Drug delivery system taking tumor vascular endothelial cells as targets and application thereof

Abstract

The invention discloses a drug delivery system taking tumor vascular endothelial cells as targets and application of the drug delivery system, and belongs to the technical field of biological medicines. The invention provides a delivery carrier taking a tumor vascular endothelial cell as a target spot. The delivery carrier comprises an exosome and MMRN2 and / or IGFBP7 protein modified at an extracellular domain of an exosome membrane protein. The invention further provides a drug delivery system with tumor vascular endothelial cells as targets, and the drug delivery system comprises the delivery carrier and tumor treatment drugs wrapped in the delivery carrier. The exosome is modified to target CD93 molecules on the surface of glioma vascular endothelial cells, so that the drug is enriched in glioma, the curative effect is enhanced, and the safety is improved; the compound can specifically activate inflammation signal channels in glioma vascular endothelial cells, induce inflammatory response in glioma and remodel tumor-associated macrophages, can directly inhibit tumor growth, can promote presentation of tumor-associated antigens, and induces strong tumor specific immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug delivery system targeting tumor vascular endothelial cells and its application. Background Technology

[0002] Gliomas are the most common malignant primary gliomas in adults, with high incidence, recurrence, and mortality rates, and extremely poor prognosis. The invasive growth of gliomas means that conventional surgical resection cannot completely cure them. Currently, the commonly used treatment method is surgery combined with radiotherapy, chemotherapy, and immunotherapy, but the prognosis is still not ideal. Generally, gliomas are divided into localized gliomas and diffuse gliomas. Localized gliomas are benign and can be cured by surgical resection; in contrast, diffuse gliomas are more malignant, and surgical resection alone is unlikely to achieve a cure. Local drug delivery to glioma tissue is both difficult and risky, therefore safer and more effective treatment methods are needed.

[0003] To address the limitations of existing treatment strategies for malignant gliomas, new treatment approaches have emerged in preclinical diagnosis and clinical treatment, such as molecular targeted therapy, immunotherapy, gene therapy, stem cell-based therapies, and nanotechnology. Due to the success of molecular targeted therapy in several other cancer types, such as melanoma, chronic myeloid leukemia, and lung cancer, this treatment strategy has brought great hope to the treatment of gliomas and has made significant progress in the past two decades. For example, in 2009, the U.S. Food and Drug Administration (FDA) approved molecular targeted therapies such as bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), for the treatment of gliomas. However, current targeted drug delivery to gliomas is limited by the blood-brain barrier (BBB), making it difficult for most chemotherapy drugs and macromolecular therapeutic agents (such as antibodies and nucleic acid drugs) to be effectively delivered to the tumor site, resulting in very low drug delivery efficiency. Furthermore, the glioma microenvironment (GME) is highly heterogeneous and immunosuppressive, including tumor-associated macrophage (TAM) pro-cancer polarization and T cell exhaustion, which limits the efficacy of immunotherapy.

[0004] Currently, targeted therapy for glioma focuses primarily on targeting tumor cells, while methods for delivering drugs through targeted vascular endothelial cells of tumors have not yet been reported. This invention, for the first time, constructs a drug delivery platform targeting glioma vascular endothelial cells by modifying exosomes, thereby achieving the goal of targeted delivery of various drugs into the glioma microenvironment. Summary of the Invention

[0005] To address the problems existing in the prior art and to solve the issue of low drug delivery efficiency in glioma treatment, this invention aims to design and provide a drug delivery system targeting tumor vascular endothelial cells and its application. This invention modifies exosomes with polyprotein 2 (MMRN2) or insulin-like growth factor binding protein 7 (IGFBP7) to provide a nanocarrier that can target and deliver chemical or immunotherapeutic drugs into the tumor microenvironment. The therapeutic efficacy and safety of this targeted delivery system for glioma are verified by delivering temozolomide and an interferon gene-stimulating protein agonist (STING signaling agonist), respectively.

