Bone-Targeted Extracellular Vesicles from Mesenchymal Stem Cells for Osteoporosis Therapy

Abstract

Yayu Wang,1,* Jie Yao,2– 4,* Lizhao Cai,2– 4,* Tong Liu,1 Xiaogang Wang,2,4 Ye Zhang,2– 4 Zhiying Zhou,2– 4 Tingwei Li,2– 4 Minyi Liu,2– 4 Renfa Lai,2– 4 Xiangning Liu2– 4 1Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, People’s Republic of China; 2Department of Stomatology Medical Center, The First Affiliated Hospital of Jinan University, Guangzhou, 510630, People’s Republic of China; 3School of Stomatology, Jinan University, Guangzhou 510632, People’s Republic of China; 4Clinical Research Platform for Interdiscipline of Stomatology, Jinan University, Guangzhou 510630, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiangning Liu; Renfa LaiThe First Affiliated Hospital of Jinan University, Guangzhou 510632, People’s Republic of ChinaTel [phone]Fax [phone]Email [email protected]; [email protected]: Current drugs used for osteoporosis therapy show strong adverse effects. Stem cell-derived extracellular vesicles (EVs) provide another choice for osteoporosis therapy. Mouse mesenchymal stem cells (mMSCs)-derived EVs promote bone regeneration; however, their clinical application is limited due to non-specific tissue targeting. Alendronate specifically targets bone tissue via hydroxyapatite. Therefore, EVs were combined with alendronate to generate Ale-EVs by “click chemistry” to facilitate EVs targeting bone via alendronate/hydroxyapatite binding.Methods: Ale-EVs were characterized based on size using dynamic light scattering analysis and morphology was visualized by transmission electron microscopy. Hydroxyapatite affinity of Ale-EVs was detected by flow cytometry. Bone targeting of Ale-EVs was tested by ex vivo fluorescent imaging. Cell viability was assessed by using WST-8 reduction assay kit for testing the ability of Ale-EVs to promote mMSCs proliferation. Alkaline phosphatase experiment was used to detect ability of Ale-EVs to promote differentiation of mouse mesenchymal stem cells in vitro. Western blotting and Q-PCR assay were used to detect the early marker of osteogenic differentiation. Antiosteoporotic effects of Ale-EVs were detected in ovariectomy (OVX)-induced osteoporosis rat model. The safety of the Ale-EVs in vivo was measured by H&E staining and serum markers assay.Results: In vitro, Ale-EVs had high affinity with hydroxyapatite. Also, ex vivo data indicated that Ale-EVs-DiD treatment of mice induced strong fluorescece in bone tissues compared with EVs-DiD group. Furthermore, results suggested that Ale-EVs promoted the growth and differentiation of mouse MSCs. They also protected against osteoporosis in ovariectomy (OVX)-induced osteoporotic rats. Ale-EVs were well tolerated and no side effects were found, indicating that Ale-EVs specifically target bone and can be used as a new therapeutic in osteoporosis treatment.Conclusion: We used the Ale-N3 to modify mouse mesenchymal stem cells-derived extracellular vesicles by copper-free “click chemistry” to generate a Ale-EVs system. The Ale-EVs had a high affinity for bone and have great potential for clinical applications in osteoporosis therapy with low systemic toxicity.Keywords: extracellular vesicles, EVs, mesenchymal stem cells, MSCs, bone-targeting, osteoporosis, click chemistry

Keywords

• extracellular vesicles (evs)
• mesenchymal stem cells (mscs)
• bone-targeting
• osteoporosis
• click chemistry