Cell Adhesion on RGD-Displaying Knottins with VaryingNumbers of Tryptophan Amino Acids to Tune the Affinity for Assemblyon Cucurbit8uril Surfaces

摘要

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV²⁺). The binding affinity of the knottins with CB[8] and MV²⁺ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaceswith a higher valency, and in addition, more pronounced focal adhesionformation was observed on the higher-valency knottin surfaces. Weattribute this effect to the enhanced coverage and the enhanced affinityof the knottins in their interaction with the CB[8] surface. Collectively,these results are promising for the development of biomaterials includingknottins via CB[8] ternary complexes for tunable interactions withcells.