Stable Protein‐Based G‐Quadruplex‐Derived Supramolecular Bioinks as Tunable ECM‐Mimetic Constructs Assembled by Combining Non‐Covalent and Covalent Strategies

Abstract

ABSTRACT G‐quadruplex hydrogels hold great promise for biofabrication owing to their dynamic supramolecular nature, provided their inherent instability under physiological conditions is overcome. Here, a bioinspired strategy that synergistically combines supramolecular self‐assembly, under macromolecular crowding conditions, with in‐bath enzymatic covalent crosslinking was employed to create stable, protein‐based G‐quadruplex‐derived hydrogels. Mimicking the crowded intracellular milieu, the addition of Ficoll enhances G‐quadruplex stability and tunes the rheological behavior, while transglutaminase‐mediated crosslinking reinforces the network, preserving its structural integrity over extended periods. This combined approach yields printable bioinks with optimal viscosity, yield stress, and shear‐thinning properties, enabling the fabrication of complex, multilayered 3D constructs that support enhanced cell viability and proliferation within an extracellular matrix (ECM)‐mimetic fibrillar environment. Moreover, the modulation of the crosslinking density allows controlling cellular responses, offering a versatile platform for tailoring the biomechanical microenvironment. This study establishes a new class of hybrid G‐quadruplex hydrogel bioinks, exhibiting unprecedented stability under physiological conditions, biofunctionality, and off‐the‐shelf availability, unlocking their potential for advanced tissue engineering and regenerative medicine strategies.