Preparation for sirna and drug co-delivery system for efficiently targeting glioma initiating cells, and use

Abstract

Preparation for an siRNA and drug co-delivery system for efficiently targeting glioma initiating cells, and a use; D VAP targeting peptide modification is performed on exosomes by utilizing a click chemistry method, siRNA and mitoxantrone are loaded into the exosomes, and construction is performed to obtain a target-modified drug carrier exosome DVAP-Exo / siRNA / MIT. The target-modified exosome co-carries chemotherapeutic mitoxantrone, which has a strong killing effect on glioma initiating cells and can trigger immunogenic cell death, and a small interfering nucleic acid (siNotch 1), which is capable of reducing glioma initiating cell stemness; targeted tracking and killing of residual glioma initiating cells and tumor cells are achieved, and a new avenue is provided for glioblastoma treatment.

Description

Preparation and application of a siRNA co-delivery system that efficiently targets glioma-initiating cells Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the preparation and application of a siRNA co-delivery drug system that efficiently targets glioma initiating cells. Background Art

[0002] Glioblastoma (GBM) is a highly malignant and invasive primary tumor. It is difficult to achieve 100% resection of tumor cells during surgery. Residual glioma-initiating cells (GIC) and glioma cells are the root causes of tumor recurrence. Therefore, finding a method that can simultaneously target and kill residual GICs and glioma cells is the key to inhibiting the recurrence of brain gliomas. The standard clinical treatment for GBM is surgical resection supplemented with postoperative radiotherapy and chemotherapy. Currently, there are some relatively mature treatment options for brain gliomas, including temozolomid (TMZ), procarbazine, lomustine and vincristine (PCV) and mTOR (mammalian target of Rapamycin, mTOR) inhibitors. Temozolomide, a small molecule alkylating agent, is a first-line chemotherapy drug for gliomas. A 2005 study demonstrated that TMZ combined with radiotherapy significantly prolonged the median survival of glioma patients compared with radiotherapy alone. However, due to the emergence of drug resistance, the median survival rate remains low. PCV, a combination of chemotherapy drugs, has shown promising therapeutic effects in the treatment of recurrent low-grade gliomas due to its potential to alleviate clinical symptoms. mTOR inhibitors directly target mTOR, inhibiting the activity of activated mTOR and its downstream proteins, thereby interfering with tumor cell growth, differentiation, and metabolism. Temsirolimus and everolimus are small molecule inhibitors that block mammalian mTOR, thereby regulating glioma growth and progression. However, due to the development of resistance mechanisms, clinical trials of mTOR inhibitors have shown very limited efficacy in gliomas. In addition to the established treatment strategies mentioned above, a number of promising emerging strategies have been developed, including tumor treating fields, immunotherapy, and tyrosine kinase inhibitors, but none have yet demonstrated promising results in clinical trials. Mitoxantrone (MIT) has a potent cytotoxic effect on GICs and glioma cells, causing cell death by interfering with nucleic acid synthesis and double-strand break repair. The Notch 1 pathway is highly activated in GICs and plays a key role in regulating their proliferation and stemness. Anticancer drugs such as doxorubicin and paclitaxel have numerous limitations, including poor solubility in aqueous solutions, nonspecific systemic distribution, and cancer cell resistance. Therefore, single-drug cancer treatment strategies are incomplete.

[0003] Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a preparation and application of a siRNA co-delivery system that efficiently targets glioma initiating cells.

[0005] The technical solution adopted by the present invention is: an exosome drug delivery system loaded with mitoxantrone and siRNA, D VAP-targeting peptide-modified exosomes loaded with siRNA and mitoxantrone (MIT).

[0006] Preferably, the siRNA sequences are shown as SEQ ID No. 1 and SEQ ID No. 2.

[0007] The method for constructing an exosome drug delivery system loaded with mitoxantrone and siRNA comprises the following steps:

[0008] Step 1: D VAP-targeted peptide modified exosomes D VAP-Exo;

[0009] Step 2: Load siRNA and MIT into D In VAP-Exo, D VAP-Exo / MIT / siRNA.

[0010] Preferably, in step 1, DBCO is first connected to the outer surface of exosomes to obtain DBCO-Exo, and then D After VAP-N3 was mixed and incubated with DBCO-Exo, D VAP-targeting peptide-modified exosomes D VAP-Exo.

[0011] Preferably, 10 μM DBCO-sulfo-NHS is dissolved in DMSO, added to exosomes resuspended in PBS, reacted at 25°C and 120 rpm for 4 h, and excess DBCO-sulfo-NHS is removed to obtain DBCO-Exo;

[0012] Modified with azide group D VAP-N3 was incubated with DBCO-Exo at 4°C and shaker at 100 rpm to eliminate free peptides and prepare D VAP-Exo.

[0013] Preferably, in step 2, the siRNA and MIT are encapsulated into the modified exosomes by a co-incubation and transfection reagent combination method.

[0014] Preferably, it specifically includes:

[0015] Step 1: Follow D VAP-Exo:MIT:siRNA=10:8:1 ratio MIT, DVAP-Exo and siRNA were mixed, and Exo-fect transfection reagent was added and incubated at 37°C for 20 minutes. Then, ExoQuick-TC reagent was added and the mixture was placed on ice for 30 minutes.

[0016] Step 2: Centrifuge at 4°C and 13000 rpm for 3 min, discard the supernatant and retain the precipitate, add appropriate amount of PBS to resuspend, and dialyze the obtained system in a dialysis bag for 10 h to obtain D VAP-Exo / MIT / siRNA.

[0017] Application of exosome drug delivery system loaded with mitoxantrone and siRNA in the preparation of anti-glioma drugs.

[0018] Preferably, the exosome drug delivery system loaded with mitoxantrone and siRNA is capable of acting on residual glioma-initiating cells and / or glioma cells.

[0019] The advantages and positive effects of the present invention are: using targeted modified exosomes to co-load mitoxantrone and siRNA, mitoxantrone is a chemotherapy drug that can trigger immunogenic cell death, and the formed drug delivery system has a strong killing effect on glioma-initiating cells, and siRNA is a small interfering nucleic acid (siNotch 1) that can reduce the stemness of glioma-initiating cells, thereby achieving targeted tracking and killing of residual glioma-initiating cells and tumor cells, providing a new strategy for the treatment of glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Exosome targeted delivery system;

[0021] Figure 2 Characterization of exosomes; (A) Transmission electron microscopy image of exosomes; (B) Particle size distribution of exosomes; (C) Westren blot image of surface-specific molecules of cells and exosomes;

[0022] Figure 3 Characterization of the exosome-targeted drug delivery system; (A) Flow cytometry analysis of targeted exosome modification; (B) Transmission electron microscopy image of the drug delivery system; (C) Particle size distribution of the drug delivery system; Scale bar: 100 nm;

[0023] Figure 4 Stability of siRNA and MIT; (A) Agarose gel electrophoresis of siRNA after incubation with RNase A for different times; (B) Release curve of MIT at pH = 7.4;

[0024] Figure 5 Confirmation of the phenotype of glioma-initiating cell spheres; (A) Western blot of specific molecules in U87 cell spheres; (B) Immunofluorescence of CD133 and Notch 1 in U87 cell spheres; Scale bar: 200 μm.

[0025] Figure 6 In vitro targeting evaluation; (A) Uptake diagram of each drug-dosing group in HA1800 cells; (B) Uptake diagram of each drug-dosing group in U87 cells; DiI labeled exosomes (red), DAPI labeled cell nuclei (blue); Scale bar: 20 μm.

