Customizable Lipid Nanoparticle Materials for the Delivery of siRNAs and mRNAs

Abstract

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next-generation delivery materials for RNA therapeutics.

Keywords: ionizable lipids; lipid nanoparticles; mRNA; nanomaterials; siRNA.

Conflict of interest statement

The authors declare no competing financial interests.

© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1.

Medicinal chemistry approaches for evaluating the effect of ionizable lipid structure on LNP function in siRNA and mRNA LNPs

Figure 2.

a) Synthesis of OF-Deg-CX derivatives, a series of degradable diketopiperazine lipids with varied tail lengths, tail geometries, and total number of unsaturations. b) Synthesis of OF-C4-Deg-Lin, an ionizable lipid with varied linker lengths.

Figure 3.

a) LNP size and entrapment efficiencies for Luciferase siRNA cargoes measured by dynamic light scattering and Quant-IT RiboGreen RNA Assay Kits. b) LNP size and entrapment efficiencies for Luciferase mRNA cargoes measured by dynamic light scattering and Quant-IT RiboGreen RNA Assay Kits c) Knockdown evaluation of a target luciferase gene in HeLa cells with 50 ng doses of OF-Deg-CX siRNA LNPs per well (n=5). d) Luciferase protein expression in HeLa cells with 50 ng doses of OF-Deg-CX mRNA LNPs per well (n=5)

Figure 4.

a) Representative in vivo expression profile of luciferase mRNA OF-C4-Deg-Lin LNPs in mice following luciferin injection and euthanasia at a 0.75 mg/kg intravenous dose (n=3) b) Quantification of luciferase expression per organ as measured by percent of total average radiance for OF-C4-Deg-Lin, cKK-E12, and Invivofectamine luciferase mRNA formulations at 0.75 mg/kg intravenous doses (n=3).