Lipid nanoparticles are traditionally composed of nucleic acids, cholesterol, and three types of lipids. Among these, PEG-lipid is included at the lowest molar proportion, although it plays a critical role in determining LNP behavior. In this study, we investigated how variations in the acyl chain length of PEG-lipids (C14, C16, C18) and their molar ratios (1%, 2.5%. 5%) affect physicochemical characteristics and in vitro efficacy of LNPs encapsulating pDNA or mRNA. LNPs were prepared using microfluidic mixing and characterized by size, polydispersity, zeta potential, encapsulation efficiency, and apparent pKa. Transfection efficiency and cytotoxicity were evaluated in four cell lines: HepG2, L929, JAWSII and RAW264.7. The size of pDNA-LNPs ranged from 89 to 217 nm, whereas mRNA-LNPs ranged from 90 to 150 nm, both exhibiting near-neutral zeta potentials. Notably, increasing PEG-lipid molar ratio led to a reduction in LNP size. In vitro studies showed that LNPs with lower PEG-lipid molar ratios (1-2.5%) achieved enhanced cellular uptake and transfection efficiency. Specifically, for pDNA-LNPs, C14 PEG-lipid formulations were more effective in L929 and JAWSII cells, while C16 PEG-lipid formulations improved transfection efficiency in HepG2 and RAW264.7 cells, all with negligible cytotoxicity. For mRNA-LNPs, the highest transfection efficiency was observed using C14 PEG-lipid formulations. Our results highlight the critical role of PEG-lipids in modulating both the physicochemical properties and in vitro performance of LNPs. Fine-tuning PEG-lipid composition represents a key parameter in optimizing LNPs for gene delivery applications.
