A Multidimensional Approach to Modulating Ionizable Lipids for High‐Performing and Organ‐Selective mRNA Delivery

Abstract

The development of lipid nanoparticles (LNPs) has enabled a successful clinical application of mRNA vaccines. However, disclosure of design principles for the core component‐ionizable lipids (ILs), improving the delivery efficacy and organ targeting of LNPs, remains a formidable challenge. Herein, we report a powerful strategy to modulate ILs in one‐step chemistry using the Ugi four‐component reaction (Ugi‐4CR) under mild conditions. A large IL library of new structures was established simply and efficiently through a multidimensional approach, allowing us to identify the top‐performing ILs in delivering mRNA via the formulated LNPs. Adjusting the skeleton of ILs has transformed the organ‐specific and robust transfection in mRNA delivery from the liver to the spleen following different administration routes. Of note, a series of isomeric ILs were prepared and we found that the isomers mattered greatly in the performance of LNPs for mRNA delivery. Furthermore, owing to the bis‐amide bonds formed in the Ugi‐4CR reaction, the ILs within LNPs may form hydrogen bonding intermolecularly, facilitating the colloidal stabilization of LNPs. This work provides clues to the rapid discovery and rational design of IL candidates, assisting the application of mRNA therapeutics.

Cationic Lipid Pairs Enhance Liver-to-Lung Tropism of Lipid Nanoparticles for In Vivo mRNA Delivery

“…To find the optimal pair strategy of CLP between the ionizable lipid and the quaternary ammonium lipid, as shown in Figure A, we chose another ionizable lipid A4I18R2C18, which had a similar skeleton to the ionizable lipid A4I18R2C18-2 but difference in the unsaturated degree of hydrophobic tails, for investigating the structure–activity relationship of CLP under four kinds of pair manners. First, we observed that the unsaturated degree of hydrophobic tails of ionizable lipids had dramatic effect on protein expression levels of liver-targeted LNPs, and the LNP containing ionizable lipids with unsaturated hydrophobic tails (DSPC/IL) showed orders of magnitude increase of mRNA delivery efficacy in the liver than that without unsaturated hydrophobic tails (DSPC/IL′) (Figure A,B), which is highly consistent with the previous finding . Then, two ionizable lipids (A4I18R2C18-2 and A4I18R2C18) with significant difference in liver-targeted mRNA delivery ability were paired with their corresponding Q 1 -subsituted quaternary ammonium lipid (Q 1 -A4I18R2C18-2 or Q 1 -A4I18R2C18) to form four types of CLP-based LNPs by different pair manners, that is Q 1 L/IL, Q 1 L/IL′ (Q 1 -A4I18R2C18-2/A4I18R2C18), Q 1 L′/IL′ (Q 1 -A4I18R2C18/A4I18R2C18), and Q 1 L′/IL (Q 1 -A4I18R2C18/A4I18R2C18-2).…”

“…Various liver-targeted LNPs were prepared according to the previously reported formulation conditions. , Briefly, the organic phase was obtained by dissolving ionizable lipid (A4I18R2C18-2 or A4I18R2C18), DSPC, cholesterol, and DMG-PEG with a molar ratio of 50:10:38.5:1.5 in ethanol, and the aqueous phase contains mRNA molecules in 10 mM pH 4 citrate buffer. Then, LNPs were prepared by rapid mixing of the organic phase and aqueous phase at the volume ratio of 1:3 through a pipette mixing method, with the weight ratio of ionizable lipid and mRNA of 5.5:1.…”

“…1-Isocyanooctadecane (I18) was synthesized according to our previously reported studies. 32,33 1-Octylnonyl-8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate (SM-102), ((4-hydroxybutyl)azanediyl)bis-(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearoylsn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol 2000 (DMG-PEG), and cholesterol were from A.V.T. (Shanghai, China).…”

“…Ionizable lipids were synthesized by our previously reported method. 33 Briefly, A4 and R2 were dissolved in methanol and stirred at room temperature for 1 h; both C18-2 (or C18) and I18 were added, and the mixture was reacted at 45 °C for 12 h. After purification with silica gel column chromatography with the methanol/dichloromethane ratio of 1:10 (v/v), two types of ionizable lipids termed as A4I18R2C18-2 and A4I18R2C18 were obtained with yields of 60−70%, respectively. Next, the quaternization reaction of tertiary amines of ionizable lipids with various haloalkanes or halohydrins was used to produce their corresponding quaternary ammonium lipids.…”

“…First, we observed that the unsaturated degree of hydrophobic tails of ionizable lipids had dramatic effect on protein expression levels of liver-targeted LNPs, and the LNP containing ionizable lipids with unsaturated hydrophobic tails (DSPC/IL) showed orders of magnitude increase of mRNA delivery efficacy in the liver than that without unsaturated hydrophobic tails (DSPC/IL′) (Figure 4A,B), which is highly consistent with the previous finding. 33 Then, two ionizable lipids (A4I18R2C18-2 and A4I18R2C18) with significant difference in liver-targeted mRNA delivery ability were paired with their corresponding Q 1 -subsituted quaternary ammonium lipid (Q 1 -A4I18R2C18-2 or Q 1 -A4I18R2C18) to form four types of CLP-based LNPs by different pair manners, that is Q 1 L/IL, Q 1 L/IL′ (Q 1 -A4I18R2C18-2/A4I18R2C18), Q 1 L′/IL′ (Q 1 -A4I18R2C18/ A4I18R2C18), and Q 1 L′/IL (Q 1 -A4I18R2C18/A4I18R2C18-2). As represented in Figure 4A,C, four kinds of CLP-based LNPs demonstrated firefly luciferase protein expression transformed from the liver to the lungs in different levels, and Q 1 L/IL as one of the pair manners exhibited the highest protein expression in the lungs as compared to the other three kinds of pair manners.…”