Glioma-related mesenchymal stem cell exosome containing cilengitide as well as preparation method and application of glioma-related mesenchymal stem cell exosome

Abstract

The invention discloses a glioma-related mesenchymal stem cell exosome containing cilengitide as well as a preparation method and application of the glioma-related mesenchymal stem cell exosome, and belongs to the technical field of biological medicines. The prepared exosome internally contains high-concentration cilengitide and specific miRNA (such as miR-124 and miR-21 inhibitors) derived from mesenchymal stem cells, and through an improved GaMSCs-Exos delivery strategy, the cilengitide is delivered to glioma cells in a tumor microenvironment and blood vessels around the glioma cells in a targeted manner, so that the influence of a blood-brain barrier on the curative effect of a medicine is effectively avoided, and the curative effect of the medicine is improved. Meanwhile, by regulating and controlling the tumor microenvironment (such as reversing the immunosuppression state), the immunotherapy effect is enhanced. The strategy not only verifies the excellent targeting delivery performance in basic research, but also provides a new treatment thought and technical support for clinical application, and has important technical originality and clinical transformation potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to glioma-related mesenchymal stem cell exosomes containing cilengitide, and a preparation method and application thereof. Background Art

[0002] Glioblastoma is a highly malignant tumor of the central nervous system. Traditional treatments such as surgery, radiotherapy and chemotherapy often fail to achieve long-term efficacy and have a very high recurrence rate. Integrin, as a cell receptor located on the surface of tumor cells, plays an important role in regulating the adhesion, migration, invasion and angiogenesis of tumor cells, and therefore becomes an important target for the development of anti-tumor drugs. By targeting integrins, the motility of tumor cells can be interfered with, thereby inhibiting their metastatic potential. In addition, inhibiting integrins can also affect the nutrient supply of tumors, further limiting the growth and spread of tumors. Therefore, as an important therapeutic target, integrin provides broad prospects for the development of new anti-cancer therapies. Cilengitide can block tumor angiogenesis and the adhesion and migration of tumor cells by specifically inhibiting integrins. Although Cientide has shown good efficacy in other tumors, more effective strategies still need to be explored in clinical trials of gliomas. At present, the results of clinical trials of cilengitide in glioma are unsatisfactory. The CENTRIC EORTC 26071-22072 study showed that the combination of cilengitide and standard treatment did not improve the survival rate of newly diagnosed glioblastoma patients. In addition, studies on high-grade gliomas in children also showed that the efficacy of cilengitide as a single drug is limited. In our previous experiments, we found that there are a large number of integrin targets in human cerebrospinal fluid, which is enough to explain why cilengitide did not show obvious anti-tumor effects after passing through the blood-brain barrier.

[0003] Mesenchymal stem cells (MSCs) are a type of stem cells with low immunogenicity, abundant sources, and damage chemotaxis. Under the action of cytokines secreted by tumor cells and their surrounding matrix, MSCs migrate to the tumor site, aggregate, proliferate and differentiate, participate in the formation of the tumor microenvironment, and participate in the tumor process. In 2014, our research group isolated glioma-associated mesenchymal stem cells (GaMSCs) from human glioma tissue specimens and completed the relevant identification (invention patent: ZL201810878554.X). Current studies have shown that GaMSCs can promote glioma proliferation and maintain the stemness of glioma stem cells through paracrine.

[0004] Exosomes are tiny vesicles naturally secreted by cells. Due to their natural biocompatibility and low immunogenicity, they can successfully cross the blood-brain barrier, making exosomes an effective tool for drug delivery to the brain. Cilengitide is an integrin inhibitor developed by Merck. It is the first peptide drug proven to target and regulate tumor cell growth, survival and invasion. As an emerging drug delivery tool, exosomes can effectively introduce cilengitide into the central nervous system (CNS), overcoming biological barriers that other carriers cannot cross. At the same time, exosomes can accurately target tumor tissues and fuse with the membranes of target cells, effectively releasing the drugs they carry into target cells, thereby exerting a targeted therapeutic effect. Studies have shown that mesenchymal stem cells (MSCs) have the ability to migrate to the tumor microenvironment, making them ideal carriers for drug or gene delivery.

