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

Abstract

The present invention discloses an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with the iRGD targeting peptide and its application in the treatment of melanoma, belonging to the field of biomedical technology. miR-150-3p is loaded into EVs and the iRGD targeting peptide is surface-modified on them to obtain iEV-150. The preparation method of iEV-150: S1, screen miR-150-3p; S2, construct a HEK293T cell line stably expressing miR-150-3p; S3, introduce ANXA2 protein; S4, culture the HEK293T cell line; S5, isolate EVs; S6, conjugate the iRGD targeting peptide to the surface of the extracellular vesicle membrane. iEV-150 can effectively deliver miR-150-3p to melanoma cells, inhibit tumor cell proliferation, DNA damage repair and epithelial-mesenchymal transition, and promote the death of melanoma cells by inducing ferroptosis.

Description

Technical Field

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

[0002] Melanoma is a highly invasive and metastatic malignant tumor and one of the most lethal skin cancers. Due to its high heterogeneity and rapid growth, current treatment methods (including chemotherapy, targeted therapy, and immunotherapy) have limited efficacy in advanced melanoma and are prone to drug resistance. Therefore, the development of new treatment strategies with high efficiency and strong targeting has important clinical significance.

[0003] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles secreted by cells, which 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 ideal drug delivery carriers. However, due to the heterogeneity, low yield, and limited targeting delivery ability of natural EVs, the development of engineered EVs has become an important development direction in recent years. Through genetic engineering or chemical modification, the functions of EVs can be optimized, and their stability, targeting ability, and drug delivery efficiency can be improved, thereby expanding their application potential in disease treatment.

[0004] miR-150 is a reported anti-tumor miRNA that can regulate multiple target genes and induce the inhibition of melanoma growth. 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. Therefore, 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 the iRGD targeting peptide and its application in the treatment of melanoma. This engineered extracellular vesicle can target and deliver miR-150-3p to melanoma cells and inhibit the growth and metastasis of melanoma by inducing ferroptosis. The present invention combines nanodelivery technology and the ferroptosis regulation mechanism, providing 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:

[0006] An engineered extracellular vesicle (iEV-150) loaded with miR-150-3p and surface-modified with the iRGD targeting peptide, characterized in that miR-150-3p is loaded into the extracellular vesicles (EVs) and the iRGD targeting peptide is surface-modified thereon to obtain an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with the iRGD targeting peptide, labeled as iEV-150; the engineered extracellular vesicle (iEV-150) can target and deliver miR-150-3p to melanoma cells and inhibit the DNA damage repair and epithelial-mesenchymal transition of melanoma cells.

[0007] Furthermore, the preparation method of the engineered extracellular vesicle (iEV-150) is as follows:

[0008] S1. Screen the target gene miR-150-3p;

[0009] S2. Construct a HEK293T cell line stably expressing miR-150-3p using the lentiviral transfection technique;

[0010] S3. Introduce the nucleic acid sequence encoding the ANXA2 protein into the HEK293T cell line that already expresses miR-150-3p;

[0011] S4. Collect and culture the HEK293T cell line to secrete extracellular vesicles (EVs) containing miR-150-3p;

[0012] S5. Isolate the extracellular vesicles (EVs) by ultracentrifugation;

[0013] S6. Couple the iRGD targeting peptide to the surface of the extracellular vesicle (EVs) membrane to obtain the engineered extracellular vesicle (iEV-150).

[0014] Furthermore, the average particle size of the engineered extracellular vesicle (iEV-150) is 150.6 ± 11 nm.

[0015] Furthermore, the miR-150-3p is hsa-miR-150-3p, and the nucleotide sequence of the hsa-miR-150-3p is shown as SEQ ID NO:1.

[0016] Furthermore, the nucleic acid sequence of the ANXA2 protein is shown as SEQ ID NO:2, and the protein sequence of the ANXA2 protein is shown as SEQ ID NO:3.

[0017] 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 the preparation of a drug for melanoma treatment.

[0018] Furthermore, the drug for melanoma treatment targets and delivers miR-150-3p to melanoma cells through the engineered extracellular vesicle (iEV-150), inducing ferroptosis in melanoma cells to inhibit the growth and metastasis of melanoma.

[0019] Furthermore, the drug for melanoma treatment regulates ferroptosis-related factors and promotes lipid peroxidation through the engineered extracellular vesicle (iEV-150), reduces the GSH / GSSG ratio, increases the levels of intracellular Fe²⁺ and MDA, as well as the mitochondrial oxidative stress response, ultimately leading to the ferroptosis of melanoma cells.

[0020] Furthermore, the ferroptosis-related factors are 4-HNE, CHAC1, ACSL4, and GPX4.

[0021] On the other hand, the present invention provides an ANXA2 protein that promotes the enrichment of miR-150-3p in extracellular vesicles (EVs). The nucleic acid sequence of the ANXA2 protein is shown as SEQ ID NO:2, and the protein sequence of the ANXA2 protein is shown as SEQ ID NO:3.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The present invention relates to a melanoma-targeted treatment method based on engineered extracellular vesicles (EVs), particularly a preparation method and application of engineered EVs (iEV-150) containing an iRGD targeting peptide and loaded with miR-150-3p. By constructing HEK293T cells stably expressing miR-150-3p and introducing the ANXA2 protein to promote the enrichment of miR-150-3p in EVs, and simultaneously modifying the surface of EVs with the iRGD peptide to enhance their melanoma cell targeting.

[0024] 2. The research 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 the death of melanoma cells 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 side effects within the tested dose range. Further mechanism studies show that iEV-150 regulates ferroptosis-related factors (4-HNE, CHAC1, ACSL4, GPX4, etc.) and promotes lipid peroxidation, reduces the GSH / GSSG ratio, increases the levels of intracellular Fe²⁺ and MDA, and enhances mitochondrial oxidative stress response, ultimately leading to the 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 applied to the targeted treatment of melanoma and other malignant tumors and has high clinical translation value.