[0006] The blood-brain barrier is composed of tightly connected endothelial cells, pericytes, and astrocytes. First, this invention targets glioma vascular endothelial cells, circumventing the blood-brain barrier penetration challenge and significantly improving drug delivery efficiency. Second, as the interface between the central nervous system (CNS) and the peripheral immune system, these endothelial cells play a crucial role in coordinating peripheral immune responses and CNS immune responses. By delivering a STING agonist via IGFBP7-modified exosomes carrying the tumor antigen PEP3, the tumor immunosuppressive microenvironment can be altered, upregulating IFNγ expression and creating a favorable environment for CD8 activation. + T cells kill tumor cells within an inflammatory microenvironment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides that the delivery vector is obtained by inserting a polypeptide sequence between the 25th and 26th amino acids of the exosome membrane protein Lamp2b; The polypeptide sequence can bind to CD93.

[0008] The polypeptide sequence is either MMRN2 protein or IGFBP7 protein; The amino acid sequence of the MMRN2 protein is shown in SEQ ID NO.1; The amino acid sequence of the IGFBP7 protein is shown in SEQ ID NO.2.

[0009] Secondly, the present invention provides the application of the delivery carrier in the preparation of medicaments for treating tumor diseases.

[0010] The application described herein refers to glioma as the tumor disease.

[0011] Thirdly, the present invention provides a drug delivery system targeting tumor vascular endothelial cells, comprising the delivery carrier and a tumor therapeutic drug encapsulated within the delivery carrier.

[0012] The aforementioned drug delivery system targets tumor vascular endothelial cells, wherein the tumor therapeutic drug is a chemotherapy drug or an immunomodulatory drug.

[0013] Fourthly, the present invention provides a drug delivery system targeting tumor vascular endothelial cells, wherein the chemotherapeutic drug is temozolomide; The immunomodulatory drug is a STING agonist.

[0014] Fifthly, the present invention provides the application of the drug delivery system described herein in the preparation of medicaments for treating tumor diseases.

[0015] The application described herein refers to glioma as the tumor disease.

[0016] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned drug delivery system and pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention engineered CD93 molecules on the surface of glioma vascular endothelial cells using exosomes to create a targeted drug delivery carrier for the glioma tumor microenvironment. This allows for the efficient enrichment of tumor therapeutic drugs into the tumor microenvironment, achieving higher drug concentrations within the tumor while reducing the dosage, thus enabling highly effective killing of tumor cells. Simultaneously, it avoids the side effects associated with high-dose medication.

