Macrophage-derived exosomes and injectable gel

Abstract

The present invention relates to macrophage-derived exosomes and injectable gels, belonging to the technical field of tumor immunotherapy drugs. The present invention constructs macrophages that overexpress Siglec-10 through genetic engineering, processes them to form exosome-like structures, and chemically modifies them to form gel factors that can form gels in situ in tumor tissue. This effectively regulates the polarized phenotype and phagocytic ability of tumor-associated macrophages, thereby simultaneously relieving local and systemic immunosuppression, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunotherapy drugs, and specifically relates to a macrophage-derived exosome, a hydrogel preparation combining the exosome with oxidized sodium alginate, and its application in the field of tumor immunotherapy. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Macrophages are key effector cells of innate immunity and have a powerful phagocytic effect. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment. It is generally believed that CD47 is the main "don't eat me" signal molecule on the surface of tumor cells, thereby avoiding recognition, killing and phagocytosis by TAMs. CD47 is an anti-phagocytic signaling protein, and the development of antibody drugs targeting CD47 has entered the clinical trial stage. However, according to existing clinical trial data, the clinical response rate of blocking CD47 is low, suggesting the existence of other anti-phagocytic molecules. The latest research results show that the CD24 protein is specifically and highly expressed on the surface of various cancer cells such as ovarian cancer and triple-negative breast cancer. CD24 is a signaling protein similar to CD47, and can bind to Siglec-10 on the surface of TAMs to activate the SHP-1 / SHP-2-mediated phagocytic inhibitory signaling pathway.

[0004] To date, no drug has been found in clinical practice that can effectively block CD24, posing a challenge to precisely regulating the CD24-Siglec-10 pathway within the tumor microenvironment. Activated TAMs primarily include M1 and M2 types. M1 macrophages can kill tumor cells and defend against pathogen invasion, while M2 macrophages primarily promote tumor growth, invasion, and metastasis. Macrophages in tumor tissue often share the phenotype and function of M2 macrophages, exerting both immunosuppressive and tumor-promoting effects. Therefore, synergistically regulating M2 macrophages in the tumor microenvironment to polarize them to the M1 phenotype is another challenge that needs to be addressed. Summary of the Invention

[0005] To overcome the above-mentioned technical problems, the present invention provides a bioactive preparation based on macrophage-derived exosomes, as well as its preparation method and application. The present invention genetically encodes Siglec-10 into the mouse macrophage cell line RAW264.7. The engineered macrophages are expanded and polarized to the M1 phenotype, which are then processed to form macrophage-derived exosomes. After chemical modification, these exosomes can serve as gelling factors for in situ gel formation in tumor tissue, effectively regulating the polarized phenotype and phagocytic ability of tumor-associated macrophages, thereby simultaneously relieving local and systemic immunosuppression. Therefore, these exosomes have excellent practical application value.

[0006] As previously mentioned, regulating TAMs faces two major obstacles. First, their phagocytic ability is severely inhibited, limiting their innate immune response. Second, TAMs primarily adopt the M2 phenotype, exerting immunosuppressive and tumor-promoting effects. Therefore, simultaneously regulating both the phagocytic ability and polarized phenotype of TAMs is crucial for improving TAM-based immunotherapy.

[0007] Recent research has found that, unlike most tumors that inhibit the phagocytic ability of TAMs by upregulating CD47, ovarian and triple-negative breast cancer cells specifically overexpress CD24 on their surfaces. CD24 is a signaling protein involved in regulating phagocytic activity and can bind to Siglec-10 on the surface of tumor-associated macrophages, activating the SHP-1 / SHP-2-mediated phagocytic inhibitory signaling pathway. Here, the present invention utilizes genetic engineering to generate exosomes derived from M1 macrophages that overexpress Siglec-10. These exosomes compete with TAMs for CD24 binding, effectively blocking the CD24-Siglec-10 pathway and restoring the phagocytic ability of TAMs. Furthermore, these exosomes derived from M1 macrophages inherit inflammatory proteins and RNA from their parent cells, enabling them to effectively polarize M2 TAMs to an M1 phenotype, exerting anti-tumor effects. This combination simultaneously modulates both the phagocytic ability and phenotype of TAMs.

