Molecular Tuning of a Vitamin E-Scaffold pH-Sensitive and Reductive Cleavable Lipid-like Material for Accelerated in Vivo Hepatic siRNA Delivery

A lipid nanoparticle (LNP) composed of a series of SS-cleavable and pH-activated lipid-like materials (ssPalm) was previously developed as a platform of a gene delivery system. A tertiary amine and disulfide bonding were employed to destabilize the endosomal membrane and for intracellular collapse. We report herein on the development of a hepatocyte-targeting siRNA carrier by the molecular tuning of the hydrophobic scaffold, and tertiary amine structures. The gene knockdown activity against a hepatocyte-specific marker (factor VII: FVII) was improved when a more fat-soluble vitamin (vitamin E) was employed as a hydrophobic scaffold. Moreover, to allow the tertiary amines to accept protons by sensing a slight change in endosomal acidification, its structural flexibility was minimized by fixing it in a piperidine structure, and the distance between the surface of the particle to the ternary amine was increased. As a result, the pK_a value was increased to the approximately 6.18 depending on its distance, while the pK_a reached plateau when the tertiary amine was linked by an excess number of linear carbon chains. The pH-dependent membrane destabilization activity, as assessed by a hemolysis assay, was increased in parallel with the pK_a value. Moreover, the gene knockdown activity was improved in parallel with hemolytic activity. Finally, further optimization of the lipid/siRNA ratio, and the use of chemically (2′-fluoro) modified siRNA synergistically improved the gene knockdown efficacy to an effective dose (ED₅₀) of 0.035 mg/kg. The developed ssPalm represents a promising platform for use as a hepatocyte-targeting siRNA carrier.