[0026] Figure 7 Uptake diagram of each drug-treated group in U87 cell spheroids; DiI-labeled exosomes (red); scale bar: 100 μm.

[0027] Figure 8: Uptake of the drug delivery system in U87 cells; (A) Uptake of each drug delivery group in U87 cells; (B) Quantitative fluorescence analysis of Cy3-siRNA and MIT in U87 cells; Scale bar: 20 μm; DAPI-labeled cell nuclei (blue), Cy3-labeled siRNA (red), and MIT fluorescence (green); ** P<0.01, *** P < 0.001, n = 3;

[0028] Figure 9 shows drug delivery system uptake in U87 cell spheroids; (A) Uptake of each drug delivery group in U87 cell spheroids; (B) Quantitative fluorescence analysis of Cy3-siRNA and MIT in U87 cell spheroids; Scale bar: 200 μm; DAPI-labeled cell nuclei (blue), Cy3-labeled siRNA (red), and MIT fluorescence (green). ** P < 0.01, n = 3;

[0029] Figure 10 Uptake mechanism of targeted modified exosomes in U87 cells; (A) Uptake mechanism of VE in U87 cells; (B) Quantitative analysis of uptake mechanism; DiI labeled exosomes (red), DAPI labeled cell nuclei (blue); Scale bar: 20 μm; *** P < 0.001, n = 3;

[0030] Figure 11 Uptake mechanism of targeted modified exosomes in U87 cell spheroids; (A) Uptake mechanism of VE in U87 cell spheroids; (B) Quantitative analysis of uptake mechanism; DiI labeled exosomes (red), DAPI labeled cell nuclei (blue); * P < 0.05, n = 3;

[0031] Figure 12 Colocalization analysis of siRNA, MIT, and lysosomes in U87 cells; (A) Confocal microscopy images of U87 cells after VEMR uptake; (B) Quantitative analysis of colocalization coefficients using ImageJ; Scale bar: 20 μm; Lysotracker labeled lysosomes (green), DiI labeled exosomes (red), DAPI labeled cell nuclei (blue), and MIT fluorescence (purple); * P<0.05, **P < 0.01, n = 3;

[0032] Figure 13 Colocalization analysis of siRNA, MIT, and lysosomes in U87 cell spheroids; (A) Confocal microscopy image of VEMR uptake in U87 cell spheroids; (B) Quantitative analysis of colocalization coefficients using ImageJ; Scale bar: 200 μm; Lysotracker labeled lysosomes (green), DiI labeled exosomes (red), DAPI labeled cell nuclei (blue), and MIT fluorescence (purple); * P<0.05, ** P < 0.01, n = 3;

[0033] Figure 14 D Evaluation of gene silencing effects of VAP-Exo / siRNA in U87 cell spheres and GL261 cells; (A) qRT-PCR detection of Notch 1 mRNA expression; (B) Western blot detection of Notch 1 protein expression; * P<0.05, ** P<0.01, *** P < 0.001, n = 3;

[0034] Figure 15 CCK-8 assay to detect the safety of exosome-targeted drug delivery system on HUVEC and U87 cells;

[0035] Figure 16 In vitro cell proliferation evaluation; (A) CCK-8 assay to detect the effect of exosome-targeted drug delivery system on U87 cell proliferation; (B) CCK-8 assay to detect the effect of exosome-targeted drug delivery system on U87 cell sphere proliferation; ns no significant difference, * P<0.05, ** P<0.01, *** P < 0.001, n = 3;

[0036] Figure 17 Evaluation of cell migration ability; (A) Images of scratches in U87 cells and quantitative analysis of distances in each drug-treated group; (B) Images of scratches in U87 cell spheroids and quantitative analysis of distances in each drug-treated group; Scale bar: 200 μm; ns: No significant difference. * P<0.05, *** P < 0.001, n = 3;

[0037] FIG18 is a flow cytometry analysis of the effects of each drug-administered group on U87 cell apoptosis;

[0038] Figure 19 is a flow cytometry analysis of the effects of each drug-administered group on apoptosis in U87 cell spheres;

[0039] Figure 20 Small animal in vivo imaging experiment; (A) Small animal in vivo imaging scan; (B) Fluorescence signal quantitative analysis; **P < 0.01, n = 5;

[0040] Figure 21 H&E staining of brain tissue sections; (A) H&E staining images of brain tissue sections of mice in each group; (B) Quantitative analysis of tumor size in each group; * P<0.05, ** P<0.01, *** P < 0.001, n = 5;

[0041] Figure 22 TUNEL staining of brain tissue sections; (A) TUNEL staining of mouse brain tissue sections; (B) Fluorescence quantitative analysis; cell nuclei (blue), apoptotic nuclear fragments (green); Scale bar: 20 μm; * P<0.05, ** P<0.01, *** P < 0.001, n = 5;

[0042] Figure 23 Changes in body weight of mice in different drug-dosing groups;

[0043] Figure 24 H&E staining of sections of major mouse organs; scale bar: 100 μm;

[0044] Figure 25 Detection of liver and kidney function indicators in mice. DETAILED DESCRIPTION

[0045] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0046] The present invention relates to the preparation and application of a siRNA co-delivery drug system that efficiently targets glioma initiating cells. D VAP-targeting peptide-modified exosomes encapsulate siRNA and MIT, forming an exosome-based drug delivery system loaded with mitoxantrone and siRNA. This drug delivery system utilizes targeted, modified exosomes to co-load the chemotherapy drug mitoxantrone, which has a potent killing effect on glioma-initiating cells and can trigger immunogenic cell death, and siNotch 1, which can reduce the stemness of glioma-initiating cells (GICs). This achieves targeted tracking and killing of residual glioma-initiating cells and tumor cells, safely and efficiently delivering siNotch 1 and MIT, thereby inhibiting the recurrence of brain gliomas and achieving the goal of combined gene and chemotherapy therapy for brain gliomas.

[0047] The method for constructing an exosome drug delivery system loaded with mitoxantrone and siRNA is to first separate the exosomes by differential ultracentrifugation and then use click chemistry to D VAP targeting peptides are covalently coupled to the surface of exosomes to form D VAP-Exo, through the co-incubation and transfection reagent combined method for drug loading, siRNA and mitoxantrone were loaded into DIn VAP-Exo, targeted modified drug-loaded exosomes were obtained ( D The preparation process of the exosome targeted drug delivery system (VAP-Exo / siRNA / MIT, VEMR) is shown in Figure 1.

[0048] The specific steps include:

[0049] Step 1: D VAP-targeted peptide modified exosomes D VAP-Exo; first connect DBCO to the outer surface of exosomes to obtain DBCO-Exo, and then D After VAP-N3 and DBCO-Exo were mixed and incubated at 4°C and 100 rpm on a shaker to eliminate free peptides, the obtained peptides were prepared. D VAP-targeting peptide-modified exosomes D VAP-Exo.

[0050] Step 2: Encapsulate siRNA and MIT into exosomes. Use the co-incubation and transfection reagent combination method to encapsulate siRNA and MIT into the modified exosomes and obtain them after purification. D VAP-Exo / MIT / siRNA.

