Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structures of these formulations are still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and the determination of the intra-LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well-characterized PEGylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size and envelope lipid composition to Doxil; however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of 14C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction imply that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilipid layers. Negative staining TEM shows 2-5 nm electron-dense spots in the LNP’s interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply cross-link-stabilized RNA fragments. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the possibility of hydrogen bonding between mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of the ionizable lipid in Comirnaty, ALC-0315, displaying free ═O and −OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These structural features of mRNA-LNP may be important for the vaccine’s activities in vivo.
Bibliographical noteFunding Information:
The financial support by the European Union Horizon 2020 projects 825828 (Expert) and 952520 (Biosafety) are acknowledged. This project was also supported by grants from the National Research, Development, and Innovation Office (NKFIH) of Hungary (OTKA K143321) and from the Ministry of Innovation and Technology of Hungary (2020-1.1.6-JÖVŐ-2021-00013 and 2020-1.1.6-JÖVŐ-202100010). B.K. was supported by the New National Excellence Program of the Ministry for Innovation and Technology (ÚNKP-22-4-I-SE-16). J.S. thanks the logistic support by the Applied Materials and Nanotechnology, Center of Excellence, Miskolc University, Miskolc, Hungary. The work in the Y.B. lab was supported by the Barenholz Fund. This fund was established with a portion of Barenholz royalties, which the Hebrew University assigned to support research in the Barenholz lab, including this study.
© 2023 The Authors. Published by American Chemical Society.
- Doxil liposomes
- atomic force microscopy
- cryo-electron microscopy
- dynamic light scattering
- lipid nanoparticles
- phospholipid membranes