Phage-nanocleavase hybrid nanorobots and methods for drug-resistant bacteria control

Abstract

The application discloses a bacteriophage-nanocatalyst hybrid nanorobot and method for drug-resistant bacteria control. The nanorobot comprises a lytic bacteriophage capable of specifically recognizing target drug-resistant bacteria, and a metal nanocatalyst with peroxidase-like catalytic activity; the metal nanocatalyst is covalently connected to the bacteriophage coat protein and fixed on the surface of the bacteriophage after being surface-modified by a polyethylene glycol connecting molecule containing an active group. The preparation method comprises the steps of bacteriophage separation and purification, nanocatalyst synthesis, introduction of an active group on the surface of the nanocatalyst, coupling with the bacteriophage and product purification. The nanorobot has the abilities of targeted adsorption and catalytic production of active oxygen, and can be used for killing drug-resistant bacteria, destroying biofilms and degrading antibiotic resistance genes in the environment, and is suitable for scenes such as sewage treatment and biofilm pollution control.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials and environmental engineering technology, specifically to a phage-nanozyme hybrid nanorobot, its preparation method and applications, which are particularly suitable for the control of drug-resistant bacterial biofilms and the removal of released antibiotic resistance genes. Background Technology

[0002] With the widespread use of antibiotics, drug-resistant bacteria and the antibiotic resistance genes they carry are constantly accumulating and spreading in the environment, becoming a significant problem in the fields of public health and ecological security. Existing antibacterial and anti-biofilm methods generally suffer from insufficient targeting, difficulty in removing biofilms, and residues of resistance genes in complex environments, making it difficult to achieve synergistic control of drug-resistant bacteria and their genetic pollution.

[0003] Currently, there is no technical solution that effectively combines the targeted recognition characteristics of bacteriophages with the catalytic antibacterial and gene degradation characteristics of nanozymes. Existing technologies cannot achieve synergistic control of drug-resistant bacteria, bacterial biofilms, and antibiotic resistance genes. Therefore, it is urgent to develop a novel antibacterial material that combines targeting, high-efficiency antibacterial properties, and resistance gene degradation capabilities to solve the technical challenges of drug-resistant bacterial contamination and resistance gene propagation in complex environments. Summary of the Invention

[0004] This invention addresses the shortcomings of existing antibacterial technologies, such as insufficient targeting and inability to synergistically eliminate drug-resistant bacteria-biofilm-resistance genes. It provides a method for preparing a structurally stable and easily prepared phage-nanozyme hybrid nanorobot and its applications in multiple fields. This method enables targeted killing of drug-resistant bacteria, efficient removal of bacterial biofilms, and degradation of antibiotic resistance genes, thereby reducing the risk of environmental transmission of drug resistance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a phage-nanozyme hybrid nanorobot for controlling drug-resistant bacteria. The nanorobot includes a lytic phage capable of recognizing and targeting drug-resistant bacteria and lysing them, and a Pd nanozyme with peroxidase activity capable of degrading antibiotic resistance genes. The Pd nanozyme is covalently linked to the capsid protein of the lytic phage through a polyethylene glycol linker containing active groups and immobilized on its surface to form a stable composite structure.

[0006] As a preferred embodiment of the first aspect above, the target drug-resistant bacteria are preferably multidrug-resistant bacteria. E. coli NDM-1.

[0007] As a preferred embodiment of the first aspect above, the polyethylene glycol linker containing active groups is mercapto polyethylene glycol active ester HS-PEG-NHS.

[0008] As a preferred embodiment of the first aspect above, the method for preparing the Pd nanozyme is as follows: One mole of K2PdCl4 aqueous solution was gradually added to a mixed aqueous solution containing 2-2.5 moles of polyvinylpyrrolidone, 1-1.2 moles of ascorbic acid and 7-8 moles of potassium bromide. The mixture was reacted at a constant temperature of 78-82 °C for 3-4 h. The precipitate was then separated, thoroughly washed to remove impurities, and dispersed in water to obtain Pd nanozyme in aqueous dispersion form.

[0009] As a preferred embodiment of the first aspect above, the nanoparticles of the Pd nanozyme have a particle size of 5~20 nm.

