Molecular Insights into Caffeine Stacking, Partitioning, and Organization in DPPC Lipid Bilayers from Microseconds-Long Molecular Dynamics Simulations.

Caffeine (1,3,7-trimethylxanthine) is a widely consumed psychoactive drug and neurostimulant, yet its molecular organization and permeation behavior in lipid membranes are not fully understood. We employ microseconds-long, united-atom molecular dynamics simulations to investigate caffeine interactions with a solvated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) bilayer at its fluid phase. Caffeine molecules initially aggregate in the aqueous phase to form ordered stackings, which successively permeate into the membrane. The stacked assemblies gradually dissolve, reaching a stable dispersed state where caffeine molecules preferentially reside near the headgroup-acyl chain interface and orient parallel to the lipid acyl chains, consistent with previous experimental and simulation studies. Simulations initiated with caffeine in the membrane hydrophobic phase converge to the same equilibrium state, indicating a preferred localization at the interface region. Present simulation findings are further supported by free-energy calculations that demonstrate caffeine's high affinity at the headgroup-acyl chain interface. Caffeine partitioning transiently and slightly reduces bilayer thickness and increases the membrane surface area while enhancing acyl chain stiffness near the hydrophilic part of the membrane. These observed trends are reproducible over different system sizes and independent simulations. Overall, this study provides atomic-level insights into the caffeine permeation process, including its effect on the lipid bilayer structure.