Metal-organic framework-based metal antibiotics and methods of making and using the same

Abstract

The application discloses a metal-organic framework metal antibiotic and a preparation method and application thereof, the metal antibiotic comprising manganese-containing nanoparticles; a PCN MOF material formed on an outer layer of the manganese-containing nanoparticles; and a hybrid membrane formed on an outer layer of the PCN MOF material, the hybrid membrane being composed of macrophages and neutrophils. The metal-organic framework metal antibiotic can activate a cGAS-STING pathway signal channel, achieve the purpose of treating diseases related to the cGAS-STING pathway, and inhibit inflammation caused by activation of the cGAS-STING pathway, especially against implant-related biofilm infection, can avoid excessive inflammation after infection clearance, promote tissue repair, and thus provide a new possibility for a new metal immunotherapy platform.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a metal-organic framework-based metal antibiotic and its preparation method, and also relating to the application of the metal-organic framework-based metal antibiotic in the preparation of drugs for treating diseases related to the cGAS-STING pathway. Background Technology

[0002] The cGAS-STING pathway is a major pathway responsible for the immune response to recognize cytoplasmic DNA. Studies have shown that invasion by various pathogens and various stressors, such as oxidative stress, metabolic disorders, and DNA damage, can lead to the accumulation of cytoplasmic DNA and Mn. 2+ Increased concentrations of STING activate the cGAS-STING pathway. Activation of the cGAS-STING pathway triggers a series of downstream signaling events, including stimulation and recruitment of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), thereby inducing the secretion and release of type I interferon (IFN-I) and pro-inflammatory factors IL-6 and TNF-α, thus mediating anti-infective / tumor immune responses. Therefore, the cGAS-STING signaling pathway plays a crucial role in combating pathogenic microbial infections, tumors, and the development and treatment of various immune-related diseases. However, STING activation can also have adverse effects; for example, excessive STING activation can lead to inflammatory responses, resulting in inflammation-related diseases.

[0003] Orthopedic implant infection is a common and catastrophic complication following various orthopedic implant surgeries, with biofilm formation being its essential characteristic. Implant-associated biofilm infections are primarily caused by multidrug-resistant bacteria. Due to the unique biological characteristics of biofilms, treatment efficacy is low and recurrence risk is high. Implant-attached bacteria such as *Escherichia coli* and methicillin-resistant Staphylococcus aureus (MRSA) reside within the biofilm, resisting antibiotics and the host's immune defense system, leading to persistent biofilm infection. Simultaneously, the evolution of bacterial resistance, along with other factors, contributes to the unsatisfactory efficacy of current treatments for implant-associated infections.

[0004] Current immunotherapies, which focus on innate immune cells such as neutrophils and macrophages, are ineffective against biofilms due to limited immune response efficiency. Worse still, mature bacterial biofilms can suppress antigen presentation by evading immune surveillance and subsequent elimination, characterized by low frequency of bacterial-associated antigen exposure, immunosuppression within the biofilm microenvironment, and even the killing of antigen-presenting cells. Furthermore, excessive activation of the pro-inflammatory immune microenvironment can lead to excessive inflammation-related diseases, hindering tissue repair at the site of infection in the later stages of treatment. Summary of the Invention

[0005] In view of this, the present invention needs to provide a metal-organic framework-based metal antibiotic that can controllably activate the cGAS-STING pathway signaling pathway to achieve the purpose of treating diseases involving the cGAS-STING pathway, and after disease treatment, inhibit the overactivation of the STING pathway to avoid excessive inflammation. In particular, it can effectively treat vegetative-associated biofilm infections, and after the infection is cleared, it can prevent excessive inflammation and promote tissue repair.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention first provides a metal-organic framework-based metal antibiotic, comprising:

[0008] Manganese-containing nanoparticles;

[0009] PCN MOF material, wherein the PCN MOF material is formed on the outer layer of the manganese-containing nanoparticles;

[0010] And a hybridization membrane formed on the outer layer of the PCN MOF material, the hybridization membrane being composed of macrophages and neutrophils.

[0011] The manganese-containing nanoparticles mentioned in this article refer to MnO2 nanoparticles or other nanoparticles containing Mn ions, wherein the nanoparticles are spherical. As a preferred embodiment of this application, the manganese-containing nanoparticles are MnO2 nanoparticles.

[0012] In a further embodiment, the particle size of the manganese-containing nanoparticles is preferably between 20 and 80 nm.

