Multicomponent thiolactone-based ionizable lipid screening platform for efficient and tunable mRNA delivery to the lungs

Abstract

Ionizable lipids are essential components of lipid nanoparticles (LNPs) for efficient mRNA delivery. However, designing them for high protein expression, endosomal escape, and organ targeting is challenging due to complex structure-activity relationships. Here, we present a high-throughput platform for screening ionizable lipids using a two-step, scalable, one-pot reaction. This enabled the synthesis and vivo screening of 91 new lipids, followed by a structure-activity study, leading to the development of CP-LC-0729, which significantly surpasses the MC3 benchmark in protein expression with preliminary studies showing no in vivo toxicity. Additionally, a one-step strategy helped to yield a permanently cationic lipid which was tested in a fifth-lipid formulation, showing a highly selective lung delivery with a 32-fold increase in protein expression in vivo, outperforming current endogenous targeting strategies. All these findings underscore the potential of lipid CP-LC-0729 and our lipid platform in advancing the efficiency and specificity of mRNA delivery systems.

Fig. 1. Design and synthesis scheme of STAAR ionizable lipids.

a Representative scheme describing STAAR lipids synthetic steps. First, the synthesis of Bj thiolactone derivative and then the one-pot tricomponent reaction. The amine opens the thiolactone ring of Bj (aminolysis) and subsequently the thiol group reacts with the acrylate to afford the final lipid with high purity in short times and at room temperature. b Scheme of the process followed for the formulation of lipids into LNPs for mRNA delivery in vivo.

Fig. 2. Screening optimization of STAAR lipids.

a Amines (Ai), Bj derivatives and acrylates (Ck) used for lipid screening. b Scheme summarizing the strategy followed for the two screening phases. c Total flux (entire mouse body) results of screening phase 1, where B was optimized by combining different acrylates and Bj derivatives while keeping polar head (A1) fixed. Mice were intramuscularly injected (i.m.) with mRNA-Luc-loaded LNPs at an mRNA dose of 0.05 mg/kg. Luminescence imaging acquisition was performed at 4 h post-treatment and total flux was quantified. In vivo mLuc expression displayed in a heat map (n = 3 biologically independent samples). Data are presented as mean values. d Total flux results table of Screening phase 2 where A and C were optimized by combining different amines and acrylates while keeping B2 fixed. Mice were i.m injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.05 mg/kg. Total flux luminescence imaging acquisition of the whole mouse body was performed at 4 h post-treatment and total flux was quantified. In vivo mLuc expression displayed in a heat map (n = 3 biologically independent samples). Data are presented as mean values. Below each table, representative luminescence images of each lipid in mice acquired using the IVIS Lumina XRMS Imaging System are shown.

Fig. 3. Assessment of the hydrophobic tails of A4B2C3 and structure-activity relationships in STAAR lipids.

a Building blocks for hydrophobic tail optimization, combining A4, branched Bj derivatives and branched acrylates. b In vivo luminescence total flux results of a structures normalized respect to MC3 benchmark LNP, with the dashed line in the y-axis representing the results of control MC3 benchmark LNP. MC3(DOPE) LNP was also used as control employing DOPE as helper lipid. Mice were i.m injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.05 mg/kg. Luminescence imaging acquisition was performed at 4 h post-treatment and total flux was quantified. In vivo mLuc expression (n = 3 biologically independent samples). Data are presented as mean values ± standard deviation (SD). c Schematic lipids set structure displaying different functional groups varying in alpha (α) and beta (β) positions. d Results of lipids in c, in vivo mLuc expression (n = 3 biologically independent samples). Data are presented as mean values ± SD. Mice were i.m injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.05 mg/kg. Luminescence imaging acquisition was performed at 4 h post-treatment and total flux was quantified. e ζ potentials and pKa of the formulated LNPs (n = 3 biologically independent samples). Data are presented as mean values. In b and d one-way ANOVA with Tukey’s correction was used.

Fig. 4. In vivo safety and cell internalization profile of CP-LC-0729 LNP.

a Representative cryo-TEM images of CP-LC-0729 LNP. Scale bar, 100 nm. b Serum alanine aminotransferase levels (ALT), serum aspartate aminotransferase levels (AST), serum alkaline phosphatase levels (ALP), urea nitrogen concentration (UREA). Mice were intravenously injected (i.v) with mRNA-Luc-loaded LNPs at an mRNA dose of 2.5 mg/kg (n = 5 biologically independent samples). Serum was collected for ALT, AST, ALP and UREA analysis at 24 h and 48 h post-treatment. Data are presented as mean values ± SD. Two-way ANOVA with Tukey’s correction was used. c Body weight change for 15 days (measured made at days 0, 1, 2, 7, 9, 13 and 15). Mice were i.v injected with mRNA-Luc-loaded LNPs at an mRNA dose of 2.5 mg/kg (n = 5 biologically independent samples). Data are presented as mean values ± SD. d Uptake inhibition of CP-LC-0729 and MC3(DOPE) LNP by different endocytosis inhibitors (n = 3 biologically independent samples). The inhibitors used were Amiloride (inhibitor of macropinocytosis), Chlorpromazine (inhibitor of clathrin-mediated endocytosis), Genistein (inhibitor of caveolae mediated endocytosis), Methyl-β-cyclodextrin (inhibitor of lipid raft mediated endocytosis). Data are presented as mean values ± SD. e Hemolysis assay of CP-LC-0729 LNP and MC3(DOPE) LNP at pH 7.4 or 6.0. RBCs were incubated with CP-LC-0729 LNP and MC3(DOPE) LNP at an mRNA concentration of 2.5 μg/mL at 37 °C for 1 h. Positive and negative controls were carried out with 0.1% Triton-X and 1× PBS, respectively. Data are presented as mean ± SD (n = 3 biologically independent samples).

Fig. 5. Lung targeting strategy using a STAAR permanently cationic lipid.

a Synthetic scheme used to prepare a permanent cationic lipid, namely (+) CP-LC-0729, from an ionizable lipid CP-LC-0729. b Schematic representation of the formulated LNPs incorporating a STAAR permanently cationic lipid as fifth component for mRNA delivery in vivo through i.v. administration. c Top to bottom: Ex vivo luminescence of major organs from mice, pie charts showing a profile of the tissue-specificity of mLuc expression in the lungs, heart, liver, intestine, spleen and kidneys, bar representation of total luminescence found in lungs and table with the formulation details of LNPs (n = 3 biologically independent samples). Mice were i.v. injected with mRNA-Luc-loaded LNPs at an mRNA dose of 0.2 mg/kg. Images were acquired at 4 h post-treatment. For the bar graph, data are presented as mean values ± SD. One-way ANOVA with Tukey’s correction was used.