[0005] Glioma-associated mesenchymal stem cells (GaMSCs) play an important role in the tumor microenvironment, and the exosomes they secrete are considered to be important mediators of glioma progression and invasion. The exosomes of tumor-associated MSCs can not only promote the proliferation and migration of tumor cells, but also may affect the tumor microenvironment by regulating the immune response. In terms of treatment, research on exosomes of tumor-associated MSCs is gradually increasing. By regulating the composition or function of exosomes, new strategies may be provided for tumor treatment. For example, using exosomes as drug delivery carriers can effectively target anti-tumor drugs into tumor cells, thereby improving the therapeutic effect. Summary of the invention

[0006] In our previous research, based on the exosome delivery platform, we developed an exosome carrier (GaMSCs-Exos) derived from glioma mesenchyme to carry Cilengitide, a new treatment strategy for targeted inhibition of glioblastoma progression. The results showed that glioma mesenchymal exosomes carrying Cilengitide can effectively solve the problems of drug passage through the blood-brain barrier and drug targeting, providing more and more effective treatment options for glioblastoma patients.

[0007] We used glioma-associated mesenchymal stem cells (GaMSCs) as the source of exosomes and have successfully isolated and identified GaMSCs-Exos. GaMSCs-Exos can achieve precise recognition and binding to glioblastoma through specific modification of membrane proteins and surface ligands, thereby significantly improving the targeting and stability of drug delivery.

[0008] As a highly effective integrin inhibitor, cilengitide can block key signaling pathways (such as PI3K / Akt and MAPK pathways) in angiogenesis and tumor cell invasion by binding to integrins on the surface of tumor cells, thereby inhibiting tumor growth and metastasis. However, due to the presence of integrin targets in the blood-brain barrier and cerebrospinal fluid, clinical trials of cilengitide in the treatment of gliomas have failed. The GaMSCs-Exos delivery system enables cilengitide to directly target glioblastoma and effectively accumulate in the lesion area for a long time, greatly enhancing its anti-tumor activity as an integrin inhibitor, breaking through the bottleneck of existing treatment strategies in drug delivery efficiency and targeting.

[0009] The present invention provides a method for preparing glioma-related mesenchymal stem cell exosomes containing cilengitide, and the preparation method comprises the following steps:

[0010] (1) Mix GaMSCs-Exos and cilengitide at a mass ratio of 6-10:0.5-1.5;

[0011] (2) Performing ultrasonic treatment to obtain glioma-associated mesenchymal stem cell exosomes containing cilengitide.

[0012] Preferably, the step (2) further comprises the step (3) of incubating at 37° C. for 0.5-1.5 hours.

[0013] More preferably, in the step (1), the mass ratio of GaMSCs-Exos to cilengitide is 8:1.

[0014] More preferably, the preparation method of GaMSCs-Exos comprises the following steps:

[0015] (1) collecting the culture supernatant of glioma-associated mesenchymal stem cells;

[0016] (2) Differential centrifugation: 300 g × 10 min, 2000 g × 10 min, 10,000 g × 30 min, collect the supernatant in turn, and 100,000 g × 70 min to collect the precipitate. The precipitate obtained is the exosome.

[0017] The present application also provides glioma-related mesenchymal stem cell exosomes containing cilengitide prepared by the above preparation method.

[0018] The present invention also provides the use of the above-mentioned glioma-related mesenchymal stem cell exosomes containing cilengitide in the preparation of a product for treating glioblastoma.

[0019] Preferably, the product is a medicine.

[0020] More preferably, the medicine further contains medically acceptable excipients.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The exosomes prepared by the present invention contain high concentrations of cilengitide and specific miRNAs (such as miR-124 and miR-21 inhibitors) derived from mesenchymal stem cells. Through the improved GaMSCs-Exos delivery strategy, cilengitide is targeted to glioma cells in the tumor microenvironment and the blood vessels around them, effectively avoiding the effect of the blood-brain barrier on the efficacy of the drug, and at the same time, by regulating the tumor microenvironment (such as reversing the immunosuppressive state), the immunotherapy effect is enhanced. This strategy not only verifies its superior targeted delivery performance in basic research, but also provides new treatment ideas and technical support for clinical applications, with important technical originality and clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Extraction and identification of GaMSCs and exosomes in Example 1. A. Cells isolated from human glioma tumor specimens according to standard procedures grew in a spindle-shaped adherent manner; B. Cell surface markers were positive for CD73, CD90, CD105, and CD44, and negative for CD14, CD31, CD45, NG2, and PDGF-β (the horizontal axis is 10 from left to right). 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , the vertical axis is 0, 20, 40, 60, 80, 100 from bottom to top); C. At the same time, the cells can differentiate into adipocytes, chondrocytes and osteoblasts under in vitro differentiation conditions; D. Construction of intracranial xenograft tumor model of U87 cells and GaMSCs: GaMSCs are not tumorigenic, while co-culture of GaMSCs and U87 will promote tumor proliferation; E. Survival analysis of intracranial U87 and U87+GaMSCs tumors in nude mice; F. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and protein immunoblotting (WB) were used to identify the morphology, size and surface markers of exosomes.

