In vivo distribution of LNPs in major organs and evaluation of in vivo stability and translation efficacy of its packaged mRNA. (A-B) Cellular uptake of DiR-loaded LNPs by DCs isolated from the liver, spleen, and lung of C57BL/6J mice 6 hours, 12 hours, and 24 hours after IV and IM injection. (C-E) The free F-Luc mRNA and F-Luc-mRNA loaded in LNPs were injected into C57BL/6J mice by the IV route to monitor the in vivo stability of mRNA and translation efficacy. Shown is the representative real-time live imaging of animals 6 hours after injection along with quantification of BLI for different treatment groups. (F-G) The in vivo tissue distribution of DiR-loaded LNPs was monitored in the liver, with quantification of liver accumulation of DiR dye at different time points, 6 hours, 12 hours, and 24 hours after IV administration of DiR-loaded LNPs. The data are shown as mean ± SD from at least 3 independent biological experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. BLI, bioluminescence imaging; DAPI, 4′,6-diamidino-2-phenylindole; DiR, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide.