A kind of mCuS@lm antibacterial nano-cage and its preparation method and application

Abstract

The present invention discloses an mCuS@lm antibacterial nanocage, its preparation method and application. The mCuS@lm antibacterial nanocage takes hollow mesoporous copper sulfide nanoparticles as the core, and through electrostatic adsorption self-assembly, a bacterial cuproptosis amplifier and a cellular cuproptosis inhibitor are encapsulated in the hollow mesoporous copper sulfide nanoparticles, and further, a biomimetic cell membrane is coated on the surface of the hollow mesoporous copper sulfide nanoparticles to form a nanocage, which is the mCuS@lm antibacterial nanocage. The biomimetic membrane on the surface of the mCuS@lm antibacterial nanocage of the present invention highly expresses proteins related to microbial recognition and uptake, and can effectively neutralize inflammatory factors in the environment, having a potential infection targeting effect. By strengthening bacterial cuproptosis with a bacterial cuproptosis amplifier and slowing down cellular cuproptosis with a cellular cuproptosis inhibitor, the killing of bacteria is improved and the antibacterial effect is enhanced while improving biological safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an mCuS@lm antibacterial nanocage, a preparation method thereof and an application thereof. Background Art

[0002] Staphylococcus aureus is a common pathogen in nosocomial and community infections, and can cause infections in multiple parts such as skin, soft tissues, lungs, bones and blood. However, the abuse of antibiotics, the evolution of bacterial drug resistance and the increase in virulence have increased the threat of pathogens. For example, methicillin-resistant Staphylococcus aureus (MRSA). At present, antibiotics such as vancomycin and linezolid are the main treatment methods for MRSA pneumonia. However, with their extensive application, vancomycin-resistant Staphylococcus aureus has emerged clinically. We are in urgent need of exploring new targets and finding new non-antibiotic treatment strategies for MRSA pneumonia.

[0003] Copper can participate in a variety of physiological reactions as a trace element. However, the increase in intracellular copper concentration caused by its homeostasis imbalance can also lead to cell damage and copper death by affecting mitochondrial metabolism. At present, copper death is mainly found in eukaryotic cells. However, copper also has natural antibacterial effects, and some mechanisms of copper death also occur in bacteria. At present, there is still little research on the development of nanomaterials based on copper death. In addition, in the actual application process, traditional pulmonary inflammatory diseases are restricted by insufficient drug concentration at the target site, difficulty in reaching the inside of bacteria by nanomaterials and the pulmonary biological barrier. At the same time, due to the lack of targeting, it is difficult for nanomaterials to quickly identify bacteria and efficiently aggregate around bacteria. This makes the treatment efficiency of traditional nanomaterials in pneumonia relatively low. When exogenous nanoparticles are unmodified, they are easily recognized and phagocytosed by macrophages of the reticuloendothelial system, lack long-circulation effects, and are difficult to reach the target site. Moreover, the reactive oxygen species generated by high-dose copper-based materials will also cause huge oxidative stress on normal tissue cells. Especially in lung tissue, excessive ROS will exceed the processing capacity of the intracellular antioxidant system, causing oxidation of proteins, lipids, DNA and carbohydrates, resulting in cell death and lung dysfunction.

[0004] Therefore, how to endow nanomaterials with bacterial targeting, and while inducing and amplifying cuproptosis in bacteria and disrupting the bacterial redox balance, weaken the oxidative stress damage to normal tissues is the current research focus. Mitoquinone mesylate (mitoQ), a mitochondria-targeted antioxidant, can activate the Nrf2 pathway, inhibit lipid peroxidation, and protect mitochondrial matrix proteins and mitochondrial DNA. The energy generated during mitochondrial respiration is generally stored as an electrochemical potential energy in the inner mitochondrial membrane. Therefore, there is an asymmetric distribution of proton and other ion concentrations on both sides of the inner mitochondrial membrane, with a positive charge in the intermembrane space and a negative charge in the matrix. Due to the existence of this potential difference, lipophilic triphenylphosphine cations can be guided to accumulate in mitochondria. MitoQ plays a mitochondria-targeted antioxidant role in eukaryotic cells, maintaining normal redox balance, and the effect is far better than that of non-targeted antioxidants. However, due to the lack of mitochondria, the respiration of bacterial cells only relies on an electron transport chain structure similar to the cell membrane, and its targeting aggregation effect is far less than that of eukaryotic cells. Quorum sensing (QS) is a communication system for bacteria to communicate and transmit information with each other. Through "cell-cell" communication, they can make collective decisions, synchronize with other populations, adapt to environmental changes, develop antibiotic resistance, form biofilms, and produce virulence factors. Bacteria with QS defects are more vulnerable to oxidative stress damage. Therefore, inducing and enhancing the oxidative stress response by inhibiting the QS system may amplify the cuproptosis effect. Luteolin from natural sources is a flavonoid compound and is considered a quorum sensing inhibitor (QSIs). It has high biosafety and can synergistically play anti-inflammatory and antibacterial roles with mitoQ and copper-based materials.

[0005] In recent years, there have been more and more studies on biomimetic nanoparticles coated with different cell membranes. The camouflage of cell membranes from natural biological sources (red blood cells, platelets, tumor cells, stem cells, immune cells, bacteria, etc.) endows nanoparticles with powerful immune escape ability and targeted delivery ability. Currently, many studies have found that nanoparticles coated with macrophage cell membranes have certain targeting abilities for inflammation and the tumor microenvironment, while membranes pretreated with bacteria have stronger bacterial adhesion efficacy, which can prolong the residence time of drugs at the infection site. Alveolar macrophages are resident on the surface of the lung mucosa, have the functions of self-renewal and maintenance, and play an important role in the innate immune response. After being trained by MRSA infection, Toll-like receptors, TNF receptors, scavenger receptor MSR, CD14, mannose receptor CD206 and other proteins mediating microbial uptake and recognition are highly expressed on the alveolar macrophage cell membrane, making it have a strong targeting effect in MRSA pneumonia; in addition, it can act as a cytokine decoy, bind pro-inflammatory factors through its homologous cytokine receptors, and decouple the signal transduction and transcriptional activation of the macrophage inflammatory cascade. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a novel preparation method and application of mCuS@lm antibacterial nanocages. Luteolin (QSIs) derived from natural plants and mitochondrial-targeted antioxidant mitoQ are encapsulated within hollow mesoporous copper sulfide nanoparticles to synthesize a novel antibacterial nanocage material, mCuS@lm. To improve the drug loading efficiency and shield the in vivo immune system response, we coat the outer surface with alveolar macrophage membranes trained by sub-lethal dose MRSA infection, which can achieve the targeting of the mCuS@lm material to MRSA pneumonia in vivo, prolong the residence time of the nanoparticles at the lesion site, and regulate the host lung immune response. On the other hand, cuproptosis is induced by copper overload in bacteria, and the release of copper ions can generate hydroxyl radicals through the Fenton-like reaction in the infected environment, synergistically interfering with the QS signals and metabolic pathways of bacteria to kill bacteria. At the same time, the antioxidant targeting mitochondria protects the mitochondrial function of normal lung cells to maintain redox homeostasis, thereby differentially regulating the cuproptosis state of bacteria and cells to exert antibacterial and anti-inflammatory effects.

