Synthesis, characterization, and evaluation of ionizable lysine-based lipids for siRNA delivery

Abstract

We report the synthesis and characterization of a series of ionizable lysine-based lipids (ILL), novel lipids containing a lysine headgroup linked to a long-chain dialkylamine through an amide linkage at the lysine α-amine. These ILLs contain two ionizable amines and a carboxylate, and exhibit pH-dependent lipid ionization that varies with lipid structure. The synthetic scheme employed allows for the simple, orthogonal manipulation of lipids. This provides a method for the development of a compositionally diverse library with varying ionizable headgroups, tail structures, and linker regions. A focused library of four ILLs was synthesized to determine the impact of hydrophobic fluidity, lipid net charge, and lipid pK(a) on the biophysical and siRNA transfection characteristics of this new class of lipids. We found that manipulation of lipid structure impacts the protonation behavior, electrostatically driven membrane disruption, and ability to promote siRNA mediated knockdown in vitro. ILL-siRNA liposomal formulations were tested in a murine Factor VII model; however, no significant siRNA-mediated knockdown was observed. These results indicate that ILL may be useful in vitro transfection reagents, but further optimization of this new class of lipids is required to develop an effective in vivo siRNA delivery system.

Figure 1

Ionizable lysine-based lipid (ILL) structures

Figure 2

Theoretical pH-dependent ionization of ILL pH

Figure 3

pH-dependent ionization of ILL liposomes (40:40:15:5 ILL:Chol:POPC:PEG-DMG)

Figure 4

Lysis of biomembrane mimicking vesicles (45:20:20:15 DOPC:DOPE:DOPG:Chol) by ILL containing liposomes (60:40 POPC:ILL)

Figure 5

siRNA mediate knockdown of Renilla luciferase in stably transfected HeLa-Luc cells. ILL lipoplexes (40:40:20 ILL:Chol:DOPE) at N:P = 10 were used for all experiments.

Figure 6

siRNA-mediated knockdown of Factor VII in vivo in a murine model. ILL-siRNA formulations were administered at 5 mg/kg via tail vein injection (15:1 w:w ILL:siRNA), and Factor VII levels were assayed 48 hours following administration. ILL liposomal formulation – 40:40:10:10 ILL:Chol:DSPC:PEG-DMG