Biologics Drug Product Development & Manufacturing, Sanofi, Framingham, Massachusetts;
Biologics Drug Product Development & Manufacturing, Sanofi, Framingham, Massachusetts.
J Vis Exp. 2021 Feb 25(168). doi: 10.3791/62226.
Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.
基于脂质的药物载体由于其体积小、生物相容性好和包封效率高,已被用于临床和商业上可获得的递药系统。使用脂质纳米颗粒 (LNP) 来包裹核酸有利于保护 RNA 或 DNA 免受降解,同时也促进细胞摄取。LNP 通常包含多种脂质成分,包括可离子化脂质、辅助脂质、胆固醇和聚乙二醇 (PEG) 连接脂质。由于存在可离子化脂质,LNP 可以很容易地包裹核酸,在低 pH 值下,该脂质带正电荷,允许与带负电荷的 RNA 或 DNA 形成复合物。在这里,使用精确的微流控混合平台,通过将脂质成分在有机相中和核酸成分在水相中的快速混合来形成 LNP,允许纳米颗粒自组装,同时保持层流。使用动态光散射 (DLS) 测量水动力粒径和多分散性。通过测量 Zeta 电位来确定 LNP 的有效表面电荷。使用荧光染料定量包裹的核酸来表征包封效率。代表性结果表明了该方法的重现性,以及不同配方和工艺参数对所开发的 LNP 的影响。