[0007] The present invention first provides an mCuS@lm antibacterial nanocage, which takes hollow mesoporous copper sulfide nanoparticles as the core, and encapsulates a bacteria / fungus cuproptosis amplifier and a cell cuproptosis inhibitor in the hollow mesoporous copper sulfide nanoparticles through electrostatic adsorption self-assembly, and further coats a biomimetic cell membrane on the surface of the hollow mesoporous copper sulfide nanoparticles to form a nanocage, which is the mCuS@lm antibacterial nanocage.

[0008] The bacteria / fungus cuproptosis amplifier refers to the added bacteria cuproptosis amplifier or fungus cuproptosis amplifier. If the prepared mCuS@lm antibacterial nanocage is used for antibacterial, the corresponding bacteria cuproptosis amplifier is added. Correspondingly, if the prepared mCuS@lm antibacterial nanocage is used for antifungal, the corresponding fungus cuproptosis amplifier is added.

[0009] Preferably, the hollow mesoporous copper sulfide nanoparticles are prepared by an ion exchange method. Specifically, copper chloride is dispersed in isopropanol, then polyvinylpyrrolidone is added and dissolved, sodium hydroxide solution is added, hydrazine hydrate is continued to be added, and finally ammonium sulfide solution is added for reaction. After the product is centrifuged, separated, washed, and purified, it is freeze-dried to obtain the hollow mesoporous copper sulfide nanoparticles. The preparation method of the hollow mesoporous copper sulfide nanoparticles is a prior art, so the specific proportions of various substances and parameter conditions can be adjusted and set according to the prior art.

[0010] Preferably, the bacterial / fungal cuproptosis amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is at least one of the following: luteolin, coumarin, curcumin. The bacterial / fungal cuproptosis amplifier can assist in killing bacteria, act synergistically with the process of copper overload-induced cuproptosis in bacteria, and promote the occurrence of bacterial cuproptosis.

[0011] The cell cuproptosis inhibitor is an antioxidant, and the antioxidant is at least one of the following: mitoQ, SKQ1, Mito-TEMPO. The cell cuproptosis inhibitor can inhibit the cuproptosis of cells, reduce the impact on host cells under the condition of copper overload, and reduce the occurrence of host cell cuproptosis.

[0012] The biomimetic cell membrane is extracted from the cell membrane after using at least one of bacteria or fungi as the training substance and immune cells as the substance to be trained for infection training. The trained cells obtain highly expressed specific receptors related to microbial recognition and uptake on their cell membrane surface, and can effectively neutralize inflammatory factors in the environment, having potential infection targeting effects.

[0013] More preferably, the bacteria used as the training substance are selected from at least one of the following: Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Escherichia coli;

[0014] The fungi used as the training substance are selected from at least one of the following: Candida albicans, Saccharomyces cerevisiae, Cryptococcus;

[0015] The immune cells are at least one of the following: macrophages, neutrophils, dendritic cells, lymphocytes, stromal cells, epithelial cells;

[0016] The multiplicity of infection during training is 5:1 to 50:1, and the training time is 6 to 48 h. Further preferably, the multiplicity of infection during training is 10:1 to 20:1, and the training time is 12 to 24 h.

[0017] The selection of the training substance is adapted to the final use of the prepared mCuS@lm antibacterial nanocage. For example, if it is finally used to prepare anti-MRSA pneumonia drugs, Staphylococcus aureus is used as the training substance. And the immune cells used to extract the cell membrane as the biomimetic membrane can contact the training substance during the training process, so as to highly express specific receptors related to the recognition and uptake of the training substance on the cell membrane surface.

[0018] Preferably, the mass ratio of the bacterial / fungal cuproptosis amplifier to the hollow mesoporous copper sulfide nanoparticles is 80:31.25 to 1000. More preferably, the mass ratio of the bacterial / fungal cuproptosis amplifier to the hollow mesoporous copper sulfide nanoparticles is 80:31.25 to 62.5.

[0019] Preferably, the mass ratio of the ferroptosis inhibitor to the hollow mesoporous copper sulfide nanoparticles is 0.125 to 1:31.25. More preferably, the mass ratio of the ferroptosis inhibitor to the hollow mesoporous copper sulfide nanoparticles is 0.25 to 1:31.25.

[0020] Preferably, the mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:0.5 to 3. More preferably, the mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:2 to 3. Most preferably, the mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:2.

[0021] The present invention also provides a method for preparing the mCuS@lm antibacterial nanocage, comprising the following steps:

[0022] (1) Prepare hollow mesoporous copper sulfide nanoparticles;

[0023] (2) Entrap the bacterial / fungal ferroptosis amplifier and the ferroptosis inhibitor in the hollow mesoporous copper sulfide nanoparticles by electrostatic adsorption self-assembly;

[0024] (3) Coat the biomimetic cell membrane on the surface of the hollow mesoporous copper sulfide nanoparticles obtained in step (2) which have entrapped the bacterial / fungal ferroptosis amplifier and the ferroptosis inhibitor to form a nanocage, namely the mCuS@lm antibacterial nanocage.

[0025] Preferably, the hollow mesoporous copper sulfide nanoparticles are prepared by an ion exchange method;

[0026] The bacterial / fungal ferroptosis amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is at least one of the following: luteolin, coumarin, curcumin;

[0027] The ferroptosis inhibitor is an antioxidant, and the antioxidant is at least one of the following: mitoQ, SKQ1, Mito-TEMPO;

[0028] The biomimetic cell membrane is a cell membrane extracted after infection training using at least one of bacteria or fungi as the training substance and immune cells as the substance to be trained.

