Fig. 2. Characterization, genome-editing activity, biodistribution and cellular uptake mechanism of exosomeRNP.
Characterization of purified exosome (A) and exosomeRNP complexes (E). (B) Biomarkers of exosome by western blotting. (C and D) DLS and TEM the image of purified exosome. The arrows show the typical exosome nanoparticles. (F and G) DLS and TEM image of exosomeRNP complexes. The arrows show the typical exosomeRNP nanoparticles. (H and I) Cytosolic delivery of Cas9-FITC into LX-2 (H) and Huh-7 (I) cells by exosomes for 4 hours. The red arrows point at the efficient translocation of RNP into the nuclei. Scale bars, 25 μm. DAPI, 4′,6-diamidino-2-phenylindole. (J) Exosome-mediated Cas9 RNP delivery for genome editing. (K) Frequency of PUMA indel mutation detected by T7E1 assay from AML-12 cells after the specified treatments. (L) Frequency of CcnE1 indel mutation detected by T7E1 assay from AML-12 cells after the specified treatments. (M) Frequency of KAT5 indel mutation detected by T7E1 assay from LX-2 cells after the specified treatments. (N) In vivo distribution of DiR-labeled exosomes in the whole mice (top) or in the dislodged organs from mice (bottom). H, heart; Lu, lung; Li, liver; K, kidney; S, spleen. (O) Schematic illustrating the procedure to isolate different hepatic cell types and determine exosomeRNP biodistribution. (P) Percentage of each hepatic cell type that is DiI-labeled exosomeRNP-positive. (Q) Relative MFI of each hepatic cell type. (R and S) Mechanism of cellular uptake of exosomeRNP nanocomplexes in LX-2 (R) and Huh-7 (S) cells by the addition of different inhibitors. The values are normalized to the control. Statistical significance was calculated by Students’ t test (mean ± SD, n = 3). **P < 0.01, ***P < 0.001, and ****P < 0.0001.