Co-Delivery of RUNX2-Targeting miRNAs and shRNAs Using Nanoparticles Composed of Dexamethasone and PEI Induces Chondrogenesis of Human Mesenchymal Stem Cells.

RNA interference (RNAi) plays important roles, and microRNAs (miRNAs) are used as biomarkers and targets in cell therapies. In this study, we fabricated miRNAs and short hairpin RNAs (shRNAs) targeting the osteogenic RUNX2 gene to induce chondrogenesis of human mesenchymal stem cells (hMSCs). pDNA harboring these miRNAs and shRNAs was complexed with nanoparticles composed of dexamethasone and tetramethlyrhodamine-labeled branched polyethyleneimine (RDtNPs). The miRNAs and shRNAs reduced RUNX2 expression in hMSCs at early (12 h) and late (72 h) time points, respectively. Co-delivery of miRNAs and shRNAs resulted in rapid and sustained RUNX2 silencing. Moreover, dexamethasone in the nanoparticles enhanced chondrogenic differentiation. Gene and protein expression of RUNX2 was lower in hMSCs transfected with RDtNPs complexed with pDNA harboring miRNAs plus shRNAs for 72 h than in control hMSCs. Moreover, delivery of these miRNAs and shRNAs increased gene and protein expression of chondrogenic SOX9. These changes in expression facilitated the chondrogenic differentiation of hMSCs, as demonstrated by analysis of markers related to mature chondrocytes. Furthermore, histological and immunohistological analyses detected specific extracellular matrix and cartilage-related proteins in cultures of hMSCs transfected with these miRNAs and shRNAs.