The excellent light scattering sensitivity of NanoFCM enables the identification of all lipid nanoparticles, based on the concurrent fluorescent detection, further evaluation of loading fraction and encapsulation efficiency can be achieved by nucleic acid staining.
After labeling with cell-permeant nucleic acid stain, some fluorescent signals that are not associated with corresponding side scatter signals are observed, and the signal intensities (FL Intensity) are generally lower than the fluorescent signals that are associated with side scatter signals. These fluorescence signals are further confirmed to attribute to single free mRNAs by employing the pure mRNA molecules as control (data not shown). By choosing FL trigger, the free mRNA and mRNA encapusulated LNPs are shown in the dotplot. The mRNA copy number of each LNP can be achived by dividing the fluorescence intensities of each LNP by the intensity of free mRNA. The copy number ranges from 1 to around 100, with median value equals to 12. The encapsulation efficiency of mRNA can then be validated, which is 80.5% in this case.
The mRNA LNP formulation mainly contains three components: mRNA encapsulated LNPs (P1), empty LNPs (P2), and free mRNA (P3). When labeled with cell-permeant nucleic acid dye, and through SS-FL dual trigger, the three components can be displayed and well resolved in the SS-FL dotplot. After treatment with nuclease, the free mRNA population disappeared, while the ratio of empty LNPs increased, which indicates the attachment of mRNA on the surface of some LNPs. This is further verified by labeling with cell-impermeant nucleic acid dye following nuclease treatment.
The table summarizes the mRNA localization assay through different labeling strategies followed by nuclease treatment, which can be referred to optimize the formulating strategy and downstream process. By doing so, maximizing the effective loading of mRNA to obtain mRNA LNPs with high purity is possible.