On the Binding of Cell-Adhesive Proteins Vitronectin, Fibronectin and Laminin to Agarose Hydrogel

Protein binding at hydrogel interfaces is a key process in biomaterials surface science, governing the biochemical response of cells. Here, we investigate the binding behavior of three cell-adhesive proteins with distinct molecular weights, namely, vitronectin, fibronectin, and laminin on a hydrogel network composed of a low-hydrophobic agarose. Binding kinetics was evaluated through a solution-depletion method. Using destabilizing agents such as the chaotropic salt magnesium perchlorate, Mg(ClO 4 ) 2 , or the non-ionic surfactant Tween 20, our results show that binding is driven by hydrophobic effects and strongly depends on protein size. Focusing on vitronectin because of its important binding profiles and relevance in the field of mechanobiology when using agarose-based substrates for cell adhesion, atomistic Molecular Dynamics simulations show that the protein rapidly attains a stable adsorbed state without evidence of global unfolding, establishing short-range weak interactions, with constant number of interfacial hydrogen bonds in the range of 10 to 20 and approximately 200 heavy-atom contacts. The favorable binding of vitronectin is also supported by theoretical considerations on the thermodynamics of water displacement at the interface. Lastly, the combination of contact angle and nano-indentation experiments allows the conclusion that proteins form a hydrated region with variable homogeneity depending on the type of protein.