Nanophage compound ZnO2-Cu-coated HA-coated Phage and application thereof

Abstract

The invention discloses a nanophage compound ZnO-Cu-HA-Phage and application thereof.The nanophage compound is prepared through the following steps that 1, ZnO-PVP is dispersed in water, a CuCl2 solution is added, stirring continues to be conducted for 1-4 h at the room temperature, centrifugation is conducted at the temperature of 4 DEG C, a product is collected, washed and dried, and ZnO-Cu is obtained; step 2, preparing ZnO-Cu and a hyaluronic acid solution, mixing the ZnO-Cu and the hyaluronic acid solution according to a volume ratio of 1: (1-3), stirring overnight at room temperature, centrifuging at 4 DEG C, washing and drying to obtain ZnO-Cu-coated HA; step 3, activating ZnO-Cu-coated HA, diluting the activated ZnO-Cu-coated HA until the concentration is 0.05-0.2 mg / mL and the phage concentration is 0.5-2 * 10 < 11 > PFU / mL, and stirring the ZnO-Cu-coated HA and the phage concentration for 0.5 And centrifuging at 4 DEG C, collecting the product, and washing to obtain the nanophage compound ZnO2-Cu-coated HA-coated Phage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a nano-phage complex ZnO2-Cu@HA@Phage and its application. Background Art

[0002] Bacterial enteritis caused by enterohemorrhagic Escherichia coli O157:H7 is one of the great challenges faced by public health. E. coli O157:H7 can usually infect animals or humans through contaminated water sources and food. After the bacteria enter the intestine, they can adhere to and invade intestinal epithelial cells through A / E lesions, and release Shiga toxin into the blood circulation, further causing damage to the body, resulting in clinical symptoms such as diarrhea, abdominal pain, and vomiting, and can be life-threatening in severe cases. Therefore, it is necessary to find new methods for effectively treating enteritis caused by E. coli O157:H7. In recent years, phage and nanomaterial-targeted drug delivery for antibacterial have become possible methods to overcome this problem.

[0003] The present invention combines phage drugs with ZnO2-Cu to construct an innovative nanozyme-armed phage system for combating bacterial infections. Summary of the Invention

[0004] Aiming at the deficiencies of the existing problems, the purpose of the present invention is to provide a nano-phage complex ZnO2-Cu@HA@Phage and its application. The present invention proves through in vitro and in vivo experiments that the nano-phage complex provided by the present invention can effectively treat enteritis caused by E. coli O157:H7.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0006] In the first aspect, the present invention protects a preparation method of a nano-phage complex ZnO2-Cu@HA@Phage, and the method includes the following steps:

[0007] Step 1, synthesis of ZnO2-Cu nanoparticles: Weigh zinc acetate (Zn(CH3COO)2) and polyvinylpyrrolidone (PVP) according to a mass ratio of 0.5~5:0.5~5, dissolve them in pure water, then add hydrogen peroxide (H2O2), stir vigorously at room temperature for 12~36h. After stirring, the solution is milky white. Centrifuge at 4°C to obtain ZnO2-PVP modified with polyvinylpyrrolidone, collect the product and wash it clean; redisperse ZnO2-PVP in pure water, add a CuCl2 solution with a concentration of 2mg / mL, continue to stir vigorously at room temperature for 1~4h to obtain a green solution, centrifuge at 4°C, collect the product and wash it clean; dry to obtain ZnO2-Cu;

[0008] Step 2, Synthesis of ZnO2-Cu@HA nanoparticles: Prepare a 1 mg / mL ZnO2-Cu solution and a 2 mg / mL hyaluronic acid (HA) solution. Add the two solutions to the reaction vessel in a volume ratio of 1:1 to 3, stir vigorously overnight at room temperature, centrifuge at 4 °C, wash thoroughly, and dry to obtain ZnO2-Cu coated with hyaluronic acid, i.e., ZnO2-Cu@HA nanoparticles;

[0009] Step 3, Synthesis of the nanoparticle-phage complex ZnO2-Cu@HA@Phage: Activate the ZnO2-Cu@HA obtained in Step 2 and dilute it to 0.05 - 2 mg / mL. The phage concentration is 0.5 - 2×10 11 PFU / mL. Mix the two gently and stir for 0.5 - 5 h; after the reaction is completed, centrifuge at 4 °C, collect the product and wash it thoroughly to obtain the nanoparticle-phage complex ZnO2-Cu@HA@Phage.

[0010] In a specific embodiment, in Step 1, the mass ratio of zinc acetate to polyvinylpyrrolidone is 0.5 - 5:0.5 - 5, preferably 1 - 3:0.5 - 5.

[0011] In a more specific embodiment, the mass ratio of zinc acetate to polyvinylpyrrolidone is 1:0.5 - 5, preferably 1:0.5 - 1.5, 1:0.8 - 2, 1:1 - 3, etc.

