Preparation method of immunomodulatory recombinant macrophage vesicles

Abstract

The invention belongs to the technical field of biomedical treatment, and discloses a preparation method of immunomodulatory recombinant macrophage vesicles, and the preparation method specifically comprises the following steps: step 1, clinical sequencing of gingival tumor tissues and pathogenic microbiota data analysis; hybridizing and fusing a macrophage membrane infected by the porphyromonas gingivalis and an exosome membrane; and 3, constructing a piezoelectric driven ROS (reactive oxygen species) to generate the immunoregulation vesicles. The invention provides an innovative nano vesicle technology, and the technology shows double advantages in tumor treatment: firstly, accurate targeting and efficient killing of key porphyromonas gingivalis in a tumor environment are realized by constructing recombinant macrophage vesicles, and meanwhile, the stability of normal flora is kept, and tumor deterioration is prevented; and secondly, the anti-tumor immune response is obviously activated by utilizing the immunoregulation capability of the bacteria-induced macrophages and exosome vesicles thereof, including converting immunosuppressed M2 type macrophages into anti-tumor M1 type macrophages, so that the growth of solid tumors is effectively inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a method for preparing immunomodulatory recombinant macrophage vesicles. Background Art

[0002] In recent years, with the deepening of research on the relationship between oral microbiota and oral tumors, scientists have discovered that specific pathogens play an important role in the tumor microenvironment. These pathogens not only promote the occurrence and development of tumors by inducing chronic inflammation, regulating cell proliferation and apoptosis, and inhibiting immune responses, but also affect the immune environment inside the tumor through complex interactions. As one of the most complex and diverse microbial ecosystems in the human body, the oral microbiota plays an important role in the occurrence and development of oral tumors. In addition to bacteria such as Porphyromonas gingivalis, Treponema denticola, Streptococcus, Candida, and viruses such as Epstein-Barr virus and human papillomavirus (HPV) that are known to be closely related to oral tumors, there may be other types of microorganisms in the oral cavity that have not been fully studied. They may jointly affect the formation of tumors through complex interaction mechanisms. For example:

[0003] Induction of chronic inflammation: Pathogenic bacteria such as Porphyromonas gingivalis can continuously stimulate the oral mucosa by producing toxins and inflammatory mediators, leading to the occurrence of chronic inflammation. Long-term chronic inflammation is an important background for tumorigenesis, which can promote cell proliferation, gene mutation and angiogenesis, providing a favorable environment for the occurrence of tumors.

[0004] Regulation of cell proliferation and apoptosis: Microorganisms such as Streptococcus may affect the cell proliferation cycle and apoptosis process through their metabolites or directly interact with cells. This imbalance of cell proliferation and apoptosis is one of the key steps in tumorigenesis.

[0005] Suppression of the immune system: Oral microbiota may also promote tumor development by affecting the host’s immune system. For example, certain microorganisms may inhibit the activity and function of immune cells, reducing the body’s ability to recognize and eliminate tumor cells, thereby facilitating the growth and spread of tumor cells.

[0006] Based on the above understanding, the development of targeted killing tools for different pathogens has become a new strategy for the precise treatment of oral tumors. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing immunomodulatory recombinant macrophage vesicles to solve the problems raised in the above background technology.

[0008] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing immunomodulatory recombinant macrophage vesicles, the preparation method specifically comprising the following steps:

[0009] Step 1: Clinical sequencing of gingival tumor tissue and analysis of pathogenic microbiome data: By obtaining pathogenic tumor microbiome data, a Porphyromonas gingivalis-specific macrophage model is established. After co-incubation of macrophages with Porphyromonas gingivalis, cells and supernatant are collected to obtain Porphyromonas gingivalis-specific targeted macrophages and macrophage exosomes;

[0010] Step 2: Hybrid fusion of macrophage membrane and exosome membrane: Using thin film extrusion method and ultrasonic technology, the extracted macrophage membrane and exosome membrane are efficiently hybridized and fused, so that the two membrane structures are mixed together to form a double hybrid recombinant macrophage vesicle that specifically targets Porphyromonas gingivalis and reprograms the immune microenvironment;

[0011] Step three, construct piezoelectrically driven ROS-generating immunomodulatory vesicles: The dual hybrid recombinant macrophage vesicles prepared above are further coated on the piezoelectric material GO / BTO to construct a new immunomodulatory tool - Porphyromonas gingivalis recombinant macrophage vesicles with piezoelectric drive to produce ROS.

