Mechanism of the Cholesterol-dependent Anchoring and Conformation of LPP-scFv on the PEGylated Liposome Surface.

Cholesterol is a key regulator of lipid nanocarrier for membrane stability and surface functionalization efficiency. Given widely application of liposomes in delivering peptide/antibody and demand of liposome upgrading, this study elucidates mechanism of cholesterol concentration on the anchoring and conformation of an LPP-scFv system on PEGylated liposome surfaces, relying on the coarse-grained molecular dynamics simulations and experiment assays. Our results demonstrate that cholesterol modulates the insertion depth and stability of the LPP anchor by altering membrane rigidity and the conformation of surface PEG chains. At an optimal concentration of Cholesterol (DOPC/DOPE-PEG/Chol = 100:7:72) among the cholesterol concentrations tested, the system adopts a three-tier optimized conformation. The LPP achieves the most stable membrane anchoring, while its hydrophilic backbone fully extends. This phenomenon ultimately promotes the scFv domain to adopt a near-perpendicular orientation relative to the membrane, increasing its solvent-accessible surface area and minimizing nonspecific contacts with the PEG layer. In vitro experiments confirm that both the membrane insertion efficiency of LPP and the physical stability of liposomes are highest under Chol = 72 conditions. This work clarifies the molecular mechanism by which cholesterol modulates the functional conformation of surface-displayed proteins on the controllable liposomal surface, providing clues for the rational design of high-performance targeted immunoliposomes.