[0013] This metal-organic framework-based metal antibiotic uses manganese-containing nanoparticles as its core, with a porphyrin porous coordination network (PCN MOF) material and a hybrid membrane composed of neutrophils and macrophages sequentially coated on the surface of the manganese-containing nanoparticles from the inside out. Due to its ability to activate the cGAS-STING pathway, it can treat pathogenic microbial infections, tumors, and various immune-related diseases, and inhibit the inflammatory response following cGAS-STING pathway activation, thus achieving controllable activation of the cGAS-STING pathway. Taking plant-associated biofilm infections as an example, this metal antibiotic can generate ROS under US irradiation, disrupting the biofilm, killing some bacteria, and releasing bacterial dsDNA. The metal antibiotic modified with hybrid cell membranes has the ability to stimulate homing responses and inflammatory chemotaxis. Once administered, the manganese-containing nanoparticles can catalyze the conversion of hydrogen peroxide (H2O2) into oxygen, further promoting the efficiency of oxygen-dependent US-driven SDT, and killing bacteria through bacterial homeostasis (such as manganese ion excess, oxidative stress dysfunction, cell membrane integrity defects, and quorum sensing system disorder). The released dsDNA, as exposed bacterial-specific antigens (BAAs), escapes from the biofilm and is phagocytosed by antigen-presenting cells (APCs) such as macrophages and dendritic cells, activating the cGAS-STING pathway. Simultaneously, the manganese-enhanced activation of the cGAS-STING pathway promotes the polarization of M1 macrophages and the maturation of dendritic cells (DCs) characterized by the secretion of type I interferon-I (IFN-I) and the release of pro-inflammatory cytokines, thereby reversing the immunosuppressive state. This amplifies APC initiation and antigen presentation, leading to a biofilm-specific T cell response (CD4+, Th17), thereby inhibiting the biofilm. If the US (ultravascular antigen) is removed, oxygen is not consumed by the SDT (suppressive dermal antigen). Instead, the sustained oxygen production increases fibroblast survival and migration, promotes the expression of angiogenic growth factors and angiogenesis, and simultaneously enhances the secretion of anti-inflammatory factors. Excessive cGAS-STING / Th17-driven inflammation can be neutralized. In summary, this metal antibiotic is a metal antibiotic composed of a sound wave sensitizer, nanozyme, and bioactive metal ions, and it can serve as a promising new sound wave metal immunotherapy platform.

[0014] This invention further provides a method for preparing a metal-organic framework-based metal antibiotic, comprising the following steps:

[0015] S1. Provides manganese-containing nanoparticles;

[0016] S2. The manganese-containing nanoparticles, soluble metal salts and porphyrin organic ligands are mixed and reacted in a strongly polar solvent to obtain MnP nanoparticles, i.e. manganese-containing nanoparticles with PCN MOF material formed on the outer layer.

[0017] S3. Encapsulate the surface of the MnP nanoparticles with a hybrid membrane composed of macrophages and neutrophils to obtain MnPM nanoparticles, i.e., MnP nanoparticles with the hybrid membrane on the outer layer.

[0018] In a further embodiment, in step S1, the manganese-containing nanoparticles are MnO2 nanoparticles, which are self-made, and the preparation method includes the following steps:

[0019] Potassium permanganate and deionized water were mixed, and formamide was added dropwise. The mixture was stirred and reacted to obtain MnO2 nanoparticles.

[0020] This application employs a method that adjusts the proportions of raw materials in the preparation process to obtain MnO2 nanoparticles with ideal morphology and distribution. In some typical embodiments of this invention, the mass-to-volume ratio of potassium permanganate, deionized water, and formamide is 425-950 mg:480 mL:5-15 mL. Preferably, the mass-to-volume ratio of potassium permanganate, deionized water, and formamide is 950 mg:480 mL:15 mL, 425 mg:480 mL:15 mL, or 950 mg:480 mL:5 mL.

[0021] The stirring reaction can be carried out using conventional mechanical stirring methods in the field, preferably magnetic stirring, with a stirring speed of 250~800 rpm.

[0022] A further approach involves forming a PCN MOF material layer on the outer layer of manganese-containing nanoparticles via step S2, thereby obtaining MnP nanoparticles. The synthesis mechanism utilizes the coordination of metal ions and the self-assembly of organic ligands, along with Ostwald ripening-mediated PCN MOF encapsulation on the surface of manganese-containing nanoparticles.

[0023] In this article, soluble metal salts refer to soluble metal chlorides or soluble metal nitrates. Specific examples include, but are not limited to, zirconium chloride, zirconium oxychloride, strontium chloride, manganese chloride, zinc chloride, copper chloride, ferric chloride, zinc nitrate, gallium nitrate, or cerium ammonium nitrate.

[0024] The porphyrin organic ligands mentioned in this article refer to tetra(4-carboxyphenyl)porphyrin or derivative molecules containing a tetra(4-carboxyphenyl)porphyrin structure.

[0025] The strongly polar organic solvents mentioned in this article refer to organic solvents that can simultaneously dissolve soluble metal salts and porphyrin organic ligands. The specific types can be adjusted according to the types of soluble metal salts and porphyrin organic ligands.

[0026] In some typical embodiments of the present invention, the soluble metal salt is zirconium chloride, the porphyrin organic ligand is tetrakis(4-carboxyphenyl)porphyrin, and the strongly polar organic solvent is a mixed solvent of N,N-dimethylformamide and ethanol.

[0027] In the mixed solvent, the volume ratio of N,N-dimethylformamide to ethanol is 3:1 to 1:3.

[0028] In a further embodiment, in step S2, the mixing reaction is carried out under stirring at 90~140℃ for 6~24h.

[0029] In a further embodiment, step S3 includes: mixing neutrophil membrane aqueous dispersion, macrophage membrane aqueous dispersion and manganese nanoparticles, and then extruding them using an extrusion device to obtain MnPM nanoparticles.

[0030] Neutrophil membranes and macrophage membranes can be collected using methods commonly used in the art, such as gradient centrifugation and gradient freeze-thaw methods. In some typical embodiments of the present invention, neutrophil membranes are collected from centrifuges isolated from animals, preferably from 6-8 week old male mice; macrophage membranes are collected from RAW264.7 cells.

