Culture method for enhancing tumor targeting of T cell exosome

Abstract

The invention discloses a culture method for enhancing T cell exosome tumor targeting, and relates to the technical field of biology, the culture method comprises the following steps: separation and culture of T cells; establishing a simulated tumor microenvironment; co-culturing the tumor-associated fibroblasts; performing tumor cell exosome stimulation; extracting the exosome and enhancing the targeting property; the targeting enhancement is as follows: the extracted exosome is compounded with magnetic nanoparticles to form a magnetic nanoparticle-exosome compound; t cells are cultured by simulating a tumor microenvironment, and a staged synergistic effect is realized by combining a magnetic nanoparticle and exosome compounding technology and regulating and controlling the dosage proportion of a compound, so that the tumor targeting property and the anti-tumor effect of the exosome are remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a culture method for enhancing the tumor targeting of T cell exosomes. Background Art

[0002] T cell exosomes are nanoscale vesicles secreted by T cells, carrying a variety of immunologically active molecules, and having great potential in tumor immunotherapy. However, the tumor targeting of T cell exosomes obtained under conventional culture conditions is limited, and it is difficult to effectively enrich at the tumor site, which limits their clinical application.

[0003] Studies have shown that the tumor microenvironment (TME) has characteristics such as hypoxia, acidity, nutrient deficiency, and immunosuppression, which can affect the function of T cells and the secretion of exosomes. Therefore, culturing T cells by simulating the TME may induce the secretion of exosomes with stronger tumor targeting.

[0004] For example, in the Chinese patent with the application number CN202210447053.2, a glioma targeting system based on magnetic nanoparticle composite engineered exosomes and its application, this invention belongs to the field of bioengineering technology, and particularly relates to a glioma targeting system based on magnetic nanoparticle composite engineered exosomes and its application. The glioma targeting system includes: magnetic nanoparticles and engineered exosomes, and the magnetic nanoparticles and the engineered exosomes are connected through antigen-antibody reaction. The composite targeting system designed in this invention combines the magnetic targeting characteristics and drug-binding ability of magnetic nanoparticles (MNPs) with the blood-brain barrier (BBB) penetration ability and small interfering RNA encapsulation ability of engineered exosomes. By jointly targeting the DHODH and GPX4 pathways and synergistically acting with the Fe 2+ ions released by Fe3O4, the ferroptosis of glioma is jointly promoted, achieving the purpose of targeted treatment of glioma, and thus having good practical application value.

[0005] Although the above solution improves the tumor targeting of exosomes through means such as genetic engineering and chemical modification, this method is often complex in operation, high in cost, and may introduce new safety risks. Although the targeted treatment of glioma is achieved, it targets a specific type of tumor, and the preparation process of engineered exosomes is complex. Summary of the Invention

[0006] Embodiments of the present application provide a culture method for enhancing the tumor targeting of T cell exosomes, which solves the problems of complex operation, high cost, and possible introduction of new safety risks in the prior art. By simulating the tumor microenvironment to culture T cells, combining the composite technology of magnetic nanoparticles and exosomes, and regulating the dosage ratio of the complex, a phased synergistic effect is achieved, thereby significantly enhancing the tumor targeting and anti-tumor effect of exosomes.

[0007] Embodiments of the present application provide a culture method for enhancing the tumor targeting of T cell exosomes, comprising the following steps:

[0008] (1) Isolation and culture of T cells;

[0009] (2) Establishment of a simulated tumor microenvironment;

[0010] (3) Co-culture with tumor-associated fibroblasts;

[0011] (4) Stimulation with tumor cell exosomes;

[0012] (5) Extraction and targeting enhancement of exosomes;

[0013] The targeting enhancement is that the exosomes extracted in step (5) are complexed with magnetic nanoparticles to form a magnetic nanoparticle-exosome complex.

[0014] Further, the simulated tumor microenvironment described in step (2) includes the following situations:

[0015] (a) Hypoxic condition: Use a hypoxic incubator (1% O2);

[0016] (b) Acidic pH: Adjust the pH value of the culture medium to 6.5 - 6.8 using a pH buffer;

[0017] (c) Nutrient deficiency: Use a low-glucose (1 g / L) culture medium;

[0018] (d) Immune inhibitory factors: Add TGF-β (10 ng / mL) and IL-10 (10 ng / mL).

[0019] Further, the co-culture with tumor-associated fibroblasts described in step (3) means co-culturing T cells and tumor-associated fibroblasts at a ratio of 1:1.

[0020] Further, the stimulation with tumor cell exosomes described in step (4) means stimulating T cells with tumor cell exosomes (10 μg / mL).

