Nano-drug for removing intracellular bacteria as well as preparation method and application of nano-drug

Abstract

The invention relates to the technical field of biological medicines, in particular to a nano-drug for removing intracellular bacteria as well as a preparation method and application of the nano-drug. The nano-drug is composed of hollow mesoporous nano-particles, an antibacterial drug loaded in the hollow mesoporous nano-particles, metabolism activation molecules grafted on the surfaces of the particles, and an immune cell fusion membrane coated on the outermost layer and pre-stimulated by inactivated bacteria. Through a synergistic effect mechanism of metabolism activation, targeted sterilization and immune awakening, the technical problems of intracellular bacterial metabolism dormancy, low drug targeted delivery efficiency and host immunosuppression microenvironment which are difficult to overcome in the prior art are effectively solved. Experiments show that the nano-drug can specifically target an infected part, reverse a bacterial metabolism dormancy state and synchronously realize efficient sterilization and immune activation, shows an excellent clearing effect in treatment of intracellular infection caused by staphylococcus aureus and the like, and can effectively establish lasting immune memory and remarkably reduce the infection recurrence rate.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanomedicine for eliminating intracellular bacteria, its preparation method, and its uses. Background Technology

[0002] Intracellular bacterial infection is a major clinical challenge. A prime example is Staphylococcus aureus, which can invade host cells such as macrophages and epithelial cells and survive long-term within the intracellular environment. This intracellular survival allows pathogens to effectively evade the host's immune system and most antibiotics. Furthermore, once inside the cell, these bacteria actively adjust to a metabolic dormant state, transforming into so-called "persistent bacteria." In this state, even if antibiotics can enter the cell, their significantly reduced bacterial metabolic activity makes them less effective at targeting the intended target, resulting in a substantial decrease in bactericidal efficacy. This directly leads to common clinical problems such as recurrent infections, prolonged treatment cycles, and even the development of chronic infections.

[0003] Current treatment options for this type of infection include systemic administration of antibiotics. However, because drug molecules have difficulty effectively penetrating cell membranes, they cannot reach effective bactericidal concentrations intracellularly. Persistent intracellular infection can lead to dysregulation of the host's immune function, particularly prompting macrophages to transform from the bactericidal M1 phenotype to the immunosuppressive M2 phenotype, creating a microenvironment unfavorable to pathogen clearance.

[0004] Recent research directions, such as using immune adjuvants alone to activate immune responses or leveraging cell membrane coating technology to enhance the targeting capabilities of nanoparticles, have shown some improvement in certain aspects, but none have systematically addressed the core issues of bacterial metabolic dormancy, low efficiency of targeted drug delivery, and suppression of the immune microenvironment simultaneously. For example, some research teams have developed cell membrane-coated nanosystems that primarily achieve targeting functions, while other studies have employed metal ion strategies that only focus on metabolic interference. These single-mechanism solutions are insufficient to achieve radical cure of intracellular infections. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of existing technologies in eradicating intracellular bacterial infections and the high recurrence rate of infections. It provides a nanomedicine for clearing intracellular bacteria, its preparation method and uses. This nanomedicine has multiple functions, including reversing bacterial metabolic dormancy, achieving precise drug delivery and rebuilding an effective immune response, thereby fundamentally solving the treatment problem of intracellular bacterial infections and achieving unexpected technical effects.

[0006] A first aspect of the present invention provides a nanomedicine for eliminating intracellular bacteria, comprising the following components:

[0007] Hollow mesoporous nanoparticles;

[0008] Antibacterial drugs are loaded into the hollow cavities and / or mesoporous channels of the hollow mesoporous nanoparticles;

[0009] Metabolic activating molecules are grafted onto the surface of the hollow mesoporous nanoparticles. The metabolic activating molecules are carbon or nitrogen sources that can be taken up by bacteria and participate in their metabolism.

[0010] And an immune cell membrane, which covers the outermost layer of the hollow mesoporous nanoparticles; wherein the immune cell membrane is a fusion membrane of macrophage membrane and dendritic cell membrane pre-stimulated by inactivated bacteria.

[0011] This invention provides a nanomedicine for eliminating intracellular bacteria. Hollow mesoporous nanoparticles serve as both drug carriers and immunoadjuvants. Antibacterial drugs are loaded into the cavities or mesoporous channels of the nanoparticles to achieve bactericidal function. Carbon or nitrogen sources that can be taken up by bacteria and participate in metabolism are grafted onto their surfaces as metabolic activating molecules. The outermost layer is coated with a fusion membrane of macrophage and dendritic cell membranes pre-stimulated by inactivated bacteria. Studies have found that this nanomedicine achieves remarkable technical effects by restoring bacterial sensitivity through metabolic activation, targeted delivery for precise bactericidal action, and synergistic activation of the host's innate and adaptive immunity through the immune membrane. This results in highly efficient elimination of latent pathogens within host cells, reversal of immunosuppression, and a significant reduction in infection recurrence rates.

[0012] Furthermore, the metabolic activating molecule is a carbohydrate or an amino acid; and / or, the antibacterial drug is an antibiotic.

