Multifunctional exosome for cancer immunotherapy and preparation method thereof

Abstract

The invention discloses a multifunctional exosome for cancer immunotherapy, the multifunctional exosome can be used for treatment of various cancer diseases, meanwhile, the invention provides a preparation method of the multifunctional exosome, and the multifunctional exosome prepared by engineering can be used for immunotherapy of various autophagy high-expression cancers, and can be used for immunotherapy of various autophagy high-expression cancers. An MEF / HEK293T cell-derived exosome stably expressing YSA-CD64-(GFLG)-Rev ARM fusion protein is subjected to engineering modification, so that active targeting of a drug to tumor cells is realized, YSA targeting polypeptide is expressed on the surface of the drug, a PD-L1 antibody is coupled, IL-7 mRNA is loaded, autophagy of the tumor cells is inhibited, expression of MHC-I on the surfaces of the tumor cells is recovered, and meanwhile, the effect of inhibiting tumor cell apoptosis is achieved. And the PD-L1 antibody blocks an immune checkpoint, reactivates the killing of T cells on tumors, and recovers cold tumors to hot tumors, thereby realizing efficient treatment of cancers.

Description

Technical Field

[0001] The present invention discloses a multifunctional exosome for cancer immunotherapy, which can be used to treat various cancer diseases. It also provides a method for preparing the multifunctional exosome, belonging to the field of biopharmaceutical production technology. Background Art

[0002] Cancer immunotherapy is a novel therapeutic approach that harnesses the body's own immune system to identify and eradicate tumor cells. Currently, approaches such as nonspecific immune stimulation, immune checkpoint blockade (ICB), and cancer vaccines have been used to modulate immune responses. First-line immunotherapy agents are primarily antibody-based protein drugs, including cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death 1 (PD-1) inhibitors, and programmed cell death 1 ligand 1 (PD-L1) inhibitors. These drugs have been approved for clinical use and have become one of the most promising first-line treatment options for advanced cancer. Although tumor immunotherapy produces significant clinical responses with minimal side effects, a significant proportion of patients do not respond to immunotherapy. Currently, only about 20% of patients achieve long-term therapeutic effects after treatment. Most patients respond initially but develop drug resistance after relapse. This is because in the tumor microenvironment (TME), a variety of immunosuppressive cells such as tumor-associated macrophages (TAMs) are highly infiltrated, anti-tumor cytokines and tumor-associated antigens are downregulated, T cell infiltration is lacking or T cell dysfunction occurs, and the physicochemical properties of cancer cells (such as hypoxia and weak acidity) and abnormal metabolic activities can promote tumor immune escape, leading to an immunosuppressive microenvironment, which is also a major obstacle to current cancer immunotherapy.

[0003] Autophagy, a crucial cyclical process within the complex tumor microenvironment (TME), is increasingly recognized for its role in tumor immunity. Studies have shown that autophagic activity can modulate tumor immunity by regulating both the innate and adaptive immune systems, including the function of various immune cells and cytokine production. Similarly, some immune cells and cytokines can influence autophagy. Studies have shown that autophagy in tumor cells can recruit MHC-I antigen complexes to lysosomes and degrade them through the ubiquitin-binding receptor NBR1, thereby interfering with T cell recognition. Secretory autophagy can also alter immune cell function, stimulating neutrophils to exert immunosuppressive functions, inhibiting the proliferation of CD4+ and CD8+ T cells, and inducing macrophage polarization toward an M2-like phenotype. Conversely, inhibition of autophagy can enhance the expression of cancer-related membrane proteins through the accumulation of p62 / SQSTM1 and activation of NF-κB, promoting immune checkpoint blockade (ICB) immunotherapy.