[0024] Figure 2Preparation of the complex of Cilengitide and exosomes in Example 2. A. Morphological images of GaMSCs-Exos without drug loading (Blank) and drug loading (Cilengitide). B. The amount of Cilengitide incorporated into GaMSCs-Exos was determined by LC-MS / MS. C. Loading efficiency and stability of GaMSCs-Exos-Cilengitide. D. Image of glioma cells taking up exosomes. When co-incubated for 60 minutes, GaMSCs-Exos were more easily taken up by glioma cells than MSCs-Exos.

[0025] Figure 3 The in vitro inhibitory effect of GaMSCs-Exos-Cilengitide and MSCs-Exos-Cilengitide on glioblastoma cells in Example 3 was evaluated. A. CCK-8 experiments showed that GaMSCs-Exos-Cilengitide could more significantly inhibit the proliferation of glioma cells (U87 and U251) than MSCs-Exos-Cilengitide. B. Scratch experiments showed that GaMSCs-Exos-Cilengitide could more significantly inhibit the migration of glioma cells (U87 and U251) than MSCs-Exos-Cilengitide. C. Clone formation experiments showed that GaMSCs-Exos-Cilengitide could more significantly inhibit the proliferation of glioma cells (U87 and U251) than MSCs-Exos-Cilengitide. D. Transwell experiments showed that GaMSCs-Exos-Cilengitide could significantly inhibit the invasion of glioma cells (U87 and U251) compared with MSCs-Exos-Cilengitide.

[0026] Figure 4 The penetration and targeting effects of GaMSCs-Exos-Cilengitide on brain glioma in Example 4. A. Schematic diagram of the in vivo treatment plan in mice after successful glioma cell implantation and tumor modeling. B. In vivo imaging tracking of GaMSCs-Exos and MSCs-Exos aggregation in vivo. GaMSCs-Exos-Cilengitide is more targeted and can target intracranial glioma lesions. C. Representative bioluminescent images and quantification of fluorescent signals of intracranial tumors in nude mice after treatment with Cilengitide and GaMSCs-Exos-Cilengitide. D. Survival analysis of tumor-bearing nude mice after treatment with Cilengitide and GaMSCs-Exos-Cilengitide. DETAILED DESCRIPTION

[0027] Example 1

[0028] 1. Glioma-related mesenchymal stem cells:

[0029] (1) Pathological diagnosis was performed according to the World Health Organization standards. Surgical specimens were collected from patients with glioma (WHO grade III and IV) who had signed informed consent. Fresh glioma specimens were stored in PBS.

[0030] (2) Transfer to a sterile culture platform within 1 hour, rinse with PBS and cut the specimen into 1-2 mm fragments, then add trypsin for digestion for 30 minutes.

[0031] (3) Filter the solution through a 30 μm nylon mesh and collect the filtrate. Centrifuge at 1500 rpm for 15 minutes to collect the supernatant.

[0032] (4) Add red blood cell lysis buffer to lyse red blood cells for 5 minutes, centrifuge at 1500 rpm for 5 minutes, and collect the supernatant.

[0033] (5) The collected supernatant was then added to the upper layer of lymphocyte separation solution at a ratio of 1:2, and the mixture was centrifuged at 1500 rpm without acceleration for 20 minutes. The middle layer suspension was collected and washed three times with PBS.

[0034] (6) Use CD133 antibody labeled with MACS magnetic beads to separate the obtained negative cells.

[0035] (7) The collected cells were added to Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 μ / ml penicillin and 100 μ / ml streptomycin, and cultured in an incubator at 37°C and 5% CO2. Figure 1 A), medium change after 4 days.

[0036] 2. Collection of Glioma Cell Conditioned Medium

[0037] (1) Prepare glioma U87 cell suspension, take 2×10 5 The cells were inoculated with 10% DMEM and cultured overnight in an incubator.

[0038] (2) Remove the culture medium from the culture flask and wash the flask three times with PBS to remove the residual culture medium.