[0012] Preferably, the method for constructing a macrophage model specific for Porphyromonas gingivalis in step 1 is as follows:

[0013] A1, Preparation of Porphyromonas gingivalis: First, culture the bacteria overnight; then collect the bacterial solution and wash it thoroughly with PBS 2-3 times to remove impurities; then resuspend the bacteria in macrophage-specific culture medium and adjust its OD value to 0.05 to ensure the appropriate bacterial concentration;

[0014] A2, macrophage culture and infection: RAW264.7 macrophage cell line was selected for culture, and when the cells grew to a confluence of about 70%, the medium was changed; then, the pre-prepared culture medium containing resuspended Porphyromonas gingivalis was added to the cell culture system and incubated for 2 to 6 hours;

[0015] A3, collection and processing: After the co-incubation of macrophages and Porphyromonas gingivalis, collect the macrophages expressing specific receptors; at the same time, collect the exosomes released into the supernatant during the immune activation process, and store the collected macrophages and supernatant under appropriate conditions for subsequent experimental use.

[0016] Preferably, the specific preparation method of the hybrid fusion of macrophage membrane and exosome membrane in step 2 is as follows:

[0017] B1. Extraction of macrophage membrane: macrophages were treated by repeated freezing and thawing or water absorption bursting method to rupture the cell membrane and release the cell contents. The mixture was centrifuged at 2000 g for 10 minutes to remove large particles and unbroken cells. The supernatant was collected by further centrifugation. The supernatant was centrifuged at 15000 g for 30 minutes. The cell membrane was ultrasonically treated at a frequency of 42 kHz and a power of 100 W for 10 minutes to further break and homogenize the cell membrane.

[0018] B2, extraction of cell exosomes: the supernatant obtained after preliminary centrifugation in B1 was centrifuged at 12,000 g for 15 min at 4°C; exosomes were collected: after centrifugation, the supernatant was discarded, and the precipitate was rich in exosome particles;

[0019] B3, hybridization of macrophage membrane and exosomes: the mixed solution was passed through a 300 nm polycarbonate membrane and repeatedly extruded at least 10 times using a micro extruder to form a uniform nanovesicle structure;

[0020] B4, Collection and storage of nanovesicles: After extrusion, collect the obtained nanovesicles; store the nanovesicles in an appropriate buffer to ensure their stability and activity during storage.

[0021] Preferably, the method for constructing the immunomodulatory Porphyromonas gingivalis recombinant macrophage vesicles with piezoelectric ROS generation in step 3 is:

[0022] C1, material preparation: weigh 10 mg of GO / BTO; prepare 1×10 8 Macrophage membrane;

[0023] C2, mixture preparation: 10 mg of GO / BTO was mixed with 1×10 8 The macrophage membrane mixture was mixed in water to ensure that the two were fully mixed.

[0024] C3, ultrasonic treatment: the mixture was ultrasonicated at a frequency of 42 kHz and a power of 100 W for 10 min using an ultrasonic processor;

[0025] C4, microextrusion: the sonicated mixture was extruded through a microextruder equipped with a 300 nm polycarbonate membrane, repeated at least 10 times;

[0026] C5, centrifugal collection: After extrusion, the mixture was centrifuged at 15,000 g for 30 minutes; by centrifugation, the macrophage vesicles loaded with GO / BTO were separated from other components that did not form vesicles; and then the loaded nanoparticles were collected.