[0021] Further, the exosome extraction method described in step (5) is the ultracentrifugation method or the kit method.

[0022] Furthermore, the core of the magnetic nanoparticles is an iron oxide particle, the surface is coated with mesoporous silica, citric acid is loaded in the mesopores, and the CD63 antibody is surface-modified by the grafting method; the loading amount of citric acid is 15-20 wt%; the mass ratio of exosomes to magnetic nanoparticles is 1:1.

[0023] Furthermore, the outer layer of the magnetic nanoparticles is also coated with calcium carbonate to form a core-shell structure of calcium carbonate-coated magnetic nanoparticles, wherein the mass ratio of iron oxide to calcium carbonate is 1:1.

[0024] Furthermore, the core-shell structure is complexed with exosomes to form a core-shell structure complex; the mass ratio of exosomes to the core-shell structure is 1:1.

[0025] Furthermore, the core-shell structure includes a high iron oxide core-shell structure and a low iron oxide core-shell structure.

[0026] Furthermore, in the high iron oxide core-shell structure, the mass ratio of iron oxide to calcium carbonate is 7:3; in the low iron oxide core-shell structure, the mass ratio of iron oxide to calcium carbonate is 3:7.

[0027] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0028] First, the present invention successfully induces the secretion of exosomes with stronger tumor targeting by culturing T cells in a simulated tumor microenvironment, and for the first time proposes a method for culturing T cell exosomes in a simulated tumor microenvironment, providing a new idea for obtaining exosomes with stronger tumor targeting. Compared with the T cell exosomes obtained under conventional culture conditions, the exosomes obtained in the present invention have the following advantages: (1) Increased secretion: Culturing in a simulated tumor microenvironment can significantly increase the secretion of T cell exosomes; (2) Altered protein expression: Culturing in a simulated tumor microenvironment can up-regulate the expression levels of proteins such as TSG101, CD63, and Alix in T cell exosomes; (3) Enhanced tumor targeting: T cell exosomes cultured in a simulated tumor microenvironment can more effectively target tumor cells; (4) Enhanced anti-tumor activity: T cell exosomes cultured in a simulated tumor microenvironment can significantly inhibit tumor growth;

[0029] Second, by introducing the magnetic nanoparticle-exosome complex, the targeting and anti-tumor performance are further improved. The magnetic property of Fe3O4 is used to drive the complex to enrich in the tumor area by an external magnetic field, and the concentration of the complex at the tumor site is increased through a physical localization mechanism to enhance the targeting; the citric acid loaded in the mesopores releases H + , locally reducing the pH. This acidification not only simulates the acidic characteristics of the tumor microenvironment but also promotes the dissolution of Fe3O4 and Fe 2+Release; through the specific binding of the CD63 antibody to the CD63 antigen on the surface of exosomes, the stability of the complex is ensured, ensuring the stability and long-acting property of the complex in vivo, promoting the enrichment and function of the complex at the tumor site; the released Fe 2+ reacts with hydrogen peroxide in tumor cells to generate hydroxyl radicals, triggering the Fenton reaction. Hydroxyl radicals can cause lipid peroxidation and destroy the ferroptosis defense system of tumor cells, thereby inducing tumor cell death and further improving the anti-tumor performance;

[0030] Thirdly, by combining the CaCO3 coating layer with citric acid-modified magnetic nanoparticles, active acidification of the tumor microenvironment and magnetic targeting guidance are achieved, further enhancing the tumor targeting and anti-tumor effects of exosomes; in the acidic tumor microenvironment (pH 6.5 - 6.9), the calcium carbonate coating layer undergoes a neutralization reaction with hydrogen ions: CaCO3 + 2H + →Ca 2+ + CO2↑ + H2O, thus achieving dynamic regulation of the local pH. In the short term, hydrogen ions are consumed at the initial stage of the reaction, briefly increasing the local pH, but the release of carbon dioxide forms microbubbles, disturbing the microenvironment and enhancing proton diffusion, thereby improving the dispersibility; in the long term, carbon dioxide dissolves in water to form carbonic acid, further releasing hydrogen ions, eventually reducing the local pH and enhancing the acidity, which promotes the cleavage of the Fe - O bond on the surface of Fe3O4, releasing more Fe 2+ ; citric acid ionizes and releases hydrogen ions in the tumor microenvironment. Citric acid forms a stable complex with Fe 3+ to prevent the precipitation of Fe 3+ and reduces it to Fe 2+ through intracellular reducing substances;