[0039] (3) Add serum-free DMEM and culture in an incubator for 48 h.

[0040] (4) Collect the culture medium in the culture flask and centrifuge at 2000 rpm for 10 minutes to remove residues and cell debris.

[0041] (5) Collect the supernatant and store at -20°.

[0042] 3. Extraction and culture of bone marrow mesenchymal stem cells:

[0043] (1) Bone marrow residual materials were collected from fracture patients. All patients had signed informed consent.

[0044] (2) Fresh specimens were stored in PBS and transferred to a sterile culture platform within 1 hour. The specimens were rinsed with PBS and cut into 1-2 mm fragments. Trypsin was added for digestion for 30 minutes.

[0045] (3) Filter through a 30-μm nylon mesh and collect the filtrate. Centrifuge at 1500 rpm for 15 minutes to collect the supernatant.

[0046] (4) Add red blood cell lysis buffer to lyse red blood cells for 5 minutes, centrifuge at 1500 rpm for 5 minutes, and collect the supernatant.

[0047] (5) The collected supernatant was added to the upper layer of lymphocyte separation solution at a ratio of 1:2, and the mixture was centrifuged at 1500 rpm without acceleration for 20 minutes. The middle layer suspension was collected and washed three times with PBS.

[0048] (6) Use CD133 antibody labeled with MACS magnetic beads to separate the obtained negative cells.

[0049] (7) The collected cells were added to Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 μ / ml penicillin and 100 μ / ml streptomycin, and cultured in an incubator at 37°C and 5% CO2. The medium was changed after 4 days.

[0050] 4. Separate and extract exosomes from cells

[0051] (1) Collect the supernatant of target cells (glioma U87 cells or glioma-associated mesenchymal stem cells or bone marrow mesenchymal stem cells).

[0052] (2) Differential centrifugation (4°C): 300 g × 10 min, 2 000 g × 10 min, 10 000 g × 30 min, 100 000 g × 70 min.

[0053] (3) The supernatant collected by differential centrifugation was mixed with PBS and centrifuged again to obtain the extracellular particles (i.e., exosomes), which were stored in a -80°C refrigerator.

[0054] 5. Observation of the extracted exosomes under electron microscope

[0055] (1) Preparation of experimental materials: extracted extracellular particle samples, ice box, tip, centrifuge tube, tweezers, copper mesh, filter paper, 2% uranyl acetate staining solution, baking lamp and transmission electron microscope.

[0056] (2) Use the pipette tip to absorb 10ul of sample suspension and place it on the copper grid.

[0057] (3) Use a pipette tip to absorb 20 μl of 2% uranyl acetate and stain for 5 minutes.

[0058] (4) Place the stained sample under a lamp for 10-15 minutes.

[0059] (5) Take photos of the samples under a transmission electron microscope.

[0060] 6. Nanoparticle Tracking Analysis (NTA)

[0061] (1) Principle: NTA determines the size of particles in a sample based on the speed of the particles during Brownian motion. The size of a single particle is calculated based on the square average of the distance a particle moves within a given time range. This technology has been successfully used to measure the distribution of particles between 30-1000nm.

[0062] (2) Take 0.25 ml of exosome sample and inject it into the observation chamber.

[0063] (3) Turn on the laser beam to illuminate the exosome solution in the observation chamber and collect the light scattering signal.

[0064] (4)NTA software analyzes the movement of particles.

[0065] (5) The radius of the measured particle is calculated using the Stokes-Einstein equation.

[0066] 7. Flow cytometry

[0067] (1) Glioma-related mesenchymal stem cells and bone marrow mesenchymal stem cells of passage 3-4 were prepared to prepare a cell suspension and the cell density was adjusted to 1.5×10 6 / ml.

[0068] (2) For each cell type, set up a blank tube, a CD105 single-labeled tube, a CD44 single-labeled tube, a CD73 single-labeled tube, a CD90 single-labeled tube, a CD34 single-labeled tube, a CD14 single-labeled tube, a CD31 single-labeled tube, a PDGFR-β single-labeled tube, a sample test tube, and a corresponding isotype control tube. Take 100 ul of the cell suspension and add it to the flow cytometry tube.

[0069] (3) Place in the flow cytometry antibody chamber to rewarm.