[0013] Furthermore, the sugar is selected from one or more of maltose monohydrate, maltotriose, maltodextrin, glucose, and fructose; and / or the amino acid is selected from one or more of lysine, arginine, histidine, glutamic acid, and leucine; and / or the antibiotic is selected from one or more of rifampin, vancomycin, cephalosporins, amikacin, and aminoglycosides.

[0014] Furthermore, the hollow mesoporous nanoparticles are selected from one of hollow mesoporous copper sulfide, hollow mesoporous manganese dioxide, hollow mesoporous silica, hollow mesoporous titanium dioxide, and metal-organic framework materials.

[0015] Furthermore, the inactivated bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

[0016] Furthermore, the hollow mesoporous nanoparticles have a particle size of 50nm-150nm.

[0017] Furthermore, the hollow mesoporous nanoparticles were prepared using the following method:

[0018] S1. Preparation of template particles: In the presence of a polymer stabilizer, metal salts are reduced by a reducing agent in an alkaline environment to form solid or sacrificial template nanoparticles;

[0019] S2. Formation of hollow mesoporous structure: Add a mesoporous structure inducer and / or sulfur source to the product of S1, and generate a mesoporous shell in situ on the surface of the template particles through a chemical reaction. Simultaneously or subsequently, the template is removed to obtain hollow mesoporous nanoparticles.

[0020] Furthermore, the polymer stabilizer is polyvinylpyrrolidone.

[0021] Furthermore, the metal salt is a copper salt, manganese salt, silicon salt, or titanium salt.

[0022] Furthermore, in step S2, the reaction is carried out at 40℃-90℃ for 0.5-5h.

[0023] A second aspect of the present invention provides a method for preparing the above-mentioned nanomedicine, comprising the following steps:

[0024] Step 1: Load the antibacterial drug into the hollow mesoporous nanoparticles;

[0025] Step 2: Graft metabolic activation molecules onto the surface of hollow mesoporous nanoparticles loaded with antibacterial drugs to obtain modified nanoparticles.

[0026] Step 3: Mix the macrophage membranes and dendritic cell membranes pre-stimulated with inactivated bacteria to obtain a fusion membrane;

[0027] Step 4: Extrude the modified nanoparticles and the fusion film together, so that the fusion film coats the surface of the modified nanoparticles, thus obtaining the nanomedicine.

[0028] Furthermore, in step 2, the mass ratio of the metabolic activating molecules to the hollow mesoporous nanoparticles is 1:1-10; when the metabolic activating molecules include sugars and amino acids, the ratio of sugars to amino acids is 1-10:1.

[0029] Furthermore, in step 4, the protein mass ratio of the fusion membrane to the modified nanoparticles is 0.5-2:1, wherein the protein mass ratio of the macrophage membrane to the dendritic cell membrane is 1-3:1.

[0030] Furthermore, in step 2, grafting is achieved through a Schiff base reaction, specifically by reacting the amination-treated nanoparticles with a sugar solution in the presence of a reducing agent.

[0031] Furthermore, in step 3, the pre-stimulation time is 2 to 6 hours.

[0032] Furthermore, in step 4, the extrusion is performed by repeatedly extruding the polycarbonate film through a pore size of 1μm, 400nm, and 200nm 200 times in sequence using an extruder.

[0033] A third aspect of the invention provides the use of the above-described nanomedicine in the preparation of a medicament for the prevention and / or treatment of intracellular bacterial infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0034] Furthermore, the drug is used to prevent recurrence of infection.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1. This invention provides a nanomedicine for eliminating intracellular bacteria. Hollow mesoporous nanoparticles serve as the drug carrier and immunoadjuvant base. Antibacterial drugs are loaded within the cavities or mesoporous channels to achieve bactericidal function. Carbon or nitrogen sources that can be taken up by bacteria and participate in metabolism are grafted onto the surface as metabolic activating molecules. The outermost layer is coated with a fusion membrane of macrophage and dendritic cell membranes pre-stimulated by inactivated bacteria. Studies have found that this nanomedicine achieves outstanding technical effects by efficiently eliminating latent pathogens within host cells, reversing immunosuppression, and significantly reducing infection recurrence rates through a synergistic mechanism of restoring bacterial sensitivity through metabolic activation, targeted delivery for precise bactericidal action, and activation of the host's innate and adaptive immunity via the immune membrane.

[0037] 2. This invention provides a method for preparing nanomedicines. First, an antibacterial drug is loaded into hollow mesoporous nanoparticles to form a bactericidal core. Then, metabolic activation molecules are grafted onto the surface of these nanoparticles to endow them with the function of activating dormant bacteria. Next, macrophage membranes pre-stimulated by inactivated bacteria are mixed with dendritic cell membranes to prepare a biomimetic membrane material with active targeting and immune activation potential. Finally, a co-extrusion technique is used to achieve complete encapsulation of the aforementioned functionalized nanoparticles by immune cell membranes, thereby constructing a structurally complete multifunctional nanomedicine. A nanomedicine with efficient intracellular targeted delivery, specific bacterial metabolic activation, and synergistic immune regulation functions was successfully obtained, ultimately achieving complete elimination of persistent intracellular bacteria and demonstrating excellent therapeutic effects in preventing infection recurrence in animal models.