[0025] 3. The engineered extracellular vesicles provided by the present invention can target and deliver miR-150-3p to melanoma cells and inhibit the growth and metastasis of melanoma by inducing ferroptosis. The present invention combines nanodelivery technology and ferroptosis regulation mechanism, providing an innovative strategy for the precise treatment of melanoma, which can effectively overcome the drug resistance of traditional therapies and improve the treatment effect. Brief Description of the Drawings

[0026] Figure 1miR-150-3p is enriched in melanoma-derived extracellular vesicles (EVs); in the figure, (A-C) are heatmaps of miRNA expression profiles in melanoma cells and their extracellular vesicles (EVs); (D) is Venn diagram analysis; (E) is transmission electron microscopy imaging showing EVs isolated from melanoma cells; (F) is nanoparticle tracking analysis to detect the particle size distribution of EVs; (G) is the result of Western blot detection of EV markers CD9, TSG101, CD81, CD63 and HSP70; (H-J) are the expression levels of miR-150-3p and miR-1246 detected by qRT-PCR in normal melanocytes and melanoma cells, melanoma cells and extracellular vesicles, and EVs derived from melanoma cells and normal cells in sequence; (K) is the detection result of miR-150-3p in EVs untreated or treated with RNase A / Triton X-100; (L) is transmission electron microscopy imaging showing EVs isolated from the plasma of melanoma patients; (M) is nanoparticle flow cytometry analysis of the particle size distribution of EVs derived from the plasma of melanoma patients; (N) is qRT-PCR analysis of the expression level of miR-150-3p in EVs derived from the plasma of melanoma patients and healthy individuals; (O-P) are the expression scores of miR-150-3p detected by in situ hybridization in tumor tissues and adjacent normal tissues in a melanoma tissue microarray cohort in sequence; (Q) is the expression score of miR-150-3p analyzed by ISH in melanoma tissues at different clinical stages; (R) is Kaplan-Meier survival analysis.

[0027] Figure 2To identify the RNA-binding proteins that interact with miR-150-3p and study the role of ANXA2 in melanoma cells; in the figure, (A) is the typical staining map of the Bio-miR-150-3p complex shown by SDS-PAGE electrophoresis; (B) is the result of GO and KEGG enrichment analysis; (C) is the result of RNA immunoprecipitation experiment; (D) is the result of Western blotting; (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 overexpression and knockout effect diagrams of ANXA2 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 the expression of miR-150-3p in melanoma cells; (K) is the result of the effect of ANXA2 overexpression on the enrichment of miR-150-3p in exosomes derived from melanoma; (L) is the result of the effect of ANXA2 KO on the expression of miR-150-3p in melanoma cells and the level of miR-150-3p in exosomes; (M) is the result of CCK-8 experiment detection; (N) is the detection result of PCNA and CCND1 protein levels.

[0028] Figure 3 Preparation of iEV-150 loaded with miR-150-3p and surface-modified with iRGD targeting peptide; in the figure, (A) is the schematic diagram of the preparation process of engineering HEK-293T cell-derived EVs for miR-150-3p delivery to melanoma cells; (B) is the transmission electron microscope imaging of iEV-150; (C) is the detection of the particle size distribution of iEV-150 by nanoparticle tracking analysis; (D) is the result of Western blot detection of markers in EV-150 and iEV-150; (E) is the detection of the expression level of miR-150-3p in EVs derived from cells in different treatment groups by qRT-PCR; (F) is the detection of the expression level of miR-150-3p in engineered exosomes iEV-NC and iEV-150 by qRT-PCR.

[0029] Figure 4For the in vivo and in vitro targeting evaluation of engineered iEV-150; in the figure, (A) is the analysis of the uptake of PKH67- and PKH26-labeled EVs by melanoma cells using laser scanning confocal microscopy imaging; (B) is the analysis of the uptake of engineered iEV-150 and EV-150 by melanoma cells using transmission electron microscopy imaging; (C) is the analysis of the uptake of FAM-labeled iEV-150 and EV-150 by melanoma cells using flow cytometry; (D) is the analysis of the distribution of DIR-labeled iEV-150 and EV-150 in a mouse melanoma model using an in vivo imaging system; (E) is the IVIS analysis of the fluorescence intensity distribution in the major organs and tumor tissues of mice 9 h after injection; (F) is the mouse melanoma group.

[0030] Figure 5 For the biosafety evaluation of iEV-150; (A) is the pathological analysis results of heart, lung, liver, kidney, and spleen tissue sections of C57BL / 6 mice after tail vein injection of 5×10¹ 0 particles of iEV-150 or PBS once every other day for 7 days; (B-D) are the level analyses of ALT, AST, and creatinine CR in mouse serum in sequence.

[0031] Figure 6 For the in vitro inhibition of melanoma cell proliferation and EMT by EV-150; (A) is the detection of DNA replication in A375 and A875 cells by EDU staining; (B) is the analysis of the cell cycle distribution of A375 and A875 cells using flow cytometry; (C) is the result display of the sphere formation experiment; (D) is the detection of cell growth by the colony formation experiment; (E) is the Western blot analysis of the expression levels of marker genes related to proliferation, EMT, and DNA damage repair in melanoma cells.

[0032] Figure 7Effect of EV-150 on inhibiting the growth and lung metastasis of murine melanoma 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; (B-D) are the representative images, tumor growth curves and tumor weights of the subcutaneous melanoma xenograft model in sequence; (E) is the representative image of TUNEL staining of the subcutaneous melanoma xenograft model; (F-I) are the representative immunohistochemical staining images of 4-HNE, Cleaved caspase-3 and GSDMD-N in the subcutaneous melanoma xenograft model in sequence; (J) is the detection of the metastatic ability of B16-F10-luc cells after treatment with iEV-150 or iEV-NC by bioluminescence imaging; (K) is the representative image of the lung metastasis model (upper), statistical analysis of the number of lung nodules (middle), and representative image of HE staining of lung tissue (lower); (L) is the survival analysis of mice in the subcutaneous xenograft model after treatment with iEV-150 and iEV-NC.

[0033] Figure 8Mechanism of iEV-150 inhibiting 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 specific inhibitors of various cell death pathways; (B) shows the results of the A375 cell sphere formation assay; (C) shows the experimental schematic diagram of the subcutaneous injection mouse model of B16-F10 melanoma cells and the tail vein injection of iEV-150 / iEV-NC and Fer-1 / DMSO; (D) shows the photos of tumors in each group of mice after tail vein injection of iEV-150 / iEV-NC and Fer-1 / DMSO; (E) shows the weights of tumors in each group of mice; (F) shows the tumor growth curves of each group of mice; (G) shows the results of Prussian blue staining, TUNEL detection, and 4-HNE immunohistochemical detection of tumor tissue sections in each group of mice; (H) shows the statistical analysis of the data; (I) shows the fluorescence images detected in the lungs of mice by recording the in vivo fluorescence signal through IVIS; (J) shows the quantitative analysis of the fluorescence intensity of mice; (K) shows the representative images and HE staining results of the mouse lung metastasis model; (L) shows the quantitative analysis of the number of lung nodules in mice; (M) shows the analysis of lipid ROS levels in A375 cells in the co-culture iEV-150 group, Erastin group, and the co-culture iEV-150 and addition of Fer-1 group; (N) shows the analysis of mitochondrial superoxide levels in each group of A375 cells; (O-R) shows the detection of Fe²⁺ levels, GSH / GSSG ratio, MDA levels, and cell death percentage in each group of A375 cells by flow cytometry; (S) shows the detection of the expression levels of 4-HNE, CHAC1, ACSL4, and GPX4 by Western blot; (T) shows the analysis of the mitochondrial membrane potential of A375 cells; (U) shows the analysis of the mitochondrial structure of melanoma cells after treatment in each group by transmission electron microscopy (TEM).