[0004] As endogenous drug delivery vehicles, exosomes can protect the stability and effectiveness of their encapsulated contents, maintain stability in the bloodstream, and possess high biocompatibility, low toxicity, low immunogenicity, and the ability to overcome natural barriers. Furthermore, exosomes possess a homing effect, targeting secreting cells and fusing with target cell membranes to deliver drugs directly into the cytoplasm, preventing their destruction by lysosomes. This, to a certain extent, can improve the limitations of traditional drug delivery systems and address drug transport issues in immunotherapy. Summary of the Invention

[0005] The present invention discloses a multifunctional exosome for cancer immunotherapy, which is prepared by a dual-stimulation electroporation system. It uses exosomes derived from normal mouse embryonic fibroblasts (MEF) or human embryonic kidney cells (HEK293T) for engineering transformation to achieve universal active targeting of drugs to tumors.

[0006] To achieve the purpose, the present invention adopts the following technical solutions: After mass culture of MEF / HEK293T cells, the IL-7-RRE-IIB mRNA plasmid was loaded into MEF / HEK293T cells stably overexpressing the YSA-CD64-(GFLG)3-Rev ARM fusion protein using a dual-stimulation electroporation system (DES). After 24 hours of culture, the cell culture supernatant was collected, and cells and debris were removed by differential centrifugation. Finally, purified drug-loaded exosomes (AT-Exo) were further obtained by ultracentrifugation. AT-Exo were incubated with PD-L1 antibodies at room temperature for 2 hours, and free antibodies were removed by ultracentrifugation to obtain multifunctional exosomes (AI-Exo) with surface-coupled PD-L1 antibodies that can actively target tumors and inhibit tumor cell autophagy.

[0007] The method for preparing multifunctional exosomes for cancer immunotherapy according to the present invention comprises the following steps: 1) Construction of a MEF / HEK293T cell line stably overexpressing the YSA-CD64-(GFLG)3-Rev ARM fusion protein (CD64-MEF / HEK293T); 2) Loading therapeutic drugs Through DES, IL-7-RRE-IIB mRNA plasmid was mixed with MEF / HEK293T cell suspension, and IL-7-RRE-IIB mRNA was loaded into MEF / HEK293T cells by electroporation and ultrasound dual stimulation, stimulating exosome secretion. After extracting the supernatant, the cells were subjected to differential centrifugation, filtration, and ultracentrifugation to obtain purified AT-Exo. The supernatant was stimulated for exosome secretion using a dual-stimulation electroporation system, filtered through a 0.22 μm filter membrane, and the filtrate was centrifuged at 100,000 g in an ultracentrifuge. 3) Surface modification PD-L1 antibody was added to the AT-Exo purified in step 2) and incubated at room temperature. The mixture after incubation was ultracentrifuged, and the unmodified PD-L1 antibody was washed to obtain AI-Exo.

[0008] The present invention provides a method for loading therapeutic drugs targeting tumors using a dual stimulation electroporation system. The drug loading process is as follows: A constant-flow syringe pump was used to simultaneously inject 100 μg / mL of IL-7-RRE-IIB mRNA plasmid and a CD64-MEF / HEK293T cell suspension into a buffer channel at a constant flow rate. The suspension was then passed through a microfluidic structure with integrated platinum electrodes under nanosecond pulses of fixed amplitude, frequency, and duration. This loaded the CD64-MEF / HEK293T cells with IL-7-RRE-IIB mRNA plasmid. Exosome secretion was then stimulated again by ultrasonic nozzles. Cells were harvested directly from a culture dish and cultured for 24 hours. The culture supernatant was centrifuged and further purified using an ultracentrifuge to obtain AT-Exosomes. The AT-Exosomes were then incubated with a PD-L1 antibody at room temperature for 2 hours and further purified by ultracentrifugation to obtain AI-Exosomes.

[0009] Furthermore, the present invention also defines multifunctional exosomes obtained by the defined method, which are cup-shaped, contain characteristic exosomal proteins such as CD63, CD81, and CD9, and have an average diameter of 140 nm. The multifunctional exosomes produced by the present invention can inhibit autophagy in tumor cells and synergize with immune checkpoint therapy to inhibit cancer proliferation.