[0051] The prepared targeted modified drug-loaded exosomes were tested and found to have intact structure, intact morphology, and slightly increased particle size. The exosomes had a good protective effect on siRNA and were not easily degraded by nucleases. The MIT in the drug-loaded system was stably released within 48 hours, with good release stability. D VAP peptide specifically binds to the receptor GRP78 protein, enhancing the uptake of targeted exosomes by glioma-initiating cells. Encapsulation of siRNA and MIT does not affect the targeting ability of the exosome drug delivery system; exosomes can carry siRNA and MIT and be successfully taken up by glioma-initiating cells. D VAP-Exo / siNotch 1 has a significant silencing effect on the Notch 1 gene; the exosome-targeted drug delivery system has a significant anti-tumor effect in vivo, and the exosome-targeted drug delivery system has good biosafety.

[0052] The exosome drug delivery system loaded with mitoxantrone and siRNA can be used to prepare anti-glioma drugs. Among them, mitoxantrone has a strong killing effect on glioma initiating cells and brain glioma cells; the Notch1 pathway is highly activated in glioma initiating cells and plays an important role in regulating the proliferation and stemness maintenance of glioma initiating cells. The small interfering nucleic acid (siNotch1) that can reduce the stemness of glioma initiating cells is carried together with mitoxantrone through exosomes to target and track glioma initiating cells and tumor cells. Among them, the siRNA sequences are shown in SEQ ID No.1 and SEQ ID No.2, D The VAP targeting peptide sequence is shown in SEQ ID No.3.

[0053] SEQ ID No.1 UGCCAAAUGCCUGCCAGAATT(sense)

[0054] SEQ ID No.2 UUCUGGCAGGCAUUUGGCATT(Antisense)

[0055] SEQ ID No. 3 D S D N D T D R D V D A D P

[0056] The present invention is described below with reference to the accompanying drawings. Experimental methods without specific operating steps are carried out in accordance with the corresponding product specifications. Unless otherwise specified, the instruments, reagents, and consumables used in the examples can be purchased from commercial companies.

[0057] Example 1: Construction and characterization of exosome-targeted drug delivery system

[0058] Mouse mononuclear macrophage RAW264.7 cells were provided by Professor Zhao Jingwen from the Department of Gastroenterology, Tianjin Medical University General Hospital, and cultured in high-glucose DMEM medium containing 10% exosome-free fetal bovine serum, 1% double-antibody (penicillin-streptomycin), and 1% non-essential amino acids.

[0059] 1.1 Acquisition and characterization of exosomes

[0060] Well-grown RAW264.7 cells were cultured. When the cell density reached 90-95%, the mouse monocyte-macrophage culture supernatant was collected and aliquoted into 50 mL sterile centrifuge tubes. The supernatant was first centrifuged at 2000 g for 20 minutes at 4°C to remove live and dead cells. Cell debris was then removed by centrifugation at 10,000 g for 30 minutes at 4°C, and the supernatant was collected. After gradient centrifugation, the supernatant was filtered through a 0.22 μm filter into an ultracentrifuge tube and centrifuged at 100,000 g for 70 minutes at 4°C. The supernatant was discarded, and after ensuring that no residual supernatant remained, the supernatant was resuspended in 150 μL of PBS. Three tubes were resuspended again in 100 μL of PBS. Finally, exosomes were collected into 1.5 mL EP tubes. Exosome concentration was determined using a BCA assay and stored at -80°C. The morphology of the exosomes was characterized using transmission electron microscopy. The results are shown in Figure 2A , which show that exosomes are typical “disc-shaped” with a double-layer membrane structure.

[0061] 5 μL of exosomes were diluted with PBS to a volume of 1 mL, and the exosome size distribution was examined using nanoparticle tracking analysis. The results, as shown in Figure 2B, showed that the exosome size was approximately 124 nm.

[0062] Exosome surface markers were determined by Western blot. First, exosomes and cells were lysed using high-efficiency RIPA tissue / cell lysis buffer in a 4°C refrigerator on a shaker for 30 minutes. The lysate was collected into an EP tube and centrifuged at 12,000 rpm for 5 minutes. The precipitate was discarded and the supernatant was collected for protein concentration determination using a BCA protein assay. The extracted cell supernatant and exosome solution were mixed with 5× Loading Buffer in a 4:1 ratio, respectively, and heated at 100°C for 5 minutes to denature the proteins. Western blot analysis of the exosome surface markers CD9, TSG101, and CD63 was performed. As shown in Figure 2C, exosomes highly expressed CD9, TSG101, and CD63. The various characterizations of the exosomes demonstrated successful extraction and isolation.

[0063] 1.2 Targeted modification and characterization of exosomes

[0064] The targeting peptide was modified onto the surface of exosomes by click chemistry reaction. First, 10 μM DBCO-sulfo-NHS was dissolved in DMSO and added to the exosomes resuspended in PBS. The reaction was carried out at 25°C and 120 rpm for 4 hours. The reaction mixture was then centrifuged in a 50 kDa ultrafiltration tube to remove excess DBCO-sulfo-NHS and obtain DBCO-Exo. DVAP-N3 and DBCO-Exo were incubated overnight at 4°C and shaken at 100 rpm. The next day, the mixture was centrifuged twice in an ultrafiltration tube with an appropriate amount of PBS to eliminate free peptides. D VAP-Exo.

[0065] To verify the successful chemical coupling of targeting peptides to exosomes, flow cytometry was used to detect TAMRA- D The positive rate of VAP was used to investigate whether the click chemistry reaction was successful. D VAP-modified exosomes (Exo-TAMRA- D VAP) was used for detection, and the exosomes without modified targeting peptide were used as control and detected by flow cytometry. The results are shown in Figure 3A, and about 88.6% of the exosomes showed fluorescence signals, indicating that D VAP targeting peptide was successfully conjugated to exosomes.

[0066] 1.3 Determination of drug-loaded exosome encapsulation efficiency

[0067] MIT was dissolved in PBS to prepare a 5 mg / mL stock solution. The stock solution was diluted to obtain 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, and 90 μg / mL MIT solutions. The absorbance of the above 9 solutions with different concentrations at 627 nm was measured using a microplate reader to draw a standard curve for MIT.

[0068] To screen the optimal drug delivery method and conditions, electroporation, co-incubation followed by transfection reagent, and co-incubation and transfection reagent combined methods were compared.

[0069] (1) Electroporation method: Gently mix exosomes (Exo), MIT and siRNA in a ratio of 10:8:1. Adjust the parameters of the electroporator and perform electroporation at 1000V and 20ms. Then incubate at 37°C for 30 minutes to restore the membrane of the exosomes. Then add ExoQuick-TC reagent, let it stand on ice for 30 minutes to allow the exosomes to settle, centrifuge at 4°C and 13000rpm for 3 minutes, discard the supernatant and retain the precipitate, add an appropriate amount of PBS to resuspend, and dialyze the obtained system in a dialysis bag for 10 hours to obtain exosomes encapsulated with MIT and siRNA (EMR). Add 0.5% TritonX-100 solution and incubate at room temperature for 15 minutes to destroy the exosome membrane structure, release MIT, and measure the encapsulation efficiency of MIT. Use an enzyme-labeled instrument to measure the absorbance of the solution at 627nm, calculate the concentration of MIT using the standard curve, and calculate the encapsulation efficiency according to Formula 1.

