A composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs

Abstract

The present invention relates to the technical fields of biomedicine and oncology, and specifically relates to a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs, and a preparation method and application thereof. The membrane surface of the composite exosome carries TRAIL protein, and the small molecule anti-tumor drug is encapsulated inside the membrane. The preparation process of the composite exosome of the present invention is mature, efficient, has good reproducibility, and low cost; the anti-tumor effect of the composite exosome in vitro and in vivo is remarkable, the drug dosage is low and there are no toxic side effects, and the biological safety is good, providing a new strategy for tumor clinical treatment.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and oncology. Specifically, it is a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs, and its preparation method and application. Background Art

[0002] Cutaneous malignant melanoma is a malignant tumor produced by melanocytes in the skin and other organs. It accounts for about 3% of all tumors, and has strong invasiveness and metastasis; it has a high incidence rate and poor prognosis. The mortality rate of advanced melanoma is as high as 70-90%, ranking first among cutaneous malignant tumors. According to the statistical data of the American Cancer Society in 2021, in 2020, there were 106,110 newly diagnosed cases of cutaneous in situ melanoma globally and 7,180 deaths, and it is increasing at a rate of 3-5% per year, becoming one of the fastest-growing malignant tumors globally. The five-year survival rate of advanced melanoma is only 25%. Currently, the main treatment methods for malignant melanoma include chemotherapy, radiotherapy, surgical resection, immunotherapy, and targeted therapy, etc. Chemotherapy is still the main method for treating melanoma currently. The main chemotherapy drugs include dacarbazine, temozolomide, carboplatin, paclitaxel, fotemustine, and cisplatin, etc. However, the single-drug or combined-drug effective rate in melanoma is not high, about 10-15%, and it damages the normal immune function of the human body and produces serious toxic and side effects. Therefore, there is an urgent need for new, effective, and safe treatment methods for melanoma.

[0003] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) belongs to the members of the tumor necrosis factor superfamily and has an apoptosis-promoting effect on a variety of human tumor cells, including melanoma, lung cancer, glioma, breast cancer, pancreatic cancer, prostate cancer, colon cancer, kidney cancer, and cholangiocarcinoma.

[0004] Death receptor 5 (DR5) is the main receptor for TRAIL, which is highly expressed on the surface of various tumor cells, but lowly expressed or not expressed in normal cells. DR5 contains a death domain (DD). After binding to TRAIL, it binds to Fas-associated death domain protein (FADD) through DD, recruits pro-caspase 8, and generates a death-inducing signaling complex (DISC). Pro-caspase 8 in DISC self-cleaves into active caspase 8, activates caspase 3 through the caspase cascade reaction and the mitochondrial-dependent pathway, thereby mediating apoptosis.

[0005] Currently, TRAIL-based therapies include recombinant human soluble TRAIL (rhTRAIL), which has been used in preclinical studies and clinical trials of various human tumors. However, rhTRAIL has poor tumor targeting, a short half-life in vivo, and is prone to cause cell drug resistance, resulting in poor efficacy. Therefore, in view of the deficiencies of rhTRAIL, it is necessary to develop an effective and safe TRAIL delivery form to avoid TRAIL resistance and improve efficacy.

[0006] As natural immune cells and antigen-presenting cells, macrophages play an important role in regulating the tumor immune microenvironment. M1 macrophages, such as Raw264.7, can specifically bind to tumor tissues and are therefore widely used in tumor-targeted therapy. The tumor-targeting ability of M1 macrophages comes from their surface membrane proteins. At the same time, the exosomes they produce have surface membrane characteristics similar to those of macrophages and retain the cell's targeting ability and tumor penetration ability. In addition, exosomes can serve as a natural endogenous nanocarrier and also have other unique advantages, such as high stability, low toxicity, low immunogenicity, and good biocompatibility; strong tumor permeability and can cross the blood-brain barrier; can deliver a variety of therapeutic drugs, such as membrane proteins, miRNAs, siRNAs, and small molecule chemical drugs. It has been found that membrane-bound TRAIL can increase its stability, improve bioavailability and targeting, and exhibit stronger pro-apoptotic activity than rhTRAIL, which may be due to the formation of oligomers in the supramolecular structure of membrane-bound TRAIL, promoting receptor clustering of DR5, enhancing the apoptosis induction efficiency and signal transduction.

[0007] Triptolide (TPL) has antioxidant, anti-inflammatory, anti-fertility, anti-rheumatoid, neuroprotective, immunosuppressive and multi-target anti-tumor properties. In addition, studies have shown that TPL is more effective than other conventional chemotherapeutic drugs. Even in highly drug-resistant cells, TPL can show strong anti-tumor activity at nanomolar concentrations. TPL has a certain anti-therapeutic effect on malignant melanoma both in vivo and in vitro. Oral hydroxy tripdiolide tablets and soluble injectable TPL derivative Minnelide have also entered the clinical trial stage.

[0008] However, there is no report on a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs. Summary of the Invention

[0009] The object of the present invention is to solve the above-mentioned deficiencies of existing TRAIL and TPL in the treatment of malignant melanoma, and to provide a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs, its preparation method and application. The composite exosome is a drug-loaded exosome with targeting and pro-apoptotic abilities. The small molecule anti-tumor drug TPL encapsulated therein can synergistically enhance the apoptosis effect of TRAIL on malignant melanoma cells without obvious toxicity to normal tissues and organs, providing a reliable theoretical basis and experimental foundation for the clinical treatment of malignant melanoma.

[0010] Exosomes modified with TRAIL can simultaneously deliver the small molecule anti-tumor drug TPL, which can not only overcome the deficiencies of TRAIL and TPL such as drug resistance, in vivo instability and low bioavailability, but also produce a synergistic anti-tumor effect through the combination of the two, enhance the targeting and promote the tumor apoptosis effect, and reduce the toxic side effects of TPL. Therefore, it has great potential for clinical application.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] In the first aspect of the present invention, there is provided a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs, wherein the membrane surface of the exosome carries the tumor necrosis factor-related apoptosis-inducing ligand, and the small molecule anti-tumor drug is encapsulated inside the membrane.

[0013] Further, the composite exosome is prepared by first transfecting a virus expression vector overexpressing the membrane-bound TRAIL gene into the donor cells of exosomes to obtain a stable cell line overexpressing TRAIL; then isolating and purifying exosomes from the culture supernatant of the donor cells overexpressing TRAIL to obtain high-purity exosomes expressing and carrying TRAIL (TRAIL-Exo), and then loading the small molecule anti-tumor drug into the exosome membrane.

[0014] Furthermore, the concentration of TRAIL protein in the composite exosomes is 205.1 ± 13.6 pg / mL; the average particle size of the composite exosomes is 100 - 200 nm; and the concentration of the small molecule anti-tumor drug is 0.01 - 60 μg / mL.

[0015] Furthermore, the viral expression vector is a lentivirus, a retrovirus or an adenovirus.

[0016] Furthermore, the method for preparing a viral expression vector overexpressing the membrane-bound TRAIL gene transfected into exosome donor cells includes: infecting exosome donor cells with a lentivirus carrying the TRAIL gene to obtain a cell line stably overexpressing TRAIL protein. In a preferred embodiment of the present invention, it includes: constructing a lentiviral packaging three-plasmid system, the lentiviral packaging three-plasmid system consists of pSPAX2, pMD2G and a shuttle plasmid carrying the TRAIL gene. Transiently transfecting 293T cells with the lentiviral packaging three-plasmid system to obtain lentiviral particles containing the TRAIL gene, and then infecting exosome donor cells with the lentiviral particles.