[0038] 3. This invention provides a novel use of the above-mentioned nanomedicine in the preparation of drugs for the prevention and / or treatment of intracellular bacterial infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus. Through the synergistic mechanism of the nanomedicine, the surface-grafted metabolic activating molecules can reverse the metabolic dormancy state of intracellular bacteria and restore their drug sensitivity. The antibacterial drug loaded in the hollow mesoporous structure can achieve intracellular targeted release and efficient bactericidal effect. The pre-stimulated immune cell membrane coating layer simultaneously endows the nanomedicine with the ability to actively target the infection site and activates the complete immune clearance pathway in the host body from macrophage M1 polarization to T cell immune response through antigen presentation and co-stimulatory signals. Ultimately, it achieves the complete elimination of intracellular Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, significantly reduces the risk of infection recurrence, and achieves the technical effect of fundamentally curing such refractory intracellular infections. Attached Figure Description

[0039] Figure 1 Characterization of the morphology and elemental distribution of hollow mesoporous copper sulfide nanoparticles.

[0040] Figure 2 Characterization of the structure and surface properties of hollow mesoporous copper sulfide nanoparticles with different modifications.

[0041] Figure 3 To detect the difference in receptor expression between pre-stimulated and unstimulated macrophages. Flow cytometry results showed that...

[0042] Figure 4 This study analyzed the expression of costimulatory molecules in pre-stimulated and unstimulated dendritic cells.

[0043] Figure 5 Morphological characterization of hollow mesoporous copper sulfide nanoparticles coated with cell membranes.

[0044] Figure 6 To evaluate the bacterial clearance effect of different treatment groups.

[0045] Figure 7 To analyze the clearance effect of different treatment groups on intracellular bacteria.

[0046] Figure 8 To analyze the regulatory effects of different treatment groups on macrophage immune phenotypes.

[0047] Figure 9 To evaluate the anti-infective effects of different treatment groups in a mouse model of chronic osteomyelitis.

[0048] Figure 10 Analysis of the regulatory effect of pM@GVCuS on mouse bone marrow immune cells. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0050] Unless otherwise specified, the use of terms such as "upper," "lower," "left," "right," "center," "inner," and "outer" to indicate orientation or positional relationships in the description of specific embodiments of the present invention is based on the orientation or positional relationships shown in the accompanying drawings, or the orientation or positional relationship in which the product / equipment / device is typically placed during use. These terms are merely for the purpose of facilitating the description of the present invention or simplifying the description in specific embodiments, enabling those skilled in the art to quickly understand the solution, and do not indicate or imply that a particular device / component / element must have a specific orientation, or be constructed and operated in a specific positional relationship. Therefore, they should not be construed as limitations on the present invention.

[0051] Furthermore, in the description of the embodiments of the present invention, "several", "more than", and "a number of" represent at least two. The number can be any number, such as two, three, four, five, six, seven, eight, nine, or even more than nine.

[0052] Existing technologies struggle to eradicate intracellular bacterial infections, and these infections are prone to recurrence.

[0053] The first aspect of this embodiment provides a nanomedicine for eliminating intracellular bacteria, comprising the following components:

[0054] Hollow mesoporous nanoparticles;

[0055] Antibacterial drugs are loaded into the hollow cavities and / or mesoporous channels of the hollow mesoporous nanoparticles;

[0056] Metabolic activating molecules are grafted onto the surface of the hollow mesoporous nanoparticles. The metabolic activating molecules are carbon or nitrogen sources that can be taken up by bacteria and participate in their metabolism.

[0057] And an immune cell membrane, which covers the outermost layer of the hollow mesoporous nanoparticles; wherein the immune cell membrane is a fusion membrane of macrophage membrane and dendritic cell membrane pre-stimulated by inactivated bacteria.

[0058] This embodiment provides a nanomedicine for eliminating intracellular bacteria. Hollow mesoporous nanoparticles serve as the drug carrier and immunoadjuvant base. Antibacterial drugs are loaded within the cavities or mesoporous channels to achieve bactericidal function. Carbon or nitrogen sources that can be taken up by bacteria and participate in metabolism are grafted onto the surface as metabolic activating molecules. The outermost layer is coated with a fusion membrane of macrophage and dendritic cell membranes pre-stimulated by inactivated bacteria. Studies have found that this nanomedicine achieves remarkable technical effects by restoring bacterial sensitivity through metabolic activation, targeted delivery for precise bactericidal action, and synergistic activation of the host's innate and adaptive immunity through the immune membrane. This results in highly efficient elimination of latent pathogens within host cells, reversal of immunosuppression, and a significant reduction in infection recurrence rates.

[0059] In some embodiments, the metabolic activating molecule is a sugar or an amino acid; and / or, the antibacterial agent is an antibiotic.

[0060] In some embodiments, the sugar is selected from one or more of maltose monohydrate, maltotriose, maltodextrin, glucose, and fructose; and / or the amino acid is selected from one or more of lysine, arginine, histidine, glutamic acid, and leucine; and / or the antibiotic is selected from one or more of rifampin, vancomycin, cephalosporins, amikacin, and aminoglycosides.

[0061] In some embodiments, the hollow mesoporous nanoparticles are selected from one of hollow mesoporous copper sulfide, hollow mesoporous manganese dioxide, hollow mesoporous silica, hollow mesoporous titanium dioxide, and metal-organic framework materials.