[0010] In a second aspect, the present invention provides a method for preparing a phage-nanozyme hybrid nanorobot as described in any of the first aspects above, comprising the following steps: S1. For the target drug-resistant bacteria that need to be controlled, select target bacteriophages from the bacteriophage library based on the screening criteria of being able to target and lyse the target drug-resistant bacteria. S2. Prepare Pd nanozymes with peroxidase activity and modify the surface of Pd nanozymes using polyethylene glycol linkers containing active groups. S3. The modified Pd nanozyme and the lysing phage were covalently coupled under low temperature conditions, and then separated and purified to obtain the phage-nanozyme hybrid nanorobot.

[0011] As a preferred embodiment of the second aspect above, the method for screening target phages from the phage library is as follows: Different phages from the phage library or municipal wastewater after filtration and sterilization rich in phages were used as phage sources and mixed with target drug-resistant bacteria in LB medium. The culture was then filtered and sterilized to obtain crude phage extract. The crude phage extract was then mixed with the target drug-resistant bacteria, and the phage capable of lysing the target drug-resistant bacteria to form lysates was screened using the double-layer agar plate method. The selected bacteriophages were amplified and purified in multiple rounds to obtain lytic bacteriophages that could infect and replicate with the target drug-resistant bacteria as host cells.

[0012] Thirdly, the present invention provides a phage-nanozyme hybrid nanorobot prepared according to the preparation method described in any of the second aspects above.

[0013] Fourthly, the present invention provides a method for controlling drug-resistant bacteria using phage-nanozyme hybrid nanorobots as described in the first or third aspect above. The phage-nanozyme hybrid nanorobots and hydrogen peroxide are added to an environmental system containing a target drug-resistant bacterial biofilm. Utilizing the specific recognition and adsorption capabilities of the lysing phage for the target drug-resistant bacteria, the Pd nanozyme is targeted and delivered to the surface of the target drug-resistant bacteria. The Pd nanozyme catalyzes the generation of reactive oxygen species from hydrogen peroxide, which, in conjunction with the lysis effect of the lysing phage, achieves targeted sterilization of the target drug-resistant bacteria, removes the biofilm of multidrug-resistant bacteria, and simultaneously degrades the antibiotic resistance genes released after the target drug-resistant bacteria lyse.

[0014] Fifthly, the present invention provides an application of the phage-nanozyme nanorobots as described in the first or third aspect above in the killing of drug-resistant bacteria, control of drug-resistant bacterial biofilms, or degradation of resistance genes.

[0015] As a preferred option for the fifth aspect mentioned above, one or more of the following applications may be adopted: 1. Targeted killing of drug-resistant bacteria: Utilizing lytic bacteriophages to target and kill drug-resistant bacteria. E. coli The specific recognition and adsorption capabilities of NDM-1 and other drug-resistant bacteria enable the targeted delivery of Pd nanozymes to the surface of drug-resistant bacteria. The Pd nanozymes catalyze the generation of reactive oxygen species, which, in conjunction with the lysis of bacteriophages, achieves efficient and targeted sterilization of drug-resistant bacteria, reducing the impact on non-target bacteria.

[0016] 2. Application in the control and removal of drug-resistant bacterial biofilms: Nanorobots can penetrate the biofilm matrix and target and adsorb drug-resistant bacteria within the membrane. Through lysis and oxidation by reactive oxygen species, they destroy the biofilm structure and kill bacteria within the membrane. At the same time, reactive oxygen species can degrade antibiotic resistance genes within the biofilm, achieving a synergistic effect of biofilm removal and resistance gene degradation.

[0017] 3. Application in the removal of antibiotic resistance genes in aquatic environments: By adding this nanorobot to domestic sewage, river water and other water bodies, it can target and remove drug-resistant bacteria in the water, while catalytically degrading free resistance genes in the water, reducing the risk of horizontal spread of resistance genes.

[0018] 4. Application in the preparation of antibacterial functional materials: Using this nanorobot as a functional component, it can be introduced into polymer materials and inorganic carriers through composite and other methods to prepare materials with targeted antibacterial and anti-biofilm functions. These materials can be applied to devices that are prone to biofilm formation, such as medical devices, sewage treatment membranes, and water pipelines, as well as environmental water purification materials.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The hybrid nanorobot of the present invention integrates the targeting ability of bacteriophages with the catalytic antibacterial and gene degradation functions of Pd nanozymes, realizing the synergistic control of drug-resistant bacteria-biofilm-resistance genes, overcoming the single-function defects of existing technologies, and for the first time achieving targeted bactericidal killing while degrading resistance genes, reducing the risk of drug resistance transmission from the source.

