Morphological Characterization of Self-Amplifying mRNA Lipid Nanoparticles

Jacob L. Thelen, Wellington Leite, Volker S. Urban, Hugh M. O’Neill, Alexander V. Grishaev, Joseph E. Curtis, Susan Krueger, Maria Monica Castellanos

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

The mRNA technology has emerged as a rapid modality to develop vaccines during pandemic situations with the potential to protect against endemic diseases. The success of mRNA in producing an antigen is dependent on the ability to deliver mRNA to the cells using a vehicle, which typically consists of a lipid nanoparticle (LNP). Self-amplifying mRNA (SAM) is a synthetic mRNA platform that, besides encoding for the antigen of interest, includes the replication machinery for mRNA amplification in the cells. Thus, SAM can generate many antigen encoding mRNA copies and prolong expression of the antigen with lower doses than those required for conventional mRNA. This work describes the morphology of LNPs containing encapsulated SAM (SAM LNPs), with SAM being three to four times larger than conventional mRNA. We show evidence that SAM changes its conformational structure when encapsulated in LNPs, becoming more compact than the free SAM form. A characteristic “bleb” structure is observed in SAM LNPs, which consists of a lipid-rich core and an aqueous RNA-rich core, both surrounded by a DSPC-rich lipid shell. We used SANS and SAXS data to confirm that the prevalent morphology of the LNP consists of two-core compartments where components are heterogeneously distributed between the two cores and the shell. A capped cylinder core-shell model with two interior compartments was built to capture the overall morphology of the LNP. These findings provide evidence that bleb two-compartment structures can be a representative morphology in SAM LNPs and highlight the need for additional studies that elucidate the role of spherical and bleb morphologies, their mechanisms of formation, and the parameters that lead to a particular morphology for a rational design of LNPs for mRNA delivery.

Original languageEnglish
Pages (from-to)1464-1476
Number of pages13
JournalACS Nano
Volume18
Issue number2
DOIs
StatePublished - Jan 16 2024

Funding

This work was done under a cooperative research and development agreement between GlaxoSmithKline Biologicals SA and the National Institute of Standards and Technology, and a nonproprietary user agreement between UT–Battelle and Corixa Corporation. J.L.T. and M.M.C. thank the Scientific Leaders Program at GSK for sponsoring this work. The authors thank C. Lowry for performing a data integrity check of the GSK data generated and compiling the results of Table 1. The authors acknowledge Nanoimaging Services for collecting the cryo-EM data. Neutron scattering experiments on Bio-SANS were supported by the Center for Structural Molecular Biology funded by the Office of Biological & Environmental Research in the Department of Energy (DOE) Office of Science project ERKP291. This research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory. This work was done under a cooperative research and development agreement between GlaxoSmithKline Biologicals SA and the National Institute of Standards and Technology, and a nonproprietary user agreement between UT-Battelle and Corixa Corporation. J.L.T. and M.M.C. thank the Scientific Leaders Program at GSK for sponsoring this work. The authors thank C. Lowry for performing a data integrity check of the GSK data generated and compiling the results of Table 1. The authors acknowledge Nanoimaging Services for collecting the cryo-EM data. Neutron scattering experiments on Bio-SANS were supported by the Center for Structural Molecular Biology funded by the Office of Biological & Environmental Research in the Department of Energy (DOE) Office of Science project ERKP291. This research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory.

FundersFunder number
Battelle and Corixa Corporation
Corixa Corporation
U.S. Department of Energy
National Institute of Standards and Technology
GlaxoSmithKline
Office of ScienceERKP291
Biological and Environmental Research
Oak Ridge National Laboratory
UT-Battelle

    Keywords

    • lipid nanoparticles
    • mRNA vaccines
    • morphology
    • scattering
    • self-amplifying mRNA

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