[0012] In a specific embodiment, in Step 1, the mass ratio of ZnO2-PVP to CuCl2 is 3 - 10:1.

[0013] In a specific embodiment, in Step 2, the volume ratio of the ZnO2-Cu solution to the hyaluronic acid (HA) solution is 1:0.5 - 2, preferably 1:1.

[0014] In a specific embodiment, in Step 3, the concentration of the activated ZnO2-Cu@HA is 0.05 - 0.5 mg / mL.

[0015] In a specific embodiment, in Step 3, the phage concentration is 0.8 - 1.5×10 11 PFU / mL.

[0016] In a specific embodiment, in Step 3, the mixing and stirring time of ZnO2-Cu@HA and the phage is 1 - 5 h, preferably 2 - 4 h.

[0017] In a second aspect, the present invention also protects the nanoparticle-phage complex ZnO2-Cu@HA@Phage prepared by the preparation method described above.

[0018] Thirdly, the present invention also protects the application of the aforementioned nano-phage complex ZnO2-Cu@HA@Phage in the preparation of a product for preventing and treating or assisting in preventing and treating bacterial enteritis.

[0019] Among them, the prevention and treatment refer to prevention and / or treatment.

[0020] In a specific embodiment, the bacterial enteritis is caused by enterohemorrhagic Escherichia coli O157:H7.

[0021] In a specific embodiment, the product is a drug.

[0022] In a specific embodiment, the drug further contains a pharmaceutically acceptable carrier or excipient.

[0023] Beneficial effects

[0024] A nano-phage complex ZnO2-Cu@HA@Phage and its application provided by the present invention have the following beneficial effects compared with the prior art:

[0025] (1) ZnO2-Cu@HA@Phage can specifically target pathogenic bacteria in the intestine rich in microorganisms, reduce the damage to other bacteria in the intestine, and is beneficial to restoring the homeostasis of intestinal microorganisms;

[0026] (2) ZnO2-Cu@HA@Phage can achieve a bactericidal effect by generating ROS, reducing the generation of bacterial drug resistance;

[0027] (3) ZnO2-Cu@HA@Phage may be able to destroy bacterial endotoxin through the generation of ROS, reduce the inflammatory response, and contribute to the restoration of the intestinal mucosa;

[0028] (4) The production process of ZnO2-Cu@HA@Phage gets rid of the dependence on the combination of nanozymes such as Au and Ag and phages, broadening the development of nano-bio conjugates. Description of the drawings

[0029] Figure 1 The morphological structure, potential and particle size of ZnO2-Cu@HA@Phage; among them, Figure 1 Figure A in : Transmission electron micrograph of ZnO2-Cu@HA@Phage, scale bar: 50 nm; Figure 1 Figure B in : Potential diagrams of ZnO2, ZnO2-Cu, ZnO2-Cu@HA@Phage, and phage; Figure 1 Figure C in : Particle size diagram of ZnO2-Cu@HA@Phage.

[0030] Figure 2 Evaluation of the antibacterial effect of nanomaterials.

[0031] Figure 3 Evaluation of the ability of ZnO2-Cu@HA@Phage to inhibit biofilm formation.

[0032] Figure 4 Evaluation of the ability of ZnO2-Cu@HA@Phage to eradicate biofilm formation.

[0033] Figure 5 Evaluation of the ability of ZnO2-Cu@HA@Phage to eradicate biofilm formation, scale bar: 200 µm.

[0034] Figure 6 Therapeutic effects of different drugs on murine enteritis caused by Escherichia coli O157:H7.

[0035] Figure 7 Plate counts of Escherichia coli O157:H7 loads in murine feces and intestines.

[0036] Figure 8 ZnO2-Cu@HA@Phage, H&E staining of intestinal tissues after phage treatment, scale bar: 200 µm.

[0037] Figure 9 Inflammatory factor staining of intestinal tissues after treatment with different nanodrugs, scale bar: 200 µm

[0038] Figure 10 ELISA detection of LPS content in murine sera. Detailed implementation manners

[0039] The present invention will be further described in detail below in conjunction with embodiments. Reagents or equipment not indicating the manufacturer are regarded as conventional products that can be purchased through the market.

