In a study published in the Journal of American Chemical Society, a research team led by Prof. CHEN Chunying and Prof. LI Jiayang from the National Center for Nanoscience and Technology (NCNST) of the Chinese Academy of Sciences, in collaboration with researchers from Jinan University, developed a chiral engineering strategy that sensitizes the self-assembly of peptide-drug conjugates (PDCs) to pH, enabling the assemblies to form precisely inside tumor cell lysosomes and evoke ferroptosis.
Supramolecular assemblies that respond to pathological cues such as pH variation have long been pursued for precise theranostics. The conventional recipe, however, involves grafting responsive motifs—enzyme-cleavable sequences, ionizable groups, or chemically labile linkages—onto the molecular backbone, a process that demands extensive modification and additional synthetic steps. The research team asked a simpler question: instead of adding new chemical parts, what if the answer lies in the handedness of the existing ones?
Amino acids come in mirror-image forms: left-handed (L), the kind found in natural proteins, and right-handed (D). The researchers synthesized all possible chiral sequences of a naphthalene-capped Phe-Phe-Lys tripeptide, conjugated each with an inhibitor of carbonic anhydrase IX (CAIX), a protein overexpressed on hypoxic tumor cells, and compared the resulting PDCs. One configuration stood out. The heterochiral conjugate with alternating D/L residues, termed DLD-ABS, proved exquisitely pH-sensitive: as the pH dropped from 7.5 to 5.5, its critical micellization concentration fell by more than 40-fold, and its morphology transformed from nanospheres into amyloid-like nanofibers. Its homochiral counterpart, by contrast, formed nanofibers regardless of pH. Mechanistic studies traced this sensitivity to protonation of the C-terminal carboxyl group, which rearranges the molecular conformation and drives β-sheet assembly—a stereochemical effect achieved with minimal interference to the backbone. A single D-amino acid substitution also shielded the conjugates from proteolytic degradation.
This molecular switch translated into precise spatial control inside cells. In CAIX-overexpressing triple-negative breast cancer cells, the homochiral PDC assembled into nanofibers on the cell surface and stayed there. The heterochiral DLD-ABS, however, was internalized as nanospheres through CAIX-mediated endocytosis and reached the lysosomes—where the acidic milieu protonated it and triggered nanofiber formation in situ. The growing fibers ruptured the lysosomal membrane, spilling protons and iron into the cytosol. Combined with CAIX inhibition and the resulting intracellular acidification, this iron release supercharged the Fenton reaction, producing a burst of reactive oxygen species and lipid peroxidation that drove the tumor cells into ferroptosis. The death pathway was confirmed by rescue experiments with the ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine, both of which abolished the cytotoxicity. Normal endothelial cells, which express little CAIX, were largely spared even at concentrations up to 400 μM.
In mouse models bearing human triple-negative breast tumors, intravenous DLD-ABS accumulated specifically in tumor tissue and significantly suppressed tumor growth over a 21-day course of treatment, with no loss of body weight and no detectable damage to major organs. Tumor analyses painted a consistent picture: proliferating (Ki67-positive) cells dwindled, tumor blood vessels (CD31) regressed, CAIX and hypoxia-inducible factor HIF-1α expression fell, while reactive oxygen species surged and the ferroptosis guardian protein GPX4 was depleted.
Beyond its therapeutic promise, this work offers a general design principle: chirality, an intrinsic property of amino acids, can serve as a minimal yet powerful lever for spatiotemporal control over supramolecular assembly—from the cell surface down to subcellular compartments. The strategy broadens the design space for responsive supramolecular systems and opens new questions about the biological effects of heterochirality.