[0008] Based on the above effects, the present invention first provides a macrophage-based derived exosome, wherein the derived exosome is a nanovesicle that overexpresses Siglec-10 and M1 macrophages.

[0009] The derived exosomes provided by the present invention are obtained by extruding M1 macrophages that overexpress Siglec-10 and are sized between 100 and 200 nm. These derived exosomes inherit the inflammatory factors and genetic material from the parent cells and are more efficient at polarizing M2 phenotype TAMs compared to existing macrophage membrane vesicles.

[0010] In a second aspect, the present invention provides the derived exosomes described in the first aspect, and the preparation method comprises the following steps: constructing macrophages overexpressing Siglec-10 by genetic engineering, screening the stably transfected cell lines for M1 polarization induction to obtain engineered cells with M1 phenotype, and extruding them into exosome-like cells.

[0011] Preferably, the genetic engineering method aims to introduce a polynucleotide encoding a target protein into a host cell via a vector. The genetic engineering method described in the present invention is not limited to the vector. As long as it can replicate in macrophages, technicians can use any vector known in the art, such as natural or recombinant plasmids, cosmids, viruses or phages.

[0012] In one embodiment provided by the present invention, the genetic engineering method is lentiviral infection. In this embodiment, macrophages overexpressing Siglec-10 are obtained by lentiviral infection, and stably transfected cell lines are screened by drugs, wherein the MOI during lentiviral infection is 25-100, preferably 50; the infection time is 8-24 hours, preferably 12 hours; the screening drug for stably transfected cell lines is puromycin, and the screening concentration is 1-8 ng / mL, preferably 6 ng / mL; the screening time is 12-48 hours, preferably 36 hours.

[0013] Preferably, the sources of the macrophages include, but are not limited to, bone marrow, peripheral blood, macrophages extracted from the peritoneal cavity, tumor-associated macrophages, or commercially available macrophage models, such as macrophage cell lines RAW 264.7 cells, ANA-1 cells, J774A.1 cells, and THP-1 cells. Based on the prior art known to those skilled in the art, it can be reasonably inferred that when the derived exosomes are applied to the human body, the macrophages can be of autologous origin. When commercially available macrophage models such as mouse macrophages are used, their primary application is to prepare a pharmaceutical research preparation.

[0014] Preferably, the M1 polarization is induced by IFN-γ and lipopolysaccharide (LPS); further, the specific steps of the polarization induction are as follows: amplifying and culturing the stably transfected cell line, replacing the culture medium containing IFN-γ and lipopolysaccharide, and continuing to culture for a period of time to obtain M1 engineered macrophages;

[0015] Furthermore, the IFN-γ concentration is 10-150 ng / mL, preferably 100 ng / mL; the lipopolysaccharide concentration is 0.5-2 μg / mL, preferably 1 μg / mL; and the culture time is 12-48 hours, preferably 24 hours.

[0016] Preferably, the exosome-like particles are extruded through a nanoformulation extrusion device, such as a liposome extruder; the pore sizes of the PC filter membranes through which the particles are extruded are 1 μm, 400 nm, and 200 nm, respectively. The number of repeated extrusions is 4-12 times, preferably 7 times.

[0017] Preferably, in the above preparation method, the extruded exosomes need to be removed by ultracentrifugation, the centrifugal speed is 50,000-200,000 g, preferably 100,000 g; the centrifugation time is 1 hour to 4 hours, preferably 2 hours; the low temperature is 0 to 4 ° C.

[0018] In addition, in order to achieve the drug-forming effect of the derived exosomes, the present invention also provides an injectable hydrogel preparation of the derived exosomes. Using the injectable hydrogel based on the derived exosomes as a carrier can, on the one hand, effectively reduce systemic side effects; on the other hand, the exosome-like vesicles act as a gel factor, and as the gel skeleton gradually degrades, it can continuously regulate the tumor microenvironment, making it more conducive to exerting a lasting therapeutic effect.

