MCuS (at) lm antibacterial nano cage as well as preparation method and application of mCuS (at) lm antibacterial nano cage

Abstract

The invention discloses an mCuS (at) lm antibacterial nano cage as well as a preparation method and application of the mCuS (at) lm antibacterial nano cage. The mCuS (at) lm antibacterial nano cage takes hollow mesoporous nano copper sulfide nanoparticles as a core, a bacterial copper death amplifying agent and a cell copper death inhibitor are entrapped in the hollow mesoporous nano copper sulfide nanoparticles through electrostatic adsorption self-assembly, and the surfaces of the hollow mesoporous nano copper sulfide nanoparticles are further coated with bionic cell membranes, so that the mCuS (at) lm antibacterial nano cage is obtained. The nanometer cage is the mCuS (at) lm antibacterial nanometer cage. The mCuS (at) lm antibacterial nano cage surface biomimetic membrane highly expresses proteins related to microbial recognition and uptake, can effectively neutralize inflammatory factors in the environment, and has a potential infection targeting effect. The bacterial copper death amplifying agent is used for enhancing bacterial copper death, and the cell copper death inhibitor is used for retarding host cell copper death, so that the bacterial killing effect and the antibacterial effect are improved under the condition that the biological safety is improved.

Description

Technical Field

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

[0002] Staphylococcus aureus (Staphylococcus aureus) is a common pathogen in hospital-acquired and community-acquired infections, which can cause infections in multiple sites such as skin, soft tissue, lung, bone, and blood. However, the abuse of antibiotics, the evolution of bacterial resistance, and the increase in virulence have increased the threat of pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Currently, antibiotics such as vancomycin and linezolid are the main treatments for MRSA pneumonia. However, with their extensive use, vancomycin-resistant Staphylococcus aureus has appeared in clinical practice. We urgently need to explore new targets and find new non-antibiotic treatment strategies for MRSA pneumonia.

[0003] Copper can be used as a trace element to participate in a variety of physiological reactions. However, the increase in intracellular copper concentration caused by its homeostasis imbalance can also cause cell damage and copper death by affecting mitochondrial metabolism. At present, copper death is mainly found in eukaryotic cells, but copper also has a natural antibacterial effect, and some mechanisms of copper death also occur in bacteria. At present, there are still few research and development of nanomaterials based on copper death. In addition, in practical applications, traditional pulmonary inflammatory diseases are limited by insufficient drug concentration at the target site, difficulty for nanomaterials to reach the inside of bacteria, and biological barriers of the lungs. At the same time, due to the lack of targeting, it is difficult for nanomaterials to quickly identify bacteria and efficiently gather around bacteria. This makes the treatment efficiency of traditional nanomaterials in pneumonia low. When exogenous nanoparticles are not modified, they are easily recognized and phagocytosed by macrophages of the reticuloendothelial system, lack long-circulation effects, and are difficult to reach the target site. The reactive oxygen species produced by high doses of copper-based materials will also produce huge oxidative stress pressure on normal tissue cells. Especially in lung tissue, excessive ROS will exceed the processing capacity of the endogenous antioxidant system of the cell, causing oxidation of proteins, lipids, DNA and carbohydrates, leading to cell death and lung dysfunction.

[0004] Therefore, how to give nanomaterials bacterial targeting and reduce oxidative stress damage to normal tissues while inducing and amplifying bacterial copper death and destroying bacterial redox balance is the focus of current research. Mitoxantrone mesylate (mitoQ), a mitochondrial-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 in the mitochondrial inner membrane as electrochemical potential energy. Therefore, there is an asymmetric distribution of proton and other ion concentrations on both sides of the mitochondrial inner membrane, with a positive charge in the membrane gap 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 mitochondrial-targeted antioxidant role in eukaryotic cells, maintaining normal redox balance, and its effect is far better than non-targeted antioxidants. However, due to the lack of mitochondria, the respiration of bacterial cells relies solely on the electron transport chain structure similar to the cell membrane, and its targeted aggregation effect is far inferior to that of eukaryotic cells. Quorum sensing (QS) is a communication system for bacteria to communicate with each other and transmit information. Through "cell-to-cell" communication, they can make collective decisions, synchronize with other populations, adapt to environmental changes, develop antibiotic resistance, form biofilms, and produce virulence factors. QS-deficient bacteria are more susceptible to oxidative stress damage. Therefore, inhibiting the QS system to induce enhanced oxidative stress response may amplify the copper death effect. Naturally derived luteolin is a flavonoid compound and is considered to be a QS inhibitor (Quorum sensing inhibitors, QSIs). It has high biosafety and can synergize with mitoQ and copper-based materials to exert anti-inflammatory and antibacterial effects.

