Fig. 4. Validation of lead LNP formulations for mRNA delivery to the lungs of female mice.

a Four lead LNP formulations were discovered from a 180-CAD lipid library after high-throughput in vitro and in vivo screening. b Whole body and ex vivo imaging of luciferase expression mediated by LNP-CAD3, 4, 9, and 10 at 6 h post-injection (0.1 mg kg⁻¹ FLuc mRNA, n = 3 mice). H: heart, Li: liver, S: spleen, Lu: lung, K: kidney. Quantification of luciferase expression in the lungs (c), liver (d), and spleen (e) using region-of-interest (ROI) analysis. f Relative luciferase expression in each measured organ. g Ai14 mice were treated with LNP-CAD9 or MC3/DOTAP LNP encapsulating Cre mRNA 3 days prior to analysis (0.3 mg kg⁻¹, n = 4 mice). Lungs were digested and stained for quantifying cell populations for tdTomato expression. PBS was injected as negative control. I.V.: intravenous. h Proportion of tdTomato⁺ cells in the lung assessed by flow cytometry. i Representative immunostaining demonstrating signal overlap between tdTomato⁺ cells and the endothelial cell marker platelet endothelial cell adhesion molecule 1 (PECAM1). DAPI was used for nuclear staining. Scale bars: 100 µm for the lung section images and 30 µm for enlarged images. g Created with BioRender.com. Statistical significance in (h) was calculated using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test. **P < 0.01; ***P < 0.001. Data are presented as mean ± s.e.m. Source data are provided in the Source Data file.