[0029] More preferably, the bacteria used as the training substance are selected from at least one of the following: Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Escherichia coli;

[0030] The fungi used as the training substance are selected from at least one of the following: Candida albicans, Saccharomyces cerevisiae, Cryptococcus;

[0031] The immune cells are at least one of the following: macrophages, neutrophils, dendritic cells, lymphocytes, stromal cells, epithelial cells;

[0032] The multiplicity of infection during training is 5:1 to 50:1, and the training time is 6 to 48 h. More preferably, the multiplicity of infection during training is 10:1 to 20:1, and the training time is 12 to 24 h.

[0033] More preferably, the mass ratio of the bacterial / fungal cuproptosis amplifier to the hollow mesoporous copper sulfide nanoparticles is 80:31.25 to 1000. More preferably, the mass ratio of the bacterial / fungal cuproptosis amplifier to the hollow mesoporous copper sulfide nanoparticles is 80:31.25 to 62.5.

[0034] The mass ratio of the cellular cuproptosis inhibitor to the hollow mesoporous copper sulfide nanoparticles is 0.125 to 1:31.25. More preferably, the mass ratio of the cellular cuproptosis inhibitor to the hollow mesoporous copper sulfide nanoparticles is 0.25 to 1:31.25.

[0035] The mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:0.5 to 3. More preferably, the mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:2 to 3. Most preferably, the mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:2.

[0036] The present invention also provides the application of the mCuS@lm antibacterial nanocage in the preparation of antibacterial and / or anti-inflammatory materials.

[0037] The present invention also provides an antibacterial and / or anti-inflammatory material comprising the mCuS@lm antibacterial nanocage.

[0038] The antibacterial target is against bacteria or fungi. The bacteria are Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae or Escherichia coli. The fungi are Candida albicans, Saccharomyces cerevisiae or Cryptococcus. The selection of the antibacterial target corresponds to the training substance.

[0039] The present invention designs an mCuS@lm antibacterial nanocage, which takes hollow mesoporous copper sulfide nanoparticles as the core, and encapsulates bacterial / fungal cuproptosis amplifiers and cellular cuproptosis inhibitors in the hollow mesoporous copper sulfide nanoparticles through electrostatic adsorption self-assembly, and further coats a biomimetic cell membrane on the surface of the hollow mesoporous copper sulfide nanoparticles to form a nanocage, which is the mCuS@lm antibacterial nanocage. The biomimetic membrane on the surface of the mCuS@lm antibacterial nanocage of the present invention highly expresses proteins related to microbial recognition and uptake, and can effectively neutralize inflammatory factors in the environment, having potential infection targeting effects. Bacterial cuproptosis is enhanced by bacterial / fungal cuproptosis amplifiers, and host cell cuproptosis is slowed down by cellular cuproptosis inhibitors, thereby improving bacterial killing and antibacterial effects while enhancing biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a detection result diagram of the training effect under different training conditions. Among them, Figure 1 a in: Detection of cell viability under different training conditions, Figure 1 b in: Expression of key proteins on the membrane surface under different training conditions.

[0041] Figure 2 It is a partial characterization result diagram of the mCuS@lm nanocage. Among them, Figure 2 a in: TEM structure and elemental analysis of hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles; Figure 2 b in: Expression of key proteins on the membrane surface before and after training; Figure 2 c in: Coomassie brilliant blue staining results of nanoparticles before and after training.

[0042] Figure 3 It is another partial characterization result diagram of the mCuS@lm nanocage. Among them, Figure 3 a in: Hydrodynamic particle size of hollow mesoporous copper sulfide nanoparticles and nanocages coated with biomimetic membranes; Figure 3 b in: Zeta potential of luteolin, mitoQ, hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles; Figure 3 c in: Particle size stability of mCuS@lm nanoparticles in aqueous solution. Figure 3 d in: Spectra of luteolin, mitoQ, hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles at 300 nm to 1000 nm. Figure 3 e in: Ability of nanoparticles to neutralize IL-1b before and after training. Figure 3 f in: Ability of nanoparticles to neutralize TNF-a before and after training. Figure 3 g in: Schematic diagram of nanoparticles neutralizing cytokine storm.

[0043] Figure 4 Detection result graph for the targeting test of mCuS@lm nano-cages. Among them, Figure 4 a in [[ ]]: The ability of nano-cages to target infected cells before and after training; Figure 4 b in [[ ]]: The ability of nano-cages to target bacteria before and after training; Figure 4 c in [[ ]]: Schematic electron microscopy image of nano-cages adhering to bacteria before and after training; Figure 4 d in [[ ]]: In vivo fluorescence imaging results of nano-cages targeting pneumonia mice before and after training; Figure 4 e in [[ ]]: Ex vivo fluorescence imaging results of nano-cages targeting pneumonia mice before and after training.

[0044] Figure 5 Detection result graph for the antibacterial ability of mCuS@lm nano-cages. Among them, Figure 5 a in [[ ]]: Agar plates of nano-cages in different groups killing MRSA bacteria at different time points; Figure 5 b in [[ ]]: Agar plates of nano-cages in different groups killing Pseudomonas aeruginosa; Figure 5 c in [[ ]]: Agar plates of nano-cages in different groups killing ciprofloxacin-resistant Pseudomonas aeruginosa; Figure 5 d in [[ ]]: Live / dead staining of nano-cages in different groups killing bacteria; Figure 5 e in [[ ]]: Proportion of dead bacteria after treatment with nano-cages in different groups.

[0045] Figure 6 Detection result graph for the antibacterial morphology and anti-biofilm ability of mCuS@lm nano-cages. Among them, Figure 6 a in [[ ]]: TEM images of nano-cages in different groups killing MRSA; Figure 6 b in [[ ]]: SEM images of nano-cages in different groups killing MRSA and Pseudomonas aeruginosa; Figure 6 c in [[ ]]: Live / dead staining of nano-cages in different groups removing MRSA biofilm; Figure 6 d in [[ ]]: Crystal violet staining of nano-cages in different groups removing MRSA biofilm.

[0046] Figure 7 Result graph for the exploration of the differential cuproptosis resistance ability of mCuS@lm nano-cages. Among them, Figure 7 a in [[ ]]: Redox state of macrophages killed and treated by nano-cages in different groups; Figure 7 b in [[ ]]: Lipid peroxidation level of macrophages killed and treated by nano-cages in different groups; Figure 7 c in [[ ]]: Copper ion concentration of macrophages killed and treated by nano-cages in different groups; Figure 7 d in [[ ]]: Redox state of bacteria killed and treated by nano-cages in different groups; Figure 7 e in [[ ]]: ROS level of macrophages killed and treated by nano-cages in different groups.