[0005] In recent years, there has been an increasing number of studies on biomimetic nanoparticles coated with different cell membranes. The camouflage of cell membranes of natural biological origin (red blood cells, platelets, tumor cells, stem cells, immune cells, bacteria, etc.) gives nanoparticles strong immune escape ability and targeted delivery ability. At present, many studies have found that nanoparticles coated with macrophage membranes have certain targeting capabilities for inflammation and tumor microenvironment, while membranes pretreated with bacteria have stronger bacterial adhesion efficiency and can prolong the retention time of drugs at the site of infection. Alveolar macrophages reside 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 MRSA infection training, the membranes of alveolar macrophages highly express Toll-like receptors, TNF receptors, scavenger receptors MSR, CD14, mannose receptor CD206 and other proteins that mediate microbial uptake and recognition, giving them a strong targeting effect in MRSA pneumonia; in addition, they can act as cytokine decoys, bind proinflammatory factors through their homologous cytokine receptors, and uncouple the signal transduction and transcriptional activation of the macrophage inflammatory cascade. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a new preparation method and application of mCuS@lm antibacterial nanocage. Natural plant-derived QSIs luteolin and mitochondria-targeted antioxidant mitoQ are encapsulated in hollow mesoporous copper sulfide nanoparticles to synthesize a new antibacterial nanocage material mCuS@lm. In order to improve the drug loading efficiency and shield the immune system response in the body, we coated the alveolar macrophage membrane after sublethal dose MRSA infection training on the outside, which can achieve the targeting of mCuS@lm material to MRSA pneumonia in the body, prolong the residence time of nanoparticles in the lesion site, and regulate the host lung immune response. On the other hand, copper overload induces copper death of bacteria, and the release of copper ions can produce hydroxyl radicals through Fenton-like reaction in the infection environment, cooperate with QSIs to interfere with bacterial QS signals and metabolic pathways to kill bacteria, and also protect the mitochondrial function of normal lung cells through mitochondrial-targeted antioxidants to maintain redox homeostasis, thereby achieving differential regulation of the copper death state of bacteria and cells to exert antibacterial and anti-inflammatory effects.

[0007] The present invention first provides a mCuS@lm antibacterial nanocage, which takes hollow mesoporous copper sulfide nanoparticles as the core, encapsulates bacterial / fungal copper death amplifiers and cell copper death inhibitors in the hollow mesoporous copper sulfide nanoparticles through electrostatic adsorption self-assembly, and further coats the surface of the hollow mesoporous copper sulfide nanoparticles with a bionic cell membrane to form a nanocage, namely the mCuS@lm antibacterial nanocage.

[0008] Bacterial / fungal copper death amplifier refers to the added bacterial copper death amplifier or fungal copper death amplifier. If the prepared mCuS@lm antibacterial nanocage is used for antibacterial, the bacterial copper death amplifier is added accordingly. Correspondingly, if the prepared mCuS@lm antibacterial nanocage is used for antifungal, the fungal copper death amplifier is added accordingly.

[0009] Preferably, the hollow mesoporous nano copper sulfide nanoparticles are prepared by an ion exchange method. Specifically, cupric chloride is dispersed in isopropanol, polyvinyl pyrrolidone is added to dissolve, sodium hydroxide solution is added, hydrazine hydrate is added, and finally ammonium sulfide solution is added to react, the product is centrifuged and washed and purified, and then freeze-dried to obtain the hollow mesoporous nano copper sulfide nanoparticles. The preparation method of hollow mesoporous nano copper sulfide nanoparticles is a prior art, so the specific proportions of each substance and parameter conditions can be adjusted and set according to the prior art.

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

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

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

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

[0014] The fungus used as the training substance is selected from at least one of the following: Candida albicans, yeast, Cryptococcus;

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

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

[0017] The selection of training substances is adapted to the final use of the prepared mCuS@lm antibacterial nanocages. For example, if it is ultimately used to prepare anti-MRSA pneumonia drugs, Staphylococcus aureus is used as the training substance. The immune cells used to extract cell membranes as biomimetic membranes can come into contact with the training substances during the training process, thereby highly expressing specific receptors related to the recognition and uptake of the training substances on the cell membrane surface.

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

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

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

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

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

[0023] (2) encapsulating the bacterial / fungal copper death amplifier and the cell copper death inhibitor in the hollow mesoporous nano copper sulfide nanoparticles through electrostatic adsorption self-assembly;

[0024] (3) coating the bionic cell membrane on the surface of the hollow mesoporous copper sulfide nanoparticles loaded with bacterial / fungal copper death amplifier and cell copper death inhibitor obtained in step (2) 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 copper death amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is at least one of the following: luteolin, coumarin, and curcumin;

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

[0028] The bionic cell membrane is a cell membrane extracted after infection training using at least one of bacteria or fungi as training materials and immune cells as trained materials.

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

[0030] The fungus used as the training substance is selected from at least one of the following: Candida albicans, yeast, Cryptococcus;

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

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

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

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

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

[0036] The present invention also provides the use of the mCuS@lm antibacterial nano cage 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 antibacterial or antifungal, the bacteria is Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae or Escherichia coli. The fungi are Candida albicans, yeast or Cryptococcus. The selection of the antibacterial target corresponds to the training material.

