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

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

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number4348
JournalScientific reports
Issue number1
StatePublished - Dec 2024


  • CARS
  • FLIM
  • Lipid nanoparticles
  • Self-amplifying mRNA

ASJC Scopus subject areas

  • General


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