Mechanistic Studies of an Automated Lipid Nanoparticle Reveal Critical Pharmaceutical Properties Associated with Enhanced mRNA Functional Delivery In Vitro and In Vivo

Abstract

Recently, lipid nanoparticles (LNPs) have attracted attention due to their emergent use for COVID‐19 mRNA vaccines. The success of LNPs can be attributed to ionizable lipids, which enable functional intracellular delivery. Previously, the authors established an automated high‐throughput platform to screen ionizable lipids and identified that the LNPs generated using this automated technique show comparable or increased mRNA functional delivery in vitro as compared to LNPs prepared using traditional microfluidics techniques. In this study, the authors choose one benchmark lipid, DLin‐MC3‐DMA (MC3), and investigate whether the automated formulation technique can enhance mRNA functional delivery in vivo. Interestingly, a 4.5‐fold improvement in mRNA functional delivery in vivo by automated LNPs as compared to LNPs formulated by conventional microfluidics techniques, is observed. Mechanistic studies reveal that particles with large size accommodate more mRNA per LNP, possess more hydrophobic surface, are more hemolytic, bind a larger protein corona, and tend to accumulate more in macropinocytosomes, which may quantitatively benefit mRNA cytosolic delivery. These data suggest that mRNA loading per particle is a critical factor that accounts for the enhanced mRNA functional delivery of automated LNPs. These mechanistic findings provide valuable insight underlying the enhanced mRNA functional delivery to accelerate future mRNA LNP product development.

Spectroscopy-Based Local Modeling Method for High-Throughput Quantification of Nucleic Acid Loading in Lipid Nanoparticles

“…Theoretically, an N/P ratio near or slightly lower than 1 (charge balance point) would ensure relatively high loading capacity while saving cargo inputs by reducing the unloaded concentrations. However, determination of the optimal formulation condition requires more evaluations of other formulation attributes and downstream efficacy outcomes. − Here, we demonstrated the LWR model can robustly predict % loading of nucleic acid cargoes in LNPs and rank their loading capacity in the 1st-tier HTS scenario, facilitating a facile and automated in-line quality assessment of LNP formulations.…”

Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach

“…This was expected as the number of full-length transcripts per particle followed the same trend, and mRNA loading per particle was previously shown to be a critical factor for enhanced mRNA functional delivery of LNPs (Supplementary Table 4). Of particular interest, protein expression was positively correlated with IL-6 cytokine release but negatively correlated with IL-18 level (Figure D,E). For instance, toll-like receptor 4 (TLR4) activation, potentially related with IL-18 release, can explain the reduced potency as it is known to block RNA translation and LNP uptake in cells. , In addition, the activation of these proinflammatory cytokines was not correlated with RNA integrity level, showcasing cellular activation likely originated from LNP properties or lipid-RNA interactions.…”

In vivo fate and intracellular trafficking of vaccine delivery systems

“…The size of LNP used for mRNA delivery is known to affect the pathway by which it enters the cell. In an in vitro experiment with 300-400 nm lipid nanoparticles, the presence of the micropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride’, was found to decrease the expression of the loaded mRNA, whereas the clathrin inhibitor, Pitstop2, had no such effect [145] . The size dependence might be due to the size of the clathrin-coated endosomal vesicles, which is reported to be around 85-100 nm [138] .…”