CN113633758A - Composite exosome of load membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug - Google Patents

Abstract

The invention relates to the technical field of biomedicine and oncology, in particular to a composite exosome of a load membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and a small-molecule antitumor drug, and a preparation method and application thereof. The membrane surface of the compound exosome carries TRAIL protein, and small-molecule antitumor drugs are encapsulated in the membrane. The preparation process of the composite exosome is mature, efficient, good in reproducibility and low in cost; the compound exosome has obvious in-vitro and in-vivo anti-tumor effects, low drug dosage, no toxic or side effect and good biological safety, and provides a new strategy for clinical tumor treatment.

Description

Composite exosome of load membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug

Technical Field

The invention relates to the technical field of biomedicine and oncology, in particular to a composite exosome loaded with a membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and a small-molecule antitumor drug, and a preparation method and application thereof.

Background

Malignant melanoma of skin is a malignant tumor produced by melanocytes of skin and other organs, accounts for about 3% of all tumors, and has strong invasiveness and metastasis; the incidence rate is high, the prognosis is poor, the death rate of the advanced melanoma is up to 70-90%, and the advanced melanoma is the first to live in skin malignant tumors. According to the statistical data of the American cancer society of 2021, 106110 new diagnosis cases and 7180 death cases of global skin in-situ melanoma increase at a rate of 3-5% per year in 2020, and become one of the fastest global malignant tumors, and the five-year survival rate of late-stage melanoma is only 25%. At present, the treatment methods of malignant melanoma mainly comprise chemotherapy, radiotherapy, surgical excision, immunotherapy, targeted therapy and the like. Chemotherapy is still the current main method for treating melanoma, and the main chemotherapeutic drugs comprise dacarbazine, temozolomide, carboplatin, paclitaxel, fotemustine, cisplatin and the like, but the effective rate of single drug or combined drug in the melanoma is not high, about 10-15%, the normal immune function of a human body is damaged, and serious toxic and side effects are generated. Thus, there is a need for new, effective and safe melanoma treatment methods.

TRAIL belongs to Tumor necrosis factor superfamily, and has apoptosis promoting effect on various human Tumor cells, including melanoma, lung cancer, glioma, breast cancer, pancreatic cancer, prostate cancer, colon cancer, renal cancer and bile duct cancer.

Death receptor 5 (DR 5) is the main receptor of TRAIL and is highly expressed on the surface of various tumor cells, but is less or not expressed in normal cells. DR5 contains a Death Domain (DD) that binds to TRAIL, followed by the binding of DD to Fas-associated Death domain protein (FADD) to recruit pro-caspase 8, generate Death-inducing signaling complex (DISC), self-cleaves pro-caspase 8 in DISC into active caspase 8, activate caspase 3 via caspase cascade and mitochondrial dependent pathways, and mediate apoptosis.

Current TRAIL-based therapies include Recombinant human soluble TRAIL (rhTRAIL) and have been used in preclinical studies and clinical trials of a variety of human tumors. However, the tumor targeting of rhTRAIL is poor, the half-life period in vivo is short, and the drug resistance of cells is easily caused, so that the curative effect is poor. Therefore, in view of the deficiencies of rhTRAIL, there is a need to develop effective and safe TRAIL delivery forms to circumvent TRAIL resistance and improve therapeutic efficacy.

Macrophages, as natural immune cells and antigen presenting cells, play an important role in regulating the tumor immune microenvironment. M1 type macrophages, such as Raw264.7, bind specifically to tumor tissues and are therefore widely used for tumor-targeted therapy. The tumor targeting ability of M1 type macrophage comes from its surface membrane protein, and the produced exosome has the surface membrane characteristic similar to macrophage and retains the cell targeting ability and tumor penetrating ability. In addition, the exosome can be used as a natural endogenous nano-carrier and also has other unique advantages, such as high stability, low toxicity, low immunogenicity and good biocompatibility; the tumor has strong permeability and can permeate blood brain barrier; a variety of therapeutic agents can be delivered, such as membrane proteins, mirnas, sirnas, and small molecule chemical drugs. The research finds that the membrane-bound TRAIL can increase the stability and improve the bioavailability and targeting property of the membrane-bound TRAIL, and shows stronger apoptosis-promoting activity than rhTRAIL, which probably enhances the apoptosis-inducing efficiency and signal conduction because the receptor clustering of DR5 is promoted by the formation of oligomerization in the supermolecular structure of the membrane-bound TRAIL.

Triptolide (TPL) has antioxidant, antiinflammatory, antifertility, antirheumatic, neuroprotective, immunosuppressive and multi-target antitumor properties. In addition, TPL has been shown to be more effective than other conventional chemotherapeutic agents, and shows potent antitumor activity at nanomolar concentrations even in highly resistant cells. TPL has certain anti-treatment effect on malignant melanoma in vivo and in vitro. Oral administration of hydroxytriptolide tablets and soluble injectable triptolide derivative, minnesolide (Minnelide), have also entered clinical trials.

However, a complex exosome loaded with a membrane-bound TRAIL and a small-molecule antitumor drug has not been reported at present.

Disclosure of Invention

The invention aims to solve the defects of the existing TRAIL and TPL in the treatment of malignant melanoma, and provides a composite exosome loaded with a membrane-bound TRAIL (tumor necrosis factor) -related apoptosis-inducing ligand and a small-molecule antitumor drug, and a preparation method and application thereof. The compound exosome is a drug-loaded exosome with targeting and apoptosis promoting capabilities, and the small-molecular antitumor drug TPL encapsulated by the compound exosome can synergistically enhance the apoptosis effect of TRAIL on malignant melanoma cells without obvious toxicity on normal tissues and organs, so that reliable theoretical basis and experimental basis are provided for clinical treatment of malignant melanoma.

The exosome modified by TRAIL delivers a small-molecular antitumor drug TPL simultaneously, so that the defects of drug resistance, in-vivo instability, low bioavailability and the like of the TRAIL and the TPL can be overcome, and meanwhile, the TRAIL and the TPL are combined to generate a synergistic antitumor effect, the targeting property is enhanced, the tumor apoptosis effect is promoted, and the toxic and side effects of the TPL are reduced, so that the TRAIL-modified exosome has great clinical application potential.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a composite exosome loaded with a membrane-bound TRAIL and a small-molecule antitumor drug, wherein the membrane surface of the exosome carries the TRAIL, and the small-molecule antitumor drug is encapsulated in the exosome.

Further, the compound exosome is obtained by transfecting donor cells of the exosome with a virus expression vector over-expressing a membrane-bound TRAIL gene to obtain a donor cell line stably over-expressing TRAIL; then separating and purifying the exosome from the culture supernatant of the donor cell over-expressing TRAIL to obtain the exosome (TRAIL-Exo) with high-purity expression carrying TRAIL, and then loading the micromolecule antitumor drug into an exosome membrane to obtain the tumor-inhibiting tumor-killing protein.

