Substituent-based Modulation of Self-Assembly and Immunogenicity of Amphipathic Peptides

Peptide-based biomaterials assembled through monomer-by-monomer self-assembly provide versatile platforms for biomedical applications due to their adjustable physicochemical properties, biocompatibility, and dynamic nature. The self-assembly process largely depends on primary sequence features, such as hydrophobicity, length, and charge, which influence the formation of various nanostructures, including fibrils and hydrogels. Amphipathic peptides, characterized by alternating polar and hydrophobic residues, are especially effective in forming supramolecular nanofibers stabilized by π–π interactions and hydrogen bonds. Chemical modifications, particularly on aromatic side chains, have proven to be a promising approach for controlling assembly morphology, stability, and biological activity. In organic chemistry, the use of chemical substituents, such as halogens, alkyl groups, or electron-donating and electron-withdrawing groups, has been widely employed to alter reactivity, stability, and molecular interactions for diverse applications, including catalysts, pharmaceuticals, and materials science. However, the influence of these substituents on peptide packing and in vivo immunogenicity remains relatively unexplored. In this study, we systematically examine how changes in the position and nature of substituents on benzyl groups attached to short amphipathic peptides affect self-assembly, fibril morphology, and immune responses. By introducing different electron-donating and withdrawing groups at the para-position of benzyl rings and modifying the chain length connecting the backbone to the aromatic moiety, we observe notable effects on fibril formation, molecular packing, and immunogenicity both in vitro and in vivo . Our results show that subtle chemical modifications are effective tools for designing tailored peptide nanomaterials with promising potential in vaccine delivery, tissue engineering, and regenerative medicine.