[0018] 2. From the perspective of immunotherapy, existing technologies also include intratumoral injection of STING agonists for tumor treatment. However, intratumoral injection of STING agonists has some disadvantages, such as causing T cell exhaustion. Vascular endothelial cells are located at the junction of the peripheral and central nervous systems and play a crucial role in coordinating peripheral and central immune responses. The drug delivery system constructed in this invention targets and delivers STING agonists to vascular endothelial cells, specifically activating STING signaling in these cells. This effectively activates the immune response in the tumor microenvironment while reducing T cell exhaustion, thereby achieving better tumor treatment results. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the exosome modification and drug loading of the present invention; Figure 2 Schematic diagram of the structure of MMRN2-Lamp2b or IGFBP7-Lamp2b plasmid; Figure 3 Images of NTA detection and electron microscopy findings in exosomes, scale bar = 100 nm; Figure 4Western blot analysis of fusion protein expression in cells and exosomes; Figure 5 For in vitro targeting assays of modified exosomes, A represents the immunostaining analysis of the transduction efficiency of modified exosomes in vitro; the dashed white box indicates the magnified area, scale bar = 50 μm. B represents the quantitative analysis of PKH26 positive cells. n = 3, one-way ANOVA. Figure 6 To assess the in vivo targeting of modified exosomes, A shows the distribution of exosomes monitored using an IVIS imaging system, with white circles indicating exosomes aggregated in the tumor region; B shows the quantitative analysis of PKH26 fluorescence in the tumor 24 hours after exosome injection, n=3, one-way ANOVA; C shows the fluorescence imaging analysis of ex vivo tissues, with white circles indicating aggregated exosomes within the tumor region. Figure 7 The efficacy of IGFBP7-modified exosomes delivering chemotherapy drugs in the treatment of glioma was evaluated. A represents the survival of GL261 cells after treatment with different doses of free temozolomide or temozolomide-loaded exosomes using CCK-8 assay (n=3, one-way ANOVA); B represents mouse body weight change curves; C represents tumor growth curves (n=5, one-way ANOVA); D represents mouse survival curves (n=8, Kaplan-Meier); and E represents HE staining results (scale bar=100 μm). Figure 8 The efficacy of IGFBP7-modified exosomes delivering chemotherapy drugs in the treatment of glioma was investigated. A represents tumor growth curves (n=5, one-way ANOVA); B represents mouse survival curves (n=8, Kaplan-Meier); C represents HE staining results (Scalebar=200 μm); and D represents tumor antigen-specific immune response detection. CD8+ T cells were isolated from tumor-infiltrating immune cells using CD8a microbeads and co-cultured with dendritic cells pre-treated with PEP-3 peptide for 48 hours. IFN-γ release was analyzed by ELISA (n=4, one-way ANOVA). Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Example 1: Purification and characterization of MMRN2-modified or IGFBP7-modified exosomes, such as Figure 1 : (1) Construction of MMRN2-modified exosomes: Using genetic engineering techniques, the MMRN2 protein was fused to the N-terminus of the exosome membrane protein Lamp2b, and a His tag was inserted into the C-terminus of Lamp2b to obtain MMRN2-Lamp2b. After transfecting cells, the supernatant was collected, and the exosomes were purified by ultracentrifugation to obtain MMRN2-modified exosomes (i.e., MMRN2-Exo).

[0022] (2) Construction of IGFBP7 modified exosomes: Using the same method as in step 1 above, except that the MMRN2 protein is replaced with the IGFBP7 protein, IGFBP7-Lamp2b is obtained. IGFBP7-modified exosomes (i.e., IGFBP7-Exo) are obtained using the same method as MMRN2-Exo.

[0023] The expression of the two fusion proteins was verified at the transcriptional and protein levels using RT-PCR and Western blotting experiments, respectively. Transmission electron microscopy and nanoparticle tracking techniques were also used to characterize the exosomes, further verifying their morphology and particle size, indicating that the modified exosomes did not alter their morphology or size.

[0024] Experimental methods: Mouse-derived MMRN2 or IGFBP7 genes were downloaded from NCBI. The mouse-derived IGFBP7 (amino acid sequence shown in SEQ ID NO.2) is as follows: : RIRRRRVNVLCLWTLCTCQLSSSAVGLSTWRNQRCLWLLSSLRRRGRALWWRRCWR WSLCSRYGVREVSQTAERQSRCCCWRTSYIGSVCVQVVSSVWQQWHHISIWLPAESCLS ESRKQRRESHHTGEQGNMRTGTKHRDTTTRHLERYWCGILELGDWHSNSSADLEQGEE GSQWCAENRAAAWRGEPGHPDTRRSREARGDRLGVGEPSVGGCWRVRVPCQQFSGPGF CRCDHRGRC SADSTEEGR RCSAGRRRVT R.

[0025] The mouse-derived MMRN2 (amino acid sequence shown in SEQ ID NO.1) is as follows: RIRRRRVNAGPRYQVLPSQARHARLETRGGYQQRPHQTELVSLPEVQASHLCSCLQNREIPGPFTAAMSTGSPLPGSQSHVSSGPEASVPGPAEGADLCGLAVLPRVPGTRLPGPQSHSKPAHRAKWTPGDLGLDGWLTWSPCPRVDGATRTTGDSAPEFC QADWAVLGLLGCLPQLPRPTQHALGHPARPETAPAALPQPLPELPRSGGKQHQPRLGEAAGHVEEREATERPRRIPEEGEASGDVYRCTRQRSGALGDRLPCGLLCRFFRRGHCSADGEVQHHIHQCGQQLLSTWLLPSSQTWRLLVCCEHYIWPRPRNGA.