[0031] Fourthly, by designing two types of particles with different iron oxide contents, high-iron oxide complexes and low-iron oxide complexes, and carrying out staged release, staged synergistic effects are achieved, optimizing the pH regulation of the tumor microenvironment and the Fe 2+ release efficiency, thereby significantly enhancing the tumor targeting and anti-tumor performance of exosomes. Specific Embodiments

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs; the terms used in the description of this invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit this invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0033] Example 1: A culture method for enhancing the tumor targeting of T cell exosomes, comprising the following steps:

[0034] (1) Isolation and culture of T cells: Isolate T cells from the peripheral blood of healthy donors or cancer patients, and culture them using RPMI-1640 medium containing 10% fetal bovine serum;

[0035] (2) Establishment of a simulated tumor microenvironment: Seed T cells in culture plates. When the cell confluence reaches 80%-90%, replace the medium with a medium containing the following components:

[0036] (a) Hypoxic condition: Use a hypoxic incubator (1% O2) or add cobalt chloride (CoCl2, 100 μM) to the medium;

[0037] (b) Acidic pH: Adjust the pH of the medium to 6.5 - 6.8 using a pH buffer;

[0038] (c) Nutrient deprivation: Use low-glucose (1 g / L) or glucose-free medium;

[0039] (d) Immunosuppressive factors: Add TGF-β (10 ng / mL) and IL-10 (10 ng / mL);

[0040] (3) Co-culture with cancer-associated fibroblasts (CAFs): Co-culture T cells with CAFs at a ratio of 1:1, or culture T cells using CAF-conditioned medium;

[0041] (4) Stimulation with tumor cell exosomes: Stimulate T cells with tumor cell exosomes (10 μg / mL);

[0042] (5) Extraction and identification of exosomes: Collect cell culture supernatants, extract exosomes using ultracentrifugation or kit methods, and identify the exosomes.

[0043] Experiments were conducted on the above examples, and the specific steps are as follows:

[0044] (I) Isolation and culture of T cells

[0045] 1. Isolate T cells from the peripheral blood of healthy donors or cancer patients;

[0046] 2. Seed T cells in culture flasks and culture them in an RPMI-1640 medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator;

[0047] 3. Replace the medium every 2 - 3 days, and passage the cells when the cell confluence reaches 80%-90%;

[0048] (II) Establishment of a simulated tumor microenvironment

[0049] 1. Seed the 3rd generation of T cells at 1×10 5Inoculate at a density of cells / holes in a 6-well plate;

[0050] 2. When the cell confluence reaches 80%-90%, replace it with a medium containing the following components:

[0051] (a) Hypoxic condition: Use a hypoxic incubator (1% O2) or add cobalt chloride (CoCl2, 100 μM) to the medium;

[0052] (b) Acidic pH: Adjust the pH value of the medium to 6.5 - 6.8 using a pH buffer;

[0053] (c) Nutrient deficiency: Use a low-glucose (1 g / L) or glucose-free medium;

[0054] (d) Immunosuppressive factors: Add TGF-β (10 ng / mL) and IL-10 (10 ng / mL);

[0055] 3. Continue culturing for 24 - 48 hours;

[0056] (III) Co-culture with cancer-associated fibroblasts (CAFs)

[0057] 1. Co-culture T cells with CAFs at a ratio of 1:1, or culture T cells using the CAF-conditioned medium;

[0058] 2. Continue culturing for 24 - 48 hours;

[0059] (IV) Stimulation with tumor cell exosomes

[0060] 1. Stimulate T cells with tumor cell exosomes (10 μg / mL);

[0061] 2. Continue culturing for 24 - 48 hours;

[0062] (V) Extraction and identification of exosomes

[0063] 1. Collect the cell culture supernatant, centrifuge at 300 g for 10 minutes to remove cell debris;

[0064] 2. Transfer the supernatant to an ultracentrifuge tube, centrifuge at 100,000 g for 70 minutes, and discard the supernatant;

[0065] 3. Resuspend the precipitate with PBS, centrifuge at 100,000 g for 70 minutes, and discard the supernatant to obtain exosomes;

[0066] 4. Identify the exosomes using methods such as transmission electron microscopy, nanoparticle tracking analysis, and Western blot;

[0067] The T cells in the control group were cultured under conventional culture conditions and did not receive treatment to simulate the tumor microenvironment; the group simulating the tumor microenvironment was Example 1.

[0068] Performance detection:

[0069] (1) Effect of simulating the tumor microenvironment on the secretion of T cell exosomes

[0070] The nanoparticle tracking analyzer was used to detect the secretion amount of T cell exosomes cultured under the simulated tumor microenvironment. The results showed that, compared with the control group, the secretion amount of T cell exosomes in the group cultured under the simulated tumor microenvironment increased significantly.