[0020] 2. The preparation method of the present invention is simple, the reaction conditions are mild (low temperature and normal pressure), no complex equipment is required, the product is easy to purify, and the reproducibility is good, making it suitable for large-scale preparation; the obtained nanorobots have a stable structure at room temperature, strong targeting, and significantly higher adsorption specificity for host drug-resistant bacteria than for non-host bacteria.

[0021] 3. The nanorobots of this invention exhibit high antibacterial efficiency. Through the synergistic effect of bacteriophage lysis and reactive oxygen species oxidation, they can reduce the number of drug-resistant bacteria below the detection limit. Simultaneously, the removal rate of bacterial biofilms can reach over 95%, and the degradation of resistant genes can achieve 2.78 log... 10 With the reduction of the copy number, the catalytic activity is less affected by the ambient pH, and the catalytic efficiency is higher in the acidic microenvironment of biofilms.

[0022] 4. The nanorobots of this invention have strong environmental adaptability and can maintain good targeting, antibacterial properties and gene degradation ability in complex water matrices such as simulated sewage and actual municipal sewage. At the same time, they can be used as functional components to prepare a variety of antibacterial functional materials, with a wide range of applications and important practical application value in the fields of environmental engineering, biomedicine, and antibacterial materials. Attached Figure Description

[0023] Figure 1 (A) image of the phage plaque and (B) TEM image of a randomly selected bacteriophage with a clear lysis plaque; Figure 2 The structure and performance characterization of Pd nanozymes: (A) XRD pattern of Pd nanozymes; (B) Transmission TEM image of Pd nanozymes; (C) HRTEM image of Pd nanozymes; (D) Histogram of particle size distribution of Pd nanozymes; (E) XPS Pd3d spectrum of Pd nanozymes; (F) ESR spectrum of hydroxyl radical generation by Pd+H2O2.

[0024] Figure 3 Assembly and characterization of N4@Pd nanorobots: (A) Coupling binding rate at different N4:Pd feed ratios; (B) Zeta potentials of phage N4, Pd nanozymes, and N4@Pd nanorobots; (C) TEM images of N4@Pd nanorobots; (D) N4@Pd nanorobots' interactions with... E. coli(E) Adsorption specificity of NDM-1; (F) Adsorption specificity of N4@Pd nanorobots for PAO1; (G) Relative enzyme activities of Pd nanozymes and N4@Pd nanorobots at different pH values.

[0025] Figure 4 The degradation effect of N4@Pd nanorobots on antibiotic resistance genes (ARG); (A) AFM images and (B) CD spectra of plasmid DNA before and after Pd+H2O2 treatment; (C) agarose gel electrophoresis images of plasmid ARG under different treatment conditions and (D) quantitative analysis results of copy number.

[0026] Figure 5 Characterization of the antibacterial activity and mechanism of N4@Pd nanorobots under different treatment conditions. E. coli NDM-1's (A) colony morphology, (B) antibacterial rate, (C) ROS level, (D) lipid peroxidation level, (E) protein leakage, and (F) ATP content.

[0027] Figure 6 For N4@Pd nanorobots E. coli The biofilm removal effect of NDM-1; under different treatment conditions E. coli (A) Crystal violet staining image of NDM-1 biofilm, (B) Biofilm biomass (OD595), (C) Polysaccharide content, (D) Protein content, (E) Quantitative analysis of ARG copy number, (F) AFM3D morphology image and (G) SEM image.

[0028] Figure 7 (A) Performance of N4@Pd nanorobots in wastewater environments; (B) Simulation of the effects of different treatment conditions on wastewater. E. coli (C) Antibacterial rate of NDM-1; (D) Biomass of biofilm (OD595) under different treatment conditions in simulated wastewater; (E) Quantitative analysis of the copy number of resistance genes under different treatment conditions in simulated wastewater. Detailed Implementation

[0029] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the present invention can be combined accordingly without mutual conflict.

[0030] It is particularly important to emphasize that the lytic phages screened in this invention are not limited to a single strain or category of phage. In fact, they only need to satisfy the function of recognizing and targeting the target drug-resistant bacteria and lysing them. Therefore, for each target drug-resistant bacterium requiring control, the screening criterion is the ability to target and lyse the target drug-resistant bacteria. Target phages meeting this criterion can be screened from natural phage libraries containing a large number of different types of phages, such as municipal wastewater and sludge, or from other artificially constructed phage libraries.