[0026] After synthesis, the fusion protein was linked to amino acids 25-26 after the N-terminal signal peptide of Lamp2b and had a 6His tag at the C-terminus for subsequent Western blotting. The structure of the fusion protein is shown below. Figure 2 As shown. The constructed plasmids were transfected into HEK293 cells. After 24 h of adherent culture, the cells were washed with PBS, replaced with exosome-free fetal bovine serum, and cultured for 48 h. The supernatant was then collected and placed in centrifuge tubes. The tubes were first centrifuged at 300 g for 10 min, then at 2000 g for 10 min to remove cell debris. Finally, the supernatant was centrifuged at 110000 g at 4 °C for 2 h. The supernatant was discarded, the centrifuge tubes were inverted, and any remaining liquid on the inner wall was allowed to dry to prevent backflow. The cells were then resuspended in 1×PBS and stored at -80 °C.

[0027] Mouse-derived Lamp2b (amino acid sequence shown in SEQ ID NO.3): MNFTITYETTNQSNKTITIAVPDKATHDGSSCGDDRNSAKIMIQFGFAVSWAVNFTKEASHYSIHDIVLSYNTSDSTVFPGAVAKGVHTVKNPENFKVPLDVIFKCNSVLTYNLTPVVQKYWGIHLQAFVQNGTVSKNEQVCEEDQTPTTVAPIIHTTAPSTTTTLTPTSTTPTPTPTPTPTVGNYS IRNGNTTCLLATMGLQLNITEEKVPFIFNINPATANFTGSCQPQSAQLRLNNSQIKYLDFIFAVKNEKRFYLKEVNVYMYLANGSAFNISNKNLSFWDAPLGSSYMCNKEQVLSVSRAFQINTFNLKVQPFNVTKGQYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYLIGRRKTYAGYQTL Experimental results: 1. Schematic diagram of plasmid construction as shown below Figure 2 As shown.

[0028] 2. Nanoparticle tracking and electron microscopy results are as follows: Figure 3 As shown, electron micrographs and NTA images of exosomes (left: NTA image; right: electron micrograph).

[0029] First, we performed NTA and electron microscopy analysis on exosomes, and the results are as follows: Figure 3 As shown, the morphology and particle size of exosomes modified with MMRN2 or IGFBP7 did not change significantly compared with control exosomes, suggesting that the modified exosomes still retain good properties and can be used as nanocarriers for drug delivery.

[0030] 3. Western blot analysis confirmed that exosomes carry MMRN2-Lamp2b or IGFBP7-Lamp2b fusion proteins: To verify whether MMRN2 and IGFBP7 were effectively carried onto exosomes, we added His tags to MMRN2 and IGFBP7, and further detected the levels of MMRN2 and IGFBP7 in cells and exosomes using Western blot. The results are as follows: Figure 4 As shown, the expression of both fusion proteins was detected in cell lysates and exosomes.

[0031] Example 2: In vitro targeting verification In in vitro experiments, PKH26-labeled exosomes were co-incubated with cell lines overexpressing mCD93.

[0032] Cells co-cultured with modified exosomes showed significant red fluorescence aggregation, while only trace fluorescence signals were detected in the unmodified exosome treatment group, verifying the efficient delivery of MMRN2-Exo or IGFBP7-Exo to CD93-overexpressing cells.

[0033] Unmodified exosomes: Native exosomes purified from HEK293 cell supernatant by ultracentrifugation and stained with PKH26.

[0034] Modified exosomes: MMRN2-Exo and IGFBP7-Exo were obtained by ultracentrifugation from the supernatant of HEK293 cell lines overexpressing MMRN2-Lamp2b or IGFBP7-Lamp2b obtained by plasmid transfection, and stained with PKH26.

[0035] These three groups of exosomes were co-incubated with HEK293 cells overexpressing mouse CD93 in vitro for 24 h, and the fluorescence distribution was observed under a microscope.

