RESUMO
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.
Assuntos
Lipossomos , Nanopartículas , Vacinas de mRNA , Animais , Cricetinae , Humanos , Pandemias , Microscopia de FluorescênciaRESUMO
The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.