[0010] The multifunctional exosomes (AI-Exo) described in the present invention can be used to prepare drugs for immunotherapy of various cancer diseases.

[0011] Since EphA2 receptors are commonly overexpressed on the surface of cancer cells, the present invention connects YSA polypeptides to the surface of exosomes and loads IL-7 mRNA therein, inhibiting autophagy of tumor cells and restoring the expression of MHC-I on the surface of tumor cells. At the same time, PD-L1 antibodies block immune checkpoints, reactivating T cells to kill tumors, and restoring "cold" tumors to "hot" tumors, thereby achieving efficient cancer treatment.

[0012] The positive effects of the present invention are: a novel dual stimulation electroporation system (DES) is disclosed that combines nanosecond pulse electroporation with ultrasound to further stimulate the secretion of exosomes while completing drug loading, thereby increasing the production of exosomes and further increasing the mRNA content contained in the exosomes; The engineered multifunctional exosomes (AI-Exo) prepared by the present invention can be used to immunotherapeuticly treat various cancers with high autophagy expression. The exosomes are derived from MEF / HEK293T cells that stably express the YSA-CD64-(GFLG)3-Rev ARM fusion protein. First, by linking the YSA polypeptide to the N-terminus of CD64, the EphA2 receptor, which is commonly overexpressed in tumor cells, is targeted, thereby achieving the purpose of precise tumor targeting of the multifunctional exosomes. The Rev ARM polypeptide linked to the C-terminus of CD64 specifically binds to the RRE-IIB RNA sequence modified at the 3' end of IL-7, thereby enhancing the loading capacity of IL-7 mRNA. A peptide (GFLG)3 that can be specifically cleaved by cathepsin B is inserted between the CD64 and Rev ARM polypeptides to promote the release of mRNA in the acidic, cathepsin B-rich tumor cytoplasm. The extracellular domain of CD64 acts as an Fcγ type I receptor and can be coupled to anti-PD-L1 antibodies. The AI-Exo of the present invention achieves active targeting of drugs to tumor cells, expresses YSA targeting polypeptide and couples PD-L1 antibody on their surface, and loads IL-7 mRNA therein, inhibiting autophagy of tumor cells and restoring the expression of MHC-I on the surface of tumor cells. At the same time, PD-L1 antibody blocks immune checkpoints, reactivates T cells to kill tumors, and restores "cold" tumors to "hot" tumors, thereby achieving efficient treatment of cancer.

[0013] The present invention increases the production of exosomes by using a dual-stimulation electroporation system and performs a series of engineering modifications on the exosomes to enable them to more accurately target the tumor site, reduce the systemic toxicity of the drug, and improve the therapeutic effect of multifunctional exosomes on cancer and their universality for multiple cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a protein immunoblot image of exosomes and AI-Exo derived from MEF cells of the present invention; Figure 2 This is a protein immunoblot image of HEK293T cell-derived exosomes and AI-Exo of the present invention; Figure 3 is a cryo-electron micrograph of AI-Exo secreted by HEK293T cells of the present invention; Figure 4 is the number of particles that stimulate exosome secretion by the dual stimulation electroporation system of the present invention compared with other methods; Figure 5 It is the morphological structure of the dual stimulation electroporation system of the present invention in comparison with other methods to stimulate exosome secretion; Figure 6 This is a flow cytometric analysis of AI-Exo uptake by non-small cell lung cancer cells of the present invention; Figure 7 This is a laser confocal image of AI-Exo uptake by non-small cell lung cancer cells of the present invention; Figure 8 This is a laser confocal image verifying the targeting of AI-Exo to non-small cell lung cancer cells; Figure 9 is the RT-qPCR result of IL-7 content in AI-Exo of the present invention; Figure 10 This is the RT-qPCR result of the present invention verifying that GFLG in AI-Exo is cleaved in response to cathepsin B; Figure 11 is a graph showing changes in tumor volume in each group of tumor-bearing models of the present invention; Figure 12 This is a graph showing changes in body weight of each group of tumor-bearing models during treatment; Figure 13 is the survival period of each group of tumor-bearing models in the present invention. DETAILED DESCRIPTION

