Engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide and application of engineered extracellular vesicle in melanoma treatment

Abstract

The invention discloses miR-150-3p-loaded engineered extracellular vesicles with the surfaces modified with iRGD targeting peptides and application of the miR-150-3p-loaded engineered extracellular vesicles in melanoma treatment, and belongs to the technical field of biological medicines.The miR-150-3p is loaded in EVs, the surfaces of the miR-150-3p are modified with the iRGD targeting peptides, and iEV-150 is obtained; the preparation method of the iEV-150 comprises the following steps: S1, screening miR-150-3p; s2, constructing an HEK293T cell line capable of stably expressing the miR-150-3p; s3, introducing an ANXA2 protein; s4, culturing an HEK293T cell line; s5, separating the EVs; s6, coupling the iRGD targeting peptide to the surface of the extracellular vesicle membrane; the iEV-150 can effectively deliver miR-150-3p to melanoma cells, inhibit tumor cell proliferation, DNA damage repair and epithelial-mesenchymal transition, and promote melanoma cell death by inducing ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide, and an application thereof in the treatment of melanoma. Background Art

[0002] Melanoma is a highly invasive and metastatic malignancy and one of the most lethal skin cancers. Due to its high heterogeneity and rapid growth, current treatments (including chemotherapy, targeted therapy, and immunotherapy) have limited efficacy in advanced melanoma and are prone to drug resistance. Therefore, the development of new, efficient, and targeted therapeutic strategies is of great clinical significance.

[0003] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles secreted by cells that can carry bioactive molecules such as proteins and miRNAs, and play an important role in intercellular communication and material transport. Due to their low immunogenicity, high biocompatibility, and ability to cross biological barriers, EVs are considered to be ideal drug delivery carriers. However, due to the heterogeneity, low yield, and targeted delivery capabilities of natural EVs, the development of engineered EVs has become an important development direction in recent years. Through genetic engineering or chemical modification, the function of EVs can be optimized, and their stability, targeting, and drug delivery efficiency can be improved, thereby expanding their potential for application in disease treatment.

[0004] MiR-150 is a reported anti-tumor miRNA that can inhibit the growth of melanoma by regulating multiple target genes and inducing it. This study found that miR-150-3p is highly enriched in melanoma EVs. However, it is difficult for natural EVs to effectively deliver miR-150-3p to the melanoma site, so how to improve the miRNA loading efficiency and tumor targeting of EVs is an urgent problem to be solved. The present invention improves the enrichment efficiency of miR-150-3p by engineering EVs, and modifies the iRGD targeting peptide on the surface of EVs to enhance its delivery and therapeutic effect in melanoma cells. Summary of the invention

[0005] To overcome the above technical problems, the present invention aims to provide an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide and its application in the treatment of melanoma. The engineered extracellular vesicle can deliver miR-150-3p to melanoma cells in a targeted manner and inhibit the growth and metastasis of melanoma by inducing ferroptosis. The present invention combines nano-delivery technology with ferroptosis regulation mechanism to provide an innovative strategy for the precise treatment of melanoma, which can effectively overcome the drug resistance of traditional therapies and improve the treatment effect, as follows: An engineered extracellular vesicle (iEV-150) loaded with miR-150-3p and surface-modified with iRGD targeting peptide, characterized in that miR-150-3p is loaded in extracellular vesicles (EVs) and iRGD targeting peptide is modified on its surface to obtain an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide, which is labeled as iEV-150; the engineered extracellular vesicle (iEV-150) can target-deliver miR-150-3p to melanoma cells, inhibit DNA damage repair and epithelial-mesenchymal transition of melanoma cells.

[0006] Furthermore, the preparation method of the engineered extracellular vesicle (iEV-150) is as follows: S1, screening of target gene miR-150-3p; S2, constructing a HEK293T cell line stably expressing miR-150-3p using lentiviral transfection technology; S3, introducing the nucleic acid sequence encoding ANXA2 protein into the HEK293T cell line expressing miR-150-3p; S4. Collect and culture HEK293T cell lines to secrete extracellular vesicles (EVs) containing miR-150-3p; S5. separating the extracellular vesicles (EVs) by ultracentrifugation; S6. The iRGD targeting peptide was coupled to the membrane surface of extracellular vesicles (EVs) to obtain engineered extracellular vesicles (iEV-150).

[0007] Furthermore, the average particle size of the engineered extracellular vesicles (iEV-150) is 150.6±11 nm.

[0008] Furthermore, the miR-150-3p is hsa-miR-150-3p, and the nucleotide sequence of the hsa-miR-150-3p is shown in SEQ ID NO:1.

[0009] Furthermore, the nucleic acid sequence of the ANXA2 protein is shown in SEQ ID NO:2, and the protein sequence of the ANXA2 protein is shown in SEQ ID NO:3.

[0010] On the other hand, the present invention provides an application of an engineered extracellular vesicle (iEV-150) loaded with miR-150-3p and surface-modified with an iRGD targeting peptide for preparing a drug for treating melanoma.

[0011] Furthermore, the melanoma treatment drug targets and delivers miR-150-3p to melanoma cells via engineered extracellular vesicles (iEV-150), thereby inducing ferroptosis of melanoma cells to inhibit the growth and metastasis of melanoma.

[0012] Furthermore, the melanoma treatment drug regulates ferroptosis-related factors and promotes lipid peroxidation through engineered extracellular vesicles (iEV-150), reduces the GSH / GSSG ratio, increases intracellular Fe²⁺ and MDA levels, and mitochondrial oxidative stress response, ultimately leading to ferroptosis of melanoma cells.

[0013] Furthermore, the ferroptosis-related factors are 4-HNE, CHAC1, ACSL4, and GPX4.

[0014] On the other hand, the present invention provides an ANXA2 protein that promotes the enrichment of miR-150-3p in extracellular vesicles (EVs), wherein the nucleic acid sequence of the ANXA2 protein is shown in SEQ ID NO:2, and the protein sequence of the ANXA2 protein is shown in SEQ ID NO:3.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention relates to a melanoma targeted therapy method based on engineered extracellular vesicles (EVs), in particular to a preparation method and application of engineered EVs (iEV-150) containing iRGD targeting peptide and loaded with miR-150-3p, by constructing HEK293T cells stably expressing miR-150-3p, and introducing ANXA2 protein to promote the enrichment of miR-150-3p in EVs, while modifying the iRGD peptide on the surface of EVs to enhance its melanoma cell targeting.

[0016] 2. The results of the present invention show that iEV-150 can effectively deliver miR-150-3p to melanoma cells, inhibit tumor cell proliferation, DNA damage repair and epithelial-mesenchymal transition (EMT), and promote melanoma cell death by inducing ferroptosis; in addition, iEV-150 can significantly inhibit tumor growth and metastasis in a melanoma mouse model and prolong the survival time of mice; biochemical and pathological analyses show that iEV-150 has no obvious liver and kidney toxicity or systemic toxic side effects within the tested dose range; further mechanism studies show iEV-150 regulates ferroptosis-related factors (4-HNE, CHAC1, ACSL4, GPX4, etc.) and promotes lipid peroxidation, thereby reducing the GSH / GSSG ratio, increasing intracellular Fe²⁺ and MDA levels, and mitochondrial oxidative stress response, ultimately leading to ferroptosis of melanoma cells; both in vitro and in vivo experiments have verified the effectiveness of iEV-150 in the treatment of melanoma; the present invention provides a precise treatment strategy for melanoma based on engineered extracellular vesicles, which can be widely used in the targeted treatment of melanoma and other malignant tumors, and has a high clinical translational value.

