N-Acetyl-l-Leucine-Polyethyleneimine-Mediated Delivery of CpG Oligodeoxynucleotides 2006 Inhibits RAW264.7 Cell Osteoclastogenesis

Introduction: CpG oligodeoxynucleotides (CpG ODN) play important roles in resisting inflammation and bone resorption. However, the inherent instability and rapid degradation hinder their wider application. This study aimed to evaluate whether N-acetyl-L-leucine-modified polyethyleneimine (N-Ac-L-Leu-PEI) could effectively deliver CpG ODN 2006 to RAW264.7 cells and and if it can regulate osteoclastogenesis in vitro.

Materials and methods: Gel retardation assay was conducted to evaluate whether N- Ac-L-Leu-PEI and CpG ODN could form a stable complex. RAW264.7 cells were divided into four groups of control group, ODN group, phosphorothioate ODN group and N-Ac-L-Leu-PEI/ODN group. Fluorescence assay was conducted to evaluate the transfection rate of ODNs in different groups. Cell viability was determined by MTT assay. Cell apoptosis was determined by live-dead cell staining and flow cytometry assay. Relative expression levels of osteoclastic differentiation factors, including Nfatc, c-fos, receptor activator of nuclear factor κB (RANK), and matrix metalloproteinase 9 (MMP9), were determined by real-time PCR and Western blot.

Results: N-Ac-L-Leu-PEI and CpG ODN could form a stable complex at a mass ratio of 1:1 (w:w). MTT assay showed that the cell viability of N-Ac-L-Leu-PEI was relatively high even at a mass ratio of 8 μg/mL. The transfection rate of N-Ac-L-Leu-PEI-ODN complex was higher than 90%. The cell proliferation and apoptosis was significantly enhanced in N-Ac-L-Leu-PEI- CpG ODN group when compared to those in phosphorothioate CpG ODN. The expression levels of Nfatc, c-fos, RANK, and MMP9 were significantly decreased in N-Ac-L-Leu-PEI/ODN complex group.

Discussion: N-Ac-L-Leu-PEI could be a potential gene vehicle for the prevention of periodontitis-mediated bone resorption.

Keywords: CpG ODN; CpG oligodeoxynucleotides; N-Ac-L-Leu-PEI; N-acetyl-L-leucine-modified polyethyleneimine; osteoclastic differentiation; proliferation.