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

Abstract

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.

Keywords: SAXS; ionizable lipid; lipid nanoparticles; lyotropic mesophases; mRNA delivery.

Fig. 1.

LNP-mediated mRNA delivery and protein expression in vivo. (A) Schematic drawing of production, internalization via endocytosis, and pH-dependent maturation of LNPs preceding endosomal release. mRNA LNP particles exhibit a core-shell structure consisting of a condensed core phase composed of mRNA, CIL, and chol, with an outer shell enriched in DSPC and a PEG corona. (B) The ionizable lipids MC3, KC2, and DD differ in head group area while sharing the same lipid tails and ionizable dimethylamino-group. (C) Mice study of ionizable lipid uptake in liver cells, (D) mRNA uptake in liver cells, and (E) in vivo protein expression per cell type showing same uptake yet unexplained order of protein expression efficacy MC ≥ KC2 > DD. Four mice in each group were used with an mRNA dose of 0.5 mg/kg. Error bars in figures (C–E) are the SEM. Significance levels are based on the Mann–Whitney method.

Fig. 2.

SAXS-based identification of pH-dependent mesophase transitions. (A) Ionizable lipid, chol, and buffer and (B) ionizable lipid, chol, buffer, and polyA. Data show the three ionizable lipids, MC3, KC2, and DD, for comparison. (C) Schematic representation of lipid phases with decreasing pH from top to bottom showing a trend from negative curvature toward zero curvature with increasing headgroup protonation: Inverse micellar fluid isotropic $L_{\mathit{II}}$ , inverse micellar cubic with P6₃/mmc symmetry, inverse micellar cubic with Fd3m symmetry, inverse hexagonal $H_{\mathit{II}}$ , and bicontinuous cubic Pn3m. In the presence of polyA (panel B), coexistence of lipid mesophases with a complexed, nucleic acid containing, phase is observed. In the pH range from 5.0 to 6.0 typically $H_{\mathit{II}}$ and $H_{\mathit{II}}^c$ coexist. See main text for further information.

Fig. 3.

MD simulations and electron density profiles of inverse phases for MC3. (A) Snapshot from an all-atom MD simulation of the $H_{\mathit{II}}$ phase for fully charged MC3 and chol. Water is shown in blue; cationic headgroup and tail of MC3 are shown in red and green, respectively. Chol is shown in yellow. (B) corresponding radial electron density profile for the individual components of a single cylindrical micelle from MD simulations. The black line indicates the total electron density; the vertical dash line is the half nearest neighbor distance (C) SAXS data are plotted as black dots, the fitted SAXS profile in blue and the form factor obtained from MD in orange. (D) Simulation of tightly packed inverse micelles for uncharged MC3 and (E) electron density profile. (F) Scattering of $L_{\mathit{II}}$ phase in red fitted in blue with the structure factor for densely packed spheres multiplied by the form factor obtained from MD. (G) Time series showing a cross-sectional view of an inverse-spherical to inverse-cylindrical micellar transition after charging the MC3 ionizable group at time t = 0. First, the MC3 headgroups swing into the water phase. Subsequently, a slower rearrangement from an inverse spherical to inverse cylindrical micelle takes place.

Fig. 4.

Scattering signature and pH-induced transitions in CIL bulk vs. LNP core phase. (A) SAXS scattering at pH 7 (red curves) of MC3/chol/water $L_{\mathit{II}}$ bulk phase, MC3/chol/water/polyA $L_{\mathit{II}}^C$ bulk phase, MC3/chol/water/mRNA $L_{\mathit{II}}^C$ bulk phase, and mRNA LNPs and corresponding SAXS data at pH 5 (black curves). The MC3/chol/water/polyA preparation at pH 7 showed time dependence with the $H_{\mathit{II}}^C$ peak dissolving over weeks. We denote the dashed line as micellar isotropic $L_{\mathit{II}}^C$ phase. The mRNA filled bulk phases and LNPs exhibit less long-range order due to mRNA secondary structure. (B) Schematic drawing of the polyA condensed $H_{\mathit{II}}^C$ phase at pH 5, the polyA condensed $L_{\mathit{II}}^C$ (polyA) phase at pH 7, and an mRNA condensed $L_{\mathit{II}}^C$ (mRNA) phase. mRNA is assumed to retain secondary structure in a disordered $L_{\mathit{II}}$ —micellar-like lipidic phase. (C) Next nearest neighbor distances as a function of pH showing pH-induced transitions in CIL bulk phases and (D) in polyA containing complexed phases. The gray area indicates the transition region with cubic phases. The reddish area indicates metastability of $H_{\mathit{II}}^c$ . (E) pH-dependent correlation distance in LNPs derived from SAXS data (F) Fluorescence anisotropy of a hydrophobic probe (DPH) as a function of pH for MC3-, KC2-, and DD-LNPs, respectively. Increasing anisotropy indicates decreasing mobility. The critical pH values of half-maximum are 6.0, 6.1, and 6.0 for MC3-, KC2-, and DD, respectively. Data shown in (C and D) are measured at room temperature, (E and F) at 37 °C.

Fig. 5.

Expression kinetics after eGFP-mRNA LNP transfection in vitro. (A) Exemplary single-cell fluorescence time courses of mRNA-Cy5 and eGFP (B) Correlation of transfection efficiency and onset time of single cells reveals delayed and less efficient expression for DD vs. MC3 and KC2. (C) eGFP-onset typically occurs 0.9 h after the event of Cy5-signal increase.