Further, the concentration of TRAIL protein in the composite exosome is 205.1 +/-13.6 pg/mL; the average particle size of the composite exosome is 100-200 nm; the concentration of the small molecular antitumor drug is 0.01-60 mug/mL.

Further, the virus expression vector is lentivirus, retrovirus or adenovirus.

Furthermore, the preparation method of the virus expression vector for over-expressing the membrane-bound TRAIL gene to transfect an exosome donor cell comprises the following steps: infecting the slow virus with TRAIL gene with the exosome donor cell to obtain the cell line with stable over-expression TRAIL protein. In a preferred embodiment of the present invention, the method comprises: constructing a lentivirus packaging three-plasmid system, wherein the composition of the lentivirus packaging three-plasmid system comprises pSPAX2, pMD2G and a shuttle plasmid carrying TRAIL genes. Transient transfection of 293T cells with the lentivirus-packaged three-plasmid system to obtain lentivirus particles containing TRAIL genes, and infection of exosome donor cells with the lentivirus particles.

Further, the donor cell is selected from one of Raw264.7 cells, natural killer cells, T cells and dendritic cells. In a preferred embodiment of the invention, the donor cells are Raw264.7 cells.

Furthermore, the micromolecular antitumor drug is a natural drug.

Furthermore, the natural medicine is triptolide TPL.

In a second aspect of the present invention, there is provided a method for preparing the above-mentioned composite exosome loaded with membrane-bound tnf-related apoptosis-inducing ligand and small-molecule antitumor drug, comprising the following steps:

s1, constructing a virus expression vector of an over-expression membrane-bound TRAIL gene, packaging the virus and transiently transferring 293T cells to obtain virus particles, infecting macrophages Raw264.7 with the purified virus particles to obtain a macrophage system of stable over-expression TRAIL, and marking the macrophage system as TRAIL-Raw264.7;

s2, separating and purifying the exosome from TRAIL-Raw264.7 cell culture supernatant by adopting a gradient ultracentrifugation method to obtain the exosome expressing and carrying TRAIL with high purity, and marking as TRAIL-Exo;

s3, loading the small-molecule antitumor drug TPL into TRAIL-Exo to obtain a composite exosome (TRAIL-Exo/TPL) loaded with the TRAIL and the small-molecule antitumor drug; the membrane surface of the compound exosome carries a tumor apoptosis promoting protein TRAIL, and a small-molecule antitumor drug TPL is encapsulated in the membrane.

Further, the method for loading the small-molecule antitumor drug TPL into TRAIL-Exo in the step S3 is an ultrasonic method, an electroporation method or a co-incubation method.

In some preferred embodiments, the preparation method comprises the following steps:

s1, constructing a lentivirus packaging three-plasmid system, and enabling the lentivirus packaging three-plasmid system to infect 293T cells to obtain the lentivirus particles with target genes.

S2, infecting Raw264.7 cells with slow virus particles containing target genes to obtain a macrophage system which stably over-expresses TRAIL and is marked as TRAIL-Raw264.7, so that the Raw264.7 cells secrete and express exosomes carrying TRAIL. Further, the composition of the lentivirus packaging three-plasmid system comprises pSPAX2, pMD2G and a shuttle plasmid carrying a target gene. Wherein, the lentivirus three-plasmid system is used for infecting 293T cells, and can be packaged to generate more lentiviruses with TRAIL genes. And infecting macrophage Raw264.7 by using the packaged lentivirus particles, so that the macrophage can pass through TRAIL protein on a membrane, and the macrophage secretes and expresses an exosome carrying TRAIL.

S3, extracting exosomes secreted by cells from TRAIL-Raw264.7 cell culture supernatant by adopting a gradient ultracentrifugation method to obtain exosomes expressing and carrying TRAIL with high purity, and marking as TRAIL-Exo;

s4, loading the small-molecular antitumor drug TPL into TRAIL-Exo to obtain a composite exosome (TRAIL-Exo/TPL) loaded with TRAIL and the small-molecular antitumor drug TPL; the membrane surface of the compound exosome expresses TRAIL carrying tumor apoptosis promoting protein, and a small-molecule antitumor drug TPL is wrapped in the membrane.

The third aspect of the invention provides an application of the composite exosome of the loaded membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and the small-molecule antitumor drug in preparing a malignant tumor treatment drug.

Further, the malignant tumor is malignant melanoma of the skin.

Compared with the prior art, the invention has the advantages that:

1. compared with the exosome only loading TRAIL or the single micromolecule antitumor drug TPL, the exosome is used for simultaneously delivering the TRAIL and the micromolecule antitumor drug, thereby enhancing the tumor targeting property of the exosome and the apoptosis promoting capability of the TRAIL, improving the tumor killing activity of the TPL, reducing the toxic and side effect on normal tissues and obviously improving the antitumor treatment effect.

2. The composite exosome of the load membrane combined TARIL and the micromolecular antitumor drug TPL can achieve the purpose of synergy, and the combined use of the two remarkably enhances the killing effect on tumor cells. The in vivo experiment result shows that the compound exosome can effectively inhibit the growth of subcutaneous malignant melanoma and has good biological safety.

3. The TRAIL and TPL can synergistically induce apoptosis by regulating an exogenous apoptosis pathway and an endogenous apoptosis pathway. In vitro experiment results show that the compound exosome can obviously inhibit tumor cell proliferation, induce tumor cell apoptosis and inhibit tumor cell invasion and migration. In vivo experiment results show that after the compound exosome is injected into tail vein, the growth of subcutaneous xenograft tumor of nude mice is obviously inhibited, tumor tissues are obviously pathologically damaged and apoptosis occurs, and no toxicity to important organs is observed.

4. The invention expounds the action mechanism of the compound exosome synergistic anti-tumor, and provides a new treatment strategy for related diseases.

5. The preparation process is mature, efficient, good in reproducibility and low in cost; the compound exosome has obvious in-vitro and in-vivo anti-tumor effects, low drug dosage, no toxic or side effect and good biological safety, and provides a new strategy for clinical treatment of melanoma.

