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. 2023 Jul 3;145(28):15171–15179. doi: 10.1021/jacs.3c01902

Figure 4.

Figure 4

(A) Effect of dilution on the nanocarrier size determined by DLS; value = 0 indicates “not detectable”. (B) Nanocarrier stability against different concentrations of heparin (0, 0.25, 0.5, 1, 2.5, and 5 IU/μg sgRNA). Ribogreen was used for the detection of free RNA. (C) Plot of median fluorescence intensities (MFI) of ATTO647N-Cas9 protein and ATTO488-sgRNA determined by flow cytometry versus xenopeptide logD7.4 values of each xenopeptide series. (D) Endocytosis pathway study with different inhibitors. Sodium azide: energy-dependent endocytosis; chlorpromazine: clathrin-mediated endocytosis; sucrose: clathrin-mediated endocytosis; nystatin: caveolae-mediated endocytosis; amiloride: macropinocytosis. Data are presented as mean ± SD (n = 3). Statistical analysis was performed by comparing each treatment group with the corresponding “no inhibitor” group. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. (E) Confocal laser scanning microscopy (CLSM) images of HeLa WT cells 4 h after treatment with the selected Cas9 RNP nanocarriers (75 nM RNP) containing 20% of ATTO647N-Cas9 and 20% ATTO488-sgRNA. Nuclei were stained with DAPI (blue). The merged channel indicates co-localization (yellow) of ATTO647N-Cas9 (red) and ATTO488-sgRNA (green). (F) CLSM images of HeLa mRuby3/gal8 cells treated with the selected Cas9 RNP nanocarriers (75 and 5 nM RNP) for 4 h. Nuclei were stained with DAPI (blue). Red punctuate mRuby3/gal8 spots indicate endosomal membrane damage.