[0040] Example 1: Synthesis of ZnO2-Cu@HA@Phage

[0041] 1. Synthesis of ZnO2-Cu nanoparticles: Weigh 5 mg of zinc acetate (Zn(CH3COO)2) and 5 mg of PVP, dissolve them in 5 mL of pure water, then add 0.5 mL of H2O2, and stir vigorously at room temperature for 24 h under a magnetic stirrer. After stirring for 24 h, the solution is milky white. Centrifuge at 12000 rpm / min and 4 °C for 10 min to obtain PVP-modified ZnO2-PVP. Collect the product and wash it 3 times with pure water. Redisperse ZnO2-PVP in 5 mL of pure water, add 1 mL of a CuCl2 solution with a concentration of 2 mg / mL, and continue to stir vigorously at room temperature for 2 h to obtain a green solution. Centrifuge at 12000 rpm / min and 4 °C for 10 min, collect the product and wash it 3 times with pure water. Finally, freeze the product overnight and then place it in a vacuum freeze dryer for freeze-drying for 24 h to obtain ZnO2-Cu.

[0042] 2. Synthesis of ZnO2-Cu@HA nanoparticles: Dissolve 1 mg of ZnO2-Cu in 1 mL of pure water, and dissolve 2 mg of hyaluronic acid (HA) in 1 mL of pure water. Add the two solutions to a round-bottom flask at a volume ratio of 1:1 and stir vigorously overnight at room temperature. Centrifuge the obtained product at 15000 rpm / min and 4 °C for 10 min, and wash it 3 times with pure water. Freeze it overnight and then place it in a vacuum freeze dryer for freeze-drying for 24 h to obtain HA-coated ZnO2-Cu.

[0043] 3. Synthesis of ZnO2-Cu@HA@Phage nanophage complex: Dissolve 2 mg of ZnO2-Cu@HA in 2 mL of pure water, add EDC (11.5 mg / mL, 100 µL) and NHS (17.25 mg / mL, 100 µL), and stir vigorously for 40 min under a magnetic stirrer. After the reaction is completed, centrifuge at 16000 rpm and 4 °C for 10 min, and wash it 3 times with pure water to obtain activated ZnO2-Cu@HA. Finally, dilute the activated ZnO2-Cu@HA to 0.1 mg / mL, and the phage PNJ1902 concentration is 1×10 11 PFU / mL. Put the two into a magnetic stirrer and stir gently for 2 h. After the reaction is completed, centrifuge at 16000 rpm and 4 °C for 10 min and wash it 3 times with pure water to obtain ZnO2-Cu@HA@Phage.

[0044] The surface of ZnO2-Cu is positively charged. Through electrostatic interaction, hyaluronic acid is coated on the outside of ZnO2-Cu. By activating the carboxyl group (—COOH) on HA, a chemical reaction occurs between it and the amino group on the phage, attaching the phage to the ZnO2-Cu nanoparticles wrapped with hyaluronic acid to form a ZnO2-Cu@HA@Phage bioconjugate (Figure A in Figure 1 . Compared with ZnO2-Cu, the ζ potential of ZnO2-Cu@HA@Phage is reduced to -12 mV (Figure B in Figure 1 , and the hydrodynamic diameter increases to 368.9 nm (Figure C in Figure 1 . Detecting the phage activity, it is found that the phage activity in Example 1 is only reduced to 10 9 -10 10 PFU / mL.

[0045] Comparative Example 1

[0046] The preparation of ZnO2-Cu is the same as that in Example 1. Phage PNJ1902 (5×10 8 PFU / ml) and ZnO2-Cu (0.1 mg / ml) are mixed at a volume ratio of 1:1, stirred vigorously at 4 °C for 3 h and 9 h, and after centrifugation and washing, the particle size and phage activity of the product are detected.

[0047] The results show that a visible precipitate appears in the product after stirring, and the average particle size is about 12 µm. By detecting the phage activity through a double-layer agar plate, it is found that the phage activity is significantly reduced to 10 4 PFU / ml. It may be that the toxicity of ZnO2-Cu is relatively strong, and the ROS generated directly damages the phage. After the phage dies, it adheres to the nanomaterial in an irregular protein form, resulting in an increase in the particle size of the nanomaterial and the formation of a visible precipitate.

[0048] Comparative Example 2

[0049] Other conditions are the same as those in Example 1, except that 3-mercaptopropionic acid (10 µL 50 mmol) is used instead of hyaluronic acid.

[0050] The results show that a visible precipitate appears after the nanomaterial and the phage are combined, and the phage activity is significantly reduced.

[0051] Comparative Example 3

[0052] The preparation of ZnO2-Cu was the same as that in Example 1, using dopamine instead of hyaluronic acid. ZnO2-Cu (1 mg / ml) and PDA (1 mg / ml) were stirred in Tris-HCl solution at pH 8.5 for 1 h, 2 h, 5 h, and 8 h. These four different PDA coating times would result in different PDA film thicknesses. The obtained ZnO2-Cu@PDA was used to prepare 4 different ZnO2-Cu@PDA@Phage materials according to the steps of Example 1, where the phage concentration was 3×10 11 PFU / ml.

