Injectable Arnebia Euchroma Polysaccharide-Based Hydrogel as CpG Oligonucleotide Delivery System with Dual Immunomodulatory Activities.

This study developed novel immunostimulatory hydrogels composed of aldehyde-modified Arnebia euchroma polysaccharide (oxidized ARP, O-ARP) and gelatin (GE) for oligonucleotide delivery. Structural characterization of three O-ARP derivatives with varying degrees of oxidation confirmed reduced molecular weight, a preserved yet altered molar ratio of monosaccharide composition, and triple-helical conformation. Hydrogels (GE-O1, GE-O2, GE-O3) were formed via a Schiff base reaction between aldehyde and amino groups. Molecular dynamics simulations showed that O-ARP and gelatin can form a stable three-dimensional (3D) network via hydrogen bonding and van der Waals interactions. In vitro studies demonstrated that both the O1-ARP derivative and GE-O1 hydrogel significantly improved RAW 264.7 macrophage viability, phagocytosis, NO production, and pro-inflammatory cytokine secretion, including IL-6, IL-1β, and TNF-α. The cationic GE-O1 hydrogel efficiently loaded anionic CpG oligonucleotides (CpG-ODN 1862) via electrostatic interaction, forming GE-O1-CpG complexes and promoting cellular uptake. Importantly, the GE-O1-CpG complex exhibited superior immunomodulatory effects compared with either GE-O1 or CpG alone, indicating a synergistic dual immunostimulatory response. In vivo studies confirmed the biosafety of GE-O1-CpG. Using OVA as a model antigen, GE-O1-CpG/OVA enhanced both humoral and cellular immune responses. These findings support GE-O1-CpG hydrogels as potential system for combined immunomodulation and nucleotide delivery.