PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development.

聚乙二醇化脂质纳米颗粒 (LNP) 通常用于递送生物活性分子，但聚乙二醇化在细胞和亚细胞水平上负载 DNA 的 LNP 相互作用中的作用仍知之甚少。在这项研究中，我们利用基因报告技术、动态光散射 (DLS)、同步加速器小角度 X 射线散射 (SAXS) 和荧光共焦显微镜 (FCS) 研究了 DNA 负载的聚乙二醇化 LNP 的作用机制。我们发现 PEG 对 DNA LNP 的尺寸或纳米结构没有显着影响，但会降低其 zeta 电位以及与阴离子细胞膜的相互作用。聚乙二醇化可提高 LNP 的结构稳定性并降低 DNA 卸载量。FCS 实验表明，聚乙二醇化的 LNP 在细胞内完整内化，并大部分穿梭至溶酶体，而未聚乙二醇化的 LNP 在质膜上经历了巨大的不稳定。这些发现可以为用于基因传递和疫苗开发的 DNA 负载 LNP 的设计、优化和验证提供信息。