Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA

The in vivo gene silencing potencies of lipid nanoparticle (LNP)-siRNA systems containing the ionizable cationic lipids DLinDAP, DLinDMA, DLinKDMA, or DLinKC2-DMA can differ by three orders of magnitude. In this study, we examine the uptake and intracellular processing of LNP-siRNA systems containing these cationic lipids in a macrophage cell-line in an attempt to understand the reasons for different potencies. Although uptake of LNP is not dramatically influenced by cationic lipid composition, subsequent processing events can be strongly dependent on cationic lipid species. In particular, the low potency of LNP containing DLinDAP can be attributed to hydrolysis by endogenous lipases following uptake. LNP containing DLinKC2-DMA, DLinKDMA, or DLinDMA, which lack ester linkages, are not vulnerable to lipase digestion and facilitate much more potent gene silencing. The superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to higher uptake and improved ability to stimulate siRNA release from endosomes subsequent to uptake.From the Clinical EditorThis study reports on the in vivo gene silencing potency of lipid nanoparticle-siRNA systems containing ionizable cationic lipids. It is concluded that the superior potency of DLinKC2-DMA compared with DLinKDMA or DLinDMA can be attributed to their higher uptake thus improved ability to stimulate siRNA release from endosome.

Graphical AbstractUptake of lipid nanoparticle (LNP) formulations of siRNA into cells. (1) LNP-siRNA associates with cell membrane followed by (2) internalization. As the pH decreases in the endosomes the ionizable cationic lipids associate with endogenous anionic lipids to destabilize the endosomal membrane of LNP-siRNA. For LNP-siRNA systems containing DLinDMA, DLinKDMA and DLinKC2-DMA siRNA is then released to the cytosol (3) leading to gene silencing. (4) LNP-siRNA systems containing DLinDAP exhibit poor gene silencing potency due to degradation of DLinDAP by endogenous lipases thus inhibiting membrane destabilization and the ability of siRNA to escape the endosome.Figure optionsDownload high-quality image (178 K)Download as PowerPoint slide