Department of Chemical and Biological Engineering, Princeton University.
Department of Chemical and Biological Engineering, Princeton University;
J Vis Exp. 2024 Aug 23(210). doi: 10.3791/67047.
Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.
脂质纳米颗粒 (LNPs) 已被证明具有作为治疗性递药载体的巨大潜力，这一点已被两种 COVID-19 信使 RNA (mRNA) 疫苗的批准和全球使用所证明。在小规模上，LNPs 通常使用微流控技术制造；然而，这些设备的局限性排除了它们在大规模上的使用。COVID-19 疫苗是使用受限冲击射流 (CIJ) 湍流混合器大量生产的。CIJ 技术可在实验室规模上进行生产，同时有信心可以将其扩展到生产规模。CIJ 混合的关键概念是，混合长度和时间尺度由混合腔中的湍流强度决定，并且纳米颗粒的形成发生在远离壁面的位置，从而消除了在表面上沉积和结垢的问题。这项工作展示了使用受限冲击射流混合器技术制造 LNPs 的过程，该技术使用两种几何形状：双射流 CIJ 和四射流多入口涡流混合器 (MIVM)。讨论了每种混合几何形状的优缺点。在这些几何形状中，LNPs 通过有机溶剂流（通常是含有可离子化脂质、共脂质和稳定的 PEG-脂质的乙醇）与水反溶剂流（含有 RNA 或 DNA 的水性缓冲液）的快速混合而形成。为了制备具有可控尺寸、ζ 电位、稳定性和转染效率的重现性 LNPs，提出了 CIJ 和 MIVM 混合器的操作参数。还介绍了与 CIJ 混合相比，混合不良（移液解决方案）制备的 LNPs 的差异。
