TiO₂ Nano‐Biopatterning Reveals Optimal Ligand Presentation for Cell–Matrix Adhesion Formation

Abstract

Nanoscale organization of transmembrane receptors is becoming recognized as critical for cellular functions, enabled by the nanoscale engineering of bioligand presentation. Previously, a spatial threshold of ≤60 nm for integrin binding ligands in cell‐matrix adhesion was demonstrated using mono‐ligand gold nanoparticles. However, the ligand geometric arrangement was limited to hexagonal arrays of mono‐ligands, while plasmonic quenching by gold limited further investigation by fluorescence‐based high‐resolution imaging. Here, these limitations were overcome with dielectric TiO2 nanopatterns, which allows fine‐tuning ligand cluster size, eliminating fluorescence quenching, thus enabling super‐resolution fluorescence microscopy on nanopatterns. By dual‐color super‐resolution imaging, high precision and consistency among nanopatterns, bioligands, and integrin nanoclusters was observed validating the high quality and integrity of both nanopattern functionalization and passivation. By screening TiO2 nanodiscs with various diameters, an increase in fibroblast cell adhesion, spreading area, and YAP nuclear localization on 100nm‐diameter compared with smaller diameters was observed, and Focal Adhesion Kinase was identified as the regulatory signal. These findings explore the optimal ligand presentation when the minimal requirements are sufficiently fulfilled in the heterogenous ECM network of isolated binding regions with abundant ligands. Integration of high‐fidelity nano‐biopatterning with super‐resolution imaging will allow precise quantitative studies to address early signaling events in response to receptor clustering and their nanoscale organization.This article is protected by copyright. All rights reserved