Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, People's Republic of China.
Department of Stomatology Medical Center, The First Affiliated Hospital of Jinan University, Guangzhou, 510630, People's Republic of China.
Int J Nanomedicine. 2020 Oct 15;15:7967-7977. doi: 10.2147/IJN.S263756. eCollection 2020.
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.
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.
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.
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.
目前用于骨质疏松症治疗的药物显示出强烈的不良反应。干细胞衍生的细胞外囊泡（EVs）为骨质疏松症治疗提供了另一种选择。小鼠间充质干细胞（mMSCs）衍生的 EV 促进骨再生；然而，由于非特异性组织靶向，其临床应用受到限制。阿仑膦酸盐通过羟磷灰石特异性靶向骨组织。因此，通过“点击化学”将 EVs 与阿仑膦酸盐结合，生成 Ale-EVs，以促进 EVs 通过阿仑膦酸盐/羟磷灰石结合靶向骨。
使用动态光散射分析对 Ale-EVs 的大小进行表征，并通过透射电子显微镜观察形态。通过流式细胞术检测 Ale-EVs 对羟磷灰石的亲和力。通过体外荧光成像测试 Ale-EVs 的骨靶向性。使用 WST-8 还原测定试剂盒评估细胞活力，以测试 Ale-EVs 促进 mMSCs 增殖的能力。碱性磷酸酶实验用于检测 Ale-EVs 体外促进小鼠间充质干细胞分化的能力。Western blotting 和 Q-PCR 检测用于检测成骨分化的早期标志物。在卵巢切除（OVX）诱导的骨质疏松症大鼠模型中检测 Ale-EVs 的抗骨质疏松作用。通过 H&E 染色和血清标志物检测评估 Ale-EVs 在体内的安全性。
体外，Ale-EVs 与羟磷灰石具有高亲和力。此外，体外数据表明，与 EVs-DiD 组相比，Ale-EVs-DiD 处理的小鼠在骨组织中诱导强烈的荧光。此外，结果表明 Ale-EVs 促进了小鼠 MSC 的生长和分化。它们还可以预防卵巢切除（OVX）诱导的骨质疏松症大鼠的骨质疏松症。Ale-EVs 耐受性良好，未发现副作用，表明 Ale-EVs 特异性靶向骨骼，可作为骨质疏松症治疗的新疗法。
我们使用无铜“点击化学”用 Ale-N3 修饰小鼠间充质干细胞衍生的细胞外囊泡，生成 Ale-EVs 系统。Ale-EVs 对骨骼具有高亲和力，具有在骨质疏松症治疗中应用的巨大潜力，且全身毒性低。
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