Effect of Lipid Composition on RNA-Lipid Nanoparticle Properties and Their Sensitivity to Thin-Film Freezing and Drying

A library of 16 lipid nanoparticle (LNP) formulations with orthogonally varying lipid molar ratios was designed and synthesized, using polyadenylic acid [poly(A)] as a model for mRNA, to explore the effect of lipid composition in LNPs on (i) the initial size of the resultant LNPs and encapsulation efficiency of RNA and (ii) the sensitivity of the LNPs to various conditions including cold storage, freezing (slow vs. rapid) and thawing, and drying. Least Absolute Shrinkage and Selection Operator (LASSO) regression was employed to identify the optimal lipid molar ratios and interactions that favorably affect the physical properties of the LNPs and enhance their stability in various stress conditions. LNPs exhibited distinct responses under each stress condition, highlighting the effect of lipid molar ratios and lipid interactions on the LNP physical properties and stability. We then demonstrated the feasibility of using thin-film freeze-drying to convert poly(A)-LNPs from liquid dispersions to dry powders while maintaining the integrity of the LNPs. Importantly, the residual moisture content in LNP dry powders significantly affected the LNP integrity.Residual moisture content of ≤0.5% or >3-3.5% w/w negatively affected the LNP size and/or RNA encapsulation efficiency, depending on the LNP composition. Finally, we showed that the thin-film freeze-dried LNP powders have desirable aerosol properties for potential pulmonary delivery. We conclude that Design of Experiments can be applied to identify mRNA-LNP formulations with the desired physical properties and stability profiles. Additionally, optimizing the residual moisture content in mRNA-LNP dry powders during freeze-drying is crucial to maintain the physical properties of the LNPs.

Aerosolization; Dry powder; Freeze-drying; Moisture content; Stability; mRNA lipid nanoparticles.