[0019] In a third aspect, the present invention provides an injectable gel, which uses the macrophage-derived exosomes described in the first aspect as an active ingredient and has alginate-modified surface.

[0020] Preferably, the alginate includes but is not limited to sodium alginate, calcium alginate, potassium alginate or oxides thereof; in an embodiment provided by the present invention with better effects, the alginate is oxidized sodium alginate.

[0021] In a fourth aspect, the present invention provides a method for preparing the injectable gel described in the third aspect, wherein the preparation method is as follows: mixing an alginate solution with the derived exosomes described in the first aspect, and then stirring the mixture under low temperature conditions for a period of time to obtain the injectable gel.

[0022] Preferably, the low temperature condition is 0-4°C.

[0023] Preferably, the reaction time is 4 to 12 hours, more preferably 6 hours.

[0024] Preferably, the alginate solution is an aqueous solution of oxidized sodium alginate, and the oxidized sodium alginate is a product of sodium alginate oxidized by sodium periodate. The specific preparation method is as follows: sodium alginate is dissolved in deionized water, sodium periodate is added, and the mixture is stirred at room temperature in the dark for a period of 3 to 5 hours, ethylene glycol is added, and the mixture is stirred and reacted for 0.5 to 1.5 hours, and then NaCl is added. Ethanol is added to the reaction system to produce precipitation, the precipitated part is redissolved, dialyzed against water, and dried to obtain sodium alginate oxide.

[0025] In a fifth aspect, the present invention provides a pharmaceutical composition comprising an active dose of the derived exosomes described in the first aspect or the injectable gel described in the third aspect.

[0026] Preferably, the derived exosomes or injectable gel further has chemical or genetic modifications, and the modifications include but are not limited to streptavidin, biotin, radioisotopes, fluorescent agents, enzymes, cytotoxic substances, anti-tumor agents, etc.

[0027] Preferably, the pharmaceutical composition includes pharmaceutically necessary carriers, which should be harmless to the subject at the dosage used. Specific types include but are not limited to buffers, antioxidants, preservatives, bactericides, chelating agents, tension regulators, surfactants, salt-forming counterions, and metal complexes.

[0028] Preferably, when the above-mentioned derived exosomes or injectable gel is used to prepare a pharmaceutical preparation for in vivo administration, the pharmaceutical preparation should be sterile, and the method for achieving its sterility can adopt conventional methods in the art, such as filtration using a sterile filter membrane.

[0029] Preferably, in the pharmaceutical composition, the derived exosomes or injectable gel should be in an effective dose, and the dose needs to be routinely determined according to the subject, the administration method and the purpose of administration; for example, based on the total mass of the pharmaceutical composition, the content of the derived exosomes or injectable gel can be in the range of about 0.01-99%, 0.1-70%, 1-30%, 0.01-0.05%, 0.05-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-1%, 1-3%, 3-5%, 5-10%, 10-20%, 20-30%, 30-50%, 50-70%, or 70-99%.

[0030] In a sixth aspect, the present invention provides an anti-tumor preparation, comprising an active dose of the macrophage-derived exosomes described in the first aspect, the injectable gel described in the third aspect, or the pharmaceutical composition described in the fifth aspect.

[0031] The anti-tumor preparations described in the sixth aspect include but are not limited to drugs used to prevent, improve or treat tumors, or model agents for the development of anti-tumor drugs, and the tumors include benign tumors and malignant tumors. At the same time, it should be noted that tumors are used in the present invention as known to those skilled in the art, and include benign tumors and / or malignant tumors. Benign tumors are defined as excessive proliferation of cells that cannot form aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular abnormalities and biochemical abnormalities that can form systemic diseases (for example, forming tumor metastases in distal organs). Malignant tumors include solid tumors, hematologic tumors, hereditary tumors, and also include primary tumors in the organs and corresponding secondary tumors (tumor metastases) in distal organs.

[0032] The solid tumors are selected from the group consisting of breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testicle, stomach, skin (such as melanoma), ureter, vagina and vulva. In a preferred embodiment of the present invention, the anti-tumor preparation is used for ovarian cancer or breast cancer, particularly triple-negative breast cancer.