[0039] The present invention designs a mCuS@lm antibacterial nanocage, which takes hollow mesoporous nano copper sulfide nanoparticles as the core, and encapsulates bacterial / fungal copper death amplifiers and cell copper death inhibitors in the hollow mesoporous nano copper sulfide nanoparticles through electrostatic adsorption self-assembly, and further coats the surface of the hollow mesoporous nano copper sulfide nanoparticles with a bionic cell membrane to form a nanocage, which is the mCuS@lm antibacterial nanocage. The bionic 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, and has a potential infection targeting effect. The bacterial / fungal copper death amplifier is used to enhance bacterial copper death, and the cell copper death inhibitor is used to slow down the host cell copper death, thereby improving bacterial killing and antibacterial effect while improving biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a graph showing the training effect test results under different training conditions. Figure 1 a in the figure: Cell viability detection under different training conditions, Figure 1 b in the figure: Expression of key proteins on the membrane surface under different training conditions.

[0041] Figure 2 This is a partial characterization result diagram of the mCuS@lm nanocage. Figure 2 a: TEM structure and elemental analysis of hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles; Figure 2 b in the figure: Expression of key proteins on the membrane surface before and after training; Figure 2 c in the figure: Coomassie Brilliant Blue staining results of nanoparticle protein before and after training.

[0042] Figure 3 This is another part of the characterization results of the mCuS@lm nanocage. Figure 3 a in the figure: hydrated particle size of hollow mesoporous copper sulfide nanoparticles and nanocages coated with biomimetic membranes. Figure 3 b: Zeta potential of luteolin, mitoQ, hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles. Figure 3 c: Particle size stability of mCuS@lm nanoparticles in aqueous solution. Figure 3 (d) Spectra of luteolin, mitoQ, hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles at 300nm to 1000nm. Figure 3 e in Figure 3: The ability of nanoparticles to neutralize IL-1b before and after training. Figure 3 f: The ability of nanoparticles to neutralize TNF-a before and after training. Figure 3 g in the figure: Schematic diagram of nanoparticles neutralizing inflammatory cytokine storm.

[0043] Figure 4 The results of the targeted test of mCuS@lm nanocage are shown in Figure 2. Figure 4 a: The ability of nanocages to target infected cells before and after training; Figure 4 b: The ability of nanocages to target bacteria before and after training; Figure 4 c: Electron microscopic diagram of bacteria adhered to nanocages before and after training; Figure 4 d in the figure: In vivo fluorescence imaging results of nanocage-targeted pneumonia mice before and after training; Figure 4 e in the figure: In vitro fluorescence imaging results of nanocage-targeted pneumonia mice before and after training.

[0044] Figure 5 The figure shows the antibacterial ability test results of mCuS@lm nanocages. Figure 5 a: Agar plates showing different groups of nanocages killing MRSA bacteria at different time points; Figure 5 b: Agar plate showing the killing of Pseudomonas aeruginosa by nanocages of different groups; Figure 5 c in the figure: agar plate showing the killing of ciprofloxacin-resistant Pseudomonas aeruginosa by different groups of nanocages; Figure 5 d in the figure: live or dead staining of bacteria killed by nanocages in different groups; Figure 5 e in the figure: The proportion of dead bacteria after treatment with nanocages in different groups.

[0045] Figure 6 The figure shows the antibacterial morphology and anti-biofilm ability test results of mCuS@lm nanocages. Figure 6 a: TEM images of different groups of nanocages killing MRSA; Figure 6 b: SEM images of different groups of nanocages killing MRSA and Pseudomonas aeruginosa; Figure 6 c: Live and dead staining of different groups of nanocages in clearing MRSA biofilm; Figure 6 d in the figure: Crystal violet staining of different groups of nanocages clearing MRSA biofilm.

[0046] Figure 7 This is the result of exploring the differential anti-copper death ability of mCuS@lm nanocages. Figure 7 a in the figure: redox state of macrophages treated with nanocage killing in different groups; Figure 7 b: Lipid peroxidation levels of macrophages treated with nanocages in different groups; Figure 7 c in the figure: copper ion concentration of macrophages treated with nanocages in different groups; Figure 7 d in the figure: Redox state of bacteria killed by nanocages in different groups; Figure 7 e in the figure: ROS levels of macrophages treated with nanocage killing in different groups.

[0047] Figure 8 This is the result of the in vivo effect test of mCuS@lm nanocage. Figure 8 a in the figure: Changes in core body temperature of mice after application of different groups of nanocages; Figure 8 b: Anatomical status of mouse lungs after application of different groups of nanocages; Figure 8 c: The wet weight / dry weight ratio of mouse lungs after application of different groups of nanocages; Figure 8 d in the figure: cell counts in bronchoalveolar lavage fluid of mice after application of different groups of nanocages; Figure 8 e in the figure: protein concentration counts of bronchoalveolar lavage fluid of mice after application of different groups of nanocages; Figure 8 f: Lung injury scores of mice after application of different groups of nanocages; Figure 8 g in the figure: HE staining results of mouse lung sections after application of different groups of nanocages.