[0017] Furthermore, the donor cells are selected from one of Raw264.7 cells, natural killer cells, T cells and dendritic cells. In a preferred embodiment of the present invention, the donor cells are Raw264.7 cells.

[0018] Furthermore, the small molecule anti-tumor drug is a natural drug.

[0019] Furthermore, the natural drug is triptolide TPL.

[0020] In the second aspect of the present invention, there is provided a method for preparing a composite exosome loaded with a membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and a small molecule anti-tumor drug, including the following steps:

[0021] S1. Construct a viral expression vector overexpressing the membrane-bound TRAIL gene, transiently transfect 293T cells through virus packaging to obtain virus particles, and then infect macrophages Raw264.7 with the purified virus particles to obtain a macrophage cell line stably overexpressing TRAIL, labeled as TRAIL-Raw264.7;

[0022] S2. Adopt gradient ultracentrifugation to isolate and purify exosomes from the culture supernatant of TRAIL-Raw264.7 cells to obtain exosomes with high purity expressing and carrying TRAIL, labeled as TRAIL-Exo;

[0023] S3. Load the small molecule anti-tumor drug TPL into TRAIL-Exo to obtain a composite exosome (TRAIL-Exo / TPL) loaded with TRAIL and the small molecule anti-tumor drug; the membrane surface of the composite exosome carries the pro-apoptotic protein TRAIL, and the small molecule anti-tumor drug TPL is encapsulated inside the membrane.

[0024] Furthermore, the method for loading the small molecule anti-tumor drug TPL into TRAIL-Exo in step S3 is the ultrasonic method, the electroporation method or the co-incubation method.

[0025] In some more preferred embodiments, the preparation method includes the following steps:

[0026] S1. Construct a lentiviral packaging three-plasmid system, infect 293T cells with the lentiviral packaging three-plasmid system to obtain lentiviral particles carrying the target gene.

[0027] S2. Infect Raw264.7 cells with the lentiviral particles containing the target gene to obtain a macrophage cell line stably overexpressing TRAIL, labeled as TRAIL-Raw264.7, so that Raw264.7 cells secrete and express exosomes carrying TRAIL. Further, the lentiviral packaging three-plasmid system consists of pSPAX2, pMD2G and a shuttle plasmid carrying the target gene. Among them, the lentiviral three-plasmid system infects 293T cells and can package and produce more lentiviruses carrying the TRAIL gene. Use the packaged lentiviral particles to infect the macrophage Raw264.7, so that the macrophage overexpresses the TRAIL protein on the membrane, thereby enabling it to secrete and express exosomes carrying TRAIL.

[0028] S3. Use gradient ultracentrifugation to extract the exosomes secreted by cells from the culture supernatant of TRAIL-Raw264.7 cells to obtain high-purity exosomes expressing and carrying TRAIL, labeled as TRAIL-Exo;

[0029] S4. Load the small molecule anti-tumor drug TPL into TRAIL-Exo to obtain a composite exosome (TRAIL-Exo / TPL) carrying TRAIL and the small molecule anti-tumor drug TPL; the membrane surface of the composite exosome expresses and carries the pro-apoptotic protein TRAIL, and the small molecule anti-tumor drug TPL is encapsulated inside the membrane.

[0030] In the third aspect of the present invention, there is provided an application of the composite exosome loaded with the membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and the small molecule anti-tumor drug as described above in the preparation of a therapeutic drug for malignant tumors.

[0031] Furthermore, the malignant tumor is cutaneous malignant melanoma.

[0032] Compared with the prior art, the advantages of the present invention are as follows:

[0033] 1. Compared with exosomes loaded only with TRAIL or the simple small molecule anti-tumor drug TPL, the present invention uses exosomes to simultaneously deliver TRAIL and the small molecule anti-tumor drug, enhancing the tumor targeting of exosomes and the apoptosis-promoting ability of TRAIL, improving the tumor-killing activity of TPL, reducing the toxic and side effects on normal tissues, and significantly improving the anti-tumor treatment effect.

[0034] 2. The composite exosomes loaded with membrane-bound TARIL and the small molecule anti-tumor drug TPL in the present invention can achieve the purpose of synergistic enhancement, and the combination of the two significantly enhances the killing effect on tumor cells. The in vivo experimental results show that the composite exosomes can effectively inhibit the growth of subcutaneous malignant melanoma and have good biosafety at the same time.

[0035] 3. In the present invention, TRAIL and TPL can synergistically induce apoptosis by regulating the extrinsic apoptosis pathway and the intrinsic apoptosis pathway. The in vitro experimental results show that the composite exosomes can significantly inhibit the proliferation of tumor cells, induce apoptosis of tumor cells, and inhibit the invasion and migration of tumor cells. The in vivo experimental results show that after tail vein injection of the composite exosomes, the growth of subcutaneous xenograft tumors in nude mice is significantly inhibited, obvious pathological damage and apoptosis occur in the tumor tissue, and no toxicity to important organs is observed.

[0036] 4. The present invention expounds the mechanism of action of the composite exosomes in synergistic anti-tumor, providing a new treatment strategy for related diseases.

[0037] 5. The preparation process of the present invention is mature, efficient, has good reproducibility, and low cost; the in vitro and in vivo anti-tumor effects of the composite exosomes are significant, the drug dosage is low and there are no toxic and side effects, and the biosafety is good, providing a new strategy for the clinical treatment of melanoma. Description of the Drawings

[0038] Figure 1 The map of the lentiviral expression vector containing the TRAIL gene constructed for Example 1;

[0039] Figure 2 The fluorescence imaging diagram (a), RT-qPCR relative expression quantification diagram (b), Western blot diagram (c), TRAIL flow cytometry detection diagram (d), and TRAIL expression concentration (e) of Raw264.7 cells overexpressing TRAIL established in Example 4 in each figure;

[0040] Figure 3The figures respectively show the transmission electron microscopy image (a), particle size distribution diagram (b), Western blot image (c), immuno-electron microscopy image (d), flow cytometry image (e) and TRAIL expression concentration (f) of TRAIL-Exo obtained in Example 5;

[0041] Figure 4 The figures respectively show the laser confocal imaging of TRAIL-Exo cell uptake (a), average fluorescence intensity of laser confocal cells (b), flow cytometry uptake map (c) and average fluorescence intensity of flow cytometry cells (d) obtained in Example 5;

[0042] Figure 5 It is the BCA protein standard curve graph in Example 7;

[0043] Figure 6 It is the TPL standard curve graph in Example 7;

[0044] Figure 7 The figures respectively show the transmission electron microscopy image (a), particle size distribution diagram (b) and TRAIL flow cytometry detection graph (c) of TRAIL-Exo / TPL prepared in Example 7;

[0045] Figure 8 It is the cell viability detection of human malignant melanoma A375 cells after being treated with different preparations for 24 h;

[0046] Figure 9 In it, (a) is the apoptosis flow cytometry image of A375 cells after being treated with different preparations; (b) is the apoptosis rate of A375 cells after being treated with different preparations;