[0027] Preferably, the mimetic treatment method based on recombinant macrophage vesicles is as follows:

[0028] 1) Applying immune-modulating recombinant macrophage vesicles to the oral cavity of a mouse model of gingival cancer for seven days;

[0029] 2) Observe the changes in mouse tumor size, bacterial killing, and immune activation of tumor tissue after seven days.

[0030] The beneficial effects of the present invention are as follows:

[0031] The present invention innovatively developed a nanovesicle technology that aims to precisely combat key Porphyromonas gingivalis in the tumor environment and simultaneously activate anti-tumor immune responses. Specifically, the technology achieves its therapeutic effect through the following two core mechanisms:

[0032] Precise targeting and killing of Porphyromonas gingivalis: First, key Porphyromonas gingivalis is used to induce macrophages, giving them specific Porphyromonas gingivalis targeting capabilities. Subsequently, the cell membrane (MacrophageMembrane, MM) is extracted from these macrophages and wrapped on piezoelectric materials to construct a unique recombinant macrophage vesicle. This vesicle can directionally identify and kill key Porphyromonas gingivalis in in situ tumors with an efficiency of up to 40%. In the process, it cleverly avoids affecting other probiotics inside the tumor, ensuring the stability of the normal flora in the tissue, thereby preventing further deterioration of the tumor that may be caused by an imbalance in the flora.

[0033] Activating anti-tumor immunity: In addition to the direct killing effect on Porphyromonas gingivalis, this technology also significantly activates the immune response inside the tumor through the immunomodulatory ability of the bacterial-induced M1 polarized macrophage membrane and its exosome vesicles (Exosoma lVes ic le). The innate immunogenicity of bacteria provides a powerful stimulation signal for these macrophages, so that this in vitro activated macrophage hybrid vesicle injected into the body can effectively resist the immunosuppressive microenvironment inside the tumor and prevent the induction and formation of M2 macrophages (usually associated with immunosuppression). More importantly, this technology can also convert existing M2 macrophages into M1 macrophages with stronger anti-tumor activity, thereby significantly inhibiting solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is a simplified flow chart of the method for preparing the recombinant macrophage vesicles of the present invention;

[0035] Figure 2 Microscope image of bacteria activating macrophages;

[0036] Figure 3This is a flow cytometric graph showing changes in the content of targeted proteins on the membrane surface after co-incubation of bacteria and macrophages;

[0037] Figure 4 Transmission electron microscope pictures of the nanomaterials before and after the targeted Porphyromonas gingivalis and immune-modulating recombinant macrophage vesicles are encapsulated by the film extrusion method of the present invention;

[0038] Figure 5 Transmission electron microscopy images of membrane-wrapped nanomaterials;

[0039] Figure 6 This is the fluorescence flow cytometric graph of the membrane before and after material wrapping;

[0040] Figure 7 This is a diagram of membrane protein content before and after material wrapping;

[0041] Figure 8 The graph shows ROS production before and after ultrasonic treatment of nanomaterials encapsulation;

[0042] Fig. 9 Transmission electron microscopy images of membrane-encapsulated nanomaterials and targeted bacteria;

[0043] Fig.10 The pathogenic microorganisms in mouse tumors were plated and counted for the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] like Figures 1 to 7 As shown, the embodiment of the present invention provides a method for preparing immunomodulatory recombinant macrophage vesicles, and the preparation method specifically comprises the following steps:

[0046] Step 1, clinical sequencing and pathogenic microbiome data analysis: By obtaining pathogenic tumor microbiome data, a Porphyromonas gingivalis-specific macrophage model is established. After macrophages are co-incubated with Porphyromonas gingivalis, cells and supernatants are collected to obtain macrophages and macrophage exosomes that specifically target Porphyromonas gingivalis. In this model, macrophages are co-cultured with the corresponding Porphyromonas gingivalis, and then the treated cells and their supernatants are collected to extract macrophages and exosomes that have specific targeting capabilities for Porphyromonas gingivalis.