[0034] Figure 9iEV-150 inhibits melanoma proliferation and metastasis by regulating NF2; (A) Heatmap showing 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 changes in the expression 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 of mRNAs enriched with miR-150-3p; (G) qRT-PCR detection of changes in the expression of candidate mRNAs after inhibiting miR-150-3p; (H) Results of miRNA pulldown experiment; (I) Overexpression of miR-150-3p increased the enrichment of NF2 mRNA in the Ago2 / RNA-induced silencing complex; (J) Luciferase reporter assay to evaluate the interaction between miR-150-3p and the 3′UTR of NF2; (K) Correlation between miR-150-3p and NF2 in melanoma tissue microarray; (L) Western blot detection of NF2 protein expression after overexpression or knockdown of miR-150-3p; (M) EDU cell proliferation assay; (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) Subcutaneous tumor images of C575BL / 6 mice; (P) Weights of mouse tumors; (Q) Growth curves of mouse tumors; (R-S) Representative images and data analysis of Matson staining, DAB-enhanced Prussian blue staining, and TUNEL staining of mouse tumor sections; (T-U) Representative images and data analysis of immunohistochemical (IHC) expression of 4-HNE, ACSL4, and CHAC1 in mouse tumor sections in sequence; (V) Lung metastasis images and HE staining after tail vein injection of cells or exosomes in different groups of mice; (W) Quantitative analysis of lung metastasis nodules in different groups of mice. Detailed implementation manners

[0035] Materials and methods:

[0036] 1. Clinical samples and tissue microarrays

[0037] Collect fresh peripheral blood samples using anticoagulant tubes containing EDTA. The blood samples were from 16 melanoma patients who had not received preoperative chemotherapy or radiotherapy, and 16 age- and sex-matched healthy controls. The samples were collected from May 2024 to December 2024 at the Cancer Hospital, 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 exosome extraction.

[0038] 2. Cell culture

[0039] Human melanoma cell lines A375 and A875 were cultured in high-glucose DMEM medium containing 10% FBS and 1 / 1000 double antibiotics. The culture medium for human normal melanocyte line PIG1 was the same as that for A375 and A875. The cell culture conditions were 37°C and 5% CO2 concentration. During the cell culture period, 9-cm diameter culture dishes were used, and the culture medium volume was 10 mL. The cells were observed twice a day. When the cell confluence reached 80%, cell passage or cryopreservation was performed. For cell passage, first, the cells were washed twice with 5 mL PBS, then 3 mL EDTA was added and the cells were digested in a 37°C incubator for 3 min. The cell digestion was observed under a microscope, 6 mL culture medium was added to terminate the cell digestion, and 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.

[0040] 3. Biodistribution of EVs in vivo

[0041] First, 5×10 6 B16-F10 cells were subcutaneously injected into C57BL / 6 mice. Five days later, when the tumors grew to 3 mm in size, EV-150 or iEV-150 was labeled with DIR red fluorescent dye, and then 5×10 5 particles of labeled EVs or iEVs were injected via the tail vein. At 3, 6, and 9 hours after injection, in vivo fluorescence images were captured using IVIS. Nine hours after injection, the tumor tissues were removed 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.

[0042] 4. miRNA real-time fluorescence quantitative PCR (qRT-PCR) detection

[0043] Extract miRNAs using the MiRNeasy Mini Kit from Qiagen, and perform reverse transcription using the reverse transcription kit from Takara. The reverse transcription system for miRNAs is as follows:

[0044] Table 1 Reverse transcription system for miRNAs

[0045]

[0046] Run the program in a PCR machine: 37°C for 1 h; 85°C for 5 min. Add 90 μL of RNase Free H2O and store at -20°C.

[0047] Perform miRNA qRT-PCR using the kit from Preomega. The primers for miR-150-3p are shown as SEQ ID NO:4, and the primers for miR-1246 are shown as SEQ ID NO:4. Using U6 as the internal reference gene, the system is as follows:

[0048] Table 2 U6 qRT-PCR system

[0049]

[0050] Table 3 miRNA qRT-PCR system

[0051]

[0052] Run the following program in a 7500 real-time fluorescence quantitative PCR machine:

[0053] Table 4 miRNA qRT-PCR reaction conditions

[0054]

[0055] 5. CCK8 assay

[0056] (1). Seed the cells into a 6-well plate first. After the cells are treated with different groups, perform trypsin digestion and cell counting.

[0057] (2). Seed 2×103 cells per well into a 96-well plate, and prepare 6 replicates for each group.

[0058] (3). Aspirate the old medium used to maintain cell growth in the 96-well plate and replace it with 100 μL of fresh medium containing 10% CCK8 reagent; add 100 μL of complete medium containing 10% CCK8 reagent to the right side of the experimental wells as a blank control, and add sterile PBS to the wells around the cells to be tested to prevent evaporation of the nutrient solution.

[0059] (4) Place the culture plate in the cell incubator and incubate it in the dark for 2 h before detection. The detection process is to use a multifunctional microplate reader to measure the absorbance value of cells in each well at 450 nm, and detect for 4 days at continuous and unified time points. The experimental data is analyzed and plotted using Graphpad prism 8 software for the detected values of each group.

[0060] 6. Colony formation assay

[0061] (1) Seed the cells into a 6-well plate first. After treating the cells with different methods for 48 h, digest and count the cells.

[0062] (2) Seed 1×10 3 melanoma cells into a 3.5 cm² cell culture dish and change the fresh high-glucose medium every 3 days.

[0063] (3) After culturing for about 2 weeks, obvious cell colonies are formed in some groups under the microscope. Wash 3 times with PBS, then add 4% paraformaldehyde to immerse the cells, and fix the cells at room temperature for 30 min.

[0064] (4) After washing 3 times with PBS, add crystal violet staining solution and stain at room temperature for 10 min. Gently wash away the unbound dye with PBS, air dry naturally, then take pictures to observe the size and number of cell colonies. When the number of cells > 50, it is recorded as a positive clone, and count the number of cell clones in all groups.