[0017] 3. The engineered extracellular vesicles provided by the present invention can target the delivery of miR-150-3p to melanoma cells and inhibit the growth and metastasis of melanoma by inducing ferroptosis. The present invention combines nano-delivery technology and ferroptosis regulation mechanism to provide an innovative strategy for the precision treatment of melanoma, which can effectively overcome the drug resistance of traditional therapies and improve the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Figure 2 shows the enrichment of miR-150-3p in melanoma-derived extracellular vesicles (EVs); (AC) are heat maps of miRNA expression profiles in melanoma cells and their extracellular vesicles (EVs); (D) is a Venn diagram analysis; (E) is a transmission electron microscopy image showing EVs isolated from melanoma cells; (F) is a nanoparticle tracking analysis to detect the particle size distribution of EVs; (G) is a Western blot to detect EV markers CD9, TSG101, CD81, CD63 and HSP70; (HJ) are qRT-PCR tests for the expression levels of miR-150-3p and miR-1246 in normal melanocytes and melanoma cells, melanoma cells and extracellular vesicles, and melanoma cells and normal cell-derived EVs; (K) is the expression of miR-150-3p in EVs without treatment or after RNase A / Triton Detection results after X-100 treatment; (L) Transmission electron microscopy imaging shows EVs isolated from the plasma of melanoma patients; (M) Nanoflow cytometry analysis of the particle size distribution of EVs derived from the plasma of melanoma patients; (N) qRT-PCR analysis of the expression level of miR-150-3p in EVs derived from the plasma of melanoma patients and healthy individuals; (OP) in situ hybridization detection of miR-150-3p expression scores in tumor tissues and adjacent normal tissues in the melanoma tissue chip cohort; (Q) ISH analysis of miR-150-3p expression scores in melanoma tissues of different clinical stages; (R) Kaplan-Meier survival analysis.

[0019] Figure 2To identify RNA binding proteins that interact with miR-150-3p and the role of ANXA2 in melanoma cells; in the figure, (A) is a typical staining image of the Bio-miR-150-3p complex displayed by SDS-PAGE electrophoresis; (B) is the results of GO and KEGG enrichment analysis; (C) is the result of RNA immunoprecipitation experiment; (D) is Western blotting results; (E) is the result of direct binding experiment using recombinant ANXA2 purified from Escherichia coli; (F) is the result of electrophoretic mobility shift assay; (G) is the result of immunofluorescence analysis; (H) is the effect diagram of ANXA2 overexpression and knockout in melanoma cells; (I) is the result of the effect of ANXA2 overexpression on exosome release; (J) is the result of the effect of ANXA2 overexpression on miR-150-3p expression in melanoma cells; (K) is the result of the effect of ANXA2 overexpression on miR-150-3p enrichment in melanoma-derived exosomes; (L) is the result of the effect of ANXA2 KO on miR-150-3p expression in melanoma cells and miR-150-3p levels in exosomes; (M) is the result of CCK-8 experiment detection; (N) is the result of PCNA and CCND1 protein level detection.

[0020] Figure 3 Preparation of iEV-150 loaded with miR-150-3p and surface modified with iRGD targeting peptide; in the figure, (A) is a schematic diagram of the preparation process of engineered HEK-293T cell-derived EVs for the delivery of miR-150-3p to melanoma cells; (B) is transmission electron microscopy imaging of iEV-150; (C) is the particle size distribution detection of nanoparticle tracking analysis of iEV-150; (D) is the result of Western blot detection of markers in EV-150 and iEV-150; (E) is qRT-PCR detection of the expression level of miR-150-3p in EVs derived from cells in different treatment groups; (F) is qRT-PCR detection of the expression level of miR-150-3p in engineered exosomes iEV-NC and iEV-150.

[0021] Figure 4The targeting evaluation of engineered iEV-150 in vivo and in vitro; in the figure, (A) is the laser scanning confocal microscopy imaging analysis of the uptake of PKH67 and PKH26 labeled EVs by melanoma cells; (B) is the transmission electron microscopy imaging analysis of the uptake of engineered iEV-150 and EV-150 by melanoma cells; (C) is the flow cytometry analysis of the uptake of FAM labeled iEV-150 and EV-150 by melanoma cells; (D) is the in vivo imaging system analysis of the distribution of DIR labeled iEV-150 and EV-150 in the mouse melanoma model; (E) is the IVIS analysis of the fluorescence intensity distribution of the main organs and tumor tissues of mice 9 hours after injection; (F) is the mouse melanoma group.

[0022] Figure 5 The biosafety of iEV-150 was evaluated. (A) C57BL / 6 mice were injected with 5×10¹ 0 Pathological analysis results of heart, lung, liver, kidney and spleen tissue sections after treatment with particulate iEV-150 or PBS every other day for 7 days; (BD) Analysis of ALT, AST and creatinine CR levels in mouse serum.

[0023] Figure 6 EV-150 inhibits melanoma cell proliferation and EMT in vitro; (A) DNA replication of A375 and A875 cells was detected by EDU staining; (B) cell cycle distribution of A375 and A875 cells was analyzed by flow cytometry; (C) spheroid formation experiment results were presented; (D) cell growth was detected by clone formation experiment; (E) Western blot analysis of the expression levels of marker genes related to proliferation, EMT and DNA damage repair in melanoma cells.

[0024] Figure 7The effect of EV-150 in inhibiting melanoma growth and lung metastasis in mice in vivo; in the figure, (A) is a schematic diagram of the experiment of subcutaneous injection of B16-F10 cells and tail vein injection of iEV-150 into C57BL / 6 mice; (BD) are representative images, tumor growth curves and tumor weights of subcutaneous melanoma transplant models; (E) is a representative image of TUNEL staining of subcutaneous melanoma transplant models; (FI) are representative immunohistochemical staining images of 4-HNE, Cleavedcaspase-3 and GSDMD-N in subcutaneous melanoma transplant models; (J) is the use of bioluminescence imaging to detect the metastatic ability of B16-F10-luc cells after treatment with iEV-150 or iEV-NC; (K) is a representative image of the lung metastasis model (top), statistical analysis of the number of lung nodules (middle), and representative images of lung tissue HE staining (bottom); (L) is the survival analysis of mice with subcutaneous transplant tumor models after treatment with iEV-150 and iEV-NC.

[0025] Figure 8The mechanism by which iEV-150 inhibits melanoma growth and metastasis by inducing ferroptosis; in the figure, (A) shows the cell proliferation of A375 cells co-cultured with iEV-150 after treatment with multiple cell death pathway specific inhibitors; (B) shows the results of the A375 cell sphere formation experiment; (C) is a schematic diagram of the B16-F10 melanoma cell subcutaneous injection mouse model and the tail vein injection of iEV-150 / iEV-NC and Fer-1 / DMSO; (D) Photos of tumors in each group of mice after tail vein injection of iEV-150 / iEV-NC and Fer-1 / DMSO; (E) is the weight of the tumor in each group of mice; (F) is the growth curve of the tumor in each group of mice; (G) is the Prussian blue staining, TUNEL detection and 4-HNE immunohistochemistry detection results; (H) is the statistical analysis of the data; (I) is the in vivo fluorescence signal recorded by IVIS and the fluorescence image detected in the mouse lung; (J) is the quantitative analysis of the mouse fluorescence intensity; (K) is the representative image of the mouse lung metastasis model and HE staining results; (L) is the quantitative analysis of the number of mouse lung nodules; (M) is the analysis of lipid ROS levels in A375 cells in the co-cultured iEV-150 group, Erastin group and co-cultured iEV-150 and Fer-1 group; (N) is the analysis of mitochondrial superoxide levels in A375 cells in each group; (OR) is the detection of Fe²⁺ levels, GSH / GSSG ratio, MDA levels and cell death percentage in A375 cells in each group by flow cytometry; (S) is Western Blot analysis was used to detect the expression levels of 4-HNE, CHAC1, ACSL4 and GPX4; (T) was the analysis of mitochondrial membrane potential of A375 cells; (U) was the transmission electron microscopy (TEM) analysis of the mitochondrial structure of melanoma cells after treatment in each group.