[0036] Experimental results: We labeled exosomes modified by different methods and control exosomes with PKH26, and then co-incubated them with CD93-HEK293 cells for 48 hours. After washing the cells three times with PBS, the transduction efficiency of different modification methods on CD93-highly expressing cells was detected by fluorescence microscopy. The results showed that MMRN2 and IGFBP7 modified exosomes were transduced into more CD93-HEK293 cells than the control group, and the IGFBP7 modified group had better results. This indicates that modified exosomes can significantly improve their transduction efficiency. Figure 5 ).

[0037] Example 3: In vivo targeting verification In vivo experiments verified that the MMRN2-Exo or IGFBP7-Exo constructed in Example 1 could efficiently accumulate in the tumor region of the brain by specifically binding to CD93 by observing the fluorescence of the brain in mice throughout the body and in isolated tissues.

[0038] Wild-type male C57BL / 6 mice aged 6-8 weeks were purchased to establish a mouse orthotopic glioma model. Using the previously constructed Luci-GL261 cell line, 1×10⁻⁶ cells were injected into the brain via stereotactic localization. 5GL261 cells overexpressing Luciferase were injected into the striatum of the right hemisphere of C57BL / 6 mice. On day 7 post-tumor implantation, tumor growth was observed using a GINN in vivo imaging system to determine tumor formation, and the mice were randomly divided into three groups. PKH26-labeled naturally secreted exosomes (Naive-Exo), MMRN2-Exo, and IGFBP7-Exo (1×10⁻⁶) were injected via tail vein. 8 The fluorescence distribution of exosomes was observed throughout the body at different times, and then the fluorescence distribution of isolated tissues (brain, heart, liver, spleen, lung and kidney) was observed at 24 hours and 48 hours after injection.

[0039] Then, brain tissue was taken, embedded in an Optimal Cutting Temperature Compound (OCT), and fixed on a cryostat. The slice thickness was 10 μm. After the brain slices were mounted on a glass slide, the red fluorescence carried by the exosomes was observed directly under a fluorescence microscope.

[0040] Experimental results: Next, we conducted an in vivo targeting validation experiment using modified exosomes. First, we established an orthotopic glioma mouse model by injecting GL261 tumor cells into the striatum region of the C57 mouse brain. On day 7 of tumor bearing, PKH26-labeled exosomes were injected via the tail vein. The dynamic distribution of exosomes in the mice was tracked using in vivo imaging at six time points: 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after injection. The results showed that the fluorescence accumulation of the two modified exosomes in the mouse brain was significantly stronger than that of the control group, indicating that MMRN2-Exo and IGFBP7-Exo modified exosomes can effectively improve the delivery efficiency to glioma vascular endothelial cells. Among them, IGFBP7-Exo showed better delivery efficiency than MMRN2-Exo, which is consistent with the targeting results validated in vitro. Figure 6 (A, B). Next, at 24 h and 48 h, we isolated six major tissues from the mouse: brain, heart, liver, spleen, lung, and kidney, and photographed and recorded the distribution of red fluorescence in different tissues. The results are as follows. Figure 6 As shown in Figure C, we found that more significant fluorescence aggregation could be observed in the brain tumor regions of mice injected with IGFBP7-Exo modified exosomes.

[0041] Example 4: The therapeutic effect of IGFBP7-modified exosomes delivering chemotherapy drugs on glioma. (1) The exosomes were loaded with the tumor chemotherapy drug temozolomide (TMZ) via ultrasound technology.

[0042] Exosome ultrasound loading TMZ a. Exosomes (i.e., 500 μg / mL) and 500 μg TMZ were incubated at 37 °C for 30 days in a total volume of 500 μL. min. b. Ultrasonic power 25 W, on for 30 s, off for 150 s, 6 cycles, operated on ice. c. Incubate the exosomes again for 1 h to stabilize the exosome membrane. d. Wash with ultracentrifugation at 120,000 g for 70 min in PBS to remove free TMZ.

[0043] HPLC quantification of the efficiency of TMZ loading on exosomes.

