Purpose: Reducing toxicity, immunogenicity, and costs of small interfering RNAs (siRNA) carrier materials are key goals for RNA interference (RNAi) technology transition from bench to bed. Recently, calcium ions (Ca²⁺) have garnered attention as a novel, alternative material for delivering siRNA to cells. However, the tolerance for Ca²⁺ concentration varies in different cell types, which has limited its applications in vivo. Bovine serum albumin (BSA) can bind to Ca²⁺ through chelation. Moreover, BSA is a favorable coating material for nanoparticles owing to its excellent biocompatibility. Therefore, we hypothesized that coating Ca²⁺-siRNA with BSA helps buffer Ca²⁺ toxicity in vivo.
Methods: BSA-Ca²⁺-siRNA nanoparticles were prepared, and the size, shape, encapsulation, and release efficiency were characterized using atomic force microscopy, scanning electronic microcopy, and gel electrophoresis. Binding nanoparticles were evaluated using attenuated total reflection-Fourier-transform infrared spectroscopy. The cellular uptake, intracellular release, cytotoxicity, and gene knockdown of nanoparticles were evaluated in periodontal ligament stem cells (PDLSCs) using laser-scanning confocal microscope, flow cytometry, and real-time quantitative polymerase chain reaction.
Results: BSA and Ca²⁺-siRNA could form a stable nano-scale complex (∼ 140 nm in diameter). The nanocomplexes could maintain siRNA release for more than 1 week in neutral phosphate-buffered saline (PBS) and could induce accelerated degradation in acidic PBS (pH 5.0). The nanoparticles were taken up by the cells, primarily through macropinocytosis, and were then released intracellularly through the acidification of endosomes/lysosomes. Importantly, the BSA-Ca²⁺ carrier had high transfection efficiency and biocompatibility both in vitro and in vivo. To demonstrate the therapeutic potential of our BSA coating-optimized Ca²⁺-siRNA technology, we showed that BSA-Ca²⁺-siWWP1 complexes strongly enhanced the osteogenic differentiation of inflammatory PDLSCs.
Conclusion: BSA-Ca²⁺ could potentially be used for siRNA delivery, which is not only highly efficient and cost-effective but also biocompatible to host tissues owing to the BSA coating.

Keywords: bovine serum albumin, calcium ions, osteogenic differentiation, small interfering RNA


中文翻译：

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牛血清白蛋白涂层优化的钙-siRNA纳米复合物可实现牙周炎治疗的便捷高效的siRNA递送

目的：降低小干扰 RNA (siRNA) 载体材料的毒性、免疫原性和成本是 RNA 干扰 (RNAi) 技术从实验室过渡到床的关键目标。最近，钙离子 (Ca ²⁺ ) 作为一种新的、可替代的用于将 siRNA 递送至细胞的材料而受到关注。然而，不同细胞类型对Ca ^{2+浓度的耐受性不同，这限制了其在体内的应用。}牛血清白蛋白（BSA）可以通过螯合与Ca ^2+结合。此外，由于BSA具有优异的生物相容性，它是一种有利的纳米颗粒涂层材料。因此，我们假设用 BSA包被 Ca ²⁺ -siRNA 有助于缓冲体内Ca ^2+毒性。
方法：制备BSA-Ca ²⁺ -siRNA纳米粒，采用原子力显微镜、扫描电子显微镜和凝胶电泳对其大小、形状、包封和释放效率进行表征。使用衰减全反射傅里叶变换红外光谱评估结合纳米粒子。使用激光扫描共聚焦显微镜、流式细胞术和实时定量聚合酶链反应在牙周膜干细胞 (PDLSC) 中评估纳米颗粒的细胞摄取、细胞内释放、细胞毒性和基因敲低。
结果： BSA 和 Ca ²⁺-siRNA 可以形成稳定的纳米级复合物（直径约 140 nm）。纳米复合物可以在中性磷酸盐缓冲盐水 (PBS) 中保持 siRNA 释放超过 1 周，并且可以在酸性 PBS (pH 5.0) 中诱导加速降解。纳米颗粒主要通过大胞饮作用被细胞吸收，然后通过内体/溶酶体的酸化释放到细胞内。重要的是，BSA-Ca ²⁺载体在体外和体内都具有很高的转染效率和生物相容性。为了证明我们的 BSA 涂层优化的 Ca ²⁺ -siRNA 技术的治疗潜力，我们发现 BSA-Ca ²⁺ -siWWP1 复合物强烈增强炎性 PDLSC 的成骨分化。
结论：BSA-Ca ²⁺可能用于 siRNA 递送，由于 BSA 涂层，这不仅高效且具有成本效益，而且与宿主组织具有生物相容性。

关键词：牛血清白蛋白，钙离子，成骨分化，小干扰RNA