[0026] Fig. 9iEV-150 inhibits melanoma proliferation and metastasis by regulating NF2; (A) Heat map showing the changes in downregulated genes in mRNA-seq after co-culture of A875 cells with iEV-150; (B) Volcano plot showing differentially expressed genes in mRNA-seq; (C) KEGG pathway enrichment analysis of differentially expressed genes; (D) RNA-seq data after co-culture with iEV-150 showing expression changes of genes related to DNA replication, DNA damage repair, cell cycle, cell migration and ferroptosis; (E) Venn diagram showing the intersection of ferroptosis-inhibiting genes and downregulated genes in RNA-seq: (F) AGO2-RIP-qRT-PCR analysis enriched miR-150-3p mRNA; (G) qRT-PCR detection of changes in candidate mRNA expression after inhibition of miR-150-3p; (H) miRNA pulldown experiment results; (I) miR-150-3p overexpression increased NF2 mRNA enrichment in the Ago2 / RNA-induced silencing complex; (J) Luciferase reporter assay to evaluate the interaction between miR-150-3p and NF2 3′UTR; (K) Correlation between miR-150-3p and NF2 in melanoma tissue microarray; (L) Western blot to detect NF2 protein expression after miR-150-3p overexpression or knockdown; (M) EdU cell proliferation analysis; (N) Western Blot analysis of the protein expression of proliferation marker genes, EMT genes and DNA damage repair genes in melanoma cells after co-culture with iEV-150 or overexpression of NF2; (O) is the image of subcutaneous tumor in C575BL / 6 mice; (P) is the weight of mouse tumor; (Q) is the growth curve of mouse tumor; (RS) is the representative images and data analysis of Matson staining, DAB-enhanced Prussian blue staining and TUNEL staining of mouse tumor sections; (TU) is the representative images and data analysis of immunohistochemical (IHC) expression of 4-HNE, ACSL4 and CHAC1 in mouse tumor sections; (V) is the image and HE staining of lung metastasis after tail vein injection of cells or exosomes in different groups of mice; (W) is the quantitative analysis of lung metastatic nodules in different groups of mice. DETAILED DESCRIPTION

[0027] Materials and Methods 1. Clinical samples and tissue microarrays Fresh peripheral blood samples were collected in EDTA-containing anticoagulant tubes from 16 melanoma patients who did not receive preoperative chemotherapy or radiotherapy, and 16 age- and sex-matched healthy controls. The samples were collected from May to December 2024 at the Cancer Hospital of the Chinese Academy of Medical Sciences. The collected blood samples were centrifuged at 3000 × g for 10 minutes at 4°C to separate the plasma supernatant. The plasma samples were then centrifuged at 16000 × g for 10 minutes at 4°C and stored at -80°C for subsequent extraction of exosomes.

[0028] 2. Cell culture Human melanoma cell lines A375 and A875 were cultured in DMEM high-glucose medium containing 10% FBS and 1 / 1000 dual antibodies. The culture medium of human normal melanoma cell line PIG1 was consistent with that of A375 and A875. The cell culture conditions were 37°C and 5% CO2 concentration. During the cell culture cycle, a 9 cm diameter culture dish was used with a culture medium volume of 10 mL. The cells were observed twice a day. When the cell confluence reached 80%, the cells were passaged or frozen. When the cells were passaged, 5 mL of PBS was used to wash the cells twice, and then 3 mL of EDTA was added to digest the cells in a 37°C incubator for 3 min. The cell digestion was observed under a microscope, and 6 mL of culture medium was added to terminate the cell digestion. Finally, the cells were centrifuged at 1500 rpm for 5 min and the supernatant was discarded. The cells were passaged at a ratio of 1:3.

[0029] 3. Biodistribution of EVs in vivo First, 5×10 6 B16-F10 cells were injected subcutaneously into C57BL / 6 mice. Five days later, when the tumors grew to 3 mm in size, EV-150 or iEV-150 were labeled with DIR red fluorescent dye and then injected via the tail vein with 5 × 10 5 After the particles were labeled with EVs or iEVs, in vivo fluorescence images were captured using IVIS at 3, 6, and 9 hours after injection. Tumor tissues were removed 9 hours after injection and then fixed in 4% PFA for 24 hours. Then, the tumor tissues were embedded and cut into 15 μm thick sections, and the sections were stained with DAPI. Fluorescence signals of DIR were captured using a fluorescence confocal microscope.

[0030] 4. miRNA real-time fluorescence quantitative PCR (qRT-PCR) detection The miRNAs were extracted using the MiRNeasy Mini Kit from Kaijie Company and reverse transcribed using the reverse transcription kit from Takara Company. The reverse transcription system of miRNA was as follows:

[0031] Table 1 miRNA reverse transcription system

[0032] Run the following program in a PCR instrument: 37°C for 1 h; 85°C for 5 min. Add 90 μL RNase Free H 2 O and stored at -20°C.

[0033] miRNA qRT-PCR was performed using a kit from Preomega, with the miR-150-3p primer as shown in SEQ ID NO:4, the miR-1246 primer as shown in SEQ ID NO:4, and U6 as the internal reference gene. The system was as follows:

[0034] Table 2 U6 qRT-PCR system

[0035] Table 3 miRNA qRT-PCR system

[0036] Run the following program in a 7500 real-time fluorescence quantitative PCR instrument: Table 4 miRNA qRT-PCR reaction conditions

[0037] 5. CCK8 Experiment (1) The cells were first seeded into a 6-well plate. After the cells were treated with different groups, they were digested with trypsin and counted.

[0038] (2) 2×103 cells / well were seeded into a 96-well plate, with 6 replicate wells for each group.

[0039] (3) Aspirate the old culture medium used to maintain cell growth in the 96-well plate and replace it with 100 μL of fresh culture medium containing 10% CCK8 reagent; add 100 μL of complete culture medium containing 10% CCK8 reagent to the right side of the experimental well as a blank control, and add sterile PBS to the wells around the cells to be tested to prevent evaporation of the nutrient solution.

[0040] (4) Place the culture plate in a cell culture incubator and culture it in the dark for 2 hours before testing. The test process uses a multifunctional microplate reader to measure the absorbance value of each well at 450 nm. The test is performed at the same time point for 4 consecutive days. The experimental data are analyzed and plotted using Graphpad prism 8 software.

[0041] 6. Plate colony formation experiment (1) The cells were first seeded into a 6-well plate. After 48 hours of different treatments, the cells were digested and counted.

[0042] (2) 1×10 3 Melanoma cells were seeded into 3.5 cm² cell culture dishes and fresh high-glucose medium was replaced every 3 days.

[0043] (3) After about 2 weeks of culture, obvious cell colony formation was observed in some groups under a microscope. After washing with PBS three times, 4% paraformaldehyde was added to immerse the cells and the cells were fixed at room temperature for 30 minutes.

[0044] (4) After washing with PBS for 3 times, add crystal violet stain and stain at room temperature for 10 min. Gently wash away the unbound dye with PBS. After drying naturally, take pictures to observe the size and number of cell colonies. A cell number >50 is recorded as a positive clone. The number of cell clones in all groups is counted.