[0071] Table 1: Effect of simulating the tumor microenvironment on the secretion amount of T cell exosomes

[0072] Group Exosome concentration (particles / mL) Control group <![CDATA[1.0×10 8 > Example 1 <![CDATA[2.0×10 8 >

[0073] (2) Effect of simulating the tumor microenvironment on the protein expression of T cell exosomes

[0074] The Western blot method was used to detect the expression levels of exosome marker proteins such as TSG101, CD63, and Alix in T cell exosomes cultured under the simulated tumor microenvironment. The results showed that, compared with the control group, the expression levels of TSG101, CD63, and Alix proteins in T cell exosomes in the group cultured under the simulated tumor microenvironment were all significantly up-regulated.

[0075] Table 2: Effect of simulating the tumor microenvironment on the protein expression of T cell exosomes

[0076] Protein Group Relative expression level TSG101 Control group 1.0 Example 1 1.5 CD63 Control group 1.0 Example 1 1.7 Alix Control group 1.0 Example 1 1.8

[0077] (3) Targeting of T cell exosomes induced by simulating the tumor microenvironment to tumor cells

[0078] The fluorescence labeling method was used to detect the targeting of T cell exosomes in the group cultured under the simulated tumor microenvironment to tumor cells. The results showed that, compared with the control group, T cell exosomes in the group cultured under the simulated tumor microenvironment could target tumor cells more effectively.

[0079] Table 3: Targeting of T cell exosomes induced by simulating the tumor microenvironment to tumor cells

[0080] Group Fluorescence intensity of tumor cells Control group 100 Example 1 200

[0081] (4) Inhibitory effect of T cell exosomes induced by simulating the tumor microenvironment on tumor growth

[0082] A mouse xenograft tumor model was used to evaluate the inhibitory effect of T cell exosomes cultured in a simulated tumor microenvironment on tumor growth. The results showed that compared with the control group, T cell exosomes in the simulated tumor microenvironment culture group could significantly inhibit tumor growth.

[0083] Table 4: Inhibitory effect of T cell exosomes induced by simulated tumor microenvironment on tumor growth

[0084] Group <![CDATA[Tumor volume (mm 3 )]]> Control group 1000 Example 1 500

[0085] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0086] In the present invention, T cells are cultured by simulating the tumor microenvironment, and exosomes with stronger tumor targeting are successfully induced to be secreted. For the first time, a method for culturing T cell exosomes by simulating the tumor microenvironment is proposed, providing a new idea for obtaining exosomes with stronger tumor targeting. Compared with T cell exosomes obtained under conventional culture conditions, the exosomes obtained in the present invention have the following advantages: (1) Increased secretion: Culturing in a simulated tumor microenvironment can significantly increase the secretion of T cell exosomes; (2) Altered protein expression: Culturing in a simulated tumor microenvironment can up-regulate the expression levels of proteins such as TSG101, CD63, and Alix in T cell exosomes; (3) Enhanced tumor targeting: T cell exosomes in the simulated tumor microenvironment culture group can more effectively target tumor cells; (4) Enhanced anti-tumor activity: T cell exosomes in the simulated tumor microenvironment culture group can significantly inhibit tumor growth;

[0087] This method is simple to operate and easy to implement, and exosomes with stronger tumor targeting and anti-tumor activity can be obtained. For the first time, a method for culturing T cell exosomes by simulating the tumor microenvironment is proposed, providing a new idea for obtaining exosomes with stronger tumor targeting; it provides a new strategy for tumor immunotherapy and has good application prospects.

[0088] Example 2: Through the establishment of a simulated tumor microenvironment in Example 1 above, T cells were successfully induced to secrete exosomes with stronger tumor targeting and anti-tumor activity, increasing the secretion amount and protein expression level of exosomes, and significantly enhancing their tumor targeting and anti-tumor activity. To further improve the tumor targeting of exosomes, further improvements were made on the basis of Example 1.

[0089] The exosomes extracted in step (5) were subjected to targeted strengthening, and the targeted strengthening means that the T cell exosomes were also complexed with magnetic nanoparticles to form a magnetic nanoparticle-exosome complex; the mass ratio of T cell exosomes to magnetic nanoparticles was 1:1;

[0090] Among them, the core of the magnetic nanoparticles is an iron oxide particle, the surface is coated with mesoporous silica, citric acid is loaded in the mesopores, and the CD63 antibody is surface-modified by the grafting method; the loading amount of citric acid is 15-20 wt%;

[0091] S1. The preparation of the magnetic nanoparticles is specifically as follows.