[0070] (4) Add anti-CD31-PE / Cy7, anti-CD34-FITC, anti-CD73-APC / Cy7, anti-CD90-PE / Cy7, anti-CD14-percp, anti-CD105-APC, anti-CD44-APC / Cy7, and anti-PDGFR-β-PE to the single-label tubes, respectively. Add the above antibodies to each sample tube, and add the corresponding isotype control antibody to the isotype control tube. After shaking and mixing, incubate at 4°C in the dark for 30 min.

[0071] (5) Add 1 ml of PBS, shake to mix, centrifuge at 1500 rpm to resuspend, and repeat twice.

[0072] (6) Add 200 μl of PBS to resuspend the cells and vortex to mix.

[0073] (7) Flow cytometry was used to detect the expression of CD105, CD44, CD73, CD90, CD34, CD14, CD31 and PDGFR-β in glioma-related mesenchymal stem cells and bone marrow mesenchymal stem cells. Figure 1 B).

[0074] 8. Induce adipogenesis, osteogenic and chondrogenic differentiation

[0075] (1) Inducing differentiation of adipocytes, osteoblasts and chondrocytes The differentiation of glioma-associated mesenchymal stem cells and bone marrow-derived mesenchymal stem cells was induced using the existing adipocyte, osteoblast and chondrocyte differentiation induction medium according to the instructions of the instructions.

[0076] (2) Stain with Oil Red O, West Red, and Alcian Blue, respectively. Figure 1 C).

[0077] 9. Construction of intracranial xenograft tumor model with U87 cells and GaMSCs

[0078] (1) After nude mice were anesthetized with 4% chloral hydrate, they were fixed with a stereotaxic apparatus to fully expose the surgical site on the head.

[0079] (2) Disinfect the corresponding parts with 0.5% iodine tincture, cut the scalp forward along the midline of both ears, with a length of about 1 cm, and use sterile forceps to separate the scalp to expose the anterior fontanelle.

[0080] (3) Calculate and use a stereotaxic apparatus to find the needle insertion point. The coordinates are approximately 1 mm in front of the anterior fontanelle and approximately 2 mm to the side. The needle insertion depth is approximately 3.5 mm.

[0081] (4) Use a micro syringe to suck out about 1*10 5After preparing a suspension of U87 or U87+GaMSCs (quantity ratio 1:1), slowly inject it at a speed of about 1ul / min. After the injection, wait for five minutes before pulling out the needle of the microinjector.

[0082] (5) Seal the injection port with artificial bone wax, disinfect and suture.

[0083] (6) After the nude mice were marked, they were placed in an SPF environment and continued to be raised. The growth and survival of the mice were dynamically observed. The results are shown in Figure 1 D. 1E.

[0084] 10. Western Blot (WB)

[0085] (1) Protein extraction and concentration measurement

[0086] Every 10 7 Add about 120 μl of protein lysis buffer (RIPA:PMSF:phosphatase inhibitor 98:1:1) to the exosomes of glioma-related mesenchymal stem cells to be tested, lyse on ice for 30 minutes, and then use ultrasonic vibration for 10 seconds. Determine the protein concentration by BCA method. Add 5× protein loading buffer to the protein supernatant, denature the protein in a 100℃ metal bath for 7 minutes, and store in a -80℃ ultra-low temperature refrigerator for later use.

[0087] (2) BCA method for protein concentration determination

[0088] Dissolve the protein standard sample (30mg BSA) in double distilled water to make it a 25mg / ml standard solution (can be frozen for long-term storage), and dilute the 25mg / ml standard solution to 0.5mg / ml when using. Mix BCA reagent A and reagent B in a volume ratio of 50:1 to prepare BCA working solution. Add 0, 1, 2, 4, 8, 12, 16, 20μl of the standard to a 96-well plate, and add RIPA to make each well up to 20μl. Add 2μl of sample to a 96-well plate and RIPA to make up to 20μl. Add 200μl of BCA working solution to each well, place it in a 37℃ incubator for 30min, and then measure the absorbance value at any wavelength between 540-595nm in an ELISA reader. The concentration of the protein is drawn based on the standard curve obtained from the absorbance value results.

[0089] (3) Protein electrophoresis

[0090] The concentration of the electrophoresis gel is determined by the molecular weight of the target protein. Assemble the plate rack and gel plate to avoid leakage, then put them into the electrophoresis tank and add electrophoresis solution. Add protein ladder and appropriate amount of sample to the sample well. Turn on the machine and start electrophoresis (120V). According to the molecular weight of the desired molecule and the electrophoresis position of the protein marker, terminate the electrophoresis at the appropriate time.