[0015] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0016] Example 1

[0017] (1) After mass culture of CD64-MEF cells, the cells were digested and centrifuged at 300 g for 5 minutes. CD64-MEF cells were collected and mixed with 100 μg / mL IL-7-RRE-IIB mRNA plasmid. Nanopulse stimulation was performed using a dual stimulation electroporation system (KDScientific, USA, model KDS 100) at an applied voltage of 140-160 V, a pulse width of 400-800 ns, and a flow rate of 1-4 mL / h. The cells were then cultured at a density of 3-6 million cells / dish for 24 hours to increase the production of exosomes. (2) The supernatant was collected and subjected to gradient centrifugation at 4°C using a high-speed refrigerated centrifuge: 300 g for 10 min to remove dead cells, 3000 g for 10 min, and 10,000 g for 20 min to remove cell debris; the supernatant was collected and filtered through a sterile 0.22 μm filter to remove larger vesicles; finally, the supernatant was centrifuged at 100,000 g for 70 min in an ultracentrifuge to precipitate the AT-Exo exosomes. The surface of the obtained AT-Exo exosomes contained CD63, TSG101, and Alix exosome marker proteins, see Figure 1 ; (3) Loading of PD-L1 antibody on the surface of exosomes: AT-Exo obtained by ultracentrifugation was incubated with PD-L1 antibody at 37°C for 2 h (protein content 1 / 0-1 / 8). The AT-Exo was then ultracentrifuged at 100,000 g for 2 h at 4°C to remove the unbound antibody to obtain AI-Exo. The obtained AI-Exo was spherical with a particle size of approximately 150 nm and good dispersion. Example 2

[0018] (1) After mass culture of CD64-HEK293T cells, the cells were digested and centrifuged at 300 g for 5 minutes. The CD64-HEK293T cells were collected and mixed with 100 μg / mL IL-7-RRE-IIB mRNA plasmid. After nano-pulse stimulation using a dual-stimulation electroporation system at an applied voltage of 140-160 V, a pulse width of 400-800 ns, and a flow rate of 2-3 mL / h, the cells were cultured at a density of 4-6 million cells / dish for 24 hours to increase the exosome production. (2) The supernatant was collected and subjected to gradient centrifugation at 4°C using a high-speed refrigerated centrifuge: 300 g for 10 min to remove dead cells, 3000 g for 10 min, and 10,000 g for 20 min to remove cell debris; the supernatant was collected and filtered through a sterile 0.22 μm filter to remove larger vesicles; finally, the supernatant was centrifuged at 100,000 g for 70 min in an ultracentrifuge to precipitate the AT-Exo exosomes. The surface of the obtained AT-Exo exosomes contained CD63, TSG101, and Alix exosome marker proteins; see Figure 2 ; (3) Loading of PD-L1 antibody on the surface of exosomes: AT-Exo obtained by ultracentrifugation was incubated with PD-L1 antibody at 37°C for 2 h (protein content 1 / 0-1 / 8), and then ultracentrifuged at 100,000 g for 2 h at 4°C to remove the unbound antibody to obtain AI-Exo. The obtained AI-Exo was spherical with a particle size of about 150 nm and good dispersion; see Figure 3 . Example 3