[0062] In some embodiments, the inactivated bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

[0063] In some embodiments, the hollow mesoporous nanoparticles have a particle size of 50 nm to 150 nm. Controlling the particle size of the hollow mesoporous nanoparticles within a reasonable range helps to effectively balance the needs of long-term circulation and tissue enrichment in vivo. This avoids excessively rapid clearance of overly small particles (<10 nm) by the kidneys and prevents excessively large particles (200 nm) from being rapidly captured by the hepatic reticuloendothelial system, thereby significantly increasing the enrichment concentration of nanomedicines at the site of infection. Furthermore, the particle size of 50 nm to 150 nm provides the ideal specific surface area and pore volume for the hollow mesoporous structure, ensuring a high loading capacity of antibacterial drugs while providing sufficient space for surface grafting of metabolic activating molecules and the outer coating of immune cell membranes. Ultimately, through the synergistic effect of optimized cellular uptake, prolonged blood circulation, and enhanced drug loading, it significantly improves the clearance efficiency of intracellular persistent bacteria and reduces the risk of infection recurrence.

[0064] In some embodiments, hollow mesoporous nanoparticles are prepared by the following method:

[0065] S1. Preparation of template particles: In the presence of a polymer stabilizer, metal salts are reduced by a reducing agent in an alkaline environment to form solid or sacrificial template nanoparticles;

[0066] S2. Formation of hollow mesoporous structure: Add a mesoporous structure inducer and / or sulfur source to the product of S1, and generate a mesoporous shell in situ on the surface of the template particles through a chemical reaction. Simultaneously or subsequently, the template is removed to obtain hollow mesoporous nanoparticles.

[0067] In some embodiments, the polymer stabilizer is polyvinylpyrrolidone.

[0068] In some embodiments, the metal salt is a copper salt, manganese salt, silicon salt, or titanium salt.

[0069] In some embodiments, in step S2, the reaction is carried out at 40°C-90°C for 0.5-5 hours.

[0070] The second aspect of this embodiment provides a method for preparing the above-mentioned nanomedicine, comprising the following steps:

[0071] Step 1: Load the antibacterial drug into the hollow mesoporous nanoparticles;

[0072] Step 2: Graft metabolic activation molecules onto the surface of hollow mesoporous nanoparticles loaded with antibacterial drugs to obtain modified nanoparticles.

[0073] Step 3: Mix the macrophage membranes and dendritic cell membranes pre-stimulated with inactivated bacteria to obtain a fusion membrane;

[0074] Step 4: Extrude the modified nanoparticles and the fusion film together, so that the fusion film coats the surface of the modified nanoparticles, thus obtaining the nanomedicine.

[0075] In some embodiments, in step 2, the mass ratio of the added metabolic activation molecules to the hollow mesoporous nanoparticles is 1:1-10; when the metabolic activation molecules include sugars and amino acids, the ratio of sugars to amino acids is 1-10:1. Studies have found that when the ratio is below 1:10, the surface-grafted metabolic molecules are too few, making it difficult to effectively reverse the metabolic dormancy state of intracellular resident bacteria; while when the ratio is above 1:1, excessive surface modification will occupy the mesoporous channels, affecting drug loading and hindering subsequent cell membrane coating. Controlling the ratio of sugars to amino acids at 1:1 to 10:1 allows for the rapid provision of energy to awaken bacteria through sugars and the supplementation of nitrogen sources required for bacterial replication through amino acids, avoiding metabolic bias or incomplete activation caused by a single nutrient source, thereby achieving broad-spectrum metabolic activation of different types of resident bacteria. Ultimately, the synergistic optimization of these two ratios ensures that, under the premise of structural stability, the nanoparticles can most efficiently restart bacterial metabolism and restore their sensitivity to antibiotics. Preferably, in step 2, the mass ratio of the metabolic activating molecules to the hollow mesoporous nanoparticles is 1:1-5; when the metabolic activating molecules include sugars and amino acids, the ratio of sugars to amino acids is 5-10:1.

[0076] In some embodiments, in step 4, the protein ratio of the fusion membrane to the modified nanoparticles is 0.5-2:1, wherein the protein ratio of the macrophage membrane to the dendritic cell membrane is 1-3:1. Studies have found that when the total membrane protein ratio is below 0.5:1, a complete and continuous coating layer cannot be formed, causing the nanoparticles' immune camouflage to fail and making them easily cleared by the body; while when the ratio is above 2:1, excessive membrane material easily forms stray vesicles, not only causing waste but also competitively inhibiting targeting and immune activation functions. Macrophage membranes and dendritic cell membranes are fused in a specific ratio of 1:1 to 3:1. The macrophage membrane endows the nanoparticles with excellent inflammatory targeting and pathogen recognition capabilities, while the dendritic cell membrane provides powerful antigen delivery and T cell co-stimulatory signals. This specific ratio ensures that the two membrane proteins and their functional molecules are distributed in an optimized conformation within the fused membrane. This avoids both the incomplete immune response caused by the functional limitations of a single membrane and the functional interference caused by the imbalance in the ratio. As a result, a complete cascade reaction is synergistically triggered, from targeted delivery and innate immune activation to the establishment of adaptive immune memory, ultimately achieving complete clearance and long-lasting protection against intracellular infections.

