In a study published in Nature Communications, a research team led by Prof. CHEN Chunying, Prof. LI Jiayang, and Prof. WANG Hui from the National Center for Nanoscience and Technology (NCNST) of the Chinese Academy of Sciences demonstrated that altering the chirality of a single amino acid residue can drive a peptide-drug conjugate (PDC) into liquid-liquid phase separation (LLPS)—and, in doing so, rewrite its behavior as a drug.
Inside living cells, membraneless organelles (MLOs) concentrate biomolecules into liquid-like droplets that orchestrate signaling and catalysis. Artificial versions of these condensates hold promise for drug delivery and biomedicine, but building them typically requires extensive backbone modification or multiple components. One fundamental feature of biomolecules has been largely overlooked in this endeavor: chirality, the handedness of their building blocks.
The team revisited a hypoxia-targeting tripeptide-drug conjugate they had previously developed, which carries a carbonic anhydrase IX (CAIX) inhibitor on a Phe-Phe-Lys backbone, and systematically reprogrammed the handedness of its three chiral centers to produce all four enantiomer pairs. Under physiological conditions, three of the pairs—including both homochiral versions—formed β-sheet-rich hydrogels through nanofiber entanglement. But the heterochiral pair with alternating D/L residues behaved entirely differently: it separated into liquid droplets that fuse, recover after photobleaching, and readily host an enzyme inside, accelerating its catalytic reaction—hallmarks of a functional artificial membraneless organelle.
Why does one residue's handedness matter so much? The answer lies in a stereochemical-ionic interplay. Elemental analysis revealed that the droplets recruit sodium ions together with ionized peptide species, while chloride is largely excluded. The positively charged sodium—or potassium, but notably not lithium, rubidium, or cesium—binds to the carbonyl and carboxyl groups of the peptide backbone, folding it into a "niche-like" structure reminiscent of the cation-binding motifs found in natural proteins. This ion bridge twists the backbone just enough to disrupt the ordered hydrogen bonding that β-sheets require, blocking fibrillization and stabilizing the liquid state. Energy landscape analysis confirmed the picture: LLPS is actually an intermediate stage on the way to gelation for all chiral variants, but the alternating D/L configuration erects the highest energy barrier against the liquid-to-gel transition, trapping the system in its liquid form under physiological conditions.
That trapped liquid state translated directly into different drug behavior. In blood serum, the heterochiral conjugate rearranges into ultrafine nanoaggregates of about 4 nm, which circulate longer and are taken up by tumor cells markedly faster than its homochiral counterpart, entering through multiple routes including caveolae-mediated endocytosis and macropinocytosis, with uptake further enhanced under hypoxia. The homochiral version, by contrast, persists as larger nanofibers that are rapidly trapped and metabolized in the liver—and its tendency to gel makes it unsuitable for intravenous injection in the first place. In two mouse tumor models, intravenously administered heterochiral conjugate accumulated at tumor sites and significantly suppressed tumor growth, with reduced CAIX expression and proliferation markers, while body weight remained steady throughout treatment.
This work establishes "chirality editing" as a minimalist design principle: rather than adding new chemical parts to a drug molecule, one can rearrange the mirror-image configuration of what is already there to gain kinetic and thermodynamic control over its assembly. Because the tripeptide represents the smallest known niche-mimicking unit, the strategy offers a blueprint for repurposing existing peptide therapeutics and points drug discovery toward a largely unexplored territory—the heterochiral mirror space.
