Date

Tuesday 18 Mar 2025

Time

15:15 - 15:45

Location

EM117

Supervisor

Sander van Kasteren

2nd reviewer

Kim Bonger

Jury

Zach Armstrong

The potential of mRNA vaccines has been demonstrated during the SARS-CoV-2 pandemic. By delivering mRNA encapsulated in lipid nanoparticles (LNPs) to cells, the expression of a desired antigen is induced, triggering an antigen-specific immune response with broad therapeutic applications. However, the mechanisms underlying mRNA delivery to the cytosol, also known as the endosomal escape, and the involvement of different immune and non-immune cells in LNP-mediated immune response remain incompletely understood, limiting the full potential of this technology.
In this study, we established a method for the intracellular detection and visualization of four distinct LNP components. The components include cholesterol, the ionizable lipids SM102, mRNA, and the fluorescent protein encoded by the mRNA. By incorporating click handles into cholesterol, SM102 and the mRNA we were able to formulate LNPs with minimal impact on LNP characteristics, allowing independent detection of these three components together with the expression of the mRNA-encoded fluorescent protein using both flow cytometry and confocal microscopy. We called these LNPs quadruple labelled LNPs. To achieve this, we determined the optimal ratio cholesterol containing a cyclopropene click handle (CpChol) to cholesterol in LNPs, ensuring minimal impact of incorporation of click handles on LNP functionality. Based on this and previous findings it was established that the ideal LNP composition for the intracellular detection of four labels consists of the cholesterol fraction in the LNPs fully substituted with CpChol and 50% of the SM102 fraction substituted with SM102-alkyne, where alkyne serves as click handle. Further analysis of the flow cytometry data revealed positive correlations between the presence of mRNA or ionizable lipid and the expression of mScarlet3. However, cells with high protein expression, appear to have lower levels of cholesterol, suggesting that mRNA and ionizable lipids may be transported between cells via extra cellular vesicles (EV’s). Additionally, confocal microscopy imaging showed that in DC2.4 cells, cholesterol and RNA are distributed in punctate structures after 24 hours of incubation with quadruple labelled LNPs, whereas fluorescent protein and ionizable lipids showed a more uniform intracellular distribution. In conclusion, we believe that our method of intracellular detection of distinct mRNA-LNP components provides a valuable tool for investigating the mechanisms behind the endosomal escape and the role of various immune and non-immune cells in LNP-mediated immune response. These findings could contribute to the optimization of RNA vaccine technology, enhancing their therapeutic potential.