The use of mRNA as a therapeutic drug class is a safe and fast alternative to viral vector or plasmid DNA therapies. Nevertheless, free mRNA will be rapidly degraded after administration to the body and only reach the cytosol of desired cells with difficulty. Lipid nanoparticles (LNP) safely deliver mRNA to cells of interest and can be used in the treatment of different diseases. Dendritic cells are the primary antigen-presenting cells and important for mRNA vaccine delivery. Efforts to increase LNP transfection of these cells are necessary and can be achieved by different approaches. Here, we present apolipoprotein E4 addition to LNP administration as one mean of increasing LNP-mediated eGFP mRNA delivery to dendritic cells. We also show some steps in the preparation method for LNP optimization using MS2 RNA as a novel model nucleic acid.

Background/Objectives: Lipid nanoparticles (LNPs) are a prominent example of delivery systems that are used to prevent the degradation of messenger ribonucleic acid (mRNA) and facilitate cell uptake. Improving LNP transfection efficiency requires careful selection of key formulation components, including the ionizable lipid and the coding sequence of the nucleic acid. Therefore, it is crucial to assess various options for the target cells, as results can differ significantly between cell types. Building on previous work investigating the effect of apolipoprotein E4 on LNP transfection of human monocyte-derived dendritic cells, we assess the impact of different ionizable lipids and compare modifications in the mRNA uridine to further optimize the delivery to these cells. Methods: LNPs containing eGFP mRNA with different uridine modifications were produced via microfluidic mixing and investigated for their in vitro transfection efficiency of human monocyte-derived dendritic cells. Transfection occurred in the presence of apolipoprotein E4 for different encapsulated mRNA concentrations. Delta mean fluorescence intensity and eGFP positive cells were measured by flow cytometry 48 h after transfection. Cell viability was assessed via AnnexinV/7-AAD staining, after comparing this method to LIVE/DEAD^TM Fixable Near-IR staining. Results: This study shows that a combination of SM-102 as the ionizable lipid with eGFP mRNA containing N1-methylpseudouridine enabled the transfection of human monocyte-derived dendritic cells with very high efficiency at low concentrations, allowed for dose sparing, and even led to the LNPs outperforming a specifically tailored electroporation protocol. Conclusions: Improvement of nucleic acid delivery to human monocyte-derived dendritic cells, known for their difficulty to be transfected, was achieved by LNP formulation tuning.