Mechanistic Insights for Glucose-Driven Coacervation of a Supramolecular Peptide and Therapeutic Protein

Abstract Biological systems leverage spontaneous and transient liquid–liquid phase separation (LLPS) of proteins and biomolecules to achieve remarkable functionality. In particular, the LLPS of intrinsically disordered proteins underpins the formation of membraneless compartments in living cells, inspiring synthetic designs based on polymers and polypeptides. While these bioinspired materials have garnered significant interest, LLPS driven by ordered supramolecular assemblies of low-molecular-weight building blocks remains less explored. The current report further investigates a glucose-triggered coacervation arising from a net-cationic supramolecular peptide amphiphile (PA) incorporating a glucose-binding motif upon its interaction with glucose at physiologically relevant concentrations in the presence of a net-anionic therapeutic protein. The phase regime for LLPS is established by varying the concentrations of each component of the system, while a systematic investigation of electrostatic, hydrophobic, and hydrogen bonding interactions performed through the use of disruptive stimuli along with structural changes to PA design reveals key molecular-scale determinants governing phase behavior. Specifically, LLPS arises through cohesive hydrogen bonding and hydrophobic interactions, with electrostatic contributions via screening from oppositely charged components and ambient salt. Interesting temperature-dependent behaviors furthermore align with established theories of complex coacervation that emphasize both enthalpic and entropic contributions. The preparation of materials that undergo LLPS through a balance of intermolecular forces enables new opportunities to design physiologically adaptive drug delivery platforms.