Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity

Abstract

One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.01 mg kg(-1)). We identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

Fig. 1. Lipidoid nanoparticle synthesis

A library of 1400 biodegradable lipidoids was synthesized combinatorially through the (a) conjugate addition of alkyl-amines (in red) to alkyl-acrylate tails (in blue). (b) A subset of the 280 amines used (complete listing in Supplementary Fig. 1) are shown here. (c) Lipidoids were formulated with cholesterol, the phospholipid DSPC, PEG2000-DMG, and siRNA to form nanoparticles. (d) A cryo-TEM image of lipidoid nanoparticles. Scale bar = 100 nm.

Fig. 2. In vitro siRNA delivery activity of lipidoid nanoparticles

(a) Relative luciferase activity values (normalized to controls) are shown for 1400 lipidoids. ~7% of the library induced >50% gene silencing (shown in red). Standard deviation (not depicted for the sake of clarity) averaged 0.05 (n = 4). (b) The tail length, (c) tail substitution number and (d) alkyl-amine composition influenced in vitro activity. Structural features that were common among efficacious materials had positive Relative Hit Rate values.

Fig. 3. In vivo siRNA delivery activity of lipidoid nanoparticles

(a) Of the ~100 lipidoids tested in mice, 15 induced high levels of Factor VII knockdown at a total siRNA dose of 5 mg/kg (data points in red). (b) The EC50 values of these top 15 lipidoids ranged from 0.05 to 1.5 mg/kg under standard formulation conditions. (c) The amount of PEG in the LNP formulation had a dramatic effect on efficacy. Data is shown for the lipidoid 304O₁₄, which was optimally formulated with 0.75 mol% PEG. (d) Dose response and Factor VII activity recovery data for the optimized 304O₁₃ LNP formulation. (e) 304O₁₃ also induced CD45 silencing in monocyte and macrophage (CD11b+) populations in the peritoneal cavity (f) as well as in dendritic cells (CD11c+) in the spleen 3 days post-injection. In panels (e) and (f), ***, **, and * correspond to p values < 0.0005, 0.005, and 0.05, respectively, by an unpaired student’s t-test when comparing to LNPs containing non-targeting siRNA. In all panels, error bars represent s.d. (n = 3).

Fig. 4. Biodistribution images for Cy5.5 labeled siRNA delivered with the lipidoid 304O₁₃

(a) IVIS and (b) Odyssey imaging show that, while naked siRNA is primarily cleared through the kidneys, 304O₁₃ mediates accumulation in the liver and spleen. (c) Confocal microscopy of 304O₁₃ – treated liver shows siRNA (red) delivery throughout the tissue, including Kupffer cells (magenta) and hepatocytes (nuclei in blue and actin cytoskeleton in green). Three animals were tested per condition for experiments depicted in panels (a) – (c), and the images shown are representative. Scale bar = 50 μm. (d) 304O₁₃ LNPs were rapidly eliminated from the bloodstream after tail vein injection. Error bars represent s.d. (n = 3).

Fig. 5. Lipidoids showed strong structure function relationships in vivo

(a) Of the 96 LNPs tested for siRNA delivery to hepatocytes in mice, 68 had 3 or more tails (blue), 44 had a tertiary amine present in the original alkyl-amine (red), and 26 had an O₁₃ tail length (yellow). 88% of the LNPs exhibiting all three structural “efficacy criteria” (overlap of circles) achieved nearly complete FVII knockdown. The probability of identifying efficacious LNPs decreased precipitously when any of the criteria were not met. (b) pKa values also significantly influenced delivery efficacy to hepatocytes in vivo. All lipidoid nanoparticles capable of mediating nearly complete Factor VII gene silencing had pKa values ≥ 5.5. Error bars represent s.d. (n = 3).

Fig. 6. A second generation library confirmed structure function relationships

Twelve second generation LNPs were made to meet structural efficacy criteria by first synthesizing custom alkyl-amines shown in (a) and reacting them with O₁₃ tails. (b) All second generation LNPs meeting structural and pKa criteria abrogated Factor VII activity in vivo (p values < 0.0005 by an unpaired student’s t-test) and (c) their EC50s under non-optimized LNP formulating conditions ranged from 0.05 to 1 mg/kg total siRNA. (d) 503O₁₃ was the most efficacious LNP upon formulation, with an EC₅₀ of 0.01 mg/kg. 503O₁₃ encapsulating control siRNA (black data point) did not result in FVII knockdown. Error bars represent s.d. (n = 3). There was a statistically significant difference between groups as determined by one-way ANOVA (F(6,14) = 86.69, p < 0.0001). Post hoc comparisons using the Tukey HSD test indicated that all tested doses of 503O₁₃ resulted in silencing levels that were significantly different than the siControl (p < 0.05).