Efficient Delivery of GSDMD‐N mRNA by Engineered Extracellular Vesicles Induces Pyroptosis for Enhanced Immunotherapy

Abstract Pyroptosis is a newly discovered inflammatory form of programmed cell death, which promotes systemic immune response in cancer immunotherapy. GSDMD is one of the key molecules executing pyroptosis, while therapeutical delivery of GSDMD to tumor cells is of great challenge. In this study, an extracellular vesicles‐based GSDMD‐N mRNA delivery system (namely EV Tx ) is developed for enhanced cancer immunotherapy, with GSDMD‐N mRNA encapsulated inside, Ce6 (Chlorin e6 (Ce6), a hydrophilic sensitizer) incorporated into extracellular vesicular membrane, and HER2 antibody displayed onto the surface. Briefly, GSDMD‐N mRNA is translationally repressed in donor cells by optimized puromycin, ensuring the cell viability and facilitating the mRNA encapsulation into extracellular vesicles. When targeted and delivered into HER2 + breast cancer cells by the engineered extracellular vesicles, the translational repression is unleashed in the recipient cells as the puromycin is diluted and additionally inactivated by sonodynamic treatment as the extracellular vesicles are armed with Ce6, allowing GSDMD‐N translation and pyroptosis induction. In addition, sonodynamic treatment also induces cell death in the recipient cells. In the SKBR3‐ and HER2 transfected 4T1‐ inoculated breast tumor mouse models, the engineered EV Tx efficiently induces a powerful tumor immune response and suppressed tumor growth, providing a nanoplatform for cancer immunotherapy.