Peptide–amphiphile complex coacervates for high-capacity genetic material delivery under physiological conditions

The clinical translation of gene therapies using non-viral delivery vectors remains constrained by challenges to reconcile high cargo capacity, biological stability, and safe intracellular release, particularly for large genetic constructs and barrier-protected tissues such as the retina in the eye. Here, we report a peptide-based complex coacervate platform that leverages liquid-liquid phase separation to overcome these limitations through a minimalistic, yet programmable molecular design. By integrating sequence-encoded peptide interactions with the biologically derived amphiphilic modulator sodium deoxycholate, we engineer coacervates that are stabilized by a combination of electrostatic, aromatic, and hydrophobic interactions. These hybrid peptide coacervates form spontaneously under physiological conditions and are compatible with a broad range of cargos, including small dyes, peptides, and multi-kilobase plasmid DNAs. Critically, the coacervates display a balance between extracellular stability and intracellular responsiveness: they remain structurally robust across wide ionic, thermal, and pH conditions, as well as serum protein environments, yet undergo controlled disassembly in response to intracellular cues, enabling efficient cytosolic release of cargo without reliance on endosomal acidification or disruptive escape agents. In biologically stringent models, including human retinal pigment epithelial monolayers, the platform preserves epithelial barrier integrity, exhibits negligible hemolysis, and achieves effective transgene expression for plasmid DNA cargos up to 9.3 kb. Beyond ocular applications, these hybrid complex coacervates provide a generalizable framework for next-generation non-viral vectors capable of bridging the translational gap between small-RNA therapeutics and emerging large-gene modalities. STATEMENT OF SIGNIFICANCE: This work presents a designed peptide-based complex coacervate platform that advances liquid-liquid phase separation (LLPS) materials from conceptual soft matter to functionally validated intracellular delivery systems. In the current work, coacervates were engineered with tailored peptide sequences and amphiphilic modulators to enhance extracellular stability, cellular uptake, and controlled cytosolic release of gene cargo, including multi-kilobase plasmid DNA, in both permissive and challenging cellular models. The system demonstrates functionality under physiologically relevant ionic strengths, serum exposure, and epithelial barriers, and enables proof-of-function expression of target genes, establishing a clear application intent beyond fundamental materials behavior.