Changes of the lipid membrane structures caused by chain‐length‐dependent doxorubicin embedment in PEGylated liposomes

Poly(ethylene glycol)‐grafted (PEGylated) liposomes receive increasingly more attention due to their practical applications in delivering vaccines, nutrients and drug molecules such as doxorubicin (DOX). PEGylated liposomes have been well documented for their capability in carrying DOX as rod‐like crystallites enclosed inside the unilamellar vesicles. This study addresses the previously unresolved question of whether DOX intercalates into liposome bilayers by employing simultaneous small‐ and wide‐angle X‐ray scattering (SWAXS), complemented by an integrated asymmetric flow field‐flow fractionation system coupled with multi‐angle light scattering, dynamic light scattering and refractive index detection. The DOX‐loaded PEGylated liposomes used are composed of phosphatidylcholine ( N :0 PC) lipids, with different lipid chain lengths N = 18, 20 and 22, and a fixed molar ratio of lipid:cholesterol:DSPE‐PEG2000 of 45:50:5. SWAXS analysis reveals that rod‐like DOX nanocrystallites—approximately 70–95 nm in length and 14 nm in diameter—are encapsulated within the PEGylated liposomes across all three lipid types, with each exhibiting distinct membrane structural responses to DOX incorporation. Notably, 22:0 PC liposomes demonstrate significant DOX‐induced disruption of lipid chain packing, accompanied by enhanced alignment of phosphate headgroups in the outer leaflet. Consistently, cryo‐EM imaging reveals pronounced faceted membrane morphologies in DOX‐loaded 22:0 PC liposomes. This faceting phenomenon is attributed to the accumulation of DOX within the excess hydrophobic core regions created by the extended aliphatic chains beyond the cholesterol saturation limit. These DOX‐enriched domains locally stiffen the membrane, promoting the formation of rigid, faceted structures.