Programmable Peptide-Based Complex Coacervate Microenvironments for Cellular Engineering

Peptide-based coacervates demonstrate remarkable potential across interdisciplinary fields of biomedicine and materials science due to their sequence programmability, dynamic self-assembly capability, and exceptional biocompatibility. Despite progress in understanding their phase behavior, the high charge density and complex intermolecular interactions present significant challenges in precisely tailoring their microenvironments and biological functions. In this study, we utilized decapeptide sequences (decaarginine R₁₀, decalysine K₁₀, and decaaspartic acid D₁₀) to explore the impact of substituting aspartic acid (D) with phenylalanine (F) in D₁₀ or lysine (K) with arginine (R) in K₁₀ on the microenvironment of coacervates. The replacement of D with F in the R₁₀/D₁₀ system led to a thermodynamic shift from enthalpy-driven (low F%) to entropy-driven (high F%) phase separation and enhanced phase separation propensity and salt resistance, while reducing internal polarity and molecular mobility. Varying R% in K₁₀/D₁₀ systems demonstrated limited impact on microdroplet viscosity and polarity compared to F% modulation, despite stabilizing droplets at R% ≥ 20%. Neither the D-to-F nor K-to-R substitutions altered the enrichment of biological macromolecules; however, the D-to-F substitution disrupted the secondary structure of double-stranded DNA. Cell coculture experiments confirmed that both R₁₀/(FD)₅ and R₁₀/D₁₀ complex coacervate microdroplets adhered to cell membranes rapidly, but R₁₀/D₁₀ exhibited stronger proliferation inhibition. This molecular-level analysis establishes a foundation for connecting the peptide sequence, condensate microenvironments, and biological functions.