Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles.

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.