[0065] 7. Immunohistochemical staining

[0066] (1) Take the prepared paraffin sections of animal tissues, place them in a drying oven at 65 °C and bake for 4 - 5 h, and then perform dewaxing treatment in the following order.

[0067] (2) Incubate with 3% H2O2 at room temperature for 30 min to inactivate endogenous enzymes.

[0068] (3) Place the sections in a glass cylinder, add immunohistochemical PBS, place the glass cylinder on a shaker and shake gently, repeat washing 3 times, 5 min each time.

[0069] (4) Immerse the sections in 0.01 M citrate buffer solution, put them in a steamer and heat with steam for 30 min for heat antigen retrieval. After cooling, wash 2 times with immunohistochemical PBS, block with 5% BSA at room temperature for 30 min, and discard the excess liquid on the sections.

[0070] (5) Precisely circle the tissue area on the sections, dilute the primary antibody with 5% BSA, and drop the primary antibody on the surface of the section tissues, place it at 4 °C and incubate overnight. Recover the primary antibody, wash the sections 3 times with immunohistochemical PBS, 5 min each time.

[0071] (6) Add biotin-labeled goat anti-mouse / rabbit IgG, incubate at 37°C for 30 min, and wash the sections 3 times with histochemical PBS for 5 min each time.

[0072] (7) Add SABC, incubate at 37°C for 30 min, and wash the sections 3 times with histochemical PBS for 5 min each time.

[0073] (8) For the DAB color development kit, take 1 mL of solution B in a brown EP tube, and add one drop of solution A (about 50 μL) to this liquid, and mix well.

[0074] (9) After adding the color developing solution to the sample area, start timing and observe the color development situation through a microscope. After the color development is completed, quickly place the sections in deionized water to terminate. The color reaction time for all groups in the same experiment must be strictly the same.

[0075] (10) Add hematoxylin to the sample area and stain for 1 min, then rinse with running water.

[0076] (11) Perform dehydration and clearing steps using the reverse operation sequence of the dewaxing to water process. After clearing, add a small amount of clear neutral resin to the central area of the tumor tissue on the section, cover with a coverslip to remove air bubbles for sealing. The sealed sections are placed in an open and ventilated place and wait for the resin to dry thoroughly for subsequent observation and photography.

[0077] 8. Western blot

[0078] Homogenize the whole cell protein extract in lysis buffer and centrifuge at 12000 × g for 15 minutes. Determine the protein concentration using the BCA method. Separate the protein lysate on a 12% SDS-polyacrylamide gel and transfer it to a polyvinylidene difluoride membrane. After the membrane is blocked in 5% bovine serum albumin and incubated at room temperature for 1 hour, the membrane is incubated with the primary antibody overnight at 4°C. After washing, the membrane is incubated with the secondary antibody conjugated to horseradish peroxidase, and the membrane signal is detected using a commercial ECL kit.

[0079] The antibodies used in this experiment are shown in Table 5.

[0080] Table 5 Antibody Information Table

[0081]

[0082] 9. Stable cell line construction

[0083] The 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. At the time of transfection, the multiplicity of infection (MOI) for A375 and A875 cells was 10, while that for HEK-293T cells was 5; after infection, the cells were screened with 5 μg / mL puromycin for 14 days and then maintained in a medium without puromycin. To generate stable cell lines, A375 and A875 cells with stable overexpression of miR-150-3p were established by puromycin selection. Similarly, HEK-293T cells with stable overexpression of miR-150-3p and ANXA2 were generated by sequential lentiviral transfection, and then puromycin selection was performed every 30 days for 5 days to maintain stable expression.

[0084] 10. Construction process of engineered extracellular vesicles

[0085] Fenghui Biotechnology (Hunan) Co., Ltd. constructed lentiviral vectors including pLV-Puro, miR-150-3p overexpression lentivirus, and ANXA2 overexpression lentivirus. Subsequently, the miR-150-3p overexpression lentivirus was transfected into HEK-293T cells at a multiplicity of infection (MOI) of 5. After infection, the cells were screened in a medium containing 5 μg / mL puromycin for 14 days and then transferred to a medium without puromycin for continued culture to establish a HEK-293T cell line with stable overexpression of miR-150-3p.

[0086] Furthermore, the ANXA2 overexpression lentivirus was transfected into HEK-293T cells with overexpression of miR-150-3p to construct a HEK-293T cell line with stable co-overexpression of miR-150-3p and ANXA2. To engineer EVs, the ExoBrooch-iRGD kit provided by Beijing Enzekangtai Biotechnology Co., Ltd. was used to conjugate Chol-PEG-iRGD to EVs loaded with miR-150-3p to obtain iEV-150.

[0087] 11. LC-MS mass spectrometry identification

[0088] Dilute trypsin to 20 ng / µl with 25 mM NH4HCO3; add an appropriate amount of trypsin to each tube, place it in an incubator at 37 °C for overnight digestion, then perform peptide purification. The peptides are dissolved in the sample dissolution solution for mass spectrometry identification. The separated peptides are directly introduced into the mass spectrometer for online detection. The original mass spectrometry files are processed and converted by the MM File Conversion software to obtain MGF format files, and then the uniprot database is searched using MASCOT.

[0089] 12. EVs Labeling and Uptake

[0090] Dilute the PKH26 / PKH67 dye in 100 μl of Diluent C (dye solution). Then, dissolve 10 μg of EVs in 20 μl of PBS, add 80 μl of Diluent C, and add the dye solution, and incubate for 5 minutes. To remove the excess dye, dilute the labeled EVs to 30 ml of PBS, recover by ultracentrifugation and resuspend in PBS. When treating cells, co-incubate 5 µg of EVs with 2×10 5 recipient cells for 24 hours. Then, wash the cells twice with PBS for 10 minutes each time, and fix them with 4% paraformaldehyde at room temperature for 10 minutes. Then, wash the cells twice again with PBS, treat them with 0.5% Triton X-100 for 5 minutes, wash them twice again with PBS. Subsequently, incubate the cells with 500 nM Phalloidin dye at room temperature for 30 minutes, wash them twice with PBS for 10 minutes each time, and finally mount the slides with DAPI blocker for microscopic imaging.