[0092] S11. Dissolve ferric chloride and oleic acid in an organic phase and prepare iron oxide nanoparticles by high-temperature thermal decomposition.

[0093] S12. Coat mesoporous silica on the surface of the iron oxide by the sol-gel method.

[0094] Among them, the pore diameter of the mesopores is 5-10 nm.

[0095] S13. Disperse the iron oxide coated with mesoporous silica in a citric acid solution, and fill the mesopores with citric acid by physical adsorption; modify the surface of the mesoporous silica with an amino silane coupling agent, and covalently connect citric acid molecules through an amide bond to form a pH-responsive acid release layer, obtaining citric acid mesoporous iron oxide nanoparticles.

[0096] Among them, the concentration of the citric acid solution is 10% w / v, and the loading amount is 15-20 wt%.

[0097] S14. Use the EDC / NHS chemical cross-linking method to immobilize the CD63 antibody on the surface of the citric acid mesoporous iron oxide nanoparticles to obtain magnetic nanoparticles.

[0098] S2. Composite of exosomes and magnetic nanoparticles: Mix the T cell exosomes extracted in step (5) with the magnetic nanoparticles at a mass ratio of 1:1, incubate at 4°C for 12 hours, and utilize the specific binding of the CD63 antibody to the CD63 antigen on the surface of the exosomes to form a magnetic nanoparticle-exosome complex.

[0099] S3. Magnetic field-guided targeting: In an animal model or clinical application, act on the tumor area through an external magnetic field to guide the enrichment of the magnetic nanoparticle-exosome complex to the tumor tissue.

[0100] Among them, the intensity of the external magnetic field is 0.5-1.5 T.

[0101] The above technical solutions are experimentally tested. Among them, the loading amount of citric acid is 17.5 wt%, and the intensity of the external magnetic field is 1 T.

[0102] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0103] By introducing magnetic nanoparticle-exosome complexes, the targeting and anti-tumor properties are further enhanced. The magnetic property of Fe3O4 is utilized to drive the enrichment of the complexes to the tumor region by an external magnetic field, and the concentration of the complexes at the tumor site is increased through a physical localization mechanism, enhancing the targeting property; citric acid loaded in the mesopores is released in the tumor microenvironment to release H + , locally reducing the pH. This acidification not only mimics the acidic characteristics of the tumor microenvironment but also promotes the dissolution of Fe3O4 and the release of Fe 2+ ; through the specific binding of the CD63 antibody to the CD63 antigen on the surface of exosomes, the stability of the complexes is ensured, ensuring the stability and long-term efficacy of the complexes in vivo, and promoting the enrichment and function of the complexes at the tumor site; the released Fe 2+ reacts with hydrogen peroxide in tumor cells to generate hydroxyl radicals, triggering the Fenton reaction. Hydroxyl radicals can cause lipid peroxidation and destroy the ferroptosis defense system of tumor cells, thereby inducing the death of tumor cells and further improving the anti-tumor property;

[0104] Magnetic field guidance increases the concentration of complexes in the tumor region and improves the homing efficiency of exosomes, promoting the more effective arrival of the complexes at the tumor site and enhancing the therapeutic effect; magnetic targeting reduces systemic exposure and decreases the toxicity to non-tumor tissues; the complexes endow exosomes with magnetic targeting ability, enabling them to break through the limitation of traditional exosome targeting that depends on microenvironment signals and achieve more precise targeted delivery; the immunomodulatory function of exosomes and Fe 2+ -mediated ferroptosis form an "immune-chemotherapy" synergistic effect to enhance the anti-tumor effect, and the acidification of citric acid enhances the membrane permeability of exosomes, promoting the release of the contents into tumor cells and improving the therapeutic effect.

[0105] Example 3: In the above Example 2, through the construction of magnetic nanoparticle-exosome complexes, combined with magnetic field-guided targeting and Fe 2+ -mediated multiple killing mechanisms, the tumor targeting and anti-tumor effects of exosomes are improved. To further enhance the tumor targeting of exosomes, it is further improved on the basis of Example 2.

