A complex for anti-tumor and its preparation method and application

Abstract

This invention discloses a complex for anti-tumor purposes, its preparation method, and its applications, relating to the field of biomedical technology. The preparation method includes the following steps: mixing senescent tumor cell exosomes with SR-717, thereby loading SR-717 into the senescent tumor cell exosomes to prepare the anti-tumor complex. This invention, by combining senescent tumor cell exosomes with the STING agonist SR-717, achieves synergistic enhancement of immunomodulatory function and immune activation signaling in the same system, thus facilitating more effective and controllable anti-tumor immunotherapy. Simultaneously, encapsulating SR-717 with exosomes helps reduce the diffusion of free drug in non-target tissues, lowering the risk of non-specific immune activation and systemic inflammatory responses.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a complex for anti-tumor purposes, its preparation method, and its application. Background Technology

[0002] The core of tumor immunotherapy lies in breaking the state of tumor immune tolerance and restoring or enhancing the body's ability to recognize and eliminate tumor cells. In recent years, cellular senescence has been recognized not only as a state of cell cycle arrest, but also as a biological process with important immune regulatory functions.

[0003] Senescent cells can significantly alter the local microenvironment by secreting various inflammatory factors, chemokines, and immunomodulatory molecules, forming the so-called senescence-associated secretory phenotype (SASP). Previous studies have shown that aging can enhance cellular immunogenicity and promote the recognition and clearance of senescent cells and adjacent abnormal cells by immune cells.

[0004] Exosomes, as important mediators of intercellular communication, have biological functions that largely depend on the physiological state of their source cells. Exosomes derived from senescent cells can carry various SASP-related bioactive molecules and induce secondary senescence in neighboring cells through paracrine signaling, thereby disseminating senescence signals within the tissue microenvironment. This process can further enhance the immunogenicity of tumor cells and provide favorable conditions for immune activation.

[0005] Meanwhile, the STING (Stimulator of Interferon Genes) signaling pathway, as an important hub of innate immunity, plays a crucial role in inducing type I interferon production, promoting dendritic cell maturation, and activating effector T cells. Small molecule STING agonists (such as SR-717) can effectively activate the STING pathway, but their immunotherapeutic efficacy remains limited in the immunosuppressive tumor microenvironment. Summary of the Invention

[0006] The purpose of this invention is to provide a complex for anti-tumor purposes, its preparation method, and its application, thereby addressing the problems existing in the prior art. This invention combines senescent tumor cell exosomes with the STING agonist SR-717, achieving a synergistic enhancement of immunomodulatory function and immune activation signaling within the same system, thus facilitating more effective and controllable anti-tumor immunotherapy.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides a method for preparing a complex for anti-tumor purposes, comprising the following steps: mixing and reacting senescent tumor cell exosomes with SR-717 to load SR-717 into the senescent tumor cell exosomes, thereby preparing the complex for anti-tumor purposes.

[0009] Furthermore, the mixing reaction process employs ultrasonic treatment.

[0010] Furthermore, the ultrasonic treatment power is 120-160W, and the time is 20-40min.

[0011] Furthermore, the mass ratio of the senescent tumor cell exosomes to the SR-717 is 1:(0.5-3).

[0012] Furthermore, the senescent tumor cell exosomes are obtained by extracting the culture supernatant of senescent tumor cells using differential centrifugation.

[0013] The senescent tumor cells were obtained by inducing tumor cell culture with doxorubicin hydrochloride.

[0014] Furthermore, the tumor cells are breast cancer cells.

[0015] The present invention also provides a complex for antitumor preparation prepared by the preparation method described above.

[0016] The present invention also provides the use of the above-described complex in the preparation of antitumor drugs.

[0017] Furthermore, when the antitumor drug is an anti-breast cancer drug, the senescent tumor cell exosomes are senescent breast cancer cell exosomes.

[0018] The present invention also provides an antitumor drug, the active ingredient of which includes the above-mentioned complex.

