Regulating surface properties of designed phase-separating peptide droplets for improved dispersibility

The liquid-liquid phase separation of biomolecules leads to the formation of liquid-like droplets that are involved in various biological functions. The design and synthesis of phase-separating droplets to reproduce biological structures and functions have a wide range of potential applications. Methods for controlling the properties of droplet interfaces must also be developed. In this study, we designed a simple phase-separating peptide, RD9, composed of cationic arginine-rich and anionic aspartic acid-rich clusters, which formed liquid-like droplets through intermolecular electrostatic interactions at the physiological pH and salt concentration. An RD9 peptide modified with a polyethylene glycol (PEG) polymer with molecular weights of 2000-20,000 Da was also synthesized. The PEG polymer lacking the RD9 moiety did not partition into the RD9 droplet, whereas PEGylated RD9 preferentially bound to its interface, similar to "amphiphilic" molecules. This surface-binding of PEGylated RD9 prevented the droplets from fusing, resulting in their enhanced dispersibility in aqueous solution. Moreover, droplet surface modification with the PEGylated peptides did not inhibit the transfer of internal molecules between the droplets. This study provides useful information for medical and engineering applications through control of the physical properties of artificial phase-separated droplets.