[0019] AI-Exo was prepared by the preparation method in Example 2, and the number and morphology of exosomes secreted by different stimulation methods were evaluated. HEK293T cells were stimulated to secrete exosomes by starvation treatment (Control), millisecond electroporation (msEP), nanosecond electroporation (nsEP), and the dual stimulation electroporation system (DES) used in the present invention. After 24 hours of culture, the supernatant was collected and the exosomes were purified using the same method as in Example 2 above. Gradient centrifugation was used to remove cells and large vesicles, and finally the exosomes were obtained by ultracentrifugation. The exosomes of the same amount of cells stimulated by different methods were analyzed by nanoparticle tracking analyzer (NTA), and the number and particle size distribution of exosome particles were obtained. The morphological structure of the exosomes stimulated by different methods was observed by cryo-transmission electron microscopy. The experimental results showed that stimulation by the dual stimulation electroporation system used in the present invention resulted in a 30-fold increase in the exosome production of HEK293T cells; see. Figure 4 , and the morphology, structure and size of exosomes secreted by different methods are similar; see Figure 5 .

[0020] The positive effects of the present invention are demonstrated by the following experimental examples:

[0021] Experimental Example 1 The AI-Exo obtained in Example 2 was stained with PKH67 and then incubated with non-small cell lung cancer cells A549. The uptake of AI-Exo by A549 was analyzed by flow cytometry. The specific steps are as follows: PKH67 dye was added to 0.5-2 mg / mL AI-Exo (100-150 μL), incubated at room temperature for 10-30 min, centrifuged at 2000 g for 20 min at 4°C using a 100 kDa ultrafiltration tube, added with 150 μL PBS, and centrifuged at 2000 g for 20 min at 4°C using a 100 kDa ultrafiltration tube. Repeat this process to obtain PKH67-stained multifunctional exosomes (PKH67-AI-Exo). PKH67-AI-Exo was added to A549 cells and incubated for 4 h. The supernatant was removed and the cells were washed twice with PBS. The cells were trypsinized and centrifuged at 300 g for 5 min. The supernatant was removed and the cells were fixed with 400 μL of paraformaldehyde solution. The uptake of PKH67-AI-Exo by non-small cell lung cancer cells was analyzed by flow cytometry. The experimental results showed that non-small cell lung cancer cells showed a high uptake of PKH67-AI-Exo. Figure 6 .

[0022] Experimental Example 2

[0023] The PKH67-AI-Exo obtained in Experimental Example 1 was co-incubated with non-small cell lung cancer cells A549, and the uptake of PKH67-AI-Exo by non-small cell lung cancer cells was analyzed by laser confocal microscopy. The specific steps are as follows: PKH67-AI-Exo was added to A549 cells and incubated for 4 h. The supernatant was removed and the cells were washed three times with PBS. The cells were fixed with 500 μL paraformaldehyde solution for 30 min and washed three times with PBS. The cells were mounted with anti-fluorescence quenching mounting medium containing Hoechst 33342 and the uptake of PKH67-AI-Exo by A549 was observed using a laser confocal microscope. The experimental results showed that A549 cells had a high uptake of PKH67-AI-Exo; see Figure 7 .

[0024] Experimental Example 3

[0025] Laser confocal microscopy experiments verified that YSA peptide-mediated AI-Exo has active tumor targeting. The specific steps are as follows: 80,000 cells / well were inoculated in a 12-well plate covered with a slide, and EphA2 antibodies and IgG were used to co-incubate with A549 cells, respectively. The specific binding of YSA to EphA2 highly expressed on the surface of tumor cells was blocked by EphA2 antibodies. After pretreatment, PKH67-labeled AI-Exo and natural Exo that do not express YSA were co-incubated with A549 cells for 4 hours. After washing away the drug with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes and washed again with PBS. The slides were then sealed with anti-fluorescence quenching mounting medium containing Hoechst 33342, and the uptake of PKH67-AI-Exo and PKH67-Exo by A549 was observed using a laser confocal microscope. The experimental results showed that blocking with the EphA2 antibody significantly inhibited the uptake of PKH67-AI-Exo by A549, indicating that active targeting was achieved by targeting EphA2 on the surface of tumor cells by YSA; see Figure 8 .

