Pore Formation: How Cubic Lipid-siRNA Constructs Escape the Endosome*

Small interfering RNA (siRNA) silences gene expression and has shown great potential in medical applications. However, intracellular delivery of siRNA remains a great challenge. Lipid nanoparticles have been one of the most successful siRNA carriers to date, but their delivery efficiency is limited due to low endosomal release. We have shown that cubic structured lipid nanoparticles (cubosomes) loaded with siRNA - cuboplexes, show greater siRNA knockdown compared to traditional lamellar structured liposomes. In this work, we aim to elucidate the reason behind this difference in delivery efficiency. We will show confocal microscopy (CF), flow cytometry and live cell imaging data demonstrating higher cellular uptake of cubosomes compared to liposomes. CF, fluorescent based fusion assays, cryogenic transmission electron microscopy and dynamic light scattering measurements indicate that compared to liposomes, cubosomes are more prone to fuse and form aggregates with endosomes, which implies a stronger pore forming capability that leads to siRNA escape. These results support our suggestion that cubosomes can lower the free energy required to promote endosome pore formation and establish a topologically active delivery mechanism.