[0047] Figure 10 In it, (a) is the Transwell invasion image of A375 cells after being treated with different preparations; (b) is the relative invasion rate of A375 cells after being treated with different preparations;

[0048] Figure 11 In it, (a) is the migration image of A375 cells after being treated with different preparations; (b) is the migration rate of A375 cells after being treated with different preparations;

[0049] Figure 12 It is the in vivo anti-melanoma effect diagram of TRAIL-Exo, TPL and TRAIL-Exo / TPL in Example 12, including the solid tumor image (a), tumor growth curve graph (b), tumor mass graph (c), tumor inhibition rate (d) and body weight change graph (e) of nude mice after tail vein injection of different preparations;

[0050] Figure 13 It is the H&E, TUNEL immunofluorescence and Ki67 immunohistochemistry images of the tumor tissues of nude mice after tail vein injection of different preparations;

[0051] Figure 14 H&E staining images of the heart, liver, spleen, lung, and kidney of nude mice after tail vein injection of different preparations;

[0052] Figure 15 Each figure in shows the Western blot of proteins related to the apoptosis pathway regulated by TRAIL-Exo / TPL (a) and the schematic diagram of its molecular mechanism for promoting tumor cell apoptosis (b). Specific implementation manners

[0053] The following describes in detail the specific implementation manners provided by the present invention in conjunction with the embodiments. However, the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0054] Example 1: Construction of a lentiviral vector overexpressing TRAIL

[0055] The nucleotide sequence of TRAIL (Homo sapiens TNF superfamily member 10, TNFSF10) (NM_003810) was synthesized onto the pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO lentiviral expression vector. The specific process is as follows: Select the vector digestion system → Digest the vector at 37°C, recover by gel electrophoresis → Recover the fragment by PCR → Place the ligation reaction system of the nucleotide sequence of TRAIL and the lentiviral expression vector in a water bath at 50°C for 20 min → Transformation (competent cell DH5α; resistance: ampicillin, 37°C, 230 rpm, 24 h) → Pick colonies from the transformed plate, shake the bacteria at 37°C and 250 rpm for 14 h → Identify the bacterial solution by PCR, and sequence the positive clone bacterial solution for overexpression.

[0056] Figure 1 is the map of the TRAIL lentiviral expression vector constructed in Example 1.

[0057] Example 2: Lentivirus packaging

[0058] Extract the constructed lentiviral expression vector and helper plasmid in large quantities using a plasmid DNA extraction kit. The plasmid concentration should be greater than 1 μg / μL, and an A260 / 280 between 1.7 and 1.8 can be used for virus packaging. Digest, centrifuge, and resuspend 293T cells, and passage them at a ratio of 1:10 into culture dishes, then continue to culture them in an incubator. When the density of 293T cells reaches 70 - 80%, transfection can be carried out. Replace the culture medium with serum-free medium and prepare the lipid transfection complex: 10 μg pSPAX2, 5 μg pMD2G, 10 μg of the shuttle plasmid containing the target gene, and 75 μL Lipofiter TM reagent. After 6 hours of transfection, replace it with complete culture medium containing 10% FBS. Collect the virus supernatant twice at 48 hours and 72 hours after transfection. When collecting the virus at 48 hours, pour the culture medium in the culture dish into a centrifuge tube, then supplement it with complete culture medium and continue to culture it in a constant temperature incubator. When collecting the virus at 72 hours, directly pour the culture medium in the culture dish into a centrifuge tube. Centrifuge the supernatant containing the virus at 1000 - 2000 × g for 5 - 10 minutes; then collect the virus stock supernatant and place it in an ultracentrifuge tube, and centrifuge it at 4°C, 80000 - 100000 × g for 60 - 120 minutes. Finally, aliquot the lentivirus ultracentrifugation solution into virus tubes and store it in a -80°C refrigerator.

[0059] Example 3: Virus titer detection

[0060] Digest and count 293T cells and dilute them to 1 - 3×10 5 / mL, add them to a 96-well plate, 100 μL per well, and prepare 6 wells for each virus. The next day, prepare 6 1.5 mL EP tubes. Add 10 μL of the virus solution to the first EP tube, and then perform 3-fold serial dilutions for a total of 6 dilution factors. On the third day, for the wells that require puromycin screening, first aspirate 100 μL of the culture medium containing lentiviral particles, and then add 100 μL of complete culture medium containing puromycin. On the fifth day, observe under a fluorescence microscope. 6 hours before observation, replace the fresh complete culture medium. Aspirate 80 μL of the culture medium from the wells, then add 80 μL of fresh complete culture medium and place it in an incubator for culture. Observe the results under a fluorescence microscope 6 hours later, and select the wells with a fluorescence percentage between 10 - 50% to calculate the virus titer. After calculation, the virus titer in Example 3 is 1.5×10 8 TU / mL.

[0061] Example 4: Establishment and identification of a macrophage cell line stably overexpressing TRAIL

[0062] 1. After the Raw264.7 cells are confluent, digest, centrifuge, and resuspend them, and adjust the cell density to 1 - 5×10 5Inoculate at a density of [[X]] cells / mL into a 6-well plate and place it in an incubator. When the cell density reaches approximately 60%, start the infection. Add 1 - 5 mL (optimally 2 mL) of the virus stock solution to each well, and simultaneously add 2 - 20 μg / mL of polybrene (optimally 5 μg / mL). After 24 h of infection, aspirate the virus stock solution and add complete medium. After 48 h of virus infection, when the cell fusion rate reaches 90%, passage the cells.

[0063] 2. Inoculate Raw264.7 cells into a 24-well plate. When the fusion rate reaches approximately 60%, replace the complete medium with a complete medium containing 0.2 - 10 μg / mL (optimally 2 μg / mL) of puromycin and treat the cells for 24 - 48 h. When the cell density reaches 70 - 80%, passage the cells and continue to culture them in the complete medium containing puromycin. After 4 days of infection, replace the medium with fresh complete medium and continue to culture for 2 more days, then perform cell passage culture to obtain the Raw264.7 macrophage cell line overexpressing TRAIL (TRAIL-Raw264.7).

[0064] 3. Total RNA extraction: After the TRAIL-Raw264.7 cells have grown to confluence in a 6-well plate, aspirate the culture medium and add an appropriate amount of Trizol reagent to each well to extract the total cellular RNA. Transfer the lysate to a centrifuge tube and let it stand at room temperature for 10 - 15 min. Add 200 μL of chloroform, mix well by vortexing, and let it stand at room temperature for 10 - 15 min. Centrifuge at 13780×g for 10 min, transfer the upper clear liquid to a centrifuge tube, add an equal volume of isopropanol, and precipitate at room temperature for 10 min. Centrifuge at 13780×g for 15 min and discard the supernatant. Wash the precipitate once with 75% ethanol. Centrifuge at 13780×g for 5 min, discard the supernatant and recover the precipitate. Air-dry the precipitate at room temperature in an inverted position for 10 min. Dissolve the precipitate with DEPC-H2O, mix well, and store at -80 °C for later use. Measure the absorbance at wavelengths of 260 nm and 280 nm using a UV-visible spectrophotometer to calculate the concentration of total cellular RNA.

