Abstract
Phase-separating peptides (PSPs) are known to undergo liquid-liquid phase separation (LLPS) to produce coacervates with applications in bioimaging, drug delivery, and theranostics. However, the structure-function relationship of the peptide building blocks remains unclear. Here, we used an aggregation-induced emission reporter to study how different β-sheet promoters impact coacervation. We synthesized an anionic Asp-peptide (D(10)) and a cationic Arg-peptide (R(10)) covalently conjugated to tetraphenylethene pyridinium (PyTPE)-an aggregation-induced emission generator (AIEgen). We included several spacer units composed of β-sheet-promoting self-assembling peptides (F(2)G(2), W(2)G(2), FFVLK, and GFFYK) between the AIEgen and R(10) and investigated their size and AIE intensities after coacervation. The properties of the coacervates vary with endogenous factors (peptide sequence, number of Arg and Asp residues, charge ratio, and total charge equivalent concentration of peptides), exogenous factors (pH, ionic strength, salts), and the β-sheet content of the constituting peptide. We observed that a minimum 10 mM phosphate buffer salt concentration, pH 4-12, total charge equivalent concentration of 0.8 mM, and charge ratio (R(10)/D(10)) of 0.5 are required for 100% peptide complexation. Peptides containing the FFVLK sequence exhibited the highest AIE (~450-fold) with the capability to form self-coacervates in phosphate buffer. The W(2)G(2) motif had negligible activation. Finally, we confirmed that the coacervates retain their shape after intracellularly labeling murine colon adenocarcinoma cells (MC38) via caveolae-mediated endocytosis with >95% cell viability.