Authors: Xiaoxia Chen, Yongfeng Ye, Mengrong Li, Taisen Zuo, Zhenhua Xie, Yubin Ke, He Cheng, Liang Hong, and Zhuo Liu
Abstract
Lipid nanoparticles (LNPs) have emerged as a versatile platform for mRNA delivery across a range of applications, including disease prevention, cancer immunotherapy, and gene editing. Structural models of mRNA-containing lipid nanoparticles (mRNA-LNPs) have also been proposed based on characterization of samples by using various advanced techniques. Among these, small angle neutron scattering (SANS) has proven essential for elucidating the lipid distribution within mRNA-LNPs, a factor crucial to both their preparation and efficacy. However, recent findings suggest that the mRNA-LNP samples prepared via commercial microfluidic techniques may contain a substantial fraction of drug-free LNPs, casting doubt on the validity of earlier structural models. In this study, we employed contrast variation SANS to characterize both drug-free LNPs and our mRNA-LNP sample, and quantified the proportion of drug-free LNPs present to be ∼30% in our mRNA-LNP sample using nano flow cytometry. By removing the contributions of drug-free LNPs from the SANS data of our mRNA-LNP sample, we were able to precisely characterize the structure of mRNA-LNPs. Consequently, we proposed structural models for both drug-free LNPs and mRNA-LNPs. Notably, our analysis revealed similar lipid distributions and shell thicknesses between the two particle types, while the solvent content in mRNA-LNPs was significantly higher, leading to a larger core size. This work not only offers a method for accurately characterizing the structure of mRNA-LNPs, but also establishes criteria for selecting appropriate analytical techniques based on the structural parameters of interest. Therefore, our findings hold significant implications for the mechanistic understanding and quality control of mRNA-based vaccines.
Fig 1. Basic characterizations of the drug-free LNP and mRNA-LNP samples. (A) Particle size distributions, (B) cryo-EM images, (C) SAXS curves, (D) encapsulation efficiency, and (E) in vitro transfection potency of the drug-free LNP and mRNA-LNP samples. HEK293T cells were transfected by the LNP and mRNA-LNP samples. Luciferase expression was quantified after 24 h of incubation (n = 3).
Fig 2. Determination of the proportion of drug-free LNPs in the mRNA-LNP sample. (A) Compiled bivariate dot-plot of the FL (fluorescence) burst area versus the SS (side scattering) burst area for drug-free LNPs (black circles) and mRNA-LNPs (green circles). (B) The mRNA copy number distribution in the mRNA-loaded LNPs.
Selected Figures
Keywords: lipid nanoparticles; mRNA vaccine; small angle neutron scattering; Flex-M
bioRxiv 2024