Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles

Abstract

Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e139; doi:10.1038/mtna.2013.66; published online 17 December 2013.

Figure 1

Plasma and tissue concentration-time profiles at different dose levels. LNPs containing 1.5 mol % PEG-C14 and trace ³H-CHE were administered to mice at doses of 0.33 (circle), 3.3 (square), and 11.1 (triangle) mg/kg of lipid, equivalent to 0.03, 0.3, and 1 mg/kg siRNA, respectively. At various times, the animals were sacrificed and the amount of radiolabel present in blood, liver, and spleen was determined as outlined in Materials and Methods section. Dose proportionality is highlighted by the inserted linear plots of AUC (ng lipid·hour/g) versus lipid dose (mg/kg). Data points represent the average of 4 animals ± 1 SD. AUC, area under the curve; LNP, lipid nanoparticles; PEG, polyethylene glycol.

Figure 2

Comparison of ¹⁴C-MC3 and ³H-CHE as LNP markers. Mice, injected with LNPs containing 1.5 mol % PEG-C14, dual labeled with ¹⁴C-MC3 and ³H-CHE, were sacrificed at various times up to a maximum of 2 hours and the amount of radiolabel present in the blood determined as outlined in Materials and Methods section. The % injected dose was determined using ³H-CHE (square) and the ¹⁴C-MC3 to ³H-CHE ratio (circle) was used to show ¹⁴C-MC3 is a stable LNP marker for at least 2 hours after injection, during which time ~90% of the injected dose distributes out of the blood compartment. Data represent the average of 4 animals ± 1 SD. LNP, lipid nanoparticles; PEG, polyethylene glycol.

Figure 3

Loss of PEG-lipid from LNPs in circulation. (a) Mice injected with LNPs containing 1.5% mol % PEG-C14, dual labeled with ¹⁴C-MC3 and ³H-PEG-C14, were sacrificed at various times and the ³H-PEG to ¹⁴C-MC3 ratio determined in blood (circle), plasma (square), and LNP isolated from plasma (triangle) as outlined in Materials and Methods section. The clearance of the LNP as measured by ¹⁴C-MC3 in the blood is shown as the percent injected dose (inverted triangle). (b) LNPs containing 1.5 mol % PEG-C14 (circle), PEG-C16 (square), or PEG-C18 (triangle), dual labeled with ¹⁴C-MC3 and the corresponding ³H-PEG-lipid were injected into mice. At various times the animals were sacrificed, plasma isolated and the amount of ³H-PEG retained with LNPs measured as outlined in Materials and Methods section. Data represent the average of 4 animals ± 1 SD. LNP, lipid nanoparticles; PEG, polyethylene glycol.

Figure 4

Effect of PEG-lipid on FVII gene silencing activity. (a) FVII siRNA LNPs containing 1.5 mol % PEG-C14 (circle), PEG-C16 (triangle), or PEG-C18 (square) were administered to mice at the indicated siRNA doses and plasma FVII protein concentrations determined 24 hours later. ED₅₀'s of 0.02, 0.03, and 0.04 mg/kg siRNA estimated for PEG-C14, PEG-C16, and PEG-C18 respectively are not significantly different (P > 0.05). (b) PEG-C14 content of 1.5 mol % (circle), 2.5% (triangle), and 3.5% (square) exhibit ED₅₀'s of 0.02, 0.03, and 0.06 mg/kg siRNA respectively, which are also not significantly different. (c) PEG-C18 content of 1.5 mol % (circle), 2.5% (triangle), and 3.5% (square) exhibit ED₅₀'s of 0.04, >0.3, and >0.3 mg/kg siRNA, respectively. The difference in activity observed at 1.5% PEG-C18 is statistically significant compared to 2.5 and 3.5% (**P < 0.005). Data points represent the average of 3–6 mice ± 1 SD. LNP, lipid nanoparticles; PEG, polyethylene glycol.

Figure 5

Effect of PEG-lipid chain length on pharmacokinetics and biodistribution. Mice were administered LNPs containing 1.5 mol % PEG-C14 (diamond), PEG-C16 (square), and PEG-C18 (triangle), dual labeled with ¹⁴C-MC3 and the corresponding ³H-PEG-lipids. At various times the animals were sacrificed and the concentration of ¹⁴C and ³H measured in blood, liver, and spleen. Data points represent the average of 4 mice ± 1 SD. LNP, lipid nanoparticles; PEG, polyethylene glycol.