Acacetin-incorporated lipid nanoparticles for inhaled mRNA vaccines

Inhaled mRNA vaccines have shown great promise for the prevention of respiratory diseases. However, their broad application is still restricted by lipid nanoparticle (LNP)-induced oxidative stress, rapid clearance of LNPs from the lung, and limited mRNA expression. Here, we show that incorporating a natural compound, acacetin, into the LNP formulation can overcome these delivery barriers and greatly improve inhaled vaccine effectiveness. A natural compound library containing 242 compounds was used in an in vitro screening experiment, and acacetin was identified as the lead compound that substantially increased LNP-mediated mRNA expression in A549 cells. We then incorporated acacetin in LNPs as a fifth component. After optimizing the formulation of LNPs in in vitro transfection assays and in vivo pulmonary delivery assays, we find acacetin-incorporated LNPs increased lung transfection by 10.82-fold. A mechanistic study showed that acacetin reshaped the intracellular environment of lung cells by activating antioxidant stress pathways and suppressing pro-inflammatory signaling, thereby maintaining high levels of mRNA translation. Inhaled administration of acacetin-incorporated LNPs, delivering mRNA encoding the SARS-CoV-2 spike protein, induced higher antigen-specific IgG (14.95-fold) and IgA (2.38-fold) levels than a conventional LNP formulation, without inducing toxicity. We demonstrate that acacetin-incorporated LNPs offer a simple and scalable strategy to improve the efficacy of inhaled mRNA vaccines.