[0070] (2) Co-incubation followed by transfection reagent method: Exo and MIT were first mixed in a ratio of 5:4, incubated at 37°C and 120 rpm on a shaker for 30 minutes, dialyzed in PBS at 4°C for 10 hours, and then siRNA and transfection reagent Exo-fect were added in a ratio of Exo:siRNA = 10:1. Incubated at 37°C for 20 minutes, ExoQuick-TC reagent was added, and the reaction was allowed to stand on ice for 30 minutes to terminate the reaction. Centrifuged at 4°C and 13,000 rpm for 3 minutes, the supernatant was discarded, and the precipitate was retained. The solution was resuspended in an appropriate amount of PBS, and the obtained system was dialyzed in a dialysis bag for 10 hours to obtain EMR. 0.5% TritonX-100 solution was added and incubated at room temperature for 15 minutes to measure the encapsulation efficiency of MIT. The absorbance of the solution at 627 nm was measured using an enzyme reader, and the concentration of MIT was calculated using the standard curve. The encapsulation efficiency was calculated according to Formula 1.

[0071] (3) Co-incubation and transfection reagent combination method: Incubate MIT, Exo, siRNA and Exo-fect transfection reagent at 37°C for 20 minutes according to the ratio of Exo:MIT:siRNA = 10:8:1, then add ExoQuick-TC reagent and let it stand on ice for 30 minutes. Then centrifuge at 4°C and 13000rpm for 3 minutes, discard the supernatant and retain the precipitate, add appropriate amount of PBS to resuspend, and dialyze the obtained system in a dialysis bag for 10 hours to obtain EMR. After dialysis, take an appropriate amount of drug-loaded system, add 0.5% TritonX-100 solution and incubate at room temperature for 15 minutes to destroy the membrane structure of exosomes and release MIT. Measure the encapsulation efficiency of MIT before and after dialysis. Use an enzyme-linked microplate reader to measure the absorbance of the solution at 627nm, calculate the concentration of MIT using the standard curve, and calculate the encapsulation efficiency according to formula 1.

[0072] In order to calculate the encapsulation efficiency of siRNA in the drug delivery system, siRNA labeled with Cy3 fluorescent dye was encapsulated into exosomes, and EMR was prepared according to the above three methods. The corresponding fluorescence value (E x =530nm,E m =570nm), and the encapsulation efficiency was calculated according to formula 1-2.

[0073] Among them, Flu pel and Flu sup are the fluorescence values ​​of siRNA in the precipitate and supernatant, respectively.

[0074] The test results are shown in Table 1;

[0075] Table 1 Encapsulation efficiency of MIT and siRNA in different encapsulation methods

[0076] The electroporation method measured MIT and siRNA encapsulation efficiencies of 11% and 42%, respectively. The co-incubation-before-transfection method measured MIT and siRNA encapsulation efficiencies of 13% and 50%, respectively. The combined co-incubation and transfection method achieved encapsulation efficiencies of 21% and 69.35%, respectively. Therefore, the combined co-incubation and transfection method was selected for subsequent MIT and siRNA encapsulation.

[0077] 1.4 Preparation and characterization of drug-loaded exosomes

[0078] According to the method in 1.2 of Example 1, click chemistry was applied to D VAP peptide was modified on the exosomes, and then siRNA and MIT were encapsulated into the exosomes according to the co-incubation and transfection reagent combination method of 1.3-(3) in Example 1 to prepare D VAP-Exo / MIT / siRNA (VEMR).

[0079] The prepared VEMRs were analyzed for morphology using transmission electron microscopy, and their particle size was analyzed using nanoparticle tracking analysis (NTA). As shown in Figures 3B and 3C, the drug-loaded exosomes were round or oval in shape, with a double-layer membrane structure. The particle size of the VEMRs was approximately 136 nm. Compared with untreated exosomes, the morphology of the drug-loaded exosomes did not change significantly, indicating that the exosomes maintained good integrity and morphology during the modification and drug loading process. The particle size also increased slightly, demonstrating that the MIT and siRNA were successfully encapsulated in the exosomes.

[0080] 1.5 Investigation of drug loading stability

[0081] EMR, EMR permeabilized with 0.5% Triton X-100, and siRNA were incubated with RNase A at 37°C for a specified period of time. The degradation of siRNA was then analyzed by agarose gel electrophoresis to assess the stability of exosome-loaded siRNA. Samples incubated with RNase A for 0, 0.5, 1, 1.5, and 2 hours were thoroughly mixed with 6× DNA loading buffer and loaded onto the wells of a 2% agarose gel. Electrophoresis was performed at 120 V in the dark for 30 minutes and imaged using a gel imaging system. The results, shown in Figure 4A, indicate that free siRNA was largely degraded by 30 minutes and completely degraded by 60 minutes. When the exosomes were permeabilized with Triton X-100 solution, the degradation of siRNA in EMR was similar to that of free siRNA. However, siRNA in EMR without permeabilization showed little degradation between 0 and 120 minutes, indicating that exosomes effectively protect siRNA and provide a certain stability, making it less susceptible to nuclease degradation.

[0082] After preparing EMR, the sample was placed in a dialysis bag and dialyzed against 3 mL of PBS at 37°C. The dialysate was collected at 12, 24, and 48 hours, and the absorbance of the MIT at 627 nm was measured using a microplate reader to assess the stability of the drug entrapment. The results, shown in Figure 4B, show that the MIT was slowly and continuously released over 48 hours, demonstrating its good stability.

[0083] Example 2: In vitro biological function evaluation of exosome-targeted drug delivery

[0084] Human normal astrocytes HA1800 were purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., and human glioma cells U87 were purchased from Wuhan Punosai Life Science Co., Ltd. Both were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% double-antibody antibodies. Luciferase-labeled mouse glioma cells GL261 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum, 1% double-antibody antibodies, 1.5 g / L sodium bicarbonate, and 0.11 g / L sodium pyruvate.

[0085] 2.1 Culture and phenotypic confirmation of glioma-initiating cell spheres

[0086] U87 cell spheres were isolated using the immunomagnetic bead method. U87 cells in good growth condition were first cultured in DMEM medium. When the cell density reached 80%, the medium was replaced with DMEM / F12 medium (20 ng / mL EGF, 20 ng / mL bFGF and B27). Culture was continued for 3-4 days, with the medium changed every two days. 0.25% trypsin was added to terminate digestion, and the cells were centrifuged at 4°C and 1000 rpm for 3 minutes. The supernatant was discarded. 1 mL of cell culture medium was added to resuspend the cells and counted, which was approximately 1×10 7 After centrifugation for 5 minutes, discard the supernatant. The cell suspension was mixed with 10μL CD133 / PE antibody (1:100) and incubated at 4°C for 10 minutes and centrifuged. After removing the unbound primary antibody, 240μL sorting solution was added to resuspend the cells, and then 60μL FCR blocker and 60μL anti-PE magnetic beads were added. After mixing, the cells were incubated at a slow speed of 15 minutes on a shaker at 4°C. Then 1mL of sorting solution was added and centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded. 500μL sorting solution was added and impurities were filtered using a cell screen. The cell suspension was added to the separation column placed on the magnetic bead separator for sorting, and the obtained CD133 + Glioma cells were cultured.

[0087] Phenotyping of glioma-initiating spheres was confirmed by Western blot. Expression of glioma-initiating sphere markers (CD133 and Nestin), Notch pathway components (Notch 1, Hes 1), and a cell differentiation marker (GFAP) were measured in U87 spheres. Phenotyping of U87 spheres was confirmed using antibodies to Notch 1, Nestin, Notch 1, Hes 1, and GFAP (1:200). Transfer was performed at 20V for 20 minutes.