[0047] Step 2: Hybrid fusion of macrophage membrane and exosome membrane: In order to enhance the targeting and immunomodulatory function, the extracted macrophage membrane and exosome membrane are efficiently hybridized and fused using thin film extrusion method and ultrasonic technology, so that the two membrane structures are mixed together to form a dual hybrid recombinant macrophage vesicle that specifically targets Porphyromonas gingivalis and reprograms the immune microenvironment; this process not only combines the advantageous characteristics of the two membrane structures, but also creates a dual hybrid recombinant macrophage vesicle that can specifically target Porphyromonas gingivalis and reshape the immune microenvironment.

[0048] Step 3: Construction of piezoelectrically driven ROS-generating immunomodulatory vesicles: The dual hybrid recombinant macrophage vesicles prepared above were further coated on the piezoelectric material GO / BTO to construct a new immunomodulatory tool - Porphyromonas gingivalis recombinant macrophage vesicles with piezoelectric drive to generate ROS (reactive oxygen species). This design not only retains the specific targeting and immunomodulatory capabilities of the vesicles, but also gives it the additional function of generating ROS through the piezoelectric effect to directly fight Porphyromonas gingivalis, providing a new strategy for the precise treatment of pathogens and the regulation of the immune microenvironment.

[0049] The method for constructing a macrophage model specific for Porphyromonas gingivalis in step 1 is specifically as follows:

[0050] A1, Preparation of pathogens: In view of the specific enrichment of Porphyromonas gingivalis in oral tumor tissues, the bacteria were first cultured overnight; then the bacterial solution was collected and thoroughly washed 2-3 times with PBS to remove impurities; the bacteria were then resuspended in a macrophage-specific culture medium and the OD value was adjusted to 0.05 to ensure a moderate bacterial concentration;

[0051] A2, macrophage culture and infection: RAW264.7 macrophage cell line was selected for culture. When the cells grew to a confluence of about 70%, the medium was changed. Subsequently, the prepared medium containing resuspended Porphyromonas gingivalis was added to the cell culture system and incubated for 2 to 6 hours. Figure 2 As shown; this step is intended to induce macrophages to specifically express receptors that bind to antigens associated with Porphyromonas gingivalis, thereby achieving pathogen-specific polarization of macrophages;

[0052] A3, Collection and processing: After the co-incubation of macrophages and Porphyromonas gingivalis, collect the macrophages expressing specific receptors; at the same time, collect the exosomes released into the supernatant during the immune activation process. These exosomes are rich in immunomodulatory factors and are of great significance for subsequent research. The collected macrophages and supernatant are stored under appropriate conditions for subsequent experiments. Figure 3 shown.

[0053] The specific preparation method of the hybrid fusion of macrophage membrane and exosome membrane in step 2 is as follows:

[0054] B1, Extraction of macrophage membrane: Collect a sufficient number of macrophages to ensure that the cells are in good condition; Use repeated freezing and thawing method or water absorption bursting method to treat macrophages to rupture the cell membrane and release the cell contents, and collect the mixed solution at this time; Centrifuge the mixture at 2000g for 10 minutes to remove large particles and unbroken cells; Collect the supernatant; Further centrifuge to collect cell membrane: Centrifuge the supernatant at 15000g for 30 minutes to precipitate the cell membrane. Remove the supernatant and retain the cell membrane precipitate. Ultrasonic treatment of cell membrane The cell membrane precipitate is ultrasonically treated at a frequency of 42kHz and a power of 100W for 10 minutes to further break and homogenize the cell membrane.

[0055] B2, extraction of cellular exosomes: centrifuge the supernatant obtained after the initial centrifugation in B1 (i.e., the supernatant after removing large particle impurities) at 12,000g (about 12,400rpm) for 15 minutes at 4°C; collect exosomes: after centrifugation, discard the supernatant, and the precipitate is rich in exosome particles; the supernatant should be aspirated as much as possible to reduce contamination by non-exosome components.

