Methods Four membrane models were constructed to reflect experimental formulations: pure DSPC, binary DSPC/cholesterol (1:3), and ternary systems with DSPC/cholesterol/ePEG at low (10:30:2) and high (10:30:10) PEG content. Simulations were performed using the Desmond engine with explicit solvent conditions across temperatures from 25 °C to -50 °C following a stepwise cooling protocol. Analysis included density profiles, area per lipid calculations, membrane thickness measurements, acyl chain tilt angles, deuterium order parameters, and leaflet interdigitation parameters. Anisotropic pressure coupling was implemented to allow independent fluctuations in membrane plane and normal dimensions.
Results Density profile analysis revealed that cholesterol incorporation expanded DSPC molecular footprint from 50.08 Å2 to 56.83 Å2 at 25 °C, creating additional space that further increased with PEG incorporation. As temperature decreased to -50°C, all systems exhibited modest compression in molecular areas while maintaining their fundamental organization. Despite dramatically reduced tilt angles from ~36° in pure DSPC to ~14° in cholesterol containing systems, membrane thickness remained constant (~46 Å) across all temperatures due to enhanced interdigitation between leaflets. Deuterium order parameters revealed contrasting temperature responses: pure DSPC showed reduced ordering at lower temperatures, while cholesterol containing membranes demonstrated enhanced acyl chain ordering upon cooling, especially in middle chain segments, with peak SCD values increasing from 0.442 at 25°C to 0.458 at -50 °C. This created distinct clustering of order parameter profiles, with subzero temperatures showing systematically higher ordering.
Conclusion The computational findings provide atomic level explanation for experimentally observed phase behavior across temperature ranges relevant to LNP storage. Cholesterol serves a critical dual role: it creates interfacial spaces in DSPC bilayers that accommodate ePEG molecules while simultaneously enhancing membrane structural integrity through temperature variations. The intensification of cholesterol's ordering effect at subzero temperatures, evidenced by increased deuterium order parameters and more sharply defined density patterns, shows its crucial role in preserving membrane organization during cold storage. The all atom molecular dynamics simulations combined with the experimental findings provide structural and mechanistic understanding of LNP membrane temperature dependent behavior, offering valuable guidance for rational design of stable therapeutic formulations.