[0053] The results showed that all four stirring schemes would increase the viscosity between the nanomaterials. The longer the stirring time, the stronger the viscosity of the nanomaterials. By detecting the ROS generation ability of the nanomaterials using TMB, it was found that the ROS generation ability was significantly reduced to 60% - 40% (the longer the stirring time, the more it decreased). At the same time, we combined ZnO2-Cu@PDA (the sample stirred for 2 h) with phage (3×10 11 PFU / ml), and the results showed that the phage concentration was significantly reduced to 1×10 3 PFU / ml.

[0054] In summary, using HA to combine with ZnO2-Cu through electrostatic interaction, compared with other materials, ZnO2-Cu@HA has more stable dispersion and relatively uniform particle size. By activating HA to combine ZnO2-Cu@HA with phage, it was found that the present invention could significantly reduce the mortality rate of phage and improve the synthesis efficiency.

[0055] Example 2: Synthesis of ZnO2-Cu@HA

[0056] 1. Synthesis of ZnO2-Cu nanoparticles: Weigh 5 mg of zinc acetate (Zn(CH3COO)2) and 5 mg of PVP, dissolve them in 5 mL of pure water, then add 0.5 mL of H2O2, and stir vigorously at room temperature for 24 h under a magnetic stirrer. After stirring for 24 h, the solution was milky white. Centrifuge at 12000 rpm / min at 4 °C for 10 min to obtain PVP-modified ZnO2-PVP. Collect the product and wash it 3 times with pure water. Redisperse ZnO2-PVP in 5 mL of pure water, add 1 mL of CuCl2 solution with a concentration of 2 mg / mL, and continue to stir vigorously at room temperature for 2 h to obtain a green solution. Centrifuge at 12000 rpm / min at 4 °C for 10 min, collect the product and wash it 3 times with pure water. Finally, freeze the product overnight and then place it in a vacuum freeze dryer for freeze drying for 24 h to obtain ZnO2-Cu.

[0057] 2. Synthesis of ZnO2-Cu@HA nanoparticles: Dissolve 1 mg of ZnO2-Cu in 1 mL of pure water, and dissolve 2 mg of hyaluronic acid (HA) in 1 mL of pure water. Add the two solutions to a round-bottom flask in a volume ratio of 1:2 and stir vigorously overnight at room temperature. The obtained product was centrifuged at 15000 rpm / min for 10 min at 4 °C, washed 3 times with pure water, frozen overnight, and then freeze-dried in a vacuum freeze-dryer for 24 h to obtain HA-coated ZnO2-Cu.

[0058] 3. The ability of ZnO2-Cu@HA to generate ROS was detected using TMB. The results showed that when ZnO2-Cu:HA reacted at a volume ratio of 1:2, the ability to generate ROS decreased by 15% compared to ZnO2-Cu, while when ZnO2-Cu:HA reacted at a volume ratio of 1:1, it only decreased by 3.5%.

[0059] Example 3: Synthesis of ZnO2-Cu@HA@Phage

[0060] 1. Synthesis of ZnO2-Cu nanoparticles: Weigh 5 mg of zinc acetate (Zn(CH3COO)2) and 5 mg of PVP, dissolve them in 5 mL of pure water, then add 0.5 mL of H2O2, and stir vigorously at room temperature for 24 h under a magnetic stirrer. After stirring for 24 h, the solution was milky white. Centrifuge at 12000 rpm / min for 10 min at 4 °C to obtain PVP-modified ZnO2-PVP. Collect the product and wash it 3 times with pure water. Redisperse ZnO2-PVP in 5 mL of pure water, add 1 mL of a CuCl2 solution with a concentration of 2 mg / mL, and continue to stir vigorously at room temperature for 2 h to obtain a green solution. Centrifuge at 12000 rpm / min for 10 min at 4 °C, collect the product and wash it 3 times with pure water. Finally, freeze the product overnight and then freeze-dry it in a vacuum freeze-dryer for 24 h to obtain ZnO2-Cu.

[0061] 2. Synthesis of ZnO2-Cu@HA nanoparticles: Dissolve 1 mg of ZnO2-Cu in 1 mL of pure water, and dissolve 2 mg of hyaluronic acid (HA) in 1 mL of pure water. Add the two solutions to a round-bottom flask in a volume ratio of 1:1 and stir vigorously overnight at room temperature. The obtained product was centrifuged at 15000 rpm / min for 10 min at 4 °C, washed 3 times with pure water, frozen overnight, and then freeze-dried in a vacuum freeze-dryer for 24 h to obtain HA-coated ZnO2-Cu.