[0031] In a further embodiment, the mass ratio of the neutrophil membrane, macrophage membrane, and MnP nanoparticles is 1~3:1~3:0.25~4. Preferably, the mass ratio of the neutrophil membrane, macrophage membrane, and MnP nanoparticles is 1:1:4, 1:1:2, 1:1:1, 1:1:0.5, 1:1:0.25, 3:1:4, 3:1:2, 3:1:1, 3:1:0.5, 3:1:0.25, 1:3:4, 1:3:2, 1:3:1, 1:3:0.5, or 1:3:0.25.

[0032] In a further embodiment, in step S3, the mixing is performed using ultrasound combined with stirring. The ultrasound duration is 5-30 minutes; the stirring speed is 400-1200 rpm, and the stirring time is 5-60 minutes.

[0033] In a further embodiment, the extrusion pore size of the extrusion equipment is 400nm and 200nm, preferably a polycarbonate porous membrane with pore sizes of 400nm and 200nm; the number of extrusions is at least 20.

[0034] The present invention further provides the use of metal-organic framework-based metal antibiotics as described above, or metal-organic framework-based metal antibiotics prepared by the aforementioned preparation method, in the preparation of medicaments for treating diseases related to the cGAS-STING pathway.

[0035] The diseases associated with the cGAS-STING pathway include pathogenic microbial infections (including but not limited to bacterial biofilm infections, planktonic bacterial infections, and fungal infections) and tumors (including but not limited to colorectal cancer, breast cancer, ovarian cancer, or cervical cancer). Preferably, the cGAS-STING pathway-related diseases are endophyte infections.

[0036] The present invention further provides a medicament for treating diseases related to the cGAS-STING pathway, including metal-organic framework-based metal antibiotics as described above or metal-organic framework-based metal antibiotics prepared by the aforementioned preparation method.

[0037] It is understood that the drug may also include pharmaceutically acceptable carriers or excipients, the specific composition of which may be selected according to the drug’s dosage form, etc.

[0038] The drug is a non-gastrointestinal dosage form.

[0039] In a further embodiment, the dosage form of the drug is selected from at least one of the following: injectable dosage form, transdermal dosage form, cavity dosage form, and mucosal dosage form.

[0040] In a further embodiment, the dosage form of the drug is preferably an injectable dosage form.

[0041] Furthermore, the metal antibiotics and drugs described in this application are particularly suitable for the treatment of vegetative-associated biofilm infections. The MnPM nanoparticles in this application can be considered as a metal antibiotic composed of a sound wave sensitizer, nanozymes, and bioactive metal ions. This allows for effective treatment of vegetative-associated biofilm infections, and after infection clearance, the continuous generation of O2 inhibits inflammation-related diseases caused by STING activation, promotes the transformation of the tissue repair-related immune microenvironment, and avoids excessive inflammation.

[0042] Therefore, in some typical embodiments, the drug is preferably an immune adjuvant, which can be used for infection treatment in patients with periprosthetic infections before primary or secondary revision surgery, reducing the incidence of intraoperative infection or the recurrence rate of infection after revision surgery.

[0043] The beneficial effects of this invention are:

[0044] The metal antibiotic MnPM in this invention can simultaneously stimulate both the innate and adaptive immune systems, effectively kill bacteria, and prevent excessive inflammation, thereby achieving controllable activation of the cGAS-STING pathway and effectively treating diseases related to the cGAS-STING pathway.

[0045] Specifically, the metal antibiotic MnPM can be used in conjunction with immunogenic sonodynamic therapy to release bacterial-specific antigens, activate the cGAS-STING signaling pathway on antigen-presenting cells for efficient antigen presentation, and activate both innate and adaptive immune systems to combat vegetative-associated biofilm infections. Furthermore, after infection clearance, the metal antibiotic MnPM can continuously generate O2 without US irradiation, thereby inhibiting inflammation-related diseases caused by STING activation, promoting the transformation of the tissue repair-related immune microenvironment, and preventing excessive inflammation.

[0046] Furthermore, this invention also provides a controllable preparation method for the metal antibiotic MnPM. This method can prepare manganese-containing nanoparticles and porphyrin-based metal-organic framework (PCN)-based metal antibiotic MnPM encapsulated by hybrid cell membranes of neutrophils and macrophages, and the size and morphology of MnPM can be controllably adjusted. This preparation method has a short production cycle, which can be completed within 4-5 days, and the process exhibits good stability and reproducibility. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the structure of the MnPM nanoparticles prepared in Example 1;

[0048] Figure 2 The image shows a TEM image of the MnO2 nanoparticles prepared in Example 1.

[0049] Figure 3 TEM images of the MnP nanoparticles prepared in Example 1;

[0050] Figure 4 The following is an illustration of oxygen generation from MnO2 nanoparticles and MnP nanoparticles in Example 1: (A) O2 generation curve; (B) Photograph of oxygen bubbles generated by MnP nanoparticles in a solution with pH=5.0 and H2O2.

[0051] Figure 5 This is a curve showing the change in the amount of singlet oxygen generated by MnP nanoparticles in a solution with pH=5.0 and H2O2 under US irradiation as a function of irradiation time in Example 1.