Drawings

FIG. 1 is a map of a lentiviral expression vector containing a TRAIL gene constructed in example 1;

FIG. 2 is a graph of fluorescence imaging (a), RT-qPCR relative expression quantification (b), Western blot (c) and TRAIL flow detection (d) of Raw264.7 cells overexpressing TRAIL, which are established in example 4, and TRAIL expression concentration (e);

FIG. 3 shows transmission electron micrograph (a), particle size distribution (b), Western blot (c), immunoelectron micrograph (d), flow chart (e) and TRAIL expression concentration (f) of TRAIL-Exo obtained in example 5;

FIG. 4 is a graph showing the laser confocal imaging (a), the laser confocal cell mean fluorescence intensity (b), the flow-through imaging (c) and the flow-through cell mean fluorescence intensity (d) of TRAIL-Exo cell uptake obtained in example 5;

FIG. 5 is a BCA protein standard graph in example 7;

FIG. 6 is a TPL standard curve in example 7;

FIG. 7 is a transmission electron micrograph (a), a particle size distribution map (b) and a TRAIL flow chart (c) of TRAIL-Exo/TPL prepared in example 7, respectively;

FIG. 8 is a cell viability assay of human malignant melanoma A375 cells treated with different agents for 24 h;

FIG. 9 (a) is an apoptosis flow chart of A375 cells treated with different agents; (b) the apoptosis rate of A375 cells treated by different preparations is shown;

FIG. 10 (a) is a diagram showing the Transwell invasion of A375 cells treated with different agents; (b) relative invasion rates of a375 cells treated with different preparations;

FIG. 11 (a) is a graph showing the migration of A375 cells treated with different agents; (b) the mobility of A375 cells treated by different preparations;

FIG. 12 is a graph showing the effects of TRAIL-Exo, TPL and TRAIL-Exo/TPL on anti-melanoma in vivo in example 12, including a graph of solid tumors (a), a graph of tumor growth (b), a graph of tumor mass (c), a graph of tumor inhibition rate (d) and a graph of body weight change (e) in nude mice after tail vein injection of different formulations;

FIG. 13 is a graph of H & E, TUNEL immunofluorescence and Ki67 immunohistochemistry of tumor tissues in nude mice after tail vein injection of different formulations;

FIG. 14 is H & E staining patterns of heart, liver, spleen, lung and kidney of nude mice after tail vein injection of different preparations;

FIG. 15 is a Western blot of TRAIL-Exo/TPL regulating the expression of apoptosis pathway related protein and its molecular mechanism of promoting tumor cell apoptosis (b).

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention, but the scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Example 1: construction of overexpression TRAIL Lentiviral vectors

A TRAIL (Homo sapiens TNF super family member 10, TNFSF10) nucleotide sequence (NM _003810) was synthesized into pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO lentivirus expression vector. The specific process is as follows: selecting a carrier enzyme digestion system → carrying out enzyme digestion at 37 ℃, carrying out gel recovery → carrying out fragment PCR recovery → placing the nucleotide sequence of TRAIL and a lentivirus expression carrier connection reaction system in a 50 ℃ warm bath for 20min → transformation (competent cell DH 5 a; resistant: ampicillin, 37 ℃, 230rpm, 24h) → carrying out PCR identification on transformed flat plate bacteria, shaking the bacteria at 37 ℃, 250rpm for 14h → bacteria liquid, and carrying out overexpression sequencing on the positive clone bacteria liquid.

FIG. 1 is a map of the TRAIL lentivirus expression vector constructed in example 1.

Example 2: lentiviral packaging

Extraction reagent using plasmid DNAThe constructed lentivirus expression vector and helper plasmid are extracted in large quantity from the cassette, the concentration of the plasmid needs to be more than 1 mug/muL, and A260/280 is between 1.7 and 1.8, so that the lentivirus expression vector and helper plasmid can be used for virus packaging. 293T cells were digested, centrifuged, resuspended in complete medium, and plated as 1: and (5) dividing the culture medium into culture dishes for 10 passages, and placing the culture medium in an incubator to continue culturing. And (5) performing transfection when the density of the 293T cells reaches 70-80%. Changing the culture solution into a serum-free culture medium, and performing lipid conversion to complete: 10. mu.g of pSPAX2, 5. mu.g of pMD2G, 10. mu.g of shuttle plasmid containing the gene of interest, 75. mu.L of Lipofilter^TMAnd (3) a reagent. After 6h of transfection, the medium was replaced with a complete medium containing 10% FBS. Viral supernatants were collected twice 48h and 72h post transfection. And when the virus is collected for 48 hours, pouring the culture medium in the culture dish into a centrifuge tube, then supplementing the complete culture medium, and placing the culture dish in a constant temperature incubator for continuous culture. When the virus is collected for 72h, the culture medium in the culture dish is directly poured into a centrifuge tube. Centrifuging the supernatant containing the virus at 1000-2000 Xg for 5-10 min; and then collecting the supernatant of the virus stock solution, placing the supernatant in an ultracentrifuge tube, centrifuging for 60-120 min at 4 ℃ and 80000-100000 Xg, and finally subpackaging the lentivirus ultracentrifuge solution into virus tubes for preservation in a refrigerator at-80 ℃.

Example 3: virus titer detection

Digesting and counting 293T cells, and diluting to 1-3 multiplied by 10⁵PermL, add to 96-well plate, 100. mu.L/well, prepare 6 wells for each virus. The next day, 6 1.5mL EP tubes were prepared, and 10. mu.L of virus solution was added to the first EP tube, followed by 3-fold gradient dilution for a total of 6 dilutions. On day three, wells requiring puromycin screening were aspirated 100 μ L of lentiviral particle-containing medium, followed by 100 μ L of puromycin-containing complete medium. On the fifth day, observation was performed under a fluorescence microscope, and 80. mu.L of the fresh complete medium was aspirated from the wells 6h before observation, and then 80. mu.L of the fresh complete medium was added and placed in an incubator for culture. And observing the result under a fluorescence microscope after 6h, and selecting the wells with the fluorescence percentage of 10-50% to calculate the virus titer. The virus titer in example 3 was calculated to be 1.5X 10⁸TU/mL。

Example 4: establishment and identification of stably over-expressed TRAIL macrophage cell line

1. Waiting for Raw264.7 cells to growAfter the cell is full, digesting, centrifuging and resuspending, and adjusting the cell density to 1-5 multiplied by 10⁵Perml, inoculated in 6-well plates, placed in an incubator until the density reaches approximately 60% and infection begins. 1-5 mL (optimally 2mL) of virus stock solution is added into each hole, and 2-20 mu g/mL polybrene (optimally 5 mu g/mL) is added at the same time. After 24h of infection, the virus stock was aspirated and complete medium was added. And (5) carrying out passage after the fusion rate of the cells reaches 90% after the cells are infected by the virus for 48 hours.

2. Raw264.7 cells are inoculated in a 24-well plate, when the fusion rate is about 60 percent, a complete culture medium containing 0.2-10 mug/mL (optimally 2 mug/mL) puromycin is replaced, and the cells are treated for 24-48 hours. And (5) when the cell density reaches 70-80%, carrying out passage, and continuously culturing by using a complete culture medium containing puromycin. After 4 days of infection, the fresh complete culture medium is replaced to continue to culture for 2 days, and cell subculture is carried out, thus obtaining the Raw264.7 macrophage system (TRAIL-Raw264.7) which over-expresses TRAIL.