[0062] 3. Synthesis of ZnO2-Cu@HA@Phage nanophage complex: Dissolve 2 mg of ZnO2-Cu@HA in 2 mL of pure water, add EDC (11.5 mg / mL, 100 µL) and NHS (17.25 mg / mL, 100 µL), and stir vigorously for 40 min under a magnetic stirrer. After the reaction, centrifuge at 16,000 rpm and 4 °C for 10 min, and wash 3 times with pure water to obtain activated ZnO2-Cu@HA. Finally, dilute the activated ZnO2-Cu@HA to 0.1 mg / mL, and the concentration of phage PNJ1902 is 1×10 11 PFU / mL. Put the two into a magnetic stirrer and gently stir for 2 h, 4 h, and 8 h. After the reaction is completed, centrifuge at 16,000 rpm and 4 °C for 10 min and wash 3 times with pure water to obtain ZnO2-Cu@HA@Phage. The phage activity of the samples stirred for 2 h and 4 h decreased to 10 9 -10 10 PFU / ml. The activity of the sample stirred for 8 h decreased to 10 7 -10 8 PFU / ml.

[0063] Example 4: Synthesis of ZnO2-Cu@HA@Phage

[0064] 1. Synthesis of ZnO2-Cu nanoparticles: Weigh 5 mg of zinc acetate (Zn(CH3COO)2) and 5 mg of PVP, dissolve them in 5 mL of pure water, then add 0.5 mL of H2O2, and stir vigorously at room temperature for 24 h under a magnetic stirrer. After stirring for 24 h, the solution is milky white. Centrifuge at 12,000 rpm / min and 4 °C for 10 min to obtain PVP-modified ZnO2-PVP. Collect the product and wash 3 times with pure water. Redisperse ZnO2-PVP in 5 mL of pure water, add 1 mL of a CuCl2 solution with a concentration of 2 mg / mL, and continue to stir vigorously at room temperature for 2 h to obtain a green solution. Centrifuge at 12,000 rpm / min and 4 °C for 10 min, collect the product and wash 3 times with pure water. Finally, freeze the product overnight and then place it in a vacuum freeze dryer for freeze drying for 24 h to obtain ZnO2-Cu.

[0065] 2. Synthesis of ZnO2-Cu@HA nanoparticles: Dissolve 1 mg of ZnO2-Cu in 1 mL of pure water, and dissolve 2 mg of hyaluronic acid (HA) in 1 mL of pure water. Add the two solutions to a round-bottom flask at a volume ratio of 1:1 and stir vigorously overnight at room temperature. The obtained product was centrifuged at 15000 rpm / min for 10 min at 4 °C, washed 3 times with pure water, frozen overnight, and then freeze-dried in a vacuum freeze dryer for 24 h to obtain HA-coated ZnO2-Cu.

[0066] 3. Synthesis of ZnO2-Cu@HA@Phage nanoparticle complex: Dissolve 2 mg of ZnO2-Cu@HA in 2 mL of pure water, add EDC (11.5 mg / mL, 100 μL) and NHS (17.25 mg / mL, 100 μL), and stir vigorously for 40 min on a magnetic stirrer. After the reaction, centrifuge at 16000 rpm and 4 °C for 10 min, and wash 3 times with pure water to obtain activated ZnO2-Cu@HA. Finally, dilute the activated ZnO2-Cu@HA to 0.1 mg / mL, and the phage PNJ1902 concentrations are 1×10 11 PFU / mL, 2×10 11 PFU / mL, 4×10 11 PFU / mL. Put the two into a magnetic stirrer and stir gently for 2 h and 4 h. After the reaction is completed, centrifuge at 16000 rpm and 4 °C for 10 min and wash 3 times with pure water to obtain ZnO2-Cu@HA@Phage. After adding 1×10 11 PFU / mL and 2×10 11 PFU / mL of phage and stirring for 2 h, the phage activity is 10 9 -10 10 PFU / ml. After stirring for 4 h, it drops to 10 8 -10 9 PFU / ml. After adding 4×10 11 PFU / mL of phage and stirring for 2 h, the phage concentration is about 10 10 PFU / ml, but obvious precipitation appears in the liquid and the dispersibility decreases. After adding 4×10 11 PFU / mL of phage and stirring for 4 h, the phage concentration is about 10 9 -10 10 PFU / ml, and obvious precipitation also appears. Therefore, it is finally decided to use the method of mixing 1×10 11 PFU / mL with ZnO2-Cu@HA and stirring for 2 h.

[0067] Detection of in vitro antibacterial effect of ZnO2-Cu@HA@Phage in Example 5

[0068] E. coli O157:H7 was cultured to the logarithmic phase. The bacterial solution was centrifuged at 3000 g for 10 min, washed twice with PBS, and the bacteria were resuspended with SIF at pH 6.5 and diluted to OD 600 = 0.05. The test groups were: phage PNJ1902, the nano-phage complex ZnO2-Cu@HA@Phage of Example 1, the final concentration of the complex material was 200 μg / mL, and the final concentration of the phage was 2×10 9 PFU / mL. In a 96-well plate, 196 μL of the bacterial solution + 4 μL of the nano-material were added to each well and mixed evenly. In the control group, 196 μL of the bacterial solution and 4 μL of pure water were added. The background group was 196 μL of simulated intestinal fluid at pH 6.5 added with 4 μL of the nano-material or phage or pure water. The 96-well plate was placed in an enzyme-linked immunosorbent assay (ELISA) reader, and the OD was detected every 30 min at 30 °C 600nm for a total of 15 h, and the growth curve of the bacteria was recorded.

