In Situ Liquid‐Liquid Phase Separation of Peptides Into Droplets Targeting Membraneless Organelles for Enhanced Cancer Chemotherapy

Abstract Liquid‐liquid phase separation (LLPS) of proteins and nucleic acids into membraneless organelles (MLOs) plays a critical role in sustaining fundamental physiological processes. However, creating artificial coacervate droplets in living cells from exogenous molecules and modulating the functions of MLOs remain challenging. To address this concern, here we reported enzyme‐induced in situ phase separation of peptides into droplets targeting MLO stress granule (SG) for enhanced cancer chemotherapy. The peptide Y SO4 F containing two sulfated tyrosine residues undergoes sulfatase‐responsive LLPS into droplets. Cellular studies confirm in situ phase separation of Y SO4 F selectively in sulfatase‐overexpressing cancer cells. By integrating with appropriate ligands, the in situ‐formed droplets d‐YF‐L SG coacervate with SGs driven by association between the ligand with SG key component protein G3BP2. Mechanistic studies illustrate that the in situ‐formed droplets enhance the cytotoxicity of sorafenib via activating caspase‐dependent apoptosis. Furthermore, animal experiments confirm that administration of the in situ‐formed droplets with sorafenib significantly inhibits tumor growth in murine models bearing tumors, accompanied by an excellent biosafety profile. The findings in this study elucidate an innovative approach for in situ formulation of coacervate droplets within tumor cells and a new material for targeting membraneless organelles, thus providing a promising new strategy for disease organelle‐targeted therapy in the future.