[0044] Liquid chromatography conditions: Mobile phase: methanol-0.5% HAc (30:70, v / v). Flow rate: 1.1 mL / min. Injection volume: 20 μL. Column temperature: 35±1 ℃. Residence time in TMZ: 3.56 min. Analytical run time: 8 min. UV wavelength: 330 nm.

[0045] (2) In vitro validation: After quantifying the drug loading efficiency by HPLC, free TMZ and exosome-loaded TMZ (50 μg / mL) were first tested in vitro with (3 x 10) 3 After 48 hours of treatment with GL261 glioma cells, the inhibitory effects of free TMZ and IGFBP7-Exo-TMZ on tumor cell proliferation were verified by CCK8 and flow cytometry apoptosis assays. The results showed that IGFBP7-Exo-TMZ and free TMZ produced the same anti-tumor effect in vitro.

[0046] (3) In vivo validation: Next, the therapeutic effect of IGFBP7-Exo-TMZ on glioma was validated in vivo. First, a mouse model bearing tumor was established using the same method as in Example 3. Mice with successfully established glioma models were randomly divided into 5 groups (PBS, IGFBP7-Exo, Free TMZ, Control-Exo-TMZ, and IGFBP7-Exo-TMZ). After tumor formation on day 7, different reagents were injected into the tail vein every 2 days for a total of 3 injections (1 mg / kg). Five days after the last treatment, the brain tissue, heart, liver, spleen, lung, and kidney tissues of the mice were isolated, embedded, and sectioned in paraffin for HE staining and TUNEL staining. During the treatment, the tumor growth, weight changes, and survival time of the mice were recorded. Combined with the monitoring of the tumor growth curve, survival time, mouse weight, and HE staining results, it was shown that IGFBP7-Exo-TMZ can inhibit tumor growth, prolong the survival time of mice, and does not cause significant damage to the normal tissues of mice.

[0047] Experimental results: We validated the therapeutic efficacy of temozolomide delivery to glioma vascular endothelial cells via modified exosomes. Temozolomide was loaded into exosomes using ultrasound, and the drug loading efficiency was analyzed by high-performance liquid chromatography (HPLC). In in vitro experiments, GL261 cells were treated with free temozolomide and temozolomide-loaded exosomes. The results showed that, at the same temozolomide dose, the inhibitory effects on tumor growth were similar for both temozolomide-loaded exosomes and free temozolomide (Figure 7A). These results further confirmed the accuracy of temozolomide loading onto exosomes and also indicated that encapsulating temozolomide in exosomes did not affect its cytotoxicity. Next, we used a glioma xenograft mouse model to test the in vivo therapeutic effect of temozolomide-loaded exosomes. The mouse body weight change curves showed that all treatments did not affect the normal growth of the mice (Figure 7B). Mice injected with modified temozolomide-loaded IGFBP7 exosomes showed significant tumor growth inhibition, while mice injected with the same dose of free temozolomide or native temozolomide-loaded exosomes exhibited tumor growth curves similar to those of the PBS-injected group (Figure 7C). IGFBP7 modification enhanced exosome accumulation in the GME, effectively increasing local tumor drug concentrations even with low-dose intravenous TMZ injection, thus producing a good anti-tumor effect. Furthermore, the tumor growth curves of the unmodified exosome (unloaded with TMZ) group did not change significantly compared to the PBS-injected group. Treatment with temozolomide-loaded IGFBP7 exosomes also significantly improved the survival rate of glioma-bearing mice (Figure 7D). Histological staining results showed that only mice treated with temozolomide-loaded IGFBP7 exosomes exhibited significant tissue damage in the tumor region (Figure 7E). In summary, IGFBP7-modified exosomes can deliver chemotherapeutic drugs to glioma vascular endothelial cells and increase drug concentration at the tumor site, thereby enhancing therapeutic efficacy and safety.