[0047] Figure 8 This is the graph of the in vivo effect detection results of mCuS@lm nano-cages. Among them, Figure 8 a in it: The change in the core body temperature of mice after applying nano-cages of different groups; Figure 8 b in it: The anatomical state of the lungs of mice after applying nano-cages of different groups; Figure 8 c in it: The wet weight / dry weight mass ratio of the lungs of mice after applying nano-cages of different groups; Figure 8 d in it: The cell count in the bronchoalveolar lavage fluid of mice after applying nano-cages of different groups; Figure 8 e in it: The protein concentration count in the bronchoalveolar lavage fluid of mice after applying nano-cages of different groups; Figure 8 f in it: The lung injury score of mice after applying nano-cages of different groups; Figure 8 g in it: The HE staining results of the lung sections of mice after applying nano-cages of different groups.

[0048] Figure 9 This is the graph of the in vivo effect detection results of mCuS@lm nano-cages. Among them, Figure 9 a in it: The pictures of bacterial plating on the lungs of mice after applying nano-cages of different groups; Figure 9 b in it: The bacterial plating count on the lungs of mice after applying nano-cages of different groups; Figure 9 c in it: The MPO immunohistochemical pictures of the lungs of mice after applying nano-cages of different groups.

[0049] Figure 10 This is the graph of the biosafety evaluation detection results of mCuS@lm nano-cages. Among them, Figure 10 a in it: The live / dead staining of cells after applying nano-cages of different groups; Figure 10 b in it: The viability of cells after applying nano-cages of different groups; Figure 10 c in it: The blood biochemical indexes of mice after applying nano-cages of different groups; Figure 10 d in it: The blood routine indexes of mice after applying nano-cages of different groups; Figure 10 e in it: The HE staining results of important organs of mice after applying nano-cages of different groups. Specific implementation manners

[0050] Unexpectedly, the present invention developed hollow mesoporous copper sulfide nanoparticles loaded with plant-derived luteolin and mitochondrial targeted antioxidant-mitoQ, and used them to fight MRSA through the membrane-coated non-antibiotic mCuS@lm nanocages with good biocompatibility after MRSA training macrophages, and applied them to acute MRSA pneumonia. The mCuS@lm nanocage has excellent targeting of the infection site, can be used as a nanobait to neutralize inflammatory factors at the infection site, and slowly releases the loaded drug with the assistance of near-infrared zone II laser, and exerts excellent antibacterial and anti-inflammatory effects by differentially regulating the copper death state of bacteria and cells at the infection site.

[0051] The present invention designs a mCuS@lm antibacterial nanocage. We self-assembled by electrostatic adsorption of plant-derived luteolin and mitochondrial targeted antioxidant-mitoQ, and synthesized hollow mesoporous copper sulfide nanocages by ion exchange method, and carried the self-assembled drugs therein. Through MRSA infection training of alveolar macrophages to obtain cell membranes with high expression of specific receptors, and the cell membranes were uniformly coated on the surface of the nanocage by thin film extrusion, thereby developing a new type of bionic nanoparticles (mCuS@lm) with high efficiency and high biosafety. The particle size of the nanoparticles is uniform, about 100-200nm, presenting a hollow pipe structure, and the surface bionic membrane highly expresses proteins related to microbial recognition and uptake, and can effectively neutralize inflammatory factors in the environment, with potential infection targeting effects. In vitro, mCuS@lm nanoparticles can efficiently kill planktonic MRSA and remove the biofilm formed by MRSA under near-infrared, destroying MRSA cell walls and cell membranes. At the same time, mCuS@lm nanoparticles can inhibit the QS system of MRSA, reduce the expression of bacterial virulence factor-related genes, and interfere with the bacterial copper transport system to reduce its pathogenicity. In addition, mCuS@lm nanoparticles have no obvious cytotoxicity, which is likely to bring hope for refractory MRSA pneumonia in vivo. In mice with acute MRSA pneumonia, mCuS@lm nanoparticles can target the site of lung infection in vivo. At the same time, mCuS@lm nanoparticles can improve the survival rate of mice with acute MRSA pneumonia, reduce the bacterial load in the lungs, improve the severity of lung injury, and reduce lung inflammatory response, which has certain clinical transformation potential. Finally, in vivo toxicity and safety tests verified that mCuS@lm nanoparticles have excellent biosafety and have no obvious toxic side effects on important organs (heart, lungs, liver, spleen, kidneys) and blood. This provides a new idea for the efficient and precise treatment of refractory MRSA pneumonia in clinical practice.

[0052] The antibacterial and anti-inflammatory effects of mCuS@lm nanocages were demonstrated by the preparation of mCuS@lm nanocages and the antibacterial and anti-inflammatory data in vivo and in vitro.

[0053] Example 1

[0054] Cultivate MRSA, stimulate mouse alveolar macrophages at different ratios of MOI for different times, explore the training effects at different MOI and different time points, collect cells from each group, detect cell viability by CCK8, and detect the expression of the typical microbial recognition receptor TLR2 and the typical cytokine storm neutralizing receptor TNFR1 by WB.

[0055] Table 1: Training effects under different training conditions

[0056]

[0057] The results are as Figure 1 shown in Table 1. When the time is 24 h and 48 h, cell viability is significantly inhibited, especially in the MOI = 50:1 group. At 24 h, when MOI is greater than 10:1, the expression of TNFR1 and TLR2 in cells of each group is significantly increased. Combining the results of cell viability and marker expression, the optimal training conditions are selected as a multiplicity of infection of 10:1 and a training time of 24 h to obtain trained cells.

[0058] Example 2

[0059] (1) Extraction of the trained biomimetic membrane:

[0060] Cultivate MRSA and stimulate the mouse alveolar macrophage MH-S cell line (ATCC, CRL-2019, purchased from the Cell Bank of the Chinese Academy of Sciences) at a ratio of MOI = 10:1 for 24 hours. Collect the cells, centrifuge (1000 rpm, 3 min, 4 °C), discard the supernatant, and wash 3 times with PBS. Then, resuspend the cells with a pre-cooled mixture containing PMSF (phenylmethylsulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), and protease and phosphatase inhibitors (broad-spectrum serine, cysteine, and acid protease inhibitors / aminopeptidase inhibitors, as well as serine / threonine, tyrosine, acid, and alkaline phosphatase inhibitors, purchased from Suzhou Xinsaimai Biotechnology Co., Ltd., product number P002), and place on ice bath for 10 - 15 min. Subsequently, repeatedly freeze-thaw the sample in liquid nitrogen and at room temperature several times until the degree of cell breakage reaches more than 70%. Then, centrifuge (700 g, 10 min, 4 °C) to remove the cell nuclei and unbroken cells, and carefully collect the supernatant into a new centrifuge tube. Centrifuge (14000 g, 30 min, 4 °C) to precipitate the cell membrane fragments.

