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

Abstract

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:

• N-acetyl-L-leucine-modified polyethyleneimine
• N-Ac-L-Leu-PEI
• CpG oligodeoxynucleotides
• CpG ODN
• proliferation
• osteoclastic differentiation

Acknowledgments

We thank Prof. Quanshun Li, Jilin University School of Life Sciences for providing N-Ac-L-Leu-PEI. This research was supported by grants from the National Natural Science Foundation of China (No.81600879, No.81970946 and No. 51972240), Medical Support Program of the Jilin province (No.20170311032YY), Science and Technology Project of Jilin Provincial Department of Finance (No. jsz2018170-12) and Wenzhou Science and Technology Project (Y20150262).

Author Contributions

Y.Q. Shen, H.N. Wang, and W.W. Yu conceived and designed the study, and wrote the manuscript. J.F. Cao, H.Y. Li, Y. Zhen, and Z. Chen acquired and analyzed the data. H.B. Lin conducted the cell tests of the N-Ac-L-Leu-PEI/CpG ODN 2006 complex. All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, agreed on the journal to which the article has been submitted, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.