[0052] Figure 6 TEM images of the MnPM nanoparticles prepared in Example 1;

[0053] Figure 7 This is a bar chart of bacterial plate counts from Example 2;

[0054] Figure 8 These are SEM images of the biofilm after treatment under different conditions in Example 2;

[0055] Figure 9 These are crystal violet-stained photographs of biofilms treated under different conditions in Example 2;

[0056] Figure 10 These are three-dimensional confocal fluorescence images of the biofilm after treatment under different conditions in Example 2;

[0057] Figure 11 This is a bar chart showing the percentage of relative Mn ion content in bacteria treated under different conditions in Example 3;

[0058] Figure 12 This is a bar chart showing the protein leakage content in bacteria after treatment under different conditions in Example 3;

[0059] Figure 13 This is a bar chart showing the bacterial membrane permeability of bacteria treated under different conditions in Example 3;

[0060] Figure 14 The bar chart shows the ROS content in bacteria after treatment under different conditions in Example 3.

[0061] Figure 15 These are the Western blot bands after different treatment conditions in Example 4;

[0062] Figure 16 The bar chart shows the ELISA results after different treatment conditions in Example 4;

[0063] Figure 17 This is a bar chart showing the cell migration rates after different treatment conditions in Example 5;

[0064] Figure 18 This is a bar chart showing the changes in scratch spacing after different treatment conditions in Example 5;

[0065] Figure 19 This is a bar chart showing the number of blood vessel rings after different treatment conditions in Example 5;

[0066] Figure 20 This is a SEM image of residual bacteria on the surface of a PEEK slide taken from an in vivo animal experiment in Example 6.

[0067] Figure 21 The bar chart shows the flow cytometry analysis and quantitative results of T cells in the in vivo animal experiment in Example 6.

[0068] Figure 22 The bar chart shows the flow cytometry analysis and quantitative results of DC cells in the in vivo animal experiment in Example 6.

[0069] Figure 23 Immunohistochemical staining for STING, IL17, and IL23r in the in vivo animal experiments of Example 7;

[0070] Figure 24 Tissue sections of the kidney, liver, lung, spleen, and heart from Example 7;

[0071] Figure 25 This is a heatmap of differentially expressed genes from the in vivo animal transcriptomics analysis in Example 7.

[0072] Specific implementation methods

[0073] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Furthermore, unless otherwise specified, methods not specifically describing conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0075] Example 1 Synthesis and characterization of MnO2, MnP and MnPM nanoparticles

[0076] The structure of the MnPM nanoparticles in this application is as follows: Figure 1 As shown in the diagram, the core is MnO2 nanoparticles, which are encapsulated by two layers: an inner layer of PCN MOF material and an outer layer of a hybridization membrane composed of neutrophils and macrophages. The synthesis of MnPM nanoparticles is detailed below with specific examples.

[0077] 1. Preparation of MnO2 nanoparticles

[0078] 950 mg of potassium permanganate (KMnO4) was mixed with 480 mL of deionized water and stirred for 5 min. Then, 15 mL of formamide was added dropwise to the mixture and stirred for another 8 h. Finally, the product was centrifuged, washed with water, and freeze-dried to obtain MnO2 nanoparticles.

[0079] The morphology of the prepared MnO2 nanoparticles was observed using TEM (JEM-2100 Plus), and elemental spectra were used to analyze the elemental composition of the material. The results are as follows: Figure 2 As shown, the prepared MnO2 nanoparticles are uniformly dispersed.

[0080] 2. Preparation of MnP nanoparticles

[0081] 140 mg MnO2 was dispersed in 20 mL of a mixed solution of N,N-dimethylformamide (DMF) / ethanol. Then, 27.8 mg zirconium chloride (ZrCl4) and 50 mg tetrakis(4-carboxyphenyl)porphyrin (TCPP, TCI, Shanghai) were added to the above mixed solution. The mixture was magnetically stirred at 90 °C for 6 h. The product was then collected by centrifugation, washed three times with ethanol, and freeze-dried to obtain MnP nanoparticles.

[0082] The morphology of the prepared MnP nanoparticles was observed using TEM (JEM-2100 Plus), and elemental spectra were used to analyze the elemental composition of the material. The results are as follows: Figure 3 As shown, the prepared MnP nanoparticles are uniformly dispersed, and a layer of translucent PCN MOF material can be seen on the outer layer of the MnP nanoparticles.

[0083] To assess oxygen generation, a dissolved oxygen meter was used to detect the oxygen generated in deionized water. For example... Figure 4 As shown, MnO2 nanoparticles can generate oxygen in a solution containing H2O2 at pH 5.0. The generation of singlet oxygen (1O2) was detected by SOSG detection using a fluorescence spectrometer. Figure 5 As shown, under US excitation in a solution with pH=5.0 and containing H2O2, MnP can produce singlet oxygen (1O2), where the US power, frequency, duty cycle, and irradiation time are 1.5 W / cm². 2 1 MHz, 50% and 8 min.

[0084] 3. Preparation of MnPM nanoparticles

[0085] In this embodiment, the MnPM nanoparticles are prepared by encapsulating them using a hybrid membrane of neutrophils and macrophages. The specific steps are as follows:

[0086] RAW264.7 cells were cultured in complete DMEM containing 10% fetal bovine serum; RAW264.7 cells (2 × 10⁶ cells per well) were cultured in 10% fetal bovine serum. 7 After centrifugation, the precipitate was resuspended in pre-cooled hypotonic buffer containing 1× protease inhibitor (pH = 7.4, 10 mM Tris, 1 mM MgCl2) and incubated on ice for half an hour.