[0056] B3, hybridization of macrophage membrane and exosome: the extracted macrophage membrane and exosome are mixed evenly in an appropriate buffer (such as PBS); the mixture is passed through a 300nm polycarbonate membrane and repeatedly extruded at least 10 times using a micro extruder; this step promotes the fusion and hybridization of the macrophage membrane and the exosome membrane by physical extrusion to form a uniform nanovesicle structure, such as Figure 4 , Figure 5 As shown;

[0057] B4, Collection and storage of nanovesicles: After extrusion, the nanovesicles are collected; these vesicles contain both the components of the macrophage membrane and the characteristics of exosomes. Protein characterization such as Figure 7 As shown, the nanovesicles may have dual functions of targeting Porphyromonas gingivalis and immunomodulation. The nanovesicles were stored in an appropriate buffer (such as PBS) to ensure that they remained stable and active during storage.

[0058] Wherein, the method for constructing the immunomodulatory Porphyromonas gingivalis recombinant macrophage vesicles with piezoelectric ROS generation in step 3 is:

[0059] C1, material preparation: Weigh 10 mg of GO / BTO (a nanomaterial that may be used for drug delivery or imaging purposes). 8 A macrophage membrane (to ensure the purity and functionality of the cell membrane source).

[0060] C2, mixture preparation: 10 mg of GO / BTO was mixed with 1×10 8 Mix the macrophage membrane mixture in water and ensure that the two are thoroughly mixed.

[0061] C3, ultrasonic treatment: The mixture was ultrasonicated at a frequency of 42 kHz and a power of 100 W for 10 min using an ultrasonic processor. This step was intended to further promote the binding of GO / BTO to the cell membrane and may help form a more homogeneous mixture.

[0062] C4, micro-extrusion: The sonicated mixture was extruded through a micro-extruder equipped with a 300 nm polycarbonate membrane, and this was repeated at least 10 times; this step rearranged the components in the mixture by physical extrusion to form vesicles of a specific size and structure.

[0063] C5, centrifugal collection: After extrusion, the mixture was centrifuged at 15,000 g for 30 minutes; by centrifugation, the macrophage vesicles loaded with GO / BTO were separated from other components that did not form vesicles; and then the loaded nanoparticles were collected.

[0064] Collection and purification: Carefully collect the supernatant or pellet after centrifugation (depending on the distribution of vesicles after centrifugation), which may contain the desired recombinant macrophage vesicles.

[0065] Optional steps: further purification of the vesicles, such as by dialysis, filtration or other methods to remove unbound impurities or residues.

[0066] Characterization and detection: Use appropriate characterization techniques (such as transmission electron microscopy, dynamic light scattering, flow cytometry, etc.) to detect the morphology, size, and surface characteristics of the prepared recombinant macrophage vesicles. Verify the functionality of the vesicles, such as drug delivery efficiency, cell targeting ability, etc.

[0067] Among them, the simulated treatment methods based on recombinant macrophage vesicles are as follows:

[0068] 1) Applying immune-modulating recombinant macrophage vesicles to the oral cavity of a mouse model of gingival cancer for seven days;

[0069] 2) Observe the changes in mouse tumor size, bacterial killing, and immune activation of tumor tissue after seven days.

[0070] Example 1

[0071] The method for preparing the hybrid of macrophage membrane and exosome membrane is as follows:

[0072] 1) The collected macrophages are subjected to repeated freeze-thaw or water absorption bursting method to rupture the cell membrane and collect the mixed solution;

[0073] 2) The mixture was centrifuged at 2000 g for 10 minutes, the supernatant was collected and centrifuged at 15000 g for 30 minutes to collect the cell membranes. The cell membranes were sonicated at a frequency of 42 kHz and a power of 100 W for 10 minutes;

