PDF(Pubmed)
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.
摘要：
COVID-19大流行引发了合成RNA疫苗平台的复苏，可扩展，低成本制造，和治疗性疫苗的安全管理。自扩增mRNA（SAM），在传递到细胞质中时自我复制，导致强烈和持续的免疫反应。此类mRNA被包封在脂质纳米颗粒(LNP)内,所述脂质纳米颗粒充当用于递送至细胞质的载体。更好地理解LNP介导的SAM在不同类型细胞中的摄取和释放机制对于设计有效的疫苗至关重要。这里,我们使用高光谱相干反斯托克斯拉曼散射(HS-CARS)显微镜和多光子激发荧光寿命成像显微镜(FLIM)研究了婴儿仓鼠肾(BHK-21)细胞中SAM-LNP制剂的细胞摄取和随后的细胞内SAM表达.开发了基于HS-CARS和FLIM特征的细胞分类管道以获得与SAM-LNP摄取相关的光谱和代谢变化的见解。我们观察到LNP处理的细胞与PBS处理的细胞中HS-CARS模式的脂质强度升高，同时荧光图像显示在处理5小时内BHK-21细胞核和细胞质内的SAM表达。在一个单独的实验中，我们观察到SAM表达与结合的NAD(P)H群体的平均荧光寿命之间的强相关性。这项工作证明了多模态光学成像技术评估SAM-LNP的细胞摄取以及疫苗表达后细胞微环境中发生的后续变化的能力和意义。
