The kinetics of endosomal disruption reveal differences in lipid nanoparticle induced cellular toxicity

Lipid nanoparticles (LNPs) are widely used to deliver mRNA therapeutics and vaccines; but treatment-related inflammation can pose safety issues for applications requiring higher doses or repeat administration. The mechanisms underlying the toxicity of LNPs are not fully understood, so improved understanding of the intracellular pathways that sense LNP entry into the cell will facilitate LNP design for successful deployment across different therapeutic applications. Here we explored how experimental conditions can influence the uptake of LNP-delivered mRNA in vitro and modulate both the expression pattern of the cargo and cytotoxicity. We found that altering serum protein concentrations in culture media can influence cellular uptake of LNPs, resulting in expression patterns that were not dose-dependent. Additionally, changing or removing the mRNA cargo did not significantly affect cytotoxicity, indicating that this response was driven by the LNP components. Using a fluorescent Galectin 9 reporter system, kinetic experiments with different serum protein concentrations were conducted to measure LNP uptake, endosomal escape and cargo expression to systematically explore how these processes are related to cytotoxicity. We demonstrate that cytotoxicity is driven by both endosomal disruption and lipid chemistry. Our finding that the rate and magnitude of endosomal disruption correlates with cytotoxicity provide a new focus for investigation. Developing LNPs with optimal mRNA release mechanisms, and a reduced rate and duration of endosomal disruption, could alleviate some of the safety concerns associated with LNP/mRNA. Finally, we found that individual cationic lipid components can also influence cytotoxicity that is independent of cargo delivery, implying that at least some of the cytotoxicity can be mitigated with chemical design.