可电离脂质纳米制剂的制备及其用于递送骨保护素mRNA的研究

随着全球人口老龄化的加剧，如何安全有效治疗骨质疏松成了临床所面临的重大挑战之一。近年来，mRNA疫苗在预防新型冠状病毒疫情传播方面取得巨大成功，mRNA技术获得了前所未有的关注。越来越多的科研工作者开始尝试使用mRNA疗法解决困扰医学界许久的难题，或者通过mRNA疗法来替代传统的治疗手段。本研究通过合成、筛选和优化制备了一种具有高递送效率的可电离脂质纳米递送系统，并尝试递送编码人骨保护素的mRNA，以探讨mRNA疗法治疗小鼠骨质疏松症的可行性。

本研究首先通过筛选不同类型的阳离子表面活性剂，鉴定出一种具有mRNA递送活性的表面活性剂。随后，通过组合设计合成了一系列兼具表面活性剂和脂样结构特征的可电离脂质化合物，并从中筛选出含有可电离仲胺和两条不对称烷基链的表面活性剂衍生的先导脂质化合物。该脂质化合物与辅助脂质通过逐级配方优化后，能够与mRNA自组装形成稳定且分散度较小的可电离脂质纳米粒。体外活性测试结果显示该脂质纳米粒拥有较好的mRNA递送活性（可将mRNA高效递送至常规转染细胞甚至是难以转染的巨噬细胞）、稳定性和生物相容性。进一步体内实验证明，负载人骨保护素mRNA的脂质纳米粒通过尾静脉注射至小鼠体内后，可在小鼠血液中检测到人骨保护素的表达，这为后续使用mRNA疗法治疗骨质疏松症奠定了基础。

总之，本研究制备了可高效负载和递送mRNA的可电离脂质纳米粒。同时，本研究通过静脉注射可电离脂质纳米粒，可将编码人骨保护素的mRNA递送至小鼠体内，并成功检测到人骨保护素的表达。这些研究证明了mRNA疗法在骨质疏松治疗领域的应用潜力。

With the increasing aging of the global population, how to treat osteoporosis safely and effectively has become one of the major challenges facing clinical practice. In recent years, with the great success of mRNA vaccines in preventing the spread of the COVID-19 pandemic, mRNA technology has received unprecedented attention. More and more researchers are trying to use mRNA therapy to solve the problems that have plagued the medical community for a long time, or to refresh the traditional treatment through mRNA therapy. This study has prepared high-efficiency ionizable lipid nanoparticles via synthesis, screening, as well as optimization, and has attempted to use such delivery system to deliver mRNA encoding human osteoprotegerin protein. We anticipate that these studies can be used to demonstrate the feasibility of mRNA therapy to treat osteoporosis in mice.

For the first step, a surfactant with mRNA delivery activity was identified through screening of different types of cationic surfactants. Subsequently, a series of ionizable lipid-like compounds with structural features of surfactants and lipid-like compounds were synthesized via combinatorial design. A surfactant-derived lead lipid with a secondary amine head group and two asymmetric alkyl chains was screened out. Through a stepwise optimization of formulation parameters, the surfactant-derived lipid could self-assemble with helper lipids into monodisperse and stable lipid nanoparticles. The in vitro results have showed that the lipid nanoparticles excellent good mRNA delivery activity (efficient delivery of mRNA to commonly used cells and even hard-to-transfect macrophages), stability, and biocompatibility. Furthermore, in vivo studies have further demonstrated that the expression of human osteoprotegerin protein could be detected in mouse plasma following the intravenous injection of lipid nanoparticles loaded with human osteoprotegerin mRNA into mice, which laid a foundation for the subsequent use of mRNA therapy for osteoporosis.

In summary, this study has prepared ionizable lipid nanoparticles that can efficiently encapsulate and deliver mRNA. Moreover, this study has successfully detected the expression of human osteoprotegerin protein in mice following intravenous injection of human osteoprotegerin mRNA-loaded ionizable lipid nanoparticles. These studies have demonstrated the therapeutic potential of mRNA therapy in the field of osteoporosis treatment.

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