[0045] 7. Immunohistochemical staining (1) Take the prepared animal tissue paraffin sections, place them in a 65℃ drying oven for 4-5 hours, and then dewax them in the following order.

[0046] (2) 3% H 2 O 2 Incubate at room temperature for 30 min to inactivate endogenous enzymes.

[0047] (3) Place the slices in a glass jar, add histochemical PBS, place the jar on a shaker and shake slowly. Repeat the washing process three times, 5 minutes each time.

[0048] (4) Immerse the slices in 0.01 M citrate buffer solution and heat them in a steamer for 30 min for heat antigen repair. After cooling, wash them twice with histochemical PBS and block them with 5% BSA at room temperature for 30 min. Shake off the excess liquid on the slices.

[0049] (5) Accurately circle the tissue area on the slice, dilute the primary antibody with 5% BSA, and add the primary antibody to the surface of the slice tissue. Incubate at 4°C overnight, recover the primary antibody, and wash the slice three times with histochemical PBS, each time for 5 minutes.

[0050] (6) Add biotin-labeled goat anti-mouse / rabbit IgG and incubate at 37°C for 30 min. Wash the sections three times with histochemical PBS, each time for 5 min.

[0051] (7) Add SABC and incubate at 37°C for 30 min. Wash the sections with histochemical PBS three times, 5 min each time.

[0052] (8) Using the DAB colorimetric kit, take 1 mL of solution B into a brown EP tube and add a drop of solution A (about 50 μL) to the solution and mix well.

[0053] (9) After the color developing solution is added to the sample area, start timing and observe the color development through a microscope. After the color development is completed, the slice is quickly placed in deionized water to terminate the reaction. The color development reaction time of all groups in the same experiment must be strictly consistent.

[0054] (10) Add hematoxylin to the sample area for 1 min and rinse with running water.

[0055] (11) Perform the dehydration and clearing steps in the reverse order of the dewaxing to water process. After clearing, add a small amount of transparent neutral resin to the center of the tumor tissue on the slice, cover with a glass slide to remove bubbles and seal the slice. Place the slice in an open and ventilated place to wait for the resin to dry thoroughly before subsequent observation and photography.

[0056] 8. Western blot Whole cell protein extracts were homogenized in lysis buffer and centrifuged at 12,000 × g for 15 minutes. Protein concentration was determined using the BCA method, and protein lysates were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% bovine serum albumin for 1 hour at room temperature, and then incubated with primary antibodies at 4°C overnight. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase, and membrane signals were detected using a commercial ECL kit.

[0057] The antibodies used in this experiment are listed in Table 5.

[0058] Table 5 Antibody information

[0059] 9. Construction of stable cell lines Lentiviral vectors pHB-U6-mir-150-3p-EF1-LUC-PURO and pLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro pHB-ANXA2-EF1-LUC-PURO were constructed by Fenghui Biotechnology (Hunan) Co., Ltd. During transfection, the infection multiplicity (MOI) of A375 and A875 cells was 10, while that of HEK-293T cells was 5; after infection, cells were selected with 5 μg / mL puromycin for 14 days, and then cells were maintained in medium without puromycin. To generate stable cell lines, A375 and A875 cells stably overexpressing miR-150-3p were established by puromycin selection. Similarly, HEK-293T cells stably overexpressing miR-150-3p and ANXA2 were generated using sequential lentiviral transfection, followed by 5-day puromycin selection every 30 days to maintain stable expression.

[0060] 10. Construction process of engineered extracellular vesicles Hunan Fenghui Biotechnology Co., Ltd. constructed lentiviral vectors including pLV-Puro, miR-150-3p overexpression lentivirus, and ANXA2 overexpression lentivirus, and then transfected the miR-150-3p overexpression lentivirus into HEK-293T cells at an infection multiplicity of MOI = 5. After infection, the cells were selected in a medium containing 5 μg / mL puromycin for 14 days, and then transferred to a puromycin-free medium for continued culture to establish a HEK-293T cell line that stably overexpresses miR-150-3p.

[0061] ANXA2-overexpressing lentivirus was further transfected into HEK-293T cells overexpressing miR-150-3p to construct a HEK-293T cell line stably co-overexpressing miR-150-3p and ANXA2. To engineer EVs, Chol-PEG-iRGD was linked to miR-150-3p-loaded EVs using the ExoBrooch-iRGD kit provided by Beijing Enze Kangtai Biotechnology Co., Ltd. to obtain iEV-150.

[0062] 11. LC-MS mass spectrometry identification Dilute trypsin to 20 ng / µl with 25 mM NH4HCO3; add appropriate amount of trypsin to each tube, place in a 37 degree incubator for enzymatic digestion overnight, and then purify the peptides. Dissolve the peptides with sample dissolving solution and perform mass spectrometry identification. The separated peptides directly enter the mass spectrometer for online detection. The original mass spectrometry file is processed and converted by MM File Conversion software to obtain MGF format file, and then MASCOT is used to search the uniprot database.

[0063] 12. EVs labeling and uptake PKH26 / PKH67 dye was diluted in 100 μl diluent C (dye solution). Then, 10 μg of EVs was dissolved in 20 μl PBS, 80 μl diluent C was added, the dye solution was added, and incubated for 5 minutes. To remove excess dye, the labeled EVs were diluted to 30 ml PBS, recovered by ultracentrifugation and resuspended in PBS. When treating cells, 5 μg of EVs was mixed with 2×10 5 The recipient cells were co-incubated for 24 hours. Then, the cells were washed twice with PBS for 10 minutes each time and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Then, the cells were washed twice with PBS, treated with 0.5% Triton X-100 for 5 minutes, washed twice with PBS again, and then the cells were incubated with 500nM Phalloidin dye for 30 minutes at room temperature, washed twice with PBS for 10 minutes each time, and finally mounted with DAPI-containing blocking agent for microscopic imaging.

[0064] 13. Animal Experimentation C57BL / 6 mice (4-6 weeks old) were obtained from Beijing Sibeifu Biotechnology Co., Ltd., China, and were housed under standard specific pathogen-free (SPF) conditions. Before the start of the experiment, the mice were acclimated to the environment for one week. In the mouse xenograft tumor experiment, 5 × 10 6 The mouse melanoma cell line B16-F10 was injected subcutaneously into C57BL / 6 mice. Starting from day 7, 5 × 10 10 After 21 days, the mice were killed and the melanomas were removed for subsequent experiments. In the tail vein transfer assay, 5 × 10 5 B16-F10-luc cells were injected into the tail vein of nude mice. After 5 days, 5 × 10 10 particles engineered iEV-NC or iEV-150; 21 days later, the nude mice were anesthetized, and fluorescent images of the mice were captured using IVIS, and the mice were killed and their lungs were removed for examination.

[0065] 14. Isolation of RISC-associated RNA Cells overexpressing miR-150-3p or NC were fixed with 1% formaldehyde, followed by chromatin fragmentation. After cell lysis using NETN buffer, the lysate was incubated with small magnetic beads coated with IgG or anti-Pan-Ago antibody. Immunoprecipitated RNA was released by proteinase K digestion, extracted with phenol / chloroform / isopropanol, and purified by glycogen ethanol precipitation. Subsequently, RNA was solubilized and treated with DNase I.

[0066] 15. Biotin RNA pull-down assay Cells were lysed with RIP buffer supplemented with RNase inhibitor (Invitrogen) and protease inhibitor cocktail. Lysates were pre-cleared with empty magnetic beads before incubation. Biotinylated RNA probes were first immobilized on streptavidin-coated magnetic beads (Invitrogen) and then incubated with pre-cleared cell lysate for 4 h at 4°C. Subsequently, the beads were washed five times with RIP buffer to remove unbound material. Finally, bound proteins and RNA were eluted with Laemmli sample buffer for further analysis. For Western blot analysis, 0.5–1 mg of cell lysate and 1–3 μg of biotinylated RNA were used. For RNA pull-down experiments using purified protein, 1–2 μg of purified protein and 1–3 μg of biotinylated RNA were sufficient.