3. Total RNA extraction: after the TRAIL-Raw264.7 cells are fully grown in the 6-well plate, the culture solution is aspirated, and a proper amount of Trizol reagent is added into each well to extract the total RNA of the cells. And transferring the lysate into a centrifuge tube, and standing at room temperature for 10-15 min. Adding 200 mu L of chloroform, shaking and mixing uniformly, and standing at room temperature for 10-15 min. 13780 Xg, centrifuging for 10min, sucking supernatant into a centrifuge tube, adding equal volume of isopropanol, and precipitating at room temperature for 10 min. 13780 Xg, centrifuge for 15min, and discard the supernatant. The precipitate was washed 1 time with 75% ethanol. 13780 Xg for 5min, discard the supernatant and recover the pellet. Air drying at normal temperature for 10 min. With DEPC-H₂Dissolving the precipitate with O, mixing, and storing at-80 deg.C. The absorbance at 260nm and 280nm wavelength was measured by UV-visible spectrophotometer, and the total RNA concentration of the cells was calculated.

4. Reverse transcription: using ReverTra

The qPCR RT Kit reverse transcription reaction Kit carries out reverse transcription on RNA. The reverse transcription reaction conditions were 37 deg.C, 20min and 95 deg.C, 5 min. The reverse transcription reaction system is as follows:

real-time qPCR (RT-qPCR): miRNA was detected using the Hanbio miRNA qPCR Detection Primer kit. TRAIL gene detection was performed using a LightCycler 96 real-time fluorescent quantitative PCR instrument and expression levels were calculated. The primer sequences are as follows:

the RT-qPCR reaction system is as follows:

the RT-qPCR reaction conditions were as follows:

western blot detection: inoculating the over-expressed TRAIL-Raw264.7 cells into a 6-well plate, after the cells adhere to the wall, removing the supernatant, washing the cells for 3 times by PBS, adding a proper amount of PMSF, and adding RIPA lysate. The cells were scraped off with a spatula and collected in a centrifuge tube. Centrifuging at 12000rpm for 10min, and collecting supernatant to obtain total protein solution. Total protein concentration was determined using the BCA kit. Protein samples are mixed according to a volume ratio of 1:1 Add 2 XLoading buffer and boil for denaturation 15 min. 12% of separation gel and 5% of concentrated gel are prepared, 20 mu L of protein sample is added into the sample loading hole, and electrophoresis is carried out for 30min. The methanol activated PVDF membrane was covered on the gel and constantly transferred to the membrane for 30min. The membrane was placed on a shaker and blocked with 5% skim milk for 1h, and anti-TRAIL primary antibody (1: 1000, Abcam) was added and incubated overnight at 4 ℃. HRP coat anti-rabbit IgG secondary antibody (1: 3000, Abcam) was added, incubated at room temperature for 1h, the membrane was added to ECL reagent and reacted for 2min before exposure. And developing and imaging the exposed film.

5. Flow detection: TRAIL-Raw264.7 cells in 6-well plates were digested, centrifuged, resuspended in PBS, and adjusted to a cell density of 1X 10⁷and/mL. 100 mu L of cell suspension is taken, 5 mu L of PE anti-human TRAIL Antibody staining solution is added, and the mixture is mixed evenly. mu.L of PE Mouse IgG1 isotype control antibody staining solution was added to the control tube, and the tube was protected from light and stained for 15 min. 2mL of PBS was added to each tube to wash the cells, centrifuged at 1000rpm for 5min, the supernatant was discarded, resuspended in 400. mu.L of PBS, transferred to a 5mL flow tube, and TRAIL positivity was determined by flow cytometry.

ELISA detection: TRAIL protein standard solutions with concentrations of 0, 15.9, 31.2, 62.50, 125.00, 250.00, 500.00 and 1000.00pg/mL are prepared in sequence. TRAIL-Raw264.7 cells are digested, centrifuged and resuspended, and then freeze-thawing is repeated for 3 times, 10000 Xg is centrifuged for 5min, and cell supernatant is collected. 100 mu L of the TRAIL protein standard solution and cell supernatant are respectively added into a 96-well enzyme label plate pre-coated with anti-human TRAIL, and the mixture is incubated for 120min at room temperature (or incubated for 90min at 37 ℃). The supernatant was discarded, and 100. mu.L of 1 × Biotinylated anti-human TRAIL antibody was added to each well and incubated at room temperature for 90min (or 37 ℃ for 60 min). The plate was washed 3 times with 1 × Wash Buffer, 100 μ L of 1 × Avidin-Biotin-Peroxidase Complex was added per well, and incubated at room temperature for 40min (or at 37 ℃ for 30 min). The plate was washed 5 times with 1 × Wash Buffer, 90 μ L of Color Developing Reagent was added to each well, and incubated at room temperature for 30min in the absence of light (or 15-25 min at 37 ℃). 100. mu.L of the termination solution was added to each well, reacted for 30min, and the absorbance value of each well was measured at a wavelength of 450nm to calculate the expression level of TRAIL in TRAIL-Raw264.7 cells.

FIG. 2 is a fluorescent microscopic picture (a) of Raw264.7 cells overexpressing TRAIL, RT-qPCR expression relative quantification of TRAIL (b), Western blot (c) of TRAIL, flow diagram (d) of TRAIL and expression concentration (e) of TRAIL, respectively, established in example 4. From the figure, Raw264.7 cells were well grown after transfection of the TRAIL-expressing lentiviral expression vector and puromycin screening (FIG. 2 (a)). The TRAIL gene was overexpressed in transfected Raw264.7 cells (FIG. 2 (b)). TRAIL was expressed in transfected raw264.7 cells (fig. 2 (c)). The transfected Raw264.7 cell surface TRAIL has higher TRAIL positive rate (figure 2(d)) and higher TRAIL expression concentration which is 125.3 +/-10.5 pg/1 mu g protein (figure 2(e)), and further proves that the TRAIL is expressed in the transfected Raw264.7 cell, which indicates that the construction of the overexpression TRAIL-Raw264.7 stable cell line is successful.

Example 5: extraction and characterization of TRAIL-Exo

1. Exosome extraction: after the TRAIL-Raw264.7 cells reach 80% confluence, the culture solution is discarded, PBS is used for washing for 3 times, complete culture solution containing 10% of exosome-free serum is added for continuous culture for 24-48 h, and the supernatant is collected. Centrifuging the supernatant at 300 Xg for 10min to remove viable cells; then, centrifuging at 2000 Xg for 10min to remove dead cells; then centrifuged at 10000 Xg for 30min to remove cell debris. The supernatant was centrifuged at 120000 Xg for 70min to obtain a pellet of hetero-proteins and exosomes. The pellet was resuspended in PBS and centrifuged again at 120000 Xg for 70min to remove contaminating proteins. The exosome pellet was finally resuspended in PBS and stored in a-80 ℃ freezer. All centrifugation procedures were performed at 4 ℃.

2. Transmission electron microscopy: resuspending the exosome by 2 percent paraformaldehyde, dripping 5-10 mu L of exosome suspension on a Formvar-carbon copper net, and standing for 10min at room temperature; PBS wash 2 times, each 3 min. Placing the copper net on 50 mu L of 0.3% uranyl acetate liquid drop for negative dyeing for 5 min; the copper mesh was washed 3 times on 100 μ L PBS droplets for 1 min. And then placed on a 2% methylcellulose drop for 10 min. And after naturally airing, observing and shooting the form of the exosome by using a transmission electron microscope at the voltage of 80 kV.

