Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

Abstract

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

“…ORNst showed a two-fold higher uptake in liver (8416 ng/mg tissue), in spleen (4012 ng/mg tissue) and lung (174 ng/mg tissue), and a slightly reduced accumulation in kidney. These findings are again consistent with reports that siRNAs conjugated to hydrophobic fatty acids are transported to the liver at the expense of kidney (26). ORNch showed seven-fold higher levels in liver (34150 ng/mg tissue), a two-fold increase in spleen (5412 ng/mg tissue) and a 60% decrease in kidney compared to ORN1; the presence of ORNch in lung tissue was below accurate detection.…”

“…This study of splice-switching oligonucleotide conjugates complements recent investigations of the pharmacokinetic and pharmacodynamic properties of antisense and siRNA conjugates (26,27). The results reinforce the notion that increased delivery does not necessarily translate to improved potency.…”

Recent Advances in the Design of Self‐Delivery Amphiphilic Drugs and Vaccines

“…Beyond the linker length and composition, the optimization of a particular amphiphilic drug also includes the amphiphilic modification that tunes the overall hydrophilic/lipophilic balance. For example, lipid composition and hydrophobicity have been shown to control the protein binding and biodistribution of lipid‐conjugate siRNA . Taken together, the above examples demonstrated the structures and physicochemical properties of amphiphilic drugs are highly tunable and can be tailored for targeted drug delivery.…”

“…Since lipid molecules exhibit strong affinity toward albumin and lipoproteins, lipid‐drug conjugates are widely used to hijack the trafficking pathways of serum proteins. The affinity and specificity of protein binding are primarily determined by the structures and hydrophobicity of the lipid …”

Selective tissue targeting of synthetic nucleic acid drugs

“…In contrast, association with HDL or apolipoprotein E promotes uptake into peripheral tissues via SR-BI. While the majority of work with cho-lesterol-siRNA conjugates was directed toward hepatic delivery, recent work demonstrated that cholesterol conjugation can deliver siRNA to muscle and other extrahepatic tissues in mice (60,61).…”