[0106] The outer layer of the magnetic nanoparticles is also coated with calcium carbonate to form a core-shell structure of calcium carbonate-coated magnetic nanoparticles;

[0107] Among them, the mass ratio of iron oxide to calcium carbonate is 1:1; the core-shell structure is complexed with exosomes to form a core-shell structure complex; the mass ratio of exosomes to the core-shell structure is 1:1;

[0108] The preparation of the core-shell structure is specifically as follows:

[0109] Disperse the citric acid mesoporous iron oxide nanoparticles obtained in step S13 in a mixed solution containing 10 mM calcium chloride and 10 mM sodium carbonate; adjust the pH to 9.0, stir at room temperature for 2 hours, and form a calcium carbonate coating layer after centrifugal washing;

[0110] Use the carbodiimide (EDC) / N-hydroxysuccinimide (NHS) chemical cross-linking method to immobilize the CD63 antibody on the surface of the calcium carbonate coating layer to form a core-shell structure of the calcium carbonate-coated magnetic nanoparticles.

[0111] The experiment of this example is carried out on the basis of Example 2. The difference between the experiment of this example and the experiment of Example 2 is that a calcium carbonate coating layer is introduced in this example.

[0112] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0113] By combining the CaCO3 coating layer with the citric acid-modified magnetic nanoparticles, active acidification of the tumor microenvironment and magnetic targeting guidance are realized, further improving the tumor targeting and anti-tumor effects of exosomes;

[0114] In the acidic tumor microenvironment (pH 6.5 - 6.9), the calcium carbonate coating layer undergoes a neutralization reaction with hydrogen ions:

[0115] CaCO3 + 2H + →Ca 2+ + CO2↑ + H2O

[0116] Thereby realizing the dynamic regulation of the local pH. In the short term, hydrogen ions are consumed at the initial stage of the reaction, and the local pH is briefly increased. However, the release of carbon dioxide forms microbubbles, disturbing the microenvironment and enhancing proton diffusion, thereby improving the dispersibility; In the long term, carbon dioxide dissolves in water to form carbonic acid, further releasing hydrogen ions, Ultimately reducing the local pH and enhancing the acidity, promoting the cleavage of the Fe-O bond on the surface of Fe3O4 and releasing more Fe 2+ ; Citric acid ionizes and releases hydrogen ions in the tumor microenvironment. Citric acid forms a stable complex with Fe 3+ to prevent the precipitation of Fe 3+ and reduces it to Fe 2+ by intracellular reducing substances;

[0117] CaCO3 dynamically adjusts the local pH through the neutralization reaction and CO2 generation, avoiding cytotoxicity caused by excessive acidification. Citric acid: continuously releases H + to maintain the acidic environment and make up for the pH rebound after the reaction of CaCO3; The decomposition of CaCO3 enhances proton diffusion, and citric acid directly provides H + Both of them jointly promote the dissolution of Fe3O4, making Fe 2+The release amount is significantly increased;

[0118] Through the synergistic effect of the CaCO3 coating layer and citric acid modification, the regulation of the tumor microenvironment is achieved, the dispersibility is improved, and thus the tumor targeting and anti-tumor effects of exosomes are further enhanced.

[0119] Example 4: In Example 3 above, through the synergistic effect of the CaCO3 coating layer and citric acid modification, the regulation of the tumor microenvironment is achieved, the dispersibility of protons is improved, and at the same time, the tumor targeting and anti-tumor effects of exosomes are enhanced. To further improve the tumor targeting of exosomes, it is further improved on the basis of Example 3.

[0120] The core-shell structure of the calcium carbonate-coated magnetic nanoparticles includes a high-iron oxide core-shell structure and a low-iron oxide core-shell structure;

[0121] Among them, the mass ratio of iron oxide to calcium carbonate in the high-iron oxide core-shell structure is 7:3; the mass ratio of iron oxide to calcium carbonate in the low-iron oxide core-shell structure is 3:7;

[0122] The preparation of the high-iron oxide core-shell structure is specifically as follows:

[0123] Iron oxide nanoparticles are prepared by the high-temperature thermal decomposition method, and the diameter of the nanoparticles is 20 nm;

[0124] The iron oxide is dispersed in a mixed solution containing 7 mM calcium chloride and 3 mM sodium carbonate, the pH is adjusted to 9.0, stirred at room temperature for 2 hours, centrifuged and washed to form a calcium carbonate coating layer with a thickness of 10 nm;

[0125] The CD63 antibody is modified by the EDC / NHS chemical cross-linking method;

[0126] 15 wt% of citric acid is loaded into the pores of calcium carbonate.

[0127] The preparation of the low-iron oxide core-shell structure is specifically as follows:

[0128] Iron oxide nanoparticles are prepared by the high-temperature thermal decomposition method, and the diameter of the nanoparticles is 20 nm;

[0129] The iron oxide is dispersed in a mixed solution containing 3 mM calcium chloride and 7 mM sodium carbonate, the pH is adjusted to 9.0, stirred at room temperature for 2 hours, centrifuged and washed to form a calcium carbonate coating layer with a thickness of 20 nm;

[0130] The CD63 antibody is modified by the EDC / NHS chemical cross-linking method;

[0131] 20 wt% of citric acid is loaded into the pores of calcium carbonate.