[0048] Fig. 9 This is the result of the in vivo effect test of mCuS@lm nanocage. Fig. 9 a in the figure: pictures of bacterial plating in the mouse lungs after application of different groups of nanocages; Fig. 9 b: Bacterial plate counts in the lungs of mice after application of different groups of nanocages; Fig. 9 c in the figure: Immunohistochemistry images of MPO in the lungs of mice after application of different groups of nanocages.

[0049] Fig.10 This is the result of the biosafety evaluation of mCuS@lm nanocages. Fig.10 a in the figure: Live or dead staining of cells after applying different groups of nanocages; Fig.10 b: The viability of cells after applying different groups of nanocages; Fig.10 c in the figure: blood biochemical indexes of mice after application of different groups of nanocages; Fig.10 d in the figure: blood routine indicators of mice after application of different groups of nanocages; Fig.10 e in the figure: HE staining results of important organs of mice after application of different groups of nanocages. DETAILED DESCRIPTION

[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] MRSA was cultured and mouse alveolar macrophages were stimulated with different MO1 ratios for different time periods to explore the training effects of different MO1 and time points. Cells in each group were collected and cell viability was detected by CCK8. The expressions of typical microbial recognition receptor TLR2 and typical cytokine storm neutralizing receptor TNFR1 were detected by WB.

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

[0056]

[0057] The results are as follows Figure 1 As shown in Table 1, cell viability was significantly inhibited at 24h and 48h, especially in the MOI=50:1 group. At 24h, when MO1 was greater than 10:1, the expression of TNFR1 and TLR2 in each group of cells was significantly increased. Combining the results of cell viability and marker expression, the optimal training conditions were selected as the multiplicity of infection of 10:1 and the training time of 24h to obtain trained cells.

[0058] Example 2

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

[0060] MRSA was cultured and stimulated with mouse alveolar macrophage MH-S cell line (ATCC, CRL-2019, purchased from the Chinese Academy of Sciences Cell Bank) at a ratio of MOI = 10:1 for 24 hours. The cells were collected, centrifuged (1000rpm, 3min, 4°C), the supernatant was discarded, and washed 3 times with PBS. Then, the cells were resuspended in 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 Xinsaimei Biotechnology Co., Ltd., catalog number P002), and placed in an ice bath for 10-15min. Subsequently, the samples were repeatedly frozen and thawed in liquid nitrogen and room temperature until the degree of cell disruption reached more than 70%. Then, the nuclei and unbroken cells were removed by centrifugation (700g, 10min, 4°C), and the supernatant was carefully collected into a new centrifuge tube. Centrifugation (14000 g, 30 min, 4°C) was performed to pellet cell membrane fragments.

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

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

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

[0064] By magnetic thermal stirring for 48 h, 0.08 mg / mL luteolin and 0.25 mg / mL mitoQ were electrostatically self-assembled into 31.25 μg / mL hollow mesoporous copper sulfide nanoparticles to synthesize CuS@lm nanocages.

[0065] Table 2: Effects of mixing cell membrane and nanoparticles at different mass ratios

[0066]

[0067] The isolated MRSA-infected trained alveolar macrophage membranes were mixed with CuS@lm at different mass ratios. The optimal ratio was explored based on TEM observation of cell membrane coating rate and particle size detection (Table 2). Finally, it was determined that the mixture was fully mixed at a mass ratio of 1:2. The mixture of cell membrane and nanoparticles was first sonicated at 200 kHz and 100 W for 20 min using an ultrasonicator, and then extruded 10 times under a 200 nm polycarbonate porous membrane using a liposome extruder. Finally, the excess cell membrane was removed by centrifugation (12000 rpm, 10 min, 4°C) to prepare synthetic mCuS@lm (where the first "m" represents 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 hollow mesoporous copper sulfide nanoparticles for killing MRSA by adding luteolin was determined by the microbroth dilution method. The hollow mesoporous copper sulfide nanoparticles were diluted in a 96-well cell culture plate in multiple ratios, and the concentrations were 1000μg / mL, 500μg / mL, 250μg / mL, 125μg / mL, 62.5μg / mL, 31.25μg / mL, 15.625μg / mL, 7.8125μg / mL, and 0μg / mL, respectively, with a drug volume of 100μL per well; MRSA was incubated in TSB medium at 37℃ overnight, and the bacterial solution was centrifuged and divided into two groups. The density of the first group was adjusted to 1×10 6 CFU / mL, and the second group was diluted to 1×10 6 CFU / mL. Subsequently, two bacterial solutions were added to the 96-well plate to make the final concentration of luteolin 80μg / mL. After culturing in a 37°C incubator for 24 hours, observe whether there is bacterial growth. When there is no bacterial growth, the concentration can be judged as MIC. The results are shown in Table 3. Luteolin can reduce the MIC value of hollow mesoporous copper sulfide nanoparticles by 8 times, from 250μg / mL to 31.25μg / mL. This shows 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 to be 31.25:80.

