Mechanistic insights into how mixing factors govern polyelectrolyte-surfactant complexation in RNA lipid nanoparticle formulation.

ABSTRACT

HYPOTHESIS: Lipid nanoparticle self-assembly is a complex process that relies on ion pairing between nucleic acids and hydrophobic cationic lipid counterions for encapsulation. The chemical factors influencing this process, such as formulation composition, have been the focus of recent research. However, the physical factors, particularly the mixing protocol, which directly modulates these chemical factors, have yet to be mechanistically examined using a reproducible mixing platform comparable to the industry standard. We here utilize Flash NanoPrecipitation (FNP), a scalable rapid mixing platform, to isolate and systematically investigate how mixing factors influence this complexation step, first by using a model polyelectrolyte-surfactant system and then generalizing to a typical RNA lipid nanoparticle formulation. EXPERIMENTS Aqueous polystyrene sulfonate (PSS) and cetrimonium bromide (CTAB) solutions are rapidly homogenized using reproducible FNP mixing and controlled flow rates at different stoichiometric ratios and total solids concentrations to form polyelectrolyte-surfactant complexes (PESCs). Then, key mixing factors such as total flow rate, inlet stream relative volumetric flow rate, and magnitude of flow fluctuation are studied using both this PESC system and an RNA lipid nanoparticle formulation. FINDINGS: Fluctuations in flow as low as ± 5 % of the total flow rate are found to severely compromise PESC formation. This result is replicated in the RNA lipid nanoparticle system, which exhibited significant differences in size (132.7 nm vs. 75.6 nm) and RNA encapsulation efficiency (34.0 % vs. 82.8 %) under fluctuating vs. steady flow. We explain these results in light of the chemical variables isolated and studied; slow or nonuniform mixing generates localized concentration gradients that disrupt the balance between the hydrophobic and electrostatic forces that drive complex formation. These experiments contribute to our understanding of the complexation stage of lipid nanoparticle formation and provide practical insights into the importance of developing controlled mixing protocols in industry.