Non-Canonical Peptide Self-Assembly for Mitochondria-Specific Cargo Delivery

Abstract Targeting mitochondria is a promising strategy for precision tumor therapy, yet ligand-free mitochondrial drug delivery remains challenging. Here, we report a minimalist, intrinsically mitochondria-targeting noncanonical tetrapeptide, X-GNNpY, comprising one glycine, two (S)-2-amino-3-(naphthalen-1-yl)-propanoic acid residues (N), and one phosphorylated tyrosine (pY). Without external targeting ligands, X-GNNpY selectively accumulates in tumor-cell mitochondria through enzyme-instructed self-assembly. Conjugating chlorambucil (CLB) yields CLB-GNNpY, which efficiently redirects CLB to mitochondria. CLB-GNNpY exhibits good cellular selectivity, induces mitochondrial stress and immunogenic cell death, promotes M1-like macrophage polarization, and markedly enhances antitumor phagocytosis. This proof-of-concept study establishes noncanonical peptide self-assembly as a ligand-free supramolecular strategy for mitochondrial drug delivery and immune modulation in tumor therapy.