[0088] The Western blot results are shown in Figure 5A , indicating that the sorted U87 cell spheres highly expressed CD133, Nestin (stem cell markers), Notch 1, and Hes 1 (Notch pathway components), but the expression level of GFAP (cell differentiation marker) in the cell spheres was low; the immunofluorescence staining results are shown in Figure 5B , showing that the U87 cell spheres expressed high levels of CD133 and Notch 1. These results indicate that the sorted U87 cell spheres meet the characteristics of glioma-initiating cell spheres and have almost no differentiation.

[0089] 2.2 In vitro targeting evaluation

[0090] Exosomes were labeled with the membrane dye DiI, and Exo and VE were co-incubated with U87 cells, U87 cell spheroids, and normal astrocytes HA1800 for 4 hours. After the cells were stained with DAPI, the fluorescence intensity in the cells and cell spheroids was detected using laser confocal microscopy to investigate the targeting of Exo, VE, and VEMR to U87, U87 cell spheroids, and HA1800. The results of laser confocal microscopy are shown in Figures 6-7. D The fluorescence signal in U87 cells and U87 cell spheroids treated with VAP-Exo was stronger than that in free Exo, indicating that the targeting peptide modification enhanced the uptake of exosomes in U87 cells and U87 cell spheroids. The fluorescence signal intensity in the VEMR group was almost unchanged from that in the VE group, indicating that the encapsulated MIT and siRNA had little effect on the targeting ability of the exosome drug delivery system.

[0091] The specificity of targeted peptide delivery was evaluated by competitively blocking the specific binding of the targeting system to GRP78 on the cell surface using free targeting peptide. The specific method is as follows: first, U87 cells, U87 cell spheres and HA1800 cells were co-incubated with free targeting peptide for 1 hour. After washing with PBS, VE was added, and the intracellular fluorescence intensity was measured using a laser confocal microscope to investigate the effect of receptor blocking on the targeting of the system. The results are shown in Figures 6-7. DAfter treatment with VAP targeting peptide and addition of VE, U87 cells, U87 cell spheres and HA1800 cells showed no fluorescent signals, indicating that the targeting peptide specifically binds to the receptor and promotes the uptake of exosomes by cells and cell spheres.

[0092] 2.3 Uptake of targeted modified drug-loaded exosomes in cells

[0093] U87 cells and U87 cell spheres in the logarithmic growth phase were digested with trypsin, resuspended by centrifugation, and seeded into 24-well plates covered with confocal slides at a rate of 5 × 10 cells per well. 4 After culturing for 24 hours in a 37°C, 5% CO2 incubator, PBS, siRNA, MIT, EMR, and VEMR were added to U87 cells and U87 cell spheroids for 4 hours. siRNA was labeled with Cy3 fluorescent dye, and MIT itself is fluorescent. The exosome concentration per well was 10 μg / mL for U87 cells, and 40 μg / mL for U87 cell spheroids. After 4 hours, the culture medium was aspirated, and the cells were washed three times with PBS. The cells were then fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI. Finally, the cells were observed and photographed under a laser confocal microscope.

[0094] The results are shown in Figure 8. In U87 cells, almost no red fluorescence and green fluorescence were detected in the free siRNA group and the free MIT group, indicating that free siRNA and free MIT were almost unable to enter the cells; a small amount of red and green fluorescence signals could be observed in the EMR group, while the VEMR group had stronger red and green fluorescence signals, and siRNA and MIT were mainly distributed in the cytoplasm, indicating that exosomes can carry siRNA and MIT successfully into U87 cells.

[0095] In U87 cell spheroids, as shown in Figure 9, in the free siRNA group and the free MIT group, almost no red fluorescence and green fluorescence were detected in the cell spheroids; a small amount of red and green fluorescence signals could be observed in the EMR group, while the VEMR group had stronger red and green fluorescence signals. In summary, this indicates that exosomes can carry siRNA and MIT and successfully enter cells and cell spheroids.

[0096] 2.4 Uptake mechanism of targeted modified exosomes in cells

[0097] U87 cells and U87 cell spheres in the logarithmic growth phase were collected after trypsin digestion, resuspended by centrifugation, counted, and seeded into 24-well plates covered with confocal slides at 5 × 10 cells per well. 4After culturing for 24 h in a 37°C, 5% CO2 cell culture incubator, cells were pretreated with different endocytosis inhibitors, including 50 μM amiloride hydrochloride, 5 mM methyl-β-cyclodextrin (M-β-CD), 10 μg / mL chlorpromazine, and 80 μM dynasore for 1 h. D VAP-Exo were incubated for 2 hours. To investigate the effect of temperature on cellular uptake, one group of cells was placed at 4°C for uptake. After 2 hours, the cells were washed three times with PBS, and the cell nuclei were stained with DAPI. Finally, the cells were observed and photographed under a laser confocal microscope.

[0098] The results are shown in Figures 10 and 11. Under 4°C, U87 cells and cell spheres D VAP-Exo was hardly taken up, and the fluorescence signal was significantly different from that of the control group, indicating that D VAP-Exo uptake is an energy-dependent pathway. The caveolin inhibitor M-β-CD and the clathrin inhibitor chlorpromazine did not cause changes in exosome uptake. Amiloride is a Na + / H + Amiloride and Dynasore are inhibitors of VE exchange and macropinocytosis. Dynasore is a GTPase inhibitor that inhibits dynamin-mediated endocytosis. Amiloride and Dynasore treatment reduced VE uptake in cells, indicating that exosome entry occurs through dynamin-mediated endocytosis and macropinocytosis. This suggests that exosome entry is associated with energy-dependent processes mediated by dynamin and macropinocytosis.

[0099] 2.5 Colocalization analysis of siRNA, MIT, and lysosomes

[0100] VEMR was prepared using Cy3-labeled siRNA and MIT for observation of the intracellular localization of MIT and siRNA. U87 cells and U87 cell spheres in the logarithmic growth phase were trypsinized, collected, centrifuged, resuspended, and counted. The cells were seeded into confocal microplates and cultured for 24 hours. VEMR was added and incubated for 4 hours. The cells were washed three times with PBS and cultured for an additional 2 or 6 hours. Lysotracker (75 nM) solution was then added and incubated for 30 minutes to stain lysosomes. The cells were washed three times with PBS, and Hoechst was added to stain the nuclei for 30 minutes. After washing three times with PBS, the fluorescence distribution was observed under a laser confocal microscope. The colocalization of MIT, Cy3-siRNA, and Lysotracker fluorescence was analyzed using Image J.

[0101] As shown in Figure 12, after system enters U87 cell, during 2h, siRNA, MIT and lysosomal green fluorescence present a large amount of overlapping, and siRNA and lysosomal colocalization coefficient is 0.972, and MIT and lysosomal colocalization coefficient is 0.64, but along with the prolongation of time, overlapping fluorescence part reduces gradually.During 8h, siRNA and lysosomal colocalization coefficient is 0.67, and MIT and lysosomal colocalization coefficient is 0.3, and siRNA and MIT have significant difference at 2h and 8h and lysosomal colocalization coefficient.In U87 cell sphere (Figure 13), siRNA, MIT and lysosomal colocalization situation and VEMR similarly show that most of siRNA and MIT first enter lysosome after being endocytosed in U87 cell sphere, and along with the prolongation of time siRNA, MIT partly escape from lysosome.

[0102] 2.6 In vitro gene silencing effect detection

[0103] Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect mRNA expression. The sequences of siNotch 1 and the negative control (siNC) are shown in Table 2. qRT-PCR was used to detect the silencing effect of siRNA on the target gene. The specific steps are as follows:

[0104] (1) Cell seeding plate: U87 cell spheres and GL261 cells were seeded into 24-well plates, with 1×10 cells per well. 5 When the cells grow to a density of about 80%, they are used for drug administration.

