Lyotropic Liquid Crystalline Phase Nanostructure and Cholesterol Enhance Lipid Nanoparticle Mediated mRNA Transfection in Macrophages

Macrophages are unique immune cells attracting growing attention as a potential candidate for cell‐based therapy for infectious diseases and cancer. Strategies that can reprogramme or gene‐edit macrophages hold potential across a spectrum of acute and chronic conditions. Herein, lipid nanoparticles (LNPs) are developed containing the ionizable lipid SM‐102, helper lipid monoolein which is known for self‐assembly in aqueous solutions into the inverse cubic lyotropic liquid crystalline mesophase, and cholesterol as an mRNA nanocarrier. The immortalized alveolar macrophage cell line (MH‐S cells) is utilized to investigate how cholesterol concentration impacts on mRNA delivery which is further validated using primary mouse alveolar macrophages isolated from the bronchoalveolar compartment and human monocyte derived macrophages. By using high‐throughput synchrotron small angle X‐ray scattering (SAXS), an acidification‐induced non‐ordered to ordered internal nanostructure transition of the formulated LNPs is observed, following the transition sequence of inverse micellar to hexagonal to cubic mesophase in the pH range from 7 to 4. Cholesterol is identified as another crucial component for superior mRNA transfection in macrophages, contributing to nanostructure transition and protein corona variation. Successful ex vivo mRNA transfection is also achieved in primary macrophages, highlighting the prospectivity of reprogramming macrophages as a cell therapy for lung diseases. This study highlights the critical roles of lipid nanostructure self‐assembly, notably the inverse cubic and hexagonal phases, and cholesterol in successful intracellular delivery and transfection of mRNAs to lung macrophages, advancing RNA technology for cell‐based therapy for lung diseases.