Enhanced Pulmonary and Splenic mRNA Delivery Using DOTAP-Incorporated Poly(β-Amino Ester)-Lipid Nanoparticles.

mRNA-based therapies hold tremendous promise for treating various diseases, yet their clinical success is hindered by delivery challenges. This study developed a library of 140 lipocationic Poly(β-amino ester)s (PBAEs) and formulated lipid-polymer hybrid nanoparticles (LPHs) with four helper lipids, including 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), to enhance mRNA delivery. Initial in vitro screening of four representative PBAEs identified the D/P4-1 formulation (DOTAP/PBAE molar ratio of 4:1) as the most effective. Further screening of the library using this formulation identified eight top-performing LPHs. In vivo experiments confirmed high luciferase expression in the spleen and lungs of mice following intravenous administration of Luc mRNA-loaded LPHs. Detailed analysis revealed that DOTAP incorporation influenced LPH properties, including apparent pK_a, surface charge, and internal hydrophobicity, enabling enhanced mRNA release and cellular uptake. This study demonstrates potent approaches to modulate PBAE-lipid nanoparticle properties by altering PBAE structures and nanoparticle composition, offering insights for designing effective hybrid carriers for mRNA therapeutics.