[0105] (2) Lipo / siNotch 1 administration: Centrifuge 1OD siRNA powder at 3000rpm for 3min to allow the powder to settle at the bottom of the tube, add 250μL DEPC water to dissolve it, and prepare a 0.01nmol / μL stock solution. First, take 3μL Lipofectamine 2000 reagent (Lipo) and add it to 9μL serum-free DMEM medium, and let it stand at room temperature for 5min; take 10μL dissolved siRNA solution and add it to 90μL serum-free DMEM medium, add the diluted siRNA solution to the Lipo solution, pipet evenly, and let it stand at room temperature for 20min. Add the prepared Lipo / siNotch 1 to the well plate, culture for 6h, then replace it with DMEM medium containing serum and double antibody, and continue to culture for 48h.

[0106] (3) VE / siNotch 1 administration: Prepare VE:siNotch 1:Exo-fect transfection reagent at a ratio of 10:1:2, add to the cell well plate in DMEM medium containing serum and double antibody, and culture for 48 hours.

[0107] (4) RNA extraction: Add appropriate amount of PBS to the well plate and wash twice, add 250 μL Trizol to each well, let it stand for 5 minutes, and then transfer it to an EP tube; add chloroform, vortex for 30 seconds, and centrifuge at 12000g for 15 minutes; aspirate the upper colorless solution into a new EP tube, add isopropanol, turn it upside down 3 times, mix it evenly, let it stand at room temperature for 10 minutes, and centrifuge it at 12000g for 10 minutes; discard the supernatant, add 75% ethanol, vortex for 10 seconds, centrifuge it at 7500g for 5 minutes, and discard the supernatant; add DEPC water, place in a metal bath at 60℃ for 5 minutes to dissolve the RNA, and use Nanodrop to measure the RNA concentration.

[0108] (5) Reverse transcription: Synthesize cDNA using a rapid reverse transcription kit. Prepare the gDNA removal reaction system according to the instructions, mix thoroughly, centrifuge briefly, and incubate at 42°C for 3 min. Prepare the reverse transcription reaction system according to the instructions (primer sequences are shown in Table 3), add it to the gDNA removal reaction solution, and mix thoroughly. Heat in a PCR amplifier at 42°C for 15 min and then at 95°C for 3 min.

[0109] (6) qPCR: qPCR reaction was performed using the SuperReal fluorescence quantitative premix kit and operated on ice in the dark. 2×SuperReal PreMix Plus, primers, cDNA, and ddH2O were melted at room temperature and placed on ice. The reaction system was prepared according to the instructions and the reaction was performed in a real-time fluorescence quantitative PCR instrument (the reaction program was set as follows: 95°C, 15 min; 95°C, 3 s; 60°C, 20 s; 40 cycles). β-actin was used as the internal reference and the reaction was performed according to 2 -△△Ct The relative expression level of Notch 1 gene was calculated by the method.

[0110] Table 2 siRNA sequences

[0111] Table 3 Primer sequences

[0112] The expression of mRNA levels was detected by qRT-PCR. The results are shown in Figure 14A. After VER treatment, the mRNA level of Notch 1 in U87 cell spheres decreased by 60%, and the Notch 1 mRNA expression level in GL261 cells decreased by 45%, which was significantly different from the negative control (free siNotch 1).

[0113] U87 cell spheres were seeded into 12-well plates at 3 × 10 cells per well. 5When the cell density reached approximately 80%, Lipo / siNotch 1 and VE / siNotch 1 were administered, and protein expression was detected by Western blot. Notch 1 protein expression in U87 cell spheres was detected (Figure 14B). Consistent with the PCR results, Notch 1 protein expression was significantly downregulated, comparable to that in the positive control group (Lipo / siNotch 1), while free siNotch 1 protein expression remained unchanged. VER can effectively silence target genes and inhibit target protein expression at the cellular level, providing data support for its subsequent in vivo application.

[0114] 2.7 In vitro cell safety evaluation

[0115] U87 cells and HA1800 cells in the logarithmic growth phase were digested with trypsin and collected by centrifugation. Fresh culture medium was added to resuspend the cells and counted. The concentration was 5×10 4 The cell suspension of 100 μL / mL was inoculated into a 96-well plate, and the volume added to each well was 100 μL. The plate was then placed in a cell culture incubator and cultured for 24 h. When the cell density reached about 80%, different concentrations of Exo, Exo / siNC, D VAP-Exo / siNC, exosome concentrations were 0, 5, 10, 20, 50, and 100 μg / mL. After incubation for 48 hours, the culture medium was discarded and culture medium containing 10% CCK-8 reagent was added. The cells were incubated at 37°C for another 40 minutes. The absorbance of each well was measured at 450 nm. The culture medium wells without cells or the drug to be tested served as the blank group, and the cell group without drug was used as the control group. The cell survival rate was calculated according to Formula 3:

[0116] Cell viability % = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%(Formula 3)

[0117] The safety of exosome-targeted siRNA on normal cell line HUVEC and human glioma cell line U87. As shown in Figure 15, the common concentration of exosomes in cell experiments is 10μg / mL. At this time, the survival rate of HA1800 cells is about 90%, and as the concentration increases to 100μg / mL, the survival rate of HA1800 cells does not change significantly. In U87 cells, as the concentration of exosomes increases, D After VAP-Exo / siNC treatment, the survival rate of U87 cells remained above 80%, with no significant difference compared with the control group, indicating that the exosome targeted drug delivery system has good safety.

[0118] 2.8 In vitro cell proliferation evaluation

[0119] The cell viability was determined using the CCK-8 assay to investigate the cytotoxicity of each preparation. Eight treatment groups were set up: PBS, MIT, siRNA, MIT+siRNA, VE, VEM, VER, and VEMR. U87 cells and U87 cell spheres were seeded in 96-well plates at 1×10 cells per well. 4 Each drug group was added to the cells, with the concentration of MIT at 5 μg / mL and the concentration of siRNA at 200 nM. The well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 48 hours. The cell survival rate was calculated according to Formula 3.

[0120] The results are shown in Figure 16. In U87 cells, due to the low expression of Notch 1 in U87 cells, there was no significant difference in cell survival between the VEMR and VEM groups. However, compared with the MIT, siRNA, and MIT+siRNA groups, the cell survival rate of the VEMR group was significantly reduced. In U87 cell spheroids, due to the high expression of Notch 1 in cell spheroids, the cell survival rates of the VER and VEM groups decreased by 30% and 60%, respectively, and the cell survival rate of the VEMR group decreased by approximately 90%. These results indicate that VEMR can significantly inhibit the proliferation of U87 cells and U87 cell spheroids.

[0121] 2.9 Evaluation of cell migration ability

[0122] The cell scratch test was used to examine the effect of the exosome targeted drug delivery system on the migration ability of U87 cells and U87 cell spheres. U87 cells and U87 cell spheres in the logarithmic growth phase were cultured at 2×10 5 The cells were seeded at a density of 100 μg / mL in a 12-well plate. When the cell density reached about 80%, a vertical line was drawn in the middle of each well using a 200 μL pipette tip, and the floating cells were washed away with PBS. The healing of the scratches was observed under a microscope at 0 h, 12 h, and 24 h after the scratches. The scratch distance was quantified using Image J software, and the scratch distance at each time point was calculated to characterize the inhibitory effects of MIT and siRNA silencing on the migration of U87 and U87 cell spheres.

