Fluorescence-Based Stopped Flow Analysis of mRNA-Lnp Degradation Kinetics

Lipid nanoparticles (LNPs) are highly effective vessels to safely deliver nucleic acids into the body and have become increasingly prevalent over the last two years as researchers have been rapidly developing and testing new mRNA vaccines to combat COVID-19. As vaccine manufacturing scales up, it is becoming increasingly important to have a method for quickly analyzing the quality of mRNA-LNPs. Current techniques (e.g., HPLC, gel electrophoresis) are time-intensive, expensive, and less effective at certain RNA lengths. To resolve these issues, the Schneider lab has pioneered the use of wormlike micelle buffers (WMBs) in capillary electrophoresis (CE) to analyze mRNA-LNPs. In order to establish CE as a trusted LNP stability assay, secondary tests are needed to corroborate its results.

This project used a stopped-flow device in conjunction with fluorescence spectrometry to obtain accurate kinetic parameters for LNP degradation. mRNA-LNPs were formed by mixing mRNA with a combination of lipids (ionizable lipid, cholesterol, lipid-anchored PEG, and a helper lipid). The mRNA-LNP solution was then combined with a buffer solution containing surfactant, which solubilizes the LNPs, and RiboGreen, a fluorescent dye which stains the mRNA upon their release from the LNPs. These solutions were mixed within a stopped flow apparatus inside a UV-Vis spectrophotometer, which allowed for measurement of fluorescence changes in real time as the LNPs were rapidly solubilized. This experiment was performed with two different LNP formulations (with and without PEG), two different surfactants (Triton X-100 and 12/8/1/0 [a wormlike micelle buffer]), and various surfactant concentrations.

Each experiment produced a fluorescence vs time curve which was linearized via a logarithmic transformation, from which three distinct kinetic constants could be determined. The time for complete LNP degradation was 10-20 seconds for all experiments, which is roughly the same time frame as is seen in similar CE-based stability assays. Comparing the average first kinetic constant (k1) from each set of experiments, no significant difference was found between the normal and modified LNPs when mixed with the 12/8/1/0 buffer. However, in the Triton X-100 experiments, k1 was found to be significantly lower for the modified PEG-less LNPs than for the normal LNPs, which makes sense based on the role of PEG in LNP formation. Overall, we can conclude that stopped flow fluorescence spectroscopy can be used as a quick, effective assay of mRNA-LNP stability and degradation time.