Abstract
This study investigates the influence of post-processing techniques on lipid nanoparticles (LNPs) designed for miRNA delivery in in vitro transfection models. We compared blank and miRNA-loaded LNPs (LNP-miRNA) in terms of size, polydispersity index, zeta potential, electrophoretic mobility, and conductivity. miRNA encapsulation increases lipid particle size by 43.6%, due to structural rearrangements. Post-processing methods, including sonication, filtration, dialysis, and thermal treatment, significantly alter particle characteristics. Sonication and filtration decrease particle size and improve uniformity, enhancing colloidal stability. Dialysis further refines the particle size but decreases its electrophoretic mobility. Non-dialyzed, sonicated, and filtered LNP-miRNA samples demonstrate the most favorable electrokinetic profile, maintaining low conductivity (0.003 mS/cm) and high electrophoretic mobility (3.16 ± 0.22 µm cm/V·s), suggesting optimal stability for gene delivery. Zeta potential measurements show that sonication and filtration increase the surface charge of LNP-miRNA formulations from +18.9 to +29.3 mV, enhancing colloidal stability, while dialysis reduces it to +1.9 mV. Although sonicated and filtered LNP-miRNA samples exhibited more favorable physicochemical properties, the dialyzed formulations modulate intracellular trafficking, resulting in earlier intracellular availability and prolonged persistence of delivered miRNA. This work establishes a framework for optimizing non-viral miRNA delivery by showing how post-processing shapes LNP stability and transfection performance.