Supplementary Materials abb4005_SM. T-Au-NP (His-tagged Au nanoparticles conjugated to NTA-NP and covered with iRGD). (G) Transfection performance of CNE-2 cells with T-CC-NPs or NT-CC-NPs. Mean SD (= 3). *** 0.001. (H) Confocal laser beam scanning microscopy (CLSM) of one CNE-2 cells transfected with T-CC-NPs or NT-CC-NPs. Nuclei (blue) had been stained using Hoechst 33324; lysosomes (crimson) were labeled with LysoTracker Reddish; Ce6 emitted reddish fluorescence. Scale bar, 10 m. (I) Circulation cytometry of cells in (B) and (C). (J) Biodistribution of T-CD-NPs and NT-CD-NPs [made up of the dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) rather than Ce6] HNRNPA1L2 in CNE-2 xenograft mice. (K) Quantification of DiR fluorescence in (E) (mean SD, = 3). eGFP protein was fused with Cas9 to its tumor cell uptake. MFI, mean fluorescence intensity. The cationic copolymer iRGD-PD was then used to neutralize the unfavorable charge of the CC-NPs and to expose the tumor-targeting ligand iRGD. The zeta potential and size of the iRGD-PDCcoated CC-NPs (T-CC-NPs) increased with the amount of iRGD-PD used (Fig. 2D). At 15 g of iRGD-PD, T-CC-NPs showed a lower unfavorable charge (?3 mV) and a smaller size (110 nm) than at 15 g of iRGD-PD (favorable for cell uptake and long blood circulation); thus, 15 g of iRGD-PD was selected for subsequent experiments. The nanoparticles were examined using transmission electronic microscopy (TEM). NTA-Ce6-NPs showed a core-shell structure with a diameter of ~65 nm. Binding of Cas9/sgRNA to form CC-NPs increased the radius by ~10 nm, roughly the diameter of Cas9 RNP (= 3). (I) TEM images and (J) dynamic light scattering (DLS) analysis of T-CC-NPs with or without 10 mM GSH. Level bar, 100 nm. To confirm that Ce6 was generating ROS in Protosappanin A response to NIR irradiation, we compared the ROS levels in cells with or without nanoparticles and NIR irradiation. Most (86.8%) cells treated with targeting T-Ce6-NPs plus NIR irradiation (T-Ce6/NIR) produced ROS; only 35.8% of cells treated with nontargeting NT-Ce6-NPs plus NIR (NT-Ce6/NIR) produced ROS (Fig. 3D); very few cells (0.41 and 0.11%) produced ROS when treated with T-Ce6-NPs without NIR irradiation or with NIR irradiation alone. In confocal laser scanning microscopy (CLSM) fluorescence imaging, we observed that ROS level appeared to be closely related to the intracellular Ce6 level (Fig. 3E). Cas9 RNP was degraded if caught in lysosomes for 24 hours but escaped from lysosomes in the presence of NIR-irradiated Ce6 (fig. S5I). These results indicated that treatment of cells with iRGD-modified Ce6-made up of nanoparticles and NIR irradiation was required for ROS production. Upon Ce6/ROS-induced lysosomal escape, Cas9 RNP must be released from nanoparticles to perform gene editing. The breakage of a disulfide bond between NTA and PEG (Fig. 3F) allowed detachment of Cas9 RNP in response to the high glutathione (reduced form) (GSH) level in the cytoplasm. GSH concentration in cells treated with T-Ce6-NPs recovered Protosappanin A to 90% of the control cells without NIR irradiation at 1 hour after NIR irradiation (fig. S6A), which was enough to trigger the breakage of disulfide bonds (= 3). ** 0.01 and *** 0.001. (F) Western blot and (G) immunohistochemistry analyses of luciferase and (H) immunofluorescence analyses of Cas9 and Ce6 in tumor tissue sections from CNE-2 xenograft mice receiving several nanoparticles. Protosappanin A Luciferase proteins had been stained brown. Range pubs, 50 m..