[0031] In the following examples, multidrug-resistant bacteria were used. E. coli NDM-1 is used as the target drug-resistant bacterium to be controlled, and municipal sewage rich in various types of bacteriophages is used as the phage source. However, it should be noted that when the drug-resistant bacterium to be controlled changes, this invention can also be applied to the control of other drug-resistant bacteria.

[0032] Example 1: Fabrication and Characterization of Nanorobots I. Isolation and purification of lysing bacteriophages 500 mL of municipal sewage sample was used as a phage library for screening target lytic phages. The sample was centrifuged at 8000 r / min for 10 min to remove solid impurities. The supernatant was filtered through a 0.22 μm filter membrane for sterilization. Then, 100 mL of the sterilized sewage and 10 mL of multidrug-resistant bacteria in the exponential growth phase were collected. E. coli Mix the NDM-1 bacterial suspension and add it to 200 mL of LB medium. Incubate at 37°C. o C. The culture was enriched by shaking at 150 rpm for 12 h; the culture was centrifuged at 8000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain crude phage extract.

[0033] Phages were screened using the double-layer agar plate method: 100 μL of crude phage extract was mixed with 100 μL of... E. coli NDM-1 bacterial suspension mixed, 37 o Incubate at 37°C for 15 min, then add to 5 mL of melted 0.7% LB soft agar, mix quickly, and pour onto a solid LB plate. o After culturing at C for 12 h, single spots with clear lysis patterns were picked from the plates (e.g., Figure 1 (A) was eluted with 1 mL of SM buffer to obtain a single-spot suspension of phage.

[0034] The phage single-spot suspension was amplified and purified through multiple rounds: the eluent and... E. coliNDM-1 bacterial culture was mixed and enriched, and after centrifugation and filtration, phage amplification solution was obtained. The amplification solution was then subjected to density gradient centrifugation and filtered through a 0.22 μm filter membrane to finally obtain a high-purity lysed phage suspension (for ease of description, this phage is referred to as N4), with a titer of 10. 12 PFU·mL -1 4 o C is stored for later use, and its form is as follows Figure 1 As shown in B in the diagram.

[0035] II. Synthesis of Pd nanozymes In this embodiment, 0.42 g of polyvinylpyrrolidone, 0.12 g of ascorbic acid, and 0.6 g of potassium bromide were dissolved sequentially in 32 mL of deionized water and stirred at room temperature until completely dissolved to form a homogeneous and transparent solution. The solution was then transferred to a three-necked flask and incubated at 80 °C. o While maintaining a constant temperature of C, slowly add 12 mL of 0.05 mol / L K₂PdCl₄ aqueous solution dropwise. After the addition is complete, continue stirring at 80°C. o The reaction was carried out at a constant temperature for 3 hours.

[0036] After the reaction was completed and cooled to room temperature, the reaction solution was centrifuged at 12000 r / min for 15 min. The precipitate was washed several times with acetone, and then precipitated with 80 mg·mL⁻¹. -1 The sample was washed twice with a mixture of NaCl aqueous solution and acetone (3:5, v / v) to remove residual polyvinylpyrrolidone, unreacted precursors, etc. Finally, the precipitate was dispersed in deionized water to obtain an aqueous dispersion of Pd nanozyme with a concentration of 1 mg·mL⁻¹. -1 Store at 4℃ for later use.

[0037] Performance characterization: Pd was characterized using XRD, TEM, XPS, etc. Figure 2 The results showed that the Pd nanozyme had a uniform structure and a particle size distribution of about 10 nm. The catalytic activity of the Pd nanozyme was detected by ESR, and the results showed that it could efficiently generate hydroxyl radicals (·OH) in the presence of H2O2. III. Fabrication of N4@Pd Nanorobots Preparation of modified Pd nanozymes: Take 1 mL of the Pd nanozyme aqueous dispersion prepared in this example (100 μg·mL⁻¹) -1 Add 100 μL of HS-PEG-NHS solution (1 mg·mL) -1 ), 0 o The modified Pd nanozyme solution was obtained by stirring the mixture at low temperature for 1 h.

