Fig. 5 ∣. Design rules and optimized LNPs for therapeutic nebulized mRNA delivery.
a, LNP delivery to the lung can be improved by carefully selecting the type and molar amount of PEG-lipid. b, NLD1 was designed with a cationic lipid and high amount of PEG-lipid. c, NLD1 forms small, stable LNPs over time, average ± s.t.d., n = 3 replicates per group. d, NLD1 delivers more mRNA to lungs at low doses in vivo than leading LNPs previously optimized for systemic mRNA delivery. ***P < 0.0007, one-way ANOVA, average ± s.e.m., n = 5 mice per group, biological replicates shown. e,f, A total of 547 inflammatory genes were examined after exposure to NLD1. NLD1 was well tolerated in vivo (e) as compared to an LPS control (f). Each grey or purple dot represents a gene, and light grey dotted vertical lines indicate >2-fold change in gene expression. The dotted horizontal line indicates a P value of 0.05. g,h, NLD1 carrying AncNanoLuc was administered at a dose of 20 μg per mouse. Protein expression was quantified (g) and imaged (h) over several days. ****P < 0.0001, *P = 0.021, one-way ANOVA, average ± s.e.m., n = 3 mice per group, biological replicates shown. i, NLD1 therapeutic treatment regimen for H1N1 study. Mice were treated with two NLD1 treatments (total dose, 100 μg per mouse) before administering 1.5× LD50 dose of PR8 virus. j,k, Mouse weights were taken daily for up to 14 d post infection. All mice treated with NLD1 recovered on the basis of weights. Out of 6 control mice treated with H1N1, 5 mice lost 15% or more of body weight and were euthanized. n = 6 mice per group, biological replicates shown.