[0065] 4. Reverse transcription: Use the ReverTra qPCR RT Kit reverse transcription reaction kit to reverse transcribe the RNA. The reverse transcription reaction conditions are 37 °C for 20 min and 95 °C for 5 min. The reverse transcription reaction system is as follows:

[0066]

[0067] 5. Real-time qPCR (RT-qPCR): Use the Hanbio miRNA qPCR Detection Primer kit to detect miRNA. Use a LightCycler 96 real-time fluorescence quantitative PCR instrument to detect the TRAIL gene and calculate the expression level. The primer sequences are as follows:

[0068]

[0069]

[0070] The RT-qPCR reaction system is as follows:

[0071]

[0072] The RT-qPCR reaction conditions are as follows:

[0073]

[0074] 4. Western blot detection: Seed the overexpressing TRAIL-Raw264.7 cells in a 6-well plate. After the cells adhere to the wall, discard the supernatant, wash the cells 3 times with PBS, add an appropriate amount of PMSF, and then add RIPA lysis buffer. Scrape the cells with a cell scraper and collect them into a centrifuge tube. Centrifuge at 12,000 rpm for 10 min, collect the supernatant to obtain the total protein solution. Determine the total protein concentration using a BCA protein assay kit. Add 2× loading buffer to the protein sample at a volume ratio of 1:1 and boil for 15 min for denaturation. Prepare a 12% separating gel and a 5% stacking gel, add 20 μL of the protein sample to the loading wells, and perform electrophoresis for 30 min. Cover the methanol-activated PVDF membrane on the gel and transfer the proteins at a constant current for 30 min. Block the membrane on a shaker with 5% non-fat milk for 1 h, add anti-TRAIL primary antibody (1:1000, Abcam) and incubate overnight at 4°C. Add HRP goat anti-rabbit IgG secondary antibody (1:3000, Abcam) and incubate at room temperature for 1 h. Add the membrane to the ECL reagent and react for 2 min, then expose it. Develop and image the exposed film.

[0075] 5. Flow cytometry detection: Digest and centrifuge the TRAIL-Raw264.7 cells in the 6-well plate, resuspend them with PBS, and adjust the cell density to 1×10 7 / mL. Take 100 μL of the cell suspension, add 5 μL of PE anti-human TRAIL Antibody staining solution, and mix well. Add 20 μL of PE Mouse IgG1 isotype control antibody staining solution to the control tube. Stain the cells in the dark for 15 min. Add 2 mL of PBS to each tube to wash the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend with 400 μL of PBS, transfer to a 5 mL flow cytometry tube, and detect the positive rate of TRAIL using a flow cytometer.

[0076] 6. ELISA assay: Prepare TRAIL protein standard solutions with concentrations of 0, 15.9, 31.2, 62.50, 125.00, 250.00, 500.00, and 1000.00 pg / mL in sequence. Digest, centrifuge, and resuspend TRAIL-Raw264.7 cells, freeze-thaw them three times repeatedly, centrifuge at 10,000×g for 5 min, and collect the cell supernatant. Take 100 μL each of the above TRAIL protein standard solutions and cell supernatant and add them to a 96-well enzyme-linked immunosorbent assay (ELISA) plate pre-coated with anti-human TRAIL, and incubate at room temperature for 120 min (or incubate at 37 °C for 90 min). Discard the supernatant, add 100 μL of 1× Biotinylated anti-human TRAIL antibody to each well, and incubate at room temperature for 90 min (or incubate at 37 °C for 60 min). Wash the plate three times with 1× Wash Buffer, add 100 μL of 1× Avidin-Biotin-Peroxidase Complex to each well, and incubate at room temperature for 40 min (or incubate at 37 °C for 30 min). Wash the plate five times with 1× Wash Buffer, add 90 μL of Color Developing Reagent to each well, and incubate in the dark at room temperature for 30 min (or incubate at 37 °C for 15 - 25 min). Add 100 μL of termination solution to each well, react for 30 min, measure the absorbance value of each well at a wavelength of 450 nm, and calculate the expression level of TRAIL in TRAIL-Raw264.7 cells.

[0077] Figure 2 The figures respectively show the fluorescence microscope image (a), relative quantitative RT-qPCR expression of TRAIL (b), Western blot of TRAIL (c), flow cytometry of TRAIL (d), and TRAIL expression concentration (e) of Raw264.7 cells overexpressing TRAIL established in Example 4. As can be seen from the figures, the Raw264.7 cells after transfection with the overexpressing TRAIL lentiviral expression vector and puromycin screening have good growth status ( Figure 2 (a)). The TRAIL gene has an overexpression effect in the transfected Raw264.7 cells ( Figure 2 (b)). TRAIL is expressed in the transfected Raw264.7 cells ( Figure 2 (c)). The positive rate of TRAIL on the surface of the transfected Raw264.7 cells is relatively high ( Figure 2 (d)), and at the same time, there is a relatively high TRAIL expression concentration, which is 125.3 ± 10.5 pg / 1 μg protein ( Figure 2(e)) further demonstrated the expression of TRAIL in transfected Raw264.7 cells, indicating the successful construction of the overexpressing TRAIL-Raw264.7 stable cell line.

[0078] Example 5: Extraction and Characterization of TRAIL-Exo

[0079] 1. Exosome extraction: After the TRAIL-Raw264.7 cells reached 80% confluence, the culture medium was discarded, and the cells were washed 3 times with PBS. Then, complete medium containing 10% exosome-free serum was added and the cells were cultured for another 24 - 48 h. The supernatant was collected. First, the supernatant was centrifuged at 300×g for 10 min to remove live cells; then centrifuged at 2000×g for 10 min to remove dead cells; and then centrifuged at 10 000×g for 30 min to remove cell debris. The supernatant was centrifuged at 120 000×g for 70 min to obtain the precipitate of miscellaneous proteins and exosomes. The precipitate was resuspended in PBS and centrifuged again at 120 000×g for 70 min to remove miscellaneous proteins. Finally, the exosome precipitate was resuspended in PBS and stored in a -80°C refrigerator. All centrifugation procedures were carried out at 4°C.

[0080] 2. Transmission electron microscopy: The exosomes were resuspended with 2% paraformaldehyde. 5 - 10 μL of the exosome suspension was dropped onto a Formvar-carbon copper grid and left at room temperature for 10 min; washed 2 times with PBS, 3 min each time. The copper grid was placed on a 50 μL droplet of 0.3% uranyl acetate solution for negative staining for 5 min; placed on a 100 μL droplet of PBS for washing for 1 min, and washed 3 times. Then placed on a 2% methylcellulose droplet for 10 min. After natural drying, the morphology of the exosomes was observed and photographed with a transmission electron microscope at 80 kV voltage.