[0091] (4) Transfer

[0092] Use a plate-shaped tool to gently lift the small glass plate, and then cut off the concentrated gel. Refer to the protein marker, cut the separation gel according to the molecular weight of the target protein, and cut the PVDF membrane. The PVDF membrane needs to be fully soaked in analytical grade methanol for 5 minutes, which is the activation step. Place the gel and PVDF membrane in the transfer clip, and avoid bubbles during the operation. Place the assembled clip in the transfer tank, paying attention to the positive and negative directions. Add transfer liquid to the transfer tank and add an ice box at the same time. After the transfer liquid overflows the upper edge of the transfer clip, cover the lid, transfer the transfer tank to a container filled with ice, and start transferring. The transfer current is 200mA and the time is 90 minutes.

[0093] (5) Blocking and primary and secondary antibody incubation

[0094] After the transfer is completed, the transfer effect is preliminarily judged by protein marker, and the PVDF membrane is soaked in 5% skim milk prepared in TBST, blocked for 1 hour (25°C), then washed with TBST and incubated with the corresponding primary antibody. The primary antibody incubation condition is incubated overnight on a shaker at 4°C. After the primary antibody incubation is completed, the membrane is washed with TBST and incubated with the secondary antibody at room temperature for 60 minutes.

[0095] (6) Development

[0096] After the secondary antibody incubation, wash the membrane thoroughly with TBST. At the same time, prepare the ECL colorimetric solution and evenly drip it on the surface of the PVDF strips, then use the Bio-Rad imager for development. The results are shown in Figure 1 F.

[0097] Example 2

[0098] 1. Exosome drug loading process:

[0099] (1) Ultrasonic treatment separated GaMSCs-Exos or MSCs-Exos were mixed with the drug Cilengitide (drug to exosome mass ratio 1:8) and then ultrasonicated using a homogenizer probe. The drug loading of the ultrasonic loading method utilizes mechanical shear force to diffuse into the exosomes through the deformed membrane. The integrity of the exosome membrane is reduced after ultrasonic treatment. Although the membrane microviscosity is significantly reduced after ultrasonic treatment, the ultrasonic treatment process does not affect the membrane-bound proteins and lipid content of the exosomes. In addition, the integrity of the exosome membrane can be restored by incubation at 37°C for 1 hour. For morphological pictures of GaMSCs-Exos without drug loading (Blank) and drug loading (Cilengitide), see Figure 2 A.

[0100] (2) High performance liquid chromatography (HPLC): The drug loading efficiency (i.e., the incorporated amount) of GaMSCs-Exos was detected to be 75%.

[0101] 2. The stability of GaMSCs-Exos-Cilengitide can be found in Figure 2 C.

[0102] 3. Immunofluorescence experimental steps for exosome phagocytosis (1) Exosome fluorescence labeling

[0103] ① Add GaMSCs-Exos or MSCs-Exos (10-100 μg) into 1 mL PBS.

[0104] ② Add an appropriate amount of fluorescent dye (such as PKH26, according to the recommended concentration in the reagent manual, usually 1:250 dilution).

[0105] ③ Mix gently and incubate at room temperature in the dark for 5-10 minutes.

[0106] ④ Remove unbound dye by ultracentrifugation (e.g., 100,000×g, 70 min) or density gradient centrifugation and recover the stained exosomes.

[0107] ⑤ Resuspend the exosomes in serum-free medium and adjust the concentration to the experimental requirements.

[0108] (2) Cell culture

[0109] The target cells U87 and U251 were seeded on coverslips in 24-well plates or 6-well plates and cultured to about 50-70% confluence. The cell culture medium was replaced with serum-free medium to reduce the interference of serum on the uptake of exosomes.

[0110] (3) Incubation of exosomes and cells

[0111] ① Add labeled exosomes to the target cells, with a final concentration of 10-50 μg / mL.

[0112] ②Incubate in a cell culture incubator at 37°C for 1 hour.

[0113] ③After incubation, gently wash the cells three times with serum-free medium to remove unbound exosomes.

[0114] (4) Fixation and staining

[0115] ① Fix the cells with 4% paraformaldehyde for 10-15 minutes at room temperature.

[0116] ② Rinse with PBS three times, 3 minutes each time.

[0117] ③Optional:

[0118] Use Triton X-100 (0.1%) to permeabilize the cell membrane (if it is necessary to observe the intracellular structure).

