Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform
Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform
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DOI：
10.3791/62226
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发表时间：
2021-02-01
影响因子：
1.2
通讯作者：
Bandekar, Amey
中科院分区：
文献类型：
--
作者：
Bailey-Hytholt, Christina M.;Ghosh, Paroma;Bandekar, Amey
基于脂质的药物载体由于其尺寸小、生物相容性和高封装效率而被用于临床和商业上可用的递送系统。使用脂质纳米颗粒 (LNP) 封装核酸有利于保护 RNA 或 DNA 免遭降解，同时还能促进细胞摄取。 LNP 通常含有多种脂质成分，包括可电离脂质、辅助脂质、胆固醇和聚乙二醇 (PEG) 缀合脂质。由于存在可电离的脂质，LNP 可以很容易地封装核酸，在低 pH 下呈阳离子性，并允许与带负电荷的 RNA 或 DNA 络合。 LNP 是通过快速混合有机相中的脂质成分和水相中的核酸成分来封装信使 RNA (mRNA) 或质粒 DNA (pDNA) 形成的。这种混合是使用精确的微流体混合平台进行的，允许纳米颗粒自组装，同时保持层流。使用动态光散射 (DLS) 测量流体动力学尺寸和多分散性。 LNP 上的有效表面电荷通过测量 zeta 电位来确定。使用荧光染料来量化包埋的核酸来表征封装效率。代表性结果证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。
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.