[0038] Coupling: Take 1 mL of modified Pd nanozyme dispersion and add 1 mL of N4 phage suspension (1×10⁻⁶) prepared in this example. 10PFU·mL -1 The feed volume ratio of bacteriophage N4 to Pd nanozyme dispersion was 1:1. o The reaction was carried out at low temperature for 2 h with stirring to achieve amidation and covalent coupling, forming the crude product of N4@Pd nanorobots.

[0039] Purification: The crude product was first centrifuged at 5000 r / min for 5 min to remove agglomerates. The supernatant was then centrifuged at 10000 r / min for 20 min, and the supernatant was discarded to remove unbound Pd nanozymes and HS-PEG-NHS. Finally, the precipitate was dispersed in 100 μL of deionized water to obtain a high-concentration purified N4@Pd nanorobot aqueous dispersion. The final N4@Pd nanorobot dispersion contained 1×10⁻⁶ Nd nanorobots. 11 PFU·mL -1 Bacteriophage N4 and approximately 500 μg·mL -1 Pd nanozymes.

[0040] Morphological characterization: The morphology of Pd nanozymes and N4@Pd nanorobots was observed using transmission electron microscopy (TEM). Figure 3 The results showed that the phage had a typical head and tail structure, and the Pd nanozymes had a uniform cubic morphology with an average particle size of 10.4 ± 2.3 nm. In the N4@Pd nanorobot, the Pd nanozymes were uniformly immobilized on the surface of the phage capsid protein, and multiple Pd nanozymes were bound to the surface of each phage, resulting in a stable structure.

[0041] Performance characterization: Zeta potential detection showed ( Figure 3 The potentials of the phage (B) were -25.3 ± 2.7 mV, the Pd nanozyme potential was -8.4 ± 1.9 mV, and the N4@Pd nanorobot potential was -15.1 ± 2.1 mV, confirming successful coupling. Adsorption specificity experiments showed that the N4@Pd nanorobot exhibited strong adsorption characteristics to... E. coli NDM-1 ( Figure 3 The adsorption capacity of D in the sample is related to the adsorption capacity of Pseudomonas aeruginosa PAO1 ( Figure 3 More than 6 times that of E in ( Figure 3 (D in the text) has excellent targeted adsorption capabilities.

[0042] This demonstrates that the N4@Pd nanorobot was successfully fabricated in this embodiment. In this robot, the Pd nanozyme is covalently linked to the capsid protein of the lytic phage via a polyethylene glycol linker containing active groups, forming a stable composite structure. The lytic phage can recognize and target drug-resistant bacteria, lysing them. The Pd nanozyme possesses peroxidase activity and can synergistically and efficiently lyse the bacteria with the lytic phage. E. coliNDM-1 degrades and cleaves antibiotic resistance genes produced by its degradation. Further examples below demonstrate its application in controlling drug-resistant bacteria.

[0043] Example 2: Detection of resistance gene degradation This embodiment is used to verify the effect of the Pd nanozyme and N4@Pd nanorobot prepared in Example 1 on the degradation of resistance genes.

[0044] First, the resistance gene plasmid was combined with the Pd nanozyme prepared in Example 1 (final concentration 50 μg·mL). -1 ) and H2O2 (final concentration 1 mM) at 37 o After incubation at C for 2 h, the structural changes of the plasmid before and after treatment were characterized by AFM and CD spectroscopy. Figure 4 As shown in Figure A, untreated plasmid DNA exhibits a continuous, smooth, fibrous chain structure, typical of intact double-stranded DNA. However, when Pd nanozyme and H2O2 act together, the DNA chains undergo numerous breaks and shortenings, losing their long, continuous structure and network connectivity, resulting in a large number of short fragments. CD spectral analysis indicates that ( Figure 4 (B) The characteristic absorption peak intensities of the treated plasmid DNA at 250 nm and 278 nm changed significantly and were accompanied by slight shifts, indicating that its secondary structure was disrupted.