[0033] Such hereditary tumors include, for example, retinoblastoma and Wilms tumor.

[0034] The hematological neoplasms may be aggressive and indolent forms of leukemia and lymphoma, i.e., non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphocytic leukemia (ALL), Hodgkin's disease, multiple myeloma and T-cell lymphoma, and also include myelodysplastic syndrome, plasmacytoma, tumoroid syndrome and cancer of unknown primary site and AIDS-related malignancies.

[0035] The beneficial technical effects of one or more of the above technical solutions are:

[0036] (1) The present invention combines CD24 blockade and macrophage polarization for anti-tumor effects, while alleviating the phagocytic inhibition of TAMs and the immunosuppression caused by the M2 phenotype, resulting in a more excellent anti-tumor effect;

[0037] (2) The present invention prepares, for the first time, a macrophage-derived exosome modified with sodium alginate oxide, which has a gelling factor function and can quickly form a gel in the presence of calcium ions;

[0038] (3) The present invention synthesized for the first time a gelling factor based on cell-derived exosomes, which is structurally different from existing gelling factors and has higher biological activity;

[0039] (4) The cell-derived exosome-based hydrogel prepared by the present invention shows good biocompatibility with normal cells and has few toxic side effects, providing the possibility of designing safer local drug delivery carriers;

[0040] (5) The present invention utilizes immunotherapy based on the bioactive preparation of cell-derived exosomes to change the phenotype of TAMs, improve their phagocytic ability of tumors, enhance the anti-tumor effect in vivo, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0042] Figure 1 These are scanning electron microscopy images of macrophage-derived exosome nanostructures before and after modification with sodium alginate oxide in Example 1;

[0043] Figure 2 This is a photo of the O-SM1Aexo gel in Example 1;

[0044] Figure 3 This is a scanning electron microscope image of the O-SM1Aexo gel in Example 1;

[0045] Figure 4 This is a characterization diagram of the in vivo anti-tumor activity experiment of O-SM1Aexo hydrogel in Example 1;

[0046] Figure 5 This is an experimental characterization diagram of the in vivo anti-tumor immune activity of O-SM1Aexo hydrogel in Example 1. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0048] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0050] Example 1: Preparation of injectable hydrogel

[0051] In this embodiment, a method for preparing macrophage-derived exosomes and an injectable hydrogel based on the derived exosomes is provided:

[0052] 1. Preparation of macrophage-derived exosomes:

[0053] 1) Construction of a Siglec-10-overexpressing mouse macrophage cell line using a lentiviral vector: Engineered macrophages overexpressing Siglec-10 were obtained by lentiviral infection at an MOI of 50. 12 hours after infection, the cells were selected with 6 ng / mL puromycin for 26 hours to obtain a stable transfected cell line.

[0054] 2) Polarizing the engineered macrophages to an M1 phenotype: Stably transfected cells were plated in 10 cm culture dishes and incubated in an incubator for 48 hours for expansion. The culture medium was then discarded and cultured in a medium containing 100 ng / mL IFN-γ and 1 μg / mL lipopolysaccharide for 24 hours to obtain engineered macrophages with an M1 phenotype.

[0055] 3) Collect engineered cells with the M1 phenotype and extrude them into exosome-like structures using a nanoextruder: Using the nanoextruder, pass the cells through PC filters with pore sizes of 1 μm, 400 nm, and 200 nm, respectively. Each pore size requires repeated extrusion seven times to obtain an exosome-like solution.

[0056] 4) Using low-temperature ultracentrifugation to remove impurities and obtain structurally homogeneous exosome-like vesicles: ultracentrifuge the exosome-like vesicle solution at low temperature at a speed of 100,000 g for 2 hours.