[0061] (2) Synthesis of hollow mesoporous copper sulfide nanoparticles:

[0062] Hollow mesoporous copper sulfide particles were synthesized by the ion exchange method. 60 mL of isopropyl alcohol and 0.1 mmol of copper chloride were stirred at room temperature with a magnetic stirrer for 48 h, 0.2 g of polyvinylpyrrolidone was added and stirring continued for 48 h until dissolved, and then 1 mmol of sodium hydroxide solution was added. Finally, 0.4 mL of hydrazine hydrate was added and stirred. The above product was washed with ultrapure water and ethanol, and 35 μL of ammonium sulfide solution was added and stirred until dark green. Centrifugation (8000 rpm, 15 min, 4 °C) and washing and purification were carried out, and freeze-drying was carried out for 48 h to finally obtain hollow mesoporous copper sulfide nanoparticles.

[0063] (3) Synthesis of mCuS@lm nanocages:

[0064] 0.08 mg / mL luteolin and 0.25 mg / mL mitoQ were coated inside 31.25 μg / mL hollow mesoporous copper sulfide nanoparticles by electrostatic self-assembly through magnetic thermal stirring for 48 h to synthesize CuS@lm nanocages.

[0065] Table 2: Mixing effects of cell membranes and nanoparticles with different mass ratios

[0066]

[0067] The alveolar macrophage cell membranes infected with MRSA after training and separated were mixed with CuS@lm at different mass ratios. The best ratio was explored according to the observation of the cell membrane coating rate by TEM and particle size detection (Table 2). Finally, it was determined that they were fully mixed at a mass ratio of 1:2. The mixture of the cell membrane and the nanoparticles was first sonicated with a sonicator at a power of 200 kHz and 100 W for 20 min, then extruded 10 times through a 200 nm polycarbonate porous membrane with a liposome extruder, and finally centrifuged (12000 rpm, 10 min, 4 °C) to remove the excess cell membrane to prepare and synthesize mCuS@lm (where the first "m" represents the cell membrane, "CuS" represents hollow mesoporous copper sulfide nanoparticles, "1" represents luteolin, and the second "m" represents mitoQ) nanocages.

[0068] Example 3

[0069] The minimum inhibitory concentration (MIC) of luteolin-added hollow mesoporous copper sulfide nanoparticles against MRSA was determined by the microbroth dilution method. The hollow mesoporous copper sulfide nanoparticles were serially diluted in a 96-well cell culture plate at concentrations of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, and 0 μg / mL, with a drug volume of 100 μL per well. MRSA was incubated overnight at 37 °C in TSB medium. After centrifugation, the bacterial solution was divided into two groups. The first group was adjusted to a density of 1×10 6 CFU / mL with TSB medium, and the second group was diluted to 1×10 6 CFU / mL with a medium containing luteolin. Subsequently, two portions of the bacterial solution were added to the 96-well plate to make the final concentration of luteolin 80 μg / mL. After culturing in an incubator at 37 °C for 24 h, the presence or absence of bacterial growth was observed. When there was no bacterial growth, this concentration could be determined as the MIC. The results are shown in Table 3. Luteolin could reduce the MIC value of hollow mesoporous copper sulfide nanoparticles by 8-fold, from a maximum of 250 μg / mL to 31.25 μg / mL. This indicates that luteolin can enhance the bactericidal effect of hollow mesoporous copper sulfide nanoparticles. Therefore, we will determine the ratio of hollow mesoporous copper sulfide nanoparticles to luteolin as 31.25:80.

[0070] Table 3: Antibacterial effects of mixtures of hollow mesoporous copper sulfide nanoparticles and luteolin at different ratios

[0071]

[0072] Example 4

[0073] The optimal ratio was determined by detecting the lipid peroxidation level of macrophages induced by different concentrations of mitoQ after infection using a fluorescence confocal microscope. First, macrophages were cultured in a six-well plate for 24 h and infected with MRSA for 2 h in each group. Subsequently, 31.25 μg / mL of hollow mesoporous copper sulfide nanoparticles and different concentrations of mitoQ were added to each group of cells and cultured for 4 - 6 h. The cells were collected, stained with a BODIPY probe, and the lipid peroxidation level of each group of cells was detected. The results are shown in Table 4, and the optimal anti-inflammatory concentration of mitoQ was finally determined.

[0074] Table 4: Anti-inflammatory effects of mixtures of mitoQ and hollow mesoporous copper sulfide nanoparticles at different ratios

[0075] mitoQ concentration (mg / mL) 1 0.5 0.25 0.125 0 Lipid peroxidation level Low Low Low Medium High

[0076] Example 5

[0077] Characterization of mCuS@lm nanocages: The hydrodynamic diameter, polydispersity index, and zeta potential were measured using a nanoparticle size analyzer (Mastersizer 3000E, UK). The stability of the mCuS@lm suspension prepared in Example 2 was tested in room temperature physiological saline for 15 days. The morphologies of CuS@lm and mCuS@lm were detected using a high-resolution 200 kV TEM (JEM-2100Plus, Japan). Briefly, before use, the nanoparticles were sonicated for 10 minutes. Next, a drop of the nanoparticles was placed on a carbon-coated copper grid. Excess liquid was blotted away with filter paper perpendicular to the copper grid. The protein composition was identified by SDS-PAGE and Coomassie Brilliant Blue staining.

[0078] The results are as Figure 2 shown in a of [reference]. The hollow mesoporous copper sulfide particles at different magnifications have a large surface area and a hollow pore mesoporous structure, are uniform in size, and have a diameter of about 150 - 200 nm. The hollow mesoporous copper sulfide particles contain Cu and S elements, indicating that mCuS@lm nanoparticles have been successfully prepared. The mCuS@1m nanoparticles cover a complete cell membrane structure with a lipid bilayer, are slightly larger in diameter than the hollow mesoporous copper sulfide particles, can enter the lungs smoothly, and are not easily blocked in the pulmonary artery leading to death. As Figure 2 shown in b of [reference], among the cell membrane proteins of MH-S cells trained with MRSA infection, the expressions of proteins related to the recognition and uptake of microorganisms such as TLR2, TNFR1, and SRA are all increased. As Figure 2 shown in c of [reference], under the same conditions, the total protein profile of mCuS@1m nanoparticles is similar to that of the alveolar macrophage membrane after simple MRSA training, while no proteins are expressed on the surface of the hollow mesoporous copper sulfide particles. This indicates that coating the biomimetic membrane on the surface of the nanoparticles does not affect their protein expression.

