As an advanced nucleic acid therapeutical modality, mRNA can express any type of protein in principle and thus holds great potential to prevent and treat various diseases. Despite the success in COVID-19 mRNA vaccines, direct local delivery of mRNA into the lung by inhalation would greatly reinforce the treatment of pulmonary pathogens and diseases. Herein, we developed lipid nanoparticles (LNPs) from degradable ionizable glycerolipids for potent pulmonary mRNA delivery via nebulization. A panel of proprietary ionizable glycerolipids with branched tails and five ester bonds were developed through a three-step esterification reaction, and LNPs formed from a lead glycerolipid identified as TG4C enabled highly efficient in vivo mRNA delivery via systemic administration with around 6-fold higher luciferase protein expression than commercial LNPs from SM102 and Dlin-MC3-DMA (MC3). Formulation screening revealed that LNPs formed at a TG4C:DOPE:cholesterol:DMG-PEG molar ratio of 50:10:38.5:1.5 (TG4C-LNPs4) had high stability against nebulization with slight changes of size distribution and mRNA encapsulation efficiency, and the nebulized TG4C-LNPs4 afforded an equivalent percentage of positive cells and a slightly lower EGFP fluorescence intensity in lung cell lines (A549, BEAS-2B). Following pulmonary delivery in mice, TG4C-LNPs4 induced efficient transfection in the majority of epithelial cells in the lung, leading to apparent bioluminescence evenly distributed in all five lung lobes. In an elastase-induced emphysema model in mice, TG4C-LNPs4 loaded with mRNA encoding hepatocyte growth factor could significantly suppress the secretion of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in bronchoalveolar lavage fluid and counteract the alveoli wall thinning. Notably, partial substitution (25%) of cholesterol with budesonide, an anti-inflammatory glucocorticoid, in TG4C-LNPs4 generated equivalent protein expression and significantly improved therapeutic efficacy. Taken together, our study provides robust and high-performing nanovehicles based on degradable ionizable glycerolipids, enabling potently local mRNA delivery to the lung for the treatment of pulmonary diseases.
