Histidine Decapeptide‐Incorporating Lipid Nanoparticles with Low Ionizable Lipids Proportion for Efficient Small Interfering RNA Delivery

The diversification of lipid compositions in lipid nanoparticles (LNPs) is crucial for expanding their clinical applications and overcoming current limitations. In this study, LNPs with varying lipid compositions are fabricated using three different mixing processes (pipette, vortex, and microfluidic mixing) for small interfering RNA (siRNA) delivery. While both siRNA and hydrophobic fluorescent dye are successfully incorporated within LNPs using pipette- and vortex-mixing, hydrophilic peptides cannot be encapsulated. Following optimization of ionizable lipid proportion via cost-efficient vortex-mixing method, LNPs with a lower ionizable lipid proportion (27.72%), termed LNP5, are selected and fabricated with histidine decapeptide (His10) during formulation via microfluidic mixing method to supplement the function of approximately half of the ionizable lipids by simple addition of His10. His10- incorporated LNP5 (LNP5H) exhibited a 1.6-fold increase in gene silencing efficiency, compared to conventional LNPs (cLNPs; ionizable lipid proportion of 47.95%). Furthermore, LNP5H maintained siRNA potency for 4 weeks when stored in a 1% sucrose solution at -70 °C. Taken together, it fabricates potent LNP5H with low proportion of ionizable lipids via fast and easy processes, which can be applied to a variety of siRNA therapeutics for their efficient intracellular delivery.