OBJECTIVE: To evaluate the efficiency and protein expression profiles of newly synthesized lipid nanoparticles (LNPs) as a nonviral vector gene delivery platform for gene editing of donor lungs for immunomodulation. METHODS: mCherry mRNA was formulated with 10 LNP candidates (5 ionizable lipids × 2 helper lipids), and gene delivery efficiency in cell lines was assessed using flow cytometry. The most promising LNP was optimized, and its protein expression was compared with that of adenoviral vectors (ADVs) using an mCherry enzyme-linked immunosorbent assay ELISA in a human precision-cut lung slice (PCLS) model. Additionally, intratracheal administration in a rat in vivo model was used to evaluate the relationship between LNP dose and protein expression using mCherry and human interleukin-10 (hIL-10) mRNA. RESULTS: An ionizable lipid ST-1 (formulated with 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) achieved high transfection rates of 91.3 ± 6.9% in human lung epithelial cells and 88.4 ± 6.3% in human monocytic cells. Reducing the DOTAP content from 39% to 7.8% further improved the transfection rate. Peak mCherry expression in human PCLS with ST-1 DOTAP (7.8%) occurred at 2 days postadministration, which was faster than with adenoviral vectors. Intratracheal administration of 500 μg/kg of ST-1 DOTAP (7.8%) with mCherry to rat left lungs caused significant lung injury; however, 50 μg/kg of hIL-10 mRNA mitigated the LNP-induced lung inflammation, achieving the target concentration of 59 pg/mg protein in the left lung. CONCLUSIONS: LNPs achieved rapid and efficient protein expression in vitro and in vivo that exceeded the performance of viral vectors; however, significant lung injury was observed with intratracheal LNP administration. This findings underscores the need to further optimize LNP-mediated gene delivery to the lung before translation to the clinic.