Transport Properties of Self-Assembling G-Hydrogels: Evidence for a Tunable Fickian Diffusivity

The mixing of Guanosine (Gua) and Guanosine 5'-monophosphate (GMP) in water in selected compositions yields highly hydrated, transparent, and self-healing self-assembled supramolecular G-hydrogels, attractive for biomedical applications. This work investigates how hydrogel composition affects solute transport, including diffusion, binding, loading, and release properties, using a set of fluorescent probes with varying size and polarity. Although small/wide-angle X-ray scattering techniques showed that no structural changes are induced by probe addition, even when intercalation into G-quadruplexes is expected, the internal mesh structure of the hydrogel, modulated by the Gua:GMP ratio, directly impacts probe diffusivity and loading. Tighter networks (e.g., 1:1) slow diffusion and enhance retention compared to looser configurations (e.g., 1:4). Moreover, UV-visible titrations revealed markedly different binding affinities (K_b ≈ 5.7 × 10⁴ M^-1 for DAPI, 8.0 × 10³ M^-1 for ThT, and 1.4 × 10² M^-1 for RhB), which are expected to result in lower diffusion coefficients and slower release, especially for DAPI and ThT. Indeed, diffusion coefficients, obtained via fluorescence recovery after photobleaching and time-resolved fluorescence spectroscopy, reach 90, 20, and 60 μm²/s for FITC-dextran, ThT, and RhB, respectively. Probe release kinetics, modeled via Weibull fitting, indicated sustained release with characteristic times (τ) between 9.6 and 23.2 h and β ≈ 1 in 1× PBS, consistent with predominantly Fickian diffusion. Remarkably, switching to 10× PBS significantly accelerated release (τ reduced by ≈ 40-50%), suggesting that ionic strength and/or pH changes critically affect not only probe-hydrogel interactions but also the internal gel architecture, altering porosity, mesh size, and network tortuosity, thus enhancing molecular mobility. Overall, the G-hydrogel system offers a structurally tunable and composition-dependent platform capable of finely regulating molecular transport and release profiles, making it highly suitable for controlled drug delivery and adaptive biomaterial applications.