Quantifying the Activation Barrier for Phospholipid Monolayer Fusion Governing Lipid Droplet Coalescence

Lipid droplet (LD) coalescence is a critical cellular process that reshapes lipid storage, drives metabolic disease progression, and dictates the stability of LD-mimetic drug carriers. However, the rate-limiting step-fusion of the phospholipid monolayers surrounding neutral-lipid cores-remains poorly quantified compared to bilayer fusion. Here, we quantitatively determine the activation barrier for LD coalescence by tracking the kinetics in protein-free adiposome models. Using a multi-technique approach combining time-resolved dynamic light scattering and small-angle X-ray scattering, we reveal that monolayer fusion is the kinetic bottleneck. We demonstrate that lipid composition is a powerful regulator of this barrier: cone-shaped lipids (e.g., dioleoylphosphatidylethanolamine) lower the barrier and promote fusion, while phosphatidylcholine-rich monolayers enhance stability. A continuum fusion model, adapted for curved monolayers, explains these results through changes in spontaneous curvature, hydration repulsion, and stalk energetics. Our findings establish composition-dependent design rules for controlling LD dynamics in metabolic health and for engineering stable or triggerable lipid-based delivery vehicles.