[0019] The present invention discloses the following technical effects:

[0020] This invention develops an anti-tumor complex by combining senescent tumor cell exosomes with the STING agonist SR-717. This achieves synergistic enhancement of immunomodulatory function and immune activation signaling within the same system, thus facilitating more effective and controllable anti-tumor immunotherapy. Senescent tumor cell exosomes can induce secondary senescence in tumor-associated cells via paracrine signals, thereby increasing the immunogenicity of tumor cells and improving the tumor immune microenvironment. SR-717 loaded into senescent tumor cell exosomes can further activate the STING signaling pathway, promoting the initiation and amplification of the innate immune response. This results in an anti-tumor immune response that is superior in both intensity and duration to the use of senescent tumor cell exosomes or STING agonists alone. Simultaneously, encapsulating SR-717 in exosomes helps reduce the diffusion of free drug in non-target tissues, lowering the risk of non-specific immune activation and systemic inflammatory responses, thereby improving the safety and controllability of the treatment process. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the cell morphology and staining results of senescent galactosidase (SA-β-Gal) after treatment with different concentrations of drugs.

[0023] Figure 2 Transmission electron microscopy (TEM) image of aging exosomes;

[0024] Figure 3 This is a graph showing the results of Western blot (WB) detection of exosome characteristic proteins.

[0025] Figure 4 The images show the results of the exosome uptake experiment; where A is a confocal microscope image and B is a flow cytometry result.

[0026] Figure 5 Figure A shows the characterization results of S-Exo@SR717; where A is the particle size distribution of S-Exo@SR717; and B is the particle size potential statistics of S-Exo@SR717.

[0027] Figure 6 The release curves of SR-717 after incubation in PBS buffer at pH 5.0 or pH 7.4 are shown.

[0028] Figure 7 SA-β-Gal staining image of cells after treatment with senescent exosomes;

[0029] Figure 8 The graph shows the results of detecting the expression level of MHC-I molecules on the surface of 4T1 tumor cells in each group; where A is the quantitative analysis graph of the mean fluorescence intensity (MFI) of MHC-I; and B is the flow cytometry detection result of the MHC-I expression level.

[0030] Figure 9 The image shows the results of the detection of the maturation level of bone marrow-derived dendritic cells (BMDCs) in each group; where A represents CD11c. + CD80 in cells + CD86 + Flow cytometry scatter plot of double-positive mature dendritic cells; B is a statistical graph of the proportion of mature dendritic cells;

[0031] Figure 10 The growth curves of tumor volume in 4T1 tumor-bearing mice in different treatment groups are shown. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Terminology Explanation:

[0038] The SR-717 involved in this invention is a non-nucleoside STING agonist, with CAS number 2375421-09-1 and molecular formula C. 15 H8F2LiN5O3, the structural formula is as follows:

[0039] .

[0040] The mouse breast cancer cells (4T1) used in the following examples were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0041] Example 1: Establishment of a tumor cell senescence model, preparation and characterization of senescent exosomes

[0042] 1. Establishment of a tumor cell senescence model

[0043] (1) Culture of tumor cells: 4T1 cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum and 1% (w / v) penicillin and streptomycin and placed in a 37°C, 5% CO2 incubator.

[0044] (2) Inducing cell senescence: When the cell density reaches 60%-70%, add 0, 25 or 50 nM doxorubicin hydrochloride for 48 hours, then wash the cells with PBS and replace with a culture medium without doxorubicin for another 48 hours to obtain senescent cells.

[0045] (3) SA-β-Gal staining: Staining was performed according to the kit instructions, and the proportion of positive cells was observed and counted under an inverted microscope.

[0046] like Figure 1 As shown, when treated with 50 nM doxorubicin hydrochloride, the proportion of SA-β-Gal staining positive cells can reach more than 80%. Subsequently, senescent exosomes were extracted from 4T1 cells treated with 50 nM doxorubicin hydrochloride.

