Peptide Coacervates with Cathepsin B-Triggered Intracellular Cargo Release

Understanding the molecular grammar of liquid–liquid phase separation has enabled rational design of peptide coacervates as intracellular therapeutic carriers with tunable viscoelasticity, controlled cargo partitioning, and enhanced cellular uptake. However, sequence-level determinants governing coacervate disassembly remain poorly characterized. Encoding protease-recognition motifs within phase-separating peptides offers controlled, localized catalytic disassembly without requiring global perturbation via macroscopic triggers such as pH, ionic strength, temperature, or redox potential. Yet, enzymatic cleavage of coacervates is spatially biased and constrained by limited enzyme partitioning, restricted substrate accessibility, and altered kinetics relative to the dilute phase. Here, we test whether the lysosomal protease cathepsin B (CatB)─often upregulated in cancer cells─acts on coacervates assembled from phase-separating peptides encoding CatB-recognition motifs to enable intracellular cargo release in cancer cells. Common CatB-recognition motifs (GFLG and EVCit), developed for linker-based delivery systems under dilute conditions, preserved phase separation but exhibited poor CatB-triggered cleavage and cargo release. In contrast, a de novo motif, FRFV, designed from CatB subsite preferences, retained catalytic activity as confirmed by mass spectrometry and enabled protease-triggered cargo release in vitro and in cancer cells. Thus, protease-recognition motifs require reevaluation within dense-phase architectures to enable efficient intracellular cargo release through coacervate disassembly.