Figure 2.

Preparation and characterization of unmodified LNP. (A) Schematic to explain microfluidics mixing for construction of LNPs, which are composed of an ionizable cationic lipid (DLin-MC3-DMA), helper lipids (cholesterol and DSPE), and DSPE-PEG₂₀₀₀. (B) Physicochemical characterization of LNP size, polydispersity index (PDI), and ζ-potential. mRNA encapsulation efficiency (EE) was determined by the RiboGreen assay. (C) Cryo-EM images of PolyA/LNP and mGFP/LNP. Scale bars are 100 nm. (D) Confocal images to show GFP expression, following LSEC incubation with 0.5 μg/mL PolyA/LNP or mGFP/LNP for 24 h. The images were obtained by staining nuclei and the surface membrane with Hoechst33342 and WGA594, respectively. Scale bar is 40 μm. (E) In vivo and ex vivo IVIS images following IV injection of 1 mg/kg DiR/mGFP/LNP. The ex vivo images of the explanted organs were used to show that most of particles are localized in the liver and spleen. The lower panel shows a digital fluorescence scanning view of a liver slice to show the sinusoidal distribution of GFP expression in relation to the more scattered distribution of Kupffer cells. GFP staining was performed with anti-GFP antibody (AF488), and Kupffer cells were detected using an anti-F4/80 antibody (AF647). Left scale bar, 500 μm; right scale bar, 200 μm, c.v., central vein (n = 3).