[0070] Table 3: Antibacterial effects of different ratios of hollow mesoporous copper sulfide nanoparticles mixed with luteolin

[0071]

[0072] Example 4

[0073] Fluorescence confocal microscopy was used to detect the lipid peroxidation levels of macrophages induced by infection with different concentrations of mitoQ to determine the optimal ratio. First, macrophages were cultured in six-well plates for 24 hours, and each group of cells was infected with MRSA for 2 hours. Subsequently, 31.25 μg / mL hollow mesoporous copper sulfide nanoparticles and different concentrations of mitoQ were added to each group of cells and cultured for 4-6 hours. The cells were collected and stained with BODIPY probe to detect the lipid peroxidation levels of each group of cells. The results are shown in Table 4, and the optimal mitoQ anti-inflammatory concentration was finally determined.

[0074] Table 4: Anti-inflammatory effects of different proportions of mitoQ mixed with hollow mesoporous copper sulfide nanoparticles

[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 hydrated particle size, polydispersity index and zeta potential were determined by a nanoparticle size analyzer (Mastersizer 3000E, UK). The mCuS@lm prepared in Example 2 was suspended in normal saline at room temperature for 15 days for stability testing. The morphology of CuS@lm and mCuS@lm was detected by high-resolution 200kV TEM (JEM-2100Plus, Japan). In short, the nanoparticles were subjected to 10 minutes of ultrasonic treatment before use. Next, a drop of nanoparticles was dropped on a carbon-coated copper grid. Excess liquid was absorbed with filter paper perpendicular to the copper grid. Protein composition was identified by SDS-PAGE and Coomassie Brilliant Blue staining.

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

[0079] from Figure 3 As can be seen from a in the figure, the average particle size of the hollow mesoporous copper sulfide nanoparticles is 159.9 nm, the average particle size of the mCuS@lm nanoparticles is 167.2 nm, and the average thickness increases by 7.3 nm, which indicates that the cell membrane trained by MRSA infection can be successfully coated on the surface. Figure 3 Figure b shows that the average Zeta potential of mitoQ is 12.32mV. Its positive charge can be widely and rapidly taken up by mitochondria driven by the mitochondrial membrane potential. Luteolin carries a negative charge on its surface and can self-assemble with mitoQ in the nanocage through electrostatic adsorption. The average Zeta potential of hollow mesoporous copper sulfide nanoparticles is -9.12mV, and the average Zeta potential of mCuS@lm nanoparticles is -9.80mV, which is in line with research expectations. Subsequently, we tested the stability of mCuS@lm nanoparticles in aqueous solution. Figure 3As shown in the figure, mCuS@lm nanoparticles can maintain a stable particle size within 15 days. Finally, we tested the UV-Vis-NIR spectra of luteolin, mitoQ, hollow mesoporous copper sulfide nanoparticles and mCuS@lm nanoparticles ( Figure 3 d), it was found that mitoQ had a characteristic absorption peak at 400nm, luteolin had a characteristic absorption peak at 350nm, and hollow mesoporous copper sulfide nanoparticles had a characteristic absorption peak at 900nm. By detecting the spectrum of mCuS@lm nanoparticles, we found that the nanoparticles were successfully loaded with two drugs. Figure 3 As shown in the figure, compared with the nanoparticles before training, the mCuS@lm nanoparticles after training can more effectively neutralize the inflammatory factors IL-1b and TNF-a.

[0080] Example 6

[0081] Targeting test of mCuS@lm nanocages: Non-trained nanoparticles labeled with Nile red staining and trained nanoparticles labeled with Nile red staining were prepared for bacterial targeting test. MRSA and Pseudomonas aeruginosa were cultured in TSB and LB medium, respectively. Then, these nanoparticles were incubated with bacteria for different times. The fluorescence signal of bacteria was detected by flow cytometry (BD Bioscience, USA). Subsequently, MH-S cells were infected with MRSA for 2h and then incubated with trained nanoparticles and untrained nanoparticles for different times. Cell fluorescence signals were recorded using a confocal fluorescence microscope (Zeiss, Germany). Mouse pneumonia targeting experiments were performed 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, lungs, liver, kidneys, spleen, stomach, intestines) were isolated, and their fluorescence signals were recorded and analyzed.

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

[0083] like Figure 4As shown in (b), at 4 h, 99.6% of MRSA bacteria could bind to the fluorescently labeled tmCuS@lm nanoparticles. In contrast, only 40.5% of MRSA bacteria could bind to the fluorescently labeled mmCuS@lm nanoparticles, which indicates that the MRSA-trained alveolar macrophage biomimetic membrane can give the nanoparticles better MRSA infection targeting ability. Figure 4 This conclusion was verified at the TEM level.