[0047] 2. Extraction of senescent exosomes

[0048] Senescent 4T1 cells induced with 50 nM doxorubicin hydrochloride were further cultured in DMEM medium containing 1% (v / v) exosome-free fetal bovine serum and incubated at 37°C in a 5% CO2 incubator for 48 h. The cell culture supernatant was collected, and exosomes derived from senescent tumor cells were extracted by differential centrifugation at 4°C. The specific steps are as follows:

[0049] (1) First, centrifuge the collected cell culture supernatant at 300 ×g for 10 min to remove residual cells;

[0050] (2) Transfer the supernatant obtained from step (1) to a new centrifuge tube and centrifuge at 2000 ×g for 20 min to remove cell debris and apoptotic bodies;

[0051] (3) Centrifuge the supernatant obtained in step (2) at 10000 ×g for 30 min to further remove large vesicles and impurities;

[0052] (4) Filter the supernatant obtained by centrifugation in step (3) through a 0.22 μm sterile filter membrane to remove residual particles and cell debris;

[0053] (5) Transfer the filtered filtrate to an ultracentrifuge tube and centrifuge at 4°C and 100,000 × g for 70 min. Discard the supernatant and collect the precipitate.

[0054] (6) The precipitate was resuspended in sterile PBS buffer and centrifuged again at 4°C and 100,000 ×g for 60 min to further remove soluble proteins and impurities.

[0055] (7) The final precipitate was resuspended in 100-200 μL of sterile PBS to obtain senescent tumor-derived exosomes (S-Exo).

[0056] The obtained S-Exo was packaged and stored at -80℃ for later use, avoiding repeated freeze-thaw cycles.

[0057] 3. Characterization of exosomes derived from senescent tumor cells

[0058] To verify that the extracted vesicles were exosomes, their morphology, particle size distribution, and characteristic protein expression were characterized. 10 μL of S-Exo suspension was dropped onto a carbon membrane copper grid and allowed to stand at room temperature for 2 min for adsorption. Excess liquid was then absorbed with filter paper. Subsequently, 2% phosphotungstic acid solution was added for negative staining for 30–60 s, and excess staining was absorbed again. After drying at room temperature, the vesicles were observed and photographed using a transmission electron microscope. The results showed that the extracted S-Exo vesicles exhibited typical round or cup-shaped vesicle structures with complete morphology and clear edges, consistent with the morphological characteristics of exosomes. Figure 2 ).

[0059] S-Exo samples were diluted to an appropriate concentration using sterile PBS, and their particle size distribution and concentration were determined using a nanoparticle tracking analyzer (NTA). The results showed that the particle size of S-Exo was mainly distributed between 30-200 nm, which is consistent with the particle size range of exosomes.

[0060] S-Exo protein concentration was determined using the BCA method. 20-30 μg of total protein was added to protein loading buffer and heated at 95℃ for 5 min to denature the protein. SDS-PAGE electrophoresis was then performed to separate the proteins, and the proteins were transferred to a PVDF membrane. After blocking with rapid blocking buffer at room temperature for 1 h, primary antibodies were added: anti-CD9 antibody, anti-CD63 antibody, anti-TSG101 antibody, anti-CD81 antibody, anti-Alix antibody, and anti-Calnexin antibody, and incubated overnight at 4℃. The next day, the membrane was washed three times with TBST for 10 min each time, followed by incubation with secondary antibody at room temperature for 1 h. After washing, the membrane was developed using ECL chemiluminescence reagent, and images were acquired. The results showed that specific expression of CD9, CD63, CD81, TSG101, and Alix proteins was detected in S-Exo. Figure 3 This further proves that the vesicles extracted in this invention are exosomes. The absence of Calnexin expression in S-Exo indicates that the obtained sample was not contaminated by endoplasmic reticulum components, further demonstrating the high purity of the extracted exosomes.

