Peptide-Based Complex Coacervates Stabilized by Cation−π Interactions for Cell Engineering

Complex coacervation is a form of liquid-liquid phase separation, whereby two types of macromolecules, usually bearing opposite net charges, self-assemble into dense microdroplets driven by weak molecular interactions. Peptide-based coacervates have recently emerged as promising carriers to deliver large macromolecules (nucleic acids, proteins and complex thereof) inside cells. Thus, it is essential to understand their assembly/disassembly mechanisms at the molecular level in order to tune the thermodynamics of coacervates formation and the kinetics of cargo release upon entering the cell. In this study, we designed histidine-rich peptides consisting of modular sequences in which we systematically incorporate cationic, anionic, or aromatic residues at specific positions along the sequence in order to modulate intermolecular interactions and the resulting coacervation stability. We show that cation-π interactions between arginine and aromatic side chains are particularly efficient in stabilizing complex coacervates, and these interactions can be disrupted in the protein-rich intracellular environment, triggering the disassembly of complex coacervates followed by cargo release. With the additional grafting of a disulfide-based self-immolative side chain, these complex coacervates exhibited enhanced stability and could deliver proteins, mRNA, and CRISPR/Cas9 genome editing tools with tunable release kinetics into cells. This capability extends to challenging cell types, such as macrophages. Our study highlights the critical role of cation-π interactions in the design of peptide-based coacervates, expanding the biomedical and biotechnology potential of this emerging intracellular delivery platform.