Mechanisms of Lipid Nanoparticle-Mediated mRNA Transport Across Lipid Membranes

Licentiate thesis, 2025

After cellular uptake, RNA-containing lipid nanoparticles (LNPs) are typically located within endosomes, membrane-delimited intracellular organelles. To exert their therapeutic effect, their RNA cargo must translocate across the endosomal membrane to reach the cytosolic translational machinery. This critical step, known as endosomal escape, remains a major bottleneck for effective delivery.

The work presented in this thesis investigates the mechanisms underlying LNP-mediated endosomal escape using a simplified model system designed to mimic key features of the endosomal environment, including an anionic supported lipid bilayer (SLB) and acidification. Time-resolved fluorescence microscopy demonstrated that acidification triggers fusion of LNPs with the anionic SLB, suggesting a potential pathway for mRNA release and translocation across the endosomal membrane. However, translocation across the SLB was not observed to be the dominant mechanism for mRNA release at fusion sites.

Following administration, such as by injection, LNPs inevitably interact with serum proteins, resulting in the formation of a protein corona that alters their physicochemical properties. Comparative experiments with pristine and serum-preincubated LNPs revealed that serum exposure promotes fusion at less acidic conditions, indicating that the protein corona does not inhibit fusion with anionic membranes. Nonetheless, serum incubation also led to partial mRNA loss from LNPs, which may compromise delivery efficiency.

Together, these findings provide deeper insight into the physicochemical processes that govern LNP-mediated mRNA delivery and highlight factors that can influence endosomal escape. This may inform the rational design of more effective LNP formulations for RNA-based therapeutics.