Fluorophore Modulates Intracellular Distribution of Enzyme‐Instructed Self‐Assembly of Short Peptides in Osteosarcoma Cells

Enzyme-instructed self-assembly (EISA) of peptides offers a versatile strategy for developing intracellular nanomedicines, yet the role of C-terminus fluorophores in modulating these assemblies remains insufficiently defined. Here four different fluorophores, NBD, DANS, DBD, and Cy5 are conjugated at the C-terminus of a phosphobiphenyl dipeptide to evaluate their influence on intracellular distribution, assembly morphology, and cytotoxicity. Confocal imaging reveals that NBD-, DANS-, and DBD-conjugated precursors predominantly localize to the endoplasmic reticulum, whereas Cy5 directed assemblies to mitochondria, highlighting the decisive effect of fluorophores on subcellular targeting of peptides. Transmission electron microscopy and confocal studies further show that the fluorophores markedly alter assembly pathways: NBD-conjugated precursors form dense yet still irregular aggregates, DANS-conjugated precursors form denser and more continuous aggregates, DBD-conjugated precursors yield fibrous networks, and Cy5-conjugated precursors exhibit minimal ordered assembly. These divergent morphologies correlate with cytotoxic profiles in Saos2 osteosarcoma cells, with DBD-conjugated precursors be the most potent. These results demonstrate that fluorophores can have a significant influence on the behavior of enzymatic self-assemblies and indicate that engineering the C-terminus of peptides is an effective approach for exploring EISA to develop nanomedicines.