(57c) Engineering Hydrogels with a Reversibly Tunable Modulus to Probe Cell Behavior

In vivo, the extracellular matrix is a complex dynamic environment that undergoes cycles of stiffening and softening during natural processes such as disease, wound healing, and development. Many traditional in vitro cell culture substrates, however, have static moduli, requiring one to replate cells to investigate the effect of changes in stiffness on cell behavior. In addition, current dynamic substrates often rely on stimuli that may alter the charge or redox state of the cellular environment, which can lead to unwarranted changes in phenotype. To address these issues, we present two strategies for the reversible control of hydrogel modulus using azobenzene-containing crosslinkers. By irradiation with either 365 nm or 405 nm light, we show that azobenzenes can modulate the structure of a peptide-based crosslinker in covalently bound PEG-based hydrogels, as well as reversibly associate with cyclodextrin units in hyaluronic acid (HA)-based supramolecular gels to control crosslinking density. In both hydrogel platforms, irradiation with 365 nm light (10 mW/cm², 5 min) leads to azobenzene isomerization to the cis conformation, which corresponds to a decrease in the crosslinking density and therefore an overall softening of the hydrogel. The reverse isomerization can be initiated with visible light (400-500 nm, 10 mW/cm², 5 min). Using this method, the modulus can be switched by up to 2-fold in a biologically relevant range (starting modulus between 100 Pa – 1000 Pa). Importantly, the azobenzene photoswitch was carefully chosen to maximize the lifetime of the cis state (half-life of 9 hours at 37°C), which enables the investigation of cell-matrix interactions such as spreading in response to changes in stiffness. This strategy is fully cytocompatible and due to the tunability and non-invasive properties of the stimulus, these innovative materials should be broadly applicable to examining the effect of modulus changes on many different cell functions and cell types.