[0067] 16. Ferroptosis index detection The intracellular iron content was measured using a total iron colorimetric kit. 5 Cells were seeded into 6-well plates at a density of 10 cells / well. After 24 hours of treatment with the experimental group, the cells were collected and lysed. After centrifugation (15,000×g, 10 minutes), the supernatant was collected. 80 μL of sample and 80 μL of color development solution were added to each well, the mixture was incubated at 37°C for 40 minutes, and the absorbance (OD) was measured at a wavelength of 593 nm using a microplate reader.

[0068] The intracellular ferrous iron content was measured using a ferrous iron colorimetric kit (E-BC-K881-M; Elabscience, Wuhan, China). 5 Cells were seeded into 6-well plates at a density of 10 cells / well. After 24 hours of treatment with the experimental group, the cells were collected and lysed. After centrifugation (15,000×g, 10 minutes), the supernatant was collected. 80 μL of sample and 80 μL of color development solution were added to each well, the mixture was incubated at 37°C for 10 minutes, and the absorbance (OD) was measured at a wavelength of 593 nm using a microplate reader.

[0069] Malondialdehyde (MDA) levels were measured using an MDA colorimetric kit (E-BC-K028-M; Elabscience, Wuhan, China). 5 Cells were seeded into 6-well plates at a density of 10 cells / well. After 24 hours of treatment with the experimental group, the cells were collected and lysed. The supernatant was collected by centrifugation. 0.1 mL of sample and 1 mL of working solution were added to each EP tube. The samples were incubated in a 100°C water bath for 40 minutes, cooled to room temperature, and centrifuged (15,000×g, 10 minutes). The absorbance (OD) of the supernatant was measured at a wavelength of 532nm using a microplate reader.

[0070] Lipid ROS levels were measured using C11-BODIPY (Cat.# D3861, Thermo Fisher) following the manufacturer’s instructions. Briefly, after 2 days of treatment, cells were incubated with 10 mmol / L C11-BODIPY at 37°C, 5% CO 2 After incubation, the cells were washed three times with PBS and the C11-BODIPY green fluorescence (484 nm / 510 nm) was measured using a flow cytometer.

[0071] Mitochondrial superoxide levels in melanoma cells were measured using MitoSOX Red mitochondrial superoxide indicator (Invitrogen). After 24 h of treatment, cells were stained with 10 mmol / L MitoSOX reagent in PBS for 10 min and incubated at 37°C, 5% CO 2 After removing the excess MitoSOX, the fluorescence of the cells was detected by flow cytometry with an excitation / emission wavelength of 510 nm / 580 nm.

[0072] GSH and GSSG levels were measured using a GSH / GSSG assay kit (Abcam, ab138881), and the GSH / GSSG ratio was calculated according to the manufacturer’s instructions.

[0073] Mitochondrial membrane potential (C2003S, Beyotime, China) detection: cells were incubated with 500 μL JC-1 staining working solution (BD, USA) at 37°C for 20 min. Then, cells were washed twice with PBS and images were taken for analysis using a fluorescence microscope or flow cytometer.

[0074] 17. Immunohistochemistry and hematoxylin-eosin staining Paraffin-embedded sections were first dewaxed with xylene, then rehydrated through an ethanol gradient, and heat-induced antigen retrieval was performed using sodium citrate buffer. Sections were treated with 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity. Next, sections were blocked with 5% BSA and incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at 37°C for 30 minutes. 3,3′-diaminobenzidine solution was used for color development and finally observed under a microscope.

[0075] Tumor tissue and lung tissue of mice were stained with hematoxylin-eosin for pathological evaluation. Paraffin sections were first dewaxed with xylene and then rehydrated through an ethanol gradient. The sections were stained with hematoxylin for 5 minutes at room temperature and then transferred to water for 2 minutes to remove floating color. The sections were then transferred to differentiation solution (1% hydrochloric acid alcohol) for staining for a few seconds to 30 seconds, followed by washing with water for 30-60 minutes. Finally, the sections were stained in eosin solution for 2 minutes and washed with water again before further observation.

[0076] Example 1 miR-150-3p is enriched in melanoma-derived extracellular vesicles (EVs) To screen for miRNAs with important biological functions in melanoma, miRNA sequencing analysis was performed on A875 melanoma cells and their extracellular vesicles (EVs). Figure 1 A total of 135 downregulated miRNAs and 143 upregulated miRNAs were identified in A875 EVs (p<0.05, |log2FC|>2). In addition, the GSE35387 dataset (differentially expressed miRNAs in A375 cells and their EVs) was screened and reanalyzed by querying the Gene Expression Omnibus (GEO) database ( Figure 1 B) and GSE125030 dataset (differentially expressed miRNAs in primary melanoma cells and their EVs) ( Figure 1 C). Comprehensive analysis of the results of the three data sets showed that both miR-150-3p and miR-1246 showed a stable upregulation trend in melanoma-derived EVs ( Figure 1 D). To further verify the characteristics of EVs, EVs were isolated from A375 and A875 cells and analyzed by transmission electron microscopy (TEM) ( Figure 1 E) Particle size distribution analysis ( Figure 1 F) and EV-specific marker detection ( Figure 1 G) characterized them. The results showed that compared with normal melanocytes (PIG1), miR-1246 and miR-150-3p were significantly upregulated in melanoma cells (A375 and A875) ( Figure 1 H). In addition, the expression levels of these two miRNAs were further increased in melanoma-derived EVs compared with parental cells ( Figure 1 I). In the comparison between normal melanocyte-derived EVs and melanoma EVs, the expression level of miR-150-3p was significantly increased, while the change of miR-1246 was relatively small ( Figure 1 J). Further studies found that the expression of miR-150-3p in EVs did not change significantly after RNase A treatment, but the expression level decreased after RNase A and Triton X-100 combined treatment, indicating that miR-150-3p was stably encapsulated inside EVs rather than adsorbed on the surface of EVs ( Figure 1 K).

[0077] To investigate the expression of miR-150-3p in melanoma patients, EVs were isolated from the plasma of 16 melanoma patients ( Figure 1 L and 1M, and EVs from healthy donor plasma were used as controls. qRT-PCR analysis showed that the expression level of miR-150-3p in plasma EVs of melanoma patients was significantly higher than that in healthy individuals ( Figure 1 N). In addition, in order to analyze the expression level of miR-150-3p in tissues, in situ hybridization (ISH) detection was performed on melanoma tissue chips. The results showed that the miR-150-3p ISH score in melanoma tissues was significantly reduced compared with that in adjacent normal tissues ( Figure 1 O and 1P). Further analysis of melanoma tissue samples of different clinical stages revealed that the miR-150-3p ISH scores of stage II-IV melanoma tissues were significantly lower than those of stage I, suggesting that the expression level of miR-150-3p may be negatively correlated with the clinical progression of melanoma ( Figure 1 Q). Kaplan-Meier survival analysis further revealed that patients with lower miR-150-3p expression had a significantly shorter overall survival than those with higher expression ( Figure 1 R), indicating that the expression level of miR-150-3p may be correlated with the prognosis of melanoma patients.

