Enzyme‐Assisted Selective Biosynthesis of Polyrotaxane Assemblies in Living Cells for Precision Supramolecular Phototherapy

Abstract In contrast to conventional bottom‐up self‐organization in an abiotic environment, it remains a formidable challenge to construct mechanically interlocked and biologically active supramolecules in cells and organisms. Notably, the in‐situ formation of polyrotaxane‐type nanoarchitectures has not been reported, and their biological implication is unexplored. Here, it is report the first selective biosynthesis of polyrotaxane assemblies in living cells for combined tumor therapy. Four‐armed porphyrins appended with lysine and glutamine residues are enzymatically coupled via overexpressed transpeptidase in malignant cells. The robust host–guest complexation between permethylated β‐cyclodextrin and porphyrin not only drives the morphological conversion from disordered polymers to 2D polyrotaxanes, but also improves the structural integrity and photophysical performance of porphyrin scaffolds. The chemical modification of the cyclodextrin skeleton further enables the biofunctional diversification of intracellularly formed polyrotaxanes, as demonstrated by the covalent incorporation of a targeting motif and photoactivable prodrug for specific cytoskeletal disruption. The obtained polyrotaxane assemblies exhibit efficient singlet oxygen generation up to 221% and prolonged tumor retention over 120 h, resulting in elevated photocytotoxicity and effective suppression of cell dissemination under light irradiation. The findings establish a unique in‐cellulo assembling evolution for attaining topologically complex supramolecules and demonstrate an in‐situ nanoformulation of biomedical agents in disease theranostics.