Nowadays, soft tissue damage are typically replaced by grafts from other body parts. However, tissue grafting can be problematic due to the need for several surgical procedures or lack of sufficient tissue material. Recently, hydrogels have emerge as an alternative treatment for tissue regeneration. Specifically guanosine-based hydrogels show unique self-assembly properties resulting in hydrogels with high water content and nanofibrilar structure mimicking the extracellular matrix. Four planar guanosine-quartet can lead to 3D nanofibrous G-quadruplex (G4) structures by π-π stacking interactions between aromatic purine rings that under certain conditions, are able to encapsulate enough water to produce G4 hydrogels. Combined with 3D printing technology, they could provide a highly hydrated and well-defined 3D environment optimal for cell survival and differentiation. However, hydrogels currently lack lifetime stability, biological activity, and printability, limiting their use and applicability. We hypothesized that using more complex boronic acid derivatives such as phenylboronic acid, 2-formilphenylboronic acid, and 2-naphtylboronic acid, in addition to boric acid, would further enhance G4 and thus hydrogel stability.
Guanosine, boronic acid derivatives, and potassium hydroxide (20-120 mM) were mixed at 80 °C for 15 minutes and cooled down for gelation. Inversion test was performed to select compositions capable of gel formation. Next, printability properties were determined by a semi-quantitative filament fusion and collapse test. The best hydrogels were then evaluated by scanning electron microscopy and dynamic step-strain sweep and peak-hold rheology assays. Additionally, hydrogel pH, degradability in the complete medium, and nutrient diffusion (FITC-Dextran) were determined. Viability of SaOS-2 cells seeded onto the printed hydrogels was also assessed using confocal laser scanning microscopy and live-cell mapping.
A total of 76 hydrogel compositions passed the initial inversion test, of which ~1/3 showed suitable properties in the filament collapse and fusion test. We then selected the best composition for each boronic acid derivative for a comprehensive analysis. SEM analysis revealed nanofibrillar structures in the hydrogel networks of all four boronic acid derivatives, evident of successful G4-quadruplex formation, and rheological testing confirmed good thixotropic properties. Importantly, while all tested hydrogels showed a pH between 7.4 and 8.3, only hydrogels obtained with boric acid were stable for up to 7 days. Furthermore, successful diffusion of FITC-Dextran molecules (70, 500 and 2000 kDa) into the hydrogel was observed, indicating that nutrients of various sizes may be transported through the scaffold, and finally, a cell viability of ~80% after 24 hours was determined for SaOS-2 cells seeded onto the printed hydrogels.
We developed a novel printable hydrogel based on guanosine and different boronic acids. Only boric acid hydrogel showed good printability and thixotropic properties and was stable in medium for up to 7 days, while no cytotoxicity was observed. Thus, this hydrogel formulation represents an excellent candidate for initial steps of angiogenesis or as potential antibacterial biomaterial for biomedical applications.
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