(T1330-07-46) Evaluation of Next-Generation Lipids with Varying Chemistries for Enhancing the Endosomal Escape and Extrahepatic Delivery Potential of mRNA-LNP Therapeutics

Purpose: Lipid nanoparticles (LNPs) are being extensively explored to enhance the delivery and effectiveness of nucleic acid therapeutics. Formulation design, stability, and safety are key determinants for building a robust and efficient mRNA-LNP drug product. A major bottleneck in the field remains the endosomal escape of mRNA-LNPs, which is vital to achieve their successful intracellular delivery for execution of the encoded functions. Our preliminary evaluation included screening of several excipients to develop a platform LNP formulation for mRNA therapeutics. Importantly, the type and chemistry of lipid components are critical for determining the endosomal escape capability of the drug product. Given this, the main objective of this study was to screen next-generation lipids (phospholipids, sterols, and ionizable lipids) with different chemistries for improved potency and endosomal escape capability, as well as identify any potential for extrahepatic delivery (Figure 1).

Methods: A stepwise screening approach was executed for next-generation phospholipids (unsaturated lipid tails with different head groups), sterols (cholesterol analogs), and ionizable lipids with varying head groups (disulfide, imidazole, alkyne, alkyl amino alcohol, branched chain structure) at multiple N/P ratios to determine optimal formulations in terms of their in vitro physicochemical characterization and potency. To assess the drug product quality during formulation optimization, a panel of quality attributes was tested including particle size, polydispersity index (PDI), zeta potential, particle concentration, optical density (OD), visual inspection, encapsulation efficiency (EE%), TNS assay for pKa measurement, and in vitro transfection efficiency in HEK-293 cells.

Results: For the screening of phospholipids, four different types with variations in head groups and unsaturated lipid tails were evaluated, including PL1-PL4 alone as well as in combination (Combinations 1 and 2). Resultant Fluc mRNA-LNP formulations exhibited ≤100 nm particle size (Figure 2a) and ≥90% encapsulation efficiency (Figure 2b). All these formulations displayed unimodal size distribution, neutral surface charge, and particle concentration in the range of 10¹³-10¹⁴ particles/mg of mRNA (data not shown). Importantly, Combination 1 resulted in significantly higher in vitro transfection efficiency compared to the other formulations, with no cytotoxicity observed when tested in HEK-293 cells (Figure 2c). This optimized phospholipid combination was used to assess different cholesterol analogs (Analogs 1-4) with variations in the head and tail groups. Amongst these, cholesterol and its Analog 1 demonstrated acceptable particle size (80-90 nm), encapsulation efficiency ( >90%) and in vitro transfection efficiency that was higher than the other analog-containing Fluc mRNA-LNP formulations (Figure 2d-f). Given the higher stability of cholesterol containing LNPs when subjected to freeze-thaw cycles (data not shown), it was selected to screen various ionizable lipids with difference in the chemistry of their head groups (IL1-IL9). Each formulation was optimized to identify the optimal N/P ratio that yielded acceptable physicochemical properties (Figure 3a-b). Selected N/P ratios for each ionizable lipids were then evaluated for their in vitro potency in HEK-293 cells. Most of the tested formulations showed higher luciferase expression as compared to the standard MC3 ionizable lipid containing mRNA-LNPs (Figure 3c). None of the tested ionizable lipids were found to be toxic in HEK-293 cells, when treated at an mRNA dose of 10 ng per well. We also evaluated the pKa values for each of these LNP formulations using the TNS assay to find values ranging between 5.8-6.8, indicating the capability to escape early/late endosomes (pH 5-7) following potential in vivo administration (Figure 3d-e). In vivo biodistribution and endosomal escape assays are currently under evaluation for these optimized LNP formulations.

Conclusion: Taken together, the results of our research indicate that lipid components with varying chemistry have a significant effect on the physicochemical characterization and potency of mRNA-LNP formulations. Specifically, lipid components with different chemical head groups will be evaluated to understand the impact on endosomal escape and extrahepatic delivery potential of LNP formulations.


Figure 1. Schematic representation for screening of various next-generation lipids to enhance the endosomal escape and extrahepatic delivery potential of mRNA-LNPs. Varying chemistries of each lipid component were screened to understand the effect on in vitro characterization and potency of mRNA-LNPs, followed by potential in vivo evaluation.


Figure 2. Results obtained from screening of different phospholipid combinations and sterol analogs in mRNA-LNPs. Physiochemical properties of mRNA-LNPs containing next-generation phospholipid combinations including (a) Particle size, (b) Encapsulation efficiency, (c) In vitro transfection efficiency. Physiochemical properties of mRNA-LNPs containing sterol analogs in terms of (d) Particle size, (e) Encapsulation efficiency, and (f) In vitro transfection efficiency in HEK-293 cells. P-values indicate statistically significant differences in comparison to Lipofectamine (positive control). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


Figure 3. Results obtained from screening of various next-generation ionizable lipids in mRNA-LNPs in terms of (a) Particle size, (b) Encapsulation efficiency, (c) In vitro transfection efficiency tested at 10 ng mRNA dose. (d) TNS assay graphs and (e) Respective pKa values. P-values indicate statistically significant differences in the transfection efficiency of the tested formulations as compared to the MC3 formulation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.