Engineered nanovesicle platform simultaneously triggers YAP-dependent ferroptosis and reprograms T-cell immunity through miR-150-3p codelivery in melanoma microenvironment.

Rationale: Melanoma remains a highly aggressive malignancy with limited effective therapies and frequent resistance to immune checkpoint blockade (ICB). Extracellular vesicles (EVs) represent a promising platform for RNA-based therapeutics, but their clinical translation is impeded by inefficient cargo loading and insufficient tumor-specific targeting. To address these limitations, we developed an engineered EV strategy integrating efficient miRNA packaging with tumor-targeting surface modifications to enhance therapeutic outcomes in melanoma. Methods: Engineered EVs (iEV-150) were generated by co-expressing miR-150-3p and Annexin A2 (ANXA2) in HEK293T cells, followed by surface modification with tumor-targeting iRGD peptides. Mechanistic insights were obtained using RNA sequencing, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and luciferase reporter assays. Ferroptosis induction was evaluated through lipid peroxidation analysis, mitochondrial membrane potential assays, and transmission electron microscopy (TEM). Therapeutic efficacy and biodistribution were assessed in vivo using subcutaneous and metastatic melanoma mouse models. Immune modulation was examined by analyzing CD8⁺ T cell activation via flow cytometry in co-cultures of patient-derived CD8⁺ T cells and melanoma cells treated with iEV-150. Results: miR-150-3p was elevated in melanoma-derived EVs, and ANXA2 was identified as a key RNA-binding protein that selectively facilitated its loading into EVs. iEV-150 exhibited enhanced uptake by melanoma cells and improved tumor-specific accumulation in vivo. Mechanistically, iEV-150 suppressed NF2 expression, disrupted the NF2-LATS1 interaction, activated YAP signaling, and subsequently upregulated ferroptosis-related genes ACSL4 and CHAC1, thereby inducing ferroptosis through the NF2-Hippo-YAP axis. In addition to its direct anti-tumor effects, iEV-150 promoted CD8⁺ T cell infiltration and activation within the tumor microenvironment, and significantly enhanced the therapeutic efficacy of ICB in melanoma models. Conclusions: iEV-150 integrates ANXA2-mediated miRNA loading, tumor-specific targeting, ferroptosis induction, and immune microenvironment reprogramming. This engineered EV strategy provides an effective RNA-based therapeutic platform to overcome ICB resistance and enhance precision treatment in melanoma.