[0057] 2. Preparation method of injectable hydrogel:

[0058] 1) Preparation of oxidized sodium alginate: Sodium alginate was dissolved in deionized water, followed by the addition of sodium periodate. The mixture was stirred in the dark at room temperature for 4 hours. Ethylene glycol was added and stirring continued for 1 hour. NaCl was then added. Ethanol was added to the reaction system to produce a precipitate. The precipitate was then separated and redissolved, and dialyzed against water for 3 days. Oxidized sodium alginate was obtained after freeze-drying.

[0059] 2) Preparation of sodium alginate oxide-modified exosome-like vesicles: An excess of oxidized sodium alginate solution was mixed with the macrophage-derived exosomes in deionized water and stirred thoroughly. After reacting at low temperature for 6 hours, the excess sodium alginate oxide was removed by ultrafiltration and centrifugation to obtain modified exosome-like vesicles, designated O-SM1Aexo hydrogel.

[0060] Example 2

[0061] In this embodiment, another method for preparing macrophage-derived exosomes and an injectable hydrogel based on the derived exosomes is provided:

[0062] 1. Preparation of macrophage-derived exosomes:

[0063] 1) Construction of a Siglec-10-overexpressing mouse macrophage cell line using a lentiviral vector: Siglec-10-overexpressing engineered macrophages were obtained by lentiviral infection at an MOI of 75. After 8 hours of infection, the cells were selected with 8 ng / mL puromycin for 15 hours to obtain a stable transfected cell line.

[0064] 2) Polarization of engineered macrophages to an M1 phenotype: Stably transfected cells were plated in 10 cm culture dishes and incubated in an incubator for 45 hours for expansion. The culture medium was then discarded and cultured for 18 hours in a medium supplemented with 150 ng / mL IFN-γ and 1.5 μg / mL lipopolysaccharide to obtain engineered macrophages with an M1 phenotype.

[0065] 3) Collect engineered cells with the M1 phenotype and extrude them into exosome-like structures using a nanoextruder: Using the nanoextruder, pass the cells through PC filters with pore sizes of 1 μm, 400 nm, and 200 nm, respectively. Each pore size requires repeated extrusion 10 times to obtain an exosome-like solution.

[0066] 4) Using low-temperature ultracentrifugation to remove impurities and obtain structurally homogeneous exosome-like vesicles: ultracentrifuge the exosome-like vesicle solution at low temperature at a speed of 200,000 g for 1 hour.

[0067] 2. Preparation method of injectable hydrogel:

[0068] 1) Preparation of oxidized sodium alginate: Sodium alginate was dissolved in deionized water, followed by the addition of sodium periodate. The mixture was stirred in the dark at room temperature for 3 hours, followed by the addition of ethylene glycol and stirring for 0.8 hours. NaCl was then added, and ethanol was added to the reaction system to cause precipitation. The precipitated portion was separated and redissolved, and then dialyzed against water for 2.5 days. The oxidized sodium alginate was obtained after freeze-drying.

[0069] 2) Preparation of sodium alginate oxide-modified exosome-like vesicles: An excess of oxidized sodium alginate solution was mixed with the macrophage-derived exosomes in deionized water and stirred thoroughly. The mixture was reacted at 2°C for 5 h, and then the excess sodium alginate oxide was removed by ultrafiltration and centrifugation to obtain modified exosome-like vesicles.

[0070] Example 3

[0071] In this embodiment, another method for preparing macrophage-derived exosomes and an injectable hydrogel based on the derived exosomes is provided:

[0072] 1. Preparation of macrophage-derived exosomes:

[0073] 1) Construction of a Siglec-10-overexpressing mouse macrophage cell line using a lentiviral vector: Engineered macrophages overexpressing Siglec-10 were obtained by lentiviral infection at an MOI of 30. After 18 hours of infection, the cells were selected with 6 ng / mL puromycin for 26 hours to obtain a stable transfected cell line.

[0074] 2) Polarizing the engineered macrophages to an M1 phenotype: Stably transfected cells were plated in 10 cm culture dishes and incubated in an incubator for 48 hours for expansion. The culture medium was then discarded and cultured in a medium containing 40 ng / mL IFN-γ and 0.5 μg / mL lipopolysaccharide for 48 hours to obtain engineered macrophages with an M1 phenotype.

