Poly-lysine-modified recombinant protein nanocages for effective delivery of small activating RNA.

Small activating RNA (saRNA) holds significant promise as a therapeutic platform for various diseases. However, the development of efficient nanocarriers that can overcome existing delivery challenges and ensure effective cellular uptake remains a critical hurdle. In this study, we aimed to address this issue by genetically modifying four lysine residues at the N-terminus of the FTH gene through gene recombinant technology, resulting in the creation of poly-lysine-H-apoferrin (4LF) vectors. These vectors were designed to efficiently deliver saRNA encoding the Sirtuin1 (Sirt1) protein to chondrocytes, thereby mitigating cartilage damage. The poly-lysine modification conferred the ability of 4LF@saRNA nanoparticles (NPs) to escape from lysosomes via proton sponge effects and to release saRNA into the cytoplasm through the pH-induced degradation of the 4LF vector, ultimately activating the target gene. To enhance the retention of NPs within the joint cavity and facilitate intra-articular delivery, we incorporated the 4LF@saRNA NPs into a thermosensitive self-healing hydrogel composed of chitosan, oxidized chondroitin sulfate (OCS), and sodium β-glycerophosphate (β-GP). Experimental results demonstrated that the chitosan/OCS/β-GP-4LF@saRNA (OCCG-4LF@saRNA) delivery system effectively delivered the 4LF@saRNA NPs to chondrocytes both in vitro and in vivo, resulting in a significant increase in Sirt1 protein expression. This upregulation led to a reduction in chondrocyte apoptosis, enhanced cell migration, and improved cartilage protection, effectively alleviating symptoms of osteoarthritis. In conclusion, our findings suggested that the 4LF-based delivery system hold considerable potential for effective intracellular saRNA delivery, demonstrating promising biocompatibility, stability, and delivery efficiency, with significant therapeutic implications for cartilage repair.