[0069] Example 6 Detection of the ability to inhibit biofilm

[0070] E. coli O157:H7 was cultured to the logarithmic phase, centrifuged at 3000 g for 10 min, washed twice with PBS, and diluted twice with 3% TSB (diluted to 10 6 CFU / mL). 196 μL of the bacterial solution was added to each well of a 96-well plate, and then 4 μL of ZnO2-Cu@HA@Phage and phage solution of Example 1 were added. The final concentration of the nano-material was 200 μg / mL, and the final concentration of the phage was 2×10 9 PFU / mL, and incubated at 37 °C for 24 h. After incubation, the biofilm was washed three times with PBS to remove planktonic bacteria. 200 μL of formaldehyde was added to each well to fix the biofilm. After drying for 5 min, 1% (w / v) crystal violet solution was added and stained for 15 min, and then washed with PBS again until the control wells were colorless. After drying at 37 °C for 20 min, finally 33% acetic acid (200 μL) was added to dissolve it, and the absorbance was measured at OD 590nm to determine the biomass of the biofilm.

[0071] Example 7 Detection of the ability to remove biofilm

[0072] E. coli O157:H7 was cultured to the logarithmic phase, centrifuged at 3000 g for 10 min, washed twice with PBS, and diluted twice with 3% TSB (diluted to 10 6CFU / mL). Add 200 μL of 3% TSB to each well of a 96-well plate, then add 10 μL of the diluted bacterial solution. After mixing, incubate at 37 °C for 48 h to form a biofilm. Wash the biofilm 3 times with PBS. Prepare ZnO2-Cu@HA@Phage (Example 1) with a concentration of 200 μg / mL using 3% TSB, and the phage concentration is 2×10 9 PFU / mL. Incubate at 37 °C for 24 h. After incubation, wash the biofilm 3 times with PBS to remove planktonic bacteria. Add 200 μL of formaldehyde to each well to fix the biofilm. After drying for 5 min, add 1% (w / v) crystal violet solution and stain for 15 min, then wash again with PBS until the control wells are colorless. Dry at 37 °C for 20 min. Finally, add 33% acetic acid (200 μL) to dissolve and measure the absorbance at OD 590nm to determine the biomass of the biofilm.

[0073] Form a biofilm in a confocal dish in the same way. After washing the biofilm, prepare ZnO2-Cu@HA@Phage with a concentration of 200 μg / mL using 3% TSB, and the phage concentration is 2×10 9 PFU / mL. Incubate at 37 °C for 24 h. Wash 3 times with PBS, add DAPI solution and incubate for 10 min. Finally, wash 2 times with PBS and scan the stained biofilm with CLSM.

[0074] Example 8 Detection of in vivo antibacterial effect of ZnO2-Cu@HA@Phage

[0075] ICR female mice at 4-6 weeks of age were randomly divided into 4 groups (n = 6): PBS+PBS group (blank control group), O157:H7+PBS group (O157:H7 group), O157:H7+ZnO2-Cu@HA@Phage (Example 1) group (ZnO2-Cu@HA@Phage group), O157:H7+phage group (phage PNJ1902 group). The mice were fasted and watered for 24 h. Before the experiment, the mice were gavaged with 100 μL of 5% NaHCO3 to neutralize gastric acid. 30 min later, the mice in the blank control group were gavaged with 200 μL of PBS, and the mice in the other groups were gavaged with 2.5×10 10 CFU / mL of Escherichia coli 200 μL, and gavaged continuously 6 times at an interval of 12 h each time to establish an enteritis model infected with Escherichia coli. After gavaging the bacterial solution continuously 6 times, it could be observed that the hair of the mice all over the body was rough, the spirit was listless, and there was a hunched back phenomenon, indicating that the model was established. Dilute ZnO2-Cu@HA@Phage to 200 μg / mL with PBS, and dilute the phage to 5×10 9CFU / mL; Each mouse was gavaged with 200 µL, gavaged three times continuously, with a 12-hour interval between each gavage. The blank control group and the O157:H7 group were gavaged with 200 µL of PBS in the same manner. After treatment, the mice were observed for 7 days, and the changes in body weight and bacterial load in the feces were recorded. Finally, the blood of the mice was collected, and the intestinal tissues of the mice were collected to detect the concentrations of inflammatory factors and LPS in the mouse serum, as well as the changes in the intestinal structure and bacterial load of the mice.