[0048] Example 5: The therapeutic effect of IGFBP7-modified exosomes delivering immunomodulatory drugs on glioma. (1) IGFBP7-Lamp2b and HBVc-PEP3 (PEP3 is a small peptide cloned from the EGFRVⅢ mutation region, which carries HBVc <hepatitis B virus core antigen> and can enhance the immunogenicity of PEP3) were sequentially linked to a lentiviral vector and infected cells. Exosomes were collected to obtain IGFBP7 modified exosomes carrying glioma antigen HBVc-PEP3. Then, the STING agonist (cGAMP, CAT# HY-12512) was loaded into the exosomes by electroporation to obtain IGFBP7-Exo-cGAMP-PEP3.

[0049] Exosome electroretransfer drugs: The STING agonist (CdGMP, catalog number HY-107780 or cGAMP, catalog number HY-12512) was loaded into exosomes using an electroporation system. The simplified procedure is as follows: 100 μL of an electroporation mixture containing exosomes (100 μg / mL) and STING agonist (100 μg / mL) was prepared and transferred to a pre-chilled 4 mm electroporation vessel, incubated at 30 °C for 30 min. After electroporation at 200 V, the sample was incubated at 37 °C for 1 h to allow membrane structure recovery. To remove unencapsulated agonist, the mixture was ultracentrifuged at 100,000 × g for 2 h at 4 °C, washed with PBS, and resuspended to obtain the purified Exo-cGAMP complex. The loading efficiency of the STING agonist was quantitatively analyzed by measuring the optical density (OD) at 245 nm.

[0050] (2) In vivo experiments: The therapeutic effect of IGFBP7-Exo-cGAMP-PEP3 on glioma was verified in vivo. First, GL261 cells overexpressing EGFRvIII and Luciferase were established by infecting GL261 cells with lentivirus carrying mouse EGFRvIII and Luciferase genes, named GL261-EGFRvIII. GL261-EGFRvIII cells were injected into the striatum of the right brain of C57BL / 6 mice to establish a tumor-bearing mouse model using the same method as in Example 3. Mice with successfully established glioma models were randomly divided into 6 groups (PBS, IGFBP7-Exo, IGFBP7-PEP3-Exo, Free cGAMP, IGFBP7-Exo-cGAMP, IGFBP7-Exo-PEP3-cGAMP). After tumor formation on day 7, different reagents (3 mg / kg) were injected into the tail vein every 3 days for a total of 3 injections. Seven days after the final treatment, brain tissue, as well as heart, liver, spleen, lung, and kidney tissues from mice were isolated, embedded, and sectioned in paraffin for HE staining. Tumor growth, weight changes, and mouse survival were recorded throughout the treatment process. The monitoring of tumor growth curves, survival time, mouse weight, and HE staining results showed that IGFBP7-Exo-cGAMP inhibited tumor growth and prolonged mouse survival compared to other control groups, without causing significant damage to normal tissues. However, IGFBP7-Exo-cGAMP-PEP3 showed better therapeutic effects. To verify whether different treatments induced tumor-specific immune responses, brain and spleen tissues were collected from each group of mice, and immune cells were isolated. CD8+ T cells were isolated from tumor-infiltrating immune cells in each group of mice. PEP3 peptides and dendritic cells were pre-conjugated, and the cells were co-cultured with tumor-infiltrating CD8+ T cells for 24 hours. The level of glioma antigen-specific immune response was detected by measuring the IFN-γ secretion level in the supernatant. The results showed that IGFBP7-Exo-cGAMP-PEP3 could better induce the generation of tumor antigen-specific T cells. These results indicate that delivery of STING agonists to glioma vascular endothelial cells via exosomes can induce a certain tumor antigen-specific immune response; however, simultaneous delivery of tumor-associated antigens can produce a more significant tumor-specific immune response.

