Fv10i

CNV lesion sizes were measured on RPE-choroid-sclera flat mounts. At 3,7, and 14 d after laser photocoagulation, mice were deeply anaesthetized and received intravenous injection of 0.5-ml phosphate-buffered saline (PBS) containing 20-mg/ml fluorescein isothiocyanate-dextran (MW = 2000 kDa, Sigma-Aldrich, St. Louis, MO, USA). Eyes were enucleated and fixed in 4% paraformaldehyde for 12 h. The entire retina was carefully dissected from the eyecup, and the eyecups were flat-mounted in Fluoromount™ (Diagnostic BioSystems, Pleasanton, CA, USA) with the RPE layer facing up. CNV lesions were observed with a fluorescence confocal microscope (FV10i, Olympus Tokyo, Japan; × 10 objective and × 3 digital zoom). The CNV-related neovascular areas were outlined and measured within the dotted line using imaging software OLYMPUS FLUOVIEW-ASW Version 02. 01 (Olympus) for FV10i with the operator that was unaware of treatment groups. Analysis included the total areas within the dotted lines.
Three days after laser treatment, the area of the POH-Rhodamine fluorescent region around CNV lesions in mice was also measured. Then 2 nmol of POH-Rhodamine was intravenously injected 6 h before FITC-dextran injection.

After treatment with various transcytosis inhibitors for 30 min, the cells were further incubated with coumarin-6 (in green)-loaded SUV-RF at 37 °C for 90 min. The cells were then rinsed using PBS. The images were taken using a confocal laser-scanning microscope (Olympus FV10i, Olympus America Inc., Center Valley, PA, USA) with excitation at 490 nm and emission at 520 nm. The cells were also stained with DAPI (in blue) in the nucleus for comparison. At least 3 photos were taken in each sample. The representative image of each treatment is exhibited.

Morphological changes in the nuclear chromatin of the apoptotic cells were identified by staining with DAPI. Cells were grown in 6-well plates at a density of 1 x 10⁵ cells per well for 48 h before treating with relevant drugs for 48 h. They were then washed with cold PBS, fixed with methanol for 30 min, rewashed and stained with 200 mL of DAPI solution (1 mg/mL) at 37°C for 30 min. After removing the staining solution, the apoptotic cells were visualized using fluorescence microscopy (Axiovert 200, ZEISS Inc., Germany and Olympus FV10i, Tokyo Inc., Japan).

For MSC detection, cells were transduced for 8 hours with a lentiviral vector (MOI 5) coding for GFP as previously reported^{75 (link)}. 72 hours after transduction, GFP expression was confirmed by confocal microscopy and flow cytometry (Supplementary Figure 2). GFP-MSCs were seeded in conventional 2D cultures or in 3D-spheroids for three days. After that, 2D cultured GFP-MSCs and GFP-MSC-spheroids were trypsinized and labeled with DiR tracer (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethyl indo tricarbocyanine Iodide, Thermo Fisher) according to supplier’s instructions. After labeling, cells were rinsed and slowly injected by the tail vein (1 × 10⁶ 2D-MSCs or 1 × 10⁶ MSC-spheroids in 300 μl of saline containing 10% rat serum). Control animals received 300 μl of vehicle. Twenty-four hours after injection, animals were anesthetized and perfused intracardially with 100 ml of 0.1 M PBS (pH 7.4), followed by 200 ml formalin solution. The brain, lungs, liver and kidneys were removed and DiR fluorescence was inspected in an In-Vivo MS FX PRO equipment, (In-Vivo Imaging Systems,Bruker, Ettlingen GE). For GFP detection, brain coronal sections (30 μm) were obtained, counterstained with 4,6 diamino-2-phenylindiol (DAPI, Invitrogen), and examined by confocal microscopy (Olympus-fv10i) at 10 and 60× magnification.

DiI (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Thermo Fisher, Waltham, MA, USA) was used as a lipophilic fluorescent probe of E, and miR-125 was labeled with FAM (carboxyl fluorescein; GenePharma). SAS cells were seeded overnight and incubated with DiI//LPN-KL or FAM-miR/SLN-KL for the indicated time. These cells were then fixed with 4% paraformaldehyde for 10 min and stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) at 37 °C to identify the nucleus. For DiI//LPN-KL intracellular localization, MitoTracker^® Green (MitoGreen), LysoTracker^® Green (LysoGreen), and an antibody against EGFR were added to monitor mitochondrial, lysosomal, and EGFR distribution. For the intracellular distribution of FAM-miR/SLN-KL (green), MitoTracker^® Red (MitoRed; Thermo Fisher), LysoTracker^® Red (LysoRed; Thermo Fisher), and an antibody against EGFR were used to detect mitochondrial, lysosomal, and EGFR localization. For both nanoparticle formulations, early endosomes were identified by immunofluorescence staining with an antibody against early endosome antigen 1 (EEA1) overnight. Images were obtained using a confocal laser scanning microscope (CLSM; Olympus FV10i; Olympus, Tokyo, Japan) with a 60× objective lens at a magnification of 1500×. The representative images are shown (n = 3).