[0079] From Figure 3 a of [reference], it can be seen that the average particle size of the hollow mesoporous copper sulfide nanoparticles is 159.9 nm, and the average particle size of the mCuS@lm nanoparticles is 167.2 nm, with an average thickness increase of 7.3 nm, indicating that the cell membrane trained with MRSA infection can be successfully coated on the surface. Figure 3 b of [reference] shows that the average Zeta potential of mitoQ is 12.32 mV, and its positive charge can be widely and rapidly taken up by mitochondria driven by the mitochondrial membrane potential. Luteolin carries a negative charge and can self-assemble with mitoQ through electrostatic adsorption inside the nanocage. The average Zeta potential of the hollow mesoporous copper sulfide nanoparticles is -9.12 mV, and the average Zeta potential of the mCuS@lm nanoparticles is -9.80 mV, which is in line with the research expectations. Subsequently, we detected the stability of the mCuS@lm nanoparticles in aqueous solution. From Figure 3As can be seen from c in [reference], the mCuS@lm nanoparticles can maintain a stable particle size within 15 days. Finally, we detected the ultraviolet-visible-near-infrared spectra of luteolin, mitoQ, hollow mesoporous copper sulfide nanoparticles, and mCuS@lm nanoparticles ( Figure 3 in d). The results showed that mitoQ had a characteristic absorption peak at 400 nm, luteolin had a characteristic absorption peak at 350 nm, and the hollow mesoporous copper sulfide nanoparticles had a characteristic absorption peak at 900 nm. By detecting the spectrum of the mCuS@lm nanoparticles, we found that the two drugs were successfully loaded into the nanoparticles. From Figure 3 as can be seen from e-g in [reference], compared with the nanoparticles before training, the trained mCuS@lm nanoparticles could more effectively neutralize the inflammatory factors IL-1β and TNF-α.

[0080] Example 6

[0081] Targeting test of mCuS@lm nanocages: Nile red-stained non-trained nanoparticles and Nile red-stained trained nanoparticles were prepared for bacterial targeting tests. MRSA and Pseudomonas aeruginosa were cultured in TSB and LB media respectively. Then, these nanoparticles were incubated with bacteria for different times. The fluorescence signals of the bacteria were detected by flow cytometry (BD Bioscience, USA). Subsequently, MH-S cells were infected with MRSA for 2 h, and then incubated with trained nanoparticles and non-trained nanoparticles for different times. Confocal fluorescence microscopy (Zeiss, Germany) was used to record the cell fluorescence signals. Mouse pneumonia targeting experiments were carried out using IR783 labeled with CuS@lm and mCuS@lm. These nanoparticles were injected into mice with acute pneumonia. After different times, the fluorescence signals were recorded using IVIS (PerkinElmer, USA). Subsequently, important organs (heart, lung, liver, kidney, spleen, stomach, intestine) were isolated, and their fluorescence signals were recorded and analyzed.

[0082] As Figure 4 shown in a in [reference], compared with the control group, the nanoparticles coated with alveolar macrophage membranes were more easily taken up by infected alveolar macrophages, which may be related to their homologous targeting ability. In addition, the nanoparticles coated with alveolar macrophage membranes trained with MRSA could better target infected macrophages, and this targeting ability became more obvious with the extension of time. At the same time, we found that the bacterial fluorescence signals in the control group were diffused inside and outside the cells, while the bacterial fluorescence signals in the treatment group were mostly concentrated in the cytoplasm, which may be related to the bactericidal effect of the nanoparticles themselves, and the nanoparticles coated with alveolar macrophage membranes trained with MRSA may promote macrophages to phagocytose bacteria.

[0083] As Figure 4As shown in b of [reference], at 4 h, 99.6% of MRSA bacteria could bind to fluorescently labeled tmCuS@lm nanoparticles. In contrast, only 40.5% of MRSA bacteria could bind to fluorescently labeled mmCuS@lm nanoparticles, indicating that the alveolar macrophage biomimetic membrane after training with MRSA could endow the nanoparticles with better MRSA infection targeting ability. Figure 4 c in [reference] verified this conclusion at the TEM level.

[0084] As Figure 4 Shown by the in vivo fluorescence imaging results in d of [reference], in the mice of the CuS@lm group, the fluorescence signal was distributed throughout the body, and with the extension of time, the fluorescence intensity gradually decreased. On the third day after injection, the fluorescence intensity of the nanoparticles was mainly concentrated in the liver. Figure 4 The ex vivo fluorescence imaging results in e of [reference] were basically consistent with the in vivo results. However, compared with the uncoated nanoparticles, on the third day after injection, the fluorescence intensity of the mCuS@lm nanoparticles was mainly concentrated in the lung, and the ex vivo fluorescence imaging results were basically consistent with the in vivo fluorescence intensity distribution. In summary, after coating the alveolar macrophage membrane trained with MRSA infection, the mCuS@lm nanoparticles could effectively target the lung after MRSA infection.

[0085] Example 7

[0086] Bacterial culture and counting: MRSA and Pseudomonas aeruginosa were cultured in TSB and LB media respectively. The bacterial suspension was centrifuged at 4000 rpm for 10 min, then the bacterial suspension was diluted twofold, incubated at 37 °C on the corresponding TSA and LB agar plates for 12 h, photographed and counted for colonies using Image J.

[0087] Bacterial live / dead detection: Bacterial suspensions were treated with different nanoparticles for 24 h, then stained with SYTO 9 and PI at 37 °C for 15 min, and imaged using a confocal microscope (Zeiss, Germany).

[0088] Biofilm formation and disruption detection: Bacterial suspensions were cultured at 37 °C for 72 h to form mature biofilms. The mature biofilms were treated with different nanoparticles for 3 h, washed after treatment, stained with SYTO 9 and PI at 37 °C for 15 min, and finally 3D images of the biofilms were taken using a confocal fluorescence microscope (Zeiss, Germany). The remaining biofilms after washing were stained with crystal violet.