[0045] Meanwhile, multiple control and experimental groups were set up to investigate drug-resistant bacteria. E. coli NDM-1 resistance gene plasmid solution (concentration 50 ng·μL) -1 Different treatment agents were added to the samples for gel electrophoresis and qPCR quantification. The specific treatment agents added to each group are as follows: Control group (PBS only), H2O2 group (1 mM H2O2 only), Pd+ H2O2 group (50 μg·mL) -1 Pd nanozyme + 1 mM H2O2), N4 group (only 1×10 10 PFU·mL -1 Lysogenic bacteriophage N4), N4 + Pd + H2O2 group (adding 1×10 10 PFU·mL -1 Lysogenic bacteriophage N4 + 50 μg·mL -1 Pd nanozyme + 1 mM H2O2 (the three components were added independently, with N4 and Pd nanozyme not pre-coupled), N4@Pd + H2O2 group (N4@Pd nanorobot aqueous dispersion prepared in Example 1 was added + 1 mM H2O2, wherein the amount of N4@Pd nanorobot aqueous dispersion added needs to be controlled to contain 1×10 in the final system). 10PFU·mL -1 Bacteriophage N4 and 50 μg·mL -1 Pd nanozymes), 6 replicates per group, incubated at 37℃ for 6 h. The concentrations of the components described in each group are the final concentrations in the final system. Agarose gel electrophoresis results confirmed that... Figure 4 As shown in C, complete plasmid DNA bands appeared in the control group, the H2O2-only treatment group, and the N4-only treatment group; however, the plasmid bands in the Pd + H2O2 group, the N4 + Pd + H2O2 group, and the N4@Pd + H2O2 group disappeared significantly. qPCR quantitative detection results showed ( Figure 4 In the D), compared with the control group, the N4@Pd+ H2O2 group bla NDM-1 The copy number of resistance genes decreased by approximately 3.2 log. 10 This achieves highly efficient inactivation of resistance genes. The above results directly demonstrate that the N4@Pd nanorobot of this invention can catalytically degrade resistance genes carried by plasmids, effectively disrupting the structural and functional integrity of drug resistance.

[0046] Example 3: Detection of antibacterial activity of N4@Pd nanorobots Escherichia coli NDM-1 was cultured to the exponential growth phase, centrifuged at 8000 rpm for 5 min, resuspended in PBS buffer, and diluted to 1×10⁻⁶. 8 CFU·mL -1 The subjects were divided into 6 groups, each receiving a different treatment agent. The specific treatment agents added to each group are as follows: control group (PBS only), H2O2 group (1 mM H2O2 only), Pd+ H2O2 group (50 ng / mL). -1 Pd nanozyme + 1mM H2O2), N4 group (only 1×10 7 PFU·mL -1 Lysogenic bacteriophage N4), N4 + Pd + H2O2 group (adding 1×10 7 PFU·mL -1 Lysogenic bacteriophage N4 + 50 ng·mL -1 Pd nanozyme + 1 mM H2O2 (the three components were added independently, with N4 and Pd nanozyme not pre-coupled), N4@Pd + H2O2 group (N4@Pd nanorobot aqueous dispersion prepared in Example 1 + 1 mM H2O2 was added, wherein the amount of N4@Pd nanorobot aqueous dispersion added needs to be controlled to contain 1×10 in the final system). 10 PFU·mL -1 Bacteriophage N4 and 50 μg·mL -1Pd nanozymes), three replicates per group, incubated at 37 °C for 6 h, and the component concentrations described in each group are the final concentrations in the final system.

[0047] After incubation, 100 μL of bacterial culture from each group was serially diluted and plated on LB agar plates. Figure 5 (A) After incubation at 37℃ for 12 h, colony counts were performed, and the antibacterial rate was calculated. Figure 5 (B in the text); simultaneously, the ROS level of each group of bacterial cultures was measured ( Figure 5 C), lipid peroxidation level (5D in the figure), protein leakage ( Figure 5 E in the content and ATP content ( Figure 5 The results showed that the antibacterial rate of the N4@Pd + H2O2 group reached 100%, and the bacterial count was reduced to the detection limit (10 CFU·mL). -1 The levels of N4@Pd nanorobots were significantly higher than those of other groups. The ROS level, lipid peroxidation level, and protein leakage of this group were the highest, while the ATP content was the lowest. This indicates that after targeted adsorption, N4@Pd nanorobots utilize the synergistic effect of phage lysis and reactive oxygen species oxidation to destroy bacterial cell membranes and induce metabolic disorders, thereby achieving highly efficient sterilization.

[0048] Example 4: Biofilm clearance by N4@Pd nanorobots Preparation of biomembranes: E. coli NDM-1 bacterial culture diluted to 1×10 6 CFU·mL -1 Add 1 mL to each well of a 24-well plate and incubate at 37°C for 24 h to form a mature biofilm. Discard the culture medium in the wells and gently rinse twice with PBS buffer to remove any unattached planktonic bacteria.

