Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging

Abstract The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong...

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Main Authors: Kajari Bera, Renán A. Rojas-Gómez, Prabuddha Mukherjee, Corey E. Snyder, Edita Aksamitiene, Aneesh Alex, Darold R. Spillman, Marina Marjanovic, Ahmed Shabana, Russell Johnson, Steve R. Hood, Stephen A. Boppart
Format: Article
Language:English
Published: Nature Portfolio 2024-02-01
Series:Scientific Reports
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Online Access:https://doi.org/10.1038/s41598-024-54697-3
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author Kajari Bera
Renán A. Rojas-Gómez
Prabuddha Mukherjee
Corey E. Snyder
Edita Aksamitiene
Aneesh Alex
Darold R. Spillman
Marina Marjanovic
Ahmed Shabana
Russell Johnson
Steve R. Hood
Stephen A. Boppart
author_facet Kajari Bera
Renán A. Rojas-Gómez
Prabuddha Mukherjee
Corey E. Snyder
Edita Aksamitiene
Aneesh Alex
Darold R. Spillman
Marina Marjanovic
Ahmed Shabana
Russell Johnson
Steve R. Hood
Stephen A. Boppart
author_sort Kajari Bera
collection DOAJ
description Abstract The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.
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spelling doaj.art-9fc1328809a0480cb6006ae1bf4fb0492024-03-05T19:03:29ZengNature PortfolioScientific Reports2045-23222024-02-0114111310.1038/s41598-024-54697-3Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imagingKajari Bera0Renán A. Rojas-Gómez1Prabuddha Mukherjee2Corey E. Snyder3Edita Aksamitiene4Aneesh Alex5Darold R. Spillman6Marina Marjanovic7Ahmed Shabana8Russell Johnson9Steve R. Hood10Stephen A. Boppart11GSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Vaccines, Rockville Center for Vaccines ResearchGSK Vaccines, Rockville Center for Vaccines ResearchGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignGSK Center for Optical Molecular Imaging, University of Illinois Urbana-ChampaignAbstract The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.https://doi.org/10.1038/s41598-024-54697-3Self-amplifying mRNACARSFLIMLipid nanoparticles
spellingShingle Kajari Bera
Renán A. Rojas-Gómez
Prabuddha Mukherjee
Corey E. Snyder
Edita Aksamitiene
Aneesh Alex
Darold R. Spillman
Marina Marjanovic
Ahmed Shabana
Russell Johnson
Steve R. Hood
Stephen A. Boppart
Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging
Scientific Reports
Self-amplifying mRNA
CARS
FLIM
Lipid nanoparticles
title Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging
title_full Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging
title_fullStr Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging
title_full_unstemmed Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging
title_short Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging
title_sort probing delivery of a lipid nanoparticle encapsulated self amplifying mrna vaccine using coherent raman microscopy and multiphoton imaging
topic Self-amplifying mRNA
CARS
FLIM
Lipid nanoparticles
url https://doi.org/10.1038/s41598-024-54697-3
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