Bioorthogonal Click Intracellular Hydrogelation to Control Cell Cycle Behavior

Hydrogel networks synthesized through efficient click reactions have become prevalent biomaterials to further our understanding into how cells interact with their surrounding environment¹. Intracellular hydrogelation is an emerging application for these materials and presents new opportunities for hydrated polymer networks. The intracellular microenvironment is highly crowded and is composed of complex biochemical networks that regulate the internal state of a cell. Through hydrogelation, intracellular macromolecular crowding can be achieved, reducing reaction rates on a cellular level, and creating gelation-induced quiescence. It is envisioned that this technique could expand routes for preservation, reducing the reliance on costly cryopreservation techniques and cold-chain transportation². Herein, we induce gelation in the cytosol of living cells with high molecular weight poly(ethylene glycol) (PEG) macromers through click reactions causing supraoptimal macromolecular crowding. PEG macromers were functionalized with either dibenzylcyclooctyne (DBCO), a ring strained alkyne, or a terminal azide to form networks through a spontaneous strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. For photocatalyzed intracellular gelation, PEG macromers were functionalized with norbornene or thiol reactive groups. With the additional of nitrobenzyl moieties (UV-degradable) into the polymer network, degradation of the network could be achieved in a spatiotemporal manner. Efficient transfection of PEG macromers into various cell lines and primary cells was achieved with lipofectamine as confirmed through flow cytometry (&gt;85%) with minimal apoptosis. Intracellular PEG uptake was quantified and confirmed higher retention of the gelled network in comparison to individual PEG macromers. Using immunofluorescence, flow cytometry, and bioactivity assays, cellular PEG uptake, structure, and proliferation were assessed. The effects of intracellular hydrogelation included decreased DNA turnover, delayed new protein synthesis, and disrupted cytoskeletal organization. To track cell-cycle progression in real time, cells with a genetically encoded fluorescent sensor for Cyclin-Dependent Kinase 2 (CDK2) activity were implemented³. CDK2 activity builds up continuously in cells that are actively progressing through the cell cycle and turns off when cells enter gelation-induced quiescence. By tracking single cells in real time, intermitotic times were demonstrated to increase with gelation, and a reduction in cellular speed indicated a decrease in motility (30 μm/hr vs. 15 μm/hr). Fluorescent correlation spectroscopy (FCS) was employed to understand changes in the cytosol’s viscosity after intracellular crosslinking⁴. With this technique, it was demonstrated that hydrogelation significantly slowed diffusion inside the cytoplasm, doubling the viscosity of the cytoplasm on a nanometer length scale when PEG macromers were crosslinked. Wound healing assays demonstrated reduction in the coordinated response of cells to repair after intracellular gelation, suggesting potential relevance for this technique for wound preservation. With the addition of UV-degradable moieties into PEG macromers, the effects of intracellular gelation were reversed through degradation of the hydrogel network. Intracellular polymerization using bioorthorgonal click reactions offers new opportunities to further explore intracellular dynamics and the effects of intracellular polymerizations. It is envisioned that this high-throughput method will enable the expansion of biomaterial-based approaches to study a facile, low-cost method of preservation.<br/><br/>1) M. W. Tibbitt, K. S. Anseth, <i>Sci. Transl. Med.</i> <b>2012</b>, <i>4</i>, (160), 160ps24-160ps24.<br/>2) L. J. Macdougall, et.al., <i>ACS Biomater. Sci. Eng.</i> <b>2021</b>, <i>7</i>, (9), 4282-4292.<br/>3) S. L. Spencer, et.al., <i>Cell.</i> <b>2013</b>, <i>155</i> (2), 369–383.<br/>4) K. Kwapiszewska, et.al., <i>J. Phys. Chem. Lett.</i> <b>2020</b>, <i>11,</i> (16), 6914–6920.