[0091] 13. Animal Experiments

[0092] C57BL / 6 mice (4 - 6 weeks old) were obtained from Beijing Speywo Bio-Technology Co., Ltd., China, and were housed under standard specific pathogen-free (SPF) conditions. One week before the experiment, the mice were allowed to acclimate to the environment. In the mouse xenograft tumor experiment, 5 × 10 6 murine melanoma cell line B16-F10 cells were injected subcutaneously into C57BL / 6 mice; starting from the 7th day, 5 × 10 10 particles of iEV-NC or iEV-150 were injected into the nude mice via the tail vein every 3 days; after 21 days, the mice were sacrificed and the melanoma tumors were removed for subsequent experiments. In the tail vein metastasis experiment, 5 × 10 5 B16-F10 -luc cells were injected into the tail vein of nude mice; 5 days later, 5 × 10 10Particle-engineered iEV-NC or iEV-150; After 21 days, nude mice were anesthetized, and IVIS was used to capture the fluorescence images of the mice. The mice were sacrificed and their lungs were removed for examination.

[0093] 14. Isolation of RISC-related RNAs

[0094] Cells overexpressing miR-150-3p or NC were fixed with 1% formaldehyde and then chromatin fragmentation was performed. After lysing the cells with NETN buffer, the lysate was incubated with small magnetic beads coated with IgG or anti-Pan-Ago antibody. The immunoprecipitated RNA was released by proteinase K digestion, extracted with phenol / chloroform / isopropanol, and purified by glycogen ethanol precipitation. Subsequently, the RNA was dissolved and treated with DNase I.

[0095] 15. Biotin RNA pull-down assay

[0096] Cells were lysed with RIP buffer, and RNase inhibitor (Invitrogen) and protease inhibitor mixture were added to the lysate. The lysate was pre-cleared with empty magnetic beads before incubation. Biotin-labeled RNA probes were first immobilized on streptavidin-coated magnetic beads (Invitrogen), and then incubated with the pre-cleared cell lysate at 4°C for 4 hours. Subsequently, the magnetic beads were washed five times with RIP buffer to remove unbound substances. Finally, the bound proteins and RNAs 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 biotin-labeled RNA were used. For RNA pull-down assays using purified proteins, 1–2 μg of purified protein and 1–3 μg of biotin-labeled RNA were sufficient.

[0097] 16. Detection of ferroptosis index

[0098] The intracellular iron content was measured using a total iron colorimetric assay kit. Cells were seeded into 6-well plates at a density of 2×10 5 cells / well. After 24-hour treatment of 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 chromogenic solution were added to each well, and the mixture was incubated at 37°C for 40 minutes. The absorbance (OD) was measured at a wavelength of 593 nm using a microplate reader.

[0099] The intracellular ferrous iron content was measured using a ferrous iron colorimetric assay kit (E-BC-K881-M; Elabscience, Wuhan, China). Cells were seeded into 6-well plates at a density of 2×10 5Cells were seeded into 6-well plates at a density of

[0100] × 10 5 cells per well. After 24 h of experimental treatment, the cells were collected and lysed. The supernatant was collected after centrifugation (15,000×g, 10 min). 80 μL of sample and 80 μL of chromogenic solution were added to each well, and the mixture was incubated at 37 °C for 10 min. The absorbance (OD) was measured with a microplate reader at a wavelength of 593 nm.

[0101] Lipid ROS levels were measured using C11-BODIPY (Cat.# D3861, Thermo Fisher) according to the manufacturer's instructions. Briefly, after 2 days of treatment, the cells were incubated with 10 mmol / L C11-BODIPY for 30 min at 37 °C in the dark under 5% CO2. After incubation, the cells were washed three times with PBS, and the green fluorescence of C11-BODIPY (484 nm / 510 nm) was measured using a flow cytometer.

[0102] The level of mitochondrial superoxide in melanoma cells was measured using MitoSOX Red mitochondrial superoxide indicator (Invitrogen). After 24 h of treatment, the cells were stained with 10 mmol / L MitoSOX reagent in PBS for 10 min and treated in the dark at 37 °C under 5% CO2. After removing the excess MitoSOX, the fluorescence of the cells was detected using a flow cytometer with an excitation / emission wavelength of 510 nm / 580 nm.

[0103] GSH and GSSG levels were measured using a GSH / GSSG detection kit (Abcam, ab138881), and the GSH / GSSG ratio was calculated according to the manufacturer's instructions.

[0104] Detection of mitochondrial membrane potential (C2003S, Beyotime, China): Cells were incubated with 500 μL of JC-1 staining working solution at 37°C for 20 minutes (BD, USA). Then, the cells were washed twice with PBS, and images were taken using a fluorescence microscope or flow cytometer for analysis.

[0105] 17. Immunohistochemistry and hematoxylin-eosin staining

[0106] Paraffin-embedded sections were first dewaxed with xylene, then rehydrated through a gradient of ethanol, and heat-induced antigen retrieval was performed using citrate buffer. The sections were treated with 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity. Subsequently, the sections were blocked with 5% BSA and incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody at 37°C for 30 minutes. Color development was performed using 3,3′-diaminobenzidine solution, and finally, observations were made under a microscope.

[0107] Tumor tissues and lung tissues of mice were subjected to hematoxylin-eosin staining for pathological evaluation. Paraffin sections were first dewaxed with xylene and then rehydrated through a gradient of ethanol. The sections were stained with hematoxylin at room temperature for 5 minutes, then transferred to water and washed for 2 minutes to remove the floating stain. The sections were then transferred to the differentiation solution (1% hydrochloric acid alcohol) and stained for several seconds to 30 seconds, followed by washing with water for 30 - 60 minutes. Finally, the sections were stained with eosin solution for 2 minutes, washed with water again, and then further observed.

[0108] Example 1 miR-150-3p is enriched in melanoma-derived extracellular vesicles (EVs)

[0109] 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). A total of 135 downregulated miRNAs and 143 upregulated miRNAs were identified in A875 EVs (p < 0.05, |log2FC| > 2). In addition, by querying the gene expression database (GEO), the GSE35387 dataset (differentially expressed miRNAs in A375 cells and their EVs) ( Figure 1 B) and the GSE125030 dataset (differentially expressed miRNAs in primary melanoma cells and their EVs) ( Figure 1 C) were screened and reanalyzed. By comprehensively analyzing the results of the three datasets, miR-150-3p and miR-1246 both 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 transmission electron microscopy (TEM) was used (Figure 1 E), particle size distribution analysis ( Figure 1 F), and detection of EV-specific markers ( Figure 1 G) were used to characterize them. The results showed that compared with normal melanocytes (PIG1), the expressions of miR-1246 and miR-150-3p were significantly up-regulated in melanoma cells (A375 and A875) ( Figure 1 H). In addition, compared with parental cells, the expression levels of these two miRNAs in melanoma-derived EVs were further increased ( 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 treatment with RNase A, but decreased after combined treatment with RNase A and Triton X-100, indicating that miR-150-3p was stably encapsulated inside EVs rather than adsorbed on the surface of EVs ( Figure 1 K).

