Coupling Lipid Nanoparticle Structure and Automated Single‐Particle Composition Analysis to Design Phospholipase‐Responsive Nanocarriers

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small‐angle X‐ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP–phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate is an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme‐responsive LNP solutions for stimuli‐responsive delivery and diseases where PLD is dysregulated. Structured lipid nanoparticles (LNPs) are characterized using Raman, X‐ray, and neutron scattering to quantify the activity of the enzyme phospholipase D (PLD). Combining single‐particle and bulk analysis for static and dynamic systems enables a label‐free dynamic monitoring of PLD activity in LNPs. This demonstrates that particle structure drives the interaction of lipid nanocarriers with PLD.