[0076] The experimental results are as follows:

[0077] 1. Detection of in vitro antibacterial effect of ZnO2-Cu@HA@Phage

[0078] By detecting the inhibitory effects of different drugs on Escherichia coli O157:H7 within 15 hours ( Figure 2 ), the results showed that ZnO2-Cu@HA@Phage could inhibit the growth of bacteria for a long time within 15 hours, while the bacteria developed obvious resistance to phage PNJ1902 at 8 hours, resulting in rapid bacterial reproduction after 8 hours. This indicates that ZnO2-Cu@HA@Phage can quickly anchor bacteria and produce antibacterial effects through the combined action of phages and nanomaterials, achieving long-term antibacterial and bacteriostatic effects.

[0079] 2. Detection of the ability to inhibit biofilm

[0080] To evaluate the ability of the nanomaterials to inhibit biofilm formation, the effects of the drugs on biofilm formation were detected by co-incubating Escherichia coli O157:H7 with different concentrations of ZnO2-Cu@HA@Phage and phages. The results showed that when the concentration of the nanomaterials was 200 µg / mL, the biofilm amount of ZnO2-Cu@HA@Phage was 7%, while the biofilm amount of the phage group was 40%. ZnO2-Cu@HA@Phage could significantly inhibit the formation of biofilm compared with the control group and the phage group, indicating that ZnO2-Cu@HA@Phage could significantly inhibit the formation of biofilm by Escherichia coli O157:H7 ( Figure 3 )

[0081] 3. Detection of the ability to remove biofilm

[0082] Escherichia coli O157:H7 can produce biofilms by aggregating and secreting extracellular polysaccharides and other substances. Biofilms are beneficial for bacterial colonization and resistance to the host's immune system and antibiotics, thus causing continuous damage to the body. Therefore, destroying biofilms is also an important part of antibacterial. By culturing Escherichia coli O157:H7 to form biofilms, and then adding different concentrations of ZnO2-Cu@HA@Phage, and a final concentration of 2×10 9PFU / mL phages were used to observe their ability to eliminate biofilms. The results showed that the eradication rate of ZnO2-Cu@HA@Phage was significantly better than that of the phage group ( Figure A in Figure 4 ). It was found by SEM observation that compared with the biofilm of the control group, ZnO2-Cu@HA@Phage could significantly eliminate the formed biofilm ( Figure B in Figure 4 ).

[0083] Finally, the effects of different nanomaterials on Escherichia coli in the DAPI-labeled biofilm were observed by confocal microscopy. The results showed that compared with the phage group, ZnO2-Cu@HA@Phage could significantly eliminate Escherichia coli in the biofilm( Figure in Figure 5 ).

[0084] 4. Detection of in vivo antibacterial effect of ZnO2-Cu@HA@Phage

[0085] The results of the survival rate of mice showed that compared with the blank control group, after 7 days of treatment, the survival rate of the phage treatment group reached 50%, while the ZnO2-Cu@HA@Phage treatment group reached 100% survival rate ( Figure A in Figure 6 ), indicating that the ZnO2-Cu@HA@Phage bioconjugate could improve the therapeutic effect of phages in mice. By observing the change of mouse body weight, it was found that the daily weight gain of mice after ZnO2-Cu@HA@Phage treatment was higher than that of the phage treatment group, and the body weight of mice on the seventh day was equivalent to that of the blank control group ( Figure B in Figure 6 ).

[0086] Through the analysis of the bacterial load in the feces and intestines of mice, the results showed that compared with the O157:H7 group, after ZnO2-Cu@HA@Phage treatment, the bacterial load in the feces of mice decreased by 4 orders of magnitude within 7 days, while the phage treatment group only decreased by 1 order of magnitude ( Figure A in Figure 7 ). After observing for 7 days, the intestines of mice were collected for bacterial load counting. The results showed that compared with the positive control group, the survival rate of Escherichia coli O157:H7 in the intestine after ZnO2-Cu@HA@Phage treatment was significantly reduced, and the bactericidal efficiency could reach 98%, while the phage group could only reach 37% bactericidal efficiency ( Figure B in Figure 7 ).

[0087] According to histopathological analysis, after infection with Escherichia coli O157:H7, the intestinal villus structure of mice was severely damaged, a large number of tissue fragments were present in the intestinal lumen, a large number of immune cells infiltrated, and severe intestinal epithelial damage occurred. After phage treatment, there was only slight damage to the intestinal structure of mice ( Figure in Figure 8). After treatment with ZnO2-Cu@HA@Phage, the intestinal villus structure of the mice was intact, close to that of normal mice. There was almost no infiltration of immune cells in the mucosa and submucosa, indicating that ZnO2-Cu@HA@Phage could protect the integrity of the intestinal mucosa and reduce the damage of bacteria and inflammatory factors to the intestinal mucosa ( Figure 8 ). By staining the inflammatory factors in the tissue sections and analyzing them with ImageJ, the results showed that the intestinal tissue structure in the positive control group was abnormal, accompanied by the release of a large amount of inflammatory factors. The intestinal tissue structure in the ZnO2-Cu@HA@Phage treatment group recovered, with low expression of inflammatory factors, approaching that of healthy mice ( Figure 9 ).