[0110] To explore 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. The results of qRT-PCR analysis showed that the expression level of miR-150-3p in plasma EVs of melanoma patients was significantly higher than that of healthy individuals ( Figure 1 N). In addition, to analyze the expression level of miR-150-3p in tissues, in situ hybridization (ISH) was performed on a melanoma tissue microarray. The results showed that compared with adjacent normal tissues, the miR-150-3p ISH score in melanoma tissues was significantly decreased ( Figure 1 O and 1P). Further analysis of melanoma tissue samples at different clinical stages found that the miR-150-3p ISH scores in stage II-IV melanoma tissues were significantly lower than those in 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 related to the prognosis of melanoma patients.

[0111] Example 2 Exosomal trafficking of miR-150-3p regulated by ANXA2

[0112] Although the miRNA profiles in extracellular vesicles (EVs) differ from their donor cells, the mechanisms underlying this difference remain unclear. RNA-binding proteins (RBPs) are known to play important roles in the exosomal sorting of miRNAs. Therefore, we attempted to identify RBPs that interact with miRNAs enriched in cancer-derived exosomes. For this purpose, we isolated biotinylated miR-150-3p complexes from A875 cells using streptavidin-coated magnetic beads, which showed a typical staining pattern of the Bio-miR-150-3p complex in SDS-PAGE electrophoresis ( Figure 2 A) for the isolation and identification of proteins interacting with miR-150-3p. After gel digestion, liquid chromatography-mass spectrometry (LC-MS) analysis was performed to identify proteins unique to the Bio-miR-150-3p complex and absent in the control biotinylated miRNA complex. GO and KEGG enrichment analyses of these identified proteins showed that ANXA2, RAB14, TMED9, VAPA, and ARF4 are key regulators of the EV transport pathway ( Figure 2 B). We verified the interaction between miR-150-3p and the RNA-binding proteins identified in the GO and KEGG analyses by RNA immunoprecipitation (RIP) experiments, using IgG as a negative control to exclude non-specific binding and the GAPDH antibody as an internal control. The results are shown as Figure 2 C. The expression of miR-150-3p in the ANXA2 precipitate was more than 13-fold higher than that in the IgG precipitate, indicating a strong and specific interaction between ANXA2 and miR-150-3p. In contrast, the expression of miR-150-3p increased in the TMED9 precipitate, while the levels in the RAB14, VAPA, and ARF4 precipitates were comparable to those in the IgG control group. The interaction between miR-150-3p and ANXA2 in the RNA pull-down experiment 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, it was 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, an electrophoretic mobility shift assay (EMSA) also confirmed a supershift phenomenon after incubation of miR-150-3p with ANXA2 ( Figure 2 F). Immunofluorescence analysis showed the co-localization of miR-150-3p and ANXA2 in melanoma cellsFigure 2 G).

[0113] To investigate the role of ANXA2 in melanoma cells, we overexpressed ANXA2 in melanoma cells ( Figure 2 H, bottom panel), and found that overexpression of ANXA2 did not enhance exosome release ( Figure 2 I). However, overexpression of ANXA2 significantly decreased miR-150-3p expression in melanoma cells ( Figure 2 J), and simultaneously significantly increased 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), and simultaneously increased the expression of intracellular miR-150-3p ( Figure 2 L, left panel). This finding was negatively correlated with the miR-150-3p level in exosomes ( Figure 2 L, right panel). Overall, these data indicate that the sorting of miR-150-3p into exosomes is ANXA2-dependent.

[0114] To further investigate the effect of ANXA2 KO on melanoma cells, we detected cell proliferation using the CCK-8 assay. ANXA2 KO significantly inhibited the proliferation of melanoma cells ( Figure 2 M). This result was consistent with the decrease in the protein levels of PCNA and CCND1 ( Figure 2 N). Notably, inhibition of miR-150-3p expression reversed the inhibitory effect of ANXA2 KO on the expression of PCNA and CCND1 ( Figure 2 N), ultimately leading to the induction of cell growth ( Figure 2 M).

[0115] Example 3 Preparation and Characterization of Engineered iEV-150

[0116] To achieve targeted therapy for melanoma, a HEK293T cell line stably expressing miR-150-3p was constructed using 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 isolated by ultracentrifugation, and the iRGD targeting peptide was conjugated to the surface of the EV membrane to construct iEV-150 that is rich in miR-150-3p and can target melanoma cells ( Figure 3 A).

[0117] The obtained 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 3 B). In addition, the results of NTA analysis 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). The 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). The qRT-PCR results showed that the miR-150-3p expression level in EVs derived from cells stably overexpressing miR-150-3p was increased by 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 found that the expression level of miR-150-3p in iEV-150 was significantly upregulated compared with that in the EV negative control (iEV-NC), indicating that miR-150-3p had been successfully loaded into the interior of EVs ( Figure 3 F).

[0118] Example 4 iEV-150 exhibits enhanced targeting ability to melanoma cells

[0119] To evaluate the targeting ability of engineered iEVs to melanoma cells, a laser scanning confocal microscope (LSCM) was used to observe the uptake of PKH26-labeled EVs by cells. The results showed that melanoma cells (A375, A875, and B16-F10) had a higher uptake rate of iEV-150 compared with unmodified EV-150 ( Figure 4 A). In addition, transmission electron microscopy (TEM) further confirmed that melanoma cells endocytosed more iEVs ( Figure 4 B). To verify whether the iRGD targeting peptide was successfully introduced into EVs, FAM-labeled Chol-PEG-CRGDKGPDC was used in the experiment, and its delivery efficiency in melanoma cells was analyzed by flow cytometry. The results showed that the FAM fluorescence signal in melanoma cells treated with iEV-150 was significantly enhanced compared with that in EV-150, indicating that the modification of the iRGD peptide improved the targeting ability of EVs to melanoma cells ( Figure 4C). To investigate the in vivo targeting ability of engineered extracellular vesicles (iEVs), DIR-labeled EV-150 and iEV-150 (5×10¹ 0 particles) were injected via the tail vein into C57BL / 6 mice bearing subcutaneous melanoma tumors. In vivo imaging system (IVIS) analysis showed that iEV-150 exhibited stronger tumor targeting ability compared to 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 a remarkable improvement in the delivery efficiency of iEVs at the melanoma site, and the fluorescence intensity reached a peak at 6 hours after administration. In addition, at 9 hours after injection, the major organs (kidney, liver, lung, spleen, heart) and tumor tissues were removed for ex vivo imaging analysis, and the results showed that the enrichment of iEV-150 at the tumor site was significantly increased compared to EV-150 ( Figure 4 E). Furthermore, the presence and spatial distribution of EVs within tumor tissues were evaluated, and further analysis of tumor sections was performed. The results showed that iEV-150 had a higher infiltration degree and stronger fluorescence signal in tumor tissues compared to unmodified EV-150 ( Figure 4 F).