[0081] 3. Immunoelectron microscopy: Resuspend exosomes with 2% paraformaldehyde. Take 5 - 10 μL of the exosome suspension and drop it onto a Formvar-carbon copper grid, then place it at room temperature for 10 min. Wash with PBS twice, 3 min each time. Transfer the grid to 50 mM glycine and incubate for 3 min. Block the grid with 1% BSA blocking buffer for 10 min. Drop 5 μL of the diluted TRAIL primary antibody (1:100) onto the grid and incubate for 30 min. Transfer the grid to PBS and wash 5 times, 3 min each time. Incubate with the diluted 5 nm colloidal gold-conjugated goat anti-mouse IgG (1:50) for 20 min. Wash the grid with PBS 8 times, 2 min each time. Place the grid in a 1% glutaraldehyde droplet and fix for 5 min. Place the grid on 100 μL of distilled water and wash 8 times, 2 min each time. Place the copper grid on 50 μL of a 0.3% uranyl acetate droplet for negative staining for 5 min; place the copper grid on 100 μL of a PBS droplet and wash for 1 min, wash 3 times. Then place it on a 2% methylcellulose droplet for 10 min. After natural drying, observe and photograph the morphology of exosomes with a transmission electron microscope at 80 kV voltage.

[0082] 4. Particle size determination: Take an appropriate amount of exosomes and dilute them with 1 mL of PBS. Place them in a cuvette and detect their particle size with a Malvern laser particle size analyzer at room temperature.

[0083] 5. Western blot detection: Add an appropriate amount of PMSF to the exosomes, then add an appropriate amount of RIPA lysis buffer and lyse thoroughly on ice. Centrifuge at 100,000×g for 1 h and take the supernatant. Prepare a 12% separating gel and a 5% stacking gel and wait for the gel to solidify. Place the gel plate in the electrophoresis tank and add the electrophoresis buffer. Take the exosome suspension, mix it with an equal volume of 2× loading buffer, and boil for 5 min for denaturation. Add 20 μg of the loading solution to each well and set a constant voltage of 70 V. When the indicator bromophenol blue enters the separating gel, change to a constant voltage of 90 V for electrophoresis. When the indicator reaches about 0.5 cm from the bottom of the gel, turn off the power supply and take out the gel plate. Activate the PVDF membrane with methanol in advance, wash the membrane with ddH2O, and soak the gel in the transfer buffer for 15 min for equilibration. Prepare the transfer "sandwich" in the order of black side (negative electrode) → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → red side (positive electrode). Transfer at a constant current of 200 mA for 70 min. After the transfer is completed, take out the PVDF membrane, block the PVDF membrane with 5% BSA at room temperature for 2 h, and wash the membrane on a shaker with TBST buffer for 5 min × 3 times. Add the diluted Anti-CD9 primary antibody (Abcam, 1:2000), Anti-CD63 primary antibody (Abcam, 1:1000), Anti-TSG101 primary antibody (Abcam, 1:1000), and Anti-TRAIL primary antibody (Abcam, 1:1000) respectively, and incubate overnight at 4°C. Then place it on a shaker and wash the membrane with TBST buffer for 5 min × 3 times. Add the HRP Goat anti-Rabbit secondary antibody (Abcam, 1:2000) and incubate at room temperature for 2 h. Then place it on a shaker and wash the membrane with TBST buffer for 10 min × 3 times. React the membrane with the ECL chemiluminescence kit for 2 min, expose the PVDF membrane, and image it with the ChemiDoc imaging system.

[0084] 6. Flow cytometry detection: Take 100 μL of the TRAIL-Exo solution, add 5 μL of the PE anti-human TRAIL antibody staining solution, and add 20 μL of the PE mouse IgG1 isotype control antibody staining solution to the control tube. After incubating in the dark at 4°C for 15 min, add 2 mL of PBS and wash 2 times, centrifuge at 120,000×g for 70 min, discard the supernatant, resuspend with 400 μL of PBS, transfer to a 5 mL flow cytometry tube, and detect with a flow cytometer.

[0085] 7. TRAIL concentration determination: Determine the expression level of TRAIL in TRAIL-Exo using an ELISA kit according to the method in Example 4.

[0086] Figure 3The figures respectively show the transmission electron microscopy image (a), particle size distribution map (b), protein immunoblotting image (c), immunoelectron microscopy image (d), flow cytometry image (e) and TRAIL expression concentration (f) of TRAIL-Exo obtained in Example 5. The extracellular vesicles separated by gradient ultracentrifugation showed a typical saucer-like or spherical structure ( Figure 3 (a)). The average particle size was 100 nm and the particle size distribution was uniform ( Figure 3 (b)). The expression of exosome-specific proteins CD9, CD63, TSG101 and TRAIL could be significantly detected ( Figure 3 (c)), and these results were all within the scope of exosome characteristics. The immunoelectron microscopy image more intuitively showed that gold nanoparticles could attach to the outer surface of the exosome membrane and the morphology of the exosome did not change significantly ( Figure 3 (d)). The positive binding rate of TRAIL-Exo and TRAIL antibody exceeded 95% ( Figure 3 (e)), and at the same time, TRAIL-Exo also had a high TRAIL expression concentration of 205.1±13.6 pg / 1 μg exosome protein ( Figure 3 (f)). These results indicated that the exosomes secreted by transfected TRAIL macrophages could express or carry TRAIL on the membrane surface, and TRAIL-Exo was successfully obtained.

[0087] Example 6: Cell uptake experiment

[0088] 1. PKH67 fluorescence labeling: Dilute the TRAIL-Exo solution with Diluent C. Take an appropriate amount of PKH67 staining solution and add it to the Diluent C dilution. Mix the diluted TRAIL-Exo and the staining solution at a volume ratio of 1:1 and place it in the dark for 1-5 min. Terminate the staining with 1% BSA solution, and use an ultrafiltration centrifugal tube (100 kDa) to separate the PKH67-labeled TRAIL-Exo.

[0089] 2. Laser confocal imaging: Seed A375 cells in a 35 mm confocal culture dish. After the cells adhered, discard the supernatant, and add PKH67-labeled TRAIL-Exo and exosomes from normal macrophages not transfected with TRAIL (Exo) respectively, and continue to culture for 6 h. Discard the supernatant, wash twice with PBS, fix with 4% paraformaldehyde for 10-15 min, stain with DAPI for 5-10 min, observe and take pictures with a laser confocal microscope, and calculate the average fluorescence intensity of cell uptake with Image J.

[0090] 3. Flow cytometry detection: Seed A375 cells in a 6-well plate. After the cells adhered to the wall, discard the supernatant, and add TRAIL-Exo labeled with PKH67 and Exo respectively. Continue culturing for 6 h. Discard the supernatant, wash 3 times with PBS, resuspend with PBS to 400 μL after digestion and centrifugation, transfer to a 5 mL flow cytometry tube, and detect with a flow cytometer.

[0091] Figure 4 The figures in [[]] are respectively the confocal laser scanning microscopy images (a), confocal average fluorescence intensity (b), flow cytometry uptake images (c), and flow cytometry average fluorescence intensity (d) of A375 cells' uptake of TRAIL-Exo and Exo labeled with PKH67 in Example 6. From Figure 4 which, it can be seen that TRAIL-Exo shows more extensive green fluorescence and stronger average fluorescence intensity on the membrane and in the cytoplasm of A375 cells, indicating that A375 cells have a higher uptake efficiency for TRAIL-Exo.

