S‐Nitrosothiols as Thiol‐Protecting Groups for Controlled Thiol‐Maleimide Crosslinking of Homogeneous Soft Hydrogels

ABSTRACT

The thiol‐maleimide Michael‐type addition is used in bioconjugation and hydrogel crosslinking for its chemoselectivity and rapid kinetics under physiological conditions. However, this same reactivity limits its use for soft hydrogels, as gelation often proceeds faster than precursor mixing, leading to spatially heterogeneous networks. Here, S‐nitrosothiols (RSNOs) are introduced as thiol‐protecting groups that suppress premature thiol‐maleimide coupling and enable homogeneous mixing of polymer precursors prior to on‐demand crosslinking. Despite common assumptions about RSNO instability, RSNO‐modified 4‐arm polyethylene glycol (PEGSNO) is stable in aqueous solution for months at 4°C protected from light. The nucleophiles sodium ascorbate or sodium thiosulfate (STS) trigger thiol regeneration and controlled crosslinking with maleimide‐modified PEG. The resulting hydrogels are homogeneous with Young's moduli tunable across a physiologically relevant range by independently varying nucleophile identity, concentration, or polymer content. In a cell‐adhesive system based on maleimide‐modified cold water fish skin gelatin, STS‐triggered gelation supports 3D cell encapsulation, with cytocompatibility depending on formulation and cell type, while matrix stiffness is tuned through polymer content and STS concentration. This work establishes RSNO chemistry as a previously unexplored strategy for controlled thiol‐maleimide crosslinking, enabling reproducible, homogeneous network formation, with potential for dual‐function materials coupling network formation with local reactive nitrogen species delivery.