Glycine Composition and Ion Valency Tune Phase Behavior and Drug Encapsulation in Designer Peptide Condensates

Nano- and microencapsulation that combines high loading capacity with stimulus-responsive release remains a challenge for therapeutic delivery. Designed peptide condensates formed by liquid-liquid phase separation offer a versatile and biocompatible platform to address this need. Here, we systematically link backbone flexibility and ion identity to phase behavior, material properties, payload encapsulation, and protease-triggered condensate disassembly using minimalistic cationic-aromatic peptides that differ in their glycine content. Moreover, we systematically studied how monovalent vs divalent anions affect phase behavior and payload encapsulation. Our results show that glycine-poor sequence forms the most highly packed condensates and that the kosmotrope divalent sulfate ions markedly increase dense-phase peptide concentration and droplet size. Glycine content, which regulates charge density and backbone flexibility, directly affects condensate dynamics, showing faster diffusion with an increasing number of glycine residues. High-performance liquid chromatography partitioning analysis of five FDA-approved small molecules demonstrates compound-specific and salt-mediated recruitment, where both the hydrophobicity/polarity and the charge state of the compounds affect their encapsulation in the dense phase. Moreover, using trypsin as a proteolytic trigger, we show how the glycine content affects condensate disassembly. Overall, these results facilitate practical design rules that show how to tune charge and aromatic density, backbone flexibility, and ion valency to regulate dense-phase packing, payload encapsulation, and release. These insights advance the rational engineering of peptide condensates for targeted sequestration and controlled therapeutic release.