Polyethylene-Glycol-Conjugated Peptide Coacervates with Tunable Size for Intracellular mRNA Delivery

Coacervate microdroplets (CMs), formed through liquid–liquid phase separation (LLPS) of biomacromolecules, hold significant potential for biomedical applications such as intracellular delivery vehicles or enhanced microcatalytic reactors. However, their micrometer size and tendency to coalesce are sometimes deemed unsuitable for those applications. Here, we introduce a strategy to control the size and stability of peptide-based coacervates derived from histidine-rich beak peptides (HB pep) by conjugating the peptides with poly(ethylene glycol) (PEG) and preparing mixtures of PEGylated and non-PEGylated HB pep . PEGylation introduces steric hindrance, stabilizing the coacervates in the nanoscale size range with controlled size distribution depending on the PEGylated-to-non-PEGylated peptide ratio, but initially affects cellular uptake and cargo recruitment of the resultant coacervate nanodroplets (CNs). By incorporating positively charged residues into the peptide sequence, mRNA recruitment and intracellular delivery abilities of CNs are restored. Furthermore, PEG-stabilized CNs exhibit improved cellular uptake and mRNA transfection at the physiological temperature of 37 °C. This approach expands the molecular design of LLPS-based delivery systems with potential for targeted in vivo applications and also highlights opportunities for adapting coacervate-based technologies in catalysis and bioreactors.