[0061] Example 2: Exosome Uptake Experiment

[0062] S-Exo was fluorescently labeled using Dir or PKH67. 4T1 cells were seeded in 6-well plates or confocal culture dishes. When cell confluence reached 60%–70%, fluorescently labeled S-Exo was added, and the cells were incubated at 37°C in a 5% CO2 incubator for 12 h. After incubation, the cells were gently washed three times with PBS to remove untaken free exosomes.

[0063] For the PKH67-labeled group, cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and the nuclei were stained with DAPI for 5 min. Finally, observation was performed using a laser confocal microscope. The results showed that the green fluorescence signal was mainly distributed in the cytoplasm and appeared as punctate clusters, indicating that S-Exo could be effectively taken up by the recipient cells and enter the cell. Figure 4 (A)

[0064] For the Dir-labeled group, cells were collected after incubation, washed with PBS, and resuspended in flow cytometry buffer. Intracellular fluorescence intensity and the proportion of positive cells were detected by flow cytometry. Results showed that, compared with the control group, the average fluorescence intensity of cells in the exosome-treated group was significantly increased, and the proportion of fluorescently positive cells was significantly increased. Figure 4 (B) This further demonstrates that S-Exo can be efficiently taken up by receptor cells.

[0065] Example 3: Preparation and Characterization of S-Exo@SR717

[0066] 1. Drug loading, particle size, and potential determination: SR-717 solution and S-Exo solution were mixed thoroughly to a final concentration of 1 mg / mL for both SR-717 and S-Exo. The mixture was then sonicated at 120 W power and 4℃ for 30 minutes, alternating between 3 minutes of sonication and 3 minutes of rest. After sonication, the mixture was incubated at 37℃ for 60 minutes to restore the exosome membrane. Excess drug was removed by ultrafiltration to prepare SR-717-loaded senescent tumor cell exosomes, named S-Exo@SR717. The particle size, potential, and polydispersity index (PDI) of S-Exo@SR717 were determined using a laser particle size analyzer. The results are shown in [Figure number missing]. Figure 5 The particle size distribution results showed that the average hydrodynamic particle size of S-Exo@SR717 was 157.4±4.7 nm, and the polydispersity index (PDI) was only 0.26±0.01. The particle size distribution showed a single peak with a narrow peak shape, indicating that the exosomes were uniform in size after drug loading, with no obvious agglomeration and good dispersion stability of the system. The potential statistics showed that the surface of S-Exo@SR717 was negatively charged, and the Zeta potential was maintained at around -19 mV.

[0067] The above results demonstrate that the S-Exo@SR717 prepared by this invention has uniform particle size, good dispersibility, and good physicochemical stability.

[0068] 2. In vitro release experiment: To evaluate the drug release behavior of S-Exo@SR717, this invention determined the in vitro SR-717 release curves of S-Exo@SR717 at pH 7.4 (physiological environment) and pH 5.0 (late endosome and lysosomal environment). S-Exo@SR717 was added to PBS at different pH values ​​and incubated in a shaker at 37°C. PBS solutions were collected at different time points, and equal volumes of PBS were added. The OD value at 290 nm was measured using a UV spectrophotometer, and the cumulative drug release of SR-717 was calculated. The results are shown below. Figure 6 The results showed that the drug release rate of S-Exo@SR717 was significantly higher in an acidic environment than in a neutral environment, exhibiting good acid-sensitive release characteristics. S-Exo@SR717 maintains good stability under physiological conditions, and upon entering an acidic intracellular environment, it can rapidly release SR-717, which is beneficial for targeted drug delivery and on-demand release, improving therapeutic efficacy and reducing systemic toxicity.

[0069] Example 4

[0070] The SR-717 solution and S-Exo solution were mixed thoroughly to achieve final concentrations of 0.5 mg / mL for SR-717 and 1 mg / mL for S-Exo. The mixture was then sonicated at 140 W at 4 °C for 40 minutes, alternating between 3 minutes of sonication and 3 minutes of rest. After sonication, the mixture was incubated at 37 °C for 60 minutes to allow the exosome membrane to recover. Excess drug was removed by ultrafiltration to prepare SR-717-loaded senescent tumor cell exosomes.