[0087] Similarly, neutrophils were isolated from the bone marrow of male mice (6-8 weeks old), and the frozen cells were thawed and washed three times with 1×PBS. The cells were then suspended in a hypotonic buffer containing 30 mM Tris-HCl (pH = 7.5, Sigma, T8230) and pre-chilled 1× protease inhibitor (pH = 7.4, 10 mM Tris, 1 mM MgCl2) and incubated on ice for half an hour.

[0088] The cells that had been decomposed after being placed on ice were centrifuged (2500 rpm, 4℃, 15 min) and the supernatant was collected. The collected supernatant was centrifuged again (100,000 g, 4℃, 120 min) to obtain the precipitate, which was the cell membrane. Then, the membranes were washed with 0.2 mM EDTA (100,000 g, 4℃, 60 min) and the neutrophil membranes and macrophage membranes were collected separately and stored for later use (short-term storage at 4℃, long-term storage at -80℃).

[0089] Finally, the extracted macrophage membrane aqueous dispersion (1 mg / mL), neutrophil membrane aqueous dispersion (1 mg / mL), and MnP nanoparticles (1 mg / mL) were mixed in deionized water; the mixture was sonicated for 10 min and stirred at 800 rpm for 30 min; then, the mixture was extruded sequentially through 400 nm and 200 nm polycarbonate porous membranes using an Avanti micro extruder, and the extrusion was repeated more than 20 times; finally, the free membrane was removed by centrifugation (13000 rpm, 4℃, 30 min), and the resulting mixed membrane-encapsulated MnP nanoparticles, abbreviated as MnPM nanoparticles, were further purified.

[0090] Fluorescence imaging of neutrophil and macrophage hybrid membranes was performed using the PKH67 Green Fluorescent Cell Linker Mini Kit and DiI (DiIC18(3)). Results are as follows: Figure 6 As shown, the prepared MnPM nanoparticles are uniformly dispersed and have an outer layer encapsulated with a hybrid membrane of macrophages and neutrophils.

[0091] Example 2: In vitro anti-biofilm evaluation

[0092] In this embodiment, *Escherichia coli* (ATCC 35218) and *MRSA* (ATCC 43300) were cultured in trypsin-soy broth (TSB) medium to evaluate the treatment effects in different groups. The specific steps are as follows:

[0093] First, 200 μL of bacterial suspension (1 × 10⁻⁶) was prepared. 7(CFU / mL) was inoculated into 96-well plates or confocal culture dishes and cultured at 37 °C for 48 h to form a mature biofilm;

[0094] Then, the mature biofilms were treated with the control group, US group, PCN group, PCN + US group, MnP group, and MnP + US group, respectively. Each treatment group was injected with PBS solution at a concentration of 30 μg / mL of the corresponding material (injection volume 200 μL); the control group was injected with an equal volume of PBS solution, without the corresponding material and without US irradiation; the power, frequency, duty cycle, and irradiation time of the US were 1.5 W / cm². 2 1 MHz, 50% and 8 min.

[0095] 1. The bacterial counts of different groups were calculated using the plate method to evaluate the in vitro anti-biofilm effect: First, the treated biofilm was resuspended in 1 mL of phosphate-buffered saline (PBS) and further diluted with PBS. Then, 100 μL of the diluted solution was spread on blood agar plates and incubated (37 ℃, 24 h), and bacterial colonies were counted. The results are as follows: Figure 7 As shown, the MnP + US treatment group had the fewest residual bacteria.

[0096] 2. Evaluation of in vitro anti-biofilm effect by scanning electron microscopy: Biofilms were cultured on titanium sheets and treated with different methods. The treated biofilms were then fixed at 4 °C for 12 h using electron microscopy fixative (Servicebio, China). The treated biofilms were then dehydrated at room temperature with different gradients of ethanol (50%, 70%, 80%, 90%, 95%, and 100%). After lyophilization, the samples were sputter-coated with gold and observed using scanning electron microscopy.

[0097] The results are as follows Figure 8 As shown, the bacterial biofilm structure was most severely damaged in the MnP + US treatment group.

[0098] 3. Evaluation of in vitro anti-biofilm effect by detecting crystal violet: When performing crystal violet detection, the biofilm retained in the plate was fixed with anhydrous ethanol and stained with crystal violet for 20 min; then the stained biofilm was photographed with a digital camera; finally, the biofilm was dispersed in 30% glacial acetic acid, and the absorbance at 595 nm was recorded using an enzyme-labeled microplate reader to evaluate the biomass of the biofilm.

[0099] The results are as follows Figure 9 As shown, the bacterial biofilm structure was most severely damaged in the MnP + US treatment group.

[0100] 4. The treated biofilm was stained using the LIVE / DEAD BacLight Bacterial Viability Kit in the dark for 30 min. The stained biofilm was then observed using CLSM. After staining, the biofilm was rinsed twice with PBS. The rinsed biofilm was then resuspended in 500 μL PBS and analyzed using flow cytometry to observe its three-dimensional structure.

[0101] The results are as follows Figure 10 As shown, the bacterial biofilm structure was most severely damaged in the MnP + US treatment group.

