Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing
Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing
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DOI：
10.3791/64810
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发表时间：
2023-01-01
影响因子：
1.2
通讯作者：
Mitchell, Michael J.
中科院分区：
文献类型：
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作者：
El-Mayta, Rakan;Padilla, Marshall S.;Mitchell, Michael J.
近年来，随着Moderna和辉瑞/BioNTech公司成功开发出COVID-19 mRNA疫苗，脂质纳米颗粒（LNPs）引起了广泛关注。这些疫苗已经证明了mRNA-LNP疗法的有效性，并为未来的临床应用打开了大门。在mRNA- lnp系统中，lnp作为递送平台保护mRNA货物不被核酸酶降解并介导其细胞内递送。LNPs通常由四种成分组成：可电离脂质、磷脂、胆固醇和脂锚定聚乙二醇（PEG）偶联物（脂质-PEG）。在这里，包裹编码萤火虫荧光素酶的mRNA的LNPs是通过含有LNP脂质成分的有机相和含有mRNA的水相的微流体混合而成的。然后在体外测试这些mRNA-LNPs，使用基于生物发光板的实验来评估它们在HepG2细胞中的转染效率。此外，通过侧尾静脉静脉注射C57BL/6小鼠，在体内评估mRNA-LNPs。全身生物发光成像是通过使用体内成像系统进行的。结果表明mRNA-LNP的特性、转染HepG2细胞的效率以及C57BL/6小鼠的总发光通量具有代表性。
Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.