Enhanced stability and sustained delivery of structurally dense DNA nanostructures via a biodegradable hydrogel platform

DNA nanostructures offer programmable architectures with precise ligand arrangement, yet their in vivo utility is often limited by structural fragility and rapid systemic clearance. Here, we present a robust platform for the sustained delivery of structurally dense, multi-helical square block DNA nanostructures (SQBs) by encapsulating them within a biodegradable hydrogel composed of thiolated hyaluronic acid (HA) and gelatin. Distinct from DNA-based hydrogels where the DNA network serves as the structural matrix, this platform employs a decoupled architecture that entraps SQBs as discrete nanoparticles within a tunable polymeric matrix. This platform is engineered to maintain an Mg2+-rich microenvironment to preserve the structural integrity of SQB cargo, as evidenced by the intact morphology of nanostructures recovered from the matrix, while allowing independent control over degradation and release kinetics. In vitro, encapsulated SQBs retained their electrophoretic stability and exhibited release profiles governed by the hydrogel's crosslinking density and gelatin content. Importantly, SQB-hydrogel hybrids demonstrated sustained intracellular uptake in RAW 264.7 macrophages for up to 5 days, whereas free SQBs were rapidly internalized and cleared within 3 days. In vivo subcutaneous administration further confirmed that the hybrid system maintained detectable fluorescence for 10 days, significantly outperforming free SQBs, which were cleared within 24 hours. These findings establish a versatile hydrogel framework that effectively serves as a sustained depot for complex DNA nanostructures, offering a generalizable strategy for their localized and long-term deployment in therapeutic applications.