[0075] 3) Collect engineered cells with the M1 phenotype and extrude them into exosome-like structures using a nanoextruder: Using the nanoextruder, pass the cells through PC filters with pore sizes of 1 μm, 400 nm, and 200 nm, respectively. Each pore size requires repeated extrusion 10 times to obtain an exosome-like solution.

[0076] 4) Using low-temperature ultracentrifugation to remove impurities and obtain structurally homogeneous exosome-like vesicles: ultracentrifuge the exosome-like vesicle solution at low temperature at a speed of 150,000 g for 2 hours.

[0077] 2. Preparation method of injectable hydrogel:

[0078] 1) Preparation of oxidized sodium alginate: Sodium alginate was dissolved in deionized water, followed by the addition of sodium periodate. The mixture was stirred in the dark at room temperature for 5 hours, followed by the addition of ethylene glycol and stirring for 1.5 hours. NaCl was then added, and ethanol was added to the reaction system to cause precipitation. The precipitated portion was separated and redissolved, and then dialyzed against water for 4 days. The oxidized sodium alginate was obtained after freeze-drying.

[0079] 2) Preparation of sodium alginate oxide-modified exosome-like vesicles: An excess of oxidized sodium alginate solution was mixed with the macrophage-derived exosomes in deionized water and stirred thoroughly. The mixture was reacted at 1°C for 6 hours, and then the excess sodium alginate oxide was removed by ultrafiltration and centrifugation to obtain modified exosome-like vesicles.

[0080] Characterization of the properties of the derived exosomes and injectable gel in Example 1:

[0081] 1. Scanning electron microscopy (SEM) identification of the nanostructure of macrophage-derived exosomes before and after modification

[0082] 10 μL of macrophage-derived exosome solution before and after modification was pipetted onto the carbon film copper grid, and excess liquid was removed with filter paper. The solution was dried at room temperature and then observed under a scanning electron microscope. Figure 1 As shown, scanning electron microscopy images can confirm that there are obvious differences in the morphology of macrophage-derived exosomes before and after modification, and the exosomes are uniform in size and well dispersed.

[0083] 2. Gelation characteristics and micromorphology of O-SM1Aexo hydrogel

[0084] For more intuitive observation and comparison, the gelling properties of the O-SM1Aexo gel factor prepared in Example 1 were photographed and observed. Figure 2 In the experiment, O-SM1Aexo gel factor can quickly form a gel in the presence of calcium ions. The microstructure of the formed gel was observed, and it can be seen that O-SM1Aexo hydrogels are all porous structures with huge drug loading potential.

[0085] 3. In vivo tumor inhibition study of O-SM1Aexo hydrogel

[0086] 1). Establishment of animal model

[0087] Female Balb / c mice aged 6 to 8 weeks were used to establish an in vivo bilateral anti-tumor model. 4T1 cell suspension (8 × 10 cells per mouse) was added. 5 cells) were subcutaneously inoculated into the right forelimb of mice to establish orthotopic tumors. Three days later, 4T1 cell suspension (8×10 cells per mouse) was added. 5 cells) were subcutaneously inoculated into the left forelimb of mice to establish distal tumors.

[0088] 2). In vivo tumor inhibition experiments in animals

[0089] In situ tumors reach ~60 mm 3Afterwards, the mice were weighed and randomly divided into two groups: saline as a control group and O-SM1Aexo hydrogel as an experimental group. The dosage, calculated as exosome-like protein, was 25 mg / kg. After a single intratumoral injection, tumor volume was measured every two days and calculated according to the following equation:

[0090]

[0091] L represents the maximum diameter (mm), and W represents the minimum diameter (mm).

[0092] After the completion of various treatments, the mice were sacrificed and the tumors were removed and weighed. The inhibition rate of the tumor in each group of mice was calculated according to the following formula:

[0093]

[0094] W c represents the average tumor weight of the saline group, and W t The final tumor weights of the other groups are indicated.

[0095] In vivo tumor inhibition experiment results Figure 5 shown.