[0123] The results are shown in Figure 17. 24 hours after the scratch, in U87 cells, compared with the PBS group and VE group, the cells in the siRNA group and VER group were almost completely fused, indicating that siRNA did not affect the normal migration of U87 cells. Compared with the MIT and MIT+siRNA groups, the "wounds" in the VEM and VEMR groups healed more slowly, indicating that the VEM and VEMR groups significantly reduced the "wound" healing speed of U87 cells.

[0124] In U87 cell spheres, 24 hours after the scratch, the cells in the siRNA group were almost completely fused compared with the PBS group, while the wounds in the MIT and MIT+siRNA groups healed more slowly. Compared with the VE group, the wounds in the VER, VEM, and VEMR groups healed more slowly, and compared with VEM and VER, VEMR significantly inhibited the healing of the wound. These results indicate that D VAP can significantly enhance the targeting of the system and improve the uptake of siRNA by U87 cells and U87 cell spheres. In addition, MIT and siRNA can jointly enhance the inhibitory effect of the exosome targeted drug delivery system on the migration of U87 cells and U87 cell spheres.

[0125] 2.10 Evaluation of cell apoptosis

[0126] U87 cells and U87 cell spheres in the logarithmic growth phase were cultured at 2×10 5 Cells were seeded at a density of 100 μL in a 12-well plate and PBS, MIT, siRNA, MIT+siRNA, VE, VEM, VER, and VEMR were added. Incubation was continued for 24 hours. Floating and adherent cells were collected, centrifuged, and the supernatant discarded. The cells were then washed three times with PBS. The cell pellet was resuspended in 100 μL PBS, stained with Annexin V-APC / DAPI, and analyzed by flow cytometry.

[0127] The results are shown in Figure 18. In U87 cells, compared with the PBS group (3.50%), the cell apoptosis rate in the siRNA group (2.73%) did not change significantly, while the cell apoptosis rates in the MIT and MIT+siRNA groups increased significantly, reaching 19.24% and 21.84%, respectively; compared with the VE group (2.20%), the cell apoptosis rate in the VER group (3.45%) did not change significantly, while the cell apoptosis rates in the VEM and VEMR groups were 30.8% and 40.63%, respectively; compared with the MIT and MIT+siRNA groups, the cell apoptosis rates in the VEM and VEMR groups increased significantly.

[0128] The results are shown in Figure 19. In U87 cell spheres, compared with the PBS group (2.80%), the cell apoptosis rate in the siRNA group (4.11%) did not change significantly, while the cell apoptosis rates in the MIT and MIT+siRNA groups increased significantly, reaching 16.32% and 19.19%, respectively. Compared with the VE group (3.67%), the apoptosis rates of VER, VEM, and VEMR cells were 10.77%, 30.8%, and 40.63%, respectively. Compared with the MIT and MIT+siRNA groups, the apoptosis rates of VEM and VEMR cells increased significantly. The above results indicate that DVAP can significantly enhance the targeting of the system and improve the uptake of siRNA by U87 cells and U87 cell spheres, and VEMR can significantly promote the apoptosis of GIC and glioma cells.

[0129] The above experiments have proved that the exosome-targeted drug delivery system can exert good gene silencing and anti-tumor effects at the cellular level, laying the foundation for in vivo anti-tumor applications.

[0130] Example 3: Evaluation of the anti-tumor effect of exosome-targeted drug delivery system in vivo

[0131] In this example, an orthotopic glioma model was established to investigate the in vivo antitumor efficacy and safety of the exosome-targeted drug delivery system. Balb / c mice (6-week-old, male, 20 ± 2 g) were purchased from the China Food and Drug Administration. All animal experiments were conducted according to protocols approved by the Tianjin Medical University Animal Ethics and Welfare Committee and in compliance with the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

[0132] 3.1 Construction of orthotopic glioma model

[0133] Digest the Luc-GL261 cells that are growing well and resuspend them in PBS to adjust the cell suspension concentration to 1×10 8 / mL, put on ice for later use, 1×10 per mouse 6 Cells. The microinjection needle was cleaned once with 75% alcohol and then twice with PBS. Surgical instruments were sterilized at high temperature before use. Mice with good vital signs were removed from the animal room and appropriately sedated. Anesthesia was induced by inhalation of a mixture of isoflurane and air. The mice were then secured to a brain positioner connected to an anesthesia machine. Anesthesia was maintained and the brain was fully exposed. A scalpel was used to gently incise the mouse's scalp 0.5 cm. A craniotomy was performed along the white sutures of the skull, 2 mm to the right and 1 mm above the cross-section. A 5 mL syringe needle was used to gently drill the opening, stopping when a puncture was felt and using a cotton ball to stop any bleeding. A microinjection syringe containing 10 μL of GL261 cells was secured to the brain positioner directly above the mouse's skull. Aligning the needle with the drilled skull, the microinjection needle was gently inserted slightly into the mouse's skull using the Z-axis of the positioner. The digital display was zeroed. After a 4 mm downward movement along the Z-axis, the needle was held for 10 seconds to ensure equilibrium within the mouse's brain. Gently rotate the Z axis upward 1mm to create space for the tumor. Within 1 minute, inject 5 μL of the cell suspension into the mouse brain. Leave it for 4 minutes to ensure brain equilibrium. After the time is up, gently remove the microsyringe. Draw up 5 μL of medical glue and first drip it into the skull wound, then adhere the scalp securely. Place the mouse on an electric blanket to allow it to recover and observe its vital signs the next day.

[0134] 3.2 In vivo imaging experiments

[0135] To evaluate the therapeutic efficacy of different formulations in a mouse glioma model, mice were randomly divided into the following six groups on day 12 of tumor development: PBS, MIT, VE, VER, VEM, and VEMR. Intratumoral injections were performed in a 10 μL volume, with MIT administered at a dose of 3 mg / kg and siRNA administered at a dose of 0.416 mg / kg. Bioluminescence imaging was performed on a randomized basis within each group. On day 19 of tumor development, brain tissue and various organs were harvested from each group for H&E (Hematoxylin and Erythrocyte Embryo) and TUNEL staining.

[0136] The therapeutic effects of different dosing groups in a mouse glioma model were compared using a small animal in vivo imaging system. On days 12, 16, and 19 of tumor growth, each mouse was injected with 200 μL of luciferase substrate solution. The mice were anesthetized, and the fluorescence signal intensity was recorded using an IVIS Spectrum small animal in vivo imaging system.

[0137] The results are shown in Figure 20. Each group of mice was administered with the drug on the 12th day after tumor implantation. The in vitro anti-tumor effect of the exosome targeted drug delivery system was investigated by bioluminescence imaging (A) and bioluminescence intensity (B) on the 12th, 16th, and 19th days after tumor implantation. On the 12th day after tumor implantation, the fluorescence value of the brain glioma of each group of Balb / c mice reached 1×10 5 The above results indicate that the in situ glioma model was successfully established. Within 7 days after administration, the fluorescence values ​​of mice in the PBS and VE groups gradually increased, indicating that the brain tumors grew rapidly. The PBS and VE groups had no significant inhibitory effect on tumor growth. On the 19th day after tumor implantation, the fluorescence values ​​of the VER and VEM groups were both above 0.5×10 5 The following results show that there is a significant difference compared with the PBS group, indicating that the exosome-encapsulated siNotch 1 and MIT can play a certain gene silencing effect and kill GIC and glioma cells in vivo; the fluorescence value of the VEMR group is the lowest, which is significantly different from that of the PBS group, indicating that the use of D VAP-Exo as a carrier for the combined delivery of siNotch 1 and MIT significantly inhibited tumor growth and had a good anti-tumor effect in vivo.