[0088] Bacterial toxins are the main virulence mechanism for Escherichia coli O157:H7 to infect the host. O157:H7 can cause an inflammatory storm in the body through LPS. However, phage treatment may cause the release of endotoxins and induce inflammation. By detecting the endotoxin content in the serum of mice after treatment with phage and ZnO2-Cu@HA@Phage, the results showed that the endotoxin content in the phage and ZnO2-Cu@HA@Phage treatment group was significantly lower than that in the control group. At the same time, the endotoxin content in the serum of mice after treatment with ZnO2-Cu@HA@Phage was significantly lower than that in the phage group and was similar to the LPS content in the normal mouse group. This indicates that ZnO2-Cu@HA@Phage can reduce the endotoxin content in the serum and is beneficial to alleviating the disease process ( Figure 10 ).

[0089] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims.

Claims

1. A preparation method of a nano-phage complex ZnO2-Cu@HA@Phage, characterized in that The method includes the following steps: Step 1, synthesis of ZnO2-Cu nanoparticles: Weigh zinc acetate and polyvinylpyrrolidone according to a mass ratio of 0.5~5:0.5~5, dissolve them in pure water, then add hydrogen peroxide, stir at room temperature for 12~36 h, centrifuge at 4 °C to obtain ZnO2-PVP modified with polyvinylpyrrolidone, collect the product and wash it clean; redisperse ZnO2-PVP in pure water, add a CuCl2 solution with a concentration of 2 mg / mL, continue to stir at room temperature for 1~4 h, centrifuge at 4 °C, collect the product and wash it clean; dry to obtain ZnO2-Cu; Step 2, synthesis of ZnO2-Cu@HA nanoparticles: Prepare a ZnO2-Cu solution with a concentration of 1 mg / mL and a hyaluronic acid solution with a concentration of 2 mg / mL, add the two solutions to the reaction vessel in a volume ratio of 1:1~3, stir overnight at room temperature, centrifuge at 4 °C, then wash clean and dry to obtain ZnO2-Cu coated with hyaluronic acid, that is, ZnO2-Cu@HA nanoparticles; Step 3, Synthesis of nano-phage complex ZnO2-Cu@HA@Phage: Activate the ZnO2-Cu@HA obtained in Step 2, dilute it to 0.05~2 mg / mL, with the phage concentration of 0.5~2×10 11 PFU / mL, mix the two and stir for 0.5~5 h; after the reaction is completed, centrifuge at 4 °C, collect the product and wash it clean to obtain the nano-phage complex ZnO2-Cu@HA@Phage.

2. The preparation method of a nano-phage complex ZnO2-Cu@HA@Phage according to claim 1, characterized in that, In the said Step 1, the mass ratio of zinc acetate to polyvinylpyrrolidone is 1~3:0.5~5.

3. The preparation method of a nano-phage complex ZnO2-Cu@HA@Phage according to claim 1, characterized in that, In the said Step 1, the mass ratio of ZnO2-PVP to CuCl2 is 3~10:

1.

4. The preparation method of a nano-phage complex ZnO2-Cu@HA@Phage according to claim 1, characterized in that, In the said Step 2, the volume ratio of the ZnO2-Cu solution to the hyaluronic acid solution is 1:0.5~2.

5. The preparation method of a nano-phage complex ZnO2-Cu@HA@Phage according to claim 1, characterized in that, In the said Step 3, the concentration of the activated ZnO2-Cu@HA is 0.05~0.5 mg / mL.

6. The preparation method of a nano-phage complex ZnO2-Cu@HA@Phage according to claim 1, characterized in that, In step 3, the phage concentration is 0.8~1.5×10 11 PFU / mL.

7. The preparation method of a nano-phage complex ZnO2-Cu@HA@Phage according to claim 1, characterized in that, In the said Step 3, the mixing and stirring time of ZnO2-Cu@HA and phage is 2~4 h.

8. The nanoparticle phage complex ZnO2-Cu@HA@Phage prepared by the preparation method according to any one of claims 1-7.

9. Use of the nanoparticle phage complex ZnO2-Cu@HA@Phage according to claim 8 in the preparation of a product for preventing and treating or assisting in preventing and treating bacterial enteritis.

10. The application according to claim 9, characterized in that, The said product is a drug.

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