[0102] The experimental results of this embodiment show that the MnPM nanoparticles prepared in this application, combined with US irradiation, can destroy the structure of bacterial biofilms, thereby achieving the purpose of removing bacterial biofilms.

[0103] Example 3: Study on in vitro anti-biomembrane mechanism

[0104] Whole RNA was extracted from treated bacterial biofilms using a bacterial RNA extraction kit, and RNA-seq analysis was performed. All gene maps were analyzed by Oebiotech Co., Ltd. (Shanghai, China). Data processing, including correlation analysis, differential gene analysis, GO analysis, and KEGG analysis, was conducted on the Orbbec Cloud Platform (https: / / cloud.oebiotech.cn / task / ).

[0105] 1. RT-PCR was performed using SYBR Green qPCR Master Mix (EZBioscience) and the QuantStudio 7 Flex system (Life Technologies) to determine the relative expression levels of typical genes in the three replicates. Gene expression levels were assessed using the 2-ΔΔCt method. The manganese ion content in the bacteria was determined by elemental mapping, and the results are shown below. Figure 11 As shown, bacteria treated with MnP + US have a higher Mn ion content.

[0106] 2. The concentration of leaked protein in the bacteria was determined using the BCA protein detection kit (Elabscience, China). The results are as follows: Figure 12 As shown, bacteria treated with MnP + US exhibited higher levels of intracellular protein leakage.

[0107] 3. The collected bacteria were cultured together with ONPG solution, and the absorbance of the mixed solution at 420 nm was recorded using a microplate reader to assess the membrane permeability of the bacteria in the biofilm via ONPG hydrolysis. Results are as follows: Figure 13 As shown, the permeability of bacteria treated with MnP + US increased.

[0108] 4. Detection of bacterial ROS (1O2) using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) detection kit (Biotech, China): 500 μL of MRSA suspension (1×10⁻⁶) was added... 6 CFU / mL was inoculated into 24-well plates and cultured at 37°C for 24 h. The biofilm was then collected and resuspended in PBS, washed twice with PBS, and then co-incubated with 10 μM DCFH-DA. Results are as follows: Figure 14 As shown, bacteria treated with MnP + US have higher ROS content.

[0109] The above studies show that the biofilm treated with MnP + US exhibited significant disruption of bacterial homeostasis, including excessive manganese ions, oxidative stress dysfunction, cell membrane integrity defects, and quorum sensing system disorder, thereby effectively killing bacteria.

[0110] Example 4: In vitro cGAS-STING assessment

[0111] For Western blot analysis, RAW264.7 and DC2.4 cells were seeded in six-well plates and treated according to different groups. Total protein was extracted from each group using RIPA lysis buffer, and its concentration was determined using a BCA protein quantification kit. Proteins of different molecular weights were separated by SDS-PAGE gel electrophoresis, and then the protein bands were transferred to a PVDF membrane. The membrane was blocked at room temperature for 1 hour, washed three times with TBST for 5 minutes each time, incubated overnight at 4°C with primary antibody against the target protein, washed three times with TBST, incubated for 2 hours at 4°C with HRP-labeled secondary antibody, washed three times with TBST, and developed using an ECL chemiluminescence kit. Figure 15 As shown, the STING axis was significantly activated in the MnP + dsDNA treatment group.

[0112] Enzyme-linked immunosorbent assay (ELISA) involves incubating cells and materials for 24 h, collecting the culture medium, and centrifuging (5000 rpm, 30 min) to obtain the supernatant. Then, an ELISA kit is used to determine the cytokine concentrations in different collected supernatants. For example... Figure 16As shown, it can be seen that STING activation-related cytokines (e.g., STING, IFN-I, IL6, etc.) were significantly upregulated in the group treated with MnP+ dsDNA.

[0113] The above experimental results show that the STING pathway in RAW264.7 and DC2.4 cells treated with MnP + dsDNA was significantly activated.

[0114] Example 5: In Vitro Tissue Repair Assessment

[0115] 1. Place 1 × 10⁶ RAW264.7 cells in each well. 5 Cells were seeded in 8 μm transwells for transwell migration assays. A solution containing the material was added to the lower chamber, and after incubation for 24 hours, cells at the bottom of the transwell were stained and imaged with crystal violet. Figure 17 As shown, more cells migrated in the MnP-treated group.

[0116] RAW264.7 cells (2 × 10⁶ cells per well) 5 Macrophage conditioned medium was co-cultured with the material in a six-well plate at 37°C for 1 day. The macrophage conditioned medium was collected by centrifugation and filtration, and then mixed with complete medium DMEM at a ratio of 1:2. Finally, the above macrophage conditioned medium was stored at 4°C for further use.

[0117] 2. When performing the scratch test, use EA.hy 926 cells (1 × 10⁶ cells per well). 5 Cells were placed in 12-well plates and incubated at 37°C to form a confluent monolayer. The scratch area of ​​each cell group was observed using an optical microscope (Nikon, ECLIPSE, Japan), and the scratch area was calculated using ImageJ / FIJI software (https: / / imagej.nih.gov / ij / ). Figure 18 As shown, the scratch spacing is the smallest in the MnP treatment group.

