Redox-Responsive Peptide Coacervates for Enhanced mRNA Delivery and Intracellular Release

Efficient and safe Messenger RNA (mRNA) delivery remains a central challenge in nucleic acid therapeutics. While lipid nanoparticles dominate clinically, their biosafety concerns and poor endosomal escape hinder broader application. Phase-separating peptides (PSPs) offer a biocompatible alternative, but often lack structural stability and precise control over intracellular release. In this study, HBpep-SS4 was developed as a chemically defined coacervate system with intrinsic redox-responsiveness encoded by tandem cysteines in its peptide sequence. This minimalist, single-component design eliminates the need for postsynthetic modifications or protein conjugations, simplifying the synthesis process and reducing potential toxicity. HBpep-SS4 forms stable coacervates capable of encapsulating >95% mRNA and retains responsiveness to glutathione, enabling cytosolic RNA release. It delivers a broad spectrum of RNA cargos─including linear, circular, and self-amplifying RNAs (∼9700 nt)─and achieves high transfection efficiency across multiple cell lines. Functionally, it supports genome editing via SpCas9 mRNA/sgRNA delivery, reaching 86.0% EGFP disruption and 72.5% editing at the HBB locus. Mechanistic studies reveal that HBpep-SS4 enters cells via phagocytosis and bypasses endosomal trafficking, disassembling in reductive environments without toxic byproducts. Building on these findings, HBpep-SS4 was designed with primary sequence-encoded environmental responsiveness, enabling integration of structure, function, and redox sensitivity within a single peptide-based system. Embedding functional reactivity into the peptide backbone supports glutathione-triggered disassembly, which may contribute to improved safety, manufacturing scalability, and potential applicability in RNA-based delivery platforms.