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

Abstract

Huining Wang,1 Wenwen Yu,2,3 Hongyan Li,4 Yi Zheng,4,5 Zhen Chen,4 Hongbing Lin,4 Yuqin Shen4,5 1Department of Periodontics, Institute of Stomatology, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou 325027, Zhejiang Province, People’s Republic of China; 2Department of Orthodontics, School and Hospital of Stomatology, Jilin University, Changchun, Jilin 130021, People’s Republic of China; 3Department of Orthodontics, Tianjin Stomatological Hospital, Nankai University, Tianjin 300041, People’s Republic of China; 4Department of Periodontics, School and Hospital of Stomatology, Jilin University, Changchun, Jilin 130021,People’s Republic of China; 5Key Laboratory of Organ Regeneration & Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, Jilin 130061, People’s Republic of ChinaCorrespondence: Yuqin ShenDepartment of Periodontics, School and Hospital of Stomatology, Jilin University, 1500 Qinghua Road, Changchun 130021, People’s Republic of ChinaTel [phone]Fax [phone]Email [email protected]: 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  

Keywords

• n-acetyl-l-leucine-modified polyethyleneimine (n-ac-l-leu-pei)
• cpg oligodeoxynucleotides (cpg odn)
• proliferation
• osteoclastic differentiation