[0026] Experimental Example 4

[0027] The specific binding of the Rev protein arginine-rich motif (Rev ARM peptide) coupled to the CD64 C-terminus and the Rev response element (RRE) mRNA sequence added to the 3' end of IL-7 mRNA can promote the loading of AI-Exo to IL-7 mRNA. The IL-7 content in AI-Exo can be verified by RT-qPCR. The specific steps are as follows: Exosomes were extracted from HEK293T cells (Exo) and CD64-HEK293T cells (CD64-Exo), as well as from HEK293T cells loaded with IL-7-RRE-IIB mRNA plasmid (IL-7-Exo) and CD64-HEK293T cells (AI-Exo). Total RNA was extracted using an mRNA extraction kit, reverse transcribed into cDNA, and amplified through denaturation, annealing, and extension cycles to generate RT-qPCR results. The results showed that the IL-7 mRNA loading in AI-Exo was amplified 8-fold; see Figure 9 .

[0028] Experimental Example 5

[0029] To effectively release IL-7 mRNA in tumor cells, a cathepsin B-cleavable peptide (GFLG) 3-linker was inserted between the CD64 and Rev ARM peptides. Activated cathepsin B in the acidic TME cleaves the phenylalanine-leucine bond, ensuring controlled mRNA release. RT-qPCR results of the released IL-7 mRNA were validated by simulating cathepsin B cleavage in the TME in vitro. The specific steps are as follows: AI-Exo containing (GFLG) 3 linker and exosomes without this peptide were extracted separately. PBS buffer with or without cathepsin B was then added to the exosomes. After 12 hours of treatment, free IL-7 mRNA was obtained by ultracentrifugation. Total RNA was extracted and subjected to RT-qPCR. The results showed that the IL-7 mRNA content was the highest in the group containing both (GFLG) 3 linker and cathepsin B; see Figure 10 .

[0030] Experimental Example 6

[0031] Construction of non-small cell lung cancer tumor model: C57BL / 6 (male, 18-20 g, 4 weeks old) were purchased from Liaoning Changsheng Co., Ltd. Lewis lung cancer cells (LLC) were stably passaged for 5-6 generations in DMEM culture medium. When the cells proliferated vigorously, the culture was terminated and the cell suspension was prepared in serum-free DMEM culture medium. The cell density was adjusted to 1×10 7Cells / mL were detected with an intraperitoneal injection of 50 mg / kg sodium pentobarbital. After anesthesia, the mice were immobilized on the operating table and the left thoracic region was disinfected. Skin and muscle incision was made on the left thoracic side, and the intercostal space was bluntly dissected to expose the left lung lobe. Using a microsyringe, 100 μL of the cell suspension was slowly injected into the left lung parenchyma (avoiding pleural penetration and pneumothorax). After injection, pressure was applied to stop bleeding and observe for leakage. The incision was sutured layer by layer. Antibiotics were administered postoperatively to prevent infection. The mice's activity and respiratory status were monitored during the recovery period.

[0032] In vivo treatment: All tumor-bearing mice were divided into five groups, each containing eight mice: the model (PBS) group, the empty exosome (Exo) group, the AT-Exo group, the AI-Exo group, and the PD-L1 antibody group. Drug administration began on day 3 after modeling and continued every three days for three weeks. The exosome dose was 2.5 mg / kg, and the PD-L1 antibody dose was 10 mg / kg. Tumor progression was quantified using an in vivo imaging system (IVIS) by measuring bioluminescent signal intensity on days 3, 7, 11, 15, 19, and 23. Body weight and survival of the tumor-bearing mice in each group were observed.

[0033] The results are as follows Figure 11 、 Figure 12 and Figure 13 As shown in the figure, the results show that AI-Exo can significantly inhibit the growth of lung cancer tumors, prolong the survival of tumor-bearing mice, and the weight of tumor-bearing mice in this group does not change much. The AI-Exo group showed better results.