[0120] Example 5 Safety Analysis of Engineered Extracellular Vesicles (iEV-150)

[0121] Toxicity assessment is an important parameter for judging whether a delivery vehicle is suitable for further application. To ensure safety, iEV-150 was injected via the tail vein into healthy C57BL / 6 mice at a dose of 5×10 10 particle numbers per injection, once 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 major organs (heart, liver, spleen, lung, and kidney) of mice in the iEV-150 treatment group ( Figure 5 A). These results indicate that iEV-150 did not cause obvious inflammatory reactions. In addition, to further evaluate the potential toxicity of EVs in mice, blood biochemical and blood routine analyses were performed. Biochemical index detection included alanine aminotransferase (ALT; Figure 5 B) and aspartate aminotransferase (AST; Figure 5 C) to evaluate liver function, and creatinine (CR; Figure 5D) To evaluate renal function. The results showed that the levels of these indicators were not significantly different from those of the PBS-treated control group, indicating that within the tested dose range, iEV-150 did not cause obvious hepatotoxicity or nephrotoxicity. In summary, these research results indicate that repeated administration of iEV-150 does not cause acute toxicity in the major organs of mice.

[0122] Example 6 iEV-150 inhibits melanoma cell proliferation, DNA damage repair, and epithelial-mesenchymal transition (EMT) in vitro

[0123] To study 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. The results of EDU staining showed that co-culture with iEV-150 could reduce the proportion of EDU-positive cells ( Figure 6 A). Cell cycle analysis further showed that co-culture with iEV-150 decreased the proportions of cells in the S and G2 / M phases, while increasing the proportion of cells in the G1 phase ( Figure 6 B). In addition, the sphere formation assay ( Figure 6 C) and colony formation assay ( Figure 6 D) both showed that co-culture with iEV-150 could inhibit melanoma cell proliferation.

[0124] At the mechanistic level, iEV-150 downregulated the expression of CDK2 and Cyclin E2, while upregulating the CDK inhibitor P21, which plays an important role in S-phase regulation ( Figure 6 E). In addition, iEV-150 also decreased the expression of PCNA, which is an important factor related to 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 expressions 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 the key DNA repair factors BRCA1, RAD51, and PALB2 decreased significantly, while the expression level of the DNA damage marker γ-H2AX increased ( Figure 6 E). These results indicate that iEV-150 can reduce the DNA repair ability of melanoma cells.

[0125] Example 7 iEV-150 inhibits the growth and metastasis of melanoma in vivo

[0126] To further study the effect of iEV-150, after subcutaneous tumor establishment, starting from the 7th day, iEV-150 or iEV-NC was injected via the tail vein once every 3 days ( Figure 7A). Compared with the iEV-NC treatment group, iEV-150 treatment significantly inhibited the growth of mouse tumors and reduced the tumor weight ( Figure 7 B-D). In addition, the results of TUNEL staining 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.

[0127] Immunohistochemical (IHC) analysis further showed that the expression of the lipid peroxidation marker 4-hydroxynonenal (4-HNE) in the tumor tissues treated with iEV-150 increased significantly, 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 the tumors treated with iEV-150 compared with those treated with iEV-NC ( Figure 7 K, 7H and 7I).

[0128] In addition, in the B16-F10-induced lung metastasis model, the number of lung nodules in the mice in the iEV-150 treatment group decreased significantly ( Figure 7 J and 7K). The results of survival analysis showed that the survival time of the mice in the iEV-150 treatment group was significantly longer than that in the iEV-NC treatment group ( Figure 7 L). Taken together, these results indicate that iEV-150 plays an important role in regulating melanoma cell proliferation, DNA damage repair, EMT and mouse survival.

[0129] Example 8 iEV-150 inhibits the proliferation and metastasis of melanoma cells by promoting ferroptosis

[0130] Multiple 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, five specific RCD inhibitors were used to block different cell death pathways in the experiment. 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 the melanoma subcutaneous xenograft model, tail vein injection of iEV-150 significantly inhibited tumor volume, growth rate, and weight. However, combined treatment with Ferrostatin-1 could partially reverse the inhibitory effect of iEV-150 on tumor volume and weight ( Figure 8C-8F). After analyzing tumor tissue sections by Prussian blue staining, it was found that the iron ion level increased in the iEV-150 treatment group, while the iron content decreased after co-treatment with Ferrostatin-1 ( Figure 8 Figures 8G and 8H). In addition, TUNEL staining showed that the cell death rate increased in the iEV-150 treatment group, but Ferrostatin-1 could attenuate this effect ( Figure 8 Figures 8G and 8H). The change trend of 4-HNE level was consistent with the above results ( Figure 8 Figures 8G and 8H). Further research found that the effect of iEV-150 in reducing the lung metastasis of melanoma cells in a mouse model was also partially reversed by Ferrostatin-1 ( Figure 8 Figures 8I-L).

[0131] To verify this conclusion, the iron death inducer Erastin and inhibitor Fer-1 were used in the experiment to detect the intracellular lipid ROS level ( Figure 8 Figure 8M), mitochondrial superoxide level ( Figure 8 Figure 8N), Fe²⁺ content ( Figure 8 Figure 8O), GSH / GSSG ratio ( Figure 8 Figure 8P), MDA level ( Figure 8 Figure 8Q) and cell death rate ( Figure 8 Figure 8R). The results showed that treatment with Erastin and iEV-150 could lead to an increase in intracellular lipid ROS, mitochondrial superoxide, Fe 2+ , MDA levels and cell death rate, while decreasing the GSH / GSSG ratio, and Fer-1 could reverse these effects. In addition, both Erastin and iEV-150 up-regulated the expression of the iron death-induced genes 4-HNE, CHAC1 and ACSL4, while down-regulating the iron death negative regulatory gene GPX4, and Fer-1 could partially reverse these changes ( Figure 8 Figure 8S). 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 treatment with Erastin or iEV-150 for 24 hours, the red / green fluorescence ratio decreased, indicating a decrease in MMP, and this effect could be partially reversed by Fer-1 ( Figure 8 Figure 8T). Subsequently, transmission electron microscopy (TEM) was used to analyze the cell ultrastructure. The results showed that in cells treated with Erastin and iEV-150, the mitochondrial volume decreased, the membrane density increased, and the cristae structure decreased, while Fer-1 could partially restore these changes ( Figure 8 Figure 8U).