[0089] Bacterial morphology detection: After incubating the bacterial suspension with different nanoparticles for 24 h, the bacteria were fixed with 2.5% glutaraldehyde, washed with PBS, and dehydrated successively with ethanol and acetone. Transmission electron microscopy (Thermo FEI, Tecnai G2 spirit, Czech Republic) and scanning electron microscopy (Thermo FEI, NovaNano 450, Czech Republic) were used to observe the bacterial morphology.

[0090] As Figure 5 shown in a of Figure 5 , almost all bacteria in the mCuS@lm and mCuS@lm+NiR treatment groups were killed within 24 h, which was attributed to the synergistic effect of CuS and QS inhibitors. The inhibitory effect of the QS system on MRSA virulence may enhance the amplification of MRSA virulence. In addition, due to more copper ion leakage, the nanocages showed better antibacterial effects under low-intensity near-infrared irradiation. The mCuS@lm and mCuS@lm+NiR treatment groups achieved almost 100% antibacterial rates against Pseudomonas aeruginosa and ciprofloxacin-evolved Pseudomonas aeruginosa, which was similar to the results of MRSA ( Figure 5 shown in b of Figure 5 and c of Figure 5 ). MRSA live / dead staining showed that the mCuS@lm+NiR treatment group exhibited the best antibacterial ability (

[0091] shown in d of Figure 6 and e of Figure 6 ). The detection results of TEM ( Figure 6 shown in a of Figure 6 ) and SEM (

[0092] shown in b of Figure 7 ) showed that the untreated group exhibited a complete rod-shaped (Pseudomonas aeruginosa) or spherical (MRSA) morphology, with a continuous and intact cell membrane. In the treatment group, however, the bacteria showed severe morphological deformities, such as shrinkage, membrane rupture, cytoplasmic leakage, and even cracking. This confirmed that the nanocages could damage the bacterial membrane and change the cell membrane permeability, which might be related to the ROS generated by mCuS@lm and copper poisoning that damaged the lipid bilayer, resulting in rapid leakage of the cell matrix. In addition, mature biofilms could be destroyed by the mCuS@lm nanocages ( Figure 7As shown in b in [reference], lipid peroxidation in cells incubated with mCuS and mCuS+NiR increased by more than 3-fold and more than 2-fold, respectively. In the mCuS@lm group and mCuS@lm+NiR group, due to the addition of the mitoQ component, the level of lipid peroxidation was lower. However, there was no significant difference in the level of copper accumulation among the groups ( Figure 7 ). Figure 7 As shown in d in [reference], most of the MRSA in the mCuS@lm+NiR group underwent lipid peroxidation, while the degree of lipid peroxidation in the mCuS and mCuS+NiR groups was lower. In addition, Figure 7 As shown in e in [reference], it was proved that MRSA experienced the most ROS attacks in the mCuS@lm+NiR group. To sum up, the QS inhibitor does not affect the cytoprotective effect of mCuS@lm, while mitoQ does not affect its bactericidal effect of cuproptosis. Compared with eukaryotic cells, prokaryotic cells have lower tolerance to oxidative stress. Bacteria can be quickly cleared by nanoparticles, and then, mitochondrial-targeted antioxidants can protect normal cells.

[0093] Example 8

[0094] Establishment of a mouse model of acute pneumonia: Male C57BL / 6 mice (25-30 g) at 6-8 weeks of age were used to establish an acute pneumonia model. The animals were housed in a standard environment, and the whole process was carried out on a sterile bench. The mice were depilated and disinfected one day in advance. After anesthesia, a long incision was made in the middle of the neck, and the muscle tissue was carefully separated. Finally, the trachea was bluntly dissected to fully expose it. 50 μL of MRSA (5×10 7 CFU) resuspended in PBS was slowly injected into the trachea of the mice, and then the mice were held upright and gently shaken to evenly distribute the bacteria in the lungs. Subsequently, the mice were randomly grouped. 2 h after infection, different nanoparticles were injected intravenously. The NiR group was irradiated with 1064 nm laser. That is, the whole chest area was irradiated from outside the chest cavity at a low dose of 0.3 W / cm2 for 10 min. The mice were sacrificed at different times. The lung tissues were isolated and photographed for subsequent experiments. In the survival rate and core body temperature studies, the mice were infected with a lethal dose of MRSA (50 μL, 5×10 8 CFU).

[0095] qPCR: The relative expression level was normalized by the Actin level. After treatment, the lung tissues were isolated and ground to be fully dissolved. RNA was reverse transcribed into complementary DNA using a reverse transcription kit. qPCR was performed using a 20 μL system, including 2 μL of cDNA, 0.4 μL of forward and reverse primers, 10 μL of SYBR, and 7.2 μL of double-distilled water.

[0096] In vivo and in vitro fluorescence imaging: The USA300 strain was transfected with the luciferase gene, and in this study it was named Luci-MRSA. An acute pneumonia model was established using Luci-MRSA. After corresponding treatments, potassium fluorescein was injected intraperitoneally into the mice. In vivo chemiluminescence signals were measured using IVIS (PerkinElmer, USA) at 3 h and 6 h respectively, and then the lungs of each group were isolated at 12 h and 24 h, and the corresponding fluorescence intensities were recorded and analyzed.

[0097] Histological staining and analysis: The lungs were collected, fixed with 4% paraformaldehyde, and embedded in paraffin. Standard methods were used for HE staining, Gram staining, and MPO immunohistochemical staining. In addition, fresh lungs were collected, embedded in optimal cutting temperature compound (OCT, Tissue-Tek, PA), and sectioned at 15 μm using a cryostat (Leica CM1950, Germany). The sections were stained with DAPI and DHE for 15 min, and images were taken using a confocal fluorescence microscope (Zeiss, Germany). The images and sections were blindly analyzed and counted using ImageJ. All scoring was independently completed by 2 professionals not involved in the animal experiments.

[0098] Toxicity and safety analysis: BEAS-2B was cultured in 96-well plates for the cytotoxicity experiment. After corresponding treatments, a CCK-8 kit and a Calcein-AM / PI staining kit were used to detect cell viability. In the in vivo experiment, normal mice were treated with different nanoparticles. After 14 days, the mice were sacrificed, and the main organs (heart, lung, liver, spleen, kidney) were isolated, fixed with 10% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with HE for histological examination. Finally, blood routine and blood biochemical examinations were performed.