[0092] Example 7: Preparation and characterization of TRAIL-Exo / TPL

[0093] 1. Preparation: Prepare a 1 mg / mL stock solution of TPL with DMSO. Take 100 - 1000 μL of TRAIL-Exo with a protein concentration of 1 mg / mL, add 10 - 100 μL of TPL solution diluted with PBS, mix well, and ultrasonically treat the mixture with an ultrasonic cell disruptor: 10 - 40% amplitude (optimal is 20%), 20 - 50 s on / off (optimal is 30 s), for a total of 4 - 8 cycles (optimal is 6 cycles), with a 2 min cooling time between each cycle. After ultrasonic treatment, place the mixture at 37 °C for 0.5 - 1 h, and then at 4 °C for 1 - 2 h. Centrifuge at 120 000×g for 60 - 90 min to precipitate exosomes, resuspend with PBS, and obtain TRAIL-Exo / TPL, which is stored at -80 °C.

[0094] 2. Concentration determination: Prepare protein standard solutions with concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL respectively. Calculate the protein concentration of TRAIL-Exo according to the BCA standard curve ( Figure 5 ). Prepare TPL standard solutions with concentrations of 10, 50, 100, 250, 500, 1000, 2000, 5000, 10000, 25000, 50000, 100000 ng / mL, and determine the concentration of TPL in TRAIL-Exo / TPL with a high performance liquid chromatograph according to the TPL standard curve ( Figure 6 ).

[0095] 3. Characterization: The prepared TRAIL-Exo / TPL was characterized by transmission electron microscopy, particle size, and flow cytometry according to the steps in Example 4.

[0096] Figure 7 The figures in it are respectively the transmission electron micrograph (a), particle size distribution diagram (b), and TRAIL flow cytometry analysis diagram (c) of TRAIL-Exo / TPL prepared in Example 7. It can be seen from Figure 7 that TRAIL-Exo / TPL still maintains the complete structure and properties of exosomes ( Figure 7 (a)), the particle size has increased ( Figure 7 (b)), and there is still a relatively high TRAIL positive rate ( Figure 7 (c)). When the mass ratio of TRAIL-Exo to TPL is 10:1, the encapsulation efficiency is 51.87 ± 4.85%, and the drug loading is 4.93 ± 0.48%. The above results indicate that sonication can load TPL into TRAIL-Exo to achieve a relatively high drug loading.

[0097] Example 8: Cytotoxicity experiment

[0098] A375 cells were seeded in 96-well plates at a density of 1×10 4 / well and cultured for 24 h. The supernatant was discarded, and different concentrations of TRAIL-Exo, TPL, and TRAIL-Exo / TPL solutions were added. The protein concentrations in the TRAIL-Exo group were 5, 10, 20, 50, 100, and 200 μg / mL in sequence; the TPL concentrations in the TPL and TRAIL-Exo / TPL groups were 5, 10, 20, 50, 100, and 200 ng / mL in sequence, and the cells were further treated for 24 h. After the treatment, 10 μL of CCK-8 solution was added to each well, the plate was shaken for 30 s, and the reaction was carried out in an incubator for 2 h. The absorbance of each well at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0099] Figure 8 It shows the toxicity detection of TRAIL-Exo, TPL, and TRAIL-Exo / TPL to A375 cells in Example 8. It can be seen from Figure 8 that the cytotoxicity produced by TRAIL-Exo, TPL, and TRAIL-Exo / TPL all shows concentration dependence. Compared with TRAIL-Exo and TPL, TRAIL-Exo / TPL shows lower cell viability at the same TPL concentration, indicating its stronger tumor cell cytotoxicity.

[0100] Example 9: Apoptosis experiment

[0101] A375 cells were seeded at a density of 2 - 5×10 5Inoculate at a density of [X] cells / mL into a 12-well plate. After adherence, add TRAIL-Exo, TPL, and TRAIL-Exo / TPL (TPL concentration is 50 ng / mL) respectively, and continue culturing for 24 h. Digest the cells with 0.25% trypsin without EDTA, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend with pre-cooled PBS and centrifuge again. Aspirate the PBS, add 100 μL of 1×Binding Buffer to resuspend the cells, and adjust the cell concentration to 1×10 6 / mL. Sequentially add 5 μL of Annexin V-APC and 10 μL of 7-AAD, mix by oscillation, stain in the dark at room temperature for 15 min, add 385 μL of 1×Binding Buffer to mix well, transfer to a 5 mL flow tube, and detect with a flow cytometer.

[0102] Figure 9 This is for detecting the apoptosis effect of TRAIL-Exo, TPL, and TRAIL-Exo / TPL on A375 cells in Example 9. As Figure 9 can be seen, compared with the control group, the TRAIL-Exo, TPL, and TRAIL-Exo / TPL groups can all induce apoptosis of A375 cells, and TRAIL-Exo / TPL shows a stronger effect of inducing tumor cell apoptosis.

[0103] Example 10: Cell invasion experiment

[0104] Inoculate A375 cells into a 12-well plate at a density of 5×10 5 / mL. After adherence, treat with TRAIL-Exo, TPL, and TRAIL-Exo / TPL (TPL concentration is 50 ng / mL) respectively for 6 h. After digestion and centrifugation, resuspend with blank medium and adjust the cell density to 1×10 5 / mL. Add 50 μL of diluted Matrigel to the upper chamber of the Transwell, place at 37 °C for 1 h to solidify the gel. Add 200 μL of the single-cell suspension treated with TRAIL-Exo, TPL, and TRAIL-Exo / TPL respectively to the upper chamber, and add 600 μL of complete medium containing 10% FBS to each lower chamber, and continue culturing for 24 h. Take out the chamber, wipe off the non-transmigrated cells on the upper layer of the membrane with a cotton swab, fix with 4% paraformaldehyde for 15 min, wash the membrane 2 times with PBS, stain with 0.1% crystal violet for 15 min, wash with PBS, air dry, and then take pictures with a fluorescence microscope and count the number of transmigrated cells in 5 random fields of view. Calculate the relative invasion rate of the cells by taking the ratio of the number of transmigrated cells to that of the control group.

[0105] Figure 10 This is for detecting the inhibitory effect of TRAIL-Exo, TPL, and TRAIL-Exo / TPL on the invasion of A375 cells in Example 10. AsFigure 10 It can be seen that, compared with the control group, TRAIL-Exo, TPL, and TRAIL-Exo / TPL can all inhibit the invasion and chemotaxis of A375 cells, and the inhibitory effect of TRAIL-Exo / TPL is the most significant.

[0106] Example 11: Cell migration experiment

[0107] A375 cells were seeded in a 12-well plate at a density of 5×10 5 / mL. After adherence, the cells were treated with TRAIL-Exo, TPL, and TRAIL-Exo / TPL (TPL concentration was 50 ng / mL) for 6 h respectively. The cells were collected and seeded into the wells of a dedicated culture insert. After 24 h of adherence, the insert was removed, and the cells were continuously cultured in serum-free medium for 48 h. The scratch distance of the cells at different time points was measured and observed under a fluorescence microscope, and the migration rate of tumor cells was calculated.

[0108] Figure 11 For the detection of the inhibitory effect of TRAIL-Exo, TPL, and TRAIL-Exo / TPL on the migration of A375 cells in Example 11. It can be seen that Figure 11 compared with the control group, TRAIL-Exo, TPL, and TRAIL-Exo / TPL can all inhibit the migration of A375 cells, and the inhibitory effect of TRAIL-Exo / TPL on cell migration is the most significant.