3. An immune electron microscope: resuspending the exosomes with 2% paraformaldehyde, dropping 5-10 μ L of exosome suspension onto a Formvar-carbon copper net, and standing at room temperature for 10 min. PBS wash 2 times, each 3 min. The grid was transferred to 50mM glycine and incubated for 3 min. The grids were blocked with 1% BSA blocking buffer for 10 min. mu.L of diluted TRAIL-primary antibody (1:100) was added dropwise to the grid and incubated for 30min. the grid was transferred to PBS and washed 5 times for 3min each. The goat anti-mouse IgG (1:50) was incubated with diluted 5nm colloidal gold for 20 min. The grid was washed 8 times with PBS for 2min each time. The grid was placed in a 1% glutaraldehyde solution drop and fixed for 5 min. The grid was placed on 100. mu.L of distilled water and washed 8 times for 2min each. Placing the copper net on 50 mu L of 0.3% uranyl acetate liquid drop for negative dyeing for 5 min; the copper mesh was washed 3 times on 100 μ L PBS droplets for 1 min. And then placed on a 2% methylcellulose drop for 10 min. And after naturally airing, observing and shooting the form of the exosome by using a transmission electron microscope at the voltage of 80 kV.

4. And (3) particle size measurement: and (3) taking a proper amount of exosome, diluting the exosome with 1mL of PBS, placing the exosome into a cuvette, and detecting the particle size of the exosome by using a Malvern laser particle sizer at room temperature.

Western blot detection: adding a proper amount of PMSF into exosome, adding a proper amount of RIPA lysate, fully cracking on ice, centrifuging at 100000 Xg for 1h, and taking supernatant. Preparing 12% separation glue and 5% concentrated glue to be solidified. The gel plate is placed in an electrophoresis tank, and electrophoresis buffer is added. The exosome suspension is taken, mixed with equal volume of 2 Xloading buffer solution, and boiled for denaturation for 5 min. 20 μ g of the sample solution was added to each well, and a constant pressure of 70V was set. When the indicator bromfenan enters the separation gel, the electrophoresis is carried out by using a 90V constant voltage electrophoresis. When the indicator reached about 0.5cm from the lower end of the gel, the power was turned off and the gel plate was removed. PVDF membrane was previously activated with methanol and ddH₂And O, washing the membrane, and soaking the gel in a transfer buffer solution for balancing for 15 min. The transfer film "sandwich" was prepared in the order of black face (negative electrode) → sponge → filter paper → glue → PVDF film → filter paper → sponge → red face (positive electrode). The flow was constant at 200mA for 70 min. After the membrane transfer was completed, the PVDF membrane was removed, blocked with 5% BSA at room temperature for 2h, and washed with TBST buffer on a shaker for 5min X3 times. 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) were added, and incubated overnight at 4 ℃. The membrane was then washed with TBST buffer 5min X3 times on a shaker. HRP coat anti-Rabbit secondary antibody (Abcam, 1: 2000) was added and incubated at room temperature for 2 h. The membrane was then washed with TBST buffer 10min X3 times on a shaker. The membrane was reacted with an ECL chemiluminescence kit for 2min, and the PVDF membrane was exposed and imaged with a ChemiDoc imaging system.

6. Flow detection: mu.L of TRAIL-Exo solution was added with 5. mu.L of PE anti-human TRAIL anti staining solution, and 20. mu.L of PE mouse IgG1 isotype control antibody staining solution was added to the control tube. After incubation at 4 ℃ for 15min in the dark, 2mL PBS was added and washed 2 times, centrifuged at 120000 Xg for 70min, the supernatant was discarded, resuspended in 400. mu.L PBS, transferred to a 5mL flow tube, and detected by an up-flow cytometer.

TRAIL concentration determination: the expression level of TRAIL in TRAIL-Exo was measured using ELISA kit according to the method in example 4.

In FIG. 3, the transmission electron micrograph (a), the particle size distribution map (b), the Western blot map (c), the immunoelectron micrograph (d), the flow chart (e), and the TRAIL expression concentration (f) of TRAIL-Exo obtained in example 5 are shown, respectively. Extracellular vesicles obtained by gradient ultracentrifugation are typically in a saucer-like or spherical structure (fig. 3 (a)). The average particle diameter was 100nm, and the particle diameter distribution was uniform (FIG. 3 (b)). The expression of the exosome-specific proteins CD9, CD63, TSG101 and TRAIL could be clearly detected (fig. 3(c)), all of which fit into the exosome characteristic category. The immunoelectron microscopy shows more intuitively that the gold nanoparticles can be attached to the outer surface of the exosome membrane, and the morphology of the exosome is not changed significantly (fig. 3 (d)). The positive binding rate of TRAIL-Exo and TRAIL antibody is over 95% (FIG. 3(e)), and TRAIL-Exo also has higher TRAIL expression concentration of 205.1 + -13.6 pg/1 μ g exosome protein (FIG. 3 (f)). These results indicate that the membrane surface of exosomes secreted by transfected TRAIL macrophages can express or carry TRAIL, TRAIL-Exo is successfully acquired.

Example 6: cell uptake assay

PKH67 fluorescent label: TRAIL-Exo solution was diluted with Diluent C. Adding a proper amount of PKH67 staining solution into Diluent C Diluent according to a volume ratio of 1:1, mixing the diluted TRAIL-Exo with a staining solution, and standing for 1-5min in a dark place. The staining was stopped with 1% BSA solution and the tube (100kDa) was centrifuged using ultrafiltration to isolate PKH 67-labeled TRAIL-Exo.

2. Laser confocal imaging: a375 cells were inoculated on a 35mm confocal culture dish, after the cells adhered to the wall, the supernatant was discarded, PKH 67-labeled TRAIL-Exo and common macrophage-derived exosome (Exo) of untransfected TRAIL were added, respectively, and the culture was continued for 6 h. Abandoning the supernatant, washing with PBS for 2 times, fixing with 4% paraformaldehyde for 10-15 min, dyeing with DAPI for 5-10 min, observing with a laser confocal microscope, taking a picture, and calculating the average fluorescence intensity of cell uptake by using Image J.

3. Flow detection: a375 cells are inoculated in a 6-well plate, after the cells are attached to the wall, the supernatant is discarded, and TRAIL-Exo and Exo marked by PKH67 are respectively added to continue culturing for 6 h. Discard the supernatant, wash 3 times with PBS, digest, centrifuge, resuspend to 400. mu.L with PBS, transfer to 5mL flow tube, and detect on the up flow cytometer.