[0099] As Figure 8 shown in a of Figure 8 , we found that at 16 h after infection, the body temperature of the control group mice decreased significantly, and the body temperature was lower than 32 °C. In contrast, the mice in the mCuS@lm+NiR treatment group could basically maintain normal rectal temperature, thus improving the survival rate of the mice. As Figure 8 shown in b of

[0100] , we took pictures of the lungs of the treated mice. The results showed that there were obvious manifestations such as pulmonary hemorrhage and pulmonary tissue consolidation such as edema in the PBS group. The degree of lung tissue inflammation in the other treatment groups was alleviated. However, in contrast, the degree of lung injury in the mCuS@lm+NiR group mice was the lightest. Moreover, the wet weight and dry weight of the lungs of the mCuS@lm+NiR group mice were the smallest after infection ( Figure 8 shown in c of

[0100] ), indicating that mCuS@lm+NiR treatment could reduce alveolar capillary endothelial cell injury and relieve pulmonary edema.

[0100] Subsequently, we performed alveolar lavage on the treated mice and studied the degree of inflammation by detecting the number of cells and protein content in the alveolar lavage fluid of the mice. AsFigure 8 d in Figure 8 and e in

[0101] As shown in Figure 8 g, after 48 h of MRSA infection, there were a large number of inflammatory cell infiltrations in the lung tissue of the control group, secretions in the alveolar cavity, thickening of the alveolar wall and destruction of the structure, and edema and necrosis in the surrounding blood vessels. In contrast, in the lung tissue sections of the mice in the mCuS@lm+NiR treatment group, we could find that the degree of lung injury was reduced, the lung tissue was basically normal, and most of the alveolar structures seen under the microscope were relatively intact, without obvious bleeding and edema. And the lung injury score also confirmed this conclusion ( Figure 8 in f).

[0102] As shown in Figure 9 a and Figure 9 b, mCuS@lm+NiR treatment could significantly reduce the bacterial CFU in the lung. Figure 9 c shows that a large number of Gram-positive bacteria were aggregated in the lung tissue of the control group mice. However, there were almost no purple-black bacterial masses in the lung tissue of the mice treated with mCuS@lm+NiR. This confirmed that our nanoparticles had excellent antibacterial effects.

[0103] As shown in Figure 10As shown, in the blank DMEM medium, LM, CuS, and mCuS@lm+NiR treatment groups, the cell survival rate was close to 100%, indicating that the mCuS@lm nanoparticles had no obvious cytotoxicity. In the BEAS-2B cell viability staining, almost all green fluorescence was observed, indicating that most cells remained viable after treatment with the mCuS@lm nanoparticles. The above results all showed that the mCuS@lm nanoparticles had low cytotoxicity and high biosafety. On the 14th day after treatment with each group, the serum and plasma of the mice were taken to test and analyze the blood biochemical and blood routine results. The results showed that the number of white blood cells (WBC), red blood cells (RBC), hemoglobin concentration (HGB), platelet count (PLT), lymphocyte ratio, neutrophil ratio, macrophage ratio, and mean corpuscular hemoglobin concentration (MCHC) of the mice were all within the normal range, indicating that the treatment of each group did not affect the blood system of the mice. In addition, the blood biochemical results of each group showed that aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) were also within the normal range. This indicated that the treatment of each group did not affect the liver and kidney functions of the mice. Finally, on the 14th day after treatment with each group, the hearts, lungs, livers, spleens, and kidneys of the mice in each group were dissected for H&E staining, as Figure 10 shown, no obvious inflammatory reactions occurred in the important organs of each group, and the normal tissue structure was still maintained. The above results indicated that the mCuS@lm nanoparticles had high biosafety in animals and were expected to be translated into clinical applications.

Claims

1. A kind of mCuS@lm antibacterial nanocage, characterized in that, With hollow mesoporous copper sulfide nanoparticles as the core, a bacterial / fungal cuproptosis amplifier and a cell cuproptosis inhibitor are encapsulated in the hollow mesoporous copper sulfide nanoparticles through electrostatic adsorption self-assembly, and a biomimetic cell membrane is further coated on the surface of the hollow mesoporous copper sulfide nanoparticles to form a nano cage, which is the mCuS@lm antibacterial nano cage; The hollow mesoporous copper sulfide nanoparticles are prepared by an ion exchange method; The bacterial / fungal cuproptosis amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is luteolin; The cell cuproptosis inhibitor is an antioxidant, and the antioxidant is mitoQ; The biomimetic cell membrane is a cell membrane extracted after using methicillin-resistant Staphylococcus aureus as the training substance and alveolar macrophages as the trained substance for infection training; The multiplicity of infection during training is 10:1, and the training time is 24 h; The mass ratio of the bacterial / fungal cuproptosis amplifier to the hollow mesoporous copper sulfide nanoparticles is 80:31.25 - 62.5; The mass ratio of the cell cuproptosis inhibitor to the hollow mesoporous copper sulfide nanoparticles is 0.25 - 1:31.25; The mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:

2.

2. A preparation method of mCuS@lm antibacterial nanocage, characterized in that, It includes the following steps: (1) Prepare hollow mesoporous copper sulfide nanoparticles; (2) Encapsulate a bacterial / fungal cuproptosis amplifier and a cell cuproptosis inhibitor in the hollow mesoporous copper sulfide nanoparticles through electrostatic adsorption self-assembly; (3) Coat the biomimetic cell membrane on the surface of the hollow mesoporous copper sulfide nanoparticles obtained in step (2) that have encapsulated the bacterial / fungal cuproptosis amplifier and the cell cuproptosis inhibitor to form a nano cage, which is the mCuS@lm antibacterial nano cage; The hollow mesoporous copper sulfide nanoparticles are prepared by an ion exchange method; The bacterial / fungal cuproptosis amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is luteolin; The cell cuproptosis inhibitor is an antioxidant, and the antioxidant is mitoQ; The biomimetic cell membrane is a cell membrane extracted after using methicillin-resistant Staphylococcus aureus as the training substance and alveolar macrophages as the trained substance for infection training; The multiplicity of infection during training is 10:1, and the training time is 24 h; The mass ratio of the bacterial / fungal cuproptosis amplifier to the hollow mesoporous copper sulfide nanoparticles is 80:31.25 - 62.5; The mass ratio of the cell cuproptosis inhibitor to the hollow mesoporous copper sulfide nanoparticles is 0.25 - 1:31.25; The mass ratio of the biomimetic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:

2.

3. Use of the mCuS@lm antibacterial nano cage described in claim 1 in the preparation of a drug for treating acute pneumonia caused by methicillin-resistant Staphylococcus aureus.

4. A drug for treating acute pneumonia caused by methicillin-resistant Staphylococcus aureus, characterized in that, It contains the mCuS@lm antibacterial nano cage described in claim 1.

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