[0132] In summary, this study demonstrated that engineered extracellular vesicles iEV-150 could induce ferroptosis and inhibit the growth and metastasis of melanoma, suggesting its potential application value in melanoma treatment.

[0133] Example 9

[0134] To investigate how iEV-150 promoted ferroptosis and inhibited the malignant phenotypes of melanoma cells, we performed RNA sequencing (RNA-seq) on A875 cells co-cultured with iEV-150 or iEV-NC. We screened for differentially expressed genes (DEGs) with |log2FC| > 1 and p < 0.05 ( Figure 9 A and 9B); KEGG pathway enrichment analysis showed that these differential genes were mainly involved in the cell cycle, DNA replication, DNA damage repair, and Hippo signaling pathway ( Figure 9 C); RNA-seq analysis revealed that iEV-150 upregulated the ferroptosis-related genes ACSL3, ACSL4, and CHAC1, while downregulating genes involved in DNA damage repair, cell migration, cell cycle progression, and DNA replication ( Figure 9 D).

[0135] To identify the potential target genes of miR-150-3p, we performed intersection analysis of the genes downregulated in the RNA-seq data of iEV-150-treated A875 cells with the ferroptosis inhibitor genes in the ferroptosis database. Eleven candidate genes were identified ( Figure 9 E); AGO2-RIP-qRT-PCR analysis found that NF2 and CISD3 were the most abundant mRNAs in the AGO2-miR-150-3p complex ( Figure 9 F); Consistently, fluorescence quantitative PCR showed (the primers used in PCR are shown in Table 6) that NF2 and CISD3 were upregulated in miR-150-3p knockout melanoma cells ( Figure 9 G); RNA pulldown experiments confirmed the strong interaction between miR-150-3p and NF2 ( Figure 9 H).

[0136] To further verify this interaction, RNA-ChIP analysis showed that overexpression of miR-150-3p increased the enrichment of NF2 mRNA in the Ago2 / RNA-induced silencing complex (RISC) ( Figure 9 I); In melanoma cells overexpressing miR-150-3p, qRT-PCR analysis confirmed higher levels of miR-150-3p and NF2 expression in the RISC ( Figure 9I). In addition, we constructed luciferase reporter plasmids containing wild-type or mutant miR-150-3p binding sites in the NF2 3'UTR. In melanoma cells, overexpression of miR-150-3p significantly reduced the luciferase activity of the wild-type NF2 3'UTR plasmid, while the mutant construct showed no significant change ( Figure 9 J). These results indicate that miR-150-3p directly targets the 3'UTR of NF2. Correlation analysis of melanoma tissue microarrays showed a negative correlation between the expression of miR-150-3p and NF2 in melanoma tissues ( Figure 9 K). In addition, overexpression of miR-150-3p inhibited the expression of NF2 at the mRNA level ( Figure 9 L); In melanoma cells overexpressing NF2, co-culture with iEV-150 was performed. EdU assay showed that overexpression of NF2 restored the reduction in cell proliferation caused by iEV-150 treatment ( Figure 9 M). This indicates that iEV-150 inhibits the proliferation of melanoma cells by targeting NF2. Western blot analysis in A375 and A875 cells showed that overexpression of NF2 reversed the downregulation of proliferation marker 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 induced by iEV-150 ( Figure 9 N). Animal experiments confirmed these findings. Overexpression of NF2 reversed the reduction in tumor volume and weight of mice induced by iEV-150 ( Figure 9 O-9Q). Masson staining showed that the increase in muscle fibers in melanoma tissues after iEV-150 treatment was reversed by overexpression of NF2 ( Figure 9 R-9S). Prussian blue staining revealed that iEV-150-induced iron accumulation in tumor tissues decreased after overexpression of NF2 ( Figure 9 R-9S). TUNEL staining confirmed that overexpression of NF2 reduced iEV-150-induced tumor cell apoptosis ( Figure 9 R-9S). IHC analysis showed that iEV-150 inhibited the ferroptosis markers 4-HNE, ACSL4, and CHAC1 in vivo by suppressing the expression of NF2 ( Figure 9 T, U). In addition, overexpression of NF2 reversed the inhibition of B16-F10 melanoma lung metastasis in mice induced by iEV-150 ( Figure 9 V, W). These results indicate that iEV-150 regulates the malignant phenotype and ferroptosis of melanoma cells by targeting NF2.

[0137] Note: Data are presented as mean ± standard deviation, *p < 0.05; **p < 0.01; ***p < 0.001.

[0138] Table 6 Primers used in real-time fluorescence quantitative PCR

[0139]

Claims

1. An engineered extracellular vesicle loaded with miR-150-3p and surface-modified with the iRGD targeting peptide, characterized in that, Load miR-150-3p into extracellular vesicles and modify the surface with the iRGD targeting peptide to obtain engineered extracellular vesicles loaded with miR-150-3p and surface-modified with the iRGD targeting peptide, 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; The preparation method of the engineered extracellular vesicles is as follows: S1. Screen the target gene miR-150-3p; S2. Construct a HEK293T cell line expressing miR-150-3p using lentiviral transfection technology; S3. Introduce the nucleic acid sequence encoding the ANXA2 protein into the HEK293T cell line that already expresses miR-150-3p; S4. Collect and culture the HEK293T cell line to secrete extracellular vesicles containing miR-150-3p; S5. Isolate the extracellular vesicles by ultracentrifugation; S6. Couple the iRGD targeting peptide to the surface of the extracellular vesicle membrane to obtain engineered extracellular vesicles.

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

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

1.

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

3.

5. Use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with the iRGD targeting peptide according to any one of claims 1-4, characterized in that, For the preparation of drugs for melanoma treatment.

6. Use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with the iRGD targeting peptide, characterized in that, The drugs for melanoma treatment target and deliver miR-150-3p to melanoma cells through engineered extracellular vesicles, inducing ferroptosis of melanoma cells to inhibit the growth and metastasis of melanoma.

7. Use of an engineered extracellular vesicle loaded with miR-150-3p and surface-modified with the iRGD targeting peptide, characterized in that, The drugs for melanoma treatment regulate ferroptosis-related factors and promote lipid peroxidation through engineered extracellular vesicles, reduce the GSH / GSSG ratio, increase the levels of intracellular Fe²⁺, MDA, and mitochondrial oxidative stress response, and ultimately lead to the ferroptosis of melanoma cells.

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

Images