Designing Thermally Stable DNA Hydrogels via Entropically-Driven Acridine Intercalation

Physically cross-linked hydrogels formed through supramolecular interactions typically relax more rapidly upon heating because reversible bond formation is often exothermic. In contrast, entropy-dominated associations can generate materials that maintain or strengthen mechanical properties with temperature. However, strategies to systematically tune entropy-driven behavior in polymer networks remain limited. Here, we investigate how environmental variables regulate reversible cross-linking in acridine-based DNA-intercalating supramolecular hydrogels (Acr-PEG DISHs). Hydrogels composed of 50 mg mL^{- 1} DNA and 4 mM bis-intercalating cross-linker were evaluated across buffer compositions with ionic strengths of I ≈ 0.004-0.17 M, salt concentrations from 0-0.75 M, different ion identities, and varied pH. Increasing ionic strength produced more elastic networks with slower relaxation dynamics, increasing relaxation times from ∼30-100 s in low-ionic-strength buffers to ∼55-625 s in PBS. At elevated salt concentrations (∼0.5 M), electrostatic screening dominated network behavior and increased transition state entropy by Eyring analysis. Although monovalent salts produced similar elastic responses, ion identity modulated dissociation kinetics (Na⁺< Li⁺< K⁺), whereas multivalent ions destabilized the network. In contrast, pH-dependent studies showed only minor effects because citrate-phosphate ionic strength masked acridine protonation. These findings identify the ionic environment as a powerful handle for tuning entropy-driven supramolecular hydrogel dynamics.