[0096] The modified exosome-like cells can quickly form gels under the physiological environment of tumors and exert long-lasting anti-tumor effects.

[0097] from Figure 4 It can be seen that in both in situ and distal tumors, compared with the saline group, O-SM1Aexo hydrogel can simultaneously relieve the local tumor microenvironment and systemic immunosuppression, thereby achieving a better anti-tumor effect. At the same time, this example studies the immune mechanism within the tumor. Figure 5 As shown in Figure 3, after hydrogel treatment, T cell infiltration in both in situ and distal tumors increased significantly, demonstrating the activation of a systemic immune response. Therefore, the combined application of CD24 blockade and polarization phenotype regulation has great potential in regulating TAMs to activate anti-tumor immune responses.

[0098] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A macrophage-derived exosome, characterized in that: The derived exosomes are nanovesicles of M1 macrophages that overexpress Siglec-10; The method for preparing derived exosomes comprises the following steps: constructing macrophages overexpressing Siglec-10 by genetic engineering, screening the stably transfected cell lines for M1 polarization induction to obtain engineered cells with M1 phenotype, and extruding the cells into exosome-like cells; The macrophages are macrophage cell line RAW 264.7 cells.

2. The exosome-derived exosome according to claim 1, wherein Macrophages overexpressing Siglec-10 were obtained by lentiviral infection, and stable transfected cell lines were identified through drug screening; The MOI of the lentivirus infection is 25-100, the infection time is 8-24 hours, the screening drug for the stably transfected cell line is puromycin, the screening concentration is 1-8 ng / mL, and the screening time is 12-48 hours.

3. The exosome-derived exosome according to claim 1, wherein The M1 polarization is induced by IFN-γ and lipopolysaccharide. The specific steps of the polarization induction are as follows: the stably transfected cell line is expanded and cultured, and the culture medium containing IFN-γ and lipopolysaccharide is replaced and cultured for a period of time to obtain M1 engineered macrophages; the IFN-γ concentration is 10-150 ng / mL; the lipopolysaccharide concentration is 0.5-2 μg / mL; and the culture time is 12-48 hours.

4. The exosome-derived exosome according to claim 1, wherein The nanoparticles are extruded through a nanoparticle extrusion device, which is a liposome extruder; the pore sizes of the PC filter membranes through which the extrusion is performed are 1 μm, 400 nm, and 200 nm, respectively, and the number of repeated extrusions is 4-12 times; Alternatively, in the preparation method, the exosome-like vesicles obtained after extrusion need to be removed by ultracentrifugation, and the centrifugation speed is 50,000-200,000 g; the centrifugation time is 1 hour to 4 hours; and the temperature is 0-4°C.

5. An injectable gel, characterized in that The injectable gel uses the macrophage-derived exosomes according to claim 1 as an active ingredient, and has oxidized sodium alginate modified on its surface.

6. The method for preparing the injectable gel according to claim 5, characterized in that: The preparation method is as follows: an aqueous solution of oxidized sodium alginate is mixed with the derived exosomes according to claim 1, and then stirred and reacted for a period of time under low temperature conditions to obtain the obtained product.

7. The method for preparing the injectable gel according to claim 6, wherein: The low temperature condition is 0~4 ℃; Or, the reaction time is 4 to 12 hours; Alternatively, the oxidized sodium alginate is a product obtained by oxidizing sodium alginate with sodium periodate. The specific preparation method is as follows: dissolving sodium alginate in deionized water, adding sodium periodate, stirring and reacting at room temperature in the dark for 3 to 5 hours, adding ethylene glycol, continuing to stir and react for 0.5 to 1.5 hours, and then adding NaCl, adding ethanol to the reaction system to produce precipitation, redissolving the precipitated portion, dialyzing it against water, and drying it to obtain sodium alginate oxide.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an active dose of the derived exosomes according to claim 1 or the injectable gel according to claim 5.

9. An antitumor preparation, characterized in that: The anti-tumor preparation comprises an active dose of the derived exosomes according to claim 1, the injectable gel according to claim 5 or the pharmaceutical composition according to claim 8.

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