Direct structural investigation of pH responsiveness in mRNA lipid nanoparticles: Refining paradigms

Using small angle X-ray scattering measurements, structural coherencies in lipid nanoparticle (LNP) formulations comprising messenger RNA (mRNA) were elucidated in response to pH changes and as a function of RNA to lipid ratio. Formulations were assembled using selected ionizable lipids with well-known activity from previous experiments, with otherwise identical composition. Several structural parameters were determined, including internal organization, fraction of ordered material, the underlying repeating distance, and the fractal dimension of the overall particles. Repeat distances increased with increasing pH, with profiles similar to the shape of pK a curves, therefore allowing to directly reveal the structural implication of the pH responsiveness inside the particles. The fractal dimension, which so far had not been in the center of attention for quality control, was correlated with biological activity. Such systematic data on structure-function coherencies in LNPs can serve as complementary methods to standard lab-based quality control measures. This can help to facilitate and accelerate the rational formulation development of new RNA therapeutics. • pH responsiveness of mRNA lipid nanoparticle structure was investigated. • The structural analogue to the pK a value of LNPs was determined by SAXS. • By changing lipid composition, the pK a value could be accurately tailored. • Structural attributes that are indicative for LNP activity were identified.