RNA post hoc loading into empty lipid nanoparticles occurs on millisecond timescales during turbulent mixing

Lipid nanoparticles (LNPs) are conventionally produced through mixing of lipids dissolved in ethanol against a buffer containing RNA. An alternative strategy offering improved cold-chain stability involves formulating empty LNPs (eLNPs), removing ethanol, and post hoc loading (PHL) RNA into the aqueous eLNPs. The kinetics of this approach remain unknown. Here, we employ a flowthrough small-angle X-ray scattering (SAXS) setup based on a confined impinging jets (CIJ) mixer to probe PHL kinetics. We show that RNA PHL in a scalable CIJ mixer is efficient and reproducible, and that SAXS data confirms that this process concludes within ∼12 ms under favorable conditions in rapid turbulent micromixing, suggesting a diffusion-limited aggregation mechanism. Favorable conditions were identified as an acidic pH 5.5 buffer combined with turbulent CIJ mixing. In contrast, PHL performed with a neutral pH 7.4 buffer using a CIJ mixer or under laminar flow with a pH 5.5 buffer resulted in inefficient PHL.