Small angle X-ray scattering reveals polyethylene glycol corona architecture in liposomes: influence of polyethylene glycol chain length, surface density and coating strategy

PEGylated liposomes are well-known for their properties as long-circulating nanocarriers in drug delivery systems, where the structure and organization of the polyethylene glycol (PEG) coating critically influence their stability, circulation lifetime, and biological interactions. Given the crucial role of PEG coating attributes in these structured nanoparticles, it becomes essential to explore advanced technologies capable of specifically addressing the surface properties of liposomes. A detailed surface characterization, combined with nanoparticle composition analysis, enables deep insights into critical quality attributes of nanomedicines, which are essential for ensuring both quality and safety. In this study, small-angle X-ray scattering (SAXS) was employed to examine the structural organization of PEGylated liposomes, alongside their composition analysis using High-Performance Liquid Chromatography coupled to a Charged Aerosol Detector (HPLC-CAD). The liposomes were formulated through commonly used formulation strategies, such as pre- and post-insertion of various molecular mass, PEG2000 (PEG2K) and PEG5000 (PEG5K)-modified lipids. SAXS profiles revealed systematic variations in scattering features with increasing PEG content, indicating progressive changes in corona density and interfacial organization. In addition, core-multishell modeling confirmed that the liposomes prepared using both strategies were unilamellar with stable bilayers. Pre-insertion PEG2K liposomes showed pronounced reorganization of the outer interface, with higher PEG levels producing thicker, denser coronas consistent with a mushroom-like configuration. In post-insertion formulations, both PEG2K and PEG5K systems were well described by four-shell models. PEG2K post-insertion liposomes displayed thinner but denser coronas, whereas PEG5K yielded thicker, more hydrated layers that compacted with increasing PEG concentration. Despite these interfacial variations, the bilayer core remained largely unaffected, though pre-insertion induced marked PEG-lipid leaflet asymmetry. Overall, these findings demonstrate that PEG chain length, surface density, and incorporation strategy each play a critical role in shaping the structure and density of the PEG corona. By quantifying these factors, this work provides detailed insight into PEG organization at the liposome interface and supports the rational design and quality control of sterically stabilized liposomes. • SAXS reveals PEGylation stategy dependent membrane asymmetry in liposomes. • SAXS resolves 5–10% variations in PEG layer density on PEGylated liposomes. • Core-multishell modeling confirms unilamellar structure with stable bilayers. • Post-insertion PEG2K/PEG5K generate distinct interfacial architectures. • SAXS is able to reveal PEG outer shell presence at low PEG-lipid contents.