[0109] Example 12: Evaluation of the anti-melanoma effect in vivo

[0110] 1. A375 nude mouse xenograft model: Male BALB / C nude mice, 4 - 6 weeks old, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Approximately 4×10 6 A375 cells were inoculated into the right axilla of the nude mice. When the tumor volume grew to approximately 100 mm 3 , they were randomly divided into a control group, a TRAIL-Exo group, a TPL group, and a TRAIL-Exo / TPL group (n = 6).

[0111] 2. In vivo administration: 0.9% normal saline, TRAIL-Exo, TPL, and TRAIL-Exo / TPL (TRAIL-Exo dose was 200 mg / kg, TPL dose was 0.6 mg / kg) were respectively injected via the tail vein every 2 days for a total of 8 times. Each time, the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the formula V = (a×b 2 ) / 2, and the body weight of the nude mice was weighed. The tumor growth curve and body weight change curve were plotted. The nude mice were sacrificed 24 h after the administration ended, and the heart, liver, spleen, lung, kidney, and tumor were taken, and the tumor weight of each group was weighed. The tumor growth inhibition rate was calculated.

[0112] Figure 12 For the in vivo anti - melanoma effect diagrams of TRAIL - Exo, TPL, and TRAIL - Exo / TPL in Example 12, including the solid tumor diagrams (a), tumor growth curve diagrams (b), tumor weight diagrams (c), tumor inhibition rate diagrams (d), and body weight change diagrams (e) of nude mice in each group after different treatments. From Figure 12 it can be seen that after single tail vein injection of TRAIL - Exo or TPL alone, the tumor growth was inhibited to a certain extent. After injection of an equal dose of TRAIL - Exo / TPL, the tumor growth was more significantly inhibited, showing the smallest and most stable tumor volume, the lightest tumor weight, and the highest tumor inhibition rate, and it also had no obvious effect on the body weight of nude mice, indicating that TRAIL - Exo / TPL has the most obvious in vivo anti - tumor effect and good tolerance.

[0113] Example 13: H&E staining of tumors and major organs

[0114] Take the tumor tissues and major organs (heart, liver, spleen, lung, kidney) of nude mice in each group and fix them with formalin overnight. After paraffin embedding, cut them into 3 - 5 μm thin sections, and dewax and hydrate the sections. Stain the sections with hematoxylin, wash them with tap water, differentiate with the differentiating solution, blue - return, then stain with eosin, and mount with transparent neutral gum.

[0115] Example 14: TUNEL immunofluorescence of tumor tissues

[0116] Add an appropriate amount of proteinase K to the paraffin sections of tumor tissues and cover them, and place them at room temperature for 15 min. Then place them on a shaker and wash them 3 times with PBS. After the sections are slightly dried, drop the permeabilization working solution to cover the tissues, place them at room temperature for 10 min, and then place them on a shaker and wash them 3 times with PBS. According to the number of sections and the size of the tissues, mix the TdT reagent and dUTP reagent in a volume ratio of 1:9, cover the tissues, place them in a wet box, and incubate at 37 °C for 2 h. Counter - stain the cell nuclei with DAPI, and incubate in the dark at room temperature for 10 min. Place the slides in PBS and wash them 3 times on a shaker. After the sections are slightly dried, mount them with an anti - fluorescence quenching mounting medium and observe them under a fluorescence microscope.

[0117] Example 15: Ki67 immunohistochemistry of tumors

[0118] Place the tumor sections in a repair box filled with citric acid antigen repair buffer, and then place it in a microwave oven for antigen repair. After cooling, place the slides in PBS and wash them 3 times on a shaker. Put the sections into 3% H2O2 and incubate them at room temperature in the dark for 30 min. Place the slides in PBS and wash them 3 times on a shaker. After the sections are slightly dried, add 3% BSA to cover and block them at room temperature for 30 min. Drop anti-Ki67 antibody diluent (1:200) on the sections, place them in a wet box and incubate overnight at 4 °C. Place the sections in PBS and wash them 3 times on a shaker, add diluted HRP-labeled goat anti-rabbit secondary antibody (1:2000) to cover the tissue, incubate at room temperature for 1 h, and place them in PBS and wash them 3 times on a shaker. After the sections are slightly dried, drop DAB chromogenic solution, control the chromogenic time under a microscope, and rinse the sections with tap water to terminate chromogenesis. Counterstain with hematoxylin for 3 min, wash with tap water, differentiate with 1% hydrochloric acid ethanol solution, wash with tap water, blue with ammonia water, and rinse with running water. Dehydrate the sections, air dry them and seal them with neutral gum, and observe them under a fluorescence microscope.

[0119] Figure 13 H&E, TUNEL immunofluorescence and Ki67 immunohistochemical analyses of tumor tissues of each group of nude mice obtained in Example 13, Example 14 and Example 15. From Figure 13 It can be seen that the H&E results show that compared with the control group, the tumor tissues of the TRAIL-Exo, TPL and TRAIL-Exo / TPL groups all showed pathological damage and necrosis characteristics, and the tumor tissue damage in the TRAIL-Exo / TPL group was the most obvious. The TUNEL results show that the green fluorescence intensity of the TRAIL-Exo, TPL and TRAIL-Exo / TPL groups increased in turn, indicating that the apoptosis rate of the tumor tissue increased in turn, and the TRAIL-Exo / TPL group had the highest apoptosis rate. The Ki67 results show that the brown-yellow granules in the cell nuclei of the TRAIL-Exo, TPL and TRAIL-Exo / TPL groups decreased in turn, indicating that the Ki67 positive rate decreased in turn; among them, the brown-yellow granules in the cell nuclei of the TRAIL-Exo / TPL group were the least, the positive rate was the lowest and lower than 10%, indicating that the tumor malignant proliferation index of this group was the lowest.

[0120] Figure 14 H&E staining analysis of major organs after different drug treatments in Example 13. From Figure 14 It can be seen that compared with the control group, there was no obvious damage to the tissues of nude mice in the TRAIL-Exo / TPL group, and no inflammation or edema was found in the liver and kidneys, indicating that TRAIL-Exo / TPL had no obvious organ toxicity and had good biosafety.

[0121] Example 16: Detection of the expression of related proteins promoting tumor cell apoptosis

[0122] A375 cells were seeded in 6-well plates at a density of 1×10 6 / mL. After the cells adhered to the wall, TRAIL-Exo, TPL or TRAIL-Exo / TPL (TPL concentration was 50 ng / mL) were added respectively to co-culture with the cells, and the control group cells were not treated with any drugs. Cells in each group were collected, lysed with RIPA lysis buffer for 1 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was collected. The total protein concentration was measured using a BCA kit. A 12% separating gel and a 5% stacking gel were prepared, and electrophoresis was carried out at a constant voltage of 75 V for 30 min. The methanol-activated PVDF membrane was covered on the gel, and transferred at a constant current of 300 mA for 30 min. The membrane was blocked with 5% skim milk for 1 h. Anti-Caspase 8 primary antibody (1:1000), Anti-Caspase 3 primary antibody (1:1000), Anti-Bax primary antibody (1:1000), Anti-Bcl 2 primary antibody (1:1000), Anti-Caspase 9 primary antibody (1:2000), Anti-Bid primary antibody (1:1000), Anti-Cytochrome c primary antibody (1:1000), NF-κB antibody (1:1000), anti-Survivin primary antibody (1:5000) and VEGF antibody (1:1000) were added to each group and incubated overnight at 4°C. The membrane was washed 3 times with TBST on a shaker. According to the species of the primary antibody, the corresponding HRP goat anti-mouse IgG (1:3000) and HRP goat anti-rabbit IgG (1:3000) secondary antibodies were added and incubated at room temperature for 1 h. The membrane was washed 3 times with TBST buffer on a decolorizing shaker, 10 min each time. In the darkroom, ECL A and ECL B reagents were mixed in equal volume and reacted with the membrane for 5 min. Using β-actin as an internal reference, imaging was performed using a gel imaging system to analyze the relative expression level of each protein.