[0074] 3) Extraction of exosomes from cells: centrifuge the supernatant at 12,000 × g (~12,400 rpm) at 4°C for 15 min, discard the supernatant, and the precipitate is rich in exosome particles (absorb as much supernatant as possible);

[0075] 4) Extruding the extracted cell membrane and exosomes through a 300 nm polycarbonate membrane using a micro extruder at least 10 times;

[0076] 5) After extrusion, we collected the nanovesicles and stored them in PBS;

[0077] The steps for preparing immunomodulatory recombinant macrophage vesicles are as follows:

[0078] 1) 10 mg GO / BTO and 1×10 8 The cell membrane mixture is fused in water;

[0079] 2) The mixture was ultrasonically treated at a frequency of 42 kHz and a power of 100 W for 10 minutes,

[0080] 3) Then extrude at least 10 times through a micro extruder using a 300 nm polycarbonate film.

[0081] 4) After extrusion, we collected the loaded nanoparticles by centrifugation at 15000 g for 30 min.

[0082] Example 2

[0083] The method for preparing the hybrid of macrophage membrane and exosome membrane is as follows:

[0084] 1) The collected macrophages are subjected to repeated freeze-thaw or water absorption bursting method to rupture the cell membrane and collect the mixed solution;

[0085] 2) The mixture was centrifuged at 2000 g for 10 minutes, the supernatant was collected and centrifuged at 15000 g for 30 minutes to collect the cell membranes. The cell membranes were sonicated at a frequency of 50 kHz and a power of 120 W for 20 minutes;

[0086] 3) Extraction of exosomes from cells: centrifuge the supernatant at 12,000 × g (~12,400 rpm) at 4°C for 15 min, discard the supernatant, and the precipitate is rich in exosome particles (absorb as much supernatant as possible);

[0087] 4) Extruding the extracted cell membrane and exosomes through a 300 nm polycarbonate membrane using a micro extruder at least 10 times;

[0088] 5) After extrusion, we collected the nanovesicles and stored them in PBS;

[0089] The steps for preparing immunomodulatory recombinant macrophage vesicles are as follows:

[0090] 1) 10 mg GO / BTO and 1×10 8 The cell membrane mixture is fused in water;

[0091] 2) The mixture was ultrasonically treated with a frequency of 50 kHz and a power of 120 W for 20 minutes.

[0092] 3) Then extrude at least 10 times through a micro extruder using a 500 nm polycarbonate film.

[0093] 4) After extrusion, we collected the loaded nanoparticles by centrifugation at 15000 g for 30 min.

[0094] Example 3

[0095] The method for preparing the hybrid of macrophage membrane and exosome membrane is as follows:

[0096] 1) The collected macrophages are subjected to repeated freeze-thaw or water absorption bursting method to rupture the cell membrane and collect the mixed solution;

[0097] 2) The mixture was centrifuged at 2000 g for 10 minutes, the supernatant was collected and centrifuged at 15000 g for 30 minutes to collect the cell membranes. The cell membranes were sonicated at a frequency of 50 kHz and a power of 120 W for 20 minutes;

[0098] 3) Extraction of exosomes from cells: centrifuge the supernatant at 12,000 × g (~12,400 rpm) at 4°C for 15 min, discard the supernatant, and the precipitate is rich in exosome particles (absorb as much supernatant as possible);

[0099] 4) Extruding the extracted cell membrane and exosomes through a 300 nm polycarbonate membrane using a micro extruder at least 10 times;

[0100] 5) After extrusion, we collected the nanovesicles and stored them in PBS;

[0101] The steps for preparing immunomodulatory recombinant macrophage vesicles are as follows:

[0102] 1) 10 mg GO / BTO and 1×10 8 The cell membrane mixture is fused in water;

[0103] 2) The mixture was ultrasonically treated with a frequency of 50 kHz and a power of 120 W for 20 minutes.

[0104] 3) Then extrude at least 10 times through a micro extruder using a 200 nm polycarbonate film.

