De novo designed peptide amphiphiles have been exemplified as building blocks for direct fabrication of biofunctional hydrogels through a photochemical approach. Herein, by incorporating diphenylalanine (FF) as a self-assembling antibacterial motif and dityrosine (YY) as a photocrosslinkable unit, the peptide sequences are interconnected through alternating hydrophilic D (L-aspartate) and hydrophobic X (ε-aminocaproic acid) residues to achieve a precise balance between non-covalent fibrillization and covalent crosslinking. Upon visible-light irradiation, these peptides undergo rapid photocrosslinking in aqueous media, yielding hydrogels with tunable gelation properties, robust mechanical stability, and adjustable functionality. The optimized 4Y6F hydrogel exhibits strong antibacterial efficacy against both Gram-positive and Gram-negative bacteria, attributed to synergistic structural interactions. In vivo wound healing studies demonstrate accelerated re-epithelialization, enhanced collagen deposition, and improved angiogenesis, significantly outperforming control groups and commercial formulations. This study presents a versatile peptide platform for the photochemical fabrication of hydrogels, advancing from de novo design to practical applications in antibacterial therapy and wound healing, and offering a promising bottom-up approach for a wide range of biomedical scenarios.

Combining the noncovalent and covalent interactions, a series of peptide amphiphiles were designed de novo and synthesized to architect functional assemblies by means of photochemistry. The strand of peptide sequence was structurally capped with photoactive tyrosine-tyrosine (YY) motifs at both termini, and the spacing was filled by alternating of hydrophilic D (L-aspartate) and hydrophobic X (ε-aminocaproic acid) structure. Upon visible-light irradiation, these de novo designed peptides underwent rapid photocrosslinking within merely 10 min. Interestingly, the modulation of alternating D–X pairs in occupying spacer would adjust molecular amphiphilicity, regulate charge distribution, and control particle size and loading capacity of peptide nanospheres (PNS) in aqueous media. With entirely peptide-based matrix, this PNS system could host cationic indicators of fluorescent rhodamine and magnetic Gd^III for exemplar near infrared (NIR) fluorescence and magnetic resonance (MR) imaging, which paves a pathway to biomaterial and biomedical applications using de novo designed peptides.