[0078] Example 2 ANXA2 regulates exosome transport of miR-150-3p Although the miRNA profiles in extracellular vesicles (EVs) differ from those in their donor cells, the mechanisms responsible for this difference remain unclear. RNA-binding proteins (RBPs) are known to play an important role in the exosomal sorting of miRNAs, so we sought to identify RBPs that interact with miRNAs enriched in cancer-derived exosomes. To this end, we isolated biotinylated miR-150-3p complexes from A875 cells using streptavidin-coated magnetic beads, showing a typical staining pattern of the Bio-miR-150-3p complex in SDS-PAGE electrophoresis ( Figure 2 A) was used to isolate and identify proteins that interact with miR-150-3p. After gel digestion, liquid chromatography-mass spectrometry (LC-MS) analysis was performed to identify proteins that were unique to the Bio-miR-150-3p complex and absent in the control biotinylated miRNA complex. GO and KEGG enrichment analysis of these identified proteins showed that ANXA2, RAB14, TMED9, VAPA, and ARF4 were key regulators of the EV transport pathway ( Figure 2 B). We validated the interaction between miR-150-3p and RNA binding proteins identified in GO and KEGG analysis by RNA immunoprecipitation (RIP) experiments, using IgG as a negative control to exclude nonspecific binding and GAPDH antibody as an internal control. Figure 2 As shown in C, the expression of miR-150-3p in ANXA2 precipitates was more than 13-fold higher than that in IgG precipitates, indicating a strong and specific interaction between ANXA2 and miR-150-3p. In contrast, the expression of miR-150-3p in TMED9 precipitates was increased, while the levels in RAB14, VAPA, and ARF4 precipitates were comparable to the IgG control group. The interaction between miR-150-3p and ANXA2 in RNA pull-down experiments was confirmed by Western blotting ( Figure 2 D). In addition, the direct binding between miR-150-3p and ANXA2 was further verified using recombinant ANXA2 purified from Escherichia coli. After incubation of GST-ANXA2 with biotinylated miR-150-3p, the cells were captured by streptavidin-coated magnetic beads, and Western blotting analysis confirmed that ANXA2 could directly and independently bind to miR-150-3p ( Figure 2 E). Similarly, electrophoretic mobility shift assay (EMSA) also confirmed that miR-150-3p showed supershift after incubation with ANXA2 ( Figure 2 F). Immunofluorescence analysis showed that miR-150-3p colocalized with ANXA2 in melanoma cells ( Figure 2 G).

[0079] To investigate the role of ANXA2 in melanoma cells, we overexpressed ANXA2 in melanoma cells ( Figure 2 H, bottom panel), and found that ANXA2 overexpression did not enhance exosome release ( Figure 2 I). However, ANXA2 overexpression significantly reduced miR-150-3p expression in melanoma cells ( Figure 2J), while significantly increasing its enrichment in melanoma-derived exosomes ( Figure 2 K). In addition, melanoma cells transfected with ANXA2 knockout (KO) sgRNA CRISPR virus showed a significant decrease in ANXA2 levels ( Figure 2 H, top panel and Figure 2 L, left panel), while the expression of miR-150-3p in cells increased ( Figure 2 L, left panel). This finding was negatively correlated with the level of miR-150-3p in exosomes ( Figure 2 L, right panel). Overall, these data indicate that sorting of miR-150-3p to exosomes is ANXA2-dependent.

[0080] To further investigate the effect of ANXA2 KO on melanoma cells, we used CCK-8 assay to detect cell proliferation. ANXA2 KO significantly inhibited the proliferation of melanoma cells ( Figure 2 M). This result is consistent with the decrease in PCNA and CCND1 protein levels ( Figure 2 N). Notably, inhibition of miR-150-3p expression reversed the inhibitory effect of ANXA2 KO on PCNA and CCND1 expression ( Figure 2 N), ultimately leading to the induction of cell growth ( Figure 2 M).

[0081] Example 3 Preparation and characterization of engineered iEV-150 In order to achieve targeted therapy for melanoma, HEK293T cell lines stably expressing miR-150-3p were constructed by lentiviral transfection technology. In addition, to further promote the enrichment of miR-150-3p in EVs, a lentiviral vector encoding ANXA2 overexpression was introduced to enhance the ability of miRNA to be selectively loaded into EVs. Subsequently, EVs were separated by ultracentrifugation, and iRGD targeting peptide was coupled to the EVs membrane surface to construct iEV-150 (enriched with miR-150-3p and capable of targeting melanoma cells). Figure 3 A).

[0082] The resulting engineered iRGD-EVs-miR-150-3p (iEV-150) and iRGD-EVs-miR-NC (iEV-NC) were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot, respectively. TEM imaging showed that iEV-150 had a typical "cup-shaped" morphology ( Figure 3B). In addition, NTA analysis results showed that the average particle sizes of EV-150 and iEV-150 were 112.5±7 nm and 150.6±11 nm, respectively ( Figure 3 C). Western blot results showed that both EV-150 and iEV-150 without iRGD targeting peptide modification expressed typical EV markers CD9, CD63, CD81 and HSP70, while the negative marker Calnexin was not detected ( Figure 3 D). qRT-PCR results showed that the expression level of miR-150-3p in EVs derived from cells stably overexpressing miR-150-3p was increased 490-fold compared with that in EVs derived from 293T cells. In addition, after stable overexpression of ANXA2, the expression of miR-150-3p in EVs was further increased ( Figure 3 E). Further analysis revealed that the expression level of miR-150-3p in iEV-150 was significantly upregulated compared with that in EV negative control (iEV-NC), indicating that miR-150-3p had been successfully loaded into EVs ( Figure 3 F).

[0083] Example 4 iEV-150 exhibits enhanced melanoma cell targeting ability

[0084] In order to evaluate the targeting ability of engineered iEVs to melanoma cells, laser scanning confocal microscopy (LSCM) was used to observe the uptake of PKH26-labeled EVs in cells. The results showed that compared with unmodified EV-150, melanoma cells (A375, A875 and B16-F10) had a higher uptake rate of iEV-150 ( Figure 4 A). In addition, transmission electron microscopy (TEM) further confirmed that melanoma cells internalized more iEVs ( Figure 4 B). In order to verify whether the iRGD targeting peptide was successfully introduced into EVs, the experiment used FAM-labeled Chol-PEG-CRGDKGPDC, and analyzed its delivery efficiency in melanoma cells by flow cytometry. The results showed that compared with EV-150, the FAM fluorescence signal in melanoma cells treated with iEV-150 was significantly enhanced, indicating that the modification of iRGD peptide improved the targeting ability of EVs to melanoma cells ( Figure 4 C). To investigate the targeting ability of engineered extracellular vesicles (iEVs) in vivo, DIR-labeled EV-150 and iEV-150 (5×10¹ 0 The iEV-150 particles were injected into C57BL / 6 mice bearing subcutaneous melanoma tumors via the tail vein. In vivo imaging system (IVIS) analysis showed that iEV-150 exhibited a stronger tumor targeting ability than unmodified EV-150 ( Figure 4 D). The fluorescence signal of iEV-150 was significantly stronger than that of EV-150 lacking the iRGD peptide, indicating that the delivery efficiency of iEVs in melanoma sites was significantly improved, and the fluorescence intensity reached a peak 6 hours after administration. In addition, 9 hours after injection, major organs (kidneys, liver, lungs, spleen, heart) and tumor tissues were removed for in vitro imaging analysis. The results showed that compared with EV-150, the enrichment of iEV-150 in tumor sites was significantly increased ( Figure 4 E). In addition, the presence and spatial distribution of EVs in tumor tissues were evaluated, and tumor sections were further analyzed. The results showed that iEV-150 had a higher degree of infiltration in tumor tissues and a stronger fluorescence signal than unmodified EV-150 ( Figure 4 F).

