Elucidation of lipid nanoparticle surface structure in mRNA vaccines

Abstract

Lipid nanoparticles (LNPs) have been used as a carrier for messenger RNA (mRNA) vaccines. Surface properties of LNPs are important to the stability and function of mRNA vaccines. Polyethylene-glycol (PEG) is a functional lipid at the surface of LNPs that improves colloidal stability, increases circulation time, and impacts cellular uptake. In this study, we explore in-depth lipid composition at the surface of mRNA-LNPs using high-field nuclear magnetic resonance (NMR) spectroscopy. Our results provide a unique surface lipid profile of intact LNPs identifying PEG chains and partial ionizable lipids are present with quantification capability. The surface PEG density is determined to reveal the brush-like conformation on the surface of mRNA-LNPs. Furthermore, we implement a diffusion NMR strategy for routine testing of formulated drug products during drug development. Comparative NMR analysis of different vaccine preparations and stability samples provides a global view of the mRNA-LNP surface structure for enhanced product knowledge.

Figure 1

Surface PEG Models and COMIRNATY mRNA Lipid Nanoparticle Composition. (A) two primary models of surface PEG conformation: mushroom and brush-like. (B) COMIRNATY lipid nanoparticle composition.

Figure 2

Surface Characterization of Intact mRNA-LNPs by 1D ¹H NMR. (A) top spectrum: intact mRNA-LNPs in aqueous phosphate buffer; Bottom four spectra: individual lipids: ALC-0315 (aqueous, PBS), ALC-0159 (aqueous, PBS), cholesterol (chloroform), and DSPC (chloroform). Proton signals in the mRNA-LNP spectrum were labeled in green for ALC-0159, and in black for ALC-0315; overlapped signals from both lipids were labeled in purple. (B) the detected surface protons were annotated in the molecular structures of ALC-0159 and ALC-0315. Spectra are not at equivalent scale with the lipid concentration.

Figure 3

1D ¹H NMR Spectra of mRNA-LNPs in PBS and a 2% Formulation (post-dilution with PBS). (A) top spectrum: the formulation buffer control; Middle two spectra: LNP sample in 2% formulation buffer with and without PGSTE; Bottom spectrum: the dialyzed LNP sample in PBS (bottom). (B) a schematic illustrating excipient (pink spheres) signals in formulated drug products are suppressed by T₂ filters used in PGSTE. (C) the expansion of spectra in panel A.

Figure 4

Surface Structural Characterization of Aged LNP by Diffusion 1D ¹H NMR and cryo-EM. (A) 1D ¹H NMR of two representative mRNA-LNP lots (stored at −80 °C) and one aged mRNA-LNP sample (−20 °C for 3 months). The increased intensity at ALC-0159 (PEG-CH₂-) peak at 3.8 ppm (bold label) is determined as the methylene with conformation variance that is not identical to that in the intact LNP. (B), (C) cryo-EM images of a representative mRNA-LNP and the aged LNP sample.