Proceedings of the National Academy of Sciences of the United States of AmericaDec 6, 2023

pH-driven shape changes in charged fats are important for lipid nanoparticle function

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Updated Jan 20, 2026

Abstract

Lipid nanoparticles containing different ionizable lipids rank in mRNA delivery efficacy as follows: MC3 ≥ KC2 > DD.

The mechanism behind pH-dependent endosomal release of lipid nanoparticles is not well understood.
Three ionizable lipids, DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD), were examined for their ability to mediate protein expression in mouse liver.
Ordered mesophases of cationic ionizable lipids and cholesterol were observed to change with lowering pH, indicating different structural responses.
A transition at pH 6 from Fd3m inverse micellar to inverse hexagonal phases was noted, with larger spacing in the DD lipid compared to MC3 and KC2.
In mRNA lipid nanoparticles, DD demonstrated larger internal spacing and a reduced level of protein expression compared to MC3 and KC2.

Simplified

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal [Formula: see text] and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid [Formula: see text] phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal [Formula: see text] transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD--mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m-[Formula: see text] transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.

Key numbers

> KC2 > DD

Higher Expression

Order of expression levels in liver cells after transfection.

6.5

pH Transition Point

pH value at which distinct structural changes occur in / mixtures.

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Funding

Competing interests

Competing interests statement:A.D., P.S., U.S., S.Ö., M.J., N.H., K.E., B.S., and L.L. are employed by AstraZeneca R&D Gothenburg.

PubMed

pH-driven shape changes in charged fats are important for lipid nanoparticle function

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