[0085] Example 5 Safety Analysis of Engineered Extracellular Vesicles (iEV-150) Toxicity assessment is an important parameter to determine whether the delivery vector is suitable for further application. To ensure safety, iEV-150 was injected at 5×10 10 The dose of particles / time was injected into healthy C57BL / 6 mice via the tail vein every other day for one week to evaluate its systemic toxicity. Compared with the PBS-treated control group, no mouse death or significant weight loss was observed in the experimental group during the study period (data not shown). The results of histopathological analysis showed that no obvious abnormalities or lesions were found in the main organs (heart, liver, spleen, lung and kidney) of mice in the iEV-150-treated group ( Figure 5 A). These results indicate that iEV-150 did not induce significant inflammatory responses. In addition, to further evaluate the potential toxicity of EVs in mice, blood biochemistry and routine blood analysis were performed. Biochemical indicators included alanine aminotransferase (ALT; Figure 5 B) and aspartate aminotransferase (AST; Figure 5 C) to assess liver function, and creatinine (CR; Figure 5 D) to assess renal function. The results showed that the levels of these indicators were not significantly different from the PBS-treated control group, indicating that iEV-150 did not cause significant hepatotoxicity or renal toxicity within the dose range tested. In summary, these findings indicate that multiple doses of iEV-150 do not cause acute toxicity in major organs of mice.

[0086] Example 6 iEV-150 inhibits melanoma cell proliferation, DNA damage repair and epithelial-mesenchymal transition (EMT) in vitro To investigate the effect of iEV-150 on melanoma, iEV-150 and iEV-NC were co-cultured with melanoma cells to evaluate their effects on cell behavior. EDU staining results showed that co-culture with iEV-150 reduced the proportion of EDU-positive cells ( Figure 6 A). Cell cycle analysis further showed that iEV-150 co-culture decreased the proportion of cells in the S phase and G2 / M phase, while increasing the proportion of cells in the G1 phase ( Figure 6 B). In addition, spheroid formation assay ( Figure 6 C) and clone formation assay ( Figure 6 D) Both showed that iEV-150 co-culture could inhibit melanoma cell proliferation.

[0087] At the mechanistic level, iEV-150 downregulated the expression of CDK2 and Cyclin E2, while upregulated the CDK inhibitor P21, which plays an important role in S phase regulation ( Figure 6 E). In addition, iEV-150 also reduced the expression of PCNA, an important factor associated with DNA replication ( Figure 6 E). In terms of the expression of EMT-related molecules, the expression of E-cadherin increased after co-culture with iEV-150, while the expression of N-cadherin and Vimentin decreased, suggesting that it can inhibit the epithelial-mesenchymal transition (EMT) of melanoma cells ( Figure 6 E). At the same time, the expression levels of key DNA repair factors BRCA1, RAD51, and PALB2 were significantly decreased, while the expression level of DNA damage marker γ-H2AX was upregulated ( Figure 6 E). These results suggest that iEV-150 can reduce the DNA repair capacity of melanoma cells.

[0088] Example 7 iEV-150 inhibits melanoma growth and metastasis in vivo To further investigate the effects of iEV-150, iEV-150 or iEV-NC ( Figure 7 A). Compared with the iEV-NC treatment group, iEV-150 treatment significantly inhibited the growth of mouse tumors and reduced tumor weight ( Figure 7 BD). In addition, TUNEL staining results showed that the number of dead cells in the tumor tissues of mice in the iEV-150 treatment group increased significantly ( Figure 7 E), suggesting that iEV-150 may induce melanoma cell death.

[0089] Immunohistochemistry (IHC) analysis further showed that the expression of lipid peroxidation marker 4-hydroxynonenal (4-HNE) was significantly increased in iEV-150-treated tumor tissues, indicating an elevated level of lipid peroxidation ( Figure 7 F and 7G). However, no significant differences were observed in the staining results of the apoptosis marker cleaved-caspase-3 or the N-terminal fragment of the pyroptosis execution factor GSDMD (GSDMD-N) in iEV-150-treated tumors compared with iEV-NC-treated tumors ( Figure 7 K, 7H and 7I).

[0090] Furthermore, in the B16-F10-induced lung metastasis model, the number of lung nodules in mice treated with iEV-150 was significantly reduced ( Figure 7 J and 7K). The results of survival analysis showed that the survival time of mice in the iEV-150-treated group was significantly longer than that in the iEV-NC-treated group ( Figure 7 L). Taken together, these results suggest that iEV-150 plays an important role in regulating melanoma cell proliferation, DNA damage repair, EMT, and mouse survival.

[0091] Example 8 iEV-150 inhibits the proliferation and metastasis of melanoma cells by promoting ferroptosis Various forms of regulated cell death (RCD) have been widely studied, including apoptosis, necrosis, pyroptosis, ferroptosis, and autophagy-dependent cell death. To further explore whether iEV-150 inhibits melanoma growth by inducing one of the RCD forms, the experiment used five specific RCD inhibitors to block different cell death pathways. The results showed that in the iEV-150 co-culture group, treatment with the ferroptosis inhibitor Ferrostatin-1 significantly reduced the cell death rate, suggesting that iEV-150 may partially inhibit the growth of melanoma cells by inducing ferroptosis ( Figure 8 A and 8B). In a melanoma subcutaneous xenograft model, tail vein injection of iEV-150 significantly inhibited tumor volume, growth rate, and weight. However, combined treatment with Ferrostatin-1 partially reversed the inhibitory effects of iEV-150 on tumor volume and weight ( Figure 8 C-8F). Prussian blue staining analysis of tumor tissue sections revealed that the iron level in the iEV-150-treated group was elevated, whereas the iron level decreased after Ferrostatin-1 co-treatment ( Figure 8 G and 8H). In addition, TUNEL staining showed that the cell death rate in the iEV-150-treated group increased, but Ferrostatin-1 could attenuate this effect ( Figure 8 G and 8H). The changing trend of 4-HNE levels is consistent with the above results ( Figure 8G and 8H). Further studies found that the effect of iEV-150 in reducing lung metastasis of melanoma cells in a mouse model was also partially reversed by Ferrostatin-1 ( Figure 8 IL).

[0092] To verify this conclusion, the experiment used the ferroptosis inducer Erastin and the inhibitor Fer-1 to detect the intracellular lipid ROS level ( Figure 8 M), mitochondrial superoxide level ( Figure 8 N), Fe²⁺ content ( Figure 8 O), GSH / GSSG ratio ( Figure 8 P), MDA level ( Figure 8 Q) and cell death rate ( Figure 8 R). The results showed that Erastin and iEV-150 treatment could lead to the increase of intracellular lipid ROS, mitochondrial superoxide, Fe 2+ , MDA levels and cell death rate, while reducing the GSH / GSSG ratio, and Fer-1 can reverse these effects. In addition, Erastin and iEV-150 both up-regulated the expression of ferroptosis-inducing genes 4-HNE, CHAC1 and ACSL4, while down-regulated the ferroptosis-negative regulatory gene GPX4, and Fer-1 can partially reverse these changes ( Figure 8 S). The red / green fluorescence ratio of JC-1-stained cells reflects the mitochondrial membrane potential (MMP). In healthy mitochondria, JC-1 forms red fluorescent aggregates, while depolarized mitochondria show green fluorescence. After 24 hours of treatment with Erastin or iEV-150, the red / green fluorescence ratio decreased, indicating a decrease in MMP, an effect that could be partially reversed by Fer-1 ( Figure 8 T). Subsequently, transmission electron microscopy (TEM) analysis of cell ultrastructure showed that in cells treated with Erastin and iEV-150, mitochondrial volume was reduced, membrane density increased, and cristae structure decreased, while Fer-1 could partially restore these changes ( Figure 8 U).

[0093] In summary, this study showed that engineered extracellular vesicles iEV-150 can induce cell ferroptosis and inhibit the growth and metastasis of melanoma, suggesting its potential application value in the treatment of melanoma.

[0094] Example 9 To investigate how iEV-150 promotes ferroptosis and suppresses the malignant phenotype of melanoma cells, we performed RNA sequencing (RNA-seq) on A875 cells co-cultured with iEV-150 or iEV-NC, and we screened for differentially expressed genes (DEGs). 2 FC|>1 and p<0.05( Fig. 9 A and 9B); KEGG pathway enrichment analysis showed that these differentially expressed genes were mainly involved in cell cycle, DNA replication, DNA damage repair and Hippo signaling pathway ( Fig. 9 C); RNA-seq analysis revealed that iEV-150 upregulated ferroptosis-related genes ACSL3, ACSL4, and CHAC1, while downregulated genes involved in DNA damage repair, cell migration, cell cycle progression, and DNA replication ( Fig. 9 D).

[0095] To identify potential target genes of miR-150-3p, we performed an intersection analysis between the genes downregulated in the RNA-seq data of A875 cells treated with iEV-150 and the ferroptosis-suppressing genes in the ferroptosis database. As a result, 11 candidate genes were found ( Fig. 9 E); AGO2-RIP-qRT-PCR analysis found that NF2 and CISD3 were the most abundant mRNAs in the AGO2-miR-150-3p complex ( Fig. 9 F); Consistently, quantitative PCR showed that NF2 and CISD3 were upregulated in miR-150-3p knockout melanoma cells ( Fig. 9 G); RNA pulldown experiments confirmed the strong interaction between miR-150-3p and NF2 ( Fig. 9 H).

[0096] To further validate this interaction, RNA-ChIP analysis showed that miR-150-3p overexpression increased the enrichment of NF2 mRNA in the Ago2 / RNA-induced silencing complex (RISC). Fig. 9 I); In melanoma cells overexpressing miR-150-3p, qRT-PCR analysis confirmed higher levels of miR-150-3p and NF2 expression in RISC ( Fig. 9 I). In addition, we constructed luciferase reporter plasmids containing wild-type or mutant miR-150-3p binding sites in NF2 3'UTR. In melanoma cells, miR-150-3p overexpression significantly reduced the luciferase activity of the wild-type NF2 3'UTR plasmid, while the mutant construct showed no significant change ( Fig. 9 J). These results suggest that miR-150-3p directly targets the 3'UTR of NF2, and correlation analysis of melanoma tissue microarrays showed that miR-150-3p was negatively correlated with NF2 expression in melanoma tissues ( Fig. 9 K). In addition, miR-150-3p overexpression suppressed NF2 expression at the mRNA level ( Fig. 9L); In melanoma cells overexpressing NF2, co-culture with iEV-150 was performed. EdU experiments showed that NF2 overexpression restored the reduction in cell proliferation caused by iEV-150 treatment ( Fig. 9 M). This suggests that iEV-150 inhibits the proliferation of melanoma cells by targeting NF2. Western blot analysis in A375 and A875 cells showed that NF2 overexpression reversed the iEV-150-induced downregulation of proliferation markers PCNA, cell cycle regulators CCNE1 and CDK2, EMT markers N-cadherin and Vimentin, DNA damage repair genes BRCA1 and RAD51, and DNA damage marker γ-H2AX ( Fig. 9 These findings were confirmed in animal experiments. NF2 overexpression reversed the reduction in tumor volume and weight induced by iEV-150 in mice ( Fig. 9 O-9Q). Masson staining showed that the increase in myofibers in melanoma tissues after iEV-150 treatment was reversed by NF2 overexpression ( Fig. 9 R-9S), Prussian blue staining revealed that iEV-150-induced iron accumulation in tumor tissues was reduced after NF2 overexpression ( Fig. 9 R-9S). TUNEL staining confirmed that NF2 overexpression reduced iEV-150-induced tumor cell apoptosis ( Fig. 9 R-9S), IHC analysis showed that iEV-150 inhibited the expression of ferroptosis markers 4-HNE, ACSL4, and CHAC1 in vivo by inhibiting the expression of NF2 ( Fig. 9 T, U). In addition, NF2 overexpression reversed the iEV-150-induced inhibition of lung metastasis in B16-F10 melanoma cells in mice ( Fig. 9 V, W). These results suggest that iEV-150 regulates the malignant phenotype and ferroptosis of melanoma cells by targeting NF2.

[0097] Note: Data are expressed as mean ± standard deviation, *p<0.05; **p<0.01; ***p<0.001.

[0098] Table 6 Primers used for real-time fluorescence quantitative PCR

Claims

1. An engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide, characterized in that: miR-150-3p is loaded into extracellular vesicles and iRGD targeting peptide is modified on its surface to obtain engineered extracellular vesicles loaded with miR-150-3p and modified with iRGD targeting peptide on its surface, which are labeled as iEV-150; the engineered extracellular vesicles will target and deliver miR-150-3p to melanoma cells, inhibiting DNA damage repair and epithelial-mesenchymal transition of melanoma cells.

2. The engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide according to claim 1, characterized in that: The preparation method of the engineered extracellular vesicles is as follows: S1, screening of target gene miR-150-3p; S2, constructing HEK293T cell line expressing miR-150-3p using lentiviral transfection technology; S3, introducing the nucleic acid sequence encoding ANXA2 protein into the HEK293T cell line expressing miR-150-3p; S4, collect and culture HEK293T cell lines to secrete extracellular vesicles containing miR-150-3p; S5. separating the extracellular vesicles by ultracentrifugation; S6. The iRGD targeting peptide is coupled to the surface of the extracellular vesicle membrane to obtain engineered extracellular vesicles.

3. The engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide according to claim 1, characterized in that: The average particle size of the engineered extracellular vesicles is 150.6±11 nm.

4. The engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide according to claim 1, characterized in that: The miR-150-3p is hsa-miR-150-3p, and the nucleotide sequence of the hsa-miR-150-3p is shown in SEQ ID NO:

1.

5. The engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide according to claim 2, characterized in that: The nucleic acid sequence of the ANXA2 protein is shown in SEQ ID NO:2, and the protein sequence of the ANXA2 protein is shown in SEQ ID NO:

3.

6. The use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide as claimed in any one of claims 1 to 5, characterized in that: Used for preparing drugs for treating melanoma.

7. The use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide as claimed in claim 6, characterized in that: The melanoma treatment drug delivers miR-150-3p to melanoma cells through engineered extracellular vesicles, thereby inducing ferroptosis of melanoma cells to inhibit the growth and metastasis of melanoma.

8. The use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide as claimed in claim 7, characterized in that: The melanoma treatment drug regulates ferroptosis-related factors and promotes lipid peroxidation through engineered extracellular vesicles, thereby reducing the GSH / GSSG ratio, increasing intracellular Fe²⁺ and MDA levels, and mitochondrial oxidative stress response, ultimately leading to ferroptosis of melanoma cells.

9. The use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with iRGD targeting peptide as claimed in claim 8, characterized in that: The ferroptosis-related factors are 4-HNE, CHAC1, ACSL4, and GPX4.

10. An ANXA2 protein that promotes the enrichment of miR-150-3p in extracellular vesicles, characterized in that: The nucleic acid sequence of the ANXA2 protein is shown in SEQ ID NO:2, and the protein sequence of the ANXA2 protein is shown in SEQ ID NO:3.

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