[0077] In some embodiments, in step 2, grafting is achieved through a Schiff base reaction, specifically by reacting the amination-treated nanoparticles with a sugar solution in the presence of a reducing agent.

[0078] In some embodiments, the pre-stimulation time in step 3 is 2 to 6 hours.

[0079] In some embodiments, in step 4, extrusion involves repeatedly extruding the polycarbonate membrane through pores of 1 μm, 400 nm, and 200 nm 20–30 times using an extruder. This sequentially decreasing pore size design allows for precise control of the encapsulation process: first, the initial 1 μm pore size allows for a preliminary and gentle binding of immune cell membrane fragments to the nanoparticle core, avoiding structural damage due to excessive stress; subsequently, the progressive reduction in pore size to 400 nm and 200 nm forces the flexible cell membrane to fully extend and encapsulate under fluid shear force, ultimately forming a complete, dense, uniform, and correctly oriented biomimetic membrane layer on the nanoparticle surface. Studies have confirmed that extrusion fewer than 20 times cannot ensure complete and continuous encapsulation of all particles, resulting in incomplete immune camouflage and rapid clearance during systemic circulation; while extrusion more than 30 times may damage the native conformation and activity of membrane proteins (such as key co-stimulatory molecules like TLR receptors, CD86, and MHC-II) due to excessive mechanical stress, weakening their immune arousal function.

[0080] The third aspect of this embodiment provides the use of the above-described nanomedicine in the preparation of a medicament for the prevention and / or treatment of intracellular bacterial infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0081] In some embodiments, the drug is used to prevent recurrence of infection.

[0082] To better understand the technical solutions of the above embodiments, the following more detailed experimental examples are provided for further explanation.

[0083] Example 1

[0084] Preparation of hollow mesoporous copper sulfide nanoparticles

[0085] 100 μL of CuCl2 solution (0.5 M) was added to 25 mL of deionized water containing 0.24 g PVP-K30, and the mixture was magnetically stirred at room temperature. Then, 25 mL of NaOH solution (pH = 9.0) was added, followed by 6.4 μL of N2H4·H2O (50%), forming a pale yellow Cu2O sphere suspension. After 5 min, 200 μL of Na2S aqueous solution (320 mg / mL) was added. -1 The solution was heated at 60°C for 2 hours. Finally, the HCuS NPs were centrifuged at 12,000 rpm for 8 minutes and washed three times with deionized water and ethanol, respectively. The product was then dried overnight under vacuum at 60°C to obtain hollow mesoporous copper sulfide nanoparticles.

[0086] The PBS dispersion of hollow mesoporous copper sulfide nanoparticles prepared in Example 1 was dropped onto an ultrathin carbon support film. After drying at room temperature, its morphology and structure were observed using a transmission electron microscope at an accelerating voltage of 200 kV. Simultaneously, elemental energy dispersive spectroscopy (EDS) analysis was performed.

[0087] TEM images clearly show that the prepared nanoparticles are regular spheres with a distinct hollow mesoporous structure and uniform particle size distribution, with an average diameter of approximately 100 nm. EDS elemental mapping results indicate that Cu and S elements are uniformly distributed on the nanoparticles, confirming the successful formation of copper sulfide and the uniformity of the structure. This structure provides a large specific surface area and storage space for subsequent drug loading.

[0088] Pre-stimulation of macrophage membranes and dendritic cell membranes

[0089] 1) J774A.1 cells and DC2.4 cells were grown to a moderate density in T175 culture dishes. Then, the cells were stimulated with UV-inactivated methicillin-resistant Staphylococcus aureus for 3 hours;

[0090] 2) Pre-cool PBS, wash the cells twice, and use a cell scraper to scrape the adherent cells into a 15ml centrifuge tube. Centrifuge at 800g for 5 minutes.

[0091] 3) Prepare hypotonic lysis buffer with the following formula: magnesium chloride (2mM) + potassium chloride (10mM) + Tris-hydrochloric acid (20mM) mixed well. Add one EDTA-free protease inhibitor tablet to every 10ml of hypotonic lysis buffer.

[0092] 4) Add 2.5 ml of the above lysis buffer to each centrifuge tube, resuspend and mix well, vortex for 5 seconds and then let stand on ice for 5 minutes;

[0093] 5) Place the cells in an ultrasonic homogenizer for 5 minutes (power: 300W; frequency: 40kHz) under ice bath conditions to fully disrupt the cells, and then aliquot them into 1.5ml centrifuge tubes;

[0094] 6) Centrifuge at 3500g for 10 minutes at 4℃, and transfer the supernatant to a new 1.5ml centrifuge tube;

[0095] 7) Centrifuge at 16900g at 4℃ for 30 minutes. You will see sediment at the bottom of the centrifuge tube. Carefully remove the supernatant and store it in a -80℃ refrigerator for later use.