FIG. 4 is a graph showing the laser confocal (a), confocal mean fluorescence intensity (b), flow-uptake (c) and flow-cell mean fluorescence intensity (d) uptake of PKH 67-labeled TRAIL-Exo and Exo by A375 cells in example 6, respectively. As can be seen from FIG. 4, TRAIL-Exo showed more extensive green fluorescence and stronger mean fluorescence intensity on the membrane and cytoplasm of A375 cells, indicating that A375 cells have higher uptake efficiency of TRAIL-Exo.

Example 7: preparation and characterization of TRAIL-Exo/TPL

1. Preparation: TPL stock solutions at 1mg/mL were prepared in DMSO. Taking 100-1000 mul of TRAIL-Exo with the protein concentration of 1mg/mL, adding 10-100 mul of TPL solution diluted by PBS, uniformly mixing, and carrying out ultrasonic treatment on the mixture by using an ultrasonic cell disruptor: 10-40% amplitude (optimally 20%), 20-50 s on/off (optimally 30s), 4-8 cycles (optimally 6 cycles), with a 2min cooling time between each cycle. After ultrasonic treatment, the mixture is placed at 37 ℃ for 0.5-1 h, and then placed at 4 ℃ for 1-2 h. Centrifuging at 120000 Xg for 60-90 min to precipitate exosomes, resuspending with PBS to obtain TRAIL-Exo/TPL, and storing at-80 ℃.

2. And (3) concentration determination: preparing protein standard solutions with the concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL respectively. Protein concentrations of TRAIL-Exo were calculated from BCA standard curves (FIG. 5). TPL standard solutions of 10, 50, 100, 250, 500, 1000, 2000, 5000, 10000, 25000, 50000, 100000ng/mL were prepared, and the concentration of TPL in TRAIL-Exo/TPL was determined by HPLC according to the TPL standard curve (FIG. 6).

3. And (3) characterization: the TRAIL-Exo/TPL prepared was characterized by transmission electron microscopy, particle size and flow as in example 4.

In FIG. 7, the transmission electron micrograph (a), the particle size distribution map (b) and the TRAIL flow analysis map (c) of TRAIL-Exo/TPL prepared in example 7 are shown, respectively. As can be seen from FIG. 7, TRAIL-Exo/TPL still maintained the intact structure and properties of exosomes (FIG. 7(a)), particle size increased (FIG. 7(b)), and still higher TRAIL-positivity (FIG. 7 (c)). The mass ratio of TRAIL-Exo to TPL is 10: when the medicine is 1, the encapsulation efficiency is 51.87 +/-4.85 percent, and the medicine loading rate is 4.93 +/-0.48 percent. The above results indicate that sonication can load TPL into TRAIL-Exo, achieving higher drug loading.

Example 8: cytotoxicity test

A375 cells at 1X 10⁴And inoculating the seeds in a 96-well plate for 24 h. The supernatant was discarded and TRAIL-Exo, TPL and TRAIL-Exo/TPL solutions of different concentrations were added. The protein concentration of TRAIL-Exo group is 5, 10, 20, 50, 100 and 200 mug/mL in sequence; TPL concentrations in TPL and TRAIL-Exo/TPL groups were 5, 10, 20, 50, 100, 200ng/mL in order, and treatment was continued for 24 h. After the treatment, 10. mu.L of CCK-8 solution was added to each well, and the plate was shaken for 30 seconds, and the reaction was carried out in an incubator for 2 hours. The absorbance of each well at 450nm was measured by a microplate reader, and the cell survival rate was calculated.

FIG. 8 is the toxicity assay of TRAIL-Exo, TPL and TRAIL-Exo/TPL on A375 cells in example 8. As can be seen from FIG. 8, the cytotoxicity of TRAIL-Exo, TPL and TRAIL-Exo/TPL was concentration-dependent. TRAIL-Exo/TPL showed lower cell viability at the same TPL concentration compared to TRAIL-Exo and TPL, indicating greater tumor cytotoxicity.

Example 9: apoptosis assay

A375 cells were treated at 2-5 × 10⁵The mixture was inoculated into 12-well plates at a density of one mL, and after adherence, TRAIL-Exo, TPL and TRAIL-Exo/TPL (TPL concentration 50ng/mL) were added, respectively, and the culture was continued for 24 hours. The cells were digested with 0.25% trypsin without EDTA, centrifuged at 1000rpm for 5min and the supernatant discarded. Resuspended in precooled PBS and centrifuged again. PBS was aspirated, 100. mu.L of 1 XBinding Buffer was added to resuspend the cells, and the cell concentration was adjusted to 1X 10⁶and/mL. Adding 5 μ L Annexin V-APC and 10 μ L7-AAD in sequence, shaking, mixing, dyeing at room temperature in dark for 15min, adding 385 μ L1 × Binding Buffer, mixing, transferring to 5mL flow tube, and detecting with an up-flow cytometer.

FIG. 9 is a graph showing the apoptotic effects of TRAIL-Exo, TPL and TRAIL-Exo/TPL on A375 cells in example 9. As can be seen from FIG. 9, TRAIL-Exo, TPL and TRAIL-Exo/TPL groups all induced apoptosis of A375 cells, and TRAIL-Exo/TPL showed a stronger apoptosis-inducing effect on tumor cells, compared to the control group.

Example 10: cell invasion assay

A375 cells at 5X 10⁵The density of/mL was seeded in 12-well plates. After adherence, TRAIL-Exo, TPL and TRAIL-Exo/TPL (TPL concentration all 50ng/mL) were treated for 6 h. After digestion and centrifugation, the cells were resuspended in a blank medium and the cell density was adjusted to 1X 10⁵and/mL. 50 μ L of diluted matrigel was added to the upper chamber of a Transwell and left at 37 ℃ for 1h to solidify the gel. 200. mu.L of TRAIL-Exo, TPL and TRAIL-Exo/TPL treated single cell suspensions were added to the upper chamber, and 600. mu.L of complete medium containing 10% FBS was added to the lower chamber, and the culture was continued for 24 hours. And taking out the chamber, wiping off non-membrane-penetrated cells on the upper layer of the membrane by using a cotton swab, fixing for 15min by using 4% paraformaldehyde, washing the membrane for 2 times by using PBS (phosphate buffer solution), dyeing for 15min by using 0.1% crystal violet, washing by using PBS (phosphate buffer solution), drying, photographing by using a fluorescence microscope, and counting the number of membrane-penetrated cells under 5 random fields. And calculating the relative invasion rate of the cells according to the ratio of the number of the transmembrane cells to the control group.

FIG. 10 is a graph showing the inhibition effect of TRAIL-Exo, TPL and TRAIL-Exo/TPL on A375 cell invasion in example 10. As shown in FIG. 10, TRAIL-Exo, TPL and TRAIL-Exo/TPL inhibited the invasion and chemotaxis of A375 cells, and the inhibition effect of TRAIL-Exo/TPL was most significant, compared to the control group.