[0049] Biofilm treatment: The 24-well plates with biofilms were divided into 6 groups, and different treatment agents were added to each group. The specific treatment agents added to each group are as follows: control group (PBS only), H2O2 group (1 mM H2O2 only), Pd+ H2O2 group (50 μg·mL⁻¹) -1 Pd nanozyme + 1 mM H2O2), N4 group (only 1×10 10 PFU·mL -1 Lysogenic bacteriophage N4), N4 + Pd + H2O2 group (adding 1×10 10 PFU·mL -1 Lysogenic bacteriophage N4 + 50 μg·mL -1Pd nanozyme + 1 mM H2O2 (the three components were added independently, with N4 and Pd nanozyme not pre-coupled), N4@Pd + H2O2 group (N4@Pd nanorobot aqueous dispersion prepared in Example 1 was added + 1 mM H2O2, wherein the amount of N4@Pd nanorobot aqueous dispersion added needs to be controlled to contain 1×10 in the final system). 10 PFU·mL -1 Bacteriophage N4 and 50 μg·mL -1 Pd nanozymes), 6 replicates per group, incubated at 37°C for 12 h, and the component concentrations described in each group are the final concentrations in the final system.

[0050] Biofilm clearance assay: Biofilm biomass was determined using crystal violet staining. Figure 6 (A) Polysaccharides and proteins were extracted from the biofilm and quantified. SEM and AFM were used to observe changes in biofilm morphology. Results showed that the biofilm biomass in the N4@Pd + H2O2 group was reduced by more than 95% compared to the control group. Figure 6 B in the polysaccharide ( Figure 6 C in the middle) and protein ( Figure 6 The content of D in the sample decreased significantly.

[0051] Resistance gene degradation detection: Total DNA was extracted from biofilms of each group and quantitatively detected by qPCR. bla NDM-1 Gene copy number. (e.g.) Figure 6 As shown in E, the N4@Pd+H2O2 group bla NDM-1 The gene copy number was reduced by 2.8 log compared to the control group. 10 This indicates that N4@Pd, agarose gel electrophoresis and qPCR were used to detect the integrity of the plasmid and the change in the copy number of the resistance gene, respectively.

[0052] Additionally, SEM ( Figure 6 F) and AFM ( Figure 6 The observations in G also showed that the biofilm structure of this group was completely destroyed, with only a few fragments remaining and no complete biofilm structure.

[0053] Example 5: Application and detection of N4@Pd nanorobots in complex water quality Simulated wastewater samples were taken and filtered through a 0.22 μm filter membrane to remove suspended solids. The samples were divided into 6 groups, each containing 50 mL of the solution. E. coli NDM-1 bacterial solution was used to achieve a concentration of 1×10⁻⁶. 7 CFU·mL -1Following the procedure for the six different treatment agents described in Example 3, the same type and concentration of treatment agent as described in Example 3 were added to the six groups of filtered simulated wastewater samples. The samples were stirred at room temperature for 6 hours, serially diluted, and spread on LB plates. After overnight incubation, the surviving colonies were counted. The results showed that the N4@Pd nanorobot achieved a drug-resistant bacteria removal rate of over 99%. Figure 7 (A) Similarly, after a mature biofilm formed in the wastewater sample, the treatment agent and concentration from Example 3 were added, and the biofilm biomass was determined by crystal violet staining. Simultaneously, qPCR was used for quantitative analysis of antagonistic genes. The results showed that N4@Pd nanorobot treatment achieved a biofilm removal efficiency of ~90% ( Figure 7 (B in the middle) bla NDM-1 Gene copy number decreased by 2.06 log. 10 ( Figure 7 (C in the text). The results show that the nanorobot can still maintain good targeting and antibacterial properties in complex aquatic environments, making it suitable for practical wastewater systems.

[0054] The embodiments described above are merely some preferred implementations of the present invention and are not intended to limit the invention. Without departing from the inventive concept, those skilled in the art can make various equivalent substitutions or modifications to the composition, structure, and preparation conditions of the phage-nanozyme hybrid nanorobots described in this invention. For example, the phage can be replaced with other lytic phages capable of specifically recognizing target bacteria; the metal nanozyme can be prepared using different methods or morphological structures while maintaining peroxidase-like catalytic activity; the linker can be polymer linking molecules with different molecular weights or functional groups; the reaction sequence, reaction conditions, and separation methods during the preparation process can be adjusted within a reasonable range. All the above equivalent substitutions or modifications should not be considered as departing from the protection scope of this invention and should fall within the scope defined by the claims of this invention.