[0118] 3. In the angiogenesis assay, EA.hy 926 cells (2 × 10⁶ cells per well) 4 Cells were seeded into 96-well plates pre-coated with Matrigel matrix (BD, Corning, USA) and various macrophage conditioned media, and cultured at 37°C. After 6 hours of culture, cells were fixed and imaged using an optical microscope (Nikon, ECLIPSE, Japan). The number of circles was counted using ImageJ / FIJI software (https: / / imagej.nih.gov / ij / ). Figure 19As shown, the MnP-treated group had the most vascular rings formed by cells.

[0119] The above experimental results show that after MnP treatment, the indicators related to promoting tissue repair, such as cell migration and angiogenesis, were significantly higher than those in other groups.

[0120] Example 6: In vivo analysis of anti-biofilm and immune response

[0121] All animal experiments were approved by the Animal Ethics Committee of the Department of Veterinary Medicine, University of Science and Technology of China (USTC) and the First Affiliated Hospital of USTC (2022-N(A)-056). All procedures and surgical processes followed the approved guidelines.

[0122] A mouse implant-associated biofilm infection model was established using BALB / c mice purchased from the College of Agriculture and Biology, Shanghai Jiao Tong University. The infected mice were divided into six groups, each receiving one of the following treatments: control group, US group, PCNM group, PCNM + US group, MnPM group, and MnPM + US group. Each treatment group was injected with PBS solution at a concentration of 30 μg / mL (injection volume 200 μL). The control group received only the same volume of PBS solution as the other treatment groups, without the corresponding material and without US treatment. The PCNM group consisted of PCN material coated with a hybridization membrane.

[0123] 1. The specific treatment process is as follows: 200 μL of MRSA bacterial suspension (1 × 10⁻⁶) 7 CFU / mL) and polyetheretherketone (PEEK, diameter = 8 mm, thickness = 0.5 mm) discs were co-cultured at 37 ℃ and 5% CO2 for 48 h. Before biofilm implantation, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and then the prefabricated PEEK discs with biofilms were implanted subcutaneously in the posterior neck region of mice. After shaving, disinfection, incision, and implantation, the skin wound was immediately sutured. On day 0, the implantation site was treated with the different treatment groups described above. Photographs of the infected area were taken on days 0, 1, 3, 7, 10, and 14 post-injection.

[0124] The results are as follows Figure 20 As shown, the number of bacteria remaining on the PEEK tablets was the lowest in the MnPM + US treatment group, indicating that the MnPM + US treatment group had the best anti-biofilm therapeutic effect.

[0125] 2. For in vivo flow cytometry analysis. Processed local skin tissue or spleen was collected, rinsed with PBS, and placed in 1 mL of 1640 medium containing digestive enzymes (1 mg / mL type I collagenase, 250 mg / mL type II collagenase, 50 mg / mL type I DNase). The tissue was minced, digested at 37 °C for 45 min, filtered through a 40 μm cell sieve, and then centrifuged at 400 g for 3 min. Then, 2 mL of erythrocyte lysis buffer was added, lysed for 3 min, and centrifuged. Cells were washed with PBS. Then, CD16 / 32 blocking antibody was added, cells were resuspended, and placed on ice for 30 min for blocking. Cells were washed with PBS. Then, fluorescein-labeled antibody was added, incubated on ice for 30 min, and cells were washed with PBS. The collected cells were resuspended in 500 μL PBS for flow cytometry. Figure 21 As shown, T cells are significantly increased. Figure 22 As shown, DC cells mature more rapidly.

[0126] The above experiments demonstrate that mice treated with MnPM + US exhibited a strong anti-biomembrane immune response.

[0127] Example 7: In vivo analysis of anti-biofilm and tissue repair effects

[0128] A mouse wound infection model was established using BALB / c mice from the College of Agriculture and Biology, Shanghai Jiao Tong University. Mice were anesthetized with sodium pentobarbital (1%), and the skin on their backs was shaved and disinfected. Wounds with a diameter of 8 mm were created on the backs of the mice, and then treated with different treatment groups as described in Example 6. Wounds in each group were photographed and recorded on days 0, 1, 3, 7, 10, and 14 until euthanasia. On day 14, wound tissue was collected and fixed with paraformaldehyde (4%). The fixed tissue was then dehydrated in ethanol, embedded in paraffin, and sectioned for histological analysis. Inflammation and bacterial residue were assessed using heme and eosin (H&E) staining and Giemsa staining. All sections were observed under an optical microscope (OLYMPUS, IX70, Japan). Data from three different images were quantified using ImageJ / FIJI software (https: / / imagej.nih.gov / ij / ). All images were converted to binary images with a fixed threshold for substrate comparison. Immunohistochemical staining images of CD31, IL17, IL23r, and STING were acquired using a microscope (Ci-s, Nikon). For example... Figure 23As shown, STING was successfully activated in vivo, leading to Th17 cell formation. Immunofluorescence co-staining images of K14 / K19 and IL6 / IL10 were observed under a fluorescence microscope (OLYMPUS, IX70, Japan). Histological examination of major organs (heart, liver, spleen, lung, and kidney) under different treatments was performed by H&E staining to assess the biosafety of these treatments. Figure 24 As shown, tissue sections from the kidney, liver, lung, spleen, and heart indicate that the material is not toxic.

[0129] For in vivo transcriptional analysis, tissues were collected after different treatments. Total RNA was extracted using Tizol (Invitrogen, California, USA). Transcriptome sequencing and analysis were performed by OE Biotech Ltd. (Shanghai, China). Data processing, including correlation analysis, differential gene analysis, GO analysis, and KEGG analysis, was performed on the Orbbec Cloud Platform (https: / / cloud.oebiotech.cn / task / ). Figure 25 As shown, STING activation-related cytokines were significantly upregulated.

