelectric material described in step A, that is, the preparation of hydroxylated barium titanate, is to dissolve the barium source and the titanium source in a solvent respectively, then transfer them into a reaction kettle, add the solvent, stir and mix, and put them into an oven for reaction; the product is repeatedly washed with deionized water and centrifuged until the pH of the supernatant is neutral, and then the product is dried in a vacuum drying oven to obtain tetragonal barium titanate; after the tetragonal barium titanate is evenly ground, 1g of barium titanate powder is added to 400mL of oxidant solution and stirred for 4h, and then the solid product is filtered and separated by filtration, and washed with deionized water and acetone three times in sequence, and the solid product is placed in a vacuum drying oven overnight to obtain hydroxylated barium titanate nanoparticles.

6. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 5, characterized in that: In step A, the titanium source is tetrabutyl titanate, the barium source is hydrated barium hydroxide, the solvent is deionized water, ethanol, ethylene glycol amine, ammonia water, the oxidant is hydrogen peroxide, and the molar ratio of the titanium source to the barium source is 2:

3.

7. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 4 or 5, characterized in that: The oven reaction temperature described in step A is 200° C. and the reaction time is 48 hours.

8. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 4, characterized in that: The phospholipids described in step B are soybean lecithin and distearoylphosphatidylethanolamine (DSPE); the solvents are chloroform and methanol; the water bath temperature during the rotary evaporation process is 55°C and the time is 1 hour; the power of the ultrasonic crusher is 80W, and the crushing is carried out in an ice bath for 10 minutes; the selected soybean lecithin: DSPE: cholesterol mass ratio is 4:1:

1.

9. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 2, 3, 4, 5 or 6, characterized in that: The average diameter of the hydroxylated barium titanate is 50-200nm; the average molecular weight of the vancomycin is about 1450Da; the average particle size of the bionic phage is 150-300nm, the hydroxylated barium titanate is wrapped inside the liposome, and the vancomycin molecules are distributed on the surface of the liposome membrane to realize the preparation of the bionic phage.

10. The method for preparing a high-efficiency antibacterial material based on bionics design according to claim 2, 3, 4, 5 or 6, characterized in that: The bionic phage prepared by the method is applied to a series of diseases caused by bacteria: skin trauma, acute and chronic bacterial infection of diabetic foot.

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