Acidification-Induced Structural Evolution Within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages

Lipid nanoparticles (LNPs) represent a revolutionary technology for mRNA delivery, offering potential in preventing and treating various diseases, including those addressed by the COVID-19 vaccines. Our recent research involves the development and characterization of LNP formulations using ionizable amino-lipids, including ALC-0315 and SM-102 from COVID-19 vaccines, along with innovative structure-forming helper lipids like monoolein and phytantriol. Using high-throughput small-angle X-ray scattering (SAXS) in Australian Synchrotron, we observed acidification-induced structural transitions in these ionizable LNPs from inverse micellar to hexagonal and cubic phases, within a pH range of 7 to 4, mimicking the endosomal environment. These transitions significantly enhanced the transfection efficiency in macrophages, with SM-102-based LNPs performing better due to their ability to form cubic structures at lower pH conditions.<br/><br/>Additionally, we explored how the components, such as cholesterol, stabilizers, and mRNA cargos, modulate these phase transitions and mRNA transfection. Notably, the cholesterol-enriched formulations contributed to nanostructure transitions and protein corona variations, enhancing transfection efficiency in both immortalized alveolar and primary macrophage cell lines. Our <i>ex vivo</i> studies confirmed the capacity of these LNPs in reprogramming macrophages, highlighting their potential for cell-based therapies in lung diseases.<br/><br/>Our findings elucidate the connection between LNP structural evolution and mRNA transfection efficiency, providing valuable insights for the design of next-generation LNP systems for therapeutic applications. This research underscores the importance of mesophase behavior in optimizing LNP formulations for targeted gene delivery and advancing the field of mRNA therapeutics.<br/><br/>References<br/>1. <u>Yu, H.</u>¹; Iscaro, J.¹; Dyett, B.¹; Zhang, Y.; Seibt, S.; Martinez, N.; White, J.; Drummond, C. J.; Bozinovski, S.; Zhai, J. Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with MRNA Transfection in Macrophages. <i>J. Am. Chem. Soc.</i> <b>2023</b>, <i>145</i> (45), 24765–24774. (SI cover)<br/>2. <u>Yu, H.</u>; Angelova, A.; Angelov, B.; Dyett, B.; Matthews, L.; Zhang, Y.; El Mohamad, M.; Cai, X.; Valimehr, S.; Drummond, C. J.; Zhai, J. Real–Time PH–Dependent Self–Assembly of Ionisable Lipids from COVID–19 Vaccines and In Situ Nucleic Acid Complexation. <i>Angew. Chemie</i> <b>2023</b>, <i>135</i> (35), e202304977.<br/>3. <u>Yu, H.</u>¹; Dyett, B.¹; Kirby, N.; Cai, X.; Mohamad, M. El; Bozinovski, S.; Drummond, C. J.; Zhai, J. PH–Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles. <i>Small</i> <b>2024</b>, <i>2309200</i>, 1–10. (Front cover)<br/>4. Iscaro, J.¹; <u>Yu, H.</u>¹; Martinez, N.; Subramaniam, S.; Joyce, P.; Wang H.; Dyett, B.; White, J.; Prestidge, C., Drummond, C. J.; Bozinovski, S.; Zhai, J. Lyotropic Liquid Crystalline Phase Nanostructure and Cholesterol Enhance Lipid Nanoparticle Mediated mRNA Transfection in Macrophages. <i>Adv. Funct. Mater.</i> <b>2024</b>, Accepted.