Sequence-Defined Short Peptide-Derived Coacervate Vesicles for Targeted Therapeutics

Compartmentalization underpins biological organization and the design of programmable therapeutic carriers.  Here, we report a sequence-defined short peptide, Biotin-Ser(-OPO₃ ^{2 -})-Phe-Phe-Arg (Biotin-SR), that undergoes physiological liquid-liquid phase separation to form lipid-free coacervate vesicles (CVs) with enhanced structural persistence.  The designer peptide integrates oppositely charged functionalities that drive pH-responsive coacervation, incorporating an alkaline phosphatase (ALP)-cleavable anionic phosphoester, a diphenylalanine motif that promotes hydrophobic packing, and a cationic guanidinium group that stabilizes the coacervate architecture while serving as a precursor for nitric oxide (NO) generation. These CVs selectively target cancer cells via biotin-mediated recognition. CVs of Biotin-SR with pronounced morphological persistence, as confirmed by restricted molecular exchange in FRAP analysis, and encapsulate glucose oxidase (GOx) within this stabilized vesicular architecture, undergo ALP-induced enzymatic dephosphorylation in ALP-rich tumor environments to trigger an irreversible loss of charge complementarity, GOx release and a synergistic therapeutic cascade integrating enzymatic starvation, oxidative stress amplification, and NO-mediated mitochondrial dysfunction. The resulting glucose depletion couples with NO generation to elevate reactive oxygen species (ROS), inducing mitochondrial dysfunction and suppressing tumor growth in both spheroids and xenograft models. This work demonstrates how minimal peptide sequences can translate liquid-liquid phase separation into stable coacervate vesicles with enzyme-responsive supramolecular function for targeted therapeutics.