Biionix (Bionic Materials, Implants & Interfaces) Cluster, Department of Medicine, University of Central Florida College of Medicine, Orlando, FL 32827, USA.
Biionix (Bionic Materials, Implants & Interfaces) Cluster, Department of Medicine, University of Central Florida College of Medicine, Orlando, FL 32827, USA; Department of Material Sciences and Engineering, University of Central Florida, Orlando, FL 32816, USA; Biomedical Engineering Program, Department of Mechanical and Aerospace Engineering, University of Central Florida, Orlando, FL 32816, USA.
Biomater Adv. 2025 Jan;166:214078. doi: 10.1016/j.bioadv.2024.214078. Epub 2024 Oct 19.
This project aimed to study the efficacy of a bone-targeted ultrasound-responsive nanobubble (NB) platform to deliver gene-silencing cathepsin K (CTSK) siRNA into the bone for osteoporosis treatment using in vitro and in vivo studies. To this end, characterization of CTSK siRNA loaded NB functionalized with alendronate (NB-CTSK siRNA-AL) was performed using transmission electron microscopy (TEM) imaging, and a release profile was obtained through fluorescent spectroscopy. In vitro studies were conducted by culturing NB-CTSK siRNA-AL with osteoclasts to evaluate siRNA uptake, CTSK expression, and the expression of tartrate-resistant acid phosphatase (TRAP). A control group and an NB-CTSK siRNA-AL treated group of ovariectomized (OVX) mice (n = 4) were tested. The OVX group that received treatment underwent weekly sessions for 4 weeks, during which they were exposed to low-intensity pulsed ultrasound (LIPUS) stimulation following administration of NB-CTSK siRNA-AL, prior to being sacrificed. Both groups underwent a series of tests to evaluate the bone targeting, safety, and efficacy of the nanoplatform. These tests included biodistribution studies conducted at 4 h and 24 h post-injection, a 3-point bending test of the femurs, nano-computed tomography analysis, as well as Hematoxylin & Eosin histological staining, Masson's Trichrome staining, and CTSK staining. The biodistribution showed the accumulation of NB-CTSK siRNA-AL in the bone and liver. Results showed that the OVX mice treated with NB-CTSK siRNA-AL had increased distal cortical bone thickness (174.4 ± 5.28 μm vs. 144.3 ± 10.66 μm, p > 0.05)) and bone volume fraction (16.5 ± 3.96 % vs. 6.55 ± 0.13 % (p > 0.05)). A reduced collagen degradation and downregulated CTSK expression were evident in the staining procedures. No adverse effects were recorded within histological assessments on the liver, kidney, and heart post-treatment. Morphology was shown to be normal and healthy within muscle cells post-LIPUS stimulation of NB-CTSK siRNA-AL. From these results, it can be concluded that an ultrasound-mediated NB-CTSK siRNA-AL can serve as a reliable, safe CTSK siRNA carrier to bone-specific targets for in vivo osteoporosis treatment.
本项目旨在研究一种骨靶向超声响应纳米泡（NB）平台，以通过体外和体内研究将基因沉默组织蛋白酶 K（CTSK）siRNA 递送至骨骼用于骨质疏松症治疗。为此，通过透射电子显微镜（TEM）成像对负载 CTSK siRNA 的 NB 进行功能化进行了表征，并用荧光光谱法获得了释放曲线。通过培养 NB-CTSK siRNA-AL 与破骨细胞进行体外研究，以评估 siRNA 摄取、CTSK 表达和抗酒石酸酸性磷酸酶（TRAP）表达。对一组去卵巢（OVX）小鼠（n=4）进行了对照组和 NB-CTSK siRNA-AL 处理组的测试。接受治疗的 OVX 组每周进行一次治疗，持续 4 周，在给予 NB-CTSK siRNA-AL 后进行低强度脉冲超声（LIPUS）刺激，然后处死。两组均进行了一系列测试，以评估纳米平台的骨靶向性、安全性和疗效。这些测试包括注射后 4 小时和 24 小时的生物分布研究、股骨三点弯曲试验、纳米计算机断层扫描分析以及苏木精和伊红组织染色、马松三色染色和 CTSK 染色。生物分布显示 NB-CTSK siRNA-AL 在骨骼和肝脏中的积累。结果表明，NB-CTSK siRNA-AL 处理的 OVX 小鼠的远端皮质骨厚度增加（174.4±5.28μm 与 144.3±10.66μm，p>0.05）和骨体积分数增加（16.5±3.96%与 6.55±0.13%（p>0.05））。染色程序显示胶原降解减少，CTSK 表达下调。治疗后肝脏、肾脏和心脏的组织学评估未记录到不良反应。肌肉细胞中经 LIPUS 刺激后的 NB-CTSK siRNA-AL 形态正常且健康。根据这些结果，可以得出结论，超声介导的 NB-CTSK siRNA-AL 可作为一种可靠、安全的 CTSK siRNA 载体，用于体内骨质疏松症治疗的骨特异性靶点。