[0071] Example 5

[0072] The SR-717 solution and S-Exo solution were mixed thoroughly to achieve final concentrations of 3 mg / mL for SR-717 and 1 mg / mL for S-Exo. The mixture was then sonicated at 160 W power and 4 °C for 20 minutes, alternating between 3 minutes of sonication and 3 minutes of rest. After sonication, the mixture was incubated at 37 °C for 60 minutes to restore the exosome membrane. Excess drug was removed by ultrafiltration to prepare SR-717-loaded senescent tumor cell exosomes.

[0073] Example 6: S-Exo@SR717 induces tumor cell senescence

[0074] 4T1 cells were seeded into 6-well plates, with 1×10⁶ cells per well. 5 Cells were cultured overnight at 37°C in a 5% CO2 incubator. When cell confluence reached 50%–60%, the medium was replaced with fresh medium, and S-Exo@SR717 was added to a final concentration of 50 μg / mL. Culture was continued for 48 h. After culture, the medium was discarded, and the cells were gently washed three times with PBS buffer for 5 min each time. Then, 4% paraformaldehyde was added for fixation at room temperature for 15 min, followed by three more washes with PBS. Working solution was prepared according to the instructions of the Senescence-associated β-galactosidase (SA-β-Gal) staining kit. An appropriate amount of staining solution was added to each well, and the cells were incubated at 37°C (CO2-free) in the dark for 16 h. After staining, the cells were observed and photographed under an inverted optical microscope to evaluate the level of cell senescence. Results are as follows: Figure 7 As shown, compared with the control group, the number of SA-β-Gal positive cells in the S-Exo@SR717 treatment group was significantly increased and the positive staining area was significantly expanded, indicating that S-Exo@SR717 can effectively induce cell senescence in tumor cells.

[0075] Example 7: Experiment on S-Exo@SR717 promoting tumor cell antigen presentation

[0076] 4T1 cells were seeded into 6-well plates, with 1×10⁶ cells per well.5 Cells were cultured overnight at 37°C in a 5% CO2 incubator. When cell confluence reached 50%–60%, cells were randomly divided into Control, Dox, SR-717, S-Exo, and S-Exo@SR717 groups. Each group was treated with the appropriate reagents added to the culture medium as shown in Table 1. After 48 h of further culture, the culture medium was discarded, cells were washed three times with PBS, digested with 0.25% trypsin, and collected. Cells were resuspended in flow cytometry buffer (PBS containing 2% FBS). Approximately 1 × 10⁶ cells from each group were collected. 6 Cells were incubated with fluorescently labeled anti-mouse MHC-I antibody at 4°C for 30 min in the dark. After incubation, the cells were washed twice with PBS, resuspended in 300 μL of flow cytometry buffer, and the expression level of MHC-I on the cell surface was detected by flow cytometry. The quantitative analysis was performed by mean fluorescence intensity (MFI) or the proportion of positive cells.

[0077] Table 1 Experimental groupings in the experiment of S-Exo@SR717 promoting tumor cell antigen presentation

[0078] Control group Add an equal volume of PBS and incubate for 48 hours. Dox Group Add 50 nM doxorubicin (Dox) and incubate for 48 h. SR-717 group Add 5 μM SR-717 and incubate for 48 h S-Exo Group Add 50 μg / mL S-Exo and incubate for 48 h S-Exo@SR717 Group Add 50 μg / mL S-Exo@SR717 and incubate for 48 h.

[0079] Note: The SR-717 loading of S-Exo@SR717 is 3.5% (w / w); the concentrations in the table are the final concentrations after each treatment reagent was added.

