Delivery of small interfering RNA through lyophilized natural lipid nanoparticles: effects of natural lipid selection

Lipid nanoparticles (LNPs) are the primary non-viral vectors for siRNA delivery. However, synthetic lipids face issues, such as low lysosomal escape efficiency and high cost. This study aimed to use three natural lipids to construct LNPs, optimize their preparation and freeze-drying processes, and evaluate their siRNA delivery efficiency <i>in vitro</i>. <i>Coix</i> seed lipid [<i>Coix lacryma-jobi</i> L. var. <i>mayuen</i> (Roman.) Stapf (Poaceae), CSL], <i>Brucea javanica</i> seed lipid [<i>Brucea javanica</i> (L.) Merr. (Simaroubaceae), BJL], and Soybean oil [<i>Glycine max</i> (L.) Merr. (Fabaceae), SO] were used to construct LNPs. The Z-average size, zeta potential, Polymer Dispersity Index, and N/P ratio of the LNPs were characterized. Transmission electron microscope was used for morphology observation and the MTS assay for cytotoxicity. Confocal laser scanning microscope assessed cell uptake, lysosomal escape, and co-localization of lipid droplets. The efficiency of siRNA knockdown was evaluated in three cells using qPCR and Western blot. The freeze-drying processes were optimized. The optimal LNPs exhibited a size of 160–180 nm, zeta of 44–50 mV, and PDI of &lt;0.2. At 200 μg/mL, the LNPs did not affect cell viability. CSL-LNPs, BJL-LNPs, and SO-LNPs reduced KRAS^G12D mRNA levels in AsPC-1 cells by 67.87 ± 3.89, 47.18 ± 7.65, and 42.52 ± 8.90%, respectively. Freeze-dried LNPs retained their basic physical properties and the three LNPs reducing KRAS^G12D mRNA levels by 58.47 ± 4.00, 51.83 ± 4.57, and 38.00 ± 4.89%, respectively. Natural lipids are promising components for LNPs construction, offering new avenues for siRNA delivery in gene therapy.