[0123] Figure 15 The figures in [Figure number] are respectively the detection results of apoptosis-related protein expression in Example 16 (a) and the schematic diagram of the molecular mechanism of synergistic anti-tumor (b). Figure 15It can be seen that, compared with the control group, the expressions of caspase-3, caspase-8, caspase-9, Bax, Bid and cytochrome c in cells treated with TRAIL-Exo or TPL increased, and the expression levels of these proteins further increased after treatment with TRAIL-Exo / TPL. After treatment with TPL, the expressions of Bcl-2, NF-κB, VEGF and survivin decreased, and the expression levels of these proteins further decreased after treatment with TRAIL-Exo / TPL. The specific mechanism is as follows: The intracellular region of DR5 contains a death domain (DD). After TRAIL-Exo / TPL binds to DR5, it can recruit Fas-associating protein with a novel death domain (FADD) and pro-caspase 8 to form a death-inducing signaling complex (DISC). DISC activates a large amount of caspase 8, and caspase 8 in turn activates downstream caspase 3. Caspase 3 acts on various substrates that can cause apoptosis, thus leading to apoptosis of cells. In addition, some activated caspase 8 can activate Bid to be converted into tBid. tBid translocates to the mitochondrial membrane, activates pro-apoptotic factors (Bax and Bak) and inhibits anti-apoptotic factors (Bcl-2 and Bcl-XL), and at the same time releases cytochrome c (Cytochrome c, Cyto c). Cyto c interacts with apoptotic protease activating facter-1 (APAF-1) to form an apoptosome to activate downstream caspase 9, thereby further activating caspase 3 to induce apoptosis of cells. TPL can also induce apoptosis of cells through the above caspase 8-mediated caspase cascade reaction and mitochondrial pathway. In addition, TPL can simultaneously down-regulate the expressions of VEGF, NF-κB and survivin to play a synergistic role.

[0124] The above results show that the TRAIL-Exo / TPL composite exosomes can significantly inhibit the development of melanoma in vivo and in vitro, have a significant synergistic anti-tumor effect, and at the same time reduce the toxic side effects of TPL, providing new methods and ideas for the clinical treatment of melanoma.

[0125] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application. Sequence Listing <110> Fudan University Shanghai Cancer Center <120> A Composite Exosome Loaded with Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand and Small Molecule Antitumor Drugs <130> / <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <400> 1 cggtgaattc gccaccatgg ctatga 26 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ccgaggatcc ttagccaact 20

Claims

1. A composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs, characterized in that, The membrane surface of the composite exosomes carries TRAIL protein, and the small molecule anti-tumor drug triptolide (TPL) is encapsulated inside; the concentration of TRAIL protein in the composite exosomes is 205.1±13.6 pg / mL; the average particle size of the composite exosomes is 100-200 nm; the concentration of the small molecule anti-tumor drug is 0.01-60 μg / mL; The preparation method of the composite exosomes loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drug includes the following steps: S1. Construct a lentiviral packaging three-plasmid system, and infect 293T cells with the lentiviral packaging three-plasmid system to obtain lentiviral particles carrying the target gene; S2. Infect Raw264.7 cells with the lentiviral particles containing the target gene to obtain a macrophage cell line stably overexpressing TRAIL, labeled as TRAIL-Raw264.7, so that Raw264.7 cells secrete and express exosomes carrying TRAIL; the lentiviral packaging three-plasmid system consists of pSPAX2, pMD2G and a shuttle plasmid carrying the target gene; among them, the lentiviral three-plasmid system infects 293T cells and can package and produce more lentiviruses carrying the TRAIL gene; use the packaged lentiviral particles to infect the macrophage Raw264.7, so that the macrophage overexpresses TRAIL protein on the membrane, so that it secretes and expresses exosomes carrying TRAIL; S3. Adopt gradient ultracentrifugation to extract the exosomes secreted by cells from the culture supernatant of TRAIL-Raw264.7 cells to obtain high-purity exosomes expressing and carrying TRAIL, labeled as TRAIL-Exo; S4. Load the small molecule anti-tumor drug TPL into TRAIL-Exo to obtain the composite exosomes TRAIL-Exo / TPL carrying TRAIL and the small molecule anti-tumor drug TPL; The method of loading the small molecule anti-tumor drug TPL into TRAIL-Exo is the ultrasonic method; after ultrasonic treatment, place the mixture at 37°C for 0.5-1 h, and then place it at 4°C for 1-2 h; centrifuge at 120 000×g for 60-90 min to precipitate the exosomes, and resuspend with PBS to obtain TRAIL-Exo / TPL, which is stored at -80°C.

2. A preparation method of the composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs as described in claim 1, characterized in that, Including the following steps: S1. Construct a lentiviral packaging three-plasmid system, and infect 293T cells with the lentiviral packaging three-plasmid system to obtain lentiviral particles carrying the target gene; S2. Infect Raw264.7 cells with lentiviral particles containing the target gene to obtain a macrophage cell line stably overexpressing TRAIL, labeled as TRAIL-Raw264.7, so that Raw264.7 cells secrete and express exosomes carrying TRAIL; the lentiviral packaging three-plasmid system consists of pSPAX2, pMD2G and a shuttle plasmid carrying the target gene; among them, the lentiviral three-plasmid system infects 293T cells and can package and produce more lentiviruses carrying the TRAIL gene; use the packaged lentiviral particles to infect macrophage Raw264.7, so that the macrophages overexpress the TRAIL protein on the membrane, thereby enabling them to secrete and express exosomes carrying TRAIL; S3. Adopt the gradient ultracentrifugation method to extract the exosomes secreted by cells from the culture supernatant of TRAIL-Raw264.7 cells to obtain exosomes with high purity expressing and carrying TRAIL, labeled as TRAIL-Exo; S4. Load the small molecule anti-tumor drug triptolide TPL into TRAIL-Exo to obtain a composite exosome TRAIL-Exo / TPL carrying TRAIL and the small molecule anti-tumor drug TPL; The method for loading the small molecule anti-tumor drug TPL into TRAIL-Exo is the ultrasonic method; after ultrasonic treatment, place the mixture at 37 °C for 0.5 - 1 h, and then place it at 4 °C for 1 - 2 h; centrifuge the exosomes at 120,000×g for 60 - 90 min to precipitate, and resuspend with PBS to obtain TRAIL-Exo / TPL, which is stored at -80 °C.

3. An application of the composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small molecule anti-tumor drugs as described in claim 1 in the preparation of a therapeutic drug for malignant tumors, wherein the malignant tumor is cutaneous malignant melanoma.

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