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

Aljabbari, A.; Binion, H.; Dasaro, S.; Mitra, H.; Bethiana, T.; Harris, G.; Zhou, X.; Baghbanbashi, M.; Barrio-Zhang, A.; Perez Herrera, D.; Figueiredo, M.; Wilson, B.; Ardekani, A. M.; Ristroph, K.

doi:10.64898/2026.06.26.732967

bioRxiv · 10.64898/2026.06.26.732967

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

Aljabbari, A.·Binion, H.·Dasaro, S.·Mitra, H.·Bethiana, T.·Harris, G.·Zhou, X.·Baghbanbashi, M.·Barrio-Zhang, A.·Perez Herrera, D.·Figueiredo, M.·Wilson, B.·Ardekani, A. M.·Ristroph, K.

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Abstract

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.

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Aljabbari, A., Binion, H., Dasaro, S., Mitra, H., Bethiana, T., Harris, G., Zhou, X., Baghbanbashi, M., Barrio-Zhang, A., Perez Herrera, D., Figueiredo, M., Wilson, B., Ardekani, A. M., Ristroph, K.. 2026-06-27. RNA post hoc loading into empty lipid nanoparticles occurs on millisecond timescales during turbulent mixing. https://doi.org/10.64898/2026.06.26.732967

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