[0105] 4) After extrusion, we collected the loaded nanoparticles by centrifugation at 15000 g for 30 min.

[0106] Example 4

[0107] The method for preparing the hybrid of macrophage membrane and exosome membrane is as follows:

[0108] 1) The collected macrophages are subjected to repeated freeze-thaw or water absorption bursting method to rupture the cell membrane and collect the mixed solution;

[0109] 2) The mixture was centrifuged at 2000 g for 10 minutes, the supernatant was collected and centrifuged at 15000 g for 30 minutes to collect the cell membranes. The cell membranes were sonicated at a frequency of 48 kHz and a power of 100 W for 20 minutes;

[0110] 3) Extraction of exosomes from cells: centrifuge the supernatant at 12,000 × g (~12,400 rpm) at 4°C for 15 min, discard the supernatant, and the precipitate is rich in exosome particles (absorb as much supernatant as possible);

[0111] 4) Extruding the extracted cell membrane and exosomes through a 300 nm polycarbonate membrane using a micro extruder at least 10 times;

[0112] 5) After extrusion, we collected the nanovesicles and stored them in PBS;

[0113] The steps for preparing immunomodulatory recombinant macrophage vesicles are as follows:

[0114] 1) 50 mg GO / BTO and 1×10 9 The cell membrane mixture is fused in water;

[0115] 2) The mixture was ultrasonically treated for 20 minutes at a frequency of 48 kHz and a power of 100 W.

[0116] 3) Then extrude at least 10 times through a micro extruder using a 300 nm polycarbonate film.

[0117] 4) After extrusion, we collected the loaded nanoparticles by centrifugation at 15000 g for 30 min.

[0118] Morphological observation, particle size and its distribution determination. After centrifugation of the targeted Porphyromonas gingivalis and the immunomodulatory recombinant macrophage vesicles, the precipitate was taken out, a small amount of distilled water was added to disperse it, and the sample was dropped on the carbon support membrane. The morphology was observed under a transmission electron microscope and photographed. Under a transmission electron microscope, it was observed that the targeted Porphyromonas gingivalis and the immunomodulatory recombinant macrophage vesicles were uniform and regular spherical particles, and their diameters were controllable within the range of 200 to 300 nm. The prepared nanocarriers are as follows Figure 5 shown.

[0119] The preparation and application of recombinant macrophage vesicles targeting Porphyromonas gingivalis and immunomodulation disclosed and proposed by the present invention can be achieved by those skilled in the art by referring to the content of this article and appropriately changing the conditions, routes and other links. Although the method and preparation technology of the present invention have been described through preferred embodiments, relevant technicians can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention to achieve the final preparation technology. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention.

[0120] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0121] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing immunomodulatory recombinant macrophage vesicles, characterized in that: The preparation method specifically comprises the following steps: Step 1: Clinical sequencing of gingival tumor tissue and analysis of pathogenic microbiome data: By obtaining pathogenic tumor microbiome data, a Porphyromonas gingivalis-specific macrophage model is established. Macrophages are co-incubated with Porphyromonas gingivalis from gingival tumor tissue, and the cells and supernatant are collected to obtain Porphyromonas gingivalis-specific targeted macrophages and macrophage exosomes; Step 2: Hybrid fusion of macrophage membrane and exosome membrane: Using thin film extrusion method and ultrasonic technology, the extracted macrophage membrane and exosome membrane are efficiently hybridized and fused, so that the two membrane structures are mixed together to form a double hybrid recombinant macrophage vesicle that specifically targets Porphyromonas gingivalis and reprograms the immune microenvironment; Step three, construct piezoelectrically driven ROS-generating immunomodulatory vesicles: The dual hybrid recombinant macrophage vesicles prepared above are further coated on the piezoelectric material GO / BTO to construct a new immunomodulatory tool - Porphyromonas gingivalis recombinant macrophage vesicles with piezoelectric drive to produce ROS.

