Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, Singapore, Singapore.
Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Essen, Germany.
Front Endocrinol (Lausanne). 2023 Feb 22;14:1101758. doi: 10.3389/fendo.2023.1101758. eCollection 2023.
Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity . For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.
磷酸钙（CaP）是骨骼、牙齿和肌腱等硬组织的无机部分，具有很高的生物相容性和良好的生物降解性。因此，经过 DNA 编码的骨合成因子功能化的 CaP 纳米颗粒是未来治疗开发的有前途的载体系统。在这里，我们在一个基因修饰的斑马鱼模型中分析了 CaP 纳米颗粒，在该模型中，通过核因子κB 配体（Rankl）的转基因表达可以诱导骨质疏松样病变。使用 Rankl 转基因斑马鱼来可视化和理解微注射功能化 CaP 纳米颗粒在调节破骨细胞活性期间的作用。为此，我们通过快速沉淀乳酸钙和磷酸氢二铵来合成多壳 CaP 纳米颗粒，然后添加编码破骨细胞生成抑制因子骨保护素-b（Opgb）的质粒 DNA。添加了一层聚（亚乙基亚胺）以增强细胞摄取。通过动态光散射、扫描电子显微镜和能量色散 X 射线光谱证实了合成纳米颗粒的完整性。将荧光标记的 CaP 纳米颗粒微注射到表达各种成骨细胞类型荧光报告基因的 Rankl 转基因斑马鱼胚胎的心脏、躯干肌肉或尾鳍中。共聚焦时程成像显示纳米颗粒在注射组织中均匀分布，并表明纳米颗粒被随后分化为骨吸收破骨细胞的巨噬细胞有效摄取。在 Rankl 诱导后，用 Opg 功能化纳米颗粒注射的鱼显示矿化基质的降解延迟或缺失，即骨质疏松样表型的发生率较低。这证明了 CaP 纳米颗粒可以用作载体，将调节化合物有效递送到破骨细胞并阻断其活性。