[0130] All data are expressed as mean ± SD. Statistical analysis between the two groups was performed using the Student's t-test. n = 3; mean ± SD; ns, no significance; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0131] In summary, the above experimental data demonstrate that MnPM can be used as a novel metal immunotherapy in the strategy of "immunogenic sonodynamic therapy combined with metal immunotherapy." It releases bacterial-specific antigens, activates the cGAS-STING signaling pathway on antigen-presenting cells for efficient antigen presentation, and activates both innate and adaptive immune systems to combat vegetative-associated biofilm infections. After infection clearance, MnPM can continuously generate O2 without US irradiation, thereby inhibiting inflammation-related diseases caused by STING activation, promoting the transformation of the tissue repair-related immune microenvironment, and preventing excessive inflammation.

[0132] Furthermore, it should be noted that the metal-organic framework-based metal antibiotic MnPM in this application is capable of activating the cGAS-STING signaling pathway and inhibiting inflammation-related diseases caused by STING activation, and therefore can treat diseases related to the cGAS-STING signaling pathway, which will not be elaborated here.

[0133] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0134] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A metal-organic framework-based metal antibiotic, characterized in that, include: MnO2 nanoparticles; Zirconium chloride and tetra(4) PCN MOF material formed by self-assembly of carboxyphenyl porphyrin, wherein the PCN MOF material is formed on the outer layer of the MnO2 nanoparticles; And a hybridization membrane formed on the outer layer of the PCN MOF material, the hybridization membrane being composed of macrophages and neutrophils.

2. The metal-organic framework-based metal antibiotic as described in claim 1, characterized in that, The particle size of the MnO2 nanoparticles is between 20 and 80 nm.

3. A method for preparing a metal-organic framework-based metal antibiotic as described in claim 1 or 2, characterized in that, Includes the following steps: S1, provides MnO2 nanoparticles; S2, the MnO2 nanoparticles, zirconium chloride and tetra(4) MnP nanoparticles, namely MnO2 nanoparticles with PCN MOF material formed on the outer layer, were prepared by reacting carboxyphenyl porphyrin organic ligands with a strongly polar solvent. S3. Encapsulate the surface of the MnP nanoparticles with a hybrid membrane composed of macrophages and neutrophils to obtain MnPM nanoparticles, i.e., MnP nanoparticles with the hybrid membrane on the outer layer.

4. The method for preparing the metal-organic framework-based metal antibiotic as described in claim 3, characterized in that, The MnO2 nanoparticles are self-made, and their preparation method includes the following steps: Potassium permanganate and deionized water were mixed, and formamide was added dropwise. The mixture was stirred and reacted to obtain MnO2 nanoparticles.

5. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 4, characterized in that, The mass-to-volume ratio of potassium permanganate, deionized water, and formamide is 425-950 mg: 480 mL: 5-15 mL.

6. The method for preparing the metal-organic framework-based metal antibiotic as described in claim 4, characterized in that, The stirring rate of the stirring reaction is 250~800 rpm.

7. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 3, characterized in that, In step S2, the highly polar solvent is capable of simultaneously dissolving zirconium chloride and tetra(4) chloride. Organic solvents for carboxyphenyl porphyrin organic ligands.

8. The method for preparing the metal-organic framework-based metal antibiotic as described in claim 7, characterized in that, The highly polar solvent is a mixture of N,N-dimethylformamide and ethanol.

9. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 8, characterized in that, In the strongly polar solvent, the volume ratio of N,N-dimethylformamide to ethanol is 3:1 to 1:

3.

10. The method for preparing the metal-organic framework-based metal antibiotic as described in claim 3, characterized in that, In step S2, the mixing reaction is carried out by stirring at 90~140℃ for 6~24 h.

11. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 3, characterized in that, Step S3 includes: MnPM nanoparticles were prepared by mixing neutrophil membrane aqueous dispersion, macrophage membrane aqueous dispersion and MnP nanoparticles and then extruding them using an extrusion device.

12. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 11, characterized in that, The mixing process involves ultrasonic stirring.

13. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 12, characterized in that, The ultrasound duration is 5-30 minutes; the stirring speed is 400-1200 rpm, and the duration is 5-60 minutes.

14. The method for preparing a metal-organic framework-based metal antibiotic as described in claim 11, characterized in that, The extrusion orifice diameter of the extrusion equipment is 400nm and 200nm; the number of extrusions is at least 20.

15. The method for preparing a metal-organic framework-based metal antibiotic according to any one of claims 11-14, characterized in that, The mass ratio of the neutrophil membrane, macrophage membrane, and MnP nanoparticles is 1~3:1~3:0.25~4.

16. The use of the metal-organic framework-based metal antibiotic as described in claim 1 or 2, or the metal-organic framework-based metal antibiotic prepared by any one of claims 3 to 15, in the preparation of a medicament for treating endophytic infections.

17. A drug for treating endophyte infection, characterized in that, Includes metal-organic framework-based metal antibiotics as described in claim 1 or 2, or metal-organic framework-based metal antibiotics prepared by any one of the preparation methods described in claims 3 to 15.

Images