Example 11: cell migration assay

A375 cells at 5X 10⁵The density of/mL was seeded in 12-well plates. After adherence, cells were treated with TRAIL-Exo, TPL and TRAIL-Exo/TPL (TPL concentration 50ng/mL), respectively, for 6 h. Collecting cells, and inoculating the cells into the special culture insert holes. The insert was removed 24h after attachment and incubation was continued with serum free medium for 48 h. And measuring and observing the scratch distance of the cells at different moments by using a fluorescence microscope, and calculating the migration rate of the tumor cells.

FIG. 11 is a graph showing the effect of TRAIL-Exo, TPL and TRAIL-Exo/TPL on the inhibition of A375 cell migration in example 11. As shown in FIG. 11, TRAIL-Exo, TPL and TRAIL-Exo/TPL all inhibited the cell migration of A375, and the cell migration inhibition effect of TRAIL-Exo/TPL was most significant.

Example 12: evaluation of in vivo anti-melanoma Effect

A375 nude mouse graft tumor model: male BALB/C nude mice, 4-6 weeks old, were purchased from Shanghai Slek laboratory animals, Inc. Will be about 4X 10⁶A375 cells were inoculated in the right armpit of nude mice until the tumor volume reached about 100mm³The control group, TRAIL-Exo group, TPL group and TRAIL-Exo/TPL group were randomly assigned (n ═ 6).

2. In vivo administration: 0.9% physiological saline, TRAIL-Exo, TPL and TRAIL-Exo/TPL (TRAIL-Exo dose is 200mg/kg, TPL dose is 0.6mg/kg) were injected into tail vein every 2 days for 8 times. The long diameter (a) and the short diameter (b) of the tumor were measured with a vernier caliper each time according to the formula (V ═ a × b)²) And/2, calculating the tumor volume, weighing the weight of the nude mice, and drawing a tumor growth curve and a weight change curve. Nude mice were sacrificed 24h after dosing was completed, heart, liver, spleen, lung, kidney and tumor were harvested, and each group was weighed. And calculating the tumor growth inhibition rate.

FIG. 12 is a graph showing the effect of TRAIL-Exo, TPL and TRAIL-Exo/TPL on anti-melanoma in vivo in example 12, including a graph of solid tumors (a), a graph of tumor growth (b), a graph of tumor mass (c), a graph of tumor inhibition rate (d) and a graph of body weight change (e) in each group of nude mice after different treatments. As can be seen from FIG. 12, after TRAIL-Exo or TPL was injected into tail vein alone, tumor growth was inhibited to some extent, while after equal doses of TRAIL-Exo/TPL were injected, tumor growth was more significantly inhibited, minimal and most stable tumor volume, minimal tumor weight and highest tumor inhibition rate were shown, and no significant effect was observed on body weight of nude mice, indicating that TRAIL-Exo/TPL had the most significant in vivo anti-tumor effect and good tolerance.

Example 13: tumor and major organs H & E

Tumor tissues and major organs (heart, liver, spleen, lung, kidney) were taken from each group of nude mice and fixed with formalin overnight. After paraffin embedding, the slices are cut into 3-5 mu m slices, and the slices are dewaxed and hydrated. Staining the sections with hematoxylin, washing with tap water, differentiating the differentiation medium, turning blue, staining with eosin, and sealing with transparent neutral gum.

Example 14: TUNEL immunofluorescence in tumor tissue

Adding appropriate amount of proteinase K to cover the paraffin section of the tumor tissue, and standing at room temperature for 15 min. The cells were washed 3 times with PBS in a decolorization shaker. After the section is slightly dried, the membrane-breaking working solution is dripped to cover the tissue, the tissue is placed for 10min at normal temperature, and then the tissue is placed on a decoloring shaker and washed for 3 times by PBS. According to the number of slices and the size of tissues, taking a TdT reagent and a dUTP reagent according to the ratio of 1: mix at 9 vol%, cover the tissue, place in a wet box, incubate for 2h at 37 ℃. DAPI counterstain nuclei, and incubate for 10min at room temperature in the dark. Slides were washed 3 times in PBS on a shaker. After the sections were slightly dried, the sections were mounted with an anti-fluorescence quenching mounting agent and observed under a fluorescence microscope.

Example 15: tumor Ki67 immunohistochemistry

And (3) placing the tumor slices in a repairing box filled with citric acid antigen repairing buffer solution, and then placing the repairing box in a microwave oven for antigen repairing. After cooling, the slides were washed 3 times in PBS on a shaker. Slicing into 3% H₂O₂And incubating for 30min at room temperature in a dark place. Slides were washed 3 times in PBS on a shaker. After the sections were slightly dried, 3% BSA was added dropwise to cover them and blocked at room temperature for 30min. An anti-Ki67 antibody diluent (1: 200) was added dropwise to the sections and incubated overnight in a wet box at 4 ℃. Sections were washed 3 times in PBS on a shaker, diluted HRP-labeled goat anti-rabbitt secondary antibody (1: 2000) was added to cover the tissue, incubated at room temperature for 1h, placed in PBS and washed 3 times on a shaker. After the section is slightly dried, DAB color developing solution is dripped, the color developing time is controlled under a microscope, and the section is washed by tap water to stop color development. Counter-staining with hematoxylin for 3min, washing with tap water, differentiating with 1% ethanol hydrochloride solution, washing with tap water, returning blue with ammonia water, and washing with running water. Dehydrating the slices, drying in the air, sealing the slices with neutral gum, and observing under a fluorescent microscope.

FIG. 13 is H & E, TUNEL immunofluorescence and Ki67 immunohistochemical analysis of tumor tissues of each group of nude mice obtained in example 13, example 14 and example 15. As can be seen from FIG. 13, the H & E results show that the TRAIL-Exo, TPL and TRAIL-Exo/TPL groups of tumor tissues all exhibited pathological lesion and necrosis characteristics compared to the control group, with the TRAIL-Exo/TPL group of tumor tissues being most significantly damaged. The TUNEL results show that the green fluorescence intensity of TRAIL-Exo, TPL and TRAIL-Exo/TPL groups is sequentially enhanced, which indicates that the apoptosis rate of tumor tissues is sequentially increased, and the TRAIL-Exo/TPL group has the highest apoptosis rate. Ki67 results show that brown yellow particles in the cell nuclei of TRAIL-Exo, TPL and TRAIL-Exo/TPL groups are reduced in sequence, which indicates that the Ki67 positive rate is reduced in sequence; among them, the TRAIL-Exo/TPL group has the least brown yellow particles in the cell nucleus, and the lowest positive rate is lower than 10%, which indicates that the malignant proliferation index of the group of tumors is the lowest.

FIG. 14 is an analysis of H & E staining of major organs after treatment with different drugs in example 13. As can be seen from FIG. 14, compared with the control group, the tissues of the TRAIL-Exo/TPL nude mice have no obvious injury, and neither liver nor kidney inflammation nor edema is found, indicating that the TRAIL-Exo/TPL has no obvious organ toxicity and has good biological safety.