[0138] 3.3 Brain tissue sections

[0139] On day 7 after administration, brain tissue was removed from randomly selected mice in each group and fixed in 4% paraformaldehyde for 48 hours. Paraffin sections were prepared and stained with H&E. Panoramic digital slide scanning microscopy was used to observe and photograph the brain tissue pathology.

[0140] The results are shown in Figure 21. Frozen sections from the different formulation groups were collected, stained with H&E, and observed using a tissue section imaging quantitative analysis system (A). The ratio of tumor area to total brain tissue area was quantitatively analyzed using Image J software (B). Both the PBS and VE groups had large, dense areas of GBM cells, and the ratio of tumor area to total brain tissue area was above 15%, indicating rapid tumor growth in both groups and no inhibitory effect on tumor growth. The tumor area ratio in the VER and VEM groups was less than 12%, indicating that exosome-encapsulated siNotch 1 and MIT can exert a certain gene silencing effect and kill GIC and glioma cells in vivo. The tumor area ratio in the VEMR group was less than 0.5%, significantly smaller than that in the other treatment groups, and there was a significant difference compared to the other groups, indicating that combined administration can significantly inhibit brain tumor growth in mice.

[0141] On the seventh day after administration, brain tissues of mice were randomly selected from each group and fixed in 4% paraformaldehyde. After fixation, the brain tissues were placed in 30% sucrose solution for sedimentation. When the tissue sank to the bottom, the sedimentation was completed. In a suitable container, OCT embedding agent was added and placed in -80°C. After the OCT was completely solidified, it was placed in a freezing microtome and sliced ​​to a thickness of 20 μm. The cut slices were spread on a glass slide, moistened with an appropriate amount of PBS, and stored at 4°C. Fluorescent staining was performed according to the TUNEL kit to observe cell apoptosis, and the images were taken with a laser confocal microscope. The results are shown in Figure 22. The cells in the PBS group and the VE group had almost no fluorescent signal and did not cause tumor cell apoptosis. Compared with VE, the green fluorescence signal of the VER group was enhanced, indicating that siNotch 1 can effectively exert the gene silencing effect in vivo. The green fluorescence signal of the free MIT group was significantly weaker than that of the VEM group. Compared with the MIT group, VEM had a stronger effect of inducing tumor cell apoptosis. Compared with the VEM and VER groups, the green fluorescence signal of the VEMR group was the strongest, with significant statistical differences, indicating that combined administration can significantly induce tumor cell apoptosis.

[0142] 3.4 In vivo safety assessment of exosome-targeted drug delivery system

[0143] The weight changes of mice in the different dosing groups were examined. On day 12 after tumor loading, the weights of the six groups of mice (PBS, MIT, VE, VER, VEM, and VEMR) were recorded daily at the same time for 1-7 days after dosing. As shown in Figure 23, after successful orthotopic glioma modeling, the weights of the mice in each group did not change significantly compared to the PBS control group within 7 days after dosing, demonstrating normal growth trends. This indicates that the exosome-targeted drug delivery system does not affect the normal growth of the mice after entry into the body.

[0144] H&E staining of the organs of each mouse group was performed. Seven days after dosing, the mice in each group were dissected, and the hearts, livers, spleens, lungs, and kidneys were removed. The organs were fixed in 4% paraformaldehyde for 48 hours, and paraffin sections were prepared and stained with H&E. Major organs of each group were removed and stained with H&E to assess histopathological changes. The results, as shown in Figure 24, showed clear organ morphology and structure in all groups. Similar to the control group, no pathological changes such as cell shrinkage, necrosis, or inflammation were observed. This demonstrates that the exosome-based targeted drug delivery system has good in vivo biosafety.

[0145] The liver and kidney function indicators of each group of mice were tested. On the 7th day after administration, blood was collected from the eyeballs of each group of mice, and serum was extracted by centrifugation. The liver and kidney function indicators were tested according to the aspartate aminotransferase (AST) test kit, alanine aminotransferase (ALT) test kit, creatinine (Cr) test kit, and urea test kit. The liver and kidney function indicators of each group of mice were tested. The results are shown in Figure 25. Compared with the PBS group, there were no significant differences in the liver function indicators: AST, ALT; renal function indicators: urea nitrogen, creatinine, in each group, indicating that the exosome targeted drug delivery system does not have a significant impact on the liver and kidney organs of mice.

[0146] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A siRNA co-delivery drug system that efficiently targets glioma initiating cells, characterized by: D VAP-targeting peptide-modified exosomes loaded with siRNA and mitoxantrone.

2. The siRNA co-delivery drug system for highly efficient targeting of glioma initiating cells according to claim 1, characterized in that: The siRNA sequences are shown in SEQ ID No. 1 and SEQ ID No.

2. D The VAP targeting peptide sequence is shown as SEQ ID No.

3.

3. The method for constructing the siRNA co-delivery drug system for efficiently targeting glioma initiating cells according to claim 1 or 2, characterized in that: The steps include: Step 1: D VAP-targeted peptide modified exosomes D VAP-Exo; Step 2: Load siRNA and mitoxantrone into D In VAP-Exo, targeted modified drug-loaded exosomes are obtained D VAP-Exo / MIT / siRNA.

4. The method for constructing the siRNA co-delivery drug system for highly efficient targeting of glioma initiating cells according to claim 3, characterized in that: In step 1, DBCO is first connected to the outer surface of exosomes to obtain DBCO-Exo, and then D After VAP-N3 and DBCO-Exo were mixed and incubated, D VAP-targeting peptide-modified exosomes D VAP-Exo.

5. The method for constructing the siRNA co-delivery drug system for highly efficient targeting of glioma initiating cells according to claim 4, characterized in that: 10 μM DBCO-sulfo-NHS was dissolved in DMSO and added to exosomes resuspended in PBS. The mixture was reacted at 25°C and 120 rpm for 4 h, and the excess DBCO-sulfo-NHS was removed to obtain DBCO-Exo. Modified with azide group D VAP-N3 was incubated with DBCO-Exo at 4°C and 100 rpm on a shaker to eliminate free peptides and prepare D VAP-Exo.

6. The method for constructing the siRNA co-delivery drug system for highly efficient targeting of glioma initiating cells according to claim 3, characterized in that: In step 2, siRNA and MIT were loaded into D VAP-Exo.

7. The method for constructing the siRNA co-delivery drug system for highly efficient targeting of glioma initiating cells according to claim 6, characterized in that: Specifically include: Step 1: Follow D VAP-Exo:MIT:siRNA=10:8:1 ratio of MIT, D VAP-Exo and siRNA were mixed, and Exo-fect transfection reagent was added and incubated at 37°C for 20 min. Then ExoQuick-TC reagent was added and placed on ice for 30 min. Step 2: Centrifuge at 4°C and 13,000 rpm for 3 min, discard the supernatant and retain the precipitate, add an appropriate amount of PBS to resuspend, and dialyze the obtained system in a dialysis bag for 10 h to obtain D VAP-Exo / MIT / siRNA.

8. Use of the siRNA co-delivery drug system for efficiently targeting glioma initiating cells according to claim 1 or 2 in the preparation of anti-glioma drugs.

9. The use according to claim 8, characterized in that: The exosome drug delivery system loaded with mitoxantrone and siRNA can act on glioma initiating cells and / or glioma cells.

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