Enhanced stability of siRNA-loaded lipid nanoparticles during lyophilization via acetyl-arginine and disaccharides

RNA-loaded lipid nanoparticles (LNPs) are highly sensitive to temperature fluctuations, which present significant challenges for storage and transportation. Lyophilization is a potential strategy to enhance storage stability; however, the freeze-drying process can subject nanoparticles to stresses that may alter their physicochemical properties and compromise RNA integrity. This study evaluated the effects of formulation composition on the stability and functionality of small interfering RNA (siRNA)-loaded LNPs after lyophilization. Among the formulations tested, a combination of 10% ( w / v ) trehalose and 50 mM N-acetyl-L-arginine (NALA) most effectively preserved particle size and encapsulation efficiency following freeze-drying. The NALA-based formulation also exhibited improved storage stability across 4 °C, 25 °C, and 40 °C compared to the corresponding arginine-containing formulation. Cryo-TEM analysis revealed that NALA-containing LNPs retained a spherical morphology with a homogeneous internal core after lyophilization, indicating improved structural preservation during freeze-drying. Additionally, reconstituted siRNA-LNPs containing NALA maintained cellular function comparable to pre-lyophilization samples. These findings demonstrate that a stabilizer system based on trehalose and NALA can enhance the stability of lyophilized siRNA-LNPs while preserving their physicochemical properties and biological activity.