[0096] Preparation of nanomedicines for eliminating intracellular bacteria

[0097] First, the hollow mesoporous nanoparticles were amination-treated. 20 mL of PEI (1 mg / mL) was added to 20 mL of hollow mesoporous nanoparticles (1 mg / mL), and after stirring and mixing for 6 hours, the nanoparticles were washed and centrifuged to obtain amination-treated hollow mesoporous nanoparticles. Then, a selected sugar solution (1 mL, 1 mg / mL) was mixed with a hollow mesoporous nanoparticle solution (1 mL, 1 mg / mL) at 70°C, and 0.01 mg of NaBH4 was added. The reaction proceeded for 6 hours, allowing a Schiff base reaction to occur between the sugar molecules and the amination-treated hollow mesoporous nanoparticles. Similarly, after washing and centrifugation, sugar-modified hollow mesoporous nanoparticles were obtained. Then, vancomycin (10 mg / mL) was added and incubated with the sugar-modified hollow mesoporous nanoparticles for 12 h. After washing and centrifugation, drug-loaded nanoparticles were obtained. Finally, pre-stimulated macrophage membranes and dendritic cell membranes were mixed 1:1. The mixed cell membranes were then mixed with the hollow mesoporous nanoparticles obtained above at a concentration of 1:2. The mixture was repeatedly extruded through a polycarbonate membrane with pore sizes of 1 μm, 400 nm, and 200 nm 20–30 times. Finally, the cell membrane-coated nanoparticles were purified by centrifugation at 14,000 g for 30 min and stored in PBS at 4 °C until further use.

[0098] In this embodiment, the mass ratio of the metabolic activating molecules (carbohydrates) to the hollow mesoporous nanoparticles is approximately 1:2. The protein mass ratio of the fusion membrane to the modified nanoparticles is 1:1. Specifically, the protein mass ratio of the macrophage membrane to the dendritic cell membrane is 1:1.

[0099] like Figure 2 As shown, nitrogen adsorption-desorption tests were performed on blank CuS, vancomycin-loaded CuS (VCuS), and CuS co-loaded with sugars and vancomycin (GVCuS) to calculate their specific surface area (BET); at the same time, the Zeta potential of the products of each step was measured using a dynamic light scattering particle size analyzer.

[0100] BET testing showed that the specific surface area and pore volume of the material decreased significantly from blank CuS to the final GVCuS, directly proving that the drug vancomycin and sugar molecules were successfully loaded and filled into the mesopores and hollow cavities of the nanoparticles. Zeta potential testing showed that the potential of the nanoparticles shifted positively after amination treatment, and the potential changed significantly again after sugar modification, verifying from an electrochemical perspective that sugar molecules were successfully grafted onto the surface of the nanoparticles.

[0101] like Figure 3 and Figure 4 As shown, J774A.1 macrophages and DC2.4 dendritic cells pre-stimulated with inactivated MRSA (experimental group) and unstimulated cells (control group) were collected separately. Specific fluorescent antibodies were used to spectroscopy the TLR2 and TLR6 receptors on the cell membrane surface. Figure 3) and CD80, CD86, MHC-II molecules ( Figure 4 The cells were stained and then quantitatively analyzed by flow cytometry. Flow cytometry results showed that, compared with unstimulated cells, the expression levels of TLR2 and TLR6 receptors on the surface of pre-stimulated macrophage membranes were significantly upregulated. Figure 3 Simultaneously, the expression of co-stimulatory molecules CD80, CD86, and MHC-II on the surface of pre-stimulated dendritic cell membranes was significantly enhanced. Figure 4 This demonstrates that the pre-stimulation step successfully confers a high level of pathogen recognition and antigen delivery potential on the immune cell membrane.

[0102] The final prepared pM@GVCuS nanomedicine was dropped onto a TEM grid and observed after negative staining. Figure 5 As shown, a clear low-electron-density halo is visible around the nanoparticles, contrasting sharply with the high-electron-density HCuS core inside. This directly confirms that the pre-stimulated immune cell membranes have successfully coated the nanoparticle surface, forming a complete core-shell structure.

[0103] Example 2

[0104] In vitro antibacterial and immunomodulatory function verification

[0105] Free vancomycin (Vanco), pM@VCuS (sugar-free), pM@GCuS (drug-free), and the pM@GVCuS of the present invention were co-incubated with MRSA, and the bactericidal effect was evaluated by (a) plate colony counting, (b) fluorescent staining of live and dead bacteria, and (c) crystal violet staining.

[0106] like Figure 6 As shown, multiple test results consistently indicate that only the pM@GVCuS group of this invention achieved a near 100% bacterial clearance rate. The pM@VCuS group (with drug but no sugar) and the Vanco group showed incomplete sterilization, while the pM@GCuS group (with sugar but no drug) had almost no bactericidal effect. This demonstrates that both carbohydrate metabolism activation and antibiotic sterilization are indispensable, and that they produce a synergistic effect in the system of this invention.

[0107] A macrophage intracellular MRSA infection model was established. The cells were treated with pM@VCuS and pM@GVCuS, respectively. The intracellular bacteria were then stained with specific fluorescent dyes and observed using a confocal microscope.

[0108] like Figure 7 As shown, almost no fluorescent signal was observed in the cells of the pM@GVCuS-treated group, indicating that intracellular bacteria were completely eliminated. However, obvious fluorescent spots of bacteria were still observed inside the cells of the pM@VCuS-treated group, proving the presence of residual bacteria. This result directly confirms the unique advantage of this invention in eliminating residual intracellular bacteria through metabolic activation.

