Figure 4.

Assessment of mannose-decorated LNP that deliver reporter-encoding GFP mRNA. The LNPs described in Figure 3 were used to perform these studies: (A, B) Flow cytometry to assess GFP expression in LSECs, exposed to 0.5 μg/mL mGFP/LNP-Man, with or without co-incubation with competitive binding PolyA/LNP-Man at ratios of 1:0, 1:1, and 1:2 for 24 h (n = 3). CD206 binding specificity was determined by including a blocking antibody. The statistically significant decline in GFP fluorescence as a result of binding competition was confirmed by assessing mean fluorescence intensity (MFI). (C, D) Flow cytometry to compare GFP fluorescence intensity in LSEC, following uptake of 0.25 and 0.5 μg/mL mGFP/LNP or mGFP/LNP-Man (n = 3) over 24 h. (E) In vivo and ex vivo IVIS imaging following IV injection of 1 mg/kg DiR-labeled nondecorated or mannose-decorated LNPs for 6 h (n = 3). Particles were synthesized as in Figure 3, with composition shown in Figure S5. (F) Average DiR radiant efficiency analysis of the main organs (n = 3). (G) Representative digital fluorescence scanning images to compare the intrahepatic distribution of DiR/mGFP/LNP and DiR/mGFP/LNP-Man. Nuclei were stained with DAPI, while GFP and LSEC were detected by anti-GFP (AF488) and anti-LYVE1 antibody (AF594), respectively. Scale bar is 200 μm. (H) Relative GFP fluorescence intensity compared using ImageJ analysis. (I) Fluorescence overlap to determine colocalization of GFP with LSEC, using ImageJ analysis (n = 3); N.S., no significant difference; * p < 0.05; ** p < 0.01; and *** p < 0.001.