[0132] Mix the high-iron oxide core-shell structure and the low-iron oxide core-shell structure with T cell exosomes at a mass ratio of 1:1 and incubate at 4°C for 12 hours to form a high-iron oxide complex and a low-iron oxide complex.

[0133] Release different amounts of iron oxide complexes in stages:

[0134] Stage 1: High-iron oxide complex (dose 5 mg / kg), apply a 1.5 T magnetic field for 6 hours;

[0135] Stage 2: After 6 hours, inject the low-iron oxide complex (dose 5 mg / kg), and reduce the magnetic field strength to 0.5 T and continue for 18 hours;

[0136] Based on Example 3, conduct the experiment of this example. The difference between the experiment of this example and the experiment of Example 3 is that the core-shell structure of calcium carbonate-coated magnetic nanoparticles in this example includes a high-iron oxide core-shell structure and a low-iron oxide core-shell structure.

[0137] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0138] By designing two kinds of particles with different iron oxide contents, namely the high-iron oxide complex and the low-iron oxide complex, and carrying out staged release, staged synergistic effects are achieved, optimizing the pH regulation of the tumor microenvironment and the Fe 2+ release efficiency, thereby significantly enhancing the tumor targeting and anti-tumor performance of exosomes;

[0139] The high-iron oxide complex has a high iron oxide content and strong magnetic responsiveness, and can be rapidly enriched to the tumor area under an external magnetic field with a magnetic field strength of 1.5 T to preferentially complete the local acidification task; the low-iron oxide complex has a low iron oxide content and weak magnetic responsiveness, and arrives at the tumor area later. Since the magnetic force of the magnetic field on the low-content iron oxide complex is small, its arrival speed at the tumor area is relatively slow. After the high-iron oxide complex completes the preliminary task, it relies on the acidic environment created by the previous complex to efficiently release Fe 2+ ; Release complexes with different iron oxide contents in stages. In the early stage, the high-iron oxide complex is rapidly enriched, and its outer layer of CaCO3 reacts with H + in the microenvironment to release CO2 and preliminarily acidify the local environment, the pH decreases, and the preliminary release of Fe 2+ is initiated; in the later stage, in the already acidified environment, the thick layer of CaCO3 of the low-iron oxide complex accelerates dissolution, releases more H + , further reduces the pH, promotes the deep dissolution of iron oxide, releases a high concentration of Fe 2+ , maximizes the Fenton reaction efficiency, thereby enhancing the tumor targeting and anti-tumor performance of exosomes;

[0140] In the composite with different proportions of iron oxide, the reaction characteristics of calcium carbonate with hydrogen ions remain unchanged. However, under the differences in the order and time of the composite with different magnetic proportions reaching the tumor area, it can form dynamic pH regulation. The high-iron-oxide composite arrives first and initiates acidification. The low-iron-oxide composite further accelerates decomposition in the acidified environment, releasing more hydrogen ions and enhancing the acidification effect. The citric acid loading is adjusted according to the iron oxide proportion, with 15 wt% loading for high iron oxide and 20 wt% loading for low iron oxide, to meet the acidification requirements at different stages, ensure the continuous release of hydrogen ions in the tumor microenvironment, maintain the acidic environment, and promote the dissolution of iron oxide and the release of Fe 2+ release;

[0141] Through the staged action of high- and low-iron-oxide composites, the pH of the tumor microenvironment is gradually reduced, avoiding the possible microenvironment rebound caused by the one-time acidification of a single composite, and achieving precise regulation of the pH of the tumor microenvironment; realizing the continuous supply of Fe 2+ to ensure the continuous progress of the Fenton reaction and enhance the ferroptosis induction effect; magnetic guidance enables high- and low-iron-oxide composites to be enriched in the tumor area, reducing the exposure of non-tumor tissues. The staged acidification design avoids the sudden drop in pH caused by the one-time release of excessive H + , thus reducing the damage to normal tissues;

[0142] By designing calcium carbonate-coated magnetic nanoparticles with two different iron oxide contents, using the strong magnetic responsiveness of the high-iron-oxide composite to rapidly enrich to the tumor area to initiate preliminary acidification and Fe 2+ release, and the low-iron-oxide composite deeply releases Fe 2+ in the acidified environment. Combining the staged pH regulation and Fe 2+ release strategy, it realizes the precise remodeling of the tumor microenvironment, significantly improves the anti-tumor effect and optimizes the safety. At the same time, it enhances the targeting and functional synergy of T cell exosomes, further enhancing the tumor targeting and anti-tumor performance of exosomes.

