Cholesterol Condensation and Phospholipid Convergence in cis-ssPalmO-phe Bilayers: A Molecular Dynamics Study

Lipid nanoparticles (LNPs) deliver nucleic acids, yet how the ionizable lipid, phospholipid, and cholesterol organise the membrane at the molecular level remains unclear. Here, we performed atomistic molecular dynamics simulations of flat bilayers of the ionizable lipid cis-ssPalmO-phe (50 mol%) with one of four phospholipids (DSPC, DSPE, DOPC, DOPE) and cholesterol at 40 mol% phospholipid and 40 mol% cholesterol, with the binding free energy of cholesterol calculated using umbrella sampling. In the cholesterol-rich systems, all four bilayers were thicker (from 4.37–5.12 to 5.14–5.80 nm) and more tightly packed (from 2.02–2.27 to 2.60–2.86 lipids nm−2) as compared to the phospholipid-rich system. Also, in cholesterol-rich systems, lateral diffusion was 30 to 40% lower; cholesterol stood more upright. Cholesterol tilt and acyl-chain order became more alike across the four phospholipids, and the spread in the binding free energy of cholesterol narrowed (about 9 to 5 kcal mol−1) while its mean rose only modestly (about 13 to 16 kcal mol−1). Total cholesterol–cis-ssPalmO-phe (IL)interaction energies and hydrogen-bond counts rose roughly three-to seven-fold, in step with cholesterol number. Because the ionizable lipid (IL) fraction was fixed, more cholesterol means less phospholipid, so these effects reflect composition and cannot be assigned to cholesterol alone. Head-group chemistry set the sign of the electrostatic potential, whereas diffusion fell in every system regardless of acyl-chain saturation. These results give a molecular