Claims

1. A phage-nanozyme hybrid nanorobot for controlling drug-resistant bacteria, characterized in that, The nanorobots include lysing phages capable of recognizing and targeting drug-resistant bacteria to lyse them, and Pd nanozymes with peroxidase activity capable of degrading antibiotic resistance genes; the Pd nanozymes are covalently linked to the capsid protein of the lysing phages through a polyethylene glycol linker containing active groups and immobilized on its surface to form a stable composite structure.

2. The phage-nanozyme hybrid nanorobot for controlling drug-resistant bacteria as described in claim 1, characterized in that, The target drug-resistant bacteria are preferably multidrug-resistant bacteria. E. coli NDM-1.

3. The phage-nanozyme hybrid nanorobot for controlling drug-resistant bacteria as described in claim 1, characterized in that, The polyethylene glycol linker containing active groups is thiol-based polyethylene glycol active ester HS-PEG-NHS.

4. The phage-nanozyme hybrid nanorobot for controlling drug-resistant bacteria as described in claim 1, characterized in that, The preparation method of the Pd nanozyme is as follows: One mole of K2PdCl4 aqueous solution was gradually added to a mixed aqueous solution containing 2-2.5 moles of polyvinylpyrrolidone, 1-1.2 moles of ascorbic acid and 7-8 moles of potassium bromide. The mixture was reacted at a constant temperature of 78-82 °C for 3-4 h. The precipitate was then separated, thoroughly washed to remove impurities, and dispersed in water to obtain Pd nanozyme in aqueous dispersion form.

5. The phage-nanozyme hybrid nanorobot for controlling drug-resistant bacteria as described in claim 1, characterized in that, The Pd nanozyme has a nanoparticle size of 5~20 nm.

6. A method for preparing a phage-nanozyme hybrid nanorobot as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. For the target drug-resistant bacteria that need to be controlled, select target bacteriophages from the bacteriophage library based on the screening criteria of being able to target and lyse the target drug-resistant bacteria. S2. Prepare Pd nanozymes with peroxidase activity and modify the surface of Pd nanozymes using polyethylene glycol linkers containing active groups. S3. The modified Pd nanozyme and the lysing phage were covalently coupled under low temperature conditions, and then separated and purified to obtain the phage-nanozyme hybrid nanorobot.

7. The preparation method according to claim 6, characterized in that, The method for screening target bacteriophages from the bacteriophage library is as follows: Different phages from the phage library or municipal wastewater after filtration and sterilization rich in phages were used as phage sources and mixed with target drug-resistant bacteria in LB medium. The culture was then filtered and sterilized to obtain crude phage extract. The crude phage extract was then mixed with the target drug-resistant bacteria, and the phage capable of lysing the target drug-resistant bacteria to form lysates was screened using the double-layer agar plate method. The selected bacteriophages were amplified and purified in multiple rounds to obtain lytic bacteriophages that could infect and replicate with the target drug-resistant bacteria as host cells.

8. A phage-nanozyme hybrid nanorobot prepared according to the preparation method described in claim 6 or 7.

9. A method for controlling drug-resistant bacteria using phage-nanozyme hybrid nanorobots as described in claim 1 or 8, characterized in that, The phage-nanozyme hybrid nanorobot and hydrogen peroxide were added to an environment containing a target drug-resistant bacterial biofilm. Utilizing the specific recognition and adsorption capabilities of the lysing phage for the target drug-resistant bacteria, the Pd nanozyme was targeted and delivered to the surface of the target drug-resistant bacteria. The Pd nanozyme catalyzed the generation of reactive oxygen species from hydrogen peroxide, which, in synergy with the lysis effect of the lysing phage, achieved targeted sterilization of the target drug-resistant bacteria, removed the multidrug-resistant bacterial biofilm, and simultaneously degraded the antibiotic resistance genes released after the target drug-resistant bacteria were lysed.

10. The application of the phage-nanozyme nanorobot as described in claim 1 or 8 in the killing of drug-resistant bacteria, control of drug-resistant bacterial biofilms, or degradation of resistance genes.

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