[0080] The results are as follows Figure 8 As shown, compared with the Control group, the Dox group significantly increased the expression of MHC-I on the surface of tumor cells, proving the successful construction of the experimental system. Among the experimental groups, the SR-717 group could increase the expression of MHC-I on the surface of tumor cells to a certain extent; the S-Exo group further enhanced the MHC-I expression level; while the S-Exo@SR717 group showed the most significant promoting effect, with its MHC-I positive rate and average fluorescence intensity being significantly higher than those of other experimental groups.

[0081] Example 8: Experiment on the promotion of dendritic cell maturation by S-Exo@SR717

[0082] (1) Preparation of bone marrow-derived dendritic cells (BMDC)

[0083] BALB / c mice aged 6–8 weeks were euthanized by cervical dislocation. The femurs and tibias were aseptically separated, the epiphyses were removed, and the bone marrow cavity was flushed with RPMI-1640 complete medium to collect bone marrow cell suspension. After removing red blood cells with erythrocyte lysis buffer, the cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum, 20 ng / mL recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF), and 10 ng / mL recombinant mouse IL-4. The cells were seeded in culture dishes and cultured at 37°C in a 5% CO2 incubator. Half the volume of fresh medium containing cytokines was replaced on days 3 and 5. On days 6–7, suspension and semi-adherent cells were collected as immature bone marrow cells (BMDCs) for subsequent experiments.

[0084] (2) BMDC treatment and maturity testing

[0085] BMDC was inoculated into 24-well plates, with 5 × 10⁶ cells per well. 5 Cells were randomly divided into four groups: Control group, LPS group (positive control), S-Exo group, SR-717 group, and S-Exo@SR717 group. Each group received the corresponding treatment reagents as shown in Table 2 and was cultured at 37℃ in a 5% CO2 incubator for 24 h. After culture, cells were collected and washed twice with PBS, and Fc receptor blocking was performed for 10 min with anti-mouse CD16 / CD32 antibody. Subsequently, fluorescently labeled antibodies CD11c, CD80, and CD86 were added and incubated at 4℃ in the dark for 30 min. After incubation, cells were washed twice with PBS, resuspended in flow cytometry buffer, and analyzed by flow cytometry, with CD11c... + CD80 + CD86 + Cell ratio is used to evaluate the maturity of dendritic cells.

[0086] Table 2 Experimental Groups in the S-Exo@SR717-Promoted Dendritic Cell Maturation Experiment

[0087] Control group Add an equal volume of PBS and incubate for 24 hours. LPS Group Add 1 μg / mL lipopolysaccharide (LPS) and incubate for 24 h. SR-717 group Add 10 μM SR-717 and incubate for 24 h S-Exo Group Add 50 μg / mL S-Exo and incubate for 24 h. S-Exo@SR717 Group Add 50 μg / mL S-Exo@SR717 and incubate for 24 h.

[0088] Note: The SR-717 loading of S-Exo@SR717 is 3.5% (w / w); the concentrations in the table are the final concentrations after each treatment reagent was added.

[0089] (3) Experimental results

[0090] The results are as follows Figure 9As shown, compared with the Control group, the LPS group significantly promoted BMDC maturation, proving the successful construction of the experimental system. Both the S-Exo and SR-717 groups increased the expression levels of CD80 and CD86 to varying degrees, while the S-Exo@SR717 group induced the most significant BMDC maturation effect, with a significantly higher proportion of mature dendritic cells than the other groups. These results indicate that the S-Exo@SR717 prepared in this invention can effectively promote dendritic cell maturation, increase the expression of co-stimulatory molecules CD80 and CD86, thereby enhancing antigen presentation capacity and promoting the activation of anti-tumor immune responses.