[0084] like Figure 4 As shown in the in vivo fluorescence imaging results in Figure d, in the CuS@lm group of mice, the fluorescence signal was distributed throughout the body, and the fluorescence intensity gradually weakened over time. On the third day after injection, the fluorescence intensity of the nanoparticles was mainly concentrated in the liver. Figure 4 The in vitro fluorescence imaging results of e in the figure are 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 lungs, and the in vitro fluorescence imaging results were basically consistent with the in vivo fluorescence intensity distribution. In summary, after coating the membrane of MRSA-infected alveolar macrophages, mCuS@lm nanoparticles can effectively target the lungs after MRSA infection.

[0085] Example 7

[0086] Bacterial culture and counting: MRSA and Pseudomonas aeruginosa were cultured in TSB and LB medium, respectively. The bacterial suspension was centrifuged at 4000 rpm for 10 min, then the bacterial suspension was double diluted and incubated on the corresponding TSA and LB agar plates at 37°C for 12 h, and photos were taken and colony counts were performed using Image J.

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

[0088] Biofilm formation and destruction detection: The bacterial suspension was cultured at 37°C for 72 h to form a mature biofilm. The mature biofilm was treated with different nanoparticles for 3 h, washed, stained with SYTO 9 and PI for 15 min at 37°C, and finally 3D images of the biofilm were taken using a confocal fluorescence microscope (Zeiss, Germany). The residual biofilm after washing was 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 with ethanol and acetone in turn. The bacterial morphology was observed using transmission electron microscopy (Thermo FEI, Tecnai G2 spirit, Czech Republic) and scanning electron microscopy (Thermo FEI, NovaNano 450, Czech Republic).

[0090] like Figure 5 As shown in a, almost all bacteria were killed in the mCuS@lm and mCuS@lm+NiR treatment groups 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 the large leakage of copper ions, the nanocages showed better antibacterial effect under low-infrared irradiation. The mCuS@lm and mCuS@lm+NiR treatment groups achieved almost 100% antibacterial rate against Pseudomonas aeruginosa and ciprofloxacin-evolved Pseudomonas aeruginosa, which was similar to the results of MRSA ( Figure 5 b and Figure 5 c). MRSA live / dead staining showed that the mCuS@lm+NiR treatment group exhibited the best antibacterial ability ( Figure 5 d and Figure 5 e) in.

[0091] TEM( Figure 6 a) and SEM( Figure 6 b) The test results in the untreated group showed that the untreated group showed a complete rod-shaped (Pseudomonas aeruginosa) or spherical (MRSA) morphology, and the cell membrane was continuous and intact. In the treated group, the bacteria showed severe morphological deformities, with wrinkling, membrane rupture, cytoplasmic leakage and even cracking. This confirms that the nanocage can destroy the bacterial membrane and change the permeability of the cell membrane, which may be related to the ROS produced by mCuS@lm and copper poisoning that destroy the lipid bilayer, leading to rapid leakage of the cell matrix. In addition, mature biofilms can be destroyed by mCuS@lm nanocages ( Figure 6 c and Figure 6 d), while the biofilm of the untreated group showed more compact characteristics, indicating that it was a more vigorous bacteria.

[0092] Compared with other nanomaterials that induce bacterial copper death, which can easily cause damage to host cells, mCuS@lm nanocages have fewer side effects on healthy cells. Figure 7 a and Figure 7As shown in (b), lipid peroxidation increased more than 3 and 2 times in cells incubated with mCuS and mCuS+NiR, respectively. However, the mCuS@lm group and mCuS@lm+NiR group had a milder lipid peroxidation level due to the addition of mitoQ components. However, there was no significant difference in the copper accumulation level between the groups ( Figure 7 in ). Figure 7 Figure d shows that most of the MRSA in the mCuS@lm+NiR group underwent lipid peroxidation, while the lipid peroxidation in the mCuS and mCuS+NiR groups was milder. Figure 7 e in the figure demonstrates that MRSA experienced the most ROS attack in the mCuS@lm+NiR group. In summary, QS inhibitors did not affect the cytoprotective effect of mCuS@lm, while mitoQ did not affect its copper-death bactericidal effect. Prokaryotic cells have a lower tolerance to oxidative stress than eukaryotic cells. Bacteria can be quickly eliminated by nanoparticles, and then mitochondria-targeted antioxidants can protect normal cells.

[0093] Example 8

[0094] Establishment of acute pneumonia model in mice: Acute pneumonia model was established in 6-8 week old male C57BL / 6 mice (25-30g). Animals were kept in a standard environment and the whole process was performed on a sterile bench. 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, blunt dissection was performed to fully expose the trachea. 50 μL of MRSA (5×10 7 CFU) were slowly injected into the mouse trachea, and then the mice were stood upright and gently shaken to evenly distribute the bacteria in the lungs. The mice were then randomly divided into groups. 2 hours after infection, different nanoparticles were injected intravenously. The NiR group was irradiated with a 1064nm laser. That is, the entire chest area was irradiated with a low dose of 0.3W / cm2 from outside the chest cavity for 10 minutes. The mice were killed at different times. The lung tissues were separated and photographed to prepare for subsequent experiments. In the survival rate and core body temperature study, mice were infected with a lethal dose of MRSA (50μL, 5×10 8 CFU) infection.

