Spatiotemporal control of chemical assembly in living cells remains challenging. We have now developed an efficient and general platform to precisely control the formation of assemblies in living cells. We introduced an O‐[bis(dimethylamino)phosphono]tyrosine protection strategy in the self‐assembly motif as the Trojan horse, whereby the programmed precursors resist hydrolysis by phosphatases on and inside cells because the unmasking of the enzymatic cleavage site occurs selectively in the acidic environment of lysosomes. After demonstrating the multistage self‐assembly processes in vitro by liquid chromatography/mass spectrometry (LC‐MS), cryogenic electron microscopy (Cryo‐EM), and circular dichroism (CD), we investigated the formation of site‐specific self‐assembly in living cells using confocal laser scanning microscopy (CLSM), LC‐MS, and biological electron microscopy (Bio‐EM). Controlling chemical assembly in living systems spatiotemporally may have applications in supramolecular chemistry, materials science, synthetic biology, and chemical biology.

Controlling the enzymatic reaction of macromolecules in living systems plays an essential role in determining the biological functions, which remains challenging in the synthetic system. This work shows that host–guest complexation could be an efficient strategy to tune the enzymatic self-assembly of the peptide. The formed host–guest complexation prevents the enzymatic kinetics of peptide assemblies on the cell surface and promotes cellular uptake of assemblies. For uptake inside cells, the host–guest complex undergoes dissociation in the acidic lysosome, and the released peptide further self-assembles inside the mitochondria. Accumulating assemblies at mitochondria induce the ferroptosis of cancer cells, resulting in cancer cell death in vitro and the tumor-bearing mice model. As the first example of using host–guest complexation to modulate the kinetics of enzymatic self-assembly, this work provides a general method to control enzymatic self-assembly in living cells for selective programming cancer cell death.

Biological components (protein, DNA, lipid rafts, etc.) self-sort to form higher-order structures with elegant modulation by endogenous stimuli for maintaining cellular functions in living cells. However, the challenge of producing self-sorted higher-order assemblies of peptides in living systems (cells and tissues) spatiotemporally has yet to be achieved. This work reports the using of a biocompatible strategy to construct self-sorted assemblies of peptides in living cells and tumorbearing mice. The results show that the designed peptides selfsort to form distinct nanostructures in living cancer cells using an endogenous trigger, as evidenced by confocal laser scanning microscopy and Bio-EM. Wound-healing experiments indicate that the in situ generation of self-sorted nanostructures exhibits a synergistic effect that significantly decreases the migration of cancer cells. In vivo experiments demonstrate that the designed peptides could self-sort in tumor-bearing mice and improve the tumor penetrating ability of the impenetrable component in tumor tissue. We can further program the formation of selfsorted materials through orthogonal triggers by introducing an exogenous trigger (light) and an endogenous trigger independently. Thus, this work provides a strategy to control multiple self-assembling processes in the context of the living system and provides a general strategy to construct self-sorted structures for the emergent properties of materials science.

Supramolecular chirality plays an indispensable role in living and synthetic systems. However, the generation and control of filament chirality in the supramolecular hydrogel of short peptides remains challenging. In this work, as the first example, we report that the heterodimerization of the enantiomeric mixture controls the alignment, chirality, and stiffness of fibrous hydrogels formed by aromatic building blocks. The properties of the resulting racemic hydrogel could not be achieved by either pure enantiomer. Cryo-EM images indicate that the mixture of L and D enantiomers forms chiral nanofibers, the percentage of which can be readily controlled through stoichiometric co-assembly of heterochiral enantiomers. 2D NOESY NMR and diffusion-ordered NMR spectroscopy reveal that heterodimerization of enantiomers plays a crucial role in the formation of chiral nanofibers. Further mechanistic studies unravel the mechanism of supramolecular chirality formation in this two-component system. Molecular dynamics simulations confirm that the intermolecular hydrogen bond and π–π interaction of heterodimers play important roles in forming a chiral hydrogel. Furthermore, regulation of the adhesion and morphology of mammalian cells is achieved by tuning the relative ratio of L and D enantiomers at the same concentration. This work illustrates a novel strategy to control the supramolecular chirality of aromatic peptide hydrogels for materials science. Graphical Abstract

Peptide-based materials hold great promise for various biomedical applications and have drawn increasing attention over the past five years. Despite the progress in fabrication and handling peptide materials in vitro, manipulating assemblies of peptides in living cells (or animals) is still in its infancy. In this contributing review, recent work is summarized using endogenous triggers to construct functional assemblies of peptides in vivo. After introducing the triggers for inducing peptide assemblies, the recent progress is highlighted of the in situ construction of assemblies for biomedical applications with emphasis on cancer therapy. Finally, a brief perspective is provided to discuss the future promises and challenges of this emerging area of supramolecular chemistry.