Intracellular Crowding by Bio‐Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis

• Published in: Advanced materials (Weinheim) Vol. 34; no. 31; pp. e2202882 - n/a
• Main Authors: Macdougall, Laura J., Hoffman, Timothy E., Kirkpatrick, Bruce E., Fairbanks, Benjamin D., Bowman, Christopher N., Spencer, Sabrina L., Anseth, Kristi S.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.08.2022
• Subjects: Alkynes; Alkynes - chemistry; Animals; Azides - chemistry; biostasis; Cell death; Cellular structure; click chemistry; Crosslinking; Cycloaddition; Hydrogels; Hydrogels - chemistry; intracellular crosslinking; Mammals; Materials science; poly(ethylene glycol); Polyethylene glycol; Polyethylene Glycols - chemistry; Polymers; Polymers - chemistry; Polysaccharides; Protein synthesis; Proteins; Vitrification; intracellular crosslinking; click chemistry; biostasis; hydrogels; poly(ethylene glycol)
• ISSN: 0935-9648; 1521-4095; 1521-4095
• DOI: 10.1002/adma.202202882

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine‐mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio‐orthogonal strain‐promoted azide–alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5‐fold. Real‐time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 h vs 19 h) and decreases in motility (30 µm h−1 vs 15 µm h−1). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho‐Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function. Intracellular crosslinking in living cells using poly(ethylene glycol) macromers is introduced. Increased macromolecular crowding in the cytosol of the cell causes crosslink‐induced quiescence and results in reduced protein translation, cellular motility, and actin dynamics while cell viability remains high. The effects can be reversed through polymer degradation with UV light.