[0034] The above is only a general description and implementation method of the present invention, and does not limit the scope of patent protection of the present invention. Any equivalent changes made based on the description and drawings of the present invention, or any direct or indirect application of the patent of the present invention to other related technical fields, shall be deemed to be within the scope of protection of the patent of the present invention.

Claims

1. A multifunctional exosome for cancer immunotherapy, characterized by: The multifunctional exosomes are cup-shaped, contain CD63, CD81, and CD9 exosome-specific proteins, and have an average diameter of 140 nm. They are engineered by exosomes derived from MEF / HEK293T cells that stably express the YSA-CD64-(GFLG)3-Rev ARM fusion protein. By linking the YSA peptide to the N-terminus of CD64, the EphA2 receptor overexpressed in tumor cells is targeted, promoting the targeting effect of multifunctional exosomes; the Rev ARM peptide linked to the C-terminus of CD64 specifically binds to the RRE-IIB RNA sequence modified at the 3' end of IL-7, enhancing the loading amount of IL-7 mRNA; a peptide (GFLG)3 that can be specifically cleaved by cathepsin B is inserted between CD64 and Rev ARM peptides to promote the release of mRNA in the acidic, cathepsin B-rich tumor cytoplasm; the extracellular domain of CD64 acts as an Fcγ type I receptor and can be coupled to anti-PD-L1 antibodies.

2. The method for preparing multifunctional exosomes for cancer immunotherapy according to claim 1, comprising the following steps: 1) Construction of a MEF / HEK293T cell line stably overexpressing the YSA-CD64-(GFLG)3-Rev ARM fusion protein (CD64-MEF / HEK293T); 2) Loading therapeutic drugs Using a dual-stimulation electroporation system, 100 μg / mL IL-7-RRE-IIB mRNA plasmid was mixed with MEF / HEK293T cell suspension. IL-7-RRE-IIB mRNA was loaded into MEF / HEK293T cells through dual stimulation of electroporation and ultrasound, stimulating exosome secretion. After extracting the supernatant, the cells were sequentially subjected to differential centrifugation, filtration, and ultracentrifugation to obtain purified drug-loaded exosomes (AT-Exo). The supernatant was stimulated for exosome secretion using a dual-stimulation electroporation system, filtered through a 0.22 μm filter membrane, and the filtrate was centrifuged at 100,000 g in an ultracentrifuge. 3) Surface modification PD-L1 antibody was added to the drug-loaded exosomes purified in step 2) and incubated at room temperature. The mixture after incubation was ultracentrifuged at 4°C, and the unmodified PD-L1 antibody was washed to obtain multifunctional exosomes.

3. The method for preparing multifunctional exosomes according to claim 2, wherein: The process of loading the targeted therapeutic drug using a dual stimulation electroporation system is as follows: A constant-flow syringe pump was used to simultaneously inject 100 μg / mL of IL-7-RRE-IIB mRNA plasmid and a CD64-MEF / HEK293T cell suspension into the buffer channel at a fixed flow rate. The suspension was then passed through a microfluidic structure with integrated platinum electrodes under nanosecond pulses with fixed pulse amplitude, frequency, and duration. The IL-7-RRE-IIB mRNA plasmid was loaded into the CD64-MEF / HEK293T cells. Subsequently, an ultrasonic nozzle was used to stimulate exosome secretion again, and the cells were directly collected using a culture dish. After culturing for 24 hours, the culture supernatant was centrifuged and further purified using an ultracentrifuge to obtain drug-loaded exosomes. The exosomes were incubated with PD-L1 antibodies at room temperature for 2 hours and further purified by ultracentrifugation to obtain multifunctional exosomes.

4. Use of the multifunctional exosomes (AI-Exo) according to claim 1 in the preparation of a drug for treating cancer.

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