[0109] M2 macrophages were co-cultured with (a) unstimulated cell membrane-coated M@GVCuS or (b) prestimulated cell membrane-coated pM@GVCuS, and the expression levels of M1 / M2 phenotypic markers (CD86, CD206, PD-L1) and MHC-II were detected by flow cytometry.

[0110] like Figure 8 As shown, compared with the M@GVCuS group, pM@GVCuS treatment significantly upregulated the expression of M1 markers CD86 and MHC-II, while downregulating the expression of M2 markers CD206 and the immunosuppressive molecule PD-L1. This indicates that the nanomedicine of the present invention, especially its pre-stimulated cell membrane component, can effectively reverse the immunosuppressive microenvironment and reprogram M2 macrophages into the M1 phenotype with bactericidal and antigen-transfer capabilities.

[0111] Example 3

[0112] Evaluation of in vivo anti-infection and recurrence prevention effects

[0113] A chronic osteomyelitis model was established in mice, and the mice were randomly divided into PBS, free vancomycin, pM@VCuS, and pM@GVCuS treatment groups. Bioluminescent signals at the infection site in the femur were continuously monitored using an in vivo imaging system to assess the degree of infection and recurrence.

[0114] like Figure 9 As shown, both the vancomycin group and the pM@VCuS group were able to suppress infection in the initial stage of treatment, but infection recurrence occurred on days 14 and 21, respectively. In stark contrast, no recurrence signal was detected in the pM@GVCuS group throughout the entire 21-day observation period, achieving radical cure of the infection. This strongly demonstrates the persistence and superior efficacy of the "metabolic activation-bactericidal-immune awakening" three-pronged strategy of this invention in preventing infection recurrence.

[0115] At the end of treatment, mouse bone marrow cells were collected and subjected to comprehensive immune cell subset analysis by flow cytometry.

[0116] like Figure 10As shown, in vivo data and in vitro results corroborate each other. pM@GVCuS treatment significantly increased the proportion of CD86+CD11c+ (activated dendritic cells) and MHC-II+CD11c+ (dendritic cells with strong antigen-transmitting capacity) in the bone marrow, and increased the proportion of CD86+F4 / 80+ (M1 macrophages) while decreasing the proportion of CD206+F4 / 80+ (M2 macrophages). More importantly, in terms of adaptive immunity, pM@GVCuS treatment significantly increased the proportion of IFN-γ+CD4+ and IFN-γ+CD8+ (effective T cells) and decreased the proportion of LAG3+CD4+ and LAG3+CD8+ (exhausted T cells). These data, from both innate and adaptive immune perspectives, fully reveal the deep mechanism by which the nanomedicine of this invention achieves complete eradication of infection and prevention of recurrence by reshaping the immune microenvironment and establishing an effective immune response.

[0117] The nanomedicine provided in this embodiment consists of biocompatible hollow mesoporous nanoparticles, a drug loaded in the hollow mesoporous spaces of the nanoparticles, sugars grafted onto the surface of the nanoparticles, and a pre-stimulated cell membrane. The hollow mesoporous nanoparticles are nanoparticles that can activate cellular immunity, such as hollow mesoporous copper sulfide or hollow mesoporous manganese dioxide nanoparticles. The drug is a drug that can treat bacterial infections, such as antibiotics rifampin or vancomycin. The sugars are monosaccharides, oligosaccharides, and polysaccharides that can be taken up and utilized by bacteria, such as maltose monohydrate, maltose, and maltodextrin. The pre-stimulated cell membrane is a macrophage membrane and a dendritic cell membrane pre-stimulated by scavenging bacteria. This nanomedicine can target and accumulate at the site of infection through chemotaxis, and enter the cell interior of the infected intracellular bacteria through membrane fusion. Through uptake and utilization by the intracellular bacteria, it further targets the intracellular bacteria and exerts metabolic activation on the intracellular bacteria, thereby effectively clearing them. Meanwhile, the membrane is derived from pre-activated macrophages and dendritic cells, and compared to ordinary cell membranes, it carries specific bacterial antigen information as well as membrane surface activation receptors and co-stimulatory molecules.

[0118] By coating the surface of nanoparticles with the membrane, the following effects can be achieved:

[0119] 1. Specific pathogen recognition: The pre-stimulated membrane surface expresses pattern recognition receptors such as TLR2 and TLR6, as well as co-stimulatory molecules such as CD80, CD86 and MHC-II, enabling nanoparticles to specifically recognize and bind to bacterial antigens.

[0120] 2. Activation of innate immunity: The membrane-modified nanoparticles can activate immunosuppressive M2 macrophages into bactericidal M1 macrophages, while enhancing antigen delivery capacity (MHC-II high).

[0121] 3. Adaptive immune enhancement: By promoting the expression of dendritic cell co-stimulatory molecules, the membrane-modified nanoparticles can activate helper T cells, increase the proportion of IFNγ+CD8+ and IFNγ+CD4+ cells, and inhibit LAG3+ suppressor T cells, thereby enhancing antigen-specific immune responses.

[0122] Comparative Example 1

[0123] Compared to Example 1, the unstimulated fusion membrane was used, but the rest of the process was the same as in Example 1.

