Figure 5.
In Vivo Spatially Targeted, Layer-Specific Delivery of MCO-II into Retina by CW Near-Infrared Laser Beam-Based NOD Using fGNRs with an SPR Peak-Matching NOD Laser Beam
(A) Schematic representation of in vivo NOD in rd10 mouse eye and subsequent steps to visualize the expression. (B and C) Fluorescence image of retina: (B) after intravitreal injection of fluorescence (Alexa Fluor 488) dye and (C) control (no dye injection). (D and E) Confocal fluorescence image of retina after intravitreal injection of fluorescence dye-labeled functionalized gold nanorods (fGNRs) with the surface plasmon resonance (SPR) peak at 1,064 nm: (D) ~1 h after injection and (E) overnight (>12 h) incubation. (F) Immunostained confocal image of NOD-treated mice retina, showing the expression of MCO-II-mCherry (red) overlaid on nuclear stain DAPI (in blue). (G and H) Higher magnification image of retinal cells: (G) DAPI and (H) mCherry. The laser dose was 600 mJ/mm2 (20 mW for 30 s, 1-mm2 area). (I) Chemiluminescent western blot of retinal cell extract after in vivo transfection of MCO-II-mCherry by NOD. The immunoprecipitated retinal lysate shows distinct mCherry. (J and K) Quantification of expression (measured by fluorescence intensity) in retina for different NOD laser doses: (J) with 1-h incubation of fGNRs and (K) overnight incubation of fGNRs. Mean + SD. Insets: representative fluorescence images of retina for control and NOD-transfected retinas. (L and M) Confocal fluorescence images of immunostained MCO-reporter (mCherry) in (L) RGC and (M) bipolar cell layers. (N) RGC layer-specific expression of MCO achieved by in vivo NOD (3D-stacked confocal image of retina).