[0119] Perform staining according to the experimental design:

[0120] Nuclei were stained with DAPI or Hoechst 33342 and incubated at room temperature in the dark for 10 minutes.

[0121] If you need to label the cell membrane or organelles, you can add specific fluorescent antibodies (such as antibodies against membrane proteins or lysosomes), incubate in the dark for 30 minutes, and then wash.

[0122] (5) Microscope observation

[0123] ① Install the corresponding filter on the microscope (such as the red channel to observe PKH26-labeled exosomes, and the blue channel to observe DAPI-stained cell nuclei).

[0124] ② Use confocal microscopy or fluorescence microscopy to take images. The results are shown in Figure 2 D, The distribution of exosomes around cells and their localization within cells can be observed.

[0125] (6) Data analysis

[0126] Evaluation of exosome uptake efficiency by microscopic images:

[0127] Whether the exosome fluorescence signal overlaps with the cell nucleus or organelle markers (successful endocytosis).

[0128] If quantitative analysis is required, the distribution of exosomes can be quantitatively analyzed in combination with fluorescence intensity measurement software (such as ImageJ).

[0129] Example 3

[0130] Add 1*10 5 GaMSCs-Exos-Cilengitide and MSCs-Exos-Cilengitide were added, and then appropriate amounts of glioblastoma cells were added and incubated for 1 hour. After the incubation, the following tests were performed:

[0131] 1. CCK-8 detection of cell proliferation level

[0132] (1) Take the treated glioma cells, digest and centrifuge the cells, discard the supernatant, and adjust the cell concentration to 1×10 5 Then use a sterile tip to draw up 100ul of the cell mixture and add it to a 96-well plate, so that the number of cells in each well of the 96 wells is 1×10 4 indivual.

[0133] (2) At different times (0h, 24h, 48h and 72h), 10ul of CCK-8 cell proliferation detection reagent was added to the wells of the 96-well plate.

[0134] (3) Continue to culture the above cells for 4 hours and gently shake the 96-well plate to mix the detection reagents in the wells, then place it on an ELISA reader for detection and record the OD450 absorbance data.

[0135] 2. Transwell assay to detect cell invasion ability

[0136] (1) Dissolve Matrigel in advance, mix Matrigel and serum-free medium in a volume ratio of 3:1, then use a sterile tip to take 40ul of the mixture and add it to the pre-cooled Transwell chamber and wait for the Matrigel to solidify.

[0137] (2) Take the treated glioma cells, digest and centrifuge the cells, discard the supernatant, and adjust the cell concentration to 1×10 5 / ml, put 600ul of serum-containing complete culture medium into the lower chamber, then put the Transwell chamber into the well with complete culture medium, and then add 100ul of serum-free cell mixture into the Transwell chamber with Matrigel matrix gel.

[0138] (3) After about 13 hours of cell culture, the chamber was removed and the Matrigel matrix gel was gently wiped with a cotton swab. Then, the cells were fixed with 4% paraformaldehyde for 10 minutes. The liquid in the chamber was sucked out with a tip pipette and washed with PBS. The cells were stained with crystal violet for 20 minutes. The liquid in the chamber was sucked out with a tip pipette and washed once with PBS. Finally, the liquid in the chamber was sucked out and the chamber was dried.

[0139] (4) Take photos and count them under a microscope.

[0140] 3. Clone formation:

[0141] (1) After trypsin digestion of cells in the logarithmic growth phase, resuspend them in complete medium (basal medium + 10% fetal bovine serum) to form a cell suspension, and count them;

[0142] (2) Cell seeding: 400–1,000 cells / well (depending on cell growth conditions, generally 700 cells / well) were seeded in each experimental group in a 6-well culture plate.

[0143] (3) Continue culturing until 14 days or until the number of cells in most individual clones is greater than 50, changing the medium every 3 days and observing the cell status;

[0144] (4) After cloning was completed, the cells were photographed under a microscope, then washed once with PBS, 1 mL of 4% paraformaldehyde was added to each well for fixation for 30-60 min, and washed once with PBS;

[0145] (5) Add 1 ml of crystal violet stain to each well and stain the cells for 10-20 minutes.

