Predictive Lung- and Spleen-Targeted mRNA Delivery with Biodegradable Ionizable Lipids in Four-Component LNPs

Abstract

Background/Objectives: Lipid nanoparticles (LNPs) are leading mRNA delivery vehicles, with ionizable lipids (ILs) as their key component. However, the relationship between the IL structure and LNP endogenous organ-targeting is not well understood. In this study, we developed a novel library of biodegradable ILs featuring beta-propionate linkers, which, when incorporated into a four-component LNP formulation, show excellent extrahepatic selectivity and high protein expression. Methods: We explored the impact of structural modifications in the hydrophobic chains and polar-head groups in the ILs while keeping the linkers unchanged. In vivo results were evaluated to examine how structural changes influence the biodistribution to spleen or lungs. LNP formulations were assessed for their protein expression levels and organ-specific targeting. Additionally, protein corona formation by the best-performing LNPs was examined to provide further mechanistic insights. Results: Organ targeting was significantly influenced by structural changes in the ILs, allowing for precise control of the biodistribution between the spleen and lungs. Branched hydrophobic chains demonstrated a higher propensity for spleen targeting, while modifications in the polar-head group could drastically shift biodistribution from the lung to the spleen. This led to the identification of LNPs' zeta potential as a key determinant of their extrahepatic targeting properties. Notably, ionizable lipid A3T2C7, also known as CP-LC-1495, displayed strong lung selectivity (97%) and high protein expression in lung tissue (1.21 × 10⁸ p/s). Similarly, several promising candidates for spleen-targeting LNPs displayed protein expression levels exceeding 1 × 10⁷ p/s (selectivity >80%). Conclusions: This study elucidates the structure-function relationships of ILs in passive organ-specific mRNA delivery, highlighting how the fine-tuning of hydrophobic chains, polar-head groups, and surface charge (zeta potential) allows for the precise control of LNP endogenous biodistribution, a mechanism influenced by protein corona formation. These findings enable the rational design of targeted LNP systems, enhancing their therapeutic potential for specific organs, such as the spleen and lungs.

Keywords: extrahepatic delivery; four-component LNPs; ionizable lipids; lipid nanoparticles; lung targeting; mRNA; protein corona; spleen targeting.

Figure 1

Retrospective lipid library analysis for the screening and selection of ionizable lipids with lung-targeting LNPs. (A) Scheme of ionizable lipid screening formulated in LNPs administered via in vivo intramuscular (i.m.) injection, where mice received mRNA-Luc-loaded LNPs at a dose of 0.05 mg/kg mRNA. (B) Graphic of the ζ potential and total flux of ~1500 ionizable lipids from the intramuscular screening. LNPs with equal or greater than zero potential are illustrated with green color. Ionizable lipids contained in LNPs within boxes were selected as potential candidates for lung targeting. (C) Illustration of the lung-targeting screening in mice. (D) Graphic of the ex vivo total bioluminescence flux quantification and selectivity in the lung for selected ionizable lipids. Mice were intravenously (i.v) injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.5 mg/kg (n = 2 biologically independent samples). Data are presented as mean values. (E) General lipid structure identified for extrahepatic targeting containing up to three beta-propionate linkers; beta-propionate linkers are highlighted in blue.

Figure 2

Combinatorial synthesis and organ-specific targeting of beta-propionate ionizable lipids. (A) Scheme describing the steps for the synthesis of the ionizable lipids. (B) Structures of the 4 thiolactone-sulfur (Ty) derivatives, 11 acrylates (Cz) and amines (Ax) used in the combinatorial synthesis. (C) Illustration of the lung and spleen targeting. Mice were i.v. injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.5 mg/kg. (D) Heat map depicting the spleen specificity of ionizable lipids, correlated with the hit rate of Cz hydrophobic tails and Ty in spleen tissue. (E) Heat map depicting the spleen specificity of ionizable lipids, correlated with the hit rate of Cz hydrophobic tails and Ty. (F) Quantified luciferase expression in spleen, liver, lung, heart, intestine and kidneys. (D,E) An “X” indicates that the LNPs precipitated and were not used in the study. Mice were i.v. injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.5 mg/kg (n = 2 biologically independent samples). Data are presented as mean values.

Figure 3

Analysis of the ionizable lipid polar-head influence on organ-specific targeting. (A) Structures of the thiolactone-sulfur (Ty) derivatives, acrylates (Cz) and amines (Ax) employed for the ionizable lipid synthesis. These ionizable lipids were incorporated into LNPs for the evaluation of organ targeting. (B) (Left) Heat map depicting the spleen specificity of the ionizable lipids. (Right) Representative ex vivo bioluminescence images of mouse organs. (C) (Left) Heat map depicting the lung specificity of the ionizable lipids. (Right) Representative ex vivo bioluminescence images of mouse organs. The organs are arranged in two rows: the top row (from left to right) includes the liver, kidneys, and lungs, while the bottom row (from left to right) includes the heart, intestines, and spleen. (D) (Top) Total flux (p/s) in the six main organs obtained for the LNP formulation of each lipid candidate upon i.v. administration and subsequent ex vivo analysis. (Bottom) Stacked bar chart depicting the percentage expression in each organ for the lipid candidates upon ex vivo analysis. Zeta potential values are shown as black dots within the graph. Mice were i.v. injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.5 mg/kg (n = 2 biologically independent samples). Data are presented as mean values.

Figure 4

Protein-corona composition of organ-targeting LNPs. (A) Schematic illustration of how protein coronas form upon entry into the bloodstream, influencing nanoparticle targeting and directing them to specific organs. (B) Normalized physicochemical properties of adsorbed proteins, including their isoelectric point, hydrophobicity (GRAVY), secondary structure elements (helix, sheet, turn), instability index, charge, and aromaticity. (C) Distribution of the isoelectric points (arrow) and (D) net charge of proteins adsorbed onto lung- and spleen-targeting LNPs. (E) Heatmap of enriched proteins in spleen-targeting and (F) lung-targeting LNP coronas.