[0095] qPCR: Relative expression was normalized by Actin level. After treatment, lung tissue was isolated and ground to fully dissolve. RNA was reverse transcribed into complementary DNA using a reverse transcription kit. qPCR was performed using a 20 μL system, including 2 μL cDNA, 0.4 μL forward and reverse primers, 10 μL SYBR, and 7.2 μL double distilled water.

[0096] In vivo and in vitro fluorescence imaging: The USA300 strain was transformed with the luciferase gene and named Luci-MRSA in this study. Luci-MRSA was used to establish an acute pneumonia model. After the corresponding treatment, the mice were intraperitoneally injected with potassium luciferin. The in vivo chemiluminescence signal was measured using IVIS (PerkinElmer, USA) at 3h and 6h, and then the lungs of each group were separated at 12h and 24h, and the corresponding fluorescence intensity was recorded and analyzed.

[0097] Histological staining and analysis: Lungs were collected, fixed with 4% paraformaldehyde, and embedded in paraffin. HE staining, Gram staining, and MPO immunohistochemical staining were performed using standard methods. 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). DAPI and DHE staining were used for 15 min, and images were taken using a confocal fluorescence microscope (Zeiss, Germany). Images and sections were analyzed blindly and counted using ImageJ. All scoring was performed independently by 2 professionals who were not involved in the animal experiments.

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

[0099] like Figure 8 As shown in a, we found that 16 hours after infection, the body temperature of mice in the control group dropped significantly and was below 32°C. In contrast, the mice treated with mCuS@lm+NiR were able to maintain normal rectal temperature, thereby improving the survival rate of mice. Figure 8 As shown in b, we took pictures of the lungs of treated mice. The results showed that the PBS group had more obvious pulmonary hemorrhage, edema and other lung tissue consolidation. The degree of lung tissue inflammation in the other treatment groups was reduced. However, in comparison, the lung damage of mice in the mCuS@lm+NiR group was the mildest. Moreover, the wet weight and dry weight of the lungs of mice in the mCuS@lm+NiR group after infection were the smallest ( Figure 8 c), indicating that mCuS@lm+NiR treatment can reduce the damage of alveolar capillary endothelial cells and alleviate pulmonary edema.

[0100] We then performed bronchoalveolar lavage on the treated mice and studied the degree of inflammation by detecting the number of cells and protein content in the bronchoalveolar lavage fluid of the mice. Figure 8 d and Figure 8 As shown in Figure e, the number of cells and protein concentration in the alveolar lavage fluid of mice in the control group were the highest, while after treatment with mCuS@lm+NiR nanoparticles, the cell infiltration in the alveoli of mice decreased and the protein concentration decreased. This shows that mCuS@lm+NiR can effectively improve the lung inflammation of mice with acute MRSA infection and reduce pulmonary edema.

[0101] like Figure 8 As shown in g, 48 hours after MRSA infection, the lung tissue of the control group was infiltrated with a large number of inflammatory cells, secretions were found in the alveolar cavity, the alveolar wall was thickened and the structure was destroyed, and the surrounding blood vessels were edematous and necrotic. In contrast, in the lung tissue sections of mice in the mCuS@lm+NiR treatment group, we can find that the degree of lung damage is reduced, the lung tissue is basically normal, and most of the alveolar structures seen under the microscope are relatively intact, without obvious bleeding and edema. The lung injury score also confirmed this conclusion ( Figure 8 f) in.

[0102] like Fig. 9 a and Fig. 9 As shown in (b), mCuS@lm+NiR treatment can significantly reduce bacterial CFU in the lungs. Fig. 9 Figure c shows that a large number of Gram-positive bacteria are accumulated in the lung tissue of the control mice. However, there are almost no purple-black bacterial clusters in the lung tissue of the mice treated with mCuS@lm+NiR. This confirms that our nanoparticles have excellent antibacterial effects.

[0103] like Fig.10As shown in the figure, in the blank DMEM medium, LM, CuS, and mCuS@lm+NiR treatment groups, the cell survival rate was close to 100%, indicating that mCuS@lm nanoparticles had no obvious cytotoxicity. In the live-death staining of BEAS-2B cells, almost all green fluorescence can be observed, indicating that most cells are still alive after being treated with mCuS@lm nanoparticles. The above results all show that mCuS@lm nanoparticles have low cytotoxicity and high biosafety. The serum and plasma of mice were taken on the 14th day after treatment in each group to test and analyze the blood biochemistry and blood routine results. The results showed that the number of white blood cells (WBC), red blood cells (RBC), hemoglobin concentration (HGB), platelet number (PLT), lymphocyte ratio, neutrophil ratio, macrophage ratio, and mean corpuscular hemoglobin concentration (MCHC) of mice were all within the normal range, indicating that the treatment of each group did not affect the blood system of 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 shows that the treatment of each group did not affect the liver and kidney function of mice. Finally, on the 14th day after each group of treatment, we dissected the hearts, lungs, livers, spleens, and kidneys of each group of mice for H&E staining. Fig.10 As shown in the figure, all the important organs in each group showed obvious inflammatory response, but still maintained normal tissue structure. The above results show that mCuS@lm nanoparticles have high biosafety in animals and are expected to be transformed into clinical practice.