[0091] Example 9: Antitumor Experiment in Tumor-Bearing Mice

[0092] The specific steps for evaluating the in vivo antitumor effects of free drug and SR-717-loaded senescent tumor cell exosomes are as follows:

[0093] Female BALB / c mice (4-5 weeks old) were injected with 1×10⁻⁶ spores into the lower right back. 6 A subcutaneous breast cancer mouse model was established using 4T1 cells. 4-5 week old female BALB / c mice were injected with 1×10T1 cells into the lower right back. 6 4T1 cells were used when the mouse tumor volume reached 100 mm. 3 At approximately 10:00 AM, the tumors were randomly divided into 6 groups, with 6 biological replicates in each group. The groups were: Control, SR-717, S-Exo, S-Exo@SR717(iv), S-Exo@SR717(sc), and S-Exo@SR717(iv+sc). The drugs were administered via tail vein or subcutaneous injection on days 6, 8, 10, 12, 14, and 16. The types and dosages of drugs are shown in Table 3. The length and width of the tumors were measured every two days using calipers, and the tumor volume was calculated using the formula: Tumor volume = 1 / 2 × length × width. 2 .

[0094] Table 3 Experimental groupings for antitumor experiments in tumor-bearing mice

[0095] Control group Intravenous injection of an equal volume of PBS SR-717 group SR-717 was administered intravenously at a dose of 0.6 mg / kg. S-Exo Group S-Exo was administered intravenously at a dose of 3 mg / kg. S-Exo@SR717(iv) group S-Exo@SR717 was administered intravenously at a dose of 3 mg / kg. S-Exo@SR717(sc) group S-Exo@SR717 was administered subcutaneously at a dose of 3 mg / kg. S-Exo@SR717(iv+sc) group Intravenous injection of S-Exo@SR717 1.5 mg / kg + subcutaneous injection of S-Exo@SR717 1.5 mg / kg

[0096] Note: The SR-717 drug loading of S-Exo@SR717 is 3.5% (w / w).

[0097] Tumor growth curves of 4T1 tumor-bearing mice in different treatment groups are as follows: Figure 10As shown in the figure, both the SR-717 group and the S-Exo group were able to inhibit tumor growth to some extent compared with the PBS group, but the tumor-suppressing effect was limited. Treatment with senescent tumor cell-derived exosomes loaded with SR-717 (S-Exo@SR717) significantly inhibited tumor growth in mice, with both the intravenous and subcutaneous injection groups showing better anti-tumor effects. Notably, the combined intravenous and subcutaneous injection group [S-Exo@SR717 (iv+sc)] exhibited the most significant tumor-suppressing effect, with significantly lower tumor volume than other groups, indicating that the combined administration strategy can further enhance the anti-tumor efficacy of S-Exo@SR717.

[0098] The above results indicate that S-Exo@SR717 has good in vivo anti-tumor activity, and that intravenous and subcutaneous injection can achieve the best anti-tumor effect by synergistically enhancing systemic immune activation and local immune response.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a complex for antitumor purposes, characterized in that, Includes the following steps: The senescent tumor cell exosomes were mixed with SR-717 to load the senescent tumor cell exosomes, thus preparing the anti-tumor complex.

2. The preparation method according to claim 1, characterized in that, The mixing reaction process employs ultrasonic treatment.

3. The preparation method according to claim 2, characterized in that, The ultrasonic treatment has a power of 120-160W and a duration of 20-40 minutes.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the senescent tumor cell exosomes to the SR-717 is 1:(0.5-3).

5. The preparation method according to claim 1, characterized in that, The exosomes of senescent tumor cells were obtained by extracting the culture supernatant of senescent tumor cells using differential centrifugation. The senescent tumor cells were obtained by inducing tumor cell culture with doxorubicin hydrochloride.

6. The preparation method according to claim 5, characterized in that, The tumor cells are breast cancer cells.

7. A complex for antitumor purposes prepared by the preparation method according to any one of claims 1-6.

8. The use of the complex as described in claim 7 in the preparation of an antitumor drug.

9. The application as described in claim 8, characterized in that, When the anti-tumor drug is an anti-breast cancer drug, the senescent tumor cell exosomes are senescent breast cancer cell exosomes.

10. An antitumor drug, characterized in that, The active ingredient includes the complex according to claim 7.

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