2. The method for preparing an immunomodulatory recombinant macrophage vesicle according to claim 1, characterized in that: The method for constructing a macrophage model specific for Porphyromonas gingivalis in step 1 is specifically as follows: A1, Preparation of Porphyromonas gingivalis: First, culture Porphyromonas gingivalis from gingival tumor tissue overnight; then collect the bacterial solution and wash it thoroughly with PBS 2-3 times to remove impurities; then resuspend the bacteria in macrophage-specific culture medium and adjust its OD value to 0.05 to ensure a moderate bacterial concentration; A2, macrophage culture and infection: RAW264.7 macrophage cell line was selected for culture, and when the cells grew to a confluence of about 70%, the medium was changed; then, the pre-prepared culture medium containing resuspended Porphyromonas gingivalis was added to the cell culture system and incubated for 2 to 6 hours; A3, collection and processing: After the co-incubation of macrophages and Porphyromonas gingivalis, collect the macrophages expressing specific receptors; at the same time, collect the exosomes released into the supernatant during the immune activation process, and store the collected macrophages and supernatant under appropriate conditions for subsequent experimental use.

3. The method for preparing an immunomodulatory recombinant macrophage vesicle according to claim 1, characterized in that: The specific preparation method of the hybrid fusion of macrophage membrane and exosome membrane in step 2 is as follows: B1, Extraction of macrophage membrane: Use the freeze-thaw method or the water swelling method to treat macrophages to rupture the cell membrane and release the cell contents. Centrifuge the mixture at 2000g for 10 minutes to remove large particles and unruptured cells. The supernatant was collected; the cell membranes were further collected by centrifugation: the supernatant was centrifuged at 15000 g for 30 minutes, and the cell membranes were ultrasonically treated at a frequency of 42 kHz and a power of 100 W for 10 minutes to further disrupt and homogenize the cell membranes; B2, extraction of cellular exosomes: the supernatant obtained after the initial centrifugation in B1 was centrifuged at 12,000 g for 15 min at 4 °C; Collect exosomes: After centrifugation, discard the supernatant and the precipitate is rich in exosome particles; B3, hybridization of macrophage membrane and exosomes: the mixed solution was passed through a 300 nm polycarbonate membrane and repeatedly extruded at least 10 times using a micro extruder to form a uniform nanovesicle structure; B4, Collection and storage of nanovesicles: After extrusion, collect the obtained nanovesicles; store the nanovesicles in an appropriate buffer to ensure their stability and activity during storage.

4. The method for preparing an immunomodulatory recombinant macrophage vesicle according to claim 1, characterized in that: The method for constructing the immunomodulatory Porphyromonas gingivalis recombinant macrophage vesicles with piezoelectric ROS generation in step 3 is: C1, material preparation: weigh 10 mg of GO / BTO; prepare 1×10 8 Macrophage membrane; C2, mixture preparation: 10 mg of GO / BTO was mixed with 1×10 8 The macrophage membrane mixture was mixed in water to ensure that the two were fully mixed; C3, ultrasonic treatment: the mixture was ultrasonicated at a frequency of 42 kHz and a power of 100 W for 10 min using an ultrasonic processor; C4, microextrusion: the sonicated mixture was extruded through a microextruder equipped with a 300 nm polycarbonate membrane, repeated at least 10 times; C5, centrifugal collection: After extrusion, the mixture was centrifuged at 15000g for 30 min; By centrifugation, the macrophage vesicles loaded with GO / BTO were separated from other non-vesicle-forming components; and then the loaded nanoparticles were collected.

5. The method for preparing an immunomodulatory recombinant macrophage vesicle according to claim 1, characterized in that: The following are the mimetic treatments based on recombinant macrophage vesicles: 1) Applying immune-modulating recombinant macrophage vesicles to the oral cavity of a mouse model of gingival cancer for seven days; 2) Observe the changes in mouse tumor size, bacterial killing, and immune activation of tumor tissue after seven days.

Images