Fig. 4: Overview of the second-round lipid virtual screening and experimental validation.

a Data representation of Model 2 predicting mRNA delivery efficiency. Compared to the representation method of Model 1, the positive criterion was set as 2-fold the delivery efficiency of the standard MC3 LNP. b External validation of Model 2, and associating Model 1 and 2 to screen the generated ionizable lipids. c The six lipids selected for experimental validation. d Female BALB/c mice (6–8 weeks old) were intravenously injected with LNPs loaded with luciferase mRNA at a dose of 5 μg per mouse, and at certain time points, total luminescence was detected after injection of D-luciferin. Time courses of the total flux of the screened six lipids. e The AUC of total luminescence of the screened six lipids. For MC3, SM-102, and other groups, n = 6, 5, 3, respectively. All data are presented as the mean ± SD. Statistical significance was analyzed by one-way ANOVA (ns, not significant; *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001. The P makers in black are results of the comparisons with MC3, and those in red are with SM-102. Source data are provided as a Source Data file). MC3 DLin-MC3-DMA, AUC area under curve, LNP lipid nanoparticle, SD standard deviation, ANOVA Analysis of Variance.