Delivery of miRNA-29b Using R9-LK15, a Novel Cell-Penetrating Peptide, Promotes Osteogenic Differentiation of Bone Mesenchymal Stem Cells

Abstract

Delivery of osteogenesis-promoting microRNAs (miRNAs) is a promising approach to enhance bone regeneration. In this study, we generated nanocomplexes comprising the novel cell-penetrating peptide R9-LK15 and miR-29b and investigated their effects on osteogenic differentiation of bone mesenchymal stem cells (BMSCs). R9-LK15/miR-29b nanocomplexes were prepared and characterized. The transfection efficiency, cell viability, and osteogenic differentiation were investigated. The results showed that R9-LK15 maintained the stability of miR-29b in serum for up to 24 h. Moreover, R9-LK15 efficiently delivered miR-29b into BMSCs; the transfection efficiency was ~10-fold higher than that achieved using Lipofectamine 2000. The Cell Counting Kit-8 assay showed that R9-LK15 and R9-LK15/miR-29b nanocomplexes had negligible cytotoxic effects on BMSCs. Delivery of R9-LK15/miR-29b nanocomplexes promoted osteogenic differentiation of BMSCs and extracellular matrix mineralization by upregulating alkaline phosphatase expression and downregulating histone deacetylase-4 expression. In general, we developed a novel miRNA delivery system that has a high transfection efficiency and promotes osteogenic differentiation.

Figure 1

Characteristics of R9-LK15/miR-29b nanocomplexes. (a) Dynamic light scattering analysis of the size and zeta potential of R9-LK15/miR-29b nanocomplexes formed by mixing R9-LK15 and miR-29b at various molar ratios. The concentration of miR-29b was 50 nM. (b) A gel retardation assay to determine the formation of nanocomplexes following mixing of R9-LK15 and miR-29b at molar ratios of 1:1–4:1. (c) A TEM image of R9-LK15/miR-29b nanocomplexes. (d) Stability of miR-29b alone and complexed with R9-LK15 upon incubation in serum at 37°C for various durations.

Figure 2

Transfection efficiency of R9-LK15/miR-29b nanocomplexes. (a) Confocal microscopy images of BMSCs treated with FAM-labeled miR-29b (50 nM) alone or complexed with Lipo or R9-LK15. Untreated cells (Con) and those treated with Lipo and R9-LK15 served as controls. Scale bar: 20 μm. (b) Flow cytometric analysis of uptake of FAM-labeled miR-29b by BMSCs. (c) Fluorescence intensity of FAM in confocal microscopy images. Data are the mean ± SD of triplicate experiments. ^∗∗∗P < 0.001.

Figure 3

Cytotoxic effects of R9-LK15/miR-29b nanocomplexes on BMSCs. (a) LIVE/DEAD assay of untreated BMSCs (Con) and those treated with miR-29b, Lipo, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes for 72 h. (b) The percentage of dead cells was calculated. Data are the mean ± SD of triplicate experiments. ^∗P < 0.05. (c) CCK-8 assay of untreated BMSCs (Control) and those treated with miR-29b, Lipo, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes for 2, 4, and 6 days. Data are the mean ± SD of triplicate experiments. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 4

Effects of R9-LK15/miR-29b nanocomplexes on ALP activity and ECM mineralization. (a) ALP activity in untreated BMSCs (Con) and those treated with miR-29b, Lipo, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes for 7 days. (b) Alizarin Red S staining in untreated BMSCs (Con) and those treated with miR-29b, Lipo, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes for 14 days. (c) Quantification of ALP activity. (d) Quantification of Alizarin Red S staining. Scale bar: 500 μm. ^∗∗P < 0.01.

Figure 5

Effects of R9-LK15/miR-29b nanocomplexes on expression of osteogenesis-related genes and proteins. (a) RT-PCR analysis of ALP mRNA expression in untreated BMSCs (Con) and those treated with miR-29b, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes for 7 days. (b) RT-PCR analysis of HDAC4 mRNA expression in untreated BMSCs (Con) and those treated with miR-29b, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes for 7 days. (c) Western blot analysis of HDAC4 protein expression in untreated BMSCs (Con) and those treated with miR-29b, R9-LK15, Lipo/miR-29b nanocomplexes, and R9-LK15/miR-29b nanocomplexes. (d) Quantification of relative HDAC4 protein expression using ImageJ. Data are the mean ± SD of triplicate experiments. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 6

Schematic illustration to explain how transfection of R9-LK15/miR-29b nanocomplexes promotes osteogenesis.