[0146] 4. Scratch test

[0147] (1) Marking: First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, using a ruler to measure, approximately every 0.5 to 1 cm, across the holes, with at least 5 lines across each hole;

[0148] (2) Plating: Cells in the logarithmic growth phase are trypsinized into a single cell suspension and inoculated into a 6-well culture plate; 6 cells are plated per well, and the inoculation principle is that the fusion rate reaches 100% after overnight, and the final total volume of culture medium per well is 2 mL;

[0149] (3) The cells were cultured in a 37°C, 5% CO2 incubator for 24 h;

[0150] (4) Scratching: On the second day, use a 200 μL pipette tip to scratch the 6-well plate cover or ruler parallel or perpendicular to the horizontal line on the back. The pipette tip should be vertical and not tilted.

[0151] (5) Washing: Wash the cells three times with PBS, remove the scratched cells, and add serum-free culture medium;

[0152] (6) Photographing: Wipe off the horizontal scratch marks on the back of the 6-well plate. Take photos under a 4x microscope, ensuring that the scratch marks are centered and vertical, and pay attention to the consistency of the background. Add the sample to be tested, set the concentration gradient, and take samples at 0 and 24 h, and take photos.

[0153] (7) Result analysis: After opening the image using Image J software, 6 to 8 horizontal lines were randomly drawn to calculate the mean of the intercellular distance or the mean of the scratch area.

[0154] The results of the above experiments can be found in Figure 3 .

[0155] Example 4 Animal Experiment

[0156] (1) After nude mice were anesthetized with 4% chloral hydrate, they were fixed with a stereotaxic apparatus to fully expose the surgical site on the head.

[0157] (2) Disinfect the corresponding parts with 0.5% iodine tincture, cut the scalp forward along the midline of both ears, with a length of about 1 cm, and use sterile forceps to separate the scalp to expose the anterior fontanelle.

[0158] (3) Calculate and use a stereotaxic apparatus to find the needle insertion point. The coordinates are approximately 1 mm in front of the anterior fontanelle and approximately 2 mm to the side. The needle insertion depth is approximately 3.5 mm.

[0159] (4) Use a microsyringe to suck up about 10 μl of glioma cell suspension and then slowly inject at a speed of about 1 ul / min. After the injection is complete, wait five minutes before pulling out the microsyringe needle.

[0160] (5) Seal the injection port with artificial bone wax, disinfect and suture.

[0161] (6) After marking the nude mice, place them in an SPF environment and continue to raise them, and dynamically observe their growth status and survival status.

[0162] (7) Prepare 1*10 9 GaMSCs-Exos-Cilengitide or an equivalent amount of Cilengitide was injected intraperitoneally every three days until the mice died. Animal red grape imaging was used to track the absorption process of exosomes loaded with drugs and tumor proliferation. The results are shown in Figure 4 .

[0163] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing glioma-related mesenchymal stem cell exosomes containing cilengitide, characterized in that: The preparation method comprises the following steps: (1) Mix GaMSCs-Exos and cilengitide at a mass ratio of 6-10:0.5-1.5; (2) Performing ultrasonic treatment to obtain glioma-associated mesenchymal stem cell exosomes containing cilengitide.

2. The preparation method according to claim 1, characterized in that: The step (2) further comprises the step (3) of incubating at 37° C. for 0.5-1.5 hours.

3. The preparation method according to claim 2, characterized in that: In the step (1), the mass ratio of GaMSCs-Exos to silengitide is 8:

1.

4. The preparation method according to any one of claims 1 to 3, characterized in that The preparation method of GaMSCs-Exos comprises the following steps: (1) Collecting the culture supernatant of glioma-associated mesenchymal stem cells; (2) Differential centrifugation: 300 g × 10 min, 2000 g × 10 min, 10,000 g × 30 min, collect the supernatant in turn, and 100,000 g × 70 min to collect the precipitate. The precipitate obtained is the exosome.

5. Glioma-associated mesenchymal stem cell exosomes containing cilengitide prepared by the preparation method according to any one of claims 1 to 3.

6. Glioma-associated mesenchymal stem cell exosomes containing cilengitide prepared by the preparation method according to claim 4.

7. Use of the glioma-related mesenchymal stem cell exosomes containing cilengitide according to claim 5 in the preparation of a product for treating glioblastoma.

8. Use of the glioma-related mesenchymal stem cell exosomes containing cilengitide according to claim 6 in the preparation of a product for treating glioblastoma.

9. The use according to claim 7 or 8, characterized in that: The product described is a drug.

10. The use according to claim 9, characterized in that: The medicine also contains medically acceptable auxiliary materials.

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