[0143] The above-mentioned examples were detected: 1. Magnetic targeting enrichment efficiency: Through in vivo fluorescence imaging: DiR (near-infrared fluorescent dye) was used to label exosomes, and FITC (green fluorescence) was used to label magnetic nanoparticles. Inductively coupled plasma mass spectrometry (ICP-MS) quantification: The iron element concentration in tumor tissues was detected (P<0.05);

[0144] 2. Fe 2+ release amount and pH responsiveness: Ferrous ion fluorescence probe (FerroOrange): The composite was incubated in a simulated tumor microenvironment (pH 6.0 and 7.0), and ferrous ions were quantified by flow cytometry. Ferrozine spectrophotometry: The absorbance of the ferrous ion-ferrozine complex (562 nm) was detected.

[0145] 3. The targeting of T cell exosomes in the simulated tumor microenvironment culture group to tumor cells was detected by fluorescence labeling method.

[0146] 4. The inhibitory effect of T cell exosomes in the simulated tumor microenvironment culture group on tumor growth was evaluated using a mouse xenograft tumor model. The detection results are shown in Table 5 below;

[0147] Table 5

[0148]

[0149] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A culture method for enhancing the tumor targeting of T cell exosomes, characterized in that It includes the following steps: (1) Isolation and culture of T cells; (2) Establishment of a simulated tumor microenvironment; (3) Co-culture with tumor-associated fibroblasts; (4) Stimulation with tumor cell exosomes; (5) Extraction and targeting enhancement of exosomes; The targeting enhancement means that the exosomes extracted in step (5) are complexed with magnetic nanoparticles to form a magnetic nanoparticle-exosome complex.

2. The culturing method for enhancing the tumor targeting property of T cell exosomes according to claim 1, wherein, The simulated tumor microenvironment described in step (2) includes the following situations: (a) Hypoxic condition: Using a hypoxic incubator (1% O2); (b) Acidic pH: Adjusting the pH value of the culture medium to 6.5 - 6.8 using a pH buffer; (c) Nutrient deficiency: Using a low-glucose (1 g / L) culture medium; (d) Immune suppressor factors: Adding TGF-β (10 ng / mL) and IL-10 (10 ng / mL).

3. The culturing method for enhancing the tumor targeting property of T cell exosomes according to claim 1, wherein, The co-culture with tumor-associated fibroblasts described in step (3) means co-culturing T cells and tumor-associated fibroblasts at a ratio of 1:

1.

4. The culturing method for enhancing the tumor targeting property of T cell exosomes according to claim 1, characterized in that, The stimulation with tumor cell exosomes described in step (4) means stimulating T cells with tumor cell exosomes (10 μg / mL).

5. The culturing method for enhancing the tumor targeting property of T cell exosomes according to claim 1, characterized in that, The exosome extraction method described in step (5) is the ultracentrifugation method or the kit method.

6. The culture method for enhancing the tumor targeting of T cell exosomes according to claim 1, wherein, The core of the magnetic nanoparticles is an iron oxide particle, the surface is coated with mesoporous silica, citric acid is loaded in the mesopores, and the surface is modified with CD63 antibody by the grafting method; the loading amount of citric acid is 15 - 20 wt%; the mass ratio of exosomes to magnetic nanoparticles is 1:

1.

7. The culture method for enhancing the tumor targeting of T cell exosomes according to claim 1, characterized in that The outer layer of the magnetic nanoparticles is further coated with calcium carbonate to form a core-shell structure of calcium carbonate-coated magnetic nanoparticles, wherein the mass ratio of iron oxide to calcium carbonate is 1:

1.

8. The culturing method for enhancing the tumor targeting property of T cell exosomes according to claim 7, wherein The core-shell structure is complexed with exosomes to form a core-shell structure complex; the mass ratio of exosomes to the core-shell structure is 1:

1.

9. The culture method for enhancing the tumor targeting of T cell exosomes according to claim 7, wherein, The core-shell structure includes a high-iron-oxide core-shell structure and a low-iron-oxide core-shell structure.

10. The culture method for enhancing the tumor targeting of T cell exosomes according to claim 1, wherein, In the high-iron-oxide core-shell structure, the mass ratio of iron oxide to calcium carbonate is 7:3; in the low-iron-oxide core-shell structure, the mass ratio of iron oxide to calcium carbonate is 3:7.

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