The PEG-lipid is the least abundant component of lipid nanoparticles (LNPs) that acts as a master regulator of their functional performance. While its role in vivo is well established, its impact under in vitro conditions, commonly used for LNP screening, remains insufficiently understood. Here, we investigate how PEG-lipids with varying acyl/alkyl chain lengths modulate the physicochemical properties, surface dynamics, and functional performance of siRNA LNPs. We formulated siRNA-loaded LNPs incorporating either the "sheddable" short-chain DMG-PEG(C14) or the "persistent" long-chain DSPE-PEG(C18), or a combination of both. Our results show that colloidal properties (size <120 nm, PDI < 0.2, and neutral Zeta potential) and encapsulation efficiency (≥ 90%) remain consistent across all LNP variants. PEG-lipid composition modulates the apparent pKa and its cooperativity, as mixed PEG-lipid systems exhibit a lower pKa and a broader transition upon pH change, reflecting a more heterogeneous interfacial environment not captured by bulk physicochemical parameters. Using a combined Förster resonance energy transfer andH NMR approach, we demonstrate that LNPs undergo pronounced, medium-dependent structural and surface remodeling, driven by PEG shedding and protein interactions. Notably, mixed PEG-lipid formulations display intermediate shedding kinetics and enhanced surface adaptability. These differences translate into formulation-dependent cellular uptake and gene silencing kinetics in HCT116 cells, with LNPreaching a functional "sweet spot" between rapid onset and sustained silencing without detectable cytotoxicity. This work highlights the importance of considering PEG shedding and medium-induced remodeling during early-stage LNP screening, providing a framework for the rational design of siRNA delivery systems with tunable functional kinetics. 2000 2000DMG/DSPE1
