pH-dependent structural transitions in cationic ionizable lipid mesophases are critical for lipid nanoparticle function

by Selleckchem | Jan 14 2024

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (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-LNP-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.

Comments:

The information you provided dives deep into the structural behavior of lipid nanoparticles (LNPs) used in mRNA-based therapies and their response to pH changes. The study explores the distinct behavior of ionizable lipids (DLin-MC3-DMA, DLin-KC2-DMA, and DLinDMA) in LNPs when exposed to varying pH levels.

The observed differences in enhanced green fluorescent protein (eGFP) expression in the mouse liver mediated by these ionizable lipids despite similar mRNA delivery per cell fraction suggest a correlation between the LNPs' efficacy and their response to pH alterations.

The study identifies a sequence of ordered CIL (cationic ionizable lipid)/cholesterol bulk phases in water as pH decreases, showing various structural configurations like isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal, and bicontinuous cubic Pn3m symmetry. Additionally, when polyadenylic acid, serving as an mRNA surrogate, is introduced to the CIL/cholesterol mix, excess lipid coexists with a condensed nucleic acid lipid phase.

Of note is the discontinuity observed in the next-neighbor distance within the excess lipid phase at the Fd3m inverse micellar to inverse hexagonal transition at pH 6. This transition exhibits distinct differences in spacing and hydration between DD, MC3, and KC2. Interestingly, in mRNA LNPs, DD displayed larger internal spacing, delayed onset, and reduced eGFP expression levels in vitro compared to MC3 and KC2.

The findings suggest that the pH-driven transition in bulk phases is a defining factor in the differences among CIL-specific mRNA LNP efficacy. This pH-dependent behavior in LNPs could impact their effectiveness in delivering mRNA payloads, potentially explaining the variations observed in eGFP expression mediated by different ionizable lipids.

This research contributes valuable insights into understanding the intricacies of LNPs, potentially aiding in the optimization and development of more effective mRNA-based therapies by manipulating the lipid components' response to pH changes.