[0051] Experimental results: Transcriptomic analysis of endothelial cells revealed a significant increase in the expression of numerous inflammatory and chemokines, as well as a significant increase in the expression of cell adhesion molecules such as ICAM1, after STING activation. This suggests that STING activation in glioma vascular endothelial cells may activate other immune cells within the tumor by secreting a large number of inflammatory factors. Simultaneously, STING-activated glioma vascular endothelial cells may also recruit peripheral immune cells by secreting chemokines and mediate their infiltration into the tumor via highly expressed adhesion molecules, thereby reshaping the tumor microenvironment and providing an ideal environment for inducing adaptive immunity. It is well known that the presence of a large number of highly immunogenic antigens is also crucial for efficiently inducing antigen-specific immune responses. Therefore, the synthetic peptide PEP-3, containing a tumor-specific mutant fragment of epidermal growth factor receptor type III (EGFRvIII), was fused with the hepatitis B virus core (HBVc) protein to enhance immunogenicity. HBVc-PEP3, along with the STING agonist, was loaded into modified exosomes and delivered to glioma vascular endothelial cells via intravenous injection. We validated the therapeutic effect of STING agonist-loaded exosomes on glioma using an orthotopic glioma model. Treatment with empty exosomes or exosomes carrying only HBVc-PEP3 had little inhibitory effect on glioma growth, indicating that tumor microenvironment remodeling is crucial for immunotherapy. Both free STING agonists and exosomes carrying STING agonists exhibited more significant tumor inhibition, with exosomes carrying STING agonists showing a more pronounced inhibitory effect. This may be because delivery of STING agonists via modified exosomes increases the concentration of STING agonists in the tumor and also enhances the stability of STING agonists in vivo. Exosomes carrying both STING agonists and tumor antigens further enhanced the inhibitory effect on gliomas. Figure 8 A), and significantly prolonged the survival of tumor-bearing mice (Figure 8B). Pathological analysis of the tumor tissue further confirmed these results, showing extensive tissue damage and massive apoptosis in the tumor tissue of mice treated with exosomes carrying STING agonist and tumor antigen (Figure 8C). This may be due to the strong immunogenicity of hepatitis B virus core antigen (HBVc) enhancing innate immunity, or due to the secretion of large amounts of tumor antigen by exosome-transduced endothelial cells, thereby enhancing the tumor-specific immune response. By co-culturing tumor-infiltrating CD8+ T cells with dendritic cells pre-incubated with PEP-3 peptide, the results showed that injection of exosomes carrying STING agonist enhanced the tumor antigen-specific immune response, and the simultaneous carrying of tumor antigen HBVc-PEP3 in the exosomes further enhanced the tumor antigen-specific immune response (Figure 8C). Figure 8 D).

Claims

1. A delivery vector targeting tumor vascular endothelial cells, characterized in that, The delivery vector was obtained by inserting a polypeptide sequence between the 25th and 26th amino acids of the exosome membrane protein Lamp2b; The polypeptide sequence can bind to CD93.

2. The delivery carrier as claimed in claim 1, characterized in that, The polypeptide sequence is either MMRN2 protein or IGFBP7 protein; The amino acid sequence of the MMRN2 protein is shown in SEQ ID NO.1; The amino acid sequence of the IGFBP7 protein is shown in SEQ ID NO.

2.

3. The use of the delivery carrier as described in claim 1 in the preparation of a medicament for treating tumor diseases.

4. The application as described in claim 3, characterized in that, The tumor is a glioma.

5. A drug delivery system targeting tumor vascular endothelial cells, characterized in that, It includes the delivery carrier as described in claim 1, and the tumor therapeutic drug encapsulated within the delivery carrier.

6. The drug delivery system targeting tumor vascular endothelial cells as described in claim 5, characterized in that, The tumor treatment drugs are chemotherapy drugs or immunomodulatory drugs.

7. The drug delivery system targeting tumor vascular endothelial cells as described in claim 6, characterized in that, The chemotherapy drug is temozolomide, and the immunomodulatory drug is a STING agonist.

8. The use of the drug delivery system as described in claim 5 in the preparation of a medicament for treating tumor diseases.

9. The application as described in claim 8, characterized in that, The tumor is a glioma.

10. A pharmaceutical composition, characterized in that, It includes the drug delivery system as described in claim 5 and pharmaceutically acceptable excipients.

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