[0124] Unstimulated fusion membrane + contains metabolic activating molecules + contains antibiotics.

[0125] Compared to the fusion membrane of unstimulated macrophages and unstimulated dendritic cells, the fusion membrane obtained by pre-stimulation treatment in this invention exhibits significantly enhanced overall performance under the same drug administration conditions: the enrichment of infection foci is increased by approximately 2.3 times, the expression of the immunosuppression-related marker PD-L1 is reduced by approximately 0.6 times, and the expression of the immune activation-related marker CD86 is increased by approximately 1.4 times. These results indicate that the pre-stimulation step can significantly enhance the fusion membrane's ability to target and recognize infection foci, and effectively enhance the awakening and activation of the host immune system, providing a key technological basis for the efficient clearance of intracellular bacteria and drug-resistant bacteria.

[0126] Comparative Example 2

[0127] Compared to Example 1, Comparative Example 2 used macrophage membranes that had been pre-stimulated with inactivated cells, and did not use dendritic cell membranes. The other processes were the same as in Example 1, forming a single-factor comparative experiment.

[0128] Pre-stimulated single macrophage membrane + metabolic activating molecules + antibiotics.

[0129] Compared to the treatment method of pre-stimulated single macrophage membrane combined with bacterial metabolic activating molecules and antibiotics, the pre-stimulated macrophage membrane-dendritic cell fusion membrane described in this invention can significantly enhance the immunomodulatory effect in vivo, with antigen presentation-related functions increased by approximately 1.6–1.9 times and adaptive immune response-related indicators increased by approximately 3.7–5.3 times. These results indicate that by fusing the pre-stimulated macrophage membrane with the dendritic cell membrane, antigen presentation and subsequent T-cell immune responses can be further amplified on the basis of innate immune recognition, thereby achieving stronger immune activation and synergistic anti-infection effects.

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanomedicine for eliminating intracellular bacteria, characterized in that, It consists of the following parts: Hollow mesoporous nanoparticles; Antibacterial drugs are loaded into the hollow cavities and / or mesoporous channels of the hollow mesoporous nanoparticles; Metabolic activating molecules are grafted onto the surface of the hollow mesoporous nanoparticles. The metabolic activating molecules are carbon or nitrogen sources that can be taken up by bacteria and participate in their metabolism. And an immune cell membrane, which covers the outermost layer of the hollow mesoporous nanoparticles; wherein the immune cell membrane is a fusion membrane of macrophage membrane and dendritic cell membrane pre-stimulated by inactivated bacteria.

2. The nanomedicine for eliminating intracellular bacteria according to claim 1, characterized in that, The metabolic activating molecule is a sugar or amino acid; and / or, the antibacterial drug is an antibiotic; and / or, the hollow mesoporous nanoparticles are selected from one of hollow mesoporous copper sulfide, hollow mesoporous manganese dioxide, hollow mesoporous silica, hollow mesoporous titanium dioxide, and metal-organic framework materials; and / or, the inactivated bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

3. The nanomedicine for eliminating intracellular bacteria according to claim 2, characterized in that, The sugar is selected from one or more of maltose monohydrate, maltotriose, maltodextrin, glucose, and fructose; and / or the amino acid is selected from one or more of lysine, arginine, histidine, glutamic acid, and leucine; and / or the antibiotic is selected from one or more of rifampin, vancomycin, cephalosporins, amikacin, and aminoglycosides.

4. The nanomedicine for eliminating intracellular bacteria according to claim 1, characterized in that, The hollow mesoporous nanoparticles have a particle size of 50nm-150nm.

5. The method for preparing nanomedicines for eliminating intracellular bacteria as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Load the antibacterial drug into the hollow mesoporous nanoparticles; Step 2: Graft metabolic activation molecules onto the surface of hollow mesoporous nanoparticles loaded with antibacterial drugs to obtain modified nanoparticles. Step 3: Mix the macrophage membranes and dendritic cell membranes pre-stimulated with inactivated bacteria to obtain a fusion membrane; Step 4: Extrude the modified nanoparticles and the fusion film together, so that the fusion film coats the surface of the modified nanoparticles, thus obtaining the nanomedicine.

6. The method according to claim 5, characterized in that, In step 2, the mass ratio of the metabolic activating molecules to the hollow mesoporous nanoparticles is 1:1-10; when the metabolic activating molecules include sugars and amino acids, the ratio of sugars to amino acids is 1-10:

1.

7. The method according to claim 5, characterized in that, In step 4, the protein ratio of the fusion membrane to the modified nanoparticles is 0.5-2:1, wherein the protein ratio of the macrophage membrane to the dendritic cell membrane is 1-3:

1.

8. The method according to claim 5, characterized in that, In step 2, grafting is achieved through a Schiff base reaction, specifically by reacting the amination-treated nanoparticles with a sugar solution in the presence of a reducing agent; and / or, in step 3, the pre-stimulation time is 2-6 hours.

9. The method according to claim 5, characterized in that, In step 4, extrusion is performed by repeatedly extruding polycarbonate films with pore sizes of 1μm, 400nm, and 200nm through an extruder 20 to 30 times.

10. Use of the nanomedicine according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of intracellular bacterial infections; wherein the intracellular bacterial infection is an infection caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

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