Strategies for accelerating osteogenesis through nanoparticle-based DNA/mitochondrial damage repair

The efficiency of gene therapy is often dictated by the gene delivery system. Cationic polymers are essential elements of gene delivery systems. The relatively cheap cationic polymer, polyethyleneimine, has high gene delivery efficiency and is often used for gene delivery. However, the efficiency of gene therapy with polyethyleneimine-pDNA polyplex (PEI) is low. Human mesenchymal stem cells transfected with polyethyleneimine and a plasmid carrying the important osteogenic differentiation gene runt-related transcription factor 2 (RUNX2) accumulated DNA double-strand breaks and mitochondrial damage proportional to the amount of polyethyleneimine, reducing viability. Genomic/cellular stabilizer mediating RUNX2 delivery (GuaRD), a new reagent incorporating RS-1 NPs developed in this study, promoted DNA repair and prevented the accumulation of cell damage, allowing the delivery of pRUNX2 into hMSCs. while maintaining genome and mitochondrial stability. DNA damage was significantly lower and the expression of DNA repair-related genes significantly higher with GuaRD than with PEI. In addition, GuaRD improved mitochondrial stability, decreased the level of reactive oxygen species, and increased mitochondrial membrane potential. Osteogenic extracellular matrix (ECM) expression and calcification were higher with GuaRD than with PEI, suggesting improved osteogenic differentiation. These results indicate that lowering the cytotoxicity of PEI and improving cell stability are key to overcoming the limitations of conventional gene therapy, and that GuaRD can help resolve these limitations.