Structural heterogeneity in mRNA-LNP subpopulations revealed by AF4-SAXS: implications for cargo loading and cell transfection.

Lipid nanoparticles are the leading platform for the delivery of nucleic acid therapeutics, yet their structural complexity remains a significant barrier to achieve rational design and predictable function. Part of this complexity arises from the non-equilibrium assemblies that are difficult to identify using ensemble average techniques given the substantial heterogeneity in all properties. Aiming to overcome the limitations of traditional characterization methods, we combined asymmetric flow field-flow fractionation with in-line small-angle X-ray scattering and spectroscopic analyses, nanoflow cytometry, and cryo-EM to construct detailed structural models of mRNA-loaded nanoparticles formulated with different amounts of mRNA loading (N/P ratios of 3 and 6). This combination of techniques revealed that microfluidic formulation produces structurally diverse nanoparticle subpopulations differing in size, anisotropy, and cargo loading. Notably, these variations extend to the particle internal organization: spheroidal geometries display densely loaded mRNA cores, whereas bleb-like morphologies exhibit reduced mRNA content relative to the lipid amount within segregated domains at the core. NanoFCM further shows that the N/P ratio modulates cargo distribution across individual nanoparticles, with N/P=6 yielding a more uniform mRNA copy number per particle across subpopulations than N/P=3. These differences resulted in higher transfection efficacies for the N/P=6 formulation, highlighting core organization and loading homogeneity as key parameters for efficacious delivery. Together, these results establish a direct link between LNP architecture, internal organization, cargo distribution, and transfection efficiency, underscoring the importance of accounting for heterogeneity in the rational design of nucleic acid delivery systems.