(4ck) Binary Combinations of Lipid-Like Materials Act Synergistically to Improve siRNA Delivery in Vitro and In Vivo

RNA interference therapeutics represent a new class of gene-suppressing drugs which have marked potential to cure a wide variety of human diseases, including viral infections, heart disease, and cancer. The primary obstacle to the clinical application of siRNA is the development of safe and effective delivery systems. Previously, lipid-like materials, termed ?lipidoids', have been synthesized in our lab, and several leading candidates have been employed for siRNA delivery applications. The vast majority of the lipidoids studied, however, were not capable of inducing gene silencing in vitro. In order to overcome the inherent limitations of single lipidoid delivery materials and to increase the material space available for therapeutic development, binary combinations of lipidoids have been assessed for improved transfection performance both in vitro and in vivo.

Thirty-six lipidoids were synthesized through the Michael addition of amines with acrylates or acrylamides, which were then combined to form 630 binary pairs. These combinations were each tested at six weight fractions each, resulting in 3,780 test formulations. The binary lipidoid formulations were screened for their ability to safely deliver siRNA into a modified HeLa cell line, and this high-throughput analysis resulted in the identification of a number of synergistic lipidoid combinations which demonstrated non-toxic, specific knockdown of firefly luciferase. Although the individual lipidoid components had no transfection ability on their own, some materials were able to achieve 90% knockdown in combination. In vivo testing was performed on the leading hits from the in vitro screen. Anti-Factor VII lipidoid-siRNA nanoparticles were injected in C57BL/6 mice, and Factor VII levels were measured in the blood serum 48 hours post-injection to quantify knockdown. Again, although the individual lipidoids were incapable of silencing the target gene on their own, several lipidoid combinations (dosed at 2 mg/kg of siRNA) produced dramatic synergistic effect in the form of near-complete Factor VII knockdown. Toxicity was preliminarily assessed by monitoring changes in mouse bodyweight, and no toxicity was detected for synergistic lipidoid combinations compared to the negative control.

Although the reason for such synergistic behavior is unclear, it is speculated that the phenomenon may involve the two-step siRNA delivery process. While certain lipidoids may be able to facilitate transport of siRNA across the cellular membrane, they may lack the ability to escape from the endosome once inside the cell. Similarly, other lipidoids may be skilled at endosomal escape, but are not able to gain cellular entry. It is speculated that the in vitro and in vivo screening analysis has enabled the identification of binary lipidoid combinations that are adept at different steps of the transfection process.