Claims

1. A mCuS@1m antibacterial nanocage, characterized in that: With hollow mesoporous copper sulfide nanoparticles as the core, bacterial / fungal copper death amplifiers and cell copper death inhibitors are encapsulated in the hollow mesoporous copper sulfide nanoparticles through electrostatic adsorption self-assembly, and a bionic cell membrane is further coated on the surface of the hollow mesoporous copper sulfide nanoparticles to form a nanocage, namely the mCuS@lm antibacterial nanocage.

2. The mCuS@lm antibacterial nanocage according to claim 1, characterized in that: The hollow mesoporous copper sulfide nanoparticles are prepared by an ion exchange method; The bacterial / fungal copper death amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is at least one of the following: luteolin, coumarin, and curcumin; The cell copper death inhibitor is an antioxidant, and the antioxidant is at least one of the following: mitoQ, SKQ1, Mito-TEMPO; The bionic cell membrane is a cell membrane extracted after infection training using at least one of bacteria or fungi as training materials and immune cells as trained materials.

3. The mCuS@lm antibacterial nanocage according to claim 2, characterized in that: The bacteria used as training substances are selected from at least one of the following: Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Escherichia coli; The fungus used as the training substance is selected from at least one of the following: Candida albicans, yeast, Cryptococcus; The immune cell is at least one of the following: macrophages, neutrophils, dendritic cells, lymphocytes, stromal cells, and epithelial cells; The infection multiplicity during training is 5:1 to 50:1, and the training time is 6 to 48 hours.

4. The mCuS@lm antibacterial nanocage according to claim 1, characterized in that: The mass ratio of bacterial / fungal copper death amplifier to hollow mesoporous copper sulfide nanoparticles is 80:31.25-1000; The mass ratio of cell copper death inhibitor to hollow mesoporous copper sulfide nanoparticles is 0.125-1:31.25; The mass ratio of the bionic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:0.5-3.

5. A method for preparing mCuS@lm antibacterial nanocage, characterized in that: The following steps are involved: (1) preparing hollow mesoporous copper sulfide nanoparticles; (2) encapsulating the bacterial / fungal copper death amplifier and the cell copper death inhibitor in the hollow mesoporous nano copper sulfide nanoparticles through electrostatic adsorption self-assembly; (3) coating the bionic cell membrane on the surface of the hollow mesoporous copper sulfide nanoparticles loaded with bacterial / fungal copper death amplifier and cell copper death inhibitor obtained in step (2) to form a nanocage, namely the mCuS@lm antibacterial nanocage.

6. The method for preparing the mCuS@lm antibacterial nanocage according to claim 5, characterized in that: The hollow mesoporous copper sulfide nanoparticles are prepared by an ion exchange method; The bacterial / fungal copper death amplifier is a quorum sensing inhibitor, and the quorum sensing inhibitor is at least one of the following: luteolin, coumarin, and curcumin; The cell copper death inhibitor is an antioxidant, and the antioxidant is at least one of the following: mitoQ, SKQ1, Mito-TEMPO; The bionic cell membrane is a cell membrane extracted after infection training using at least one of bacteria or fungi as training materials and immune cells as trained materials.

7. The method for preparing the mCuS@lm antibacterial nanocage according to claim 6, characterized in that: The bacteria used as training substances are selected from at least one of the following: Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Escherichia coli; The fungus used as the training substance is selected from at least one of the following: Candida albicans, yeast, Cryptococcus; The immune cell is at least one of the following: macrophages, neutrophils, dendritic cells, lymphocytes, stromal cells, and epithelial cells; The infection multiplicity during training is 5:1 to 50:1, and the training time is 6 to 48 hours.

8. The method for preparing the mCuS@lm antibacterial nanocage according to claim 5, characterized in that: The mass ratio of bacterial / fungal copper death amplifier to hollow mesoporous copper sulfide nanoparticles is 80:31.25-1000; The mass ratio of cell copper death inhibitor to hollow mesoporous copper sulfide nanoparticles is 0.125-1:31.25; The mass ratio of the bionic cell membrane to the hollow mesoporous copper sulfide nanoparticles is 1:0.5-3.

9. Use of the mCuS@lm antibacterial nanocage according to any one of claims 1 to 4 in the preparation of antibacterial and / or anti-inflammatory materials.

10. An antibacterial and / or anti-inflammatory material, characterized in that: It comprises the mCuS@lm antibacterial nanocage described in any one of claims 1 to 4.

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