Example 16: detection of expression of related protein for promoting tumor cell apoptosis

A375 cells at 1X 10⁶The cells were inoculated in 6-well plates, and after the cells were attached, TRAIL-Exo, TPL or TRAIL-Exo/TPL (TPL concentration was 50ng/mL) were added, respectively, to co-culture with the cells, without any drug treatment for the control cells. Collecting each group of cells, adding RIPA lysate to lyse the cells for 1min, centrifuging at 12000rpm for 10min, and collecting the supernatant. Total protein concentration was determined using the BCA kit. Preparing 12% separation gel and 5% concentrated gel, and performing constant-pressure 75V electrophoresis for 30min. The PVDF membrane activated by methanol is covered on the gel, 300mA constant flow membrane is carried out for 30min, the membrane is sealed by 5% skimmed milk powder for 1h, 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-kB antibody (1: 1000), Anti-Survin primary antibody (1: 5000) and VEGF antibody (1: 1000) are respectively added into each group, and the mixture is incubated overnight at 4 ℃. The membrane was washed 3 times with TBST on a shaker. Corresponding secondary HRP coat anti-mouse IgG (1: 3000) and HRP coat anti-rabbitIgG (1: 3000) antibodies were added to the respective antibodies and incubated at room temperature for 1 h. Using TBST buffer solution in dehydrationThe membrane was washed 3 times for 10min each time on a color shaker. Mixing ECL A and ECL B reagents in equal volumes in a dark room, reacting with a membrane for 5min, taking beta-actin as an internal reference, and imaging by using a gel imaging system to analyze the relative expression level of each protein.

FIG. 15 is a graph showing the results of detection of the expression of apoptosis-related proteins (a) and a schematic diagram of the molecular mechanism of synergistic anti-tumor therapy (b) in example 16. As can be seen from FIG. 15, the expression levels of caspase-3, caspase-8, caspase-9, Bax, Bid, and cytochrome c of TRAIL-Exo or TPL-treated cells were increased, and the expression levels of these proteins were further increased after TRAIL-Exo/TPL treatment, as compared to the control group. After TPL treatment, the expression of Bcl-2, NF-kappa B, VEGF and survivin is reduced, and the expression level of the proteins is further reduced after TRAIL-Exo/TPL treatment. The specific mechanism is as follows: the DR5 intracellular region contains Death Domains (DD), after TRAIL-Exo/TPL is combined with DR5, Fas Death domain related protein (FADD) and pro-caspase 8 can be collected to form a Death-inducing signaling complex (DISC), the DISC activates a large amount of caspase 8, the caspase 8 activates downstream caspase 3, and the caspase 3 acts on various substrates which can cause apoptosis, thereby causing apoptosis; in addition, partially activated caspase 8 can activate Bid to tBod, tBod is transferred to mitochondrial membrane, pro-Apoptotic factors (Bax and Bak) are activated, anti-Apoptotic factors (Bcl-2 and Bcl-XL) are inhibited, and Cytochrome c (Cytochrome c, cytoc) is released, and the cytoc interacts with Apoptotic enzyme activating factor (apoptosis protease activating factor-1, APAF-1) to form an Apoptotic complex to activate downstream caspase 9, thereby further activating caspase 3 to induce apoptosis. TPL can also induce apoptosis through the caspase 8 mediated caspase cascade and mitochondrial pathways described above. In addition, TPL can simultaneously reduce the expression of VEGF, NF-kB and survivin to play a synergistic role.

The results show that the TRAIL-Exo/TPL composite exosome can obviously inhibit the development of melanoma in vivo and in vitro, has obvious synergistic antitumor effect, reduces the toxic and side effect of TPL, and provides a new method and thought for clinically treating the melanoma.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Sequence listing

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Claims (10)

1.A composite exosome carrying a membrane-bound TRAIL protein and a small-molecule antitumor drug is characterized in that the membrane surface of the composite exosome carries the TRAIL protein, and the small-molecule antitumor drug is encapsulated in the membrane.

2. The composite exosome loaded with the membrane-bound TRAIL-related apoptosis-inducing ligand and the small-molecule antitumor drug according to claim 1, wherein the composite exosome is obtained by transfecting a donor cell of an exosome with a virus expression vector overexpressing a membrane-bound TRAIL gene to obtain a donor cell line stably overexpressing TRAIL; then separating and purifying from the culture supernatant of donor cells over expressing TRAIL to obtain exosomes expressing TRAIL with high purity, marking as TRAIL-Exo, and loading the micromolecule antitumor drug into the membrane of TRAIL-Exo to obtain the product.

3. The complex exosome loaded with membrane-bound TRAIL-related apoptosis-inducing ligand and small-molecule antitumor drug according to claim 1, wherein the concentration of TRAIL protein in the complex exosome is 205.1 ± 13.6 pg/mL; the average particle size of the composite exosome is 100-200 nm; the concentration of the small molecular antitumor drug is 0.01-60 mug/mL.

4. The complex exosome of loaded membrane-bound TRAIL and small-molecule antitumor drug according to claim 2, wherein the viral expression vector is lentivirus, retrovirus or adenovirus.

5. The complex exosome loaded with the membrane-bound TRAIL and small-molecule antitumor drug according to claim 2, wherein the donor cell is Raw264.7 cell, natural killer cell, T cell or dendritic cell.

6. The complex exosome of loaded membrane-bound TRAIL and small molecule antitumor drug according to claim 1, wherein the small molecule antitumor drug is a natural drug.

7. The complex exosome of loaded membrane-bound TRAIL and small-molecule antitumor drug according to claim 7, wherein the natural drug is triptolide TPL.

8. The method for preparing the composite exosome of the loaded membrane-bound TRAIL and small molecule antitumor drug according to claim 1, comprising the following steps:

s1, constructing a virus expression vector of an over-expression membrane-bound TRAIL gene, packaging the virus, transiently transferring 293T cells to obtain virus particles, infecting macrophages Raw264.7 with the purified virus particles to obtain a macrophage system of stable over-expression TRAIL, and marking the macrophage system as TRAIL-Raw264.7;

s2, extracting an exosome from TRAIL-Raw264.7 cell culture supernatant by adopting a gradient ultracentrifugation method to obtain an exosome expressing TRAIL with high purity, and marking as TRAIL-Exo;

s3, loading the small-molecule antitumor drug into TRAIL-Exo to obtain a composite exosome loaded with TRAIL and the small-molecule antitumor drug; the membrane surface of the compound exosome carries a tumor apoptosis promoting protein TRAIL, and a small-molecule antitumor drug is encapsulated in the membrane.

9. The method of claim 8, wherein the method for loading the small-molecule antitumor drug into TRAIL-Exo in step S3 is an ultrasonic method, an electroporation method or a co-incubation method.

10. Use of the complex exosome of the loaded membrane-bound TRAIL and small-molecule antitumor drug according to any one of claims 1 to 7 in the preparation of a therapeutic drug for malignant tumor, which is malignant melanoma of the skin.

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